Background
Nerve regeneration is a complex biological phenomenon. In the peripheral nervous system, nerves can spontaneously regenerate without any treatment if nerve continuity is maintained (axonotmesis) whereas more severe type of injuries must be surgically treated by direct end-to-end surgical reconnection of the damaged nerve ends [
1‐
3]. Unfortunately, the functional outcomes of nerve repair are in many cases unsatisfactory [
4] thus calling for research in order to reveal more effective strategies for improving nerve regeneration. However, recent advances in neuroscience, cell culture, genetic techniques, and biomaterials provide optimism for new treatments for nerve injuries [
5‐
17].
The use of materials of natural origin has several advantages in tissue engineering. Natural materials are more likely to be biocompatible than artificial materials. Also, they are less toxic and provide a good support to cell adhesion and migration due to the presence of a variety of surface molecules. Drawbacks of natural materials include potential difficulties in their isolation and controlled scale-up [
11]. In addition to the use of intact natural tissues, a great deal of research has focused on the use of purified natural extracellular matrix (ECM) molecules, which can be modified to serve as appropriate scaffolding [
11]. ECM molecules, such as laminin, fibronectin and collagen have also been shown to play a significant role in axonal development and regeneration [
12,
18‐
27]. For example, silicone tubes filled with laminin, fibronectin, and collagen led to a better regeneration over a 10 mm rat sciatic nerve gap compared to empty silicone controls [
9]. Collagen filaments have also been used to guide regenerating axons across 20-30 mm defects in rats [
23‐
27]. Further studies have shown that oriented fibers of collagen within gels, aligned using magnetic fields, provide an improved template for neurite extension compared to randomly oriented collagen fibers [
28,
29]. Finally, rates of regeneration comparable to those using a nerve autograft have been achieved using collagen tubes containing a porous collagen-glycosaminoglycan matrix [
30‐
32]. Nerve regeneration requires a complex interplay between cells, ECM, and growth factors. The local presence of growth factors plays an important role in controlling survival, migration, proliferation, and differentiation of the various cell types involved in nerve regeneration [
12‐
14,
33]. Therefore, therapies with relevant growth factors received increasing attention in recent years although growth factor therapy is a difficult task because of the high biological activity (in pico- to nanomolar range), pleiotrophic effects (acting on a variety of targets), and short biological half-life (few minutes to hours) [
34]. Thus, growth factors should be administered locally to achieve an adequate therapeutic effect with little adverse reactions and the short biological half-life of growth factors demands for a delivery system that slowly releases locally the molecules over a prolonged period of time. Employment of biodegradable membranes enriched with a cellular system producing neurotrophic factors has been suggested to be a rational approach for improving nerve regeneration after neurotmesis [
11].
The aim of this study was thus to verify if rat sciatic nerve regeneration after end-to-end reconstruction can be improved by seeding
in vitro differentiated N1E-115 neural cells on a type III equine collagen membrane and enwrap the membrane around the lesion site. The N1E-115 cell line has been established from a mouse neuroblastoma [
35] and have already been used with conflicting results as a cellular system to locally produce and deliver neurotrophic factors [
12‐
14,
36,
37].
In vitro, the N1E-115 cells undergo neuronal differentiation in response to dimethylsulfoxide (DMSO), adenosine 3', 5'-cyclic monophosphate (cAMP), or serum withdrawal [
38‐
43,
36,
37,
12‐
14]. Upon induction of differentiation, proliferation of N1E-115 cells ceases, extensive neurite outgrowth is observed and the membranes become highly excitable [
38‐
43,
36,
37,
12‐
14]. The interval period of 48 hours of differentiation was previously determined by measurement of the intracellular calcium concentration ([Ca
2+] i). At this time point, the N1E-115 cells present already the morphological characteristics of neuronal cells but cell death due to increased [Ca
2+] i is not yet occurring as described elsewhere [
38‐
43,
36,
37,
12‐
14].
Discussion
Transected peripheral nerves can regenerate provided that a connection is available between the proximal and distal severed stumps and, when no substance loss occurred, surgical treatment consists in direct end-to-end suturing of the nerve ends [
1‐
3,
62,
63]. However, in spite of the progress of microsurgical nerve repair, the outcome of nerve reconstruction is still far from being optimal
4. Since during regeneration axons require neurotrophic support, they could benefit from the presence of a growth factors delivery cell system capable of responding to stimuli of the local environment during axonal regeneration.
In the present study, we aimed at investigating the effects of enwrapping the site of end-to-end rat sciatic nerve repair with equine type III collagen nerve membranes either alone or enriched with N1E-115 pre-differentiated into neural cells in the presence of 1.5% of DMSO. The rationale for the utilization of the N1E-115 cells was to take advantages of the properties of these cells as a neural-like cellular source of neurotrophic factors [
12‐
14,
36,
37].
Results showed that enwrapment with a collagen membrane, with or without neural cell enrichment, did not lead to any significant improvement in most of functional and stereological predictors of nerve regeneration that we have assessed. The only exception was represented by motor deficit recovery which was significantly improved after lesion site enwrapment with membrane enriched with neural cells pre-differentiated from N1E-115 cell line.
