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BACKGROUND:Osteopontin, a kind of extracellular matrix glycoprotein, has been found to participate in synthesis and catabolism of osteoarthritic chondrocyte extracellular matrix. However, the effect of osteopontin on the proliferation ability of osteoarthritic chondrocytes is little reported. OBJECTIVE:To investigate the effect of osteopontin on the chondrocyte proliferation in human knee osteoarthritis. METHODS: Cartilage samples were obtained from the patients with knee osteoarthritis undergoing knee arthroplasty at the Xiangya Hospital from January 2012 to June 2012. The chondrocytes were isolated and cultured, and then divided into four groups: blank control, osteopontin, Con-shRNA and osteopontin-shRNA groups. The cell proliferation was detected by MTT and BrdU assays. RESULTS AND CONCLUSION: After transfection of osteopontin-shRNA lentivirus, the infection rate was up to 80%. Compared with the blank control group, osteopontin group showed a significant increase in the absorbance value of osteoarthritic chondrocytes, while after osteopontin-shRNA lentivirus transfection, the absorbance value was significantly decreased (bothP < 0.05). Additionally, after osteopontin-shRNA transfection, the expression level of osteopontin was significantly downregulated (P < 0.05). To conclude, osteopontin can promote the proliferation of osteoarthritic chondrocytes, which is considered as a new treatment target for osteoarthritis.
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BACKGROUND: SOX-9 plays an important role in occurrence and development of cartilaginous tissues,enhances agglutination of mesenchymal cells,has structural domains of transcriptional activation,and can directly regulate the transcription,OBJECTIVE: To construct pDC316-SOX-9 plasmid for transfection of rabbit bone marrow stromal cells (BMSCs) using SOX-9 gene,and to investigate the effects of SOX-9 gene on growth characteristics of BMSCs and product expression. DESIGN,TIME AND SETTING: The cell gene engineering in vitro experiment was performe,d at the Beijing Institute of Genome, Chinese Academy of Scienees from June 2006 to January 2007.MATERIALS: Eight SPF New Zealand rabbits aged 6 weeks were used for culture of BMSCs.METHODS: pDC316-SOX-9 plasmid was used for transfection of rabbit BMSCs by liposome mediated method,Cell transfection included a SOX-9 plasmid transfection group,a blank plasmid group and a blank control group (only treatment of liposome). MAIN OUTCOME MEASURES: Cell morphology; growth activity; The SOX-9 protein expression in rabbit BMSCs were detected by immunohistochemistry,hematoxylin-eosin staining,reserve transcriptase-polymcrase chain reaction (RT-PCR) and enzyme-labeled immunosorbent assay (ELISA). RESULTS: Some cell colonies were detected at 4 days after pDC316-SOX-9 plasmid transfection.Spindle-shaped cells were collected after clone amplification.Cells in the blank control group gradually died over time.Cell activities in the SOX-9 plasmid transfeetion group and the blank plasmid group significantly prolonged,reached a peak at 2 weeks of transfection,and then gradually decreased.At 6 days,BMSCs were yellow in the SOX-9 plasmid transfection group following immunohistochemistry,expressing SOX-9 protein.Hematoxyliu-eosin staining showed many dikaryocytes,rich cell proliferation,similar to chondroblasts.No SOX-9 protein expression and unproductive cell proliferation in the blank plasmid group.SOX-9 mRNA was detected in the SOX-9 plasmid transfection group,but SOX-9 mRNA was not detected in the blank plasmid group and blank control group.SOX-9 levels were significantly higher in the SOX-9 plasmid transfection group than in the blank plasmid group and blank control group at 24,48 and 72 hours,1,2 weeks (P＜ 0.01).CONCLUSION: Rabbit BMSCs were successfully transfected with pDC316-SOX-9 plasmid to enhance cell growth activity and to persistently stably secrete SOX-9 protein,resulting in the differentiation of BMSCs into cartilages.
