Objective To explore the effect of tri pterygium glycoside (TG) on the skeletal muscle atrophy and apoptosis after nerve allograft. Methods Twenty Wistar male rats were adopted as donors, weighing 200-250 g, and the sciatic nerves were harvested. Fifty SD male rats were adopted as recipients, weighing 200-250 g. Fifty SD rats were made the models of10 mm right sciatic nerve defect randomly divided into five groups (n=10): group A, group B, group C, group D and group E.groups A and B received fresh nerve allograft, groups C and D received sciatic nerve allograft pretreated with TG, and group E received autograft. The SD rats were given medicine for 5 weeks from the second day after the transplantation: groups A and E were given physiological sal ine, groups B and D TG 5 mg/ (kg·d), and group C TG 2.5 mg/ (kg·d). At 3 and 6 weeks, respectively, after nerve transplantation, general observation was performed; the structure of skeletal muscles was observed by HE staining; the diameter of skeletal muscles was analyzed with Image-Pro Plus v5.2; the ultrastructure of skeletal muscles was observed by TEM; the expressions of Bax and Bcl-2 were detected by immunohistochemical staining; and the apoptosis of skeletal muscles was detected by TUNEL. Results All rats survived to the end of the experiment. In general observation, the skeletal muscles of SD rates atrophied to different degrees 3 weeks after operation. The muscular atrophy in group A was more serious at 6 weeks, and that in the other groups improved. The wet weight, fiber diameter and expression of Bcl-2 in group A were significantly lower than those in groups B, C, D and E (P lt; 0.01)；those in groups B, C and D were lower than those in group E (P lt; 0.05); and there were no significant differences among groups B, C and D (P gt; 0.05). The apoptosis index and expression of Bax in group A were significantly higher than those in groups B, C, D and E (P lt; 0.01)；those in groups B, C and D were higher than in groupE (Plt; 0.05); and there were no significant differences among groups B, C and D (P gt; 0.05). Three weeks after nerve allograft, under the l ight microscope, the muscle fibers became thin; under the TEM, the sarcoplasmic reticulum was expanded. Six weeks after nerve allograft, under the l ight microscope, the gap of the muscle fibers in group A was found to broaden and connective tissue hyperplasia occurred obviously; under the TEM, sarcomere damage, serious silk dissolution and fragmentary Z l ines were seen in group A, but the myofibrils were arranged tidily in the other groups, and the l ight band, dark band and sarcomere were clear. Conclusion TG can decrease the skeletal muscle atrophy and apoptosis after nerve allograft. The donor’s nerve that is pretreated with TG can reduce the dosage of immunosuppressant for the recipient after allograft.
OBJECTIVE: To define how to preserve the severed limbs to prolong the period of replantation. METHODS: The original articles about preservation of severed limbs in recent years were reviewed, it was suggested that the period of replantation was determined by the injury of skeletal muscle. RESULTS: When the environment of severed limbs was changed, the injures of skeletal muscle could be decreased. CONCLUSION: After the severed limbs are reasonably preserved, the period of replantation may be prolonged.
Objective To observe whether the motor nerve babysitter could improve the delayed nerve anastomosisand promote the functional recovery. Methods Sixteen SD rats weighing 200-250 g were randomly divided into 2 groups.In group A, the left musculocutaneous nerve was transected to make the model of biceps brachii denervation and anastomosed to its proximal end 6 weeks later; In group B, the musculocutaneous nerve was transected and the distal end was coapted to the purely motor medial pectoral nerve immediately (nerve babysitter) and the musculocutaneous nerve was separated from the medial pectoral nerve, and reanastomosed to its proximal end 6 weeks later. In the animal model, the left l imbs served as experimental sides, the right l imbs as control sides. After 6 and 12 weeks of the second surgery, behavioral test (grooming test) was performed and the degree of the biceps brachii atrophy was observed, the latent period and the ampl itude of the maximun action potentials of the biceps brachii were detected, the wet muscle weight, muscle fiber cross-section area and the activity of Na+-K+-ATPase of the biceps brachii were measured. Results After 4 weeks of the second surgery, grooming behavior was found in group B, while few grooming behavior was seen in group A till 6 weeks after the secondary surgery. After 6 weeks of the second surgery, the recovery rate of the latent period and the ampl itude, the wet muscle weight, muscle fiber cross-section area and the enzymatic activity of Na+-K+-ATPase of the biceps brachii in group A was 187.25% ± 1.97%, 46.25% ± 4.63%, 55.14% ± 1.99%, 49.97% ± 1.71%, and 65.81% ± 2.24%, respectively, which was significantly different from that in group B (155.96% ± 3.02%, 51.21% ± 2.13%, 74.18% ± 1.82%, 55.05% ± 1.64% and 71.08% ± 1.53%, respectively, P lt; 0.05). After 12 weeks of the second surgery, the recovery rate of the latent period and ampl itude, the wet muscle weight, muscle fiber cross-section area andthe enzymatic activity of Na+-K+- ATPase of the biceps brachii in group A was 145.36% ± 3.27%, 51.84% ± 5.02%, 77.92% ± 1.73%, 61.04% ± 2.68% and 71.94% ± 1.65%, respectively, which was significantly different from that in group B (129.83% ± 8.36%, 75.22% ± 2.78%, 84.51% ± 1.34%, 78.75% ± 3.69% and 84.86% ± 1.81%, respectively, P lt; 0.05). Conclusion Motor nerve babysitting could reduce muscular damage after denervation, improve the effect of delayed nerve repair and promote the functional recovery of musculocutaneous nerve.
