Differential Expression Profiles and Functional Predication of Circular Ribonucleic Acid in Traumatic Spinal Cord Injury of Rats.

Recent studies indicate that circular ribonucleic acids (circRNAs) are involved in a variety of human diseases. The roles of circRNAs in traumatic spinal cord injury (SCI) remain unknown, however. We performed RNA-seq to analyze the circRNA expression profile in rat spinal cord after SCI and to investigate the relevant mechanisms. In all, 150 circRNAs were significantly differentially expressed in rat spinal cord after SCI by a fold-change ≥2 and p value ≤0.05. Among these, 99 circRNAs were upregulated, while 51 were downregulated. Gene ontology, Kyoto Encyclopedia of Genes and Genomes pathway analyses, and circRNA/miocroRNA (miRNA) interaction networks were conducted to predict the potential roles of circRNAs in the process of SCI. In addition, the expression levels of six selected circRNAs were verified successfully by quantitative real-time polymerase chain reaction. Further study identified circRNA_07079 and circRNA_01282 as being associated with SCI, and they may participate in the pathophysiology of SCI through circRNA-targeted miRNA-messenger RNA axis. In summary, the results of our study revealed the expression profiles and potential functions of differentially expressed circRNAs in traumatic SCI in rats; this may provide new clues for studying the mechanisms underlying SCI and also present novel molecular targets for clinical therapy of SCI.

Rifampin suppresses osteoclastogenesis and titanium particle-induced osteolysis via modulating RANKL signaling pathways.

Wear particles liberated from the surface of prostheses are considered to be main reason for osteoclast bone resorption and that extensive osteoclastogenesis leads to peri-implant osteolysis and subsequent prosthetic loosening. The aim of this study was to assess the effect of rifampin on osteoclastogenesis and titanium (Ti) particle-induced osteolysis. The Ti particle-induced osteolysis mouse calvarial model and bone marrow-derived macrophages (BMMs) were used. Rifampin, at dose of 10 or 50 mg/kg/day, was respectively given intraperitoneally for 14 days in vivo. The calvariae were removed and processed for Further histological analysis. In vitro, osteoclasts were generated from mouse BMMs with receptor activator of nuclear factor-κB ligand (RANKL) and the macrophage colony stimulating factor. Rifampin at different concentrations was added to the medium. The cell viability, tartrate-resistant acid phosphatase (TRAP) staining, TRAP activity and resorption on bone slices were analysis. Osteoclast-specific genes and RANKL-induced MAPKs signaling were tested for further study of the mechanism. Rifampin inhibited Ti-induced osteolysis and osteoclastogenesis in vivo. In vitro data indicated that rifampin suppressed osteoclast differentiation and bone resorption in a dose-dependent manner. Moreover, rifampin significantly reduced the expression of osteoclast-specific markers, including TRAP, cathepsin K, V-ATPase d2, V-ATPase a3, c-Fos, and nuclear factor of activated T cells (NFAT) c1. Further investigation revealed that rifampin inhibited osteoclast formation by specifically abrogating RANKL-induced p38 and NF-κB signaling. Rifampin had significant potential for the treatment of particle-induced peri-implant osteolysis and other diseases caused by excessive osteoclast formation and function.

Deferoxamine released from poly(lactic-co-glycolic acid) promotes healing of osteoporotic bone defect via enhanced angiogenesis and osteogenesis.

