By, Tanner Jones, Andrew Gloe, Michael Grabarits, Hoi Wai Chau, and Sarah Bradner

University of Gothenburg, The Sahlgrenska Academy, Institute of Biomedicine, Hakan Nygren Cecilia Eriksson Katrin Richter Karin Ohlson

Elos Medical AB, Backendalsvagen Nicklas Billerdahl Mattias Johansson

PhD. Histology; Histologiska Institutionen at Gothenburg University Thesis: Immunoenzyme methods;.

Head of the Imaging Mass Spectrometry Research Group at University of Gothenburg as of 2008 Focus on Histological Analysis by use of TOF-SIMS

Ph.D Advisor of Cecelia Eriksson and Katrin Richtor

Doctoral Degree: Medicine/Histology Thesis: Interactions between whole blood and

TiO2 surfaces with focus on adhesion and activation of polymorphonuclear granulocytes

Post-Doctoral Work(2003): The University of Gothenburg

Most Current(2011) : Head Life Sciences Deptartment, Biomedicine, University of Skövde

16 peer reviewed publications

Masters of Science in Biology at the University of Rostock, Germany

Doctorate: University of Gothenburg

5 Peer Reviewed Publications

Not one of Professor Nygren’s doctoral students.

Has no citations on either PubMed or Wiley Online Library Could be a lab tech or a just the result of

translational butchery.

Elos Medtech- self described as “one of Europe’s leading development and production partners for medical technology products and components.” Based in Timersdala Sweden

Appears their involvement in the project was concerned with the design and supply of the experimental materials.

No conflict of Interest statement

TOF-SIMS (Time of Flight Secondary Ion Mass Spectrometry): A method of imaging, which allows for the characterization of a specimen’s chemical: composition distribution depth profile. ToF-SIMS is particularly useful in that does not depend on probes

or antibodies which would impose their own unique physical and chemical limitations on what can be imaged. TOF-SIMS imaging limited only by what can be ionized in a single sample analysis session.

The great challenge lies in sample preparation Imaging must performed under ultra high vacuum

conditions. Samples most be freeze dried or freeze fractured to keep

them as close to native conditions as possible

http://www.youtube.com/watch?v=8wzZcsNk_80

Cortical bone: High density, mature osseous tissue. Cortical bone facilitates support of the whole body and protection of the organs while also providing levers for movement

Passivation: The process of intentionally producing a layer of corrosion on the surface of a biomaterial for the purpose of reducing its surface reactivity.

Bone Resorption: The process by which osteoclasts break down bone into its constituent minerals.

Anodic oxidation:An electrolytic passivation method in which the treated material forms the negative terminal of an electric circuit.

Mallory’s Trichrome Stain: Commonly Used for the identification of connective tissue

Cell Members affected: NucleiRed CytoplasmPale Red ErythrocytesOrange Collagen FibersDeep

Blue

More Specifically : Keratin Orange CartilageBlue Bone MatrixDeep Blu Muscle FibersRed

Post fracture Bleeding, blood coagulation, hematoma

Inflammation

Soft Callus Formation

Hard Callus Formation

Bone Remodeling

Occurs immediately after injury

Extravascular blood cells form a blood clot

All the cells within the blood clot degenerate and die

Thrombin and Growth Factors are released by activated leukocytes Activate fibroblasts aggregate and form

granulation tissue

Platelets in the hematoma serve as chemotaxins for osteogenic cells

Filled with vascular endothelial growth factor (VEGF) Involved in angiogenesis and bone

t

Stabilizes the fractured area with granulation tissue and fibrocartilage

Spongy material

Callus will keep expanding until fracture is stabilized

Internal and External callus

Once stabilized blood vessels will invade the callus

Very narrow compact region found in the fracture union

Internal callus has high cellular density

Very compact region

Found adjacent to the fibrin clot (hematoma)

Contains cells of endosteal origin

Large quantities of Fibrin and cartilage

External callus is larger, but low cellular density

External callus is adjacent to bone marrow

Cells are derived from progenitor cells found in the periosteum

Polymorphic MSC and osteoblasts are responsible for early synthesized bone matrix

Primarily made of woven bone and cartilage

Vascular density in the callus increases

Endochondral ossification of spongy bone into woven bone

Vesicles are released by osteoblasts Initiates tissue mineralization Release hydroxyapatite crystals

Organic components of bone are mineralized Type I collagen fibrils and

noncollagenous matrix proteins

Convert less stable spongy bone into stronger woven bone

Over laps with the hard callus formation

Hard callus is still bulky and needs to be remodeled into previous uninjured state

Woven bone is replaced over time with compact lamellar bone

Bone becomes more organized in parallel fibers

VEGF are the growth factors that regulate remodeling Attracting endothelial cells and osteoclasts Stimulates osteoblast differentiation

Osteoclasts remove woven bone, and osteoblasts lay down lamellar bone

Bone healing is a process that does not result in scaring

Insertion of implants leads to complete healing

Poorly inserted implants can lead to instability and eventually failure Instability causes fibrous

encapsulation instead of implant bone contact

Implants that extend into the marrow cavity cause bone tissue to remain in the marrow cavity This is not observed in normal

fracture healing

Why does the presence of a titanium plate placed in the fractured union lead to the formation of bone tissue in the marrow cavity?

