2. Bone Tissue Engineering & Regenerative Medicine The Approach to Save Lives Nasrin Lotfibakhshaiesh, MD, PhD Assistant Professor Department of Tissue Engineering School of Advanced Technologies in Medicine Tehran University of Medical Sciences 6 th October 2014 The 5 th International Congress on Recent Research Achievements in Medical Sciences

3. Strontium containing bioactive glass coatings Motivation Characterisation (Glass powder/coated implants) Glass design criteria Glass synthesis Bioactivity test Animal study Cell Culture studies Outline

4. Introduction (Motivation) Demand for tissue or organ replacement / regenerative strategies Some tissues: limited capacity of spontaneous regeneration; regenerative capacity decreases after extensive damage Existing therapies: limitations. Eg.: Organ transplantation: - organ shortage - immunosuppression associated problems Increase in life expectancy (aging population); - organ failure - traumatic injury - aging-associated diseases 4

5. TE is an interdisciplinary field that applies the principles of engineering and the life sciences towards the development of biological substitutes that restore, maintain, or improve tissue function. Langer and J. Vacanti “Tissue Engineering”. Science, 260: 920-6, 1993 Tissue Engineering (TE)

6. Regenerative Medicine Designing “smart” materials to control cellular behaviour Growth factors Biomimetic materials Cells makes use of: Current challenges: Proper nutrient supply and waste removal from constructs (blood vessels); Control and direct cell differentiation (combination of factors). 4 6

7.  130,000 Joint Replacement operations per annum in the UK  Greater physical demands on implants  Younger patients  Longer life expectancy 7 http://images.wellcome.ac.uk/ Increasing clinical need for prosthetic implants: Joint Replacement Surgery

8.  Implants fail  Aseptic Loosening  Pain  Periprosthetic Fracture  Significant cause of morbidity and mortality  Expensive +++ 8 Joint Replacement Surgery

9.  Aseptic loosening still occurring with HA  Oral treatment of osteoporosis has poor compliance  local incorporation of anti- osteoporosis medication may reduce peri- prosthetic fracture risk 9 Current Need

10. 2 µm 200 µm 300 µm Powder Scaffold Coating  Bonds to bone  Bone repair and bone regeneration  Need to tailor bioactive glasses to specific applications Bioactive Glasse (BG) 10

11. A drug used for treatment and prevention of osteoporosis (EU approved, FDA pending) Stimulates bone formation Inhibits bone resorption Gentleman E, Lotfibakhshaiesh N, et al. Biomaterials 31, 3949-3956 (2010) Strontium ranelate 11

12. Aim to develop a bioactive glass coating:  Thermal expansion coefficient (TEC) similar to Ti-alloy  Large sintering window  Amorphous coatings  Strong interface adhesion with Ti-alloy implant 12 BG Coatings

13. Glass composition: SiO 2 ; P 2 O 5 ; CaO; Na 2 O; SrO; MgO; ZnO and K 2 O Synthesis via melt-quench route Reduces TEC Suppresses crystallization MgO Suppresses crystallization Bactericidal properties ZnO Stimulates bone formation Inhibits bone resorption SrO Increasing phosphate content 1.07 P 9.87 2.14 P 10.01 4.28 P 10.40 6.42 P 10.74 BG ( 10% Sr) TEC (×10 -6 ° C -1 ) TEC Ti6Al4V = 9.5 - 10.5 X 10 -6 ° C -1 BG synthesis 13

14. Heat Flux (mCal/s) Temperature ( °C ) TgTg T po Sintering window 520 °C 585 °C 580 °C800 °C Sintering window 10%Sr 45S5 T po TgTg BG characterization: Differential scanning calorimetry (DSC) 14

15. X-ray Diffraction: Apatite crystallisation increase for BG with high P 2 O 5 content after immersion in SBF for 4 weeks in compare with the BG with low P 2 O 5 content Bioactivity test results 15

16. Adding P 2 O 5 to the glass composition prevents extreme pH rises. pH changes in SBF 16

17. Schematic representation of the enamelling technique process Ethanol + BG particles BG coating Porcelain furnace 750 °C 30 min under vacuum BG coating 17

18. Cell culture study (in vitro) 18

19. A) Metabolic activity : MTT activity was also significantly greater (p<0.01) in cells treated with dissolution ions from 4.28 and 6.24 mol% P 2 O 5 BGs as compared to controls at day 28. Cell culture results 19

20. B) Viability: LIVE/DEAD staining images of Saos-2 cells at day 14 on BG coatings indicated that all coating materials were not cytotoxic. C) Attachment: SEM micrograph of Saos-2 cell demonstrated that the BG coating encouraged cell attachment. Scale bars = 200µm SEM image of Saos-2 cell on 4.28P BG coating at lower magnification 2kx. Cell culture results 20

21. With increasing P 2 O 5 content in the series of Sr-substituted BG the glass becomes more bioactive High phosphate content Sr-substituted glass can enhance osteoblast metabolic activity Conclusions (in vitro) 21

22. 22 Aim: To compare the degree of osseointegration of a novel strontium- substituted BG coating with a commercial hydroxyapatite coating In vivo Study

23. 23 Method and Surgery

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25. Investigation was involved:  Mechanical testing (push-out) 25  Tibia carefully cut parallel to base of implant  Screw-driven Instron used to push-out implant parallel to the long axis  Maximal force measured and shear stress value calculated  Histology (bone-implant contact, bone volume etc.)  Femurs dissected free of soft tissue and placed in 10% formalin for 1 week then 70% ethanol  Samples dehydrated in graded ethanol and embedding in resin  15µm sections produced using a diamond saw  3 stained (Acid Fuchsine and Methylene Blue)  Imaging (SEM, SEM-EDX) 200 µm 100 µm Bone Ti alloy BG coating Investigation of Osseointegration

26. Sr-substituted BG coated implants showed a trend for increasing maximal shear strength with a statistically significant difference evident at twenty-four weeks. 26 Push-out testing

27. The ratio of bone volume to total volume. 27 Bone volume/ total bone volume

28. Light microscopy images of the bone-implant interface and peri-implant bone. 28 Histology Evaluation

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30. 30 SEM-EDX

31.  This novel Sr-substituted BG coating produces enhanced fixation of implants in bone, compared to HA, principally through stimulation of increased peri-implant bone formation.  Of particular interest would be applications in patients with reduced bone mineral density. 31 Conclusions

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33. Classification of publications according to clinical application

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35. Glass Coatings Alloy Interface Sigmoid curve presents a classical diffusion profile in both alloy and glass coatings Lotfibakhshaiesh, et al. Journal of Non-Crystalline Solids, PNCS-12-065R1 (2010) 35

36. Glass Ti6Al4V Well attached Glass Ti6Al4V Gap and cracks Cross-section of 100% Sr glass coating Cross-section of 10% Sr glass coating Glass Ti6Al4V 30 µm 36

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