Characterization of human osteoblastic cells: Influence of the culture conditions

Abstract

Human osteoblastic cells were isolated enzymatically from adult human spongy bone and grown in MEM-Ham F12 1:1 medium supplemented with 2% Ultroser (USM). They were subcultured and examined for osteoblast features by morphological, histological, and biochemical approaches. The cells had a characteristic polyhedral morphology and produced a high level of alkaline phosphatase (ALKP). Confluent cultures were uniformly stained for ALKP and flow cytometry analysis with fluorescein diphosphate gave a single peak signal, reflecting a highly positive population, distinct from cultures of fibroblasts. The ALKP activity was stimulated by 1,25 (OH)2 vitamin D3. CD 44 was strongly expressed in these cultures, although osteoblasts are negative in vivo and osteocytes are positive. The main collagen synthesized was type I collagen and osteocalcin was produced after stimulation by vitamin D3. 10 mM betaGP induced mineralization and microprobe analysis of the crystals showed a composition close to hydroxyapatite. Changing the culture conditions to MEM-10% calf serum acted on cell behavior: it reduced the production of these biochemical markers of osteoblasts and the morphology became fibroblastlike with more rapid cell multiplication. The parameter most affected by the change in culture medium was ALKP, which was selected as the determinant criterion for defining an osteoblast culture. ALKP activity was then used to characterize a culture of cells seeded in a collagen gel.

Mineralization and Alkaline Phosphatase Activity in Collagen Lattices Populated by Human Osteoblasts

“…Bone fragments were taken during surgical hip replacement from subjects aged 50-80 years and digested with trypsin and collagenase [16]. Cells collected from the second and the third digestions were grown in Earle's Minimum Essential Medium/MEM-Ham F12 (1:1) supplemented with 1 mM pyruvate, 2 mM glutamine, 50 g/ml ascorbic acid, 5 M non-essential aminoacids, 50 g/ml penicillin and streptomycin, and 2% Ultroser (Gibco BRL).…”

“…The present study uses an in vitro system that closely mimics a tissue-like environment with a three-dimensional culture in a type I collagen gel. Adult human osteoblastic cells that retained their osteoblastic phenotype during the first few passages were used [16]. We first characterized cell proliferation pattern and viability within collagen gels as well as cell ability to retract the matrix.…”

Bone tissue engineering in a rotating bioreactor using a microcarrier matrix system

“…3,5 Researchers in our laboratory and others have investigated biodegradable scaffolds for in vitro bone engineering, which possess a suitable 3-D environment for the cell function together with the capacity for gradual resorption and replacement by host bone tissue. [6][7][8][9][10][11][12][13][14][15][16][17][18] This 3-D matrix milieu provides the necessary microenvironment for cell-cell and cell-matrix interaction, and is sufficient for the production of limited amounts of mineralized bone matrix in static culture. To demonstrate clinical feasibility of tissueengineered bone and to sufficiently match the intrinsic properties of autogenous bone-graft material, rapid mineralization of osteoid tissue grown in vitro must be achieved.…”

In vitro osteoblast-like cell metabolism in spondylodesis-a tool that may predict fusion capacityA prospective study in 50 patients with a 1-year follow-up

“…While age-related reductions in metabolic activity (Chavassieux et al 1990, Fedarko et al 1992, Pfeilschifter et al 1993, Sutherland et al 1995, Christensen et al 1999) and proliferative ability (Termine 1990, Fedarko et al 1992, Pfeilschifter et al 1993, DʼIppolito et al 1999) have been reported, other studies report no age-related effect (Chavassieux et al 1990, Evans et al 1990, Katzburg et al 1999. Uncontrolled basic experimental conditions, such as the number of passages, confluence stages, various sources of bone tissue and small sampling sizes in some studies may have contributed to these discrepancies (Chavassieux et al 1990, Matsuyama et al 1990, Pfeilschifter et al 1993, Schmidt and Kulbe 1993, Kassem et al 1997, Rattner et al 1997, Siggelkow et al 1999b). …”

“…Osteoblast-like cell cultures have been investigated extensively and their osteoblast-like characteristics described-i.e., osteocalcin, alkaline phosphatase, and collagen type I production and their response to osteotropic growth factors and hormones (Bellows et al 1990, Fedarko et al 1990, Lian and Stein 1995, Kassem et al 1997, Rattner et al 1997, Siggelkow et al 1999a.…”