Objective To explore the role and mechanism of stress stimulation in the repair process of the rotator cuff. Methods The supraspinatus tendon was cut off at the osteotendinous junction (OTJ) and then sutured in situ (Carpenter's model). The experimental group started treadmill running after one week immobility, and the control group were free in the cage. All animals were sacrificed at 4 time points (2, 4, 8 and 16 weeks after operation). Histological changes, immunohistology of yon Willebrand factor and tenascin-C at OTJ were compared between the 2 groups. Results There was no significant difference between the 2 groups in the early repair period. Significant differences between the 2 groups in histology and angiogenesis appeared 8 weeks after operation, and significant differences in expression Of Tenascin-C appeared 16 weeks after operation. Conclusion As stress stimulation can promote angiogenesis and expression of Tenascin-C at OTJ, facilitating the reconstruction of bone and tendon, it plays a critical role in the repair process of rotator cuff tear.

[Objective] To examine the specific gene and protein expression of primary and passaged chondrocytes in normal oxygen tension and hypoxia.[Methods]Articular chondrocytes were isolated from limb joint cartilage of C57BL/6 mice(3~5 days).Primary chondrocytes(P0)and passaled chondrocytes(P1,P2)were cultured in normal oxygen tension and hypoxia respectively for two days.The mRNA levels of collagen II,aggrecan,sox9,Indian hedgehog(ihh),parathyroid hormone-related protein(PTHrP),bone morphogenetic protein(BMP)-4 and wnt5a were examined with RT-PCR,and protein levels of collagen II,aggrecan were examined with immunohistochemistry and toluidine blue staining.[Results]During the culture of primary chondrocyte,the expression of collagen II and aggrecan increased under hypoxia,mRNA levels of collagen II,wnt5a increased and ihh decreased at the same time.The mRNA levels of special genes and differentiation related genes of passaged chondrocytes were not altered under hypoxia.[Conclusion]Protein and mRNA level of Collagen II of primary chondrocyte under hypoxia increased.It may be regulated by chondrocyte differentiation gene ihh,and wnt5a.The chondrocyte phenotype could not be resumed under hypoxia through short-term monolayer culture in vitro.

Objective To observe the tissus implanted into the damaged articular cartilage of rabbits with induced autogeneic mesenchymal cells. Methods The autologous mesenchymal cells derived from bone marrow of New Zealand rabbits were harvested. With basic fibroblast growth factor(bFGF， 25 ng/ml) and transforming growth factor beta 1(TGF-? 1， 2 ng/ml)， the cells were induced and expanded in cell culture. The induced cells with absorbable gelatin sponge as a carrier were then implanted into the damaged articular cartilage in rabbits as experimental group. The absorbable gelatin sponge without cells were served as control. Specimens were harvested at the end of 4, 12 and 24 week after implantation, and were stained with toluidine blue. Results By RT- PCR, it was confirmed that there was expression of typeⅡ procollagen mRNA in the induced mesenchymal cell. After implantation, it was difficult to macroscopically distinguish the repaired tissues from the normal cartilaginous tissue in the experimental group in 24 weeks. While the defect of articular cartilage was filled with white and swampy tissue in the control group at the same time. Histologically, the defective area of the articular cartilage was replaced by the formation of neo- cartilage which showed positive staining of toluidine blue in 4 weeks in the experimental group. The neo- cartilage was modeled to normal cartilage tissues in 12 weeks and was similar to the surrounding cartilage in 24 weeks. But in the control group, the defect of articular cartilage was not repaired by cartilage tissue at every stage and were replaced by fibrocartilage which was shown weakly positive staining of toluidine blue in 24 weeks. Conclusion The transplant of the induced autogeneic mesenchymal cells derived from bone marrow might promote repair of articular cartilage, and restore its structure and function.

