Expression Of Osteoblastic Phenotype Research Articles

Tissue compatibility of biomaterials: assessment of bioactivity concerning the osteogenic response to the materials

When porous hydroxyapatite ceramics or rat bone marrow cells were implanted at subcutaneous sites, bone formation could not be detected at the sites. However, when composites of the ceramics and bone marrow were implanted, bone formation appeared at 3 weeks post implantation on the ceramic pore surface. Thus, the bone formed bonded to the ceramic. Bone Gla protein (BGP) is synthesized exclusively by bone or dentin-forming cells. Therefore, we analyzed gene expression of BGP in the composites by the northern blotting method. Total RNAs were extracted from the harvested implants, transferred to the nylon membranes, and hybridized to redialabeled probe. The probe used was oligonucleotides (46 mer) of rat BGP DNA. The BGP mRNA could not be detected in the bone marrow cells before the implantation or in the ceramic implantation without marrow cells. However, high level of the mRNA comparable with that in rat cancellous bone was found at 4 weeks post implantation of the composites. Thus, osteoblastic phenotype expression of undifferentiated cells on the surface of hydroxyapatite was confirmed. These results suggest that bioactive materials support the osteogenic differentiation on the surface and the differentiation finally results in tissue integration (bone bonding) with the materials.

L-ascorbic acid 2-phosphate promotes osteoblastic differentiation of MC3T3-E1 mediated by accumulation of type I collagen.

L-Ascorbic acid 2-phosphate (Asc-P: a stable ascorbic acid derivative) markedly stimulated synthesis of marker proteins for osteoblastic differentiation such as alkaline phosphatase and osteocalcin in a murine osteoblastic cell line, MC3T3-E1, suggesting that Asc-P could promote osteoblastic differentiation. L-Azetidine 2-carboxylate (AzC) diminished the stimulatory effects of Asc-P on the synthesis of alkaline phosphatase and osteocalcin due to its inhibitory effects on mature collagen secretion. Growing cells on the dishes coated with type I collagen resulted in an increased expression of osteoblastic phenotypes even in the presence of AzC. Coating with fibronectin, however, failed to promote differentiation. These results suggest that the promotion of cell differentiation caused by Asc-P is mediated by the accumulation of mature collagen.

Human osteogenic protein-1 induces both chondroblastic and osteoblastic differentiation of osteoprogenitor cells derived from newborn rat calvaria.

Osteogenetic protein-1 (OP-1), a member of the TGF-beta superfamily, induces endochondrial bone formation at subcutaneous sites in vivo and stimulates osteoblastic phenotypic expression in vitro. Primary cultures of newborn rat calvarial cells contain a spectrum of osteogenic phenotypes ranging from undifferentiated mesenchymal osteoprogenitor cells to parathyroid hormone (PTH)-responsive osteoblasts. We examined whether treatment of this cell population with recombinant human osteogenic protein-1 could induce chondrogenesis in vitro. Markers of chondroblastic versus osteoblastic differentiation included alcian blue staining at pH 1, alkaline phosphatase-specific activity, osteocalcin radioimmunoassay, and expression of collagen mRNAs. 6 d of treatment (culture days 1-7) with 4-100 ng OP-1/ml caused dose-dependent increases in alcian blue staining intensity and alkaline phosphatase activity (4.7- and 3.4-fold, respectively, at 40 ng/ml), while osteocalcin production decreased twofold. Clusters of round, refractile, alcian blue-stained cells appeared by day 3, increased in number until day 7, and then became hypertrophic and gradually became less distinct. Histochemically, the day 7 clusters were associated with high alkaline phosphatase activity and became mineralized. mRNA transcripts for collagen types II and IX were increased by OP-1, peaking at day 4, while type X collagen mRNA was detectable only on day 7 in OP-1-treated cultures. Delay of OP-1 exposure until confluence (day 7) amplifies expression of the normal osteoblastic phenotype and accelerates its developmental maturation. In contrast, early OP-1 treatment commencing on day 1 strongly amplifies chondroblastic differentiation. In the same protocol, TGF-beta 1 alone at 0.01-40 ng/ml fails to induce any hypertrophic chondrocytes, and in combination with OP-1, TGF-beta 1 blocks OP-1-dependent chondroinduction. OP-1 is believed to act on a subpopulation of primitive osteoprogenitor cells to induce endochondrial ossification, but does not appear to reverse committed osteoblasts to the chondrocyte phenotype.

Osteogenic differentiation of marrow stromal stem cells in porous hydroxyapatite ceramics

Bioactive materials such as hydroxyapatite ceramics (HA) are known to show a stable interfacial bond with tissues (the bone bonding property has been especially well documented). However, the biological phenomenon on the material surface that evoke bone bonding is not well documented. To address this issue, composites of porous HA and rat marrow cells were implanted into ectopic (subcutaneous) sites. At 3 weeks after implantation, active osteoblasts lining the HA surface initiated bone formation, primarily as mature bone (lamellar type). Gene expression study using Northern blot analysis of the composites showed that alkaline phosphatase (ALP) and bone Gla protein (BGP) mRNAs began to appear at 2 weeks and the level of BGP mRNA at 4 weeks was comparable to that of cancerous bone. However, HA implantation without marrow cells did not show bone formation and neither BGP nor ALP mRNA were detected. These results indicate that the HA surface is able to support cell differentiation (osteoblastic phenotype expression of marrow stromal stem cells), as well as cell coordination resulting in mature bone formation that bonds to the HA.

