Temporal and spatial gene expression of major bone extracellular matrix molecules during embryonic mandibular osteogenesis in rats

It is not known how gene expression of bone extracellular matrix molecules is controlled temporally and spatially, or how it is related with morphological differentiation of osteoblasts during embryonic osteogenesis in vivo. The present study was designed to examine gene expressions of type I collagen, osteonectin, bone sialoprotein, osteopontin, and osteocalcin during mandibular osteogenesis using in situ hybridization. Wistar rat embryos 13–20 days post coitum were used. The condensation of mesenchymal cells was formed in 14-day rat embryonic mandibles and expressed genes of pro-(I) collagen, osteonectin, bone sialoprotein and osteopontin. Cuboidal osteoblasts surrounding the uncalcified bone matrix were seen as early as in 15-day embryonic mandibles, while flat osteoblasts lining the surface of the calcified bone were seen from 16-day embryonic mandibles. Cuboidal osteoblasts expressed pro-1(I) collagen, osteonectin and bone sialoprotein intensely but osteopontin very weakly. In contrast, flat osteoblasts expressed osteopontin very strongly. Osteocytes expressed the extracellular matrix molecules actively, in particular, osteopontin. The present study demonstrated the distinct gene expression pattern of type I collagen, osteonectin, bone sialoprotein, osteopontin and osteocalcin during embryonic mandibular osteogenesis in vivo.     

Growth on type I collagen promotes expression of the osteoblastic phenotype in human osteosarcoma MG-63 cells.

Using MG-63 cells as a model system capable of partial osteoblastic differentiation, we have examined the effect of growth on extracellular matrix. MG-63 cell matrix and purified type I collagen induced a morphological change characterized by long cytoplasmic processes reminiscent of those seen in osteocytes. Concurrent biochemical changes involving bone marker proteins included increased specific activity of cell-associated alkaline phosphatase and increased secretion of osteonectin (up to 2.5-fold for each protein); all changes occurred without alterations in the growth kinetics of the MG-63 cells. The increase in alkaline phosphatase activity was maximal on days 6-8 following seeding; increased osteonectin secretion was most prominent immediately following seeding; all changes decreased as cells reached confluence. Growing cells on type I collagen resulted in an increased induction of alkaline phosphatase activity by 1,25(OH)2D3 (with little change in the 1,25(OH)2D3 induction of osteonectin and osteocalcin secretion), and increased TGF-beta induction of alkaline phosphatase activity as well (both TGF-beta 1 and TGF-beta 2). Both the 1,25(OH)2D3 and TGF-beta effects appeared to be synergistic with growth on type I collagen. These studies support the hypothesis that bone extracellular matrix may play an important role in osteoblastic differentiation and phenotypic expression.     

Matrix proteins associated with bone calcification are present in human vascular smooth muscle cells grownin vitro

Summary Atherosclerotic lesions are composed of cellular elements that have migrated from the vessel lumen and wall to form the cellular component of the developing plaque. The cellular elements are influenced by various growth-regulatory molecules, cytokines, chemoattractants, and vasoregulatory molecules that regulate the synthesis of the extracellular matrix composing the plaque. Because vascular smooth muscle cells (VSMC) constitute the major cellular elements of the atherosclerotic plaque and are thought to be responsible for the extracellular matrix that becomes calcified in mature plaques, immunostaining for collagenous and noncollagenous proteins typically associated with bone matrix was conducted on VSMC grownin vitro. VSMC obtained from human aorta were grown in chambers on glass slides and immunostained for procollagen type I, bone sialoprotein, osteonectin, osteocalcin, osteopontin, decorin, and biglycan. VSMC demonstrated an intense staining for procollagen type I, and a moderately intense staining for the noncollagenous proteins, bone sialoprotein and osteonectin, two proteins closely associated with bone mineralization. Minimal immunostaining was noted for osteocalcin, osteopontin, decorin, and biglycan. The presence in VSMC of collagenous and noncollagenous proteins associated with bone mineralization suggest that the smooth muscle cells in the developing atherosclerotic plaque play an important role in the deposition of the extracellular matrix involved in calcification of developing lesions.     

