in ultra high - university of toronto t-space...isolated using hi& performance liquid...
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IN VITRO DEGRADATION
OF ULTRA HIGH MOLECULAR WEIGHT POLYETaYLENE (UEMWPE)
BY OXIDATIVE AND/OR aYDROLYTIC PROCESSES
Angela Wai-Wai Lee
A thesis submitted in conforrnity with the requirements for the degree of
MASTlERS OF APPLIED SCIENCE
Graduate Department of Chemicai Engineering and Applied Chemistry
University of Toronto
O Copyright by Angela Wai-Wai Lee, 1998
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Inr V~mo DEGRADATION OF ULTRA EIGH MOLECULAR W E I G ~ POLYETHYLENE
(UHMWPE) BY O~~>ATIV]E AWOR HYDROLYTIC PROCESSES
MAS TER^ OF APPLED SCIENCE, 1998
ANGELA WAI-WAI LEE
UNIVERSITY OF TORONTO
~ s T R A c T
The Wear of UHMWPE implants generates polymer and metai particulates which can be
phagocytosed by human macrophages. This thesis focused on the study of polyethylene
particdate degradation by modeiing the oxidative and hydrolytic processes that are associated
with phagocytic activities, specifically H202 and cholesterol esterase. As well, synergistic effects
on UHMWPE degradation by metal contsiminants and H D 2 were examined. Oxidation was
measured using Fourier transform i n h e d spectroscopy (FTIR). Degradation products were
isolated using hi& performance liquid chromatography and identified using mass spectroscopy,
attenuated total reflectance FTIR and nuclear magnetic resonance. An initial assessrnent of the
effect of cobalt-treated HDPE particles on human monocyte/macrophage cytokine @-1 P, IL-6
and TNF-a) release was performed.
The chernical oxidation of LEIMWi?E particles was observed on particles that were pre-
sensitized using themal and y-irradiation treatments. Several oligomeric products, including
compounds that contained alkanes, alkenes and hydroxyls, were isolated. CeU viability studies
with particles showed increased necrosis with increased severity of environmentai oxidation
conditions. Cytokine data were inconclusive due to donor culture variability and very low values
after 48 hours of incubation.
This thesis was the accumulation of the efforts of many individuals. Their wisdom and
assistance made the completion of this thesis possible.
I wodd like to thank Dr. LP. Santerre, my supervisor, for di the time and effort he invested in
me. His continued enthusiasrn and encouragement gave me a giimpse to the makings of a tnre
researcher.
Other individuais who need to be mentioned for their kindness and expertise are: Dr. Erin
Boynton, Stuart Rae, Dr. Xijia Gu, Dr. T h Burrows and Dr. Kimberly Dwyer.
The following individuals picked me up during the times when it would never end. Lisa Weiler,
Yi-wen Tang, Christopher McCloskey, Jeannette Ho, Greg Woo, and Frank Wang. They kept
te lhg me that 1 couid do it.
Finally, 1 would like to thank my family aod Mt. Ali Ansari. With their love and patience, 1 was
able to know the meaning of generosity.
AAS
EDAX
ELISA
ATR-F'TrR
CE
F m
HDPE
HMDM
HP1
HPLC
IL- 1 p IL-6
LDPE
MS
NMR
0.1.
f MMA
PTFE
SEM
SrMS
THR
TNF-a
uJ3MwPE
XPS
Atomic absorption spectrometry
Energy dispersive analysis X-Ray
Enzyme-Linked immunosorbent assay
Attenuated total reflectance FTIR
C holesterol esterase
Fourier transform infrared spectroscopy
High density polyethylene
Human monocyte-derived macrophages
Howmedica Product Monnation
High performance Liquid chromatography
hterleukin- 1 cytokine
hterleukin-6 cytokine
Low density polyethy lene
Mass spectroscopy
Nuclear magnetic resonance spectroscopy
Oxidation index
PolymethylmethacryIate
Teflono
Scanning electron microscopy
Secondary ion mass spectroscopy
Total hip replacement
Tumot necrosis factor a cytokine
Ultra high tnolecular weight polyethylene
X-ray photoelectron spectroscopy
A~STRACT
ACKNO WLEDGMENTS
ABBREVIA~ONS AND NOMENCLATURE
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF APPENDICES
2.1 History of Hip Implants
2.2 UHMUTPE: The Material
2.3 Manufacturing of UHMWPE
2.3.1 Poiymerization
2.3.2 Processing Of UHMWPE
2.3.3 Sterilization of UHMWPE
2.4 Wear and MecMcal Failure
2.5 Oxidation of UHMWPE
2.5.1 The Effect of Sterilization and the Environment
2.5.2 In Vivo Oxidation
2.5.3 Storage
2.5.4 Thennal Oxidation
2.5.5 Mechanism of UHMWPE Degradation
2.6 Biocompatibility, Biomaterial Interactions & Formation of Superoxides
2.6.1 Definition o f Biocompatibility
2.6.2 Bioactive, Biotolerant & Bioinert
2.6.3 Bulk versus Particdates: Effect on Infianunatory Response
2.6.4 Phagocytic Reaction to Particles
2.6.5 Monocytes/Macrophages, Neutrophils & the Inflamrnatory Response
ii
iii
iv
v
viii
ix
xi
1
3
3.1 Material Selection
3.2 Test Sample Preparation
3.2.1 Preparing Films from Bar Stock
3.2.2 Sterilization of Samples
3.2.3 Coating of Particles with Cobalt Chloride
3 -3 Characterization of Test Specimens
3.3.1 Surface Morphology of Polyethylene
3.3.2 Chernical Composition of Polyethylene
3.3.2.1 X-Ray PhotoeIectron Spectroscopy 0 8 s ) 3.3.2.2 Energy Dispersive Analysis X-Ray (EDAX)
3.4 Oxidation of WMWPE
3.4.1 Oxidation by Themial Treatment
3 A.2 Chernicd Oxidation of UHMWPE
3.4.2.1 Determination of Sodium Hypochlorite Activity
3.4.2.2 Determination of Hydrogen Peroxide Activity
3 A2.3 Half-life Study for Hypochlorous Acid and Hydrogen Peroxide
3.4.3 incubation Experiments
3.5 Characterization of UHMWPE Oxidation
3.5.1 Fourier Transform Innared Spectroscopy (FTIR)
3.5.2 Sulphur Dioxide Staining
3.6 Incubation of UHMWPE under Hydrolytic Conditions
3.7 Extraction of Degradation Products
3.8 High Performance Liquid Chromatography (HPLC)
3.9 C haracterization of Iso lated Biodegradation Products
3.9.1 Mass Spectroscopy
3.9.2 Attenuated Total Reflectance FTIR (ATR-FTIR)
3.9.3 Nuclear Magnetic Resonance Spectroscopy (NMFt)
3.10 In Vitro Studies of HDPE Particles with Human Monocytes
3.1 0.1 Preparation of Particles
3.10.2 Preparation of Ce11 Culture
3.10.3 Anaiysis of DNA, Ce11 Viability and Cytokines
4.0 RESULTS . . . . . . , . . , . . . . . , , . , . . . . . , . . ,.. , , . . . , . . . . . . . . . . . . . . . - . . . . . . . . , . . . . , . , . . . , . . . . . . . . . . , ,. 4.1 Characterization of Polyethylene Samples
4.1.1 Surface Morphology
4.1.2 Chemical Composition of Polyethylene
4.2 Themial Oxidatiori of Particle Samples
4.2.1 Sulphur Dioxide Staining
4.2.2 Cobalt Treatment of Thermaily Oxidized UHMWPE and HDPE
4.3 Chemical Oxidation of UHMWPE
4.3.1 Assessment of HOC1 Induced Oxidation
4.3.2 Assessment of Hydrogen Peroxide Induced Oxidation
4.4 Hydrolytic Degradation of Oxiciized UHMWPE
4.4-1 Characterization of Acid-Treated UHMWPE Particles
4.4.2 HPLC Analysis of Hydrochlonc Acid incubation Solutions
4.4.3 HPLC Analysis of Cholesterol Esterase Incubation Solutions
4.4.4 Mass Spectroscopy Analysis
4.4.5 Chemical Characterization of the HPLC Products
4.5 In Vitro Study of HDPE Particles with Human Monocytes
4.5.1 Viability Study Results
4.5.2 Cytokine Results
5.0 Drscussro~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Chemical Characterization of Test S pecimens
5.1.1 Irradiation
5.1.2 Cobalt-Treated Samples
5.2 Thermal and Chernical ûxidation of UHMWPE Samples
5.2.1 Thermal Oxidation
5.2.2 Chemical Oxidation
5.3 Hydrolysis of Oxidized UHMWPE
5.4 In Vitro Study of HDPE Particles with Human Monocytes
6.0 SUMMARY AND CONCLUSIONS.. ... ... . . . . ... ... ... .. . . . . . .. ... . .. .. .... . .. ... . . . ... .. . , . . .. ... 7 -0 RECOMMENDATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 8.0 REFERENCES . . . . . . . . . . . . . . . . . . . , . . . . . , . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . Appendices
vii
Table 2.1
Table 2.2
Table 2 3
Table 2.4
Table 2.5
Table 2.6
Table 2.7
Table 2.8
Table 3.1
Table 3.2
Table 3.3
Table 3.4
Table 3.5
Table 3.6
Table 3.7
Table 3.8
TabIe 3.9
Cornparison of Selected Physical Properties of UHMWPE and HDPE
Selected UHMWPE Resins and Their Melting Points and Crystaalinity
Standard Properties of UHMWPE Powder
Current Source of UHMWPE for Commercial Implants on the Market
Properties of UHMWPE Fabricated Fom
Measurable Free Radicals after Different Methods of Sterilizaîion
Radical Produced fiom the y-Irradiation of UHMWPE
Secretory Products of Mononuclear Phagocytes
Material Properties for the UHMWPE and HDPE Particles
Material Properties of UHMWPE Bar Stock
Experimental Conditions for üHMWPE Particle Samples
Experimental Conditions for UHMWPE Film Samples
Chernical Treatment of Film Samples
Sources for Chemicais Used in HOC1 Assay
Chemicais for Hydrogen Peroxide Assay
Processing Conditions for Particle Samples
Program One Gradient R u for HPLC
Table 3.10 Program Two Gradient Run for HPLC
Table 3.1 1 Particle Treatments for Particle-Monocyte Experiments
Table 4.1 XPS Results of Elemental Composition of UHMWPE Films and Particles
Table 4.2 Oxidation Indices of UHMUrPE Films and Resin
Table 4 3 XPS Results for Cobalt-Coated Samples
Table 4.4 Oxidation Indices of Cobalt Chloride Treated m E
Table 4.5 Oxidation Indices of Hydrolytically Treated Samples
Table 4.6 Molecular Weights of the Isolated HPLC Products
Table 5.1 Chemicai Structure of Fragmented Ions Related to Peak 1
Table 5.2 Oxidation Indices of TherrnaUy Oxidized HDPE
viii
Figure 2.1
Figure 2 3
Figure 2 3
Figure 2.4
Figure 3.1
Figure 3.2
Fipre 3 3
Figure 3.4
Figure 3.5
Figure 3.6
Figure 3.7:
Figure 3.8:
Fipre 3.9:
Figure 4.1
Figure 4.2
Structure of Polyethylene
Crystalline Regions and Amorphous Regions in UHMWPE
Free Radical Polymer Degradation
Environment in Phagolysosome for Monocytes and Neutrophils
Direction of Carbon Sputtering on the Surface
A Typicd FTIR Spectra of Oxidized UHMWPE
Schematic of Sulphur Dioxide Staining Apparatus
Preparation of HPLC Samples
HPLC Block Diagram
Quadrupole Arrangement for Mass S pectrometer
Schematic of Triple Quadrupole Mass Spectrometer
ATR-FTIR Light Path
Separation Gradient of Whole Blood
Scanning Electron Micrographs of UHMWPE Resin Particles
Scanning Electron Micrographs of HDPE Resin Particles
Fipre 4.3 Oxidation of UHMWPE Particles Post y-Irradiation
Fipre 4.4 Fluorescence micrograms of Sulphur Dioxide Treated Films
Figure 4.5 Fluorescence micrograms of Sulphur Dioxide Treated UHMWE Particles
Fipre 4.6 FTIR Results for Cobalt Chloride Treated UHMWPE
Figure 4.7 EDAX Results for Cobalt Chloride Coated UHMWPE and HDPE
Figure 4.8 Consumption of HOC1 with Different Substrates
Figure 4.9 Various Treatments of UHMWPE Film Samples
Figure 4.10
Figure 4.1 1
Figure 4.12
Figure 4.13
Fipre 4.14
Figure 4.15
Figure 4.16
Cornparison of the Oxidation Indices of UHMWPE Films and Particles
Effect of Incubation of Pre-Heated UHMWPE Particles
FTIR Results of Various Chernical Treatments of Irradiated Particles
FTIR Result for Hydrochloric Acid Treated UHMWPE Samples
HPLC Results on the Effect of Hydrochloric Acid on UHMWPE
HPLC Results: Incubation of UHMWPE Particles with CE and Buffer
Resolution of Peak 4 Ushg P r o g m Two
Figure 4.17
Figure 4.18
Figure 4.19
Figure 4.20
Figure 4.21
Figure 4.22
Fipre 4.23
Figure 4.24
Figure 4.25
Figure 4.26
Figure 4.27
Figure 4.28
Figun 4.29
Figure 430
Figure 4.31
Figure 4.32
Figure 4.33
Figure 434
EPLC Resuits Effect of Heat Oxidation of UHMWPE Particles in BufTer 83
Effect of Heat Oxidation on Enzyme-Treated üHMWPE Particles 84
HPLC of Peak Analyzed with Mass Spectroscopy 85
Mass Spectrogram of the HPLC Product, Peak 3 86
MS-MS of Parent Peak = 340.6 From Peak 3 86
MS-MS Resdt for Parent Pe-79.4 Peak 3 87
MS-MS of Peak 1, Parent Peak m/z=371.4 88
MS-MS of Peak 2, Parent Peak d z 4 5 3 . 2 88
MS-MS of Peak 4A- 10, Parent Peak h l 72.8 88
ATR-FTIR Result of WLC Isolate at 44 Minutes 90
FTIR Standard of Dichloromethane 91
Proton NMR Result of Peak 3 93
Carbon- 13 NMR Result of Peak 3 93
Necrosis of Non-Adherent Human Monocytes 94
SEM Micrographs of Human Monocytes and Treated HDPE 95
IL- 1 f3 Release from Human Monocytes 96
IL-6 Release fiom Human Monocytes 97
TNF-a Release fkom Human Monocyte Supernatant 98
Figure 5.1 SEM Micrograph of Cobalt Treated UHMWPE and Non-Treated UKMWPE 102
Figure 5.2 SEM Micrograph of UWMWPE Particles and Film 1 06
Figure 5.3 Cornparison of the W Absorbance Spectra of Peaks 2 and 3 110
Figure 5.4 UV Absorbance of the Product Associated with Peak 3 111
Figure 5.5 ATR-FTIR Resultant of a Subtraction of Peak 3 and Dichloromethane 113
Appendix A
Appendix B
Appendix C
Appendix D
Appendix E
Appendix F
Appendix G
Appendix H
Phosphate B a e r Recipe and Chernicals for H2& A s s y
Caiculation of UHMWPE Amount based on Surface Area
Calibration Curves for HOC1 and H202
C holesterol Esterase Assay
Mass Spectra of Peak 4 Products
Mass Spectra of Stearic Acid
Caiibration Curves for Cytokine Andysis
ATR-FTIR Data for Peak 2
Each year, a significant number of patients are diagnosed with joint diseases. The pathogenesis
of these diseases often leads to loss of articular cartilage and the synovial fiuid that lubricates the
bone-bone interface. The best option for treatment for these diseases is to replace the joint with a
biomedical implant. In North Arnerica aione, it was estimateci that 400,000 total joint
replacements were performed in 1993 &i et al, 19941. However, many of these implants have
been shown to be mechanicaily destabilized or loosened. Movement at the implant-bone
interface or at the implant-implant interface can produce Wear particles [Howie et al., 19931.
These particles have been show to migrate to the bone-implant interface and cause M e r
prosthetic loosening and bone loss due to the extreme inflammatory response to the particles.
This necessitates the need for revision surgery [Hardinge, 19831.
Amficial hip implants are composed of bal1 and socket configurations. The socket or the
acetabular cup is composed of ultra high molecular weight polyethylene (UHMWPE), metal
ailoys or ceramic while the head of the femur is composed of ceramics or metai alloys such as
titanium or cobalt-based, stainless steel. The type discussed in this thesis is the polyethylene-
metal implant. Through normal use, metai and polymer particles are generated. These particles
have been shown to migrate to the bone-implant interface and stimulate a cellular response to the
particles. This response has been shown to be associated with bone resorption around the
implant and causes a further destabilization of the implant [Howie et al.. 19931.
Destabilization of the implants usually involves the resorption of bone at the implant-bone
interface. The proposed mechanism for this observation begins with the Wear particles eliciting
an infiammatory response where macrophages/fibroblasts/giant body cells attempt to
phagocytose the Wear particles. This results in the production of various cytokines that activate
osteoclasts to resorb the adjacent bone and increase bone loss [Jasty, 19931. When this occurs,
the mechanical integrity of the implant is lost. Most research to date has investigated the effect
of polymer and metal m e n & on their ability to induce cytokines and other ceU mediators to
signal inflammatory cells (i. e. monocytes and macrophages) to the bone-implant interface. What
these studies have failed to address is the effect of the inaammatory environment on the particles
foiiowing ceii phagocytosis or the stimulation of other inflammatory responses to these particles.
The infiammatory environment contains oxidative compoimds, namely hydrogen peroxide and
hypochlorous acid, as weli as other hydrolytic agents. These oxidative compounds rnay M e r
degrade UHMWPE particles in the presence of metal particles that may catalyze these reactions.
The aim of this study is to address this issue and determine the effects of these oxidîzed
UHMWPE particles on their ability to stimulate bone resorptive cytokine release by human
monocytes. It has been hypothesized that oxidized LTHMWPE can be hydrolyzed with
cholesterol esterase. As well, another hypothesis of this thesis is that there may be an eEect of
the UHMWPE degradation products, fiorn both oxidative and hydrolytic processes, on the
stimulation of the idammatory processes observed around the implant [Santerre et al., 19971.
Specifically, the cytokines which have been linked to bone resorption will be assessed.
The objectives of this study are:
1. Develop a mode1 to oxidize polyethylene films and particles using thermal and
chernical oxidization.
2. Hydrclyze the oxidized polyethylene with hydrochlonc acid and a macrophage-
derived lysosomal enzyme, cholesterol esterase.
3. Isolate and identiQ the dominant degradation products fkom the hydrolysis reactîons.
4. Perform an initial assessrnent of the effect of oxidized cobalt-treated polyethylene
particles on human monocyte/macrophage cytokine production.
2.1 History of Hip Implants
The need for an adficial hip became apparent when other surgical techniques failed to relieve
the pain associated with degenerative diseases of the joint. Materials used at the beginning of
this cenhiry included skui, pig's bladder and gold foi1 [Hardinge, 19831. By the 1950's the need
for better materials was apparent. The precursor to the modern hip implant originated in England
and was introduced by McKee [Hardinge, 19831. It was composed of a combination of
metdmetai cobaltchrome alloy for hip replacement. The major problem associated with this
type of implant was high fiction between metai intediaces, resulting in high Wear [Semlitsch,
19901.
in 1958, Sir John Charnley of Wrightington Hospital developed a low friction hip system by
combining a polymer socket of ~eflon@ with a stainless steel metai bail [Semlitsch, 19901.
Between 1958 and 1961, Charnley implanted about 300 implants. M e r an implantation period
of three years, it was observed that there was displacement of the femoral head. In addition,
radiographically, the PTFE components showed a 6-7 mm loss of material through Wear. The
surrounding tissue was loaded with PTFE particles and exhibited severe granulomatous foreign-
body reactions P I , Semlitsch, 19901. In 1962, Chamley changed the material of the articular
component to UHMWPE and coupled it with stems composed of 3 16L stainless steel, Co-Cr-Mo
alloy, or Ti-6Al-4V alloy [Davidson et al., 19921. The UHMWPE had a higher resistance to
Wear and creep compared to ~eflon? It becarne popular in the 1970's for hip endoprostheses as
well as for artificial knee, ankle, shoulder, elbow and wrists joints [Semlitsch, 19901.
Other altemative articulating surfaces were investigated during the 1970's and 1980's included
polyacetal (polymethylene oxide; specifically Delrin 100, D e h 150, DeLrin 100 and Celcon) in
the Christian hip prosthesis and Poly Two (carbon-fiber-reinforced ultrahigh molecular weight
polyethylene by Zimmer) ['Li2 et al.. 19941. However, these materials were found to have a
significantly higher rate of failure cornpared to the Chamley implants ~ i * et d. 19941. The
failure of the latter system was attributed to poor adhesion between the fibers and the UHMWPE
matrix ~i~ et al.. 19941. The use of polyester(polyethy1ene terephtalate) (PETP) was also
investigated as a potential material for the acetabuiar cup wttlemier, 19861. It had excellent
physicai properties compaxed to UHMWPE, but it degradeci in vivo with the generation of
microparticles with a mean size of 0.5 to 20 p. Presently, all materials create Wear debris of
approximately 100,000,000 particles per day within the range of 1 p to 200 p [Clarke, 199 11.
Many more materials have been hied and have failed. Thus, UHMWPE has remained the
material of choice despite the generaton of particle debris.
The fuhire of endoprostheses of the joints lies in the development of new materials supenor to
UHMWPE. These materials mut have the following attributes:
easier processing capabilities
fke f?om impurities
higher wear-resistance
ease of stenlization without adverse or oxidation effects.
2.2 UHMWPE: The Material
UHMWPE is composed of repeating ethylene units (Figure 2.1). The molecular weight of
UHMWPE cm go up to 6 million compared to other types of polyethylene resins which have
rnolecular weights in the range of 10,000 to 500,000 @PI]. The significantly higher molecuiar
weight is important. In general, the physical properties of polyethylene change proportionally
with iocreasing molecular weight, until a molecular weight of 1 million is reached. At this point,
there is a sudden increase in melt viscosity where the material's processibility changes and hence
the characteristics of the finai product @3irnkl.aut, 1 9901.
n= 2.5 x 105
Figure 2.1: Chernieal Structure of Poiyethylene
The polymer is semi-crystauine; the mimstnicture consists of crystaiiine regions and amorphous
regions as seen in Figure 2.2. In the amorphous region, the long chahs hinder the ability of
molecules to order themselves into crystalline arrays, thus limiting the percenage of the
material's crystallinity as determineci by differentid scanning calorimetry WrJ. The strands are
held together by random mechanical entanglements and the occasional chemical crosslinks (in
irradiated samples) [A. wang', 19971. The crystalline regions are composed of unit cells called
IameIiae. These c m be m e r organized to form larger structures cailed spherulites. The
IarnelIae in UHMWPE are folded polyethylene chahs consisting of 150 CH2 groups in an
orthorombic shape arrangement. The mistance to the propagation of fatigue cracks was found to
improve as the crystalIinity increased [I,i2 et ai., 1 9941.
Smounding the crystalline regions and comecting the separate crystals are bridging tie
molecuies. These molecules act as crosslinks between the crystaUine domains and provide load
bearing' and stress transfer as weii as physical and chemical strength p u . The number of tie
molecules is higher in UHMWPE compared to high density polyethylene (HDPE) WI]. The
effect of this higher molecular weight can be seen in a cornparison of physical properties between
I D P E and UHMWPE (Table 2.1).
Table 2.1: Cornparison of Selected Physical Properties of
UAMWPE and HDPE [ ~ i ' et aL, 19941
Property r HDPE IUHMWPE I Molecular Weight (million g/mole) Melting Point (OC) Density (kg&) Teasile Yield M a )
0.05 to 02
130 to 137 0.952 to 0.965 26.2 to 33.1
200 to 350 ,
0.8 to 1.5 Elongation at Break (%) Tensile Modulus GPa) I
2 to 6
125 to 135 0.930 to 0.945 19.3 to 23
1 O to 1200 0.4 to 4.0
Izod Impact (J/m) Shore-D Hardness
21 to 210 66 to 73
> 1070 no break 60 to 65
Figure 2.2: CrystPlline and Amorphous Regions in UAMWPE p u
While the mechanical properties of UHMWPE are provided by this unique microstructure,
property dif5erences Vary fiom one formulation of UHMWPE to the other dependhg upon:
crystallinity; number of tie molecules; number and nature of entanglements and crosslinks; and
the presence of orientation of the polymer chah [A.Wang, 19971. The qualities that make
UHWMPE suitable for implant applications are:
low fiction coefficient
high abrasion resistance
hi& impact resistance
high ductility and biocompatibility
high resistance in vivo
0 hydrophobic and resistant to aggressive media
23 Manufacturing of UHMWPE
It was estimatecl that more than 41,000 tonnes of UHMWPE were produced in 1993. The main
industrial uses for this material are skis, cutting boards and coal-chute liners and other
applications which require hi& Wear resistance and better physicd pmperties than Teflona [Li2
et al., 19941. For the 400,000 total joint replacements perfonned in North Amenca in 1993,O. 1 1
kg of UHMWPE was w d for each implant This accounts for over 45,000 kg of UHMWPE
implanted which represents about 0.1 % of the total annuai production of UHMWPE [Li2 et al.,
1 9941.
2.3.1 Polymerization
UHMWPE is polymerized in an inert gas atmosphere ~irnkraut, 19901. Pure ethylene gas is
suspended in a hydrocarbon solvent (Le. hexane) fiee of polar impurities in the presence of a
catdyst p i 2 et al., 19941. The catalyst is a moisture and air-sensitive Ziegler-Nana coordination
catalyst composed of titanium tetrachioride and an aluminum allcyl compound WI]. Polymerization occurs on the catalyst sUTface at temperatures between 66 to 80°C at pressures
between 4 to 6 bar [Li2 et al. .. 19941. The resultant polymer is a fine granular white powder. The
molecular weight is controlled by varying the temperature [HPI]. On average, 100,000 ethylene
molecules are added onto an active catalyst center until the growing polymer totally encloses the
catdyst and forms a molecule with a molecdar weight of 3 million pirakraut, 19901. The
molecular weight distribution is controlled by the ability of ethylene molecules to access active
catalyst sites. Each resin manufacturer has slightly dZferent polymerization conditions which are
contained in proprietary information. A listing of different resins dong with their melting points
znd crystallinity is provided in Table 2.2.
