James B. Graham

Chondroitinase treatment increases the effective length of acellular nerve grafts

Acellular nerve allografts have been explored as an alternative to nerve autografting. It has long been recognized that there is a distinct limit to the effective length of conventional acellular nerve grafts, which must be overcome for many grafting applications. In rodent models nerve regeneration fails in acellular nerve grafts greater than 2 cm in length. In previous studies we found that nerve regeneration is markedly enhanced with acellular nerve grafts in which growth-inhibiting chondroitin sulfate proteoglycan was degraded by pretreatment with chondroitinase ABC (ChABC). Here, we tested if nerve regeneration can be achieved through 4-cm acellular nerve grafts pretreated with ChABC. Adult rats received bilateral sciatic nerve segmental resection and repair with a 4 cm, thermally acellularized, nerve graft treated with ChABC (ChABC graft) or vehicle-treated acellularized graft (Control graft). Nerve regeneration was examined 12 weeks after implantation. Our findings confirm that functional axonal regeneration fails in conventional long acellular grafts. In this condition we found very few axons in the distal host nerve, and there were marginal signs of sciatic nerve reinnervation in few (2/9) rats. This was accompanied by extensive structural disintegration of the distal graft and abundant retrograde axonal regeneration in the proximal nerve. In contrast, most (8/9) animals receiving nerve repair with ChABC grafts showed sciatic nerve reinnervation by direct nerve pinch testing. Histological examination revealed much better structural preservation and axonal growth throughout the ChABC grafts. Numerous axons were found in all but one (8/9) of the host distal nerves and many of these regenerated axons were myelinated. In addition, the amount of aberrant retrograde axonal growth (originating near the proximal suture line) was markedly reduced by repair with ChABC grafts. Based on these results we conclude that ChABC treatment substantially increases the effective length of acellular nerve grafts.

Metalloproteinase-Dependent Predegeneration In Vitro Enhances Axonal Regeneration within Acellular Peripheral Nerve Grafts

Injury to peripheral nerve initiates a degenerative process that converts the denervated nerve from a suppressive environment to one that promotes axonal regeneration. We investigated the role of matrix metalloproteinases (MMPs) in this degenerative process and whether effective predegenerated nerve grafts could be producedin vitro. Rat peripheral nerve explants were cultured for 1–7 d in various media, and their neurite-promoting activity was assessed by cryoculture assay, in which neurons are grown directly on nerve sections. The neurite-promoting activity of cultured nerves increased rapidly and, compared with uncultured nerve, a maximum increase of 72% resulted by 2 d of culture in the presence of serum. Remarkably, the neurite-promoting activity of short-term cultured nerves was also significantly better than nerves degeneratedin vivo. We examined whether in vitrodegeneration is MMP dependent and found that the MMP inhibitorN-[(2R)-2(hydroxamidocarbonylmethyl)-4-methylpantanoyl]-l-tryptophan methylamide primarily blocked the degenerative increase in neurite-promoting activity. In the absence of hematogenic macrophages, MMP-9 was trivial, whereas elevated MMP-2 expression and activation paralleled the increase in neurite-promoting activity. MMP-2 immunoreactivity localized to Schwann cells and the endoneurium and colocalized with gelatinolytic activity as demonstrated by in situ zymography. Finally,in vitro predegenerated nerves were tested as acellular grafts and, compared with normal acellular nerve grafts, axonal ingressin vivo was approximately doubled. We conclude that Schwann cell expression of MMP-2 plays a principal role in the degenerative process that enhances the regeneration-promoting properties of denervated nerve. Combined with their low immunogenicity, acellular nerve grafts activated by in vitropredegeneration may be a significant advancement for clinical nerve allografting.

Chondroitinase Applied to Peripheral Nerve Repair Averts Retrograde Axonal Regeneration

Antegrade, target-directed axonal regeneration is the explicit goal of nerve repair. However, aberrant and dysfunctional regrowth is commonly observed as well. At the site of surgical nerve coaptation, axonal sprouts encounter fibrotic connective tissue rich in growth-inhibiting chondroitin sulfate proteoglycan that may contribute to misdirection of axonal regrowth. In the present study, we tested the hypothesis that degradation of chondroitin sulfate proteoglycan by application of chondroitinase at the site of nerve repair can decrease aberrant axonal growth. Adult rats received bilateral sciatic nerve transection and end-to-end repair. One nerve was injected with chondroitinase ABC and the contralateral nerve treated with vehicle alone. After 28 weeks, retrograde axonal regeneration was assessed proximal to the repair by scoring neurofilament-immunopositive axons within the nerve (intrafascicular) and outside the nerve proper (extrafascicular). Intrafascicular retrograde axonal growth was equivalent in both control and chondroitinase treatment conditions. In contrast, chondroitinase treatment caused a pronounced (93%) reduction in extrafascicular retrograde axonal growth. The decrease in axon egress from the nerve was coincident with an increase in antegrade regeneration and improved recovery of motor function. Based on these findings, we conclude that chondroitinase applied at the site of nerve transection repair averts dysfunctional extrafascicular retrograde axonal growth.