The regeneration capacity of osteoporotic bones is generally lower than that of normal bones. Current methods of osteoporotic bone defect treatment are not always satisfactory. Recent studies demonstrate that activation of the hypoxia inducible factor-1α (HIF-1α) pathway, by genetic methods or hypoxia-mimicking agents, could accelerate bone regeneration. However, little is known as to whether modulating the HIF-1α pathway promotes osteoporotic defect healing. To address this problem in the present study, we first demonstrated that HIF-1α and vascular endothelial growth factor expression levels are lower in osteoporotic bones than in normal bones. Second, we loaded poly(Lactic-co-glycolic acid) (PLGA) with the hypoxia-mimetic agent deferoxamine (DFO). DFO released from PLGA had no significant effect on the proliferation of mesenchymal stem cells (MSCs); however, DFO did enhance the osteogenic differentiation of MSCs. In addition, DFO upregulated the mRNA expression levels of angiogenic factors in MSCs. Endothelial tubule formation assays demonstrate that DFO promoted angiogenesis in human umbilical vein endothelial cells. Third, untreated PLGA scaffolds (PLGA group) or DFO-containing PLGA (PLGA + DFO group) were implanted into critically sized osteoporotic femur defects in ovariectomized rats. After treatment periods of 14 or 28 days, micro-CT, histological, CD31 immunohistochemical, and dynamic bone histomorphometric analyses showed that DFO dramatically stimulated bone formation and angiogenesis in a critically sized osteoporotic femur defect model. Our in vitro and in vivo results demonstrate that DFO may promote the healing of osteoporotic bone defects due to enhanced angiogenesis and osteogenesis. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2515-2527, 2016.

mTORC1 maintains the tumorigenicity of SSEA-4(+) high-grade osteosarcoma.

Inactivation of p53 and/or Rb pathways restrains osteoblasts from cell-cycle exit and terminal differentiation, which underpins osteosarcoma formation coupled with dedifferentiation. Recently, the level of p-S6K was shown to independently predict the prognosis for osteosarcomas, while the reason behind this is not understood. Here we show that in certain high-grade osteosarcomas, immature SSEA-4(+) tumor cells represent a subset of tumor-initiating cells (TICs) whose pool size is maintained by mTORC1 activity. mTORC1 supports not only SSEA-4(+) cell self-renewal through S6K but also the regeneration of SSEA-4(+) TICs by SSEA-4(-) osteosarcoma cell dedifferentiation. Mechanistically, active mTORC1 is required to prevent a likely upregulation of the cell-cycle inhibitor p27 independently of p53 or Rb activation, which otherwise effectively drives the terminal differentiation of SSEA-4(-) osteosarcoma cells at the expense of dedifferentiation. Thus, mTORC1 is shown to critically regulate the retention of tumorigenicity versus differentiation in discrete differentiation phases in SSEA-4(+) TICs and their progeny.

Activation of HIFa pathway in mature osteoblasts disrupts the integrity of the osteocyte/canalicular network.

The hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α, are the central mediators of the homeostatic response that enables cells to survive and differentiate in low-oxygen conditions. Previous studies indicated that disruption of the von Hippel-Lindau gene (Vhl) coincides with the activation of HIFα signaling. Here we show that inactivation of Vhl in mature osteoblasts/osteocytes induces their apoptosis and disrupts the cell/canalicular network. VHL-deficient (ΔVHL) mice exhibited a significantly increased cortical bone area resulting from enhanced proliferation and osteogenic differentiation of the bone marrow stromal cells (BMSCs) by inducing the expression of ß-catenin in the BMSC. Our data suggest that the VHL/HIFα pathway in mature osteoblasts/osteocytes plays a critical role in the bone cell/canalicular network and that the changes of osteocyte morphology/function and cell/canalicular network may unleash the bone formation, The underlying mechanism of which was the accumulation of ß-catenin in the osteoblasts/osteoprogenitors of the bone marrow.

Bisphosphonates and risk of subtrochanteric, femoral shaft, and atypical femur fracture: sensitivity and trim and fill studies.

OBJECTIVE: This study carried out sensitivity analysis and trim-fill analysis between bisphosphonates and subtrochanteric, femoral shaft, and atypical femur fracture. METHODS: A random-effects model was used finally. Sensitivity, trim and fill, and publication bias analyses were done. RESULTS: Under a random-effects model (I(2)=87.535), the Z-value=5.672, p-value of test of null<0.001. Bisphosphonate exposure was associated with an increased risk of atypical femur fracture (3.243 [95% CI 2.160-4.870]). When any study is removed, the remaining sensitivity analysis results are still significant. Trim and fill results show that two studies were missed. After filling them, a funnel plot of precision by log risk ratio was more symmetrical. CONCLUSION: This study suggests that (1) there is an increased risk of subtrochanteric, femoral shaft, and atypical femur fracture in bisphosphonate users; (2) any single study does not influence the total sensitivity; (3) two studies have been lost, theoretically.

