Bone Sialoprotein mRNA Expression Research Articles

Natural Mineralized Scaffolds Promote the Dentinogenic Potential of Dental Pulp Stem Cells Via the Mitogen-Activated Protein Kinase Signaling Pathway

The selection of a suitable scaffold material is important for dentin tissue regeneration, as the characteristics of biomaterials can potentially influence cell proliferation and differentiation. We compared the effects of different scaffolds on dentin regeneration based on dental pulp stem cells (DPSCs) and investigated the regulatory mechanisms of odontogenic differentiation of DPSCs by these scaffolds. Five different scaffolds were tested: demineralized dentin matrix (DDM), ceramic bovine bone (CBB), small intestinal submucosa (SIS), poly-L-lactate-co-glycolate, and collagen-chondroitin sulfate-hyaluronic acid. DPSCs cultured on DDM and CBB exhibited higher levels of alkaline phosphatase (ALP) activity and mRNA expression of bone sialoprotein, osteocalcin, dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) than those cultured on the other three scaffolds. Further, the phosphorylation levels of mitogen-activated protein kinase (MAPK) ERK1/2 and p38 in DPSCs cultured on DDM and CBB were also significantly enhanced compared with the other three scaffolds, and their inhibitors significantly inhibited odontogenic differentiation as assessed by ALP activity and mRNA expression of DSPP and DMP-1. The implantation experiment confirmed these results and showed a large amount of regular-shaped dentin-pulp complex tissues, including dentin, predentin, and odontoblasts only in the DDM and CBB groups. The results indicated that natural mineralized scaffolds (DDM and CBB) have potential as attractive scaffolds for dentin tissue-engineering-promoted odontogenic differentiation of DPSCs through the MAPK signaling pathway.

Cytotoxic effect of eugenol on the expression of molecular markers related to the osteogenic differentiation of human dental pulp cells

The cytotoxic effect of eugenol on the expression of molecular markers related to the osteogenic differentiation of human dental pulp cells such as collagen synthesis and the expression of two osteogenesis-related genes, alkaline phosphatase (ALP) and bone sialoprotein (BSP), was studied using human dental pulp cells (D824 cells). Cellular growth and survival were decreased by treatment of cells with eugenol in a concentration-dependent manner. The incorporation rate of [(3)H] proline into the acid-insoluble fraction and the synthesis of type I-V collagens were also reduced by treatment of cells with eugenol in a concentration-dependent fashion. The mRNA expression of ALP was scarcely affected in cells exposed to eugenol, whereas the mRNA and protein expression of BSP was down-regulated depending on the concentrations of eugenol. The results suggest that because collagen synthesis and BSP expression play a critical role in hard tissue formation, eugenol used for endodontic treatment may give rise to cytotoxic effects to the normal function of stem cells reported to exist in human dental pulp tissue and periodontal ligament.

Inhibition of metastasis-associated gene 1 expression affects proliferation and osteogenic differentiation of immortalized human mesenchymal stem cells

MTA1 is known to be responsible for independent nucleosome remodelling and deacetylase complexes with ability to regulate divergent cellular pathways. However, additional biological functions have, up to now, remained largely unexplored. The present study was initiated to investigate involvement of MTA1 in osteogenic differentiation of immortalized human mesenchymal stem cells (MSCs). MSCs were examined for expression of MTA1 and stably transfected clones expressing shRNA to MTA1 were generated. Cells were grown under osteogenic and non-osteogenic conditions. Effects of silencing on cell proliferation, calcium deposition and alkaline phosphatase (ALP) activity were studied. mRNA expression of bone sialoprotein (BSP), osteopontin (OSP), runt-related transcription factor 2 (Runx2), osteocalcin (OC), collagen type I (Col1A) and ALP were analysed. Transfected cells showed reduction in proliferation and significant increase in calcium deposition and expression of osteogenic marker genes, BSP, OSP, Runx2, OC and Col1A, when they were grown under osteogenic conditions. Under non-osteogenic conditions, expression of BSP and OSP were also markedly upregulated, whereas expression of osteogenic marker genes, Runx2, OC and Col1A, was almost unaffected. Expression of ALP was slightly suppressed under non-osteogenic conditions but significantly increased under osteogenic differentiation conditions, as assessed by enzyme activity and mRNA expression assays. Our data collectively suggest that endogenously produced MTA1 constrains osteogenic differentiation of MSCs and that targeting of this molecule may provide a novel strategy for enhancing bone regeneration.

