Directing the differentiation of human dental follicle cells into cementoblasts and/or osteoblasts by a combination of HERS and pulp cells.

It is known that the dental follicle (DF) consists of progenitor cells that give rise to the cementum, periodontal ligament, and alveolar bone; but little information is available about the regulation of DF cell differentiation into either cementogenic or osteogenic cell lineages for the regeneration of diseased periodontal tissue. Here, we investigated the roles of DF, Hertwig's epithelial root sheath (HERS), and pulp cells in the cementum and during alveolar bone formation. We cultured these cells; transplanted them alone or in combination into immunocompromised mice; and observed their effects at 6 and 12 weeks. Histological and immunohistochemical results revealed that DF cells formed cementum-like tissues with immunoreactivity to cementum-derived attached protein, bone sialoprotein, type I collagen, and alkaline phosphatase. In addition, HERS cells played a role in the induction and maturation of cementum-like tissues formed by DF cells. In contrast, implants of DF cells in the presence of pulp cells led to the formation of bone-like tissues. Interestingly, in the presence of both HERS and pulp cells, DF cells formed both cementum-like and bone-like tissues. We demonstrated that while HERS cells are able to induce DF cell differentiation into cementoblasts and promote cementum formation, pulp cells could direct DF cell differentiation into osteoblasts and enhance alveolar bone formation. These results suggest that the combined use of DF, HERS, and pulp cells could direct DF cell differentiation into cementoblasts and/or osteoblasts in vivo, thus providing a novel strategy for the successful repair and regeneration of diseased periodontal tissue.

Nuclear factor I-C is essential for odontogenic cell proliferation and odontoblast differentiation during tooth root development.

Our previous studies have demonstrated that nuclear factor I-C (NFI-C) null mice developed short molar roots that contain aberrant odontoblasts and abnormal dentin formation. Based on these findings, we performed studies to elucidate the function of NFI-C in odontoblasts. Initial studies demonstrated that aberrant odontoblasts become dissociated and trapped in an osteodentin-like mineralized tissue. Abnormal odontoblasts exhibit strong bone sialoprotein expression but a decreased level of dentin sialophosphoprotein expression when compared with wild type odontoblasts. Loss of Nfic results in an increase in p-Smad2/3 expression in aberrant odontoblasts and pulp cells in the subodontoblastic layer in vivo and primary pulp cells from Nfic-deficient mice in vitro. Cell proliferation analysis of both cervical loop and ectomesenchymal cells of the Nfic-deficient mice revealed significantly decreased proliferative activity compared with wild type mice. In addition, Nfic-deficient primary pulp cells showed increased expression of p21 and p16 but decreased expression of cyclin D1 and cyclin B1, strongly suggesting cell growth arrest caused by a lack of Nfic activity. Analysis of the pulp and abnormal dentin in Nfic-deficient mice revealed an increase in apoptotic activity. Further, Nfic-deficient primary pulp cells exhibited an increase in caspase-8 and -3 activation, whereas the cleaved form of Bid was hardly detected. These results indicate that the loss of Nfic leads to the suppression of odontogenic cell proliferation and differentiation and induces apoptosis of aberrant odontoblasts during root formation, thereby contributing to the formation of short roots.

A histomorphometric study on collagen-apatite composite as a graft material: the influence of gap size at the titanium-bone interface in animal model.

The purpose of this study was to evaluate the healing process of collagen-apatite composite (CAC) at the titanium-bone interface in animal model. Small gaps (0.5 or 1.0 mm-sized wells) were prepared in the epoxy-resin block implants coated with pure titanium. The gaps were filled with CAC or demineralized freeze-dried bone (DFDB). The titanium-coated epoxy-resin block implants were inserted in the tibia of rabbit for 4 weeks or 8 weeks. The microscopic features of bony healing process in the grafted gaps were examined and analyzed. In the histomorphometric analysis, CAC group showed higher fraction of newly-formed bone than DFDB group in both 0.5 and 1.0 mm gap subgroup at 4-week specimen (P < 0.05). In the transmission electron microscopic examinations, osteoblasts of the newly-formed bone of CAC group showed more cellular activity than that of DFDB group. From the results, it was expected that CAC had more beneficial property on early bony healing process than DFDB at the titanium-bone interface.

