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PubMed2000

Functional characterization of osteoblasts and osteoclasts from alkaline phosphatase knockout mice.

Tissue nonspecific alkaline phosphatase (TNAP) knockout (ko) mice manifest defects in bone mineralization that mimic the phenotypic abnormalities of infantile hypophosphatasia. In this article, we have searched for phenotypic differences between calvarial osteoblasts and osteoclasts in wild-type (wt), heterozygous and homozygous TNAP null mice. In vitro release of 45Ca from calvarial bones, with and without stimulation with parathyroid hormone (PTH), revealed no functional difference between osteoclasts from the three TNAP genotypes. Studies of primary cultures of TNAP+/+, TNAP+/-, and TNAP-/- calvarial osteoblasts revealed no differences in the rate of protein synthesis or in the expression levels of messenger RNAs (mRNAs) for osteopontin (OP), osteocalcin (OC), collagen type I, core binding factor alpha1 (Cbfa 1), N-cadherin, Smad 5, and Smad 7. Release of interleukin-6 (IL-6) from calvarial osteoblasts under basal conditions and after stimulation with PTH, tumor necrosis factor alpha (TNF-alpha) or IL-1beta was similar in all genotypes. The amount of cyclic adenosine monophosphate (cAMP) accumulation also was comparable. However, although cultures of primary TNAP-/- osteoblasts were able to form cellular nodules as well as TNAP positive osteoblasts do, they lacked the ability to mineralize these nodules in vitro. Mineralization also was delayed in TNAP+/- osteoblast cultures compared with cultures of wt osteoblasts. Incubation with media supplemented with recombinant TNAP, but not with enzymatically inactive TNAP, restored mineralization in ko osteoblast cultures. Our data provide evidence that osteoblasts in TNAP null mice differentiate normally but are unable to initiate mineralization in vitro. The fact that even heterozygous osteoblasts show delayed mineralization provides a rationale for the presence of bone disease in carriers of hypophosphatasia.

Alkaline Phosphatase↗

PubMed2000

A role for N-cadherin in the development of the differentiated osteoblastic phenotype.

Cadherins are a family of cell surface adhesion molecules that play an important role in tissue differentiation. A limited repertoire of cadherins has been identified in osteoblasts, and the role of these molecules in osteoblast function remains to be elucidated. We recently cloned an osteoblast-derived N-cadherin gene from a rat osteoblast complementary DNA library. After in situ hybridization of rat bone and immunohistochemistry of human osteophytes, N-cadherin expression was localized prominently in well-differentiated (lining) osteoblasts. Northern blot hybridization in primary cultures of fetal rat calvaria and in human SaOS-2 and rat ROS osteoblast-like cells showed a relationship between N-cadherin messenger RNA expression and cell-to-cell adhesion, morphological differentiation, and alkaline phosphatase and osteocalcin gene expression. Treatment with a synthetic peptide containing the His-Ala-Val (HAV) adhesion motif of N-cadherin significantly decreased bone nodule formation in primary cultures of fetal rat calvaria and inhibited cell-to-cell contact in rat osteoblastic TRAB-11 cells. HAV peptide also regulated the expression of specific genes such as alkaline phosphatase and the immediate early gene zif268 in SaOS-2 cells. Transient transfection of SaOS-2 cells with a dominant-negative N-cadherin mutant (NCADdeltaC) significantly inhibited their morphological differentiation. In addition, aggregation of NCTC cells derived from mouse connective tissue stably transfected with osteoblast-derived N-cadherin was inhibited by either treatment with HAV or transfection with NCADdeltaC. Together, these results strongly support a role for N-cadherin, in concert with other previously identified osteoblast cadherins, in the late stages of osteoblast differentiation.

Animals↗

PubMed2001

Tensile stress induces bone morphogenetic protein 4 in preosteoblastic and fibroblastic cells, which later differentiate into osteoblasts leading to osteogenesis in the mouse calvariae in organ culture.

