Objective To observe the histomorphology and the biocompatibil ity of acellular nerve prepared by different methods, to provide the experimental evidence for the selection of preparation of acellular nerve scaffold. Methods Forty-eight adult Sprague Dawley rats, male or female, weighing 180-220 g, were selected. The sciatic nerves were obtained from 30 rats and were divided into groups A, B, and C (each group had 20 nerves). The acellular sciatic nerves were prepared by the chemical methods of Dumont (group A), Sondell (group B), and Haase (group C). The effect to remove cells was estimated by the degree of decellularization, degree of demyel ination, and intergrity of nerve fiber tube. The histocompatibil ity was observed by subcutaneous implant test in another 18 rats. Three points were selected along both sides of centre l ine on the back of rats, and the points were randomly divided into groups A1, B1, and C1; the acellular nerve of groups A, B, and C were implanted in the corresponding groups A1, B1, and C1. At 1, 2, and 4 weeks after operation, the rats were sacrificed to perform the general observation and histological observation. Results The histomorphology: apart of cells and the dissolved scraps of axon could be seen in acellular never in the group A, and part of Schwann cell basilar membrane was broken. In group B, the cells in the acellular never were not removed completely, the Schwann cell basilar membrane formed bigger irregular hollows, part of the Schwann cell basilar membrane was broken obviously. But in the group C, the cells were completely removed, the Schwann cell basilar membrane remained intactly. Group C was better than group A and group B in the degree of decellularization, degree of demyel ination, integrity of nerve fiber tube and total score, showing significant differences (P lt; 0.05). The subcutaneous implant test: there were neutrophils and lymphocytes around the acellular nerve in 3 groups at 1 week after implant. A few of lymphocytes were observed around the acellular nerve in 3 groups at 2 weeks after implant. The inflammation was less in groups A1, B1, and C1 at 4 weeks after implant, part of the cells grew into the acellular nerve and arranged along the Schwann cell basilar membrane. The reaction indexes of the inflammational cells in group A1 and group B1 were higher than that in group C1 at 1, 2, and 4 weeks after implant, showing significant differences (P lt; 0.01), but there was no significant difference between group A1 and group B1 (P gt; 0.05). Conclusion The acellular sciatic nerves prepared by Haase method has better acellular effect and the histocompatibil ity than those by the methods of Dumont and Sondell.
Objective Poly (propylene carbonate) (PPC), a newly reported polymer, has good biodegradabil ity and biocompatibil ity. To explore the feasibil ity of using electrospinning PPC materials in nerve tissue engineering, and to observe the effect of al igned and random PPC materials on axonal growth of rat dorsal root gangl ions (DRGs) in vitro. Methods Either al igned or randomly oriented sub-micron scale polymeric fiber was prepared with an electrospinning process. DRGs were harvested from 3 newborn Sprague-Dawley rats (female or male, weighing 4-6 g), and were incubated into 12-pore plate containing either al igned (the experimental group, n=6) or randomly oriented sub-micron scale polymeric fiber (the control group, n=6). The DRGs growth was observed with an inverted microscope; at 7 days immunofluorescent staining and scanning electronic microscope (SEM) observation were performed to quantify the extent of neurite growth andSchwann cells (SCs) migration. Results Either al igned or random fibers were fabricated by an electrospinning process. The diameter of the individual fiber ranged between 800 nm and 1 200 nm. In al igned PPC material, 90% fibers arranged in long axis direction, but the fibers in random PPC material arranged in all directions. The DRGs grew well in 2 PPC materials. Onthe al igned fiber film, the majority of neurite growth and SCs migration from the DRGs extended unidirectionally, parallel to the al igned fibers; however, neurite growth and SCs migration on the random fiber films oriented randomly. The extents of neurite growth were (2 684.7 ± 994.8) μm on the al igned fiber film and (504.7 ± 52.8) μm on the random fiber films, showing significant difference (t= —5.360, P=0.000). The distances of SCs migration were (2 770.6 ± 978.4) μm on the al igned fiber film and (610.2 ± 56.3) μm on the random fiber films, showing significant difference (t= —5.400, P=0.000). The extent of neurite growth was fewer than the distances of SCs migration in 2 groups. Conclusion The orientation structure of sub-micron scalefibers determines the orientation and extent of DRGs neurite growth and SCs migration. Al igned electrospinning PPC fiber is proved to be a promising biomaterial for nerve regeneration.