Table 2.2: Selected UIIMWPE Resins and Their Melting
Points and Crystallinity pi2 et al, 19941
Type of Resin Melting Crystailinity Point (OC) (%)
1900 145 75 412 143 60 413 143 67 415 143 58
Table 23: Standard Properties of UEMWPE Powder
1 1 s~ecifïed in ASTM D 4020 1
Properties Type of Powder
1 Molecular Weight 1 Relative solution viscosity > 1
ASTM F 648-84 Homopolymer of ethylene as
Number of particles of 2.30 (ASTM D 4020) I
< 25 particles per 300 gm con tamhants Trace Elements
After polyrnerization, the solvent suspension goes through a series of centrifiiging, stripping, and
Max Content @pm) AliuninuIn 100 Titanium 300 Calcium 1 O0 Chloride 120
Particle Size
drying steps where UHMWPE is separated fiom the suspendhg agents and other residues. The
al l powder shall pass through a No. 16 (1.18 mm) sieve
powder is passed through a 500 pm filter to remove large particdates and yields a product with a
mean particle size of 100 p. The dry resin powder is separated into a silo for homogenization
and bagged with the addition of small amounts of phannaceutical-grade calcium stearate to
inhibit the yellowing of the product during processing p u . The regdation that guides the
degree of purity of UHMWPE and the specification of material properties is in accordance with
ASTM F 648-84 for the UHMWPE (Table 2.3). Three grades of UHMWPE used in orthopaedic
implants are 421, 41 5 GUR (Hoechst/Celanese) and 1900 (Himont) [ ~ i ~ et al, 19941. The
polymer is pre-formed into sheets or bar stock before fabrication into implants. The UHMWPE
used in orthopedic devices found on the market today is illustrated in Table 2.4 ['Dwyer1, 19961.
2.3.2 Processing Of UHMWPE
Processing of UHMWPE resin is difficult because of its high viscosity PI]. Cornmon
processing methods use hi& pressures and temperatures to consolidate UHMWPE and include
direct molding, ram extrusion and compression molding.
Direct molding entails resin powder compression in a mold which is heated at temperatures
between 200 and 250°C [Kellersohn, 19901. Devices made by this method have no extemal
machining lines and exhibit a high gloss s d a c e pi2 et ai., 19941. Ram-extrusion and
compression molding are two-step processes. Ram-extrusion involves high pressures to extrude
a cylindricd bar stock [I,i2 et al., 19941 while compression molding involves moldiog the
polymer into a sheet [Li2 et al.. 19941. M e r the bar stock or sheet is made, an implant can be
M e r machined. As a final processing step, some manufacturers anneal the component to
remove any residual stresses [I,,i2 et ai., 19941. However, there have k e n reports [Zhu et ai.,
19951 that annealhg UHMWPE causes deformaton of the crystallites. For purposes of medical
implants, the final fabricated forrn must also adhere to the ASTM F 648-84 standard as listed in
Table 2.5.
Lncomplete consolidation of the resins greatly shortens the life of the implant F i et ai., 1995,
~ w ~ e ? et al., 19961. Implants that have been directly molded have been show to be less
susceptible to particle debris production compared to implants machined fkom extruded bar stock
[Bankston et ni., 1995, Rentfiow et ai., 19961. The ram-extmded polymer contains voids which
may increase Wear [Learmonth et al., 1997. These authors showed that machined cups produced
an mual linear Wear rate of O. 1 lm while direct molded cups only resulted in 0.05mm. The
discrepancy was attributed to inconsistencies in polyethylene fabrication by different methods
[Windau et al.. 1 9961.
Table 2.5: Properties of UHMWPE Fabricated Form mu
l Number of light patches
Mechanical Requirements (minimum): Temile yield strength Vitimate tende strength Elongation at break izod impact strength (double notch)
1 Hardness (Shore D)
2.3.3 Sterüization of UESMWPE
ASTM F 64û-84 Standards No stabiiizers or processing aids to be added No particle > 300 p; no more than 10 particies of 300 pm or less. No Iight patch > than 3 0 0 p in a 400cm2 samale Between 0.930 and 0.944dcmJ
19.3 x 106 N/m2 27.58 x 106 ~ / m ~ 200% 1070 J/m 2% after 90 min. recovery (6.895 x 1 o6 N/m2 for 24 hours)
Any material implanted into the body must be sterilized. Since UHMWPE is thermolabile, the
methods available for sterilizing of these implants are: ethylene oxide gas, irradiation, and gas
plasma sterilization. Since the 1970's, the &est method for the sterilization of UHMWPE has
been with the use of gamma radiation fiom "cobalt, at a minimum dose of 25 kGy or 2.5 Mrad.
The t h e varies fkom 1.5 to 1 8 hours depending on the radiation source [~treicher'~, 19881.
Changes in structure and mechanical properties have been associated with the irradiation of
UHMWPE. Specifically, these changes were noted when the process was carried out in an air
environment as opposed to irradiation in a vacuum environment [Gsell et al.. 1996, St. John et
al., 19971. The altered properties were in part explained by an increased susceptibility to
polymer oxidation (see fùrther discussion in Section 2.4).
Ethylene oxide sterilization has been commonly used to sterilize heat-sensitive medical devices.
Ethylene oxide reacts with the bacteria and renders it inactive [Collier et al., 19961. The efficacy
of ethylene oxide stenlization is quite high. It has high penetrability, but does not alter surface
properties [Tabrizian et al., 19971. The method involves purging the system with ethylene oxide
gas for a period of tirne, after which, the ethylene oxide must be removed fkom the system [Ries1
et a . , 19961 because o f its toxic residue and potentidy carcinogenic byproducts [Ramer et
al., 19961. However, in ment years, the elhination of its use has k e n advocated due to the
presence of toxic residue [Ratner et al.. 19961.
Ln the past few years, cold gas plasma sterilization has gained prominence. Many of these
techniques involve the use of oxidative chernicals such as hydrogen peroxide vapour at low
temperatures to inactivate microorganisms and remove the peroxide residues mui, 19971. This
process works by oxidizing bacteriai cellular components [Collier et al., 19961. One problem
encountered is the slight oxidation of polymer samples. However, it is not as extreme as that
exhibited using y-irradiation wui, 1 9971.
A cornparison of different stedization methods and their generation of detectable arnounts of
fiee radicais using electron spin resonance is shown in Table 2.6 [Coilier et al.. 19961. Data
show that gamma irradiation under air and vacuum exhibited a meamble amount of fiee
radicals while ethylene oxide and gas plasma sterilization are fiee fiom their presence. These
fiee radicals were primary fiee radicals, which occurred with the cleavage of the backbone, and
peroxide radicals, which occurred in the presence of oxygen.
TabIe 2.6: Measurable Free Radicals after Different
Methods of Sterilization [Collier et al , 19961
Free Rsdicals?
1 Gas Plasma I no I Ethylene Oxide
1 y-Irradiation (air) I Yes l
no
2.4 Wear and Mechanical Failure
As previously noted, most prosthetics are made of a metallic core mch as titanium, stainless
steel, or cobalt-chromium-chromium alloys, while the articular surface is made of UHMWPE.
y-Irradiation (vacuum) YeS
Cernented implants are stabilized by polymethylmethacryiate (PMMA) cements. The
incorporation of these materials has its own biocompatibility implications and wiU not be m e r
addressed here.
The early concem of the bearing surfaces in joint arthroplasty was the limited life expectancy of
the prosthesis as a result of wear Friedman et al., 19931. Now the present concem centers on the
amount of Wear debris produced £iom the bearing nirfaces. The Wear debris causes many
biological interactions that are linked to the loosening of the joint In the cases of metal-backed
acetabdar cups, there are two bearing surfaces present: the interface between the cup and the
femoral head and the interface between the cup and the metai backing that is attached to the
bone. Learmonth and colieagues beiieve that contributing factors to Wear in clinical practice are:
matenal polymorphism, poor design features and ke-body Wear [Learmonth ef al., 19971.
Material polymorphism occurs fiom the different methods of processing the polymer which may
lead to incomplete consolidation of the resin. Many authors have identified other factors besides
three-body Wear which contribute to the loss of material [Wright et al.. 19901.
Wright and colleagues have identified various modes of surface damage to polyethylene surfaces
in joint implants [Wright et al., 19901.
Surface deformation: permanent defonnation occurring on or around articulating
surfaces.
Scratching: indentation lines found in the dominant direction of motion . Blrnllshing: areas which have become highly polished.
a Abrasion: shredded or tufted appearance, attributed to direct sliding contact with
either bone or polymethyl methacrylate.
Embedded PMMA debns: can be recognized by the colour and texture differences
between PMMA and polyethylene and verified using energy dispersive spectroscopy.
Pitting: voids o c c m h g in an articulating surface, ususually irregularly shaped and 2 to
3 mm across and 1 to 2 mm deep.
Delamination: large "sheets" of polyethylene have been separated and removed, with
evidence of subsurface failure mechanisms occurrllig parailel to the articulating
surfaces.
Of these damage modes, Wright and coiieagues identifieci that pitting and delarnination release
the largest arnount of polyethylene debris to the surroundhg tissue. Al1 of these surface damages
are related to long-terni problems. Other authors disagree [Friedman et al., 1993, McKellop et
al.. 1 9951. They associate Wear debris generation to other mechanisms including adhesion,
abrasion and fatigue. They also suggest Wear debris acts as a stress concentrator and produces
third-body wear. Regardless of the damage modes, the production of particulates is a serious
factor in the long-term stability of total joint replacements.
Adhesive wear occurs when interatomic forces between coupling wear surfaces are greater than
the intrinsic forces between the molecules of the bulk material Friedman et al., 19931. Thus,
material adheres to the opposite d a c e of the Wear couple. UHMWPE transfers its surface to
the harder materials and subsequently sheds into the joint space. This is similar to the
delanilnation process proposed by Wright and colleagues. Another aspect of adhesion occurs
when a passive layer of oxide from the metal adheres to the polyethylene. This roughens the
metal surface and increases Wear debris.
Abrasive Wear depends on contact stresses, surface hardness and surface finish (roughness).
When a soft surface is abraded with a hard surface, debris resuits. Hardness plays a role because
the harder the material, the longer a smooth surface finish is maintained. Surface hardness can
be increased by nitriding or ion implantation. Fatigue or pitting is another factor in debris
generation. High contact stresses in UHMWPE cm cause subsurface stress that exceeds the
maximum fatigue strength of the polyethylene. Multiple stress cycles can propagate crack
formation and produce Wear debris.
Not ody is the amount of Wear debris generated of great concem, but the size of the Wear debris
is also of importance. McKellop et al. [1995] have shown that subrnicron particles are the result
of adhesive, abrasive and microfatigue mechanism while larger particles (tens of microns) were
related to third-body Wear. Submicron and micron size debris have been associated with
idammatory periprosthetic bone loss which has been W e d to aseptic loosenuig of total joint
replacements [McKellop el al., 19951. Further discussion is found in Section 2.6.4.
One such implication of Wear debris is believed to be the activation of manophages and the
development of bone resorption leading to the aseptic loosening of the implant. This
phenornenon is not well elucidated but follows several documented phases [Basie et al, 19961.
Local ce11 damage occurs adjacent to cernent layer related by chemicai, mechanical
and thennal phenornena
Abrasion and corrosion fiom fiction between materiais and micromotion between
implant and tissue produce Wear particles.
Wear particles induce a local infiammatory response and further include formation of
a fibrohistiocytic membrane a d o r bone resorption.
Leads to prosthetic loosening.
Hence, the challenge to the continued use of UHMWPE is to prevent the elevated levels of
polyethylene Wear particles. This is important not only because of the danger in wearing out the
implant, but also because there are inflammatory responses to the particdate debris that prevent
the implant's long term use.
2.5 Oxidation of UHMWPE
Oxidation is believed to contribute to changes in the mechanical properties of UHMWPE
m a c 1 et al., 1994, Ries2 et al., 1996, Flynn et al.. 1996, Pascaud et al., 19971 and as a result,
increases the material's susceptibility to Wear [Greer et al., 1996, Li et al.. 19961. The oxidation
of UHMWPE has been shown to occur in the amorphous structure of the materiai because it is
more accessible to oxygen diffusion [Scheir et al., 19911. The oxidation in this region
propagates non-unifody because of the coils present in the arnorphous structure. The rate of
oxidation is diffusion controlled since the permeation of oxygen is the slowest step. Mer
oxygen diffusion occurs, the oxidation reaction can propagate throughout the matenal in an
autocatalytic process. Another factor that may contribute to the oxidation is the presence of
metai ions [Zhao et al., 19951. These metal ions cm act as a catalyst for the oxidation reaction.
Chemical radicals have been shown to be generated in the material after irradiation. These
radicals can react with oxidative species upon implantation. It has also been estabiished that the
properties of UHMWPE change upon gamma sterilization, différent storage environments,
thermal environments, and under photooxidation and implantation conditions. These pro-
changes include: increased embrittlement, decreased ciamping ability, increased hardness
~ y e r e ? et al., 19841; decreased creep and Wear because of higher crystaUinity ~ y e r e ? et al.,
19841, and decreased abrasion resistance that will accelerate debris production [Betts, 19931. In
addition, there are potentiai toxic eEects of lower-molecular-weight constituents pyere? et al.,
1 9841.
2.5.1 The Effect of Steriliwition and the Environment
Since the 1970 '~~ the most common method for the sterilization of UHMWPE has been gamma
radiation fiom 60~obalt. However, irradiation of UHMWPE has been show to cause changes to
the materiai's chernical and mechanical properties [~treicher", 19881. Upon gamma irradiation
of a hear hydrocarbon in air, several events occur simultaneously:
cleavage of carbon-carbon bond in the backbone
generation of uasaîurated groups
a formation of radicds and the extraction of hydrogen.
Al1 of these can cause chain scission, crossluiking and oxidation or peroxidation [~treicher',
1988, R.imnac3 et al., 19941. The primary formation of radicais occurs when the irradiation
breaks the covalent bonds between the carbon and hydrogen to produce a combination of alkyl
and allyl type groups. This lowers the average rnolecular weight of the material. ~ ~ e r e ? et al.
(1984) found that 35% of soluble constituents of an unused, sterilized UHMWPE cup had an
average rnolecular weight of 210,000 g/mol. It has been reported that when irradiation occurs in
air, a smdl fiaction of peroxy radicals are formed [Man et al., 19911. Common radicals
identified in the presence and absence of oxygen after irradiation are shown in Table 2.7.
Table 2.7: Radicai Produced from the y-Imdiation of UHMWPE [Jahan et a&, 19911
In the Absence of 0 2
H H H
H H H
In the Presence of O2
Chain
Chain
Scission
When UHMWPE undergoes 60~o-irradiation in a nitrogen environment, the polyethylene
oxidation is reduced and thus Wear is also reduced [Streicher', 19881. However, under this inert
environment, crosslinking can occur which may effect the mechanical properties, depending on
the degree of crosslinking [I2imnac3 et al., 19941. This crosslinking is seen as beneficiai by
A. Wang and Sun [A. Wang et al., 1996, Sun et al., 19961 since hip simulation tests have shown
less Wear with these polymers.
The severity of the oxidation is dependent on the irradiation source, the absorbed dose and the
amount of oxygen accessible to the generated radicals [Streicher ' , 1 9881. Numerous authors
[streicherl, 1988, ~ i m n a c ~ et al., 1994 Sutula et al., 1994, Jahan et al., 199 11 have observed
relationships between decreased mechanicd properties and oxidation of UHMWPE induced by
irradiation. Sutula et al. (1994) concluded that high oxidaîion areas showed a significant
correlation with clinical Wear modes of cracking and delamination @=0.01) and can effect the
dinical performance of the implant.
Premnath and colleagues (1996) have summarized the following observations on the pst-
irradiation oxidation of UHMWPE implants.
(i) Within the first weeks after irradiation, the degree of oxidation, as measured by the
concentration of carbonyl groups, decreases with increased distance away fiom the implant
d a c e . However, after 120 days, oxidation is higher Imm f?om the surface compared to
amount of oxidation found at the surface.
(ii) Components irradiated and stored in air have oxidation following the surface contour profile
of the implant.
(iii) nie width of the peak in the oxidation profile and extent of oxidation increased with time.
(iv) Increased density areas have increased oxidation. M e r a few years, the material is very
brittle.
(v) The amount of oxidation noted in retrieved implants (afler removal of fatîy acids) can be
simulated in accelerated oxidation experiments at higher temperatures and in the presence of
oxygen.
(vi) These changes in the mechanical properties limit the longevity of total joint arthroplasties.
(vii) The FTTR spectra were similar for shelf-aged and retrieved samples, but varied in intensity.
Al1 spectra showed a prominent ketone-aidehyde peak at 1720-1 730 cm*', shoulder of
carboxylic acids at 1697- 1740 cm-', and esters at 1 738- 1740 cm-'.
(viii) n i e effects of irradiation on mechanical properties are a consequence of chah scission due
to oxidation and crosslinking. On a molecular level, this results in changes in crystallinity,
crystallioe morphology, decreases in entandement density and in the number of tie
molecules. In tu~ii, changes occur in the tensile behavior, visco-elastic properties, fracture
strength, fatigue and finaily Wear.
With the difficulties encountered with sterilhtion by irradiation, alternative methods are being
investigated.
2.5.2 In Vnto Oxidation
Numerous authors have found that many UHMWPE devices are already oxidized at the time of
implantation [Eyerer et al., 19841. Property changes such as increased density, increased number
of extractabie components, decreased tensile strength and elongation have aiso been reported. In
Eyerer's study (1984), s e v d loosened cups were removed 3 weeks to I l years after
implantation. They found that the density of the UHMWPE cups increased with implantation
time and it was dependant upon implant position and loading conditions. As well, the amount of
extractable constituents increased with time [Eyerer et al., 19841. As early as 1955, reports
[Eyerer et al., 19841 indicated residual monomer was found in the urinary excretion of rats after
26 weeks of implanted radiolabeled polyethylene in rats.
The implantation environment has been implicated as a cornponent of the mechanism of
degradation. M e r implantation, the UHMWPE is befieved to be susceptible to oxidation
because of the presence of oxidants such as hypochiorous acid, hydrogen peroxide, nitric acid
and the saline present in the synovial fluid Weiss, 19871. These reagents act to M e r degrade
the implant, therefore resulting in increased density and decreased average molecular weight
[ ~ ~ e r e ? et al., 19841. These property changes have been long recognized for their effect on
aging and ultimate failure of UHMWPE [ ~ u m a c ~ e t al., 1 994, Kurtz et ai. .. 1 994 and Streicher ' , 19881.
It has been hypothesized [Betts et al., 1994, 19931 that the transition metal salts (metal implants)
and sodium chlonde may play a role in catalyzing the oxidation. Betts found that physiological
concentrations of sodium chlonde produced oxidation and reduced crystailinity of üHMWPE
comparable to the measured values for retrieved components, implanted for 3-5 years. However,
he found that the presence of metal did not produce significant amounts of oxidation. Eyerer et
al. (1984) have presented FTIR results of several failed acetabular cups that may elucidate the
mechanism of in vivo oxidation. They found that a new hip joint cup showed a C=O band curved
due to gamma radiation. After one year of implantation, the C=O band remained unchanged but
the aldehyde band @CHO) began to shift. M e r nine years of implantation, there were
noticeable shifts in both bands.
However, there is a significant debate whether in vitro oxidation of the UHMWPE cups occurs.
Contrary to the above findings, ~ostrom' et al. (1994) did not find any evidence of increased
density in retrieved components (less than 2.5 years follow-up). James et al. (1993) showed that
the technique of FTIR spectroscopy can show "oxidation" when absorbed esterified fatty acids
were present at the d a c e . This did not necessarily prove the presence of oxidative groups
incorporated into the polyethylene.
2.5.3 Storage
A further consequence of the irradiation process is that non-reacted fkee radicals remain trapped
within the polymer, m d y within the crystalline regions. Over time, the fiee radicals migrate to
arnorphous regions causing pst-irradiation oxidation with the atrnosphenc oxygen [~treicher',
1988, ~treiche?, 1988, Jahan et al., 199 11. If inert gas or a vacuum is used as a storage medium
instead of air, the irradiation does not cause oxidation [streicher' , 19881. Subsequent exposure to
an ambient environment resumed the degradation process [Rirnnac3 et al., 19941.
At temperatures well below the rnelting point of the polymer, the life span of the radicals is
measured in years [Jahan et al., 19911, thus the oxidative degradation process can be initiated
well before the time of implantation. It has been shown that afier irradiation, the mechanicd
properties of UHMWPE deteriorate upon storage. Runnac et al. [1994] performed a study to
determine the tirne-course of oxidative degradation and the extent to which the degradation
proceeded through the bulk of UHMWPE following irradiation and storage on the shelf. They
found marked changes in the density and the inhred spectnim was consistent with continuing
oxidative degradation occurring throughout the year of shelf storage. As well, most of the
oxihtion was found on the surface. Other authors suggest that most of the changes in materiai
properties, and consequentiy the contact stresses, occur durhg the first 20 months after
irradiation [Kurtz et al., 1 9941.
~treiche? (1988) performed a storage experiment with irradiated samples in air and nitrogen at
2 1 OC and in water at 37OC. His results corresponded to others [ ~ ~ l u i a c ' , et al.. 19941. The most
oxidation was found in samples stored in water while the least oxidation was found in samples
stored in a nitrogen atmosphere. He also found evidence that crosslinking was present in the
UHMWPE samples stored in nitrogen and concluded that the crosslinking irnproved mechanicd
and triboiogical properties. This was contrary to RUnnacis conclusions conceming crosslinking
[Rirnnacl et al., 1 9941.
Oxidation has also k e n found following aging in saline solution [Hastings, 19861. For non-
irradiated samples of low density polyethylene, it was reported that oxidation levels were greater
for 0.1M sodium chloride, than for 0.01M sodium chionde, while water showed lower levels
than the latter, and air samples had the l es t oxidation [~imnac' et al., 19941. The effect of
storage in water at 37OC versus storage in air was also confinned by others [streicherl, 19881.
With the incorporation of oxygen in the hydrophobie polyethylene, hydrophilic groups are
produced to create higher surface energy [streicherl, 19881. Thus, the ability for the diffusion of
water molecules is increased &er irradiation and increased oxidation is observed [streicherl,
1 9881.
2.5.4 Thermal Oxidation
Themai oxidation of polyethylene is a well-documented phenornenon pawkins et al-, 1 97 1 1. Due to the processing temperatures of over 200-250°C for UHMWPE, the susceptibility of
polymer oxidation is of major concem. However, thermal oxidation is also a concem in vivo. It
has been proposed that f?ee radical reaction is accelerated due to fictional heating and stress in
the loading zones [Jahan et al., 19911. T'us, it has been reported that there is uneven oxidation
found throughout the explanted specimen [Sutula et al., 19941. This fictional heating is
consistent with observations of temperature rise in acetabular cups during in vitro fictional Wear
stress tests and in vivo telemeûy observations [Jahan et al., 199 1, I3avidson2 et al.. ! 9881. A
raise in temperature between 5 to 15°C [Davidson et al.. 1987, ~avidson' et al., 1988, ~ a v i d s o n ~
et al., 19881 was found in other shidies. Other studies found a raise in temperature of 4 to 7°C in
the overall materiai dependhg on the type of articulating surfaces and an increase of up to 50°C
at the articulating surface Fanzer et al., l992].
2.5.5 Mechanism of UHMWPE Degradation
The mechanism for fiee-radical induced degradation of UHMWPE in the absence of oxygen [Ali
et al., 19941 involves the following steps: initiation, propagation and termination (see Equations
2.1 to 2.3). In the initiation reaction, energy is absorbed from an extemal source, causing the
scission of a covalent backbone or cross-luik (see Equation 2-1). The activation energy needed
to cleave a chah varies fiom 30 to 90 kcaVmol Pigger et al., 19921. Such reactions generally
require heat, U V Iight [Bigger et al., 19921, or high-energy radiation, preferably in the presence
of oxygen, to proceed [Ali et al., 1 9941.
Several different radicais cm be fonned and a few are listed in Table 2.7. M e r radicals include
the hydrogen radical formed by the cleavage of C-H bond. The ability of these radicals to
propagate the fiee radical reaction depends on their mobility and the available fiee volume within
the matetid (i.e. crystalline areas do not ailow for chah mobility). Also, the accessibility of fiee
radicals to other fke radicals and interactions with other reactive species, such as oxygen,
lpremnath et al., 19961 will depend on the fke volume. Propagation occurs by converthg the
polymer entirely to monomer or by the radicals abstracting a n e i g h b o ~ g hydrogen atom so that
the radical is transferred to another chain or M e r down the same chah (Equation 2-2).
Propagation is important in fkee-radical degradation because in the physiological environment, it
is believed that there is an abundance of fiee radicals [Ali et al., 19941. Termination occurs by
the bimolecular homo or cross reaction of the radicals as shown in Equation 2-3 [Fodor et al..
19911.
Initiation Step:
Propagation:
Termination:
Figure 2.3: Free Radical Poiymer Degradation [Ah et aL, 1994, Fodor et aL, 19911
In the presence of oxygen, Fodor et ai. (1991) presented the following mechanism for the
oxidation of polyethylene to fom carboxylic acid groups. In the initiation step (Equation 2-4),
the -CH - - radicals are formed during propagation, these radicals are oxidized to form c h a h
carrying peroxyl radicals (Equation 2-5). The reaction of the peroxyl radicals with the substrate
results in two pathways as seen in Equations 2-6 and 2-7. The first is polymer hydroperoxide
formation and the second is the formation of carboxylic groups with simultaneous chah scission.
It has been observed that 80% of the polymeric hydroperoxides are converted into end products
(Equation 2-6). The alkoxyl radicals with peroxyl radicals are the precursors of carboxylic acids
in polyethylene oxidation. Aldehydes c m aiso be active intermediate products of the process
because their oxidation reaction is rapid.
Initiation of a fiee radical:
The fiee radicals react with oxygen to form a peroxyi radical:
The peroxyl radicals propagate the reaction or fom allcyi radicals (chah ends):
OOH -CH- ' 1
L ' OH
l OH
-c- --c- l I I
- c + CH* (2-7)
OOH 0' II O
There is a significant body of evidence that supports the hypothesis put forth by I2irnnac2 et al.
(1994) which states that "the mechanical properties and physicd properties of irradiated
polyethylene are affected by both physiological Ioading and environment and that these effects
are interactive". Not only are there complex issues regarding the oxidative degradation of
UHMWPE as a bulk material, there are compiex issues involving the stability of UHMWPE
Wear particles that have not been addressed in the literahire. In general, the effect of particles on
biocompatibility has been stuclied, but there is no literaîure that discusses the effect of
chemicdly-altered (oxidized) UHMWPE particles on biocompatibility.
2.6 Biocompatibility , Biomaterial Interactions & Formation of Superoxides
2.6.1 Defmition of Biocompatibüity
The concept of biocompatibility has long been a subject of debate [Ratner, 1993, Boss et al.,
1995, Williams, 1986, 19871. Williams (1 986) proposed that the definition of biocompatibility is
"the state of affairs when a biomateriai exists within a physical environment, without either the
material adversely and significantiy affecting the body, or the environment of the body adversely
and signiticantly affecthg the material". Williams (1987) also proposed another definition as the
following: "the ability of a material to perform with an appropriate host response in a specific
application". Ratner proposed a dennition in terms of the expectations of the biomaterial as "the
exploitation by materials of the proteins and cells of the body to rneet a specifïc performance goal"
[Ratner, 1 9931. By the very nature of all these definitions, the term biocompatibility is elusive.
No material can be completely biocompatible, but it can be more biocompatible than another
[Boss et al., 19951. Therefore, Boss and colleagues proposed that biocompatibility is not just a
chemicai composition property of the biomaterials, but also depends on physical attributes.
