BACKGROUND:Transforming growth factor-βhas been shown to exert an obvious induction effect on the differentiation of bone marrow mesenchymal stem cells into chondrocytes. Cyclical tensile strain simulates mechanical environment of chondrocytes in the body, and plays an important regulatory role in cellproliferation and differentiation. OBJECTIVE:To discuss the synergy of transforming growth factor-βand cyclical tensile strain in inducing the differentiation of bone marrow mesenchymal stem cells into chondrocyte-like cells. METHODS:A total of 10 2-month-old New Zealand rabbits were selected. Bone needle was used to penetrate the medul ary cavity of bone. 3.0-4.0 mL of bone marrow was extracted for isolation and culture of bone marrow mesenchymal stem cells. Passage 3 cells were randomly assigned to four groups:blank, transforming growth factor-β, cyclical tensile strain and cyclical tensile strain+transforming growth factor-βgroups. After 1, 3 and 6 days, cells were obtained. General morphology was observed using safranin O staining. Glycosaminoglycan levels were detected by alcian blue staining. Matrix metal oproteinase-13 and tissue inhibitor of metal oproteinase-1 levels in supernatant were measured using ELISA. Type II col agen, matrix metal oproteinase-13 and tissue inhibitor of metal oproteinase-1 mRNA relative expression was detected using RT-PCR. RESULTS AND CONCLUSION:Safranin O staining showed fusiform or irregular triangular cells. cellnumber
and matrix secretion increased in each experimental group than in blank group. Glycosaminoglycan levels in the supernatant were greater in the transforming growth factor-βand cyclical tensile strain+transforming growth factor-βgroups than in the blank group (P<0.05). Type II col agen mRNA relative expression was higher in the cyclical tensile strain+transforming growth factor-βgroup than in the blank group (P<0.05). Results indicated that transforming growth factor-βand cyclical tensile strain could induce the differentiation of bone marrow mesenchymal stem cells into chondrocytes, showing an apparent cooperative action.

BACKGROUND: A lot of important organs are worthless for clinical application because they are hard to store for a long time. In addition, tissues or organs which are dealt with cryopreservation also attack ischemia/reperfusion injury with the recovery of blood flow; especially, skeletal muscle is the most involved tissue.OBJECTIVE: To observe the protective influence of edaravone on cellular membrane and mitochondria of replanted rat extremities following ischemia/reperfusion injury due to cryopreservation and rewarming.DESIGN: Randomized contrast animal study.SETTING: Basic Medical College of Southern Medical University; Department of Hand and Foot Surgery, Shandong Provincial Hospital.MATERIALS: The experiment was carried out in the Cryopreservation Laboratory, Shandong Provincial Hospital from April to November 2006. A total of 36 healthy adult male Wistar rats were provided by Experimental Animal Center of Medical College of Shandong University. All rats were randomly divided into control group, cryopreservation group and edaravone group with 12 in each group.METHODS: Femoral artery and vein of rats in control group were exposured, but extremities were not blocked. Rats in other two groups were used to establish ischemia/reperfusion injury models of replanted extremities. Before cryopreservation, their right hindlimbs were cut off and maintained in liquid nitrogen container for 1 month. After the operation mentioned above, the broken limbs were rewarmed, perfused with routine eluant and replanted. Four hours later, blood supply of extremities was recirculated and the samples were selected. Eluant in edaravone group contained 0.5 mg/kg edaravone. Samples of skeletal muscle were selected at the same time point to establish cellular membrane and extract mitochondria. Furthermore, fluorescence polarization of cellular membrane (reflecting liquidity in cellular membrane lipid area), malondialdehyde (MDA) content of mitochondria, superoxide dismutase (SOD) activity and respiratory controlling rate were measured; meanwhile, mitochondrial ultrastructure of skeletal muscle was observed under transmission electron microscope.MAIN OUTCOME MEASURES:①Fluorescence polarization of cellular membrane, MDA content of mitochondria, SOD activity and respiratory controlling rate of skeletal muscle; ②mitochondrial ultrastructure of skeletal muscle.RESULTS: All 36 rats were involved in the final analysis without any loss. ①SOD activity and respiratory controlling rate of mitochondria in skeletal muscle: The values of these two items were higher in edaravone group that those in cryopreservation group (P＜0.05).②Fluorescence polarization of cellular membrane and MDA content of mitochondria in skeletal muscle: The values of these two items were lower in edaravone group than those in cryopreservation group (P＜0.05). ③Mitochondrial ultrastructure of skeletal muscle: Injured degree of skeletal muscle was milder in edaravone group than that in cryopreservation group.CONCLUSION: Edaravone can relieve ischemia/reperfusion injury of skeletal muscle and protect cellular membrane and mitochondria of skeletal muscle. Its mechanism may be related to directly inhibiting hydroxy free radicals, increasing SOD activity of skeletal muscle, reducing generation of MDA and promoting normal oxidative phosphorylation.

