budapest university of technology and economics ...porous polymeric scaffolds for bone tissue...

POLYMERS FOR ADVANCED TECHNOLOGIES

Polym. Adv. Techno!. 2006; 17: 889-897

Published online 3 October 2006 in Wiley InterScience (www.interscience.wiley.com). 001: 10.1002/pat.768DDI

Synthesis, structural and mechanical properties of porouspolymeric scaffolds for bone tissue regeneration basedon neat polY(8-caprolactone) and its composites withcalcium carbonatet

Laszlo Olah 1,2*, Katarzyna Filipczak1, Zbigniew Jaegermann3, Tibor Czigany2,Lajos Borbas4, Stanislaw Sosnowski4, Piotr Ulanski1 and Janusz M. Rosiak11 Institute of Applied Radiation Chemistry, Technical University of Lodz, Wroblewskiego 15, 93-590 Lodz, Poland2Department of Polymer Engineering and Center of Biomechanical Studies, Budapest University of Technology and Economics,Muegyetem rkp 3, 1111 Budapest, Hungary31nstitute of Glass and Ceramics, Postepu 9, 02-676 Warsaw, Poland4Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland

Received 30 November 2005; Revised 7 March 2006; Accepted 3 April 2006

The aim of the present study was to develop new materials which could be applicable as bone

substitutes or be used in bone tissue engineering. Two types of porous scaffolds based on poly(e-caprolactone) (PCL) were investigated. Type 1 scaffolds were prepared by solvent casting/particulate

leaching technique, using NaCI with the grain size 250-500 /-Lmas a porogen. In the case of Type 2scaffolds, the biodegradable polymer was blended with calcium carbonate, which, in contrast to

NaCl, is not leached out from the product during manufacture, either in the form of calcite powder or

aragonite (needle-like crystals). Influence of manufacturing technique and initial substrate compo-

sition on product properties was investigated. The tests involved porosity measurements, structureanalysis by optical and scanning electron microscopy and mechanical studies (determination of

compression strength and modulus). The results indicate the important role of the phase exchangeprocess in the formation of micropores. In this process PCL precipitated from its acetone solution in

the presence of water creating microporous three-dimensional polymer structures. The Type 1

scaffolds possessed both micropores and macropores. Good interconnectivity between the pores

was observed for samples of the initial porogen content higher than 33%. Microporous samplescontaining inorganic filler have lower porosity and higher compression strength. For Type 2 scaffolds

the shape of filler particles has an important influence on mechanical properties-replacing powderwith needle-like crystals (in the same weight amount) results in a three- to five-fold increase in

compression modulus. cg 2006 John Wiley & Sons, Ltd.

KEYWORDS: composites; biomaterials; mechanical properties; macroporous polymers; synthesis

INTRODUCTION

The need for bone grafting to replace skeletal defects oraugment bony reconstruction has become more prevalentrecently. There are many bone graft options available for thesurgeon, including autografts, allografts or xenografts, eitherof a cortical or cancellous structure of bones (Fig. 1).Cancellous bone, because of its large surface area, has greaterpotential for forming new bone than cortical bone does.l The

*Correspondence to: 1. Olah, Department of Polymer Engineeringand Center of Biomechanical Studies, Budapest University ofTechnology and Economics, Muegyetem rkp 3, 1111 Budapest,Hungary.E-mail: [email protected]'8th International Symposium on Polymers for Advanced Tech-nologies 2005 (PAT 2005), Budapest, 13-16 September, 2005, Part 2.

biological activity of bone grafts is a result of two functions:osteogenesis and mechanical support.

The recently emerged new discipline, defined as tissueengineering, combines aspects of cell biology, engineering,material science and surgery with the outcome goal toregenerate functional skeletal tissues as opposed to replacingthem. Repair and regeneration of skeletal tissues arefundamentally different processes. In many situations, scar,which is the result of rapid repair, can function satisfactorily,such as in the early phases of bone restoration. Regenerationis a relatively slow process that ultimately results in aduplication of the tissue that has been lost. Biomatrices,scaffolds, or delivery vehicles are important components of

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890 L. Olah et al.

