Philip J. Johnson

Controlled Release of Neurotrophin-3 and Platelet-Derived Growth Factor from Fibrin Scaffolds Containing Neural Progenitor Cells Enhances Survival and Differentiation into Neurons in a Subacute Model of SCI:

A consistent problem with stem/neural progenitor cell transplantation following spinal cord injury (SCI) is poor cell survival and uncontrolled differentiation following transplantation. The current study evaluated the feasibility of enhancing embryonic stem cell-derived neural progenitor cell (ESNPC) viability and directing their differentiation into neurons and oligodendrocytes by embedding the ESNPCs in fibrin scaffolds containing growth factors (GF) and a heparin-binding delivery system (HBDS) in a subacute rat model of SCI. Mouse ESNPCs were generated from mouse embryonic stem cells (ESCs) using a 4–/4+ retinoic acid (RA) induction protocol. The ESNPCs were then transplanted as embryoid bodies (EBs, 70% neural progenitor cells) into the subacute model of SCI. ESNPCs (10 EBs per animal) were implanted directly into the SCI lesion, encapsulated in fibrin scaffolds, encapsulated in fibrin scaffolds containing the HBDS, neurotrophin-3 (NT-3), and platelet-derived growth factor (PDGF), or encapsulated in fibrin scaffolds with NT-3 and PDGF with no HBDS. We report here that the combination of the NT-3, PDGF, and fibrin scaffold (with or without HBDS) enhanced the total number of ESNPCs present in the spinal cord lesion 2 weeks after injury. In addition, the inclusion of the HBDS with growth factor resulted in an increase in the number of ESNPC-derived NeuN-positive neurons. These results demonstrate the ability of fibrin scaffolds and the controlled release of growth factors to enhance the survival and differentiation of neural progenitor cells following transplantation into a SCI model.

Fibrin-based tissue engineering scaffolds enhance neural fiber sprouting and delay the accumulation of reactive astrocytes at the lesion in a subacute model of spinal cord injury.

The purpose of this study was to evaluate the effects of fibrin scaffolds on subacute rat spinal cord injury (SCI). Long-Evans rats were anesthetized and underwent a dorsal hemisection injury; two weeks later, the injury site was re-exposed, scar tissue was removed, and a fibrin scaffold was implanted into the wound site. An effective method for fibrin scaffold implantation following subacute SCI was investigated based on the presence of fibrin within the lesion site and morphological analysis 1 week after implantation. Prepolymerized fibrin scaffolds were found to be present within the lesion site 1 week after treatment and were used for the remainder of the study. Fibrin scaffolds were then implanted for 2 and 4 weeks, after which spinal cords were harvested and evaluated using markers for neurons, astrocytes, and chondroitin sulfate proteoglycans. Compared with untreated control, the fibrin-treated group had significantly higher levels of neural fiber staining in the lesion site at 2 and 4 weeks after treatment, and the accumulation of glial fibrillary acidic protein (GFAP) positive reactive astrocytes surrounding the lesion was delayed. These results show that fibrin is conducive to regeneration and cellular migration and illustrate the advantage of using fibrin as a scaffold for drug delivery and cell-based therapies for SCI.

“Controlled release of neurotrophin-3 from fibrin-based tissue engineering scaffolds enhances neural fiber sprouting following subacute spinal cord injury”

This study investigated whether delayed treatment of spinal cord injury with controlled release of neurotrophin‐3 (NT‐3) from fibrin scaffolds can stimulate enhanced neural fiber sprouting. Long Evans rats received a T9 dorsal hemisection spinal cord injury. Two weeks later, the injury site was re‐exposed, and either a fibrin scaffold alone, a fibrin scaffold containing a heparin‐based delivery system with different concentrations of NT‐3 (500 and 1,000 ng/mL), or a fibrin scaffold containing 1,000 ng/mL of NT‐3 (no delivery system) was implanted into the injury site. The injured spinal cords were evaluated for morphological differences using markers for neurons, astrocytes, and chondroitin sulfate proteoglycans 2 weeks after treatment. The addition of 500 ng/mL of NT‐3 with the delivery system resulted in an increase in neural fiber density compared to fibrin alone. These results demonstrate that the controlled release of NT‐3 from fibrin scaffolds can enhance neural fiber sprouting even when treatment is delayed 2 weeks following injury. Biotechnol. Bioeng. 2009; 104: 1207–1214.

