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.

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.

Tissue engineering and peripheral nerve regeneration (Ⅲ)——Sciatic nerve regeneration with PDLLA nerve guide

The biodegradation rate and biocompatibility of poly(d, / -lactide) (PDLLA) in vivo were evaluated. The aim of this study was to establish a nerve guide constructed by the PDLLA with 3-D microenvironment and to repair a 10 mm of sciatic nerve gap in rats. The process of the nerve regeneration was investigated by histological assessment, electrophysiological examination, and determination of wet weight recovery rate of the gastrocnemius muscle. After 3 weeks, the nerve guide had changed from a transparent to an opaque status. The conduit was degraded and absorbed partly and had lost their strength with breakage at the 9th week of postoperation. At the conclusion of 12 weeks, proximal and distal end of nerves were anastomosed by nerve regeneration and the conduit vanished completely. The results suggest that PDLLA conduits may serve for peripheral nerve regeneration and PDLLA is a sort of hopeful candidate for tissue engineering.

Spatiotemporal microRNA profile in peripheral nerve regeneration:miR-138 targets vimentin and inhibits Schwann cell migration and proliferation

While the peripheral nervous system has regenerative ability,restoration of sufficient function remains a challenge.Vimentin has been shown to be localized in axonal growth fronts and associated with nerve regeneration,including myelination,neuroplasticity,kinase signaling in nerve axoplasm,and cell migration;however,the mechanisms regulating its expression within Schwann cell（SC） remain unexplored.The aim of this study was to profile the spatial and temporal expression profile of micro RNA（mi RNA） in a regenerating rat sciatic nerve after transection,and explore the potential role of mi R-138-5 p targeting vimentin in SC proliferation and migration.A rat sciatic nerve transection model,utilizing a polyethylene nerve guide,was used to investigate mi RNA expression at 7,14,30,60,and 90 days during nerve regeneration.Relative levels of mi RNA expression were determined using microarray analysis and subsequently validated with quantitative real-time polymerase chain reaction.In vitro assays were conducted with cultured Schwann cells transfected with mi RNA mimics and assessed for migratory and proliferative potential.The top seven dysregulated mi RNAs reported in this study have been implicated in cell migration elsewhere,and GO and KEGG analyses predicted activities essential to wound healing.Transfection of one of these,mi RNA-138-5 p,into SCs reduced cell migration and proliferation.mi R-138-5 p has been shown to directly target vimentin in cancer cells,and the luciferase assay performed here in rat Schwann cells confirmed it.These results detail a role of mi R-138-5 p in rat peripheral nerve regeneration and expand on reports of it as an important regulator in the peripheral nervous system.

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.

Stimulating effect of thyroid hormones in peripheral nerve regeneration:research history and future direction toward clinical therapy

Injury to peripheral nerves is often observed in the clinic and severe injuries may cause loss of motor and sensory functions.Despite extensive investigation,testing various surgical repair techniques and neurotrophic molecules,at present,a satisfactory method to ensuring successful recovery does not exist.For successful molecular therapy in nerve regeneration,it is essential to improve the intrinsic ability of neurons to survive and to increase the speed of axonal outgrowth.Also to induce Schwann cell phenotypical changes to prepare the local environment favorable for axonal regeneration and myelination.Therefore,any molecule that regulates gene expression of both neurons and Schwann cells could play a crucial role in peripheral nerve regeneration.Clinical and experimental studies have reported that thyroid hormones are essential for the normal development and function of the nervous system,so they could be candidates for nervous system regeneration.This review provides an overview of studies devoted to testing the effect of thyroid hormones on peripheral nerve regeneration.Also it emphasizes the importance of combining biodegradable tubes with local administration of triiodothyronine for future clinical therapy of human severe injured nerves.We highlight that the local and single administration of triiodothyronine within biodegradable nerve guide improves significantly the regeneration of severed peripheral nerves,and accelerates functional recovering.This technique provides a serious step towards future clinical application of triiodothyronine in human severe injured nerves.The possible regulatory mechanism by which triiodothyronine stimulates peripheral nerve regeneration is a rapid action on both axotomized neurons and Schwann cells.