Figure 1. Macroscopic features of bone structure: (A) can-cellous bone; (8) cortical bone.

tissue engineering strategies. Both synthetic and naturalmaterials have been used as delivery vehicles for cytokines orcells or both. It is still not clear what the ideal physical,chemical, and mechanical characteristics of the carrier

should be for each specific tissue application?Bones of the skeletal system provide the supporting

structure for the body, as it was mentioned in the firstparagraph. Bone is a structural composite composed ofcollagen fibers with biological apatite precipitated along thecollagen fibrils (Figs. 1 and 2).3 The high elastic modulushydroxyapatite mineral comprises approximately 70% of thedry bone mass and contributes significantly to the bonestiffness.

Ceramics, polymers of lactic and glycolic acid, collagengels, and other natural and synthetic polymers have beenused to fabricate delivery vehicles and have been tested bothin vitro and in vivo.The optimal properties of biomatrices foreach implantation site must be defined and include theirbiodegradability?

In a number of studies it has been shown that purepolymers seem to be too flexible and too weak on their own tomeet the mechanical demands of certain applications, e.g. asimplants in orthopedic surgery. They may swell, warp orrelease undesired products (such as monomers, fillers,plasticizers, antioxidants), depending on the usage andapplication.3

43%

I ~waterEJ Apatite mineral III Organic matrix IFigure 2. Composition of the bone in volume percent.


laDCeramic materials are known for their good biocompat-

ibility (in some cases bioactivity), corrosion resistance, highcompression strength and high hardness. At the same timethey have also some drawbacks such as low fracturetoughness and high stiffness. The elastic modulus of

ceramics is at least 10-20 times higher than those of hardtissues. One of the major problems in orthopedic surgery isthe significant difference between the stiffness of the boneand ceramic implants. The bone is insufficiently loadedcompared to the implant, and this phenomenon is called"stress-shielding" or stress protection. Polymer-ceramic orpolymer-inorganic composites could be the alternative wayto overcome many short-comings of materials mentionedearlier. The literature gives a fair show about polymer-ceramic composites4-8 for biomedical purposes. Hydroxya-patite [CalO(PO4)6(OHhJ is one of the best known, widelystudied inorganic material, well known for its biocompat-ibility, bioactivity, high osteoconductivity and relatively highstrength and modulus.4,5.9.10Moreover, since its biologicalanalogue is the major component of bone, hydroxyapatiteseems to be the most relevant additive to bone scaffolds.

Certainly, there are many other filler materials, such asalumina and zirconia.5.6 By definition, osteoblasts derivefrom bone-lining cells and are responsible for the formationof the bone. They initially lay down the collagenous matrix,osteoid, in which mineral is deposited, and they probablyalso have a role in its mineralization.ll Basically,Ca2+is oneof the most remarkable ions in the bone, that is why theexpectation is that it helps osteoblasts to mineralize the bonytissue.

In this work polY(E-caprolactone) (PCL) is used as a

polymer scaffold and matrix material for the compositereinforced with calcium carbonate in the forms of calcite

powder and aragonite crystals. PCL is a linear resorbablealiphatic polyester, which is subjected to biodegradationbecause of the susceptibility of its aliphatic ester linkage tohydrolysis.12 The products generated are either metabolizedvia the tricarboxylic acid (TCA) cycle or eliminated by directrenal secretionY Application of PCL might be limited in thefield of drug delivery or resorbable sutures due to its slowdegradation and resorption kinetic, but this slow degra-dation could be beneficial for bone tissue engineering. Itgives time for osteoblasts to build up the bony tissue and inparallel to the bone regeneration the implant will slowlydegrade. During this investigation calcium carbonate is used,which is easily available, but has not been yet widely testedfor biomedical purposes, although recently there were somearticles about the possibility of using CaCO3 in the field ofbone filling. 13-15Advantageous properties of ceramics-basedcomposites are well-known, such as reducing shrinkage,good dispersibility, non-toxicity, increasing the impactstrength, modulus of elasticity and also the compressivestrength.16 Calcium carbonate filler is available in various