Effects of Vitamin E on the Osteoblast Differentiation

Vitamin E is thought to affect bone formation and bone remodeling. In this study, we investigated the effects of vitamin E (alpha-tocopherol and delta-tocopherol) on the osteoblasts isolated from rat calvariae. At 4 and 7 days (Day 4 and 7) after induction of osteoblastic differentiation, treatment of alpha-tocopherol (100 and 200 microM) and delta-tocopherol (2 and 20 microM) for 3 days significantly decreased alkaline phophatase activity of the cultured osteoblasts. At Day 14, however, no significant change was detected in ALP activity and expression of bone sialoprotein mRNA in the osteoblasts treated with alpha-tocopherol or delta-tocopherol for 3 days. Expression of osteocalcin mRNA was decreased by treatment of alpha-tocopherol (100 and 200 microM) and delta-tocopherol (2 and 20 microM) at Day 4 and 7. At Day 14, expression of osteocalcin mRNA was decreased only with treatment of 200 microM alpha-tocopherol. In addition, the noncalcified nodules were decreased by treatment of alpha-tocopherol (200 microM) and delta-tocopherol (20 microM) at Day 7. However, treatment of alpha-tocopherol and delta-tocopherol showed no significant change of formation of calcified nodules at Day 14. These results indicate that vitamin E inhibits differentiation of osteoblasts especially from early stage to osteoid-producing stage.

Zonal Responsiveness of the Human Intervertebral Disc to Bone Morphogenetic Protein-2

In vitro experiment using bone morphogenetic protein-2 (BMP-2) and cells from the nucleus pulposus (NP), transitional zone (TZ), and anulus fibrosus (AF) of the human intervertebral disc (IVD). To demonstrate the differential effect of BMP-2 on DNA synthesis, proteoglycan synthesis, and osteocalcin mRNA expression in human IVD cells from the NP, TZ, and AF, respectively. BMP-2 has been proven to be effective in stimulating proteoglycan synthesis in articular chondrocytes and IVD cells from the NP. Nevertheless, the effect of BMP-2 on cells from different regions of the IVD has not yet been thoroughly elucidated. Human IVDs were harvested from surgical disc procedures and tissue from the NP, TZ, and AF was obtained. Disc tissue was enzymatically digested, and IVD cells were cultured three-dimensionally in alginate beads. Then IVD cell cultures from the NP, TZ, and AF were exposed to BMP-2. DNA synthesis and newly synthesized proteoglycan were measured. Reverse transcription-polymerase chain reaction for mRNA expression of bone sialoprotein, DLX5, osteocalcin, and collagen type I, was performed. Cells from the AF responded to BMP-2 with mitogenesis. There was no significant increase in DNA synthesis in cultures from the NP and TZ treated with BMP-2. Only cells from the NP showed a significant increase in newly synthesized proteoglycan in response to BMP-2. IVD cells from all zones demonstrated no significant expression of bone sialoprotein, DLX5, osteocalcin mRNA after treatment with BMP-2. BMP-2 clearly exerted a mitogenic effect on AF cells, and stimulated proteoglycan synthesis in NP cells. However, BMP-2 did not have an osteogenic effect in any IVD region. Taken together, these results confirm that BMP-2 can be used as an anabolic agent for mitogenesis in AF cells and NP cell matrix regeneration without the possibility of osteogenesis.