OBJECTIVE To observe the ultrastructural changes and number of satellite cells in different muscles with different denervation interval and investigate the mechanism of denervation atrophy. METHODS Muscles of different denervation interval were harvested, which were 6 biceps brachii and 6 abductor digiti minimi. The ultrastructure of the samples were observed under transmission electron microscope. The number of nucleus and satellite cells were counted to calculate the percentage content of satellite cells. RESULTS In early stage of denervation, the myofilament and sarcomere of the majority were well oriented. The nucleoli of some muscle cell nucleus were enlarged and pale. Vacuolarization was also seen in some mitochondria. There was no obvious proliferation of collagen fiber around myofibers. After denervation of half a year, rupture and disorientation of myofilament was seen. The nucleus became smaller, dark stained, and some of them were condensed. There was proliferation of fibroblasts, adipose cells and collagen fibers around myofibers. Motor endplate was not recognized one year after denervation. In the early stage of denervation, satellite cell percentage of the two muscles was relatively high. It then declined with time. One year after denervation, satellite cells were scarcely detected. Comparison of the curves for satellite cell declination in two muscles revealed that the declination of the abductor digiti minimi was faster than that of biceps brachii. Decrease of the former started 3 months after denervation, while the latter started after 6 months. CONCLUSION Disappearing of motor endplate and proliferation of collagen fibers are main factors that affect the treatment outcome in late cases. Decrease of satellite cell number is another cause. The correlation of less satellite cell in abductor digiti minimi and poorer recovery of hand intrinsic muscles indicates that increment of satellite cells in long-term denervated muscles may be one of the effective measures to improve treatment outcome.
Objective To explore the in vitrodifferentiation of the rat mesenchymal stem cells (MSCs ) into the skeletal muscle cells induced by the myoblast differentiation factor (MyoD) and 5-azacytidine. Methods The MSCs were taken from the rat bone marrow and the suspension of MSCs was made and cultured in the homeothermia incubator which contained 5% CO2at 37℃. The cells were observed under the inverted phase contrast microscope daily. The cells spreading all the bottom of the culture bottle were defined as onepassage. The differentiation of the 3rd passage of MSCs was induced by the combination of 5-azacytidine, MyoD, transforming growth factor β1, and the insulin like growth factor 1. Nine days after the induction, the induced MSCs were collected, which were analyzed with the MTT chromatometry, theflow cytometry, and the immunohistochemistry. Results The primarily cultured MSCs grew as a colony on the walls of the culture bottle; after the culture for 5-7 days, the cells were shaped like the fibroblasts, the big flat polygonal cells, the medium sized polygonal cells, and the small triangle cells; after the culture for 12 days, the cells were found to be fused, spreadingall over the bottle bottom, but MSCs were unchanged too much in shape. After the induction by 5-azacytidine, some of the cells died, and the cells grew slowly. However, after the culture for 7 days, the cells grew remarkably, the cell volume increased gradually in a form of ellipse, fusiform or irregularity. After theculture for 14 days, the proliferated fusiform cells began to increase in a great amount. After the culture for 18-22 days, the myotubes increased in number and volume, with the nucleus increased in number, and the newly formed myotubes and the fusiform myoblst grew parallelly and separately. The immunohistochemistry for MSCs revealed that CD44 was positive in reaction, with the cytoplasm ina form of brown granules. And the nucleus had an obvious border，and CD34 was negative. The induced MSCs were found to be positive for desmin and specific myoglobulin of the skeletal muscle. The flow cytometry showed that most of the MSCs and the induced MSCs were in the stages of G0/G1，accounting for 79.4％ and 62.9％，respectively; however, the cells in the stages of G2/S accounted for 20.6％ and 36.1％. The growth curve was drawn based on MTT，which showed that MSCs weregreater in the growth speed than the induced MSCs. The two kinds of cells did not reach the platform stage，having a tendency to continuously proliferate.ConclusionIn vitro,the rat MSCs can be differentiated into the skeletal muscle cells with an induction by MyoD and 5-azacytidine, with a positive reaction for the desmin and the myoglobulin of the skeletal muscle. After the induction, the proliferation stage of MSCs can be increased, with a higher degree of the differentiation into the skeletal muscle.