Objective To review the research progress of electrospun nanofibers scaffold in nerve tissue engineering. Methods The related l iterature on electrospun nanofibers scaffold in nerve tissue engineering was extensively reviewed and analyzed. Results A variety of material nanofibers scaffolds can be fabricated through electrospinning. The chemical and physical properties of the scaffold can be modified and it was suitable for neuron. The scaffold can bridge the defect of peripheral nerve and partial function can be restored. Conclusion Electrospun nanofibers scaffold has broad appl ication prospects in nerve tissue engineering.
Objective To review the fundamental research and the experimental study in the nerve tissue engineering of self-assembl ing peptide nanofiber scaffold (SAPNS). Methods The l iterature concerning basic and experimental studies on SAPNS in the nerve tissue engineering was extensively reviewed. Results SAPNS can promote the neural stem cell adhesion,prol iferation, differentiation and neuron axon outward growth and extension, promote extracellular matrix synthesis and inhibit gl ial cell adhesion and differentiation, and simulate the environment of a cell in the body. Conclusion SAPNS is an ideal matrix material and provides a new way for the repair of nerve tissue injury.
ObjectiveTo study the effect of the loaded concentration gradient of nerve growth factor (NGF) immobilized conduit on rat peripheral nerve defect repair. MethodsThe peripheral nerve conduits made of poly (ε-caprolactone)-block-poly (L-lactide-co-ε-caprolactone) were prepared with uniform loads or concentration gradient loads by combining differential absorption of NGF/silk fibroin (SF) coating, and the gradient of NGF was immobilized in the nerve conduits. ELISA method was used to exam the NGF release for 12 weeks in vitro. Twenty-four male Sprague Dawley rats (weighing, 220-250 g) were selected to establish the right sciatic nerve defect model (14 mm in length) and randomly divided into 4 groups according to repair methods. The transected nerve was bridged by a blank conduit without NGF in group A, by a conduit containing uniform loads of NGF in group B, by a conduit concentration gradient loads of NGF in group C, and by the autogenous nerve segment in group D. The gross observation, electrophysiological examination, histological observation, and transmission electron microscope observation were carried out to assess the nerve regeneration at 12 weeks after surgery. ResultsThe cumulative release amount of NGF was (14.2±1.4) ng/mg and (13.7±1.3) ng/mg in gradient of NGF loaded conduits and uniform NGF loaded conduits respectively at 12 weeks, showing no significant difference (t=0.564, P=0.570). All the animals survived to completion of the experiment; plantar ulcers occurred at 4 days, which healed at 12 weeks; groups C and D were better than groups A and B in ulcerative healing. At 12 weeks after surgery, the compound muscle action potential of group A was significantly lower than that of groups B, C, and D (P<0.05), and group B was significantly lower than groups C and D (P<0.05), but no significant difference was found between groups C and D (P>0.05). The axon density of group C was significantly higher that of groups A, B, and D (P<0.05); group D was significantly higher than groups A, B, and C, and group C was significantly higher than groups A and B in the axon number, axon diameter, and area of muscle fiber (P<0.05); the thickness of myelin sheath of groups C and D was significantly larger than that of groups A and B (P<0.05), but no significant difference was found between groups C and D (P>0.05). ConclusionGradient of NGF loaded nerve condnits for rat sciatic nerve defect has similar results to autogenous nerve, with a good bridge, which can promote the sciatic nerve regeneration, improve the myelinization of the regenerating nerve, and accelerate the function reconstruction of the regenerating nerve.
ObjectiveTo summarize the applications of Schwann cells (SCs), stem cells, and genetically modified cells (GMCs) in repair of peripheral nerve defects. MethodsThe literature of original experimental study and clinical research related with SCs, stem cells, and GMCs was reviewed and analyzed. ResultsSCs play a key role in repair of peripheral nerve defects; the stem cells can be induced to differentiate into SCs, which can be implanted into nerve conduits to promote the repair of peripheral nerve defect; genetically modified technology can enhance the function of SCs and different stem cells, which has been regarded as a new option for tissue engineered nerve. ConclusionAlthough great progress has been made in tissue engineered nerve recently, mostly limited to the experimental stage. The research of seed cells in application of tissue engineered nerve need be studied deeply.