Multiple signaling pathways involved in stimulation of osteoblast differentiation by N-methyl-D-aspartate receptors activation in vitro.

AIM: Glutamate receptors are expressed in osteoblastic cells. The present study was undertaken to investigate the mechanisms underlying the stimulation of osteoblast differentiation by N-methyl-D-aspartate (NMDA) receptor activation in vitro. METHODS: Primary culture of osteoblasts was prepared from SD rats. Microarray was used to detect the changes of gene expression. The effect of NMDA receptor agonist or antagonist on individual gene was examined using RT-PCR. The activity of alkaloid phosphotase (ALP) was assessed using a commercial ALP staining kit. RESULTS: Microarray analyses revealed that 10 genes were up-regulated by NMDA (0.5 mmol/L) and down-regulated by MK801 (100 µmol/L), while 13 genes down-regulated by NMDA (0.5 mmol/L) and up-regulated by MK801 (100 µmol/L). Pretreatment of osteoblasts with the specific PKC inhibitor Calphostin C (0.05 µmol/L), the PKA inhibitor H-89 (20 nmol/L), or the PI3K inhibitor wortmannin (100 nmol/L) blocked the ALP activity increase caused by NMDA (0.5 mmol/L). Furthermore, NMDA (0.5 mmol/L) rapidly increased PI3K phosphorylation, which could be blocked by pretreatment of wortmannin (100 nmol/L). CONCLUSION: The results suggest that activation of NMDA receptors stimulates osteoblasts differentiation through PKA, PKC, and PI3K signaling pathways, which is a new role for glutamate in regulating bone remodeling.

[Effects of rat serum containing Chinese herbal medicine Sangen Decoction on osteoclastogenesis and bone resorption of osteoclasts induced by polymethylmethacrylate particles].

OBJECTIVE: 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. METHODS: Macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) were used to induce differentiation of bone marrow-derived macrophages (BMMs) towards osteoclasts. BMMs and polymethylmethacrylate particles with ratio of 1:3 were added to the 24-well plate and 96-well plate with bone slices respectively. A total of 50 male SD rats were divided into 5 groups randomly with each group containing 10 rats. After being treated with different drugs, serum samples of rats in each group were extracted, i.e., the blank serum, Western medicine (ibandronate) serum and high-, medium-, and low-dose Sangen Decoction serum and were added to the medium respectively. The tartrate-resistant acid phosphatase (TRAP) staining was used to identify the differentiation of BMMs and for counting of osteoclasts. Area of lacuna induced by osteoclast bone resorption on the bone slices was measured by computer image processing. RESULTS: Numbers of osteoclasts of treatment groups were less than that of blank group by TRAP staining (P<0.05); numbers of osteoclasts of positive control group and high-dose Sangen Decoction group were much lower than those of medium- and low-dose Sangen Decoction groups (P<0.05), and no difference was found between Western medicine group and high-dose Sangen Decoction group (P>0.05). In bone resorption assay, area of lacuna of blank group was larger than those of treatment groups (P<0.05); areas of lacuna of Western medicine group and high-dose Sangen Decoction group were much smaller than those of medium- and low-dose Sangen Decoction groups (P<0.05), and no difference was found between Western medicine group and high-dose Sangen Decoction group (P>0.05). CONCLUSION: Sangen Decoction can inhibit osteoclastogenesis induced by polymethylmethacrylate particles as well as bone resorption function of osteoclasts.

[In vitro and in vivo study of chondrogenesis on the hybrid scaffold from fibrin modified PLGA and adipose-derived stem cells].