Effect of microfibrillar collagen on growth and differentiation of osteoblast-like MC3T3-E1 cells

Effect of microfibrillar collagen on the proliferation and differentiation of osteoblastic cells was studied using MC3T3-E1 (E1) cells. In order to achieve direct contact of microfibrillar collagen with E1 cells, they were embedded in denatured collagen gel, and DNA content, [3H] thymidine incorporation, alkaline phosphatase activity, and45Ca accumulation were examined after long-term culture. Microfibrillar collagen embedded with E1 cells increased DNA content and stimulated DNA synthesis of E1 cells in a dose-dependent manner. In vitro mineralization induced by E1 cells was also stimulated by microfibrillar collagen in a dose-dependent manner: 1mg/ml of microfibrillar collagen stimulated45Ca accumulation by about 3 fold, and 10 mg/ml by 5 fold. Alkaline phosphatase activity was not affected by the presence of microfibrillar collagen. Because the interaction of specific RGD tripeptide recognition site on collagen fiber with cell surface adhesion receptors is proposed to affect the proliferation and differentiation of various cells, it is suggested that the interaction of osteoblastic cells with collagen fibers plays an important role in the regulation of proliferation and the expression of osteoblastic phenotype in these cells.

Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development.

We examine clonal murine calvarial MC3T3-E1 cells to determine if they exhibit a developmental sequence similar to osteoblasts in bone tissue, namely, proliferation of undifferentiated osteoblast precursors followed by postmitotic expression of differentiated osteoblast phenotype. During the initial phase of developmental (days 1-9 of culture), MC3T3-E1 cells actively replicate, as evidenced by the high rates of DNA synthesis and progressive increase in cell number, but maintain a fusiform appearance, fail to express alkaline phosphatase, and do not accumulate mineralized extracellular collagenous matrix, consistent with immature osteoblasts. By day 9 the cultures display cuboidal morphology, attain confluence, and undergo growth arrest. Downregulation of replication is associated with expression of osteoblast functions, including production of alkaline phosphatase, processing of procollagens to collagens, and incremental deposition of a collagenous extracellular matrix. Mineralization of extracellular matrix, which begins approximately 16 days after culture, marks the final phase of osteoblast phenotypic development. Expression of alkaline phosphatase and mineralization is time but not density dependent. Type I collagen synthesis and collagen accumulation are uncoupled in the developing osteoblast. Although collagen synthesis and message expression peaks at day 3 in immature cells, extracellular matrix accumulation is minimal. Instead, matrix accumulates maximally after 7 days of culture as collagen biosynthesis is diminishing. Thus, extracellular matrix formation is a function of mature osteoblasts. Ascorbate and beta-glycerol phosphate are both essential for the expression of osteoblast phenotype as assessed by alkaline phosphatase and mineralization of extracellular matrix. Ascorbate does not stimulate type I collagen gene expression in MC3T3-E1 cells, but it is absolutely required for deposition of collagen in the extracellular matrix. Ascorbate also induces alkaline phosphatase activity in mature cells but not in immature cells. beta-glycerol phosphate displays synergistic actions with ascorbate to further stimulate collagen accumulation and alkaline phosphatase activity in postmitotic, differentiated osteoblast-like cells. Mineralization of mature cultures requires the presence of beta-glycerol phosphate. Thus, MC3T3-E1 cells display a time-dependent and sequential expression of osteoblast characteristics analogous to in vivo bone formation. The developmental sequence associated with MC3T3-E1 differentiation should provide a useful model to study the signals that mediate the switch between proliferation and differentiation in bone cells, as well as provide a renewable culture system to examine the molecular mechanism of osteoblast maturation and the formation of bone-like extracellular matrix.

Immunohistochemical localization of a ∼66 kD glycosylated phosphoprotein during development of the embryonic chick tibia

Localization of a approximately 66 kD glycosylated phosphoprotein during morphogenesis of the embryonic chick tibia has been accomplished using immunohistochemistry. Although initial expression of the tibial osteoblast phenotype is detected as early as stage 28.5, with the deposition of osteoid matrix beginning at stage 30, little or no immunoreactivity against the approximately 66 kD glycosylated phosphoprotein is observed in pre-osteoblasts, osteoblasts, osteocytes, or in the uncalcified osteoid matrix during the early events of tibia development. Immunoreactivity was first observed at stage 32 when mineralization of the osteoid matrix is initiated. At this and all later stages, the phosphoprotein is located almost exclusively in the extracellular matrix at the mineralization front with essentially no detectable staining in the adjacent unmineralized osteoid matrix. Similarly, no cellular staining is observed when even the lightly mineralized extracellular matrix is strongly immunoreactive. Only scant immunostaining is present over the heavily mineralized regions, although demineralization of these areas with EDTA exposes a low intensity, punctate staining pattern. Additionally, cryosections of developing calvaria stained with this antiserum only display reactivity in regions of bone matrix undergoing mineralization. These localization studies support the hypothesis that this phosphoprotein is intimately associated with the process of bone matrix mineralization in the developing chick long bone.

