TISSUE ENGINEERING.pptx

INTRODUCTION
KEY ELEMENTS IN TISSUE ENGINEERING
- Progenitor cells
- Scaffold
- Signalling molecules
DESIRED PROPERTIES AND WAYS TO ENHANCE THE REGENERATIVE CAPACITY OF SCAFFOLDS
4. GENE THERAPY IN PERIODONTAL TISSUE ENGINEERING
5. RECENT DEVELOPMENTS
6. CRITICAL ANALYSIS OF PRESENT STATUS OF TISSUE ENGINEERING FOR PERIODONTICS.
4. CONCLUSION
5. REFERENCES

1. TISSUE ENGINEERING Dr. BEENA VIJAYAN PARVATHY 3rd YEAR POST GRADUATE Dept. of Periodontology and Oral Implantology

2. CONTENTS INTRODUCTION KEY ELEMENTS IN TISSUE ENGINEERING - Progenitor cells - Scaffold - Signalling molecules DESIRED PROPERTIES AND WAYS TO ENHANCE THE REGENERATIVE CAPACITY OF SCAFFOLDS 4. GENE THERAPY IN PERIODONTAL TISSUE ENGINEERING 5. RECENT DEVELOPMENTS 6. CRITICAL ANALYSIS OF PRESENT STATUS OF TISSUE ENGINEERING FOR PERIODONTICS. 4. CONCLUSION 5. REFERENCES

3. INTRODUCTION DEFINITION Tissue engineering is the branch of biology where tissues are produced in culture by cells seeded (grown) in various porous absorbable matrices by using biological principles. Langer M et al; 1993 Tissue engineering is defined as the science of fabrication of new tissues for replacement and regeneration of lost tissues or defined tissues. Baum et al; 2000

4. First proposed by Langer & Vacanti ; 1993. The primary aim of this therapy is to deliver biologically active elements which get integrated into the host tissues and result in 3D regeneration of the lost tissue which is structurally and functionally similar to the tissue which was lost. TE is a regenerative treatment of periodontal defects with an agent, or procedure, requires that each functional stage of reconstruction be grounded in a biologically directed process. Bartold et al, 2000

5. KEY ELEMENTS IN TISSUE ENGINEERING CONDUCIVE ENVIRONMENT VASCULAR SUPPLY

6. APPROACHES USED TO REGENERATE TISSUES EX VIVO APPROACH IN VIVO APPROACH Tissue created in a lab by culturing cells on a biodegradable scaffold in the presence of molecular factors required for growth and then transferred into the body. All components required for regeneration are placed in the tissue defect and an environment which is conducive to maximum regeneration is created to achieve favorable regeneration

7. PROGENITOR CELLS/STEM CELLS These cells can differentiate into different types of end cells and can form the desired structural components of the lost tissue. Vats et al; 2002 Criteria to achieve effective, long–lasting repair of damaged tissues : 1. Adequate number of cells must be produced to fill the defect. 2. Cells must be able to differentiate into desired phenotypes. 3. Cells must adopt appropriate 3D structural support / scaffold and produce ECM. 4. Minimal associated biological risks. 5. Cells must integrate with native cells and overcome the risk of immunological rejection.

8. SOURCE AUTOGENIC FROM ANOTHER MEMBER OF SAME SPECIES. ADV : UNIFORMITY, STANDARDISATION, COST EFFECTIVE, QUALITY CONTROL. ALLOGENIC IDEAL SOURCE FROM THE PATIENT LOW IMMUNE COMPLICATIONS XENOGENIC FROM OTHER SPECIES GREAT RISK OF IMMUNOLOGICAL ACTION .

