Objective To investigate the effect of Hyaluronic acid(HA) on the expression of matrix metalloproteinases(MMPs)-3,9,13 and tissue inhibitor of MMPs(TIMP)-1 mRNA stimulated by interleukin (IL)-1β in chondrocytes from arthritis model in vitro.Methods Different levels of Sodium Hyaluronate(10,20,40μg/ml)were added to culture medium of rat chondrocytes.The ehondrocytes were isolated from rat osteoarthritis model.One hour later,10 ng/ml IL-1β were added to each sample and co-cultured for 24 hours.The expressions of MMP-3,9,13 and TIMP-1 mRNA were detected bv RT-PCR.The concentration of nitric oxide (NO) in the supernatants were measured by Griess reaction.Results HA could down-regulate MMP-3,9,13 mRNA expression but had no effect on TIMP-1 mRNA expression.Moreover,it also had a dose-dependent down regulate effect on nitric oxide synthesis.Conclusion This study demonstrates that HA can down-regulate the TIMP-1/MMPs expression by inhibiting NO production.

BACKGROUND: Biomechanics of bone in vivo is concerned by the scholars who work for orthopaedic and medical bioengineering all the time. The application of strainmeter in this measurement has been studied for many years, but the long-term and successive measuring problems have not been solved. A new installing method for analyzing biomechanics of bone in vivoshould be considered to be explored. OBJECTIVE: To investigate the biochemical change of external fixator at both external and internal medullary cavity of anxial compressing fracture end invivo when external fixator is used DESIGN: Randomized and controlled animal experiment SETTING: Laboratory of Orthopaedic Department, Renmin Hospital, Wuhan University MATERIALS: Totally 18 healthy white rabbits, of rather gender, with the body mass of 3.6 to 4.2 kg , were chosen. METHODS: This experiment was conducted at the laboratory of Or thopaedic Department, Renmin Hospital, Wuhan University in November 2004. The chosen 18 healthy white rabbits were randomly divided into 2groups. External measuring group (Group A): the strain guage was affixed to the tibia external cortex by 502 gauge. Internal measuring group (Group B): strain guage coated with bone cement was installed on the tibia internal cortex. Group A and Group B were divided into two subgroups A1, A2 and B1, B2, respectively according to the pressure 0.5 time body mass and 1 time body mass. Strain voltage change and attenuation coefficient after compressing were measured with scaler. Statistical comparison was performed among the groups. MAIN OUTCOME MEASURES: ① The data of scaler curve. ② Attenuation coefficient of different compressions.RESULTS: ① The scaler curves of Group A changed greatly during early stage. After reaching stable stage, trendline of internal and external cortex went consistent but the former value was higher than that of the latter . The time of compression reaching stable was shorter in the Group B than in the Group A, and the absolute value of strain was smaller in the Group B than in the Group A. ② Before reaching stable after compression, the attenuation coefficient was low in the Group A1 as compared with Group A2. There was the same result between Group B1 and Group B2 group. When comparison was conducted between Group A and Group B, the curve of Group A decreased firstly, then ascended, but there was no ascending tendency in the Group B. After reaching stable, there were descending tendency in the Group A1 group and Group A2. It descended fast in the Group A2, while it kept at normal level in the Group B1 group. Fluctuation appeared in the Group A2. CONCLUSION: Strain of external medullary cavity is significantly larger than that of internal medullary cavity. It is easier to reach stable in the inter nal medullary cavity than in the external medullary cavity. 0.5 time of body mass is suitable at the initial period in treating fracture with external fixator.

