OBJECTIVE: To investigate the preparation of bone acellular extra-cell matrix(AECM) and to analyze its component. METHODS: With low-osmosis theory and method of cell extraction by detergent, bone acellular extra-cell matrix was prepared. We observed morphologic changes with HE, Mallory-Heidenhain rapid one-step dyeing and Alcian blue dyeing and examined fibronectin(FN) and laminin(LN) with immunohistochemistry. RESULTS: Light microscope showed that the collagen fibers arranged regularly in AECM with blankness of bone lacunas by HE, Mallory-Heidenhain rapid one-step dyeing and that the region around bone lacunas was stained different degrees of blue-green by Alcian blue dyeing. The result of immunohistochemistry showed there are positive markers of FN and LN in ECM. CONCLUSION: This method for preparation of bone acellular extra-cell matrix is effective, and it can keep natural structure of collagen fibers and maintain components of ECM, such as proteoglycan, FN and LN.
Objective To evaluate the feasibility of poly-L-lactide(PLLA)/porcinederived xenogeneic bone(PDXB) composite as a scaffold for the bone tissue engineering. Methods The film and the scaffold of the PLLA-PDXB composite were respectively prepared by a solution casting method and a solution casting-particle leaching method. The composite film and scaffold were further treated by the surface alkaline hydrolysis. The surface morphology of the composite was observed by the scanning electron microscopy, and hydrophilicity degree of the composite was measured. The OCT-1 osteoblastlike cells were cultured and amplified in vitro as the seeding cells, which werethen implanted on the film and scaffold. The adherence rate, adherence shape,proliferating activity, and growing morphology of the OCT-1 osteoblastlikecells were observed on the film. Results The PDXB particle 50 μm in diameter on average had a similar phase structure to that of hydroxyapatite. But its Ca/P ratio was lower than that of hydroxyapatite. After the surface alkaline hydrolysis, the PDXB particle could be exposed on the surface of the PLLA-PDXB composite. The surface roughness and hydrophilicity of the PLLAPDXB composite were obviously enhanced. The cell adherence rate and the cell proliferation activity of the PLLAPDXB composite were higher than those of the pure PLLA material. The cells tended to grow on the exposed surface of the PDXB particles. The cells seeded on the composite scaffold could migrate to the inside of the composite scaffold and grew well. Conclusion The PLLA-PDXB composite has a good cell affinity, and this kind of composite can hopefullybecome a new scaffold material to be used in the bone tissue engineering.
Objective To evaluate the osteogenic potential of human bone marrow mesenchymal stem cells (MSCs) transferred with human bone morphogenetic protein 2(BMP 2) gene by adenovirus. Methods The MSCs were isolated from human bone marrow and cultured in vitro. They were divided into 3 groups: Adv hBMP 2 transduced group; Adv βgal transduced group; untransduced group. Western immunoblot analysis, alkaline phosphatase(ALP) staining, Von Kossa staining, and a quantitative ALP activity assay were performed. Nine unde mice received injection into a thigh muscle to test the osteoinductivity of the three types of cells. Results In the Adv-hBMP-2 transprotein; most MSCs were stained positively for ALP activity 9 day after transduction; the MSCs reached the peak of ALP activity 12 day after transduction; the calcified nodes formed 21 days after transduction. The ectopic bones formed in the thigh muscles of the nude mice. Little bone formation was observed in the other groups 4 weeks after cell injection. Conclusion Adenovirus mediated hBMP-2 gene transfection can induce osteogenesis of human bone marrow mesenchymal stem cells.
OBJECTIVE To study the feasibility of constructing tissue engineered cartilage by differentiated rabbit bone marrow mesenchymal stem cells(MSC) cultured in vitro and in vivo. METHODS The MSC were isolated from the nucleated cells fraction of autologous bone marrow by density gradient centrifuge, and then induced into chondrogenic differentiation by adding dexamethasone, transforming growth factor-beta 1 (TGF-beta 1) and ascorbic acid in vitro. After 3 weeks, some cells turned to round shape and secreted metachromatic matrix. The cartilaginoid grafts composed of chondrogenic MSC. Bovine type I collagen and human fibrin were cultured within the chondrogenic medium for 2 weeks in vitro or transplanted subcutaneously adjacent to the knee joint for 3 weeks in vivo. RESULTS The most cells in the grafts were degenerated and disappeared after cultured in vitro. But the residual cells were survival and secreted metachromatic staining proteoglycan with toluidine blue, which was characteristic cartilage matrix. The grafts developed into matured cartilage tissue assessed by histological examination after 3 weeks of transplantation in vivo. CONCLUSION MSC can be used as functional cells to constructing tissue engineered cartilage.
