Creating 3D constructs with cranial neural crest-derived cell lines using a bio-3D printer.

OBJECTIVES: The development of bio-three-dimensional (bio-3D) printers has led to significant advances in regenerative medicine. Three-dimensional constructs, including spheroids, are maintained by extracellular matrix proteins secreted by cells so that the cells can be cultured in conditions closer to the physiological environment. This study aimed to create a useful 3D construct as a model of the dentin-pulp complex. METHODS: We examined the expression patterns of extracellular matrix proteins and cell proliferation areas in a 3D construct created using O9-1 cells derived from cranial neural crest cells of mice. The 3D construct was created by sticking the spheroid cultures onto a needle array using a bio-3D printer. RESULTS: Cell proliferation areas along with characteristic expression of tenascin C and DMP1 were evaluated. The expression of tenascin C and DMP1 was significantly enhanced in the spheroids compared to that in two-dimensional cultures. Moreover, cell proliferation regions and tenascin C expression were confirmed in the outer layer of spheroids in the embryonic stem cell medium, with insignificant DMP1 expression being observed. Interestingly, in a 3D construct cultured in calcification-induction medium, DMP1 expression was promoted, and DMP1-positive cells existed in the outermost layer without overlapping with tenascin C expression. CONCLUSIONS: The extracellular matrix proteins, tenascin C and DMP1, were expressed in a polarized manner in spheroids and 3D constructs, similar to the findings in the dental papilla. Therefore, these 3D constructs show potential as artificial models for studying odontogenesis.

Conditional knockout of transient receptor potential melastatin 7 in the enamel epithelium: Effects on enamel formation.

Transient receptor potential melastatin 7 (TRPM7) is a unique ion channel connected to a kinase domain. We previously demonstrated that Trpm7 expression is high in mouse ameloblasts and odontoblasts, and that amelogenesis is impaired in TRPM7 kinase-dead mice. Here, we analyzed TRPM7 function during amelogenesis in Keratin 14-Cre;Trpm7fl/fl conditional knockout (cKO) mice and Trpm7 knockdown cell lines. cKO mice showed lesser tooth pigmentation than control mice and broken incisor tips. Enamel calcification and microhardness were lower in cKO mice. Electron probe microanalysis (EPMA) showed that the calcium and phosphorus contents in the enamel were lower in cKO mouse than in control mice. The ameloblast layer in cKO mice showed ameloblast dysplasia at the maturation stage. The morphological defects were observed in rat SF2 cells with Trpm7 knockdown. Compared with mock transfectants, the Trpm7 knockdown cell lines showed lower levels of calcification with Alizarin Red-positive staining and an impaired intercellular adhesion structures. These findings suggest that TRPM7 is a critical ion channel in enamel calcification for the effective morphogenesis of ameloblasts during amelogenesis.

Inhibition of retinoid X receptor improved the morphology, localization of desmosomal proteins and paracellular permeability in three-dimensional cultures of mouse keratinocytes.

Retinoic acid (RA) plays an important role in epithelial homeostasis and influences the morphology, proliferation, differentiation and permeability of epithelial cells. Mouse keratinocytes, K38, reconstituted non-keratinized stratified epithelium in three-dimensional (3D) cultures with serum, which contains retinol (a source of RA), but the morphology was different from in vivo epithelium. The formed epithelium was thick, with loosened cell-cell contacts. Here, we investigated whether the inhibition of RA receptor (RAR)/retinoid X receptor (RXR)-mediated signaling by an RXR antagonist, HX 531, improved K38 3D cultures in terms of morphology and intercellular junctions. The epithelium formed by 0.5 µM HX531 was thin, and the intercellular space was narrowed because of the restoration of the layer-specific distribution of desmoglein (DSG)-1, DSG3 and plakoglobin (PG). Moreover, the levels of desmosomal proteins and tight junction proteins, including DSG1, DSG2, DSG3, PG, claudin (CLDN)-1 and CLDN4 increased, but the adherens junction protein, E-cadherin, did not show any change. Furthermore, CLDN1 was recruited to occludin-positive cell-cell contacts in the superficial cells and transepithelial electrical resistance was increased. Therefore, K38 3D cultures treated with 0.5 µM HX531 provides a useful in vitro model to study intercellular junctions in the non-keratinized epithelium.

