B. Conjunctiva synthesis in vivo (regeneration) 1. Structure and function of conjunctiva. 2. Clinical effects of conjunctival scarring. 3. Anatomically well-defined defect. 4. Synthesis of conjunctival stroma, followed by re-epithelialization. article handed out Inhibition of Conjunctival Scarring and Contraction by a Porous Collagen-Glycosaminoglycan Implant Hsu et al., in Investigative Ophthalmology and Visual Science 41:2402-2411 1. Structure and function of the conjunctiva The conjunctiva covers the exposed part of the sclera (opaque part of eye) and the inner surface of the eyelids. The epithelial tissue is stratified and contains goblet cells in the surface layers. Goblet cells synthesize and secrete mucus that contributes to the protective and lubricating layer on the exposed surface of the eye. Underneath the epithelial tissue is the conjunctival stroma, a loose vascular supportive tissue. 2. Clinical effects of conjunctival scarring ? Spontaneous healing of deep wounds in the conjunctiva occurs by contraction, scar formation and re-epithelialization. ? Conjunctival scarring is common endpoint for several opthalmic disorders, resulting from infection (trachoma), traumatic (chemical burns) and surgical (pterygium) causes. Anatomy of the conjunctiva Fornix Eyelid Cornea Sclera Epithelium Substantia Propria Conjunctival stroma 3. Anatomically well-defined defect Defect: Excise complete epithelia and stroma. Spontaneous healing: Full-thickness defect in conjunctiva heals by contraction and scar synthesis. Conjunctiva wound model Conjunctiva Tenon’s capsule Sclera Sclera Scaffold DRT Sutures 4. Synthesis of conjunctival stroma, followed by re- epithelialization Sequential synthesis (as for skin): Synthesize stroma using dermis regeneration template (DRT). Epithelial tissue spontaneously synthesized over new stroma. Effect of DRT on contraction kinetics of conjunctival defect. It is experimentally convenient to study contraction of the fornix, a tissue attached to the conjunctiva. ungrafted grafted with scaffold DRT 30 % Fornix Shor tening 15 0 15 300 Days Hsu et al., 2000 14 days epithelialization (red) of scar (blue) ungrafted Image removed due to copyright considerations. See Figure 6 in [Hsu 2000] Image removed due to copyright consi erations. See Figure 6 in [Hsu 2000] epithelialization (red) of incipient new dermis (blue) grafted with DRT Test of synthesis of conjunctival stroma (use microscope polarizing stage to study orientation of collagen fibers) Images removed due to copyright considerations. See [Hsu 2000] I r d due t c right c iderations. See [Hsu 2000] synthesized conjunctival stroma conjunctival scar normal conjunctiva C. Nerve synthesis in vivo (regeneration) (Ch. 6) 1. Structure of peripheral nerve. 2. Experimental parameters for study of induced regeneration. 3. Synthesis of myelinated axons and BM (nerve fibers) 4. Evidence (?) of synthesis of an endoneurium. 5. Synthesis of a nerve trunk (including summary of kinetics of synthesis). 6. Comparative regenerative activity of various reactants. Nervous system = central nervous system (CNS) + peripheral nervous system (PNS) Image removed due to copyright considerations. Image removed due to copyright consi erati ns. Nervous System Chamberlain et al., 1998 CNS PNS Image removed due to copyright considerations. Image removed due to copyright consi erations. 1. Structure of a peripheral nerve. I Nerve fibers comprise axons wrapped in a myelin sheath, itself surrounded by BM (diam. 10-30 μm in rat sciatic nerve). Axons are extensions (long processes) of neurons located in spinal cord. They comprise endoplasmic reticulum and microtubules. 1. Structure of a peripheral nerve. II Myelinated axons (diam. 1-15 μm) are wrapped in a myelin sheath; nonmyelinated axons also exist. They are the elementary units for conduction of electric signals in the body. Myelin formed by wrapping a Schwann cell membrane many times around axon perimeter. No ECM inside nerve fibers. Myelin sheath is a wrapping of Schwann cell membranes around certain axons. 