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PubMed1989

Connective tissue-associated proteins in crevicular fluid: potential markers for periodontal diseases.

Tests for periodontal disease that are able to detect both ongoing and future loss of clinical attachment would be valuable assets in determining the diagnosis and treatment of periodontal diseases. We hypothesized that connective tissue-associated proteins could be detected in crevicular fluid and would reflect the biochemical activity of the periodontium in health and disease. To test this hypothesis, crevicular fluid samples obtained from patients with various states of periodontal disease were analyzed for the presence of several connective tissue-associated proteins using a dot blot assay. Two such proteins, osteonectin and N-propeptide alpha I type I collagen, were detected in crevicular fluid samples of patients with periodontal disease. Furthermore, the amount of these proteins detected in crevicular fluid appeared to increase with increased probing depth at the sampled site. These studies indicate that measurements of connective tissue-associated proteins in crevicular fluid may prove to be a valuable tool for diagnosing periodontal diseases.

Biomarkers↗
PubMed1997

Expression of extracellular matrix proteins in human periodontal ligament cells during mineralization in vitro.

Periodontal regeneration is a complex process that requires coordinated responses from several cell types within the periodontium. It is generally accepted that the periodontal ligament (PDL) has a heterogeneous cell population, where some of the cells may be capable of differentiating into either cementoblasts or osteoblasts. Thus, it has been hypothesized that PDL cells play a role in promoting periodontal regeneration. However, definitive evidence to support this concept is lacking. Previously, we reported that PDL cells induce biomineralization as determined by Von Kossa histochemistry and transmission electron microscopy. To further determine the osteoblast-like properties of PDL cells, human PDL cells were exposed to dexamethasone (DEX) in order to promote an osteoblast phenotype, and then cell activity monitored during mineral nodule formation in vitro. For mineralization studies, cells were cultured in DMEM containing 10% FBS and a) vehicle only; b) ascorbic acid (50 micrograms/ml) and beta-glycerophosphate (10 mM); or c) ascorbic acid, beta-glycerophosphate and DEX (100 nM) for 30 days. In addition, the effects of DEX on PDL cells in non-mineralizing media were determined. Cells were stained weekly to evaluate mineral-like nodules, using the Von Kossa method. Northern blot analyses for mRNA steady state levels for several bone-associated proteins, i.e., osteopontin (OPN), bone sialoprotein (BSP), alkaline phosphatase (ALP), osteocalcin (OCN), alpha 2(1)(type 1) collagen and osteonectin (ON), were performed. DNA levels were also determined during the 30-day mineralization period. Under phase contrast microscopy, PDL cells in non-mineralizing media treated with DEX exhibited a more spindle-shaped morphology when compared with similar cells not exposed to DEX. Mineralizing conditions were required to induce mineral nodule formation. However, in this situation, mineral induction was independent of DEX; and furthermore, DEX-treated cells did not exhibit a different morphological pattern when compared with non-DEX treated cells. Mineral-like nodules were first seen at day 15, in concert with an increase followed by a decrease in expression of type I collagen and ON mRNA in both DEX-treated and non-treated cultures. Using Northern blot analysis for detection of specific proteins, we found that PDL cells did not express OPN, BSP, OCN, or ALP under any of the conditions used in this study. DEX did not alter DNA content in the cultures during the mineralization period. These results confirm that human periodontal ligament cells can be induced to mineralize in vitro and indicate that dexamethasone does not significantly alter the extent and pattern of mineralization.

Alkaline Phosphatase↗
PubMed2003

Immunohistochemistry of matrix markers in Technovit 9100 New-embedded undecalcified bone sections.

