Tissue-engineered Nerve Conduits Research Articles

Morphological Methods to Evaluate Peripheral Nerve Fiber Regeneration: A Comprehensive Review.

Regeneration of damaged peripheral nerves remains one of the main challenges of neurosurgery and regenerative medicine, a nerve functionality is rarely restored, especially after severe injuries. Researchers are constantly looking for innovative strategies for tackling this problem, with the development of advanced tissue-engineered nerve conduits and new pharmacological and physical interventions, with the aim of improving patients' life quality. Different evaluation methods can be used to study the effectiveness of a new treatment, including functional tests, morphological assessment of regenerated nerve fibers and biomolecular analyses of key factors necessary for good regeneration. The number and diversity of protocols and methods, as well as the availability of innovative technologies which are used to assess nerve regeneration after experimental interventions, often makes it difficult to compare results obtained in different labs. The purpose of the current review is to describe the main morphological approaches used to evaluate the degree of nerve fiber regeneration in terms of their usefulness and limitations.

A hyaluronic acid granular hydrogel nerve guidance conduit promotes regeneration and functional recovery of injured sciatic nerves in rats.

A hyaluronic acid granular hydrogel can promote neuronal and astrocyte colony formation and axonal extension in vitro, suggesting that the hydrogel can simulate an extracellular matrix structure to promote neural regeneration. However, in vivo experiments have not been conducted. In this study, we transplanted a hyaluronic acid granular hydrogel nerve guidance conduit to repair a 10-mm long sciatic nerve gap. The Basso, Beattie, and Bresnahan locomotor rating scale, sciatic nerve compound muscle action potential recording, Fluoro-Gold retrograde tracing, growth related protein 43/S100 immunofluorescence staining, transmission electron microscopy, gastrocnemius muscle dry/wet weight ratio, and Masson's trichrome staining results showed that the nerve guidance conduit exhibited similar regeneration of sciatic nerve axons and myelin sheath, and recovery of the electrophysiological function and motor function as autologous nerve transplantation. The conduit results were superior to those of a bulk hydrogel or silicone tube transplant. These findings suggest that tissue-engineered nerve conduits containing hyaluronic acid granular hydrogels effectively promote the morphological and functional recovery of the injured sciatic nerve. The nerve conduits have the potential as a material for repairing peripheral nerve defects.

Repair of peripheral nerve injuries using a prevascularized cell-based tissue-engineered nerve conduit

One of the major challenges in the development of a larger and longer nerve conduit for peripheral nerve repair is the limitation in oxygen and nutrient diffusion within the tissue after transplantation preventing Schwann cell and axonal migration. This restriction is due to the slow neovascularization process of the graft starting from both nerve endings. To overcome this limitation, we propose the design of a living tissue-engineered nerve conduit made of an internal tube with a three-dimensional structure supporting axonal migration, which is inserted inside a hollow external tube that plays the role of an epineurium and is strong enough to be stitched to the severed nerve stumps. The internal tube is made of a rolled living fibroblast sheet and can be seeded with endothelial cells to promote the formation of a network containing capillary-like structures which allow rapid inosculation with the host nerve microvasculature after grafting. Human nerve conduits were grafted in immunodeficient rats to bridge a 15 mm sciatic nerve gap. Human capillaries within the pre-vascularized nerve conduit successfully connected to the host circulation 2 weeks after grafting. Twenty-two weeks after surgery, rats transplanted with the nerve conduits had a similar motor function recovery compared to the autograft group. By promoting rapid vascularization of the internal nerve tube from both ends of the nerve stumps, this endothelialized nerve conduit model displays a favorable environment to enhance axonal migration in both larger caliber and longer nerve grafts.

