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PubMed1998

Immunocytochemical study of the maxilla and maxillary sinus during human fetal development.

We quantitatively examined the distribution of the proliferating cell nuclear antigen (PCNA), which is associated with cell division, and of components of the ECMs (collagen types I and III, tenascin and osteonectin) in the immature zones at three sites: palatine, inferior and vertical surface region of the fetal human maxilla as well as the maxillary sinus (MS) at 12, 16, 20, 24, and 28 weeks gestation. The percentage of PCNA-positive cells was the highest at 16 weeks in the immature zones of maxilla. Tenascin and fibrillar collagens (collagen types I and III) were especially present in the cellular zone linked to the bone and cartilage matrices of the immature zones of the maxillary bone at 20-24 weeks gestation. The osteonectin was detected on the maxillary bone from 24 weeks. These distributions of ECMs revealed the specific and contrasting profiles of development in the human maxillary bone and might reflect the formation of paranasal sinus as MS.

Cell Count↗
PubMed1990

[The effects of transforming growth factor beta 1 on mouse osteoblastic cell line MC3T3-E1--especially on gene expression of differentiation markers].

Regulation of alpha 2(I) collagen and osteonectin/SPARC gene expressions in mouse osteoblastic MC3T3-E1 cells by TGF-beta 1 was studied. TGF-beta 1 regulated those differentiation markers of osteoblast phenotypes, although the effects were inconsistent depending on serum concentrations. Exposure of MC3T3-E1 cells to TGF-beta 1 in low serum culture caused stimulation of alpha 2(I) collagen and osteonectin expressions but no stimulation in high serum culture. These inconsistent responses may be due to the presence of some component(s) in serum. bFGF also suppressed stimulation by TGF-beta 1 on alpha 2(I) collagen and osteonectin expressions. Serum factors as well as locally derived growth factors may interact with TGF-beta 1 and modulate the actions of TGF-beta 1 in osteoblastic cells. The present result explains the complexity of TGF-beta 1 actions on bone cells in vitro and also suggests that the actions on bone formation are modified by other factors in vivo.

Animals↗
PubMed2000

Quantitative analysis of circulating tumor cells in peripheral blood of osteosarcoma patients using osteoblast-specific messenger RNA markers: a pilot study.

Metastasis is a major cause of mortality and morbidity in osteosarcoma (OS) patients. To monitor tumor dissemination, we assessed the circulating tumor burden in OS patients by semiquantitative reverse transcription-PCR using osteocalcin, osteonectin, osteopontin, and type I collagen (COLL) mRNAs as molecular markers. We distinguished levels of the mRNAs in peripheral blood between OS patients and healthy subjects using an OS-derived cell line (Saos-2) as a reference standard. We prospectively analyzed 40 peripheral blood samples from 11 OS patients at diagnosis and 29 healthy subjects. In all 29 (100%) healthy subjects, we detected osteocalcin, osteonectin, and osteopontin mRNAs that were most likely attributed to illegitimate transcription in normal hematopoietic cells. In contrast, we found low COLL mRNA levels in only 35% (10 of 29) of healthy subjects, but significantly higher COLL mRNA levels in 91% (10 of 11) of OS patients (P < 0.0001). The reverse transcription-PCR assay for COLL mRNA was sensitive down to the detection of 10 Saos-2 cells among 10(6) normal peripheral blood nucleated cells. The upper limit of COLL mRNA determined among the healthy subjects was found exceeded by six OS patients. The substantially elevated COLL mRNA levels in peripheral blood seemed to originate from circulating malignant cells in these six OS patients, all of whom subsequently developed clinical metastases within 12 months of diagnosis (P = 0.002). Conversely, no metastases were detected in the remaining OS patients with normal COLL mRNA levels. Quantification of COLL mRNA may prove valuable for diagnosing OS micrometastasis and assessing prognosis.

Adolescent↗
PubMed2002

Differential gene expression analysis in a rabbit model of osteoarthritis induced by anterior cruciate ligament (ACL) section.