AIM: To investigated the effect of the presence of fibrin in the PLGA scaffold on the differentiation of adipose-derived stem cell (ASCs) into chondrocytes in the chondrogenic media. METHODS: ASCs were prepared by colagenase I digestion of fat from rabbits. The PLGA scaffolds were prepared by LDM technology. The hybrid scaffold was fabricated by a freeze-drying method. Isolated ASCs were cultured in the PLGA without and with fibrin up to 14 days in specific chondrogenic medium. The surface property of the scaffold was observed by SEM. Cell attachment was evaluated, and glycoaminoglycans (GAGs) content was tested by biochemical method. RESULT: When ASCs were seeded within fibrin modified PLGA scaffold in vitro, enhanced cellular attachment and differentiation were observed compared to unmodified PLGA scaffold. The study from articular cartilage defect repaired showed that the group from the autologous ASCs seeded on fibrin-PLGA scaffold had better chondrocyte morphology, tissue integration, continuous subchondral bone, and much thicker newly formed cartilage layer as compared with other groups. CONCLUSION: Such modification of PLGA may ultimately enhance the efficacy of tissue engineered scaffolds for cartilage tissue engineering using ASCs.

Biomechanical effects of the Coflex implantation on the lumbar spine. A nonlinear finite element analysis.

OBJECTIVE: To evaluate the effect of the stabilization of the Coflex device on the biomechanical behavior of the instrumented and adjacent spinal segments. METHODS: The study was carried out at the Department of Orthopedics, Shanghai Institute of Traumatology and Orthopedics, Ruijin Hospital, Shanghai, China between September 2009 and May 2010. Upon validation, a finite element model of L3-S1 segment was developed to simulate and analyze the biomechanics of the intact and Coflex implanted states subjected to simulate loading of flexion, extension, lateral bending, and axial rotation. RESULTS: This study predicted that the segmental motion, intradiscal pressure, and facet contact force at the levels adjacent to the Coflex implanted level were not significantly affected by the implantation of Coflex device. There was a significant decrease in range of motion in extension at the instrumented level of the Coflex implanted model relative to the intact model. Furthermore, the level implanted with Coflex device showed a significant decrease in intradiscal pressure in extension and a decrease in facet contact force in extended, lateral bending and axial rotational conditions, compared with the intact model. CONCLUSION: This study suggests that the Coflex device has the potential of effectively unloading the disc in extension and the facet joints in extension, lateral bending, and axial rotation at the Coflex implanted level, without deleterious effects on the adjacent segments under the simulated physiological condition.

Effect of erythropoietin on the survival of retinal neurocytes in culture upon serum withdrawal.

OBJECTIVES: To clarify whether erythropoietin (EPO) could substitute for the serum component in cultured retinal neurocytes suffering from serum withdrawal. METHODS: The study was performed in the Shanghai Institute of Traumatology and Orthopedics, Shanghai, China between April 2008 and March 2009. A total of 160 postnatal 2-3 day-old Sprague-Dawley rats were used for this study. After the retinal neurocytes were cultured for 48 hours, the culture media was replaced with serum-free media, and the cells were exposed to 1 U/ml, 3 U/ml, and 6 U/ml EPO for another 24 or 48 hours, the cell body diameter was then assessed using a computerized image-analysis system, and the survival and apoptosis rates of those cells were estimated by method of transcription and translation assay and flow cytometry. Immunocytochemistry was used to detect EPO and erythropoietin receptor (EPOR) expression. RESULTS: The retinal neurocytes had obvious EPO/ EPOR expression. The early (p = 0.002) and total (p = 0.049) apoptosis rates of retinal neurocytes cultured with serum withdrawal were significantly higher than that of neurocytes cultured with serum, and the cell viability of neurocytes cultured with serum withdrawal was significantly lower than that of neurocytes cultured with serum (p = 0.047). The EPO had no effect on the cell body diameter of cultured retinal neurocytes. The cell viability and the apoptosis rates of retinal neurocytes were not significantly different from that of simple serum-withdrawal culture at any EPO concentration. CONCLUSION: As the addition of EPO immediately after serum withdrawal had no effect in preventing retinal neurocytes apoptosis induced by serum withdrawal, EPO cannot substitute for the serum component.

[Expression of hypoxia inducible factor-1alpha in long bone development].

