The recent success of acellular nerve allografts in investigational studies and clinical reports suggests an impending paradigm shift in clinical management of short gap, small diameter nerve injuries. In our current clinical practice, commercially available acellular nerve grafts have largely replaced nerve conduits as the preferred alternative to nerve autografts for surgical management of short gap injuries in noncritical, small diameter peripheral nerve defects. The basis for this shift lies in the superior regenerative capacity of processed acellular allografts compared to available nerve conduits; this was highlighted in a recent study in which commercially available processed nerve allograft significantly outperformed commercially available conduits. 23
The presence of native extracellular matrix and intact SC basal laminae within acellularized grafts supported greater numbers of regenerating axons and more successful guidance of regenerating axons compared to empty conduits. 23,27
Despite the promise of acellular nerve allografts as an effective nerve substitute, experimental studies to date have only investigated small diameter small gap defects. The currently available acellular nerve grafts possess reduced regenerative capacities compared to fresh nerve isograft and will only support nerve regeneration over limited distances.23
Comprehensive investigations capable of identifying the limitations of contemporary acellular nerve grafts have yet to occur. One of the primary impediments that limits such studies remains the identification of a low-cost acellular nerve graft that is both comparable to commercially available acellular nerve grafts and conducive to laboratory use. Our study was designed to comparatively assess the regenerative capacities of three established acellular nerve graft models, examine the effect of processing technique on nerve graft efficacy, and to identify an investigational surrogate to commercially available Avance® processed nerve allografts.
Cold-preserved, detergent-processed, and AxoGen®-processed nerve allografts represent the most prevalent and widely studied acellular nerve graft models. Despite arising from a similar appraisal of the molecular bases of nerve graft immunogenicity, each model employs significantly different methods of processing donor nerve tissue. 14,13,15,24,28
Specific decellularization techniques applied within each model largely determine both the percentage of antigenic cellular constituents removed from the donor nerve, and the quality of preserved nerve extracellular matrix. A lack of comparative studies within prior investigations has prevented direct examination of the effect of processing on axonal regeneration. In this study, differentially processed acellular nerve grafts were directly compared in vivo
to determine the effect of processing techniques on nerve regeneration.
The regenerative capacity of acellular nerve grafts and empty nerve conduits was evaluated by assessing axonal regeneration and functional recovery following nerve repair. Histomorphometric analysis, a well established technique useful in quantifying pertinent ultra-structural characteristics of nerve tissue, was utilized to examine acute axonal regeneration through implanted nerve grafts and conduits. 25
Measurement of evoked force production in reinnervated distal musculature was simultaneously utilized to evaluate the chronic functional sequelae of nerve regeneration through implanted grafts/conduits. Close correlation was observed between histomorphometric and electrophysiological assays throughout the study. Successful axonal regeneration through implanted grafts/conduits largely predicted chronic functional recovery following nerve repair, suggesting the regenerating axons maintained the ability to functionally reinnervate distal end organs. Examination of both metrics revealed that acellular nerve allografts (cold-preserved, detergent-processed, and AxoGen®-processed nerve allografts) supported significant increases in functional nerve regeneration compared to empty silicone nerve guidance conduits. Poor regeneration through empty conduits was expected due to the poor regenerative micro-environment provided by silicone conduits and the critical length of the nerve defects. Specifically, silicone conduits have previously been shown to be unable to support axonal regeneration across nerve defects greater than 10 mm in length.29,30
The lack of successful nerve regeneration confirmed silicone nerve conduits as an effective negative control and demonstrated that axonal regeneration observed in this study was not influenced by spontaneous regeneration (i.e. “blow-through”) that is commonly observed in rodent models of nerve injury/repair.6
Evaluation of axonal regeneration and functional recovery through acellular nerve grafts revealed that processing technique modulates nerve graft efficacy in vivo
. In this study, detergent-processed nerve allografts were observed to support superior nerve regeneration and functional recovery compared to both cold-preserved and AxoGen®-processed nerve allografts. Increased nerve regeneration through detergent-processed nerve allografts may result from more successful removal of cellular debris and preservation of native nerve micro-structure within donor nerve tissue. Hudson et al.
previously described the superiority of the optimized detergent-processing technique over alternative methods of nerve decellularization.14
Yet, AxoGen®-processed nerve allografts, which are known to employ similar methods of nerve decellularization, did not demonstrate equivalent levels of nerve regeneration in our study.
Alternatively, improved nerve regeneration through detergent-processed nerve grafts may be a result of shorter lengths of time between explantation of donor nerve and implantation of processed nerve. The detergent-processing technique employed in the current study required four days of post-processing prior to successful decellularization of donor nerve tissue, where AxoGen®-processing and cold-preservation techniques require longer lengths of time. Extended processing times have been shown to adversely affect the integrity of the endoneurial microstructure within nerve grafts.14
Inferior preservation of nerve ECM in grafts that undergo lengthy processing techniques may yield poor regenerative support for regenerating axons extending through the implanted graft and result in poor functional recovery post-operatively.
