ObjectiveTo investigate the effect of human adipose-derived stem cells (hADSCs) on pressure ulcers in mouse.MethodsThe subcutaneous adipose tissue from voluntary donation was harvested. Then the hADSCs were isolated and cultured by mechanical isolation combined with typeⅠcollagenase digestion. The 3rd generation cells were identified by osteogenic, adipogenic, chondrogenic differentiations and flow cytometry. The platelet rich plasma (PRP) from peripheral blood donated by healthy volunteers was prepared by centrifugation. The pressure ulcer model was established in 45 C57BL/6 mice by two magnets pressurized the back skin, and randomly divided into 3 groups (n=15). The wounds were injected with 100 μL of hADSCs (1×106 cells) transfected with a green fluorescent protein (GFP)-carrying virus, 100 μL human PRP, and 100 μL PBS in hADSCs group, PRP group, and control group, respectively. The wound healing was observed after injection. The wound healing rate was calculated on the 5th, 9th, and 13th days. On the 5th, 11th, and 21st day, the specimens were stained with HE staing, Masson staining, and CD31 and S100 immunohistochemical staining to observe the vascular and nerve regeneration of the wound. In hADSCs group, fluorescence tracer method was used to observe the colonization and survival of the cells on the 11th day.ResultsThe cultured cells were identified as hADSCs by induced differentiation and flow cytometry. The platelet counting was significantly higher in PRP group than in normal peripheral blood group (t=5.781, P=0.029). General observation showed that the wound healing in hADSCs group was superior to those in PRP group and control group after injection. On the 5th, 9th, and 13th days, the wound healing rate in hADSCs group was significantly higher than those in PRP group and control group (P<0.05). Histological observation showed that compared with PRP group and control group, inflammatory cell infiltration and inflammatory reaction were significantly reduced in hADSCs group, collagen deposition was significantly increased, and skin appendage regeneration was seen on the 21st day; at each time point, the expression of collagen was significantly higher in hADSCs group than in PRP group and control group (P<0.05). Immunohistochemical staining showed that the number of neovascularization and the percentage of S100-positive cells in hADSCs group were significantly better than those in PRP group and control group on the 5th, 9th, and 13th days (P<0.05). Fluorescent tracer method showed that the hADSCs could colonize the wound and survive during 11 days after injection.ConclusionLocal transplantation of hADSCs can accelerate healing of pressure ulcer wounds in mice and improve healing quality by promoting revascularization and nerve regeneration.
Objective To investigate the effects of icariin and mixed prescri ption of icariin, radix hedysari polysaccharide, and l iquid extracted from earthworm on peri pheral nerve regeneration. Methods Twenty male SD rats weighing (200 ± 10) g were selected and randomized into four groups (n=5 per group): sham operated group (group A), model group (group B), icariin group (group C), and mixed l iquid group (group D). In group A, the left sciatic nerves of the rats were only exposed, and treated at fixed time from the following day with the NS (2 mL/d). In groups B, C, D, the models were made by clamping sciatic nerve and treated with NS, icariin and mixed l iquid, respectively (2 mL/d). The general state ofanimals was observed after the treatment daily. The nerve function index, motor nerve conductive velocity and the morphous and number of myel inated sciatic nerve fibers were measured at 21 days. Results Animals in various groups were all in good state. After 21 days, the weights of rats in groups A, B, C and D were (366.9 ± 14.0), (370.1 ± 16.3), (373.3 ± 19.6) and (374.0 ± 11.4) g, respectively, and there was no significant difference among these groups (P gt; 0.05). For sciatic function index, there was no significant difference between group A and group D (P gt; 0.05), between group B and group C (P gt; 0.05), while there was significant difference between group B and group D (P lt; 0.05). For tibial function index, there was significant difference between group A and groups B, C, D (Plt; 0.05), there was no significant difference between group B and groups C, D (Pgt; 0.05). For peroneal function index, there was no significant difference between group A and groups C, D (P gt; 0.05), between group B and groups C, D (P gt; 0.05). The sciatic motor nerve conductive velocities of group A, B, C and D were (45.0 ± 2.9), (8.0 ± 2.6), (13.4 ± 6.8), and (19.6 ± 9.3) m/s, respectively, there was no significant difference between group B and group C (P gt; 0.05), and there was significant difference between group A and groups B, C, D and between group B and group D (P lt; 0.05). The size of individual myel inated sciatic nerve fibers of regenerated nerves in groups B, C, and D was significantly smaller than that in group A. Comparing with group A, the number of myel inated sciatic nerve fibers in groups B, C, and D was 93.3% ± 35.6%, 90.6% ± 37.1%, and 115.4% ± 40.6%, respectively, but there was no significant difference among four groups (P gt; 0.05). Conclusion Icariin and mixed prescription are safe. The improving peripheral nerve regeneration effect of mixed prescription is more obvious than that of icariin, indicating the comprehensive study of modified formula radixhedysari is necessary to find the effective part or mixture of effective compounds with fixed percentage.
