Transepithelial channels for leukocytes in the junctional epithelium.

BACKGROUND AND OBJECTIVES: The junctional epithelium (JE) has been recognized as a defensive organ rich in polymorphonuclear leukocytes (PMNs). However, the migration of PMNs through the JE has not been clearly documented. For mucosal defense, PMNs migrate outwards over the epithelium to defend the intestinal or respiratory tract on the epithelial surface. With this background, the present study investigated whether there is any structural evidence showing the transepithelial migration of PMNs through the JE in gingival mucosa. METHODS: Three-dimensional modeling of gingiva surrounding mouse molars at varying ages was performed by array tomography. Images of the serial sections for array tomography at the 800 nm thickness were obtained using back scattered electron (BSE) detector equipped in the field-emission scanning electron microscopy (FESEM). Expressions of neutrophil marker or CD47 were immunohistochemically examined on the frozen sections. RESULTS: Array tomography using FESEM and 3-dimensional modeling clearly showed that a system of epithelial channels developed between keratinocytes and generally ran along the long axis of the JE. Most PMNs were found inside the channels, rather than being scattered throughout the JE. The channels could be traced from the base of the JE to the bottom of the gingival sulcus, although some channels were fragmented and interrupted with short intercellular gaps. CONCLUSIONS: These findings suggest that the JE may be an organ for transepithelial migration of PMNs to the bottom of the gingival sulcus through epithelial channels, as occurs in the epithelial lining of other organs such as the intestinal or respiratory tract.

Characterization of junctional structures in the gingival epithelium as barriers against bacterial invasion.

BACKGROUND AND OBJECTIVE: Adherens junctions (AJs) and tight junctions (TJs) are known to play a crucial role in maintaining the physical barrier function of the epithelium. Here, we aimed to characterize the distribution of AJs and TJs throughout the gingival epithelium and to obtain insights into the physiological importance of these junctional structures. METHODS: Sections of mouse gingival tissue were examined using transmission electron microscopy (TEM) and bio-high voltage electron microscopy tomography. The gingival sections were stained for E-cadherin and JAM-A as markers of AJs and TJs, respectively, and examined using confocal microscopy and lattice structured illumination microscopy. Bacteria within the gingival epithelium were examined using in situ hybridization. RESULTS: Junctional structures, including desmosomes, AJs, and TJs, were observed throughout the gingival epithelium. The expression levels of E-cadherin were particularly low in the granular/keratinized layers of the oral epithelium (OE), while extremely low JAM-A levels were detected in the granular/keratinized layers of the sulcular epithelium (SE). The three-dimensional rendering of the junctional structures revealed that both AJs and TJs in the gingival epithelium formed discontinuous short bands or patches. Interestingly, strong bacterial signals were observed at the granular/keratinized layers of both SE and OE, but a few bacteria were detected within the junctional epithelium (JE) and the basal/spinous layers of the SE and OE. CONCLUSIONS: AJs and TJs form a discontinuous barrier throughout paracellular passage in the gingival epithelium; nevertheless, they seem to play an important role in defending against invading bacteria.

JNK-associated scattered growth of YD-10B oral squamous carcinoma cells while maintaining the epithelial phenotype.

Cell scattering of epithelial carcinoma cancer cells is one of the critical event in tumorigenesis. Cells losing epithelial cohesion detach from aggregated epithelial cell masses and may migrate to fatal organs through metastasis. The present study investigated the molecular mechanism by which squamous cell carcinoma cells grow scattered at the early phase of transformation while maintaining the epithelial phenotype. We studied YD-10B cells, which are established from human oral squamous cell carcinoma, because the cells grow scattered without the development of E-cadherin junctions (ECJs) under routine culture conditions despite the high expression of functional E-cadherin. The functionality of their E-cadherin was demonstrated in that YD-10B cells developed ECJs, transiently or persistently, when they were cultured on substrates coated with a low amount of fibronectin or to confluence. The phosphorylation of JNK was up-regulated in YD-10B cells compared with that in human normal oral keratinocyte cells or human squamous cell carcinoma cells, which grew aggregated along with well-organized ECJs. The suppression of JNK activity induced the aggregated growth of YD-10B cells concomitant with the development of ECJs. These results indicate for the first time that inherently up-regulated JNK activity induces the scattered growth of the oral squamous cell carcinoma cells through down-regulating the development of ECJ despite the expression of functional E-cadherin, a hallmark of the epithelial phenotype.

