Osteoblast differentiation of bone marrow stromal cells cultured on silica gel and sol-gel-derived titania.

Primary cultures of osteogenic precursor cells derived from rat bone marrow stroma were performed on commercially available pure titanium discs (Ti c.p.) and surface modified Ti c.p.using a sol-gel technique (Ti sol). In separate repeated experimental runs, cell behavior and in vitro mineralization were compared with cultures on silica gel bioactive glass discs (S53P4). All substrates were incubated in simulated body fluid prior to the experiment. Overall, variable effects between experimental runs were seen. Apparently, this was due to the heterogeneous nature of the used cell population. Therefore, only careful conclusions can be made. Initial cell adhesion and growth rates between 3 and 5 days of culture--analyzed by cell numbers--were in general comparable for the two titanium substrates, while initial growth up to day 3 is suggested to be higher in Ti c.p. compared to Ti sol. Although initial cell adhesion on the S53P4 glass discs was lower than the titanium substrates, cell growth rates appeared to be higher on the silica gel compared to the two titanium substrates. Further, there were some indications that the early and late osteoblast differentiation markers, alkaline phosphatase and osteocalcin, monitored up to day 24, were elevated in Ti c.p cultures compared to Ti sol cultures. There were no differences observed in in vitro mineralization between the titanium groups. S53P4 seemed to display a substantially higher differentiating capacity for both osteogenic cell markers as well as in vitro mineralization compared to the two titanium substrates.

Analysis of integrin expression in U2OS cells cultured on various calcium phosphate ceramic substrates.

Earlier we observed that calcium phosphate (Ca-P)-coated implant substrates stimulated the differentiation of osteoblast-like cells compared to uncoated substrates. This suggests that this difference in osteogenic induction is due to the chemical composition of the substratum. We hypothesized that Ca-P coatings modulate integrin expression patterns, because those receptors are the sensors of the cell. Therefore, in the present study we quantitatively analyzed integrin expression of osteosarcoma cells and their proliferation behavior on various well-defined Ca-P substrates. For this study we used the osteosarcoma cell line U2OS. Five groups of substrates were used: thermanox (Th), uncoated titanium (Ti), dense sintered hydroxyapatite (HA), and two Ca-P-coated titanium discs (TiHA-O% and TiHA-5%). At day 5, cell numbers were significantly lower (p < 0.05) for both types of Ca-P-coated titanium substrates compared to the other substrates. There were no significant differences between HA and uncoated titanium. From day 5 to 8, accumulated cell number was ranking highest to lowest HA > Th = Ti > TiHA-0% > TiHA-5%. Integrin expression at day 5 and day 8 of incubation was analyzed by flow cytometry for integrin subunits beta 1, alpha 3, alpha 4, alpha 5, alpha 6, and alpha v. Fluorescence-activated cell sorting (FACS) analysis showed that the cells express high levels of beta 1, low levels of alpha 4, alpha 5, and alpha 6, and moderate levels of alpha 3 and alpha v integrin subunits on the various biomaterial substrates. Minor differences in integrin expression between the various substrates were seen. Therefore, the observed differences in proliferation between the coatings may reside in modulating the functional properties of integrins.

Differential display of human marrow stromal cells reveals unique mRNA expression patterns in response to dexamethasone.

Human bone marrow stromal cells (hBMSC) are pluripotent cells that have the ability to differentiate into bone, cartilage, hematopoietic-supportive stroma, and adipocytes in a process modulated by dexamethasone (DEX). To characterize changes in hBMSC in response to DEX, we carried out differential display experiments using hBMSC cultured for 1 week in the presence or absence of 10(-8) M DEX. When RNA from these cells was used for differential display, numerous cDNA bands were identified that were up-regulated and down-regulated by DEX. The cDNA bands were reamplified by PCR and directly used to screen an hBMSC cDNA library. Seven clones were isolated and characterized by DNA sequencing and found to encode the following genes: transforming growth factor-beta-induced gene product ((beta)ig-h3), calphobindin II, cytosolic thyroid-binding protein, 22-kDA smooth muscle protein (SM22), and the extracellular matrix proteins osteonectin/SPARC, type III collagen, and fibronectin. To confirm that these genes were regulated by DEX, the cells were treated continuously with this hormone for periods ranging from 2 to 30 days, and steady-state mRNA levels were measured by Northern blot analysis. All genes showed some level of regulation by DEX. The most profound regulation by DEX was observed in the (beta)ig-h3 gene, which showed a relative 10-fold decrease in mRNA levels after 6 days of treatment. Interestingly, (beta)ig-h3 expression was not altered by DEX in fibroblasts from other human tissues, including thymus stromal fibroblasts, spleen stromal fibroblasts, and foreskin fibroblasts. In summary, differential display of DEX-treated hBMSC revealed unique patterns of gene expression and has provided new information about phenotypic changes that accompany the differentiation of hBMSC toward osteogenesis. J. Cell. Biochem. 76:231-243, 1999. Published 1999 Wiley-Liss, Inc.