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BACKGROUND: The study of isolation, purification, culture, cell labeling, inducing factors, effects of gene transfection on cytobiology, cell carrier construction, and time window for back transplantation of cell compound pertaining to marrow stromal cells (MSCs) is still in its infancy. OBJECTIVE: To search for an in vitro culture method that can be simply and effectively obtained. DESIGN, TIME AND SETTING: The present cytological in vitro experiment was performed at the Beijing Institute of Genome, Chinese Academy of Sciences between June 2006 and July 2007. MATERIALS: Eight specific pathogen-free New Zealand rabbits, aged 6 weeks, were provided by the Laboratory Animal Center, Institute of Genetics and Development, Chinese Academy of Sciences. METHODS: Under sterile condition, 1 mL rabbit bone marrow was taken and diluted with D-Hanks solution. Following centrifugation and subsequent supernatant removal, bone marrow was re-suspended using dulbecco's modified eagle's medium (DMEM) for single cell suspension. Next, single cell suspension was dropped onto the liquid surface of equal-volume lymphocyte separation medium (density: 1.077). Subsequent to centrifugation, cloudlike mononuclear cell layer was collected and re-suspended with DMEM containing 20% fetal bovine serum. The cells were inoculated at lxl0/cm2 and purified by adherent method. When 70%-80% of flask bottom was covered, cell digestion and passage was performed.MAIN OUTCOME MEASURES: Cell growth was observed with an operating microscope. Surviving cells were counted by Trypan blue viability test. Cell identification was performed by hematoxylin-eosin staining. Through the use of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, cell viability was detected to observe the cellular resuscitation of the cultured cells following cryopreservation. RESULTS: Twenty-four hours after inoculation, cells began to adhere to the wall, exhibiting short shuttle- or triangle-shaped appearance with different sizes of cellular processes. Three days later, adherent cells began to divide, and cell clusters could be found in some areas; One week later, most of cells exhibited scattered fibroblast-like growth; After passage, cells were evenly distributed with long shuttle-shaped appearance and arranged orderly. Following 3 passages, there wound be 5×106-1×107 adherent cells in 1 mL MSC suspension. Approximately 90% of MSCs survived and identified as mononuclear cells. Cells vigorously grew at days 1-6 after inoculation and reached a peak level at day 8, followed by a viability decline. After 56 days of resuscitation, frozen cells displayed a good and rapid growth. CONCLUSION: Highly purified MSCs can be acquired by gradient centrifugation of lymphocyte separation medium. Enough seeded cells can be obtained by in vitro culture and the cellular viability does not change after frozen preservation and resuscitation.
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BACKGROUND: Some scholars have used finite element analysis to simulate spinal biodynamics. But there are few reports on finite element imitation of lumbar biodynamics system before and after artificial disc replacement. OBJECTIVE: This study aimed to do finite element analysis by establishing new three dimensional finite element models of SB-Chaite Ⅲ lumbar disc replacement. DESIGN, TIME AND SETTING: The observational experiments were performed at the Laboratory of Orthopaedics of Xiangya Hospital of Central South University from December 2003 to August 2004. PARTICIPANT: A healthy male volunteer served as simulation. His T12-S1 underwent continuous CT scanning. There were altogether 264 images with 2 mm in thickness each. Three-dimensional images were reconstructed every 15? in order to obtain the data for three-dimensional model. METHODS: The CT images and human anatomical data were processed by 3DSMAX software to establish three-dimensional L4-5 model of normal Chinese males. MAIN OUTCOME MEASURES: It was transformed to finite element model after processed by SAP2000 software together with SB-Chaite Ⅲ disc prosthesis model. RESULTS: The three-dimensional model and finite element model of lumbar spine were successfully established. The finite element model of SB-Chaite Ⅲ disc replacement in L4-5 spine was established. The total nodes were 2 542, including 1 924 Solid units, 592 Area units and 50 Link units. CONCLUSION: The finite element model of artificial disc replacement can be established by CT scanning, digital processor and computer aided design.