Drosophila Atlastin regulates the stability of muscle microtubules and is required for synapse development.

Hereditary spastic paraplegia (HSP) is an inherited neurological disorder characterized by progressive spasticity and weakness of the lower extremities. The most common early-onset form of HSP is caused by mutations in the human gene that encodes the dynamin-family GTPase Atlastin-1 (Atl-1). Recently, loss of the Drosophila ortholog of Atl-1 (Atl) has been found to induce locomotor impairments from the earliest adult stages, suggesting the developmental role of atlastin-subfamily GTPases. Here, we provide evidence that Atl is required for normal growth of muscles and synapses at the neuromuscular junction (NMJ). Atl protein is highly expressed in larval body-wall muscles. Loss-of-function mutations in the atl gene reduce the size of muscles and increase the number of synaptic boutons. Rescue of these defects is accomplished by muscular, but not neuronal expression of Atl. Loss of Atl also disrupts ER and Golgi morphogenesis in muscles and reduces the synaptic levels of the scaffold proteins Dlg and alpha-spectrin. We also provide evidence that Atl functions with the microtubule-severing protein Spastin to disassemble microtubules in muscles. Finally, we demonstrate that the microtubule-destabilizing drug vinblastine alleviates synapse and muscle defects in atl mutants. Together, our results suggest that Atl controls synapse development and ER and Golgi morphogenesis by regulating microtubule stability.

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.

Rho-associated kinase (ROCK) inhibition reverses low cell activity on hydrophobic surfaces.

Hydrophobic polymers do not offer an adequate scaffold surface for cells to attach, migrate, proliferate, and differentiate. Thus, hydrophobic scaffolds for tissue engineering have traditionally been physicochemically modified to enhance cellular activity. However, modifying the surface by chemical or physical treatment requires supplementary engineering procedures. In the present study, regulation of a cell signal transduction pathway reversed the low cellular activity on a hydrophobic surface without surface modification. Inhibition of Rho-associated kinase (ROCK) by Y-27632 markedly enhanced adhesion, migration, and proliferation of osteoblastic cells cultured on a hydrophobic polystyrene surface. ROCK inhibition regulated cell-cycle-related molecules on the hydrophobic surface. This inhibition also decreased expression of the inhibitors of cyclin-dependent kinases such as p21(cip1) and p27(kip1) and increased expression of cyclin A and D. These results indicate that defective cellular activity on the hydrophobic surface can be reversed by the control of a cell signal transduction pathway without physicochemical surface modification.

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.

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.

Vitamin D maintains E-cadherin intercellular junctions by downregulating MMP-9 production in human gingival keratinocytes treated by TNF-α.

