Peripheral nerve regeneration through nerve conduits evokes differential expression of growth-associated protein-43 in the spinal cord

Growth-associated protein 43 plays a key role in neurite outgrowth through cytoskeleton remodeling.We have previously demonstrated that structural damage of peripheral nerves induces growth-associated protein 43 upregulation to promote growth cone formation.Conversely,the limited regenerative capacity of the central nervous system due to an inhibitory environment prevents major changes in neurite outgrowth and should be presumably associated with low levels of growth-associated protein 43 expression.However,central alterations due to peripheral nerve damage have never been assessed using the growthassociated protein 43 marker.In this study,we used the tubulization technique to repair 1 cm-long nerve gaps in the rat nerve injury/repair model and detected growth-associated protein 43 expression in the peripheral and central nervous systems.First,histological analysis of the regeneration process confirmed an active regeneration process of the nerve gaps through the conduit from 10 days onwards.The growth-associated protein 43 expression profile varied across regions and follow-up times,from a localized expression to an abundant and consistent expression throughout the regeneration tissue,confirming the presence of an active nerve regeneration process.Second,spinal cord changes were also histologically assessed,and no apparent changes in the structural and cellular organization were observed using routine staining methods.Surprisingly,remarkable differences and local changes appeared in growth-associated protein 43 expression at the spinal cord level,in particular at 20 days post-repair and beyond.Growth-associated protein 43 protein was first localized in the gracile fasciculus and was homogeneously distributed in the left posterior cord.These findings differed from the growth-associated protein 43 pattern observed in the healthy control,which did not express growth-associated protein 43 at these levels.Our results revealed a differential expression in growth-associated protein 43 protein not only in the regenerating nerve tissue but also in the spinal cord after peripheral nerve transection.These findings open the possibility of using this marker to monitor changes in the central nervous system after peripheral nerve injury.

Tissue-engineered constructs for peripheral nerve repair:current research concepts and future perspectives

Traumatic injuries resulting in peripheral nerve lesions lead to important morbidity with devastating social and economic consequences.When the lesioned nerve cannot be sutured directly,a nerve graft is generally required to bridge the gap.Although autologous nerve grafting is still the first choice for reconstruction,it has the severe disadvantage of the sacrifice of a functional nerve.Research in tissue engineering and nerve regeneration may have a dramatic impact on clinical and surgical treatment of such nerve lesions.The authors review the latest concepts in tissue engineering for nerve repair,including scaffold engineering of neural guides,biomaterial modification,cell therapy,growth factors delivery,and electrical stimulation.Recent literature is reviewed in detail,pointing out the most interesting present achievements and perspectives for future clinical translation.Electronic search of the literature was performed using MEDLINE,Embase,and the Cochrane Library to identify research studies on peripheral nerve regeneration through tissue-engineered conduits.The following medical subject headings were used to carry out a systematic search of the literature:“nerve regeneration”,“stem cells”,“biomaterial”,“extracellular matrix”,“functional regeneration”,“growth factors”and“microchannels”.Included literature was published between 1991 and 2014.The reference lists from the retrieved articles were also reviewed for additional articles.In total,76 articles were included in this study.

Biological and artificial nerve conduit for repairing peripheral nerve defect

OBJECTIVE: Recently, with the development of biological and artificial materials, the experimental and clinical studies on application of this new material-type nerve conduit for treatment of peripheral nerve defect have become the hotspot topics for professorial physicians. DATA SOURCES: Using the terms "nerve conduits, peripheral nerve, nerve regeneration and nerve transplantation" in English, we searched Pubmed database, which was published during January 2000 to June 2006, for the literatures related to repairing peripheral nerve defect with various materials. At the same time, we also searched Chinese Technical Scientific Periodical Database at the same time period by inputting " peripheral nerve defect, nerve repair, nerve regeneration and nerve graft" in Chinese. STUDY SELECTION: The materials were firstly selected, and literatures about study on various materials for repairing peripheral nerve defect and their full texts were also searched. Inclusive criteria: nerve conduits related animal experiments and clinical studies. Exclusive criteria: review or repetitive studies. DATA EXTRACTION: Seventy-nine relevant literatures were collected and 30 of them met inclusive criteria and were cited. DATA SYNTHESIS: Peripheral nerve defect, a commonly seen problem in clinic, is difficult to be solved. Autogenous nerve grafting is still the gold standard for repairing peripheral nerve defect, but because of its application limitation and possible complications, people studied nerve conduits to repair nerve defect. Nerve conduits consist of biological and artificial materials. CONCLUSION: There have been numerous reports about animal experimental and clinical studies of various nerve conduits, but nerve conduit, which is more ideal than autogenous nerve grafting, needs further clinical observation and investigation.

