Regeneration of periodontal tissue is the most promising method for restoring periodontal structures. To find a suitable bioactive three-dimensional scaffold promoting cell proliferation and differentiation is critical in periodontal tissue engineering. The objective of this study was to evaluate the biocompatibility of a novel porcine acellular dermal matrix as periodontal tissue scaffolds both in vitro and in vivo. The scaffolds in this study were purified porcine acellular dermal matrix (PADM) and hydroxyapatite-treated PADM (HA-PADM). The biodegradation patterns of the scaffolds were evaluated in vitro. The biocompatibility of the scaffolds in vivo was assessed by implanting them into the sacrospinal muscle of 20 New Zealand white rabbits. The hPDL cells were cultured with PADM or HA-PADM scaffolds for 3, 7, 14, 21 and 28 days. Cell viability assay, scanning electron microscopy (SEM), hematoxylin and eosin (H&E) staining, immunohistochemistry and confocal microscopy were used to evaluate the biocompatibility of the scaffolds. In vitro, both PADM and HA-PADM scaffolds displayed appropriate biodegradation pattern, and also, demonstrated favorable tissue compatibility without tissue necrosis, fibrosis and other abnormal response. The absorbance readings of the WST-1 assay were increased with the time course, suggesting the cell proliferation in the scaffolds. The hPDL cells attaching, spreading and morphology on the surface of the scaffold were visualized by SEM, H&E staining, immnuohistochemistry and confocal microscopy, demonstrated that hPDL cells were able to grow into the HA-PADM scaffolds and the amount of cells were growing up in the course of time. This study proved that HA-PADM scaffold had good biocompatibility in animals in vivo and appropriate biodegrading characteristics in vitro. The hPDL cells were able to proliferate and migrate into the scaffold. These observations may suggest that HA-PADM scaffold is a potential cell carrier for periodontal tissue regeneration.
Absorbable Implants ; Acellular Dermis ; Animals ; Biocompatible Materials ; chemistry ; Cell Adhesion ; physiology ; Cell Culture Techniques ; Cell Movement ; physiology ; Cell Proliferation ; Cell Shape ; physiology ; Cell Survival ; physiology ; Durapatite ; chemistry ; Fibrosis ; Humans ; Microscopy, Electron, Scanning ; Muscle, Skeletal ; surgery ; Necrosis ; Periodontal Ligament ; cytology ; surgery ; Rabbits ; Regeneration ; physiology ; Swine ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry

OBJECTIVESTo evaluate the expression profile of myoD microRNA-29 (miR-29) family in L6 myoblast differentiated to myotube of L6 myotube treated by glucose and insulin, and to further probe the molecular mechanism of myoD regulating the expression of miR-29 clusters.

METHODSThe expression of myoD and miR-29 family was detected by using real-time PCR and Western blot analysis. The potential promoter and transcription factors binding sites of miR-29 clusters were predicted by Promoter scan and transcriptional factor search. The promoter sequence of miR-29b1-a and miR-29b2-c cluster was cloned into a luciferase reporter plasmid and the regulatory effect of myoD was analyzed by using dual luciferase reporter assay. Electrophoretic mobility shift assay was further conducted to indicate the binding of myoD on specific sequence. Moreover, overexpression of myoD was achieved by a recombinant adenovirus system (Ad-myoD). L6 cells were infected with Ad-myoD and real-time PCR was conducted to analyze the expression of miR-29b and miR-29c.

RESULTSThe expression levels of myoD, miR-29a, miR-29b, and miR-29c were increased in L6 myoblast differentiated to myotube. The expression of myoD, miR-29b, and miR-29c was up-regulated in L6 myotube treated with glucose and insulin, but miR-29a depicted no significant change. Dual luciferase reporter gene assay showed that myoD functioned as a positive regulator of miR-29b2-c expression and myoD could bind to the specific sequence located at the promoter region of miR-29b2-c cluster. Enforced expression of myoD led to a marked increase of miR-29b and miR-29c levels in L6 cells.

CONCLUSIONMyoD might act as a crucial regulator of myogenesis and glucose metabolism in muscle through regulating the expression of miR-29b2-c.

