The Collagen Scaffolds Loaded with Engineered Basic Fibroblast Growth Factor Facilitate the Recovery of Sciatic Nerve Transection
|School||Southern Medical University,|
|Keywords||CBD-bFGF Linear ordered collagen scaffold (LOCS) Collagen tube Targeting repair Sciatic nerve regeneration|
Background and object:Peripheral nerve injury, which results in poor recovery of function and subsequent impaired quality of life for the people, is a common problem in society today. It may lead to the loss of the sensory and motor function as a result of birth trauma, accidents, and acts of violence. After injury, axons could regenerate from neurons. Despite its own capacity for repair, the functional recovery is not satisfactory. According to extent and severity of the injuries and the distance and time required for axons to regenerate, the functional recovery is different. In humans and animals, axon regeneration is at low speeds of1or3mm/day. Based on nerve fiber injury and subsequent sparing or loss of nerve continuity, the first clinical classification scheme includes three types:(1). Nerve compression with or without demyelination;(2). Axon transection with both perineurium and epineurium remaining.(3). Nerve transection where the continuity of the epineurium is disrupted. Remyelination and axon regeneration may contribute to functional recovery after injuries.Artificial nerves are typically developed to elicit axon regeneration to facilitate of functional recovery. Various nerve conduits including biodegradable and nonbiodegradable materials have been used to promote the nerve regeneration in the past few years and achieved some progress. However, their disadvantages have inspired the search for other strategies that are more effective for peripheral nerve reconstruction.Collagen material is one of the best-characterized scaffolds in tissue engineering. Known to its low antigenecity and excellent biocompatibility, it is widely used for tissue regeneration. In this work, a collagen tube was produced to serve as a physical bridge to the lesion between the proximal and distal stumps of the disconnected nerve. Previous study showed that the nerve regenerate in a misdirected way may result in loss of function. Linear ordered collagen scaffold (LOCS), another collagen material, was proved to be a good nerve guidance material in our previous work. Besides that, linear ordered collagen scaffold could also be used to bind neurotrophic factors or as a back-bone to construct a drug delivery system.On the other hand, growth factors are also important in promoting the nerve growth in the peripheral nervous system (PNS). Among them, basic fibroblast growth factor (bFGF) is one of the important factors that have been reported to promote neurite extension. With regard to the effects of bFGF on Schwann cells, it was previously demonstrated from in vitro and in vivo studies that this molecule could stimulate Schwann cell proliferation.However, simple delivery of bFGF solution is far from satisfactory because of its short half-life and rapid diffusion in the flushing of body fluids. In order to maintain an appropriate concentration at the injury site, periodic injection or large doses is usually applied which may result in infection and serious side effects. Previous research showed that the collagen-binding domain (CBD) could bind in a saturable fashion to the native collagen. Therefore, we fused CBD to native(NAT) bFGF and created the CBD-bFGF which showed notable collagen-binding ability without impacting the cytokine activity. Afterwards, it was loaded on the linear ordered collagen scaffold to control its release rate.Object:In this research, a natural biological functional scaffold was constructed by collagen tube filled with CBD-bFGF loaded-LOCS with an aim to promote the regeneration in a5mm long rat sciatic nerve transection model.Methods and materials(1) Preparation of protein CBD-bFGF and NAT-bFGF.The CBD-bFGF was produced as previous described with simply modified. Gene of CBD-bFGF was amplified using polymerase chain reaction (PCR) and inserted to the expression vector pET-28a. The constructed expression vector for bFGF with CBD (pET-CBD-bFGF) was transformed into BL21(DE3) strain of E.coli. After that, the expression of the proteins were induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and cultured. Then the nickel chelation chromatography was used to purify the targeted proteins from supernatants. The NAT-bFGF without collagen-binding domain was also constructed in the same way. Bradford method was used to quantify the concentration of protein solution.