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Stimulatory effects of distinct members of the bone morphogenetic protein family on ligament fibroblasts

K Bobacz¹, R Ullrich², L Amoyo¹, L Erlacher¹, J S Smolen¹, W B Graninger¹

¹ Department of Internal Medicine III, Division of Rheumatology, Medical University of Vienna, Austria
² Department of Pathology, Medical University of Vienna, Austria

Correspondence to:
Dr K Bobacz
Department of Internal Medicine III, Division of Rheumatology, Allgemeines Krankenhaus, Waehringer Guertel 18-20, A-1090 Vienna, Austria; klaus.bobacz{at}univie.ac.at

Objective: To investigate effects of cartilage derived morphogenetic protein-1 and -2 (CDMP-1, CDMP-2), bone morphogenetic protein (BMP)-7 and BMP-6 on metabolism of ligament fibroblasts and their osteogenic or chondrogenic differentiation potential.

Methods: Ligament fibroblasts were obtained from 3 month old calves, plated as monolayers or micromass cultures, and incubated with or without CDMP-1, CDMP-2, BMP-7, and BMP-6. Expression of the indicated growth factors was assessed by RT-PCR and western immunoblotting. The presence of their respective type I and II receptors, and lineage related markers, was investigated in stimulated and unstimulated cells by RT-PCR and northern blotting. Biosynthesis of matrix proteoglycans was assessed by [³⁵S]sulphate incorporation in monolayers. Alcian blue and toluidine blue staining was done in micromass cultures.

Results: CDMP-1, CDMP-2, BMP-7, and BMP-6 were detected on mRNA and on the protein level. Type I and II receptors were endogenously expressed in unstimulated ligament fibroblasts. The growth factors significantly stimulated total proteoglycan synthesis as assessed by [³⁵S]sulphate incorporation. Toluidine blue staining showed cartilage-specific metachromasia in the growth factor treated micromass cultures. Transcription analysis of stimulated ligament fibroblasts demonstrated coexpression of chondrocyte markers but no up regulation of osteogenic markers.

Conclusion: CDMP-1, CDMP-2, BMP-7, and BMP-6 and their receptors were expressed in ligament tissue. These growth factors induced matrix synthesis in fibroblasts derived from bovine ligament. The preferential expression of cartilage markers in vitro suggests that CDMP-1, CDMP-2, BMP-7, and BMP-6 have the potential to induce differentiation towards a chondrogenic phenotype in ligament fibroblasts. Thus, fibroblasts from ligaments may serve as a source for chondrogenesis and tissue repair.

Abbreviations: AP, alkaline phosphatase; BM, basal medium; BMP, bone morphogenetic protein; CDMP, cartilage derived morphogenetic protein; FBS, fetal bovine serum; GDF, growth and differentiation factor; PBS, phosphate buffered saline; RT-PCR, reverse transcriptase-polymerase chain reaction; TGFß, transforming growth factor ß

Keywords: bone morphogenetic proteins; cartilage derived morphogenetic proteins; cartilage; fibroblasts; ligaments

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