Animals ; Cell Differentiation ; drug effects ; physiology ; Cell Line ; Gene Expression Regulation ; drug effects ; physiology ; Glucose ; pharmacology ; Hypoglycemic Agents ; pharmacology ; Insulin ; pharmacology ; Mice ; MicroRNAs ; biosynthesis ; genetics ; Multigene Family ; physiology ; Muscle Fibers, Skeletal ; cytology ; metabolism ; MyoD Protein ; genetics ; metabolism ; Myoblasts ; cytology ; metabolism ; Sweetening Agents ; pharmacology

Myostatin (Mstn) is a member of the transforming growth factor-beta superfamily that functions as a negative regulator of skeletal muscle growth and differentiation in mammals. The transcriptional regulation of Mstn is controlled by multiple genes including MEF2, which raise the importance of identifying the binding sites of MEF2 on myostatin promoter region and mechanisms underlying. In this study, we investigated the transcriptional regulation of MEF2 on porcine Mstn promoter activity in C2C12 cells. Sequence analysis of the 1 969 bp porcine Mstn promoter region revealed that it contained three potential MEF2 motifs. Using a serial deletion strategy, we tested the activity of several promoter fragments by luciferase assay. Overexpression of MEF2C, but not MEF2A increased Mstn promoter activity in all the promoter fragments with MEF2 motifs by two to six folds, in both C2C12 myoblasts and myotubes. When we transfected exogenous MEF2C, Mstn mRNA level was also upregulated in C2C12 cells, but the protein level was only significantly increased in myotubes. Thus, we propose that MEF2C could modulate and restrain myogenesis by Mstn activation and Mstn-dependent gene processing in porcine. Our research also provided potential targets and an effective molecule to regulate Mstn expression and gave a new way to explore the functional performance of Mstn.
Animals ; Cells, Cultured ; Gene Expression Regulation ; MEF2 Transcription Factors ; Mice ; Muscle, Skeletal ; metabolism ; Myoblasts ; cytology ; Myogenic Regulatory Factors ; genetics ; physiology ; Myostatin ; genetics ; physiology ; Promoter Regions, Genetic ; Swine

The present study was aimed to investigate the effects of hypoxia on iron metabolism of skeletal muscle. Rat L6 skeletal muscle cells were randomly divided into three groups which were exposed to hypoxia (1% O(2)) for 0, 12, 24 h, respectively. Iron isotope tracing method was used to determine iron uptake and release. Iron content of labile iron pool (LIP) was investigated by flow cytometry, and the expressions of transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), hypoxia-inducible transcription factor-1 (HIF-1) in L6 cells were observed by Western blot. The results showed that, compared with 0-hour hypoxia group, 12-hour hypoxia group exhibited significantly increased iron uptake and LIP (P < 0.05), as well as decreased iron release (P < 0.01). Not only iron uptake and release, but also LIP in 24-hour hypoxia group were significantly decreased, compared with those in 0- and 12-hour hypoxia groups (P < 0.01). The expressions of HIF-1, DMT1 (IRE), DMT1 (non-IRE) and TfR1 in 12-hour hypoxia group were significantly increased compared with those in 0-hour hypoxia group (P < 0.01). On the contrary, the expressions of DMT1 (IRE), DMT1 (non-IRE) and FPN1 in 24-hour hypoxia group were significantly decreased compared with those in the other two groups. However, TfR1 expression in 24-hour hypoxia was higher than those in 0- and 12-hour hypoxia groups (P < 0.05 or P < 0.01). These results suggest hypoxia plays an important role in iron metabolism of skeletal muscle cells. Moderate hypoxia can increase iron uptake and decrease iron release, resulting in higher LIP, but a prolonged hypoxia induces a disordered iron metabolism of skeletal cells.
Animals ; Cation Transport Proteins ; metabolism ; Cell Hypoxia ; Cell Line ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Iron ; metabolism ; Muscle, Skeletal ; cytology ; metabolism ; Oxygen ; physiology ; Rats ; Receptors, Transferrin ; metabolism ; Time Factors

OBJECTIVETo investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) into myocytes and their expression of dystrophin/utrophin after transplantation in mdx mice.

METHODSBrdU-labeled fifth-passage rat MSCs were transplanted in mdx mice with previous total body gamma irradiation (7 Gy). At 4, 8, 12 and 16 weeks after the transplantation, the mice were sacrificed to detect dystrophin/BrdU and utrophin expressions in the gastrocnemius muscle using immunofluorescence assay, RT-PCR and Western blotting. Five normal C57 BL/6 mice and 5 mdx mice served as the positive and negative controls, respectively.

