Chen Anmin

Biocompatibility studies on fibrin glue cultured with bone marrow mesenchymal stem cells in vitro.

SummaryBy culturing bone marrow mesenchymal stem cells of rabbits with fibrin gluein vitro, the biocompatibility of fibrin glue was investigated to study whether this material can be used as scaffolds in bone tissue engineering. After 2-months old New Zealand rabbits had been anesthetized, about 4–6 ml of bone marrow were aspirated from rabbit femoral trochanter. The monocytes suspension was aspirated after bone marrow was centrifuged with lymphocyte separating medium and cultured primarily. Then the cells were divided into two groups: one was cultured with complete medium and the other with induced medium. The cells of the two groups were collected and inoculated to the culture plate containing fibrin glue. In the control group, cells were inoculated without fibrin glue. The implanted cells and materials were observed at different stages under a phase-contrast microscope and scanning electron microscope. MTT and alkaline phosphatase (ALP) were measured. Bone marrow mesenchymal stem cells grew on the surface of fibrin glue and adhered to it gradually. Cells light absorption value (A value) and the ALP content showed no significant difference. Fibrin glue had no inhibitory effect on cell morphology, growth, proliferation and differentiation. It has good biocompatibility and can be used as scaffold materials for bone marrow mesenchymal stem cells in bone tissue engineering.

Effects of icariin on expression of OPN mRNA and type I collegen in rat osteoblastsin vitro

SummaryTo study the effects of Icariin on expression of osteopontin (OPN) mRNA and type I collagen in rat osteoblastsin vitro and to explore its possible mechanisms in preventing osteoporosis. OB was isolated from calvaria of new-born new-born fetal Sprague-Dawley (SD) rats by means of modified sequential collagenase digestion and incubated in MEM medium and the cell morphology was observed under inverted phase contrast microscope, OB was identified by alkaline phosphatase (ALP) staining. Different concentration (0.1 μg/mL, 1.0 μg/mL, 10 μg/mL) of Icariin was added to the OB and incubated. The effect of Icariin on the proliferation and osteogenesis of OB was monitored by MTT analysis. The expression of type I collagen was estimated with immunohistochemistry techniques. The expression levels of mRNA of OPN in the cells in every group were examined by reverse-transcriptase polymerase chain reaction (RT-PCR). The expression of OPN mRNA and type I collagen was strengthened gradually with the increase of Icariin concentration and peaked with 10 μg/mL Icariin on the 5th day. Icariin could significantly promote the expression of OPN mRNA and type I collagen in rat osteoblastsin vitro. The levels of expression of OPN mRNA and type I collagen were changed with different concentration of Icariin. Icariin could effectively prevent and treat osteoporosis and promote the bone formation.

Effects of co-grafts mesenchymal stem cells and nerve growth factor suspension in the repair of spinal cord injury

SummaryTo investigate effect of the transplantation of mesenchymal stem cells (MSCs) in combination with nerve growth factor (NGF) on the repair of spinal cord injury (SCI) in adult rats, spinal cord of adult rats (n=32) was injured by using the modified Allens method. One week after the injury, the injured cords were injected with Dubecco-modified Eagles medium (DMEM, Group I), MSCs (Group II), NGF (Group III), and MSCs plus NGF (Group IV). One month and two months after the injury, rats were sacrificed and their injured cord tissues were sectioned for the identification of the transplanted cells. The axonal regeneration and the differentiation of MSCs were examined by immunocytochemical staining. At the same time, rats were subjected to behavioral tests by using the open-field BBB scoring system. Immunocytochemical staining showed that axonal regeneration and the transplanted cells partially expressed neuron-specific nuclear protein (NeuN) and glial fibrillary acidic protein (GFAP). At the same time, significant improvement in BBB locomotor rating scale (P<0.05) were observed in the treatment group. More importantly, further functional improvement were noted in the combined treatment group. MSCs could differentiate into neurons and astrocytes. MSCs and NGF can promote axonal regeneration and improve functional recovery. There might exist a synergistic effect between MSCs and NGF.