forms: calcite, vaterite or aragonite (needle-like crystals).The calcite has a trigonal and the aragonite has anorthorhombic crystal structure. The latter has typicallyhigher elasticity modulus than the powder, betterdispersibility due to the surface treatment, increasedimpact strength, tensile strength and elongation atbreak. 16

Polym. Adv. Techno/. 2006; 17: 889-897

DO!: 10.1002/pat

OBIThe scope of the present study was to increase the

mechanical strength of the highly porous PCL scaffolds,because the good biocompatibility of PCL as a biodegrad-able, aliphatic polyester has been proven, ]2,17,18but itsporous samples have low mechanical strength. In this workbiodegradable composites of PCL and CaCO3 have beenprepared by mixing the powder or needle-like crystals ofcalcium carbonate (i.e. calcite or aragonite) with acetonesolutions of PCL. During the investigation a generalcharacterization was made of the processed samples, theporosity, the mechanical properties and the surface proper-ties were tested using a scanning electron microscope. Theeffect of added calcite or aragonite on the mechanicalproperties and porosity of resulted scaffolds was comparedwith PCL-based scaffolds prepared via solvent casting/particulate leaching technique.

EXPERIMENTAL

Materials

PCL of a nominal number-average molecular weightMn = 8.0X 104 Da and weight-average molecular weightMw = 1.3X105Da was purchased from Aldrich. It was driedunder reduced pressure at 40°C before use.

Calcite filler-CaCO3-in the form of calcite powder waspurchased from POCh Gliwice (Poland) and underwent wetgrinding (the mass median diameter of the powderdo.5= 3.0fLm).The shape of calcite particles is shown inFig. 3.

Aragonite filler-CaCO3-in the form of aragoniteneedle-like crystals was manufactured in Bioceramic Depart-ment of The Institute of Glass and Ceramics (Warsaw,

Poland) by the precipitation method (the mass mediandiameter of the powder do.5= 7.9 fLm).The shape of particlesis shown in Fig. 4.

Sodium chloride and acetone were of analytical grade andwere used as received.

Scaffold processingBasically, two kinds of PCL scaffolds were synthesized:highly porous scaffolds prepared by solvent-casting/particulate leaching technique (Type 1) and scaffolds

Figure 3. SEM picture of the structure of calcite filler atmagnification 1OOOx.

Copyright ~ 2006 John Wiley & Sons, Ltd.

Porous polymeric scaffolds for bone tissue regeneration 891

Figure 4. SEM picture of the structure of aragonite filler atmagnification 1000 x.

reinforced with calcite powder or aragonite (Type 2). Inpreparation of the latter, both solvent casting and phaseexchange methods were tested.

Preparation of the type 1 (highly porous/ not reinforced)scaffoldsSodium chloride was used in this technique as the water-leachable pore-forming component. NaCl in the range ofgrain size between 250 < c/J< 500 fLm was used, because inearlier studies researchers pointed out that this is the idealpore size range for bone scaffolds.s,B Scaffolds synthesizedusing grains of this size were shown to have the bestbiocompatibility and best mechanical properties.B NaClgrains were added to the PCL solution to form a slurry. Theslurry consisting of different amounts of NaCl and polymerat a constant PCL concentration in acetone (20% by weight),was poured into stainless steel cuboid-shaped molds(15mm x 15mm x 10mm size) with two opposite sidesopen, and immersed in water. After 12hr the formedsemi-products were removed from the molds. To removeNaCI, water extraction at room temperature was continuedfor 14 days (it was found that the specimens after the firstweek had released over 99%of added salt,with the exceptionof samples of too low initial NaCl content to provide poreinterconnectivity, see later). The obtained porous materialwas dried under reduced pressure at 40°C.