Mechanical Tensile Stress Effects on the Expression of Bone Sialoprotein in Bovine Cementoblasts

To develop a new cementoblast culture method and to detect bone sialoprotein (BSP) expression in response to high and low mechanical tensile stress in cementoblast in vitro. Cementoblasts were collected from the roots of newborn bovine teeth and were identified with cementum-derived attachment protein (CAP) antibody 3G9. Cell proliferation was evaluated by MTT [3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, and mineralization was confirmed by von Kossa staining. Mechanical tensile stress was applied in vitro to the cementoblast with the use of a uniaxial four-point bending system with 2000 or 4000 microstrains, at a frequency of 0.5 Hz for 3, 6, 12, 24, or 36 hours. BSP mRNA level was quantified by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). A large amount of cementoblast was observed to be expressing CAP. Cementoblasts had a proliferation tendency similar to that of osteoblasts but different from that of periodontal ligament (PDL) cells. Cementoblasts had the ability to become mineralized between osteoblasts and PDL cells. The mechanical tensile stress significantly up-regulated BSP mRNA expression, which reached a peak at 24 hours in both 2000 and 4000 microstrain groups (P < .01) and was tenfold and sixfold higher than that of controls, respectively. BSP expression dropped toward baseline levels at 36 hours in both groups. Mechanical tensile stress up-regulated the expression of BSP. Low mechanical tensile stress induced earlier and more intensive up-regulation of BSP mRNA; this might represent the optimal stimuli for cementoblast activity.

Regulation of human bone sialoprotein gene transcription by platelet-derived growth factor-BB

Platelet-derived growth factor (PDGF) is produced by mesenchymal cells and released by platelets following aggregation and is synthesized by osteoblasts. In bone, PDGF stimulates proliferation and differentiation of osteoblasts. PDGF also increases bone resorption, most likely by increasing the number of osteoclasts. Bone sialoprotein (BSP) is thought to function in the initial mineralization of bone, selectively expressed by differentiated osteoblast. To determine the molecular mechanisms PDGF regulation of human BSP gene transcription, we have analyzed the effects of PDGF-BB on osteoblast-like Saos2 and ROS17/2.8 cells. PDGF-BB (5 ng/ml) increased BSP mRNA and protein levels at 12 h in Saos2 cells, and induced BSP mRNA expression at 3 h, reached maximal at 12 h in ROS17/2.8 cells. Transient transfection analyses were performed using chimeric constructs of the human BSP gene promoter linked to a luciferase reporter gene. Treatment of Saos2 cells with PDGF-BB (5 ng/ml, 12 h) increased luciferase activities of all constructs between – 184LUC to – 2672LUC including the human BSP gene promoter. Effects of PDGF-BB abrogated in constructs included 2 bp mutations in the two cAMP response elements (CRE1 and CRE2), activator protein 1(3) (AP1(3)) and shear stress response element 1 (SSRE1). Luciferase activities induced by PDGF-BB were blocked by protein kinase A inhibitor H89 and tyrosine kinase inhibitor herbimycin A. Gel mobility shift analyses showed that PDGF-BB increased binding of CRE1, CRE2, AP1(3) and SSRE1 elements. CRE1- and CRE2-protein complexes were supershifted by CREB1 and phospho-CREB1 antibodies. Notably, AP1(3)-protein complexes were supershifted by c-Fos and JunD, and disrupted by CREB1, phospho-CREB1, c-Jun and Fra2 antibodies. These studies, therefore, demonstrate that PDGF-BB stimulates human BSP transcription by targeting the CRE1, CRE2, AP1(3) and SSRE1 elements in the human BSP gene promoter.

Effects of Ultrasound on the Proliferation and Differentiation of Cementoblast Lineage Cells

The purpose of this study was to investigate the effects of low-intensity pulsed ultrasound (LIPUS) stimulation on the proliferation and differentiation of cementoblast lineage cells. An immortalized human periodontal ligament cell line (HPL) showing immature cementoblastic differentiation was used. Cultured HPL cells were subjected to LIPUS exposure (frequency = 1 MHz; pulsed 1:4; intensity = 30 mW/cm(2)) or sham exposure for 15 minutes per day. Expression levels of alkaline phosphatase (ALP), type I collagen (Col-I), runt-related gene 2 (Runx2), bone sialoprotein (BSP), osteocalcin (OCN), and osteopontin (OPN) mRNA were analyzed with real-time polymerase chain reaction analysis. Furthermore, ALP activity, collagen synthesis, and protein level of Runx2 were examined after 6 days of LIPUS exposure. mRNA and protein levels of ALP, Col-I, and Runx2 were significantly increased by LIPUS exposure compared to controls, whereas BSP, OCN, and OPN mRNA expression could not be detected in HPL cells, irrespective of LIPUS exposure. LIPUS enhanced ALP activity, collagen synthesis, and Runx2 expression of HPL cells, which provides important insight into the promotion of early cementoblastic differentiation of immature cementoblasts.