2.6.2 Bioactive, Biotolerant & Bioinert
In the context of host-implant interactions, biomaterials are classified as biotolerant, bioinert, or
bioactive Furlong et al., 19911. In terms of orthopedic prostheses, implant materiais can be
considered biotolenint when there is ody an intervening fibrous layer formed at the host-implant
interface. It can be bioinert when the bone achieves direct contact as seen with titanium; or
bioactive when there is true coalescence at an atornic level. The three types of fixation are
determïned by surface characteristics that c m be termed distance osteogenesis, contact
osteogenesis and bonding osteogenesis, respectively Furlong et al., 1 99 1 1.
It is known in reconstnictive orthopaedic surgery that al1 implants cause an inflanmatory
response Poss et al., 19951 on implantation following surgical trauma Imrnediately after
implantation, hydrophobie materials acquire a layer of host protein [Tang et al., 19951. Plasma
or interstitiai fluid proteins rapidly coude with the matenai surface and bind strongly to the
sufface [Tang et al., 19951. Therefore, the implant is spontaneously coated with a random layer
of denatured and pattiaily denatured proteins. This protein layer influences M e r responses to
the implant and determines m e r biocompatibility [Tang et al., 1 9951.
2.6.3 Bulk versus Particulates: Effect on Inflammatory Response
The effect of the geometry of a biomaterid on the host response is well documented in the
literature. Modem biomaterials in bulk form with distinctive geometry are generally well
accepted by the tisse environment [Cioodman2 et al., 19921 with an inert fibrous encapsulation
present around the bulk of the implant [~oodman' et al., 19921. UHMWPE, as a bulk material,
can be classified as biotolerant based on its chemical composition [Boss et al., 19951. It has been
shown to produce littie inflammatory response in vivo marchant et ai., 19861 compared to the
respoase seen with particles. Not only are dflerent material particles engulfed by cells, but in
general, their breakdown products cause inflammatory and grandornatous reactions [Boss et al.,
1995, Santavirta et al., 19931. An inflammatory response for particles composed of UHMWPE,
titaniurn and cobalt chrome alloys is elicited according to Howie et al., 1988 [~oodman~ et al.,
19901. However, Goodman and colleagues (1990) found that metal particles did not elicit an
inflamrnatory response. To the contrary, polyethylene and PMMA particles incited a foreign
body response similar to that seen surroundhg faiied joint arthroplasties in humans [~oodrnan~
et al., 19901. Other events at the host-implant interface are affected by the chemical,
topographical and geometric nature of the surface of the device and the amount of deposited Wear
particles [Boss et al., 19951. These factors cari induce tissue reactions, which can be followed by
prosthetic failure.
One such tissue response is the formation of a synovial-like membrane at the interface between
the loose, non-septic failed total hip replacement. This membrane had the histological and
histochernical characteristic of the synovium (îe. the membrane lining the joint containing
lubncant). The significance of this membrane is its ability to produce prostaglandin E2 and
collagenase. These biomolecules have k e n implicated in bone resorption around the implant
which leads to progressive loosening of the implants and clinical failure of the device. [Goldring
et al., 19831. Previously, loosening had been attributed ody to mechanical factors such as poorly
supported acetabular components and hadequate distribution of cernent around the femur.
The inflammatory reaction to total hip replacement Wear particles is well-documented [Tang et
ai., 1995, Schrnalzried et al., 19921. This problem has been associated with an elevated rate of
implant failure and re-operation &er 10 years (between 10% and 30%). 0- linear (diffuse)
andl or lytic (localized) areas of periprosthetic bone loss have been identified at the sites of failed
implants [Schmaizried et al., 19921. Furthemore, the degree of bone lost has been indicative of
the local concentrations and distribution of debns [Schmalzried et ai., 1 9921.
2.6.4 Phagocytic Reaction to Particles
One estimate of the average Wear rate of UHMWPE is 0.1 mm per year which resdts in 20
million particles produced per &y or 7 biiiion particles per year [Tang et ai., 19951. ui addition
to the concern of wearing out the lining of the implant, there is a concem of the amount of Wear
debns produced of phagocytosable size (i. e. less than 10 jm, r(roronov, et ai., 19971). The size
of the particle determines in part the nature of the infiammatory response. Large Wear particles
(up to several hundred microns) induce fibrous or giant ce11 reactions [~oodman' et al., 1992,
Amstutz et al., 19921 while small particles (<MO p) are phagocytosed by macrophages [Tang
et al., 19951. Phagocytosis is initiated by binding to surface recepton of particles greater than
0.5 p in size, otherwise picocytosis (îe. absorption of small particles without the formation of a
pseudopodia or a membrane extension) occurs [shanbhaglet al., 19941. There is a range of
partich sizes found at the site of the implant. Some authors [Kobayahi et al., 19971 have found
that the particles range fkom 0.40 to 1.15 pn while others [Bosco et ul., 1994, Mahoney et al..
1 9951 have reported them in the range of 1 00 p in size.
in addition to the size, other factors such as chernical composition, concentration [shanbhag2 et
al., 19941 and geometry watlaga et al., 19761 of the particles induce different inflammatory
responses. The diEerent materials in a total hip implant al1 cause different degrees of
inflammatory response. Earlier cesearchers thought that the cernent material (methyl
methacrylate) used in cemented total hip replacement was responsible for initiating bone
destruction based on histological hdings [Goodman1 et al., 1992, Amstutz et al., 19921. It was
hypothesized that the mechanical forces at the interface led to localized cernent fractures, which
generated particdate PMMA. These particles then initiated the foreign body response which led
to bone resorption. Upon revision arthroplasty procedures, surgeons commonly found a thick
membrane at the bone-cernent interface [Goodman1 et al., 19921. Upon M e r examination, the
membrane was composed of fibrous tissue stroma with histiocytes, and giant cells surrounding
and engulfing particdate PMMA, polyethylene and metal debris [Goodman1 et al., 19921. The
presence of PMMA particles promoted the use of cementless implants [Schrnaizried et al, 19921,
however, there has been no clinical or experirnentd evidence to suggest that the use of
cementless THR prostheses with polyethylene sockets has prevented the adverse biological host
response [Santavirta et al., 19931.
Several studies [Santavirta et al., 1993, Schmalzried et al., 1992, Davidson et al., 19931 have
shown that d l paaiculate materials fiom the implant (PMMA, UHMWPE, titanium, cobalt-
chromium, A1203 and 2k02 ) can induce a foreign body reaction to varying extents. Studies
show that cementless implants still release UHMWPE and metal particles fiom the articulahg
surfaces, and these materials may be the primas, trigger for macrophage-induced osteolysis
[Amstutz et al., 19921. Several groups have M e r suggested that polyethyiene Wear debris is a
main factor in triggering the innammatory response [Boynton et al., 199 1 , Schmalmed et al.,
19921. They found that polyethylene activates the osteolytic membrane containing macrophages
and foreign-body giant cells around implants. Boynton and colleagues have advocated a
mechanism for the activation of white blood cells and bone Iysis which follows the following
sequence of events:
Micromotion contributes to the formation of metal and polyethylene debris in
excessive amounts.
This debns activates the macrophages located within the membrane and stimulates
the production of foreign-body giant cells.
These cells attempt to destroy the polyethylene debris and simultaneously secrete
prostaglandin E2, collagenase and other pro-inflammatory indictors.
These cell processes in turn activate and accelerate local bone resorption and
perpetuate the cycle of loosening.
The particle concentration found at the implant site has been identified as another important
factor in initiating the inflammatory response. Some researchers [Davidson, 1993, Murray et al.,
1990, Boynton et al., 1991, Schmalzried et ai.., 19921 have proposed that there is a threshold
level of particdates that the body is able to tolerate and is dependent on the type, size, and
geometry of the debns. Above this level, the degree of macrophage activation results in the
activation of the bone resorption process. Thus, minimizing the rate of Wear is needed to
improve both the long-terni stability of the prosthesis and the local bone structure.
2.6.5 Monocytes/Macrophages, Neutrophils & the Idammatory Response
Neutrophils and monocytes are the most mobile and active phagocytic leukocytes of the
idammatory response. Although the neutrophils are unable to ingest particles larger than
bacteria (1 to 3 p), monocyte-derived macrophages can enguif relatively large objects (3 to 10
p). Both types of phagocytes contain numerous lysosomes that are filled with digestive
enzymes capable of breaking down various organic molecules [Hole, 19873. Monocytes are the
precurson of tissue macrophages which are seen in intenacial membranes (bonekement or bone
/prosthesis interfaces) arouad the components of failed total joint replacements [Shanbhag et al.,
1 9951.
Mammation mediated by activated macrophages is one of several hypotheses that have been
proposed to explain the mechanism of aseptic loosening with endoprostheses [Kossovsb et al.,
19911. Dating back to the t h e of Sir John C h d e y , it was noted that "the presence of
macrophages at the interface is a tissue response that no implant surgeon can lightly dismiss"
[Amstutz et al., 19921. The evidence which irnplicated macrophages as the causative agent of
bone loss and thus failed implants was outlined by Amstutz and colleagues:
Macrophages are inevitably present in the tissue around loosened implants, usually
with an abundance of intracellular polymeric and metallic Wear debris.
Macrophages are hown to secrete products that can cause bone lysis, and they cm be
induced to produce these agents after phagocytosis.
Tiny scalloped edges are observed in the bone that correspond in size to the adjacent
macrophages and that are seen in the absence of osteoclasts.
Schrnalzried and colleagues found that the number of macrophages had a direct relationship to
the degree of bone resorption found in prosthetic replacements [SchmalPied et al., 19921.
When the idammatory response is triggered, macrophages residing in the connective tissue
become mobilized and begin to attempt phagocytosis of the foreign matter. These macrophages
appear on the surface of most implants [BelIon et al-, 19941 and are known to excrete products
such as superoxides that can cause bone lysis [Kossovsky et al., 199 1, ~oodman' et al., 19921 or
bone resorption when they phagocytose particles [Murray, et aL, 19901. The inflamed tissue
releases cytokines and other ce11 mediators that are carried away by the blood and stiniuiate the
release of many white blood cells nom the bone marrow. Within a few hours, large numbers of
neutrophils migrate into the innamed tissues and act as phagocytes. During phagocytosis,
neutrophils produce a variety of oxidants which cause injury to the surrounding healthy tissues
~ l i s s ' et al., 199 11. As the inflammation subsides, macrophages clean up the cellular debns by
phagocytosis [Hole, 19871. Studies have shown macrophages at the implant and tissue interface
are still present 12 weeks d e r implantation peiion et d , 19941.
During phagocytosis, chemicals secreted within the ceUs and into the surrounding environment
may M e r induce degradation [Boss et al., 19951. Phagocytes release proteins such as
lysozymes, peroxidases, elastase as well as oxidants such as superoxides, hydrogen peroxide,
hypochlorous acid, and hydroxyl radicais mosen et al., 1995, Lasser, 19831 as seen in Table 2.8.
These highly reactive species are the main mediators of tissue damage during infiammation
including lipid peroxidation, DNA scission, and protein oxidation ~ossovsky et ai., 199 11.
Table 2.8: Secretory Products of Mononuclear Phagocytes passer, 19831
A. E n y m u Lysozyme
Ncutrai protcascs Plasminogcn activator Collagrnase Elastase Protcugiycan dcgrading protcase Angiotcnsin
Acid hydrolasts Protcases Estcrasts Lipases Sulfatascs Ribonucleases Phosphatases Glycosidases Caîhcpsins
B. Complement Compoaenîs C. Enzyme Inhibitors
Plasmin inhibitor u-M8~t0giobulin
D. Binding Protcin Transfmin Ferritin
Transcobalamin ï i Fibmneain
E Oxygcn Metabolites Supcroxidts Hydrogcn pcroxide Hydroxyl radical Singlct oxygcn
F. Biorctivc Lipids hostaglandins Thromboxant Ltukotritnes Platclet activating factors
G. Endogenotu Pyrogcn H. Activating Factor
Colony-stimulating factor Lymphocyte activating factor Of crythroid p~cufsors Of fibmblasts Of microvascuianirt
I. Inhibiting Factors Inttrfmns Of aunor Of IP~hocytEs Of LUteria monocytogenes
Neutrophils are leukocytes that con& of the "fint line of defense" against infectious agents or
"nonself" substances that penetrate the body's defense [Smith, 19941. Theu role is to
phagocytose and destroy infectious agents [Smith, 19941. During phagocytosis, cytosolic
granules fuse with the invaginating plasma membrane to form a phagolyosome into which they
release their contents, thereby creating a highly toxic environment [Smith, 19941 as seen in
Figure 2.4. The highly toxic environment includes the production of three main oxidants (Table
2.8):
superoxide anion
hydrogen peroxide
hypochlorous acid
The most toxic of these oxidants has k e n identified as hypochlorous acid ~ l i s s ' et al., 199 11. 1 t
cm cause direct oxidation and inactivation of cellular proteins and mediate indirect oxidation
with transitional metals such as ~ e ~ + or cu2+ which can undergo reduction and oxidation
reactions to produce reactive hydroxyl fiee radicals ~ l i s s " et al., 199 11 which have unlimited
reactivity toward biomolecules [Rosen et al., 19951. However, some particles are too large to be
enguifed, and this leads the macrophages to undergo a state of "hstrated phagocytosis" in which
no phagosome is formed [Smith, 19941. The steps of '"fnistrated phagocytosis are as follow. The
ce11 tries to engulf a particle which is too big. This leads to ce11 death and the production of
necrotic ce11 debris and particles. This ce11 debns is enguifed by other macrophages and can lead
to ce11 death. In this case, there is an extracellular release of products which can lead to tissue
damage [Weiss, 1 9891.
The oxidative burst of neutrophils and macrophages is triggered by phagocytosis and results in
the sequential production of a variety of microbiostatic and microbicidal reactive oxygen species
as seen in Figure 2.4. The superoxide (O2? is fonned by the univalent WcCord, 19831 reduction
of molecular oxygen by single electrons from NADPH. Other more potent reactive oxygen
species formed by the superoxides are hydrogen peroxide, hypochlorous acid and the hydroxyl
radical (OH-) ~os sovsky et al., 199 11. At physiological pH, superoxides rapidly dismutate to
hydrogen peroxide [Rosen et d, 19951 with or without the presence of an enzyme ~ c ~ o r d l ,
19741. Hypochlorous acid is formed by the reaction of hydrogen peroxide with chloride ions
fiorn the extracellular fluid catalyzed with myeloperoxidase Weiss, 1989, Rosen et al., 1995,
Kettie et al., 19891 in monocytes and neutrophils, but macrophages do not produce this enzyme
[Rosen et ai., 19951. As weil, secondary chlorinated amine compounds are generated with
reactions between HOC1 and nitrogen-containing compounds Weiss, 1989, Smith, 19941. It was
found that 30 to 70 % of the hydrogen peroxide can be converted to HOCl depending on the
experimental systern in use [Smith, 19951.
O2
lysozyme
pro teasese
azurocidin
de fensins
Degradation Product
Figure 2.4: Environment in phagolysosome [Smith, 19941 for both monocytes and
neutrophik (Rosen et aL, 19951.
The stimulation of human monocytes has been show to be a function of the composition and
concentration of the particles [Shanbhag et al., 19951. When experiments were carried to study
the effect of several orthopedic material-derived particles (less than 1 p) on their ability to
induce monocytic response, it was found that titanium-6% aluminum- 4% vanadium (TiAiV)
induced the greatest release of inflammatory mediators; pure titanium and fabncated UHMWPE
caused a moderate effect and particles of UHMWPE retrieved fiom intefiacial membranes of
failed uncemented THR were less stimulatory in short term in vih-O experiments.
Other cellular products secreted by the macrophage are grouped into the broad category of
cytokines. They encompass colony-stimulating factors, intederom, growth factors and
interleukins. Cytokines are defined as "a soluble glycoprotein, non-immunoglobuiin in nature,
released by living cells of the host, which act non-e~lzymatically in picomolar to nanomolar
concentrations to regdate host ce11 function" [Gowen, 19941. The cytokines of interest are
interleukin-1 P, interleukin-6 and -or necrosis factor-a. In aseptic loosening of implants, these
cytokines have been implicated to help stimulate the resorption of bone [Horowitz et al., 1 9951.
ln summary, it has been shown that UHMWPE is susceptible to oxidative degradation. This
degradation, through fiee radical reactions, serves to change the mechanical properties of
UHMWPE by increased density, decreased fatigue strength, embrittlement and finally increased
the susceptibility of UHMWPE to Wear mechanisms. The Wear mechanisms produce particles of
UHMWPE dong with other metal particdates that have been shown to induce inflammatory
responses. The generated particles are by-products of oxidative degradation through increased
Wear susceptibility. Hence, the particles that interact with inflammatory cells likely exist as
pda l ly oxidized particles. There is no literature to date that has confirmed this. In addition,
investigatoa have concentrated on determining the effect of unaltered resin on the inflammatory
response. As well, there is no literature to date which addresses the combined relationship
between the effect of trace metals and üHMWPE on the inflanmatory response. However, there
is much literature which addresses the macrophage/monocyte response to UHMWPE and metal
particulates separately. Thus, it is an objective of this study to determine the effect of
chemically-altered polyethylene on the secretion of ce11 mediators (cytokines) that have been
suspected to cause bone resorption.
It has also been shown in general for other biomaterials that d e r phagocytosis of particles,
degradation products can M e r induce inflammatory responses. This has not been s h o w for
UHMWPE. The determination of oxidative and hydrolytic degradation products of UHMWPE
particles will be the main focus of this study. Future studies will determine the effect of these
degradation products on their ability to induce inflammatory responses.
3.0 MATERIALS AND ~METHODS
3.1 Material Selection
The two types of polyethylene used in this study were ultra-hi& molecular weight polyethyiene
(UHMWPE) and hi& density polyethylene (HDPE). UHMWPE was studied as a paaiculate
form and as a film fonn. The UHMFKPE particles were obtauied fiom the Hospital for Speciai
Surgery (HSS), New York, NY and the HDPE particles were obtained fkom Shamrock
Technologies Inc., Newark, NJ. The properties of these particulates are listed in Table 3.1. Film
experiments were also conducted with ram-extnided UHMWPE bar (HSS). The bar stock HSS
was cylindrical in shape with a diameter of 7.5 cm. Its processing history and properties are
listed in Table 3.2.
Table 3.1 Material Propehes for the UIMWPE
and HDPE Particles
1 Resin Lot f N/A
Property
Resin Resin Manufacturer
Note: This is not medical grade polyetf
Shamrock* HDPE S-395 (N2 md) Sharnrock
-
HSS UHMWPE 4150 HP Hoechst Celanese
Table 33: Material Properties of UHMWPE bar stock
I HSS, PolyHi Solidur, Hoechst Celanese Reference UHMWPE
Property Resin Resin Manufacturer Processor Form
I lot # 1 337x I
RSS 4150 HP Hoechst Celanese PolyHi Solidur Rod
1 Processing 1 Ram-Extruded 1
ïhe specific gravity was performed relative to water which had a specific gravity of 1.00 at 25°C.
Meltiog Temp. Density, R/mL
135.6"C 0.93
35
UHMWPE was selected as the primary matenal of study since this type of polyethylene is
commody used in the fabrication of aiticular cornponents in total joint replacements. Both the
particles and films were used in the in vitro biodegradation experiments. However, the ce11
studies reported in this thesis required that the particles be d l enough to allow for ce11
phagocytosis of the particles to occur. Since UHMWPE was not readily available in this size
range, this aspect of the work required an alternative. HDPE was selected for this purpose
because it could be commercially procured and was available in 4 to 10 p size distribution.
While the HDPE was not medicd grade, other studies have used this material to investigate cell-
particle interactions and have not reported any toxic behavior [ ~ o o d m a n ' ~ et al, 19961.
Furthemore, Voronov poronov et al.. 19981 found no endotoxins present and have shown that
the material had similar chemical characteristics with UHMWPE, as measured by both X-ray
photoelectron spectroscopy (XI'S) and Fourier transform h f k e d spectroscopy (FTIR) analysis.
3.2 Test Sample Preparation
3.2.1 Preparing Fiims from Bar Stock
n ie bar stock was first sectioned with a band saw into usable blocks (1.5 cm x 2 cm x 1 cm).
Films with the dimensions of 2 x i cm and a thickness of 2 0 p were sliced using a Reichert-
Jung 2050 Microtome with a tungsten-carbide steel blade. Mer films were sliced, they were
placed in a 37°C water bath to uncurl. The samples were clamped between two glass slides
overnight to flatten the sample. Al1 the film sample preparation was performed using facilities at
the Department of Pathology, Mt. Sinai Hospital, Toronto, Ontario, Canada.
3.2.2 Sterilization of Samples
Sterilization by irradiation is the most common method used to sterilize UHMWPE cornponents.
Other methods such as ethylene oxide treatment are also in use, but there has been a cal1 by the
FDA to phase out its use due to the toxic residues which remain following sterilization [Hui et
al., 19971. Samples were y-irradiated with a 2.5 Mrad cobalt-60 source in the presence of air at
the Department of Chernical Engineering, University of Toronto. Sterilization under nitrogen
atrnosphere was not available at these facilities. The selected dosage of radiation is standard for
the sterilization of al1 medical implant systems [streicher12, 19881.
36
There have been snidies wbich suggest that storage thne after irradiation increases the
mceptibility of UHMWPE oxidation [Kurtz et al., 19941. Thus, al1 samples within a set of
experiments were irradiated together and stored at room temperature for sllnilar perïods pnor to
use in experiments.
3.2.3 Coating of Particles with Cobalt Chloride
Experiments were carried out to determine the effect of metal ions on the oxidation of
UHMWPE. Cobait ion was of pmticular interest since analytical studies of UHMWPE have
shown the presence of cobalt ions at the interface between the UHMWPE cup and the cobalt-
chromium bail in total hip replacements Prodner et al. 1997. In other studies, cobalt ions have
k e n shown to catalyze the oxidation of polymers [Zhao et al., 19951. Cobalt chioride was
selected as a source of cobalt ion for the work in this thesis.
The mass ratio of cobalt chioride to UHMWPE particles was 89100. The cobalt chloride (ASC
reagent grade, BDH Inc., Toronto, Ont.) was added to the polymer in two 50 mi, centrifuge tubes
and approximately 80 mL of HPLC grade ethanol (Sigma-Aldrich, St. Louis, MO) was used to
dissolve the cobalt chloride. The tubes were vigorously stirred using a Vortex-Genie mixer
[Scientific Industries Inc., Bohemia, NY] and the solutions were poured into twelve glass petri
plates. KimwipesB were secured to the top of the petri plates to prevent contamination of the
samples. Care was taken to ensure the Kimwipe@ was not saturated with the solution. The peûi
plates were left in the fume hood for two days until the alcohol was completely evaporated.
The powder was scraped off the petri plates into a mortar and evenly mixed using a pestle. The
powder mixture was then washed using 20 mL of HPLC-grade water and a glass rod. The water
was filtered fiom the powder in a filtration system with a 0.5 p Teflon filter, # FHüP04700
fiom MiIlipore Corporation (Bedford, MA). This was performed three times. The Hter was
used to collect the particles. The filter which supported the powder was placed in a covered petri
plate and left to completely dry in a 37°C oven overnight.
The amount of cobalt chlonde on the surface was analyzed by Scanning Electron Microscopy
(SEM), Energy Dispersive Analysis X-Ray (EDAX) combined with X-Ray Photoelectron
Spectroscopy W S ) and Fourier Transform uifirared (F'TR) Spectroscopy.
3 3 Characterization of Test Specimens
3.3.1 Surface Morphology of Polyethylene
Estimates of the particle shape and sue range were determined by scauning electron microscopy
(SEM) using a Hitachi (mode1 2500) scanning electron microscope (Faculty of Dentistry,
University of Toronto). The instrument was operated by Mr. Robert Chemecky, Department of
Biomatenals, Faculty of Dentistry. The samples were mounted onto cylindncal sample holders
(stubs) using double-sided carbon tape and sputter-coated with a 3.0 m layer of platinum using a
POLARON SC5 15 SEM coatiag system (Polaron Instruments Inc., Doylestown, PA). Sputter-
coating was carried out in order to inhibit charging, reduce thermal damage and improve
secondary election emission.
3.3.2 Chernical Composition of Polyethylene
The chernical composition of the polyethylene samples was determined by X-ray Photoelectron
Spectroscopy (XPS), Fourier Transforrn Infrared Spectroscopy @TIR) and Energy Dispersive
Analysis X-Ray (EDAX). The FTIR analysis protocols will be discussed in section 3.4.3.1.
3.3.2.1 X-Ray Photoelectron Spectroscopy (XPS)
X-ray photoelectron spectroscopy is a common method used to determine the elemental
composition of the surface of a material. It can detect up to a depth of 10 nm depending upon the
take-off angle between the x-ray source gun and the sample surface.
Prior to analysis, the poiymer films were washed with 1,1,2 trichlorotrifluoroethane (Sigma-
Aldrich, St.Louis, MO) to remove any silicon-containhg contamination, rinsed well with double-
distilled water, covered and placed in a vacuum oven ovemight at 37OC to remove my water.
Both films and particdate samples were mounted on copper tape to hold the sample in place at
the tirne of analysis.
The XPS instrument was operated by Dr. Rana Sodhi. The instrument consisted of a Leybold
MAX 200 XPS system with an unmonochromated MgKa x-ray source (Centre for Biomaterials,
University of Toronto). Survey, low resolution and hi& resolution scans were perfonned for
carbon and oxygen as well as silicon-based contaminates. The sensitivity factors for each
compound were deterrained empincally by the maaufacturer. The scans were calibrated to the
38
carbon-carbon bond located at 285.0 eV. Data analysis was performed in Dr. Santerre's
labonitory using the program ESCA Tools interfaced with MatLABQ for Windows, Version
4.2b.
3322 Energy Dispersive Anaiysis X-Ray (EDAX)
EDAX is used to determine the elemental metal compositions of elements with a greater
molecular weight than sodium. Hence, the presence of cobalt and chlonde on cobalt chloride
treated particle samples codd be detected. EDAX spectra were obtained by Mr. Fred Neub (Pratt
Building, University of Toronto) ushg a Hitachi Scanning Electron Microscope operating with a
15eV x-ray beam coupled with a LZ-5 Link Analytical attachrnent (S-570 Link ANI O, England).
The cobalt chloride coated particles were mounted onto steel stubs using carbon tape. Since the
particles were not flat, they were sputter-coated on three sides (Figure 3.1) with carbon using an
Edwards Coating System E306A, Britain. Triple coating was applied to obtain an even coat in
al1 directions.
Figure 3.1: Direction of carbon sputtering on the surface of the sample mounted on stud.
3.4 Oxidation of UEIMWPE
There is concern of the degradation mode by which UHMWPE occurs since it is the most
common articular components used in total joint replacements. Oxidative degradation has been
identified as the prevalent method of UHMWPE degradation &i2 et al., 19941. Many studies
have shown that the oxidation of UHMWPE in total joint replacements can promote
destabilization of the implant. The oxidation of UHMWPE arises fiom many sources. Themal
oxidation is of concem during the processing of the implant B. Wang et al., 19881 and during
normal articulation [Jahan et al., 199 11. m e r methods of oxidation include y-irradiation that
has been show to cause the production of fiee radicals in the system and help to propagate the
chah scission. Chernical oxidation is also of concern due to the presence of salts and oxidants
39
such as hydrogen peroxide in the physiology environment. The piapose of this study was to
oxidize m E particles and to isolate and identify the degradation products produced by
both oxidative and hydrolytic methods. Film samples were initidy used to determine the
protocols. In addition, the synergistic effect of chernical oxidation and cobalt treatment of
TJHMWPE on the particles was assessed.
