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PubMed2004

Ultrastructural characterization of the implant/bone interface of immediately loaded dental implants.

Primary stability and an optimized load transfer are assumed to account for an undisturbed osseointegration process of implants. Immediate loaded newly designed titanium dental implants inserted in the mandible of minipigs were used for the characterization of the interfacial area between the implant surface and the surrounding bone tissue during the early healing phase. Histological and electron microscopical studies were performed from implant containing bone specimens. Two different load regimens were applied to investigate the load related tissue reaction. Histological and electron microscopical analysis revealed a direct bone apposition on the implant surfaces, as well as the attachment of cells and matrix proteins in the early loading phase. A striking finding of the ultrastructural immunocytochemical investigations was the synthesis and deposition of bone related proteins (osteonectin, fibronectin, fibronectin receptor) by osteoblasts from day one of bone/biomaterial interaction. Calcium-phosphate needle-like crystallites were newly synthesized in a time-related manner directly at the titanium surface. No difference in the ultrastructural appearance of the interface was found between the two loading groups. Our experimental data suggest that loading of specially designed implants can be performed immediately after insertion without disturbing the biological osseointegration process.

Animals↗
PubMed2004

The modulation of osteogenesis in vitro by calcium titanium phosphate coatings.

Calcium phosphate coated titanium and titanium alloy are widely used as dental and orthopaedic implants. This study examines the effect of novel calcium titanium and calcium titanium zirconium phosphates suitable for plasma-spraying onto titanium substrata on the expression of bone-related genes and proteins by human bone-derived cells (HBDC) and compares this behavior to that on native titanium and hydroxyapatite-coated titanium. Test materials were an acid etched and sand-blasted titanium surface (Ti-DPS), a plasma-sprayed hydroxyapatite coating (HA), and five materials which were created from CaTi(4)(PO(4))(6) (CTP) and CaZr(4)(PO(4))(6) (CZP): sintered CaTi(4)(PO(4))(6) (CTP-S1), sintered 46CaO.23TiO(2).31P(2)O(5) (CTP-S2), sintered CaTiZr(3)(PO(4))(6), (CTZP-S1), sintered 46CaO.23ZrO(2).31P(2)O(5) (CTZP-S2) and sintered 55CaO.20TiO(2).31P(2)O(5) (CTP-S3). HBDC were grown on the substrata for 3, 7, 14 and 21 d, counted and probed for various mRNAs and proteins (type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase and bone sialoprotein). All substrates significantly affected cellular growth and the temporal expression of an array of bone-related genes and proteins. At 14 and 21 d, cells on CTP-S3 displayed significantly enhanced expression of all osteogenic mRNAs. Surfaces of CTP-S1 and CTP-S3 had the most effect on osteoblastic differentiation inducing a greater expression of an array of osteogenic markers than recorded for cells grown on Ti-DPS and HA, suggesting that these novel materials may possess a higher potency to enhance osteogenesis.

Alkaline Phosphatase↗
PubMed2006

Dexamethasone's enhancement of osteoblastic markers in human periodontal ligament cells is associated with inhibition of collagenase expression.

Although dexamethasone (Dex) substantially enhances the osteoblastic phenotype in osteogenic cells, including human periodontal ligament (PDL) cells, the basis for this response remains poorly understood. Since the accretion of a collagenous matrix is important for an osteoblastic response and dexamethasone is known to decrease collagenase expression, we examined whether osteoblastic differentiation mediated by Dex is linked to a decrease in collagenase expression in PDL cells. Early passage human PDL cells were exposed to Dex, or ascorbic acid (AA) or beta-glycerophosphate (betaGP) alone, or in various combinations in serum-free media for 3 or 5 days. Cells exposed to Dex alone or any combinations of treatments that included Dex demonstrated increased core binding factor alpha 1 (Cbfa1), alkaline phosphatase (AP), osteonectin (ON), osteopontin (OP), bone sialoprotein (BSP) and collagen I (alpha1) expression when compared to control cells or those exposed to AA or betaGP. The induction of these osteoblastic markers was accompanied by a decrease in collagenase-1 expression. Collagenase activity showed a statistically significant strong negative relationship to Cbfa1 (Pearson's r=-0.97), AP (r=-0.87), OP (r=-0.95) and BSP (r=-0.82) in 5-day cultures, and moderately strong relationship to ON (r=-0.74) from 3 days culture. Dex also produced a dose-dependent increase in AP that was paralleled by a decrease in collagenase activity (r=-0.98). Addition of collagenase inhibitors increased AP expression while concomitantly suppressing collagenase activity. Conversely, addition of exogenous collagenase decreased the AP phenotype of the cells, which was more marked in the absence then in the presence of Dex. The findings indicate that Dex enhances specific markers of osteoblastic differentiation in PDL cells by decreasing collagenase expression, and suggest that endogenous collagenase may regulate osteoblastic differentiation of these cells.