Transforming growth factor beta modulates proliferation of osteoblastic cells: relation to its effect on receptor levels for epidermal growth factor.

The effect of transforming growth factor beta (TGF-beta) on cellular proliferation of osteoblastic MC3T3-E1 cells was studied with particular emphasis on its effect on modulation of epidermal growth factor (EGF) receptors. In other cells, TGF-beta has been reported to augment EGF receptors. Exposure of MC3T3-E1 cells to TGF-beta initially increased cell surface EGF receptor levels and decreased the rate of DNA synthesis. The initial elevation of EGF receptor levels was due to increased receptor number per cell, not to changes in binding affinity. On the contrary, prolonged exposure (longer than 40 h) resulted in a decrease in EGF receptor and an increase in the rate of DNA synthesis. Thus, the effects of TGF-beta on these cells appears to be biphasic, reflecting complex mechanisms of action; the early effects of TGF-beta may be consistent with cellular differentiation to the osteoblastic phenotype with decreased cellular proliferation, whereas chronic exposure of these cells to TGF-beta stimulated cellular proliferation and inhibited osteoblastic phenotype expression. It is not likely that stimulation of cellular proliferation was through elevation of EGF receptor levels, because TGF-beta did not enhance the stimulatory effect of EGF on cellular proliferation. Thus, we conclude that TGF-beta possesses a stimulatory effect on the cellular proliferation of osteoblastic MC3T3-E1 cells independent of its modulative effect on EGF receptor level.

First bone formation and the dissection of an osteogenic lineage in the embryonic chick tibia is revealed by monoclonal antibodies against osteoblasts

Detailed studies of the origin and differentiation of osteogenic cells can be facilitated by cell-specific markers. To this end, we immunized mice with a heterogeneous population of chick embryonic bone cells and subsequently generated and selected for monoclonal antibodies against cell surface determinants. Supernatants from growing hybridoma colonies were screened immunohistochemically against frozen sections of embryonic stage 35 (day 9.5) chick tibiae. Three cell lines, SB-1, SB-2, and SB-3, which each secrete a different antibody against osteogenic cells, have been successfully cloned, stabilized, and immortalized. Antibody SB-1 reacts with a family of cells in embryonic bone, liver, kidney, and intestine, which are identically stained by the histochemical stain for alkaline phosphatase. The SB-2 antigen is present only on osteoblasts, while the SB-3 antigen is expressed on the surface of osteoblasts, ependymal cells and ventricular myoblasts. Studies on the developmental progression of osteoblasts in the embryonic tibia indicate that the determinants recognized by SB-1, SB-2, and SB-3 are temporally coupled to the appearance of the pre-osteoblast marker alkaline phosphatase. Detailed morphologic analyses reveal that SB-1 reacts with a large family of osteogenic cells residing between the surface of newly formed bone and the overlying periosteal osteoprogenitor cells. By contrast, SB-2 and SB-3 appear to react with those mature osteoblasts involved in the secretion and mineralization of osteoid. Cells which are imprisoned within bone matrix (osteocytes) of the developing tibia are not recognized by these antibodies, but are immunostained by an osteocyte-specific monoclonal antibody which does not react with SB-1, SB-2 or SB-3 positive cells. The results reported here suggest the existence of an osteogenic cell lineage which is characterized by a series of discrete cell states prior to the overt expression of the Secretory Osteoblastic phenotype. We propose that the emergence and abatement of phenotypically distinct osteogenic cell surface antigens follows a precise spatial and temporal sequence which reflects the position of cells within the osteogenic lineage.

Stimulation of skeletal-derived cell cultures by different electric field intensities is cell-specific

Pulsed electric stimulation, coupled capacitively to different cell cultures of skeletal origin, caused immediate changes in the cellular levels of cyclic AMP and a later enhanced DNA synthesis. Changes both in cyclic AMP level and DNA synthesis were correlated with the strength of the applied electric field. Cultures of calvaria bone cells which contain mainly two cell types, parathyroid hormone responsive cells (osteoblast-like) and prostaglandin E 2 responsive cells (fibroblast-like), respond to both low (13 V/cm) and to high (54 V/cm) electric field strength, with no response at intermediate (24 V/cm) field strength. Rat epiphyseal cartilage responded like bone cells both to low and high field intensities, while rat condylar cartilage responded only to the intermediate field strength. Moreover, subcultures of calvaria bone cells, which lost their osteoblastic phenotype expression during subculturing, were responsive only to low field strength. On the other hand, osteoblast-enriched cultures, derived from calvaria bone grown in low calcium, were responsive only to the high field strength. These findings suggest that the response to various electric field intensities is cell-specific and might be used as an additional parameter to characterize cell types. Our study points to the possibility that when exposing a whole organ to an electrical stimulation it is possible to affect specifically only one cell population out of the many cell types existing in the organ.