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BACKGROUND: During recent years, mononucleotide polymorphism of some genes is possibly related to affectability of osteoarthritis (OA). However, previous researches mainly compare the gene expression of synoviocytes between OA and rheumatoid arthritis (RhA); therefore, the correlation of gene expression between synovioblast and fibroblast in other tissues should be further studied as compared with OA.OBJ ECTIVE: To observe the differences of gene expression between OA synovioblast and skin fibroblast.DESIGN: Observational contrast analysis.SETTING: People's Hospital of Shenzhen City.PARTICIPANTS: Synovium tissue was derived from OA patients who received replacement of knee joint in the Department of Orthopaedics, People's Hospital of Shenzhen City. All OA patients met the diagnostic criteria of osteoarthritis established by American College of Rheumatology in 1995. Three patients including 1 male and 2 females aged more than 65 years old and they did not have cardiac and pulmonary disease and diabetes mellitus. Three male normal volunteers who aged 25 to 35 years did not have rheumatic disease, osteoarthritis and dermatosis. All subjects provided a confirmed consent. The main reagents were detailed as follows: RPMI1640 culture medium, fetal bovine serum and TRIZOL agent (Invitrogen Life Technologies Company, USA); pGEM-T pUC (Progema Company, USA);Display PROFILE-BASIC and Display PROFILE Probe kits (Qbiogen Company, USA).METHODS: The experiment was carried out in People's Hospital of Shenzhen City from January to June 2005. Synovium of OA patients were treated with primary culture to obtain synovioblast; meanwhile, skin fibroblast treated with primary culture from normal subjects was regarded as the control group. Restricted enzyme section differential display was used to separate the different-expressed genes of synovioblast and skin fibroblast in OA patients. In addition, blast technique was used to compare the resulted ranks with Genbank ranks.MAIN OUTCOME MEASURES: Differences of gene expression between synovioblast and skin fibroblast in OA patients.RESULTS: Gene expressions of superoxide dismutase (SOD), TFPI2, CXCL2, CXCL6 and transforming growth factor (TGF) were high in synovioblast of OA patients as compared with those in skin fibroblast of normal subjects.CONCLUSION: Gene expressions of SOD, TFPI2, CXCL2, CXCL6 and TGF are high in synovioblast of OA patients as compared with those in skin fibroblast of normal subjects. This suggests that gene may play a certain role in onset of OA.
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BACKGROUND:Different methods and biomaterials have been applied in animal experiments and clinical practice to prevent the formation of epidural scars,Biodegradable and sticky semi-fluid gels are the most often used material.Salvia miltforrhiza radix (SMR) and carbomer have been clinically confirmed to be the safe and effective drugs and gel agents. OBJECTIVE:To observe the effect of SMR-gel on preventing epidural adhesion after laminectomy.DESIGN:A complete randomized grouping design, a controlled experiment. SETTING:Department of Orthopaedics,Shenzhen People's Hospital (Second Clinical College of Jinan University). MATERIALS:Thirty-six healthy pure New Zealand rabbits were used,either male of female,clean degree,2-3 years of age. They were randomly divided into four groups with 9 rabbits in each group:blank control group,gel contro group, HA group and SMR-gel group. Carbomer934 powder (Shanghai People's Pharmaceutical Factory, batch number: 20000510) , hyaluronic acid (HA) [Shandong Bausch & Lomb Freda Pharaceutical, Co., Ltd.,No.H10960136,2 mL (20 mg)].METHODS:The experiments were carried out in the animal laboratory of Shenzhen People's Hospital from April 2002 to August 2003.①Preparing SMR-gel:SMR was prepared into extract powder.Carbomer934 powder was added by water for dissolving and swelling and stayed overnight,then SMR-gel was prepared by dipping with triethanolamine,adding with SMR extract powder (2 g),then adding with purified water till 100.0 g and stirring uniformly.②The rabbits were anesthetized. and the lamina of vertebra was totally resected at L3 and L6 (reserving superior and Inferior articular processes).then defects of 10 mm×5 mm were made to expose the dura mater.The vertebral defects were added with 1 mL carbomer gel, 1 mL HA (20 g/L) and 1 mL SMR-gel in the gel control group,HA group and SMR-gel group respectively.whereas nothing was added in the blank control group.