PURPOSE: Despite the well-known anti-inflammatory effects of vitamin D in periodontal health, its mechanism has not been fully elucidated. In the present study, the effect of vitamin D on strengthening E-cadherin junctions (ECJs) was explored in human gingival keratinocytes (HGKs). ECJs are the major type of intercellular junction within the junctional epithelium, where loose intercellular junctions develop and microbial invasion primarily occurs. METHODS: HOK-16B cells, an immortalized normal human gingival cell line, were used for the study. To mimic the inflammatory environment, cells were treated with tumor necrosis factor-alpha (TNF-α). Matrix metalloproteinases (MMPs) in the culture medium were assessed by an MMP antibody microarray and gelatin zymography. The expression of various molecules was investigated using western blotting. The extent of ECJ development was evaluated by comparing the average relative extent of the ECJs around the periphery of each cell after immunocytochemical E-cadherin staining. Vitamin D receptor (VDR) expression was examined via immunohistochemical analysis. RESULTS: TNF-α downregulated the development of the ECJs of the HGKs. Dissociation of the ECJs by TNF-α was accompanied by the upregulation of MMP-9 production and suppressed by a specific MMP-9 inhibitor, Bay 11-7082. Exogenous MMP-9 decreased the development of ECJs. Vitamin D reduced the production of MMP-9 and attenuated the breakdown of ECJs in the HGKs treated with TNF-α. In addition, vitamin D downregulated TNF-α-induced nuclear factor kappa B (NF-κB) signaling in the HGKs. VDR was expressed in the gingival epithelium, including the junctional epithelium. CONCLUSIONS: These results suggest that vitamin D may avert TNF-α-induced downregulation of the development of ECJs in HGKs by decreasing the production of MMP-9, which was upregulated by TNF-α. Vitamin D may reinforce ECJs by downregulating NF-κB signaling, which is upregulated by TNF-α. Strengthening the epithelial barrier may be a way for vitamin D to protect the periodontium from bacterial invasion.

Substrate roughness induces the development of defective E-cadherin junctions in human gingival keratinocytes.

PURPOSE: The entry of bacteria or harmful substances through the epithelial seal of human gingival keratinocytes (HGKs) in the junctional epithelium (JE) is blocked by specialized intercellular junctions such as E-cadherin junctions (ECJs). However, the influence of roughened substrates, which may occur due to apical migration of the JE, root planing, or peri-implantitis, on the development of the ECJs of HGKs remains largely unknown. METHODS: HGKs were cultured on substrates with varying levels of roughness, which were prepared by rubbing hydrophobic polystyrene dishes with silicon carbide papers. The activity of c-Jun N-terminal kinase (JNK) was inhibited with SP600125 or by transfection with JNK short hairpin RNA. The development of intercellular junctions was analyzed using scanning electron microscopy or confocal laser scanning microscopy after immunohistochemical staining of the cells for E-cadherin. The expression level of phospho-JNK was assessed by immunoblotting. RESULTS: HGKs developed tight intercellular junctions devoid of wide intercellular gaps on smooth substrates and on rough substrates with low-nanometer dimensions (average roughness [Ra]=121.3±13.4 nm), although the ECJs of HGKs on rough substrates with low-nanometer dimensions developed later than those of HGKs on smooth substrates. In contrast, HGKs developed short intercellular junctions with wide intercellular gaps on rough substrates with mid- or high-nanometer dimensions (Ra=505.3±115.3 nm, 867.0±168.6 nm). Notably, the stability of the ECJs was low on the rough substrates, as demonstrated by the rapid destruction of the cell junction following calcium depletion. Inhibition of JNK activity promoted ECJ development in HGKs. JNK was closely associated with cortical actin in the regulation of ECJs in HGKs. CONCLUSIONS: These results indicate that on rough substrates with nanometer dimensions, the ECJs of HGKs develop slowly or defectively, and that this effect can be reversed by inhibiting JNK.

Antioxidant alpha-lipoic acid inhibits osteoclast differentiation by reducing nuclear factor-kappaB DNA binding and prevents in vivo bone resorption induced by receptor activator of nuclear factor-kappaB ligand and tumor necrosis factor-alpha.

The relationship between oxidative stress and bone mineral density or osteoporosis has recently been reported. As bone loss occurring in osteoporosis and inflammatory diseases is primarily due to increases in osteoclast number, reactive oxygen species (ROS) may be relevant to osteoclast differentiation, which requires receptor activator of nuclear factor-kappaB ligand (RANKL). Tumor necrosis factor-alpha (TNF-alpha) frequently present in inflammatory conditions has a profound synergy with RANKL in osteoclastogenesis. In this study, we investigated the effects of alpha-lipoic acid (alpha-LA), a strong antioxidant clinically used for some time, on osteoclast differentiation and bone resorption. At concentrations showing no growth inhibition, alpha-LA potently suppressed osteoclastogenesis from bone marrow-derived precursor cells driven either by a high-dose RANKL alone or by a low-dose RANKL plus TNF-alpha (RANKL/TNF-alpha). alpha-LA abolished ROS elevation by RANKL or RANKL/TNF-alpha and inhibited NF-kappaB activation in osteoclast precursor cells. Specifically, alpha-LA reduced DNA binding of NF-kappaB but did not inhibit IKK activation. Furthermore, alpha-LA greatly suppressed in vivo bone loss induced by RANKL or TNF-alpha in a calvarial remodeling model. Therefore, our data provide evidence that ROS plays an important role in osteoclast differentiation through NF-kappaB regulation and the antioxidant alpha-lipoic acid has a therapeutic potential for bone erosive diseases.