Mechanical stress is an important factor controlling bone remodeling, which maintains proper bone morphology and functions. However, the mechanism by which mechanical stress is transduced into biological stimuli remains unclear. Therefore, the purpose of this study is to examine how gene expression changes with osteoblast differentiation and which cells differentiate into osteoblasts. Tensile stress was applied to the cranial suture of neonatal mouse calvaria in a culture by means of helical springs. The suture was extended gradually, displaying a marked increase in cell number including osteoblasts. A histochemical study showed that this osteoblast differentiation began in the neighborhood of the existing osteoblasts, which can be seen by 3 h. The site of osteoblast differentiation moved with time toward the center of the suture, which resulted in an extension of osteoid. Scattered areas of the extended osteoid were calcified by 48 h. Reverse-transcription polymerase chain reaction (RT-PCR) revealed that tensile stress increased bone morphogenetic protein 4 (BMP-4) gene expression by 6 h and it remained elevated thereafter. This was caused by the induction of the gene in preosteoblastic cells in the neighborhood of osteoblasts and adjacent spindle-shaped fibroblastic cells. These changes were evident as early as 3 h and continued moving toward the center of the suture. The expression of Cbfa1/Osf-2, an osteoblast-specific transcription factor, followed that of BMP-4 and those cells positive with these genes appeared to differentiate into osteoblasts. These results suggest that BMP-4 may play a pivotal role by acting as an autocrine and a paracrine factor for recruiting osteoblasts in tensile stress-induced osteogenesis.

Alkaline Phosphatase↗

PubMed2002

Comprehensive microarray analysis of bone morphogenetic protein 2-induced osteoblast differentiation resulting in the identification of novel markers for bone development.

Osteoblasts are cells responsible for matrix deposition during bone development and although temporal expression of many genes has been related to osteoblast differentiation, a complete description of osteoblast-specific gene regulation will lead to a better understanding of osteoblast function. In this study, microarray technology was used to analyze gene expression on a broad scale during osteoblast differentiation. Expression analysis of 9596 sequences revealed 342 genes and expressed sequence tags (ESTs) to be modulated differentially during a time course experiment in which murine C2C12 mesenchymal progenitor cells were induced to differentiate into mature osteoblasts by treatment with bone morphogenetic protein 2 (BMP-2). By means of hierarchical clustering, these genes were grouped by similarities in their expression profiles, resulting in subsets of early, intermediate, and late response genes, which are representative of the distinct stages of osteoblast differentiation. To identify new bone markers, the bone specificity of the late response genes was determined by comparing BMP-induced expression in C2C12 and MC3T3 osteoblasts with that in NIH3T3 fibroblasts. This resulted in the identification of nine novel genes and ESTs that were induced specifically in osteoblasts, in addition to the well-known markers ALP and osteocalcin. For at least one of these novel genes, Wnt inhibitory factor 1, and two of the ESTs, expression in developing bone was verified in vivo by in situ hybridization of E16.5 mouse embryos. In conclusion, by a combination of in vitro and in vivo screening approaches, a set of new genes related to osteoblast differentiation and skeletal development has been identified.

3T3 Cells↗

PubMed2003

Human adult chondrocytes express hepatocyte growth factor (HGF) isoforms but not HgF: potential implication of osteoblasts on the presence of HGF in cartilage.

HGF is increased in human OA cartilage, possibly from Ob's. RT-PCR shows HGF isoforms are differently regulated between chondrocytes and Ob. A paracrine cross-talk between subchondral bone and cartilage may occur during OA. Recently, hepatocyte growth factor (HGF) has been identified by immunohistochemistry in cartilage and more particularly in the deep zone of human osteoarthritic (OA) cartilage. By investigating HGF expression in cartilage, we found that chondrocytes did not express HGF; however, they expressed the two truncated isoforms, namely HGF/NK1 and HGF/NK2. Because the only other cells localized near the deep zone are osteoblasts from the subchondral bone plate, we hypothesized that they were expressing HGF. Indeed, we found that HGF was synthesized by osteoblasts from the subchondral bone plate. Moreover, OA osteoblasts produced five times more HGF than normal osteoblasts and almost no HGF/NK1, unlike normal osteoblasts. Because prostaglandin E2 (PGE2) and pro-inflammatory cytokines such as interleukin (IL)-1 and IL-6 are involved in OA progression, we investigated whether these factors impact HGF produced by normal osteoblasts. PGE2 was the only factor tested that was able to stimulate HGF synthesis. However, the addition of NS398, a selective inhibitor of cyclo-oxygenase-2 (COX-2) had no effect on HGF produced by OA osteoblasts. HGF/NK2 had a moderate stimulating effect on HGF production by normal osteoblasts, whereas osteocalcin was not modulated by either HGF or HGF/NK2. When investigating signaling routes that might be implicated in OA osteoblast-produced HGF, we found that protein kinase A was at least partially involved. In summary, this study raises the hypothesis that the HGF found in articular cartilage is produced by osteoblasts, diffuses into the cartilage, and may be implicated in the OA process.

