Morphological and biochemical characterization of mineralizing primary cultures of avian growth plate chondrocytes: Evidence for cellular processing of Ca²⁺ and Pi prior to matrix mineralization

Abstract

Advances in the culture of mineralizing growth plate chondrocytes provided an opportunity to study endochondral calcification under controlled conditions. Here we report that these cultures synthesize large amounts of proteins characteristically associated with mineralization: type II and X collagens, sulfated proteoglycans, alkaline phosphatase, and the bone-related proteins, osteonectin and osteopontin. Certain chondrocytes appeared to accumulate large amounts of Ca2+ and Pi during the mineralization process: laser confocal imaging revealed high levels of intracellular Ca2+ in their periphery and X-ray microanalytical mapping revealed the presence of many Ca(2+)- and Pi-rich cell surface structures ranging from filamentous processes 0.14 +/- 0.02 microns by 0.5-2.0 microns, to spherical globules 0.70 +/- 0.27 microns in diameter. Removal of organic matter with alkaline sodium hypochlorite revealed numerous deposits of globular (0.77 +/- 0.19 micron) mineral (calcospherites) in the lacunae around these cells. The size and spatial distribution of these mineral deposits closely corresponded to the Ca(2+)-rich cell surface blebs. The globular mineral progressively transformed into clusters of crystallites. Taken with earlier studies, these findings indicate that cellular uptake of Ca2+ and Pi leads to formation of complexes of amorphous calcium phosphate, membrane lipids, and proteins that are released as cell surface blebs analogous to matrix vesicles. These structures initiate development of crystalline mineral. Thus, the current findings support the concept that the peripheral intracellular accumulation of Ca2+ and Pi is directly involved in endochondral calcification.

Pivotal Role of Bcl-2 Family Proteins in the Regulation of Chondrocyte Apoptosis

“…It was reported that both intracellular and extracellular calcium and phosphate levels increase in the hypertrophic area where terminally differentiated chondrocytes mineralize the surrounding matrix and subsequently die (11,30,33). In vitro studies have also made clear that P i induces the mineralization and cell death of chondrocytes (11,12).…”

Bioactive Glass 13-93 as a Subchondral Substrate for Tissue-engineered Osteochondral Constructs: A Pilot Study

“…However, previous studies have shown calcium is known to have an effect on the extracellular matrix deposition of cartilage [35,43,44]. The intracellular concentration of calcium channels plays an important role in regulating chondrocyte proliferation and maturation in monolayer [44], and therefore high concentrations of calcium could be of benefit for cartilage tissue engineering.…”

Endochondral Bone Formation as Blueprint for Regenerative Medicine

“…Bone healing by endochondral ossification is influenced by many regulatory mechanisms. However, while interaction of Indian hedgehog (Ihh) and Parathyroid hormone related protein (PTHrP) is one of the best known regulatory mechanism in the growth plate, such an interplay is yet unknown for fracture healing (Wu, Ishikawa et al 1995;Vortkamp, Lee et al 1996;Volk and Leboy 1999). The role of growth factors during bone healing processes is better studied.…”