Natural tissues possess several advantages when compared to synthetic materials, when use to reconstruct peripheral nerves after injury. Natural materials are more likely to be biocompatible than artificial materials, are less toxic, and provide a support structure to promote cell adhesion and migration. Drawbacks, on the other hand, include potential difficulties with isolation and controlled scale-up. In addition to intact acellular tissues, a great deal of research has focused on the use of purified natural ECM proteins and glycosaminoglycans, which can be modified to serve as appropriate scaffolding. ECM molecules, such as laminin, collagen, and fibronectin, have been shown to play a significant role in axonal development and repair in the body [
19,
24]. There are a number of examples in which the ECM proteins laminin, fibronectin, and collagen have been used for nerve repair applications [
12,
18‐
27]. For example, silicone tubes filled with laminin, fibronectin, and collagen show improved regeneration over a 10 mm rat sciatic nerve gap compared to empty silicone controls [
9]. Collagen filaments have also been used to guide regenerating axons across 20-30 mm defects in rats [
23,
26,
27]. Further studies have shown that oriented fibers of collagen within gels, aligned using magnetic fields, provide an improved template for neurite extension compared to randomly oriented collagen fibers [
28,
29]. Rates of regeneration after neurotmesis comparable to those using a nerve autograft have been achieved using collagen tubes containing a porous collagen-glycosaminoglycan matrix [
31,
32].
Results of this study contribute to the lively debate about the employment of cell transplantation for improving post-traumatic nerve regeneration [
64,
65]. Actually, a great enthusiasm among researchers and especially the public opinion has risen over the last years about cell-based therapies in Regenerative Medicine [
66‐
68] and there seems to be widespread conviction that this type of therapy is not only effective but also very safe in comparison to other pharmacological or surgical therapeutic approaches. By contrast, recent studies showed that cell-based therapy might be ineffective for improving nerve regeneration [
66‐
69], and results of the present study are in line with these observations. Recently, it has even been shown that N1E-115 cell transplantation can also have negative results by hindering the nerve regeneration process after tubulisation repair [
12]. Of course, the choice of the cell type to be used for transplantation is very important for the therapeutic success and use of another cell type could have led to better results, especially when the cellular system of choice is derived from autologous or heterologous stem cells1 [
1,
12,
15‐
17,
64,
70]. Moreover, the construction of more appropriate tube-guides with integrated growth factor delivery systems and/or cellular components could improve the effectiveness of nerve tissue engineering. In fact, single-molded tube guides may not give sufficient control over both the mechanical properties and the delivery of bioactive agents. More complex devices will be needed, such as multilayered tube guides where growth factors are entrapped in polymer layers with varying physicochemical properties or tissue engineered tube guides containing viable stem cells [
1,
12,
15‐
17,
64,
70]. The combination of two or more growth factors will likely exert a synergistic effect on nerve regeneration, especially when the growth factors belong to different families and act via different mechanisms. Combinations of growth factors can be expected to enhance further nerve regeneration, particularly when each of them is delivered at individually tailored kinetics [
11,
12,
15‐
17,
64,
70,
71]. The determination and control of suitable delivery kinetics for each of several growth factors will constitute a major hurdle both technically and biologically with the biological hurdle lying in the compliance with the naturally occurring cross talk between growth factors and cells. A solution to this problem may be the use of autologous stem cells because they can synthesize several growth factors and differentiate into Schwann cells which are critical for very long gaps [
11,
12,
15‐
17,
64,
70,
71].
Previous work already published by other research groups, point out a very interesting source of stem cells for nerve regeneration of peripheral nerve and spinal cord. They developed hair follicle pluripotent stem cells (hfPS) and have shown that these cells can differentiate to neurons, glial cells
in vitro, and other cell types, and can promote nerve and spinal cord regeneration
in vivo. These cells are located above the hair follicle bulge (hfPS cell area) and are nestin and CD34 positive, and keratin 15 negative [
72‐
75]. The mouse hfPS cells were implanted into the gap region of the severed sciatic and tibial nerve of mice. These cells, after 6-8 weeks, transdifferentiated largely into Schwann cells. Also, blood vessels formed a network around the joined sciatic and tibial nerve. Function of the rejoined sciatic and tibial nerve was confirmed by contraction of the gastrocnemius muscle upon electrical stimulation and by walking track analysis [
73‐
75]. hfPS cells can promote axonal growth and functional recovery after peripheral nerve injury, offering an important opportunity for future clinical application. These hfPS cells, in contrast to Embrionic stem cells, N1E-115 cells after
in vitro differentiation and induced pluripotent stem cells, do not require any genetic manipulation, are readily accessible from any patient, and lack the ethical issues, do not form tumors.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SA, APV and ASPV carried out the kinematic collecting data and the kinematic data analysis, participated in the functional data analysis, JMR and ALL carried out the animal surgeries, euthanasia, preparation of samples for histological and stereological analysis and participated in the functional evaluation analysis, PASADS carried out all the statistical analysis, the interpretation of kinematic data and participated in the paper draft, MV, AGand MJS performed the functional evaluation and analysis and were responsible for keeping the experimental animals, MF, SR, and SG performed the histological and stereological analysis, ACM carried out the animal surgeries euthanasia and preparation of samples for histological and stereological analysis. ACM together with SG and PASADS designed and coordinated the study, elaborated the manuscript and were responsible for the funding acquisition. All the authors read and approved the final manuscript.