Osteoarthritis (OA) is the most common of all joint diseases to affect mankind and is characterized by the degradation of articular cartilage. The low availability of normal and pathologic human cartilage and the inability to study the early stages of the disease in humans has led to the development of numerous animal models of OA. The aim of our study was to establish gene expression profiles during the progression of a rabbit model of OA induced by anterior cruciate ligament (ACL) section. Semiquantitative RT-PCR was used to follow expression of several relevant molecules (type II and X collagens, aggrecan, osteonectin, betaig-h3, BiP, TIMP-1, MMP-1, -3, -13, aggrecanase-1, -2) during development of OA in articular cartilage. In parallel, we monitored the activities of collagenase, caseinase, phospholipase A2 and glycosyltransferases (xylosyl-, galactosyl-, glucuronyl- and N-acetyl-galactosaminyl-transferase). Novel cDNA clones for rabbit type X collagen, aggrecanase-1 and -2, osteonectin and BiP were constructed to obtain species-specific primers. Ours result show that MMP-13 (collagenase-3) gene expression increased dramatically early after ACL surgery and remained high thereafter. An increase in MMP-1 (collagenase-1) and MMP-3 expression was also noted with an absence of variation for TIMP-1 expression. In addition, the global MMPs activities paralleled the MMP gene expression. These data together characterize at the molecular level the evolution of OA in this rabbit model. Furthermore, we have undertaken a search for identifying differentially expressed genes in normal and OA cartilage in this model, by differential display RT-PCR. We present here preliminary results with the determination of the best technical conditions to obtain reproducible electrophoresis patterns of differential display RT-PCR.

Aggrecans↗
PubMed2003

Mineralised deposits in the uterine glands of mares with chronic endometrial degeneration.

Chronic degenerative disease of the mare's endometrium is characterised by changes in the uterine glands, including cystic dilation, hyperplasia and periglandular fibrosis. Endometrial biopsies were taken from 23 mares with different grades of endometrial degeneration. Solid structures were identified within the lumina of the uterine glands and shown to be calcified by histochemical staining. Most of them were not homogenous but composed of a mixture of mineral and organic substances. Further examinations of these mineralised structures by immunohistochemical methods revealed the presence of the non-collagenous matrix proteins osteopontin, osteonectin and bone sialoprotein, which are known to be involved in calcification processes such as urolithiosis. Osteopontin and bone sialoprotein were identified within the calculi, frequently arranged in concentric layers. Osteonectin was the only matrix protein that was also present in the glandular epithelium. Osteocalcin was not found in either the calculi or the glandular epithelial cells.

Animals↗
PubMed2004

[Re-growth of induced-osseous tissue encapsulated in muscular fasciae for prefabrication of an osteo-musculo-cutaneous flap].

OBJECTIVE: To improve the survival of mesenchymal stem cells (MSCs) for prefabrication of an osteo-musculo-cutaneous flap. METHODS: In a mouse model, the compound of MSCs and p(3HB-co-3HH) were embeded in the latissimus dorsi muscle as an experimental group and the muscle pocket of the buttock as the control. The examinations of the HE staining, hybridization in situ of osteonectin mRNA and Von kossa staining were used to evaluate the results. RESULTS: The expression of osteonectin mRNA and the Von kossa staining showed that the latissimus dorsi muscle group was superior to the control in 2 weeks and 4 weeks after the surgery in vivo. CONCLUSION: The results indicate that the above-mentioned technique may be a good alternative for the prefabrication of the osteo-musculo-cutaneous flap.

Animals↗
PubMed2005

[Study on culture and in vitro osteogenesis of blood-derived human mesenchymal stem cells].

OBJECTIVE: To establish a method of isolating and culturing adult human blood-derived stem cells(MSCs) and to investigate their osteogenic potential in vitro. METHODS: Thirty peripheral blood collected from 30 adult volunteers (15 ml per person). Adult human MSCs derived from peripheral blood from the lymphocyte separation fluid fraction of mononuclear cells, cultured in alpha-Modified Eagle's Medium glucose containing 20% fetal bovine serum, and proliferated through a process of subculturing. The phenotype was analyzed with flow cytometry. For in vitro osteogenic differentiation, MSCs from the second passage presence of osteogenic supplements (100 nmol/L dexamethasone,10 mmol/L beta-glycerophosphate, 50 micromol/L and 10 nmol/L 1,25-2-hydroxide vitamin D3). In the fifth passage cells, the activity of alkaline phosphatase, expression level of collagen type I, osteocalcin and osteonectin were determined. And the calcium tubercle would be examined after the continual one-month culture of the fifth passage. RESULTS: MSCs existed in blood of adult human. And the clone forming efficiency of blood-derived MSCs was 0.27 +/- 0.22/10(6) mononuclear The MSCs expressed CD44,CD54,CD105,and CD166, but did not CD14, CD34, CD45,and CD31. Under osteogenic supplements, the MSCs were found to be higher activity of alkaline phosphatase and higher expression of collagen type I , osteocalcin and osteonectin. And the calcium tubercle formation was examined through fluorescence labeling method. CONCLUSION: The isolation and culture conditions established for adult human select a distinct population of peripheral blood-derived adherent cells. Adult human blood-derived osteogenic potential in vitro, and may be used as seed cells for bone tissue engineering.

