MECKELS CARTILAGE CHONDROCYTES IN ORGAN-CULTURE SYNTHESIZE BONE-TYPE PROTEINS ACCOMPANYING OSTEOCYTIC PHENOTYPE EXPRESSION

We examined whether Meckel's cartilage of embryonic mice, 17 days in u tero, undergo the cellular transformation into the osteocyte-like phen otype under organ culture conditions. Explants were grown by our origi nal pithole method modified Trowell-type cultures for up to 4 weeks at 37 degrees C under 5% CO2 in air. Specimens were examined using histo logical procedures including immunostaining and electron microscopy. I n addition, the effects of beta-glycerophosphate on matrix calcificati on were also examined in cultures with or without beta-glycerophosphat e. Addition of beta-glycerophosphate induced calcification at a higher level, but calcium mineral deposition occurred regardless of the addi tion of beta-glycerophosphate to the culture medium. Light and electro n microscopic analyses showed that freshly isolated chondrocytes prior to cell culture had typical hypertrophic morphology, but shortly afte r commencement of culture, they showed morphological modifications. Th e cells showing chondrocytic phenotypes became basophilic elliptical c ells, and eventually transformed into flattened osteocyte-like cells. Bone-like features for cellular elements were characterized by spindle -shaped cells with elongated processes accompanying bone-specific thic k-banded collagen fibrils. Immunostaining showed that at 2 weeks in cu lture, type I and type II collagens coexisted in the matrix, but subse quently type II collagen synthesis ceased and was replaced by type I c ollagen synthesis. Immunofluorescent labeling for osteocalcin was note d first in the peripheral cells by 1 week, but at 3 weeks this reactio n spread to the central zone in explants. Alkaline phosphatase activit y (ALPase) was expressed on the cells in the central zone prior to cal cium mineral deposition as shown by von Kossa's reaction at 3 weeks in culture. These results showed that Meckel's cartilage chondrocytes in organ culture synthesize bone-type proteins accompanying osteocytic p henotype expression.