Preparation of the type 2 (reinforced) scaffoldsIn the case of calcite- or aragonite-filled scaffolds, twofabrication methods were tested. The initial procedure wasbased on solvent casting. PCL solutions in acetone, withdifferent amounts of filler added, were dried at 40°C.Thisprocedure did not yield materials of expected properties (seelater). Therefore, this process was modified by replacingsolvent evaporation by phase exchange. The slurry obtainedby mixing PCL solution in acetone (20 or 25% by weight) withfiller was placed in open molds and immersed in water for12hr. After that time, the solidified scaffolds were removed

from the molds. They were immersed in water for a further12hr then dried at 40°Cat atmospheric pressure. Solutionsofdifferent concentrations and with different amounts of filler

Polym. Adv. Technol. 2006; 17: 889-897

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892 1. Olah et al.

were used, and the influence of these parameters on the

properties of the scaffolds was checked.

The amounts of porogen or filler are given as their weight

percent in the initial substrate mixture.

Analytical proceduresSurface morphology of porous scaffolds was studied using

an optical microscope (Euromex) equipped with a Canon

EOS 50 camera. Furthermore, scanning electron microscopy(SEM) pictures of the scaffolds were made at various

magnifications to investigate the surface morphology of

PCL based samples. Samples were stuck to specimen stubs

using two-sided sticky tape, sputtered with gold, and viewed

using a 5500LV Scanning Electron Microscope (JEOL, Japan).

The mechanical properties of PCL based scaffolds wereanalyzed with a Zwick BZ2.5/TNIS universal mechanical

testing machine by using cuboid specimens (with 10mm height

and 7.5 x 7.5mm cross-section), at an upper load limit of 1kNand a crosshead speed of 1 mm/min. The strength was

calculated at 10% of elongation and the compressive modulus

was estimated according to the ISO 604:2002 standard and theZWICK user manual, from the stress-strain curve, where the

slope was fitted to the curve at 2 and 10% of elongation.

The porosity of the sample was determined from weightand volume measurements, the densities were interpolated,

the theoretical weight estimated, and finally, the total

porosity of the specimens was calculated.

The samples were sterilized by gamma rays in a cobaltchamber (IARC, Lodz, Poland) with a standard dose of

25 kGy, at a dose rate of 0.4 kGy /hr.For determination of each data point, a minimum number

of five samples and in average six samples were tested, and

in the case of mechanical testing and evaluating porosity thestandard deviation was less then 10% of the measured value.

RESUL TS AND DISCUSSION

Highly porous PCL scaffolds prepared usingNaCl porogenScaffolds (Type 1) were prepared by solvent casting andparticulate leaching technique, as described earlier. First, theinfluence of the NaCl content in the initial mixture on the

porosity, structure and mechanical properties (compressivestrength and modulus) of the products was investigated.Finally, biocompatibility testing of these scaffolds wasperformed.

Theporositydata of PCLscaffolds:salt-leached(Type1)andcomposite (Type 2), are shown in Fig. 5, as a function ofporogen content (for Type 1 scaffolds) or filler content (for Type2 scaffolds). Salt-leached scaffolds have macropores formed bythe salt grains (Figs. 6-8) and, in general, their porosity is higher

. than that of samples containingno porogen.Due to the phaseexchange process involved in fabrication of the materials,microporeswerealsopresentin thescaffoldevenin theabsenceof porogen, and their formation accounts for the porosityobservedin the samplesmade of pure PCL(containingneithersalt nor the filler) according to the same procedure (Fig. 9).Interconnected pores are needed to assure that there is noresidual salt inside the specimen/bone graft. The interconnec-tivityof the samples is well-grantedin most cases,in order to

Copyright (jJ 2006 John Wiley & Sons, Ltd.

