Altered Gene Expression in Articular Chondrocytes of Smad3^(ex8/ex8) Mice, Revealed by Gene Profiling Using Microarrays

It has been previously reported that small mother against decapentaplegic 3 （Smad3） gene knockout （Smad3^ex8/ex8） mice displays phenotypes similar to human osteoarthritis, as characterized by abnormal hypertrophic differentiation of articular chondrocytes. To further clarify the crucial target genes that mediate transformation growth factor-β （TGF-β）/Smad3 signals on articular chondrocytes differentiation and investigate the underlying molecular mechanism of osteoarthritis, microarrays were used to perform comparative transcriptional profiling in the articular cartilage between Smad3^ex8/ex8and wild-type mice on day five after birth. The gene profding results showed that the activity of bone morphogenetic protein （BMP） and TGF-β/cell division cycle 42 （Cdc42） signaling pathways were enhanced in Smad3^ex8/ex8 chondrocytes. Moreover, there was altered gene expression in growth hormone/insulin-like growth factor 1 （Igfl） axis and fibroblast growth factor （Fgf） signaling pathway. Notably, protein synthesis related genes and electron transport chain related genes were upregulated in Smad3^ex8/ex8 chondrocytes, implying that accelerated protein synthesis and enhanced cellular respiration might contribute to hypertrophic differentiation of articular chondrocytes and the pathogenesis of osteoarthritis.

GROWTH DIFFERENTIATION FACTOR-5 STIMULATES THE GROWTH AND ANABOLIC METABOLISM OF ARTICULAR CHONDROCYTES

Objective To observe the effect of growth differentiation factor-5 (GDF-5) on the growth and anabolic metabolism of articular chondrocytes. Methods The articular chondrocytes isolated from rats were treated with various concentrations of rmGDF-5, and the growth of chondrocytes measured by MTT assay, the cellular cartilage matrices formation detected sulfated glycosaminoglycan by Alcian blue staining and type Ⅱcollagen by RT-PCR. Results After 7 days culture, MTT assay showed that GDF-5 enhanced the growth of chondrocytes in a dose-dependent manner, RT-PCR showed that GDF-5 clearly induced the synthesis of type Ⅱ collagen because of the col2a1 mRNA band more and more strong in a dose-dependent. Chondrocytes were cultured with GDF-5 for 14 days, the intensity of Alcian blue staining was greatly enhanced, especially, at a high concentration of 1000ng/mL, and GDF-5 enhanced the accumulation of the Alcian blue-stainable material in a concentration-dependent manner and in a does-dependent manner. Conclusion GDF-5 enhanced the growth of mature articular chondrocytes, and stimulated the cellular cartilage matrices formation in mono-layer culture.

Transfection of Articular Chondrocytes with rhBMP7 Gene and Its Expression

In order to investigate the possibility of expression of exogenous gene in transduced articular chondrocytes, plasmid pcDNA3 rhBMP7 was delivered to cultured chondrocytes. Through immunohistochemical staining and RT PCR assay, the expression of rhBMP7 gene was detected. And the bioactivity of transgene expression product was detected through MTT assay as well. It was confirmed that exogenous gene could be expressed efficiently in transduced chondrocytes and the transgene expression product had obvious bioactivity. The present study provided a theoretical basis for gene therapy on the problems of articular cartilge.

Serum-free media for articular chondrocytes in vitro expansion

Background In vitro chondrocyte expansion is a major challenge in cell-based therapy for human articular cartilage repair. Classical culture conditions usually use animal serum as a medium supplement, which raises a number of undesirable questions. In the present study, two kinds of defined, serum-free media were developed to expand chondrocytes in monolayer culture for the purpose of cartilage tissue engineering. Methods Bovine chondrocytes were expanded in serum-free media supplemented with fibroblast growth factor-2 and platelet-derived growth factor or fibroblast growth factor-2 and insulin-like growth factor. Expansion culture in a conventional 10% fetal bovine serum （FBS） medium served as control. Fibronectin coating was used to help cell adhesion in serum-free medium. Next, in vitro three-dimensional pellet culture was used to evaluate the chondrocyte capacity. Cell pellets were expanded in different media to re-express the differentiated phenotype （re-differentiation） and to form cartilaginous tissue. The pellets were assessed by glycosaminoglycans contents, collagen II, collagen I and collagen X immunohistological staining. Results Chondrocytes cultured in serum-free media showed no proliferation difference than cells grown with 10% FBS medium. In addition, chondrocytes expanded in both serum-free media expressed more differentiated phenotypes at the end of monolayer culture, as indicated by higher gene expression ratios of collagen type II to collagen type I. Pellets derived from chondrocytes cultured in both serum-free media displayed comparable chondrogenic capacities to pellets from cells expanded in 10% FBS medium. Conclusion These findings provide alternative culture approaches for chondrocytes in vitro expansion, which may benefit the clinical use of autologous chondrocytes implantation.

Immortalization of human articular chondrocytes and induction of their phenotype

Objective To immortalize human articular chondrocytes ( HACs) using gene transfection and to maintain stable phenotype of transformed HACs after induction.Methods HACs were transfected with the retroviral vector pLXSN encoding human papillomavirus 16E7 (HPV16E7), and the transformed clones were sorted and proliferated. Karyotype analysis, clone forming tests and nude mice tumor forming tests were applied to check the characteristics of the transformation. Type II collagen of transformed chondrocytes was inducted with free serum medium (FSM) supplemented with nutridoma-sp and ascorbate.Results Immortalized HACs were isolated with fifty passages achieved. The HPV16E7 transformed cells were confirmed to be benign. Induction of FSM with nutridoma-sp and ascorbate promoted type II collagen of transformed chondrocytes to the high levels of normal chondrocytes.Conclusion HACs transformed with HPV16E7 survive for long periods in vitro, and type Ⅱ collagen can maintain stability after induction.

Novel perspectives on leptin in osteoarthritis:Focus on aging

t Osteoarthritis(OA)is a common chronic joint disease characterized by articular cartilage degeneration,subchondral sclerosis,synovitis,and osteophyte formation.OA is asso-ciated with disability and impaired quality of life,particularly among the elderly.Leptin,a 16-kD non-glycosylated protein encoded by the obese gene,is produced on a systemic and local basis in adipose tissue and the infrapatellar fat pad located in the knee.The metabolic mech-anisms employed by leptin in OA development have been widely studied,with attention being paid to aging as a corroborative risk factor for OA.Hence,in this review,we have attempted to establish a potential link between leptin and OA,by focusing on aging-associated mechanisms and proposing leptin as a potential diagnostic and therapeutic target in aging-related mecha-nisms of OA that may provide fruitful guidance and emphasis for future research.