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Objective:To observe the effect of hypoxia on the dedifferentiation process of chondrocytes in vitro and explore the role of collagen prolyl 4-hydroxylase (C-P4Hs).Methods:Chondrocytes were treated with COCl 2 for different concentrations, and selecting the optimal COCl 2 concentration for hypoxia inductionwas100 μmol/L, the mouse costal chondrocytes were divided into the normal oxygen group and the hypoxia group, and the indexes of the 3rd generation 0.5-72 h and the 1st, 3rd, 5th and 7th generation at 72 h were detected respectively. The proliferation rate was determined by CCK8 method and cell count, and the dynamic changes of HIF-1α, P4Hα1, P4Hα2 and Col II in each group were detected by RT-qPCR, IF and Western blot. Results:Costal chondrocytes were cultured under different concentration of COCl 2 for 48 h. When COCl 2>150 μmol/L, the proliferation rate ( P<0.05) was significantly decreased. Normal oxygen and hypoxia induced rib chondrocytes for 0-72 h, and RT-qPCR showed significant increases in P4Hα2 and Col II mRNA in hypoxia group ( P<0.05). IF showed that HIF-1α and P4Hα2 accumulated in the nucleus under hypoxia, and P4Hα2 gradually entered the cytoplasm from the nucleus. Westernblot analysis showed that HIF-1α and P4Hα2 protein expressions were significantly increased in hypoxia group ( P<0.05). The expression of Col II protein in hypoxia group ( P<0.05) increased at the induction stage. CCK8 and cell count results showed that the proliferation rate and cell number of each generation in the hypoxic group were significantly increased ( P<0.05), and there was still potential for proliferation when the cells were transferred to the 6-7 generation. RT-qPCR showed that hypoxia group each generation cells P4Hα2, Col II mRNΑ were significantly increased ( P<0.05). Westernblot results showed that HIF-1α, P4Hα2 and Col II protein expressions were increased in each generation of hypoxia group ( P<0.05). ConcIusion:Increased expression of P4Hα2 through hypoxia induced HIF-1α can accelerate post-translational modification of Col II in chondrocytes and increase synthesis and accumulation of Col II. P4Hα2 may be responsible for increased proliferation rate and delayed dedifferentiation of chondrocytes cultured in vitro under hypoxia condition.
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Objective To compare the effect of closed reduction internal fixation and open reduction and internal fixation on the recovery of double ankle joint function and the degree of pain in patients with comminuted double malleolus fracture after operation.Methods From June 2015 to March 2017,78 patients with comminuted malleolus fracture in the Department of Orthopedics from Changzhi People's Hospital Affiliated to Shanxi Medical University were randomly divided into two groups according to the digital table ,with 39 cases in each group.The control group was treated with open reduction and internal fixation ,and the study group was treated with closed reduction and internal fixation.The amount of bleeding ,operation time,time of hospitalization were compared between the two groups.Six months after operation,the ankle function and pain score were used to evaluate the clinical efficacy.Results All the operations of 78 patients were successfully performed.The operative blood loss,operative time and hospitalization time in the study group were significantly lower than those in the control group (t＝4.65,14.63,7.83,all P＜0.05).Six months after operation,the excellent and good rate of the double ankle function in the study group (97.44%) was significantly higher than that in the control group (74.36%)(χ2＝6.77,P＝0.01).The pain scores of the two groups were significantly lower than those before treatment (t＝24.84,33.81,all P＜0.01).Six months after treatment,the pain score in the study group[(1.74 ±0.55)points]was significantly lower than that of the control group [(2.78 ± 0.80)points] (t＝6.69,P＜0.01).Conclusion Closed reduction and internal fixation for the patients with commi-nuted malleolus fracture after the operation of the joint without reduction ,are beneficial to promote the recovery of the double ankle joint function and reduce the pain degree ,and have the advantages of small wound ,less bleeding in the operation,short operation time and short hospitalization time.The clinical application should be popularized.