3.4.1 Oxidation by Thermal Treatment
Oxidation by heat-treatment was performed to determine if a relationship existed between
oxidation index and incubation time at dif5erent temperanires. Samples of UHMWPE were
placed in an air-flow oven for various t h e periods and temperatures (i.e.. 37OC, 60°C, 80°C and
10S°C). The 37OC was chosen because it is the human body temperature. The temperatures of
60°C, 80°C and 105OC were chosen because they were used in previous accelerated aging
experiments for UHMWPE [Poggie et al., 1997, McKeilop et al., 1997, Dwyer 19961. The
samples consisted of both films and particles with some samples subjected to 2.5 Mrad of
irradiation. A listing of the heat-treated samples and their nomenclature is given in Tables 3.3
and 3.4, respectively.
Table 33; Experimental Conditions for UHMWPE Particle samples* I
- Al1 samples were performed in triplicate.
Ternperahires
irradiated control
non-hdiated control
37°C
Sample Series
C2
C 1 1
100 -106
Table 3.4: Experimental Conditions for UHMWPE Film Samples'
l Temperature I 1 Time @OUR) 1 60°C 1 80°C 1 105°C 1
1 O (irradiated) l c2 l c2 l c2 l 1 120 (5 days) 1 N/A 1 N/A 1 fWd-1, 2,3 ( 864 (36 days), irradiated
864 (36 days), non-irradiated
11440 (60 days) non-irradiated 1 f60n%-13,3 1 Bon%-1 2 ,3 1 NIA 1
f60J92,3
1
1440 (60 days) irradiated If60%-1,2,3
3.4.2 Chernical Oxidation of UHMWPE
The particles and films were subjected to two oxidants, sodium hypochlorite and hydrogen
peroxide. Studies have shown that these two chernicals are present in signincant amounts during
the inflamrnatory response at the site of the implant [Weiss, 19891. The sodium hypochlorite
concentration used was 7.6 mM, which is withùi the concentration range found at the site of
macrophages and neutrophils pliss et d, 1 99 1, Weiss, 1 989, and Sutherland et al., 19931. The
hydrogen peroxide concentration chosen was 10 w/wO/o. Miile this concentration is very high as
compared to what may be expected physiologically, it was chosen simply to obtain preliminary
data that reflected the effect of chernical oxidation. It did not necessarily reflect specific
intercellular conditions. Future work will M e r explore the importance of concentration on the
process and the model. Similar concentrations have been used by others in the study of oxidation
of UHMWPE implant components [RUnnac et al., 1994 and Pascaud et al., 19961. The
nomenclature for the samples and their treatments are given in Table 3.5.
f6OnJ,2,3
Al1 sampies were formed in triplicate. N/A experiinents were not performed
fû0-1,273
BO%-1,2,3
NIA
f80-1,2,3
N/A
N/A
Table 3.5: Chernicd Treatment of FiIm Samples
Treatment of Film Samples
1 positive control: 5 &y heated irxadiated sample at 105°C 1 1 36 days irradiated sample in 10 w/w% H2& I
H202n 1 36 days non-irradiated sample in 10 w/w% H2@ 1
3.4.2.1 Determination of Sodium Hypochlorite Activity
A spectrophotometric assay for chlorine-containhg compounds was obtained by Dr. Rosalind
Labow (University of Ottawa, Ottawa, ON) and was adapted fiom the methods of four research
articles [Weiss et ai., 1982, Shacter et al., 199 1, Kukreja et al.. 1989 and Thomas, 19791. The
method was based on the reaction of taurine with HOCVOCI- to form a long-lived compound,
taurine N-monochIoramine (TaUNHCl), as defined below:
HOC1 + HzN-CH2CH2SOsH - CNH-CH2CH2S03H + H20 Taurine Taurine chloramine
Equation 3- 1
Taurine chloramine was able to oxidize potassium iodide to iodine. The resultant iodine reacts
with excess potassium iodide to form periodide as seen in the following reaction:
Equation 3-2
The penodide ion can be detected spectrophotometrically at 3 50 nm [Alexander, 19621.
Table 3.6: Sources for Chernicals Used in HOC1 Assay
The chernids used in this assay and their sources are given in Table 3.6. The assay was carried
out using a spectrophotometer (LKB Biochrom, model: Ultmspec 11, No.# 4050, Cambridge,
England) with a 3 mL quartz cuvette. To 500 pL of a pH 7.4 phosphate buf5er solution (see
preparation in Appendix A), the following aliquots were added: 150 pL of 65mM taurine
solution, 250 pL of 120 mM potassium iodide solution, and 1 mL of the test sarnple containing
HOCI. The cuvette was covered with a cuvette cap and mked well. A calibration curve was
determined by varying the concentration of sodium hypochlorite present in the cuvette and
meaniring the corresponding absorbance (see Appendix C).
3-4-22 Determination of Hydrogen Peroxide Activity
Hydrogen peroxide activity was determined using a method based on the specîrophotometric
detemination of 1-3 formed when hydrogen peroxide is added to a solution of iodide (13
rneasured at a wavelength of 3 5 1 nm [Klassen et al.. 1 9941. The reaction proceeds as follows:
EIzo2+21-+2H' - Iz+2HzO Equation 3-3
and the iodideliodine species are in equilibrium:
The chemicals for this assay are located in Appendix A. Solution A was made in a 500 mL
volumetric flask and contained: 33g of potassium iodide, lg of sodium hydroxide, and O.lg of
ammonium molybdate tetrahydrate dissolved in filtered water. This solution was stirred for 10
minutes to dissolve the molybdate and poured into a foil-covered bottle to prevent photochemical
oxidation of I'.
Solution B contained 10 g of potassium hydrogen phthalate dissolved in 500 mL of water in a
volumetric flask. A calibration curve was created using equal amounts (by weight) of solutions
A and B and varying the concentration of hydrogen peroxide (see Appendix C). The absorbance
of the resultant solution was then rneasured at 35 1 nm. The chemicals and their suppliers are
given in Table 3.7.
43
Table 3.7: Chernicals for Hydrogen Peroxide hsay
1 potassium hydrogen phthalate, 99.95%, 1 Aldrich Chernical Company, Milwaukee, W 1
Chernicd Supplier
potassium iodide, 99.99+%
sodium hydroxide, 99.99%
ammonium molybdate tetrahydrûte, ACS
reagent grade
3.433 HaifWe Study for Aypochlorous Acid and Hydrogen Peroxide
A half-life study was performed to detemine the activity of the oxidant over the incubation
period when in the presence of the polymer. The half-life value was chosen to be the time at
which the samples would be replenished.
Aldrich Chernical Company, Milwaukee, WI
Aldrich Chernical Company, Milwaukee, WI
Aldrich Chemical Company, Milwaukee, WI
hydrogen peroxide, 30 w/wO/o,
Using a Mettler-Toledo analytical balance (Greifensee, Switzerland), approxirnately 0.240 g of
UHMWPE particles was weighed, placed in a glass French bottle (VWR Scientific, #363 19-760,
Mississauga, ON), and placed on its side. The lid of the bottle was Teflon@-lined. A 15 mL
aliquot of either 4mM sodium hypochlorous acid or 10 w/w?? hydrogen peroxide solution in
phosphate bufTer (pH 7.4) was added to the polymer. The concentration of the oxidant was read
in triplicate at penodic intervals (e-g., t = 0, 2, 4, 6, 12, 24, 48 hours). For purposes of
cornparison, the half-life of hypochlorous acid was also determined using both the UHMWPE
film and a polyurethane (synthesized fiom toluene diisocyanatel polycaproIactone/ ethylene
diamine). This polyurethane was used as a positive control as it is readily oxidized by
hypochlorous acid [McCloskey, 19981. A sample containhg only hypochlorous acid was used as
a negative control.
Aldrich Chemical Company, Milwaukee, WI
3.4.3 Incubation Experimenb
The low density of polyethylene made it difficult to completely expose the particles to the
oxidation solutions. To overcome this problem, attempts were made to maximize the particle
exposure by using a high surface area container that ailowed for maximum particle/oxidant
interactions. As it was also desired that the container be inert relative to the oxidants, glass petri
plates were tried; however, it was found that the solutions had a tendency to evaporate within a
44
few days. A wide-mouth French square giass bonle with the dimensions of 5.5 cm by 5.5 cm by
1 1.4 cm (VWR Scientific, Mississauga, ON) was used with a Tefion@-lined lid. The surface
area available at the particle-solution interface was 52.80 cm2. The m w of particles used to
form a monolayer in this surface area was 0.230 g. This includes 10% excess since it was
assumed that 10% of the particles would be lost during replenishment. The estimation was
calcdated based the particle sizes seen in the SEM measurement (see Appendix B for the
calculation) .
The polyethylene particles were weighed and placed into the bottles that were laid on their side.
For the film samples, three films were placed in the bottles in a similar manner. An aliquot of 15
mL of oxidant or buffer control solutions was carefully added to the bottle to ensure that a
monolayer was floating on the d a c e of the solution. The bottles were capped tightly and
covered with aluminum foi1 because the oxidants were light-sensitive. AI1 çamples were
incubated at 37°C for 30 days. n i e solutions of HOCl were totally replaced every day while the
solutions of hydrogen peroxide were replaced weekly (see Appendix C).
In order to avoid a significant increase in volume of hydrogen peroxide or HOCl in the
incubation samples, the solutions were completely replaced weekly or daily depending on the
oxidant. An aspirator system was used with either a 20.5 G needle or a glass pipette on the end
of a hose. The needle was carefidly placed in the corner of the bottie under the monolayer and
the bottie was tilted to the same side in order to withdraw the solution. The collected solution
was discarded. Fresh hydrogen peroxide solution or HOC1 was then added to the botîle, ensuring
that the particles remained in a monolayer.
The experiments were texminated by separating the oxidation solution from the particles. This
was done using a filtration system with a 0 . 5 ~ TeflonB membrane filter. The particles were
coilected on the fiiter, placed in a covered petri dish and left to dry in a 37°C oven for two days
or until the residual water evaporated. The incubation solutions were prepared for HPLC
analysis (see Section 3.7) and the particles were d y z e d using FTIR (see Section 3.5.1).
45
3.5 Characterization of WBMWPE Oxidation
The degree of UHMWPE oxidation was quantifieci using FTIR and visuaily characterized using a
sulphur dioxide staining technique.
3.5.1 Fourier Transfom Infrared Spectroscopy @TIR)
Fourier tmnsform infhred spectroscopy is an analytical technique that is used to identifi the
various chernical groups present in a substance. The theoretical principal is based on the ability
of chemicai groups to absorb energy that will be used to increase the vibrational motion of the
bonds pavia, 19791. However, only chemical groups that contain a dipole moment can absorb
infirared radiation. These different groups c m absorb energy to increase the vibrational motion at
a characteristic wavelength or wavenumber. IR spectroscopy can be used to f i nge rp~ t a specific
compound and the types of vibrational motion can be used to elucidate the chemical structure of
the material.
in this study, there were two methods of FTIR applied to quanti& the amount of oxidation found
in UHMWPE: transmission FTIR and Diffuse Reflectance FTiR @RIFT) spectroscopy. In
transmission FTIR, a spectrum is generated fiom the vibrational energy that is detected when the
infkared radiation passes through a sample. However, DRIFT generates an FTIR s p e c t . by
using the reflectance of the radiation fiom the surface of the sample. Reflectance consists of
regular reflection and diffuse reflectance. D i f i e reflectance results when light enters a
substance where it is partidly absorbed and the rays emerge fkom the substance afler it has been
scatîered [Svehla, 19761. The reading obtained using the diffuse reflectance mode is Kubelka-
Munk (K-Munk). These units are linearly proportional to the sample concentration
[GRAMSI386 User's Guide] and are andogous to transmission in normai transmission FTIR. K-
Munk can be converted fkom transmission data using the following relation:
[l - tansm mission^ K - Munk = [2 x ~rammission]
The particles were d y z e d using the reflectance mode because the sample preparation was
quicker and easier, and the data tended to be more reproducible than results acquired by
transmission FTIR. The resdts obtained by transmission FTIR contained much variability in the
sample preparation. The particle samples were diluted with FTIR grade potassium bromide
46
(Aidnch Chernical Company, Milwaukee, WI) in a 1 5 ratio (mass of particles to mass of KBr).
The samples were ground together using a marble mortar and pestle and placed in a sample
holder (1 cm diameter, 0.5 cm depth). The surface of the sample was hand-pressed and flatteneci
using a spatula The samples were scanned using a nitrogen purge to remove most of the water
and carbon dioxide present in the sample chamber. Ail film samples were carried out in
absorbance mode while the particle samples utilized the refiectance mode. The reflectmce mode
gave an indication of what hct ional groups were present within the upper 10 pn of the surface,
depending upon the ability of the materials to absorb IR radiation. If the sample is 100%
reflective, 10 pm of the surface depth wi11 been seen [communication with Dr. Xijia Gu].
ûtherwise, less of the surface is seen.
The experiments were cmied out using a Bomen Hartmann & Braum, MS-Series FT-Raman
iostniment and GRAMS/386 software at the Photonics Research of Ontario, University of
Toronto. Al1 samples were scanned in the mid-IR region (350 to 3500 cm-').
A normal FTIR spectrum of UHMWPE is seen in Figure 3.2. It contains a sharp assymetric
stretching at 2915 cm-', a sharp symmetric stretching at 2848 cm-', methylene in-plane
deformation at 1466 cm", a slight methylene umbrella deformation at 1375 cm-' and a strong in-
phase rock at 722 cm-' [James et al.. 19931. In order to assess the amount of oxidation in the
experirnents, a convenient measure of this value was needed. Various authors have chosen
different definitions of the oxidation index that involve heights and areas of various peaks petts
et aL. 1993, Silverstein et al., 1 993, A. Wang et al., 1996, Goldman et al., 19971. The oxidation
index chosen to be used in this study involved taking the ratio of the area for peaks containhg
the carbonyl groups relative to the area associated with methylene groups (see oxidized sarnple in
Figure 3.2). Reporting the ratio of areas accounts for variations in intensities due to variability in
sarnple preparation, and hence provides a means of normalizing the oxidation levels for each
measurement. The specific wavenumber range used was defined in the following equation:
area l6~Ocm" to 1850 cm" O. 1. =
area 1400cm-' ro 1550 cm-' Equation 3 6
This definition was used by Muratoglu ~ u r a t o g i u et al.. 19961. This definition was chosen
47
because it encompassed the greatest range in the oxidative area that included aldehydes, esters
and carbxylic acids. Since extent of oxidation was not known, this definition proved to be the
most general. This method made no assumptions about the type of oxygen-associated group and
requires no peak-fitting. Unfortunately, the primary disadvantage of this approach is contained
within the definition itself (i.e. it invoived the ratio of peak areas). Upon severe oxidation, the
region between 1400 to 1550 cm" can be deconvoluted into at least six different peaks. This is a
problem because when oxidation increases, the area under the methylene group (1400 to 1550
cm") is subject to change because the baseline is no longer flat. As weli, with the addition of the
other peaks, this area camot be well resolved and may result in a decreased peak area for the
methylene peak This artificially increases the oxidation index. Other authors compared the
oxidative peaks in a narrower methylene peak area which only consisted of one peak 1450 cm-'
(Brossa et al., 1996) and 1370 cm-' (Greer et al., 1996). By narrowing the range of the
methylene peak, a more accurate measure of oxidation should be found. However, this method
entails curve-fitting the peak, which may in itself introduce errors. To overcome this problem,
other investigaton have chosen to use another CHÎ base peak altogether. Goldman et aQ1996)
have chosen the methylene peak at 20 1 0 cm-'.
3970 3470 2970 2470 1970 1470 970 470
Wavenumber (cm-')
- (hQdized Sanpk - Non-Oxïciized Sampie
Figure 3.2: Typical FïIR Spectra of Non-Oxidized and Oxidized UBMWPE. The oxidized
sample was heated in an airflow oven at 10J°C for fwe days.
48
3.5.2 Suiphnr Dioxide Staining
Since gaseous suiphur dioxide reacts specincally with the hydroperoxy groups praent in
oxidized polyethylene [Scheirs, 19911, the degree of oxidation on the surface of the test materiai
was assessed by dphur dioxide staining. Hydroperoxides are readily converted to alcohols and
ketones Fessenden et al., 19901. However, this staining has a limited ability to assess the full
degree of UHMWPE oxi&tion because it only reflects residual hydroperoxy groups and not
other more stable groups that would have formed as a result of oxidation. The reaction of S02
with a peroxy group is s h o w in Equation 3-7. When the polymer sample is heated in the
presence of S02, the conjugated double bonds formed during the reaction, appear as darker
regions using fluorescence microscope. It has been shown that the increase in the intensity of the
darker regions increases with polymer hydroperoxide concentration [Scheirs, 199 11. As well, the
production of sulphwic acid during the heat treatment causes charring that intensifies the staining
effect.
Oxidized polyethylene samples were placed in a seaied reaction kenle. Sulphur dioxide gas
(CANOX, Toronto, Ont.) was introduced into the kettle up to a pressure of 345 kPa. The gas
flowed through the reaction vessel for 12 hours. Non-reacted S02, flowing fiom the reactor, was
directed into a double-stage sodium hydroxide trap filled with enough sodium hydroxide pellets
to react with the residual sulphur dioxide. This reaction fomed water and sodium sulphate. Any
non-reacted suiphur dioxide was M e r diluted in a water bath. The schematic of the apparatus
is given in Figure 3.3. At the completion of the treatment, nitrogen was used to purge the
reaction vessel and the samples were heated at 1 15OC in an air-flow oven for 40 minutes.
02 - CH2 -CH2 - CH2 - CH2 - + --CH2 - CH - CH -- CH2 --
A OOH OOH
Equation 3-7: Reaction of Oxidized Polymer with Sulphur Dioxide [Scbeir, 19911. The
. oxidation of the hydrocarbon produces hydroperoxide that readiiy reacts
with S02.
49
The S a treated samples were mounted onto giass microscope slides with DPX Momtant for
Histology (Fluka Chemical Corp., Ronkonkoma, NY). This mounting agent was used because it
does not reflect nor absorb in ultraviolet radiation or excite using fluorescence. The Nikon
Eclipse E600 fluorescence microscope, with a Y-FL EPI attachent and mercury lamp was used
to andyze the stained samples at the Photonic Research of Ontario, University of Toronto.
*
b
Sulphur Double-Stage Water Bath dioxide NaOH Trap cylinder
Polyethy lene Sample
Figure 33: Schematic of Sulphur Dioxide Strining Apparatus.
3.6 Incubation of UHMWPE under Hydrolytic Conditions
Oxidized UHMWPE particle samples were incubated with hydrochlonc acid or a hydrolytic
enzyme (cholesterol esterase, CE) to determine if the oxidation process rendered the materials
susceptible to hydrolytic degradation. The innuence of enzymes on the oxidized materials would
potentially reflect a mechanism of degradation associated with partictes phagocytosed by hurnan
monocyte-derived macrophages. Specifically, CE has been s h o w to be produced by monocyte-
derived macrophages [Labow et al.. 1 9981.
The hydrochlonc acid used in the studies consisted of a 4 N solution, which was diluted fiom a
12 N HCI solution (Fisher Scientific, Nepean, Ont.) using HPLC grade water. The source of CE
used was bovine pancreas, E.C. 3.1.1.13 (Genyme Diagnostics, Cambridge, MA, # 1 O8 1). A
unit of CE activity was dehed as the production 1 nmoVmin of p-nitrophen01 fiorn the
hydrolysis of pnitrophenyl acetate in the presence of CE at pH 7.0 at 25°C. This is represented
by the following reaction:
CE p-nittmphenyl acetate + water - pnitrophenol + acetic acid Equation 3-8
Full details of the assay are given in Appendix D. The hydrolysis experiment was initiated with a
15mL aliquot of a 40 U/mL stock solution of cholesterol esterase and was replenished daily with
200pL of a 320 U/mL solution of CE. Since the susceptibility to hydrolytic attack of the
oxidized polyethylene was unknown, the initial concentration was chosen to be the same
concentration as was determined by previous work with a polyetherurethane system pee, 19951.
The oxidized polymer sample was placed in a clean, sterile French bottie that was lying on its
side. Aliquots of either cholesterol esterase or hydrochloric acid (15 mL) were added to the
polymer. Aseptic technique was used. The reactor vessels were capped with Tenon@ -lined lids
and placed in the 37°C oven for either 15 days or 30 days at which point the experiment was
terxninated. The particles were isolated fiom the solutions using a stenle filter system (cat#
25935-200, from Coming Incorporated, Coming, NY) with a 0 . 4 5 ~ nylon, Iow extractable
grade membrane. The polymer was dried in a 37°C oven and stored at room temperature until
FTIR analysis. The incubation solutions were kept for analysis of the degradation products.
3.7 Extraction of Degradation Products
in order to prepare the incubation solution samples for high performance liquid chromatography
(HPLC), the enzymes and salts were removed fiom the solutions, the solutions were concentrated
and then dissolved into a suitable mobile phase. Al1 solutions were processed in the same
manner, regardless if enzymes were present. Triplicate solutions from the same reaction
conditions were pooled in order to increase the intensity of the HPLC and mass spectroscopy
signals. The preparation scheme for the HPLC samples is shown in Figure 3.4. Thtee extracts
f?om the incubation samples were analyzed for degradation products: samples extracted from the
particles, samples from the organic phase of chlorofomilbufTer extraction and the methanol
extract of the fieeze-dried aqueous phase fiom the choroform/bufTer extraction.
The particles were extracted with chloroform to remove any products bound to the polymer.
These samples are denoted with "X-" before the sample name (e.g, #41 X-30d CE 11). The
incubation solution was filtered using a 5,000 NWK centrifuge filter unit (Millipore, UF-CL) to
remove any traces of enyme. This Eilter unit was appropriate for removing the enzyme since the
CE had a molecular weight of approximately 130,000 [Sonnenbom, 19821.
The solution extracts were prepared in the following marner. A 10 mL aliquot of HPLC grade
chloroform (Caledon Laboratories Ltd., Georgetown, ON) was added to the incubation solutions.
The chlorofonn/aqueous mixture was vortexed for 5 minutes and then centrifuged to separate the
two phases. The chloroform was then removed with a Pasteur pipette. The chloroform was
evaporated with a stream of highly pure, dry nitrogen (Grade 4.8, BOC Gases, Mississauga, ON)
until a residue rernained. The incubation solutions were extracted with chloroform three times
and pooled to retrieve the degradation products, which were soluble in chloroform. These
samples were labeled without any prefk (e.g.. #4 1 30d CE 1 1).
Particles &
l 1 Incubation Solution 1
dissohred in mobile phase '-E
I 1 1
Figure 3.4: Preparation of HPLC Samples
-
The aqueous portion of the incubation solution was fiozen in liquid nitrogen and fieeze-dried
(#FDX-54A, FTS Systems inc., Stone Ridge, NY). The recovered solids were extracted with 90
v / P ? methanol-water solution. The water was used to dissociate any products that were
combined with salts. The methanol solution was subsequently evaporated, using a stream of
Parüdes ' Incubation Solution
I r I
extracteci with chomfonn / îibr with 5 K filter
1 Organic Phase I chlorofon extraction
Aqueous Phase 1 freeze-d ried
I I
52
Grade 4.8 nitrogen, to recover the product. These samples have the symbol "fd:" denoted before
the samplename (e.g.. #41 fd: 30dCE 11).
FUially, the residues nom al1 three extracts were separately dissolved with the starting HPLC
mobile phase (100 fi of methanol and 900pL of ammonium acetate buffer, pH 7.0, see section
3.8). These solutions were finally filtered using a Millipore 5,000 NWK cut-off centrifuge filter
(Millipore, UF-CL) and stored at 4°C until ready for HPLC analysis. A list of the samples and
their processing conditions is given in Table 3.8.
Table 3.8: Processing Conditions for Particle Samples:
Controls
N/A are samples which were not perfonned.
15 days with CE
30 days in b a e r
30 days with CE
3.8 High Performance Liquid Chromatography (HPLC)
High performance liquid chromatography is a comrnon technique used in analytical chemistry to
separate various compounds fiom a mixture. The separation is based upon the a£€inity of the
compound for either the stationary phase (column) or the mobile phase (solvent system). A
block diagram of the HPLC system used in this study is described in Figure 3.5.
Heated for 6
days at 1 0 5 ~ ~ '
A w a t e p HPLC system was used in the separation of biodegradation products. It was
composed of a 600E multisolvent delivery system and used methanol, ammonium acetate b d e r
(pH 7.0) and water as the solvents making up the mobile phases. The solvents were sparged with
heliurn to remove bubbles that couid interke with the function of the column. Prior to use, dl
the solvents were filtered to remove particdate contamination, and sonicated to degas the
solvents.
15d CE only
30d buf only
30d CE only
Al1 nonsontrol samples were prr-oxidized with hydrogen peroxide before incubating with a hydrolytic solution.
Heated for 1 Z
days at 1 OSOC'
CoC12 + heated for
1 1 days at 1050~'
15d CE 6
30d buff 6
30d CE 6
15dCE 11
30d buff 11
30d CE 11
N/ A
UH(Co) 30 buff
UH(Co) 30 CE
53
A UK6 injector (watersm) delivered the sample through an in-line coiumn filter (SS, 0.22 p),
past a guardpack column filter (pbondapak, C18, watersTM) and through a ~ a t e e
pBondapakTM Cl8 steel column (4.6 by 250 mm) packed with dimethyloctodecylsilyl bonded
amorphous silica. The io-line column and guardpack filters protect the CI8 column fiom
particulates and hi& column loading. The products are eluted off the column at different
retention times and are detected using a photodiode amy detector (watersm PDA, mode1 996).
The pump/system controller (watersTM 600 controller) delivered and mixed various solvents at
specified compositions and flow rates. Miiiennium 20 10 software was used to control the system
and acquire and process the data.
mimplsystem controf Ier
b
Solvent V ! In- line Rexrvoirs - Pump - Auto-injecter + filter/guard _c/ (2 or 3) column
1
Figure 3.5: HPLC block diagram wtersTM Manual: Guide to Successful Operation of
Your LC Systeml.
The retention time and the degree of separation can be varied depending upon the solvent system
used (polar or non-polar), the type of system run (isocratic or gradient) and the type of column
used (polar or non-polar) WcMaster, 19941. In the following experiments, the stationary phase
had a lower polarity than the mobile phase. The mobile phase was a mixhire of methanol and 2
m M ammonium acetate buffer (pH 7.0).
54
Samples that contain many products, simila. in chernical nature, are often very W c u l t to isolate
ushg a simple isocratic systern. For this study, the samples were run using two mobiie phase
gradient methods. The fïrst gradient program (Table 3.9) was run for al l samples. The second
program (Table 3.10) was nui if further separation of the more polar products in the sample was
required and was run only on samples that were collected within the first 10 minutes of "Program
One".
