Objective To investigate the effects of sodium hyaluronate solution on the proliferation and differentiation of myoblasts. Methods The 3rd subculture myoblasts from muscle of infant SD rat were cultured in four growth media, in which the concentrations of sodium hyaluronate were 0.05% (group A) , 0.1%( group B), 0.2% (group C)and 0 (group D, control group), respectively. The proliferation rate of myoblasts in each medium was observed through growth curves by means of count and MTT. At the same time, the subculture myoblasts were cultured in differentiated media in which the concentrations of sodium hyaluronate were 0 and 0.1%. The capacity of fusion of myoblasts was compared between two kinds of differentiated media. Results There were the nearly same proliferation curse in Groups A, B and C: increasing by logarithm at 2 days and reaching peak value at 4 days. The myoblasts in Group D increased slowly: increasing by logarithm at 3 days, doubling at 5 days and reaching peak value at 6 days. MTT has the similar curse to counting. The myoblast proliferation of Group B was more than that of the other groups. The peak value of myoblast fusion was 35% at 6 days in common differentiated media; slowly reached 11.7% at 7 days in the differentiated media in which the concentrations of sodiumhyaluronate was 0.1%.Conclusion Sodium hyaluronate at certain concentration can be a decent media for myoblasts, it can accelerate proliferation and differentiation of myoblasts.
Objective To establish a model of the human marrow mesenchymal stem cells (hMSCs) cultured under the hypoxic condition in adults and to investigate the biological features of MSCs under hypoxia.Methods The bone marrow was obtained by aspiration at the posterior superior iliac spine in 3 healthy adult subjects. hMSCs were isolated by the gradient centrifugation and were cultured in the DMEM-LG that contained 20％ fetal bovine serum. The serial subcultivation was performed 10-14 days later. The second passage of the hMSCs were taken, and they were divided into the following 4 groups according to the oxygen concentrations and the medium types: the normoxic group（20％O2, DMEM-LG, Group A）, the hypoxic group（1％O2, DMEM-LG,Group B）, the normoxic osteoblast induction group（20％O2, conditioned medium, Group C）, and the hypoxic osteoblast induction group（1％O2, conditioned medium, Group D）. The biological features of the cultured hMSCs under hypoxia were assessed bythe cell count, the MTT method, the colony forming unit-fibroblast, the real-time RT-PCR, and the alkaline phosphatase (ALP) activity, and the alizarinred staining. Results The hMSCs cultured in the Group B and Group D had a significantly higher proliferation rate than those in the Group A (Plt;0.01), and the culture effect was not influenced by the medium type. The hMSCs in the Group B had a significantly higher level of the colony-forming unit capability than the hMSCs cultured in the Group A(Plt;0.01). After the induction, hMSCs in the Group B had a decreasednumber of the osteoblasts than hMSCs in the Group C. The hMSCs in the Group D had a gradually-increasedactivity of ALP, which was significantly lower than that in the Group C(Plt;0.01). The RT-PCR examination revealed that ALP,osteocalcin, and mRNA expressions of collagen type Ⅰ and osteonectin in the Group Csignificantly increased （P＜0.01). By comparisonamong the 3 groups, after the 4-week culture the obvious calcium salt deposit and the red-stained calcium nodus could be observed.ConclusionHypoxia can promote the proliferation rate of hMSCs, enhance the colonyforming ability and inhibit the differentiation of the osteoblasts.