Stimulation of osteoblasts with Emdogain increases the expression of specific mineralization markers

The purpose of this study was to determine the effects of enamel matrix derivative on mRNA expression of markers related to periodontal healing. Murine osteoprogenitor cells (MC3T3-E1) were grown for 12 and 16 days in mineralization media and stimulated with 100 microg/mL Emdogain (EMD). Cell cultures treated with 2% and 10% fetal calf serum (FCS) served as control. The mRNA expression of bone sialoprotein (BSP), osteopontin (OPN), and runt-related protein 2 (Runx2) was analyzed by real-time polymerase chain reaction. One-way analysis of variance was used for statistical analysis. Stimulation with EMD significantly (P < .01) enhanced mRNA expression of BSP up to 13.9-fold and of OPN up to 3.2-fold at day 16 compared with the 2% FCS control. The expression of mRNA for transcription factor Runx2 was not significantly changed. The beneficial effects seen in periodontal regeneration after treatment with EMD may be related to an increase of the mineralization markers BSP and OPN at mRNA level.

Osteoblast proliferation and maturation on bioactive fiber-reinforced composite surface

The objective of this study was to evaluate the proliferation and osteogenic potential of bone-marrow derived osteoblast-like cells on fiber-reinforced composite (FRC) substrates with and without bioactive glass surface modification. Three FRC materials were fabricated for the study: (a) grit-blasted FRC, (b) grit-blasted FRC with bidirectional net reinforcement and (c) FRC with bioactive glass (BAG) coating. Rat bone-marrow derived osteoblast-like cells were harvested and cultured on experimental material plates and on cp. titanium plates (control) for 21 days. The materials' surfaces were characterized by roughness testing and scanning electron microscopy. Cell growth and differentiation kinetics were subsequently investigated by evaluating proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) and bone sialoprotein (BSP) production. On day 14, the cell proliferation was significantly lower (P<0.05) on FRC-BAG than on titanium and FRC. The proliferation on the other three materials was equal throughout the experiment. The maximal ALP activities on FRC, FRC-Net, and titanium were observed on day 21, whereas FRC-BAG had already reached the maximal level on day 14. Expression of osteoblastic markers (OC, BSP) indicates that the fastest osteogenic differentiation takes place on FRC after 7 days. In contrast, a slower differentiation process was observed on titanium than on any other tested material (P<0.015) at 21 days, as was confirmed by increased mRNA expression of OC and BSP. It can be concluded that the proliferation and maturation of osteoblast-like cells on FRC appears to be comparable to titanium. Presence of BAG enhances cell maturation.

Cellular responses of rat periodontal ligament cells under hypoxia and re‐oxygenation conditions in vitro

The aim of this study was to investigate the responses of periodontal ligament cells under hypoxia and re-oxygenation conditions in vitro. Periodontal ligament fibroblasts were isolated from rat incisors. In the hypoxia group, cells were incubated in 2% O(2) for 1-3 d. In the re-oxygenation group, cells were first incubated under the same conditions as the hypoxia group for 24 h and then were returned to normoxic conditions and cultured for 1-2 additional days. Proliferation ratios increased in all groups in a time-dependent manner. Proliferation ratios in both the hypoxia and re-oxygenation groups were significantly higher than in the control group on days 2 and 3. Alkaline phosphatase activity was significantly higher in the hypoxia group than in the control and the re-oxygenation groups. The expression of bone sialoprotein mRNA was significantly higher in the hypoxia group than in the control group on days 1 and 2. The expression of vascular endothelial growth factor mRNA was significantly higher in the hypoxia group than in the control group on days 1 and 2. In the re-oxygenation group, the level of expression of bone sialoprotein mRNA and vascular endothelial growth factor mRNA were similar to those of the control group. The expression of heat shock protein 70 mRNA in the hypoxia group was similar to that in the control group, whereas in the re-oxygenation group it was statistically higher than in the other groups. These results suggest that periodontal ligament cells maintain their osteogenic ability in hypoxia and re-oxygenation conditions in vitro.