Table 3.9: Program One Gradient Run for HPLC
1 Time 1 Flow rate 1 Port A' (%) 1 Port B '(%) 1
"Program One" has a 10% methanol isocratic systern in the beginning to allow rnany of the polar
contamination products to be separated fiom the sample. The gradient over 50 minutes allows
for separation of the non-polar peaks found in the rest of the sample. The 100% methanol
isocratic gradient at the end of the run allows for the elution of any non-polar products that are
strongly bonded to the column.
Table 3.10: Program Two Gradient Run for HPLC
"Program Two" was run in order to isolate the products found at the beginning of "Program
One". This was only used d e r the products fiom "Program One" were collected and M e r
concentrated. It consisted of a slow gradient fiom 100% b a e r to 10% methanol after which an
isocratic run occurred at 100% methanol to remove any products still bound to the column.
- -- - -----
t Port A consisted of 100% methanol. Port B coosisted of 2 m M ammonium acetate buffer at pH 7.0.
AN HPLC chromatograms were displayed at 210 nm because at this absorbance, the products of
interest were clearly differentiated. The W absorbance spectnim allowed for the determination
of the purity of each peak by rnatching the spectra to various sections of that peak. The peaks of
interest were collected and prepared for mass spectrum analysis, attenuated total reflectance
FTIR and NMR
3.9 Characterization of Isolated Biodegradation Products
3.9.1 Mass Spectroscopy
Mass spectroscopy is a technique used to determine the molecular weight of a pure sample. Ion
spray mass spectroscopy bombards a sample with a high energy bearn of electrons to resuit in
hgments of positive ions which are produced fiom molecules by the removal of an electron
Favia, 19791. The ions are accelerated in an electnc field and separated according to their mass-
to-charge ratios. Finally, ions with a particular mass-to-charge ratio are detected. The mass
spectnun is recorded as a measurement of relative intensity versus mass-to-charge ratio (m./@,
where the relative intensity is compared to the nurnber of counts of the most intense peak, which
is labeled as the rnolecular ion (m. The hgmentation pattern is characteristic for each
compound. Multiple peaks can be associated with the same molecular ion when combined with
different salts, which are present in the mobile phase or the sample (e.g. MN^', MK', MN&).
By piecing together the fÎagments, a general structure of the compound can be elucidated.
Further fkgmentation of selected mass hgments can occur using tandem mass spectroscopy
(MS-MS). This provides a mass spectrum with minimal contamination.
Figure 3.6: Quadrupole Arrangement for a M a s Spectroscope pavia, 1979)
56
Mass spectrometry was canied out on a . API-III triple quadrupole mass spectrometer (MS-MS)
(Perkin-Eher/Sciex, Concord, ON) at the Laborittory of the Carbohydrate Research Centre,
University of Toronto. A quadrupole consists of four solid rods arranged in pardel to the
direction of the ion beam (Figure 3.6). A direct-current and radiofkquency are applied to the
rods. An oscillating electrostatic field is genenited between the rods, which r e d t s in oscillating
ions. Ions with the "correct" fiequency pass through the rods to the detector while ions having
the incorrect fiequency hit the rods and are undetected. This method selects the range which the
mass-to-charge ratio can be focused. The nrst quadrupole detects the initial molecular ions
[Pavia, 19791. The second quadrupole is the reaction region for the ionic collisions of a selected
molecular or parent ion by the first quadrupole in MS-MS. The third quadrupole is used as a
molecular weight analyzer of fragments fiom both MS and MS-MS. Ions fragmented fiom the
parent ion are named daughter ions. The pressure of collision gas (argon) in the second
quadruple cm Vary the degree of Wenta t ion . The arrangement of the triple quztdrupole mass
spectrometer is seen in Figure 3.7.
The samples collected from HPLC were fiozen in liquid nitrogen and fieeze-dried using a
vacuum purnp. The samples were dissolved in 40pL of HPLC grade methaool (Mallinckrodt
Baker Inc., Paris, KY) and 20 pL of the sample was injected though a 7125 injector (Rheodyne).
The methanol mobile phase had a flowrate of 0.02 mL/min. A 5.00 kV voltage was applied to
the tip of the ion spray needle while 80 V was applied to the entrance of the spectrometer. Mass
to charge ratios from 100 to 2000 a.m.u typically have a standard deviation of i 0.5 a.m.u.
Figure 3.7: Schematic of Triple Quadrupole Mass Spectrometer
&
3.9.2 Attenuated Total Retlectance FïIR (ATR-FlrIR)
ATR-FTIR is a method based upon FTIR (see section 2.5.1). The attenuated total refiectance
apparatus gives a more sensitive rneasurement of the chernical cornponents dissolved within a
~-{Thirdb["....i Quadruple Second @adnipole ' First
Quadmpole Ionization Chamber
Electric Field --*
57
solvent. Light passes through a high refiactive index crystal and reaches the interface between
the sample and prism. The radiation penetrates a few microns into the sample and then is totaily
reflected back into the prism [Svehla, 19791 as seen in Figure 3.8.
Angle of / Incidence
Figure 3.8: ATR-F"MR Light Path
For the chemicai characterization of the biodegradation products, several HPLC passes were
required to obtain enough products. The samples were fiozen in liquid nitrogen and the
methanol in the sample was evaporated with a vacuum pump. The remaining aqueous sample
underwent a liquid-liquid extraction using dichloromethane.
The ATR-FTIR (Graseby Specac Ltd, ûrpington, Kent, UK) apparatus was located at the
Photonics Research of Ontario, University of Toronto. It consisted of a zirconium selicade
crystal with a solvent trough located at the top surface. A pure dichloromethane sample was
analyzed as well as the sample dissolved in the solvent. One hundred scans were taken of the
sample in order to get a strong enough signal to distinguish the sample nom noise.
3.9.3 Nuclear Mapetic Resonance Spectroscopy (NMR)
Nuclear magnetic resonance provides information about the type of hydrogen present in each
molecule. It is based upon the absorption of radio waves by certain nuclei when they are in a
strong magnetic field Fessenden, 19901. The nuclei of compounds either have paired (do not
have a spin) or unpaired electrons (has a spin). When a nucleus has a spin, a s m d magnetic field
results and cm be detected. Hydrogen and carbon-13 among other atoms exhibit this
characteristic. The immediate electmnic environment surrounding it affects each hydrogen atom.
The position of a proton depends on the net strength of the applied magnetic field and the
58
induced moleçular field around the proton. If the induced field around a proton is strong, it
opposes the applied field more strongly and thus a greater applied field is needed to resonate the
proton. This phenornenon is caiied shielding and the si& will be seen upfield in the spectra
The reference for the chernical shifts was the solvent peak. This gives an accuracy of 0.1 ppm.
The benzene solvent peak is placed at 7.15 ppm and the shifts for al1 protons are reported relative
to this reference. The difference between these &equencies is caiied a chernical shift. Chernical
shifts are reported in 6 values, which are expressed as ppm of the applied radiofkquency. At a
fiequency of 500 MHz, 1 .O ppm has a fkquency of 500 mHz.
NMR samples were run in the Nuclear Magnetic Resonance Laboratory at the Lash Miller
Chemistry Building, University of Toronto by Dr. Timothy Burrow. A Unity 500 MHz
Spectrometer (Varian Association, Bello Alto, CA) with a 3 mm direct probe was used. The
collection of sample for NMR was identical to those described previously for ATR-FTIR.,
however the samples were dissolved in deuterated benzene (Cambridge Isotope Laboratories,
Andover, MA) instead of dichloromethane. Benzene was chosen because it contained a very
clean spectrum that would prevent overlapping with the peaks detected in the sample.
3.10 In Vitro Studies of HDPE Particles with Human Monocytes
A preliminary study of the interaction of monocytes with treated polyethylene particles may lend
insight to the mechanism of polyethylene particle-induced bone los. The cytokines IL-IP, IL-6
and TNFa have been linked to bone loss in particle-induced inflammation [Horowitz et al.,
19951. Based on time limitations, only the particles listed in Table 3.1 1 were assessed. These
particles were phagocytosed by human monocyte-derived macrophages (HMDM) and the amount
of cytokine production of IL4 P, IL-6 and TNF-a was assessed using an in vitro model. Al1 the
ce11 culture work descnbed in this section was carried out by Mr. Stuart Rae at the Centre for
Biomaterials. These experirnents were based on protocols develo ped by Mrs. Irina Voronov
[Voronov et al., 1997 and were adapted for human monocytes.
Table 3.11: Particle Treatments for Particle-Monocyte Experiments
1 HDPE ody 1 HDPE
3.10.1 Preparation of Particles
HDPE-Co
HDPE-Co-Hz&
CollagedDMSO only
The particles were mixed in a collagen solution in order to allow them to adhere to coverslips.
This procedure effectively overcame the problem of particles, having a lower density than water,
to interact with the HMDM [Voronov et al., 19971. Bnefly, one gram of particles was initially
suspended in 0.5 mL of dimethyl sulphoxide (BDH Laboratories, Toronto, ON) and then mixed
with 14.5 mL of 0.01% collagen type 1 monomer solution nom calf-skin (C-8919, Sigma, St.
Louis, MO). The final concentration of particles was adjusted to IO' particles/ml. The solution
was coated on sterile coverslips. The collagen matrix was crosslinked upon dryuig at room
temperature. In this rnanner, the particles were trapped within a collagen matrix and adhered to
the coverslip. The coverslips were placed in six-well tissue culture plates (Falcon, Fisher
Scientific, Whitby, Ont.) and UV-sterilized in a laminar flow hood for one hou before use.
1 1 day heated HDPE coated with CoCh
11 day heated HDPE + CoCh + 30 day hydrogen peroxide treated
no HDPE: coliagen + DMSO
3.10.2 Preparation of Cell Culture
Human whole blood was collected (120 mL) in vacutainers (Becton Dickenson, Franklin Lakes,
NJ) fkom healthy volunteers. Four parts whole blood were layered on three parts stenle Ficoll-
Paque Research Grade Solution (Pharmacia Biotech, Baie D'Urfe, PQ) in a 50 mL centrifuge
tube. The mixture was centrifbged at 1 750 rpm for 40 minutes at 18OC poronov et al., 19981.
Ficoll-Paque medium is a practical means of isolathg monocytes fiom other cellular components
in the mixture. The resultant gradient containhg the S e m , buffy coat, and red blood cells, is
illustrated in Figure 3.9. n i e buffy coat layer that contained monocytes and lymphocytes was
collected, washed with PBS buEer (without calcium or magnesium), and resuspended in the
media, RPMI-1640 (Sigma, S t Louis, MO) with 10% heated and activated fetal calf serum
(Gibco BRL, Burlington, ON), 1% penicillin-streptomycin (Gibco BRL, Burlington, ON) and 2
rnL of L-glutamine (Gibo BRL, Burlington, ON).
- Buffy Coat (monocytes & lymphocytes) H'
Figure 3.9: SeparPtion Gradient of Whole Blood
The cells were plated on the collagen/particles treated coverslips at a concentration of 1 x 1 o6 ceiIs
in 2 mL of media per well. Samples were incubated at 37OC in a 5% COz, 100% humidity
atmosphere for two hours after which the non-adherent cells were removed. A 2 mL aliquot of
fiesh media was added to the cells and the Cels were incubated for 24 and 48 hour intervals.
Twenty-four hours pnor to the termination of the experiment, the media was replaced with 2 mL
of fiesh media to determine the cytokine production produced in the final 24 hours. At the end of
each incubation time period, the adherent cells were washed with PBS, lysed with a solution of
Triton-X and EDTA and analyzed for the amount of DNA present. Floating cells were
centrifuged and resuspended in PBS and checked for ceIl viability. The media was collected for
the analysis of the following cytokines: interleukin- 1 (IL4 B), interleukin-6 (IL-6) and tumor
necrosis factor a (TNF-a).
3.10.3 Analysis of DNA, Cell Viability and Cytokines
DNA analysis was performed to determine the number of adhered cells present in the sample
using a spectrofluorometric method. The method is outlined by Labow et al.. (1998) and was
perfomed by Mr. Stuart Rae in Dr. J. Callahan's laboratory at the Hospital for Sick Children,
Toronto. Briefly, it entailed the lysing of the cells using a 0.4% Triton -X/lOmM EDTAfand
phosphate b a e r saline solution (without calcium or magnesium). A standard caiibration curve
using a fluorometric method was determined The calibration curve was performed using known
amounts of DNA (calf thymus) and obtaining the correspondhg fluorescence values (Appendix
G)*
6 1
The viability of the monocytes was determined ushg a standard m a n blue exclusion test
Freshney, 19941. Equal volumes of the suspended cells and 0.4% trypan blue stain (GIBCO
BRL, Grand Island, NY) were ssdded to a 32-weii plate. The mOaure was placed on a
hemocytometer and under a Iight microscope to determine the number of cells which took up the
blue stain compared to those who did not. Dead cells stained blue because the breakdown of the
ceil membrane allowed the uptake of the dye Freshney, 19941.
ELISA kits were used to quanti@ the amount of IL- 1 P, IL-6 and T N F a cytokines. The kits
were as foUows: Cytoscreenw Irnmunoassay Kit (KHC0012) for human IL-If! (Biosource
Intemational, Camariiio, CA), Human IL-6 Duoset (80-3548-00) and Human TNF-a Duoset (80-
3933-00) nom Genyme Diagnostics (Cambridge, MA). The quantity of cytokines was
normaiized by the DNA values in order to account for potential ciifferences in the ceU numbers.
4.0 RESULTS
4.1 Characterization of Polyethylene Samples
The characterization of test specimens, prior to chemicai and thermal treatment, included the
examination of the surface morphology and determination of the chernical composition of the
specimens.
4.1.1 Surface Morphology
The surface rnorphology was studied using SEM. Figures 4.1 and 4.2 show the range of sizes
and shapes for UHMTKPE and HDPE particle samples, respectively. The average size of the
WHMWPE as compared to the HDPE particles was 250 pu vernis 5 p. The UHMWPE shows
a cauliflower arrmgement which is composed of fuie particle bundles held together with fibril
strands. The HDPE appears as one solid mass throughout its siructure.
A. B.
Figure 4.1: Scanning Electron Micrographs of UHMWPE Resin Particles at a magnincation of
A. lOOx and B. 5,000~
Figure 4.2: Scanning Electron Micrographs of HDPE Resin Particles at a magnincation of A.
1,500~ and B. 6,000~.
4.1.2 Chernical Composition of Polyethylene
The chemical composition of the virgin m E was determined using XPS and FTIR while
the d y s i s of HDPE was previously reported by Voronov and colleagues [Voronov et al.,
19981.
The XPS elemental chemical composition for UHWMPE film and particle is given in Table 4.1.
Traces of silicon were associated with silicone contaminates (-SiO(CH&). In the final
presentation of results for elemental characterization, carbon, oxygen and silicon that were
associated with the silicone were subtracted fiom the original data. Silicon levels were less than
6% for the nIm samples and less than 1% for the particle samples. n ie film samples were
analyzed at take-off angles of 90° and 1 5 which pro bed the sarnples at approximate depths of 1 0
nrn and 3 nrn, respectively. It should be noted that it was not possible to obtain measurements at
64
XPS angles less than 90° for particle samples since the specimen shape did not provide a large
enough flat surface area to match the spatial resolution of the instrument As well, it should be
noted that no standard deviation was reported for the irradiated particle samples in Table 4.1
because the adys i s was oniy performed on samples nom one batch of irradiated treated
particles.
Table 4.1 : XPS Results of Elementd Composition of UHMWPE Films and Particles
1 l S O Take-off Angle 1 90" Take-off Angle
Sample Cls 0 1 s Sap Cls
film: îrradiated 95.76 & 3.17 2 1.06 + 92.40 + 1.31 1 .O3 0.35 1.68
film: non-irradiated 97.22 2 2.50 + 0.27 + 94.42 + 0.93 0.71 0.2 1 0.39
particles: irradiated N/A N/A 97.9
particles: non- N/A N/A 98.61 2
irradiated 0.88
Note: Data are reported in tems o f mean + standard deviation.
NIA is not applicable; Units of samples are relative atomic percentage,
n=3 for al1 samples except for the particles: irradiated samples (n= I )
Al1 UHMWPE materials contained some oxygen at a depth of 10 nm, aithough the filrn samples
appear to contain slightly more (1%) oxygen than the non-irradiated particle samples. However,
including the errors, the amount of oxygen seen for filrn and particle samples are essentially the
sarne at about 2 atornic percent. As well, there was no statistical difference @<O.OS) between the
non-irradiated and irradiated fiim samples in terms of the amount of oxygen present at either the
3 or 10 nm depth.
FTIR was found to be more sensitive to different oxidation leveis than XPS. The oxidation index
was defined as the ratio of the area under the carbonyl groups (1650cm-' to 1850 cm") to the
methylene group (1400 cm-' to 1550 cm-'). The oxidation indices for irradiated and non-
irradiated UHMWPE resin and films are given in Table 4.2. There was no signincant ciifference
@ < 0.05) between the irradiated and non-irradiated film samples. However, between the
65
irnidiated and non-llradiated particle samples, there is a significant ciiffierence (p < 0.05).
Furthemore, there was a signincant difference between the film and particles samples.
Table 4.2: Oxidation Indices of UHMWPE Films and Resin, n=3
1 Sample 1 Oxidation Indes 1
1 film: non-irradiated 1 0.099 + 0.005 1 film: irradiated
1 particles: inadiated 1 0.05 1 + 0.008 I
mean + standard deviation O. 104 + 0.023
1 particles: non- inadiated 1 0.034+ 0.007 1
4.2 Thermal Oxidation of Particle Samples
UHMWPE particles were heated in an air-fiow oven for vaqkng time periods at one of the
following temperatures: 37, 60, 80 and 105OC. The particle samples were analyzed using FTIR
and the oxidation index was determined. A plot of oxidation index vernis t h e for the various
temperatures is shown in Figure 4.3.
- -
O 5 0 100 1 5 0
T ime (hours)
+ 3 7 O C - 6 0 O C - 8 0 O C - 105 O C
Figure 4.3: Oxidation of UEIMWPE Particles Post y-Irradiation. Heat oxidation was
performed at 37OC, 60°C, 80°C and 105OC. (Error bars are the standard
deviation of the samples and n=3)
66
In generai, the oxidation index did not show any signincant ciifference between any of the
samples for the fïrst 100 hours of incubation (Figure 4.3). Foliowing this period, the samples
treated at 105°C underwent a signifîcant increase in oxidation. This increase was carried out to
170 hours. However. samples treated at 37.60 and 80°C showed no significant ciifferences in the
oxidation values between each other throughout the 170 hour oxidation period.
It was originally expected that changes in oxidation indices would be observed for several
temperatures and that sirnplified mode1 could be used to predict time/temperahue relationships.
However, due to the obvious lack of differences in oxidation indices, at temperatures other than
105°C. this was not further pursued.
4.2.1 Sulphur Dioxide Staining
Sulphur dioxide staining was used to provide a pictonal representation of the oxidized
UHMWPE samples. No quantification was performed using this analysis since the method did
not reflect al1 the chernical groups generated by oxidation (see Section 3.5.2). Film and particle
samples were heated for 6 and 1 1 days at 105OC and stained with sulphur dioxide.
Figure 4.4 shows the result of heat oxidized films d e r 6 days of heating at 105OC. The top
panels show the oxidized films while the bottom panels exhibit the non-oxidized films. The
oxidized films clearly show the grain boundaries as seen by the solid dark green lines. This is
where the greatest amount of oxidation is found. These boundaries were also previously
observed by Dwyer [Dwyer, 19961. It was noted that there were no distinguishing grain
boundaries present in the non-oxidized films. However, a few regions of dark green were
present, which indicate some oxidation.
Figure 4.5 shows the sulphur dioxide staining for particle samples. A clear picture was difficult
to obtain because the three-dimensional nature of the particles prevented a clear field of view in
the microscope. The 250 pm mean size of the particles make it difficult to focus on the entire
particle. Both the 11 day heated and the irradiated control particle samples exhibited signs of
fluorescence while neither the non-irradiated control and the non-stained sample (Figures 4.5 A
and B) showed any fluorescence. This indicated that oxidation by irradiation is readily seen by
this staining technique.
Figure 4.4: Fluorescence Micrograms of Sulphur Dioxïde Treated Films. A. 10x oxidized
nIm (6 day heat treatment at 105OC), B. 20x oridized f h , C. 10x control
film (non-heated and non-irradiated), and D. 20x control film.
Figure 4.5: Wuorescence Micrograms of Sulphar Dioxide Treated UHMWPE Particles. AU
samples were observed at a magnification of 20s A. Non-irradiated and not
heated, B. Irradinted, but not stained, C. Oxidized for 11 days at 10S°C, and D.
Irradiation Only,
4.2.2 Cobalt Treatment of Thermaiiy Oxidized UHMWPE and HDPE
The chemical composition of particles that were treated with cobalt ion samples was studied by
X P S , FT'U& SEM and EDAX. In general, the amount of cobalt contained in the particles was
difficult to quanti@ since data nom the three methods used ta analyze the presence of CoC12
were conflicting. The XPS resuits are given in Table 4.3. Since the proportion of cobait to
69
chloride should be 1:2, it was expected that this ratio should be seen at the surface of the
particles. However, this was aot observed since the ratio of Co to Cl was 9:l for HDPE and 25
for UHMWPE as shown in Table 4.3,
Table 43: XPS Results (Atornic Percentages) for Cobalt-Coated Samples of hdiated
UHMWPE and HDPE Resin Particles Using a 90° Take-Off Angle.
The FTIR results (Figure 4.6) showed that there was no ciifference between the UHMWPE
samples treated or not treated with cobalt chlonde. However, both samples showed the presence
of oxidation peaks a s a result of their thermal treatment at 105OC for 11 days. This test was
perfomed to veri@ that both materials had similar oxidation post-treatment in order to test the
synergistic effect of CoClz in the presence of chernical oxidants, thus reflecting the in vivo
accumulation of metal ions.
Figure
-- 3
- - 2 . 5
-- 2
3500 3000 2500 2000 1500 1 O00 500
W a v e n u r n b e r ( c m - ' )
4.6: FTIR Results for Cobalt Chloride Treated UaMWPE Particles. A. Therrnally
Oxidized and Cobalt Chloride Treated Sample. B. Thermal Oxidized ControI.
Only a qualitative description of the cobalt codd be determined using EDAX. Since the energy
of the x-rays is higher in EDAX than that of XPS, there is a deeper penetration of the rays into
the sample. Hence, the method is much less sensitive for surface atiaIysis. The EDAX results
70
(see Figure 4.7) also showed the presence of both cobalt and chloride on the both the UHMWPE
and HDPE samples. Since the CoC12 was only introduced as a trace metal to the d a c e , it was
observed that the EDAX signals for Co and Cl were very weak relative to the background signal
(Figure 4.7).
Y&9? KrO 10-0 > FS= IK ch 2 F b 98 cts met1 : UHP)IPE-C
Y.897 KtV tO.0 > F tK c h - 106 cts lqEw2t eimcieer
Figure 4.7: EDAX Results for Cobalt Chloride Coated UHMWPE and ADPE. A.
UHMWPE not coated with cobalt chloride. B. UHMWPE coated with cobalt
chloride. C. HDPE not coated with cobalt chloride, and D. HDPE coated with
cobalt chloride
4.3 Chernical Oxidation of UEMWPE
4.3.1 Assessrnent of HOCl Indnced OQdation
A prelirninary investigation of the chernical-induced oxidation of UHMWPE was carried out
using hypochlorous acid. The amount of hypochlorous acid consumed by the polymer substrate
was used as an indicator of the matenal's oxidation (see Figure 4.8) because it has been shown
that HOCl changes the colour of the polymer upon incubation. Both irradiated UHMWPE fiIm
and particles were studied in addition to controls consisting of non-irradiated UHMWPE film
and a polyesternethane (TDY PCUED) which has been shown to readily consume HOCl
WcCloskey, 19981. The initial concentration of HOCl used was 7.6 mM. Al1 samples were
perfomed in triplicate. A cdibration curve of HOC1 concentration and absorbance (Appendix
C) was used to determine the HOC1 concentration.
O 5 10 15 20 25 30 35
Time (hours)
+PEU (positive control) -I)-Non-lrradiated films -1rradiated films lrradiated particles +HOCI only -
Figure 4.8: : Consumption of HOC1 with Dinerent Substrates: PEU is a polyesternethane
(positive control) sample, irradiated and non-irradiated UHMWPE films and
irradiated UHMWPE particles. The error bars are the standard deviations with n=3.
73
M e r 30 hours of incubation at 37OC, the polyurethane positive control sample showed the
greatest consumption of HOCI. This confirms that degradation occurs. In the presence of this
material, there was approximately 8% of the original HOCl activity remaining d e r the test
period. The least amount of hypochlorous acid degradation was exhibited by the pure HOCl (no
substrate) sample and the non-irradiated UHMWPE nIms. There was a slight increase in the
degradation of the oxidant when the UHMWPE films were irradiated. The irradiated UHMWPE
particles showed the same degree of HoCl collsumption as the i d a t e d UHMWPE films. Both
of the i d a t e d üKMWPE samples showed a statistical difference using a snident t-test
@<O.OS) when compared to the HOCl control sample. However, there was no statistical
difference between the non-irradiated UHM7KPE samples and the HOCl alone sample.
4.3.2 Assessrnent of Hydrogen Peroxide Induced Oxidation
Film samples were subjected to a 10 w/w% hydrogen peroxide solution and compared with heat
and buffer treatments. Oxidation indices for the film samples were determined from
transmission FTIR spectca in the mid-infmred region. These oxidation indices were used as a
relative measure of the amount of oxygen incorporated into the chemistry of the polyethylene.
The data are reported for treated UHMWPE films in Figure 4.9.
The non-irradiatedhon-treated film sample had a baseline oxidation index of slightly under 0.1.
The non-irradiatedlbuffer-treated sarnple also had a similar oxidation index. The greatest effect
of oxidation was seen with the irradiated and heated sampie, even though it was only subjected to
the treatment for 5 days as compared to 30 days for the chernical treated samples. The films that
were thermally treated were more brittle than the other treated films. The irradiated and non-
irradiated hydrogen peroxide samples also exhibited increases in the oxidation index as
compared to the baseline control, with the irradiated sample showing a higher level of oxidation.
The oxidation index was dso determined for particle samples using DRIFT, as detailed in section
3.5.1. A cornparison of the effect of various treatments for the films and particles is given in
Figure 4.10. The base-line oxidation indices for non-irradiated films and particles are 0.10 and
0.02, respectively. When these two samples were heated for six days at 10S°C, there was a
significant increase of the oxidation index for the film sample, but not for the particles. The
74
effect of thermal uiduced degradation on polyethylene is well documented wawkins et al., 1971,
Winslow, et al., 19961.
It was interesting to note that while the irradiation of the particles resulted in a direct increase of
the oxidation index, the same was not observed for the film samples. However when both
irradiated particle and film sarnpies were treated with chernical or thermal oxidation conditions,
an increase in the oxidation index was observed relative to non-irradiated, non-treated with
chernicals and non-heated samples.