BACKGROUND: Many scholars adopt allograft interbody fusion for vertebral body resection and reconstruction, bone fusion time is better than autologous bone graft's, and its integration provides an early support and stabilizing, but the preparation of allogeneic bone graft material is easy to destroy b0ne-inducing factor in matrix, which is not conducive to bone growth. OBJECTIVE: To innovatively design and verify the ability of reconstructing rabbit cervical vertebrae with the compound of humeral cortical ring allograft (HCA) packed with red bone marrow (RBM) and autogenous cancellous bone (ACB). DESIGN, TIME AND SETTING: Randomized controlled animal experiment was performed at the laboratory of Orthopedic Department in Renmin Hospital of Wuhan University, between October 2004 and March 2006. MATERIALS: Sixty healthy adult New Zealand white rabbits, of either sex, body mass of 2.0-2.5 kg, were involved in this study. Twelve rabbits were used for HCA preparation, while the remaining 48 rabbits were randomly divided into 3 groups with sixteen rats in each group. Autologous RBM was extracted from the anterior superior lilac spine through puncture; ACB was obtained from td-cortical bone of rabbit iliac crest. Autologous RBM and ACB were compounded and filled in the self-made HCA. METHODS: Models of the fourth cervical vertebrae defect were created by surgery to simulate tumor resection in New Zealand white rabbits, which were divided into 3 groups randomly. Combined transplant group was treated with the compound of RBM+ACB+HCA; autologous bone transplant group with autogenous lilac crest; HCA transplant group with HCA. MAIN OUTCOME MEASURES: Vertebral reconstructions were evaluated by X-ray, histopathological observation and scanning electron microscope, as well as measurement of serum alkaline phosphates at different periods postoperatively. RESULTS: Eight weeks post-surgery, graft materials fused with the upper and lower cervical fusion, a large number of bone callus were observed in combined transplant group and autologous bone transplant group; HCA transplant group was present with a small amount of callus growth and poor fusion. Serum alkaline phosphatase levels were elevated in all groups, significantly higher in combined transplant group and autologous bone transplant group compared with HCA transplant group (P < 0.01 ). There were no significant differences of serum alkaline phosphatase levels between combined transplant group and autologous bone transplant group at 4 weeks or among 3 groups 8 weeks (P > 0.05). Histological analysis exhibited numerous mature bone matrix, bone trabecula and bone marrow cavity formed in combined transplant group and autologous bone transplant group. Scanning electron microscopy showed that many new bone formations in combined transplant group and autologous bone transplant group.CONCLUSION: The compound of RBM+ACB+HCA and autogenous lilac crest transplantation can efficiently reconstruct cervical vertebrae, RBM+ACB can improve the reconstruction efficiency of HCA, and could use as a matedal in cervical reconstruction.

BACKGROUND: Vertebral reconstruction is stil a chal enge for spinal surgeons. Ideal reconstruction materials should have good osteogenesis ability, reliable support performance, low price and simple operating steps.
OBJECTIVE:To investigate the effect and feasibility of al ogenic cortical ring packed with autogenous cancellous bone in the reconstruction of rabbit cervical vertebra defect.
METHODS:Cervical vertebra defect models were established by resection of the fourth cervical vertebra in rabbits. Frozen-dried humeral cortical ring of rabbits was made to prepare the al ogenic cortical ring which was packed with autogenous cancellous bone. Then, al ogenic cortical ring packed with autogenous cancellous bone, single al ogenic cortical ring and autogenous iliac bone were used to repair rabbit cervical vertebra defects.
RESULTS AND CONCLUSION:The X-ray, histopathological examination, and scanning electron microscope examinations al showed that the bone union was most significant in group reconstructed with al ogenic cortical ring packed with autogenous cancellous bone. The alkaline phosphates activity in serum was higher in groups reconstructed with al ogenic cortical ring packed with autogenous cancellous bone and with autogenous iliac bone than in group reconstructed with only al ogenic cortical ring at 2 and 4 weeks after operation (P<0.01), but there was no difference between the former two groups. The biomechanical examination showed that the stability of al ogenic cortical ring packed with autogenous cancellous bone was higher than that of autogenous iliac bone at 1 month after operation (P<0.01). These findings indicate that the effect of al ogenic cortical ring packed with autogenous cancellous bone in the reconstruction of rabbit cervical vertebra defects is satisfactory, with a good biocompatibility, which is a promising method for the reconstruction of vertebral defects.