Material advancement in tissue-engineered nerve conduit

Abstract Peripheral nerve injuries resulting from various traumatic events can cause mobility problems and sensory impairment, jeopardizing patients’ life quality and bringing serious economic burdens. Due to the shortcomings of autologous nerve grafts, such as limited tissue sources, unmatched size, and loss of innervation at the donor site, tissue-engineered nerve grafts using both natural and synthetic materials have been employed in the treatment of peripheral nerve defect and to promote nerve regeneration. Apart from traditional advantages such as good biocompatibility and controllable degradation, the development of fabrication technology and the advancement in material science have endowed tissue-engineered nerve conduits with upgraded properties such as biomimetic surface topography, extracellular matrix components, neurotrophic factors, and cell seeding, or a conduit with micropores on the surface for substance exchange and/or with fillers inside for microenvironment simulation. This article reviews recent progress in the biomaterials employed in fabricating tissue-engineered nerve conduits, in vitro characterization, and their applications in nerve repair in animal studies as well as in clinical trials.

Bioabsorbable nerve conduits three-dimensionally coated with human induced pluripotent stem cell-derived neural stem/progenitor cells promote peripheral nerve regeneration in rats

Peripheral nerve regeneration using nerve conduits has been less effective than autogenous nerve grafts. To overcome this hurdle, we developed a tissue-engineered nerve conduit coated with mouse induced pluripotent stem cell (iPSC)-derived neurospheres, for the first time, which accelerated nerve regeneration in mice. We previously demonstrated the long-term efficacy and safety outcomes of this hybrid nerve conduit for mouse peripheral nerve regeneration. In this study, we investigated the therapeutic potential of nerve conduits coated with human iPSC (hiPSC)-derived neurospheres in rat sciatic nerve defects, as a translational preclinical study. The hiPSC-derived quaternary neurospheres containing neural stem/progenitor cells were three-dimensionally cultured within the nerve conduit (poly l-lactide and polycaprolactone copolymer) for 14 days. Complete 5-mm defects were created as a small size peripheral nerve defect in sciatic nerves of athymic nude rats and reconstructed with nerve conduit alone (control group), nerve conduits coated with hiPSC-derived neurospheres (iPS group), and autogenous nerve grafts (autograft group) (n = 8 per group). The survival of the iPSC-derived neurospheres was continuously tracked using in vivo imaging. At 12 weeks postoperatively, motor and sensory function and histological nerve regeneration were evaluated. Before implantation, the hiPSC-derived quaternary neurospheres that three-dimensional coated the nerve conduit were differentiated into Schwann-like cells. The transplanted hiPSC-derived neurospheres survived for at least 56 days after implantation. The iPS group showed non-significance higher sensory regeneration than the autograft group. Although there was no actual motor functional nerve regeneration in the three groups: control, iPS, and autograft groups, the motor function in the iPS group recovered significantly better than that in the control group, but it did not recover to the same level as that in the autograft group. Histologically, the iPS group demonstrated significantly higher axon numbers and areas, and lower G-ratio values than the control group, whereas the autograft group demonstrated the highest axon numbers and areas and the lowest G-ratio values. Nerve conduit three-dimensionally coated with hiPSC-derived neurospheres promoted axonal regeneration and functional recovery in repairing rat sciatic nerve small size defects. Transplantation of hiPSC-derived neurospheres with nerve conduits is a promising clinical iPSC-based cell therapy for the treatment of peripheral nerve defects.

Biosynthesis, characterization and evaluation of the supportive properties and biocompatibility of DBM nanoparticles on a tissue-engineered nerve conduit from decellularized sciatic nerve.

In this study, we examined the supporting effects of nano-demineralized bone matrix on the cultivation of Wharton's jelly stem cells on acellularized nerve scaffold. Demineralized bone matrix nanoparticles were prepared and characterized by several experiments. Decellularized sciatic nerve scaffolds were prepared and their efficiency was evaluated using histological stainings and biomechanical testing. Results of histological staining indicated that the integrity of the extra cellular matrix components was preserved. Also, the growth and viability of WJSCs on the scaffolds were significantly higher in DBM nanoparticle groups. We conclude that supportive properties of nano-DBM groups showed better cell viability and a suitable microenvironment for proliferation, retention, and adhesion of cells compared with other groups.

