The osteogenc potential of bone marrow has been proved by experiment. To investigate more in details, bone marrow was obtained from the trochanteric region of femur of NewZealand rabbit in 4 to 8 weeks old. After being cultured in vitro for one week, the hematopoietic component of the bone marrow had disappeared, thus the stromal cells were obtained. Then the stromal cells were subcultured in cultural fluid containing dexamethasone (10-8 mol/L) and natrium glycerophosphate (10mmol/L). Under the phasecontrast microscope, it was found that being cultured for 15 days. The stromal cells were lined up in one layer and late the secretion activity was increased and gradually transformed into multilayer structure and was congregated into diffused opaque clusters in twenty days. During culture, the cells were examined by tetracycline fluorescence label, histochemistry stains, transmission electron microscopy, scanning electron microscopy and energy dispersive X-ray microanalysis. The results showed that the morphological and biological characteristics of the cultured stromal cells derived from the bone marrow were similiar to those of osteoblasts and could synthesized mineralized new bone tissue in vitro.
ObjectiveTo investigate the regulatory effect of simvastatin on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) at middle/late stages by p38MAPK pathway under condition of osteoinductive environment. MethodsThe bone marrow of bilateral femur and tibia were harvested from 20 4-week-old female Sprague Dawley rats. BMSCs were isolated and cultured with whole bone marrow culture method; the second generation of cells were randomly divided into 5 groups: control group (complete medium, CM), simvastatin group (simvastatin medium, SIM), osteogenic induction group (osteogenic induction medium, OM), simvastatin and osteogenic induction group (simvastatin+osteogenic induction medium, OM+SIM), and blocker group (SB203580+simvastatin+osteogenic induction medium, OM+SIM+SB). MTT assay was used to detect the cell activity in CM group and SIM group at 2, 3, 4, 5, and 6 days, ELISA method to measure the content of alkaline phosphatase (ALP) in OM group and OM+SIM group at 7 and 14 days. The mRNA and protein expressions of osteocalcin (OCN) were detected by real-time quatitative PCR and Western blot after 1, 12, and 24 hours of osteogenic induction at 21 and 28 days. The protein expressions of phospho-p38 (p-p38) and p38 in OM group, OM+SIM group, and OM+SIM+SB group were detected by Western blot at the best induction time of simvastatin. ResultsMTT assay showed that no significant difference was found in absorbance (A) value between CM group and SIM group at each time point (P > 0.05), indicating no effect of 1×10-7 mol/L simvastatin on cell viability. ELISA results showed that ALP content significantly increased in OM+SIM group when compared with OM group at 7 and 14 days; the ALP content was significantly higher at 7 days than 14 days in OM group and OM+SIM group (P < 0.05). OCN mRNA and protein expressions at 12 hours were significantly higher than those at other time points in each group (P < 0.05), and the expressions of OM+SIM group was significantly higher than those of OM group (P < 0.05). The best induction time of simvastatin was 12 hours. At 12 hours after blocking intervention, the p-p38/p38 in OM+SIM+SB group was significantly lower than that in OM group and OM+SIM group (P < 0.05), and the p-p38/p38 in OM+SIM group was significantly higher than that in OM group (P < 0.05). ConclusionSimvastatin can increase the mRNA and protein expression levels of OCN and the protein of p-p38 in osteogenic differentiation of BMSCs at middle/ late stages, and its best induction time is 12 hours.
Objective To clarify the trends of expression levels of several up-regulated micro RNA (miRNA) in tissues of atrophic bone nonunion and mRNAs and proteins of their related target genes in osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and to explore their biological functions. Methods The hBMSCs were isolated from bone marrow of il iac bone by gradient centrifugation, and cultured. Osteogenic culture medium was used for osteogenic differentiation of the 4th generation of hBMSCs. The changes of corresponding miRNAs, mRNA and protein expression levels of related target genes were observed at 0 hour, 12 hours, 1 day, 2 days, 4 days, 7 days, and 14 days, by quantitative real-time PCR and Western blot. Results In the process of hBMSCs osteogenic differentiation, the mRNA and protein expression levels of osteoblastic target genes [alkal ine phosphatase l iver/bone/kidney (ALPL), bone morphogeneticprotein 2 (BMP-2), and platelet-derived factor alpha polypeptide (PDGF-A)] at most time points increased significantly whencompared with the values at 0 hour except that of BMP-2 decreased at 12 hours and 1 day, with maximum changes at 1 to 7 days. The miRNA expression levels, mRNA and protein expression levels changed significantly at different time points, while the trends of hsa-miRNA-149 and hsa-miRNA-654-5p changes were negatively correlated with the trends of ALPL and BMP-2 mRNA and protein expression changes respectively (P lt; 0.05). There was no obviously negative correlation between the trends of hsa-miRNA-221 change and PDGF-A change (P gt; 0.05). Conclusion In the osteogenic differentiation process of hBMSCs, hsa-miRNA-149 and hsa-miRNA-654-5p are closely related with the mRNA and protein regulation of ALPL and BMP-2, respectively.