OBJECTIVE: To observe dynamically the development of fetal long bone and detect the expression and distribution of HIF-1alpha,to investigate the expression pattern and possible effects of hypoxia inducible factor-1alpha (HIF-1alpha) in fetal long bone development of mouse. METHODS: E12.5, E13.5, E14.5, E15.5, E16.5 and E17.5 pregnant C57BL6 mice were sacrificed. After sacrifice, the embryos were delivered by caesarean section. The development of fetal long bone was dynamically observed by stereoscopic microscope, and the distributional expression of HIF-1alpha protein was detected by using method of immunohistochemistry. The expression of HIF-1alpha mRNA and osteoblast marker gene at various stage were also detected by using methods of reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The cartilaginous long bone began to form and joints outline arised at E13.5, then the primary ossification center was observed at E14.5, showing opaque ossification under stereoscopic microscope,and then the osteogenesis expanded and extended to both sides. Immunohistochemistry demonstrated lots of HIF-1alpha protein positive chondrcytes in the center of primary ossification at E14.5, then they decreased dramatically. HIF-1alpha mRNA expressed at high level from E13.5 to E15.5, and then decreased to low level. CONCLUSION: Fetal long bone development pattern appeared to be endochondral osteogenisis process, existing hypoxia microenviroment may increase HIF-1alpha mRNA expression and thus initiate the cascade of endochondral osteogenisis.

Prolyl hydroxylase inhibitors increase neoangiogenesis and callus formation following femur fracture in mice.

Skeletal trauma and impaired skeletal healing is commonly associated with diminished vascularity. Hypoxia inducible factor alpha (HIF-1) is a key transcription factor responsible for activating angiogenic factors during development and tissue repair. Small molecule inhibitors of the prolyl hydroxylase enzyme (PHD), the key enzyme responsible for degrading HIF-1, have been shown to activate HIF-1, and are effective in inducing angiogenesis. Here we examined the effects of several commercially available PHD inhibitors on bone marrow mesenchymal stromal cells (MSCs) in vitro and in a stabilized fracture model in vivo. Three PHD inhibitors [Desferrioxamine (DFO), L-mimosine (L-mim), and Dimethyloxalylglycine (DMOG)] effectively activated a HIF-1 target reporter, induced expression of vascular endothelial growth factor (VEGF) mRNA in vitro, and increased capillary sprouting in a functional angiogenesis assay. DFO and DMOG were applied by direct injection at the fracture site in a stabilized murine femur fracture model. PHD inhibition increased the vascularity at 14 days and increased callus size as assessed by microCT at 28 days. These results suggest that HIF activation is a viable approach to increase vascularity and bone formation following skeletal trauma.

NMDA enhances stretching-induced differentiation of osteoblasts through the ERK1/2 signaling pathway.

Activation of the excitatory neurotransmitter N-methyl-d-aspartate (NMDA) and stretching both increase Ca(2+) influx in osteoblastic cells. We postulated that NMDA would enhance the osteoblastic cell's response to stretching. The goal of this study was to investigate, in the presence of the neurotransmitter NMDA, the effect of mechanical loading on osteoblast's stage of differentiation and the mitogen-activated protein kinase (MAPK) signaling pathway associated with it. Rat primary osteoblastic cells were subjected to cyclic, equibiaxial stretch for 48 h in the presence or absence of NMDA. Pretreatment with 0.5 mM NMDA significantly enhanced the stretching magnitude-dependent increase in osteogenesis markers. MK801, an antagonist of NMDA receptors, abolished those responses. To further study the mechanism of this response, osteoblastic cells were stretched for 5, 15, or 60 min in the absence of NMDA. Cyclic stretch induced a rapid increase in extracellular signal-regulated kinase ERK1/2 phosphorylation with the peak at 15 min, but no changes were noted in p38 and JNK pathway signaling. NMDA could enhance ERK1/2 phosphorylation stimulated by stretching. U0126, an inhibitor of ERK1/2, blocked the increase in osteogenesis markers. In conclusion, the current study demonstrates that there is a synergistic effect between mechanical stimulation and NMDA in osteoblasts. ERK1/2 signaling may be the common pathway in the increased response to stretching in the presence of NMDA in osteoblastic cells.

Hypoxia regulation of facilitated glucose transporter-1 and glucose transporter-3 in mouse chondrocytes mediated by HIF-1alpha.