Functional roles of the tissue inhibitor of metalloproteinase 3 (TIMP-3) during ascorbate-induced differentiation of osteoblastic MC3T3-E1 cells

The tissue inhibitor of the metalloproteinase-3 (TIMP-3) gene was isolated as a gene involved in the process of ascorbate-induced differentiation of mouse MC3T3-E1 cells by the differential display method. The functional roles of TIMP-3 were characterized by establishing stable cell lines, which constitutively expressed the TIMP-3 gene. The TIMP-3 transfectants produced type I collagen at the same level as that of normal cells in response to ascorbic acid 2-phosphate (AscP). However, the expression of the other osteoblastic marker proteins such as alkaline phosphatase (ALPase), osteopontin (OP), osteocalcin (OC), osteonectin (ON) and matrix metalloproteinases (MMPs) remained at a low level even in the presence of AscP. Furthermore, no mineralization of the extracellular matrix (ECM) occurred with the transfectants. Remodeling ECM through TIMPs and MMPs is concluded to be required for osteoblastic differentiation.     

Confocal imaging and timing of secretion of matrix proteins by osteoblasts derived from avian long bone

Primary osteoblasts derived from avian long bone have been evaluated in terms of spatial and temporal expression of known osteoblastic marker proteins during the early phases of cell culture. Confocal imaging of matrix proteins revealed that osteocalcin, bone sialoprotein, osteopontin, and osteonectin were restricted to the cell interior at day 4 of culture; secretion and deposition into the extra-cellular matrix of bone sialoprotein and osteopontin was evident at 8 and 12 days of culture. Osteocalcin and osteonectin were not deposited in the matrix within the timeframe of the study. Total collagen levels produced and alkaline phosphatase activity were substantial by day 4 of culture, and increased from that point 4.0- and 5.5-fold, respectively, by culture day 12. The expression of type I collagen, PTHrP receptor, osteopontin, bone sialoprotein and osteocalcin was followed by Northern blot analysis. Type I collagen and osteopontin mRNA were expressed at constant levels throughout the culture period. Over the 12 days of culture both PTH/PTHrP receptor and bone sialoprotein mRNA expression were found to increase by 2.3- and 2.5-fold, respectively. In contrast, the expression of osteocalcin message decreased by 2.5-fold by day 8 of culture.     

Differential expression of dentin matrix protein 1, type I collagen and osteocalcin genes in rat developing mandibular bone

The expression of dentin matrix protein 1 (Dmp1) mRNA has been compared with that of type I collagen and osteocalcin mRNAs during bone formation in the rat mandible, using in situ hybridization. At embryonic day 15 (E15), type I collagen and osteocalcin mRNAs were expressed by the majority of newly-differentiated osteoblasts attached to unmineralized bone matrices, whereas Dmp1 mRNA expression was confined to only a few osteoblasts. Expression of these genes increased as the number of osteoblasts increased in specimens from E16 to E18. At E20, expression of Dmp1, type I collagen and osteocalcin was also observed in osteocytes. Dmp1 expression continued in osteocytes as they matured up to the 90-day-old specimens, whereas type I collagen and osteocalcin expression in osteocytes almost disappeared at 30 days of postnatal life. In contrast, osteoblasts continued to express type I collagen and osteocalcin in 90-day-old rats, but transiently expressed Dmp1 mRNA, which was seen in the minority of osteoblasts at 14 days of postnatal life. These data show that the developmental expression patterns of Dmp1 in osteogenic differentiation differ from those of type I collagen and osteocalcin, and Dmp1 appears to be expressed by osteocytes throughout ossification in the skeleton. These observations indicate that Dmp1 may serve unique biological functions in osteocyte and bone metabolism.     

Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures

We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or 1 microM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25-dihydroxyvitamin D3 to these dexamethasone-treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25-dihydroxyvitamin D3-treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone-treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the osteoblast phenotype in cultures of rat marrow stromal fibroblasts.     