ObjectiveTo investigate the differentiation of rat adipose-derived stem cells (ADSCs) into neuronlike cells by indirect co-culture with Schwann cells (SCs) in vitro so as to look for the ideal seed cells for tissue engineering. MethodsSCs were isolated from sciatic nerves of 1-2 days old Sprague-Dawley rats with enzymatic digestion method. Immunofluorescence staining was used to identify SCs with the marker S-100. ADSCs were isolated from the epididymal fat pads of adult male Sprague-Dawley rats by means of differential attachment. And the cell phenotypes (CD29, CD34, CD45, CD73, CD90, and CD105) of ADSCs at passage 3 were determined by flow cytometry analysis. Primary SCs and ADSCs at passage 3 were co-cultured at a ratio of 2:1 in Transwell culture dishes (experimental group), and ADSCs cultured alone served as control group. Immunofluorescence and flow cytometry were adopted to investigate the neural differentiation of ADSCs at 14 days. The expression differences for neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP2), neuronal nuclei protein (NeuN), and glial fibrillary acidic protein (GFAP) were detected, and the percentage of positive cells was calculated. ResultsADSCs were successfully extracted and can passage in a considerable large amount. Flow cytometry analysis showed that ADSCs at passage 3 were positive for CD29, CD90, CD73, and CD105 expression, but negative for CD34 and CD45 expression. The ADSCs of the experimental group showed contraction of nucleus, increasing of soma refraction, and several long and thick protrusions of cell body. The cell shape had no obvious change in the control group. Both immunofluorescence and flow cytometry analysis results showed the expressions of MAP2, NSE, NeuN, and GFAP at 14 days after co-cultured with SCs, and the positive cell ratios were significantly higher than those in the control group (P<0.01). ConclusionCo-culture with SCs not only can promote the survival regeneration of ADSCs, but also can induce the differentiation of ADSCs into neuron-like cells.
Objective To review the research progress of graphene and its derivatives in repair of peripheral nerve defect. Methods The related literature of graphene and its derivatives in repair of peripheral nerve defect in recent years was extensively reviewed. Results It is confirmed by in vitro and in vivo experiments that graphene and its derivatives can promote cell adhesion, proliferation, differentiation and neurite growth effectively. They have good electrical conductivity, excellent mechanical properties, larger specific surface area, and other advantages when compared with traditional materials. The three-dimensional scaffold can improve the effect of nerve repair. Conclusion The metabolic pathways and long-term reaction of graphene and its derivatives in the body are unclear. How to regulate their biodegradation and explain the electric coupling reaction mechanism between cells and materials also need to be further explored.
Objective To explore a green route for the fabrication of thermo-sensitive chitosan nerve conduits, improve the mechanical properties and decrease the degradation rate of the chitosan nerve conduits. Methods Taking advantage of the ionic specific effect of the thermo-sensitive chitosan, the strengthened chitosan nerve conduits were obtained by immersing the gel-casted conduits in salt solution for ion-induced phase transition, and rinsing, lyophilization, and 60Co sterilization afterwards. The nerve conduits after immersing in NaCl solutions for 0, 4, 12, 24, 36, 48, and 72 hours were obtained and characterized the general observation, diameters and mechanical properties. According to the above results, the optimal sample was chosen and characterized the microstructure, degradation properties, and cytocompatibility. The left sciatic nerve defect 15 mm in length was made in 20 male Sprague Dawley rats. The autologous nerves (control group, n=10) and the nerve conduits (experimental group, n=10) were used to repair the defects. At 8 weeks after operation, the compound muscle action potential (CMAP) was measured. The regenerated nerves were investigated by gross observation and toluidine blue staining. The gastrocnemius muscle was observed by HE staining. Results With the increased ionic phase transition time, the color of the conduit was gradually deepened and the diameter was gradually decreased, which showed no difference during 12 hours. The tensile strength of the nerve conduit was increased gradually. The ultimate tensile strength showed significant difference between the 48 hours and 12, 24, and 36 hours groups (P<0.05), and no significant difference between