Influence of TGF-β1 on the expression of BSP, DSP, TGF-β1 receptor I and Smad proteins during reparative dentinogenesis

Reparative dentin has a wide variety of manifestations ranging from a regular, tubular form to an irregular, atubular form. However, the characteristics of reparative dentin have not been clarified. This study hypothesized that the level of bone sialoprotein (BSP) expression will increase if the newly formed reparative dentin is bone-like but the dentin sialophosphoprotein (DSPP) level will decrease. In order to test this hypothesis, the expression of BSP and DSP was examined by immunohistochemistry and the expression of BSP was measured by in situ hybridization in an animal model. The pulps of 12 maxillary right first molars from twelve male rats were exposed and capped with MTA. In addition, in order to understand the role of transforming growth factor-beta 1 (TGF-beta1) during reparative dentinogenesis, the expression of BSP and DSPP mRNA was analyzed by RT-PCR in a human dental pulp cell culture, and the transforming growth factor-beta 1 receptors (TbetaRI) and Smad 2/3 were examined by immunofluorescence in an animal model. DSP was expressed in the normal odontoblasts and odontoblast-like cells of the reparative dentin. Interestingly, BSP was strongly expressed in the odontoblast-like cells of reparative dentin. The level of the TbetaRI and Smad 2/3 proteins was higher in the reparative dentin than in the normal dentin. TGF-beta1 up-regulated BSP in the human pulp cell cultures. This suggests that reparative dentin has both dentinogenic and osteogenic characteristics that are mediated by TGF-beta1.

Behavior of Human Dental Pulp Cells Exposed to Transforming Growth Factor-Beta1 and Acidic Fibroblast Growth Factor in Culture

The development of methods for regenerative endodontic procedures requires an understanding of the factors regulating the development of odontoblasts from adult cell populations such as pulpal cell lines. In this study, we exposed cultures of human pulp cells (7th passage) to growth factors including transforming growth factor-beta1 (TGF-beta1, at 1 or 5 ng/mL), acidic fibroblast growth factor (aFGF, 5 ng/mL), or a combination of the 2 growth factors and evaluated cellular morphology and markers of cell phenotype including alkaline phosphatase activity, osteocalcin, bone sialoprotein (BSP), and dentin sialophosprotein (DSPP). The mean number of nucleoli in the 1 ng/mL TGF-beta1 group was significantly higher than with 5 ng/mL aFGF. Alkaline phosphatase activity was significantly greater with 1 ng/mL TGF-beta1 versus 5 ng/mL TGF-beta1 + 5 ng/mL aFGF (P < .05). Osteocalcin mRNA was expressed in all samples. The cells exposed to 1 ng/mL TGF-beta1 were stimulated; however, exposure to growth factors for 8 days was not sufficient for expression of BSP and DSPP mRNA. Cells treated with 1 ng/mL TGF-beta1 exhibited higher activity, whereas 5 ng/mL aFGF-treated cells were inhibited. Although osteocalcin was observed in all cultures, suggestive of the potential for odontoblast formation, under the present conditions, the exposure to TGF-beta1 and aFGF was not sufficient to induce expression of the dentin matrix components BSP and DSPP.

Effects of quercetin and quercetin 3‐glucuronide on the expression of bone sialoprotein gene

