Tissue Engineering.pptx for study purpose

Medical education

1. PRINCIPLES OF TISSUE ENGINEERING DR ROHAN SINGH BHADHORIYA MS GENERAL SURGERY JR1 VIMS MEDICAL COLLEGE ,GAJRAULA

2. Definition The term “tissue engineering” as it is nowadays used was introduced in1987. “Tissue Engineering is the application of the principles and methods of engineering and life sciences toward the fundamental understanding of structure-function relationships in normal and pathologic mammalian tissue and the development of biological substitutes to restore, maintain, or improve function.”

3. Definition Regenerative Medicine - "process of replacing, engineering or regenerating human or animal cells, tissues or organs to restore or establish normal function". This field holds the promise of engineering damaged tissues and organs by stimulating the body's own repair mechanisms to functionally heal previously irreparable tissues or organs

4. The goal To assemble functional constructs that restore, maintain, or improve damaged tissues or whole organs. Growth of cell in three dimensional systems Delivery systems for protein therapeutics Cell cultivation methods for culturing recalcitrant cells Transgenic protein expression in transplantable cells Vehicles for delivering transplantable cells Avoiding immunogenicity in transplantation systems Development of markers for tracking transplanted cell Developing in vivo and ex vivo biosensors for monitoring cell behaviour during tissue production.

5. Tissue engineering Tissues can be viewed as a composite of: cells (both parenchymal and stromal) matrix and blood vessels. Cell maintenance and behavior including growth and regeneration are influenced by biochemical and biomechanical interplay. The development of a functional tissue must be vascularized to ensure survival of the neotissue. Each of these components is the purview of the tissue engineer.

6. Components of TE

7. Cell sources for tissue engineering Cells used for TE, Autologous - preferred due to the lack of immunogenicity Heterologous, Xenogeneic and each type may be mature differentiated precursor stem cell form.

8. Cell sources for tissue engineering Chondrocytes for cartilage Osteocytes for bone Schwann cells for nerves Fibroblasts for ligament and tendon engineering All these have significant proliferative potential in vitro Adult cardiomyocytes, hepatocytes, and adipocytes Challenge - difficult to culture and expand in vitro

9. Cell sources for tissue engineering A second challenge Collection of cells – biopsy; uncomfortable and impossible due to diseased state of the tissue Solution Utilize stem cells; expanded and differentiated ex vivo. Multiple types of stem cells exist E.g. embryonic, adult, and induced pluripotent stem cells. Discuss embryonic stem cells Focus on stem cell types most relevant to the plastic surgeon; sources of cells, advantages and disadvantages, use in TE

10. Cell sources for tissue engineering

11. Embryonic stem (ES) cells Totipotent Infinitely proliferative Differentiate into all tissue types Are also unstable and form teratomas Ethical and legal concerns - sourcing and utilization Successful differentiation protocols have been found to induce ES cells along specific lineage pathways from all germ layers towards many specific tissues and organs. These cells are probably immunogenic and ethical issues will persist. Regulatory and organizational issues

12. Adult stem cells Multipotent Limited in their proliferation capacity and differentiation potential. Collected and expanded from tissue biopsies through a process referred to as the colony forming unit (CFU) assay Adult stem cells in bone marrow: Hematopoietic stem cells (HSCs), which differentiated into the white blood cell population and Mesenchymal stem cells (MSCs), Progenitors of bone, cartilage, fat, and muscle. Endothelial progenitor cells (EPCs) have been isolated and cultured from adult peripheral blood.

13. Adult stem cells MSCs and EPCs also present in fat tissue associated with the microvasculature Known as adipose-derived stem cells (ASCs). Relevant stem cells in Plastics Mesenchymal stem cell Adipose-derived stem cell Endothelial progenitor cell.

14. Mesenchymal stem cells Do not express MHC class II markers Showed to be immune-privileged and may be used as allografts.