Alkaline Phosphatase↗
PubMed2005

Characterization of the non-collagenous proteins in avian cortical and medullary bone.

Northern blotting, RT-PCR, and Western blotting techniques were used to characterize the matrix constituents of avian cortical and medullary bone. Extracts of bone tissue were found to contain multiple isoforms of bone sialoprotein (BSP), osteopontin (OPN), osteonectin (ON), osteocalcin (OC), and dentin matrix protein-1 (DMP-1). Only single transcripts were observed with Northern blotting; therefore it was concluded that the isoforms were due to differences in post-translational modifications. Since medullary bone is rich in keratan sulfate (KS), RT-PCR was used to investigate the expression of known keratan sulfate-containing proteoglycans (KSPGs). Although this tissue was found to express lumican and osteoglycin/mimecan, there was little evidence to suggest that these proteoglycans were a major source of the keratan sulfate glycosaminoglycans. Treatment of medullary bone extracts with keratanase resulted in the appearance of a BSP immunoactive band of approximately 59 kDa. However, it was not possible to isolate and identify the intact keratan sulfate proteoglycan.

Animals↗
PubMed2005

Polymerized bonding agents and the differentiation in vitro of human pulp cells into odontoblast-like cells.

OBJECTIVES: Odontoblasts are highly differentiated post-mitotic cells, which under pathological conditions such as carious lesions and dental injuries may degenerate and be replaced by other pulp cells. We have recently shown that this physiological event can be reproduced in an in vitro assay system, but is highly modified by the presence of unpolymerized resinous monomers. Our hypothesis was that the photopolymerization of the monomers in the bonding agents might abolish these negative effects. The purpose of this study was to evaluate the effects of polymerized dentin bonding agents, through dentin slices, on odontoblast differentiation in vitro. METHODS: Pulp cells were obtained from human third molars. They were used to study the effects of four dentin bonding agents through 0.7 mm dentin slices which served as a barrier between the bonding agents and the culture medium. The media containing the bonding agents' extracts were added at non-toxic concentrations onto the cultured cells. Immunohistochemistry was performed to study the differentiation of pulp fibroblasts into odontoblasts under these conditions by evaluating the expression of several odontoblast specific genes. RESULTS: Pulp fibroblasts cultivated under these conditions synthesized type I collagen, osteonectin, dentin sialoprotein and nestin at the same level as in control cultures. Moreover, pulp cells synthesized a mineralized nodular extracellular matrix. Expression of these proteins was higher in the cells contributing to the nodule formation. In addition, except nestin, all these proteins were expressed in the mineral nodules. SIGNIFICANCE: This work shows the lack of effects of photopolymerized bonding agents, through dentin slices, on cytodifferentiation of secondary odontoblasts.

Adolescent↗
PubMed2007

Initial osteoblast-like cell response to pure titanium and zirconia/alumina ceramics.

OBJECTIVES: Outstanding mechanical properties, resistance to scratching and high biocompatibility make zirconia/alumina ceramics interesting for dental applications. To solve the problem of the well-known low temperature degradation and to provide stable mechanical properties a novel zirconia alloy ((Y,Nb)-TZP/alumina) was developed. The aim of this study was to investigate the initial bone cell response to this new zirconia/alumina composite ceramic. METHODS: HOS cells were cultured on zirconia/alumina composite (Zc) and pure titanium (Ti) discs. Surface topography was examined by atomic force microscopy (AFM), cell morphology by scanning electron microscopy (SEM). Cell proliferation (MTS) and alkaline phosphatase activity was measured at 1, 4 and 8 days. The mRNA expression of Cycline D1, the cell cycle regulating gene, integrin beta 1, osteonectin (ON) and beta-actin were evaluated by RT-PCR analysis after 12, 24 and 48 h. RESULTS: Both substrates showed a very smooth character with R(a)-values in the range of 0.002-0.113 microm supporting a continuous cellular growth. After 8 days, cell proliferation on Zc was higher than on Ti. The mRNA expression of cyclin D1 showed similar activity after 48 h on both surfaces, ALP activity was higher on Zc after 8 days. ON expression however showed no difference between the two groups. SIGNIFICANCE: Our data demonstrate that this new zirconia composite ceramic showed at least equivalent or slightly better biological response of osteoblast-like HOS cells than pure titanium during a short-time cell culture period.

