Nerve Regeneration Chamber Research Articles

Produit d’ingénierie tissulaire comme chambre de régénération nerveuse – vaisseau ombilical traité, déshydraté et stérile

Umbilical vein and arteries are located in the umbilical cord and surrounded by a dense extracellular matrix named Wharton's jelly. Due to their origin, these tissues are highly available. Nerve lesions or sections are common pathologies + healing can be accelerated by the use of a chamber sleeved or wrapped around the nerve. The goal is to develop from umbilical vessels a biologic conduit, secured and dehydrated to be used as nerve regeneration chamber. The umbilical cord is sampled according to directive 2006 17 CE (clinical and biological donor criteria). The vessels are extracted and inverted to expose to have an internal dense Wharton's jelly that takes part in the tissue holding. The vessels are treated with a disinfectant and virally inactivating chemical process. They are freeze-dried and finally gamma sterilized. Viral and microbiological security of the process are evaluated by spiking 10exp6 viruses strain in the Wharton's jelly and determining their destruction after process application. Biocompatibility tests are carried out according to ISO 10993 standard. Viability and attachment of mesenchymal stem cells (MSC) are conducted. Structural properties of the vessels are determined by staining the main proteins and components. Its physical characteristics are evaluated through its tensile mechanical resistance. In vivo implantation of the product on 1 cm sciatic nerve gap in a rat model evaluates its efficacy as a nerve regeneration chamber. The chemical treatment applied reduces the viral load of enveloped and non-enveloped viruses by more than 4 logs. No strain is recovered after the gamma sterilization. The product meets ISO 10993 requirements. MSC are viable and attached all along the product surface. The process applied enables the preservation of the product structure and the maintenance of the main components, collagen IV, elastin, laminin I and hyaluronic acid. The tensile mechanical resistance of the product is 100 kPa. The in vivo study shows effective nerve regeneration after 8 weeks with presence of early myelinated mature axons from the proximal to the distal stumps and a complete integration of the well-tolerated product. The process applied on the umbilical vessels is secured. The final product presents a physiologic structural integrity and an excellent biocompatibility. The tissue engineering product from inverted umbilical vessels is secured and dehydrated. Due to its physical and biological properties, it is an innovative alternative as nerve regeneration chamber, available in different sizes.

Complement components of nerve regeneration conditioned fluid influence the microenvironment of nerve regeneration.

Nerve regeneration conditioned fluid is secreted by nerve stumps inside a nerve regeneration chamber. A better understanding of the proteinogram of nerve regeneration conditioned fluid can provide evidence for studying the role of the microenvironment in peripheral nerve regeneration. In this study, we used cylindrical silicone tubes as the nerve regeneration chamber model for the repair of injured rat sciatic nerve. Isobaric tags for relative and absolute quantitation proteomics technology and western blot analysis confirmed that there were more than 10 complement components (complement factor I, C1q-A, C1q-B, C2, C3, C4, C5, C7, C8β and complement factor D) in the nerve regeneration conditioned fluid and each varied at different time points. These findings suggest that all these complement components have a functional role in nerve regeneration.

Biological conduit small gap sleeve bridging method for peripheral nerve injury: regeneration law of nerve fibers in the conduit.

The clinical effects of 2-mm small gap sleeve bridging of the biological conduit to repair peripheral nerve injury are better than in the traditional epineurium suture, so it is possible to replace the epineurium suture in the treatment of peripheral nerve injury. This study sought to identify the regeneration law of nerve fibers in the biological conduit. A nerve regeneration chamber was constructed in models of sciatic nerve injury using 2-mm small gap sleeve bridging of a biodegradable biological conduit. The results showed that the biological conduit had good histocompatibility. Tissue and cell apoptosis in the conduit apparently lessened, and regenerating nerve fibers were common. The degeneration regeneration law of Schwann cells and axons in the conduit was quite different from that in traditional epineurium suture. During the prime period for nerve fiber regeneration (2–8 weeks), the number of Schwann cells and nerve fibers was higher in both proximal and distal ends, and the effects of the small gap sleeve bridging method were better than those of the traditional epineurium suture. The above results provide an objective and reliable theoretical basis for the clinical application of the biological conduit small gap sleeve bridging method to repair peripheral nerve injury.