OBi100

1:: 90'"u 80~Co 70c:';:' 60~ 500

~40" 30'"1:0 20

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Amount of filler in percent70 80

Figure 5. Total porosity of the processed scaffoldas a func-tion of the amount of differentfiller(forsamples synthesizedusing NaCI as a porogen, the amount of filler should beunderstood as the amount of soluble additive).

prove it optical and SEM pictures illustrating the structure ofthe scaffolds were taken. In Fig. 6, the structure of a scaffold canbe seen, where the porogen-content was 50%. The thick walls,thin polymer layers and interconnected pores can be seen. Thescaffold, shown in Fig. 7, contained 83% sodium chloride. It isevident that walls are thinner and have more precise contours;there is no thin polymer film due to the less amount of polymer.In both Figs. 6 and 7, interconnected pores can be seen.Figures 8 and 9 contain the SEM pictures. The samples weremade of different materials. The sample in Fig. 8 was obtainedfrom 20% PCL solution in acetone, mixed with salt (66% of

porogen). Both the macropores (resulted from salt-leaching)and micropores (resulted from solvent casting method) arevisible. The sizes of macropores correlate well with the size of

the used fraction of sodium chloride. The next sample (Fig. 9)was synthesized from pure PCL solution, without salt, andmicropores in the polymer matrix can been seen.

On the next two plots (Figs. 10 and 11), the strength andmodulus values are shown for salt-leached scaffolds. The

mechanical strength curve had a critical point approximatelyat 33% porogen content. At this ratio the compressivestrength was significantly higher, which feasibly is a result ofthe (small amount) residual salt. Apart from the result ofthe specimen (with 33% sodium chloride content), the

Figure 6. Optical microscopic picture of the cross-sectionmorphology of salt-leached scaffold, at NaCI content 50%,made of 20% PCl solution.

Polyrn. Adv. Techno!.2006; 17; 889-897

001; 10.1002/pat

.- ....,-+-.+....._-+- -...

"""- L - .- ""--

- -+- Salt-leached

........Calcite-powder filled&20% solution-+-Aragonite filled&20% solution f----

-IF- Calcite-powderfilled&25% solution f----......- Aragonite filled&25% solution

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Figure 7. Optical microscopic picture of the cross-sectionmorphology of salt-leached scaffold at NaCI-content 83%made of 20% PCl solution.

Figure 8. SEM picture of salt-leached scaffold made of 20%

PCl solution, at a NaCI content of 66%, magnification 75x.

compressive strength decreases slowly in the beginning, then

at about 66% it starts decreasing faster, and at 83% of NaCl,

the strength was about 0.09 MPa. These are low values, but it

must be remembered that at 83% salt filling the porosity wasalmost 90%. In parallel with the compressive strength, also

Figure 9. SEM picture of the surface morphology of the pure

PCl scaffold (made of 20% PCl solution), magnification 1OOx.



0,0020 30 40 50 60 70

Amount of filler in percent80 90

Figure 10. Compressive strength of salt-leached, porous

scaffolds, made of 20% PCl solution, at 10% of elongation.

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3.5..Q.:E 3.0.5:g 2.5:;..,~ 2.0

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Amount 01 filler in percent80 90

Figure 11. Compressive modulus of highly porous PCl scaf-folds, made of 20% PCl solution.

the modulus is relatively high at 33% porogen content, and itundergoes a pronounced decrease with increasing saltcontent in the initial mixture.

PCL based composite scaffolds prepared bysolvent casting and phase exchange techniqueComposite scaffolds made of PCL and calcium carbonate

filler (Type 2) were synthesized either by solvent casting orby phase exchange, as described earlier.

SEM analysis of samples obtained by both these methodswere performed. In Figs. 12 and 13, samples made by solvent

Figure 12. SEM pictures on the cross-section morphology of

calcite-filled composite scaffold prepared by solvent casting,at calcite powder content 33% and solution concentration

20%, at magnification 100 x.