Objective To investigate the effect of astragalus polysaccharides(AP) on chitosan／polylactic acid(AP／C／PLA)scaffolds and marrow stromal cells(MSCs)tissue engineering on periodontal regeneration of horizontal alveolar bone defects in dogs． Methods MSCs were isolatedfrom the bone marrow and then cultured in conditioned medium to be induced to become osteogenic．The MSCs were harvested and implanted into AP／C／PLA and C／PLA scaffolds．A horizontal alveolar bone defect(5 mm depth, 2 mm width)were produced surgically in the buccal side of the mandibular premolar 3 and 4 of 10 dogs．The defects were randomly divided into 4 groups(n=10)：Group A, root planning only(blank contro1); group B, AP／C／PLA with conditioned medium(medium contro1);group C, C／PLA with MSCs(scaffolds contro1); and group D, AP／C／PLA with MSCs(experimental group)．Eight weeks after surgery， block sections of the defects were collected for gross, histological and X-ray analysis. Results MSCs induced in vitro exhibited an osteogenic phenotype with expressingcollagen I and alkaline phosphatase． X-ray film observation showed that the bone density and height had no changes in group A; in group B, the bone density was increased to a certain extent and furcation area reached a few height, but no height was increased in interdental septum; in group C，the bone density was increased and furcation area nearly reached the native height,but interdental septum reached a few height;in group D,the bone density was increased significantly and furcation area and interdental septum reached the native height. Histological evaluation showed that there was greater tissue formation in group D than that in groups A, B and C, in which new alveolar bone, new cementum, periodontal ligament with Sharpey’s fibers, and new bone tissue was similar to native periodontal tissues. Ingroup A，B, C and D respectively, the amount of new alveolar bone regeneration was 0.83±0.30, 1.46±0.55, 2.67±0.26 and 2.90±0.41 mm; new cementum regeneration was 0.78±0.45，1.30±0.60，2.29±0.18 and 2.57±0.22 mm; the amount of connective tissue adhesion was 0.80±0.22，1.33±0.34，2.23±0.42 and 2.64±0.27 mm; all showing significant differenecs between group D and groups A, Band C (Plt;0.05)．Conclusion The technology of tissue engineering with AP／C／PLAscaffolds and induced MSCs may contribute to periodontal regeneration．
Objective To introduce the latest advances of research on repair of the degenerative intervertebral disc with gene transduction.Methods The recentlypublished articles about the treatment of degenerative disc with gene transduction were reviewed, especially the articles published during the recent 5 years about the application of this therapy to regulating the synthesisand degradation of the extracellular matrix of the degenerative intervertebral disc.Results The shape and function of the normal intervertebral disc were reported to be closely related to the synthesis and degradation of the extracellular matrix of the intervertebral disc. The extracellular matrix of the intervertebral disc was a target for the gene transduction to repair the degenerative intervertebral disc. There was a great development of the treatment with gene transduction, especially in vector choice, target gene transduction, and transgene regulation and safety. Conclusion The advances of the research have indicated that repair of the degenerative intervertebral disc with gene transduction is a keyto curing the disease of the degenerative intervertebral disc.
Objective To study the feasibility of core-binding factor α1 (Cbfa1) gene modified marrow mesenchymal stem cells (MSCs) composed with porcine acellular bone extracellular matrix in repairing the radial defects. Methods Radial defects of 1.2 cm in length were created in 40 Japanese white rabbits and they were divided into four groups. In group A, MSCs isolated from homogeneous rabbits were infected with Cbfa1 recombinant adenovirus and implanted into acellular bone exteracellular matrix, and then the complexes were implanted into defects. In group B, the complexes including the MSCs without Cbfa1 gene-modified and scaffoldmaterial were implanted into defects. In group C, only the scaffold material was implanted. In group D, defects were not treated as the control. The macroscopic, X-ray and histologic analysis were performed to evaluate the repair effect at 4, 8 and 12 weeks postoperatively. The repaired radius were examined by biomechanical test at 12 weeks postoperatively. Results By gross examination，mature hard new bone formed at grafted areas at 12 weeks postoperativelyin group A, osteotomized ends connected by much callus in group B and less callus in group C at grafted areas. In contrast, bone nonunion formed in group D. X-ray and histological examination showed that the repaired results of defects in the group A were better than those in others groups evidently in extracellular matrix degradation, new bone remodeling and marrow cavity rebuilding at 4 and 8 weeks postoperatively. At 12 weeks postoperatively, the cortical bone became mature lamellar bone, new bone remolding was complete and marrow cavity was smooth in group A. Only proximal end of defects showed that marrow cavity was remolded partially in group B. The continuous callus could be observed in bone defect, and no obvious marrow cavity remolding was observed in group C. Lots of fibrous connective tissue filled in defect and bone nonunion was shown in group D. There was no significant difference in the damage compress loading of repaired radius between groups A, B and D (Pgt;0.05), but there was significant difference between groups C and D(Plt;0.01).Conclusion These results demonstrate that Cbfa1 gene modified MSCs combined with acellular bone extracellular matrix can be used to repair rabbit radial defects.