Identification of proteins in fluid collected from nerve regeneration chambers

We examined whether there are novel neurotrophic factors (NTFs) in nerve regeneration conditioned fluid (NRCF). Nerve regeneration chamber models were established in the sciatic nerves of 25 New Zealand rabbits, and NRCF was extracted from the chambers l week postoperatively. Proteins in NRCF were separated by native polyacrylamide gel electrophoresis (PAGE), and Western blot and ELISA were used to identify the proteins. A novel NTF was identified in a protein fraction corresponding to 220 kDa.

Human umbilical cord mesenchymal stem cell-loaded amniotic membrane for the repair of radial nerve injury.

In this study, we loaded human umbilical cord mesenchymal stem cells onto human amniotic membrane with epithelial cells to prepare nerve conduits, i.e., a relatively closed nerve regeneration chamber. After neurolysis, the injured radial nerve was enwrapped with the prepared nerve conduit, which was fixed to the epineurium by sutures, with the cell on the inner surface of the conduit. Simultaneously, a 1.0 mL aliquot of human umbilical cord mesenchymal stem cell suspension was injected into the distal and proximal ends of the injured radial nerve with 1.0 cm intervals. A total of 1.75 × 10(7) cells were seeded on the amniotic membrane. In the control group, patients received only neurolysis. At 12 weeks after cell transplantation, more than 80% of patients exhibited obvious improvements in muscular strength, and touch and pain sensations. In contrast, these improvements were observed only in 55-65% of control patients. At 8 and 12 weeks, muscular electrophysiological function in the region dominated by the injured radial nerve was significantly better in the transplantation group than the control group. After cell transplantation, no immunological rejections were observed. These findings suggest that human umbilical cord mesenchymal stem cell-loaded amniotic membrane can be used for the repair of radial nerve injury.

English

The aim of this study was to investigate the function of human insulin-like growth factor-1 (hIGF-1) transgene in vivo towards the regeneration of peripheral nerves. Thirty (30) male Wistar rats were randomly grouped after the sciatic nerve regeneration chamber model was set up. The hIGF-1 Treatment Group (TG) was injected the mixed solution of pcDNAhIGF-1 and LipfectAmine (hIGF-1 DNA was 4 ug) immediately at the spot of crush injury. The Treatment Model Group (MG) was injected 10 ul of the mixed solution of pcDNA3.1, LipfectAmine and physiological saline, while the control group (CG) was not injected any solution. The functional indexes of sciatic nerve were tested after 8 weeks of the injection. At the same time, electrophysiology of the regenerated nerve fibers, histology, morphology and ultrastructural organization observation were carried out. Compared with MG and CG, TG showed significant improvements in the fields of nerve conductive velocity of the regenerated nerve fibers, the greatest amplitude and electric potential of complex musculation electric potential (P<0.01). The axon diameter, medulla sheath thickness and medullated nerve fibers counts of the regenerated nerve fibers of TG were also higher than those of MG and CG (P<0.01). The degree of maturity of the regenerated nerve fibers of TG was better in the ultrastructural organization observation. Furthermore, other functional indexes were improved. hIGF-1 transgene in vivo could promote the regeneration of the injured peripheral nerves.   Key words: Peripheral nerve, insulin-like growth factor-1, electrophysiology, histology.

The experimental study on repair of peripheral nerve rupture using a nerve regeneration chamber created by autologous epineurium small gap coaptauon