9. PROGENITOR CELL PRODUCTION IN PERIODONTAL TISSUES PROGENITOR CELLS PDL derived progenitor cells Periosteal cells Bone marrow derived MSCs Adipose derived SCs Gingival fibroblasts PDL derived mesenchymal stromal cells 1 st evidence for presence of undifferentiated mesenchymal cells within periodontal tissues. MuCulloch & co-workers; 1987

10. PDL DERIVED PROGENITOR CELLS Capacity to produce cementum and periodontal ligament like structures contributes periodontal tissue repair . PDL stem cells differentiates into cementoblast like cells and collagen forming cells . Nakahara et al; 2004 GTR  based on  these cells allowed to proliferate in the area of periodontal defect to differentiate into cells required for regeneration .

11. PDL DERIVED MESENCHYMAL STROMAL CELLS Initially identified in adult bone marrow . Seo et al; 2004 A clogenic clusters of adherent fibroblastic-like cells or fibroblastic colony forming units with the potential to undergo extensive proliferation . Have capacity to differentiate into different stromal cell lineages . Friedstein et al; 1970,1976 & 1987 Consists of multipotency , clonogenic ability , high proliferation and the expression of the putative stem cell marker STRO-1 & perivascular cell marker CD146 .

12. PERIOSTEAL CELLS Differentiates into osteoblastic lineage , also express PDL related genes . Cells are clonogenic , displayed long tolomers and expressed markers of mesenchymal stem cells (MSC’s). De Bari et al; 2006

13. BONE MARROW-DERIVED MESENCHYMAL STEM CELLS (BM-MSC’s) Capable of producing bone , cartilage , adipose tissue , muscle and periodontal tissues . PRP + BM-MSC’s  Periodontal regeneration .

14. ADIPOSE DERIVED STEM CELLS (ADSC’s) Used in periodontal regeneration . Available in abundant . PRP + ADSC’s  Periodontal regeneration .

15. GINGIVAL FIBROBLATS Root coverage  Recession  cell transplantation therapy using gingival fibroblast. Gingival fibroblast were seeded onto sponges of human Type I or III recombinant collagen . After culturing , vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) released in culture media  ↑ fibroblast proliferation.

16. SCAFFOLD OR SUPPORTING MATRIX Scaffolds are natural or synthetic materials used to carry biologically active molecules to the site of regeneration. Primary requirements: 1. Biocompatible 2. Biodegradable 3. Simple and predictable manufacturing process 4. Porous, mechanically stable and 3D structure.

17. SCAFFOLD NATURALLY DERIVED SYNTHETICALLY DERIVED Fibrin Collagen Chitosan Alginate Hyaluronic acid Polyanhydrides Polyorthoesters Poly ( α – hydroxyesters )

18. ADVANTAGE OF SYNTHETICALLY DERIVED Biocompatible Biodegradable Bioresorbable Easily forms 3D structural matrices Poly ( lactide -co- glycolide )( PLGA) copolymers  controlled degradation behavior & mechanical properties can be adjusted according to the requirements. DISADVANTAGES OF SYNTHETICALLY DERIVED Degrade  Produce acidic by products  hamper process of regeneration.

19. SCAFFOLD FABRICATION Fiber bonding Emulsion freeze drying High pressure processing Electrospinning Rapid prototyping Solvent casting / particulate leaching Thermally induced phase separation Gas foaming / particulate leaching

20. FIBER BONDING PGA PLA Disadvantage : Matrix porosity achieved cannot be precisely controlled .

21. EMULSION FREEZE DRYING An emulsion solution containing a dispersed water phase and an organic continuous phase is freeze dried . It results in formation of a porous scaffold with various pore size and interconnectivities . Using this technique, up to 95% porous scaffold with a pore size, up to 200 µm has been prepared. Nuber et al; 1995

22. SOLVENT CASTING / PARTICULATE LEACHING This involves use of a water soluble porogen , such as salt . In this technique, the polymer (PLLA or PLGA) is first dissolved in chloroform or methylene chloride and then is casted onto a petri dish filled with porogen . Once the solvent evaporates , the polymer / salt composite is leached in water for two days to remove the porogen . The amount of porosity depends on the quantity of salt added and the pore size depends on the crystal size of the salt particles .