Adult↗

PubMed2002

Regulation of human cranial osteoblast phenotype by FGF-2, FGFR-2 and BMP-2 signaling.

The formation of cranial bone requires the differentiation of osteoblasts from undifferentiated mesenchymal cells. The balance between osteoblast recruitment, proliferation, differentiation and apoptosis in sutures between cranial bones is essential for calvarial bone formation. The mechanisms that control human osteoblasts during normal calvarial bone formation and premature suture ossification (craniosynostosis) begin to be understood. Our studies of the human calvaria osteoblast phenotype and calvarial bone formation showed that premature fusion of the sutures in non-syndromic and syndromic (Apert syndrome) craniosynostoses results from precocious osteoblast differentiation. We showed that Fibroblast Growth Factor-2 (FGF-2), FGF receptor-2 (FGFR-2) and Bone Morphogenetic Protein-2 (BMP-2), three essential factors involved in skeletal development, regulate the proliferation, differentiation and apoptosis in human calvaria osteoblasts. Mechanisms that induce the differentiated osteoblast phenotype have also been identified in human calvaria osteoblasts. We demonstrated the implication of molecules (N-cadherin, Il-1) and signaling pathways (src, PKC) by which these local factors modulate human calvaria osteoblast differentiation and apoptosis. The identification of these essential signaling molecules provides new insights into the pathways controlling the differentiated osteoblast phenotype, and leads to a more comprehensive view in the mechanisms that control normal and premature cranial ossification in humans.

Apoptosis↗

PubMed2000

Autocrine signals promote osteoblast survival in culture.

We have studied the survival requirements of osteoblasts to test the hypothesis that osteoblasts undergo programmed cell death (PCD) or apoptosis unless they are continuously signalled by other cells not to do so. Osteoblasts survived for 6 days in culture at high cell density in the absence of other cell types, serum or exogenous proteins, but they died with the morphological features of apoptosis in these conditions at low cell density. Osteoblast survival was enhanced during the first 2 days of culture by the addition of the sulphydryl compound, cysteine to the culture medium which was converted intracellularly to the antioxidant glutathione. Catalase, an enzyme decomposing hydrogen peroxide, also protected the cells, whereas superoxide dismutase had no effect. Therefore, osteoblasts in culture are sensitive to toxic compounds derived from molecular oxygen, i.e. hydroxyl radicals or hydrogen peroxide spontaneously generated in CMRL medium containing ascorbate and ferrous ions. Conditioned medium from high density cultures prevented osteoblast apoptosis in low density cultures, as long as antioxidants were also present. The enhancing effect of conditioned medium on osteoblast survival was prevented by neutralizing antibodies to insulin-like growth factor-I (IGF-I) and IGF-II but not by antibodies to either platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF). These results suggest that in addition to regulating cell growth and differentiation, IGF-I and IGF-II also function as survival factors for osteoblasts. Our data also indicate that antioxidants are required for osteoblast survival and that they enhance growth factor mediated osteoblast survival.

Animals↗

PubMed2001

Changing RANKL/OPG mRNA expression in differentiating murine primary osteoblasts.