Immortalization and characterization of bone marrow stromal fibroblasts from a patient with a loss of function mutation in the estrogen receptor-alpha gene.

A male patient with abnormal postpubertal bone elongation was shown earlier to have a mutation in both alleles of the estrogen receptor, resulting in a nonfunctional gene. Marrow stromal fibroblasts (MSFs) derived from this patient were called HERKOs (human estrogen receptor knock outs), and in order to obtain continuous HERKO cell lines, they were immortalized using a recombinant adenovirus-origin-minus SV40 virus. MSFs are unique cells because they support hematopoesis and contain a mixed population of precursor cells for bone, cartilage, and fat. Three established cell lines (HERKO2, HERKO4, and HERKO7) were characterized and compared with the heterogeneous population of nonimmortalized HERKOs for their osteogenic potential. We performed Northern analysis of matrix genes implicated in bone development and metabolism and an in vivo bone formation assay by transplanting the cells subcutaneously into immunodeficient mice. All three HERKO lines expressed high amounts of collagen 1A1, osteopontin, osteonectin, fibronectin, decorin, biglycan, and alkaline phosphatase. Except for osteopontin, expression of these genes was slightly lower compared with nonimmortalized HERKOs. In the in vivo bone formation assay, the heterogeneous population of nonimmortalized HERKOs formed bone with high efficiency, while the HERKO lines induced a high-density, bone-like matrix. Finally, all HERKO cell types secreted high levels of insulin-like growth factor I and interleukin-6 into the culture medium relative to cells of normal human subjects. In summary, these lines of HERKO cells retain several of the phenotypic traits of MSFs after immortalization, including matrix and cytokine production, and provide a valuable source of a unique human material for future studies involving estrogen action in bone and bone marrow metabolism.

The influence of transforming growth factor beta 1 on the development of embryonic mouse long bones.

Transforming growth factor beta (TGF-beta) is an important regulator of bone metabolism, and is found in large quantities in embryonic and adult bone tissue. The influence of TGF-beta 1 on chondro-osteogenesis was studied. In organ cultures of developing long bone rudiments of embryonic mice, growth and development of the various cartilaginous and osseous compartments were investigated by morphometric analysis and autoradiography after [3H]-thymidine labelling. TGF-beta 1 (1 ng/ml) inhibited both chondrogenesis and osteogenesis, and also inhibited matrix calcification. The effect was greatest in cell populations with the highest proliferation rate. It was noticed that the bone collar formation was inhibited. This may be due to an inhibition of osteoblast proliferation or differentiation, but it seems more likely to be an inhibition of the manufacture of matrix substance. These data suggest that TGF-beta 1 may be an important regulator of embryonic bone development.

Decreased mineralization and increased calcium release in isolated fetal mouse long bones under near weightlessness.

Mechanical loading plays an important role in the development and maintenance of skeletal tissues. Subnormal mechanical stress as a result of bed rest, immobilization, but also in spaceflight, results in a decreased bone mass and disuse osteoporosis, whereas supranormal loads upon extremities result in an increased bone mass. In this first in vitro experiment with complete fetal mouse cartilaginous long bones, cultured under microgravity conditions, we studied growth, glucose utilization, collagen synthesis, and mineral metabolism, during a 4-day culture period in space. There was no change in percent length increase and collagen synthesis under microgravity compared with in-flight 1x gravity. Glucose utilization and mineralization were decreased under microgravity. In addition, mineral resorption, as measured by 45Ca release, was increased. These data suggest that weightlessness has modulating effects on skeletal tissue cells. Loss of bone during spaceflight could be the result of both impaired mineralization as well as increased resorption.

Opposite effects of osteogenic protein and transforming growth factor beta on chondrogenesis in cultured long bone rudiments.