Characterisation of the temporal sequence of osteoblast gene expression during estrogen-induced osteogenesis in female mice

Osteoblast differentiation under in vitro conditions is associated with increased expression of non-collagenous bone proteins including osteocalcin, osteopontin, and osteonectin, the exact function of which remain poorly understood. To determine whether these proteins play an important role in the formation of mineralised bone matrix by osteoblasts in vivo, we analysed the time-course of their expression during estrogen-induced osteogenesis in female mice, and compared this with the formation of new cancellous bone. Female mice were sacrificed prior to or following treatment with 17beta-estradiol for up to 32 days (500 microg/animal/week). Total RNA was extracted from femurs, and changes in expression of genes for a range of osteoblast-derived proteins assessed by Northern blot analysis. In parallel experiments, the time course of cancellous bone formation was determined by measuring bone mineral density (BMD) of the distal femur. Estrogen led to a rapid increase in BMD, which reached significance by Day 16. This was preceded by three-fold increases in expression of alkaline phosphatase (ALP) and type I collagen (COL I) at Days 8 and 12 respectively. In contrast, osteocalcin, osteopontin, and osteonectin expression showed no change during this initial period, although modest increases were observed at later times (i.e., Days 20 and 24). Our results suggest that osteocalcin, osteopontin, and osteonectin are not involved in the initial phase of the osteogenic response to estrogen, suggesting that these non-collagenous bone proteins do not play a direct role in the formation of mineralised bone matrix by osteoblasts in vivo.     

Fetal bovine bone cells synthesize bone-specific matrix proteins

We isolated cells from both calvaria and the outer cortices of long bones from 3- to 5-mo bovine fetuses. The cells were identified as functional osteoblasts by indirect immunofluorescence using antibodies against three bone-specific, noncollagenous matrix proteins (osteonectin, the bone proteoglycan, and the bone sialoprotein) and against type 1 collagen. In separate experiments, confluent cultures of the cells were radiolabeled and shown to synthesize and secrete osteonectin, the bone proteoglycan and the bone sialoprotein by immunoprecipitation and fluorography of SDS polyacrylamide gels. Analysis of the radiolabeled collagens synthesized by the cultures showed that they produced predominantly (approximately 94%) type I collagen, with small amounts of types III and V collagens. In agreement with previous investigators who have employed the rodent bone cell system, we confirmed in bovine bone cells that (a) there was a typical cyclic AMP response to parathyroid hormone, (b) freshly isolated cells possessed high levels of alkaline phosphatase, which diminished during culture but returned to normal levels in mineralizing cultures, and (c) cells grown in the presence of ascorbic acid and beta-glycerophosphate rapidly produced and mineralized an extracellular matrix containing largely type I collagen. These results show that antibodies directed against bone-specific, noncollagenous proteins can be used to clearly identify bone cells in vitro.     

Immunohistochemical investigations on the differentiation marker protein E11 in rat calvaria, calvaria cell culture and the osteoblastic cell line ROS 17/2.8

 Until now, many extracellular matrix proteins, e.g. osteopontin and osteonectin, have been used to determine a cell’s osteogenic maturation. The disadvantage in evaluation of these proteins is their relative wide-ranging appearance throughout the osteogenic differentiation process. Thus, the aim of this study was to establish an immunohistochemical setup using E11, a marker that binds selectively to cells of the late osteogenic cell lineage. In addition, the histochemical expression of the bone matrix proteins osteonectin, osteopontin and fibronectin was compared to that of E11 using monoclonal antibodies. For light microscopical detection of osteogenic markers in cultured cells we developed a simple paraffin technique using a fibrin glue as embedding medium. This allows the handling of cultured cells such as a tissue sample and includes the use of stored biological specimens for further immunohistochemical experiments. We used newborn rat calvariae for whole tissue preparations and for isolation and cultivation of bone cells. In addition, we included the rat osteosarcoma cell line ROS 17/2.8 in this study. For the first time, we have localised E11 in osteocytes of rat calvaria preparations at the electron microscopical level. E11 was detected at plasma membranes of osteocytes and their processes, but not at those of osteoblasts. Accompanying experiments with cultured newborn rat calvaria cells and ROS 17/2.8 cells revealed E11 reactivity on a subset of cells. The results obtained confirm the suitability of the differentiation marker E11 as a sensitive instrument for the characterisation of bone cell culture systems. Accepted: 25 August 1998     

