Genomic chondrocyte culture profiling by array-CGH, interphase-FISH and RT-PCR.

OBJECTIVE: In vitro expansion is an important step to acquire sufficient cells in human tissue engineering technologies. The high number of chondrocytes needed for human articular cartilage implants requires in vitro expansion of the primary cells, bearing a theoretical risk of in vitro induced changes in the genomes. To gain more insights into this situation, model cultures were prepared and analyzed. DESIGN: 25 chondrocyte cell DNA samples from nine donors were analyzed by array comparative genomic hybridization (aCGH) on whole genome level and 28 chondrocyte cell samples from 16 individuals were analyzed by fluorescence in situ hybridization (FISH) on single cell level. The expanded cells were further characterized upon the chondrocytic mRNA phenotype by reverse-transciptase polymerase chain reaction (RT-PCR). RESULTS: The molecular karyotyping results revealed autosomal stability, but all male samples analyzed by aCGH displayed a variable loss of the Y-chromosome. These data were confirmed by FISH-experiments and suggest an age dependant effect toward the loss of the Y-chromosome in cultured chondrocytes. RT-PCR data for the mRNAs from collagen types I, II, and aggrecan and the pro-inflammatory cytokine interleukin-1ß (IL-1ß) did not reveal any correlation of transcriptional activity in cultures with Y-chromosome losses, nor were there statistically significant differences between cells from female and male donors. CONCLUSIONS: While cells of male origin may suffer from an age-related loss of the Y-chromosome, there was no indication of a functional impairment. The data suggest some caution toward applying proliferative steps when considering chondrocytes from elderly male patients for tissue engineering approaches.

[Ankle chondrocytes are more resistant to Interleukin-1 than chondrocytes derived from the knee]. / Sprunggelenkchondrozyten besitzen eine höhere Interleukin-1-Resistenz als Kniegelenkchondrozyten.

BACKGROUND: The incidence of degenerative changes and osteoarthritis is lower in the ankle than in the knee joints. This cannot be explained exclusively with differences in anatomy and biomechanical properties of these two synovial joints. Previous studies have indicated distinct differences in the biochemical composition of the extracellular matrix of articular cartilage from knee and ankle joints. The aim of this study was to identify potential metabolic differences between knee and ankle joint chondrocytes using isolated cells to distinguish the secondary effects of the resident extracellular matrix from the primary matrix-independent effects of cellular differentiation. METHOD: Isolated knee and ankle chondrocytes from the same human donor were cultured in alginate beads and subsequently exposed to a three-day pulse of the catabolic cytokine interleukin-1 (IL-1) as a model of an inflammatory episode. The metabolism of proteoglycans (PG's) was analyzed as expressed changes in 35S-sulfate incorporation into glycosaminoglycans (GAG's). RESULTS: The presence of IL-1 induced an inhibition of PG synthesis in knee and ankle articular chondrocytes. The 50% inhibitory concentration (IC50) of IL-1 was about 5 times lower for knee than for ankle chondrocytes. CONCLUSION: Ankle chondrocytes are more resistant to IL-1 induced inhibition of PG synthesis than chondrocytes from the knee.

Introduction: molecular and biomechanical basis of osteoarthritis.

Osteoarthritis has developed into the most common chronic disease in the highly industrialized nations. Moreover, because of the prevalence of the disease in the elderly, this trend occurs worldwide as a consequence of increasing longevity due to the overall improvement in living conditions and health status. In contrast, research on osteoarthritis is still financially marginalized within biomedical research, so that the molecular and biophysical bases for disease initiation and progression are largely unmapped. The following sequence of five reviews highlights a remarkable change in that body of knowledge taking place at the beginning of the World Health Organization (WHO) 'Bone and Joint Decade 2001-2010'. The data and ideas presented in these articles reflect to some extent the guidelines set up by the WHO and by the National Institutes of Health of the USA and therefore allow a glimpse into the directions that research in osteoarthritis will take in the future.

Recombinant human osteogenic protein 1 is a potent stimulator of the synthesis of cartilage proteoglycans and collagens by human articular chondrocytes.

OBJECTIVE: To study the effects of recombinant human osteogenic protein-1 (rHuOP-1; bone morphogenetic protein-7) on proteoglycan and collagen synthesis by human articular chondrocytes. METHODS: Articular chondrocytes from fetal, adolescent, and adult human donors were cultured in alginate beads for 4 days in a mixture of Ham's F-12, Dulbecco's modified Eagle's medium, 10% fetal bovine serum (FBS), then for an additional 3-10 days in the presence and absence of rHuOP-1, with and without FBS. Chondrocyte synthetic activity was measured as the amount of incorporation of 35S-sulfate into proteoglycans and 3H-proline into hydroxyproline. Sieve chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were performed to identify specific proteoglycans and collagens. RESULTS: Recombinant human OP-1 markedly stimulated the synthesis of proteoglycans (mostly aggrecan) and collagens (predominantly type II) by all chondrocyte preparations. This did not require the presence of FBS and was associated with continued expression of the chondrocyte phenotype. CONCLUSION: Recombinant human OP-1 is a more potent stimulator of the synthesis of cartilage-specific molecules by human articular chondrocytes than are other factors tested for comparison, including TGF beta 1 and activin A.