Objective To investigate the effects of chitosan/pCDNA3.1 (+) CrmA on matrix metalloproteinase (MMP)-1 and tissue inhibitor of metalloproteinase (TIMP)-1 expression of chondrocytes induced by interleukin-1β (IL-1β) in mRNA and protein levels.Methods Rabbit chondrocytes were isolated and cultured.Chondrocytes were treated with PBS,10 μg/ml chitosan (CS) and chitosan/ pCDNA3.1 (+)CrmA respectively for 6 hours.Then 10 ng/ml IL-1β was added into the culture medium.After 48 hours,real time polymerase chain reaction (real time PCR) and Western blot assay were used to examine the changes of MMP1 and TIMP-1 in mRNA and protein levels.Results In CS/pCDNA3.1 (+)CrmA treated group (0.44±0.04),the messenger RNA expression of MMP-1 in chondrocytes was significantly suppressed compared with corresponding samples of PBS treated group (1.00±0.05) and CS treated group (0.76±0.07),There was significant difference between three groups (F=106.93,P＜0.01).The MMP-1 protein expression of chondrocytes in CS/pCDNA3.1 (+)CrmA treated group (0.28±0.03) was lower than that of PBS treated group (0.73±0.06) and CS treated group (0.46±0.05)(F=59.66,P＜0.01).No significant difference of TIMP-1 expression among the three groups was observed in mRNA and protein levels.Conclusion CS/pCDNA3.1(+) CrmA can significantly inhibit the mRNA and protein expressions of MMP-1 of chondrocytes induced by IL-1β,which leads to up-regulation the ratio of TIMPs to MMPs of IL-1β induced chondrocytes.It may be a part of the mechanisms of the therapeutic effects of CS/pCDNA3.1 (+)CrmA on osteoarthritis.

Objective To study the effect of interleukin (IL)-1β on mitochondria of chondrocytes and reactive oxygen species.Methods Rat chondrocytes were isolated and cultured in vitro,10 ng/ml IL-1β was added for establishing model of osteoarthritis.Then,ratio of apoptosis was surveyed by Annexin V-FITC and PI flow-cytometry.After Hoechst 33342 dyeing,morphology of nucleus was observed by fluorescence microscope.After rhodamine-123 luorescent staining applied,change of mitochondria membrane potential was observed by confocal microscopy.We evaluated the ability of ATP synthesizing in mitochondria by luciferase reaction.Content of active oxygen was observed by confocal microscopy.T test was used for statistical analysis.Results IL-1β could obviously reduce the mitochondrial membrane potential of chondrocytes and its ability to synthesize ATP,and could promote the expression of reactive oxygen species in cells.The values were changed to (24±4) U,(4.1±0.8) pmol/106 cells and (89±7) U from (86±10) U,(13.3±3.0) pmol/106 cells and (17±3) U.The difference had statistical significance.Conclusion IL-1β can induce chondrocyte differentiation by destroying the mitochondrial membrane integrity of chondrocytes and promoting the expression of reactive oxygen species.It can also promote the articular cartilage degeneration.