③Gross samples:Three rabbits were killed 4,6 and 8 weeks postoperatively in each group.vertebraI ventral fascia were stripped to remove the spinal segments (L3,L6) for operation completely,and totally 24 samples for each time.One sample was selected in each group 4 weeks postoperatively. and the samples were observed under H-600 transmission electron microscope (Hitachi). ④The adhesion compactness of scar tissue with dura mater was evaluated in the 24 samples of the 4 groups at 8 weeks postoperatively:There were 4 grades:No obvious adhesion between dural sac and scar tissue for grade O:Extensive and compact adhesion between dural sac and scar tissue. impossible blunt dissection between dural sac and scar tissue.incomplete dural sac after sharp dissection for grade Ⅲ.Each spinal segment was cut into 4 parts equally,and all were prepared into sections and stained,then the thickness of epidural scar was determined with Tiger2000 image analyzer. ⑤The rank sum test was used in the scar adhesion compactness grading evaluated with naked eyes,whereas analysis of variance.and two-two comparison were used in analyzing the thickness of epidural scar.P＜0.05 was considered as significant difference.MAIN OUTCOME MEASURES:①Results of gross scar adhesion compactness grading at 8 weeks and comparison of the thickness of epidural scar at 4.6 and 8 weeks;②Results of gross observation,histological examination and ultrastructure.RESULTS: All the 36 rabbits were involved in the analysis of results. ①Results of gross observation and pathohistological examination:There was compact adhesion at each time point in the blank control group,part adhesion in the gel control group and HA group, and no obvious adhesion in the SMR-gel group.②Results of quantitative analysis:The rabbits with lower scores of scar adhesion compactness grading In the blank control group,gel control group and HA group were obviously fewer than those in the SMR-gel group (W=45-52,P＜0.05-0.01).The scar thickness at 4 and 8 weeks in the SMR-gel group was obviously less than that in the other 3 groups(F=128.657,152.246,80.891,P＜0.01).③Results of observation under transmission electron microscope:The proliferation of fibroblasts at 4 week was active in the blank control group,gel control group and HA group,but inactive in the SMR-gel group.CONCLUSION:①SMR can inhibit the fibroblasts to proliferate,differentiate and synthetize into secretory collagens,and then inhibit the formation of epidural scar adhesion.②HA can be absorbed by organs very early,which reduces its role in preventing adhesion.Whereas carbomer gel can stay longer, and it plays a role in inhibiting and blocking adhesion in the whole process of wound repairing.
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BACKGROUND: The bone marrow stromal cells in red bone marrow (RBM) are the non-specific bone growth factors,and the target cells of bone morphogenetic protein (BMP), and they have the activities of osteoinduction and osteogenesis.Injectable calcium alginate gel (CaAG) is a degradable biomaterial with good histocompatibility,it provides scaffold for the proliferation and adhesion of osteoblasts and chondroblasts,and it is good for the proliferation of new capillaries.OBJECTIVE:To prepare CaAG-BMP-RBM gel compound,and observe its osteoinductivity.DESIGN:A randomized controlled animal trial.SETTINGS:Department of Orthopaedics,Shenzhen People's Hospital;Department of Orthopaedics,Union Hospital affiliated to Tongji Medical College,Huazhong University of Science and Technology.MATERIALS:The experiments were carried out in Shenzhen People's Hospital from February 2002 to February 2003.Twenty-seven pure SD rats, either male or female,weighing (200±20) g,were purchased from the Animal Experimental Center of Southern Medical University (No.SCXK2002-99).BMP was bought from Xijing Hospital affiliated to the Fourth Military Medical University of Chinese PLA.METHODS:The rats were randomly divided into three groups:CaAG-BMP-RBM group(n=9),CaAG.BMP group (n=9) and CaAG group (n=9).In the CaAG-BMP-RBM group,the rats were anesthetized,then 0.1 mL RBM collected from trochanteric section of fetour was added into the prepared CaAG-BMP compound (0.4 mL),sufficiently mixed,and then injected into posterior femoral muscle.The rats in the CaAG-BMP group and CaAG group were implanted with CaAG-BMP and CaAG into bilateraI posterior femoral muscles respectively.①The conditions of becoming conscious after anesthesia, incision healing, diet and activities were observed, the size and consistency of the implants and distribution of vessels were also observed.②The indexes were detected at 1,2 and 4 weeks after model establishment respectively,and 3 rats were used for each time point.The sections were observed under light microscope.and the activity of alkaline phosphatase (ALP) was determined.MAIN OUTCOME MEASURES: ①General conditions of animals and gross observation of the implants;②Histopathological observation;③ALP activities.RESULTS: All the 27 SD rats were involved in the analvsis of results.The rats became conscious at 4.6 hours after anesthesia,and they could eat normally;Hematom at incision disappeared.and the rats could move normally at 24 hours;No sign of infection at incision was observed at 72 hours;The incisions healed completely and suture shed after 2 weeks.The incisions were stage Ⅰ healing in all the 27 rats.