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The aim of this study is to investigate the repressive effects of enzyme-digested edible bird's nest (EBND) on the combination of arid environment and UV-induced intracellular oxidative stress, cell death, DNA double-strand breaks (DSBs) and inflammatory responses in human HaCaT keratinocytes and three-dimensional (3D) epithelium equivalents. An oxygen radical antioxidant capacity assay showed that EBND exhibited excellent peroxyl radical scavenging activity and significantly increased cellular antioxidant capacity in HaCaT cells. When EBND was administered to HaCaT cells and 3D epitheliums, it exhibited significant preventive effects on air-drying and UVA (Dry-UVA)-induced cell death and apoptosis. Dry-UVA markedly induced intracellular reactive oxygen species (ROS) generation in HaCaT cells and 3D epitheliums as quantified by CellROX® Green/Orange reagents. Once HaCaT cells and 3D epitheliums were pretreated with EBND, Dry-UVA-induced intracellular ROS were significantly reduced. The results from anti-γ-H2A.X antibody-based immunostaining showed that EBND significantly inhibited Dry-UVA-induced DSBs in HaCaT keratinocytes. Compared with sialic acid, EBND showed significantly better protection for both keratinocytes and 3D epitheliums against Dry-UVA-induced injuries. ELISA showed that EBND significantly suppressed UVB-induced IL-6 and TNF-α secretion. In conclusion, EBND could decrease arid environments and UV-induced harmful effects and inflammatory responses in human keratinocytes and 3D epithelium equivalents partially through its antioxidant capacity. [ABSTRACT FROM AUTHOR]

To elucidate the mechanism underlying the failure of root formation after irradiation, we established a method of local irradiation of the molar tooth germ and demonstrated that radiation directly affected dental root development. In the current study, to locally irradiate the lower first molars of 5-day-old C57BL/6J mice, we used lead glass containing a hole as a collimator. We confirmed that our local irradiation method targeted only the tooth germ. The irradiated root was immature in terms of apical growth, and dentin formation was irregular along the outside of the root apices. Moreover, calcified tissue apically surrounded Hertwig's epithelial root sheath, which disappeared abnormally early. This method using a local irradiation experimental model will facilitate research into radiation-induced disorders of dental root formation. [ABSTRACT FROM AUTHOR]

Elucidating the mechanisms underlying human pain sensation requires the establishment of an in vitro model of pain reception comprising human cells expressing pain-sensing receptors and function properly as neurons. Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells and a promising candidate for producing human neuronal cells, however, the functional properties of differentiated hDPSCs have not yet been fully characterized. In this study, we demonstrated neuronal differentiation of hDPSCs via both their expression of neuronal marker proteins and their neuronal function examined using Ca2+ imaging. Moreover, to confirm the ability of nociception, Ca2+ responses in differentiated hDPSCs were compared to those of rat dorsal root ganglion (DRG) neurons. Those cells showed similar responses to glutamate, ATP and agonists of transient receptor potential (TRP) channels. Since TRP channels are implicated in nociception, differentiated hDPSCs provide a useful in vitro model of human peripheral neuron response to stimuli interpreted as pain. [ABSTRACT FROM AUTHOR]

The spontaneous dominant mouse mutant, Elbow knee synostosis (Eks), shows elbow and knee joint synosotsis, and premature fusion of cranial sutures. Here we identify a missense mutation in the Fgf9 gene that is responsible for the Eks mutation. Through investigation of the pathogenic mechanisms of joint and suture synostosis in Eks mice, we identify a key molecular mechanism that regulates FGF9 signaling in developing tissues. We show that the Eks mutation prevents homodimerization of the FGF9 protein and that monomeric FGF9 binds to heparin with a lower affinity than dimeric FGF9. These biochemical defects result in increased diffusion of the altered FGF9 protein (FGF9Eks) through developing tissues, leading to ectopic FGF9 signaling and repression of joint and suture development. We propose a mechanism in which the range of FGF9 signaling in developing tissues is limited by its ability to homodimerize and its affinity for extracellular matrix heparan sulfate proteoglycans. [ABSTRACT FROM AUTHOR]

Synthetic biomaterials have been developed and used for bone grafting. Here, we developed a biodegradable sponge composite for bone tissue engineering by combining β-tricalcium phosphate (β-TCP) and collagen. In addition, we sought to determine the optimal β-TCP granules/collagen ratio by evaluating and bone formation in vivo. Porous β-TCP granules were mixed with atelocollagen hydrochloride solution at various ratios -- 0.02, 0.05, 0.1, and 0.2 g/mL. The resultant mixtures were freeze-dried and subjected to dehydrothermal treatment in vacuo. The final composites obtained were designated β-TCP/collagen sponge composites (β-TCP/CS). Through compression testing, it was found that the stress values for β-TCP/CS (0.2 g/mL) were higher than those of the other three composites over the whole strain range. Histological evaluation at four weeks after implantation revealed that the collagen sponge had degraded and newly formed bone was present on the surface of the β-TCP granules. At 12 weeks, the β-TCP granules were completely degraded and remodeling of the lamellar bone was observed. [ABSTRACT FROM AUTHOR]