BACKGROUND:Diabetic osteoporosis is gaining public attention.However,few studies have reported the effect of a high-glucose environment on the osteogenic differentiation of human umbilical cord mesenchymal stem cells and the corresponding therapeutic strategies. OBJECTIVE:To investigate whether vitamin D3 can restore the osteogenic differentiation potential of human umbilical cord mesenchymal stem cells in a high-glucose environment. METHODS:The viability of human umbilical cord mesenchymal stem cells was detected by CCK-8 assay to screen the appropriate vitamin D3 intervention concentration.Under the high-glucose environment,RT-qPCR,western blot assay,immunofluorescence,JC-1 mitochondrial membrane potential,alizarin red staining,and β-galactosidase staining were used to evaluate the osteogenic differentiation potential,intracellular reactive oxygen species accumulation,mitochondrial membrane potential alteration,and cell senescence of human umbilical cord mesenchymal stem cells after vitamin D3 intervention.The underlying mechanism was also discussed. RESULTS AND CONCLUSION:(1)Vitamin D3 significantly promoted the proliferation of human umbilical cord mesenchymal stem cells in the range of 0.1 μmol/L to 1 mmol/L.(2)High-glucose environment down-regulated the mRNA and protein level expressions of osteogenic-related genes α1-I collagen,alkaline phosphatase,Runt-associated transcription factor 2,and osteocalcin in human umbilical cord mesenchymal stem cells,which induced oxidative stress and cellular senescence.(3)Vitamin D3 at an intervention concentration of 10 μmol/L significantly restored the osteogenic phenotype of human umbilical cord mesenchymal stem cells under high-glucose conditions and attenuated intracellular oxidative stress and cellular senescence by activating the Nrf2/HO-1 signaling pathway.(4)These findings suggested that the osteogenic differentiation ability of human umbilical cord mesenchymal stem cells was reduced in the high-glucose environment,and vitamin D3 could partially improve their osteogenic differentiation ability and reduce cell damage.

BACKGROUND:Osteoporosis has a high incidence,leading to fracture and other complications.However,existing drugs have great side effects and are difficult to meet the clinical application. OBJECTIVE:To explore the effect and potential mechanism of fucoxanthin on osteoporosis induced by glucocorticoid. METHODS:Primary rat osteoblasts were inoculated in 6-well plates.When the cell fusion reached 80%,the cells were divided into four groups:the control group was cultured alone for 24 hours,the glucocorticoid group was intervened with dexamethasone for 24 hours,the fucoxanthin group was intervened with fucoxanthin for 24 hours,and the glucocorticoid + fucoxanthin group was intervened with dexamethasone and fucoxanthin at the same time for 24 hours.After intervention,cell proliferation,apoptosis,intracellular reactive oxygen species level,and protein expression of apoptosis-related proteins,bone formation-related proteins,and nuclear factor erythroid-2-related factor 2 were detected. RESULTS AND CONCLUSION:Cell counting kit-8 results showed that the cell viability was decreased in the glucocorticoid group compared with the control group(P＜0.05)but increased in the glucocorticoid+fucoxanthin group compared with the glucocorticoid group(P＜0.05).JC-1 mitochondrial membrane potential staining and flow cytometry assay showed that the percentage of apoptosis increased in the glucocorticoid group compared with the control group(P＜0.05)but decreased in the glucocorticoid+fucoxanthin group compared with the glucocorticoid group(P＜0.05).Western blot assay showed that compared with the control group,the protein expression of BAX and cleaved poly(ADP-ribose)polymerase was elevated in the glucocorticoid group(P＜0.05),and the protein expression of BCL2,type Ⅰ collagen α1 peptide chain,alkaline phosphatase,osteocalcin,and RUNX2 was decreased in the glucocorticoid group(P＜0.05).Compared with the glucocorticoid group,the protein expression of BAX and cleaved poly(ADP-ribose)polymerase was decreased(P＜0.05),and the protein expression of BCL2,type Ⅰ collagen α1 peptide chain,alkaline phosphatase,osteocalcin,and RUNX2 was elevated(P＜0.05)in the glucocorticoid+fucoxanthin group.Fluorescent probe assay showed an increase in reactive oxygen species level in the glucocorticoid group compared with the control group(P＜0.05)and a decrease in reactive oxygen species level in the glucocorticoid+fucoxanthin group compared with the glucocorticoid group(P＜0.05).Immunofluorescence staining and western blot assay showed that the protein expression of nuclear factor erythroid-2-related factor 2 in the glucocorticoid group was decreased compared with that in the control group(P＜0.05);and the protein expression of nuclear factor erythroid-2-related factor 2 in the glucocorticoid+fucoxanthin group was elevated compared with that in the glucocorticoid group(P＜0.05).To conclude,fucoxanthin can improve glucocorticoid-induced osteoblast apoptosis and the expression of bone formation-related molecules by activating nuclear factor erythroid-2-related factor 2.