the 48 hours and 72 hours groups (P>0.05). As a result, the nerve conduit after ion-induced phase transition for 48 hours was chosen for further study. The scanning electron microscope (SEM) images showed that the nerve conduit had a uniform porous structure. The degradation rate of the the nerve conduit after ion-induced phase transition for 48 hours was significantly decreased as compared with that of the conduit without ion-induced phase transition. The nerve conduit could support the attachment and proliferation of rat Schwann cells on the inner surface. The animal experiments showed that at 8 weeks after operation, the CMAPs of the experimental and control groups were (3.5±0.9) and (4.3±1.1) m/V, respectively, which showed no significant difference between the two groups (P<0.05), and were significantly lower than that of the contralateral site ［(45.6±5.6 m/V), P>0.05］. The nerve conduit of the experimental group could repair the nerve defect. There was no significant difference between the experimental and control groups in terms of the histomorphology of the regenerated nerve fibers and the gastrocnemius muscle. Conclusion The green route for the fabrication of thermo-sensitive chitosan nerve conduits is free of any toxic reagents, and has simple steps, which is beneficial to the industrial transformation of the chitosan nerve conduit products. The prepared chitosan nerve conduit can be applied to rat peripheral nerve defect repair and nerve tissue engineering.
ObjectiveTo observe the changes in the structure and function of the retina in diabetic patients, and preliminarily explore the changes in the characteristics of neuropathy and microvascular damage in different degrees of diabetic retinopathy (DR). MethodsA prospective controlled study. From May to December 2020, 63 eyes of 63 patients with type 2 diabetes who were recruited from the Department of Ophthalmology of Shandong Provincial Hospital and 40 healthy volunteers with age and sex matching in the same period (control group) were included in the study. All subjects underwent optical coherence tomography angiography (OCTA) and portable non-mydriatic visual electrophysiological diagnosis system RETeval. OCTA was used to measure the thickness of the retinal nerve fiber layer (pRNFL) around the optic disc, the blood flow density of theradial peripapillary capillary (RPC) around the optic disc, and the thickness of the macular ganglion cell complex (GCC). The "DR evaluation plan" mode of the RETeval device was used to perform flash electroretinogram examination, and the "DR evaluation score" measured by the system was recorded. According to the DR grading standard established in the early treatment of DR research, DR was classified. Diabetic patients were divided into non-DR (non-DR) group, mild to moderate non-proliferative DR (mNPDR) group, and severe non-proliferative DR (sNPDR) group , Proliferative DR (PDR) group, with 12, 16, 18, and 17 eyes respectively. The comparison of pRNFL thickness, GCC thickness, RPC blood flow density and "DR assessment score" between groups was performed by one-way analysis of variance; the correlation between pRNFL thickness and RPC blood flow density was analyzed by Pearson correlation analysis. ResultsCompared with the control group, the overall, upper and lower thickness of the macular GCC of the affected eyes in different degrees of DR groups were significantly thinner, and the difference was statistically significant (F=13.560, 15.840, 5.480; P<0.05). Compared with the control group, the overall pRNFL (F=6.120), upper part (F=6.310), lower part (F=5.330), upper nose (F=7.350), lower nose (F=2.690), the upper nasal side (F=4.780), the upper temporal side (F=3.710), and the lower temporal side (F=3.750) became thinner, the difference was statistically significant (P<0.05). Correlation analysis results showed that the whole optic disc, upper part, lower part, upper nose, upper nasal side, lower nasal side, and lower temporal RPC blood flow density were positively correlated with pRNFL thickness (r=0.260, 0.256, 0.275, 0.489, 0.444, 0.542, 0.261; P<0.01). The "DR evaluation scores" of the eyes in the control group, non-DR group, mNPDR group, sNPDR group, and PDR group were 12.71±5.62, 22.18±3.77, 24.68±2.41, 24.98±2.78, 29.17±7.98 points; the DR lesions were more severe, the evaluation score were higher, and the difference was statistically significant (F=1.535, P<0.01). ConclusionCompared with the control group, the macular GCC, pRNFL thickness and RPC blood flow density of diabetic patients are significantly reduced; the "DR evaluation score" is increased, and it is related to the severity of DR.