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BACKGROUND:With the development of sports medicine and research of radiologic imaging techniques, Blumensaat line (the radio-opaque line at the roof of the intercondylar notch) has been paid increasing attention. Blumensaat line is considered as measurement indexes of knee diseases. Taking advantage of the Blumensaat line, many surgeons and radiography physicians are trying to diagnose some knee diseases. OBJECTIVE:According to the knowledge about Blumensaat line in auxiliary diagnosis of knee disease, we hoped that it wil have a wide application in clinic. METHODS:A computer-based online search of CBM, CNKI, Wangfang Database and PubMed between 2000 and 2015 was performed for articles addressing Blumensaat line. We summarized its application as different diagnostic indicators. The key words were patel a alta, Blumensaat line, anterior cruciate ligament (ACL) injuries and ACL reconstruction. Thirty-nine studies were accorded with the inclusion criteria. RESULTS AND CONCLUSION:Blumensaat line represents the tangential y hit part of the roof in the intercondylar fossa. The line can be used for diagnosing ACL injuries and directing ACL restruction. (1) Blumensaat line and patel a alta:Patel a heights can be measured with the use of Blumensaat method, modified Blumensaat method and modified Blumensaat ratio. Modified Blumensaat ratio was found by Japanese researchers in 2014 and it is efficient. (2) Blumensaat line and ACL injuries:Blumensaat angle is formed by Blumensaat line and ACL. If this angle is negative or it is greater than 15°, we can draw a conclusion that the ACL was hurt. (3) Harner’s method can be used for choosing an accurate isometric point and a perfect bone tunnel’s angel in ACL reconstruction.
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BACKGROUND:Proximal femoral nail anti-rotation has good biomechanical basis, and has obvious advantages for intertrochanteric fracture in aged patients, but there are some problems in the clinic, because of improper handling of material matching and operation details, which can impact therapeutic effects and functional recovery. OBJECTIVE:To analyze the efficacy and issues of proximal femoral nail anti-rotation in the treatment of intertrochanteric fracture in patients at more than 60 years old. METHODS:From July 2011 to July 2012, proximal femoral nail anti-rotation was used to treat 56 cases of intertrochanteric fractures. Clinical data bank was established to analyze intraopeative problems and postoperative complications. At 1, 3, 6, 9 and 12 months postoperatively, outpatient and telephone folow-up were carried out to evaluate therapeutic effects and functional recovery of hip joint. RESULTS AND CONCLUSION:Four patients died within 1 year. Seven patients lost within a year for other reasons. The remaining 45 patients were folowed with the time from 12 to 24 months, with an average time of 18.2 months. Harris score was (85.00±6.75) points. There were excelent in 26 cases, good in 15 cases, average in 3 cases and poor in 1 case, with an excelent and good rate of 91%. 18 cases were not satisfied with the position of fracture fragments. In 9 cases, proximal femur was not match with the proximal femoral nail anti-rotation. Seven cases were not satisfied because of the location and length of the spiral blade. Seven cases affected lateral cortex fracture. One case experienced postoperative pulmonary embolism. One case suffered from cardiovascular and cerebrovascular diseases. Nine cases suffered from local sweling. 13 cases experienced hip pain. Five cases affected the healing of fracture extended. Results showed that proximal femoral nail anti-rotation for intertrochanteric fracture in aged patients obtained good outcomes, but we should improve the separation of fracture fragments and reduce intraoperative and postoperative complications.
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BACKGROUND:The main component of cartilage, type Ⅱ col agen gene expression in chondrocyte is positively correlated with SOX9 concentration in a dose-dependent manner. OBJECTIVE:To observe the variation of SOX9 and type Ⅱ col agen mRNA content at different periods in the differentiation process (osteogenic, chondrogenic, adipogenic induction) of mesenchymal stem cel s, and to explore the correlation of SOX9 expression and type Ⅱ col agen. METHODS:Bone marrow mesenchymal stem cel s were isolated from 4-week-old Kunming mice, and cultured in vitro to passage 3. The cel phenotype was identified with flow cytometry. Cel s were divided into three groups and subjected to three kinds of induction conditions favorable for adipogenic, chondrogenic and osteogenic differentiation, and each group was observed at three time points. In addition, the non-induced cel s were used as a control group. The total RNA of cel s was extracted at 3, 7, 14 days after induction, and SOX9 and type Ⅱ col agen mRNA was quantified with reverse transcription-polymerase chain reaction. The induced cel s were stained by immunofluorescence to observe the differentiation and perform statistical analysis. RESULTS AND CONCLUSION:Passage 3 bone marrow mesenchymal stem cel s grew wel , and cel phenotype was confirmed as stem cel s by flow cytometry. The staining results showed that, the cel s differentiated into chondrocytes, adipocytes and osteoblasts. The SOX9 mRNA levels in the induced cel s were the highest in chondrogenic differentiation group, then in osteogenic differentiation group, and the lowest in adipogenic differentiation group. Type Ⅱ col agen mRNA levels in the induced cel s were the highest in chondrogenic differentiation group, then in adipogenic differentiation group, and the lowest in osteogenic differentiation group. SOX9 expression in chondrogenic differentiation group increased at 3 and 7 days, and then decreased at 14 days. While type Ⅱ col agen expression increased at 3, 7, 14 days. SOX9 mRNA levels increased as the osteogenic differentiation, while type Ⅱ col agen expression gradual y decreased. There was no significant difference in the SOX9 mRNA expression between adipogenic differentiation group and control group (P>0.05), while type Ⅱ col agen expression was not regularly changed. Experimental findings suggest that, critical effect of SOX9 in chondrogenic differentiation is better than that in osteogenic and adipogenic differentiation. SOX9 is associated with type Ⅱcol agen, which may alter along with the SOX9 in the early chondrogenic differentiation;SOX9 may play a fine-tuning role in the process of chondrogenic and osteogenic differentiation.