Quercetin is a typical flavonol-type flavonoid and is present in a variety of vegetables, and their antioxidant effect implies their possible role in the prevention of oxidative stress related chronic diseases. Bone sialoprotein (BSP) is a noncollagenous protein of the extracellular matrix in the mineralized connective tissues that has been implicated in the nucleation of hydroxyapatite crystals. Previously, we reported that isoflavone (genistein) activated BSP gene transcription is mediated through an inverted CCAAT box in the proximal BSP gene promoter. The present study investigates the regulation of BSP transcription in a rat osteoblast-like cell line, ROS 17/2.8 cells, by quercetin and its conjugated metabolite quercetin 3-glucuronide. Quercetin and quercetin 3-glucuronide (5 microM) increased the BSP mRNA levels at 12 h and quercetin upregulated the Cbfa1/Runx2 mRNA expression at 12 h. From transient transfection assays using various sized BSP promoter-luciferase constructs, quercetin increased the luciferase activity of the construct (pLUC3), including the promoter sequence nucleotides -116 to -43. Transcriptional stimulations by quercetin were almost completely abrogated in the constructs that included 2 bp mutations in the inverted CCAAT and FRE elements whereas the CCAAT-protein complex did not change after stimulation by quercetin according to gel shift assays. Quercetin increased the nuclear protein binding to the FRE and 3'-FRE. These data suggest that quercetin and quercetin 3-glucuronide increased the BSP mRNA expression, and that the inverted CCAAT and FRE elements in the promoter of the BSP gene are required for quercetin induced BSP transcription.

Bone Sialoprotein Gene Transfer to Periodontal Ligament Cells May Not Be Sufficient to Promote Mineralization In Vitro or In Vivo

To improve regenerative therapies, it is important to understand the cells and factors modulating periodontal tissues. Our group has focused on bone sialoprotein (BSP), a mineralized tissue-selective protein considered to be involved in the initiation of cementogenesis and osteogenesis. In this study, we examined whether gene transfer of BSP into periodontal ligament (PDL) cells would result in an increased ability of PDL cells to promote mineralization in vitro and in vivo. PDL cells obtained from CD-1 mice were immortalized using simian virus (SV) 40 large T antigen (TAg) and designated SV-PDL cells. SV-PDL cells were infected in vitro with LacZ gene-expressing control adenovirus vector. A 1,000 plaque-forming unit (pfu) titer was selected (based on X-gal staining) and cells were infected with mouse BSP-expressing replication-deficient adenoviral vector to determine the mRNA expression and protein level of BSP. Total RNA was isolated from cells on days 2, 4, and 6. Media were obtained on days 3, 5, and 7 for protein determination. Northern blot analysis was performed for mRNA expression and Western blot analysis for protein expression. To test the effect of BSP gene transfer on the mineralization of PDL cells, in vitro (von Kossa) and in vivo (severe combined immunodeficiency [SCID] mice) experiments were performed. Under normal conditions, PDL cells do not express BSP transcripts and do not promote significant mineralization. SV-PDL cells infected with a BSP viral vector expressed and secreted substantial levels of BSP as confirmed by Northern and Western blot analysis. BSP mRNA and protein levels were strong on day 2 and still apparent on day 6, although not as great. However, no mineral nodule formation was noted either in vitro or in vivo. Although BSP is an important and necessary protein for mineralization, it may not be sufficient for promoting mineralization without the addition or removal of other factors. Further studies will help to clarify the specific factors required for promoting mineralization, a required step for designing predictable periodontal regenerative therapies.

歯根膜細胞におけるＯｓｔｅｒｉｘ転写因子の発現に関する研究

Periodontal ligament (PDL) has a heterogeneous cell population, where some of the cells may be capable of differentiating into either cementoblasts or osteoblasts. Recently, C 2 H 2 zinc finger transcription factor Osterix has been reported. Osterix is one of the master regulators of bone cell differentiation and it has two different isoforms. According to a recent report, osteogenic differentiation of murine embryonic stem cells can be induced by overexpression of Osterix. The purpose of this study was to investigate about the expression of Osterix on human PDL (hPDL), and whether the osteogenic differentiation of hPDL cells can be induced by overexpression of Osterix. hPDL cells were obtained from healthy human teeth indicated for extraction for orthodontic treatment. All procedure used in this study was approved by the local ethical committee of Tokyo Medical and Dental University. To investigate expression of Osterix mRNA in hPDL tissues and cells, RT-PCR experiments were performed. Two different isoform Osterix expression vectors were made and transiently transfected into hPDL cells. Osteogenic differentiation was assessed by RT-PCR for genes associated with the osteoblast lineage such as Osteopontin, Osteocalcin, and Bone Sialoprotein. RT-PCR analyses showed that osterix mRNA was expressed in both hPDL tissue and cells. The expression of Osterix short isoform was higher than that of the long isoform. Overexpression of Osterix induced upregulated expression of Bone Sialoprotein mRNA. In expression levels of Osteopontin and Osteocalcin mRNA, compared to the control, no difference was observed. In conclusion, Osterix plays important roles in the osteoblastic differentiation in hPDL cells and modulates the mineralization.