Extracellular matrix protein mediated regulation of the osteoblast differentiation of bone marrow derived human mesenchymal stem cells

The biomimetic approach of tissue engineering exploits the favorable properties of the extracellular matrix (ECM), to achieve better scaffold performance and tissue regeneration. ECM proteins regulate cell adhesion and differentiation through integrin mediated signal transduction. In the present study, we have examined the role of ECM proteins such as collagen type I, fibronectin, laminin and vitronectin in regulating the proliferation and osteogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs were grown on selected ECM protein treated tissue culture plates. The growth kinetics was assessed by calculating the doubling time of the cells on different ECM treated plates. The cells were directed to osteoblast lineage by growing them in osteogenic induction media for 21 day. Differentiation was evaluated at different time points by osteoblast differentiation associated gene expression, alkaline phosphatase (ALP) activity, histochemical staining for mineralized matrix and calcium quantification. The doubling time of hMSCs cultured on collagen type I was significantly low, which was followed by laminin and fibronectin treated plates. However, doubling time of hMSCs cultured on vitronectin treated plate was not significantly different than that of the untreated control. High ALP gene (ALPL) expression and associated enhancement of mineralization were observed on collagen type I, fibronectin and vitronectin treated plates. Collagen type I showed early onset of mineralization with high ALP activity and up-regulation of osteopontin, ALPL, bone sialoprotein and osteocalcin genes. Vitronectin also up-regulated these genes and showed the highest amount of calcium in the secreted mineral matrix. Therefore, we conclude that, ECM proteins indeed modified the growth patterns and induced the osteoblast differentiation of hMSCs. Our findings have significant implication for bone tissue engineering applications.     

Biochemical comparison of fibroblast populations from different periodontal tissues: characterization of matrix protein and collagenolytic enzyme synthesis

To compare the expression of extracellular matrix components by fibroblasts from different periodontal tissues, rat molar periodontal ligament fibroblasts (RPL) and rat gingival fibroblasts (RGF) were isolated and cultured from individual animals. Pulse-chase experiments using [35S]methionine as a precursor revealed that confluent populations of early passage cells of both cell types synthesized similar amounts of collagen, fibronectin, and SPARC/osteonectin. Qualitative and quantitative differences were apparent in the relative proportions of type III collagen, in the rates of procollagen processing, and in the synthesis of a small number of unidentified proteins observed by sodium dodecyl sulphate--polyacrylamide gel electrophoresis. Collagen constituted 24-26% of the radiolabelled proteins secreted by both cell types, type I being the predominant collagen, with lower amounts of type III (3-8% RGF, 8-18% RPL) and type V (approximately 1%) collagens. Procollagen processing in the culture medium of RPL cells was more rapid than for RGF cells, but was increased in multilayered cultures of both RPL and RGF. In multilayered cultures, collagen TCA fragments, indicative of tissue collagenase activity, were also identified. Active and latent tissue collagenases and a latent form of a novel collagenolytic enzyme (matrix metalloendoproteinase-V) that cleaves native TCA fragments were demonstrated in these cultures. Addition of either concanavalin A (10(-6) M) or retinoic acid (10(-5) M) to the culture medium stimulated the secretion of the latent collagenolytic enzymes. Collagenase inhibitor was also synthesized by both RGF and RPL cells. SPARC/osteonectin, a 40-kilodalton glycoprotein, represented 0.5-1.0% of the secreted radiolabelled proteins of both cell types.     