Aluminum Oxide↗
PubMed2004

Osteofibrous dysplasia and adamantinoma: correlation of proto-oncogene product and matrix protein expression.

To investigate the relationship between osteofibrous dysplasia (OFD) and adamantinoma, we analyzed the expression of several proto-oncogene products and extracellular matrix proteins by immunohistochemistry and correlated our results with histological and ultrastructural findings. C-fos and c-jun, but not c-Met, were observed in OFD and in the fibrous and epithelial components of differentiated and classical adamantinomas. Staining for collagen IV, laminin and galectin-3, a laminin binding protein was seen in OFD and around cell nests in adamantinoma. E-, P-, and N-cadherin expression was found in all cases of classical adamantinoma, but not in differentiated adamantinoma or OFD. Osteonectin was detected in both the epithelial and fibrous components of adamantinomas, but osteopontin and osteocalcin were not seen in classical adamantinomas. The results show common expression of a number of oncoproteins and bone matrix proteins in adamantinoma and OFD, some of which are associated with mesenchymal-to-epithelial cell transformation. These findings would be in keeping with the hypothesis that OFD represents a precursor lesion of adamantinoma. Differential expression of a number of bone matrix protein in adamantinoma may also be of diagnostic use in distinguishing these 2 lesions immunohistochemically.

Adolescent↗
PubMed2006

Effects of proinflammatory cytokines on the expression of mineralization markers and heme oxygenase-1 in human pulp cells.

The roles of IL-1alpha and TNF-alpha in early pulp inflammation were investigated by determining the alkaline phosphatase (ALP) activity, the osteonectin (ON), osteocalcin (OC), bone sialoprotein (BSP), and heme oxygenase-1 (HO-1) expression using an immunoblot method. Primary cultured dental pulp cells were treated with IL-1alpha, TNF-alpha, or both for 3, 7, and 14 days. The pulp cells treated with IL-1alpha for 3 days showed elevated ALP activity and increased ON, OC, and HO-1 expression, whereas TNF-alpha treatment did not increase the ALP activity and no BSP was expressed until day 14. The pulp cells treated with both IL-1alpha and TNF-alpha for 3 days showed increased HO-1 expression compared with that of the control. These data suggest that IL-1alpha and TNF-alpha produced in the early inflammatory reaction have different functions in human pulp cells. IL-1alpha induces ALP, ON, and OC in tooth mineralization and it may play a role in the cytoprotection of pulp cells via HO-1 expression, while long-term treatment of TNF-alpha may inhibit the tooth mineralization.

Alkaline Phosphatase↗
PubMed2003

Bone tissue engineering by primary osteoblast-like cells in a monolayer system and 3-dimensional collagen gel.

PURPOSE: To engineer living bone tissue in vitro, bone cells must be multiplied and differentiated in cell culture. Osteoblasts are known to be the crucial cells responsible for the bone modeling process. Periosteal-derived osteoblasts were therefore cultured for up to 3 weeks in Petri dishes as well as in a 3-dimensional collagen gel. METHODS: Proliferation, migration, and differentiation of cells as well as the synthesis of extracellular matrix proteins were monitored during the culture period by histology, electron microscopy, and immunohistochemistry. Mineral formation was investigated by electron diffraction studies and element analysis. RESULTS: Osteoblasts proliferated and migrated in Petri dishes as well as in the collagen gel without loss of viability during the whole experimental period. They demonstrated a mature osteoblast phenotype as indicated by the synthesis of a bone-like extracellular matrix. They formed an extracellular matrix containing osteocalcin, osteonectin, and newly synthesized collagen type I in both environments. Mineral formation was seen in colocalization with the bone-like extracellular matrix proteins in Petri dishes. Microanalytical investigations revealed a matrix vesicle-mediated mineral formation at early stages of culture. CONCLUSIONS: Our cell culture confirmed the ability to multiplicate differentiated and viable osteoblast-like cells in 2- and 3-dimensional space. Additionally, bone-like mineralization can be induced by primary osteoblasts in monolayer culture. The data suggest that this approach can be used as a tool in bone tissue engineering.

