[Treatment of early avascular necrosis of femoral head by core decompression and transplantation of human hepatic growth factor gene-modified osteoblasts: experiment with rabbits].

OBJECTIVE: To evaluate the effects of transplantation of human hepatocyte growth factor (hHGF) gene-modified osteoblasts combined with core decompression in treatment of avascular necrosis of femoral head (ANFH). METHODS: The plasmid pcDNA3.1(+)-hHGF containing hHGF gene was constructed. Osteoblasts were isolated from fetal rabbits, cultured, and transfect3d with the plasmid pcDNA3.1(+)-hHGF or blank plasmid pcDNA3.1(+), or used as controls. Thirty-six adult New Zealand rabbits were made into ANFH models, underwent core decompression, and were randomly divided into 3 groups. Group A, transplanted with osteoblasts transfected with pcDNA3.1(+)-hHGF plasmid, Group B, transplanted with osteoblasts not transfected with pcDNA3.1(+)-hHGF plasmid, and Group C, injected with PBS medium. 2, 4, and 8 weeks later samples of femoral head were obtained to undergo CT, histological examination, and capillary ink infusion so as to observe the angiogenesis and osteogenesis. RESULTS: The pcDNA3.1(+)-hHGF transfected osteoblasts showed stable expression of hHGF. The numbers of newly formed vessels of the femoral heads of the group transfected with pcDNA3.1(+)-hHGF-transfected osteoblasts 2 and 4 weeks later were (29.47 +/- 1.64) and (34.02 +/- 1.72)/cm2 respectively, both significantly higher than those of the group transfected with blank plasmid-transfected osteoblasts [(20.61 +/- 1.91) and (25.57 + 2.20)/cm2 respectively, both P <0. 01]. Eight weeks later the numbers of mature trabecular bone and bone marrow of Groups A and B were significantly higher than those of Group C. CONCLUSION: Core decompression combined with transplantation of HGF gene-modified osteoblasts promotes angiogenesis, enhances bone formation, and improves the restoration of avascular necrosis of femoral head.

[Biocompatibility of combined deproteinized bone coated with hepatocyte growth factor as scaffold for osteoblasts in vitro in fetal rabbits].

OBJECTIVE: To determine the cellular compatibility of combined deproteinized bone(DPB) coated with hepatocyte growth factor (HGF), and to observe the adherent effect of osteoblasts in response to HGF. METHODS: Osteoblasts were isolated from fetal rabbits. Osteoblasts were cultured with DPB coated with HGF and deproteinized bone as experimental group and contral group, respectively. The proliferation and alkalinephosphatase activity were tested. Their growth was examined by inverted phase contrast microscope and scanning electronmicroscope. RESULTS: The osteoblasts were attached to the outside and inside surfaces and grew well. HGF/DPB could stimulate the alkalinephosphatase activity of the osteoblasts and improve the proliferation of the osteoblasts. CONCLUSION: HGF/DPB has good biocompatibility and bone induction. HGF could improve the adherent effect of DPB on osteoblasts, and it could be used as scaffold material for the bone tissue engineering.

[Construction and expression of recombinant plasmid pcDNA3.1(+) -hHGF available in osteoblast].

OBJECTIVE: To construct a plasmid carrying human hepatocyte growth factor (hHGF) gene and determine the effects of the hepatocyte growth factor (HGF) gene on proliferation and differentiation of osteoblast in vitro. METHODS: The full length cDNA of hHGF, which was amplified from human liver mRNA by RT-PCR, was cloned into pcDNA3.1(+) vector to construct pcDNA3.1(+) -hHGF recombinant plasmid. With lipofectamine, the recombinant plasmid pcDNA3.1(+) -hHGF was used to transfect the culture osteoblasts. After pcDNA3.1(+) - hHGF transfection, the positive cell clones were selected with G418. The stable transfection and expression of hHGF in the osteoblasts were measured by immunohistochemical staining and RT-PCR respectively. The effect of pcDNA3.1(+) -hHGF transfection on osteoblast proliferation was measured by MTT colorimetric assay. Flow cytometer was used to determine the effect of pcDNA3.1(+) -hHGF transfection on cell cycle of osteoblast. The quantitive detection of hHGF expression was performed through ELISA. Alkaline phosphatase (AP) was also detected using enzyme kinetics. RESULTS: The recombinant plasmid pcDNA3. 1(+) -hHGF was identified by restriction endonuclease digestion and nucleotide sequencing. Osteoblasts could be effectively transfected by pcDNA3.1(+) -hHGF with lipofectamine in vitro. The stable expression of hHGF in pcDNA3.1(+) -hHGF transfection osteoblast was confirmed. Human HGF protein was detected by immunohistochemical staining and RT-PCR in osteoblasts 4 weeks after cell clone selected with G418. pcDNA3.1(+) -hHGF gene transfer responsible to improve the proliferation was proved by MTT assay, flow cytometer showed cellular proportion in "S" phase obviously increased. AP activity in transfected cells was increased significantly. CONCLUSIONS: Osteoblasts which express stable and high-level hHGF was established successfully, exogenous hHGF gene could stimulate the proliferation, differentiation and function of osteoblast and could be applied further to gene therapy.

[Platelet and endothelial cell-derived microparticles in steroid-induced osteonecrosis of the femoral head of rabbit model].

OBJECTIVE: To explore the relationship between the alterations of platelet-derived microparticles (PMPs) and endothelial cell-derived microparticles (EMPs) and steroid-induced osteonecrosis of the femoral head. METHODS: Forty-four adult Japanese white rabbits were randomly divided into 2 groups: experimental group (n = 44), injected intramuscularly once with 20 mg/kg of methylprednisolone acetate, and control group (n = 28) injected with normal saline of the same dose. Blood samples were collected from the auricular vein while the animals were in a fasting state in early morning prior to experiment (week 0), and 2, 4, and 8 weeks after the injection. The levels of prothrombin time (PT), activated partial prothrombin time (APTT), and anti-thrombokinase-III (AT-III) were examined. Plasma was ultra-centrifuged and the circulating platelet and EMPs were isolated to undergo flow cytometry to detect the amounts of CD31, CD42a, CD54, and CD62E. 1, 2, 4, and 8 week after the injection 6 rabbits from the experimental group and 4 rabbits from the control group were sacrificed respectively and their femoral heads were taken out to undergo histopathologic examination with HE staining and microthrombus and elastic fiber staining. RESULTS: The PT, APTT, and AT-III levels increased significantly 1 to 8 weeks after the injection. The numbers of EMPs and PMPs were also increased markedly after steroid administration. Histopathologic examination demonstrated an accumulation of bone marrow cell debris, presence bone empty lacunae in the trabeculae 1 week after the steroid injection. 2, 4, and 8 weeks after the steroid injection the bone necrosis was remarkable, fatty degeneration of bone marrow cells occurred, the diameter of fat cells increased, and the area ratio of fatty cells became higher. MSB staining was positive. CONCLUSION: High dose glucocorticosteroid increases the levels of PMPs and EMPs that may be related to hypercoagulability and thrombosis in microcirculation and play an important role in steroid-induced osteonecrosis. MP can be considered a true target in the pharmacological control of steroid-induced osteonecrosis.