Objective To investigate the feasibility and superiority of a nerve regeneration chamber createded by autologous epineurium small gap coaptation in repairing peripheral nerve rupture.Methods A total of 40 Sprague Dawley(SD)rats weighing 250 to 350 g were randomly divided into two groups with 20 rats each.In the experimental group,the sciatic nerve was transected and repaired by autologous epineurium small gap coaptation.In control group,the sciatic nerve was transected and repaired by epineurium neurorrhaphy.At postoperative 2,4,6,8,10,12,14 and 16 weeks,2 SD rats from each group were deeply anesthetized for observation of autonomic activities,the appearance of the regeneration chamber and coaptation sites under the surgical microscope.The structural changes of the sciatic nerve were observed under microscope and transmission electron microscopy.Results The experimental rats recovered autonomic activities sooner.There were minimal adhesions at the outer surface of the epineurial tube to the surrounding tissue.The morphologic changes of the sciatic nerve showed connective tissue hyperplasia was significantly reduced in the small gap group,and the nerve fibers were more orderly arranged.Transmission electron microscope showed that the layers of myelin sheath were arranged compactly and regularly in concentric circles,and Schwann cell hyperplasia was obvious in the small gap group.No such changes were observed in the control group.Conclusion The nerve regeneration chamber created by autologous epineurium small gap coaptation in repairing peripheral nerve rupture is feasible and superior to in situ epineurium neurorrhaphy.This can be a new method to repair nerve ruptures. Key words: Sciatic nerve; Epineurium; Animal experimentation; Small gap bridging

Effect of near-infrared light-emitting diodes on nerve regeneration

BackgroundPhotobiomodulation by red to near-infrared light-emitting diodes (LEDs) has been reported to accelerate wound healing, attenuate degeneration of an injured optic nerve, and promote tissue growth. The purpose of this study was to investigate the effect of LEDs on nerve regeneration. A histological study as well as a measurement of antioxidation levels in the nerve regeneration chamber fluid was performed. MethodsFor the histological study, the bilateral sciatic nerves were transected, and the left proximal stump and the right distal stump were inserted into the opposite ends of a silicone chamber, leaving a 10-mm gap. Light from an LED device (660 nm, 7.5 mW/cm2) was irradiated for 1 h per day. At 3 weeks after surgery, regenerated tissue was fixed and examined by light microscopy. For the antioxidation assay of chamber fluid, the left sciatic nerve and a 2-mm piece of nerve from the proximal stump were transected and inserted into opposite sides of a silicone chamber leaving a 10-mm gap. LEDs were irradiated using the same parameters as those described in the histological study. At 1, 3, and 7 days after surgery, antioxidation of the chamber fluid was measured using an OXY absorbent test. ResultsNerve regeneration was promoted in the LED group. Antioxidation of the chamber fluid significantly decreased from 3 days to 7 days in the control group. In the LED group, antioxidation levels did not decrease until 7 days. ConclusionsChamber fluid is produced from nerve stumps after nerve injury. This fluid contains neurotrophic factors that may accelerate axonal growth. Red to near-infrared LEDs have been shown to promote mitochondrial oxidative metabolism. In this study, LED irradiation improved nerve regeneration and increased antioxidation levels in the chamber fluid. Therefore, we propose that antioxidation induced by LEDs may be conducive to nerve regeneration.

Effects of electro-acupuncture on remyelination of the transected sciatic nerve in rats

Objective To investigate the effects of electro-acupuncture (EA) on remyelination after sciatic nerve was transected in rats, so as to explore the possible mechanism of EA in treatment of peripheral nerve injury. Methods After transection of rat's sciatic nerve, the nerve regeneration chamber was built,then the rats were given EA stimulation at frequencies of 5 Hz and 100 Hz, respectively, and Solochrome Cyanine staining, HE staining of nerve fibers, immunohistochemical staining and semi-quantitative image analysis technique were employed to observe the remyelination of the nerve fibers and S-100 protein expression of Schwann cells. Results EA could significantly promote remyelination of the nerve fibers, elevate S-100 protein expression of Schwann Cells in repairing process of transected sciatic nerve of rats. The therapeutic effect of 5 Hz EA group was better than that of 100 Hz EA group. Conclusion EA is an effective way for the treatment of peripheral nerve injury. It can promote proliferation of Schwann cells and nerve fiber myelination. The therapeutic effects of EA are frequency-depondent. Key words: Electro-acupuncture; Peripheral nerve injury; Remyelination; S-100 protein

Effect of electro-acupuncture on S-100 protein expression of nerve fibers in the repair process of transected sciatic nerve in rats