RESULTSFour weeks after MSC transplantation, less than 1% of the muscle fibers of the mdx mice expressed dystrophin, which increased to 15% at 16 weeks. Donor-derived nuclei were detected in both single and clusters of dystrophin-positive fibers. Some BrdU-positive nuclei were centrally located, and some peripherally within myofibers. Utrophin expression decreased over time after transplantation.

CONCLUSIONThe myofibers of mdx mice with MSC transplantation express dystrophin, which is derived partially from the transplanted MSCs. Dystrophin expression from the transplanted MSCs partially inhibits the upregulation of utrophin in mdx mouse muscle, showing a complementary relation between them.

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Dystrophin ; genetics ; metabolism ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred mdx ; metabolism ; Muscle Fibers, Skeletal ; cytology ; metabolism ; Muscular Dystrophy, Animal ; metabolism ; therapy ; Rats ; Utrophin ; metabolism

BACKGROUNDMyoglobin is expressed exclusively in striated skeletal muscles and has been implicated in nitric oxide scavenging. Accumulating data suggest a critical role for nitric oxide in both the endogenous and therapeutic angiogenic response to ischemia. A clear role for myoglobin in ischemic skeletal muscle is uncertain. We hypothesized that myoglobin overexpression has an adverse impact on the angiogenic response to ischemia.

METHODSMuscle-specific myoglobin over-expressing transgenic mice (MbTG, n = 11), wild type littermates (WT, n = 23) underwent unilateral femoral artery ligation and excision. Laser doppler perfusion imaging was used to monitor changes in hindlimb perfusion before surgery and weekly after surgery up to 28 days. Tissue ischemia was assessed by a necrosis incidence. Upon termination of the experiment (28 days after surgery), skeletal muscles (gastrocnemius, and tibialis anterior) were harvested, the distal part of the muscle was frozen and embedded for histology study, the proximal part was used either to detect vascular endothelial growth factor (VEGF) level with enzyme-linked immunosorbent assays (ELISA) or to determine the proliferation (proliferating cell nuclear antigen (PCNA)) and apoptosis (Bax, and Bcl-2) condition in ischemic muscle by Western blotting. Capillaries were stained with endothelial phosphate alkaline staining and vascular density was expressed in capillaries/fiber.

RESULTSThe recovery of perfusion in MbTG mice was similar to that of WT mice on day 7 (0.485 +/- 0.095 vs 0.500 +/- 0.084) but was significantly less on day 14 (0.536 +/- 0.086 vs 0.623 +/- 0.077, P < 0.05), day 21 (0.588 +/- 0.082 vs 0.684 +/- 0.068, P < 0.01) and day 28 (0.606 +/- 0.079 vs 0.733 +/- 0.093, P < 0.01). The necrosis incidence was higher in MbTG than in WT (54.5% vs 21.6%). Vascular density was less in MbTG compared with that in WT (gastrocnemius 0.19 +/- 0.08 vs 0.30 +/- 0.08, P < 0.05; tibialis anterior 0.22 +/- 0.11 vs 0.33 +/- 0.04, P < 0.05). With ischemic injury, the VEGF level was increased in both MbTG and WT (45.2% and 20.4%, respectively). Western blotting showed that after hindlimb ischemia the proliferation was similar in both MbTG and WT, however, apoptosis was increased in MbTG relative to WT, shown as more expression of Bax and less expression of Bcl-2.

CONCLUSIONAn increase in expression of myoglobin protein in skeletal muscle reduces the endogenous perfusion recovery following surgically induced hind-limb ischemia.

Animals ; Apoptosis ; genetics ; Blotting, Western ; Cell Proliferation ; Enzyme-Linked Immunosorbent Assay ; Hindlimb ; blood supply ; metabolism ; Immunohistochemistry ; Ischemia ; therapy ; Mice ; Mice, Transgenic ; Muscle, Skeletal ; blood supply ; cytology ; metabolism ; Myoglobin ; genetics ; physiology

OBJECTIVETo evaluate the compositional variation of fibrous callus in the fracture site and the joint cavity and joint cartilage after being transplanted in the muscle pouch.

