Jea Seung Ko

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).

Thin film of low-crystalline calcium phosphate apatite formed at low temperature ☆

Surface modification of biomaterials to improve biocompatibility without changing their bulk properties is desired for many clinical applications and has become an emerging technology in biomaterial research and industry. In the present study, a simple method of coating the solid surfaces of metals, organic tissue matrices, glasses, inorganic ceramics as well as organic polymers with a thin film of low-crystalline apatite crystals (LCA) was developed. Acidic solution containing calcium and phosphate ions was neutralized with alkaline solution to form calcium phosphate precipitates at low temperature. Precipitates of solid calcium phosphate particles were, then, removed by filtration. Concentration of free ions in the filtered ion solution which were not involved in the formation of calcium phosphate precipitate was high enough to induce the heterogeneous nucleation on the solid surfaces at low temperature. Thin layers of calcium phosphate crystals were formed on the surfaces of metals, glasses, inorganic ceramics, organic polymers including hydrophobic ones, and biological tissue matrices with this solution. The thin layer of crystals consisted of poorly crystalline calcium phosphate apatite crystals which contain high amount of labile ions like bone crystals and did not dissolve in the physiologic solutions. Various cells attached to this crystal layer and proliferated well.

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.

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.

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.

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.

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. (J Histochem Cytochem 57:469–476, 2009)

Dissolution of poorly crystalline apatite crystals by osteoclasts determined on artificial thin-film apatite.

Poorly crystalline apatite (PCA) crystals introduced into bone tissue should be stable for a definite period before they are dissolved as a result of a host response. In this report, the dissolution of PCA crystals by the action of osteoclasts was studied on artificial thin films. These consisted of PCA crystals having similar crystallographic properties to bone crystals which were developed for assaying the osteoclast activity in vitro. The dissolution of minerals by osteoclasts decreased along with the decreased amount of labile phosphate and hydrogen phosphate domains of apatite crystals, which were caused by the crystal maturation temperature. A profound effect on mineral dissolution by pH in the culture medium was also shown. Low acidity considerably increased mineral dissolution, whereas a slight alkalinity totally blocked mineral dissolution. There was little difference in the mineral dissolution behavior of osteoclasts near the physiologic pH. In addition, it was determined whether mineral dissolution by osteoclasts was dependent on the destruction of the organic matrix. Nocodazole was introduced to inhibit the secretion of hydrolytic enzymes, and acetazolamide was added to inhibit acid production by the osteoclasts. There was no significant change as a result of nocodazole addition on mineral dissolution or by the addition of acetazolamide on degradation of collagen. These results indicate that small changes in the physicochemical properties of apatite crystals can decrease resorption by osteoclasts, which can be highly activated at low pH. These results also suggest that mineral dissolution and organic degradation by osteoclasts are self-regulating.

Kinetic Model for the Simulation of Hen Egg White Lysozyme Adsorption at Solid/Water Interface

A simulation model for adsorption kinetics of hen egg white lysozyme (HEW) adsorption to hydrophilic silica is proposed. The adsorption kinetic data were monitored by usingin-situ ellipsometry. The model is based on an irreversible adsorption mechanism allowing two different adsorbed states. The adsorbed states were differentiated based on binding strengths resistant to the concentration gradient exerted by rinse. Molecules desorbing and remaining upon rinse were identified as loosely bound (state 1) and tightly bound (state 2) states, respectively. The adsorption rate constants were assumed to be a time-dependent nonlinear function in order to account for the change in surface properties originating from the protein layer formed on the surface. The parameters of adsorption rate constants were evaluated by using adsorption kinetic data at different protein concentrations, and the relationships between the adsorption parameters and protein concentration were established which eventually demonstrated a linear relationship. The established relations between the adsorption parameters and concentration elucidated the effect of protein concentration on adsorption to hydrophilic silica.