OBJECTIVES: Chondrocytes reside in a hypoxic environment; they utilize glucose as the main energy source. Facilitated glucose transport represents the first rate-limiting step in glucose metabolism. RESULTS: Here we demonstrated that hypoxia induced an elevated expression level of GLUT-1 and -3 in chondrocytes. Also elevated was hypoxia inducible factor-1alpha (HIF-1alpha) protein level. On the contrary, deletion of HIF-1alpha led to complete loss of the hypoxia-induced gene expression of GLUT-1 and 3. CONCLUSION: These results suggested that chondrocytes had the capacity to detect and respond to low oxygen availability with changes in expression of oxygen-regulated genes, and that hypoxia regulation of facilitated GLUT-1 and -3 was mediated by HIF-1alpha.

[The regulation of hypoxia inducible factor-1alpha on osteoblast function in postmenopausal osteoporosis].

OBJECTIVE: To study the regulation of hypoxia inducible factor-1alpha (HIF-1alpha) on osteoblast function in postmenopausal osteoporosis. METHODS: From October 2004 to May 2006, Cre-Loxp recombinase was used to create mice which the HIF-1alpha gene in osteoblasts was conditional knock-out, 24 female wild-type (WT) mice and 24 female conditional knock-out (CKO) mice of 3 months old were operated on ovariotomy. At 0,4,8 weeks after operation, bone histomorphometry parameters were measured with computer image analysis in HE stain sections and in tetracycline bone double labeling fluorescence sections; Bone density and the trabecular bone architecture parameters were measured by Micro-CT; The mRNA expression of vascular endothelial growth factors (VEGF), RunX2, OC, ALP were detected with quantitative RT-PCR; The protein expression of VEGF and RunX2 were detected with Western-blotting. RESULTS: In CKO mice, the trabecular number, volume, thickness, bone density, mineral apposition rate (MAR), the expression of VEGF, RunX2, OC, ALP on mRNA level and the expression of VEGF, RunX2 on protein level decreased significantly compared with WT mice especially in 8 weeks after operation. CONCLUSIONS: The bone formation ability of osteoblasts in CKO mice was reduced compared with WT mice after ovariotomy. HIF-1alpha can regulate the bone formation ability of osteoblasts in postmenopausal osteoporosis.

[Regulation of hypoxia inducible factor-1alpha on osteoblast function in osteogenesis].

OBJECTIVE: To explore the regulation of hypoxia inducible factor-1alpha (HIF-1alpha) on osteoblast function in osteogenesis. METHODS: Skull-cap bone of HIF-1alpha Loxp/Loxp and VHL Loxp/Loxp C57/BL6 mice were taken out and cultured so as to obtain osteoblasts which were infected with the recombinant adenovirus Ad-Cre so as to conditionally knock out the HIF-1alpha gene and its up-stream gene for von Hippel-Lindau disease (VHL) using Cre-Loxp recombinase technique. Then the osteoblasts were cultured under 2% O2 for 48 hours. Real-time PCR and Western-blotting were used to detect the mRNA and protein expression of vascular endothelial growth factor (VEGF), core binding factor al (RunX2), alkaline phosphatase (ALP) and osteocalcin (OC). Transgenic FVB mice mated with C57/BL6 mice with both HIF-1alpha and VHL alleles to obtain the mice with the osteoblasts with the HIF-1alpha and VHL genes conditionally knocked-out. At the age of 3 months the distal femurs of HIF-1alpha/VHL conditionally knocked-out mice and wild type mice were obtained to undergo hematoxylin-eosin staining and micro-CT to evaluate the bone histomorphometry and bone mineral density (BMD). RESULTS: The mRNA and protein expressions of VEGF, RunX2, ALP, and OC of the HIF-1alpha conditionally knocked-out osteoblasts were all decreased and the mRNA and protein expressions of VEGF, RunX2, ALP, and OC of the VHL conditionally knocked-out osteoblasts were all increased. The values of bone histomorphometry and bone mineral density (BMD) of the HIF-1alpha conditionally knocked-out mice were both significantly lower than those of the wild-type mice, whereas the values of bone histomorphometry and BMD of the VHL conditionally knocked-out mice were both significantly higher than those of the wild-type mice (all P < 0.01). CONCLUSION: Under both the physiological and pathological hypoxia environment in bone tissues HIF-1alpha can promote the bone formation ability of osteoblast.