Objective To investigate the ability of the biodegradable polycaprolactone (PCL) material to repair bone defect and to evaluate the feasibility ofusing the PCL as the scaffold in tissue engineering bone. Methods The bone defect models of 4.5 mm×12 mm were made in the bilateral femoral condyle of 65 NewZealand white rabbits. The PCL cylinder was implanted into the right side of defect(experimental group, n=60), the high dense crystality hydroxyapatite was implanted into the left side of defect (control group, n=60), and the incision was sutured without any implants (blank group, n=5). The samples were harvested and observed by examinations of gross, X-ray, bone density,99mTc-MDP bone scanning, γ-display ratio and scanning electron microscope (SEM) after 3, 6, 9 and 12 months of operation. The results were compared between the experimental group and control group. Results At 3, 6, 9 and 12 months after operation, the gross and X-ray examinations indicated that the bone defect filled with the new bone onthe PCL-tissue surface, and no delayed inflammatory reaction appeared. The average bone mineral density was greater in the experimental group than that in the control group, and the difference had statistical significance(P＜0.05). Theresults of 99mTc-MDP bone scanning and γ-display ratio showed that thenuclide uptake was more in the PCL group than that in the control group. The SEM result proved that the new compact bone formed on the PCL migrating surface as the PCL degraded gradually，but the collagen fiber sheathe formed around the hydroxyapatitein the control group. Conclusion PCL possesses good biocompatibility and high bone inductive potentiality, it can be used to repair bone defect.
Objective
To construct the lentiviral vector containing homo sapiens forkhead box C2 (Foxc2) gene and to detect its expression in bone marrow mesenchymal stem cells (BMSCs) of rabbits.
Methods
Human Foxc2 gene coding region fragment was obtained by RT-PCR and then cloned into the plasmid of LV-green fluorescent protein (GFP) to prepare Foxc2 lentiviral plasmid. Foxc2 lentiviral plasmid, pGC-LV, pHelper1.0, and pHelper2.0 were co-transfected into 293T cells to obtain recombinant virus containing Foxc2 gene. The lentiviral titer was detected. BMSCs were isolated from bone marrow of rabbit and infected with Foxc2 recombined lentiviral, then the optimum multiplicity of infection (MOI) was determined by detecting the intensity of fluorescence expression. The expression of Foxc2 in the infected BMSCs was determined at 1, 3, and 7 days after transfection by inverted fluorescence microscope and Western blot. After osteogenic induction, Alizarin red staining was done to observe the formation of mineralized nodule.
Results
The Foxc2 recombinant lentiviral vector was constructed and was confirmed by restriction enzyme digestion and sequencing analysis. It could efficiently transfect 293T cells and express in 293T cells. The lentiviral titer was 2 × 108 TU/mL. The optimum MOI was 200. The inverted fluorescence microscope observation showed that the Foxc2 gene expressed in 84.5% ± 4.8% of infected BMSCs at 3 days after transfection. The expression of Foxc2 in infected BMSCs was stable and high, and increased gradually within 7 days after transfection by Western blot. At 2 weeks after osteogenic induction, Alizarin red staining showed that there were a large number of red calcified matrix deposition in the cytoplasm.
Conclusion
Foxc2 recombined lentivirus with high viral titer is successfully constructed and packaged, and the Foxc2 gene can be transfected into BMSCs with stable and high expression of Foxc2 in infected cells, and these cells may be applied for gene therapy of avascular necrosis of the femoral head.
Repair of bone defect by compound of bone morphogenic protein (BMP) and its prior bone matrix gelatin (BMG) was compared with repair by BMP with hydroxyapatite(HA). The results showed that the BMP/BMG group was found fibrous callus in the bone defect in 4th week. In 8th week a large quantity of osseos trabecula was found. In 12th week the BMP was absorbed completely and was replaced by newly formed bone. In 16th week the recanalization appeared in the bone cavity. While in the BMP/HA group, although the fibrous callus was appeared in the 4th and 8th weeks, the HA was not absorbed. In the 12th and 16th weeks the change was similar to that in the 8th week and no recanalization of bone marrow cavity. It was suggested the BMP/BMG compound might be an ideal material to repair the bone defect.
OBJECTIVE: To study the effect of simvastatin on the expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphates (ALP) activity in the primary cultured bone marrow stromal cells, and to elucidate the mechanism of the anabolic osteogenetic effect of simvastatin. METHODS: Bone marrow stromal cells in femur and tibia of adult mouse were cultured in vitro. after treated with different concentrations of simvastatin (0, 0.1, 0.2, 0.5 and 1.0 mumol/L) or recombinant human BMP-2 for 72 hours, ALP activity of bone marrow stromal cells was determined. BMP-2 expression of bone marrow stromal cells was analyzed by using immunocytochemistry and Western blotting. RESULTS: After treated with simvastatin for 72 hours, BMP-2 expression increased, while little BMP-2 expression could be observed in the control group. ALP activity also increased in a dose-dependent manner; t-test showed that ALP activity in the group which concentrations of simvastatin were 0.5 mumol/L (t = 2.35, P = 0.041), 1.0 mumol/L (t = 2.348, P = 0.041) had significant difference when compared with control group. CONCLUSION: Simvastatin lead to high expression of BMP-2 in bone marrow stromal cells, via the increased auto- or para-crine of BMP-2, and ALP activity increased. These may be parts of the mechanism on the anabolic osteogenetic effect of simvastatin.