Osteoblast-osteoclast coordination is critical in the maintenance of skeletal integrity. The modulation of osteoclastogenesis by immature cells of the osteoblastic lineage is mediated through receptor activator of NF kappa B (RANK), its ligand RANKL, and osteoprotegerin (OPG), a natural decoy receptor for RANKL. Here, the expression of OPG and RANKL in primary mouse osteoblastic cultures was investigated to determine whether the osteoclastogenic stimulus depended on the stage of osteoblastic differentiation and the presence of the calciotrophic hormone 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)). OPG mRNA expression was increased in osteoblastic cultures after the onset of mineralisation relative to less mature cultures, but did not alter in response to 1,25-(OH)(2)D(3) treatment. In contrast, basal RANK L mRNA expression did not change during differentiation but was significantly enhanced by 1,25-(OH)(2)D(3) treatment at all times. The stimulatory effects of 1,25-(OH)(2)D(3) on RANKL were lessened in more mature cultures, however. The RANKL/OPG ratio, an index of osteoclastogenic stimulus, was therefore increased by 1,25-(OH)(2)D(3) treatment at all stages of osteoblastic differentiation, but to a lesser degree in cultures after the onset of mineralisation. Thus the 1,25-(OH)(2)D(3)-driven increase in osteoclastogenic potential of immature osteoblasts appears to be mediated by increased RANKL mRNA expression, with mature osteoblasts having relatively decreased osteoclastogenic activity due to increased OPG mRNA expression. These findings suggest a possible mechanism for the recently proposed negative regulatory role of mature osteoblasts on osteoclastogenesis and indicate that the relative proportions of immature and mature osteoblasts in the local microenvironment may control the degree of resorption at each specific bone site.

Analysis of Variance↗

PubMed2001

Temporal pattern of stimulation of osteoblast-associated genes during mechanically-induced osteogenesis in vivo: early responses of osteocalcin and type I collagen.

Mechanical loading is an essential environmental factor in skeletal homeostasis, but the response of osteoblast-associated genes to mechanical osteogenic signal is largely unknown. This study uses our recently characterized in vivo osteoinductive model to analyze the sequence of stimulation and the time course of expression of osteoblast-associated genes in mechanically loaded mouse periodontium. Temporal pattern of regulation of osteocalcin (OC), alkaline phosphatase (ALP), and type I collagen (collagen I) was determined during mechanically-induced osteoblast differentiation in vivo, using a mouse tooth movement model earlier shown to induce bone formation and cell-specific regulation of genes in osteoblasts. The expression of target genes was determined after 1, 2, 3, 4, and 6 days of orthodontic movement of the mouse first molar. mRNA levels were measured in the layer of osteoblasts adjacent to the alveolar bone surface, using in situ hybridization and a relative quantitative video image analysis of cell-specific hybridization intensity, with non-osseous mesenchymal periodontal cells as an internal standard. After 24 hours of loading, the level of OC in osteoblasts slightly decreased, followed by a remarkable 4.6-fold cell-specific stimulation between 1 and 2 days of treatment. The high level expression of OC was maintained throughout the treatment with a peak 7-fold stimulation at day 4. The expression of collagen I gene was not significantly affected after 1 day, but it was stimulated 3-fold at day 2, and maintained at a similar level through day 6. The ALP gene, which we previously found to be mechanically stimulated during the first 24 hours, remained enhanced from 1.8- to 2.2-fold throughout the 6 days of treatment. Thus, in an intact alveolar bone compartment, mechanical loading resulted in a defined temporal sequence of induction of osteoblast-associated genes. Stimulation of OC 48 h after the onset of loading (and 24 h prior to deposition of osteoid) temporally coincided with that of collagen I, and was preceded for 24 h by an enhancement of ALP. Identification of OC as a mechanically responsive gene induced in functionally active osteoblasts in this study is consistent with its potential role in limiting the rate of mechanically-induced bone modeling. Furthermore, these results show that temporal progression of mechanically-induced osteoblast phenotype in this in vivo model occurs very rapidly. This suggests that physiologically relevant mechanical osteoinductive signal in vivo is targeting a population of committed osteoblast precursor cells that are capable of rapidly responding by entering a differentiation pathway and initiating an anabolic skeletal adaptation process.

Animals↗

PubMed1993

Interaction of osteoblasts with extracellular matrix: effect of mast cell chymase.