Modification of tubular chitosan-based peripheral nerve implants: applications for simple or more complex approaches

Surgical treatment of peripheral nerve injuries is still a major challenge in human clinic.Up to now,none of the well-developed microsurgical treatment options is able to guarantee a complete restoration of nerve function.This restriction is also effective for novel clinically approved artificial nerve guides.In this review,we compare surgical repair techniques primarily for digital nerve injuries reported with relatively high prevalence to be valuable attempts in clinical digital nerve repair and point out their advantages and shortcomings.We furthermore discuss the use of artificial nerve grafts with a focus on chitosan-based nerve guides,for which our own studies contributed to their approval for clinical use.In the second part of this review,very recent future perspectives for the enhancement of tubular(commonly hollow)nerve guides are discussed in terms of their clinical translatability and ability to form three-dimensional constructs that biomimick the natural nerve structure.This includes materials that have already shown their beneficial potential in in vivo studies like fibrous intraluminal guidance structures,hydrogels,growth factors,and approaches of cell transplantation.Additionally,we highlight upcoming future perspectives comprising co-application of stem cell secretome.From our overview,we conclude that already simple attempts are highly effective to increase the regeneration supporting properties of nerve guides in experimental studies.But for bringing nerve repair with bioartificial nerve grafts to the next level,e.g.repair of defects>3 cm in human patients,more complex intraluminal guidance structures such as innovatively manufactured hydrogels and likely supplementation of stem cells or their secretome for therapeutic purposes may represent promising future perspectives.

Gene expression profiles of human adipose-derived mesenchymal stem cells dynamically seeded on clinically available processed nerve allografts and collagen nerve guides

It was hypothesized that mesenchymal stem cells(MSCs) could provide necessary trophic factors when seeded onto the surfaces of commonly used nerve graft substitutes. We aimed to determine the gene expression of MSCs when influenced by Avance■ Nerve Grafts or Neura Gen■ Nerve Guides. Human adipose-derived MSCs were cultured and dynamically seeded onto 30 Avance■ Nerve Grafts and 30 Neura Gen■ Nerve Guides for 12 hours. At six time points after seeding, quantitative polymerase chain reaction analyses were performed for five samples per group. Neurotrophic [nerve growth factor(NGF), glial cell line-derived neurotrophic factor(GDNF), pleiotrophin(PTN), growth associated protein 43(GAP43) and brain-derived neurotrophic factor(BDNF)], myelination [peripheral myelin protein 22(PMP22) and myelin protein zero(MPZ)], angiogenic [platelet endothelial cell adhesion molecule 1(PECAM1/CD31) and vascular endothelial cell growth factor alpha(VEGFA)], extracellular matrix(ECM) [collagen type alpha I(COL1A1), collagen type alpha III(COL3A1), Fibulin 1(FBLN1) and laminin subunit beta 2(LAMB2)] and cell surface marker cluster of differentiation 96(CD96) gene expression was quantified. Unseeded Avance■ Nerve Grafts and Neura Gen■ Nerve Guides were used to evaluate the baseline gene expression, and unseeded MSCs provided the baseline gene expression of MSCs. The interaction of MSCs with the Avance■ Nerve Grafts led to a short-term upregulation of neurotrophic(NGF, GDNF and BDNF), myelination(PMP22 and MPZ) and angiogenic genes(CD31 and VEGFA) and a long-term upregulation of BDNF, VEGFA and COL1A1. The interaction between MSCs and the Neura Gen■ Nerve Guide led to short term upregulation of neurotrophic(NGF, GDNF and BDNF) myelination(PMP22 and MPZ), angiogenic(CD31 and VEGFA), ECM(COL1A1) and cell surface(CD96) genes and long-term upregulation of neurotrophic(GDNF and BDNF), angiogenic(CD31 and VEGFA), ECM genes(COL1A1, COL3A1, and FBLN1) and cell surface(CD96) genes. Analysis demonstrated MSCs seeded onto Neura Gen■ Nerve Guides expressed significantly higher levels of neurotrophic(PTN), angiogenic(VEGFA) and ECM(COL3A1, FBLN1) genes in the long term period compared to MSCs seeded onto Avance■ Nerve Grafts. Overall, the interaction between human MSCs and both nerve graft substitutes resulted in a significant upregulation of the expression of numerous genes important for nerve regeneration over time. The in vitro interaction of MSCs with the Neura Gen■ Nerve Guide was more pronounced, particularly in the long term period(> 14 days after seeding). These results suggest that MSC-seeding has potential to be applied in a clinical setting, which needs to be confirmed in future in vitro and in vivo research.

The reasons for end-to-side coaptation:how does lateral axon sprouting work?

Nerve fibers are attracted by sutureless end-to-side nerve coaptation into the recipient nerve. Opening a window in the epineurium enhances axon attraction and myelination. The authors analyze the features of nerve repair by end-to-side coaptation. They highlight the known mechanisms of axon sprouting and different hypotheses of start up signals(presence or absence of an epineurial window, role of Schwann cells, signaling from the distal trunk). The clinical literature is also presented and differences between experimental and clinical applications are pointed out. The authors propose their point of view and perspectives deriving from recent experimental and clinical experiences.