Adult↗
PubMedDate not supplied

Effect of titanium surface roughness on human osteoblast proliferation and gene expression in vitro.

PURPOSE: Cell proliferation and extracellular matrix formation are primary events in bone formation. At the dental implant-tissue interface, implant surface roughness modulates osteoblast functions. The aim of the present in vitro study was to investigate the effect of varying surface roughness of titanium implant material on cell proliferation and mRNA expression of specific markers of osteoblast phenotype. MATERIALS AND METHODS: Primary cultures of osteoblasts derived from human mandibular bone were cultured on titanium surfaces. Three titanium surfaces were studied: machined titanium, microsandblasted titanium, and macro-sandblasted titanium (average surface roughnesses of 0.5 and 3 microm, respectively). Cell morphology was estimated by scanning electron microscope analysis and cell proliferation by measuring the amount of 3H-thymidine incorporation into DNA. mRNA expression of osteonectin, osteopontin, bone sialoprotein (BSP), and Runx2, which are markers of osteoblastic phenotype, were determined by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. RESULTS: Human osteoblasts cultured on machined titanium spread more and were flatter than cells cultured on rough titanium. All blasted surfaces showed significantly higher DNA synthesis than the machined surfaces. Osteonectin mRNA expression was similar on all surfaces. Other mRNA transcripts were increased in osteoblasts cultured on rough titanium surfaces, particularly the macrosandblasted surface. CONCLUSIONS: An average surface roughness of 3 microm (macro-sandblasted titanium) is more suitable than an average surface roughness of 0.5 microm (micro-sandblasted titanium) in favoring osteoblast differentiation in vitro.

Analysis of Variance↗
PubMed1987

Characterization of a human osteosarcoma cell line (Saos-2) with osteoblastic properties.

This study examines the osteoblastic properties of the established human osteosarcoma cell line Saos-2. Saos-2 cells inoculated into diffusion chambers, which were implanted i.p. into nude mice, produced mineralized matrix in 4 of 6 chambers at 8 weeks. In 5 of 6 chambers there was a strong positive alkaline phosphatase reaction. In culture the alkaline phosphatase levels increased with time and cell density, reaching very high levels at confluence: 4-7 mumol/mg protein/min. The cells show a sensitive adenylate cyclase response to parathyroid hormone, 50% effective dose = 2.8 nM, which increases with cell density and is further raised by dexamethasone treatment. They also exhibit typical binding of 1-25-dihydroxyvitamin D3 to 3.2S receptor protein with an apparent Kd of 0.21 nM; the numbers of sites per cell were 3,300 at 50,000 cells/cm2 and 1,800 at 280,000 cells/cm2. The presence of osteonectin was visualized with a monoclonal antibody which revealed a reticular pattern on the cell surface. Osteonectin was also detected in the medium by Western blots, migrating at around Mr 40,000 in nonreduced gels and Mr 44,000 in reduced gels. The Saos-2 cells thus possess several osteoblastic features and could be useful as a permanent line of human osteoblast-like cells and as a source of bone-related molecules.

Adenylyl Cyclases↗
PubMed1995

Expression of matrix genes during incorporation of cancellous bone allografts and autografts.