Objective: To observe the effects of electro-acupuncture on S-100 protein expression of nerve fibers on the regeneration process of transected sciatic nerve of rats, so as to explore the partial mechanism of electro-acupuncture treatment for peripheral nerve injury.Methods: Following rat's sciatic nerve transection, the nerve regeneration chamber model was established, then the rat was given electro-acupuncture stimulation at the frequency of 5 Hz and frequency of 100 Hz, and the immunohistochemical staining method and image analyzing semi-quantitative technique were adopted to determine the percentage of S-100 protein area,hence to observethe effect of electro-acupuncture on the repair process of transected sciatic nerve of rat and the S-100 protein expression. Results: Electro-acupuncture could significantly potentiate the S-100 protein expression of Schwann Cells (SC) in the repair process of transected sciatic nerve. At the 14th week, 17th week and 20th week after operation, the percentages of S-100 protein area were more obviously increased in 5 Hz electro-acupuncture group and 100 Hz electro-acupuncture group than in model group, with significant differences (P＜0.05). Especially, the better effect was obtained in 5 Hz electro-acupuncture group. Judging from the dynamic treatment, it is very vital to be treated early. Conclusion: Electro-acupuncture is an effective method for the treatment of peripheral nerve injury, and its therapeutic effect is related to the improvement of SC proliferation.

Study of in vivo differentiation of rat bone marrow stromal cells into schwann cell-like cells

In order to raise an abundant and accessible reservoir for Schwann cells (SCs), which are used as seed cells for constructing tissue-engineered nerve grafts, we investigated the feasibilty of in vivo differentiation of bone marrow stromal cells (MSCs) into SC-like cells. In this study, MSCs were harvested from adult rats' bone marrow, culture-expanded, and characterized. Subcultured MSCs were then labeled with Hoechst 33342, followed by transplantation into the nerve regeneration chamber, which was made of a silicone tube bridging the sciatic nerve defect of the rats. Four weeks after surgery, some of the differentiated MSCs turned into SC-like cells immunopositive to S-100 protein, accompanied by myelination of the regenerated nerve fibers. Walking-track analyses provided evidence that transplantation of MSCs contributed to reconstruction of the sciatic nerve and reinnervation of target tissues. The experimental results suggest that MSCs are capable of differentiating into SC-like cells in vivo, making them a promising candidate for cell transplantation in peripheral nerve repair.

An injectable nerve regeneration chamber for studies of unstable soluble growth factors

Modern surgical techniques cannot guarantee functional recovery following peripheral nerve injuries. Research into factors that may influence nerve regeneration has therefore assumed a prominent potential therapeutic role. We report here on the development of an approach to allow for direct manipulation of the microenvironment of regenerating peripheral nerve axons. We show that solutions can be delivered directly to this local milieu in vivo and that such a delivery can be performed multiple times over an extended period, potentially facilitating studies of multiple molecular players that act locally. We also demonstrate that the bundle of regenerated axons are amenable to morphological analysis by 21 days and that the injection system remains patent for at least 21 days.

VEGF enhances intraneural angiogenesis and improves nerve regeneration after axotomy

Whilst there is an increased understanding of the cell biology of nerve regeneration, it remains unclear whether there is a direct interrelationship between vascularisation and efficacy of nerve regeneration within a nerve conduit. To establish this is important as in clinical surgery peripheral nerve conduit grafting has been widely investigated as a possible alternative to the use of nerve autografts. The aim of this study was to assess whether vascular endothelial growth factor (VEGF), a highly specific endothelial cell mitogen, can enhance vascularisation and, indirectly, axonal regeneration within a silicone nerve regeneration chamber. Chambers containing VEGF (500-700 ng/ml) in a laminin-based gel (Matrigel) were inserted into 1 cm rat sciatic nerve defects and nerve regeneration examined in relation to angiogenesis between 5 and 180 d. Longitudinal sections were stained with antibodies against endothelial cells (RECA-1), axons (neurofilament) and Schwann cells (S-100) to follow the progression of vascular and neural elements. Computerised image analysis demonstrated that the addition of VEGF significantly increased blood vessel penetration within the chamber from d 5, and by d 10 this correlated with an increase of axonal regeneration and Schwann cell migration. The pattern of increased nerve regeneration due to VEGF administration was maintained up to 180 d, when myelinated axon counts were increased by 78 % compared with plain Matrigel control. Furthermore the dose-response of blood vessel regeneration to VEGF was clearly reflected in the increase of axonal regrowth and Schwann cell proliferation, indicating the close relationship between regenerating nerves and blood vessels within the chamber. Target organ reinnervation was enhanced by VEGF at 180 d as measured through the recovery of gastrocnemius muscle weights and footpad axonal terminal density, the latter showing a significant increase over controls (P < 0.05). The results demonstrate an overall relationship between increased vascularisation and enhanced nerve regeneration within an acellular conduit, and highlight the interdependence of the 2 processes.