Transforming Growth Factor-Beta and Sonic Hedgehog Signaling in Palatal Epithelium Regulate Tenascin-C Expression in Palatal Mesenchyme During Soft Palate Development.

During palatogenesis, the palatal shelves first grow vertically on either side of the tongue before changing their direction of growth to horizontal. The extracellular matrix (ECM) plays an important role in these dynamic changes in palatal shelf morphology. Tenascin-C (TNC) is an ECM glycoprotein that shows unique expression in the posterior part of the palatal shelf, but little is known about the regulation of TNC expression. Since transforming growth factor-beta-3 (TGF-ß3) and sonic hedgehog (SHH) signaling are known to play important roles in palatogenesis, we investigated whether TGF-ß3 and SHH are involved in the regulation of TNC expression in the developing palate. TGF-ß3 increased the expression of TNC mRNA and protein in primary mouse embryonic palatal mesenchymal cells (MEPM) obtained from palatal mesenchyme dissected at embryonic day 13.5-14.0. Interestingly, immunohistochemistry experiments revealed that TNC expression was diminished in K14-cre;Tgfbr2 fl/fl mice that lack the TGF-ß type II receptor in palatal epithelial cells and exhibit cleft soft palate, whereas TNC expression was maintained in Wnt1-cre;Tgfbr2 fl/fl mice that lack the TGF-ß type II receptor in palatal mesenchymal cells and exhibit a complete cleft palate. SHH also increased the expression of TNC mRNA and protein in MEPM cells. However, although TGF-ß3 up-regulated TNC mRNA and protein expression in O9-1 cells (a cranial neural crest cell line), SHH did not. Furthermore, TGF-ß inhibited the expression of osteoblastic differentiation markers (osterix and alkaline phosphatase) and induced the expression of fibroblastic markers (fibronectin and periostin) in O9-1 cells, whereas SHH did not affect the expression of osteoblastic and fibroblastic markers in O9-1 cells. However, immunohistochemistry experiments showed that TNC expression was diminished in the posterior palatal shelves of Shh-/+ ;MFCS4 +/- mice, which have deficient SHH signaling in the posterior palatal epithelium. Taken together, our findings support the proposal that TGF-ß and SHH signaling in palatal epithelium co-ordinate the expression of TNC in the posterior palatal mesenchyme through a paracrine mechanism. This signal cascade may work in the later stage of palatogenesis when cranial neural crest cells have differentiated into fibroblast-like cells. The spatiotemporal regulation of ECM-related proteins by TGF-ß and SHH signaling may contribute not only to tissue construction but also to cell differentiation or determination along the anterior-posterior axis of the palatal shelves.

The reduced susceptibility of mouse keratinocytes to retinoic acid may be involved in the keratinization of oral and esophageal mucosal epithelium.

Keratinocytes take up serum-derived retinol (vitamin A) and metabolize it to all-trans-retinoic acid (atRA), which binds to the nuclear retinoic acid receptor (RAR). We previously reported that serum-affected keratinocyte differentiation and function; namely, it inhibited keratinization, decreased loricrin (LOR) and claudin (CLDN) 1 expression, increased keratin (K) 4 and CLDN4 levels, and reduced paracellular permeability in three-dimensional (3D) cultures of mouse keratinocytes (COCA). Contrarily, RAR inhibition reversed these changes. Here, we aimed to examine whether atRA exerted the same effects as serum, and whether it was involved in the differential oral mucosa keratinization among animal species. Porcine oral mucosal keratinocytes, which form non-keratinized epithelium in vivo, established keratinized epithelium in 3D cultures. Both mouse and porcine sera induced non-keratinized epithelium at 0.1% in COCA 3D cultures. Although atRA caused the same changes as serum, its effective concentration differed. atRA inhibited keratinization at 0.1 nM and 1 nM in porcine or human keratinocytes and COCA, respectively. Furthermore, atRA upregulated CLDN7 in the cytoplasm but not in cell-cell contacts. These atRA-induced changes were reverted by RAR inhibition. The results indicate that serum-induced changes are probably due to the effect of serum-derived atRA, and that mouse keratinocytes require higher atRA concentrations to suppress keratinization than porcine and human keratinocytes. We propose that the lower susceptibility of mouse keratinocytes to atRA, rather than a lower retinol concentration, is a possible reason for the keratinization of mouse oral mucosal epithelium.