(2) Preparation of LOCS and collagen tube. LOCS which derived from bovine aponeurosis were prepared according to previous report. Collagen tube was derived from collagen membranes. After scrolled on molds, collagen membranes were cross-linked. Then the collagen tube was washed using NaH2PO4and distilled water followed by freeze-drying. LOCS in5mm long and collagen tube in7mm long were sterilized by irradiation. CBD-bFGF, NAT-bFGF and PBS were loaded on LOCS respectively.(3) Surgical procedure. All rats were randomized into3groups containing:nerve gap bridged by collagen tube+LOCS+CBD-bFGF group, nerve gap bridged by collagen tube+LOCS+NAT-bFGF group, nerve gap bridged by collagen tube+LOCS+PBS group. Appropriate anaesthesia depth was met by the intraperitoneal injection of sodium pentobarbital for each rat. After exposure of right sciatic nerve on the right side, a3-mm long segment was transected to create a5-mm-long defect due to the retraction of the nerve ends. Then the7mm long collagen tube which filled with a bunch of LOCS fibers carrying CBD-bFGF, NAT-bFGF or PBS was sutured by9/0monofilament nylon interrupted sutures to the proximal and distal ends of the transected sciatic nerve. Six weeks and twelve weeks post-transplantation, animals were sacrificed using an overdose injection of sodium pentobarbital.(4) Walking track analysis. Sciatic function index (SFI) value was calculated using the method proposed by Bain et al. The leaving footprints on white paper were recorded to calculate sciatic function index value.(5) Electrophysiological evaluation. Twelve weeks after surgery, the rats were anaesthetized as mentioned above. After re-expose the regenerated sciatic nerve, the crook-shaped bipolar stimulating electrode was placed on the proximal end of the nerve while another two record electrodes were placed on the proximal and distal end of the nerve. Nerve conduction velocity (NCV) was recorded by an electromyography system.(6) FG retrograde tracing. At weeks10, the rats were sedated under conditional anesthesia. The sciatic nerve was re-exposed as described above. Fluorogold was injected at the site of the distal end. After closed in layers, the rats were allowed to survive for two weeks. The rats were sacrificed at the12th week, the dorsal root ganglia (DRG) at L4-L6was dissected out and cut with a cryostat The fluorescent neurons with bright golden-color were counted in each section under a fluorescence microscope.(7) Histological investigation and morphometric analysis. At6and12weeks following implantation, the newborn nerve at the injured site was harvested. After post-fixed, the regenerated nerve was embedded in paraffin and cut on a cryostat. Each section was submitted to immunohistochemistry using anti-neurofilament(NF) and anti-S100. The percentage of NF and S-100(positive staining area/total area) were calculated. Some sections were stained with Luxol fast blue. The number and diameter of the newborn myelin sheath were evaluated by Image-Pro Plus software. The thickness of the remyelinated nerves was observed by transmission electron microscope. Some newborn nerves were stained with hematoxylin and eosin(HE) to detect the linear ordered structure.(8) Muscle-mass ratio and Masson trichrome staining. The weight ratio of the gastrocnemius muscles was measured. Immediately after the rats were sacrificed, the two sides of gastrocnemius muscles were harvested and weighed on electronic balance while they were still wet. Muscle-mass ratio was calculated by dividing the experimental-side muscle mass weight by the contralateral-side muscle mass weight. After that, the mid-belly of the muscle samples was submitted to Masson trichrome staining. The percentage of muscle (positive staining area/total area) was calculated.Statistical analysisAll statistical analysis was performed with one-way analysis of variance (ANOVA) and S-N-K post hoc test for multiple comparison using SPSS13.0software. The data were presented as means±SEM. P values<0.05or<0.01was considered statistically significant and expressed as*P<0.05or**P<0.01. Results:(1) General conditions of nerve grafting. Twelve weeks post-graft implantation, the gap at the injury site was bridged continuously. The collagen tube was absorbed and replaced by a tissue with nerve-like appearance. There was no serious adhesion of regenerated nerve to the tissue around.