①Results of gross observation of the implants:At 1 week after implantation.the size of implant did not decrease and vessels in-grew in both the CaAG-BMP-RBM group and CaAG-BMP group;At 2 weeks,lhe quality was hard, and the section appeared as gray and white with deposition of bone-like tissues;At 4 weeks,a great amount of vessels in-grew,and there were many depositions of hard bone-like tissues.②Results of the histopathological observation under light microscope:In the CaAG-BMP.RBM group.many mesenchymal cells aggregated, osteoblasts and chondroblasts proliferated actively al 1 week;The chondrocytes tended to mature with cartilage-like and bone-like matrixes at 2 weeks; Many osteocytes were observed and fibrous bone formed at 4 weeks.which were more then those in the other two groups.③Results of ALP activity:At 1.2 and 4 weeks after implantation.the ALP activities in the CaAG group [(0.179±0.018),(0.058±0.017).(0.027±0.018)IU/g] were lower than those in the CaAG-BMP-RBM group[(0.922±0.226),(1.169±0.249),(0.431±0.081)IU/g,P＜0.01);At 1 and 4 weeks,the ALP activities in the CaAG-BMP group[(0.447±0.015),(0.276±0.081)IU/g]were lower than those in the CaAG-BMP-RBM group (P＜0.05).CONCLUSION:The compound of CaAG-BMP-RBM possesses stable and lasting osleoinductivity.
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BACKGROUND:There is lack of an objective and standardized animal model for assessing the therapeutic effect of physical and medication treatment on bone defoct, the effectiveness of operation, as well as the role of bone substitute in the repairing of bone defects.DESIGN:Verified study on the experimental model of bone nonunion in rabbitsSETTING: Department of Orthopaedics in Shenzhen people' s Hospital MATERIALS:Twenty common grade pure New Zealand rabbits of either gender were selected with body mass of (2.5±0.5)kg,aged 6 to 8 months.METHODS :This experiment was carried out at the experimental animal center of Shenzhen people's Hospital between May and August 1999. 1.5cm bone segment (including periosteum)was cut off in the middle of forearm radius in 20 common grade pure NewZealand rabbits,the broken ends were covered with bone wax, 10 weeks later, the bone nonunion status was assessed by macropathological observation, pathohistological and X-ray examination.MAIN OUTCOME MEASURES:Observations on rabbit forearm radius defects by macropathological observation, pathohistological and X-ray examination.RESULTS :Twenty rabbits(40 side radius)were enrolled in this study and weeks later, bone defect region was found filled with fibrous cicatricial tissue without osseous connection ,bone wax was not absorbed, capitulum was ossified with medullary cavity blocked,a small amount of callus formed at both broken ends of fractural bone ,length of bone defect ranged from 0.8 to blocked under optical microscope,chondrocyte and osteocyte could be observed arranging disorderly and covered with fibrous membrane,defect reosseous connection could be detected at defect region at week 10,broken end was ossified and medullary cavity was blocked ,there was small amount of callus appeared at both broken ends displaying irregular shape.CONCLUSION:Bone nonunion experimental animal was successfully established on rabbits in this study, with pathological changes meeting the need of bone nonunion and displaying typical properties,which can be used as reliable and feasible experimental animal model.
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Objective To discuss clinical results of treatment of trimalleolar fractures by minimally invasive surgical osteosynthesis. Methods From January 2002 to Doctober 2005, twenty-eight cases (mean age: 38.7 years) of trimalleolar fracture were treated by minimally invasive surgical osteosynthesis. Their lateral and posterior ankle joints were exposed through the Gatellier-Chastang incision. The sequence of reduction and fixation of ankle fracture was firstly the posterior ankle, then the medial and lateral malleolus, and distal tibiofibular syndesmosis lastly. Postoperatively, all the patients were fixed externally from 3 to 4 weeks with plaster splint. Results Follow-ups of 18 months on average revealed that all the cases healed. The healing time ranging from 2.8 to 4.5 months averaged 3.2 months. According to the Baird-Jackson scoring system, the results were rated as excellent in 16 cases, good in eight cases, moderate in three cases, and poor in one case, with the good-excellent rate being 85.7% . Conclusions The anatomical reduction and firm internal fixation are key factors in treatment of trimalleolar fractures. The minimally invasive surgical osteosynthesis is a good method due to the minimal invasion, a high rate of union, and fewer complications it results in.
