Isolation of Cells from Ovine Fetal Long Bone and Characterization of their Osteoblastic Activities duringin vitroMineralization

Abstract

Studies about bone formation and regulation are complex due to a close relationship between bone cells. Primary cell cultures allow to understand osteoblastic function. We isolated cells from the cortical metacarpal bone of 85 or 120 day-old ovine fetuses by an enzymatic method. After first passage and cell amplification, the growth medium (DMEM, ascorbic acid and fetal calf serum 10%) was replaced at confluence by a mineralization medium (MM: DMEM, ascorbic acid, beta-glycerophosphate, insulin). Alkaline phosphatase (ALP) activity in cell-matrix layer increased after 4 days of cultures in MM and maximized at day 6. We also measured osteocalcin, ALP and IGF-I secretion simultaneously during mineralization. PTH, PTHrP and 1.25(OH)2D3 decreased ALP activity in cell-matrix layer after 4 days of treatment in MM without fetal calf serum (FCS). Cells from 120 day-old fetuses were cultivated in MM with 10% FCS during 32 days to induce mineralization. Inorganic phosphorus concentration increased in medium between days 5 and 12, Ca concentration decreased in medium after 12 days of culture. Mineralization started at day 12, in the same time ALP activity appeared in medium. Osteocalcin secretion increased between days 6 and 12, decreased at day 14 and increased from day 16 until day 32. Ovine fetal bone cells produced IGF-I until first days of culture in MM. Such ovine osteoblast phenotype cells having the capacity to differentiate and mineralize in vitro would be a model to study the endocrine regulation of osteoblastic function in large mammals.

Effect of Wnt6 on Human Dental Papilla Cells In Vitro

“…This enzyme is bound to the membrane of osteoblasts and functions to enhance osteogenesis by degrading pyrophosphate that inhibits crystallization at the calcification site and organic phosphate esters to increase the inorganic phosphate concentration. Therefore, ALP is considered an important marker for biomineralization and an early marker for osteoblasts or odontogenic marker genes (31). As indicated by the cytochemistry results, the ALPase activity was consistently increased in hDPC/Wnt6, with the ALP mRNA remaining at a relatively high level during the 12 days of culture.…”

In search of an osteoblast cell model for in vitro research

“…The available literature focusing on osteoblastic activities of ovine cells in vitro indicate these cells are capable of mineralisation (Collignon et al, 1997). Foetal ovine osteoblast cells stimulated post-proliferatively with ascorbic acid, β-glycerophosphate and CaCl 2 exhibited the highest ALP activity after 6 days culturing and formation of mineralisation centres from day 12.…”

“…Culture medium analysis demonstrated a rapid increase in ALP concomitant with initiation of mineralisation. Furthermore, ovine osteoblast cells produced osteocalcin in a time-dependent manner from day 6 (Collignon et al, 1997). Interestingly, a more recent study has shown that trabecular bone from ovine specimens classed as middle-aged (3-5 year old) behaved similarly to human cells isolated from the femoral condyle (donor age not known).…”

Isolation and characterization of osteoblast cultures from normal and osteopenic sheep for biomaterials evaluation

“…In the literature, however, many investigators have suggested that sheep can also be used for the study of osteoporosis. [12][13][14][15][16][17] Nevertheless, to our knowledge, very few in vitro experiments have been conducted on sheep osteoblasts [18][19] and, therefore, we undertook the present study.…”