1 3.051J/20.340J Lecture 23 Tissue Engineering II Case Examples 1. Diabetes Treatment by Cell Encapsulation Diabetes Mellitus (Type I or insulin-dependent): pancreatic disorder in which pancreas ceases to produce insulin • 100M cases worldwide • 15M cases in U.S. ⇒ $94B in treatment annually • Symptoms: tiredness, weight loss, extreme thirst Note: In Type II diabetes, insulin is manufactured but not used. Traditional Therapy: Daily insulin injections Problem: abnormal release pattern generates long-term complications • Blindness • Loss of circulation in limbs (frequently requires amputation) • Kidney failure What is Insulin? • hormone (MW ~6 kD) released in response to blood glucose levels • assists cells in glucose absorption3.051J/20.340J 2 Pancreas: an endocrine gland, incorporating: ¾ Pancreatic Acini: release digestive enzymes to the duodenum ¾ Islets of Langerhans (1-2% of pancreas vol.): release insulin to bloodstream Cell Encapsulation Therapy • 1st reported in mid-1970’s (W.L. Chick, Joslin Res. Lab., Boston) ⇒ glucose homeostasis achieved in rats • Tubular implants in pancreatectomized dogs, early 1990’s (R.P. Lanza) Pancreas Islet of Langerhans PVC-PAN membrane Islets ~5 µm acrylic housing3.051J/20.340J 3 - Implanted in peritoneal (abdominal) cavity - Membrane connected to vasculature by PTFE grafts - No exogenous insulin required for > 10 weeks - Little fibrosis up to 30 weeks (biocompatible acrylic) - Mechanisms of failure: a) membrane rupture (80-90% of devices by 5-7 mo.) b) thrombosis c) infection d) loss of islet function, islet necrosis • Human trials 1994 (Sharp & Lacy) - Implanted subcutaneously - PAN-PVC hollow fiber (1.5 cm length) - Human islets in alginate matrix - 90-95% islet viability after 2 weeks Limitation: Insulin quantity—several meters of fiber for therapeutic # of cells Possible solutions: a) “super” cell lines—high insulin output, low nutrient needs b) advances in angiogenesis References R.H. Li, “Materials for cell encapsulation”, Adv. Drug Delivery Rev. 33 (1998) 87-109. R.P. Lanza, R. Langer, W.L. Chick, Principles of Tissue Engineering, R.G. Landes Co.: Austin, TX, 19974 3.051J/20.340J 2. In Vitro Cartilage Regeneration Basics of Cartilage: • incorporates a single cell type (chondrocytes) • low vascularization (limits in vivo regeneration capability) • provides joint lubrication (knee) • basis of soft structural members (nose, ears) Conditions: • torn cartilage (athletic injury) (~0.5M/yr in U.S.) • rheumatoid arthritis (enzymes from phagocytes degrade cartilage) • birth defects/cosmetic (28,000/yr in U.S.) Joint fluid Cartilage menisci In vitro Chondrogenesis • Materials: nonwoven PGA fiber mesh or salt-leached; ~95% porous • Procedure: scaffold prewet with culture medium & seeded • PGA matrix replaced over time by cells, collagen & GAGs Salt-leached scaffold made by 3.082 students 10% polylactide5 3.051J/20.340J Limitations: a) Low solids content: poor mass transfer in culture b) Irregular tissue morphology: lack of flow field in vitro Revised Approach: Bioreactors (R. Langer, MIT) Spinner Flask medium magnetic stirbar tissue constructs Rotating Vessel fluid replacement tissue constructs (free-floating)6 3.051J/20.340J Cells GAG Collagen H2O Static 4 10 15 Rotating 7 30 19 88 Natural 4 38 42 75 Bioreactor-Grown Tissues: • tissue dimensions close to original scaffold • higher solids content than static • histology mimics natural cartilage ~ 30 µm capsule flat cells & collagen: gives stiffness uniform mix of cells, GAG, collagen Commercial Status: Genzyme (Cambridge, MA) FDA-approved for autologous knee cartilage repairs Remaining Challenges: a) scale-up of tissue dimensions b) improved mechanical properties3.051J/20.340J 7 2. In Vivo Nerve Regeneration Central Nervous System (CNS): Brain & Spinal cord Spinal cord: neurons coated by oligodendrocytes—secretions inhibit regeneration Peripheral Nervous System (PNS): nerve branches that process information from the environment; some regenerative capacity Nerve: several fasicles encased in epinurium fasicles: axon bundles surrounded by connective tissue dendritesaxon (up to 1 m long) Schwann cells: - generate myelin (insulator) - secrete neutrophic factors neuron synapse8 3.051J/20.340J Condition: Severed nerve • loss of support (neurotrophic factors) • scar ingrowth • displacement of resprouting axons Nerve Guidance Channels: use regenerative capacity of PNS History WWI - Rubber tubes used to guide axon growth - low biocompatibility 1960’s - Silastic trials (crosslinked silicone rubber) Today - Limited clinical use - Silicone & PGA devices - largest bridgeable gap ~ 1 cmdistal stump (to organ) proximal stump (to spine)9 3.051J/20.340J Case Study: Severed rat sciatic nerve in silicone guide Time elapsed Morphology hours influx of serum, fibrin, neurotrophic factors one week longitudinally oriented fibrin coalesces to bridge stumps cells invade: macrophages, fibroblasts, Schwann, endothelial axons sprout from proximal stump four weeks axon sprouts reach distal stump (~ 1cm) Regenerated Nerves • less axons & thinner sheath • slower signal conduction • lower amplitude signal10 3.051J/20.340J Channel Design Considerations Resorbable vs. Nonresorbable resorbable—larger inflammatory response nonresorbable—susceptible to compression injury Impermeable vs. Porous impermeable—poor nutrient, waste and O2 transport porous—interference by wound healing mechanisms, poor orientation semipermeable—MW cutoff 50-100 kD enables transport & sequesters GF Schwann cell-seeded gels - secrete neurite-promoting basal lamina, NGFs - can be genetically engineered to secrete neurotrophins - evidence of CNS regeneration support (optic nerve regen. demonstrated) Remaining Challenges: a) large-gap repair b) CNS regeneration (spinal cord work at MIT) Important Remaining Challenges in Tissue Engineering 1. angiogenesis/mass transport limitations 2. delivering appropriate signals to cells 3. providing appropriate mechanical stimulus for