Animals↗
PubMed2005

Analysis of cell-seeded 3-dimensional bone constructs manufactured in vitro with hydroxyapatite granules obtained from red algae.

PURPOSE: Bone tissue engineering is a promising approach for the treatment of defective or lost bone in the maxillofacial region. Biocompatible and biodegradable scaffolds seeded with living cells are used to create functional tissue for load-bearing bone reconstruction. The aim of this study was to manufacture cell-seeded 3-dimensional bone constructs based on hydroxyapatite ceramic granule calcified from red algae and mesenchymal cambial-layer precursor cells. The ability of these cells to grow on hydroxyapatite ceramic was quantitatively investigated to evaluate 3-dimensional bone constructs for their potential use in bone tissue engineering. MATERIALS AND METHODS: Mesenchymal cambial-layer precursor cells were isolated from mandibular periosteum biopsy samples of 3 patients. To manufacture 72 bone constructs, these cells and hydroxyapatite ceramic granules (C GRAFT/Algipore; Clinician's Preference LLC, Golden, CO) were cultivated under osteogenic differentiation conditions in a rotating wall vessel system. After 6 and 21 days, histologic examination and scanning electron microscopy were performed. The absolute DNA content, protein synthesis, and alkaline phosphatase activity were also quantified. The osteoblastic phenotype of the constructs was confirmed by the expression of bone-specific genes (osteocalcin, osteonectin, osteopontin, and core binding factor alpha1) using semiquantitative reverse transcription-polymerase chain reaction and Western blot analysis. RESULTS: Cells within the constructs showed good viability, which was evidenced by an increase in DNA content over the culture period. The decrease in alkaline phosphatase-specific activity could be an indicator of the maturation of cells and the induction of mineralization. The osteoblastic phenotype of the constructs was demonstrated on protein and at the RNA level over the entire culture period. CONCLUSION: We observed a positive effect of hydroxyapatite ceramic granules on mesenchymal cambial-layer precursor cell behavior in cell-seeded 3-dimensional bone constructs, indicating the potential applicability of C GRAFT/Algipore composites in bone tissue engineering.

Blotting, Western↗
PubMed2005

Gamma-Linolenic acid regulates the expression and secretion of SPARC in human cancer cells.

SPARC (secreted protein acidic and rich in cystein), also known as osteonectin and BM40, is a secreted glycoprotein. It confers matrix adhesion of cells including cancer cells, thus promoting cell migration. SPARC has been shown to be involved in the aggressive nature of cancer. The current study investigated the role of a n-6 polyunsaturated fatty acid, gamma linolenic acid (GLA) on the expression and secretion of SPARC from cancer cells. Human breast cancer cell line MCF-7 and MDA-MB-231, human colon cancer cells HT115 and HRT-18 were used in the study. Cancer cells were treated with GLA or other fatty acids over a range of concentrations. Presence of SPARC in the supernatant and in the cell lysate were analysed using Western blotting. Cellular SPARC was also assessed using immunocytochemistry. SPARC transcript in these cells were studied using RT-PCR. Cell-matrix adhesion was determined using a cell-matrix adhesion assay and cell migration analysis. Treatment of MDA-MB-231 and HT115 cells with GLA, at non-toxic levels, resulted in reduction of SPARC in supernatant as well as in the cell lysate. In contrast, there were little changes in the supernatant SPARC in MCF-7 and in HRT-18 cells. Cellular SPARC, as revealed by immunocytochemistry, also demonstrated a similar trend of changes as seen with protein blotting. Analysis of the SPARC transcript using RT-PCR has shown an up-regulation of SPARC mRNA by the fatty acid. GLA reduced cell-matrix adhesion in these cancer cells. It is concluded that GLA is a regulator of SPARC secretion and expression in cancer cells. It reduces the secretion of SPARC into surrounding environment, which may contribute to the reduction of cancer cells adhesion to the extracellular matrix and cell motility.