The healing process of cylindrical metaphyseal bone defects filled with fresh autogeneic or frozen allogeneic cancellous bone grafts was studied using histology, biomechanical testing, and molecular biology. The results were compared with the healing pattern observed in nongrafted defects, where a strong initial expression of Type III collagen was followed by slowly starting expression of osteonectin and Type 1 collagen genes. Autogeneic or allogeneic cancellous bone grafts considerably increased the healing rate of the defects. The sequence of healing was similar with both types of grafts, but the maximal levels of Type III and Type I collagen messenger ribonucleic acids (mRNAs) were delayed by 1 week when allografts were used. Histologically, during the initial phase the grafts had an invasion of progenitor mesenchymal tissue within 2 weeks followed by intramembranous bone formation and by a gradual decline in Type I collagen expression. Osteonectin mRNA was present throughout the healing process, but no expression of Type II collagen was seen. Indentation testing showed no significant biomechanical differences between the autografts and allografts. Histology and molecular biology testing results revealed that the production of extracellular matrix starts slower in allogeneic cancellous bone grafts than in autografts, suggesting a basic difference in the initial host tissue response between autogeneic and allogeneic bone grafts.

Animals↗
PubMed1993

Gene expression at graft-host interfaces of cortical bone allografts and autografts.

The healing processes of autogenous and frozen allogeneic cortical bone grafts were compared in rats. Two unicortical defects created in the proximal metaphysis of the tibia were autografted or allografted with blocks of cortical bone. The biomechanical properties of the graft-host junction and the time-related changes of the expression of genes coding for Type I, II, III, and X collagens and osteonectin were determined at one, two, four, and eight weeks. The strength of the allograft-host bone union was lower than that of autografts at the early stage of incorporation but the difference diminished by eight weeks. Northern analysis of graft mRNAs demonstrated a strong expression of Type I collagen and osteonectin genes at the beginning of autograft incorporation. The allografts showed a lower expression of the corresponding genes at one and two weeks, but the difference diminished thereafter. The gene coding for Type III collagen showed a temporary expression at two weeks both in allografts and autografts, probably because of the formation of inflammatory fibrovascular stroma provided by the host bone at the graft-host interface. No expression of cartilage-specific Type II and Type X collagens were observed, demonstrating that the healing in both grafts proceeded through primary bone healing. Compared with cortical-cortical junctions of autografts, the graft-host interfaces of allografts exhibited a reduced osteoinductive process and a slower increase of union strength.

Animals↗
PubMed1993

Regulation of extracellular matrix genes during fracture healing in mice.

The expression of several collagens, cartilage proteoglycan core protein (aggrecan), and osteonectin was investigated during healing of standardized fractures of the mouse tibia. Total RNA was extracted from fracture calluses and normal bone, and analyzed by Northern hybridization using specific cDNA probes. Based on the predominant fibrillar collagen type expressed, three phases of fracture healing were observed. Type III collagen expression characteristic for the inflammatory stage increased first, followed by Type II collagen expression indicative of the cartilaginous reparative phase, and by Type I collagen production, characteristic for the ossification and remodelling stages. Type IX collagen and aggrecan expression coincided with Type II collagen expression during the chondrogenesis. The mRNA for Type X collagen, a marker for hypertrophic chondrocytes during endochondral ossification, occurred somewhat later than that of other cartilage-specific genes. Osteonectin mRNA was present throughout the healing process and peaked during rapid new bone growth. This study confirms that the pattern of gene expression in the murine system is similar to, but somewhat faster than, other species analyzed (rat, rabbit, human).

Aggrecans↗
PubMed1996

Matrix proteins with high affinity for calcium ions are associated with mineralization within the elastic fibers of pseudoxanthoma elasticum dermis.

Ultrathin sections from the dermis of five normal subjects and from 10 patients suffering from pseudoxanthoma elasticum (PXE) were analyzed by immunoelectron microscopy with the aim of identifying and localizing proteins associated with the mineral precipitates within the altered elastic fibers. Serial sections were processed by indirect immunogold cytochemistry using primary antibodies against human fibronectin, vitronectin, bone sialoprotein, alkaline phosphatase, osteonectin, and osteopontin. In the latter two cases, antibodies against synthetic peptides were also used. The results indicate that normal elastic fibers contained osteopontin, and that this protein was associated with the apparently normal elastin as well as with the needle-shaped mineral precipitates in the elastic fibers of patients. On the contrary, significant amounts of vitronectin, alkaline phosphatase and, less, of bone sialoprotein were associated with the polymorphous mineral precipitates inside the elastic fibers. Large amounts of osteonectin and fibronectin, together with vitronectin, were localized on the microfilament aggregates, which were often associated with altered elastic fibers in PXE dermis and were never visualized in the dermis of control subjects. The results seem to indicate once more that PXE is a complex disorder of the fibroblast synthetic control. Elastic fiber mineralization might be considered a secondary event, which could depend on the abnormal synthesis and accumulation within the elastic fibers of proteins that are normally involved in mineralization processes.