1. Structure of a peripheral nerve. III Nonmyelinated axons (diam. <1 μm) function in small pain nerves. Although surrounded by Schwann cells, they lack myelin sheath; Schwann cells are around them but have retained their cytoplasm. Basement membrane (tubular) encases the myelin sheath. Structure similar to that of skin BM. 1. Structure of a peripheral nerve. IV Nerve fibers are embedded in endoneurium: a delicate packing of loose vascular supporting tissue that is rich in collagen fibers. Definitely ECM! Many nerve fibers with their associated endoneurium are packed in a collagenous layer, the perineurium. This forms a fascicle. Multifascicular nerves encased in a collagenous layer, the epineurium. Cylindrical symmetry of peripheral nerve structure Summary of nerve trunk structure proceeding radially from the center out: [axon ? myelin sheath ? BM] ? endoneurium ? perineurium ? epineurium. [ …. ] = “nerve fiber” Rat sciatic nerve (nerve trunk). One fascicle. Several thousand axons. Image removed due to copyright considerations. See Figure 10.7 (upper left) in Yannas, I. V. Tissue and Organ Regeneration in Adults. New York: Springer-Verlag, 2001. Image removed due to copyright consi erati ns. See Figure 10.7 (upper left) in Yannas, I. V. Tissue and Organ Regeneration i Adults. New York: Springer-Verl g, 2001. (idealized) nerve trunk nerve fiber Rat Sciatic Nerve Cross Section Individual Axon Longitudinal view of nerve fiber Image removed due to copyright considerations. Image removed due to copyright consi erati ns. Chamberlain, 1998 2. Experimental parameters for study of regeneration A. Anatomically well-defined defect – Designate experimental volume – Delete nonregenerative tissue(s) – Anatomical bounds – Containment of exudate B. Timescale of observations – Short-term (<20 wk) and long-term (>20 wk) assays The tissue triad in skin and nerves Replace w/ Artist redraws Regenerative similarity of tissues in skin and nerves Skin The epidermis is a regenerative tissue. After excision, it regenerates spontaneously. Reversible injury. No contraction. The dermis is a nonregenerative tissue in the adult. After excision, it does not regenerate spontaneously. Irreversible injury. Contraction occurs with scar formation. Skin spontaneous healing of full-thickness skin excision by contraction and scar formation The injured myelin sheath regenerates spontaneously Myelin sheath Axoplasm Injured axoplasm and myelin. BM and endoneurium intact. Regenerated myelin Neuroma formation. The endoneurium does not regenerate. Transected nerve. Both myelin and endoneurium are severely injured. Neuroma forms at each stump by contraction and scar formation. Intact nerve fiber Image removed due to copyright considerations. See Figure 2.5 in [Yannas]. Image removed due to copyright consi erati ns. See Figure 2.5 in [Yannas]. healing Spontaneously healed nerve fiber 2. Experimental parameters (cont.) C. Assays of configuration – Correction for experimental gap length. – Correction for animal species. – Critical axon elongation, L c . Relation to defect closure rule. – Shift length, ?L. Characterization of devices. – Long-term: fidelity of regeneration. C. Assays of configuration (cont.) Use corrected values of frequency of reinnervation (%N) across tubulated gaps. This correction allows comparison of %N data from studies with different gap lengths and different species. Critical axon elongation, L c , the gap length above which %N drops below 50% (or the gap length where the odds of rein- nervation are even). Data from several investigators have shown that L c = 9.7 ± 1.8 mm for the rat sciatic nerve and 5.4 ± 1.0 mm for the mouse sciatic nerve. L c = 9.7 ± 1.8 mm for the rat sciatic nerve and 5.4 ± 1.0 mm for the mouse sciatic nerve Characteristic curve defines critical axon elongation, L c , at %N = 50% Image removed due to copyright considerations. See Figure 6.1 in [Yannas]. Image removed due to copyright consi erations. See Figure 6.1 in [Yannas]. Data from rat and mouse superpose when plotted vs. reduced length, L/L c Use single data point to determine L c for unknown device Image removed due to copyright considerations. See Figure A.1 in [Yannas]. Image removed due to copyright consi erati ns. See Figure A.1 in [Yannas]. Relation between L c , ?L and C, S, R terms in defect closure rule Image removed due to copyright considerations. See Table 6.2 in [Yannas]. Image removed due to copyright consi erati ns. e 6.2 in [Yannas]. 2A. Synthesis of myelinated axons [NB: Neuron in culture provides spontaneous outgrowth of axons that serve as “substrate” for synthesis of myelin and BM. Schwann cells also obtained in culture from a neuron.] A myelin sheath around axons has been synthesized in vitro in the presence of Schwann cells, with or without presence of an ECM component. 