Thickness: 1mm Diameter: 2.5mm Threaded hole with 0.8mm diameter Grade 1 Unalloyed titanium, low oxygen. Grade 2 Unalloyed titanium, standard oxygen. Grade 2H Unalloyed titanium (Grade 2 with 58 ksi minimum UTS). Grade 3 Unalloyed titanium, medium oxygen. .. . . Grade 38

Passivated discs in 4.9M HNO3 for 20 min

Washed in alcohol

Anodic Oxidation to grow porous oxides

Platnium band (cathode) titanium+discs (anode)

HF (hydrofluoric acid) + H2SO4 (Sulfuric acid)= strong oxidizing agent B11

HF (hydrofluoric acid) + H2SO4 (Sulfuric acid) + H3PO4 (phosphoric acid) G4 and G1

Rinsed in deionized water alcohol based washing

Auger Electron Spectroscopy (AES): provides elemental analysis of surfaces by measuring energies of backscattered electrons. -very sensitive -can monitor surface cleanliness -compositional analysis of specimens in surface region

Time-of-flight secondary ion mass spectrometry -positive and negative spectra recorded

http://www.cem.msu.edu/~cem924sg/Topic10.pdf

Surfaces were photographed

SEM images segmented

Measured mean pore diameter, #pores/µm2, and surface porosity

Male Sprague

Dawley rats (350-500g)

Anesthesia with Isofluran

Baxter

Shaving and cleaning of calves with

iodine Muscle and

bone exposed by 2cm-long

lateral incision

Muscularis tibialis anterior

aside and periosteum

open

1mm diameter

Hole drilled in facies

lateralis of tibia

Incision, rinse and Implant

placed in each tibia

Skin sutured Buprenorphin below dermis

and epidermis

Free post-op movements

Post-op

The surgical procedure used to insert the implant consisted of drilling a 1mm diameter hole in the facies lateralis of the tibia with a low speed drill. How could drilling method detrimentally impact the rate of implant healing and osseointegration?

Incisions made in bone

Left to heal and no implant

Animals sacrificed at 4,7 and 14 days

Bone site of implantation was extracted

Samples fixed in PBS for 3 days

Decalcified for 2 weeks in 0.5% paraformaldehyde in PBS (makes bone flexible and easier to analyze)

Samples were rinsed in water for 15 min

Samples dehydrated in graded series ethanol

Imbedded in Histowax imbedding medium

Cut and mounted on Superfrost plus glass slides

Stained with Mallory’s trichrome

Stain tissue photographed with microscope

Area measured

Percent of bone contact with implant relative to blood and connective tissue measured

Thickness of bone in contact was not measured

ANOVA post hoc test: examining of data after the experiment to look for patterns. Statistical test performed once pattern is found.

Significance set to p<0.05

B11 was processed using H2PO4 Contains low P component

compounds

G4 and G1 were processed using H2PO4 andH2SO4

Phosphorus is the second abundant mineral in the bone. Used for development and

maintenance of healthy bones

How can the different in surface compound affects the implant healing results?

How might the long-term effects vary among the four surface properties control, B11, G4 and G1. If these surfaces were studied long-term, what may be another useful variable to quantify besides bone-to-implant contact?

Figure 4. (a-e) Normal healing after (a) 0 days, (b) 4 days, (c) 4 days (close-up), (d) 7 days, (e) 14 days.

4 Days Formation of soft callus and new bone

7 days Formation of woven bone and hard callus Bone resorption with in the marrow

14 days Woven bone has been replaced with more

mature bone

Figure 4. (f-i) Implant healing of the control surface after (f) 4 days, (g) 7 days, (h) 14 days.

4 Days New bone formation adjacent to the

endosteum of the cortical bone

7 Days Woven bone surrounding the implant

14 Days Woven bone on the implant surface has been

replaced by lamellar bone

Within 14 days, bone formation, resorption, and maturation had taken place in both fracture and implant healing

- overtime, bone and marrow will be completely restored in fracture healing

- Osseointegration is necessary for implant stability (imbedding in layer of bone good, fibrous tissue formation around implant bad)

- Excessive bone resorption also bad

REFERENCE STUDY THIS STUDY Hanawa et al. in a similar rat Ti implant study found: - Initial bone formation in the marrow followed by resorption - After 18 days, bone stayed in a thin line around implant

- After 7 days, some bone found in close contact with implant surface + bone formation in marrow space around implant

- Bone resorption in marrow between 7-14 days

- After 14, bone stayed close to implant

Ushida et al. drilled holes in bones similar to this study but no implants inserted: - Bone formation in marrow after 5-7 days - After day 11, bone gradually replaced with marrow (resorption)

- “small islands of bone” (bone formation) seen after 4 days in both implant/fracture healing

- Resorption at 14 days

Takeshita et al. - Ti implants in rat tibia studied after 28 and 730 days. - Found bone thickness increases

after implant but established early in

- After resorption of callus bone, implant surrounded by thin incomplete layer of bone after 2 weeks same layer seen in

Reference Study THIS STUDY Medard et al. found: - the amount of bone in rats

decreased where implant exposed to marrow between 7 & 21 days

- mature bone stayed close to surface

- After 14 days, initially formed bone resorbed

- Mature bone remained close to surface

- This study in accord with previous research regarding healing process

- Resorption is very important for strong implant attachment

** “Reducing Desorption” of bone early on could be an asset for developing implants that integrate better

http://www.intechopen.com/source/html/29733/media/image2.jpg

• Other studies have shown more porous implants integrate better with bone in the long term (6-12 weeks) in rabbits •This study showed implant healing was not significantly affected by implant porosity since all implants had similar bone contact

Dhert et al. agrees: “biology rather than implant properties” is main factor in early implant healing

Fractures and implant injuries both heal in similar ways in terms of structure and rate

After 14 days, the implant was enveloped in lamellar (strong) bone and the marrow restored

Porosity of titanium implant did not affect bone integration after only 7 days