Trabecular bone is routinely analysed by histomorphological-histometrical and immunohistochemical techniques as means of assessing the differentiation status of bone deposition and growth. Currently few embedding resins exist for which both morphological and immunohistochemical analyses can be performed on mineralised tissue. Paraffin, the standard embedding medium for bone enzyme and immunohistochemistry, can only be used on demineralised tissue, but then trabecular structure may be badly preserved. Methyl methacrylate (MMA), the resin of choice for undecalcified bone histology can only be used for bone immunohistochemistry if the usual, highly exothermic polymerisation procedure is avoided which destroys tissue antigenicity. Consequently, most current practices involve cutting samples in half to be processed in separate resins when more than one type of analysis is required. Technovit 9100 New is a low temperature MMA embedding system that is purported to significantly improve tissue antigenicity preservation allowing polymerisation at -20 degrees C. In this study, Technovit 9100 New-embedded undecalcified trabecular bone samples (adult human, young bovine and ovine) yielded immunolabelling with several bone matrix markers and preserved morphological features in 7 microm sections when stained with Masson-Goldner, von Kossa, or toluidine blue. Bone samples from all resins used were immunolabelled with antibodies against osteocalcin, alkaline phosphatase, osteopontin, osteonectin, bone sialoprotein and procollagen type I amino-terminal propeptide. Technovit-embedded bone yielded more reliable immunolabelling of the matrix proteins when compared with heat or cold-cured LR White or standard embedded MMA samples. Technovit 9100 New provided better routine histology than LR White, and was comparable to MMA. Results demonstrated that Technovit 9100 New can be used as a low-temperature acrylic resin embedding method for routine undecalcified bone histology, as well as for immunohistochemistry.

Acrylic Resins↗
PubMed2006

Culture medium modulates the behaviour of human dental pulp-derived cells: technical note.

In vitro approaches have extensively been developed to study reparative dentinogenesis. While dental pulp is a source of unidentified progenitors able to differentiate into odontoblast-like cells, we investigated the effect of two media; MEM (1.8 mM Ca and 1 mM Pi) and RPMI 1640 (0.8 mM Ca and 5 mM Pi) on the behaviour of human dental pulp cells. Our data indicate that MEM significantly increased cell proliferation and markedly enhanced the proportion of alpha-smooth muscle actin positive cells, which represent a putative source of progenitors able to give rise to odontoblast-like cells. In addition, MEM strongly stimulated alkaline phosphatase activity and was found to induce expression of transcripts encoding dentin sialophosphoprotein, an odontoblastic marker, without affecting that of parathyroid hormone/parathyroid hormone related protein-receptor and osteonectin. In conclusion, these observations demonstrate that not only proliferation but also differentiation into odontoblast-like cells was induced by rich calcium and poor phosphate medium (MEM) as compared to RPMI 1640. This study provides important data for the determination of the optimal culture conditions allowing odontoblast-like differentiation in human pulp cell culture.

Alkaline Phosphatase↗
PubMed1992

Characterization of mineral-binding 40-kDa glycoprotein extracted from young adult rabbit alveolar bone.

A forty-kilodalton (40-kDa) protein was extracted from alveolar bone of young adult rabbit with 0.5 M EDTA after extraction with 4 M GuHCl, and purified by gel-filtration, anion-exchange and hydroxyapatite columns using a high-pressure liquid chromatography system under denaturing conditions. The purified 40-kDa protein was not susceptible to bacterial collagenase and thrombin, but was cleaved by cyanogen bromide. The protein was stained blue with Stains-all. Among various lectins, concanavalin A and lentil lectin agglutinin bound to this protein, but peanut agglutinin, Ricinus communis agglutinin, phytohemagglutinin-E and wheatgerm lectin agglutinin did not. Lectin binding assays showed that the protein is a glycoprotein containing large amounts of mannose and/or glucose residues, but is not a fragment of proteoglycan. The amino acid composition of the protein shows a characteristically high content of acidic amino acids. Therefore, the mineral-binding 40-kDa glycoprotein is considered to be osteonectin/secreted protein acidic and rich in cysteine (SPARC), in terms of similarities to bovine and porcine osteonectins with regard to molecular weight and contents of glycoses and amino acids.

Alveolar Process↗
PubMed1989

Expression of genes for non-collagenous proteins during embryonic bone formation.

In situ hybridization has been used to localize RNAs for a variety of non-collagenous proteins during embryogenesis of the mouse, with particular reference to bone formation. Transcripts of the Sparc (osteonectin) gene are first detected around 14.5d post coitum (p.c.) in the periosteal layer around the cartilage model of long bones and in membrane bone in the head. By 15.5d p.c. transcripts are seen in hypertrophic cartilage and later in cells within the bone closely associated with new matrix. By contrast, RNA for 2ar (osteopontin) is not seen in the periosteal layer but is confined to cells in the marrow region. TIMP (tissue inhibitor of metalloproteinase) transcripts show a very similar distribution to those for Sparc.

