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.
ObjectiveTo review the effects and mechanisms of various myokines secreted by skeletal muscle on various bone tissue cells.MethodsLiterature related to myokines and their regulation of bone tissue cells was reviewed and analyzed comprehensively in recent years.ResultsBone and skeletal muscle are important members of the motor system, and they are closely related in anatomy, genetics, and physiopathology. In recent years, it has been found that skeletal muscle can secrete a variety of myokines to regulate bone marrow mesenchymal stem cells, osteoblasts, osteoclasts, and bone cells; these factors mutual crosstalk between myoskeletal unit, contact each other and influence each other, forming a complex myoskeletal micro-environment, and to some extent, it has a positive impact on bone repair and reconstruction.ConclusionMyokines are potential targets for the dynamic balance of bone tissue cells. In-depth study of its mechanism is helpful to the prevention and treatment of myoskeletal diseases.
Objective Extracellular matrix is one of the focus researches of the adi pose tissue engineering. To investigate the appropriate method to prepare the porcine skeletal muscle acellular matrix and to evaluate the biocompatibility of the matrix. Methods The fresh skeletal muscle tissues were harvested from healthy adult porcine and were sl iced into2-3 mm thick sheets, which were treated by hypotonic-detergent method to remove the cells from the tissue. The matrix was then examined by histology, immunohistochemistry, and scanning electron microscopy. The toxic effects of the matrix were tested by MTT. Human adi pose-derived stem cells (hADSCs) were isolated from adi pose tissue donated by patients with breast cancer, and identified by morphology, flow cytometry, and differentiation abil ity. Then, hADSCs of passage 3 were seeded into the skeletal muscle acellular matrix, and cultured in the medium. The cellular behavior was assessed by calcein-AM (CA) and propidium iodide (PI) staining at 1st, 3rd, 5th, and 7th days after culturing. Results Histology, immunohistochemistry, and scanning electron microscopy showed that the muscle fibers were removed completely with the basement membrane structure; a large number of collagenous matrix presented as regular network, porous-like structure. The cytotoxicity score of the matrix was grade 1, which meant that the matrix had good cytocompatibil ity. The CA and PI staining showed the seeded hADSCs had the potential of spread and prol iferation on the matrix. Conclusion Porcine skeletal muscle acellular matrix has good biocompatibility and a potential to be used as an ideal biomaterial scaffold for adi pose tissue engineering.
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 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 study the protective effects of ischemic preconditioning(IP) duration against ischemic reperfusion injury of skeletal muscle. Methods Thirty-six Wister rats were made amputation-like models, which underwent temporary amputation at the level of the femur, excluding the femoral vessels. They were divided into 6 groups(n=6) according to different treatments before ischemiareperfusion: group A(4 hours of ischemiareperfusion); groups B, C, D, E(5, 10,15, 20 minutes of ischemia and 5, 10, 15, 20 minutes of reperfusion respectively, for 3 cycles, 4 hours ischemiareperfusion ); group F (no ischemia-reperfusion). The malondialdehyde(MDA), the extent of edema and necrosis of skeletal muscle were measured to observe protective effects of different ischemic preconditioning duration. Results Five minutes of ischemic preconditioning（IP5）could protect skeletal muscle of ischaemia against necrosis and the survival area of the muscle was 82.47%.The effects of IP10 and IP 15 were significantly superior to that of IP5 and the survival areas of the muscle were 89.03% and 89.49%. The effect of IP20(78.27%) was significantly inferior to that IP5. IP5 could reduce edema of skeletal muscle, the effect of IP10 was significantly superior to that of IP5. IP5, IP 10,and IP 15 could decrease the level of MDA, but IP20 did not decrease it. Conclusion The trend of protective effect of IP on ischemia-reperfusion injury of themuscle in rats first rise to the peak and then go down,10minutes ofIPis optimal.
Objective To investigate the effect of various concentration of platelet-rich plasma (PRP) on osteogenic differentiation of rabbit skeletal muscle-derived stem cells (SMSCs) cultured in vitro. Methods Blood drawn from the central ear arteries of 9 one-year-old New Zealand white rabbits weighing 2.5-3.0 kg (male and female) was used to prepare PRP (Landesberg method). Full blood count and platelet count in PRP were tested. Soleus muscle of right hindl imb in rabbit was obtained and used to culture SMSCs in vitro. The cells at passage 3 were randomly divided into different groups: the experimental groups in which the cells were treated by conditioned culture media with various concentrations of autologousPRP (6.25%, 12.50%, 25.00%, 50.00%), and the control group in which the cells were treated with the media without PRP. At different time points after intervention, osteogenetic activity of the cells was detected by ALP staining observation, ALP activity detection was conducted, al izarin red staining for calcium nodules and immunofluorescence staining for osteocalcin were performed, and core binding factor α1 (Cbfα1) of osteogenic gene expression was tested by RT-PCR. Results The full blood PRP count and the platelet count in PRP was (3.06 ± 0.46) × 105/μL and (18.08 ± 2.10) × 105/μL, respectively. ALP staining: the cells in all the experimental groups were positive for the staining with many black sediment particles in cytoplasm; the cells in the control group were negative staining. ALP activity: all the experimental groups were higher than the control group (P lt; 0.05), the experimental group at 12.50% was superior to other experimental groups at each time point (P lt; 0.05). Al izarin red staining: at 14 days after culture, orange-red calcium nodules were evident in all the experimental groups; no orange-red calcium nodules were observed in the control group with a mineral ization rate of zero; there were significant difference between the experimental groups and the control group in terms of mineral ization rate (P lt; 0.05), the experimental group at 12.50% had a higher mineral ization rate than other experimental groups (P lt; 0.05). Immunofluorescence staining for osteocalcin: at 7 days after culture, the experimental groups were positive for the staining with yellow fluorescence in cytoplasm, and the result of the control group was negative. RT-PCR detection: no obvious changes of the gene expression were noted at 4, 12, and 24 hoursafter culture in the control group; the gene expression in all the experimental groups was significant superior to that of control group, especially at 12 hours, and the expression in the experimental group at 12.50% was the highest. Conclusion PRP can obviously promote the osteogenic differentiation of SMSCs cultured in vitro in a concentration-dependent manner, and the 12.50% is proved to be the ideal concentration.