Role of Bovine Bone Morphogenetic Proteins in Bone Matrix Protein and Osteoblast-Related Gene Expression During Rat Bone Marrow Stromal Cell Differentiation

Bone morphogenetic proteins (BMPs) are known to promote osteogenesis, and clinical trials are currently underway evaluating the ability of certain BMPs to promote bone graft and fracture healing. To observe the mechanism of osteoinductive and bone formation, 100 microg of bovine BMP was tested during osteogenic differentiation of rat bone marrow stromal cells (MSCs) and C2C12 line culture for 14 and 28 days. We examined alkaline phosphatase (ALP) by assay, immunohistochemical studies for bone matrix proteins, and mRNA expression of bone matrix proteins and osteoblast-related analysis by reverse-transcription polymerase chain reaction. ALP activity in MSC cultures was elevated by bovine BMP by two to fivefold (P < 0.05-0.001). DNA and protein content increased over 14 days. BMP significantly increased the mRNA expression of type I collagen, ALP, osterix, osteocalcin, osteopontin, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-A, and parathyroid hormone receptor time dependently during the osteoblastic differentiation. There was no markedly enhanced mRNA expression of bone sialoprotein (BSP) and glyceraldehyde-3-phosphate dehydrogenase compared with that of control. Immunohistochemical results also showed BMP increased immunoreactive positivity of type I collagen, osteocalcin, osteonectin, osteopontin, and BSP during the C2C12 differentiation. These data indicated that BMP enhances our ability to stimulate the differentiation of osteoblast-like cells and increases osteoinductivity, bone matrix protein formation and mineralization, angiogenesis, and chondrogenesis during osteoblast progenitor cell differentiation in vitro and that the role of chondrogenic is weak.

An In Vivo Model to Study Osteogenic Gene Regulation: Targeting an Avian Retroviral Receptor (TVA) to Bone With the Bone Sialoprotein (BSP) Promoter

To study bone development in vivo, a transgenic mouse model was established in which an avian retroviral receptor (TVA) gene driven by the BSP promoter was selectively expressed in skeletal tissues. The model was validated by showing suppressed BSP expression and delayed bone and tooth formation after infection with a virus expressing a mutated Cbfa1/Runx2 gene. Tissue-specific expression of the avian retroviral (TVA) receptor can be used to efficiently target ectopic expression of genes in vivo. To determine the use of this approach for studies of osteogenic differentiation and bone formation at specific developmental stages, transgenic mice expressing the TVA receptor under the control of a 5-kb bone sialoprotein (BSP) promoter were generated. The mice were first analyzed for tissue-specific expression of the TVA gene and then, after infection with a viral construct, for the effects of a dominant-negative form of the Cbfa1/Runx2 transcription factor on bone formation. We first generated transgenic mice (BSP/TVA) in which the TVA gene was expressed under the control of a 4.9-kb mouse BSP promoter. The tissue-specific expression of the TVA gene was analyzed by RT-PCR, in situ hybridization, and immunohistochemistry and compared with the expression of the endogenous BSP gene. A 396-bp fragment of mutated Cbfa1/Runx2 (Cbfa1mu) encoding the DNA-binding domain was cloned into a RCASBP (A) viral vector, which was used to infect neonatal BSP/TVA mice. Expression of the TVA receptor mRNA and protein in the transgenic mice was consistent with the expression of endogenous BSP. Four days after systemic infection with the Cbfa1mu-RCASBP (A) vector, RT-PCR analyses revealed that the expression of BSP mRNA in tibia and mandibles was virtually abolished, whereas a 30% reduction was seen in calvarial bone. After 9 days, BSP expression in the tibia and mandible was reduced by 45% in comparison with control animals infected with an empty RCASBP vector, whereas BSP expression in the membranous bone of calvariae was decreased approximately 15%. However, after 4 and 8 weeks, there was almost no change in BSP expression in any of the bone tissues. In comparison, a reduction in osteopontin expression was only observed 9 days after viral transfection in the three bones. Histomorphological examination revealed that bone formation and tooth development were delayed in some of the mice infected with mutated Cbfa1. These studies show that BSP/TVA transgenic mice can be used to target genes to sites of osteogenesis, providing a unique system for studying molecular events associated with bone formation in vivo.

