ABSTRACT
Cartilage oligomeric matrix protein (COMP) is a protein present in the cartilage matrix and is expressed more abundantly in osteoarthritis cartilage than in healthy cartilage. The present study was designed to investigate the effect of transforming growth factor ß (TGFß) on COMP deposition and the influence of COMP on collagen biochemistry in a long-term 3-dimensional culture. Bovine chondrocytes in alginate beads were cultured with or without 25 ng/mL TGFß2 for 21 or 35 days. COMP was overexpressed in bovine chondrocytes using lentiviral transfection. COMP gene expression, COMP protein production, collagen and proteoglycan deposition, and collagen fibril thickness were determined. Addition of TGFß2 resulted in more COMP mRNA and protein than the control condition without growth factors. Lentiviral transduction with COMP resulted in elevated gene expression of COMP and increased COMP levels in the alginate bead and culture medium compared to untransfected cells. Overexpression of COMP did not affect the deposition of collagen, collagen cross-linking, proteoglycan deposition, or the mechanical properties. Stimulating COMP production by either TGFß2 or lentivirus resulted in collagen fibrils with a smaller diameter. Taken together, COMP deposition can be modulated in cartilage matrix production by the addition of growth factors or by overexpression of COMP. Inducing COMP protein expression resulted in collagen fibrils with a smaller diameter. Because it has been demonstrated that the collagen fibril diameter is associated with mechanical functioning of the matrix, modulating COMP levels should be taken into account in cartilage regeneration strategies.
ABSTRACT
BACKGROUND: Damage to articular cartilage is one of the features of osteoarthritis (OA). Cartilage damage is characterised by a net loss of collagen and proteoglycans. The collagen network is considered highly important for cartilage function but little is known about processes that control composition and function of the cartilage collagen network in cartilage tissue engineering. Therefore, our aim was to study the contribution of collagen amount and number of crosslinks on the functionality of newly formed matrix during cartilage repair. METHODS: Bovine articular chondrocytes were cultured in alginate beads. Collagen network formation was modulated using the crosslink inhibitor beta-aminopropionitrile (BAPN; 0.25mM). Constructs were cultured for 10 weeks with/without BAPN or for 5 weeks with BAPN followed by 5 weeks without. Collagen deposition, number of crosslinks and susceptibility to degradation by matrix metalloproteinase-1 (MMP-1) were examined. Mechanical properties of the constructs were determined by unconfined compression. RESULTS: BAPN for 5 weeks increased collagen deposition accompanied by increased construct stiffness, despite the absence of crosslinks. BAPN for 10 weeks further increased collagen amounts. Absence of collagen crosslinks did not affect stiffness but ability to hold water was lower and susceptibility to MMP-mediated degradation was increased. Removal of BAPN after 5 weeks increased collagen amounts, allowed crosslink formation and increased stiffness. DISCUSSION: This study demonstrates that both collagen amounts and its proper crosslinking are important for a functional cartilage matrix. Even in conditions with elevated collagen deposition, crosslinks are needed to provide matrix stiffness. Crosslinks also contribute to the ability to hold water and to the resistance against degradation by MMP-1.
Subject(s)
Aminopropionitrile/pharmacology , Chondrocytes , Extracellular Matrix/chemistry , Fibrillar Collagens/metabolism , Regeneration/physiology , Tissue Engineering/methods , Alginates , Animals , Biomechanical Phenomena , Cartilage, Articular/pathology , Cattle , Cell Count , Cells, Cultured , Extracellular Matrix/drug effects , Extracellular Matrix/enzymology , Gene Expression , Matrix Metalloproteinases/pharmacology , Osteoarthritis/pathology , Permeability , Phenotype , Stress, Mechanical , Time FactorsABSTRACT
OBJECTIVE: Examine effects of insulin-like growth factor 1 (IGF1), transforming growth factor beta2 (TGFbeta2) and fibroblast growth factor 2 (FGF2) on proteoglycan and collagen network and biomechanical properties of the newly formed cartilage matrix. METHODS: Bovine articular chondrocytes were cultured in alginate beads for 3 weeks with or without FGF2, TGFbeta2 or IGF1 in the presence of 10% FCS. Proteoglycan content, collagen content, hydroxylysylpyridinoline cross-links and overall matrix metalloproteinase (MMP) activity in the culture medium were measured. Alginate disks cultured for 5 weeks were used to evaluate the effect of growth factors on mechanical properties of the construct by determining the equilibrium aggregate modulus and secant modulus. RESULTS: IGF1 increased collagen and proteoglycan deposition. FGF2 mainly decreased collagen deposition and TGFbeta2 proteoglycan deposition. A decrease in cross-links was observed in matrix produced by chondrocytes cultured in the presence of TGFbeta2. IGF1 and FGF2 had no influence on the number of cross-links per collagen molecule. Overall MMP activity was significantly higher in culture medium of cells cultured with FGF2. TGFbeta2 and IGF1 had no effect on MMP activity. After 35 days of culture, the matrix produced under influence of IGF1 had a lower permeability and a trend to increase stiffness. FGF2 showed a trend to lower both properties. TGFbeta2 had no effect on these parameters. CONCLUSION: IGF1, TGFbeta2 and FGF2 had differential effects on collagen network formation. Of the three growth factors tested, IGF1 seems to be best in promoting the formation of a functional collagen network since it increased proteoglycan and collagen deposition and improved the mechanical properties.