Polym. Adv. Techno/. 2006; 17: 889-897

DOr: 10.IO02/pat

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894 L. Olah et al.

Figure 13. SEM pictures on the cross-section morphology ofcalcite-filled composite scaffold prepared by solvent casting,at calcite powder content 33% and solution concentration20%, at magnification 1500x.

casting can be seen. The material consists of large particlesmade of polymer-linked calcite grains, while there is almostno continuous polymer matrix between the particles. Theparticles are much larger than the original single calcitegrains (the size of the latter was about l/Lm). Lack ofinterconnection between the particles in the materialprepared by this method resulted, as it could be expected,in very poor mechanical properties. Actually, the materialwas too weak to be tested for compressive strength under thestandard experimental conditions.

In the case of the phase exchange technique themorphology is totally different. Instead of non-homogenousstructure a continuous polymer-mineral matrix wasobtained, which is shown in Fig. 14. No formation ofseparate large particles was observed, the calcite grains aremore uniformly dispersed in the polymer matrix and theinterfacial connection in these scaffolds is better than

previously shown. On the micron scale (Fig. 15) the structureis similar as in the previous case, and the porous structure ofthe sample can be clearly seen. In consequence of improved

Figure 14. SEM pictures on the cross-section morphology ofcalcite-filled scaffold prepared by phase exchange, at calcitepowder content 33% and solution concentration 20%, atmagnification 100x.

Copyright <D2006 John Wiley & Sons, Ltd.

OBi

Figure 15. SEM pictures on the cross-section morphology ofcalcite-filled scaffold prepared by phase exchange, at calcitepowder content 33% and solution concentration 20%, atmagnification 1000 x.

coherency the mechanical properties of these scaffolds aremuch better (see later).

Figures 16 and 17 illustrate the structure of samples madeby phase exchange using the needle-like aragonite crystals.The difference between the samples containing both types offiller is easily seen, especially at higher zoom rates. At 100xzoom both of them are homogenous; the needle-likegeometry of aragonite crystals can be seen. The stringystructure of the aragonite-polymer system shows itself, andthe porous characteristic of the composites is apparent, too.

It can be seen that both calcite and aragonite are randomlydistributed in the composite, and the aragonite crystals arenot oriented. Which resulted in relatively homogenousmechanical and shrinkage properties.

The synthesized samples are microporous. It was foundthat the porosity of calcite- and aragonite-filled scaffolds areonly slightly dependent on the filler content (Fig. 5). Thevalues were in the range 50-60%, i.e. even somewhat lowerthan in filler-free specimens obtained by the same method. Itseems that the presence of filler influences the phase

Figure 16. SEM pictures on the cross-section morphology ofaragonite-filled scaffold prepared by phase exchange, ataragonite content 33% and solution concentration 20%, atmagnification 100x.


001: 10.1002/pat

UBI

Figure 17. SEM pictures of the cross-section morphology of

aragonite-filled scaffold prepared by phase exchange, ataragonite content 33% and solution concentration 20%, at

magnification 500x.

exchange process to some extent. Despite the fact that most ofthe pores in Type 2 scaffolds apparently result from thephase exchange, some increase in porosity with the fillercontent seems to indicate that the presence of the filler itselfat higher concentrations does contribute to pore formation,probably due to the formation of free spaces between thefiller particles which are not penetrated by the PCL solutionand remain void of polymer after the process is completed(d. Figs. 14-17). Nevertheless, the overall porosity of thesesamples is lower than observed for Type 1 scaffolds, whereboth micropores and macropores are formed (Fig. 5).