OBJECTIVE: To observe the changes of heme oxygenase-1 (HO-1) expression in the skeletal muscle after ischemia-reperfusion of hind limb in rats. METHODS: A model of hind limb ischemia was made by clamping femoral artery with a microvascular clip. Soleus muscle was obtained from the animals received sham operation, 4 h ischemia without reperfusion and 2 h, 4 h, 8 h, 16 h, 24 h reperfusion after 4 h ischemia. Soleus histology and malondialdehyde (MDA) content were measured. The levels of HO-1 mRNA and protein were measured in different time by Northern blotting, Western blotting and immunohistochemistry technique. RESULTS: After ischemia-reperfusion of limb, HO-1 mRNA increased at the 2nd hour, reached a peak at the 8th hour, and returned toward baseline at the 24th hour. The change of protein level was essentially in agreement with that of mRNA. Immunohistochemical results showed that HO-1 expressed primarily in skeletal muscle cytoplasma. There were no positive signals of mRNA and protein in sham group and in ischemia group. After limb reperfusion, MDA contents in the soleus muscle increased significantly when compared with that in the sham group (P lt; 0.05). MDA content of the 8th after reperfusion decreased significantly when compared with that of the 4 h after reperfusion (P lt; 0.05). CONCLUSION: Ischemia-reperfusion can induce HO-1 expression in skeletal muscle in rats, which may provide protection for injured tissue.
Objective To investigate the pathological changes in the neuromuscular junction during ischemiareperfusion(IR) in the skeletal muscle. Methods Forty-eight healthy adult Wistar rats (24 male, 24 female) were equally randomised into the following 6 groups: Group A (control group): no ischemiareperfusion; Group B: ischemia by clamping the blood vessels of the right hindlimb for 3 hours; Group C: ischemia by clamping for 4.5 hours;Group D: ischemia by the clamping for 4.5 hours followed by reperfusion for 1.5hours; Group E: ischemia for 4.5 hours followed by reperfusion for 24 hours; and Group F: ischemia for 4.5 hours followed by reperfusion for 2 weeks. Then, the medial head of the gastrocnemius muscle flap model was applied to the right hindlimb of each rat. The medial head of the gastrocnemius muscle was isolated completely,leaving only the major vascular pedicle, nerve and tendons intact.The proximal and distal ends (tendons) were ligated while the vessel pedicle was clamped. And then, Parameters of the muscle (performance,contraction index,colour,edema,bleeding) were observed. The muscle harvested was stained with gold chloride(AuCl3) and the enzymhistochemistry assay (succinate dehydrogenase combined with acetylcholine esterase) was performed. Morphology and configuration of the neuromuscular junction were observed during the ischemiareperfusion injury by means of the AuCl-3 staining. The result of the enzymhistochemical reactions was quantitatively analyzed with the computer imageanalysis system. And then, additional 5 rats were prepared for 3 different models identical with those in Groups A, C and E separately. The specimens were harvested from each rat and were stained with HE and AuCl-3, and they were examined under the light microscope. Results During the period of ischemia, the skeletal muscle of Group B showed the colour of purple and edema.The colour and edema became worse in Group ,while dysfunction of elasticity and contraction appeared obviously with plenty of dark red hemorrhagic effusion at the same time.After reperfusion,the color and edema of muscle in Group D became improved while the elasticity and function of contraction was not improved. Hemorrhagic effusion of Group D turned clearer and less than Group C.Group E was similar to Group D in these aspects of muscle except for much less hemorrhagic effusion. Skeletal muscle in Group F showed colour of red alternating with white, adhesion,contracture of muscle, exposure of necrotic yellow tissue and almost lost all its functions. The AuCl3 staining showed that during IR, necrosis of the myocytes was followed by degeneration of their neuromuscular junctions, and finally the nerve fibers attached to these neuromuscular junctions were disrupted like the withering of leaves. The enzymhistochemistry assay showed thatthere was no significant difference in the level of acetylcholine esterase between the ischemic group (Groups B and C) and the control group (Group A) (Pgt;0.05). However, the level of acetylcholine esterase in all the reperfused groups (Groups D, E and F) decreased significantly when compared with the control group（Group A）and the ischemic groups (Groups B and C) (Plt;0.01). Conclusion The distribution of the nerve fibers and the neuromuscular junctions in the mass of the muscles is almost like the shape of a tree. The neuromuscular junction seems to be more tolerant for ischemia than the myocyte. Survival ofthe neuromuscular junction depends on its myocytes alive. Therefore, an ischemiareperfusion injury will not be controlled unless an extensive debridement of the necrotic muscle is performed.
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.