Acellular nerve allografts in peripheral nerve regeneration: A comparative study

Introduction: Processed nerve allografts offer a promising alternative to nerve autografts in the surgical management of peripheral nerve injuries where short deficits exist. Methods: Three established models of acellular nerve allograft (cold‐preserved, detergent‐processed, and AxoGen‐processed nerve allografts) were compared with nerve isografts and silicone nerve guidance conduits in a 14‐mm rat sciatic nerve defect. Results: All acellular nerve grafts were superior to silicone nerve conduits in support of nerve regeneration. Detergent‐processed allografts were similar to isografts at 6 weeks postoperatively, whereas AxoGen‐processed and cold‐preserved allografts supported significantly fewer regenerating nerve fibers. Measurement of muscle force confirmed that detergent‐processed allografts promoted isograft‐equivalent levels of motor recovery 16 weeks postoperatively. All acellular allografts promoted greater amounts of motor recovery compared with silicone conduits. Conclusion: These findings provide evidence that differential processing for removal of cellular constituents in preparing acellular nerve allografts affects recovery in vivo. Muscle Nerve, 2011

Limited regeneration in long acellular nerve allografts is associated with increased Schwann cell senescence

Repair of large nerve defects with acellular nerve allografts (ANAs) is an appealing alternative to autografting and allotransplantation. ANAs have been shown to be similar to autografts in supporting axonal regeneration across short gaps, but fail in larger defects due to a poorly-understood mechanism. ANAs depend on proliferating Schwann cells (SCs) from host tissue to support axonal regeneration. Populating longer ANAs places a greater proliferative demand on host SCs that may stress host SCs, resulting in senescence. In this study, we investigated axonal regeneration across increasing isograft and ANA lengths. We also evaluated the presence of senescent SCs within both graft types. A sciatic nerve graft model in rats was used to evaluate regeneration across increasing isograft (~autograft) and ANA lengths (20, 40, and 60 mm). Axonal regeneration and functional recovery decreased with increased graft length and the performance of the isograft was superior to ANAs at all lengths. Transgenic Thy1-GFP rats and qRT-PCR demonstrated that failure of the regenerating axonal front in ANAs was associated with increased levels of senescence related markers in the graft (senescence associated β-galactosidase, p16(INK4A), and IL6). Lastly, electron microscopy (EM) was used to qualitatively assess senescence-associated changes in chromatin of SCs in each graft type. EM demonstrated an increase in the presence of SCs with abnormal chromatin in isografts and ANAs of increasing graft length. These results are the first to suggest that SC senescence plays a role in limited axonal regeneration across nerve grafts of increasing gap lengths.

Nerve endoneurial microstructure facilitates uniform distribution of regenerative fibers: a post hoc comparison of midgraft nerve fiber densities.

Despite their inferiority to nerve autograft, clinical alternatives are commonly used for reconstruction of peripheral nerve injuries because of their convenient off-the-shelf availability. Previously, our group compared isografts with NeuraGen(®) (Integra, Plainsboro, NJ) nerve guides, which are a commercially available type I collagen conduit and processed rat allografts comparable to Avance(®) (AxoGen, Alachua, FL) human decellularized allograft product. From this study, qualitative observations were made of distinct differences in the pattern of regenerating fibers within conduits, acellular allografts, and isografts. In the current post hoc analysis, these observations were quantified. Using nerve density, we statistically compared the differential pattern of regenerating axon fibers within grafts and conduit. The conduits exhibited a consistent decrease in midgraft density when compared with the isograft and acellularized allografts at two gap lengths (14 mm and 28 mm) and time points (12 and 22 weeks). The decrease in density was accompanied by clustered distribution of nerve fibers in conduits, which contrasted the evenly distributed regeneration seen in processed allografts and isografts. We hypothesize that the lack of endoneurial microstructure of conduits results in the clustering regenerating fibers, and that the presence of microstructure in the acellularized allograft and isografts facilitates even distribution of regenerating fibers.

Peripheral nerve injury after local anesthetic injection.