We studied the effect of mast cell chymase on the interaction between osteoblasts and extracellular matrix. Chymase was purified from mast cell lysate by anion exchange chromatography. Osteoblasts were isolated from rat calvarias by collagenase digestion. Incubation of osteoblasts with mast cell lysate (40-170 micrograms/ml) or purified chymase (8-32 micrograms/ml) resulted in changes in cell-matrix interaction and cell morphology. Osteoblasts treated with chymase also showed a gradual detachment from the artificial substrata and from the biomatrix (collagen-digested rib fragment). A similar effect of mast cell chymase on the osteoblasts was found in vitro on endosteum of an intact parietal bone. Neutral protease inhibitors abolished the effect of both crude and purified enzyme preparations on the cell-matrix interaction. Mast cell chymase had no effect on osteoblast viability. The effect of enzyme on osteoblast proliferation was studied with lower concentrations of enzyme (0.2 micrograms/ml) in order to avoid cell detachment; there was no effect on either the metaphase index or on the number of cells after 5 days of incubation with chymase. Osteoblast attachment and cell spreading on different matrix proteins (fibronectin, vitronectin, extract of noncollagenous matrix proteins from rat bone) were significantly altered by their pretreatment with chymase. Matrix fibronectin of osteoblasts in culture as well as soluble vitronectin and fibronectin were digested by rat mast cell chymase. Our data suggest that mast cells through action of neutral protease chymase may alter molecules in extracellular matrix that are important in osteoblast adhesion, cell spreading, maintenance of cell morphology, and, most likely, cell function.

Animals↗

PubMed2000

[Mitogenesis of platelet-derived growth factors to human osteoblasts modulated by basic fibroblast growth factor].

OBJECTIVES: To investigate the mechanism by which basic fibroblast growth factor modulates the mitogenesis of platelet-derived growth factor to human osteoblasts. METHODS: The osteoblasts isolated from human fetal calvaria were incubated with PDGF-AB (100 ng/ml) or bFGF(10 ng/ml) combined with PDGF-AB (100 ng/ml); the growth curve was plotted. The(3)H-TdR incorporation of the osteoblasts was measured after the cells were incubated with different combination of bFGF and PDGF-AA or PDGF-BB. After incubated with bFGF (10 ng/ml) for 24 hours, the number of PDGFR-alpha and PDGFR-beta on the membrane of the osteoblasts was detected by fluoroimmunoassay. RESULTS: Four days after PDGF-AB added into the medium, the population of the osteoblasts was larger than that of the control (P < 0.05). The number of the osteoblasts incubated with PDGF-AB (12.1 x 10(4)) was 1.8 times as large as the control (6.8 x 10(4)) in the 10th day (P < 0.05), and that of the osteoblasts incubated with both bFGF and PDGF-AB increased more quickly than the cells only incubated with PDGF-AB. The incorporation of (3)H-TdR into the osteoblasts cultured with bFGF combined with PDGF-AA (533.6 +/- 13.1) was more than that cultured only with PDGF-AA (435.4 +/- 14.8, P < 0.01), so was the incorporation of (3)H-TdR of those cells cultured with bFGF and then PDGF-AA (633.8 +/- 51.5). bFGF up-regulated PDGFR-alpha and down-regulated PDGFR-beta on the surface of the human osteoblasts. CONCLUSION: bFGF elevates the mitogenesis of PDGF-AA or -AB to human osteoblasts by up-regulating PDGFR-alpha.

Cell Proliferation↗

PubMed2004

[Study of osteoblasts transfected with gfp in vitro and traced in vivo].