Animals↗
PubMed1989

Type beta transforming growth factor regulates expression of genes encoding bone matrix proteins.

TGF beta modulates the growth and differentiation of various cell types, in part by regulating the production of extracellular matrix proteins. In rat osteoblast-like cells TGF beta stimulates the production of collagen, osteopontin and osteonectin. On the other hand, TGF beta inhibits the production of osteocalcin, one of the most abundant non-collagenous bone matrix proteins, which is only expressed in osteoblasts. Inhibition of osteocalcin expression by TGF beta in the rat osteoblastic osteosarcoma, ROS 17/2.8 cells, occurs at least in part through transcriptional control.

Animals↗
PubMed1993

Noncollagenous proteins in heterotopic ossification. Immunohistochemical analysis in 15 paraplegies.

We used immunohistochemical techniques to investigate the distribution pattern of osteonectin, osteocalcin, bone sialoprotein II and the small proteoglycans decorin and PG 100 during different stages of heterotopic ossification (HO) in pressure sores of paraplegic patients. All these noncollagenous proteins (NCPs) accumulated in fibroblasts and preosteoblasts, predominantly in the activity centers of early osteogenetic areas. Mature types of HO showed a more discrete expression pattern for this protein group, with weaker reactions in the narrow osteoblastic rims. Decorin was detected predominantly in the stroma of HO. Our results indicate that the NCPs are important components during the pathogenesis of HO and that fibroblasts may serve as osteoprogenitor cells.

Adult↗
PubMed1998

Incorporation of cortical bone allografts and autografts in rats: expression patterns of mRNAs for the TGF-betas.

Healing of bone grafts is dependent on the rate of new bone formation. To understand better the regulation of new bone formation in the graft we have studied local production of TGF-beta1, 2 and 3, and of the small proteoglycans by determining their mRNA levels in a rat bone graft model. These mRNA levels were compared to the healing rates of autografts and allografts, as determined by histology, UV-microscopic evaluation of tetracycline-labeled new bone formation, microradiography and mechanical testing at 1, 2, 4 and 8 weeks of healing. Analyses showed that, analogous to slower bone formation in allografts, the induction of TGF-beta1 gene expression was slower than in allografts, when compared with autografts. A similar delay was seen in decorin gene expression. The results agree with the suggested role of TGF-beta1 in induction of type I collagen and osteonectin production. Our findings thus support the view that locally produced TGF-beta1 plays a role in normal graft incorporation, while local production of TGF-beta3, and particularly TGF-beta2, may be less important in this respect.

Animals↗
PubMed1998

Evidence that breast cancer associated microcalcifications are mineralized malignant cells.

Microcalcifications are often associated with both benign and malignant human breast lesions. Around 40% of mammary carcinoma present such ectopic mineralization and frequently, they are the only mammographic feature that indicate the presence of a tumoral lesion. Microcalcifications associated with breast cancer are usually composed of hydroxyapatite, the bone specific mineral. The mechanisms responsible for the formation of such crystals within breast malignant tissue have not been elucidated. A possible clue could be provided by the recent demonstration that breast cancer cells express several bone matrix proteins including osteonectin, osteopontin and bone sialoprotein (BSP). This latter phospho-protein is involved in the initiation of hydroxyapatite crystallisation and its expression in breast cancer has been associated to the presence of hydroxyapatite microcalcifications. We examined 10 human breast cancer lesions which were characterized by the presence of microcalcifications and high expression of BSP. Histological examination of the lesions suggested, in most of the cases, that the microcalcifications were breast cancer cells which became mineralized. Hydroxyapatite stained in blue by hematoxylin appears concentrated around single of associated cancer cells. Staining of these tissue sections with 4',6 diamidino-2-phenylindole which specifically labels DNA led us to demonstrate that the mineralizated structures contain cells. These data are the first direct demonstration that breast microcalcifications are fossils of cancer cells. The mechanisms for such a phenomenon remain to be demonstrated. We speculate that the high expression of BSP could create an appropriate microenvironment for the crystallisation of calcium and phosphate into hydroxyapatite.

