Effects of proteoglycan modification on mineral formation in a differentiating chick limb-bud mesenchymal cell culture system

Abstract

In the presence of 4 mM inorganic phosphate, differentiating chick limb-bud mesenchymal cells plated in micromass cultures form a mineralized matrix resembling that of chick calcified cartilage. To test the hypothesis that cartilage proteoglycans are inhibitors of cell mediated mineralization, the synthesis, content, and turnover of proteoglycans were altered in this system, and the extent of mineralization and properties of the mineral crystals examined. In all cases where the proteoglycan synthesis or proteoglycans present were modified to provide fewer or smaller molecules, mineralization was enhanced. Specifically, when proteoglycan synthesis was blocked by treatment with 10(-10) M retinoic acid, extensive mineral deposition occurred on a matrix devoid of both proteoglycans and cartilage nodules. The crystals, which formed rapidly, were relatively large in size based on analysis by X-ray diffraction or FT-1R microspectroscopy, and were more abundant than in controls. When 2.5 or 5 mM xylosides were used to cause the synthesis of smaller proteoglycans, the extent of mineral accretion was also increased relative to controls; however, the matrix was less affected, and the extent of mineral deposition and the size of the crystals were not as markedly altered as in the case of retinoic acid. Modification of existing proteoglycans by either chondroinase ABC or hyaluronidase treatment similarly resulted in increased mineral accretion (based on 45Ca uptake or total Ca uptake) relative to cultures in which the proteoglycan content was not manipulated. Crystals were more abundant and larger than in control mineralizing cultures. In contrast, when proteoglycan degradation by metalloproteases was inhibited by metal chelation with o-phenanthroline, the Ca accretion at early time points was increased, but as mineralization progressed, Ca accumulation decreased. These data provide evidence that in this culture system, proteoglycans are inhibitors of mineralization.

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].…”

“…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.…”

Evaluation of bioreactor-cultivated bone by magnetic resonance microscopy and FTIR microspectroscopy

“…The utility of FTIR microspectroscopy in studying mineralized tissues is well established (33)(34)(35)(36). The technique combines histology-like spatial localization with the quantitative capability of bulk chemical analysis and it is used routinely in such specimens to assess the spatial distribution of mineral and collagen (from the phosphate and amide peaks, respectively) (37,38).…”