15. Mesenchymal stem cells Paracrine-growth factor hormonal- cytokine immune-modulatory effects probably account for the benefits seen with these stem cells. E.g., Ischemia increases homing of these cells to the injured site MSCs release high levels of vascular endothelial growth factor (VEGF) This modulates the repair of capillaries. MSCs injected intravenously in cardiac infarct models do not implant in the heart nor become heart tissue Lodge in the lung Activated to secrete the anti-inflammatory protein TSG-6 Probably the anti-inflammatory factor that induces the beneficial effects.

16. Adipose-derived stem cells Abundant Ease of harvest by liposuction Preferred autologous stem cell source Similar properties to bone marrow-derived stem cells More easily cultured Grows more rapidly Cultured for longer periods than bone marrow stem cells before senescence Richer source of stem cells One gram of adipose tissue can yield 5000 stem cells ASC population may also have low immunogenicity

17. Endothelial progenitor cells Incorporation of a functional vasculature network in the neotissue is important Endothelial progenitor cell (EPC) most promising First identified in 1997 by Asahara et al., Present in adult circulation Isolated and expanded from peripheral blood collected through simple venipuncture. Two distinct EPC populations that participate in vascular repair and angiogenesis via different mechanisms. Circulating angiogenic cells (or colony forming unit–Hill cells) – support via paracrine signalling Endothelial colony forming cells (ECFCs) – regenerate an endothelial population

18. Challenges associated with adult stem cells Advantages An autologous and/or non-immunogenic source of cells. Limitations Patient-to-patient variations in their prevalence, proliferative capacity, and differentiation potential Additionally, their utility is also a factor of age and disease state of the donor Exit the cell proliferation cycle (prematurely senesce) or prematurely lose differentiation potential during ex vivo expansion Schipper B, Marra K, Zhang W, et al. Regional anatomic and age effects on cell function of human adipose-derived stem cells. Ann Plast Surg. 2008;60:538–544

19. Induced pluripotent stem cells (iPS) Unlimited proliferation capacity Ability to differentiate into cells from all germ layers both in vitro/vivo Major problem - Requires genetic manipulation of the cells Two of the genes used in this process (c-Myc and KLF4) are oncogenes Zhou et al. (2009) delivered the 4 proteins that the above-mentioned genes code for directly into the cell Protein-induced pluripotent stem cells (piPSCs) Bypasses the need for viral or plasmid transfection and reduces the risks of cancer formation. Drawback - efficiency of the protein induction is very low Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell (2006) 126(4):663–76. doi:10.1016/j.cell.2006.07.024

20. Induced pluripotent stem cells (iPS) Challenge Not immune privileged requiring collection, induction, expansion, and differentiation of autologous cells. Costly and time-consuming Solution Create a bank of iPS cells that can be HLA matched to the patient

21. Cellular interactions with their environment Many cell types are exquisitely sensitive to stimuli present in the environment. Stimuli include; Soluble molecules Molecular recognition sites present in the solid phase (ECM or biomaterial) Interactions with other cells Substrate stiffness and the micro/nanostructure of the surroundings

22. Soluble signals Soluble biomolecules eg Metalloproteins Growth factors Chemokines Play vital roles locally and systemically in repair and tissue development. Understand the appropriate soluble signaling molecules to maintain cell viability both during culture and in vivo, to maintain cellular phenotype, to drive lineage specific differentiation of stem cells

23. Matrix signals Cells also possess receptors such as integrins and syndecans that bind to a variety of ligands present in the extracellular matrix (ECM) Many cell types are adhesion dependent Also the number and strength of bonds with the external matrix affects a wide variety of cellular behaviors ranging from adhesion, focal adhesion formation and migration to morphology. Additionally, the ECM binds and sequesters growth factors that also drive cellular function.

