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.
【Abstract】 Objective To investigate the effect of massage on quadriceps femoris repair and the expressions of Desmin and α-Actin in rabbits so as to explore the possible molecular mechanisms of massage in repair of muscle injury. Methods Twenty-seven New Zealand white rabbits, weighing (2.0 ± 0.5) kg, were randomly divided into 3 groups: groups A (n=3), B (n=12), and C (n=12). In group A, the rabbits were not treated as controls; in groups B and C, the rabbit models of quadriceps femoris injury were prepared by self-made beater. In group B, no massage therapy was given as nature recovery controls; in group C, RT-N2 intelligent massage device was used for massage therapy at 8 days after injury, at 3 000-3 100 r/min for 15 minutes, every day for 7 days or for 14 days. The quadriceps femoris specimens were taken from 6 rabbits of groups B and C at 14 days and 21 days, respectively. HE staining was employed to detect the histomorphological change. Immunohistochemistry staining and Western blot were used to detect Desmin and α-Actin expressions. The massage therapy effect was evaluated by the histomorphological change and Desmin and α-Actin expressions. Results All rabbits survived to the end of experiment in groups B and C. No histological change was found with regular order of muscle fibers and no connective tissue in group A; obvious tissue necrosis was seen with broken muscular fibers, muscle atrophy, and irregular order in group B; and in group C, the skeletal muscle morphology and muscle atrophy were obviously improved with regenerated muscle fibers when compared with group B. Immunohistochemistry staining showed that the Desmin and α-Actin expressions obviously reduced in groups B and C, which were significantly weaker than that in group A (P lt; 0.05); the Desmin and α-Actin expressions were significantly ber in group C than in group B (P lt; 0.05), and at 21 days than at 14 days in group C (P lt; 0.05). Western blot results showed that the Desmin and α-Actin expressions were significantly higher in group A than in groups B and C (P lt; 0.05), and the expressions were lowest at 14 days in group B. Conclusion The histomorphology and cytoskeletal structure can be significantly improved after massage, which may help to repair muscle injury by up-regulation of Desmin and α-Actin expressions.
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 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.
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 reveal morphologic features and physiological function in compartments of human forearm muscles, and investigate the possibil ity of transplantation of neuromuscular compartments. Methods Sihler’ s neural staining technique was used to study the nerve branches distribution of forearm skeletal muscles in 5 human cadavers (aging26-39 years), including flexor carpi radial is, flexor carpi ulnaris (FCU), extensor carpi radial is brevis, extensor carpi ulnaris, palmaris longus (PL), flexor poll icis longus, pronator teres (PT). According to Wickiewicz’s methods, Ulnar compartment and radial compartment of forearm skeletal muscles above mentioned from 10 human cadvers were used to study the muscle architectural features. Results Each nerve branches run into the ulnar compartment and radial compartment respectively. There was statistically significant difference between the two physiological cross section areas (PSCA) of each neuromuscular compartment from forearm muscles(P lt; 0.05). Among them, PSCA of ulnar compartment of FCU was the largest. The PSCA of ulnar compartment of PT was the smallest. There was no statistically difference between the ratio (PSCA/muscle wet weight) of each neuromuscular compartment from forearm muscles (P gt; 0.05). As the ratio of PSCA to the muscle fiber length, the ulnar compartment of PT and the two compartments of PL had the highest one while the ulnar compartment of FCU had the smallest; and there was no statistically difference among the other neuromuscular compartments (P gt; 0.05). Conclusion Each of forearm muscles be divided into ulnar compartment and radial compartment and they have their own nerve supply. And there are significant differences in the physiological function in compartments of forearm muscles, which can be references in muscular compartment transplantation.
Objective To investigate an optimal method for SD rat skeletal muscle decellularization. Methods Sixteen SD rats (male and female) weighing 180-200 g were used. Thirty-six skeletal muscle bundles obtained from 10 rats were randomly divided into 3 groups: normal group (group A, n=4) received non-decellularization; time group (group T, n=16) andconcentration group (group C, n=16) underwent decellularization using hypotonic-detergent method. Concentration of sodium dodecyl sulfate (SDS) was 1.0% for T group, which was subdivided into groups T1, T2, T3 and T4 (n=4 per subgroup) according to different processing durations (24, 48, 72 and 96 hours). Group C was treated for 48 hours and subdivided into groups C1, C2, C3 and C4 (n=4 per subgroup) according to different SDS concentrations (0.5%, 1.0%, 1.5% and 2.0%). The muscle bundles of each group underwent HE staining observation and hydroxyproline content detection in order to get the optimal decellularization condition. Seven of 14 complete skeletal muscle bundles obtained from 6 SD rats were treated with the optimal decellularization condition (experimental group), and the rest 7 muscle bundles served as normal control (control group). The muscle bundles of each group were evaluated with gross observation, Masson staining and biomechanical test. Results HE staining: there was no significant difference between groups T1, T2, C1, C2 and C3 and group A in terms of muscle fiber; portion of muscle fibers in group C4 were removed; muscle fibers in group T3 were fully removed with a complete basement membrane structure; muscle fibers of group T4 were fully removed, and the structure of basement membrane was partly damaged. Hydroxyprol ine content detection: there was no significant difference between group A and groups C1, C2, C3, T1 and T2 (P gt; 0.05); significant difference was evident between group A and groups C4, T3 and T4 (P lt; 0.05); the difference between group C4 and groups T3and T4 was significant (P lt; 0.05); no significant difference was evident between group T3 and group T4 (P gt; 0.05). The optimal decellularization condition was 4 , 1.0% SDS and 72 hours according to the results of HE staining and hydroxyproline content detection. Gross observation: the muscle bundles of the experimental group were pall id, half-transparent and fluffier comparing with the control group. Masson staining observation: the collagen fibers of the experimental group had a good continuity, and were fluffier comparing with control group. Biomechanics test: the maximum breaking load of the experimental group and the control group was (1.38 ± 0.35) N and (1.98 ± 0.77) N, respectively; the maximum extension displacement of the experimental group and the control group was (3.19 ± 3.23) mm and (3.56 ± 2.17) mm, respectively; there were no significant differences between two groups (P gt; 0.05). Conclusion Acellular matrix with intact ECM and complete removal of muscle fibers can be obtained by oscillatory treatment of rat skeletal muscle at 4℃ with 1% SDS for 72 hours.
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 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 summarize the recent progress in research on the mechanism of denerved skeletal muscle atrophy. Methods The recently-publ ished l iteratures at home and abroad on denerved skeletal muscle atrophy were reviewedand summarized. Results The mechanism of denerved skeletal muscle atrophy was very complex. At present, the studyof the mechanism was based on the changes in histology, cytology and molecules. Fiber thinning and disorderly arrangement of denerved skeletal muscles were observed and apoptotic bodies were detected. Apoptosis-promoting genes expressed upregulatedly and apoptosis-restraining genes expressed down-regulatedly. Muscle satell ite cells increased after denervation, but then they decreased and disappeared because they could not differentiate to mature muscle fibers. The structural change of cytomiscrosome and down-regulation of metabol ism-related enzymes induced cell metabol ism disorder. Conclusion The histological change of skeletal muscle fibers, the change of the number of muscle satell ite cells and differentiation, the structural change of cytomiscrosome and the change of apoptosis-related and metabol ism-related gene expressions contribute to denerved skeletal muscle atrophy.