OBJECTIVE: To observe the osteoblasts transfected with green fluorescent protein (GFP)by adenovirus vector expressed in vitro and traced it in vivo in order to research the feasibility of GFP as a tracer of seeding cells for tissue engineering. METHODS: CFP were transfected into the osteoblasts which derived from adult human bone marrow stromal cell (hBMSc) by adenovirus vector after being packed in 293A cells. The nontransfected hBMSc was used as the control group. The osteoblasts in each group were observed under an inverted phase contrast microscope and fluorescence microscope. The expressive efficiency of GFP was examined by flow cytometry,and alkaline phosphatase (ALP) activities and osteocalcin (OCN) synthesis. After eight days of the transfection,the osteoblasts were implanted into the muscle of nude mouse thigh while the non-transfected osteoblasts were also implanted as a control. Four and eight weeks after the operation, the nude rats were killed and the continuous tissue sections were examined using fluorescence microscopy after adjacent sections were performed by immunohistochemistry or routine HE staining. RESULTS: The green fluorescence was shown the transfected osteoblasts which derived from bone marrow. The rate of positive expression was over 75%. After eight days of the transfection, the marker proteins of the surface of the osteoblasts showed extremely efficient expression of CD29 and CD44, but the CD34 expressed negative. Either ALP or OCN of the osteoblasts was no significant difference between the two groups (P > 0.05) four and eight days after the transfection. The GFP were obviously expressed in nude mouse both at four and eight weeks, meanwhile it did not harm on the morphology and function of the transfected osteoblasts whose immunohistochemistry examination showed positive reaction. CONCLUSIONS: GFP could be transfected osteoblasts effectively in vitro and traced in vivo in nude mouse. It may be an optimal tracer for living cells on tissue engineering research.

Adult↗

PubMed2006

Nicotine induced proliferation and cytokine release in osteoblastic cells.

Smoking has deleterious effects on osteoporosis and periodontitis both characterized by bone loss. Smoking also interferes with the protective effect that hormone replacement therapy (HRT) has on bone loss. Our study investigated two mechanisms by which smoking may affect bone metabolism: nicotine-induced proliferation and nicotine-induced cytokine secretion in osteoblasts. Two osteoblastic cell models were used: mouse osteoblasts derived from mouse calvaria and human osteoblasts. Thymidine incorporation and immunoassays were used to evaluate proliferation, interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) secretion. Parametric and nonparametric statistical analyses were used for comparisons. The results showed that nicotine induced stimulation and inhibition of proliferation in both osteoblastic cell models. In human osteoblasts, the proliferative and inhibitory effects were also donor dependent. Il-6 secretion showed different patterns in mouse and human osteoblasts. In mouse osteoblasts, nicotine significantly increased IL-6 secretion and estradiol significantly inhibited the nicotine-induced IL-6 release. In human osteoblasts, cells derived from one subject did not respond to nicotine. However, in the second sample, nicotine increased secretion of Il-6 but estradiol did not oppose this effect. In human osteoblasts, nicotine also induced an increase in the TNF-alpha secretion and estradiol opposed this increase. These results suggest that nicotine affects bone metabolism by modulating proliferation, and Il-6 and TNF-alpha secretion. These studies provide a possible explanation for differences in bone loss among subjects who smoke and offer a possible mechanism for the oppositional effect of smoking on HRT in subjects with bone loss.

Animals↗

PubMed2006

Avocado/soybean unsaponifiables prevent the inhibitory effect of osteoarthritic subchondral osteoblasts on aggrecan and type II collagen synthesis by chondrocytes.

OBJECTIVE: To determine the effects of avocado/soybean unsaponifiables (ASU) on osteoblast-induced dysregulation of chondrocyte metabolism. METHODS: Human chondrocytes were isolated from osteoarthritis (OA) cartilage and cultured in alginate beads for 4 or 10 days in the absence or presence of osteoblasts isolated from nonsclerotic (NSC) or sclerotic (SC) zones of OA subchondral bone plate in monolayer. Before co-culture, osteoblasts were incubated or not with 10 microg/ml ASU for 72 hours. Aggrecan, type II collagen, matrix metalloproteinase-3 (MMP-3) and MMP-13, tissue inhibitor of metalloproteinase (TIMP-1), transforming growth factor-beta1 (TGF-beta1) and TGF-beta3, inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) mRNA levels in chondrocytes were quantified by RT-PCR. Aggrecan, osteocalcin, TGF-beta1, interleukin 1beta (IL-1beta), and IL-6 production were assayed by immunoassays. RESULTS: In co-culture, SC osteoblasts induced a significant inhibition of matrix protein production and a significant increase of MMP synthesis by chondrocytes. In contrast, SC osteoblasts did not modify TIMP-1, TGF-beta1 and TGF-beta3, iNOS, or COX-2 mRNA levels in chondrocytes. The pretreatment of SC osteoblasts with ASU fully prevented the inhibitory effects of SC osteoblasts on matrix component production, and even significantly increased type II collagen mRNA level over the control (chondrocytes alone) value. In contrast, pretreatment of SC osteoblasts with ASU did not significantly modify the expression of MMP, TIMP-1, TGF-beta1, TGF-beta3, iNOS, or COX-2 gene by chondrocytes. CONCLUSION: ASU prevent the osteoarthritic osteoblast-induced inhibition of matrix molecule production, suggesting that this compound may promote OA cartilage repair by acting on subchondral bone osteoblasts. This finding constitutes a new mechanism of action for this compound, known for its beneficial effects on cartilage.