23. HIGH PRESSURE PROCESSING A gas such as CO2 is applied at high pressure to the dry polymer . Results in the formation of a single phase polymer / gas solution . After the formation of this single phase polymer / gas solution, the pressure is reduced , which creates thermodynamic instability of the dissolved CO2 and results in nucleation and growth of gas cells to generate pores within the polymer matrix . Mooney DJ et al; 1996

24. GAS FOAMING / PARTICULATE LEACHING A binary solution of PLA – solvent gel containing dispersed ammonium bicarbonate salt particles . The mixture was casted in a mold and subsequently immersed in hot water . Due to increased temperature , ammonia and CO2 gas are formed along with the leaching out of ammonium bicarbonate particulates from the solidifying polymer matrix . Results in formation of a highly porous structure with high inter connectivity . Park et al; 2001

25. THERMALLY INDUCED PHASE SEPARATION This is by thermodynamic demixing of a homogenous polymer – solvent solution into a polymer – rich phase and a polymer – poor phase . Liquid – liquid phase separation or emulsification / freeze – drying method is used to separate the two phases . The polymer solution is quenched below the freezing point of the solvent and subsequently freeze dried . Results in formation of a highly porous structure .

26. ELECTROSPINNING Most widely used method for preparation of nano fiber non – woven matrices . In this technique, a polymer solution is pumped at a constant rate through a syringe with a small diameter needle that is connected to a high – voltage source . When this voltage source is turned on , an electric field is created Under the strong electric field , electric charge overcomes the surface tension of the polymer solution droplet . Then a polymer jet is sprouted from the nozzle followed by solvent evaporation which forms nanofibers . Thus a highly porous 3D scaffold is formed.

27. RAPID PROTOTYPING A computer aided design ( CAD ) with pre – decided 3D architecture is formed in a layer – by – layer manner with precise control over its morphological characteristics . Most recent introduction. Advantage : Scaffold with a predetermined size, shape, porosity, chemical composition and desired mechanical properties can be fabricated.

28. DESIRABLE PROPERTIES OF SCAFFOLDS USED FOR PERIODONTAL REGENERATION Cell-cell & cell-matrix interaction Hold growth factors for desirable duration Biocompatible Allow proliferation Should not induce environmental changes

29. RECENT ADVANCES IN SCAFFOLDS MULTIPHASIC SCAFFOLDS

30. 3D-PRINTED SCAFFOLDS c

31. GELS AND HYDROGELS c

32. SMART SCAFFOLDS - Biomimetic scaffolds & bionic smart scaffolds -Immune sensitive smart scaffold -Shape memory smart scaffold -Electromechanical-stimulus smart scaffold

33. BIOMIMETIC SCAFFOLDS & BIONIC SMART SCAFFOLDS Developed using inspiration from nature . Elicit specified cellular responses mediated by interactions with scaffold – tethered peptides , especially incorporating of cell – binding peptides into biomaterials via chemical or physical modification. Biomimetic porous poly ( lactide -co- glycolide )(PLGA). Mittal et al; 2010 Scaffold fabricated by computer – generated design to mimic surface morphology and pore size distribution. Thus regenerative potential enhanced.

34. IMMUNE SENSITIVE SMART SCAFFOLD Scaffold should have immunomodulatory properties , directing the host response towards tolerance of the foreign scaffolds or regulating immunological microenvironments to promote cell survival. IL-4 has been incorporated in the scaffolds to enable their immunomodulatory capability. Incorporation of nanofibrous heparin-modified gelatin microspheres in the scaffold can spatiotemporally deliver the anti-inflammatory cytokine IL-4 to polarize the proinflammatory M1 macrophages into an anti-inflammatory M2 phenotype . It improves the osteogenic potential of the scaffold. Hu Z et al; 2018