Objective:To determine the median effective dose (ED 50) of 0.5% ropivacaine based on femoral nerve cross-sectional area for ultrasound-guided femoral nerve block. Methods:Patients of both sexes, aged 18-64 yr, of American Society of Anesthesiologists physical status I or Ⅱ, with body mass index of 20-30 kg/m 2, scheduled for elective open reduction and internal fixation for patella fracture or removal of patella fracture by internal fixation, were enrolled in this study.Ultrasonic localization of femoral nerve was performed for measurement of the femoral nerve cross-sectional area, and 0.5% ropivacaine was injected based on the area.ED 50 was determined by Dixon′s up-and-down sequential method.The initial dose was 0.22 ml/mm 2, and the difference between the two successive doses was 0.02 ml/mm 2.The effective block was defined as complete loss of pain sensation in the areas of anterior skin of knee joint, skin on the inner side of the calf and dorsal medial skin of the foot and the degree of motor block was in stages 1-3 assessed using Brunnstrom motor function within 30 min after nerve block.Nerve block was considered ineffective if pain occurred in any nerve distribution area mentioned above.The study was terminated if 7 effective and ineffective alternating waves occurred.ED 50 and 95% confidence interval (CI) were calculated using Probit analysis. Results:Twenty-seven patients were enrolled in the study with the femoral nerve cross-sectional area (75±5) mm 2.ED 50 (95%CI) of 0.5% ropivacaine for ultrasound-guided femoral nerve block was 0.106 (0.069-0.125) ml/mm 2. Conclusion:ED 50 of 0.5% ropivacaine based on femoral nerve cross-sectional area for ultrasound-guided femoral nerve block is 0.106 ml/mm 2.

Objective:To investigate the differences in gene expression levels in knee chondrocytes and chondrons in vitro.Methods:The chondrocytes and chondrons were isolated from full thickness of the 8-months ( n=5) rabbit knees cartilage. Chondrons from right knee were enzymatically isolated using 0.3% dispase and 0.2% collagenase-2 with shaking for 3 hours. Chondrocytes were isolated by 0.4% Pronase and 0.025% collagenase-2 from left knee. The mRNA levels in chondrocytes and chondrons were analyzed by quantitative real-time PCR, including matrix proteins [aggrecan(Agg), collagen(Col-2), Col-6A6, Col-10, Col-11], MMPs and inhibitors (MMP-1, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, TIMP-3), cytoskeletal proteins (Sox-9, vinculin, tubulin, actin), cytokines (IL-β, TNF-α). Analysis was performed using SPSS 16.0 statistical software, and the two-group comparisons were considered as significant by t-test at P<0.05. Results:Compared to the chondrocytes, the Agg [(5.78±0.90) vs (1.89±0.27), t=9.26, P<0.001], Col-2 [(6.29±0.76) vs (3.06±0.60), t=7.46, P<0.001], Col-6A6 [(0.89±0.18) vs (0.22±0.06), t=7.90, P<0.001], Col-10 [(3.83±0.76) vs (1.00±0.26), t=7.88, P<0.001] and TIMP-1 [(1.98±0.85) vs (1.03±0.34), t=2.32, P=0.049], TIMP-2[(3.46±1.50) vs (1.52±1.06), t=2.36, P=0.046], TIMP-3 [(3.96±0.50) vs (1.36±0.18), t=10.94, P<0.001], Sox-9 [(7.09±2.93) vs (3.24±0.77), t=2.84, P=0.022], vinculin [(3.42±1.69) vs (1.46±0.68), t=2.41, P=0.043], tubulin[(9.34±0.71) vs (2.35±0.80), t=14.61, P<0.001] showed higher expression in the chondrons. Compared to the chondrocytes, the MMP-1 [(1.02±0.30) vs (2.67±0.45), t=6.91, P<0.001], MMP-3 [(1.21±0.32) vs (2.52±0.79), t=3.44, P=0.009], MMP-13 [(1.23±0.34) vs (3.42±0.86), t=5.30, P=0.007], IL-1β [(1.02±0.14) vs (2.70±0.49), t=7.37, P<0.001], TNF-α [(0.99±0.08) vs (3.15±0.54), t=8.85, P<0.001] showed lower expression in the chondrons. There were no difference between chondrons and chondrocytes for Col-11, MMP-9, actin ( P>0.05). Conclusion:The gene expression of extracellular matrix components are higher and the gene expression levels of inflammatory factors and MMPs are decreased in chondrons compared with the chondrocytes, suggesting the chondrons have more multiplication potential as seeding cells for tissue-engineered cartilage.