BACKGROUND:Mechanical signal has close correlation with the growth, development, repair and reconstruction of the skeletal system and the development of disease, the effect and the mechanism on bone marrow mesenchymal stem cel s is worthy to concern.
OBJECTIVE:To explore the effect and mechanism of hydrostatic pressures on the differentiation of bone marrow mesenchymal stem cel s.
METHODS:Short-term experiment:the human bone marrow mesenchymal stem cel s were incubated into the normal Dulbecco’s modified Eagle’s medium, osteogenic medium or the Dulbecco’s modified Eagle’s medium containing extracel ular signal-regulated kinase 1/2 inhibitor U0126, respectively. Homemade pressure loading system was used to impose 0, 40 and 80 kPa hydrostatic pressure for 1 and 4 hours. Long-term experiment:human bone marrow mesenchymal stem cel s were incubated into the normal Dulbecco’s modified Eagle’s medium or osteogenic medium respectively, and then 40 kPa hydrostatic pressures was loaded for 4 hours per day, and lasted for 14 days. The cel s without hydrostatic pressure were regarded as the control group.
RESULTS AND CONCLUSION:Real-time quantitative reverse transcription PCR results showed that after osteogenic induction and simulated with 40 kPa hydrostatic pressure for 4 hours, the mRNA expressions of core binding factorα1 and osteocalcin in the bone marrow mesenchymal stem cel s were increased, while the mRNA expressions of peroxisome proliferator-activated receptorγ2 and adipsin were decreased, and the 80 kPa hydrostatic pressure did not cause such reactivity. The osteogenic induction effect of 40 kPa hydrostatic pressure could be partial antagonized with U0126. Histochemical staining showed that after simulated with 40 kPa hydrostatic pressure for 7 days, the expression and activity of alkaline phosphatase of bone marrow mesenchymal stem cel s were increased;after lasted for 14 days, the mRNA expressions of peroxisome proliferator-activated receptorγ2 and adipsin were increased. Certain intensity and duration of hydrostatic pressure stimulation can regulate the differentiation of bone marrow mesenchymal stem cel s, and the mechanism is only partly mediated by the extracel ular signal-regulated kinase 1/2 signaling pathway.

OBJECTIVE: To examine estrogen receptor (ER) in osteoblasts from adult human and to elucidate the mechanism of estrogen in modulating bone metabolism. METHODS: The cultured osteoblasts were harvested from bone chips by modified sequential digestive enzyme release and immunohistochemical assay of ER in osteoblasts were carried out in three groups of female adults: normal control (group 1), patients with moderate osteoporosis (group 2) and patients with serious osteoporosis (group 3). The percentages of ER-positive osteoblasts from the three groups were compared by t test. RESULTS: The brown marks that indicate ER were found in nuclei and plasma of the osteoblasts, and the percentages of ER-positive osteoblasts among three groups were significantly different. CONCLUSION: ERs exist in nuclei and plasma of the osteoblasts. Estrogen may modulate bone metabolism through binding ER in nuclei and plasma of the osteoblasts. The reduction of ER of osteoblasts may play an important role in the pathogenesis of postmenopausal osteoporosis.

BACKGROUND:Vascular endothelial growth factor (VEGF) is a crucial factor for regulating bone marrow mesenchymal stem cells in microenvironment, which can promote endothelial differentiation of bone marrow mesenchymal stem cells. But there is no report about VEGF effect on regulating the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells. OBJECTIVE:To investigate the regulatory role of VEGF in the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells. METHODS:The bone marrow mesenchymal stem cells were isolated and cultured from mouse long bones. cellCounting Kit-8 was used to test the proliferation of bone marrow mesenchymal stem cells cultured with different concentrations of recombinant VEGF proteins. The appropriate concentration of VEGF recombinant protein was selected to test their effects on the osteogenic differentiation of bone marrow mesenchymal stem cells. The mRNA and protein levels of Osterix, Runx2, alkaline phosphatase, osteocalcin and heme oxygenase-1 in bone marrow mesenchymal stem cells under induction of VEGF were detected by molecular biology methods. RESULTS AND CONCLUSION:(1) VEGF promoted the proliferation of bone marrow mesenchymal stem cells dose-dependently, and 100μg/L was the optimum concentration. (2) VEGF promoted the expression of Osterix, Runx2, alkaline phosphatase and osteocalcin in bone marrow mesenchymal stem cells under osteogenic induction. Similar results were obtained by alizarin red staining and quantification of numbers of calcium nodules. (3) VEGF induced the expression of heme oxygenase-1 in bone marrow mesenchymal stem cells at mRNA and protein levels. These findings indicate that VEGF can induce the expression of heme oxygenase-1 i in bone marrow mesenchymal stem cells, and promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.