Non-lrrad Non-lrrad lrrad Heat lrrad H202 Non-lrrad Control Buffer HZ02
Treatment of Film Samples
Figure 4.9: Various Treatments of UHMWPE Film Samples. The Control sample is the
virgin UHMWPE film sample. 'Won-Irrad" denotes a sarnple that was not
irradiated, while the "Irrad" refers to a sample irradiated with 2.5 Mrad of y-
radiation. "Heat" treated samples were heated for 5 days at 10S°C. "BufTer" and
"K202" samples were incubated at 37OC with either phosphate buffer (pH 7.4) or
10 w/w% hydrogen peroxide for 30 days. The error bars were the standard
deviations and n=3
The variability in oxidation values was highest for the hydrogen peroxide treated samples. This
Iarger error is believed to be associated with the dinicuky in exposing the particles to the
hydrogen peroxide solution throughout the experiment. The low density and the hydrophobic
character of the particles tended to drive particles to coalesce at the surface of the solution and
75
thus rninimirre the exposure of the UHM\KPE samples. In order to minimize the effects of
density, the nIm samples were flipped over every &y; however, the control of this problem was
more difficult to manage with the particle samples. The cumbined effect of heat and irradiation
proved to be the most potent combination in terms of oxidative degradation for the UHMWPE
films while both thermal and peroxide treatments with irradiation showed the strongest effect on
the particles.
FILMS PARTICLES Figure 4.10: Cornparison of the Oxidation Indices of UAMWPE Films and Particles with
various combinations of heat, irradiation, chernical treatments.
The effect of prolonghg the thermal oxidation penod for the UHMWPE particles is shown
Figure 4.1 1. Increasing the oxidation period fiom 6 days to 1 1 days caused a great increase in the
oxidation index. When 6 day heated sampies were placed in a phosphate b e e r solution, there
was an increase in the oxidative degradation. This was consistent witb the literature petts et al.,
19941 which showed that salts increase the oxidative susceptibility of polyethylene. When the
pre-heated samples were placed in a solution of hydrogen peroxide and phosphate buffer, there
was yet a M e r increase in the oxidation index value over that of samples that were treated in
phosphate b d e r alone. The samples incubated in the hydrogen peroxide solution were s h o w to
yield the greatest oxidation index values.
76
The magnitude of increase in oxidative degradation is almost eight-fold when increasing the
incubation t h e fkorn 6 days to 11 days. Another observation was that the difference in the
oxidation index values for hydrogen peroxide vernis buffier-trwited samples was greater at 6 days
versus 11 days. This suggest a saturation state for the oxidation process, where no more oxygen
can react with the available fiee radicals, is king approached. This will be fûrther discussed in
the next section.
CONTROL 30 DAYS BUFFER
30 DAYS H202
5 6 days heated 0 11 days heated
Figure 4.11: Effect of Incubation of Pre-Heated UHMWPE Particles in Phosphate Buffer
@A 7.4) and 10 w/w% Hydrogen Peroxide at 37OC. The UHMWPE particles
were pre-treated by heating them in an air-flow oven at 105°C for 6 days and 11
days. There was an n-value of 3 and the error bars represented the standard
deviations.
The FTIR spectni for the different chernical treatments of UHMWPE particles are given in
Figure 4.12. The spectra clearly show that with increased oxidation, the presence of oxygen-
containing fiinctional groups appear in the regions of 1650 to 1850 cm-' and 1000 to 1300 cm-'.
While the carbonyl peaks (1650 to 1850 cm-') are well resolved, the region between 1000 to
1300 cm-' (associated with C-O stretch [Pavia et al.. 19791) consists of many unresolved peaks.
77
The oxidation index data for the buffet alone and the peroxide solution treatments with cobalt
chloride coated particles are presented in Table 4.4. AU the UHMWPE samples were pre-heated
for 11 days at 10S°C before incubating in either hydrogen peroxide or buffer. There was no
statisticd difference between ai l three samples at a 95% confidence Ievel. The presence of cobalt
chloride did not enhance the degree of oxidation beyond the oxidation index value of
4000 3500 3000 2500 2000 1500 1 O00 500
Wavenumber (cm-')
Figure 4.12: FllR Results of Various Chemical Treatments of Irradiated Particles. A.
Combined 6 day thermal treatment with 30 &y incubation in 10 w/w% Hz02, B.
Combined 6 day thermal treatment with 30 day incubation in phosphate buffer
@H 7.4), C. 6 day themal treatment, D. Control (no heating or incubation).
Table 4.4: Oxidation Indices of Cobalt Chloride Treated UEIMWPE Samples, n=3
1 Sample 1 Oxidation Index 1
CoClz-Peroxide
Control (no CoC12)
1.80 5 0.08
1.72 + 0.02
78
4.4 Hydrolytic Degradation of Oxidized UHMWPE
In this thesis, it is postulated that the oxidation process will genemte a certain number of
hydrolysable groups at the surface of the UHMWPE materiai. Hence, it should be possible to
vaiidate this hypothesis by investigating the hydrolytic stabiiity of the particles. Hydrolytic
degradation studies of the UHMWPE particle samples were performed on the
thermally/chemically oxidized UHMWPE samples. Ail the hydrolytic samples were pre-
oxidized under the foiiowing conditions: heated for either 6 or 11 days at 10S°C, and incubated
for 30 days with either phosphate bufZer (pH 7.4) or 10 w/w% hydrogen peroxide unless
otherwise inàicated.
4.4.1 Characterization of Acid-Treated UEMWPE Particles
Hydrolytic degradation midies were performed with incubation solutions of either 4N
hydrochlonc acid or cholesterol esterase, a hydrolytic enzyme. The samples treated with
hydrochloric acid were qualitatively analyzed by FTIR in order to determine if there were any
changes in fiinctional groups found in the materiai. These spectra are s h o w in Figure 4.12 and
oxidation indices are given below in Table 4.5. Direct FTIR analysis of the samples treated with
the enzyme was not carried out since the presence of adsorbed enzymes would interfere with the
ability to effectively analyze differences in the intensity of the carbonyl peaks for the materials.
Table 4.5: Oxidation Indices of Hydrolytically Treated ~ a m ~ l e s '
Sample Oxidation Index
A. HCI, heated, irradiated 1.80
B. HCI, not-heated, irradiated O. 12
C. HCl, not heated, not irradiated
( E. Heated, irradiated (oxidized control) 1 0.32 I
0.02
D. BufTer, heated, irradiated
Al1 samples in Table 4.5 and Figure 4.12, except for those that did not undergo thermal treatment
of irradiation, showed signs of elevated oxidation. Both the carboxyl peak seen between 1650 to
1850 cm-' and the C-O peaks are visible in these samples. The spectrograms of the heat heated
1.73
' These samples were not performed in aiplicate.
samples (Figure 4.13 A and D) also showed a broadening of peaks fiom 925 to 1400 cm-' which
represents the C-O region for the dcohols, ethers, esters, and carboxylic acids [Pavia, 19791.
These d t s indicated thaî the acidic conditions on their own (sample C) do not produce a
measurable chernical change in the UHMWPE material. Furthemore, it is not clear if the HC1
has enhanced the hydrolytic nature of the b a e r solution itself since there is only a small
difference in the oxidation index values for the two samples (A and D). There was an increase in
magnitude of the oxygen-related peaks (i.e. carbonyl group) when an oxidized sample was
treated with both the hydrochloric acid and the buffer. This was evident when comparing the
oxidation indices for dl the samples as seen in Table 4.7. Both the hydrochloric acid and buEer
samples increased the oxidation index five-fold over the control condition E.
3950 3450 2950 2450 1950 1450 950 450 Wavenumber (cm-')
Figure 4.13: FTIR Result for Hydrochloric Acid Treated ZlAMWPE Samples. A.
Irradiated, heated for 5 days at 105°C and treated for 7 days with 4 N HCl; B.
Irradiated and treated with HCl (not heated); C. Treated with HCI (not heated
nor irradiated); D. Irradiated, heated and treated with phosphate bufTer (pH 7.4);
and E. hdiated and heated.
4.4.2 HPLC Analysis of Hydrochlonc Acid incubation Solutioas
The HPLC results for the incubation solutions are given in F i w s 4.14 to Figures 4.1 9. Figure
4.14 shows the effect of hydrochloric acid on different combinations of irradiation and heat-
treated UHMWPE samples. The purpose of this experiment was to detennine if pre-oxidation
made a Merence for the production of hydrolytic products of UHMWPE.
O I O 20 30 40 50 60 Time (min.)
Figure 4.14: APLC Results on the Effect of Hydrochloric Acid on UHMWPE. A. irradiated
UHMWPE heated for 5 days at 105OC and incubated with 4 N HCl for 7 days at
37°C; B. Irradiated UHMWPE incubated with HCI with no heating; C. non-
irradiated non-heated UHMWPE, incubated with HCl; D. hdia ted UHMWPE
heated for 6 days at 10S°C and incubated in bder, and E. HCl only.
Chromatograms A and D in Figure 4.14 show that pre-oxidation of the polymer produced
different degradation products such as those found at 7,43, 48 and 50 minutes compared to non-
oxidized samples (Chrornatograms B, C and E). However, Chromatograms A and D differed
within the region between O to 10 minutes. Chromatogram A has several new peaks in this
region which are believed to be specifically associated with the hydrolytic environment produced
by the acid. Chromatograph B shows some changes which Iikely reflect the synergistic action of
81
irradiation and the acidic environment. It is interesthg to note that there is a peak associated
with the hydrochloric acid control (E), which appears in all HCl treated samples with the
exception of the polymer that underwent thermal oxidation. The disappearance of the peaks fiom
the latter suggests an interaction between the material and the acid solution.
4.43 HPLC Analysis of Cholesterol Esterase Incubation Solutions
Al1 the chromatograms presented in this section were produced by the freeze-dried method of
sample preparation (refer to Section 3.7). Al1 other samples produced by the choroform
extractions did not produce any peaks and are not reported in the thesis. Figure 4.15 shows the
eEect of incubating UHMWPE particle samples with cholesterol esterase (CE) and phosphate
b d e r (pH 7.4) for 15 days. The chromatopms for the enyme treated oxidized UHMWPE and
the enzyme ody solution are labeled as A and B, respectively. It was noted from the control that
despite the filtering step that was carried out to remove protein residue, there remained several
enzyme-associated products in the HPLC sample. The enzyme control had a dominant peak at 15
minutes that was not found in chrornatogram A. Chromatogram A shows the presence of a peak
located at 7 minutes (designated as Peak 4). This peak is not found in any of the control samples
and was suspected to be associated with the enzymatic degradation of UHMWPE. Upon M e r
HPLC analysis, it was detemiined that Peak 4 was able to be resolved into one peak with a
shoulder using "Program One" of the HPLC gradient method (refer to Table 3.9 in Section 3.8).
These two peaks were collected separately and labeled 4A for the peak collected from 6.57 to
7.27 minutes and 4B for the peak collected from 7.27 to 8.07 minutes. Upon using "Program
Two" gradient method (Table 3.10, Section 3.8), the 4A and 4B peaks were separated into
several distinct peaks (see Figure 4.16). The peaks labeled with an asterisk were M e r analyzed
by mass spectroscopy since these peaks gave the strongest UV signals. Time limitations
prevented M e r characterization of the other products.
Chromatogram D in Figure 4.15 shows the isolated products of the bufEer ody sample. The peak
at 50 minutes is labeled as an unknown impurity peak and is associated with al1 chromatognims
seen in Figures 4.1 5,4.17 and 4.1 8. Chromatogram C in Figure 4.1 5 shows the chromatognun of
the buf5er treated UHMWPE following oxidative treatment. T'here are three peaks found here
that are specifically related to the oxidativd bufEer treatment process. They are located at 33,40,
82
and 45 minutes and were designateci as Peaks 1,2, and 3, respectivefy. AU t h peaks were also
found in chromatogram A which was treated with cholesterol esterase. These peaks were M e r
coiiected and analyzed by mass spectmscopy. However, due to thne restraints, ody Peak 3 was
further anaiyzed using ATR-FTIR and NMR in order to attempt product identification. This
peak was chosen because no enzyme contaminates appeared to be present and it appeared to be
quite pure according to its absorbance spectnim. The other peaks were not M e r andyzed due
tirne constraints.
1 O 20 30 40 50 60 70 Time (min.)
Figure 4.15: HPLC Results for the Incubation of UfIMWPE Particles (11 day thermal
treatment) with Cholesterol Esterase and Buffer. A. UHMWPE incubated
with CE for 15 days; B. 15 days incubation with CE only; C. UHMWPE
incubated with buffet for 15 days; and D. 15 days incubation with buf5er ody.
O 5 10 15 20 25 30 Time (min.)
Figure 4.16: HPLC Spectrum of Resoived Peak 4 Using 'Program Two" Gradient Method.
4A and 4B. Peaks denoted with au asterisk were collected for mass spectroscopy.
O 10 20 30 40 50 60 70
Time (min.)
Figure 4.17: Effect of Thermal Oxidation on UaMWPE Particles in Buffer. A. Thexmdly
treated for 1 1 days and then incubated in buffer for 15 days; B. Thermally treated
for 6 days and then incubated in b a e r for 15 days; C. Thermally treated for 6 days
and then incubated in buffer for 30 days.
84
Figure 4.17 shows the effect of thermal oxidation and buffier incubation time on the release of
products 1,2 and 3. Chromatograms A and B in Figure 4.17 were thermally treated at 1 Os0C for
11 days and 6 days, respectively, and then incubated for 15 days in buffer solutions. Comparing
the two chrornatograms shows that heating the UHMWPE for longer periods increases the
number of detectable products. The only common product to both the A and B chromatograms
was the compound isolated under Peak 2.
Chromatograms B and C in Figure 4.17 show the effect of iacreasing the bufEer incubation tune
fiom 15 days to 30 days on pre-oxidized UHMWPE particles (6 days at 10S°C). Peaks 1,2 and 3
are found in the 30 day incubation samples. Hence, it would appear that the potential for saline
buffers to enhance oxidation as a hc t ion of time is similar to that of thermal treatments.
Figure 4.18 shows the effect of thermal oxidation period on the subsequent enzymatic
degradation of UHMWPE particles. Chromatograms A and B were incubated with 40 U/mL of
cholesterol esterase for 15 days, while chromatogram C was incubated for 30 days. These
chromatograms m e r in the pre-oxidation period. It was noted that the amount of product
associated with Peak 4 shows an increase for the sample which had undergone a longer pre-
oxidation period or a longer incubation period with enzyme.
O 10 20 30 4 0 5 0 6 0 7 0 Tim e (rn in,)
Figure 4.18: Effect of Heat Oxidation on Enzyme-Treated UHMWPE Particles. A.
Thermally treated for 1 1 days and incubated for 15 days with CE; B. Thermally
treated for 6 days and incubated for 15 days CE; and C. Thermaiiy treated for
1 1 days and incubated for 30 days with CE.
85
4.4.4 Mass Specîroscopy Analysis
Products were isoiated h m the HPLC samples of either: 30 &y CE 11 for Peak 4 or 30 day
buffer 1 1 for Peaks 1,2 and 3 (see Figure 4.19). The products were chosen fiom these satnples
because they contained the greatest amount of product. The notation for Peak 4 corresponds
either to "A" or " B" depending whether it was obtained fiom the first parent peak (6.57 to 7.27
minutes) or the second shouider peak (7.27 to 8.07 minutes) fiom the HPLC "Program One".
The numbers correspond to the order the peaks came off the column in the HPLC "Program
Two". Only the peaks with the strongest signals were anaiyzed using m a s spectroscopy.
However, the samples from "Program Two" were found to contain many trace amounts of
contaminants, which made finding the molecular ion impossible.
Tim e (m in .)
Figure 4.19: HPLC Peaks Further Anaiyzed with Mass Spectroscopy. A. UHMWPE
heated at 105OC for 11 days and incubated for 30 days with CE. B. WHMWPE
heated at 105°C for 1 1 days and incubated with b&er for 30 days.
The mass spectnim for Peak 3 is given in Figure 4.20. This mass spectnun contains two
dominant parent molecular ions. The molecular ions are 340.6 and 679.8, respectively. The
cluster of peaks near the protonated mass ions included the respective sodium analogs (Le. 362.6
and 701.6). The peaks at 242 and 35 1 are unknown compounds, which have been labeled as
con taminants since they do not contain the paired sodium analog which results nom exposure to
the incubation buBen. An MS-MS was performed on both of the molecular ions suspected to be
related to UHMWPE in order to M e r elucidate the chernical make-up of the products by
identiwg their respective molecdar fragments (Figures 4.2 1 and 4.22). Figure 4.2 1 shows the
fhgmentation pattern for the 340.6 molecular ion. The highest mass to charge ratio on this
spectrum was 343. The proximity of peaks on the original parent spectra made it difscdt to
specincaiiy target the mdcharge ratio of 340.6 and hence the mass to charge range analyzed for
MS-MS was fiom 340 to 343. Therefore, the fkagmentation pattern seen in Figure 4.2 1 is a
mixture of ions associated with between the 340.6 and 343 parent peaks. Due to this problem,
there are mass ions in this spectnim which are not associated with the dominant product.
Figure 4.20: Mass Spectrogram of the HPLC Product, Peak 3.
Figure 4.21: MS-MS of Parent Peak mlz = 340.6 from Peak 3.
87
Figure 4.22 shows the fragmentation pattern for the 679.4 molecular ion. The mass to charge
ratio of this ion was noted to be roughly double that of the 340.6 ion. As weli, a cornparison of
Figure 4.2 1 and Figure 4.22 revealed a similar hgmentation pattern which may suggest that the
two ions are closely related in structure.
ml2
Figure 4.22: MS-MS Result for Parent Peak, mlz = 679.4 from Peak 3.
Figures 4.23 to 4.25 show the MS-MS resdts for peaks 1,2 and 4A-10, respectively. Figure 4.2.
shows the molecular ion is 371.4. The molecular weight differences between the of hgments
45.0, 89.0, 133.2 and 177.2 suggest the presence of polyethylene glycol segments (Le. [-
CH2CH20-] with a molecular weight of 44). Peak 2 (Figure 4.24) has a similar hgmentation
pattern to the products in Peak 3 and is suspected to be similar in chernical nature. Many
products fiom Peak 4 contain low molecular fragments that are suspected to be arnino acids. The
mass spectra of the other Peak 4 products are given in Appendix E
O 50 1 O0 150 200 250 300 350 400
mlz
Figure 4.23: MS-MS of Peak 1, Parent peak m/z=371.4
Figure 4.24: MS-MS of Peak 2, Parent peak d453.2
Figure 4m25: MS-MS of Peak 4A-10, Parent peak m/z=172.8.
89
A listing of selected isolated products and rnolecular weights (when possible) is given in Table
4.6. Some of the products could not be analyzed for the molecular ion because they contained
too many con taminants. Table 4.6 shows that the degradation products identified to date have
relatively high molecular weights whether they are enzyme associated or buffet associated.
Table 4.6 Molecular Weights of the Isolated HPLC Products
1 1 40 minutes 1 Program 1 1 453.2
I 1 44 minutes 1 Program 1 1 340.6 ( 679.4
1 4A-11 1 20 minutes 1 Program 2 1 Multiple species
4A- 10 18 minutes
4A- 13
4B-3
4B-5
4B-10
Program 2
23 minutes
8 minutes
20 minutes
25 minutes
1
172.8 1 684.6 1 758.4
Program 2
Program 2
Program 2
Program 2
Multiple species
388.2 437.0
Multiple species
Multiple species
90
4.4.5 Cbemical Characterization of the EiPLC Prodncts
The mass spectnim resuits alone couid not provide definite structures for the d o m products
and hence, M e r chemical analysis was required. ATR-FTIR spectroscopy of the samples was
performed in order to obtain iaformation on the nature of chemical groups found in the unknown
compounds. Due to Limitations in t h e , this work focused specifically on Peak 3 which
contained two molecular ions (340.6 and 679.4) of which the highest molecular weight species
dominated the sample.
The ATR-FTIR spectrum for products fiom Peak 3 is shown in Figure 4.26. The sarnple was
scanned at a resoiution of 4 cm-' using 250 scans and the analysis solution was prepared using
dichloromethane as the solvent. The double peaks seen at about 2300 cm-' are associated with
the carbon dioxide peaks from the ambient air. Since the FTIR spectrogram is compared to a
background scan, the amount of carbon dioxide in the ambient air varied between the time of
collection for the background scan and the test specimen. Therefore, a negative peak was
obtained for the solvent control upon subtraction of the background spectrograrn. A reference
spectrum fiom the Merck FTIR Atlas (GR Pachler Klaus, 1992) of dichioromethane was also
compared with the sample, and is shown in Figure 4.27. Peaks associated with the degradation
product are found at 1000 cm-', 2850 cm-', and 2950 cm-'. As well, there appears to be a
shoulder on the 1450 cm-' solvent peaks which appears at approximately 1475 cm-'.
3300 2800 2300 1800 1300 800
Wavenumber (cm-1)
Figure 4.26: ATR-FTIR Result of HPLC Isolate et 44 Minutes. A. Coliected Sample fiom
Peak 3 B. Dichloromethaae
Figure 4.27: FIlR Standard of Dichlommethane. Peaks are found at 3055,2988,2686, 2306, 1422 1265,896,747, and 706 cm-' werck FT-IR Atlas, 19921.
Proton NMR was also used to try to deduce the chernical structure of the products in Peak 3 (see
Figure 4.28). The peak located near 7.15 pprn represented the benzene carrier solvent. Ail other
peaks were shifted with respect to this peak. The shift at 0.4 pprn has been assigned to be
protons related to silicon grease contaminants [Gottieb et al.. 1997. The groups located between
0.8 to 1.6 pprn are not well resolved. As indicated by the high integration ratio (1 IO), these
peaks make-up the buik of the sample. It is suspected that the polyrneric nature of the molecule
is responsible for this observation. A similar occurrence for peaks located at 1.6 to 2.2 was
observed. A small contribution by triplet peaks located at 2.3 and 2.6 (totaled 1.46 integration
ratio) was noted. A triplet is caused by a proton having two neighbouring protons such as two
methylene groups adjacent to each other. This would suggest that the chah is heterogeneous and
substantiates the presence of hydroxyl groups which was indicated fiom the IR spectnun (Figure
4.26). The peaks located at 4.0 to 4.3 pprn indicate a triplet followed by two singlets and is
characteristic of double bonds. Once again, there is a small contribution of this structure to the
overail bulk sample.
Carbon-13 NMR was performed to determine the structural components of these peaks (Figure
4.29). The large peak Iocated at 128 pprn is the carrier solvent benzene. The other major peaks
present are located in the 25 to 30 pprn region which is indicative of the alkane groups. Further
analysis was not performed due to the extreme noise found in the sample which was caused by
too little sample.
Figure 428: Proton NMR of Peak 3. A. Wboie spectrogram from O to 11 ppm. B.
Sïice of spectrogram from O to 2.8 ppm. C. Slice of spectrogram from
3.6 to 5.8
Figure 4.29: Carbon-13 NMR of Peak 3.
4.5 In Vitro Study of HDPE Particles with Hamao Monocytes
4.5.1 Viabiiity Study Results
The viability results for the monocyte samples containing collagenlDMS0, non-treated HDPE,
cobalt-treated HDPE in a collagen ma&, and cobalt-treated and peroxide treated HDPE in a
collagen matrix are shown in Figure 4.30. It should be noted that ail cobalt treated samples ( ie. ,
HDPE-Co, and HDPE-Co-H202 ) were first heated at 105°C for 1 1 days, then coated with cobalt
sdt and then incubated for 30 days in 10 w/w?% peroxide solution. Significant numbers of
floating cells were observed in the monocyte cultures. Floating cells fiom two donors were
coilected, pooled and stained with trypan blue. The percentage of trypan blue positive cells was
interpreted to be the percentage of necrotic or dead floating cells found after 24 and 48 hours.
The cells for both 24 and 48 hours were pooled fiom two different donors. The cells treated with
cobalt and cobalt/peroxide were more irreguiarly shaped and smaller compared to the control
sample containing only the collagen/DMSO maeix (see SEM micrographs in Figure 4.31).
There was a difference in the cells treated with cobalt compared to those treated with cobalt and
hydrogen peroxide. However, there was no SEM of the monocytes with non-treated HDPE.
100 - collagenlDMS0 only
'- . . HDPE only
24 hours 48 hours
Incubation Period
Figure 430: Necrosis of Non-Adherent Human Monocytes, n=2 for PU simples but 1
Co-H202, where n=l. (The error bars represented the standard deviation of
the samples.)
The sample treated with cobalt chloride and hydrogen peroxide does not have error bars because only one study
(single donor) was performed with this treatment.
95
M e r 24 hours, there was a slight increase in the number of necrotic ceUs between the control
and the other samples as weil as between the HDPE and the cobalt and the cobalt/peroxide
treated samples (14% to 19% to 24% to 26%). However, d e r 48 hom, the treatments by HDPE
ody, cobalt and cobaWperoxide induced a significantly higher level (p<0.05) of necrosis by
increasing the percentage of dead cells to 45%, 58% and 79%, respectively. The amount of
necrosis for the control sample at 48 hours increased slightly nom the 24 hour control sample.
Figure 4.31: SEM Micrographs of Euman Monocytes and Treated HDPE. A.
CollagenDMSO without Particles. B. Cobalt-Treated Particles. C. Cobalt-
Treated Particles hcubated in Hydrogen Peroxide.
96
4.53 Cytokine Resuits
The amount of DNA in the adherent cells was detennined fiom a calibration curve that related
fluorescence to the concentration of DNA (ng/mL) (given in Appendix G). Subsequent cytokine
results (1 L-1 P, 1 L-6 and W u ) were normdized based on the concentration of DNA as seen in
Figures 4.32 to 4.34. The amount of cytokine produced represents the amount generated within a
24 hour period.
I collagenlDM SO - I HDPE only
24 hours 48 hours
lncu bation Period
Figure 4.32: IL-lp Release from Human Monocytes. Collagen/DMSO was the control sample
which contained a collagedDMS0 matrix, HDPE only was the polyethyiene
control which contained HDPE particles embedded in a collagen/DMSO rnatrix,
HDPE-Co had cobalt chionde coated HDPE (thennaily oxidized) in the same
matrix. HDPE-Co-H202 had cobalt chloride treated HDPE (thennally oxidized)
samples which were oxidized in 10 w/w% hydrogen peroxide for 30 days. Al1
samples were done twice in two culture wells (n=4). nie error bars were the
standard deviations of the samples.
The amount of IL-1 P released in the adherent monocyte culture decreased slightly compared to
the control sample after the first 24 hours when the particles were treated with the cobalt salt,
with and without hydrogen peroxide (see Figure 4.32). The samples which contained
DMSO/collagen matrix produced the most IL-1P (about 900 p g / d per pg of DNA sample),
followed by the non-treated HDPE particles (about 600 pglmL per pg of DNA). At 48 hours,
97
there was a decrease in the amount of IL-le cytokine produced nom all sample preparations
w0.05). There was no statistical difference w0.05) between the control and cobalt-treated
samples. However, data for the peroxide treated sample showed that the amount of IL-IP was
reduced by almost half of compared to all other samples after 48 hours.
The amount of IL6 released in the adherent monocyte culture was similar for al1 four samples
after 24 hours (see Figure 4.33). Once again, after 48 hours, there was a significant decrease in
the amount of measured cytokines. The level of IL-6 cytokine decreased fiom 1000 pg/mUpg
DNA to roughly 300 p g l d p g DNA for the cobalt chlonde treated sample. This sample after 48
hours produced the greatest amount of IL-6 but showed a very high variability.