Disruption of Nfic causes dissociation of odontoblasts by interfering with the formation of intercellular junctions and aberrant odontoblast differentiation.

We reported previously that Nfic-deficient mice exhibit short and abnormal molar roots and severely deformed incisors. The objective of this study is to address the mechanisms responsible for these changes using morphological, IHC, and RT-PCR analysis. Nfic-deficient mice exhibited aberrant odontoblasts and abnormal dentin formation in molar roots and the labial crown analog of incisors. The most striking changes observed in these aberrant odontoblasts were the loss of intercellular junctions and the decreased expression of ZO-1 and occludin. As a result, they became dissociated, had a round shape, and lost their cellular polarity and arrangement as a sheet of cells. Furthermore, the dissociated odontoblasts became trapped in dentin-like mineralized tissue, resembling osteodentin in the overall morphology. These findings suggest that loss of the Nfic gene interferes with the formation of intercellular junctions that causes aberrant odontoblast differentiation and abnormal dentin formation. Collectively, these changes in odontoblasts contributed to development of molars with short and abnormal roots in Nfic-deficient mice.

The effect of odontoblast conditioned media and dentin non-collagenous proteins on the differentiation and mineralization of cementoblasts in vitro.

OBJECTIVE: Cementum is an important mineralized tissue in root formation, however, the precise mechanism of cementum formation remains undetermined. The purpose of this study was to evaluate the effect of odontoblast conditioned media (CM) and dentin non-collagenous proteins (dNCPs) on the differentiation and mineralization of cementoblastic OCCM-30 cells. METHODS: The CM of ameloblastic ALCs, odontoblastic MDPC-23 and OD-11, osteoblastic MG-63, and fibroblastic NIH3T3 cells were transferred to OCCM-30 cells. dNCPs were extracted directly from porcine and human dentin and applied to OCCM-30 cells. The results were evaluated through the analysis of the morphologic appearance, expression of cementum matrix genes, and the formation of mineralized nodules in vitro. RESULTS: dNCPs hardly influenced proliferation, cell cycle modification, and chemotaxis of cementoblasts. Mineralization of cementoblasts was accelerated with dNCPs and CM from odontoblastic MDPC-23 and OD-11. RT-PCR analysis revealed the earlier and stronger expression of bone sialoprotein (BSP), alkaline phosphatase (ALP), and osteocalcin (OC) mRNAs in the MDPC23- and OD11-CM-treated OCCM-30 cells than those in the control OCCM-30 cells. The level of gene expression was also significantly higher in the dNCP-treated group than the control group. CONCLUSION: These results suggest that dentin matrix proteins, or the secreted products of odontoblasts, induced cementoblast differentiation and mineralization. These findings may contribute to the development of a periodontal treatment that includes cementum regeneration.

The role of cell signaling defects on the proliferation of osteoblasts on the calcium phosphate apatite thin film.

The intracellular signal transduction controlling the proliferation of osteoblastic cells on a thin film of poorly crystalline calcium phosphate apatite crystals (PCA) was studied in vitro. The PCA thin film was prepared on polystyrene culture dishes or cover glasses using phosphate-buffered calcium ion solution, which was made oversaturated by heating an undersaturated solution prepared at low temperature. The PCA thin film was used for cell culture without additional surface treatment. Several differences were found between the cells plated on a cell culture dish and the cells cultured on the PCA surface. Entry into S-phase of the cell cycle was markedly delayed and there was a low proliferation rate of osteoblast. On the PCA thin film, the cells spread in a more slender shape. Also, the formation of focal adhesions and stress fibers, examined using immunocytochemical staining, was strikingly weaker in the cells on PCA. The activation of focal adhesion kinase (FAK) was low as well. Expression of cyclins D1 and E was also lower. The Ras-Extracellular signal-regulated kinase (ERK)-MAP kinase signaling pathway was weakly activated by stimulation with serum. These results demonstrate that the low cell proliferation on the PCA surface appears to be due to insufficient activation of signaling that forces the cell cycle to progress and this may be due to weak adhesion signaling in cells on that surface.