Objective To investigate the possibility of theadipose tissue-derived stromal cells(ADSCs) to differentiate into the neuron-like cells and to explore a new cell source for the transplantation related to the central nervous system. Methods Adipose was digested by collagenase, cultured in the fetal bovine serum containing a medium. Trypse was used to digest the cells and the cell passage was performed. The 3rd to the 9th passage ADSCs were used to make an induction. Isobutylmethylxanthine, indomethacin, insulin, and dexamethasone were used to induce the ADSCs to differentiate into the neuron-like cells and adipocytes. Sudan black B and immunocytochemistry were used to identify the cells. Results A population of the ADSCs could be isolated from the adult human adipose tissue, they were processed to obtain a fibroblast-like population of the cells and could be maintained in vitro for an extendedperiod with the stable population doubling, and they were expanded as the undifferentiated cells in culture for more than 20 passages, which indicated their proliferative capacity. They expressed vimentin and nestin, and characteristics of the neuron precursor stem cells at an early stage of differentiation. And the majority of the ADSCs also expressed the neuron-specific enolase and βⅢ-tubulin, characteristics of the neurons. Isobutyl-methyxanthine, indomethacin, insulin, and dexamethasone induced 40%-50% of ADSCs to differentiate into adipocytes and 0.1%0.2% of ADSCs into neuron-like cells. The neuron-like cells had a complicated morphology of the neurons, and they exhibited a neuron phenotype, expressed nestin, vimentin, neuron-specific enolase and βⅢ-tubulin, but some neuron-like cells also expressed thesmooth muscle actin (SMA), and the characteristics of the smooth muscle cells; however, the neurons from the central nervous system were never reported to express this kind of protein. Therefore, the neuron-like cells from the ADSCs could be regarded as functional neurons. Conclusion Ourresults support the hypothesis that the adult adipose tissue contains the stem cells capable of differentiating into the neuron-like cells, and they can overcome their mesenchymal commitment, which represents an alternative autologous stemcell source for transplantation related to the central nervous system.
Objective To investigate the feasibility of a new kind of porous β tricalcium phosphate (β-TCP) as a scaffold for the bone tissue engineering Methods The inverted phase contrast microscope was used to observe the growth of the marrow mesenchymal stem cells (MSCs) in the experimentalgroup and the control group at 10 days.In the experimental group, the MSCs were cultured with β-TCP(3 mm×3 mm×3 mm) in the 24-hole cultivation board, and in the control to control group, only MSCs were cultivated. The scanning electron microscope was used to observe growth of MSCs at 6 days. Cultivated with β-TCP at 3, 6, 9, 12 days, the MTT assay was used to judge the biocompatibility. The cytotoxicity was analyzed with the method that used the different density(100%， 50%， 10%， 1%，0%) leaching liquor gained from β-TCP to raise MSCs. MSCs were induced into the osteoblasts and were mixed with β-TCP, and the composite was used to repair a large radius bone defect in the rabbit. The specimens were made at 2,6,12 weeks. The histology imageology, and the radionuclide bone scan were used to analyze the bone formation. Results Some MSCs had a good adherence 4 hours after MSCs were inoculated and had a complete adherence at 12 hours. The cells were shaped like polyangle, spindle or converge monolayer after 8-10 days. The cells in the two groups had no difference. The cell adhesion was good, when observed by the inverted phase contrast microscope and the scanning electron microscope at 6 days. MTT showed that the absorbance (A)was not statistically different between the experimental group and the control group (P＞0.05); the different density leaching liquor had no cytotoxicity at the different time points. Histology, X-ray, and CT tomograph showed that itcould repair the large radius bone defect in the rabbit and its in vivo degradationrate was the same as the bone formation rate. Conclusion The new porous β-TCP has a unique three dimensional (3D) stereochemical structure and superordinary physicochemical property, and so it is a good scaffold for the bone tissue engineering.