1400 HDPE only 2 1200
-r
O HDPE-CO - O HDPE-CO-MO2
V
24 hours 48 hours
Incubation Period
Figure 4.33: IL4 Relerse from Human Monocytes. ( n 4 and the eror bars are the
standard deviations.)
Figure 4.34 shows the release of TNF-a fiom the human monocyte culture after 24 and 48 hours.
In general, there is very little of this cytokine released as compared to IL-1 f3 and IL-6. As well,
the error bars were greater for this cytokine than the other cytokines. M e r 48 hours, there was
little TNF-a found in the supernatant.
24 hours 48 hours
lncu bation Period
350
Figure 4.34: TNFa Release from Human Monocyte Supernatant (CI=&, the error bars are
the standard deviations of the samples.)
300 - collagenIDMS0
-7 I HDPE only
99
5.0 DISCUSSION The piirpose of this thesis was two-fold. The fïrst objective was to oxidize and hydrolyze
UHM\KPE particles under conditions that could simulate degradation events associated within
the phagolysosomes of inflammatory ceiis, which are found in vivo near failed total joint
replacements. The second objective was to probe the hypothesis that degraded UHMWPE
particles combined with metallic ions and their associated degradation products play an
important role in inducing specific cytokine responses fkom human monocytes. To accomplish
the second objective, it was desired to first isolate and identiS, the nature of the
oxidativehydrolytic degradation products.
The need for this study &ses fiom the problem of polyethylene Wear particulates at the site of
failed total joint replacements [Campbell et al.. 19931 and their suspected role in the induction of
bone resorption at the interfaces of the failed implants. To date, the scientific literature has
focused on three areas of study concerning the importance of Wear particles. Many studies have
characterized the size and shape of UHMWPE Wear particles fiom retrieved implants [Bade et
al.. 1996, Kobayashi, et al., 1997, Matlaga et al.. 1976, shanbhagl et al.. 1994, Campbell et al..
19961. They have also attempted to assess the level of cellular response of macrophages in the
presence of polyethylene particles [S hanbhag et al., 1 995, Horowitz et al.. 1 9971 and the effect of
cobalt, chrornium and the cornbined effect of cobdt-chromium on cells [Allen et al., 1997 and
Lacy et al.. 19961. Other studies have injected polyethylene particles at the site of the
articulating surfaces in order to elicit an inflammatory response [Howie et al.. 1988 and
~oodmanl'*~ et al., 19961. However, to date, there have been no -dies that have critically
assessed the importance of the polyethylene particulates' surface chemistry on the initial response
of human monocytes. This study will be used to attempt to mode1 changes in particdate sinface
chemistry in a rnanner that reflects the idluence of elements from the implant environment.
5.1 Chernical Characterization of Test Specimens
5.1.1 Irradiation
"A material's treatment prior to implant may predispose it to stable or unstable end-use
behaviour" [Ratner et al., 19961. This has been true for the irradiation of UHMWPE implants
and has been validated again in this thesis. Before the samples were subjected to chernical
100
oxidation, they were irradiated with 30 Mrad of 60~obalt y-irradiation in accordance with
industry standards [~treicher ", 1 9881. If particle sample were not irradiated prior to incubation
or thermal experiments, there was no detectable oxidation present after these processes. This
result is very similar to many studies which showed secondary effects of irradiation such as
changes in mechanical properties. Specifically, the oxidative effect of y-irradiation on
UHMWPE has been well established and has been docurnented to cause chab scission, changes
in the density, fatigue strength and other mechanical properties phambri et al.. 1997, Brossa et
al.. 1 996, Choudhury et al.. 1996, Collier et al.. 1 996, Pascaud et al.. 19971.
XPS and FTIR r e d t s (Tables 4.1 and 4.2 and Figure 4.10) for the UHMWPE resin and film
samples show that there was no significant ciifference between the irradiated and non-irradiated
film samples. However, there was a significant ciifference in the amount of oxygen and the
degree of oxidation for analogous particle samples @<0.05). A possible explanation for these
resdts is that in order for the oxidation to be detected, fke radicals must be able to react with
oxygen species to produce oxygen-containhg groups. The processing of the film samples
consolidates the resin and aligns them in a particular direction pankston et al., 1995, Rentfiow
et al.. 19961. This removes some of the amorphous regions and limits the propagation of the
oxygen species through the bulk of the polyrner. This e x p l d o n is supported fkom studies by
Poggie et al. (1997) which have shown that more consoiidated UHMWPE specimens exhibited
less oxidation, even after treatment by y-irradiation and aging at elevated temperatures (60°C and
70°C). Since the particles have not been processed, the arnorphous regions remain intact and
easily allow for the diffusion of oxygen throughout amorphous regions. Future work could use
electron spin resonance (ESR) to determine the amount of free radicals remaining in the particles
versus the films. It would be anticipated that the particles would have fewer Eee radicals since
the diffusion of oxygen throughout the matenal would eliminate the fiee radicals following the
oxidation reaction. This technique has been previously used to determine the fiee radical
distribution in UHMWPE articulating sufaces [Jahan et al.. 199 11.
Whiie irradiation is an important factor in increasing the polymer's susceptibility to oxidaîion, it
is not the only factor. The effect of processing the resin samples into bar stock plays a role in
rendering the m E susceptible to oxidation. This is seen in Figure 4.10 which shows the
amount of initial oxidation in the non-irradiated fih samples was greater than the non-irradiated
101
resin samples. ûther studies have shown that processing the UHMWPE not only makes it more
susceptible to oxidation, but that ram-extrusion makes UHMWPE more susceptible to oxidation
than compression molded implants [Deng et al., 19971. This was attributed to the incomplete
consolidation of the resin particles and hence, there is more void volume to allow for the
diffusion of oxygen.
5.1.2 Cobalt-Treated Samples
Cobalt-chromium metal is comrnoniy used for the stem portion of total joint replacements. As
such, traces of cobalt ions have been found in retrieved implants bound with the particdate wear
debris weldnim et ai., 19931. Cobalt metal ions were also found at the site of uiflammatory
tissues arouud loosened implants weldnun et ai., 19931. It was s h o w that cobalt can accelerate
the oxidation of UHMWPE by acting as a catalyst in oxidation reactions [Zhao et al., 19951.
More specifically, cobalt chlonde in the presence of hydrogen peroxide creates hydroxyl radicals
(OH), molecular oxygen (O2) and superoxides (-O-*) which simulate oxidative products
generated in the in vivo environment.
A study by Brodner et al. (1997) has show that the concentration of cobalt found in the s e m of
patients with metal on metal cobalt implants was found to be 1.1 ppb while patients with
ceramic-on polyethylene implants had cobalt levels below the detection limit of 0.3 ppb.
Another study fiom retrieved polyethylene cups of Co-Cr-UHMWPE systems, found that after 12
years of articulation, 64 + 2 1 ppb of cobalt was found to be associated with the UHMWPE
articulating surface peldnim et al., 19931.
In the curent study, the treatment of UHMWPE with cobalt ions was used as a mode1 for cobalt-
containing particles present at the site of aseptic loosened implants. XPS characterization of the
particles (see Table 4.3) showed that less thao 0.10 atomic % of cobalt ions was found within the
top 10 nm of the surface. Since this represents the Iimit of detection for WS, it was very
difficult to quant@ the amount of cobalt present at the surface. It was observed that the particles
had a rough topography, and it could be possible that most of the cobalt chloride resided in the
crevices of the sample (as seen by the SEM micrograph, Figure 5.1). Therefore, surface
quantification by XPS would be M e r hindered. As well, investigations using EDAX analysis
also showed trace amounts of cobalt present (Figure 4.7).
Figure 5.1: SEM Micrograph of Cobalt Treated UHMWPE and Non-Treated UEMWPE.
A. Cobalt Treated Sample, B. Non-Treated Sample
The presence of trace amounts of the cobalt was also visible to the eye since the treated samples
retained a pink stain following the coating and washing procedure. To overcome the limitations
of quantification in the above techniques, it would be recommended that future characterization
of these particles be canied out using atomic absorption spectrophotometry or secondary ion
mass spectroscopy (SIMS) [Ratner et al.. 1 9961. Absorption spectrophotometry has a detection
limit of 0.3 pg/L or 0.3 ppb @Meldrum et al.. 19931 while SIMS can detect the presence of ions at
the outermost 1 m of the surface. Both of these techniques should be employed to determine
the total concentration of cobalt ions present at the surface of the sample.
It has been reported that cobalt ions in elevated concentrations (i. e., 10 to 40 ppm) are toxic to
human gingival fibroblast cells [Lacy et al., 19961. Other studies have shown that cobalt in
concentrations of O to 1.0 mg/mL proved toxic to human osteoblast-like cell iines and inhibited
the production of collagen type-1, osteocalcin and alkaline phosphatase. When cobalt chromium
and chromium alone were incubated with this cell line, there was little effect [Allen et al., 19971.
Therefore, it is important to consider the type of cobalt used in these experiments. The LD50-
LC50 (oral rat) was 766 mglkg or 766 ppm according to MSDS documents [J.T. Baker
103
Company]. Considering the amount of cobalt present in our samples was below the detection
limits for both methods used in this study, it was assumed that the levels of cobalt are probably
under the toxic level; however, a more quantitative analysis of the amount of cobalt will be
required in fbture work.
5.2 Thermal and Chemical Oxidation of UHMWPE Samples
5.2.1 Thermal Oxidation
Preliminary experiments showed that it was very difficult to oxidize UHMWPE particles at
temperatures below 80°C. In order to achieve measurable Merences in the oxidation index
values within a reasonable time h e , the temperature was increased to 105°C. Sulphur dioxide
staining (Figures 4.5) showed that at this temperature, oxidation was visible. This temperature
was chosen so that oxidation of the particles could be achieved without melting the UHMWPE
(melting temperature is about 136°C fiom MSDS). Thermal oxidation studies (see Figure 4.3)
showed that it took 1 00 hours at 1 Os0C in order to induce a substantial increase in the oxidation
index. At this tirne and temperature, the change in the oxidation index value was exponential in
nature. This suggested that a threshold condition had been achieved which allowed for an
accelerated oxidation reaction. This condition could be attributed to the sample attalliing a
critical number of fkee radicals which initiated the oxidation reaction throughout the materiai.
This oxidation reaction could be andogous to free radical polymerization reactions where the
reaction rate is known to be exponentiai in nature woung et al., 1 9951.
When comparing the differences in oxidation index for films and resin particles with different
treatments, it was evident that the film samples were more susceptible to thermal oxidation
(Figure 4.10). This was observed despite the efforts made to normalize the surface area for the
two samples. The elevated oxidation value baseline for the film samples indicated that pnor to
thermal treatment, these specimens had a greater number of reaction sites (i-e., oxygen-
containhg sites) compared to the particle sample. The presence of reactive oxygen species in
these regions (Le. carboxyl, carbonyl, and hydroxyl groups) will aiiow for oxidation to propagate
throughout the sample when M e r exposed to 6ee radicals [Ali et aï., 1994, Bigger et al.,
19921. Other studies have found that different methods of processing affect the susceptibility of
UHMWPE degradation mindau et al., 19961. Hence, in the same manner that irradiation has
pre-sensitized the UHhlTKPE to oxidation, so does the processing history.
104
5.2.2 Chemical Oxidation
Many investigators have found it difficuit to work with UHMWPE in biological systems because
its low density and hydrophobicity make it incompatible with aqueous media Hence,
consideration was given to this issue when optiminng an experimental apparatus for exposing
the particles to oxidizing and enzymatic solutions. To ensure that a maximum contact between
polyethylene with the various media was achieved, a closed, large dace-area container was
used as the reactor vessel. The system had to be closed in order to prevent the evaporation of
incubation solutions. A concem that arose with the use of this systern was the discoloration of
the Teflono-lined caps for the botties used. Some discoloration of the lining occumd after long
incubation periods, both in the presence and absence of oxidants. However, since there was
minimal contact of the incubation solutions with the cap liners, the discoloration is believed to be
the resdt of ambient gases generated by the incubation medium. To ensure that this does not
e t the composition of the degradation products found in the system, hture work should
consider testing the discolored lids for the presence of leachable components.
Preliminary experiments with film samples incubated with a relatively stmng oxidant,
hypochlorous acid, to determine the ability of the chemical oxidant to degrade the UHMWPE
substrate. Samples of UHMWPE were placed in solutions of 7.6 mM sodium hypochlonte for
24 hours. In the absence of irradiation, the HOCI activity for the films was similar to the non-
treated control samples. Again, this provided M e r evidence of the difficulty in oxidizing
UHMWPE. However when the UHMWPE was exposed to y-irradiation, the UHMWPE became
more susceptible to chemical oxidation by sodium hypochlonte (see Figure 4.8). This
observation suggests that sterilization by irradiation is not the ideal method to sterilize the
polyethylene implant materials because it renden the polymer susceptible to oxidative attack by
chemical oxidants.
Even though some oxidation of the UHMWPE occurred with HOCI, the levels were significantly
less than the observed values for the polyurethane control (Figure 4.9). It has been reported that
HOC1 can easily oxidize and cleave substituted nitrogen-containing groups such as amides, ureas
and urethanes which are present in polyurethmes mtner et al., 1 9961. Since vitgin polyethylene
does not contain any of these groups, it was difficult to oxidize. However, the irradiated film and
105
particdate samples contain a number of oxygen groups and fke radicals [Jahan et al., 19911
which can react with the oxidants.
The importance of irradiation versus non-irradiation and their eEect on chemical oxidation was
mer supported with the FTR data presented in Figures 4.9, 4.10 and 4.1 1 for hydrogen
peroxide treatment of films and particles. When both the particle and film samples were
incubated in hydrogen peroxide, an interesting result was obsewed (Figure 4.10). While there
was a signincant increase in oxidative degradation, there was no signifïcant clifference @ < 0.05,
n=3) between the two types of UHMWPE samples. However, in compathg the magnitude of
change of an irradiated sample to an irradiated and chemically treated sample, the effect of
chemical oxidation on the particle sample was pa te r because the baseline value of the particles
was smdler compared to the film's baseline value. It should be noted that the amount of
particles and film had been adjusted to yield similar surface areas.
Three possible expianations couid be considered in order to explain these results. First, the
chemical oxidation of UHMWPE may not be dependent upon the amount of oxidation initially
present in the material and hence it occurs by a diEerent mechanism as compared to thermal
oxidation and oxidation by y-irradiation. However, this is not Likely because it has been show
that in order to chemically oxidize W E , the material must be pre-sensitized with either
thermal oxidation or irradiation. Therefore, 6ee radicals and susceptible oxygen-groups are
needed in order for signincant oxidation to occur with chemical oxidants. The second
explanation entails that chemical oxidation of UHh4WPE has reached a plateau level for an
oxidation index value of 0.35. Unfortunately, Figure 4.1 1 refutes th is because an oxidation index
of 1.5 was achieved for samples that had undenvent thermal and chemical oxidation. The final
explanation assumes that chernical oxidation is diffusion limited. The susceptible fiee radicals
are present within the bulk of the sample which is floating on top of the chemical oxidant. If
oxidant is not able to enter the arnorphous regions of the polymer, then oxidation would be
delayed. For the particle sample, it would be expected tbat the clumping of the polymer resin
wouid decrease the ability of the oxidant to react with the fkee radicals. However, it is believed
that the open structure of the particles (seen in Figure 5.2A) offsets the effect of clumping and
allows the oxidant to diffuse throughout the sample. In the case of the film sample, the SEM
(Figure 5.2B) shows a smooth surface. This indicates close packing of the polymer chains and
1 O6
wouid r e d t in Limited diffiision of oxidant into the bulk of the sample where the fke radicals
exist,
Figure 5.2: SEM Micrograph of A. m E particle sample, and B. UHMWPE film
sample.
In general, there were large error bars associated with chernical oxidation treatment of particles
(Figure 4.10). This was beiieved to be associated with the n o n - d o m nature of the particle
sample. The crevices present in the particle samples appeared random (Figure 5.2A) and would
account for an uneven distribution of oxidant throughout the sample. Further experiments are
needed in order to increase the number of samples beyond the triplkate performed in this
experiment .
It was noted that if a sample underwent extended thermal oxidation, the oxidation index was not
sensitive to the presence of chernical oxidants (Figure 4.1 1). Two explanations exist. First, it is
possible that the amount of oxidation may reach a saturation point where the oxidation reaction
has reached the termination step of the fiee radical reaction. If this was the case, there would be
no fiee radicals to react with the susceptible groups and no more oxidation could occur. Another
1 O7
explanation is that limitations in the definition of the oxidation index (as discussed in Chapter 2)
could have prevented the detection of oxidation associated with the chemical reaction beyond a
certain oxidation index value. Therefore, it would be of interest to f.urther compare the oxidation
indices and choose a more suitable dekition (see discussion on defuitions in 3 S. 1).
When the heated film samples were placed in a phosphate bufEer solution, there was an increase
in the oxidative degradation as seen in Figure 4.1 1. This result is consistent with the literature
[Betts et al., 1994, Henry et al.. 19901 which has shown that salts can increase the polyethylene's
susceptibility to oxidation by acting as a catalyst for the oxidation reaction. It would be expected
that a simila. reaction should be present in the physiological environment because of al1 the saits
present in body fiuids.
When increasing the heating tune of the samples incubated in solutions (Figure 4.1 l), there was a
dramatic increase in the observed oxidation index for al1 samples. This included the control
films which were not exposed to the buf5er or the oxidant. Since the oxidation index values
appear to have reached a plateau value, it may be suggested that d e r 11 days of heating, the
sarnples have al1 approached a saturation point in oxidation. Despite this, there remains a small
but significant difference between the control and buffer treated groups, with the peroxide treated
samples (at a 95% confidence level), but not between the control and bufTer treated samples.
These data highlight the role of processing on the oxidation process and are relevant to the
physiological environment since pre-oxidized particles from Wear debris at inflammatory sites
will be engulfed by phagocytic cells which contain a harsh oxidative and hydrolytic environment
[Smith, 19941. One of the key oxidative agents present in the ce11 and secreted into the
inflammatory environment is hydrogen peroxide. If the particdate is able to be M e r oxidized
by hydrogen peroxide, after it is engulfed by the cell, then degradation products couid be
generated that may stimulate the secretion of biological agents that could play a role in the
degradation of the implant.
5.3 Hydrolysis of Oxidized UHMWPE
To date, there are no studies that have reported on the nature of biodegradation products
associated with UHMWPE particles, whether derived fkom physiologicaily relevant oxidants or
hydrolytic agents. Products generated fkom in vitro studies of this nature may be relevant to ce11
108
responses observed foilowing the phagocytosis of UHMWPE particles at sites of 6 c i d joint
implants. One of the objectives of this thesis was to isolate and identiQ some of these
degradation products.
It should be considered that hydrolysis may be hindered by high crystallinity and hydrophobicity
[Ramer et ai., 19961. While UHMWPE is known to contain some crystallinity, it is reported to
contain amorphous regions [A. Wang, 19971. Ln addition, hydrophobicity is decreased with the
introduction of oxygen-containing groups through thermal and chemical oxidation. Susceptible
hydrolytic chemical groups are fiinctional groups which have carbonyls bonded to heterochain
elements of oxygen, nitrogen or sulphur [Coury et ai., 19961. Thus, ody samples that showed
signs of oxidation (i.e., containhg carbonyl and carboxyl groups) were hydrolyzed with
hydrochloric acid cholesterol esterase enzyme or buffer. By hydrolyzing oxidized samples, it
was anticipated that hydrolysis would be more likely to cleave iow molecuiar weight oligomers
of the polyethylene.
The isolation of products associated with UHMWPE, after it had been chernically and themially
oxidized and hydrolyzed, was successfully carried out by HPLC analysis. Interestingly enough,
there were three products that were associated with bufTer-rnediated degradation, afler thermal
and chemical oxidation took place (see Figure 4.15C). This result suggested that hydrolysis of
the samples had taken place in the presence of the buffer solution or that these products were
leached fiom the oxidized matenal. The analysis of HPLC results for enymatic degradation
products was shown in Figures 4.1 5 and 4.16. Although Figure 4.16 showed that many products
were generated through this hydrolytic degradation, it was suspected that some of these products
were achiaily associated with protein contaminants fiom the enzyme or with hydrolytic
degradation products complexed with some of these contaminants. Studies in our laboratory
have show that the cholesterol esterase enyme contains 90% of non-active protein
contaminants (communication with Mrs. Yi-wen Tang). These con tambants were of low
molecular weight since a 5000 molecuiar weight cut-off filter was used in the preparation of al1
HPLC samples. In order to confirm whether these peaks are related to con taminants or actual
hydrolytic products associated with the polyethylene, purified enzyme should be used in fuhue
studies.
1 O9
The breakdown m e n t a t i o n pattemu from the mass spectroscopy redts for Peaks 1, 2, 3
(Figures 4.20,4.23,4.24) and 4A-10 (Figure 4.25) showed interesthg cbcteristics. From the
MS-MS of Peak 1 (Figure 4.23), it was proposed that the product was a polyethylene glycol
derivative and contained at least four repeat ethylene oxide uni& as seen in Table 5.1. It had a
molecular weight of 370. Oxidation should be a random process that cleaves the backbone of the
polyethylene. The probability that a product was genenited in which every second methylene
group contained an oxygen inserted into the backbone is too coincidental. Ethylene glycol
denved compounds are common laboratory contaminants and are usually found in lubricating
grease [Gottieb et al., 19971. It was not clear why this product was only found in the oxidized
samples; however, at this tirne this product is assurned to be a contamination product and no
m e r analysis was d e d out.
Table 5.1: Chemicai Structure of Fragmented Ions Related to Peak 1 (Derived from Figure
4.23)
m/z A m/z Ion Chemical Structure
Other peaks analyzed by mass spectroscopy were listed Table 4.6. It was found that the isolated
products fiom the b e e r treated samples had relatively hi& molecdar weights. For example, the
products associated with Peak 3 had molecular weights of 340 and 678. This indicates that the
oxidation and hydrolysis of UHMWPE occurs quite infrequently dong the methylene backbone.
Due to thne constraints, Peak 2 was analyzed with ATR-FTIR (see Appendix J) and Peak 3 (see
Figure 4.20) was further analyzed using both NMR and ATR-FTIR. Peak 3 was chosen because
is was simpler to analyze than peaks which contained enyme products (Peaks 4A and 4B senes)
and it was seen to have a similar mass spectrum fragmentation pattern to Product 2. Some
common hgrnented ions to the three MSMS spectra (Figures 4.21, 4.22 and 4.24) were
m/~55 ,70 , 83, 100, 1 1 1, 128, 182,2 10,226,326,43 5, and 453. If the structure of the products
in peak 3 could be elucidated, those in peak 2 codd also be easily defineci, since its molecdar
weight is less than 678, the value associated with the dominant product under peak 3.
110
A concem considered during the d y s i s of the degradation products was that there may be
additives incorporated during the manufacturing process of UHMWPE PI], such as calcium
stearate, which could be identified as a degradation product. Mass spectroscopy was perfonned
on a sarnple of stearic acid (see Appendix G) and the hgmentation pattern was compared to the
degradation products found in this study. There was no matching product found for stearic acid.
A cornparison of the W absorbance of Peak 2 and 3 (Figure 5.3) shows both UV spectra have
the inflection point at slightly more than 200 nrn. The difference between the wo spectra lies in
small increases of absorbance between 250 and 300 nm, where there is absorbance for Peak 3 but
not for Peak 2. This result M e r suggests that the two products associated with Peaks 2 and 3
are similar in nature.
Peak 2 Peak 3
hluiura
Figure 5.3: Cornparison of the W Absorbance Spectra of Peaks 2 and 3. A. W Spectra
for Peak 2, and B. UV Spectra for Peak 3.
A pur@ test was performed on Peak 3 (Figure 5.4). Absorbance was detemiiaed at several
points dong the peak. The same absorbance spectra were not found throughout the sarnple.
Figures 5.4A and 5.4B showed very sirnilar UV spectra with the iaflection found slightly under
200 m. However, Figure 5.4C showed a shifi in the inflection point about 205 m as well as
some small absorbance signals in the higher W region 260 nm and 295 nm. Absorbance
between 200 to 400 nm is indicative of double bond sequences or aromatic groups mssenden,
11 1
19901. It is highly doubtful that ammatics are present in our samp1e since none of the oxidation
pathways (see Section 2.55) reported in the literature have suggested such a product except under
extreme combustion conditions [Pacakova et al.. 19911. This was confirmed by mass
spectroscopy anaiysis which found that Peak 3 contained two molecular ions with a mass-to-
charge ratio of 341 and 679. Since the two products had the same retention t h e , the structures
must be relatively similar. It is suspected that one product may be a fragment of the other. Since
there was some difficulty in isolating the MW 341 sarnple for the MS-MS analysis and the 678
MW product seemed to be present in larger quantities (see MS intensity for parent ions in Figure
4.20), fiirther analysis of the structure was only pursued with respect to the higher molecdar
weight product-
A L O U . U O
MLuioa
Figure 5.4: W Absorbance of the Product Associated with Peak 3 to Determine Purity of
Sample. A, B, C, D, and E are different points on the curve and correspond to
different absorbance spectni.
For purposes of illustration, if the dominant peak contained a molecular weight of 678 MW, and
assuming only CH2 groups were present, then approximately 48 CH2 repeat units wouid make up
the molecde. Of course, this scenario is not possible, shce oxygen groups and some double
bonds are thought to be present based on the ATR-FTIR spectrum (Figure 4.26). There was a
hgment found in the MS-MS data of the high molecular weight product in Peak 3 (Figure 4.22).
The MS analysis showed that the last three fragments were very similar. The differences fiom
679.8 to 661.6 to 643.0 is approximately 18 which is water. This could be made up of an "OH"
fiagrnent nom an alcohol and an "H" fiagrnent from the same compound or fiom the
environment. Free protons are very cornmon during MS-MS process and it would be quite easy
to pick up extra protons. The ciifference of 1 8 is also found between 435.2 to 452.0. As well,
another cornmon fragment was 17 found between pairs of ions (Le., 309 to 326). This could be
the "OH" fragment nom an alcohol group.
Other patterns shown by the MS analysis is the difference of 100 between pairs of fragments (i.e..
452.0 to 552.2 and 435.2 to 534.6). The MS analysis showed that a Merence of 226 was
commonly repeated between several pairs of ions (569.6 to 343.2, 552.2 to 326.4, and 534.6 to
309.4). in general there were large fragments of 100 and 226 removed fiorn the molecular ion
and rnany water molecules which were attributed to the cleavage of hydroxyl groups.