BACKGROUNDChondrocytes' phenotype and biosynthesis of matrix are dependent on having an intact cytoskeletal structure. Microfilaments, microtubules, and intermediate filaments are three important components of the cytoskeletal structure of chondrocytes. The aims of this study were to determine and compare the effects of the disruption of these three cytoskeletal elements on the apoptosis and matrix synthesis by rabbit knee chondrocytes in vitro.

METHODSChondrocytes were isolated from full-thickness knee cartilage of two-month-old rabbits using enzymatic methods (n = 24). The isolated cells were stabilized for three days and then exposed to low, medium, and high doses of chemical agents that disrupt the three principal cytoskeletal elements of interest: colchicine for microtubules, acrylamide for intermediate filaments, and cytochalasin D for actin microfilaments. A group of control cells were treated with carrier. Early apoptosis was assessed using the Annexin-FITC binding assay by flow cytometry on days 1 and 2 after exposure to the disrupting chemical agents. The components and distribution of the cytoskeleton within the cells were analyzed by laser scanning confocal microscopy (LSCM) with immunofluorescence staining on day 3. The mRNA levels of aggrecan (AGG) and type II collagen (Col-2) and their levels in culture medium were analyzed using real-time PCR and enzyme-linked immunosorbent serologic assay (ELISA) on days 3, 6, and 9.

RESULTSIn the initial drug-dose-response study, there was no significant difference in the vitality of cells treated with 0.1 µmol/L colchicine, 2.5 mmol/L acrylamide, and 10 µg/L cytochalasin D for two days when compared with the control group of cells. The concentrations of colchicine and acrylamide treatment selected above significantly decreased the number of viable cells over the nine-day culture and disrupted significantly more cell nuclei. Real-time PCR and ELISA results showed that the mRNA levels and medium concentrations of AGG and Col-2 were significantly decreased for cultures treated with colchicine and acrylamide when compared with untreated cells at three, six, and nine days, and this inhibition was correlated with higher matrix metalloprotease-13 expression in these cells. Cellular proliferation, monolayer morphology, and matrix metabolism were unaffected in cytochalasin D-treated cells when compared with control cells over the nine-day culture period.

CONCLUSIONSThe disruption of the microtubulin and intermediate filaments induced chondrocyte apoptosis, increased matrix metalloprotease expression, and decreased AGG and Col-2 expression in rabbit knee chondrocyte cultures. Our findings suggest that microtubulin and intermediate filaments play a critical role in the synthesis of cartilage matrix by rabbit knee chondrocytes.

Animals ; Cartilage, Articular ; cytology ; metabolism ; Chondrocytes ; cytology ; Collagen ; metabolism ; Cytoskeleton ; metabolism ; Knee Joint ; cytology ; metabolism ; Microscopy, Confocal ; Rabbits