Aged↗

PubMed1996

Monoclonal antibodies as tools for studying the osteoblast lineage.

Knowledge of the number and kinds of differentiation steps characterizing cells of the osteoblast lineage is inadequate. To analyze further osteoblast differentiation, a number of labs have generated monoclonal antibodies to osteogenic cells, derived from both normal bone and osteosarcomas. A variety of immunolabelling patterns on primary cell cultures, cell lines, and tissue sections has been reported, including cell surface, cytoplasmic, and extracellular matrix-associated patterns. Most of the antibodies selected recognize predominantly the mature osteoblast and osteocyte; in addition, however, antibodies have been generated that recognize pre-osteoblasts. Some recognize cells of both the osteoblast and chondroblast lineages and may contribute to a better understanding of the lineage and phenotypic relationships between these two cell types. In addition to recognition in vivo of cell subpopulations of discrete maturational stages, changes in the immunolabelling patterns in vitro have also documented a differentiation sequence in cells undergoing osteogenesis in cell and tissue cultures. In at least two cases, the antibodies have been used to isolate subpopulations of cells from bone, including relatively pure populations of osteocytes. With the exception of several antibodies that are against alkaline phosphatase or known matrix proteins including osteocalcin, the nature of the macromolecular species recognized by most of the antibodies generated to date are unknown. Recently, however, one antibody was used to clone the cDNA for the beta-galactoside-binding lectin, galectin 3 or epsilon binding protein (epsilon BP; IgE-binding protein; Mac-2), from a lambda gt11 osteoblast expression library; another was used to clone from an ROS 17/2.8-COS cell expression library the cDNA for OTS-8, a putative target gene of early response genes stimulated in response to phorbol esters in MC3T3-E1 cells. Neither of these macromolecules had previously been identified in bone cells, but the recent molecular and cellular analyses have shown them to be developmentally and/or hormonally regulated in osteoblastic cells. These antibodies extend the available markers and support earlier observations that a variety of molecules are differentially expressed by cells at different stages of the osteoblast lineage. This chapter will not be an exhaustive survey of all immunocytochemical and immunohistochemical analyses of osteogenic cells and tissues but will focus on the approach of eliciting novel monoclonal antibodies by the injection of osteogenic cells or crude bone extracts and its potential for establishing new markers of the osteoblast lineage. We have not included a large number of studies documenting the use of antibodies raised against several known bone matrix proteins; while these have been crucial in developing our current understanding of osteogenic differentiation, we sought rather to highlight the potential of the "random" injection approach.

Animals↗

PubMed1998

Design and function of novel osteoblast-adhesive peptides for chemical modification of biomaterials.

Proactive, "next generation" dental/orthopedic biomaterials must be designed rationally to elicit specific, timely, and desirable responses from surrounding cells/tissues; for example, such biomaterials should support and enhance osteoblast adhesion (a crucial function for anchorage-dependent cells). In the past, integrin-binding peptides have been immobilized on substrates to partially control osteoblast adhesion; the present study focused on the design, synthesis, and bioactivity of the novel peptide sequence Lys-Arg-Ser-Arg that selectively enhances heparan sulfate-mediated osteoblast adhesion mechanisms. Osteoblast, but not endothelial cell or fibroblast, adhesion was enhanced significantly (p < 0.05) on substrates modified with Lys-Arg-Ser-Arg peptides, indicating that these peptides may be osteoblast- or bone cell specific. Blocking osteoblast cell-membrane receptors with various concentrations of soluble Arg-Gly-Asp-Ser peptides did not inhibit subsequent cell adhesion on substrates modified with Lys-Arg-Ser-Arg peptides, providing evidence that osteoblasts interact with Arg-Gly-Asp-Ser and with Lys-Arg-Ser-Arg peptides via distinct (i.e., integrin- and proteoglycan-mediated) mechanisms, each uniquely necessary for osteoblast adhesion. The present study constitutes an example of rational design/selection of bioactive peptides, confirms that osteoblast adhesion to substrates can be controlled selectively and significantly by immobilized peptides, and elucidates criteria and strategies for the design of proactive dental/orthopedic implant biomaterials.