The composites have been tested for mechanical proper-ties. Similarly as for salt-leached scaffolds, compressive testswere performed, as it is the most relevant stress type forproducts intended as bone scaffolds. In Figs. 18 and 19 theresults of calcite powder- and aragonite-reinforced scaffoldsare presented. For comparison, the data on salt-leached areshown as well. The calcite-grain filled scaffolds have astrength of about 0.6-1.0 MPa, and neither amount of fillingnor the concentration of solution did influence the results

significantly. But it has to be mentioned that for 20% PCLconcentration in acetone solution there is a local maximum (a

reproducible feature as tested on three independentlysynthesized series of samples), the value of this maximumis 2 MPa. The aragonite-reinforced scaffolds have strength at

3.0

t:. 2.5::0.50= 2.0go..~ 1.5..>;;::: 1.0Q.E:3 0.5

. .- Calc'e-powderfilled&20%PCL

Aragooi'et;lled&20%PCL

-- ,..- ---" , .-- "" ,

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20

. .. -.. '... .. ". ----.. --.40 50 60 70

Amount 01 filler I" percent

60 9030

Figure 18. Compressive strength of scaffolds obtained byphase separation method at 10% elongation.

Copyright C!;:)2006 John Wiley & Sons, Ltd.

.,.

Porous polymeric scaffolds for bone tissue regeneration 89530,0

t:. 25,0::0c.~ 20.0:;"~ 15,0...~::: 10,0Q.E0()

_. Calcite-powderfilled&20%PCL

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filled&25%PCL

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Amount ot filler In percent9{)

Figure 19. Compressive modulus of scaffolds obtained by

phase separation method.

a 2 MPa level. In this case, no local maximum was observed,

although some dependence on filler content is evident.During daily activities tibial bones are subjected to stress ofapproximately 4 MPa/ so these results indicate that thestrength of these materials are close to the desired values. Theincrease of the strength can be subjected to the curb ofthe movement of molecular chains due to the filler. Possiblythe local maximum value observed for calcite filled samplesobtained from 20% PCL solutions at 20% of filler was the

optimum and at higher filler content it started to aggregate.This observation is parallel to a similar recent study made byChen and coworkers.17

The main difference between aragonite-reinforced andcalcite-powder filled scaffold is visualized on Fig. 19. It canbe seen that the aragonite-reinforced scaffolds have highermodulus, about 15-25 MPa, which is on average 3-5 timeshigher than the typical modulus of calcite-grain filledsamples (only at specific conditions the latter may reach16MPa). It is evident that the structure of the filler has a

pronounced influence on the mechanical properties offormed composites, the fibrous calcium carbonate providingstronger reinforcement than the same mineral in the form ofgrains. Aragonite has a form of elongated, needle-likecrystals of polygonal cross-sections, produced syntheticallyunder controlled conditions. Due to their structure, they havehigher compressive strength which results in the highercompressive strength of the composite,18 It can be alsoexpected that better surface to volume ratio in comparisonwith spherical grains would provide better adhesionbetween the needle-like crystals and polymer matrix.

The effect on the modulus is more significant. At thebeginning of the measurement, the polymer matrix deforms,that is why at 2% of deformation the difference in stressbetween the calcite- and aragonite-filled composite isnegligible. But, at 10% of deformation, the filler hasconsiderable influence on the strength, leading to moresignificant differences between the samples containing eachtype of filler. These two effects result in more remarkabledifferences in moduli than in the material strength.

Calcium carbonate has been already tested as a filler incomposites with biodegradable polymers intended for bonescaffolds, although the number of such studies is limited sofar. More studies have been undertaken on using hydro-xyapatite (HA) as a filler.

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896 L. Olah et al.