Anisomycin, a JNK and p38 activator, suppresses cell-cell junction formation in 2D cultures of K38 mouse keratinocyte cells and reduces claudin-7 expression, with an increase of paracellular permeability in 3D cultures.

Keratinocytes in the oral mucosal epithelium, which is a non-keratinized stratified epithelium, are exposed to various stimuli from the oral cavity. JNK and p38 are stress-activated mitogen-activated protein kinases (MAPKs) that are phosphorylated by various stimuli and are involved in the assembly and disassembly of tight junctions (TJs) in keratinocytes. Therefore, we investigated the effects of stress-activated MAPKs on TJs in a mouse keratinocyte cell line during cell-cell junction formation in two-dimensional (2D) cultures or stratification to form non-keratinized epithelium in 3D cultures. In 2D cultures, calcium induced zipper-like staining for ZO-1 at 2 h and string-like staining for ZO-1 at 12 h, which indicated immature and mature cell-cell junctions, respectively. Anisomycin (AM), a JNK and p38 activator, inhibited formation of string-like staining for ZO-1, whereas inhibition of JNK, but not p38, after AM treatment restored string-like staining for ZO-1, although claudins (CLDNs) 4, 6, and 7 did not completely colocalize to ZO-1-positive sites. In 3D cultures, AM treatment for 2 weeks activated only p38, suppressed flattening of the superficial cells, removed CLDN7 from ZO-1-positive spots on the surface of 3D cultures, which represent TJs, and decreased transepithelial electrical resistance. Thus, short-term AM treatment inhibited maturation of cell-cell junctions by JNK, but not p38, activation. p38 activation by long-term AM treatment affected morphology of stratified structures and paracellular permeability, which was increased by CLDN7 removal from TJs. Various chronic stimuli that activate stress-activated MAPKs may weaken the keratinocyte barrier and be involved in TJ-related diseases.

CO2 laser therapy accelerates the healing of ulcers in the oral mucosa by inducing the expressions of heat shock protein-70 and tenascin C.

The treatment of ulceration or stomatitis with laser therapy is known to accelerate healing and relieve pain, but the underlying biological mechanism is not fully understood. The present study used a mouse model of ulceration to investigate the molecular mechanisms by which CO2 laser therapy accelerated the wound healing process. An ulcer was experimentally created in the palatal mucosa of the mouse and irradiated with light from a CO2 laser. Compared with controls (no irradiation), laser irradiation induced the proliferation of epithelial cells and faster re-epithelialization of the wound area. Immunohistochemistry experiments showed that heat shock protein-70 (HSP70) was expressed mainly in the epithelium of normal palatal tissue, whereas there was little tenascin C (TnC) expression in the epithelium and mesenchyme under normal conditions. Laser irradiation induced HSP70 mRNA and protein expression in the lamina propria as well as TnC expression in the mesenchyme underlying the renewing epithelium. Epithelial cells and fibroblasts were exposed to heated culture medium or laser irradiation to establish whether hyperthermia mimicked the effect of laser irradiation. Culture of fibroblasts in heated medium increased the expressions of both TnC and TGF-ß1, whereas laser irradiation induced only TnC expression. The present study indicates that CO2 laser irradiation exerts a photobiogenic effect to up-regulate TnC expression without inducing TGF-ß1 expression. We suggest that CO2 laser therapy has an advantage over thermal stimulation.