Nerve grafts with various sensory and motor fiber compositions are equally effective for the repair of a mixed nerve defect

Autologous, cellular nerve grafts are commonly used to bridge nerve gaps in the clinical setting. Sensory nerves are most often selected for autografting because of their relative ease of procurement and low donor site morbidity. A series of recent reports conclude that sensory isografts are inferior to motor and mixed nerve isografts for the repair of a mixed nerve defect in rat. The aim of the present study was to determine if the disparity reported with cellular graft subtypes exists for detergent decellularized, chondroitinase ABC processed nerve grafts. We hypothesized that processing removes or neutralizes the inferior properties attributed to sensory nerve grafts. Saphenous (cutaneous branch), femoral quadriceps (muscle branch) and tibial (mixed trunk) nerve grafts 5 mm in length were used in tensionless reconstruction of syngenic rat tibial nerves. Nerve regeneration through the grafts and into the recipient distal nerve was evaluated 21 days after grafting by two methods, toluidine blue staining of semi-thin sections (myelinated axons) and neurofilament-immunolabeling (total axons). Contrary to previous reports using this grafting scheme, we found no significant difference in the myelinated axon counts for the three cellular graft subtypes. Moreover, total axon counts indicated cellular saphenous nerve grafts were more effective than the quadriceps and tibial nerve grafts. A similar though less pronounced trend was found for the decellularized processed grafts. These findings indicate that nerve graft composition (sensory and motor) has no substantial impact on the short-term outcome of nerve regeneration in a mixed nerve repair model.

Chondroitinase C Selectively Degrades Chondroitin Sulfate Glycosaminoglycans that Inhibit Axonal Growth within the Endoneurium of Peripheral Nerve

The success of peripheral nerve regeneration is highly dependent on the regrowth of axons within the endoneurial basal lamina tubes that promote target-oriented pathfinding and appropriate reinnervation. Restoration of nerve continuity at this structural level after nerve transection injury by direct repair and nerve grafting remains a major surgical challenge. Recently, biological approaches that alter the balance of growth inhibitors and promoters in nerve have shown promise to improve appropriate axonal regeneration and recovery of peripheral nerve function. Chondroitin sulfate proteoglycans (CSPGs) are known inhibitors of axonal growth. This growth inhibition is mainly associated with a CSPGs glycosaminoglycan chains. Enzymatic degradation of these chains with chondroitinase eliminates this inhibitory activity and, when applied in vivo, can improve the outcome of nerve repair. To date, these encouraging findings were obtained with chondroitinase ABC (a pan-specific chondroitinase). The aim of this study was to examine the distribution of CSPG subtypes in rodent, rabbit, and human peripheral nerve and to test more selective biological enzymatic approaches to improve appropriate axonal growth within the endoneurium and minimize aberrant growth. Here we provide evidence that the endoneurium, but not the surrounding epineurium, is rich in CSPGs that have glycosaminoglycan chains readily degraded by chondroitinase C. Biochemical studies indicate that chondroitinase C has degradation specificity for 6-sulfated glycosaminoglycans found in peripheral nerve. We found that chondroitinase C degrades and inactivates inhibitory CSPGs within the endoneurium but not so much in the surrounding nerve compartments. Cryoculture bioassays (neurons grown on tissue sections) show that chondroitinase C selectively and significantly enhanced neuritic growth associated with the endoneurial basal laminae without changing growth-inhibiting properties of the surrounding epineurium. Interestingly, chondroitinase ABC treatment increased greatly the growth-promoting properties of the epineurial tissue whereas chondroitinase C had little effect. Our evidence indicates that chondroitinase C effectively degrades and inactivates inhibitory CSPGs present in the endoneurial Schwann cell basal lamina and does so more specifically than chondroitinase ABC. These findings are discussed in the context of improving nerve repair and regeneration and the growth-promoting properties of processed nerve allografts.

Implantation methodology development for tissue-engineered-electronic-neural-interface (TEENI) devices

Regenerative peripheral-nerve interfaces are a novel method for integrating with the peripheral nervous system. These devices have the potential to isolate and transduce both afferent (sensory) and efferent (motor) neural signals to produce fine control of advanced prosthetics. We have developed a novel regenerative device comprised of microfabricated polyimide electrode threads supported by a hydrogel scaffold containing methacrylated hyaluronic acid, collagen I, and laminin to enable intimate contact with regenerating axons. While this advanced device holds theoretical promise for establishing a stable chronic neural interface, it also requires a novel surgical approach in comparison to current existing methods of peripheral neural interface technologies. Here we describe the development of the surgical methodology required for successful chronic implantation of the TEENI device in the rat sciatic nerve.

Histological evaluation of chronically implanted tissue-engineered-electronic-neural-interface (TEENI) devices

Neural-interface devices have the potential to isolate and transduce both afferent (sensory) and efferent (motor) neural signals of the peripheral nerve to and from electrical signals in instrumentation for stimulation and recording to produce fine control of advanced prosthetics. In order to potentiate the full spectrum of possible applications, the persistent foreign-body response needs to be addressed. Here we describe the cellular and extracellular components of chronically implanted polyimide threads suspended within a tricomponent hydrogel. The results of these experiments will contribute to design modifications for future fabrications of tissue-engineered-electronic-nerve-interface (TEENI) devices.

Design, fabrication, and characterization of a scalable tissue-engineered-electronic-nerve-interface (TEENI) device

In this study, we describe a novel peripheral-nerve interface which makes use of highly flexible multi-electrode arrays that are integrated into hydrogel-based scaffolds to form a hybrid tissue-engineered electronic construct. This tissue-engineered electronic nerve interface (TEENI) is designed to be scalable to high channel counts using multiple polyimide-based “threads” that are evenly distributed through a volume of the nerve equal to its diameter times the distance between one or more nodes of Ranvier. Such scalability could greatly increase the precision and resolution of motor-control and sensory-feedback signals exchanged between amputees and advanced upper-limb prosthetic devices.