Optimal compressive force induces bone formation via increasing bone sialoprotein and prostaglandin E 2 production appropriately

Although orthodontic tooth movement can promote bone formation, the molecular mechanism that underlies this phenomenon is not fully understood. The purposes of this study were to determine how mechanical stress affects the osteogenic response of human osteoblastic cells (Saos-2), and also examine the optimal compression for osteogenesis in vitro. Saos-2 cells cultured with or without continuously compressive force (0.5∼3.0 g/cm 2). The expression of bone sialoprotein (BSP), osteopontin, and cyclooxygenase-2 (COX-2) were measured using real-time PCR, Western blot analysis and immunoassay. The calcium content in the mineralized nodules was determined using Calcium C-Test kit. Only one loading with 1.0 g/cm 2 of compressive force significantly increased the expression of BSP mRNA and protein, COX-2 mRNA expression and PGE 2 synthesis. Indomethacin, an inhibitor of PGE 2 synthesis, inhibited the compression-induced above phenomenon. Moreover, the conditioned medium from 1.0 g/cm 2 of compressive force apparently stimulated calcium content in mineralized nodules. This study demonstrates that an optimal compressive force stimulates in vitro mineralization by BSP synthesis through the autocrin action of PGE 2 production.

In vitro and in vivo evaluation of differentially demineralized cancellous bone scaffolds combined with human bone marrow stromal cells for tissue engineering

Mineralized and partially or fully demineralized biomaterials derived from bovine bone matrix were evaluated for their ability to support human bone marrow stromal cell (BMSC) osteogenic differentiation in vitro and bone-forming capacity in vivo in order to assess their potential use in clinical tissue-engineering strategies. BMSCs were either seeded on bone-derived scaffolds and cocultured in direct cell-to-scaffold contact, allowing for the exposure of soluble and insoluble matrix-incorporated factors, or cocultured with the scaffold preparations in a transwell system, exposing them to soluble matrix-incorporated factors alone. Osteoblast-related markers, alkaline phosphatase (ALP) activity and bone sialoprotein (BSP) and osteopontin (OP) mRNA expression were evaluated in BMSCs following 14 days of cocultivation in both systems. The data demonstrate that BMSCs from some donors express significantly higher levels of all osteoblast-related markers following cocultivation in direct cell-to-scaffold contact with mineralized scaffolds in comparison to fully demineralized preparations, while BMSCs from other donors display no significant differences in response to various scaffold preparations. In contrast, BMSCs cocultured independently with soluble matrix-incorporated factors derived from each scaffold preparation displayed significantly lower levels of ALP activity and BSP mRNA expression in comparison to untreated controls, while no significant differences were observed in marker levels between cells cocultured similarly with different biomaterial preparations. In addition, BMSCs were seeded directly on mineralized and partially or fully demineralized biomaterials and implanted in subcutaneous sites of athymic mice for 8 weeks to evaluate their in vivo bone-forming capacity. The ex vivo incorporation of BMSCs into all bone-derived scaffold preparations substantially increased the mean extent and frequency of samples containing de novo bone formation over similar nonseeded controls, as determined by histological and histomorphometrical analysis. No statistically significant differences were observed in the extent or frequency of bone formation between various scaffold preparations seeded with BMSCs from different donors. These results demonstrate that the in vivo osteoinductivity of bone-derived scaffolds can be modulated by ex vivo incorporated BMSCs and the extent of scaffold demineralization plays a significant role in influencing in vitro osteogenic differentiation of BMSCs depending on the coculture system and BMSC donor.