AIM: To observe the enhanced green fluorescent protein (EGFP) gene expression and cytotoxicity in human adipose derived adult stem cells (hADSCs) by pEGFP-N1, Ad5-EGFP and rAAV-2/1-EGFP, and investigate the suitable gene-transferred vehicle. METHODS: The experiment was conducted at Chinese National Human Genome Center and the Third Hospital of Peking University from January to July 2006. ①After the patients and their relative were informed consent, the subcutaneous adipose tissue was obtained from the patients undergoing routine total hip joint replacement in Department of Orthopaedics, Third Hospital of Peking University. pEGFP-N1 was provided by Clotech Company, Ad5-EGFP and rAAV-2/1-EGFP by Vector Gene Technology Company. ②hADSCs were cultured in vitro after isolated from the adipose tissue after dissected and digested with type I collagenase. ③hADSCs of passage 3 were infected with pEGFP-N1, Ad5-EGFP and rAAV-2/1-EGFP and the EGFP expression and the cell toxicity were observed. ④Twenty-four hours after being transfected, 5?104 cells were reseeded in a 24-well plate and the solution was changed three times every week. The growth curves of each group were drawn. Normal non-transfected cells served as control. The influence of different transfection ways on the growth of hADSCs was observed. RESULTS: ①Comparison of transfected efficiency with different ways: pEGFP-N1 transfection showed a higher cytotoxicity and lower efficiency of 10.5%; Ad5-EGFP could efficiently transfect hADSCs (multiplicity of infection=5?102, 82.5%); when MOI was 0.05); however, the growth capability of hADSCs was decreased significantly in the pEGFP-N1 transfection group compared with the control, and the differences were significant at day 3-10 after transfection (P

Objective To compare retrospectively the clinical outcome of total knee replacement with and without patellar replacement. Methods From January 1994 to December 2000, 109 knees in 86 patients treated for osteoarthritis and rheumatoid arthritis were evaluated postoperatively using a questionnaire and physical examination. There were 17 males and 69 females with the age ranging from 37 to 80 years (average 65.7 years). The osteoarthritis was 8 knees and rheumatoid arthritis 69 knees. Forty-two knees underwent patellar replacement, and 67 knees with reserved patella. The patients were scored using HSS Score for knees and Feller Score for patella. AP and lateral views of the knee as well as 30? and 90? axial views of the patella were taken in radiography .The data were analyzed using SPSS software. Results Replacement and non-replacement groups showed no obvious difference in post-operative knee function and the incidence of complication. The two groups showed statistical significant differences in scoring on climbing and descending stairs and rising from sitting position, the results suggested the replacement group scored slightly better than the nonreplacement group. Post-operative anterior knee pain occurred more commonly in the non-replacement group and showed significant difference (P

Objective In cartilage tissue engineering,it was a difficult problem to keep chongdro-cytes phenotype for long time.The aim was made to investigate the dedifferentiation of human articular chon-drocytes,and to verify the inhibition to dedifferentiation by transfection with recombinant pcDNA3.1(+)/TGF-? 2 in vitro.Methods Free chondrocytes were isolated from healthy articular cartilage harvested from6adult patients underwent operation because of other condition.The recombinant pcDNA3.1(+)/TGF-? 2 con-structed in advance was transferred by liposome mediated transfection to monolayer cultured articular chon-drocytes.The first,sixth and ninth generation chondrocytes being transfected and non-transfected were as-sayed by histological examination of HE and Safranine-O staining to observe their morphological changes.RT-PCR,ELISA,immunohistochemistry analysis and prime hybridization were performed to these cells to i-dentify the expression and biosynthesis of TGF-? 2 and cartilage-associated genes and proteins.Results The non-transfected articular chondrocytes in monolayer culture demonstrated the tendency of dedifferentia-tion,and lost the characteristics of their phenotype progressively in subculture.The expression of TGF-? 2 ,collagen typeⅡand proteoglycan in non-transfected cells were decreased,but expression of collagen typeⅠincreased gradually with time going on.On the contrary,as for the transfected chondrocytes,the trans-ferred gene was expressed in cells of all examined generations,and the expression levels were higher than that of non-transfected cell of same generations.The transfected chondrocytes maintained their phenotype and characteristics of articular cartilage.They expressed and produced collagen typeⅡand proteoglycan in a higher level,and collagen typeⅠin a lower level than that of non-transfected cells.Conclusion Human articular chondrocytes in monolayer culture have the tendency of dedifferentiation.The results suggested that chondrocytes tranfected by pcDNA3.1(+)/TGF-? 2 could produce endogenous TGF-? 2 .Transfection of pcD-NA3.1(+)/TGF-? 2 is able to provide transient and long lasting expression of TGF-? 2 in chondrocytes,and may be a feasible method to inhibit dedifferentiation of articular chondrocytes in vitro.