2B. Synthesis of nerve BM A BM has been synthesized in vitro in presence of neurons and Schwann cells. However, neurons were not required to be present when fibroblasts were cultured with Schwann cells. Even fibroblasts not required when laminin added to neuron-free Schwann cell culture. 3. Evidence (?) for synthesis of an endoneurium Structure. Endoneurial microenvironment surrounding each nerve fiber comprises blood vessels coursing through space filled with fluid and thin collagen fibers (51-56 nm diam.). Fluid outside blood vessels is maintained under small, positive hydrostatic pressure. Endoneurial blood vessels comprise cells that are bound by tight junctions and constitute a permeability barrier. Function. Endoneurial environment protects nerve fibers from changes in ionic strength and from pathogens in blood vessels that might modify conductivity (“blood-nerve barrier”). Endoneurium Image removed due to copyright considerations. See Figure 6.2 (top) in [Yannas]. Image removed due to copyright consi erations. See Figure 6.2 (top) i [Yannas]. Evidence (?) for synthesis of endoneurium (cont.) In vitro. No evidence for synthesis of endoneurial stroma. In vivo. Nerve trunks have been synthesized but endoneurial structures have rarely been studied. Is endoneurium present? Using silicone tube to bridge 10 mm gap between stumps did not yield a functional endoneurium. Occasionally, collagen fibers, 25-35 nm, or even 40 nm, reported outside BM, were smaller than endoneurial collagen fibers. 5. Synthesis of a nerve trunk (including kinetics) Structure. A nerve trunk comprises one or more fascicles. Each fascicle comprises several thousand nerve fibers. If monofascicular, it is covered by perineurium; if multifascicular, it is covered by epineurium. A fascicle comprises the perineurium with its bundle of thousands of nerve fibers. Some nerves comprise many fascicles, each with its own perineurial sheath; these fascicles are wrapped in a collagenous tissue, the epineurium. Function. Conducts strong nerve signals (about 10 mV) at 70 m/s. Tubulation model. Gap length variable. Image removed due to copyright considerations. See Figure 6.2 (top) in [Yan Image removed due to copyright consi erations. Tubulation model. Gap length variable. Image removed due to copyright considerations. Diagram of implant configuration. A look inside the gap sequence: Schwann cells + Fibroblasts > Nonmyel. Axons > Blood vessels > Myel. axons Image removed due to copyright considerations. See Figure 10.6 in [Yannas]. Image removed due to copyright consi erations. See Figure 10.6 i [Yannas]. axon elongation→ proximal stump distal stump Columns of Schwann cells form even in absence of axons Image removed due to copyright considerations. See Figure 10.8 in [Yannas]. Image removed due to copyright consi erations. See Figure 10.8 i [Yannas]. Contractile cell zone surrounds regenerating nerve contractile cells original stump surface regenerated nerve Image removed due to copyright considerations. Image removed due to copyright consi erations. Spilker and Seog, 2000 Cell capsule around regenerated nerves 4-mm gap Normal rat sciatic nerve Image removed due to copyright considerations. See Figure 10.7 in [Yannas]. Image removed due to copyright consi erations. See Figure 10.7 i [Yannas]. 8-mm gap Regenerated across 0-mm gap 8 weeks Image removed due to copyright considerations. See Figure 6.4 in [Yannas]. Image removed due to copyright consi erations. See Figure 6.4 in [Yannas].Regenerated nerves typically comprise several minifascicles 39 weeks article by Chamberlain et al. handed out Chamberlain, L.J. et al. “Collagen-GAG Substrates Enhances the Quality of Nerve Regeneration through Collagen Tubes up to Level of Autograft.” Experimental Neurology 154: 315-329 (1998) ain, L.J. et al. “Coll ubstrates Enhances the Quality of Nerve neration thro h Coll s u to Level of Autograft.” Experimental Neurol y 154: 315-329 (1998) KINETICS OF NERVE SYNTHESIS 30 weeks Image removed due to copyright considerations. See Figure 6.5 in [Yannas]. Image removed due to copyright consi erations. See Figure 6.5 in [Yannas]. 60 weeks Normal Y-axis: Amplitude (strength) of transmitted electric signal X-axis: Time following stimulation (at 0 ms) Electrophysiological behavior of normal (light line) and regenerated nerve (dark line) Regenerated nerve is weaker (lower peak amplitude) and slower (delayed peaking) Image removed due to copyright considerations. See Figure 6.6 in [Yannas]. Image removed due to copyright consi erati ns. See Figure 6.6 in [Yannas]. 