Preparation of TiO2 thin film and its antibacterial activity

TiO2 nanometer thin films with photocatalytic antibacterial activity were prepared by the sol-gel method on fused quartz and soda lime glass precoated with a SiO2 layer. The thin films were characterized by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and X-ray diffraction (XRD). The results show that sodium and calcium diffusion into nascent TiO2 film is effectively retarded by the SiO2 layer precoated on the soda lime glass. The antibacterial activity of the films was determined. The crystalline of TiO2 nanometer thin film has important effects on the antibacterial activity of the film.

Extraction, purification and identification of bone morphogenetic protein in conditioned medium of osteosarcoma cell (MG-63)

Objective: To find out a method of extraction and purification of bone morphogenetic protein (BMP) from osteosarcoma cell conditioned medium, and evaluate the biological activity of BMP.Methods: Conditioned medium of osteosarcoma cell lines (MG-63) was collected, concentrated and dialyzed. The concentrated protein was purified through gel chromatography on Sephcryl-S-100. The purified protein was tested by BMP monoclonal antibody (McAb), its molecular weight (MW) was determined by SDS-PAGE and its biological activity was demonstrated by heterotopic ossification.Results: The purified protein was proved to be BMP by BMP McAb, had a satisfactory heterotopic ossification, and its MW was about 21 kD.Conclusion: BMP existed in the conditioned medium of osteosarcoma cell and had a satisfactory biological activity after purification. Because osteosarcoma cell can be cultured and grew for a long timein vitro, this method will be helpful to a vast extraction of BMP and clinical application.

Effect of rhBMP-2 and osteogenic revulsants on proliferation and differentiation of bone marrow stromal cells in rats

The effects of recombinant human bone morphogenetic protein-2 (rhBMP-2) and osteogenic revulsants alone or in combination at different time points and in different dosages on proliferation and osteogenesis of bone marrow stromal cells (BMSCs) in SD rats were investigated. Rat BMSCs were cultured in vitro and induced by rhBMP-2 in different dosages (10, 50, 100 and 200 μg/L) alone or in combination with osteogenic revulsants. MTT colorimetric assay was used to evaluate The proliferation, activity of alkaline phosphoric (ALP) and osteocalcin were measured at 3rd, 6th, 9th, 12th day respectively. The results showed that rhBMP-2 and osteogenic revulsants could promote the differentiation of BMSCs towards osteoblast phenotype. The proliferation of BMSCs could be enhanced by rhBMP-2 in a dose-dependent manner. The expression of osteoblast phenotype was significantly higher by using both of them than by using them alone, which was verified by the activity of ALP and osteocalcin. It was suggested that the combined use of rhBMP-2 and osteogenic revulsants could promote the proliferation and simultaneously induce and maintain the expression of osteoblast phenotype of BMSCs in rats.

Effect of Stathmin decoy-oligodeoxynucleotides on the proliferation and differentiation of precartilainous stem cells

By using decoy-oligodeoxynucleotides (decoy-ODNS) technique, the effects of Stathmin gene on the proliferation and differentiation of in vitro cultured precartilainous stem cells (PSCs) were investigated. The Stathmin decoy-ODNs were transfected into PSCs in rats by using gene transfection technique. Under the induction of cortisol (1 μmol/L), electrophoretic mobility shift assay was used the inhibitory effects of decoy-ODNS on Stathmin gene. MTT and cytometry were used to test the cell proliferation. The expression of collagen II and V and Stathmin protein was detected by using Western blot. The results showed that Stathmin decoy-ODNs inhibited the Stathmin activity in a dose-dependent manner. When the concentration of decoy-ODNs was 10 times of standard concentration, the proliferation of PSCs was obviously suppressed and the differentiation happened. Compared to the control group, the difference was significant (P<0.05). It was concluded that decoy-ODNs could inhibit the proliferation and promote the differentiation of PSCs by antagonizing Stathmin activity.