Ultraviolet-A (UVA) irradiation induces harmful effects on skin cells and accelerates skin aging through oxidative stress. In this study, the effects of a hydrogen-generating silica material named ULH-002 against UVA injuries in human cells and 3D skin equivalents were investigated. The oxygen radical absorption capacity (ORAC) assay showed that both freshly prepared ULH-002 solutions and 7-day-old solutions exhibited equal peroxyl radical (ROO·) scavenging activities concentration-dependently. CellROX® green/orange staining showed that ULH-002 could reduce UVA-induced oxidative stress in human keratinocytes HaCaT and human gingival fibroblasts (HGFs). ULH-002 significantly prevented UVA-induced apoptotic/necrotic cell death and cell-viability decline in HGFs and keratinocytes, as shown by Annexin V/PI apoptosis assay and PrestoBlue assay, respectively. Immunostaining showed that ULH-002 prevented the UVA-induced deterioration of expression of both type IV and I collagens in the 3D skin equivalents, and similarly in monolayer HGFs. UVA-enhanced melanogenesis was observed in human melanocytes HMV-II and HMV-II cell-containing 3D skin equivalents, but markedly prevented by ULH-002 as demonstrated by Fontana–Masson's staining. In conclusion, our data suggested that ULH-002 could protect human keratinocytes and fibroblasts from UVA-induced injuries, prevent the loss of type IV and I collagens, as well as reduce melanogenesis. ULH-002 might be developed as a skin care reagent in the cosmetic industry. [ABSTRACT FROM AUTHOR]

Background: Currently, ex-vivo handling of stem cells, including transport after harvest and therapeutic preparation, is generally done in culture media containing fetal bovine serum (FBS), which promotes cell attachment, proliferation, and differentiation. However, because of safety concerns associated with the use of FBS, including potential transmission of zoonotic agents and transplant rejection because of the incorporation of foreign proteins into the stem cells, there is a need for xenogeneic serum-free culture media for clinical handling of stem cells. Methods: Dental pulp stem cells were derived from wisdom teeth donated by eight healthy volunteers and cultured in xenogeneic serum-free culture medium (XFM) or xenogeneic serum-containing culture medium (SCM). Cells were subjected to morphological, proliferation, karyotype, differentiation, marker expression, cryopreservation, and cytotoxic susceptibility analyses in vitro, as well as transplantation in vivo. Results: In primary culture, XFM cells showed lower adhesion and slightly different morphology, although the single-cell size was similar to that of SCM cells. XFM cells exhibited typical mesenchymal stem cell (MSC) characteristics in vitro and in vivo, including marker gene/protein expression, trilineage differentiation potential, and hard, osteo-dentin tissue formation. Additionally, XFM cells maintained a normal karyotype in vitro and nontumorigenic potential in vivo; however, XFM cells were more susceptible to H2O2 and ultraviolet cytotoxic stimuli. XFM cells formed a multilayered structure showing excessive cell death/division in contrast to the monolayered structure of SCM cells when reaching overconfluence. Proliferation was disrupted in overconfluent XFM cells, and these cells could not be subcultured. Dimethyl sulfoxide-free cryopreserved XFM cells yielded similar results in all of the experiments. Conclusions: This study is the first reporting successful isolation and expansion of an MSC population from donor-derived tissue (dental pulp) under xenogeneic serum-free culture conditions, as well as the application of cryopreservation, using a research strategy based on clinically oriented in-vitro and in-vivo experiments. [ABSTRACT FROM AUTHOR]

Cell culture inserts offer an in vivo-like microenvironment to investigate cell-cell interactions between co-cultivated cells. However, it is unclear if types of inserts affect cell crosstalk. Here, we developed an environment-friendly cell culture insert, XL-insert, which can reduce plastic waste with lower cost. We compared XL insert with two types of commercial disposable culture inserts, Koken® insert with atelocollagen membrane (Col-inserts) and Falcon® inserts with plastic membrane (PET-inserts) on cell-cell interactions in co-cultivated THP-1 macrophages and OP9 adipocytes. Scanning electron microscope, immunoassay and imaging analysis showed that among three types of inserts, XL-inserts allowed cytokines from co-cultivated macrophages and adipocytes to diffuse freely and offered preferable in vivo-like microenvironment for cell-cell interactions. PET-inserts showed limitations for intercellular communication due to some pores being blocked by somas on the membrane that caused much lower permeability for cytokines passing through. Col-inserts blocked large sized cytokines but allowed small sized molecules to permeate resulting in improved lipid accumulation and adiponectin secretion in OP9 adipocytes. Taken together, our data demonstrated that membrane type and pore size on the membrane affect the cross-talk between co-cultivated cells very differently. Some previous co-culture studies might have different results if the inserts were changed. • Crosstalk between macrophage and adipocyte is affected by types of culture inserts. • The handmade insert, XL insert, is easy to make, environment-friendly and low cost. • XL insert provides an in vivo like microenvironment for cell-cell interactions. • The collagen membrane insert improves lipid accumulation and adiponectin secretion. [ABSTRACT FROM AUTHOR]