Peripheral nerve allograft:how innovation has changed surgical practice

The landscape of available technology and surgical technique has changed over the last several decades,thus leading to changes in the peripheral nerve repair surgical algorithm.Neurorrhaphy is a common procedure;however,it is well recognized that nerve repair should be performed tensionless,thus preventing the ability to perform direct repair with a nerve gap.Historically,nerve gaps were repaired with autograft.However,autograft surgery has been associated with complications such as numbness and chronic pain,which left surgeons searching for alternatives.Nerve allografts were first utilized in the 1800s but failed due to the immune response.In the modern era,they were again utilized in the 1980s,but did not gain popularity because of the need for the use of immunosuppressants.It was evident through the 1990s that continued innovation in peripheral nerve repair was needed,as studies showed that only approximately 50% of patients with nerve gap repair achieved good or excellent outcomes.In the 2000s,the advent of an engineered nerve allograft(Avance■Nerve Graft)changed the landscape of peripheral nerve repair.Early clinical evaluation of Avance showed that adequate sensation was able to be achieved in nerve gaps up to 30 mm,providing an alternative to autografts.As engineered nerve allograft use became more conventional,studies showed 87.3% meaningful recovery in nerve gaps up to 50 mm.Furthermore,recent studies have shown that gaps between 50-70 mm have shown 69% meaningful recovery.While technology and surgical technique continue to improve,these results are promising for large nerve gap repair.

3D printing of functional nerve guide conduits

Nerve guide conduits(NGCs),as alternatives to nerve autografts and allografts,have been widely explored as an advanced tool for the treatment of peripheral nerve injury.However,the repairing efficiency of NGCs still needs significant improvements.Functional NGCs that provide a more favorable microenvironment for promoting axonal elongation and myelination are of great importance.In recent years,3D printing technologies have been widely applied in the fabrication of customized and complex constructs,exhibiting great potential for tissue engineering applications,especially for the construction of functional NGCs.In this review,we introduce the 3D printing technologies for manufacturing functional NGCs,including inkjet printing,extrusion printing,stereolithographybased printing and indirect printing.Further,we summarize the current methods and strategies for constructing functional NGCs,such as designing special conduit architectures,using appropriate materials and co-printing with different biological cues.Finally,the challenges and prospects for construction of functional NGCs are also presented.

Polylysine-decorated macroporous microcarriers laden with adipose-derived stem cells promote nerve regeneration in vivo

Cell transplantation is an effective strategy to improve the repair effect of nerve guide conduits(NGCs).However,problems such as low loading efficiency and cell anoikis undermine the outcomes.Microcarriers are efficient 3D cell culture scaffolds,which can also prevent cell anoikis by providing substrate for adhesion during transplantation.Here,we demonstrate for the first time microcarrier-based cell transplantation in peripheral nerve repair.We first prepared macroporous chitosan microcarriers(CSMCs)by the emulsion-phase separation method,and then decorated the CSMCs with polylysine(pl-CSMCs)to improve cell affinity.We then loaded the pl-CSMCs with adipose-derived stem cells(ADSCs)and injected them into electrospun polycaprolactone/chitosan NGCs to repair rat sciatic nerve defects.The ADSCs-laden pl-CSMCs effectively improved nerve regeneration as demonstrated by evaluation of histology,motor function recovery,electrophysiology,and gastrocnemius recovery.With efficient cell transplantation,convenient operation,and the multiple merits of ADSCs,the ADSCs-laden pl-CSMCs hold good potential in peripheral nerve repair.

Low-Intensity Pulsed Ultrasound Promote Schwann Cells Pro-Myelination Activities and Neurite Outgrowth of Dorsal Root Ganglion Neurons

In this study,we investigate how Schwann cells and dorsal root ganglion(DRG)neurons response to direct low-intensity pulsed ultrasound(LIPUS)stimuli in vitro.Primary Schwann cells and DRG were isolated from rat sciatic nerve and spine,respectively.LIPUS with varied dose of intensity(low:250 mW/cm2,medium:500 m W/cm2,high:750 m W/cm2)were applied 5 min per time for every other day,and pro-myelination indicators of Schwann cell as well as neurite outgrowth of dorsal root ganglion neurons were analyzed.Our results demonstrated that LIPUS promoted Schwann cells activity and proliferation from day 3 at the highest intensity,and day 5 at all intensities.In addition,LIPUS boosted pro-myelination activities of Schwann cells,as evidenced by increased cell population that positive for immunohistochemical staining against S100,nerve growth factor receptor(NGFR)p75,glial fibrillary acidic protein(GFAP),myelin protein zero(P0),as well as up-regulation of GFAP,Protein 0,nerve growth factor(NGF),and brain derived neurotrophic factor(BDNF)genes.Furthermore,LIPUS significantly enhanced the neurite outgrowth of DRG,with the highest intensity exhibiting longest neurite outgrowth.Taken together,our results strongly improve the understanding of cellular mechanisms of ultrasonic therapies for peripheral nerve repair.