35. SHAPE MEMORY SMART SCAFFOLD They can return from a deformed shape to their original shape by an external stimulus , such as temperature change, an electric or magnetic field and light . Scaffolds are fabricated by utilizing 3D and 4D printing technologies. Field of minimally invasive surgical therapy (MIST). The scaffold with small size is placed in the bone defect using minimally invasive means with the least damage to host tissues . With time the scaffold regains its actual size and precisely fills the bone defect . BMP2-loaded shape-memory porous nanocomposite scaffold was placed in bone defects in the rabbit model . The porous scaffold displayed shape-memory recovery from the compressed pores of 33 μ m in diameter to recover its original porous shape of 169 μ m in diameter , under both invitro & invivo conditions . Promoted bone regeneration in mandibular bone defects . Liu X et al; 2014

36. ELECTROMECHANICAL-STIMULUS SMART SCAFFOLD The piezoelectric property of certain materials can be utilized to enhance the regenerative potential of the scaffolds . The piezoelectric effect is the ability of a material to generate an electric charge in response to applied mechanical stress . Piezoelectric poly ( vinylidene fluoride- trifluoroethylene ) (PVDF- TrFE ) has been used to fabricate flexible, 3D fibrous scaffolds . Damaraju SN et al; 2017

37. WAYS TO ENHANCE THE REGENERATIVE CAPACITY OF SCAFFOLDS Addition of growth factors By soaking the scaffold in a solution of GF By encapsulation into scaffolds By covalent immobilization for controlled & extended release By incorporation into seeded cells via molecular & genetic modification

38. SIGNALING MOLECULES Secreted from various cells in response to stimulus and they act on same, neighboring or distant cells to cause specific effects. GF  stimulates synthesis of ECM by cells such as fibroblast, osteoblast etc. Recombinant growth factor , commercial use: Platelet derived growth factor (PDGF; GEM 21) Bone morphogenic protein-2 (BMP-2; Infuse) Fibroblast growth factor (FGF-2) GROWTH FACTORS BONE MORPHOGENETIC PROTEIN

39. STUDIES EVALUATING EFFICACY OF VARIOUS SCAFFOLDS USED WITH SIGNALLING MOLECULES IN ACHIEVING PERIODONTAL REGENERATION.

40. c

41. FDA APPROVED MATRICES WITH GROWTH FACTORS 1. rhBMP-2 incorporated type-I bovine collagen sponge/ scaffold ( InFuse , Medtronic Sofamor Danek ). 2. rhPDGF -BB with β -TCP scaffold ( GEM 21S , BioMimetic Therapeutics). 3. rhBMP-7 incorporated collagen sponge ( Osigraft , Stryker Biotech, Ontario, Canada).

42. FUNCTIONS OF GROWTH FACTORS PDGF  Increases chemotaxis of PMNs & monocytes . Fibroblast proliferation, ECM synthesis. ↑ chemotaxis , proliferation of endothelial cells Fibroblast to myofibroblasts . Proliferation of mesenchymal progenitor cells TGF β  Increases chemotaxis of PMNs & monocytes . Autocrine expression- generation of TNF α , IL1 β , PDGF & chemokines Stimulates epithelial proliferation & migration. Fibroblast proliferation, ECM synthesis. Inhibits proteases & ↑ inhibitor production. BMP  BMP2-4 ↑ mesenchymal progenitor cell migration. BMP7 ↑ osteoblast differentiation.

43. GENE THERAPY IN PERIODONTAL TISSUE ENGINEERING The principle of gene therapy is to transfer desirable genes to the target cell which then synthesizes a protein of interest. Transfer of gene to target cells; The gene can be introduced; VIRAL VECTORS NON – VIRAL VECTORS Retroviruses Adenoviruses Adeno associated viruses Plasmid DNA polymer complexes DIRECTLY INDIRECTLY

44. PROCEDURES Harvesting the selected cell population Expanding this population Genetically transducing the genetic material Re implanting cells in target area

45. RIBONUCLEIC ACID MEDIATED SILENCING Fire and Mello; 2006 discovered RNA interference gene silencing by double stranded DNA . It is a biological process in which RNA molecules inhibit the expression of certain genes which are detrimental to the tissue regeneration by causing the destruction of specific mRNA molecules . RNA interfernce is executed by 2 types of small RNA molecules : 1. microRNA 2. small interfering RNA RNAs are the direct products of genes , and these small RNAs can bind to other specific messenger RNA molecules .