Research of a novel tissue-engineered nerve graft for sciatic nerve repair in rats

Objective To observe the effect of novel tissue engineered nerve grafts, which combined silk fibroin/collagen scaffold with a co-culture of Schwann cells (SCs) and adipose-derived stem cells (ADSCs) , to repair rat sciatic nerve defects. Methods From February, 2015 to August, 2016, the culture and purify SCs and ADSCs were isolated. And co-cultured at a ratio of 2∶1 and introduced into a silk fibroin (SF)/collagen scaffold to construct a tissue-engineered nerve conduit (TENC), which were transplanted to bridge 10 mm long sciatic nerve defects in rats. The experiment was divided randomly into 4 groups (10 rats/group): those bridged with plain SF/collagen scaffolds(Scaffold group), those bridged with TENCs (TENC group), those bridged with autografts (Autograft group) and those unoperated side (Normal group). The mechanical properties were examined using a universal testing machine(Instron 5865). Scanning electron microscopy was performed to observe the structure of the SF/collagen scaffold and the cells' growth. A series of electrophysiological examinations and morphological analyses were performed 12 weeks after surgery to evaluate the effect of the TENC on peripheral nerve regeneration. And One-way ANOVA was used to analyze the data. If the differences between groups were statistically significant, the Turkey's method was further applied for comparison. Results The plain SF/collagen scaffold showed appropriate pore size and good intercommunicating of holes. The cells were tightly attached to and partly coiled about the scaffold and exhibited either a spindle or a spherical shape. The results of the mechanical measurement revealed that the maximum and average Young’s moduli of the SF/collagen scaffold were (10.80 ± 0.30) MPa and (8.14 ± 0.20) MPa, respectively. The mechanical properties ensure that the scaffold could resist muscular contraction and maintain its shape unchanged for a considerable period of time after grafting. All rats in each group had achieved nerve defect regeneration in varying degrees. But in terms of the effect of the repaired nerve, those treated with TENC were similar to those with autologous nerve grafts but superior to those with plain SF/collagen scaffolds. Conclusion The TENC that combined silk fibroin/collagen scaffold with a co-culture of SCs and ADSCs had normal nerve-like structure, and can bridge sciatic nerve defect and promote nerve growth. Key words: Silk fibroin; Collagen; Schwann cells; Adipose-derived stem cells; Co-culture; Tissue engineered nerve

The Differentiation Stage of Transplanted Stem Cells Modulates Nerve Regeneration

In regenerative medicine applications, the differentiation stage of implanted stem cells must be optimized to control cell fate and enhance therapeutic efficacy. We investigated the therapeutic potential of human induced pluripotent stem cell (iPSC)-derived cells at two differentiation stages on peripheral nerve regeneration. Neural crest stem cells (NCSCs) and Schwann cells (NCSC-SCs) derived from iPSCs were used to construct a tissue-engineered nerve conduit that was applied to bridge injured nerves in a rat sciatic nerve transection model. Upon nerve conduit implantation, the NCSC group showed significantly higher electrophysiological recovery at 1 month as well as better gastrocnemius muscle recovery at 5 months than the acellular group, but the NCSC-SC group didn’t. Both transplanted NCSCs and NCSC-SCs interacted with newly-growing host axons, while NCSCs showed better survival rate and distribution. The transplanted NCSCs mainly differentiated into Schwann cells with no teratoma formation, and they secreted higher concentrations of brain-derived neurotrophic factor and nerve growth factor than NCSC-SCs. In conclusion, transplantation of iPSC-NCSCs accelerated functional nerve recovery with the involvement of stem cell differentiation and paracrine signaling. This study unravels the in vivo performance of stem cells during tissue regeneration, and provides a rationale of using appropriate stem cells for regenerative medicine.

Scaffoldless tissue-engineered nerve conduit promotes peripheral nerve regeneration and functional recovery after tibial nerve injury in rats.

