Hypoxia-inducible factor (HIF)-1alpha knockout accelerates intervertebral disc degeneration in mice.

INTRODUCTION: The abnormality of nucleus pulposus (NP) plays a critical role in intervertebral disc (IVD) degeneration, in which NP cells show apoptosis and fibrosis, leading to the ability of the disc to transfer and distribute loads between the vertebrae is decreased. Considering that hypoxia inducible factor-1α (HIF-1α) is abundantly expressed in NP and that it mediates cell proliferation, migration and apoptosis in various cell types, we hypothesized that NP-HIF-1α plays an important role in NP and evaluate whether NP-HIF-1α is involved in IVD degeneration. MATERIAL AND METHODS: Sonic Hedgehog-Cre+/- mice were crossed with HIF-1αflox/flox mice to generate NP specific HIF-1α-deficient (HIF-1α-/-) mice. Magnetic resonance imaging (MRI) study was used to evaluate NP dehydration and X-ray study was used to acquire the changes of disc height. Histological changese, content of glycoproteins and the in situ expression of aggrecan were evaluated by hematoxylin & eosin (H&E) staining, safranin-O/fast green staining and immunohistochemistry assay, respectively. Western bloting was used to detect the change of extracellular matrix in IVD. RESULTS: Firstly, the results of in situ hybridization confirmed that HIF-1α in NP was successfully knocked out in HIF-1α-/- mice. Next, for HIF-1α deficiency mice, imaging study shows IVD was narrowed in X-ray and signal intensity of NP was decreased in MR T2-weight imaging. Accordingly, the size and cell number of NP and proteoglycan content was decreased in NP-HIF-1α-/- mice. Finally, Western bloting shows that protein level of collagen II and aggrecan, two main matrix in disc, were both decreased in NP-HIF-1α-/- mice. CONCLUSIONS: The present study demonstrates that HIF-1α is essential for NP development and homeostasis and the deficiency of NP-HIF-1α leads to IVD degeneration in mice.

[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.

[Osteogenesis of rabbit skin fibroblast transfected with core binding factor a1/osteoblast specific transplanting factor-2 gene].

OBJECTIVE: To study osteoblastic phenotype expression of New Zealand rabbit skin fibroblasts transfected with mouse core binding factor a1/osteoblast specific transplanting factor-2 gene (Cbfa1/Osf2). METHODS: Cbfa1/Osf2 gene, engineered into eukaryotic expression vector pSG5, was introduced into New Zealand rabbit skin fibroblasts with catholyte liposomes-Lipofectamine 2000. Meanwhile, those transfected pSG5 and un-transfected were set the control groups. The expression of Cbfa1 gene, osteocalcin (OCN) gene, alkaline phosphatase (ALP) gene and pre-peptide 2 alpha gene of collagen type I were detected by RT-PCR assay. Cbfa1 protein was detected by Western-Blot assay, in-cell ALP activity by p-nitrophenyl phosphate (PNPP) assay and OCN content in the supernatant by radio-immunity method. The ossification nodules was detected by Alizarin-Red staining and scanning electron microscope. RESULTS: Cbfa 1mRNA and Cbfa1 protein were expressed in New Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 from the first day to the fifth day, but they were not detected in the control groups. In the pSG5-Cbfa1/Osf2 transfected group, the expression of ALP gene and OCN gene were respectively induced from the third day and the forth day, pre-peptide 2 alpha gene of collagen type I was enhanced from the third day. From the sixth day, ALP activity greatly increased, OCN strongly secreted, and they were maintained at a high level for about 4 weeks, and the difference was significant compared with the control group (P < 0.05). On the forty-second day, ossification nodules were found on the surface of pSG5-Cbfa1/Osf2 gene transfected cells. CONCLUSIONS: New Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 can express osteogenesis-related genes and proteins, and form ossification nodules on their surface.