ObjectiveTo investigate the effect of cells in the epimysium conduit (EMC) on the regeneration of sciatic nerve of mice.MethodsThe epimysium of the 8-week-old male C57BL/6J enhanced green fluorescent protein (EGFP) mouse was trimmed to a size of 5 mm×3 mm, and prepared in a tubular shape (ie, EMC). Some epimysia were treated with different irradiation doses (0, 15, 20, 25, 30, 35 Gy) to inhibit cells migration. Then the number of migrating cells were counted, and the epimysia with the least migrating cells were selected to prepare EMC. Some epimysia were subjected to decellularization treatment and prepared EMC. HE and Masson staining were used to identify the decellularization effect. Twenty-four C57BL/6J wild-type mice were used to prepare a 3-mm-long sciatic nerve defect of right hind limb model and randomly divided into 3 groups (n=8). EMC (group A), EMC after cell migration inhibition treatment (group B), and decellularized EMC (group C) were used to repair defects. At 16 weeks after operation, the midline of the regenerating nerve was taken for gross, toluidine blue staining, immunofluorescence staining, and transmission electron microscopy.ResultsAt 15 days, the number of migrating cells gradually decreased with the increase of irradiation dose. There was no significant difference between 30 Gy group and 35 Gy group (P>0.05); there were significant differences between the other groups (P<0.05). The epimysium after treatment with 35 Gy irradiation dose was selected for thein vivo experiment. After the decellularization of the epimysium, no nucleus was found in the epimysium and the epimysium could be sutured to prepare EMC. At 16 weeks after operation, the nerves in all groups were recanalized. The sciatic nerve was the thickest in group A, followed by group B, and the finest in group C. Immunofluorescence staining showed that the EGFP cells in group A were surrounded by regenerated axons. Toluidine blue staining and transmission electron microscopy observation showed that the number of regenerated axons and the thickness of regenerated myelin sheath in group A were significantly better than those in groups B and C (P<0.05). There was no significant difference between groups B and C (P>0.05).ConclusionThe cellular components of the epimysium participate in and promote the regeneration of the sciatic nerve in mice.
OBJECTIVE: To explore the mechanism of tissue specificity of neurotropism in peripheral nerve regeneration, we investigated the biological characteristics of the nerve regeneration conditioned fluids(NRCF) on motoneuron of SD rats cultured in vitro. METHODS: Silicon chambers were sutured respectively to the distal stumps of motorial branch of femoral nerve and saphenous nerve to collect NRCF, namely MD-NRCF and SD-NRCF. The rats cortex motoneuron were divided into 4 groups and cocultured with MD-NRCF, SD-NRCF, b-FGF and serum-free medium respectively. The cultured cells were photoed under phase-contrast microscope, their longest neurites and cell-body areas were measured by cell image processing computer system. MTT automated colorimetric microassay was also adopted to quantify the activation of cultured motoneurons in each group. RESULTS: Cells of MD-NRCF group had longer neurites than those of the other three groups, and their activation was also superior to those of the other groups. CONCLUSION: The results suggest that MD-NRCF has more significantly neurite-promoting and neurobiological effects on motoneuron than SD-NRCF and b-FGF.