Serum affects keratinization and tight junctions in three-dimensional cultures of the mouse keratinocyte cell line COCA through retinoic acid receptor-mediated signaling.

Vitamin A, which is found in serum, is known to affect keratinocyte proliferation, epidermal differentiation, and keratinization. In mice, stratified epithelia in the oral cavity, esophagus, and forestomach are keratinized; however, these epithelia are not keratinized in humans. Several studies have reported that three-dimensional (3D) cultures of human keratinocytes in serum-containing medium could form keratinized epithelia. Here, we evaluated the effects of serum on the morphology, expression, and localization of differentiation markers and tight junction proteins, and paracellular permeability in 3D cultures of mouse keratinocytes. We found that only 0.1% calcium-depleted serum inhibited keratinization and induced a change in the expression of differentiation marker proteins from loricrin to keratin 4; the inhibition of retinoic acid receptor-mediated signaling reversed these changes. Furthermore, the serum reduced claudin-1 protein expression and prevented its localization at occludin-positive spots on the surface of 3D cultures. On the other hand, the serum increased the protein expression of claudin-4, occludin, zonula occludens-1, and E-cadherin. These changes may contribute to the reduction of the transepithelial electrical resistance by approximately half. In conclusion, mouse keratinocytes derived from the epidermis formed non-keratinized structures in 3D cultures in response to vitamin A in serum. The results suggest that retinoic acid receptor-mediated signaling may be inhibited in the mouse epithelia in the oral cavity, esophagus, and forestomach as well as the epidermis, leading to the keratinization of these epithelia.

The crucial role of the TRPM7 kinase domain in the early stage of amelogenesis.

Transient receptor potential melastatin-7 (TRPM7) is a bi-functional protein containing a kinase domain fused to an ion channel. TRPM7 is highly expressed in ameloblasts during tooth development. Here we show that TRPM7 kinase-inactive knock-in mutant mice (TRPM7 KR mice) exhibited small enamel volume with opaque white-colored incisors. The TRPM7 channel function of ameloblast-lineage cells from TRPM7 KR mice was normal. Interestingly, phosphorylation of intracellular molecules including Smad1/5/9, p38 and cAMP response element binding protein (CREB) was inhibited in ameloblasts from TRPM7 KR mice at the pre-secretory stage. An immunoprecipitation assay showed that CREB was bound to TRPM7, suggesting that direct phosphorylation of CREB by TRPM7 was inhibited in ameloblast-lineage cells from TRPM7 KR mice. These results indicate that the function of the TRPM7 kinase domain plays an important role in ameloblast differentiation, independent of TRPM7 channel activity, via phosphorylation of CREB.

Hertwig's epithelial root sheath cells contribute to formation of periodontal ligament through epithelial-mesenchymal transition by TGF-ß.

In tooth root development, periodontal ligament (PDL) and cementum are formed by the coordination with the fragmentation of Hertwig's epithelial root sheath (HERS) and the differentiation of dental follicle mesenchymal cells. However, the function of the dental epithelial cells after HERS fragmentation in the PDL is not fully understood. Here, we found that TGF-ß regulated HERS fragmentation via epithelial-mesenchymal transition (EMT), and the fragmented epithelial cells differentiated into PDL fibroblastic cells with expressing of PDL extracellular matrix (ECM). In the histochemical analysis, TGF-ß was expressed in odontoblast layer adjacent of HERS during root development. Periostin expression was detected around fragmented epithelial cells on the root surface, but not in HERS. In the experiment using an established mouse HERS cell line (HERS01a), TGF-ß1 treatment decreased E-cadherin and relatively increased N-cadherin expression. TGF-ß1 treatment in HERS01a induced further expression of important ECM proteins for acellular cementum and PDL development such as fibronectin and periostin. Taken together, activation of TGF-ßsignaling induces HERS fragmentation through EMT and the fragmented HERS cells contribute to formation of PDL and acellular cementum through periostin and fibronectin expression.