46. IMPLANTATION OF LIVE CELLS TO ACHIEVE REGENERATION McGuire and Scheyer ; 2007 Minimally invasive papilla priming procedure To augment open interproximal spaces Implanted autologous fibroblast Results were significantly better in test sites than in placebo sites Interdental papillary height Subject visual analog scale

47. Bowsma and D’souza ; 2005 Expanded autologous fibroblast Injected into periodontal pocket Pocket depth reduction Bowsma O, D’souza R, Meyerat BS. Treatment of deep periodontal pockets with autologous fibroblasts or placebo. J Dent Res 2005;84:107.

48. IMPLANTATION OF TISSUE ENGINEERED HUMAN FIBROBLAST DERIVED DERMAL SUBSTITUTE Human fibroblast derived dermal substitute ( HF-DDS ) is tissue engineered living tissue derived from dermal fibroblasts. One study compared the safety and effectiveness of a living HF-DDS to a CTG for root coverage on Miller class I or II bilateral facial recession. The results of the study showed that HF-DDS may offer a potential substitute to the CTG for root coverage .

49. APPLICATION OF BI-LAYERED CELL THERAPY Tissue engineered bi-layered skin substitutes from human foreskin  Type I collagen & viable allogenic human fibroblasts , keratinocytes Substitute to palatal tissue . Momose et al;2002 , estimated levels of vascular endothelial growth factor(VEGF), transforming growth factor(TGF α & β ), epidermal growth factor(EGF) in human cultured gingival epithelial sheets(HCGES). These GF’s influences the surrounding environment in favour of healing. Used in root coverage, increase width of keratinized gingiva and in mucogingival surgeries .

50. CRITICAL ANALYSIS OF PRESENT STATUS OF TISSUE ENGINEERING IN PERIODONTICS Various growth factors act through intracellular signalling mechanism once they attach to their corresponding surface receptors . Our knowledge of these intracellular mechanisms is still incomplete . Presently , we have insufficient data for the authentication of clinical safety and effectiveness of various newer regenerative techniques by tissue enginering . The exact mechanism by which the growth factors enhance periodontal regeneration yet remains to be proven in vivo . Although tritiated thymidine and proline labelling studies would yield valuable information regarding in vitro effects of PGDF/IGF-1 , more research is required in this field.

51. Cost effective application , and easy availability still need to be sorted out. Ideally, once delivered at the site of interest , the growth factors should act on their target cells to produce desired effects . However, there are many mechanisms which neutralize or deactivate these growth factors when placed in the biological environment. Cell culture media of xenogenic products always carry a risk of disease transmission . Newer identification techniques are required to authenticate the safety of xenogenic products. There are always possibilities of immune rejection of the implanted cell line when allogenic and xenogenic sources of the cell line are used. Need to be well investigated for any immunological reaction.

52. REFERENCES Baumm BJ, Mooney DJ. The impact of tissue engineering on dentistry. J Am Dent Assoc 2000 ; 131( 3) : 309 – 18. Langer R. tissue engineering. Science J 1993 : 260. Advanced reconstructive technologies for periodontal tissue repair. Perio 2000. A novel approach to periodontal tissue regeneration with mesenchymal stem cells and platelet rich plasma using tissue engineering technology. A clinical case report 2006. Yamamiya , J. Periodontal 2008 , Tissue engineered cultured periosteum used with platelet rich plasma and hydroxyapatite in treating human osseous defects. Nakahara T et al, in situ tissue engineering of periodontal tissues by seeding with periodontal ligament derived cells, 2004. Bartold PM, Xiao Y, Principles and applications of cell delivery systems for periodontal regeneration, Perio 2000 – 2006.