(2) Walking track analysis. The SFI value ranged from-100to0, nearly0represented normal nerve function and nearly-100represented completely loss of nerve function. Before the fourth week, there was no significant difference between three groups. From week5, the SFI value exhibited significant difference between the three grafted groups, SFI value in LOCS+NAT-bFGF group was superior to that in the PBS group but inferior to that in the LOCS+CBD-bFGF group.(3) Electrophysiological evaluation. We noted that the operated rats of each group had recovered at different level at the12th week. The NCV in LOCS+CBD-bFGF group and LOCS+NAT-bFGF group showed significant restoration compared with the LOCS+PBS group. And there was significant difference between LOCS+CBD-bFGF group and LOCS+NAT-bFGF group.(4) FG retrograde labeling. At week12after injury, FG-labeled sensory neurons in the dorsal root ganglia were displayed Golden-color under UV excitation (Fig.5A). The labeled cells were recorded for each group. The statistical results indicated that the group treated with LOCS+CBD-bFGF exhibited significantly better sensory nerve fibers recovery compared with the LOCS+NAT-bFGF group and LOCS+PBS group. The number of labeled cells was higher in LOCS+NAT-bFGF group than that in the LOCS+PBS group.(5) Histological and morphometric analysis of regenerated nerve. From the HE staining, we could see that the regenerated nerve guided by LOCS in the three groups (LOCS+CBD-bFGF group, LOCS+NAT-bFGF group and LOCS+PBS group) all exhibited good linear ordered structure. To evaluate the number and distribution of axons in the newborn nerve, we stained sections with anti-NF antibody. As shown in the figure, The LOCS+CBD-bFGF group displayed the best axonal recovery while the PBS group displayed only sparsely NF-positive area at week6and week12after the injury. The difference was statistically significant among three groups. S-100staining was used for assessment of the proliferation of Schwann cell in the regenerated nerve which was important for the successful axonal regeneration after injury. Quantitative analysis showed that the S-100positive area in the LOCS+CBD-bFGF group was significantly higher than that in LOCS+NAT-bFGF group and LOCS+PBS group at week6and week12. Moreover, in contrast to the LOCS+PBS group, the S-100positive area in the LOCS+NAT-bFGF group was significantly higher.(6) Remyelination analysis. After the staining by Luxol fast blue, the number and the diameter of the remyelinated axons were evaluated. The number and the diameter of the remyelinated axons exhibited progressive increase from LOCS+PBS group to LOCS+NAT-bFGF group to LOCS+CBD-bFGF group at week6and week12except that the difference was not statistically significant between LOCS+NAT-bFGF group and LOCS+PBS group at week6. The thickness of regenerated myelin sheaths was measured by transmission electron microscope (TEM)(Fig.7D). The LOCS+NAT-bFGF group showed significant thinner sheath than that in the LOCS+PBS group but smaller than that in the LOCS+CBD-bFGF group.(7) Muscle mass measurement and Masson trichrome staining. In contrast to the PBS group and LOCS+NAT-bFGF group, the weight ratio was higher in LOCS+CBD-bFGF group. However, there was no significant difference in the weight ratio between the LOCS+NAT-bFGF group and LOCS+PBS group. The percentage of muscle fibers positive area was calculated. There was significant difference between LOCS+PBS group, LOCS+NAT-bFGF group, LOCS+CBD-bFGF group. The percentage in the normal muscle was the highest while the percentage in the LOCS+PBS group was the lowest. The percentage in LOCS+CBD-bFGF group was higher than that in LOCS+NAT-bFGF group.ConclusionIn this study, the collagen tube, LOCS and engineered bFGF were combine used as a natural biological functional scaffold to exert synergistic effect to bridge a5-mm-long gap in the rat sciatic nerve transection model. Retained at a higher concentration compared with NAT-bFGF at the injury site, CBD-bFGF loaded on the collagen scaffolds could promote nerve regeneration and functional recovery more effectively. It is expected that the natural biological functional scaffolds could serve as a promising approach for the regeneration of peripheral nerve defects.