Damage to peripheral nerve tissue may cause loss of function in both the nerve and the targeted muscles it innervates. This study compared the repair capability of engineered nerve conduit (ENC), engineered fibroblast conduit (EFC), and autograft in a 10-mm tibial nerve gap. ENCs were fabricated utilizing primary fibroblasts and the nerve cells of rats on embryonic day 15 (E15). EFCs were fabricated utilizing primary fibroblasts only. Following a 12-week recovery, nerve repair was assessed by measuring contractile properties in the medial gastrocnemius muscle, distal motor nerve conduction velocity in the lateral gastrocnemius, and histology of muscle and nerve. The autografts, ENCs and EFCs reestablished 96%, 87% and 84% of native distal motor nerve conduction velocity in the lateral gastrocnemius, 100%, 44% and 44% of native specific force of medical gastrocnemius, and 63%, 61% and 67% of native medial gastrocnemius mass, respectively. Histology of the repaired nerve revealed large axons in the autograft, larger but fewer axons in the ENC repair, and many smaller axons in the EFC repair. Muscle histology revealed similar muscle fiber cross-sectional areas among autograft, ENC and EFC repairs. In conclusion, both ENCs and EFCs promoted nerve regeneration in a 10-mm tibial nerve gap repair, suggesting that the E15 rat nerve cells may not be necessary for nerve regeneration, and EFC alone can suffice for peripheral nerve injury repair.

A Silk Fibroin/Collagen Nerve Scaffold Seeded with a Co-Culture of Schwann Cells and Adipose-Derived Stem Cells for Sciatic Nerve Regeneration.

As a promising alternative to autologous nerve grafts, tissue-engineered nerve grafts have been extensively studied as a way to bridge peripheral nerve defects and guide nerve regeneration. The main difference between autogenous nerve grafts and tissue-engineered nerve grafts is the regenerative microenvironment formed by the grafts. If an appropriate regenerative microenvironment is provided, the repair of a peripheral nerve is feasible. In this study, to mimic the body’s natural regenerative microenvironment closely, we co-cultured Schwann cells (SCs) and adipose-derived stem cells (ADSCs) as seed cells and introduced them into a silk fibroin (SF)/collagen scaffold to construct a tissue-engineered nerve conduit (TENC). Twelve weeks after the three different grafts (plain SF/collagen scaffold, TENC, and autograft) were transplanted to bridge 1-cm long sciatic nerve defects in rats, a series of electrophysiological examinations and morphological analyses were performed to evaluate the effect of the tissue-engineered nerve grafts on peripheral nerve regeneration. The regenerative outcomes showed that the effect of treatment with TENCs was similar to that with autologous nerve grafts but superior to that with plain SF/collagen scaffolds. Meanwhile, no experimental animals had inflammation around the grafts. Based on this evidence, our findings suggest that the TENC we developed could improve the regenerative microenvironment and accelerate nerve regeneration compared to plain SF/collagen and may serve as a promising strategy for peripheral nerve repair.

Neuroregenerative effects of olfactory ensheathing cells transplanted in a multi-layered conductive nanofibrous conduit in peripheral nerve repair in rats.

BackgroundThe purpose of this study was to evaluate the efficacy of a multi-layered conductive nanofibrous hollow conduit in combination with olfactory ensheathing cells (OEC) to promote peripheral nerve regeneration. We aimed to harness both the topographical and electrical cues of the aligned conductive nanofibrous single-walled carbon nanotube/ poly (L-lactic acid) (SWCNT/PLLA) scaffolds along with the neurotrophic features of OEC in a nerve tissue engineered approach.ResultsWe demonstrated that SWCNT/PLLA composite scaffolds support the adhesion, growth, survival and proliferation of OEC. Using microsurgical techniques, the tissue engineered nerve conduits were interposed into an 8 mm gap in sciatic nerve defects in rats. Functional recovery was evaluated using sciatic functional index (SFI) fortnightly after the surgery. Histological analyses including immunohistochemistry for S100 and NF markers along with toluidine blue staining (nerve thickness) and TEM imaging (myelin sheath thickness) of the sections from middle and distal parts of nerve grafts showed an increased regeneration in cell/scaffold group compared with cell-free scaffold and silicone groups. Neural regeneration in cell/scaffold group was very closely similar to autograft group, as deduced from SFI scores and histological assessments.ConclusionsOur results indicated that the tissue engineered construct made of rolled sheet of SWCNT/PLLA nanofibrous scaffolds and OEC could promote axonal outgrowth and peripheral nerve regeneration suggesting them as a promising alternative in nerve tissue engineering.

Ultrasound imaging of chitosan nerve conduits that bridge sciatic nerve defects in rats.