Osteoblastic cell response to thin film of poorly crystalline calcium phosphate apatite formed at low temperatures.

The response of osteoblastic cells to a thin film of poorly crystalline calcium phosphate apatite crystals (PCA) was examined in vitro. The PCA thin film was prepared on polystyrene culture dishes using highly metastable calcium phosphate ion solution at low temperatures. The PCA thin film was formed through fusion and transformation of granular calcium phosphate particles, which had initially formed on the surface, into a film of calcium phosphate apatite crystal. The PCA thin film was used for cell culture without additional surface treatment. The osteoblastic cell behaviors including adhesion, proliferation, expression of the marker genes, and calcified matrix formation were examined on the PCA thin film using primary osteoblasts or MC3T3-E1 cells. The cells were well attached and had spread in a slender shape over the PCA thin film. The extent of cell proliferation on the PCA thin film is as much as on the plain dishes. In addition, a much larger number of calcified nodules had formed on the PCA thin film than on the plain dish. The expression of the marker genes such as alkaline phosphatase, osteocalcin, osteopontin, osteonectin was apparent. These results demonstrate that the osteoblasts exhibit a full spectrum of cellular activity including the adequate differentiation on the PCA thin film. Therefore, a PCA thin film can be used as a coating material for biomaterials where the surface is not adequate for inducing the full activity of bone cells.

Porous ZrO2 bone scaffold coated with hydroxyapatite with fluorapatite intermediate layer.

Highly porous zirconia (ZrO(2)) bone scaffolds, fabricated by a replication technique using polymeric sponge, were coated with hydroxyapatite (HA). To prevent the chemical reactions between ZrO(2) and HA, an intermediate fluorapatite (FA) layer was introduced. The strength of the porous ZrO(2) was higher than that of pure HA by a factor of 7, suggesting the feasibility of ZrO(2) porous scaffolds as load-bearing part applications. The coated HA/FA layer, with a thickness of about 30 microm, was firmly adhered to the ZrO(2) body with a bonding strength of 22MPa. The osteoblast-like cells were attached and spread well on the coating layer throughout the porous scaffolds. The alkaline phosphatase activity of the proliferated cells on the HA/FA coated ZrO(2) was comparable to that on pure HA and higher than that on pure ZrO(2).

MG63 osteoblastic cell adhesion to the hydrophobic surface precoated with recombinant osteopontin fragments.

The hydrophobicity of biomaterials has been recognized as a limitation to the adequate function of anchorage-dependent cells when hydrophobic biomaterials are used for tissue engineering. This is due to flawed solid-state signals from cell adhesion. In this study, a recombinant osteopontin (rOPN17-169) fragment containing the cell adhesion motifs was expressed in E. coli and was precoated on the hydrophobic surface prior to osteoblastic MG63 cell culture. Precoating the hydrophobic surface with rOPN17-169 improved osteoblastic cell adhesion, which was blocked by soluble RGDS. The adhesion of MG63 cells to rOPN17-169 pre-coated surface-activated mitogen-activated protein kinases (MAPK) such as extracellular signal-receptor kinase 1/2, p38, and c-Jun N-terminal kinase (JNK). In addition, p38 MAPK was activated in response to a soluble factor of transforming growth factor-beta in the cells adhered to the hydrophobic surface via rOPN17-169. This suggests that rOPN17-169 precoated on the hydrophobic surface can allow osteoblastic cells to generate adhesion signals sufficient for cell adhesion, MAPK activation, and the cytokine activation of osteoblastic cells.

Leptin receptor isoform expression in rat osteoblasts and their functional analysis.