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Objective To discuss the results of treatment of proximal humeral fractures in elderly patients with a locking proximal humerus plate. Methods 35 aged patients with proximal humeral fracture were treated with a locking proximal humerus plate from January 2001 to January 2004. Early rehabilitation after operation was performed. Results 35 cases were followed up for 13.2 months on the average. The mean time for bony union was 8.3 (7 to 12) weeks. 1 patient developed an avascular necrosis of the humeral head. According to the Constant Score, the average for all fractures was 81.4 (39 to 95). The excellent and good rate was 85.7%. Conclusion The locking proximal humeral plate proves to be safe and can be recommended for the treatment of proximal humeral fracture in elderly osteoporotic patients.
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Objective To investigate the clinical effects of bioglue compound and anatomic plate in treatment of tibial plateau fractures. Methods 28 cases of tibial plateau fractures were treated by means of open reduction and internal fixation with bioglue compound and anatomic plate. The intervals between operation and injury ranged from 5 to 10 days. The amounts of bioglue compound implanted ranged from 3 to 8 grams. Results All the patients were followed up for 6 to 22 months. All the fractures healed satisfactorily without sunken joint surface. According to Mechant criteria, the result was excellent in 13 cases, good in 11 cases, moderate in 3 cases and poor in 1 case. The total excellent and good rate was 85.3 %. Conclusion Internal fixation with bioglue compound and anatomic plate can result in good effects in treatment of tibial plateau fractures, because the bioglue compound possesses high bone inductive potentialities to repair bone defects.
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Objective To investigate the ability of repairing bone defect with the compound of hydroxyapatite(HA),red bone morrow (RBM), bone morphogenic protein(BMP)and fibrin sealant (FS),and the feasibility with the compounds as bone substitute material. Methods The animal models of bilateral radius bone defect were created by surgery in the New Zealand white rabbits, and were treated with the compound of HA, RBM, BMP and FS, by autograft and no implant as control.The effect were observed by gross, histopathological and X-ray examinations, and were determined by biomechanics at 2,4,8 and 12 weeks after operation. Results By gross, histopathological and X-ray examinations, the effect indicated that the bone defect were perfectly repaired with autograft and the compound of HA, RBM, BMP and FS at 12 weeks, but not with the no implant group. The effect of biomechanics had no statistically significant difference between the autograft and the compound of HA, RBM, BMP and FS. Conclusion The compound of HA, RBM, BMP and FS possesses much high bone inductive potentialities and the ability of rebuilding bone defect and can serve as an autograft substitute material.
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Objective To investigate the effect of fibrin sealant cefazoline sodium implant on prevention and treatment of orthopaedic infection. Methods The fibrin sealant antibiotic implant was made by 10 ml (100 mg/ml) profibrin (Human) mixed with 2.0 g cefazoline sodium, after which 5 ml (200 PE/ml) prothrombin complex concentrate (Human) was added. A total of 24 female New Zealand white rabbits were recruited in the study and divided into three groups, ie, Group A that was implanted with the fibrin sealant antibiotic implant, Group B that was implanted with antibiotic and Group C that was as control. The model of bone infection was made by injecting 0.2 ml(1?10~6 CFU/ml) staphylococcus aureus into the bone hole in tibia of two legs. The histologic and radiologic bone changes were evaluated 2, 4 and 8 weeks after therapy. And in vitro release test was performed. Results There was not evidence of osteomyelitis in Group A, but the osteomyelitis was observed in Groups B and C. Concentration of cefazoline sodium about (1 043.94?0.20) ?g per piece was released from fibrin sealant cefazoline sodium implant within the first 24 hours by in vitro diffusion test but felt to (7.21?0.02) ?g per piece at the 35th day. Conclusion The fibrin sealant cefazoline sodium implant is simple to make and effective in preventing and treating orthopaedic infection as well as in improving repair of the bone defects.