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.

Nuclear magnetic resonance spin-spin relaxation of the crystals of bone, dental enamel, and synthetic hydroxyapatites.

Studies of the apatitic crystals of bone and enamel by a variety of spectroscopic techniques have established clearly that their chemical composition, short-range order, and physical chemical reactivity are distinctly different from those of pure hydroxyapatite. Moreover, these characteristics change with aging and maturation of the bone and enamel crystals. Phosphorus-31 solid state nuclear magnetic resonance (NMR) spin-spin relaxation studies were carried out on bovine bone and dental enamel crystals of different ages and the data were compared with those obtained from pure and carbonated hydroxyapatites. By measuring the 31P Hahn spin echo amplitude as a function of echo time, Van Vleck second moments (expansion coefficients describing the homonuclear dipolar line shape) were obtained and analyzed in terms of the number density of phosphorus nuclei. 31P magnetization prepared by a 90 degree pulse or by proton-phosphorus cross-polarization (CP) yielded different second moments and experienced different degrees of proton spin-spin coupling, suggesting that these two preparation methods sample different regions, possibly the interior and the surface, respectively, of bone mineral crystals. Distinct differences were found between the biological apatites and the synthetic hydroxyapatites and as a function of the age and maturity of the biological apatites. The data provide evidence that a significant fraction of the protonated phosphates (HPO4(-2)) are located on the surfaces of the biological crystals, and the concentration of unprotonated phosphates (PO4(-3)) within the apatitic lattice is elevated with respect to the surface. The total concentration of the surface HPO4(-2) groups is higher in the younger, less mature biological crystals.

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.

Preparation of a bioactive and degradable poly(epsilon -caprolactone)/silica hybrid through a sol-gel method.

A bioactive and degradable poly(epsilon -caprolactone)/silica hybrid was synthesized for the application as a bone substitute. Triethoxysilane end capped polyepsilon -caprolactone) was prepared by the reaction with alpha,omega-hydroxyl poly(epsilon -caprolactone) and 3-isocyanatopropyl triethoxysilane using 1,4-diazabicyclo[2,2,2,]octane as a catalyst. It was then co-condensed with tetraethyl orthosilicate and calcium nitrate tetrahydrate via a sol-gel method. The bioactivity of the poly(epsilon -caprolactone)/silica hybrid was assessed using simulated body fluid and low crystalline apatite was successfully formed on its surface after soaking for 1 week at 36.5 degrees C. Its biodegradability was evaluated in the phosphate buffered saline and the degradability was mostly come from the poly(epsilon -caprolactone) phase in the hybrid. It means that this hybrid is likely to be applicable to a bioactive and degradable bone substitute.

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.

Effect of CaF2 on densification and properties of hydroxyapatite-zirconia composites for biomedical applications.

Hydroxyapatite (HA) composites with zirconia (ZrO2) up to 40 vol% were fabricated with the addition of CaF2. The sinterability of the composites was found to be enhanced markedly by the addition of small amounts of CaF2 (< 5 vol%). Decomposition of HA to beta-TCP was suppressed due to the substitution of F- for OH-, consequently forming fluor-hydroxyapatite (FHA) solid solution. This suppression of decomposition allowed the production of a fully dense body, which retained both high flexural strength and fracture toughness. The osteoblast-like cell (MG63) response to these F- ion-containing composites displayed comparable cell viability to pure-HA by in vitro proliferation test.

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).

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.