Osteogenic protein-1 (OP-1, also called BMP-7) is a bone morphogenetic member of the TGF-beta superfamily. In the present study, we examined the effect of recombinant human OP-1 on cartilage and bone formation in organ cultures of metatarsal long bones of mouse embryos and compared the OP-1 effects with those of human TGF-beta 1 and porcine TGF-beta 1 and beta 2. Cartilage formation was determined by measurement of longitudinal growth of whole bone rudiments during culture and by the incorporation of 35SO4 into glycosaminoglycans. Mineralization was monitored by 45Ca incorporation in the acid-soluble fraction and by measuring the length of the calcifying center of the rudiment. Toluidine blue-stained histologic sections were used for quantitative histomorphometric analysis. We found that OP-1 stimulated cartilage growth as determined by sulfate incorporation and that it increased remarkably the width of the long bones ends compared with controls. This effect was partly caused by differentiation of perichondrial cells into chondrocytes, resulting in increased appositional growth. In contrast to OP-1, TGF-beta 1 and beta 2 inhibited cartilage growth and reduced the length of whole bone rudiments compared with controls. In the ossifying center of the bone rudiments, both OP-1 and TGF-beta inhibited cartilage hypertrophy, growth of the bone collar, and matrix mineralization. These data demonstrate that OP-1 and TGF-beta exhibit opposite effects on cartilage growth but similar effects on osteogenesis in embryonic mouse long bone cultures. Since both OP-1 and TGF-beta have been demonstrated in embryonic cartilage and bone, these results suggest that they act as autocrine or paracrine regulators of embryonic bone development.

Effects of TGF-beta 2 on mineral resorption in cultured embryonic mouse long bones; 45Ca release and osteoclast differentiation and migration.

To study the effects of transforming growth factor beta 2 (TGF-beta 2) on bone resorption, cultures of 17-day-old foetal mouse metatarsal long bones were used. The long bone rudiments were cultured for 5 days in medium supplemented with 10% rat serum. The effects of TGF-beta 2 were studied at concentrations of 1, 4, and 10 ng/ml. At all concentrations TGF-beta 2 caused a significant reduction in osteoclastic resorption measured as release of 45Ca from prelabelled bones. The same long bones were subsequently used for histological evaluations. Pre-osteoclasts and osteoclasts were identified as tartrate resistant acid phosphatase (TRAP) positive cells in the mineralized diaphysis, the periosteum around the diaphysis, and the perichondrium around the cartilaginous ends. The distribution of TRAP-positive cells over the three compartments showed that TGF-beta 2 inhibited the migration of TRAP-positive cells from the periosteum into the mineralized diaphysis in a dose dependent manner. In addition, TGF-beta 2 had a biphasic effect on TRAP cell differentiation, as 1 ng/ml increased, but 4 ng/ml and higher decreased TRAP cell numbers. We conclude that TGF-beta 2 is a potent regulator of osteoclastic bone resorption, by modulating both osteoclast migration and osteoclast differentiation.

Inhibiting and stimulating effects of TGF-beta 1 on osteoclastic bone resorption in fetal mouse bone organ cultures.

The effects of TGF-beta 1 on osteoclastic resorption of fetal mouse calvaria and long bones at various stages of development was studied in organ culture. In resorbing calvariae and long bones with an established marrow cavity TGF-beta 1 (4-10 ng/ml) had a stimulating effect on 45Ca release that was partially inhibited by indomethacin. In primitive long bones, however, which were explanted before osteoclast invasion and excavation of a marrow cavity had started, TGF-beta 1 (1-4 ng/ml) inhibited 45Ca release by an indomethacin-insensitive mechanism. Histomorphometry of long bones after staining for tartrate-resistant acid phosphatase (TRAP) revealed that TGF-beta 1 treatment inhibited the migration of TRAP-positive cells from periosteum to developing marrow cavity and inhibited cell fusion. However, the formation of (mononuclear) TRAP-positive cells in the periosteum-perichondrium was strongly enhanced. These data suggest that TGF-beta 1 modulates various steps in the cascade of osteoclast development, recruitment, and activation in different ways, involving both prostaglandin-mediated and prostaglandin-independent pathways. Therefore the net effect of exogenous TGF-beta 1 on osteoclastic resorption in bone organ cultures depends on the relative prevalence of osteoclast progenitors, precursors, and mature osteoclasts in the tissue under study.