24. Intercellular signals Cells also interact with neighboring cells in both native tissue and in ex vivo culture Two main methods: >Direct contact via receptors such as cadherins >Soluble signaling through paracrine factors In traditional cell culture, single populations of cells are grown in isolation, implanted and rely on recruitment of supporting cellular structures and matrix to evolve into a stable, functional tissue However, co-culturing cells with other types prior to implantation can facilitate their survival and function. E.g. Endothelial cells for vasculature MSCs for paracrine effects or for their ability to incorporate into developing tissues in vivo like blood vessels Beating cardiomyocytes with ASCs in vitro to differentiate into cardiac lineage

25. Mechanics and structure of the environment Cells respond to the mechanical properties and dimensionality of their environment. Response of cells to environmental stiffness and dimensionality is a tool that can be used to direct cell function. E.g. chondrocytes change morphology and lose their chondrogenic capacity in 2D monolayer culture but can maintain these features in 3D culture Uses; Distraction osteogenesis Periods of stretch promote proliferation and migration Relaxation incites cells to cluster together and terminally differentiate into bone

26. Mechanics and structure of the environment 3D cultures on biomaterial supports such as Porous scaffolds Hydrogels Microspheres Under conditions designed for the desired cell attachment, migration, proliferation, and differentiation Development of new biomaterials with tailored properties Direct the fabrication of these materials into three-dimensional scaffolds to maximize the healing process

27. Biomaterials used in tissue engineering Components of solid tissues Cells Extracellular matrix - structure and biochemical signals to the cells When cells are expanded outside the body, they grow in monolayers, not the intricate patterns of a fully realized 3D tissue Thus, cells are seeded onto a 3D scaffold Biomaterial scaffolds can be thought of as artificial ECMs

28. Biomaterials used in tissue engineering Early tissue engineering used known materials. Polymers used in degradable sutures Natural materials such as coral, alginate and collagen. Recently, numerous alternatives have been formulated Biomaterials for TE Naturally occurring in the body From other natural or synthetic sources Ceramics Polymers Hydrogels Composites of these Decellularized tissues

29. Biomaterials used in tissue engineering The physical form of biomaterials can also vary to suit the application Solid materials Porous scaffolds Microspheres Hydrogels Injectable materials that may cross-link in situ etc. Simplicity - facilitate regulatory approval and translation into clinical application Selection depends on the specific requirements of the tissue being targeted

30. Biodegradable materials Most are biodegradable, to be replaced by neotissue Rate of degradation and loss of integrity will depend on; Type of biomaterial Site of implantation Properties of the biomaterial construct such as surface area to volume ratio, size, and surface chemistry Challenges - Prevention of sudden loss of physical integrity - Rapid degradation - excessive concentrations of the degradation products and can cause adverse tissue reactions.

31. Natural biomaterials Chemically similar or identical to molecules in the body Readily degraded in vivo Interact with cells on a molecular level Difficult to obtain and purify Vary in properties between batches Difficult to sterilize Alter their properties during storage, Elicit significant immunogenic responses.

32. Natural biomaterials Examples; Proteins (e.g., collagen, gelatin, silk) Polysaccharides (e.g., chitosan, hyaluronic acid) Polynucleotides Extracts of ECM components Increased interest - Decellularized extracellular matrix (dECM) In decellularization, cells are removed from allografts or xenografts to reduce immunogenicity but much of the complex composition and architecture of the ECM may be retained

33. Decellularization is the process used in biomedical engineering to isolate the extracellular matrix (ECM) of a tissue from its inhabiting cells, leaving an ECM scaffold of the original tissue.

34. Polymeric biomaterials 1. Hydrophobic polymers Biodegradable polymers that could be used in TE The polyesters poly(glycolic acid) (PGA) poly(lactic acid) (PLA) poly(ε-caprolactone) (PCL) and their copolymers such as poly(lactide-co-glycolide) (PLGA) Mechanical strength & degradation rate – altered by changing the polymer properties (molecular weight, composition, molecular architecture, crystallinity, hydrophobicity)

35. Polymeric biomaterials 2. Hydrogels Water-swollen cross-linked polymer networks which can absorb up to thousands of times their dry weight of water. Advantage of being more like most natural tissues and allowing mass transport to and from cells. Naturally derived Collagen Gelatin Hyaluronic acid Alginate Synthetic Poly(ethylene glycol) (PEG)-based polymers

36. Ceramic biomaterials Ceramic biomaterials are primarily utilized in tissue engineering of hard tissues. Calcium phosphates, such as hydroxyapatite, and bioactive glasses have been developed as bioceramics for bone tissue engineering. Characteristics Bioceramics are brittle but have high compressive strength, can bond strongly to bone, and can be osteoinductive.