Retinoic acid effects on an SV-40 large T antigen immortalized adult rat bone cell line

Clonal cell lines were established from adult rat tibia cells immortalized with SV-40 large T antigen. One clone (TRAB-11), in which retinoic acid (RA) induced alkaline phosphatase (AP) activity, was selected for further study. The TRAB-11 cells express high levels of type I collagen mRNA, type IV collagen, fibronectin, practically no type III collagen, little osteopontin, and no osteocalcin. RA stimulates proliferation of TRAB-11 cells (starting at 10 pM) and survival (starting at 100 pM). TRAB-11 cells synthesize fibroblast growth factor-2 (FGF-2), which has potent autocrine mitogenic effects on these cells and acts synergistically with RA. TRAB-11 cells attach better to type IV collagen than to fibronectin or laminin. Cell attachment to type IV collagen is increased by RA and decreased (65%) by an antibody directed against alpha1beta1 integrin. RA up-regulates steady-state levels of alpha1, mRNA without affecting beta1 mRNA expression. In conclusion, we report the establishment of a clonal cell line from the outgrowth of adult rat tibiae which is highly sensitive to RA in its growth and survival in culture, apparently as a result of integrin-mediated cell interaction with extracellular matrix proteins.     

Expression of mRNA of murine bone-related proteins in ectopic bone induced by murine bone morphogenetic protein-4

To determine whether a system of ectopic bone formation induced by osteosarcoma-derived bone-inducing substance (bone morphogenetic protein-4) can be used as a model of developing bone at the molecular level, we studied the expression of bone-related protein mRNAs in the process of ectopic bone formation using non-radioisotopic in situ hybridization. Osteonectin mRNA was detected in fibroblast-like cells, which are similar to periosteal cells from the early to middle stages of bone development. The proportion of osteonectin mRNA-expressing cells was greater than that of osteopontin mRNA-expressing cells in hypertrophic chondrocytes and osteoblast-like cells. In contrast, osteopontin mRNA was localized in a limited population of hypertrophic chondrocytes, a single layer of osteoblast-like cells adjacent to the bone trabeculae in the middle stage of bone formation, and in a limited subset of osteocytes in the late stage. A strong osteocalcin mRNA signal was detected in osteoblast-like cells from the middle to late stages and in a limited subset of osteocytes in the late stage of bone development. Since the sequential gene expression pattern of bone-related proteins in the present system is comparable to that in embryonic osteogenesis, this system may be useful as a model for studying gene expression in osteogenesis.     

In situ hybridization of bone matrix proteins in undecalcified adult rat bone sections.

We have developed a method for in situ hybridization of adult bone tissue utilizing undecalcified sections and have used it to histologically examine the mRNA expression of non-collagenous bone matrix proteins such as osteocalcin (bone Gla protein, BGP), matrix Gla protein (MGP), and osteopontin in adult rats. Expression was compared with that in bone tissues of newborn rats. In the adult bone tissue, osteocalcin mRNA was strongly expressed in periosteal and endosteal cuboidal osteoblasts but not in primary spongiosa near the growth plate. Osteopontin mRNA was strongly expressed in cells present on the bone resorption surface, osteocytes, and hypertrophic chondrocytes, but not in cuboidal osteoblasts on the formation surface. Osteopontin and osteocalcin mRNAs were expressed independently and the distribution of cells expressing osteopontin mRNA corresponded with acid phosphatase-positive mononuclear cells and osteoclasts. Expression of MGP mRNA was noted only in hypertrophic chondrocytes. In newborn rat bone tissues, expression of osteocalcin mRNA was much weaker than in adult rat bone tissues. These results clearly indicate the differential expression of mRNAs of non-collagenous bone matrix proteins in adult rat bone tissues.     

