ObjectiveTo observe the genes expression of hypoxia inducible factor 1α (HIF-1α) and HIF-2α by inducing chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) so as to provide a fundamental basis for HIF involving in the mechanism of chondrogenesis. MethodsHigh density pellet of hBMSCs was obtained by centrifugation and cultured with H-DMEM medium containing 2% fetal bovine serum (control group) and with chondrogenic medium (chondrogenic induction group) under hypoxia (2%O2) for 3 weeks. Immunohistochemistry staining was utilized to identify extracellular proteoglycan and collagen type Ⅱ at 3 weeks after culture. Western blot was applied for measuring HIF-1α and HIF-2α protein levels at 1 week after culture. Real-time quantitative PCR was performed to detect the genes expressions of HIF-1α, HIF-2α, Sox-9, collagen type Ⅱ, collagen type X, and Aggrecan at 1, 2, and 3 weeks after culture. ResultsToluidine blue staining showed sparse nucleus in the control group, and dense nucleus in the chondrogenic induction group;extracellular matrix staining was deeper in the chondrogenic induction group than the control group. Immunohistochemical staining for collagen type Ⅱ was positive in cytoplasm;when compared with the chondrogenic induction group, the control group showed sparse and light-coloured nucleus. At 1 week after culture, the protein expression levels of HIF-1α and HIF-2α in the chondrogenic induction group were significantly lower than those in the control group (t=8.345, P=0.001;t=7.683, P=0.002). When compared with control group, the HIF-1α mRNA expression was significantly down-regulated at 1 week and significantly up-regulated at 2 weeks in chondrogenic induction group (P<0.05), but no significant difference was found at 3 weeks between the 2 groups (P>0.05). And the mRNA expression of HIF-2α was significantly down-regulated and mRNA expression of Sox-9 was significantly up-regulated after chondrogenic differentiation when compared with the control group (P<0.01). The mRNA expressions of collagen type Ⅱ and collagen type X were significantly up-regulated at 2 and 3 weeks after chondrogenic differentiation when compared with the control group (P<0.05). And the mRNA expression of Aggrecan was significantly up-regulated at each time point after chondrogenic differentiation (P<0.05). ConclusionHIF-1α may involve the hBMSCs chondrogenic differentiation under hypoxia, while HIF-2α expression is depressed throughout the period and may have negative effect on differentiation.
ObjectiveTo investigate the effect of body mass index (BMI) on the outcome of posterior 360° fusion for single-level lumbar degenerative diseases. MethodsA retrospective study was carried on 302 cases of singlelevel lumbar degenerative diseases treated with posterior 360° fusion between September 2009 and September 2013. All patients were divided into 3 groups according to BMI: normal weight (BMI<24 kg/m2) in 105 cases (group A), overweight (24 kg/m2≤BMI< 28 kg/m2) in 108 cases (group B), and obese (BMI≥28 kg/m2) in 89 cases (group C). There was no significant difference in gender, age, disease duration, disease patterns, affected segments, preoperative Japanese Orthopaedic Association (JOA) score and Oswestry disability index (ODI) among 3 groups (P>0.05). The operation time, intraoperative blood loss, postoperative hospital stay, and complications were recorded. The lumbar function was assessed by JOA score and ODI at pre- and post-operation (at 3, 6, and 24 months). ResultsThe operation time, intraoperative blood loss, and postoperative hospital stay of group C were significantly more than those of groups A and B (P<0.05), but no significant difference was found between group A and group B (P>0.05). The patients were followed up 24-45 months. Postoperative JOA score and ODI showed significant improvements in each group when compared with preoperative ones (P<0.05), but there was no significant difference among groups at each time point after operation (P>0.05). There was no significant difference in the incidence of total complications among 3 groups (χ2=3.288, P=0.193). The incidence of incision-related complications (infection and poor healing) in group C was significantly higher than that of groups A and B (P<0.05), but no significant difference was shown between group A and group B (P>0.05). However, there was no significant difference in cerebrospinal fluid leak, pseudarthrosis formation, and revision among 3 groups (P>0.05). ConclusionPosterior 360° fusion for single-level lumbar degenerative diseases can obtain good effectiveness in patients with different BMI, but patients whose BMI was ≥28 kg/m2 have longer operation time, more intraoperative blood loss, longer hospital stay, and higher incidence of postoperative incision-related complications.
ObjectiveTo explore the effect of H2O2-actived RAW264.7 macrophages on the migration, proliferation, and osteogenesis gene expression of MC3T3-E1 in mice. MethodsMC3T3-E1 cells and RAW264.7 cells were cultured to the 7th generation. RAW264.7 macrophages were stimulated with 0, 25, 50 and 100 μmol/L H2O2, the cell proliferation rate was detected by MTS at 1, 3, and 6 hours after stimulated, and superoxide dismutase (SOD) content by SOD assay kit at 1 hour after stimulated. The appropriate concentration and action time of H2O2-actived RAW264.7 were obtained. The supernatant of RAW264.7 macrophages stimulated by H2O2 or not was collected at 24 hours. Then, the supernatant was used to culture MC3T3-E1 cells in groups B (not stimulated by H2O2) and C (stimulated by H2O2), and DMEM was used as a control in group A. The migration of MC3T3-E1 cells was detected at 12 and 24 hours by cell scratch test, the proliferation of MC3T3-E1 cells at 24, 48, and 72 hours by MTS assay. MC3T3-E1 cells were cultured with only complete medium in blank control group, with complete medium containing 50 μg/mL vitamin C + 10 nmol/L β sodium glycerophosphate in positive group, normal control group (adding the supernatant not stimulated by H2O2), and experimental group (adding the supernatant stimulated by H2O2). At 3, 7, and 14 days, RT-PCR was used to determine the osteogenesis related mRNA expressions of alkaline phosphatase (ALP), Runx2, osteopontin (OPN), osteocalcin (OC), bone sialoprotein (BSP), and collagen type I (COL-I). ResultsThe results of MTS and SOD assay showed that the appropriate concentration and action time of H2O2-actived RAW264.7 macrophages were 25 μmol/L and 1 hour, respectively. MTS assay showed that the proliferation rate of MC3T3-E1 cells was significant higher in groups B and C than group A (P < 0.05), in group B than group C, and significant difference was shown between groups at 2 and 3 days (P < 0.05). The cell scratch test indicated that the migration of MC3T3-E1 cells was significantly faster in groups B and C than group A, and in group C than group B at 12 hours (P < 0.05); many migrated cells were observed in all scratch sites of groups B and C at 24 hours. When compared with positive control group, the mRNA expressions of ALP, Runx2, OC and BSP in experimental group were significantly down-regulated at 7 and 14 days (P < 0.05). When compared blank control group, the mRNA expressions of OPN and COL-I in experimental group were significantly down-regulated at 7 and 14 days (P < 0.05). ConclusionThe appropriate concentration and action time of H2O2-actived RAW264.7 macrophages are 25 μmol/L and 1 hour. The H2O2-actived RAW264.7 cells can promote MC3T3-E1 cells migration, and suppress MC3T3-E1 cells proliferation and expressions of osteogenesis related genes.