Objective To evaluate the adhesion, prol iferation and osteogenic differentiation of rabbit BMSCs after cultured on freeze-dried demineral ized bone matrix (FDBM) modified with type II cadherin ectodomain (Cad- II). Methods BMSCs isolated from 10 Japanese white rabbits (male and female, 4-week-old, 0.61-0.88 kg) were cultured. The second generation of BMSCs (cell density 1 × 106 /mL) were seeded onto the Cad-II modified allogenic FDBM (experimental group) and only FDBM (control group) respectively, and then cocultured in vitro. The densities of seeded cells, the adhesion rate and their ALP activity were measured. The complex was observed through inverted phase contrast microscope and scanning electron microscope to evaluate the interaction between cells and FDBM. Another group of second generation of BMSCs (cell density 5 × 105 /mL) were seeded onto the Cad-II modified FDBM (experimental group) and only FDBM (control group) respectively, and then cocultured in vitro too. The ALP activity and osteocalcin immunohistochemical was measured. Results There was no significant difference in cell prol iferation between experimental group and control group. The adhesion rate of cells in the experimental group was 87.41% ± 5.19%, higher than that in the the control group 35.56% ± 1.75% (P lt; 0.01); the densities of seeded cells reached 5.0 × 105, showing significant difference compared with the control group (2.6 × 104, P lt; 0.05). Inverted phase contrast microscope showed that in the experimental group, more cultured BMSCs pasted in the hole and edge of the scaffold than that in the control group. HE staining showed the densities of seeded cells in the experimental group was higher than that in the control group. Scanning electron microscope showed that in the experimental group, a lot of cultured BMSCs adhered, spreaded in the scaffold, in the control group only a few BMSCs unevenly distributed in the scaffold. After 7 days of culture, the cultured BMSCs on modified FDBM expressed higher ALP activity; after 14 days of culture, the ALP activity (29.33 ± 1.53) was higher than that cultured on unmodified FDBM (18.31 ± 1.32), the positive rates of osteocucl in were 83% ± 7% in the experimental group and 56% ± 7% in the control group, showing significant difference (P lt; 0.01). Conclusion Cad-II enhanced cell adhesion to FDBM and promoted BMSCs differentiate to osteoblast, but no obvious effects were observed in cell prol iferation.
ObjectiveTo review the research progress of induced osteogenesis of bone marrow mesenchymal stem cells (BMSCs) transfected by double-gene. MethodsThe recent literature concerning the comparative research of induced osteogenesis of BMSCs transfected by double-gene was extensively reviewed. The characteristics of BMSCs, the advantage and effect of synergistic inductive osteogenesis, the application prospect and problems of BMSCs transfected by double-gene were summarized. ResultsThe effect of induced osteogenesis concerning BMSCs transfected by double-gene is far superior to single gene transfection and the activity of osteoblast is also significantly increased. The research used in bone tissue engineering experiment also obtain good effect. ConclusionInduced osteogenesis of BMSCs transfected by double-gene is able to make up for the lack of a single gene transfection and has great development prospects in the orthopaedic field.
ObjectiveTo investigate the effect of KLD-12 polypeptide complexed with recombinant human bone morphogenetic protein 2 (rhBMP-2) on osteogenic activity of rabbit bone marrow mesechymal stem cells (BMSCs). MethodsBone marrow was harvested from 3-month-old New Zealand white rabbit, and density gradient method was used to isolate and culture BMSCs. The third generation BMSCs were used for three-dimensional culture of KLD-12 polypetide/rhBMP-2 in vitro (experimental group) and KLD-12 polypeptide (control group). The morphology of the cells in the gel was observed by inverted phase contrast microscope at 7 days; alkaline phosphatase (ALP) and osteocalcin protein content were dectected at 3, 7, 10, 14, and 21 days; collagen type I immunofluorescence staining was done and real-time fluorescent quantitative PCR was performed to detect the relative expression of collagen type I and osteocalcin gene at 14 days. ResultsUnder the inverted phase contrast microscope, the BMSCs in the gel of the experimental group and the control group showed circular growth, and the distribution was uniform at 7 days. There was no significant difference in the expressions of ALP and osteocalcin protein content between 2 groups at 3 and 7 days (P > 0.05); the above indexes in experimental group were significantly higher than those in the control group at 10-21 days (P < 0.05). Laser scanning confocal microscope observation showed that immunofluorescence staining for collagen type I was positive in the experimental group, and the expression was higher than that in the control group at 14 days. Real-time fluorescence quantitative PCR detection showed that the collagen type I and osteocalcin gene expressions were significantly higher than those in the control group (t=15.902, P=0.000; t=12.998, P=0.000). ConclusionBMSCs can normally grow and proliferate in the KLD-12 polypeptide, and KLD-12 polypeptide/rhBMP-2 has good biological activity to induce BMSCs differentiation into osteoblasts.
