Type I collagen influences cartilage calcification: An immunoblocking study in differentiating chick limb-bud mesenchymal cell cultures

Abstract

Chick limb-bud mesenchymal cells, plated in high-density micro-mass culture, differentiate and form a matrix resembling chick epiphyseal cartilage. In the presence of 4 mM inorganic phosphate or 2.5 mM beta-glycerophosphate mineral deposits upon this matrix forming a mineralized tissue that, based on electron microscopy, x-ray diffraction and Fourier Transform Infrared microspectoscopy, is like that of chick calcified cartilage. In this culture system the initial mineral deposits are found on the periphery of the chondrocyte nodules. During differentiation of the cells in the high-density micro-mass cultures there is a switch from expression of type I collagen to type II, and then to type X collagen. However, type I collagen persists in the matrix. Because there is some debate about whether type I collagen influences cartilage calcification, an immunoblocking technique was used to determine the importance of type I collagen on the mineralization process in this system. Studies using nonspecific goat anti-chick IgG demonstrated that 1-100 ng/ml antibody added with the media after the cartilage nodules had developed (day 7) had no effect on the accumulation of mineral in the cultures. Nonspecific antibody added before day 7 blocked development of the cultures. Parallel solution based cell-free studies showed that IgG did not have a strong affinity for apatite crystals, and had no significant effect on apatite crystal growth. Type I collagen antibodies (1-200 ng/ml) added to cultures one time on day 9 (before mineralization started), or on day 11 (at the start of mineralization), slightly inhibited the accumulation of mineral. There was a statistically significant decrease in mineral accretion with 100 or 200 ng/ml collagen antibody addition continuously after these times. Fab' fragments of nonspecific and type I collagen antibodies had effects parallel to those of the intact antibodies, indicating that the decreased mineralization was not attributable to the presence of the larger, bulkier antibodies. The altered accumulation of mineral was not associated with cell death in the presence of antibody (demonstrated by fluorescent labeling of DNA) or with increased apoptosis (TUNEL-stain). In the immunoblocked cultures, EM analysis demonstrated that mineral continued to deposit on collagen fibrils, but there appeared to be fewer deposits. The data demonstrate that type I collagen is important for the mineralization of these cultures.

Modulation of extracellular matrix protein phosphorylation alters mineralization in differentiating chick limb-bud mesenchymal cell micromass cultures

“…As previously reported [18][19][20][21][22][23][24][25][26], the mesenchymal cells differentiated in culture and formed alcian blue positive chondrocyte nodules. At day 14 (figure 1a), the cultures began to show trace amounts of von Kossa positive staining.…”

“…Chick limb-bud mesenchymal cells plated in high-density micromass cultures differentiate to form a mineralizable matrix that resembles the growth plate in the chick [18][19][20][21][22][23][24][25][26]. Our earlier studies using 2,3-diphosphoglycerate and heparin, both non-specific inhibitors of CK2, showed inhibition of mineralization when protein phosphorylation was blocked [18].…”

“…Immunoblocking Studies-Chick specific antibodies directed against BSP, OPN, or ON were individually added to the cultures with 1:3000 or 1:1000 dilution as previously described [22]. These concentrations were selected based on pilot experiments that determined saturated binding.…”

“…Accumulation of 45 Ca per micromass spot was calculated and data normalized to the accumulation in the mineralizing control culture at day 21 or 23. While historically we had corrected for 45 Ca uptake by the non-mineralizing controls [18][19][20][21][22][23][24][25][26], in initial experiments it was noted that this correction caused a difference of less than 1%; hence data are presented without this correction. However it should be noted that a single set of experiments for each inhibitor or blocking agent was performed to validate the fact that the controls treated with the agent in question were not binding more calcium than the untreated controls.…”

“…It is our hypothesis that it is the phosphorylation of the anionic matrix proteins that is important, rather than the ability of enzymes such as alkaline phosphatase to release inorganic phosphate from these proteins. To test this hypothesis mineralization was monitored in a culture system that mimics endochondral ossification [19][20][21][22][23][24][25][26]. The cultures were challenged with inhibitors of CK2 and of phosphoprotein phosphatases, and with blocking antibodies to selected phosphorylated proteins.…”