Ultrasound imaging of chitosan nerve conduits that bridge sciatic nerve defects in rats.

Induced pluripotent stem cells for neural tissue engineering

Induced pluripotent stem cells (iPSCs) hold great promise for cell therapies and tissue engineering. Neural crest stem cells (NCSCs) are multipotent and represent a valuable system to investigate iPSC differentiation and therapeutic potential. Here we derived NCSCs from human iPSCs and embryonic stem cells (ESCs), and investigated the potential of NCSCs for neural tissue engineering. The differentiation of iPSCs and the expansion of derived NCSCs varied in different cell lines, but all NCSC lines were capable of differentiating into mesodermal and ectodermal lineages, including neural cells. Tissue-engineered nerve conduits were fabricated by seeding NCSCs into nanofibrous tubular scaffolds, and used as a bridge for transected sciatic nerves in a rat model. Electrophysiological analysis showed that only NCSC-engrafted nerve conduits resulted in an accelerated regeneration of sciatic nerves at 1 month. Histological analysis demonstrated that NCSC transplantation promoted axonal myelination. Furthermore, NCSCs differentiated into Schwann cells and were integrated into the myelin sheath around axons. No teratoma formation was observed for up to 1 year after NCSC transplantation in vivo. This study demonstrates that iPSC-derived multipotent NCSCs can be directly used for tissue engineering and that the approach that combines stem cells and scaffolds has tremendous potential for regenerative medicine applications.

Characterization of Nerve Conduits Seeded with Neurons and Schwann Cells Derived from Hair Follicle Neural Crest Stem Cells

In this study a tissue-engineered nerve conduit for repair of peripheral nerve defects was devised and characterized in vitro. To construct the nerve conduits, beagle sciatic nerves were acellularized with lysolecithin and seeded with neurons and Schwann cells, which were induced from rat hair follicle neural crest stem cells. The nerve constructs were cultured in vitro and characterized by multiple methods, including immnohistochemistry, electron microscopy, and electrophysiology at 1, 3, and 8 weeks. The same scaffolds injected with phosphate-buffered saline were used as control. We found that hair follicle neural crest stem cell-derived neurons could survive in the nerve constructs as long as 8 weeks, and the nerve constructs showed desirable electrophysiological features. This nerve construct could work as an alternative for the current standard autologous nerve transplantation, especially in peripheral nerves with large defects.

A tissue-engineered bioabsorbable nerve conduit created by three-dimensional culture of induced pluripotent stem cell-derived neurospheres

We previously reported a bioabsorbable nerve conduit coated with Schwann cells for the treatment of peripheral nerve defects. Since there have been dramatic developments in induced pluripotent stem (iPS) cells in recent years, the purpose of the present study was to create a tissue-engineered nerve conduit coated with iPS cell-derived neurospheres. Such a conduit was constructed by three-dimensional (3D)-culture of these cells using a bioabsorbable polymer conduit as a scaffold. The nerve conduit was composed of a mesh of poly L-lactide, and a porous sponge of 50% poly L-lactide and 50% poly ε-caprolactone. The primary and secondary neurospheres (PNS and SNS, respectively) induced from iPS cells were suspended in individual conduits. The conduits were incubated for 7 or 14 days in vitro and then evaluated using immunohistochemistry. All of the 7- and 14-day differentiated PNS and SNS were observed to have adhered to the inner surface of the conduits and to have migrated into the inner porous sponge. The engrafted cells were positive for anti-Tuj1, -S-100 and -GFAP antibodies, indicating that their pluripotent ability to form neural or glial cells was maintained. These findings indicate the feasibility of creating nerve conduits coated with a 3D-culture of iPS cell-derived neurospheres for the treatment of peripheral nerve defects.