Objective Mechanical stimulation and inductive factors are both crucial aspects in tissue engineered cartilage. To evaluate the effects of mechanical stimulation combined with inductive factors on the differentiation of tissue engineered cartilage. Methods Bone marrow mesenchymal stem cells (BMSCs) were isolated from newborn porcine (aged7 days and weighing 3-6 kg) and expanded in vitro. The BMSCs at passage 2 were seeded onto a scaffold of poly (lactic-coglycol ic acid) (PLGA) in the concentration of 5 × 107/mL to prepare cell-scaffold composite. Cell-scaffold composites were cultivated in a medium with chondrocyte-inducted factors (group A), in a vessel with mechanic stimulating only (group B), or mechanic stimulating combined with chondrocyte-inducted factors (group C) (parameters of mechanics: 1 Hz, 0.5 MPa, and 4 hours/day). Cell-scaffold composite and auto-cartilage served as positive control (group D) and negative control (group E), respectively. After 4 weeks of cultivation, the thickness, elastic modulus, and glycosaminoglycan (GAG) content of composites were measured. Additionally, BMSCs chondrogenic differentiation was assessed via real-time fluorescent quantitative PCR, immunohistochemistry, and histological staining. Results The thickness, elastic modulus, and maximum load in group C were significantly higher than those in groups A and B (P lt; 0.05). In groups A, B, and C, cartilage lacuna formation, GAG expression, and positive results for collagen type II were obsersed through HE staining, Safranin-O staining, and immunohistochemistry staining. The dyeing depth was deeper in group A than in group B, and in group C than in groups A and B; group C was close to group E. The GAG content in group C was significantly higher than that in groups A and B (P lt; 0.05). Real-time fluorescent quantitative PCR revealed that mRNA expressions of collagen type I, collagen type II, and GAG in group C were significantly higher than those in groups A and B (P lt; 0.05), and in group A than in group B (P lt; 0.05). Conclusion Mechanical stimulation combined with chondrocyte inductive factors can enhance the mechanical properties of the composite and induce higher expression of collagen and GAG of BMSCs.
Objective To explore the osteogenesis and angiogenesis effect of bone marrow mesenchymal stem cells (BMSCs) derived osteoblasts and endothelial cells compound with chitosan/hydroxyapatite (CS/HA) scaffold in repairing radialdefect in rats. Methods The BMSCs were isolated from Sprague Dawley rats and the 3rd generation of BMSCs were induced into osteoblasts and endothelial cells. The endothelial cells, osteoblasts, and mixed osteoblasts and endothelial cells (1 ∶ 1) were compound with CS/HA scaffold in groups A, B, and C respectively to prepare the cell-scaffold composites. The cell proliferation was detected by MTT. The rat radial segmental defect model was made and the 3 cell-scaffolds were implanted, respectively. At 4, 8, and 12 weeks after transplantation, the graft was harvested to perform HE staining and CD34 immunohistochemistry staining. The mRNA expressions of osteopontin (OPN) and osteoprotegerin (OPG) were detected by RT-PCR. Results Alkal ine phosphatase staining of osteoblasts showed that there were blue grains in cytoplasm at 7 days after osteogenic induction and the nuclei were stained red. CD34 immunocytochemical staining of the endothelial cells showed that there were brown grains in the cytoplasm at 14 days after angiogenesis induction. MTT test showed that the proliferation level of the cells in 3 groups increased with the time. HE staining showed that no obvious osteoid formation, denser microvessel, and more fibrous tissue were seen at 12 weeks in group A; homogeneous osteoid which distributed with cord or island, and many osteoblast-l ike cells were seen in groups B and C. The microvessel density was significantly higher in groups A and C than group B at 3 time points (P lt; 0.05), and in group A than in group C at 12 weeks (P lt; 0.05). The OPN and OPG mRNA expressions of group A were significantly lower than those of groups B and C at 3 time points (P lt; 0.05). In groups B and C, the OPN mRNA expressions reached peak t8 and 12 weeks, respectively, and OPG mRNA expressions reached peak at 4 weeks. Conclusion BMSCs derived steoblasts and endothelial cells (1 ∶ 1) compound with CS/HA porous scaffold can promote bone formation and vascularization in bone defect and accelerate the healing of bone defect.