Breast Neoplasms↗
PubMed1999

Development of the osteoblast phenotype in primary human osteoblasts in culture: comparison with rat calvarial cells in osteoblast differentiation.

In rat osteoblast-like cells, a time-dependent sequence of growth and differentiation-dependent genes has been identified and a model of osteoblast differentiation in culture suggested. We investigated the expression of the bone matrix-associated proteins osteonectin and procollagen I and of the bone cell phenotype-related proteins alkaline phosphatase and osteocalcin during cell culture in primary human osteoblast like cells. Primary human explant cultures from nine young healthy donors were established under highly standardized conditions. Cells in the second passage were analyzed on different days from day 1 to 32, comparing cells growing under the influence of ascorbate with controls. Gene expression was determined by Northern blot analysis or polymerase chain reaction. Osteocalcin expression was also investigated after 1,25-(OH)(2)D(3) stimulation. On the protein level, newly synthesized collagen I, alkaline phosphatase activity, and secretion of osteocalcin were analyzed at all time points. On comparing our findings to the pattern of gene expression suggested for the rat calvarial osteoblast system, we found a similar developmental sequence for the so-called "proliferation" as well as a similar, but lengthened, sequence for the "matrix maturation stage." During "matrix maturation," we found an ongoing proliferation despite increased alkaline phosphatase and decreased procollagen I gene expression. Our study, therefore, shows that in pHOB the gene expression profile proceeded to the "matrix maturation stage," as defined by Owen and colleagues, independent of ongoing proliferation. We were unable to observe the mineralization period as demonstrated by the missing increase of osteocalcin expression and lack of nodule formation in our human osteoblast model. In contrast to the rat system, we found a proliferation stimulating influence of ascorbate, suggesting species-specific differences in response to differentiation factors. From these data, we conclude that general considerations on physiology and pathophysiology of bone cell differentiation have to be confirmed in the human osteoblastic cell system.

Adult↗
PubMed2000

Differential expression of osteonectin/SPARC during human prostate cancer progression.

The precise mechanism(s) involved in invasion and metastasis of prostate cancer (CaP) is poorly understood. Osteonectin [ON (also known as SPARC or BM-40)] is an antiadhesive protein known to be involved in cell-matrix interactions, migration, and angiogenesis. In this report, we studied the expression of ON in human prostate cell lines, primary tumors, and metastatic foci of CaP. Reverse transcription-PCR and nonradioactive in situ hybridization (ISH) techniques were used to determine ON gene expression. Immunohistochemistry was carried out using the polyclonal antibody LF37 and/or the monoclonal antibody ON-mAb. Low to moderate levels of ON mRNA and protein were observed in glandular epithelial cells of normal tissue as well as a few primary CaPs. However, high levels of ON mRNA and protein were observed in most of the CaP metastatic foci, both osseous and nonosseous. This correlated well with our findings that multiple different CaP cell lines including four CaP cell lines derived from metastases show high levels of ON gene expression. Furthermore, ISH analyses and cell-specific reverse transcription-PCR evaluation showed that both the luminal and basal cells express the ON gene. We conclude that the differential pattern of ON expression suggests that it may play an important role in the progression of CaP.

Antibodies, Monoclonal↗
PubMed2001

Effects of parathyroid hormone on bone formation in a rat model for chronic alcohol abuse.