Adhesiveness↗

PubMed2000

Chitosan supports the expression of extracellular matrix proteins in human osteoblasts and chondrocytes.

The search for biocompatible materials that can support the growth and phenotypic expression of osteoblasts and chondrocytes is a major challenge in the application of tissue engineering techniques for the repair of bone and cartilage defects. Chitosan, a copolymer of glucosamine and N-acetylglucosamine, may provide an answer to this search. Chitosan is the deacetylated product of chitin, a ubiquitous biopolymer found in the exoskeleton of insects and marine invertebrates. Little is known about the utility of chitosan in propagating human osteoblasts and chondrocytes. In this study, we test the hypothesis that chitosan promotes the survival and function of osteoblasts and chondrocytes. Chitosan (4%, w/v in 2% HAc) was coated onto plastic coverslips that had been fitted into 24-well plates. Human osteoblasts and articular chondrocytes were seeded on either uncoated or chitosan-coated coverslips at 1 x 10(5)/cells per well. Cultures were incubated at 37 degrees C, 5% CO(2) for a period of 7 days. Cell viability was assessed at that time using a fluorescent molecular probe. The phenotypic expression of osteoblasts and chondrocytes was analyzed by reverse transcriptase-polymerase chain reaction and immunocytochemistry. Osteoblasts and chondrocytes appeared spherical and refractile on chitosan-coated coverslips. In contrast, greater than 90% of cells on plastic coverslips were elongated and spindle shaped after 7 days of culture. Similar to cells propagated on uncoated control wells, greater than 90% of human osteoblasts and chondrocytes propagated on chitosan remained viable. Human osteoblasts propagated on chitosan films continued to express collagen type I whereas chondrocytes expressed collagen type II and aggrecan, as shown by reverse transcriptase-polymerase chain reaction analysis and immunostaining. The present in vitro work demonstrates the biocompatibility of chitosan as a substrate for the growth and continued function of human osteoblasts and chondrocytes. Chitosan may have potential use as a tissue engineering tool for the repair of osseous and chondral defects.

Aggrecans↗

PubMed1994

Osteoblast function on synthetic biodegradable polymers.

Rat osteoblasts were cultured on films of biodegradable poly(L-lactic acid) (PLLA), 75:25 poly(DL-lactic-co-glycolic acid) (PLGA), 50:50 PLGA, and poly(glycolic acid) (PGA) for up to 14 days. Osteoblasts attached equally well to all the polymer substrates after 8 h in culture. By day 4 in culture, osteoblasts had exceeded confluency numbers, and their proliferation leveled off by day 7. An increase in alkaline phosphatase (ALP) activity from 1.92 (+/- 0.47) x 10(-7) for day 7 to 5.75 (+/- 0.12) x 10(-7) mumol/cell per min for day 14 was reported for osteoblasts cultured on 75:25 PLGA, which was comparable to that observed for tissue culture polystyrene (TCPS) controls. The ALP activities expressed by osteoblasts cultured on PLLA, 50:50 PLGA, and PGA films did not significantly increase over time. Collagen synthesis for osteoblasts cultured on all polymer substrates was similar to that of TCPS and did not vary with time. The morphology of cultured osteoblasts was not affected by the continuous degradation of the polymer substrates. These results demonstrate that poly(alpha-hydroxy esters) can provide a suitable substrate for osteoblast culture and hold promise in bone regeneration by osteoblast transplantation.

Alkaline Phosphatase↗