Anterior Lumbar Intervertebral Fusion with Artificial Bone in Place of Autologous Bone

SummaryThe feasibility of anterior lumbar intervertebral fusion with artificial bone in place of autogenous bone was investigated. Porous hydroxyapatite (HA)/ZrO2 ceramics loading bone morphogenetic protein (BMP) were implanted after removal of lumbar vertebral disc in rabbits. The adjacent intervertebral discs were also removed by the same way and autogenous illic bone was implanted. SEM observation and biomechanical test were carried out. Compound bone had a bit lower osteoinductive activity than autogenous bone by SEM (Osteoindutive activity of artificial bone in 12 weeks was the same as that of autogenous bone in 9 weeks). Biomechanical test revealed that compound bone had lower anti-pull strength than autogeneous bone (P<0.001), but there was no significant difference in anti-pull strength between compound bone at 12th week and autogenous bone at 9th week (P>0.05). It was concluded that compound bone could, be applied for anterior spinal fusion, especially for those patients who can’t use autogenous bone.

Inhibition of Proliferation of Human Osteosarcoma Cells Transfected with PIN1 Antisense Gene

Objective: To evaluate the inhibition of proliferation of human osteosarcoma cells transfected with Pin1 anti-sense gene. Methods: Different doses of antisense Pin1 gene (0, 20, 50, 100, 200, 250 µL) were transfected into osteosarcoma MG-63 cells. The cells and culture supernatant before and after transfection were collected. The curve of cell growth was made by MTT method. The cell growth cycle and apoptosis were detected by FCM. The expression of Pin1 was detected by Western-blot and that of Pin1 mRNA by polymerase chain reaction (RT-PCR) respectively. Results: MTT and FCM assays indicated that the transfection by antisense Pin1 gene could inhibit MG-63 proliferation and induce apoptosis. Western-blot assays revealed that the antisense Pin1 gene-transfected MG-63 cells had weaker staining than those without transfected with antisense Pin1 gene, and staining intensity was negatively related with doses. The cells transfected by different doses of gene (0, 20, 50, 100, 200, 250 µL) had different absorbance rate: 0.854±0.136, 0.866±0.138, 0.732±0.154, 0.611±0.121, 0.547±0.109, 0.398±0.113, 0.320±0.151 respectively, with the difference being significant by F and q test (P<0.05). The expression of Pin1 mRNA had the similar results and its absorbance rate was 0.983±0.125, 0.988±0.127, 0.915±0.157, 0.786±0.125, 0.608±0.124, 0.433±0.130, 0.410±0.158 respectively (P <0.05). Conclusion: The expression of Pin1 mRNA in MG-63 cells could be inhibited by antisense Pin1 gene, so to reduce the expression of Pin1 and depress the proliferation of human osteosarcoma cells MG-63.

Relationship between the Expression of MTA1 Gene and Invasion and Metastasis of Human Osteosarcoma Cells

AbstractObjectiveTo compare the expression level of metastasis associated-1 (MTA1) in the higher and lower metastasis sublines of human osteosarcoma cells (MG63), and to investigate the relationship between the expression level of MTA1-EGFP and in vitro invasion and metastasis of human osteosarcoma cells.MethodsThe expression level of MTA1 in two sublines of MG63 cells was detected by semi-quantitative RT-PCR, and cell invasion assay and cell proliferation assay were used to evaluate the invasive capacity in vitro in two sublines. The lower metastasis line of MG-63 cells were transfected with MTA1-EGFP full-length cDNA expression plasmid by lipofectamine. The changes of the MTA1-EGFP expression and in vitro invasion potential were measured after transfection.ResultsM8 subline expressed significantly higher level of MTA1 than that of M6 subline by RT-PCR. The invasive potentials of low metastasis MG63 cell line were increased after MTA1 gene transfection.ConclusionThere may be a relationship between MTA1 and invasive potentials of human osteosarcoma cells, and MTA1 may play a role in the molecular mechanism of tumor metastases and be a potential target for gene therapy of osteosarcoma. Further studies of MTA1 in human ostersarcoma cell metastasis are needed.