To investigate the effects of Sangen Decoction, a compound Chinese herbal medicine, on osteoclastogenesis and bone resorption function of osteoclasts induced by polymethylmethacrylate particles in vitro.

To establish a stable, useful culture system for human osteoclasts and to investigate the effect of osteoblasts on the differentiation, proliferation and activation of osteoclasts so as to provide a base for the studies on prevention and treatment of osteolysis and osteoporosis.

OBJECTIVETo investigate the probabilities of core-biding factor a1 (Cbfa1) expression by human skin fibroblasts induced in vitro.

METHODSThe fibroblasts were isolated, purified from human skin, and were grown in incubation in the media of TNF-alpha, BMP-2, and combined TNF-alpha and BMP-2 at certain concentrations, respectively. The changes in biological features of these fibroblasts correlated with osteogenesis were detected by immunohistochemistry and RT-PCR assay.

RESULTSTNF-alpha could switch phenotype of collagen in fibroblasts from Type I and III to Type I and induce fibroblasts to express Ras and BMP type I receptor (BMPR-IA). TNF-alpha in combination with BMP-2 could induce fibroblasts to express Cbfa1 and osteocalcin mRNA.

CONCLUSIONHuman skin fibroblast could be induced into pro-osteoblast expressing Cbfa1, an osteoblast-specific transcription factor and a regulation of osteoblast differentiation, and combined use of TNF-alpha and BMP-2 was one of the regulating factors.

Bone Morphogenetic Protein 2 ; Bone Morphogenetic Protein Receptors, Type I ; Bone Morphogenetic Proteins ; pharmacology ; Cells, Cultured ; Collagen ; biosynthesis ; Core Binding Factor Alpha 1 Subunit ; Core Binding Factors ; Fibroblasts ; metabolism ; Humans ; Neoplasm Proteins ; Osteocalcin ; biosynthesis ; Protein-Serine-Threonine Kinases ; biosynthesis ; RNA, Messenger ; analysis ; Receptors, Growth Factor ; biosynthesis ; Skin ; cytology ; Transcription Factors ; biosynthesis ; genetics ; Transforming Growth Factor beta ; Tumor Necrosis Factor-alpha ; pharmacology

Objective To investigate the role of hypoxia inducible factor-1α (HIF-1α) and von Hippel-Lindau (VHL) in murine endochondral ossification. Methods The knockout of HIF-1α or VHL gene in murine osteoblasts was accomplished by conditional knockout technique at 4th, 8th and 12th week, and the differences between wild-type group and knock-out group in endochondral ossification were detected by HE staining, micro-CT scanning, trabecular bone area measurement, calcium content measurement, tetracycline fluorescence labeling, Real-time PCR and Western blotting. Results After knockout of HIF-1α gene in osteoblasts, the expression of vascular endothelial growth factor ( VEGF) reduced, the rate of new bone formation stepped down, the content of calcium became less, and the trabecular bone volume decreased (P <0.05) . After knockout of VHL gene in osteoblasts, the expression of VEGF increased, the rate of new bone formation stepped up, the content of calcium became more, and the trabecular bone volume was promoted (P < 0.001). Conclusion During murine endochondral ossification, VHL/HIF-1α signal pathway promotes angiogenesis through the stimulation of VEGF expression, which subsequently accelerates osteogenesis.