[Experimental study of core binding factor a1 gene-modified rabbit skin fibroblasts enhance bone defect repair].

OBJECTIVE: To investigate bone defect healing by true bone ceramic complex carrying core binding factor a1 (Cbfa1) gene modified rabbit skin fibroblasts. METHODS: Transfect rabbit skin fibroblasts (RSF) with both eukaryotic expression vector pSG5 which could express Cbfa1 gene and pSG5. After being cultured for 48 h, the transfected RSF were seeded into true bone ceramic (TBC) of 2 cm in length and 4 mm in diameter to construct pSG5-Cbfa1/RSF/TBC complex and pSG5/RSF/TBC complex. Forty-eight bone defect model rabbits were randomized into four groups, each has 6 rabbits (12 radius), due to different treatment. group I: with pSG5-Cbfa1/RSF/TBC complex, group II: with pSG5/RSF/TBC complex, group III: with TBC, Group IV: empty control. After being seeded and cultured for about 24 h the complexes were implanted into 2 cm long bone defects in the middle of bilateral radius of rabbits. The radius were inspected by X-ray and then the specimens were collected at the end of the fourth and twelfth weeks after operation. Then, the specimens were decalcified and histologically investigated with Hematoxylin eosin staining and Masson staining methods. Newly synthesized trabecular bone was inspected by image analysis system and the strength of bone defect area treated with graft-implantation was tested with biomechanical method-three point bending test. RESULTS: In group I, trabecular bone was actively synthesized to generate a great amount of trabecular bone and osteon. Preliminary union and bone defect healing were completed with good biomechanical characteristics. There were no newly synthesized trabecular in the other three groups, and bone defect healing were not discovered. The amount of newly synthesized trabecular bone and the results of biomechanical testing differed significantly between group I and the other three (P < 0.01). The efficacy of group I was significantly better than that of the other three groups. CONCLUSION: True bone ceramic complex composed with Cbfa1 gene modified rabbit skin fibroblasts can effectively heal bone defect in rabbits.

[Expression of hypoxia inducible factor-1alpha in the fetal vertebra development: experiment with mice].

OBJECTIVE: To explore the expression pattern and effects of hypoxia inducible factor-1alpha (HIF-1alpha) in the fetal vertebra development. METHODS: Fetuses at different developmental stages were obtained from C57BL6 mice. The vertebrae of the fetuses were isolated and the development of vertebra was observed by stereoscopic and light microscope. The mRNA expression of HIF-1alpha at different time points was detected by using reverse transcription-polymerase chain reaction (RT-PCR) and the protein expression of HIF-1alpha was detected by immunohistochemistry. RT-PCR was used to detect the mRNA expression the gene of vascular endothelia growth factor (VEGF), the Col2a1, gene for the chondroblast marker collagen type 2, and the gene of the osteoblast marker, osteocalcin (OCN). RESULTS: The cartilaginous spine column began to form at E13.5. The primary ossification center was observed at E15.5, and then the osteogenesis expanded and extended to both sides. HIF-1alpha mRNA began to express at E13.5, and more significantly at E14.5 (P < 0.05), then decreased. The VEGF mRNA was expressed coincidently with HIF-1alpha. while the HIF-1alpha protein expression was observed at E14.5 and lasted a little longer till the time when the fetus was to be born. The Col2a1 mRNA expression was high at E13.3 - E15.5 and decreased at e16.5 - E18.5, however, the OCN mRNA expression was low at E13.5 - E15.5 and became high at E16.5 - 18.5. CONCLUSION: The vertebra development pattern appears to be an endochondral osteogenesis process. There exists a hypoxia microenvironment in the vertebra that may increase the mRNA and protein expression of HIF1alpha and the expression of VEGF, its downstream gene. It shows that HIF1alpha activates the downstream genes and initiates the cascade of endochondral osteogenesis.