Cell Line, Tumor↗
PubMed2005

Early gene response to low-intensity pulsed ultrasound in rat osteoblastic cells.

The aim of the current research was to quantify the changes in gene expression in rat bone marrow derived stromal cells (BMSC) to low intensity pulsed ultrasound (LIPUS) during early time points after the ultrasound application. LIPUS at 1.5 MHz, 30 mW/cm(2) was applied to BMSC for a single 20 min treatment. Real-time PCR was carried out to quantify the expression of early response genes and bone differentiation marker genes 0.5, 1, 3, 6 and 12 h after the end of the LIPUS treatment. Compared with the controls, LIPUS treatment resulted in elevated transient expression of early response genes (c-jun, c-myc, COX-2, Egr-1, TSC-22) as well as the bone differentiation marker genes, osteonectin and osteopontin, at 3 h. This induction of early response genes as well as extracellular matrix genes associated with cell proliferation and differentiation may represent the effect of LIPUS to cells of osteoblastic lineage.

Animals↗
PubMed2004

Gene expression in human osteoblastic cells from normal and heterotopic ossification.

Heterotopic ossification (HO), a possible complication of head injury, develops in sites where it is not normally present like at the vicinity of joints. It may cause pain, decrease motion and in severe cases complete joint ankylosis requiring surgical intervention. To our knowledge, no study has been made to analyze HO at the molecular level on human biopsies, whereas its etiology remains to be determined. We defined a procedure of cell fractionation from bone resections and developed quantitative RT-PCR to compare genetic expression patterns between human normal osteoblasts and heterotopic ossification forming cells. This quantitative study demonstrated a specific and strong overexpression of osteocalcin mRNA in HO-isolated cells associated with a significant upregulation of type 1 collagen and osteonectin mRNA while histological analysis showed only small cellular variations. Our results give a first molecular characterization of heterotopic ossification and we conclude that such overexpressions in HO-isolated cells could be associated with the high activity of this pathological bone.

Adult↗
PubMed2002

The induction of c-fos mRNA expression by mechanical stress in human periodontal ligament cells.

The periodontal ligament is subjected to mechanical loading during occlusion and mastication. Although internuclear transcription factors are associated with the regulatory pathway that converts these extracellular mechanical stimuli into a cellular response, there are no reports on these in human periodontal ligament fibroblasts. In this study, the amounts of c-fos mRNA in human periodontal ligament fibroblasts were investigated shortly after subjecting them to a cyclic tension force in vitro. The mRNA of alkaline phosphatase and the matrix proteins type I collagen, type III collagen, matrix Gla-protein, osteonectin, osteopontin, and osteocalcin were also examined. A significant, rapid, transient increase in c-fos mRNA was detected, which peaked 30 min after the application of mechanical force. However, there was no significant change in the mRNA for alkaline phosphatase or the matrix proteins. These results provide evidence that periodontal ligament fibroblasts and c-fos may play a critical part in the response of periodontal tissue to mechanical stimulation.

1-Carboxyglutamic Acid↗
PubMed2003

Effect of micromechanical stimulations on osteoblasts: development of a device simulating the mechanical situation at the bone-implant interface.

Many experimental models have been developed to investigate the effects of mechanical stimulation of cells, but none of the existing devices can simulate micromotions at the cellular-mechanical interface with varying amplitudes and loads. Osteoblasts are sensitive to mechanical stimuli, so to study the bone-implant interface it would be important to quantify their reaction in a situation mimicking the mechanical situation arising at that interface. In this study, we present the development of a new device allowing the application of micromotions and load on cells in vitro. The new device allowed the cells to be stimulated with sinusoidal motions of amplitudes comprised between +/- 5 and +/- 50 microm, frequencies between 0.5 and 2 Hz, and loads between 50 and 1000 Pa. The device, with a total length of 20 cm, was designed to work in an incubator at 37 degrees C and 100% humidity. Expression of various bone important genes was monitored by real-time RT-PCR. Micromotions and load were shown to affect the behavior of osteoblasts by down-regulating the expression of genes necessary for the creation of organic extracellular matrix (collagen type I) as well as for genes involved in the mineralization process (osteocalcin, osteonectin). The developed device could then be used to simulate different mechanical situations at the bone-implant interface.