Schiller et al.19developed a process for preparing implantswith special geometry based on polY(L-lactic acid) (PLLA)and poly(o, L-Iactic acid) (PDLLA) composites]. Thecomposites were made of calcium carbonate (CaCO3) andcarbonated-amorphous calcium phosphate (ACP). In thestudy they pointed out that the calcium carbonate helps tocompensate the possible foreign body reactions during thedegradation of polylactide. Even more the composite(PLLA/PDLLA/ ACP) scaffold had the best mechanical

properties; the elastic modulus was 9-10GPa which is closeto the value of cortical bone. In parallel to their experiencesand conclusions, similar results are observed in this study;the inorganic filling increases the compressive strength andmodulus significantly. In another study, Oliva and cow-orkers4 investigated the influence of natural (powder) andsynthetic (whisker) HA filling on PCL. They tested bothmechanical properties and biocompatibility of these compo-sites. In their paper they concluded that however thewhisker-filled PCL had higher strength and modulus thanthe original PCL, it was still lower then the powder-filledcomposite. The explanation for the phenomenon was theweak interfacial connection which was indicated by the SEMpictures. It was found that the maximum gain in strength wasachieved at 10% powder filling, while the highest increase inmodulus was observed at 20% of filler content. Theseobservations are in some contrast with the results in this

study, where the needle-like crystal reinforcement wassignificantly better than the powder. In the case of thearagonite-filled samples, the strength and the modulus werehighest at 40% of filler content (Figs. 18 and 19).The observeddifference underlines the fact that the filler physical andchemical structure has strong influence on the mechanicalproperties of the composite.

In the next step, the products will be tested forbiocompatibility by assessing the vitality of human osteo-blasts cultured on the scafold samples.

CONCLUSIONS

This study, aimed at testing two manufacturing methods forsynthesizing porous PCL materials being of potential interestas scaffolds for bone tissue engineering, resulted in thefollowing observations.

Phase exchange process (acetone/water), when applied inmanufacturing of porous PCL samples, results in asignificant level of microporosity.

Scaffolds obtained by solvent casting/particulate leachingtechnique with NaCl as the porogen are microporous andmacroporous, while the size of macropores corresponds tothe size of salt particles. Overall porosity and interconnec-tivity depends on (but is not proportional to) the salt content.If the porogen content is 33%, the apparent porosity isapproximately 60%. This value is lower than the original one(in the absence of salt), which makes it possible that theinterconnectivity is not assured and there is some residualsalt in the scaffold. If the salt content is higher than 33%, thepores are well interconnected, providing the possibility offully removing porogen from the product. Mechanicalstrength of these materials (in terms of compression loads)

Copyright IQ 2006 John Wiley & Sons, Ltd.

If&D-t

is moderate and depends clearly on initial porogen contents,and, as a result, on the final porosity.

Synthesis of calcite-filled PCL samples by simple solventcasting does not lead to the formation of products ofacceptable mechanical properties, due to the formation ofpoorly interconnected large grains. Much better results areobtained by using phase exchange. This technique leads tomicroporous, reinforced materials of considerable strength,which depends on the initial composition, but, first of all, onthe shape of filler particles. Usage of calcium carbonateneedle-like crystals (aragonite) results in 3-5-fold increase incompression modulus when compared to calcite powder atthe same weight content.

The data seem to indicate that PCL-based materials

enhanced with calcium carbonate, due to their goodmechanical properties, could potentially be applicable asbony tissue replacements (resorbable at long timescales).One might expect that slow dissolution of calcium carbonatefilling in biological conditions would provide in situ pHbalance to compensate for acidic products of PCL biode-gradation, and would lead to the formation of larger pores,accessible for osteoblasts, thus effectively they may act asslowly resorbable scaffolds. The salt-leached PCL porousmaterials, of lower mechanical strength but higher elasticity,could possibly be applied in bone tissue engineering formaxillofacial surgery and other non-load bearing applicationor in soft-tissue engineering.

AcknowledgmentsOne of the authors, Laszlo Olah, pays tribute to the EuropeanCommission for the grant in Marie Curie Fellowship Associ-ation (HPMT-GH-01-00228-06). This work has been sup-ported by the Ministry of Science and Information SocietyTechnologies, Poland (projects 05/PBZ-KBN-082/T08/2002,3 T08E 066 29 and 6/6.PR UE/2006/7), as well as by theInternational Atomic Energy Agency (POL/6/007).

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