Objective To study whether human amniotic fluid colony derived stem cells (hAFCSCs) are involved in regeneration of injured muscles in mice and to investigate the method and feasibil ity of hAFCSCs-based cytotherapy in the treatment of injured muscles. Methods Human second-trimester amniotic fluid was collected through ultrasound-guided amniocentesis, hAFCSCs were isolated from second-trimester amniotic fluid and cultured, and the cells at 6th-8th passages were spared. The mRNA was extracted to identify the stem cell related genes by RT-PCR. The muscular injury model of bilateral tibial is anterior muscle was establ ished by cardiotoxin and X-ray irradiation in 16 Nod/Scid mice (aged 6-8 weeks, and weighing 20-24 g). The hAFCSCs (3.3 × 107/mL, 30 μL) were injected into the right injured tibial is anterior muscles as the experimental group, while the same volume of complete medium (α-MEM containing 15%FBS, 18%Chang B, 2%Chang C, 1% penicill instreptomycin, and 1% L-glutamine) was injected into the left injured tibial is anterior muscles as the control group. At 2 and 4 weeks after cell transplantation, the immunofluorescence staining of tibial is anterior muscles was performed to detect hepatocyte growth factor receptor (c-Met), myogenic regulatory factor (Myf-5), Laminin, Desmin, and human specific nuclear mitotic apparatus protein (NuMa). Results The clone formation was observed at 5-7 days of primary hAFCSCs culture; after 8-10 days, the clones with homogeneous morphology were selected for subculture. Adequate stem cells were available after 6th-8th subculture. RT-PCR analysis showed that hAFCSCs expressed mRNA of the stem cell related genes. The immunofluorescence double-staining showed that NuMa expressed in tibial is anterior muscles of the experimental group and no myogenic phenotype expressed at 2 weeks after cell transplantation, and that single cell co-expressed NuMa and c-Met or Myf-5 at 4 weeks after cell transplantation. In some myofibers, NuMa and Laminin or Desmin were also co-expressed. No NuMa positive hAFCSCs were detected in the control group at 2 and 4 weeks after cell transplantation. Conclusion hAFCSCs can participate in the regeneration of injured mouse muscle.
Objective To study the effect of motor nerve implantation after ectopic transplantation of skeletal muscle on nerve regeneration in rat. Methods Sixty Sprague-Dewley male 8 monthold rats were randomly divided into 3 groups: control group,in situ implantation group and ectopic transplantation group. In control group, obturator nerve controlling right gracilis was cut off. In in situ implantation group, the right gracilis was cut off and replanted to its original site, and the obturator nerve was implanted to the muscle. In ectopic transplantation group, the right gracilis was cut off and transplanted to the muscle of the left leg, and the obturator nerve was implanted to the muscle. After 25 weeks, the neurophysiological information was collected through electromyography and the weight of the muscle was measured. Results The potentialwithout control of the nerve existed in control group. There were no significant differences in latency, amplitude and conduct velocity betweenin situ implantation group and ectopic transplantation group(Pgt;0.05).The atrophy of gracilis was dominant incontrol group, the weight of the muscle was 158.0±19.3 mg. The weights of the muscle were 509.6±14.5 mg in ectopic transplantation group and 516.8±12.7 mg in in situ mplantation group, showing no significant difference (P＞0.05). The weights of the muscle in in situ implantation and ectopic transplantation group were larger than that in control group, showing significant difference(P＜0.05). Conclusion Motor nerve implantation after ectopic transplantation of skeletal muscle could prevent the atrophy of the muscle and resume partial function of nerve.