The genetic defect in producing the adipose hormone leptin results among others in a drastic increase of bone mass. The current understanding is that under normal circumstances, osteoblast activity is indirectly suppressed by a hypothalamic relay induced by leptin-signalling in the brain. To investigate whether leptin might also regulate osteoblast activity in a direct manner, expression of leptin receptors in rat osteoblasts was determined and their functionality was analyzed upon recombinant leptin treatment. Reverse transcription-PCR confirmed the expression of four among the six currently described receptor isoforms, which were also able to transduce cell signalling as shown by STAT3 phosphorylation after activation.

Macrophage colony-stimulating factor promotes the survival of osteoclast precursors by up-regulating Bcl-X(L).

Macrophage colony-stimulating factor (M-CSF) is known as one of the factors essential for osteoclast development. In the present study, we examined effects of M-CSF on the apoptotic pathway of osteoclast precursors and their underlying molecular mechanisms. Osteoclast precursors underwent apoptosis in the absence of M-CSF, even in the presence of receptor activator of NF-kappakB ligand (RANKL). Active caspase-3 and -9 were detected in the osteoclast precursors and treatments of precursors with their specific inhibitors (Z-DEVD-FMK and Z-LEHD-FMK) decreased the apoptosis. M-CSF decreased apoptosis in a dose-dependent manner with decreasing in active caspases-3 and -9 levels and up-regulating Bcl-X(L). Those effects of M-CSF on inhibiting apoptosis of osteoclasts precursor by regulating anti-apoptotic signals was more effective when combined with RANKL. These results demonstrate that M-CSF acts as a survival factor for the osteoclast precursors. Furthermore, it is believed that the apoptosis of osteoclast precursors may be involved in the activation of caspase-9 and that M-CSF may promote their survival through Bcl-X(L)-induced inhibition of caspase-9 activation.

Osteoprotegerin is present on the membrane of osteoclasts isolated from mouse long bones.

Osteoprotegerin (OPG), a member of the tumor necrosis factor receptor superfamily, is known to inhibit osteoclastogenesis by acting as a soluble decoy receptor for the receptor activator of NF-kappaB ligand (RANKL). We report the presence of OPG on the membrane of osteoclasts and the possibility of the direct action of OPG on them. Highly pure osteoclast precursors were isolated from mouse long bones and induced to differentiate into mature osteoclasts by M-CSF and soluble RANKL (sRANKL). The presence of OPG on the membrane of these cells was confirmed by western blotting and immunostaining. Furthermore, sRANKL was found to be bound to the OPG on the osteoclast precursors. These results suggest that OPG might have a new role during the differentiation of osteoclasts beyond its role as a soluble decoy receptor. The mechanism of the existence of OPG on osteoclast precursors remains to be found.

Effect of electrostatic interaction on the adsorption of globular proteins on octacalcium phosphate crystal film.

The electrostatic effect on the adsorption of globular proteins, such as bovine serum albumin (BSA), hen egg white lysozyme (LZM), and beta-lactoglobulin (beta-Lg), on octacalcium phosphate (OCP)-like crystal thin films was investigated. A poorly crystalline thin film was synthesized on a tissue culture polystyrene (TCP) surface and used as a model surface in this study. The solution pH clearly affected the electrostatic properties of both proteins and surface. The adsorbed amounts obtained at quasi-steady state were readily related to the solution pH for each protein. The adsorption rate is fast during the initial period and levels off gradually. The maximum adsorbed mass occurred at pH 7 for BSA and at pH 9 for LZM. beta-Lg adsorbed similar amounts at pHs lower than 9, but the adsorbed mass decreased at pHs higher than 9 where electrostatic repulsion exists. The pH values where the maximum adsorbed mass occurred may be considered as the conditions where electrostatic attraction is most favorable. The adsorbed mass of beta-Lg was the greatest among the proteins of interest while BSA adsorbed the least despite its greater molecular mass. LZM falls into the intermediate region. According to these observations, BSA has undergone conformational changes that prevent further adsorption to a greater extent than the others. A simple relationship between the adsorption rate and the electrostatic properties was not established. However, the order of magnitude of the adsorption rate at the initial period tends to be the same as that of maximum adsorbed mass for each protein.