Shorter post-processing times may also result in greater retention or preservation of soluble and/or bound growth factors within the decellularized nerve tissue.31,32
Prior studies have demonstrated that growth factors such as vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor beta (TGF-β) are all released from ECM during active reorganization of the ECM microstructure. 33,31,34-36
Prolonged chemical or physical alteration of nerve tissue may therefore have increased the removal of embedded neurotrophic cues and progressively decreased the neuroregenerative potential of processed grafts. Extending processing techniques, such as cold-preservation and AxoGen®-processing, may also directly reduce the activity of these neuroregenerative cues through progressive chemical denaturation. Comprehensive, comparative analysis of the micro-structural composition of processed nerve grafts will be required to examine the synergistic effects processing time and processing technique have on preserving the natural neuroregenetive capacity of native nerve ECM.
In this study, AxoGen®-processed nerve grafts were unexpectedly observed to facilitate lesser degrees of functional nerve regeneration compared to detergent-processed nerve allografts. Despite our use of a similar method of nerve decellularization developed by Hudson et al.14
, detergent-processed nerve allografts supported increased axonal regeneration compared to AxoGen®-processed nerve allografts. Reduced regenerative capacity of AxoGen®-processed nerve grafts may result from optimization of the AxoGen® decellularization techniques for use with human, rather than rodent, nerve tissue. This species difference needs to be considered in consideration of the current results. Previous tissue processing techniques designed to maintain extracellular matrix integrity have been demonstrated to have significantly different effects on tissue originating from separate species. 37,38
Despite the fact that the detergent-processed and AxoGen®-processed grafts both utilize SB-10, SB-16, and Triton X-200 detergents in the decellularization of donor nerve tissue, differences in washing time could have differential affects on the integrity of the ECM within donor nerves.
Additional processing steps utilized in the AxoGen® processing technique, including gamma irradiation and flash-freezing, may also affect resulting acellular nerve graft material. While the effect of gamma irradiation on nerve microstructure has not been studied in depth, protocols using repeated episodes of freezing/thawing have been shown to have a significant negative effect on endoneurial microstructure.39,40
Due to the proprietary nature of AxoGen® decellularization techniques, little information exists as to what additional techniques are applied to each decellularized nerve graft. Despite this lack of knowledge, small differences in the processing technique utilized to prepare AxoGen®-processed and detergent-processed nerve grafts may contribute to the reduced regenerative capacity observed in AxoGen®-processed grafts.
Application of chondroitinase ABC (ChABC) to donor nerve tissue during preparation of AxoGen®-processed acellular nerve grafts may also contribute to the differences observed between AxoGen®-processed and detergent-processed nerve allografts. CSPGs are extracellular matrix molecules that decrease the ability of axons of the central and peripheral nervous system to regenerate following injury. In the peripheral nervous system, CSPG are present in the distal stump after nerve injury41-43
and are removed during normal Wallerian degeneration by Schwann cells and macrophages.44,45
Prior studies demonstrate that treatment with ChABC effectively removes CSPG from donor nerve tissue and enhances nerve regeneration through resulting acellular nerve grafts.21
Following decellularization AxoGen®-processed nerve allografts are known to undergo ChABC treatment to improve axonal regeneration through resulting graft material. Despite numerous studies investigating the positive effect of ChABC on axonal regneration, ChABC treatment may have a detrimental effect on axonal regeneration in some instances. Excessive removal of CSPG from peripheral nerve tissue may eliminate natural inhibitory cues which are utilized to guide extending neurites.46
Additionally, the enzymatic activity of ChABC may have a negative effect on the integrity of the ECM within the donor nerve. However, several studies that evaluate the effect of ChABC treatment on the regenerative capacity of peripheral nerve suggest no negative effects of ChABC treatment on nerve regeneration.47,21,48,49
The AxoGen®-processed allografts used in this study were derived from Brown Norway rats, while the detergent-processed and cold-preserved allografts were derived from Sprague Dawley rats. Despite the fact that both rat strains (Brown Norway and Sprague Dawley) used for donor allografts have a major histocompatibility complex mismatch with Lewis rats, a difference in immunological response between donor strains could have contributed to regenerative differences observed between AxoGen®-processed and detergent-processed nerve allografts. Investigations have shown that, while differences in major or minor histocompatibility complex mismatch can result in difference in regeneration across a nerve allograft50
, different donor strains that are both major histocompatibility complex mismatch for the recipient facilitate similar neural regeneration.51
While there is no guarantee that immunological differences between the two donor strains contributed to the differences in axonal regeneration seen in the grafts, the added factor that the cellular components of each donor graft were removed decreases the likelihood of an effect. Allograft processing with both of these procedures has been shown to reduce the immune response to an allograft to the level of an isograft.23,20
These data suggest that it is unlikely that immunological differences significantly affected regeneration across each graft. In addition to immunologic differences, genetic differences in allograft architecture or extracellular matrix composition may also have affected nerve regeneration. Previously we have compared several nerve regeneration paradigms (crush, transection, and conduit repair) across five rat strains (Sprague Dawley, ACI, Wistar-Furth, Lewis, and Brown Norway). Upon examination of axonal regeneration using histomorphometry and function recovery using walking track analysis at two different end points (6 and 13 weeks), no statistically significant differences between strains were noted, regardless of endpoint evaluation. The final conclusion from this study was that uniform conclusions about nerve regeneration in the rat may be drawn regardless of strain used.52
Similarly, it is not likely that any differences in axonal regeneration were minimally affected by genetic differences between donor allografts.