Real-time quantitative polymerase chain reaction and flow cytometric analyses of cell adhesion molecules expressed in human cell–multilayered periosteal sheets in vitro

Background aimsCultured human periosteal sheets more effectively function as an osteogenic grafting material at implantation sites than do dispersed periosteal cells. Because adherent cell growth and differentiation are regulated by cell-cell and cell–extracellular matrix contacts, we hypothesized that this advantage is a result of the unique cell adhesion pattern formed by their multiple cell layers and abundant extracellular matrix. To test this hypothesis, we prepared three distinct forms of periosteal cell cultures: three-dimensional cell-multilayered periosteal sheets, two-dimensional dispersed cell cultures, and three-dimensional hybrid mock-ups of cells dispersed onto collagen sponges.MethodsPeriosteal cells were obtained from human alveolar bone. Cell adhesion and extracellular matrix molecules were quantitatively determined at the messenger RNA and protein levels by means of real-time quantitative polymerase chain reaction and flow cytometry, respectively.ResultsReal-time quantitative polymerase chain reaction analysis demonstrated that regardless of culture media α1 integrin, vascular cell adhesion molecule-1, fibronectin and collagen type 1 were substantially upregulated, whereas CD44 was strongly downregulated in periosteal sheets compared with dispersed cell monolayers. With increased thickness, stem cell medium upregulated several integrins (β1, α1 and α4), CD146, vascular cell adhesion molecule-1, fibronectin and collagen type 1 in the periosteal sheets. Flow cytometric analysis revealed that the active configuration of β1 integrin was substantially downregulated in the stem cell medium–expanded cell cultures. The cell adhesion pattern found in the mock-up cultures was almost identical to that of genuine periosteal sheets.ConclusionsIntegrin α1β1 and CD44 function as the main cell adhesion molecule in highly cell-multilayered periosteal sheets and dispersed cells, respectively. This difference may account for the more potent osteogenic activity shown by the thicker periosteal sheets.     

Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix.

The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periods--proliferation, extracellular matrix maturation, and mineralization--and 2) two restriction points to which the cells can progress but cannot pass without further signals--the first when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle- and cell growth-regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid-induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.     

Analysis of heterogeneity of human keratinocytes interacting with immobilized fibronectin and collagenes I and IV types

The concept that stem cells form an independent subpopulations of somatic cells assumes the heterogeneity of cellular populations in adult tissues. As skin keratinocytes have natural affinity to extracellular matrix proteins, we made an attempt to reveal subpopulations of these cells depending on the time of their adhesion to substrates of collagen I and IV types and fibronectin. After selection for 10, 20 and 30 min the keratinocytes were cultivated for 24 h. The area of cell projection on a substrate and the spreading coefficient were measured (Kuzminykh, Petrov, 2004; Petrov et al., 2007). In 24 h statistically reliable morphological differences between the cells depending on the substratum were found. The size of the cells growing on collagen I type was twice as large as that of the cells cultivated on collagen IV type or in fibronectin. Irrespective of the substratum, up to 60-65% of the cells had a rounded form. The cultivation on collagens revealed the heterogeneity of keratinocytes both in the control cultures and under selection by adhesion time, while the cells grown on fibronectin behaved as a homogeneous population. These results suggest that, contrary to fibronectin, collagens stabilize some physiological states of keratinocytes corresponding to their interaction with extracellular matrix proteins in the organism.     

Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation

The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation can be examined in primary diploid cultures of fetal calvarial-derived osteoblasts by the combination of molecular, biochemical, histochemical, and ultrastructural approaches. Modifications in gene expression define a developmental sequence that has 1) three principal periods: proliferation, extracellular matrix maturation, and mineralization; and 2) two restriction points to which the cells can progress but cannot pass without further signals. The first restriction point is when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle and cell growth regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which an enhanced expression of alkaline phosphatase occurs immediately after the proliferative period, and later an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited; and 3) enhanced levels of expression of the osteoblast markers when collagen deposition is promoted, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and development of the osteoblast phenotype. The loss of stringent growth control in transformed osteoblasts and in osteosarcoma cells is accompanied by a deregulation of the tightly coupled relationship between proliferation and progressive expression of genes associated with bone cell differentiation.