Tissue Engineering

1. Tissue Engineering • Tissue Engineering is the in vitro development (growth) of tissues or organs to replace or support the function of defective or injured body parts. • Research is presently being conducted on several different types of tissues and organs, including: • Skin • Cartilage • Blood Vessels • Bone • Muscle • Nerves • Liver • Kidney • etc. etc. etc.

2. Tissue Organization • Before a tissue can be developed in vitro, first we must understand how tissues are organized. The basic tenant here is that: • “all tissues are comprised of • several levels of structural hierarchy” • These structural levels exist from the macroscopic level (centimeter range) all the way down the molecular level (nanometer range) • there can be as many as 7-10 distinct levels of structural organization in some tissues or organs

3. Organization of the Tendon

4. Organization of the Kidney

5. Cellular Communications • Soluble Signals: • small proteins (15-20 kDa) which are chemically stable with long half-lives (unless specifically degraded) • growth factors, steroids, hormones, cytokines, chemokines • bind to membrane receptors usually with high affinity (low binding constants: 10-100 pM) • can diffusion long distances

6. Cellular Communications • Cell-to-Cell Contact: • some membrane receptors are adhesive molecules • adherent junctions and desmosomes • other serve to create junctions between adjacent cells allowing for direct cytoplasmic communication • gap junctions • 1.5-2 nm diameter and only allow transport of small molecules ~1 kDa

7. Cellular Communications • Cell-ECM Interactions: • ECM is multifunctional and also provides a substrate that cells can communicate • since cells synthesize the ECM, they can modify the ECM to elicit specific cellular responses • several specialized receptors that allow for cell-ECM interactions • integrins, CD44, etc. • also a mechanism by with cells respond to external stimuli (“mechanical transducers”)

8. Tissue Engineering Scaffolds • Biomaterial Scaffolds Materials: • polymeric • chitosan, alginate, etc. • foams, hydrogels, fibres, thin films • natural • collagen, elastin, fibrin, etc. • hydrogels • ceramic • calcium phosphate based for bone tissue engineering • porous structures • permanent versus resorbable • degradation typically by hydrolysis • must match degradation rate with tissue growth • Chemical and Physical Modifications: • attachment of growth factors, binding sites for integrins, etc. • nanoscale physical features

9. Tissue Engineering Scaffolds

10. Culturing of Cells • Types of Cell Culture • monolayer (adherent cells) • suspension (non-adherent cells) • three-dimensional (scaffolds or templates)

11. Culturing of Cells • Sterilization Methods • ultra-violet light, 70% ethanol, steam autoclave, gamma irradiation, ethylene oxide gas • Growth Conditions • simulate physiological environment • pH 7.4, 37°C, 5% CO2, 95% relative humidity • culture (growth) media replenished periodically • Culture (Growth) Media • appropriate chemical environment • pH, osmolality, ionic strength, buffering agents • appropriate nutritional environment • nutrients, amino acids, vitamins, minerals, growth factors, etc.

12. Cell Sources • Since the ultimate goal of tissue engineering is to develop replacement tissue (or organs) for individuals, the use of autologous cells would avoid any potential immunological complications. • Various classifications of cells used in tissue engineering applications: • primary cells • differentiated cells harvested from the patient (tissue biopsy) • low cellular yield (can only harvest so much) • potential age-related problems • passaged cells • serial expansion of primary cells (can increase population by 100-1000X) • tendency to either lose potency or de-differentiate with too many passages • stem cells • undifferentiated cells • self-renewal capability (unlimited?) • can differentiate into functional cell types • very rare

13. Stem Cells • Stem cells naturally exist in some tissues (especially those that rapidly proliferate or remodel) and are present in the circulation. • There are two predominant lineages of stem cells: • mesenchymal • give rise to connective tissues (bone, cartilage, etc.) • although found in some tissues, typically isolated from bone marrow • hematopoietic • give rise to blood cells and lymphocytes • isolated from bone marrow, blood (umbilical cord) • Stem cells are rare; bone marrow typically has: • a single mesenchymal stem cell for every 1,000,000 myeloid cells • a single hematopoietic stem cell for every 100,000 myeloid cells

14. Stem Cells (Mesenchymal)

15. Stem Cells (Hematopoietic)

16. Colony-Forming Units (CFUs)

17. Bioreactors • a) Spinner Flask: • semi-controlled fluid shear • can produce turbulent eddies which could be detrimental • b) Rotating Wall • low shear stresses, high mass transfer rate • can balance forces to stimulate “zero gravity” • c) Hollow Fibre • used to enhance mass transfer during the culture of highly metabolic cells • d) Perfusion • media flows directly through construct • e) Controlled Mechanics • to apply physiological forces during culture

18. Bioreactors