BACKGROUND: Alcoholism is a risk factor for osteoporosis and it is not clear whether the detrimental effects of alcohol on bone are reversible. Parathyroid hormone (PTH) is a potent stimulator of bone matrix synthesis and is being investigated as a therapeutic agent to reverse bone loss. The present investigation was designed to determine the effects of PTH on bone formation in a rat model for chronic alcohol abuse. METHODS AND RESULTS: Alcohol was administered in the diet of female rats (35% caloric intake) for 2 weeks. Human (1-34) PTH (80 microg/kg/day) was administered subcutaneously during the second week of the study. Alcohol resulted in a transient reduction in steady-state mRNA levels for the bone matrix proteins type 1 collagen, osteocalcin, and osteonectin compared with rats that were fed an alcohol-free (control) diet. As expected, alcohol decreased and PTH increased histologic indices of bone formation. Additionally, two-way ANOVA demonstrated that alcohol antagonized PTH-induced bone formation. Despite antagonism, bone formation and mRNA levels for bone matrix proteins in alcohol-fed rats treated with PTH greatly exceeded the values in rats fed the control diet. CONCLUSIONS: The results of this study contribute to a growing body of evidence that alcohol-induced bone loss is primarily due to reduced bone formation. We conclude that alcohol does not prevent the stimulatory effects of PTH on bone formation. This is evidence that the effects of alcohol on the skeleton are reversible. Additionally, the positive effects on bone formation in rats that consumed high concentrations of alcohol suggested that PTH may be useful as an intervention to treat alcohol-induced osteoporosis.

Alcoholism↗
PubMedDate not supplied

Characterization of human bone cells in culture.

Osteoblast-like cells isolated from human bone bioptic specimens were established in culture. Their osteoblast-like phenotype was studied by biochemical, histochemical and immunohistochemical methods and by electron microscopy examination. Third-passage cell cultures exhibited high level of alkaline phosphatase activity and the exposure to human parathyroid hormone produced an increase of intracellular cAMP. Cultured cells were immunoreactive for type I and type III collagen, osteonectin, and fibronectin; when ascorbic acid and beta-glycerophosphate were added, they synthesized a rich extracellular matrix. This characterization ensures the reliability of osteoblast-like cultures when they are used as experimental models.

Adult↗
PubMed2001

Analysis of the gene expression of SPARC and its prognostic value for bladder cancer.

PURPOSE: We analyzed the gene expression of the glycoprotein termed secreted protein, acidic and rich in cysteine (SPARC), also called osteonectin and BM40, in bladder cancer and its relationship with conventional clinical-histopathological manifestations, evaluated its prognostic value for patient outcome and determined the possible mechanism underlying the effect of SPARC on bladder cancer progression. MATERIALS AND METHODS: Tissue samples from 63 patients with bladder cancer were used for analysis. Gene expression levels of SPARC and matrix metalloproteinase-2 were analyzed using reverse transcription-polymerase chain reaction. Correlations of the expression of SPARC with histopathological findings or patient outcome and with matrix metalloproteinase-2 were evaluated. RESULTS: Significantly higher expression of SPARC was observed in grades 3 and 2 than in grade 1 tumors (p <0.001 and <0.05, respectively). Stage T2 or greater invasive tumors expressed a significantly higher level of SPARC than stages T1 or less superficial tumors (p <0.0001). Patients in whom the lesions showed high SPARC expression had a significantly worse prognosis than those with low SPARC expression disease (p <0.0001). Even in those with invasive bladder cancer high SPARC expression was associated with significantly worse survival than low expression (p <0.01). Moreover, gene expression of SPARC significantly correlated with matrix metalloproteinase-2 gene expression (p <0.0001), implying that regulation of matrix metalloproteinase-2 expression may be a possible mechanism underlying the effect of SPARC on bladder cancer progression. CONCLUSIONS: A significant correlation was detected of the gene expression level of SPARC with histological grade, pathological stage and bladder cancer prognosis. SPARC may have an important role in bladder cancer progression and provide some additional information in patients with bladder cancer.

Aged↗
PubMed2002

Characterization of the osteoblast-like cell phenotype under microgravity conditions in the NASA-approved Rotating Wall Vessel bioreactor (RWV).