BACKGROUND:A well-known complication of peripheral nerve block is peripheral nerve injury, whether from the needle or toxicity of the medication used. In this study, we sought to determine the extent of damage that results from intrafascicular injection of various commonly used local anesthetics (LAs). METHODS:Sixteen Lewis rats received an intrafascicular injection of saline (control) or 1 of 3 LAs (bupivacaine, lidocaine, or ropivacaine) into the sciatic nerve (n = 4). At a 2-week end point, the sciatic nerves were harvested for histomorphometric and electron microscopic analysis. RESULTS:Animals that received intrafascicular LA injections showed increased severity of injury as compared with control. In particular, there was a significant loss of large-diameter fibers as indicated by decreased counts (P < 0.01 for all LAs) and area (P < 0.01 for all LAs) of remaining fibers in severely injured versus noninjured areas of the nerve. There was a layering of severity of injury with most severely injured areas closest to and noninjured areas furthest from the injection site. Bupivacaine caused more damage to large fibers than the other 2 LAs. In all groups, fascicular transection injury from the needle was observed. Electron microscopy confirmed nerve injury. CONCLUSIONS:Frequently used LAs at traditional concentrations are toxic to and can injure the peripheral nerve. Any combination of motor and/or sensory sequelae may result due to the varying fascicular topography of a nerve.

Reverse End-to-Side Nerve Transfer: From Animal Model to Clinical Use

PURPOSE Functional recovery after peripheral nerve injury is predominantly influenced by time to reinnervation and number of regenerated motor axons. For nerve injuries in which incomplete regeneration is anticipated, a reverse end-to-side (RETS) nerve transfer might be useful to augment the regenerating nerve with additional axons and to more quickly reinnervate target muscle. This study evaluates the ability of peripheral nerve axons to regenerate across an RETS nerve transfer. We present a case report demonstrating its potential clinical applicability. METHODS Thirty-six Lewis rats were randomized into 3 groups. In group 1 (negative control), the tibial nerve was transected and prevented from regenerating. In group 2 (positive control), the tibial and peroneal nerves were transected, and an end-to-end (ETE) nerve transfer was performed. In group 3 (experimental model), the tibial nerve and peroneal nerves were transected, and an RETS nerve transfer was performed between the proximal end of the peroneal nerve and the side of the denervated distal tibial stump. Nerve histomorphometry and perfused muscle mass were evaluated. Six Thy1-GFP transgenic Sprague Dawley rats, expressing green fluorescent protein in their neural tissues, also had the RETS procedure for evaluation with confocal microscopy. RESULTS Nerve histomorphometry showed little to no regeneration in chronic denervation animals but statistically similar regeneration in ETE and RETS animals at 5 and 10 weeks. Muscle mass preservation was similar between ETE and RETS groups by 10 weeks and significantly better than negative controls at both time points. Nerve regeneration was robust across the RETS coaptation of Thy1-GFP rats by 5 weeks. CONCLUSIONS Axonal regeneration occurs across an RETS coaptation. An RETS nerve transfer might augment motor recovery when less-than-optimal recovery is otherwise anticipated. TYPE OF STUDY/LEVEL OF EVIDENCE Therapeutic I.

NERVE ALLOGRAFTS SUPPLEMENTED WITH SCHWANN CELLS OVEREXPRESSING GLIAL-CELL-LINE-DERIVED NEUROTROPHIC FACTOR

We sought to determine whether supplementation of acellular nerve allografts (ANAs) with Schwann cells overexpressing GDNF (G‐SCs) would enhance functional recovery after peripheral nerve injury.

A transgenic rat expressing green fluorescent protein (GFP) in peripheral nerves provides a new hindlimb model for the study of nerve injury and regeneration

BACKGROUND In order to evaluate nerve regeneration in clinically relevant hindlimb surgical paradigms not feasible in fluorescent mice models, we developed a rat that expresses green fluorescent protein (GFP) in neural tissue. METHODS Transgenic Sprague-Dawley rat lines were created using pronuclear injection of a transgene expressing GFP under the control of the thy1 gene. Nerves were imaged under fluorescence microscopy and muscles were imaged with confocal microscopy to determine GFP expression following sciatic nerve crush, transection and direct suturing, and transection followed by repair with a nerve isograft from nonexpressing littermates. RESULTS In each surgical paradigm, fluorescence microscopy demonstrated the loss and reappearance of fluorescence with regeneration of axons following injury. Nerve regeneration was confirmed with imaging of Wallerian degeneration followed by reinnervation of extensor digitorum longus (EDL) muscle motor endplates using confocal microscopy. CONCLUSION The generation of a novel transgenic rat model expressing GFP in neural tissue allows in vivo imaging of nerve regeneration and visualization of motor endplate reinnervation. This rat provides a new model for studying peripheral nerve injury and regeneration over surgically relevant distances.