BACKGROUND: Bone marrow mesenchymal stem cells(MSCs) have been investigated initially. Because both fat tissue and bone marrow are tissues originated from mesoderm, so whether MSCs could be obtained from fat tissue as well, which also have multi-lineage differentiation potential?OBJECTIVE:To investigate the basic biological characteristics of adipose MSC and its differentiation into osteoblast under given culture condition for the exploration of its feasibility as seed cell in bone tissue engineering.DESIGN: A randomized and controlled study based on cells.SETTING: Department of Orthopaedics of an Affiliated Hospital of a university.MATERIALS: The study was conducted in the Department of Orthopaedics of the Third Hospital of Peking University. Adipose MSC extracted from the fat tissue of Lewis rats was used as subject.METHODS: Adipose MSCs were obtained from inguinal fat pads of Lewis rat after digestion, which were induced into adipocytes and osteoblasts with adipose and osteogenesis induced culture mediums. The differentiations were examined with cytochemical staining, immuncytochemical staining and Western blotting.MAIN OUTCOME MEASURES: Morphological and biological characteristic of adipose MSCs, and the specific mark of osteoblast after induction.RESULTS: Adipose MSCs were obtained from rat adipose tissue culture,which appeared fibroblast-likely in the culture in vitro, and could stably proliferate and passage in vitro. Primary adipose MSCs could differentiate into adipocytes spontaneously, and passaged cells could form fat drop under the reaction of insulin and dexamethasone, and then differentiate towards adipocytes after the expression of peroxidase proliferation activated receptor ?(PPAR-?) enhanced. There was significant difference between induction group and control group in alkaline phosphatase(ALP) activity detection under the induction of dexamethasone, ascorbic acid, and ?-sodium glycerophosphate(P ＜ 0.01) . Calcium node appeared in yon Kossa staining. Result of osteopontin (OPN) immunocytochemcial staining was positive,and OPN expression was detected by Western blotting after induction.CONCLUSION: MSCs with multi-lineage potential can be obtained from rat adipose tissue, and differentiate into adipocytes and osteoblasts after inductions. Therefore, adipose MSCs can possibly be served as one of optimal seed cells for bone tissue engineering.

BACKGROUND: It is still controversial whether antibiotic-loaded bone cement can prevent wound deep infection in the primary hip arthroplasty. OBJECTIVE: To retrospectively observe the effect of antibiotic-loaded bone cements in primary hip arthroplasty. DESIGN, TIME AND SETTING: A retrospective case analysis was performed for the patients undergoing primary hip arthroplasty at Department of Orthopedics, Peking University Third Hospital from February 2004 to January 2007. PARTICIPANTS: 227 consecutive patients (233 hips) underwent primary hip arthroplasty with the same antibiotic-loaded bone cement, including 69 male and 115 female, and 184 cases (191 hips) were followed up for 3-46 months. METHODS: Fifty-four patients over 71 years old with femoral neck fracture were treated with bipolar femoral head replacement. 130 patients underwent total hip arthroplasty. Seventeen patients with massive acetabular bone defect were reconstructed with impaction autogenous and heterogenous bone grafting plus mesh; 3 patients underwent acetabular structural bone grafting. Both acetabular and femoral side prosthesis were antibiotic-loaded bone cements (Refobacin~-Palacos~R 40 or Cemex~ Genta). MAIN OUTCOME MEASURES: Deep infection after operation. RESULTS: 227 patients (233 hips) did not develop early deep infection after surgery. 184 cases (191 hips) did not occur deep infection during the follow up. However, 15 cases developed swelling on the affected site or skin temperature increase, or pain surrounding joint, and underwent blood sedimentation and C-reactive protein examinations; 12 cases had normal blood sedimentation and 3 had increased blood sedimentation including 1 with rheumatoid, 1 with senile chronic bronchitis, and 1 with undetermined cause. All the 3 patients restored one month later. Fourteen patients had C-reactive protein within normal scope, and 1 with increased C-reactive protein caused by rheumatoid arthritis, but restored 6 weeks later. CONCLUSION: Antibiotic-loaded bone cements in primary hip arthroplasty can reduce incidence of deep infection.

The biocompatibility and tribological characteristics of PVA-hydrogel are excellent, but it is very difficult to make the artificial cartilage material(PVA-hydrogel) attach to the underlying bone. In this study, PVA-hydrogel is attached to the metal fibre mesh by means of micro-mechanical interlock methods at first, then the surface of metal fibre mesh is bonded to the underlying bone by the bone cement(PMMA). In this way, the artificial cartilage can be firmly attached to the underlying bone(or metal substrate ).Microstructure analysis and mechanical tests show that the attachment between artificial cartilage and the metal substrate is firm.

Abstract:Objective To assess whether interleukin-10 (IL-10) is chondroprotective in vitro. Methods Chondrocytes were isolated from femoral cartilage of rats (7-10 days) by digestion with collagenase Ⅱ. The first passage cells were grown in 24- well plates with DMEM, supplemented with 10% fetal bovine serum, for 2-4 days. The cells were then cultured in 0.1% fetal bovine serum DMEM medium, and given respectively interleukin-1 (IL-1) 100?μ/ml, IL-1 100?μ/ml+recombinant murine interleukin-10 (rmIL-10) 20?ng/ml, rmIL-10 20?ng/ml, and cultured for 48 hours. Scanning electron morphology and immunohistochemical study of nitric oxide synthase 2 and matric metalloproteinase 3 mRNA in situ hybridization were performed. Cell proliferation and morphology were observed under inverted microscope from the beginning of cell culture for three weeks. Results IL-1 stimulated granule production in the cytoplasma of chondrocytes, and the cells died in the second and third weeks of culture. IL-10 antagonized IL-1, protected the cells from death and maintained chondrocyte proliferation. Scanning electron morphology showed that IL-1 stimulated the formation of numerous microvilli on the cell surface, while thin and less numerous microvilli were found in cultures with IL-10. Immunohistochemical study and in situ hybridization showed that IL-10 inhibited NOS2 and MMP3 expression.Conclusion IL-10 not only inhibits the synthesis of inflammatory cytokines, but also directly protects chondrocytes by antagonizing IL-1.