Drug Loaded, Biodegradable Nerve Conduits for the Simultaneous Chemical and Electrical Stimulation of Neural Cells as a Therapeutic Approach for Peripheral Nerve Regeneration

Restoring peripheral nerve trauma is an important research field in regenerative medicine. One therapeutical approach is to use tissue engineered nerve conduits consisting of biodegradable polymers. These materials can be designed to include active agents to further stimulate or influence proliferation, maturation, differentiation or migration of specific neuronal cell in these nerve guides. We have developed a method to electrically deposit and immobilize neuronal cells and extracellular matrix proteins on self structured micro electrodes. These electrodes also present a feasible methodology to investigate electrical stimulation of nerve cells. In our approach, poly-D,L-lactide-co-glycolides (PLGA) were investigated as possible substrate for these electrodes, while further allowing for the integration of model substances in a drug release concept. In a first approach, caffeine was used due to its well known effect of both stimulating and inhibiting effects on certain neuronal cells, while also allowing easy incorporation into PLGA via chemical means. A Plackett-Burman experimental design was used to find the optimum composition among different parameters such as drug concentration, polymer concentration, type of solvent and film-drying condition. The optimized drug loaded polymer films were tested for their release and degradation profile, and their behavior in cell culture. Finally, we are currently establishing an integrated experimental setup, combining caffeine modified PLGA film substrates with the manufacturing of the electrode structures to investigate cell deposition via electrical means and stimulation/ inhibition via chemical release.

Recent advances in tissue-engineered nerve conduits

Recent advances in tissue-engineered nerve conduits

Biomaterials and Strategies for Nerve Regeneration

Nerve regeneration is a complex biological phenomenon. Once the nervous system is impaired, its recovery is difficult and malfunctions in other parts of the body may occur because mature neurons do not undergo cell division. To increase the prospects of axonal regeneration and functional recovery, researches have focused on designing "nerve guidance channels" or "nerve conduits." When developing ideal tissue-engineered nerve conduits, several components come to mind. They include a biodegradable and porous channel wall, the ability to deliver bioactive growth factors, incorporation of support cells, an internal oriented matrix to support cell migration, intraluminal channels to mimic the structure of nerve fascicles, and electrical activities. This article reviews the factors that are critical for nerve repair, and the advanced technologies that are explored to fabricate nerve conduits. To more accurately mimic natural repair in the body, recent studies have focused on the use of various advanced approaches to create ideal nerve conduits that combine multiple stimuli in an effort to better mimic the complex signals normally found in the body.

TISSUE ENGINEERING FOR NERVE REPAIR

Nerve regeneration is a complex biological phenomenon. Once the nervous system is impaired, its recovery is difficult and malfunctions in other parts of the body may occur because mature neurons don't undergo cell division. To increase the prospects of axonal regeneration and functional recovery, researches have focused on designing “nerve guidance channels” or “nerve conduits”. For developing tissue engineered nerve conduits, four components come to mind, including a scaffold for axonal proliferation, supporting cells such as Schwann cells, growth factors, and extracelluar matrix. This article reviews the nervous system physiology, the factors that are critical for nerve repair, and the advanced technologies that are explored to fabricate nerve conduits. Furthermore, we also introduce a new method we developed to create longitudinally oriented channels within biodegradable polymers, Chitosan and PLGA, using a combined lyophilizing and wire-heating process. This innovative method using Ni-Cr wires as mandrels to create nerve guidance channels. The process is easy, straightforward, highly reproducible, and could easily be tailored to other polymer and solvent systems. These scaffolds could be useful for guided regeneration after transection injury in either the peripheral nerve or spinal cord.

Alternatives to autologous nerve grafts.

The use of autologous nerve grafts for bridging defects in nerve continuity requires the sacrifice of healthy nerves. Development of alternatives to autologous nerve grafts is therefore well motivated. Such "bioartificial nerve grafts" can be tissue-engineered on the basis of the use of a stroma/matrix/scaffold acting in concert with cells and neurotrophic factors. Such a scaffold can be of synthetic or biological nature and can be resorbable or non-resorbable. It should act together with Schwann cells which can be pre-cultured or acutely dissociated. Neurotrophic factors, stimulating axonal growth, can be incorporated in the scaffold and can also be supplied by cells which are seeded into the stroma. So far, tissue-engineered nerve conduits have been used mainly for experimental purposes in experimental animals. However, an increasing amount of experience from the clinical use of alternatives to nerve grafts now exists, mainly tubes made of silicone or poly-glycolic acid (PGA), veins, collagen and muscle basal laminae.