Bone and Bones↗
PubMed1996

Characterization of IGFBP-3, PAI-1 and SPARC mRNA expression in senescent fibroblasts.

The RNA species encoded by IGFBP-3 (insulin-like growth factor binding protein-3), PAI-1 (plasminogen activator inhibitor-1) and SPARC (secreted protein-acidic and rich in cysteine; a.k.a. osteonectin) are overexpressed in senescent human diploid fibroblasts (HDF). Their extracellular products have the ability to modulate cell growth in culture and have been shown to have inhibitory effects on DNA synthesis and/or cell growth. This overproduction may contribute to a number of features of aging, including osteoporosis, atherosclerosis and diabetes mellitus type II. Based on analysis of steady-state mRNA levels, which showed similar patterns for all three along with overexpression in senescent cells, we further investigated their transcription rates and stability to determine reasons for their overexpression and to determine if coordinate gene regulation was involved. Characterization of the rates of transcription and the levels of message stability of these genes in early passage (young) versus late passage (old) HDF revealed that IGFBP-3, PAI-1 and SPARC are coordinately overexpressed but not regulated by a unique or simple mechanism encompassing all three transcripts. Only PAI-1 shows an increase in the rate of transcription, while all three show evidence that their overexpression is due to an increase in the stability of RNA. Thus, the overexpression of these genes in senescent fibroblasts involves interactions not only at the transcriptional level but also with protein factors involved in determining the stability and the degradation of RNA.

Blotting, Northern↗
PubMed2001

Glass reinforced hydroxyapatite for hard tissue surgery--part II: in vitro evaluation of bone cell growth and function.

Hydroxyapatite (HA)-based materials are considered to be potentially useful as bone implant materials, particularly those reinforced with glass to improve mechanical strength. However, the precise effects of glass-reinforced HA on the growth and functions of bone cells are still unclear. The present study has therefore examined the response of human osteoblast-like cells to HA and HA reinforced with two different proportions of glass, namely 2.5% and 5%. All materials enabled the cells to attach and proliferate during 7 days in culture and, although the growth was less than on control plastic surfaces, there was no deleterious effect of the 5% glass composite compared with HA alone. Flow cytometry analysis showed that there was no effect on cell size and granularity, but there were marked and highly selective changes in the expression of certain connective tissue proteins. Thus, while bone sialoprotein and osteonectin were down-regulated on HA alone, the expression of these antigens was relatively enhanced on the composite materials, and collagen type I was also up-regulated on the glass-reinforced HA. Thus, modulation of the glass composition of HA materials could be used to produce not only improved mechanical strength, but also enhanced biocompatibility.

Biocompatible Materials↗
PubMed2004

The influence on gene-expression profiling of osteoblasts behavior following treatment with the ionic products of sintered beta-dicalcium pyrophosphate dissolution.

Sintered dicalcium pyrophosphate (SDCP) is biocompatible to bone tissue both in the in vivo and in vitro model. However, the molecular mechanisms that mediated these processes have yet to be identified. In this study, we investigated the influence of SDCP ions on in vitro osteoblasts behavior. The powder of sintered beta-dicalcium pyrophosphate (SDCP) was dissolved by HCl and then diluted into different concentration of solutions by culture medium used in the osteoblast cell culture. The effects of various concentration of SDCP on bone cell activities were evaluated by using MTT assay. For the differentiation of osteoblasts, alkaline phosphatase (AP) staining, von Kossa stain for mineralized nodules and bone markers messenger ribonucleic acid (mRNA) isolation and identification were performed at 3h, days 1, 3, 7 and 14. In the presence of 10(-8)M SDCP for 14 days, the osteoblasts population was still significantly higher than that of control. In the qualitative analysis for the formation of AP staining colonies and mineralization nodules formation were not affected by SDCP ions. When osteoblasts cultured in the presence of 10(-8)M SDCP ions, the osteocalcin mRNA expression was up-regulated; while the collagen, osteonectin and osteopontin mRNA expression were down-regulated. In this study, we demonstrated that the elevated concentration of calcium and pyrophosphate ions can activate genes of the bone cells. This study will contribute to a better understanding of cell/biomaterial interactions and mechanisms that SDCP affect the bone cells.

Animals↗