Comparatively, cold-preserved nerve allografts exhibited comparable performance to commercially available AxoGen®-processed nerve grafts. Cold-preserved allografts and AxoGen®-processed nerve allografts supported statistically indistinguishable levels of axonal regeneration and functional recovery in vivo. The similarity in total number of regenerating fibers and functional output of cold-preserved and AxoGen®-processed nerve grafts suggests that, within the context of rodent models of nerve injury and repair, cold preserved nerve allografts offer a suitable substitute for commercially available AxoGen® processed nerve allografts. The results of this study therefore suggest that cold-preserved nerve grafts may be utilized in future research studies to model the behavior of commercially-available acellular nerve grafts. Utilization of cold-preserved nerve grafts is anticipated to eliminate the prohibitive cost and limited distribution associated with AxoGen®-processed nerve allografts and facilitate increased numbers of studies that examine the preparation and implementation of acellular nerve allografts.
Further evaluation of implanted acellular nerve allografts revealed that only detergent-processed nerve allograft matched the regenerative capacity of fresh nerve isograft. Unlike cold-preserved and AxoGen®-processed nerve allografts, detergent-processed nerve allografts were statistically indistinguishable from nerve isograft in supporting axonal regeneration and functional recovery in vivo
. The superior performance of detergent-processed nerve grafts suggests that this simple decellularization technique may merit clinical implementation. However, it should be emphasized that the AxoGen® Avance® is the only U.S. Food and Drug Administration approved acellular graft for clinical peripheral nerve repair. Increasingly rigorous trials will still be needed to fully characterize the potential of detergent-processed allografts to facilitate functional nerve regeneration across larger nerve defects (>30 mm) and longer nerve diameters. Prior studies confirm that larger peripheral nerve defects pose an increased challenge to nerve substitutes, as the implanted substitute must support axonal regeneration independent of both the proximal and distal nerve stumps.23
While the detergent-processed graft performed similarly to the isograft in the 14mm nerve gap model, detergent-processed grafts are not expected to match the ability of fresh nerve isograft to promote functional nerve regeneration across larger, clinically relevant nerve defects (> 30mm).53
The anticipated failure of all processed nerve allografts at this critical length highlights the need for further investigation and development of more effective nerve substitutes capable of promoting functional nerve regeneration at or above the level of fresh nerve isograft.
This study provides further evidence that different processing techniques used to remove the cellular components of acellular nerve allografts affect the regeneration of axons through the graft. For the first time, this effect on axonal regeneration has been shown to translate into differences in functional recovery. Based on our results and the results of others20
, we hypothesize that these differences are symptomatic of the variable preservation of endoneurial microstructure provided by each processing technique. Further, we demonstrated that detergent-processed grafts, optimized for rats, outperformed AxoGen®-processed grafts, optimized for humans, despite the use of similar detergents to process the graft. This suggests that for clinical use each processing technique must be optimized for human nerve. If the detergent-processing employed in this study were compared with AxoGen®-processed human grafts for human nerve regeneration, we predict that we would see the inverse of our results (specifically that AxoGen®-processed human grafts would outperform the detergent-processed human grafts). This final point has significance in a clinical setting where the use of commercially available acellular allografts or conduits are cost prohibitive, and the use of custom processed allografts is not prohibited by regulation. In this setting where a physician can prepare his/her own acellular nerve allografts for clinical use, it is imperative to understand that processing techniques described in the literature as effective in rodents will likely need to be optimized for human nerve.
Further, it establishes a low cost processing technique (cold-preservation) that mimics the regenerative performance of the clinically available acellular nerve allograft. Investigations into the limit of axonal regeneration in acellular nerve allografts will help define the clinical limitations and limit negative outcomes. An example of potential negative outcomes is the over-enthusiasm for nerve conduit use that has resulted in clinical failures in large diameter nerves and long gap injuries54
. We have shown the superiority of acellularized nerve allografts over empty conduits 23
and now anticipate similar expansion of this autograft substitute with potential deleterious results if the length size parameters are not clearly defined in the laboratory.