Objective To study the method of inducing human marrow mesenchymal stem cells (MSCs) into osteoblasts directionally and to identify osteogenesis characteristics. Methods MSCs were isolated from adult marrow using density gradient separation method and were cultured in conditioned medium containing Dex 10 -8 mol/L，β-GP 10 mmol/L，and AA 50 μg/ml. The MSCs attachment formed soon and passage 3 cells were chosen to check osteogenesis characteristics, including alkaline phosphatase assay with modified calcium-cobalt staining method, type Ⅰ collagen assay with immunohistochemistry, osteopontin and osteonectin assay with in situ hybridization and calcium nodes assay with Von Kossa staining. Results Passage 3 MSCs had typical appearance of osteoblasts and could be passaged continuously till passage 10. The rate of ALP expression was 85%. The expressions of collagen type Ⅰ, osteopontin and osteonectin were positive and calcium nodeswere seen by Von Kossa staining. Conclusion We have successfully induced human MSCs into osteoblasts; the induced cells have typical osteogenesis characteristics.
OBJECTIVE: To prevent the senescence of ’seed cells’ for tissue engineering, the life span of human fibroblasts is extended by reconstitution of telomerase activity, and the osteogenic potential of these fibroblasts are tested. METHODS: The pGRN145 plasmids encoding human telomerase reverse transcriptase (hTERT) were introduced into the normal human primary fibroblasts by electroporation. Telomerase activity was analyzed by TRAP-PCR assay. The beta-galactosidase stain was used to indicate the signs of cell senescence. The hTERT positive fibroblasts were then induced to form bone nodules. The bone nodules were stained by tetracycline and Alizarin Red S. RESULTS: Stable telomerase activity could be detected in the transfected fibroblasts and no signs of cell senescence were found in the fibroblasts cultured for more than 50 doublings. The hTERT positive fibroblasts could form bone nodules when they were cultured in vitro induced by bone morphogentic protein 2 and tumor necrosis factor-alpha. CONCLUSION: The fibroblasts with reconstituted telomerase activity reserve their osteogenic potential.
To study the method of isolating and culturing synovium-derived MSCs (SMSCs), and to investigate its multiple differentiation potential in vitro. Methods Three 2-month-old Changfeng hybrid swines weighing 8-10 kg (male and female) were used. SMSCs were harvested from the synovium of swine knee joints and cultured in vitro. When the SMSCs at passage 3 reached confluence, basic culture medium was removed, and the multi ple differentiationpotential of SMSCs was demonstrated in specific induction media (experimental group). The cells at passage 3 cultured with basic culture medium served as control group. After 21 days of chondrogenic differentiation, the cells underwent toluidine blue staining, immunohistochemistry staining and real-time fluorescence quantitative PCR detection. After 10 and 21 days of osteogenic differentiation, the cells underwent ALP staining and Al izarin red staining, respectively. After 21 days of adipogenic differentiation, the cells underwent Oil red O staining. Results SMSCs displayed long and thin or polygonal morphology 24 hours after culture. They prol iferated fast 48 hours after culture and presented large number of spindle-shaped cells with few globular cells 72 hours after culture. For the experimental group 21 days after chondrogenic induction, the cells were positive for toluidine blue staining with the formation of Aggrecan outside the cells; the immunohistochemistry staining revealed the expression of Col II; the real-time fluorescence quantitative PCR detection showed that the expressions of Col II A1, Aggrecan and SOX9 mRNA of the experimental group were greater than that of control group (P lt; 0.05). The cells were positive for ALP staining 10 days after osteogenic induction, and positive for Al izarin red staining 21 days after osteogenic induction, with the formation of calcium nodules. Oil red O staining displayed the formation of l i pid droplets inside the cells 21 days after adi pogenic induction. For the control group, the results of all the staining assays were negative except the ALP staining presenting with sl ight positive result. Conclusion SMSCs can be isolated from knee joint of swine and proliferate and differentiate into osteogenic, adi pogenic and chondrogenic cells in vitro. SMSCs may be a promising source of seed cells for tissue engineering.
Objective To review the progress and clinical application of malleable bone paste/putty. MethodsRecent literature about malleable bone paste/putty was reviewed and analyzed. ResultsThe preparation and clinical application of malleable bone paste/putty have become increasingly mature. Many kinds of malleable bone paste/putty have been applied extensively and the good clinical results have been achieved in the treatment of the irregular bone defects. The materials and methods for preparing malleable bone paste/putty are different. Then they have different bone repair abilities. ConclusionMalleable bone paste/putty provides effective method to treat irregular bone defects. But the malleable bone paste/putty still has some shortage, so further researches should be carried out.