Adolescent↗
PubMed1996

A novel technique for quantitative detection of mRNA expression in human bone derived cells cultured on biomaterials.

A nonisotopic and quantitative in situ hybridization technique was adapted to investigate the effect of biomaterials on the cellular expression of mRNA from human bone derived cells (HBD cells). HBD cells were cultured for 24 or 48 h on tissue culture plastic, alumina, and ion modified alumina. Osteocalcin, osteopontin, alkaline phosphatase, type I collagen alpha 1, and type I collagen alpha 2 mRNAs were quantified. Protein expression for collagen types I, III, and V, and for anti-human macrophages CD68 (DAKO-CD68, KP1) and CD68 (PG-M1), and anti-human myeloid/histiocyte antigen (DAKO-MAC 387) were determined immunohistochemically using monoclonal antibodies. At 24 and 48 h, levels of mRNA for alkaline phosphatase and osteonectin were greater than mRNA levels for osteopontin, osteocalcin, collagen type I alpha 1, and collagen type I alpha 2 for cells grown on the three substrata. However, at 48 h mRNA levels for alkaline phosphatase and osteonectin were significantly higher on the modified ceramic substrata relative to the native alumina. HBD cells appear to express CD68-KP1 when cultured for 24 h. The techniques provide a sensitive and reproducible assay to evaluate gene and protein expression of cells grown on different substrata.

Alkaline Phosphatase↗
PubMed2002

Influence of resinous monomers on the differentiation in vitro of human pulp cells into odontoblasts.

Odontoblasts are highly differentiated postmitotic cells, which under pathological conditions such as carious lesions and dental injuries may degenerate and be replaced by other pulp cells. A recent work showed that this physiological event can be reproduced in an in vitro assay system. The purpose of the present study was to evaluate the effects of resinous monomers on odontoblast differentiation in vitro. Pulp cores from extracted human third molars were cultured with beta-glycerophosphate (2 mM) and used to evaluate the effects of TEGDMA, HEMA, UDMA, and Bis-GMA on the differentiation of pulp fibroblasts into odontoblasts. The effect of the monomers was studied by evaluating the expression of several odontoblast specific genes. In the absence of monomers, mineral nodule formation was observed. Pulp cells contributing to the nodule formation synthesized type I collagen, osteonectin, and dentin sialoprotein (DSP). In addition, Fourier transform infrared microspectroscopy showed that the mineral and organic composition of the nodules were characteristic of dentin. When the monomers were added at nontoxic concentrations, the effects of HEMA and Bis-GMA were more evident than that of TEGDMA and UDMA on collagen 1, osteonectin, and DSP expression. However, all monomers significantly decreased DSP expression and completely inhibited the mineral nodule formation.

Acrylates↗
PubMed2003

Molecular biologic comparison of new bone formation and resorption on microrough and smooth bioactive glass microspheres.

In a recent in vitro study, chemical microroughening of a bioactive glass surface was shown to enhance attachment of MG-63 osteoblastic cells to glass. The current study was designed to delineate the effects of microroughening on the gene expression patterns of bone markers during osteogenesis and new bone remodeling on bioactive glass surface in vivo. With the use of a rat model of paired comparison, a portion of the medullary canal in the proximal tibia was evacuated through cortical windows and filled with microroughened or smooth bioactive glass microspheres. The primary bone-healing response and subsequent remodeling were analyzed at 1, 2, and 8 weeks, respectively, by radiography, pQCT, histomorphometry, BEI-SEM, and molecular biologic analyses. The expression of various genes for bone matrix components (type I collagen, osteocalcin, osteopontin, osteonectin) and proteolytic enzymes (cathepsin K, MMP-9) were determined by Northern analysis of the respective mRNAs. Paired comparison showed significant differences in the mRNAs levels for specific bone matrix components at 2 weeks: osteopontin was significantly higher (p =.01) and osteonectin significantly lower (p =.05) in bones filled with microroughened microspheres than in those filled with smooth microspheres. Bones filled with microrough microspheres also showed significantly increased ratios of cathepsin K and MMP-9 (both markers of osteoclastic resorption) to type I collagen (p =.02 and p =.02, respectively) at 2 weeks and a significantly increased expression of MMP-9 at 8 weeks (p =.05). The pQCT, histomorphometric, and BEI-SEM analyses revealed no significant differences in the pattern of bone-healing response. Based on these results, microroughening of a bioactive glass surface could trigger temporal changes in the expression of specific genes especially by promoting the resorption part of new bone-remodeling processes. Future studies are needed to evaluate if the observed changes of gene expression are directly related to the microrough surface of any biomaterial or are biomaterial specific.