METHODSThirty 2 month old New Zealand white rabbits (weighing 1-1.5 kg) were randomly divided into two groups: a callus transplantation group (Group A, n=15) and a cartilage transplantation group (Group B, n=15). In Group A, closed radius fracture was made and the autologous fibrous callus was transplanted in the right knee joint cavity at 12 days postoperatively. In Group B, the right knee joint cartilage of the animals was transplanted in the autologous back muscle pouches under anesthesia. Then all the animals were killed by overdose anesthetic 3 weeks after transplantation. And the transplanted fibrous callus, the healed bones in the fracture sites and the transplanted joint cartilage were obtained for assessment of compositional variation.

RESULTSPure fibrous composition was found in the callus at the fracture sites in Group A at 12 days postoperatively. And for 11 out of the 15 animals, the fibrous callus was transformed into cartilaginous tissues after 3 weeks of transplantation, but the fibrous callus was absent in the other 4 animals. The fibrous calluses at the original site and the fracture locus were differentiated into bony tissues. Bony tissue transformation was found in the transplanted joint cartilages in the muscle pouch of all the animals in Group B.

CONCLUSIONSThe fracture sites or joint cavity may facilitate callus differentiation in different ways: the former is helpful for osteogenesis while the latter for the development and maintenance of cartilages, and the muscle pouch is inclined to induce the osteogenic phenotype for cartilages.

Animals ; Bony Callus ; cytology ; transplantation ; Cartilage, Articular ; cytology ; transplantation ; Cell Differentiation ; Fracture Healing ; physiology ; Knee Joint ; Male ; Muscle, Skeletal ; Rabbits ; Radius Fractures ; physiopathology

OBJECTIVETo study the effect of exercises of different intensities on rat skeletal muscle cell apoptosis.

METHODSRat models of exhaustive exercise-induced skeletal muscle injury was established using tread mill exercises at different speeds, and the skeletal muscle cell apoptosis was detected using propidium iodide (PI) staining and flow cytometry.

RESULTNo obvious gastrocnemius cell apoptosis was observed in rats with normal exercise (P>0.05), but the cell apoptosis index was statistically significant in moderate and exhaustive exercise groups (P<0.05), reaching the highest level in exhaustive exercise group after exercising. The gastrocnemius cell apoptosis index increased obviously on days 1 and 3 of exercise, and stabilized in moderate and exhaustive exercise groups.

CONCLUSIONExercises can evoke cell apoptosis and accelerate apoptotic cell clearance, and the imbalance between the two events during exercises may contribute to skeletal muscle injury.

Animals ; Apoptosis ; physiology ; Flow Cytometry ; Male ; Muscle, Skeletal ; cytology ; physiopathology ; Physical Conditioning, Animal ; physiology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Time Factors

The dielectric spectroscopy of the skeletal muscle cells is approached by extracellular perfusion, using curve fitting and numerical calculation with nonlinear Cole-Cole equation, and the dielectric parameter of skeletal muscle cells is compared after perfusion, The results show: (1) the permittivy at low frequency (epsilon1) and the conductivity at hight frequency (kappa(h)) reduces in keeping with extracellular perfusate; (2) the effect of extracellular perfusion on the characteristic frequency (f(c1),f(c2)) is small; (3) the extracellular perfusion mostly influences the peaks of deltaepsilon" and tgdelta for the skeletal muscle cells.
Animals ; Bufo bufo ; Electric Conductivity ; Electricity ; Electrophysiology ; Extracellular Fluid ; physiology ; Muscle, Skeletal ; cytology ; physiology ; Spectrum Analysis ; methods

In order to elucidate muscle functional changes by acute reloading, contractile and fatigue properties of the rat soleus muscle were investigated at three weeks of hindlimb suspension and the following 1 hr, 5 hr, 1 d, and 2 weeks of reloading. Compared to age-matched controls, three weeks of unloading caused significant changes in myofibrillar alignments, muscle mass relative to body mass (-43%), normalized tension (-35%), shortening velocity (+143%), and response times. Further significant changes were not observed during early reloading, because the transitional reverse process was gradual rather than abrupt. Although most of the muscle properties returned to the control level after two weeks of reloading, full recovery of the tissue would require more than the two-week period. Delayed recovery due to factors such as myofibrillar arrangement and fatigue resistance was apparent, which should be considered for rehabilitation after a long-term spaceflight or bed-rest.
Animals ; Hindlimb Suspension ; Lactic Acid/metabolism ; Microscopy, Electron ; Muscle Contraction/*physiology ; Muscle Fatigue/*physiology ; Muscle, Skeletal/cytology/*physiology ; Myofibrils/ultrastructure ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't ; Weight-Bearing/physiology