Motoneurotrophins derived from limb buds protect the motoneurons in anterior spinal cord after nerve injury and promote nerve regeneration

The effects of limb bud-derived motoneurotrophins (LBMNTs) as seen in the motoneurons in the anterior spinal cord and sciatic nerve regeneration of adult rats, were evaluated in the present study. A nerve regeneration chamber with a nerve gap of 9 mm was created by suturing the proximal and distal ends of a random sciatic nerve into a silicone tube after removal of a 5 mm piece of nerve in the distal end, The chamber of the experimental group was filled with 34.34 μg LBMNTs and PBS (0.01 mol/ml, pH 7.0),and the control group with PBS only. At 1 day, 4 days, 1 week, 2 weeks, 4 weeks and 6 weeks post surgery, the content of acetylcholine esterase (AchE) and acid phosphatase (ACP) of the anterior spinal cord (injured side) was quantified, and the corresponding motoneuron's ultrastructure and the existant ratio were also examined. Meanwhile, the regenerated nerve from within the silicone tube was examined at 2, 4 and 6 weeks post surgery for histological studies at both the light microscopic and ultrastructural levels. The experimental group showed a smaller decrease of AchE and an increase of ACP, a larger existant ratio of motoneurons, better ultrastructure and a more mature regenerated nerve based on a larger diameter of the regenerated nerve trunk, a greater number of axons and thicker myelin sheaths than the control group. So it was concluded that LBMNTs had a high activity of protecting motoneurons in the anterior spinal cord after nerve injury and promoting nerve regeneration, and it may be a new source of neurotrophic factors (NTFs).

A diffusion-reaction model of nerve regeneration

The process of peripheral nerve regeneration has been modeled using 5 populations of mathematical variables to represent the biological activities of Wallerian degeneration, fibrin matrix development, Schwann cell activity, elongating neurites, and neovascularization. The mathematical model provided simulations of nerve regeneration following transection and crush injuries that correspond with growth behaviors quantified in biological experiments. Neovascularization was spatiotemporally quantified in nerve regeneration chambers and following nerve crush injury in order to test the simulations of the mathematical model. The vasculature in both the chamber and following nerve crush responded as predicted by the model, increasing beyond normal levels to a peak only to decrease back to normal. This behavior appeared as a traveling wave in the proximal-distal direction preceding the major thrust of neuritic outgrowth suggesting that development of the vasculature is a rate-limiting step in nerve regeneration.

Multiple neurotrophic factors including NGF-like activity in nerve regeneration chamber fluids

Silicone nerve regeneration chambers were implanted between the cut ends of the sciatic nerve of adult rats. Neurotrophic activities in cell-free fluids collected from the chambers were determined using bioassays for survival of embryonic chick ciliary and sympathetic neurons in culture. Separation by molecular exclusion HPLC of the components of fluids collected l, 2 or 3 days after implantation revealed the presence of a multitude of neurotrophic factors differing in their molecular weights, specificity towards the two types of neurons, and time course. Antiserum to nerve growth factor partially blocked sympathetic activity of fluids collected at 1 day. Affinity purified antibody was also effective and completely eliminated bioactivity of HPLC fractions corresponding to the molecular weight of nerve growth factor. The presence in the fluids of 13–18 and 20–32 kD components active towards ciliary neurons is consistent with the release of fibroblast growth factor and ciliary neurotrophic factor respectively. The stimulation of sympathetic neurons by the 13–18 kD material, and also by 4–6 and 7–11 kD components cannot be entirely accounted for by known factors. This study demonstrates that a number of neurotrophic factors, which differ in their specificity towards sympathetic and parasympathetic neurons, are made available to the region of axonal regrowth over the first few days of regeneration. Contrary to earlier reports, nerve growth factor-like activity was shown to be present in nerve regeneration chambers.