ObjectiveTo investigate the effects of exosomes from adipose-derived stem cells (ADSCs) on peripheral nerve regeneration, and to find a new treatment for peripheral nerve injury. MethodsThirty-six adult Sprague Dawley (SD) rats (male or female, weighing 220-240 g) were randomly divided into 3 groups (n=12). Group A was the control group; group B was sciatic nerve injury group; group C was sciatic nerve injury combined with exosomes from ADSCs treatment group. The sciatic nerve was only exposed without injury in group A, and the sciatic nerve crush injury model was prepared in groups B and C. The SD rats in groups A and B were injected with PBS solution of 200 μL via tail veins; the SD rats in group C were injected with pure PBS solution of 200 μL containing 100 μg exosomes from ADSCs, once a week and injected for 12 weeks. At 1 week after the end of the injection, the rats were killed and the sciatic nerves were taken at the part of injury. The sciatic nerve fiber bundles were observed by HE staining; the SCs apoptosis of the sciatic nerve tissue were detected by TUNEL staining; the ultrastructure and SCs autophagy of the sciatic nerve were observed by transmission electron microscope. ResultsGross observation showed that there was no obvious abnormality in the injured limbs of group A, but there were the injured limbs paralysis and muscle atrophy in groups B and C, and the degree of paralysis and muscle atrophy in group C were lighter than those in group B. HE staining showed that the perineurium of group A was regular; the perineurium of group B was irregular, and there were a lot of cell-free structures and tissue fragments in group B; the perineurium of group C was more complete, and significantly well than that of group B. TUNEL staining showed that the SCs apoptosis was significantly increased in groups B and C than in group A, in group B than in group C (P<0.01). Transmission electron microscope observation showed that the SCs autophagosomes in groups B and C were significantly increased than those in group A, but the autophagosomes in group C were significantly lower than those in group B. ConclusionThe exosomes from ADSCs can promote the peripheral nerve regeneration. The mechanism may be related to reducing SCs apoptosis, inhibiting SCs autophagy, and reducing nerve Wallerian degeneration.
OBJECTIVE: To study the effects of Schwann cell cytoplasmic derived neurotrophic proteins (SDNF) on the regeneration of peripheral nerve in vivo. METHODS: Ninety adult SD rats were chosen as the experimental model of degenerated muscle graft with vascular implantation bridging the 10 mm length of right sciatic nerve. They were divided randomly into three groups, 30 SD rats in each groups. 25 microliters of 26 ku SDNF (50 micrograms/ml, group A), 58 ku SDNF (50 micrograms/ml, group B) and normal saline(group C) were injected respectively into the proximal, middle and distal part of the degenerated muscle grafts at operation, 7 and 14 days postoperatively. The motorial function recovery assessment was carried out every 15 days with the sciatic nerve function index(SFI) after 15 days to 6 months of operation. Histological and electrophysiological examination of regenerating nerve were made at 1, 3 and 6 months postoperatively. RESULTS: There were significant statistic differences between the both of experimental groups(group A and B) and control group(group C) in the respects of the histological, electrophysiological examination and SFI(P lt; 0.01). CONCLUSION: The 26 ku SDNF and 58 ku SNDF can improve the regeneration of the injured peripheral nerve in vivo.