The ATR-FTIR analysis (Figure 4.26) showed that there were no aromatics present in this
product. This is difTerent fkom other thermal oxidation studies of polyethylene that showed the
presence of aromatics when low density polyethylene was placed in a combustion chamber
pacakova et al.. 19911. However, the latter study also showed the presence of 1-alkenes and
aikanes. The presence of these groups is suspected to be present based on the ATR-FTIR and
NMR shifts (Figures 4.26 and 4.28) in our study. Furthemore, their presence was suggested by
m a s spectroscopy and W absorbance results. However, the weak W, IR and NMR signals
suggest that the double bond contribution is very srnail as compared to the other functional
groups present in the product. If a double bond was present, it would be located near 1640 cm"
or 3000 to 3 100 cm-'. None was detected in the 1640 cm-', but a weak shouider peak is found in
the 3000 to 3 100 cm-' (Figure 4.26). Upon subtraction of the two spectra in Figure 4.26, it was
possible to see the presence of the IR signal in the 30 10 cm'' region which indicates the presence
of a double bonds (Figure 5.5). However, due to extremely low amount of sample used in this
study, the peak is very weak. Future work should consider repeating the measurement with a
greater concentration of sample.
M e r fiinctional groups confïrmed by the ATR-FTIR spectrogram (Figure 5.5) include the
presence of a large concentration of methylene groups (CH2) located around 1450 cm'' and
alkane stretches in the 3000 to 2820 cm" range. At 1000 cm-', there is a strong peak which has
been assigned to a hydroxyl group. At 1275 cm-' there was the presence of a very strong peak
1 I3
that was associated with the solvent (see Figure 4.27). Upon subtraction, this peak was not able
to be removed h m the spectra. As weli, Appendix H shows the ATR-FTIR subtraction of Peak
2. It contained identicai IR peaks to those found in Peak 3. This M e r supports the hypothesis
that the products associated with Peak 3 are similar to the product associated with Peak 2.
m e r oxidation studies have investigated the degradation of polyethylene films in the presence
of bacterial and liver homogenates masserbauer et al., IWO]. The homogenate was a source of
both oxidative and hydrolytic agents and hence it mirrored the existing system studied in this
thesis. In the studies by Wasserbauer et al-(1990), there were several chemical groups identified
which included temiinal hydroxyl groups (1 020- 1 030 cm-'), other hydroxyl groups ( 1 040- 1 1 70
cm-'), and double bonds (1640 cm-'). Therefore, the presence of these groups provides support
for the nature of chemical groups proposed for the biodegradation product associated with Peak
3. Unfortunately, the presence of proteins was not assessed in the study by Wasserbauer (1990)
and hence protein contaminants in the spectra could not be d e d out.
3300 2800 2300 1800 1300 800
Wavenumber (cm")
Figure 5.5: ATR-FTIR Resultant of a Subtrnction of Peak 3 and DichIoromethane (Figure
4.26). The region between 2200 to 2400 cm-' was removed. This area represented
the carbon dioxide peak.
I I 4
The NMR analysis did not show conclusive results on its own (see Figures 4.28 and 4.29) for the
structure of the product; however, it supported the presence of methylene, methyl and vinyl
groups. The specific assignment for hydroxyl protons could not be made since it is believed to
be masked by the large number of methylene groups located in the 1 to 1.5 ppm region. The
carbon-13 NMR (Figure 4.29) could only confirm the presence of alkanes at the 25 to 30 ppm
shift. It was suspected that there was a chernical shift that corresponded to an hydroxyl group,
but the majority of the sample was indeterminable in the diphatic region. Again, the amount of
sample available to be analyzed was very small (pg) and too much noise was present in a carbon-
13 spectrum. In the future, more product must be produced by changing the experimental
apparatus to increase the d a c e area of the reaction.
In summary, the structure could not be elucidated at this t h e . However, the product associated
with Peak 3 contains a majority of methylene groups, some methyl and CH groups with some
double bonds but there are fewer of these than hydroxyl groups. There are no other groups
present in this structure. It also has a large molecular weight of 680 which can determine that
about 45 carbon atoms, 96 hydrogen atoms and about at least four oxygen atoms are present in
the sample. Based on the mass spectra results, there are many very stable long chain hgments
with mass to charge ratios of 100 and 226. These may be straight hydrocarbon chah that were
cleaved in close proximity to double bonds or may contain double bonds themselves. Based on
the loss of many hgments with a m/z of 17 or 18, there were many hydroxyl groups present that
may have been pendant groups in a long hydrocarbon chain. Based on this information, M e r
work will concentrate on fùlly elucidating this structure.
The hydrolytic process resulted in many degradation products of interest. Future work will focus
on M e r isolating and determining the chernical nature of these products. However, to achieve
this objective, more degradation sample must be produced to obtain cleaner results. Other fiiture
work will use these degradation products or similar derivatives to study their influence on human
monocytes. Of specific interest is to determine whether they cm stimulate human monocytes to
produce inflammatory mediators that have been implicated in bone resorption around an implant.
115
5.4 In Wro Study of HDPE Particles with Human Monocytes
The morphology and the relative sizes for Wgin UHMWPE and HDPE particle test specirnens as
seen using SEM were described in Figures 4.1 and 4.2. The HDPE was used as a substitute
material for the cell studies due to its smaller size which permitted the monocytdmacrophage to
engulf the particles as explained in Section 3.1. The eff& of changing this material is not
known. The ciifference between the two materials lies essentidy in the increased crystallinity of
the HDPE as compared to the UHMWPE, since the chernical composition itself has k e n shown
to be similar for both materials woronov, 19971. The amorphous regions of the UHMWPE has
been shown to allow for the diffusion of k e radicals throughout the bulk of the system WI]
and hence is suspected to be a contributory factor in the oxidation of the whole material. Since
HDPE does not have a dominant amorphous structure, the degree of oxidation will not be
appropriately simulated with the HDPE particles. Table 5.2 clearly shows that there is little
oxidation present in these HDPE particles, with both the irradiated control samples and the
thermally treated samples at 10S°C for 11 days. Thus, the effect of chernically-altered
polyethylene on the stimulation of monocytic release of cytokines codd be different for both the
HDPE and UHMWPE samples. Interestingly, it has been docurnented that the oxidation of
UHMWPE generates shorter chah lengths and that the material can take on the characteristics of
HDPE [Li2 et al., 19941. Most studies interchange the two materials in investigations that
examine the effect of polyethylene Wear particles in animal models [Goodman et al.. "1 995,
12-'1996, Howie et al., 19881 and thus the use of HDPE is not out of line with current studies.
However, in light of the lack of oxidation of the HDPE particles, it may be considered for friture
work to use either UHMWPE particles or another polyethylene which is stnichirally more similar
to UHMWPE in terms of its dominant amorphous content, such as a highly branched low density
polyethylene.
Table 5.2: Oxidation Indices of Thermaiiy Oxidïzed HDPE at 105OC for 11 days.
Both samples were irradiated and had n=3
HDPE (control)
mean + standard deviation
HDPE (heated)
mean + standard deviation
116
The celi incubation experiments of this thesis were carried out as an initial assessrnent of the
eEect of modified HDPE particles on human monocytes since the premise of the thesis was
based on the hypothesis that chernicd changes in the polyethylene particies and its associated
products can influence ce11 fiinction. The investigation included viability studies, time-dependant
cytokine secretion studies and cornparisons between SEM rnicrographs of the cells using
different treatments of the particles. Due to limitations in resources, only 24 and 48 hour time
points were investigated.
In this time interval, it has been noted that most of the monocytes have not yet differentiated into
macrophages [Boynton, 199q and that differentiation would not occur until after 72 hours. The
SEM micrographs of the cells and particles (Figure 4.3 1) showed that both the human monocytes
and the particles are approximately 4 pin in size. While these ceus barely had enough cytoplasm
to cover the particles, the SEM micrographs (Figures 4.3 1) showed that the monocytes were able
to engulf the HDPE particles. The cells incubated with HDPE particles that were treated with
cobalt chloride were sickly in appeanuice (Figure 4.31 B ). The cytoplasm in these cells was
stretched thinly over the particles and much ce11 debris was present. When treated with the
hydrogen peroxide, the cells appeared more hgrnented (Figure 4.31 C). While there could be
concem that residuai hydrogen peroxide may be responsible for this result, precautions were
taken to avoid excess hydrogen peroxide. The particles treated with hydrogen peroxide were
rinsed several times with distilled water and dried in a 37OC oven for two &ys until use. This
procedure removed any residual hydrogen peroxide werck Index], therefore residual hydrogen
peroxide could not have contributed to cell death. Hence, it is believed that the modified surface
chemistry rnay have played a role in the death of the monocytes. These observations were also
supported by high level of ce11 death (approximately 80%) seen in samples treated with cobalt
and incubated with hydrogen peroxide (Figures 4.30) in the floating cells. The cobalt itself was
not responsible for necrosis because both samples of cobalt-treated HDPE and HDPE alone only
had 50% necrotic cells after 48 hours (Figure 4.30). Thus, the surface chemistry must be the
factor which caused ce11 death. The mechanism for this observation is unlaiown at this tirne.
A concem associated with the reported amount of cytokine is that there couid be an unknown
contribution fiom dead ceils that have adhered to the coverslip, as seen in the SEM micrographs
(Figure 4.3 1). These cells would also have DNA that would be used to normalize the amount of
117
cytokine release and thus reduce the amount of cytokine reported. As well, some of the floating
ceUs are viable and secrete cytokines. These cells would not be included in the DNA count and
may amficially increase the reported amount of cytokine release per amount of DNA. To
overcome this difficulty, a live-dead stain must be done on both the adherent and floating ceils to
determine the contribution of the cells on the amomt of DNA and the cytokine release.
Nonetheless, the cytokine data do refiect some changes relative to the different particle
treatments, but clearly this aspect of the work needs M e r study before any conclusions can be
made.
The cytokines investigated were IL-1 i3, DL-6 and T N F a and are shown in Figures 4.32 to 4.34.
In general, al1 cytokines were detected afler 24 hours; however, the levels were very low d e r 48
hours. The IL4 P production was inhibited the most by the cobaltmydrogen peroxide sample and
the least with the HDPE particles relative to the control. For the IL-6, the amounts of cytokine
secreted were similar for al1 particle treatments. However, for the TNF-a, the peroxide treated
samples and the collagen/DMSO sample showed the greatest secretion of this cytokine after 24
hom. Other particle/human monocyte studies showed this phenomenon of decreased cytokine
release with titanium particles plaine et al., 19961. They found that there was an initial burst of
cytokine release in the fbst 16 hours and after 24 hours, there was a decrease in the cytokine
release of IL-6 and TNF-a. This pattern may be sirnilar to the cytokine release found in this
study. Shanbhag et al.. 1995 reported the release of cytokines fiom human monocytes after a 24
hour incubation period with polyethylene particles. Longer experiments were not carried out.
They determined the e ffect of retrieved polyethy lene particles and fabricated pol yethy lene
particles on their ability to stimulate ce11 mediators fkom human monocytes. The fabricated
particles showed a greater release of cytokines compared to the retrieved particles. I f the
hypothesis in our study holds me, then based on the IL-1P data, the retrieved polyethylene
particles would be analogous to the oxidized particles in our study.
The production of al1 the cytokines decreased d e r 48 hours. An explanation for this resdt is
based on the fact that the monocytes had not difkrentiated into macrophages, and hence, the
macrophages can not be activated to produce more cytokines. It is well docurnented that
activated macrophages produce a greater amount of cytokines than monocytes [Johnston, 19881.
It is possible to stimulate monocytes to produce increased cytokine levels, as shown by Shanbhag
et al. (1995), who used the stimulant, phorbol 12-myristate acetate. As weU, studies in our
118
laboratory poynton et al., 19971 have shown in vivo that Tceiis c m ampli@ the inflammatory
response to polyethylene particles. It has dso been noted by the same authors that once the T-
ceiis are gone, the ce11 stimulus is no longer present Labow et al. (1998) found that they needed
to initiaüy activate human monocyte-derived macrophages by plating them on polystyrene cell
culture dishes before incubating them with polyurethane that underwent ceii-induced
biodegradation. In this thesis, there was no deliberate stimulation of the cells, and hence, Little
production of cytokines was observed in the period following 24 hours. Thus, to see an increase
in the production of cytokines, stimulation of the monocytes is needed.
Another important consideration is that the periprosthetic tissue response to particles is more
complicated than what has been modeled in in vitro experiments [Blaine et al., 19961. However,
most studies ody moael the effect of particdates on a particular cell population nich as
monocytes [Shanbhag et al., 1995, Rogers et al., 19971, osteoblasts [Men et al., 1997,
macrophages [Glant et al., 19931 to the particles. It has been shown that cells are able to
communicate with other cells to elicit a response. Studies [Horowitz et al., 19971 showed that
macrophage and osteoblasts interact with each other and that the interaction of macrophages with
polyethylene particles generates the release of W u and prostaglandin Ez. in addition, CO-
cultures of macrophages and osteoblasts that were incubated with polyethylene particles showed
increased levels of prostaglandin Ez and the production of IL-6. As weli, it has k e n shown that
the production of T N F s by macrophages stimulates the release of iL-6 by osteoblasts in the
presence of polymethylmethacrylate particles. While these results were shown with a mouse
ceIl-line rather than human cells, they emphasize the importance of introducing other factors to
make the experimental model more complete. To Mly model the clinical environment, a greater
understandhg of how cells signal other cells is needed. Thus, a more complex ce11 mode1 than
the one introduced in this study is needed to understand the relationship between different ceils,
polyethylene particles and their involvement in bone resorption.
6.0 SWMMARY AND CONCLUSIONS
1. Oxidation
In general, UHMWPE was relative raistant to both chemical and thermal oxidation.
Oxidation of UHM7KPE by chemical means was only observed if it was pre-
sensitized with thermal andor y-irradiation treatments. The effkct of thermal
treatments could be inherent within the materials as a r e d t of processing, although
they would not be as severe as those generated with the conditions shown in this
thesis (i.e.. 11 days at 1OS0C)
The effect of cobalt chloride on the oxidation of UHMWPE is not clear because the
amount of oxidation generated by pre-sensitizing the material with thermal treatments
produced a sanirated oxidation level.
2. Hydrolysis
Hydrolysis of UHMWPE was only observed with oxidized UHMWPE materials.
Degradation products were found with the hydrolysis of oxidized UHMWPE particles
following incubation in buffer solution, 4N hydrochlonc acid or 40 U/mL of
cholesterol esteme.
fncreasing the thermal oxidation time and incubating the sample in buffer or
hcreasing the hydrolytic incubation periods resulted in production of many
degradation products with similar HPLC retention times.
Hydrolytic degradation with 4 N hydrochloric acid or 40 U/mL cholesterol esterase
solution resulted in the production of degradation products that had similar retention
times.
The UHMWPE-derived degradation products consisted of high molecular weight
oligomers (ranging from 340 to 680). Due to the small amounts of products and
Iimited time, isolation and identification of al1 products were not possible.
The structure of the dominant product associated with Peak 3 was not able to be NIy
identified. However, it is proposed that it contains many alkane groups, with a few
hydroxyl groups and possibly some double bonds. Its molecdar weight is 679 5
a.m.u.
CelV HDPE particle studies were carried out using human monocytes and the
particles were iatroduced into the cells using methods adapted nom a mouse
macrophage cell-line model.
Increased chernical treatment on the particles (i.e. HDPE only > t h e d l y oxidized
HDPE coated with cobalt > thermally oxidized HDPE coated with cobalt and
incubated with 10 w/wO/o hydrogen peroxide), increased the amount of necrosis of the
cells.
il-le and Il-6 cytokines were detectable in the incubation medium of human
monocytes d e r 24 hours and 48 hours for al1 three treated particles: 1. HDPE; 2.
themially treated and cobalt treated HDPE, and 3. themially treated, cobalt treated and
incubated in 10 w/w% hydrogen peroxide. However, T N F a was detectable after 24
hours, but not after the 48 hour incubation period.
M e r 24 hours, there was no statistical difference among the three particle
preparations on the secretion of IL-IP. However, der 48 hours, there was a
significant decrease for the HDPE treated incubated with hydrogen peroxide
compared to the other sample preparations.
M e r 24 hours of incubations, the IL-6 samples showed no difference in the cytokine
release. However, after 48 hours, the amount of IL-6 released fiom the cells
containing HDPE treated with cobalt was significantiy higher than the other sample
preparations.
The TNF-a samples showed that the greatest release of this cytokine occurred for
cells treated with cobalt and hydrogen peroxide after 24 hours. Mer 48 hours, no
cytokines was detected.
1. This thesis used very harsb oxidation conditions to illustrate the effect of irradiation, thermal
and chemical treatments on UHMWPE. Thexmal treatments for up to 11 days at 105OC and
chemicai treatments with 7.6 mM HOC1 and 10 w/w?h hydrogen peroxide were used. Even
the hydrolytic conditions were very harsh using 4 N hydrochloric acid or 40 U/mL of
cholesterol esterase as hydrolytic agents. These conditions need to be changed to determine
the effect of these factors on UHMWPE in a physiologically relevant environment. This may
be accomplished by using cobalt chloride to lower the temperature of the thermai treatments
and the concentrations of oxidants and hydrolytic agents, since metal ions are known to be
found in Wear debris and can act as a catalyst for oxidation reactions.
Other methods such as secondary ion mass spectrometry (SIMS) or atomic absorption
spectroscopy ( U S ) are needed to determine the exact concentration of cobalt on the surface
of the material. AAS has a detection limit of 0.3 ppb, which is far supenor to the capabilities
of the SEM-EDAX system used in this study. SiMS has a spatial resolution of 50 nm, can
observe the outemost 1 to 2 nm of the surface, and provides depth profiling s u e s up to
1 p into the sample [Cooke et al., 19961.
It was odd that no low molecular weight products were found in this study. It is possible that
lower molecular weight products were not detected in the incubation solution but were
associated with the particles. Chioroform extraction was used to assess this hypothesis,
however, no products were found. Using an acetone extraction with the particles, it may be
possible to detemine their presence because many low molecular weight hydrocarbons are
more soluble in acetone than chioroform.
A better experimental design is needed to oxidize UHMWPE particles in order to generate
more product. The lirnit of detection of many of the analfical techniques is in the
microgram level. The existing protocol yields products in the nanogram or microgram level.
There were several degradation products found under the HPLC peaks that are believed to be
associated with the enzyme. Many of these products may have been complexed with the
UHMWPE degradation products. By using purified cholesterol esterase, these protein
contaminants could be eliminated and would thus make the isolation of the products easier.
It is of interest to refhe the HPLC method to decrease the run the . More effort should be
charmeled towards this concern.
122
To date, ody one product peak has been identified which contains hydroxyl groups. The
Literature review has proposed that some of the degradation products would contain some
carboxylic acid groups (see Section 2.5.5 of the literature review) since it was anticipated that
the hydrolytic conditions were cleaving ester-type linkages. To date, no carboxylic or
carbonyl groups have been identified with the isolated degradation products. Molecules with
carboxylic acid groups would be more hydrophilic than those without these groups and wodd
have been eluded faster fiom the HPLC column. There were many products that had low
retention times and they may contain some acid groups. Thus, M e r isolation and
identification of these products are required.
A new macrophage mode1 should be used which would include other celis present in the
innamrnatory environment of bone implants such as osteoblasts and neutrophils. Many of
these cells are needed to communicate with each other in order to stimulate the release of
cytokines.
The use of a "pruning" factor for the human monocytes may be needed to stimulate the cells
before detemilliing the actual secretion patterns of the cytokines.
10. The ceIl culture should be nin for longer time periods to determine the chronic effect of the
particles on macrophage ceIis rather than monocytes.
1 1. An experiment that should be done to compare the effect of thennaily oxidized HDPE and
non-oxidized HDPE on ce11 viability. This control sample was needed to determine the effect
of thermal oxidation on the viability and cytokine release since both the cobalt treated HDPE
samples were thexmally oxidized before they were incubated in the various solutions.
12. The particles used in ce11 -dies should be UHMWPE instead of HDPE since the latter does
not oxidize under the same conditions as UHiMWPE. To date, the only available UHMWPE
in the micron range was produced by a cryogenic milhg process [Shanbhag et al., 19961.
They produced a yield of lg for every 250g of starting resin. Other work should be carried
out to increase the yield. As well, other polyethylene grades with more amorphous structure
than HDPE may be considered as a potentid substitute for UHMWPE if availability of
UHMWPE particles in the micron range remains an issue.
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Bufier Used in HOC1 Assav
Buffer: 40 m M NaH2P04 (Mallinckrodt), FW Na&P04- HzO = 137.99 274 rnM NaCl (Sigma), FW= 58.44 2 mM MgCl2 (Aldrich), F W = 95.22 10 mM KCl (Malhcicrodt), FW= 74.55 adjust to pH 7.4
Taurine solution 65 rnM in ddH20 (Aldrich), FW= 125.15 Potassium iodide 120 mM in dd&û (Matheson Coleman & Beii), FW= 166.0 1 NaOCl solution (Aldrich), p= 1.097 g/rnL, FW= 74.44 (4.5g of chlorine per l OOg of solution)
Chemicais Needed For Hvdropen Peroxide Assay
Ammonium molybdate tetrahydrate (ACS reagent)
Potassium iodide, KI (99.99+%)
Sodium hydroxide, NaOH (99.99%)
Potassium hydrogen phthalate, KHP (99.95%)
Hydrogen peroxide (30%), stabilizer-fke
Water (double-distiiled and filtered though a 0 . 2 2 ~ filter)
AIcanox
Preparation:
Clean d gassware in an ultrasonic cleaner using a solution of Alcanox in hot water. Clean
cuvettes using concentrated s u h i c acid or chromic acid cleaning solution and rime very well with
distilled water before using.
APPENDIX B:
CALCULAT~~ON OF AMOUNT OF m E NEEDED.
Amomt of UHMWPE needed for experiment
Surface area of bottle: 52.8 cm2
Particle diameter for UHMWPE: range h m 100400 p. Choose 250 p (fiom SEM micrograph, Figure 4.1) as the mean diameter.
Surface area for one particle = 4 il R~ -7 2 = 1.963 x 10 m
No. of particles needed for a monolayer: Surface area of boale Surface area of a particle
= 0.00528 m2 -7 2 1.963 x 10 m
= 26897.6 particles needed to form a monolayer
Volume of one particle: 4/3 rI R~ 12 3 =8.818x 10 rn Iparticle
Mass of particles needed = (volume of particle) x (number of particles) x ( density of one particle) = 0.2090 g
Therefore 0.2090g is need to form a monolayer in the reaction bottle. Because particles are lost during the replenishment process, 10% excess particles are used. Thus, 0.230g of particles are used for each reaction vessel.
APPENDIX C:
CALIBRATION CURVES FOR HOCL AND EYDROGEN PEROXIDE ACTMTY
Calibration Curve for Sodium Hypochlorite for Active Chlorine
Concentration (m M)
Calibration Cuwe for Hydrogen Peroxide (KI, 99% pure)
0.02 0.04 0.06 0.08 O. 1
Concentration of H20, (nmol/mL)
CHOLESTEROL ESTERASE ACITVTTY ( WIUTIEN BY DR. FRAM( G.B. WANG)
De£inition of CE Activity: CE cataiysis
pnitrophenyl acetate @ M A ) + FI20 - pnitrophenol (ye11ow) + acetic acid
A unit of CE activity was defined as the production 1 nmoVmin of pnitmphenol from the hydrolysis ofpnitrophenyl acetate in the presence of CE at pH 7.0 at 25OC.
Details: Amount ofpnitrophenol formed is monitored by the increase in UV absorbane at 4 1 O nm. The molar extinction coefficient ofpnitrophenol (E) at pH 7.0 is 16,000 UmoVcm. The thickness of sample ceil (l) is u d y 1 cm. The reasonable range of UV absorbance increase (A) is 0.01 O.DJmin. based on previous experiments. C is the concentration ofpniaolphenol (moVL). Using Beer's Law (A=&), C- 625 moVUmin, If you are using a 1.5 mL cuvette, pnitrophenol amount = C x 1.5 x 1 o-~L= 0.93 75 nmol 1 nrnoVrnin. Ifyou are using a 3.0 mL cuvette, pnitrophenol amount = Cx 3 . 0 ~ 1 o J ~ = 0.9375 nmol = 2nnioVmin.
k Subatrate Preparation: 1. p-Nitrophenyl Acetate @NPA) Preparation
Weigh 22.0 mg ofpNPA Dissolve in t mL of methanol.
U. Preparation of 100 mi, of Sodium Acetate (0.1 M) Weigh 0.8g of sodium acetate (0.82g is using anhydrous sodium acetate) Dissolve in distiiied water. Adjust to pH 5.0 using HCI. Adjust volume to 100 mL.
m. Addition of 1 + II Add 99.0 mL of solution II to solution 1. The solution is light-sensitive; cover container with foil Heat gently and stir solution for 0.5 to 3 hours until solute is completely dissolved.
B. Phosphate BPner Preparation (not PBS) 0.5 M Sodium Phosphate Buffer, pH 7.0 Add 0.0 195 moles of Na&P04 Add 0.0305 moles of Na2HPO4 Adjust to pH 7.0 with either HCI or NaOH
C. Cholesterol Esterase Preparntion Prepare 100 mL of 2 unit/mL solution based on activity supplied by manufacturer. Fiiter the solution ushg a Miilex GV filter (0.22 pn). **Use this solution within 10 to 15 minutes because it is heat sensitive, else store in the fndge.
T m on spectrophotometer 20 minutes before use. Adjust to 410 nm wavelength. Deuterium is OFF and Tungsten is ON.
3.0 mL Cuvette Blank: - Test: 1 .O mL ofpNPA 1 .O mL ofpNPA 2.0 mL of phosphate buffer 2.0 mL of phosphate b& O. 10 pL phostphate buffefl 0.10 pL of CE
Before adding the enzyme to the test group, zero the spectrophotorneter. Add enzyme and measure absorbance. Take absorbance measurement every 30 seconds for 5 minutes When the dope of the graph is 0.01 O.Dfminute, the activity of 0.1 mL of the CE solution is 2 mits (20 units/mL). The value is acceptable if the dope varies between 0.008 < slop4.0012. This refers to the activity between 8 and 12 U12its/mL, respectively. If a higher value was obtained, then the relationship between the slope and the activity is not linear. Thus the solution must be diluted. This process is repeated until the slope falls between the specified range.
APPENDIX E:
Mass Spectra of Peak 4 Products: ( Peaks 4A-11,4A-12 and 4A-13)
A'.1 1
Mnss SpeetrP of Peaks 4B-3,4B-9 and 4B14
APPENDIX F:
Mass Spectra of Stearic Acid
Stearic Acid (BDH #50943)
Stearic Acid, MW 284 fiom BDH Chemicds, Toronto. Cat # 50943
A P P ~ I X G: Caiibrition Ciuves for Cytolone AnaIysis
Standard Cumes for DNA analysis: y= 0.7222~ + 26.501, where y= fluorescence and x =ng/mL DNA RL 0.9874
IL- 1 B Standard Curve for Elisa: y= 0.066~ + 0.035, where y= absorbance at 450 nm and x- pg/mL of IL-1 /3
IL-6 Standard Curve for Elisa : y= 0.0015~ + 0.0025, where y= absorbance at 450 mn and x= pg/rnL of IL4 R' = 0.9996
W-a Standard Curve for Elisa: y= 0.0026~ + 0.0664, where y = absorbance at 450 nm and x = pg/mL of RIFa R~ = 0.9833
APPENDIx W: ATR-FTIR Data for Peak 2
ATR-FTIR Substraded Spectrum of Peak 2
I m
Wavenumber (cm-')
IMAGE NALUATION TEST TARGET (QA-3)
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