The vascular response to nerve transection: neovascularization in the silicone nerve regeneration chamber

The rat sciatic nerve regeneration chamber was used to study the spatial and temporal response of the endoneurial vasculature during regeneration. Proximal and distal stumps of a transected rat sciatic nerve were placed in opposite ends of a silicone tube and allowed to regenerate for periods of 2, 3, 4 or 52 weeks after the surgery. Serial, transverse sections of nerve were studied at each time-point to quantitate the number of vessels, capillary density and the vessel luminal perimeter per nerve area. The results indicate that the vascular growth relative to the existing tissue in the chamber increases to a peak beyond normal levels and later decreases to values associated with control tissue. While this growth occurred from both proximal and distal stumps, it appeared predominately as a traveling wave in the proximal-distal direction preceding the major thrust of neuritic outgrowth from the proximal stump. Morphologic measurements of angiogenesis were paralleled in other animals by measurements of nerve blood flow using laser Doppler flometry at corresponding time-points. These data differ somewhat from previous reports of angiogenesis following nerve crush injury and are useful in formulating a general mathematical model of regeneration in the peripheral nervous system.

Experimental studies on peripheral nerve regeneration enhanced by nerve growth factor.

The effect of exogenous nerve growth factor (NGF) examined on the neural repair of adult rabbit sciatic nerve was evaluated in the present study. A nerve regeneration chamber was created by suturing the proximal and distal ends of a transected sciatic nerve into a silicone chamber. A gap of 6 mm in chamber was left after removal of a 3 mm piece of nerve in the distal ends and insertion of the proximal and distal stumps into the chamber. Animals were operated on bilaterally, one side of the chamber was filled with a 1 mg/ml NGF/normal saline (NS) (experimental) and the contralateral side with NS (control). The regenerated nerves from within the silicone chamber were dissected and fixed 1 to 5 weeks following surgery for histological studies at both the light microscopic and ultrastructural levels. The NGF chamber showed a more mature regenerated nerve based on a larger diameter of the regenerated nerve trunk, a great number of axons, and thicker myelin sheaths.

Inflammatory cells and mediators in the silicone chamber model for nerve regeneration

In the present study the inflammatory response was quantitatively evaluated during peripheral nerve regeneration. The fluid from silicone nerve regeneration chambers, inserted in rats, was collected during the early period of regeneration of transected sciatic nerves (6 h-7 d) and analysed with respect to inflammatory cells and mediators (leukotriene B 4, LTB 4, and interleukin-1α, IL-1α). Leucocytes were detected during the entire period (up to 7d after implantation). The highest concentration was detected after 24 h. PMNG (polymorphonuclear granulocyte) was the predominant cell type in the chamber fluid during the initial 5d of regeneration. Analysis of the concentration of LTB 4 demonstrated two peaks (at 24 h and 5d). The IL-1α concentration displayed an early and relatively smaller peak after 24 h and a second and much larger peak after 7d, concomitant with an increase of the number of mononuclear cells.

Blot and culture analysis of neuronotrophic factors in nerve regeneration chamber fluids

The fluid accumulating in silicone nerve regeneration chambers implanted between the cut ends of rat sciatic nerve contains neuronotrophic activities towards embryonic chick ciliary and sympathetic neurons. The blot and culture technique of Carnow et al. was used to determine if part of the neuronotrophic activities is due to ciliary neuronotrophic factor, which supports the survival of both types of neurons in vitro. The technique involves separating the fluid proteins by SDS-polyacrylamide gel electrophoresis, Western transfer, and then culturing of purified neurons on the nitrocellulose blots. After 24 hr surviving neurons are restricted to regions of the blot where neuronotrophic factor is present. Analysis of 1 and 2 day fluids showed that a multitude of factors are present, particularly in the 19-30 kD molecular weight range, with discrete peaks of activity at molecular weights consistent with those reported for ciliary neuronotrophic factor. There were several other peaks of activity present in the fluids in addition to these.