ObjectiveTo construction the telmisartan/collagen/polycaprolactone (PCL) nerve conduit and assess its effect on repairing sciatic nerve defect in rats. Methods The 60% collagen/hexafluoroisopropanol (HFIP) solution and 40% PCL/HFIP solution were prepared and mixed (collagen/PCL solution). Then the 0, 5, 10, and 20 mg of telmisartan were mixed with the 10 mL collagen/PCL solution, respectively. Telmisartan/collagen/PCL nerve conduits were fabricated via high voltage electrospinning technology. The structure of nerve conduit before and after crosslinking was observed by using scanning electron microscope (SEM). The drug release efficiency was detected by in vitro sustained release method. RAW264.7 cells were cultured with lipopolysaccharide to induce inflammation, and then co-cultured with nerve conduits loaded with different concentrations of telmisartan for 24 hours. The mRNA expressions of inducible nitric oxide synthase (iNOS) and Arginase 1 (Arg-1) were detected by using real-time fluorescence quantitative PCR. Forty adult Wistar rats were randomly divided into 4 groups (n=10). After preparing 15-mm-long sciatic nerve defect, the defect was repaired by cross-linked nerve conduits loaded with 0, 5, 10, and 20 mg telmisartan in groups A, B, C, and D, respectively. After operation, the general condition of rats was observed after operation; the sciatic function index (SFI) was tested; the bridging between the nerve conduit and sciatic nerve, and the integrity of nerve conduit were observed; the tissue growth in nerve conduit and material degradation were observed by HE staining; the expressions of CD86 (M1 macrophage marker), CD206 (M2 macrophage marker), myelin basic protein (MBP), and myelin protein 0 (P0) in new tissues were also observed by immunohistochemical staining; the expressions of neurofilament 200 (NF-200) and S-100β in new tissues were assessed by immunofluorescence staining. Results The general observation showed that the inner diameter of the nerve conduit was 1.8 mm and the outer diameter was 2.0 mm. After cross-linking by genipin, the nanofiber became thicker and denser. The drug release test showed that the telmisartan loaded nerve conduit could be released gradually. With the increase of telmisartan content in nerve conduit, the iNOS mRNA expression decreased and the Arg-1 mRNA expression increased; and the differences between 20 mg group and other groups were significant (P<0.05). In vivo experiment showed that all animals in each group survived until the completion of the experiment. The SFI was significantly higher in groups C and D than in groups A and B at different time points (P<0.05) and in group D than in group C at 6 months after operation (P<0.05). HE staining showed that there were significantly more new tissues in the middle of the nerve conduit in group D after operation than in other groups. Immunohistochemical staining showed that CD86 and CD206 stainings were positive in each group at 1 month after operation, among which group D had the lowest positive rate of CD86 and the highest positive rate of CD206, and there were significant differences in the positive rate of CD206 between group D and groups A, B, and C (P<0.05); the MBP and P0 stainings were positive in groups C and D at 6 months, and the positive rate in group D was significantly higher than that in group C (P<0.05). Immunofluorescence staining showed that the NF-200 and S-100β expressions in group D were significantly higher than those in other groups. ConclusionTelmisartan/collagen/PLC nerve conduit can promote the sciatic nerve defect repair in rats through promoting the polarization of M1 macrophages to M2 macrophages, and the nerve conduit loaded with20 mg telmisartan has the most significant effect.
ObjectiveTo review recent research progress in the use of auxiliary components of nerve conduits for the treatment of peripheral nerve injuries. MethodsAn extensive review of recent domestic and international literature was conducted to evaluate the role of auxiliary components in nerve conduits for peripheral nerve repair, with a focus on their effects and underlying mechanisms. ResultsBy incorporating auxiliary components such as bioactive molecules, therapeutic cells, and their derivatives, nerve conduits can create a more biomimetic regenerative microenvironment. This is achieved by providing neurotrophic support, modulating the immune microenvironment, improving blood and oxygen supply, and offering directional guidance for nerve regeneration. Consequently, the nerve conduit is transformed from a simple physical scaffold into an active, bio-functional repair system, which enhances the effectiveness for PNI. ConclusionWhile nerve conduits augmented with auxiliary components demonstrate improved effectiveness, further advancements are required in drug delivery systems and the integration of cellular components. Moreover, most current studies are based on animal or in vitro experiments. Randomized controlled clinical trials are necessary to validate their clinical effectiveness.