Objective To investigate the gene expression of p38mitogen-activated protein kinase (p38MAPK) and its upstream signaling molecule (mkk3 and mkk6) in fetal skin at different developmental stages and postnatal skin and its potential biological significance. Methods The fetal skin biopsies were obtained from human embryo of spontaneous abortion at gestational ages from 13 to 32 weeks and postnatal skin specimens were collected from patients(4-16 years) undergoing plastic surgery. After the morphological characteristics of skins at different developmental stages were detected with pathological methods, the gene expressions of p38MAPK, mkk3 and mkk6 in skins were examined with reverse transcriptionpolymerase chain reaction analysis (RT-PCR). Results The gene expressions of p38MAPK, mkk3 and mkk6 could all be detected in fetal and postnatal skins. In fetal skins, these 3 genes were bly expressed. Along with fetal growth and development, the gene expressions of p38MAPK and its upstream signaling molecules were faded gradually. In postnatal skin, the mRNA contents of these 3 genes were significantly decreased in comparison with those in fetal skin (Plt;0.01). Conclusion p38 MAPK mediated signal pathways might be involved in the skin developmentat embryonic stage and in the determination of cutaneous structure and function, and also in wound healing at postnatal stage. The relative increment of these gene transcription in younger fetal skin might be one of the reasons why cutaneous cells proliferate rapidly and the wounds heal without scar.
Objective To study the effect of the allogeneic bone sheet that has been treated by the freezedrying and radiation sterilization in preventing the epidural adhesion after laminectomy in sheep. Methods Laminectomy was performed on L3,4and L4,5 of 12 adult male sheep. Afteroperation, one site of L3,4 or L4,5was covered by the allogeneicbone sheet in “H” shape after the freeze-drying and radiation sterilization treatment; and the other site was used as a control. The sheep were killed and the specimens were retrieved at 4，8，12，16，20 and 24 weeks after operation to observe the scar formation process. X-ray andCT scan were performed in the segments of L3,4and L4,5at 4 and 24weeks. Results Four weeks after operation, the position and the shape of the allogeneic bone sheet were good in condition, and no lumbar spinal stenosis or compression of the dura was found in the experimental sections. Twentyfour weeks after operation theexamination on the experimental sections revealed that the vertebral canal reconstruction was completed, the allograft was absorbed almost completely, and inosculation with the lamina of the vertebra and the shape of the lumbar spine were good, with no fibroid tissues found in the epidural area. Compared with the control segment, the dura adhesion degree in the laminoplasty segment was significantly decreased (Plt;0.01), and the dura sac remained in a good shape. There was no obvious compression on the dura. Conclusion The allogeneic bone sheet after the treatment of freeze-drying and radiation sterilization can effectively reduce the scar formation after laminectomy and prevent postoperative recurrence of the spinal stenosis.
Objective To study the feasibility of using mice marrow stromal stem cells(MSCs) as seed cells for tissue engineering cartilage to embed the seed cells in acellular cartilage matrix of human auricle. Methods Acellular cartilage matrix was made from human auricle cartilage. The MSCs were isolated from the nucleated cells fraction of mice marrow by centrifuge.The MSCs were embedded in acellular cartilage matrix. After 10 day’s combined culture, the specimens were observed with optical and electrical microscope.Results The MSCs could well proliferate in the acellular cartilage matrix. The cells were not well-distributed in acellular cartilage matrix. There were more cells in the peripheral part of the matrix than in the central part of the matrix. Most of the cells were in cartilaginous lacunae. There were 1 or 2 cells in every cartilaginous lacunae.Conclusion The MSCs can be used as seed cells of tissue engineering and can well proliferate in the acellular cartilage matrix and become tissue engineering cartilage.