6. Comparative Rregenerative activity of various devices (Table 6.1, pp. 147-8) What does each of these device features contribute to the quality of regeneration? Compare values of L c and ?L. ? Tubulation ? Tube wall composition ? Tube wall permeability ? Fillings: Schwann cells, solutions of proteins, gels based on ECM components, insoluble substrates Device components PNS regeneration. Length shift, ?L, measures regenerative advantage of device relative to silicone tube standard. e.g., ?L>0 is better than standard. Tubulation Tube wall Image removed due to copyright considerations. See Table 6.1 in [Yannas]. Image removed due to copyright consi erations. e 6.1 in [Yannas]. Tube wall permeability Filling: Schwann cells Filling: protein solutions Filling: ECM-based gels Filling: insoluble substrates PNS regeneration (cont.). Length shift, ?L, measures regenerative advantage of device relative to silicone tube standard. e.g., ?L>0 is better than standard. Filling: insoluble substrates Image removed due to copyright considerations. See Table 6.1 in [Yannas]. Image removed due to copyright consi erations. e 6.1 in [Yannas]. Tubulation. Tube wall composition and permeability ? Bridging the two stumps with a tube, almost any kind of tube, greatly improves quality of regeneration. ? Tube wall composition is critically important. Silicone tubes are greatly inferior to collagen tubes. ? Increase of cell (but not protein) permeability improved quality. Silicone tube Partly regenerated rat sciatic nerve. Tubulated in silicone tube. cross-section shows thick sheath of contractile cells Image removed due to copyright considerations. See Figure 4.5 in [Yannas]. Image removed due to copyright consi erations. See Figure 4.5 in [Yannas]. Silicone tube near original proximal stump Contractile cells (brown) ensheathe regenerating stump of transected rat sciatic nerve Image removed due to copyright considerations. See Figure 4.6 in [Yannas]. Image removed due to copyright consi erati ns. See Figure 4.6 in [Yannas]. near original distal stump Tube fillings ? Schwann cells, growth factors (aFGF and bFGF) and several insoluble substrates increased quality of regeneration, sometimes greatly. ? NGF had no effect. ? Gels based on ECM components (collagen, fibronectin, laminin) had no effective or impeded regeneration. Regeneration across a 10 mm gap bridged by a silicone tube filled unfilled Image removed due to copyright considerations. See Fig. 10 in Yannas, I. V. "Biologically Active Analogues of the Extracellular Matrix: Artificial Skin and Nerves." Angew. Chem. Int. Ed. Engl. 29 (1990) 20-35. Effect of degradation rate of tube filling based on a porous ECM analog (NRT) ? Undegraded ECM analog physically impeded axon elongation. ? Optimal quality of regeneration obtained with ECM anlog that degraded at an intermediate rate. Effect of pore diameter and degradation rate on inverse conduction velocity (latency) Image removed due to copyright considerations. See Figure 10.9 in [Yannas]. Image removed due to copyright consi erations. See Figure 10.9 i [Yannas]. 1. pore structure (ligand density) 2. macromolecular structure (ligand duration) Structural features of ECM analogs used as tube fillings in nerve regeneration 3. chemical composition (ligand identity) 4. orientation of pore channel axes Image removed due to copyright considerations. See Figure 6.2 (top) in [Yan Image removed due to copyright consi erations. Image removed due to copyright considerations. See Figure 6.2 (top) in [Yan Image removed due to copyright consi erations. Long-term electro- physiological properties of various regenerated nerves Image removed due to copyright considerations. See Tables 6.1 and 6.3 in [Yannas]. Image removed due to copyright consi erations. es 6.1 and 6.3 in [Yannas]. Summary of results ? Tube presence was essential ? Tube wall composition: collagen > degradable synthetic polymer > silicone. ? Tube wall permeability: cell-permeable > protein permeable > impermeable. ? Tube fillings: --- suspensions of Schwann cells --- solution of either aFGF or bFGF (not NGF!) --- crosslinked ECM networks > ECM gels --- thin polymeric filaments oriented along tube axis --- highly porous, insoluble ECM analogs with appropriately small pore diameter, axial orientation of pore channel axes and critically adjusted degradation rate.