Objective Inducing human amniotic membrane mesenchymal stem cells (hAMSCs) to Schwann cells-like cells (SCs-like cells) in vitro, and to evaluate the efficacy of transplantation of hAMSCs and SCs-like cells on nerves regeneration of the rat flaps. Methods hAMSCs were isolated from placenta via two-step digestion and cultured by using trypsin and collagenase, then identified them by flow cytometry assay and immunofluorescence staining. The 3rd generation of hAMSCs cultured for 6 days were induced to SCs-like cells in vitro; at 19 days after induction, the levels of S-100, p75, and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence staining, Western blot, and real-time fluorescence quantitative PCR (qPCR). The levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were measured by ELISA in the supernatant of the 3rd generation of hAMSCs cultured for 6 days and the hAMSCs induced within 19 days. In addition, 75 female Sprague Dawley rats were taken to establish the rat denervated perforator flap model of the abdominal wall, and were divided into 3 groups (n=25). The 3rd generation of hAMSCs (1×106 cells) in the proliferation period of culturing for 6 days, the SCs-like cells (1×106 cells), and equal volume PBS were injected subcutaneously in the skin flap of the rat in groups A, B, and C, respectively. At 2, 5, 7, 9, and 14 days after transplantation, 5 rats in each group were killed to harvest the flap frozen sections and observe the positive expression of neurofilament heavy polypeptide antibody (NF-01) by immunofluorescence staining. Results The cells were identified as hAMSCs by flow cytometry assay and immunofluorescence staining. The results of immunofluorescence staining, Western blot, qPCR showed that the percentage of positive cells, protein expression, and gene relative expression of S-100, p75, and GFAP in SCs-like cells group were significantly higher than those in hAMSCs group (P<0.05). The results of ELISA demonstrated that the expression of BDNF and NGF was significantly decreased after added induced liquid 1, and the level of BDNF and NGF increased gradually with the induction of liquids 2 and 3, and the concentration of BDNF and NGF was significantly higher than that of hAMSCs group (P<0.05). Immunofluorescence staining showed that the number of regenerated nerve fibers in group B was higher than that in groups A and C after 5-14 days of transplantation. Conclusion The hAMSCs can be induced into SCs-like cells with the proper chemical factor regulation in vitro, and a large number of promoting nerve growth factor were released during the process of differentiation, and nerve regeneration in flaps being transplanted the SCs-like cells was better than that in flaps being transplanted the hAMSCs, which through a large number of BDNF and NGF were released.
Objective To investigate the expression change of endogenous Spastin after sciatic nerve injury in rats, and to discuss the role and significance in the peripheral nerve regeneration. Methods Thirty-six adult male Sprague Dawley rats weighing 180–220 g were randomly divided into the experimental group (n=30) and the control group (n=6). Sciatic nerve compression damage model was established in the experimental group, and the sciatic nerve was only exposed in the control group. The L4-6 spinal cord tissue was obtained to detect Spastin mRNA and protein levels by real-time fluorescence quantitative PCR and Western blot at 1, 3, 7, 14, and 28 days after operation in the experimental group (n=6) and at 7 days in the control group. Meanwhile, the sciatic nerve at 5 mm distal to the injured site was obtained to observe the ultrastructure of the distal axon by transmission electron microscope (TEM). Results The expression trends of Spastin gene and Spastin protein in L4-6 spinal cord tissue of 2 groups were basically identical. In the experimental group, the expressions of Spastin gene and protein decreased at the beginning, and then increased; the expressions reduced to the minimum at 7 days after operation, and came back to the initial level at 28 days. The expression levels of Spastin mRNA and protein at 3, 7, and 14 days were significantly lower in the experimental group than the control group (P<0.05), but no significant difference was noted between 2 groups at 1 and 28 days (P>0.05). The expression levels of Spastin mRNA and protein at 3, 7, and 14 days were significantly lower than those at 1 and 28 days in the experimental group (P<0.05), but no significant difference was noted between at 1 day and 28 days (P>0.05). At 1, 3, and 7 days after operation, the myelin damage was observed by TEM; at 14 days, there were regenerating Schwann cells; at 28 days, a large number of myelinated nerve fibers were seen, which were closed to normal form. Conclusion In the process of sciatic nerve regeneration after injury, a complex succession of changes take place in the expression of endogenous Spastin protein in rats, indicating that Spastin protein plays an important role in the process.