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.

“all tissues are comprised of several levels of structural hierarchy” 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

Organization of the Tendon

Organization of the Kidney

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

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

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”)

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

Tissue Engineering Scaffolds

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

Culturing of Cells Sterilization Methods Growth Conditions 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.

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

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

Stem Cells (Mesenchymal)

Stem Cells (Hematopoietic)

Colony-Forming Units (CFUs)

Bioreactors a) Spinner Flask: b) Rotating Wall c) Hollow Fibre 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

Bioreactors