Objective To review the current researches of scaffold materials for skeletal muscle tissue engineering, to predict the development trend of scaffold materials in skeletal muscle tissue engineering in future. Methods The related l iterature on skeletal muscle tissue engineering, involving categories and properties of scaffold materials, preparative techniqueand biocompatibil ity, was summarized and analyzed. Results Various scaffold materials were used in skeletal muscle tissue engineering, including inorganic biomaterials, biodegradable polymers, natural biomaterial, and biomedical composites. According to different needs of the research, various scaffolds were prepared due to different biomaterials, preparative techniques, and surface modifications. Conclusion The development trend and perspective of skeletal muscle tissue engineering are the use of composite materials, and the preparation of composite scaffolds and surface modification according to the specific functions of scaffolds.
ObjectiveTo explore an optimized protocol of decellularization to fabricate an ideal scaffold derived from porcine skeletal muscle acellular matrix. MethodsSerial-step protocol of homogenating-milling-detergent method was used to fabricate decellularized porcine muscle tissue (DPMT) derived from native porcine skeletal muscle tissue from adult pig waist. Histological method was used to assess the effects of decellularization and degreasing. Sirius red staining was used to analyze collagen components. Scanning electron microscopy, BCA assay, and PicoGreen assay were used to evaluate the ultrastructure, total protein content, and DNA content in DPMT. The adipose derived stem cells (ADSCs), NIH3T3 cells, and human umbilical vein endothelial cells (HUVECs) were cultured in extraction liquor of DPMT in different concentrations for 1, 3, and 5 days, then the relative growth rate was calculated with cell counting kit 8 to assess the toxicity in vitro. Live/dead cell staining was used to evaluate the cytocompatibility by seeding HUVECs on the surface of DPMT and co-cultured in vitro for 3 days. For in vivo test, DPMT was subcutaneously implanted at dorsal site of male specific-pathogen free Sprague Dawley rats and harvested after 3, 7, 14, and 28 days. Gross obersvation was done and transverse diameter of remained DPMT in vivo was determined. HE staining and immunohistochemical staining of CD31 were used to assess inflammatory response and new capillary rings formation. ResultsDecellularization of the porcine skeletal muscle tissue by homogenating-milling-detergent serial steps protocol was effective, time-saving, and simple, which could be finished within only 1 day. The decellularizarion and degreasing effect of DPMT was complete. The main component of DPMT was collagen type I and type IV. The DNA content in DPMT was (15.902±1.392) ng/mg dry weight, the total protein content was 68.94% of DPMT dry weight, which was significantly less than those of fresh skeletal muscle tissue[(140.727±10.422) ng/mg and 93.14%] (P<0.05). The microstructure of DPMT was homogeneous and porous. The result of cytocompatibility revealed that the cytotoxicity of DPMT was 0-1 grade, and HUVECs could stably grow on DPMT. In vivo study revealed DPMT could almost maintain its structural integrity at 14 days and it degraded completely at 28 days after implantation. The inflammatory response peaked at 3 days after implantation, and reduced obviously at 7 days. Difference was significant in the number of inflammatory cells between 2 time points (P<0.05). Neovascularization was observed at 7 days after implantation and the number of new vessels increased at 14 days, showing significant difference between at 7 and 14 days (P<0.05). ConclusionThe homogenating-milling-detergent serial-steps protocol is effective, time-saving, and reproducible. The DPMT reveals to be cell and lipid free, with highly preserved protein component. DPMT has good biocompatibility both in vitro and in vivo and may also have potential in promoting neovascularization.
OBJECTIVE: To study the influence of the electric stimulation of denervated muscle atrophy. METHODS: Sixteen SD rats were made the model of denervated skeletal muscle in two lower limbs by cutting off the sciatic nerve and femoral nerve. The right gastrocnemius muscle was stimulated with JNR-II nerve amp; muscle recovery instrument by skin as the experimental side and the left was not treated as the control side. The muscle histology, ultrastructure, fibrillation potential amplitude, Na(+)-K(+)-ATPase and Ca(2+)-ATPase activities were observed 2 weeks and 4 weeks after operation. RESULTS: Electric stimulation could protect mitochondria and sarcoplasmic reticulum from the degeneration. The reduction rates of muscle cell diameter and cross section in the experimental side were slower significantly than those in control side. There was no influence on fibrillation potential amplitude in the both sides after electric stimulation. The reduction rates of Na(+)-K(+)-ATPase activity in the experimental side were slower 15.59% and 27.38% respectively than those in the control side. The reduction rates of Ca(2+)-ATPase activity in the experimental side were slower 4.83% and 21.64% respectively than those in the control side. CONCLUSION: The electric stimulation can protect muscle histology, electrophysiology and enzymic histochemistry of denervated skeletal muscle from the degeneration. The electric stimulation is an effective method to prevent and treat muscle atrophy.