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Objective To observe the effect of Sox9 gene overexpressian on chondrogenesis of esenchymal stem ceils (MSCs) in vitro. Methods Rabbit MSCs were obtained and purified by gradient centrifuge and adhesion culture in vitro. MSCs were transfected by recombinant Sox9 plasmid. RT-PCR and Western blot analysis were used for transfection confirmation. Chondrogenic molecules such as type I1 collagen,aggrecan and Sox9 were detected by immunohistology, RT-PCR and western blot. Cell proliferation was tested by MTT assay. Results MSCs were successfully transfected with Sox9 gene and verified by RT-PCR and Western blot analysis. The overexpression of Sox9 had no effect on the proliferation of MSCs. Transfeeted cells expressed more chondrogenic markers than the control groups. Conclusion Sox9 gene overexpression can enhance chondrogenic differentiation of MSCs.
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BACKGROUND: Different isolation and culture methods for bone marrow mesenchymal stem cell (BMSCs) will result in varying cell activity and purity, which will influence repair effect of tissue engineering. OBJECTIVE: To investigate the optimized methods of isolation and culture of BMSCs in vitro and validate the efficacy of cell acquisition. DESIGN, TIME AND SETTING: In vitro cytology trial was performed at the laboratory of Department of Orthopedics, Second Hospital of Shanxi Medical University from October to December 2007. MATERIALS: Two 3-month-old New Zealand rabbits were provided by Shanxi Institute of Livestock. METHODS: Lymphocyte isolation solution was added to DMEM culture solution containing fresh bone marrow. BMSCs were obtained and purified by gradient centrifuge and adhesion culture in vitro. Non-attached cells were moved to new culture flask every 8 hours. The solution was changed firstly after 5 days of culture. Trypsinization was conducted at cell confluence of 90%. The first, third and fifth passages of cells were harvested. MAIN OUTCOME MEASURES: The morphology of BMSCs was observed with phase contrast microscope; the growth curve was drawn to determine doubling time. The percentage of the wel1 growth P2 cells were identified by CD44 staining by flow cytometry. RESULTS: Primary cultured BMSCs were oval, spindle-shaped or polygonal, and adhered to plastic surface within 48 hours, exhibiting spindle-shaped or polygonal with clear nucleus, and reached 90% confluence within 14 days. The first, third and fifth passages of BMSCs showed S-type, and were in latent phase at 1-3 days, in log phase 3 days later, and cells came into platform phase at 7-8 days. The mean doubling time was 24.22 hours. CD44-positive cells were found in the second passage, with positive rate of 93.0%. CONCLUSION: BMSCs are easily isolated and cultured in vitro with good growth and high purity by gradient centrifuge and adhesion culture with lymphocyte isolation solution.
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Objective To observe the change of collagen in osteoarthritis and study the pathogenesis of osteoarthritis.Methods The right hind limb of twenty-four adult New Zealand White rabbits were immobilized with plaster cast in extension position for 10,20,30 and 40 days,respectively.Six animals without immobilization served as control.The articular cartilage of the medial femur condyle was harvested for transmission electron microscopy,in-situ hybridization of collagenⅡ,and immunohistochemistry of collagenⅠ,Ⅱ,Ⅲ.Results Transmission electron microscopy showed articular cartilage was destructed from the fine collagen fiber network of tangential zone.The fine collagen fiber network did not contain chondrocyte.Immunohistochemistry and in-situ hybridization showed that in earlier period of osteoarthritis,the collagen typeⅡand its gene expression firstly increased,then decreased with destruction of ultrastructure,and chondrocytes enhanced type Ⅱ collagen expressing and synthesizing mainly in transition zone and upper deep zone.In articular cartilage of osteoarthritis there was type Ⅲ collagen,instead of typeⅠcollagen.Conclusion In osteoarthritis,articular cartilage degenerated from tangential layer,in which collagen can not be repaired after destruction,this may contribute to the chondral degeneration.