Weightlessness induces bone loss in humans and animal models. We employed the NASA-approved Rotating Wall Vessel bioreactor (RWV) to develop osteoblast-like cell cultures under microgravity and evaluate osteoblast phenotype and cell function. Rat osteoblast-like cell line (ROS.SMER#14) was grown in the RWV at a calculated gravity of 0.008g. For comparison, aliquots of cells were grown in conventional tissue culture dishes or in Non-Rotating Wall Vessels (N-RWV) maintained at unit gravity. In RWV, osteoblasts showed high levels of alkaline phosphatase expression and activity, and elevated expression of osteopontin, osteocalcin, and bone morphogenetic protein 4 (BMP-4). In contrast, the expression of osteonectin, bone sialoprotein II and BMP-2 were unaltered compared to cells in conventional culture conditions. These observations are consistent with a marked osteoblast phenotype. However, we observed that in RWV osteoblasts showed reduced proliferation. Furthermore, DNA nucleosome-size fragmentation was revealed both morphologically, by in situ staining with the Thymine-Adenine binding dye bis-benzimide, and electrophoretically, by DNA laddering. Surprisingly, no p53, nor bcl-2/bax, nor caspase 8 pathways were activated by microgravity, therefore the intracellular cascade leading to programmed cell death remains to be elucidated. Finally, consistent with an osteoclast-stimulating effect by microgravity, osteoblasts cultured in RWV showed upregulation of interleukin-6 (IL-6) mRNA, and IL-6 proved to be active at stimulating osteoclast formation and resorbing activity in vitro. We conclude that under microgravity, reduced osteoblast life span and enhanced IL-6 expression may result in inefficient osteoblast- and increased osteoclast-activity, respectively, thus potentially contributing to bone loss in individuals subjected to weightlessness.

Alkaline Phosphatase↗
PubMed2001

[Cellular and molecular biology of fibrous dysplasia].

Several recent studies reflect major progress on the molecular biology and on the mechanisms of the lesions of fibrous dysplasia (monostotic and polyostotic forms) as well as the McCune-Albright syndrome. This review includes a detailed histological description of fibrous dysplasia: disorganized collagen fibers, woven bone formation, immature cytology of the osteoblasts and preosteoblastic cells. Histological lesions are also associated with an alteration of bone proteins: such as an increase in the expression of osteonectin, decrease of osteopontin and bone sialoprotein. Cell cultures of dysplasic osteoblastic cells have shown that the bone lesions are the result of abnormalities of the proliferation and differentiation of bone forming cells with a rise in CAMP levels. Poorly differentiated cells are associated with the formation of disorganized collagen fibers and with a trouble of synthesis of bone proteins. All these findings are linked to mutations of the Gs alpha (sub-unit alpha) protein. Various activating mutations are described as well as the molecular mechanisms of different forms of fibrous dysplasia, involving specially the c-fos proto-oncogene. Therapeutic implications are quoted, specially calcitonin and bisphosphonates.

Cell Differentiation↗
PubMed2002

[The effects of intermittent hydromechanics on the differentiation and function of bone marrow stromal derived-osteoblasts in porous calcium phosphate ceramics].

OBJECTIVE: To observe the effects of the intermittent hydromechanical stimulation on the differentiation and function of the bone marrow stromal derived-osteoblasts in the porous calcium phosphate ceramics. METHODS: Rat bone marrow stromal derived-osteoblasts were seeded into porous calcium phosphate ceramic scaffolds at cells density 1 x 10(6) cells/cm(3). The cells-ceramics contrusts were cultured under rotary condition for 1 hour at 4 hours interval. After 4, 7 and 14 days cultivation, the osteoblastic phenotype markers (ALP Activity, Type I Collagen, Osteocalcin, Osteopontin, Osteonectin, and Bone Sialoprotein mRNA expression levels) were analyzed by biochemistry measurement and quantitative RT-PCR technique. Static cell culture as control. RESULTS: Under rotary cell culture condition, the ALP activities and expressions of Type I collagen mRNA increased markedly and reached the peak levels at 7 days. The expressions of other four markers mRNA occurred at 4 days and reached the peak levels at 7 days, then the expressions were down-regulated at 14 days. Under static cell culture as control, the ALP activities and expressions of Type I collagen mRNA increased gradually and reached the peak levels at 14 days. The expressions of other four markers mRNA occurred at 7 days and reached the peak levels at 14 days. CONCLUSION: The intermittent hydromechanical stimulation could promote the bone marrow stromal derived-osteoblasts differentiation and function which cultured in the porous calcium phosphate ceramics in vitro.

Alkaline Phosphatase↗