37. Advanced biomaterials for tissue engineering 1. Tailored delivery systems Growth factors, anti-inflammatory peptides, and drugs may be incorporated into biomaterial delivery vehicles for release at the desired time during tissue development. Release systems are designed to deliver multiple molecules over different timescales via continuous or pulsatile delivery, which may be programmed or triggered by some change in the local environment. Fabricated from biodegradable polymers in the form of micro or nanoparticles capsules, within walls/surfaces of scaffolds or hydrogels

38. Advanced biomaterials for tissue engineering 2. Smart Polymers Changes in environmental conditions - changes to the molecular conformation of many materials. Environmentally-induced changes may be harnessed, thus smart polymers. Used to >encapsulate and release payloads of cells or drugs, >form gels upon injection in vivo >for cell sheet engineering. Example - Thermo-responsive polymer N-isopropylacrylamide (NIPAM) Used to grow confluent cell sheets and then to detach the intact sheet along with the ECM that the cells have deposited.

39. Advanced biomaterials for tissue engineering 3. Non Fouling Materials Successful strategies - use of chemical surface modifications Initial stage of FBR is the adsorption of a complex layer of biomolecules from body fluids that can be denatured and lead to an immune reaction. Non-fouling materials (or stealth materials) resist the adsorption of these proteins. New generations of non-fouling materials - active area of research Zwitterionic polymers Mixed charged polymers Polyoxazolines

40. Advanced biomaterials for tissue engineering 4. Biofunctionalized materials Is based on a “blank slate” from the non fouling materials/surfaces. Decorated with bioactive molecules, through covalent immobilization. These biofunctionalized materials interact with receptors on the cell surface and drive cellular behavior with biological specificity. Most common strategy - materials with ligands that engage specific integrin receptors. Thus, only cells that express the appropriate integrin are able to adhere to the material.

41. Tissue engineering constructs These biomaterials are fabricated into a tissue scaffold to support regeneration. Structures Porous scaffolds and hydrogels Meshes or microspheres Techniques Polymer phase separation Particle or foam templating Cryogelation, Electrospinning Rapid prototyping methods like 3D-printing.

42. Scaffold morphologies produced by thermally induced phase separation

43. Scaffold morphologies produced by thermally induced phase separation

44. Scaffold morphologies produced by thermally induced phase separation

45. Scaffold morphologies produced by Particulate leaching

46. Scaffold morphologies produced by Electrospinning

47. Scaffold morphologies produced by Rapid prototyping

48. Scaffolds Natural scaffolds Collagen, Fibrin, Starch, Matrigel ®,Decellularized matrix & Silk fibrion Synthetic materials Polyethylene glycol, Poly (lactic glycolic) acid and Polyurethane Clinically approved dermal substitutes such as Integra® and Matriderm ® have also been used for prevascularization purposes by seeding them with endothelial colony forming cells (ECFCs) in association with either human dermal fibroblasts (hDFs) or bone marrow-derived mesenchymal stem cells (BMSCs).

49. Growth factors VEGF-A - most important in angiogenesis. Acts mainly by binding to VEGF receptor 1 (VEGFR1) expressed in endothelial cells, hematopoietic stem cells and inflammatory cells, VEGF receptor 2,(VEGFR2) expressed mainly in endothelial cells. FGF-2 (aka basic fibroblast growth factor, bFGF) Mostly involved in angiogenesis Produced by a number of differentiated cells e.g. keratinocytes, mast cells, fibroblasts, endothelial cells and smooth muscle cells, as well as by adult mesenchymal stem cells derived from bone marrow, adipose, and dermal tissue.

50. Growth factors FGF-2 - Stimulating migration and proliferation of endothelial cells in vivo, Mitogenesis of smooth muscle cells and fibroblasts, which induces the development of large collateral vessels with adventitia. FGF-2 - In the prevascularization of scaffolds - Faster inosculation of the scaffold in vivo PDGF-B released by endothelial cells - Angiogenesis process mainly by attracting pericytes that will subsequently provide stability and structural support to the newly formed vessel.

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