Animals↗
PubMed1992

Identification of proteins secreted by human osteoblastic cells in culture.

To better understand the biochemistry of matrix-forming cells, we developed a simple and reproducible procedure for the isolation and identification by N-terminal sequencing of proteins secreted by cells into culture medium and applied this procedure to the analysis of the major Coomassie blue-staining proteins under 100 kD that are secreted from three different human osteoblastic cell cultures. The major proteins secreted by normal human osteoblasts from adult trabecular bone were identified by N-terminal sequencing to be gelatinase, osteonectin, the C-terminal propeptides of the alpha 1 and alpha 2 chains of type I collagen, tissue inhibitor of metalloproteinase 1 (TIMP-1), and beta 2-microglobulin. The amounts of each of these proteins secreted into medium over a 24 h interval did not change over the 7 consecutive days of culture under serum-free conditions, which indicates that this pattern of protein secretion is not significantly affected by the serum-free conditions needed for protein identification by this method. In addition, radioimmunoassay for bone gla protein (BGP), a marker for osteoblast phenotype, revealed that BGP secretion remained high over 7 days of culture under serum-free conditions and was comparable to the rate of BGP secretion in control cultures with 10% serum. The major proteins secreted by MG-63 cells were identified by N-terminal sequencing to be gelatinase, a novel 40 kD human bone protein we termed YKL-40, TIMP-1, the recently discovered TIMP-2, and beta 2-microglobulin. Further studies revealed that YKL-40 is the only protein detectable by Coomassie staining of SDS gels of MG-63 media proteins that is induced by extended time at confluence or by treatment with 1,25-(OH)2D3. The apparent absence of detectable Coomassie-stained bands corresponding to the C-terminal propeptides of collagen in the medium of MG-63 cells suggests that these transformed cells may not be a good model for bone matrix formation. The major proteins secreted by normal fetal osteoblastic cells were identified by N-terminal sequencing to be osteonectin and the C-terminal propeptides of the alpha 1 and alpha 2 chains of type I collagen. Gelatinase and TIMP could not be detected among the conditioned medium proteins by these methods. These observations indicate that fetal osteoblasts primarily express proteins that are matrix constituents and adult human osteoblasts secrete, in addition to these, proteins that could function in matrix turnover.

Amino Acid Sequence↗
PubMed2001

Cellular and molecular properties associated with osteosarcoma cells.

Osteosarcoma cells are recognized by abnormal function that causes a primary bone tumor. Osteosarcoma cells U(2)OS and SAOS-2 were analyzed for the expression of cell surface markers. High expression was quantified for hyaloronidase receptor (CD-44) > moderate for integrins (CD-51 and -61), > and lower for selectins (CD-62). High mitotic capacity were demonstrated by gene expression (measured by RT-PCR) and the protein level (measured by FACS) for cFOS, cMYC, and cJUN. The basic definition of osteosarcoma is excessive production of pathological osteoid. Expression of mRNA for matrix genes osteocalcin, osteonectin, and biglycan was studied. Osteocalcin and osteonectin were detected in RNA from primary cultured marrow stromal, trabecular bone cells, and osteosarcoma cell lines (U(2)OS, SAOS-2). mRNA for biglycan was detected only in primary cells and MG-63 cell line and was undetectable in RNA from U(2)OS, SAOS-2 osteosarcoma cell lines and by RNA extracted from bone biopsies of osteosarcoma patients. The absence of biglycan message observed in osteosarcoma samples provides evidence for the alterations in the extra cellular matrix which result with non-mineralized osteoid produced by the osteosarcoma cells.

Adolescent↗