Abstract Tooth formation is accomplished under strict genetic programs. Although patients with chromosome 12q14 aberration shows tooth phenotype including the size and eruption timing with bone growth anomaly, its etiology is uncertain. Here, we examined expression of Hmga2, which is encoded at chromosome 12q14, in mouse tooth germs and analyzed the involvement in lower first molar (M1) and mandibular bone development. Hmga2 expression was immunohistochemically detected at enamel organ and the surrounding mesenchyme of the M1 germs. The expression was dynamically changed with gestation and rapidly decreased in postnatal mice. In Hmga2 −/− mice, the M1 germs and crowns were diminished in size, and formation and eruption of molars were delayed with mandibular bone growth retardation. Hmga2 cDNA or siRNA transfection showed that Hmga2 transcriptionally up-regulates expression of stem cell factors, Sox2 and Nanog. They were co-localized with Hmga2 in the germs, but differentially distributed at enamel organ and mesenchyme in Hmga2 −/− mice. These results demonstrate that Hmga2 expressed in tooth germs regulates the growth, sizing and eruption and stem cell factor expression in different compartment of the germ and associates with mandibular bone growth. Although future studies are needed, the present study demonstrates HMGA2 regulation of tooth genesis with skeletal development. Highlights • HMGA2 gene aberrations link with tooth genesis but the backgrounds are unknown. • Hmga2 expression was dynamically changed during mouse molar germ development. • Molar formation was hindered in Hmga2 knockout mice. • Hmga2 affected stem cell factor expression in different compartments of tooth germ. • Tooth formation is genetically regulated by HMGA2 and stem cell factors. [ABSTRACT FROM AUTHOR]

As human-origin cells, human dental pulp stem cells (hDPSCs) are thought to be potentially useful for biological and medical experiments. They are easily obtained from lost primary teeth or extracted wisdom teeth, and they are mesenchymal stem cells that are known to differentiate into osteoblasts, chondrocytes, and adipocytes. Although hDPSCs originate from neural crest cells, it is difficult to induce hDPSCs to differentiate into neuron-like cells. To facilitate their differentiation into neuron-like cells, we evaluated various differentiation conditions. Activation of K + channels is thought to regulate the intracellular Ca 2+ concentration, allowing for manipulation of the cell cycle to induce the differentiation of hDPSCs. Therefore, in addition to a conventional neural cell differentiation protocol, we activated K + channels in hDPSCs. Immunocyto-chemistry and real-time PCR revealed that applying a combination of 3 stimuli (high K + solution, epigenetic reprogramming solution, and neural differentiation solution) to hDPSCs increased their expression of neuronal markers, such as β3-tubulin, postsynaptic density protein 95, and nestin within 5 days, which led to their rapid differentiation into neuron-like cells. Our findings indicate that epigenetic reprogramming along with cell cycle regulation by stimulation with high K + accelerated the differentiation of hDPSCs into neuron-like cells. Therefore, hDPSCs can be used in various ways as neuron-like cells by manipulating their cell cycle., (2020 THE BIOPHYSICAL SOCIETY OF JAPAN.)

Mesenchymal stem cells (MSCs) as a source for regenerative medicine are now the subject of much clinical attention. There are high expectations due to their safety, low tumorigenic risk, and low ethical concerns. MSC therapy has been approved for acute graft-versus host diseases since 2015. Tooth-derived MSCs are known to have a great potential in their proliferation and differentiation capacities, even when compared with bone-marrow-derived MSCs. In particular, stem cells from human exfoliated deciduous teeth (SHEDs) are the best candidates for personal cell banking (dental pulp cell bank), because they can be obtained less invasively in the natural process of individual growth. SHEDs are known to differentiate into hepatocytes. There have been several studies showing the effectiveness of SHEDs on the treatment of liver failure in animal models. They may exert their effects either by repopulation of cells in injured liver or by paracrine mechanisms due to their immune-regulatory functions. Moreover, it may be possible to use each individuals' dental pulp cells as a future source of tailor-made differentiated hepatocytes in the context of a bioartificial liver or liver-on-a-chip to screen for drug toxicity., Competing Interests: Conflict-of-interest statement: The authors do not have any commercial affiliation or consultancy that could be construed as a conflict of interest.

Calcium hydrogen phosphate with a hydroxyapatite-like surface (CHP-HA) is a novel synthesized compound designed to overcome the limitations of bioactive ceramics. It was originally applied as nano-sized HA strips covering core plates to enhance the degree of interfacial attachment. The objective of the present study was to examine the cellular attachment, proliferation, and osteogenic differentiation of periodontal ligament stem cells (PDLSCs) on a CHP-HA substrate in comparison with conventional nanohydroxyapatite (NanoHA). The PDLSCs were cultivated with either CHP-HA or NanoHA for cellular attachment, proliferation, and osteogenic differentiation assay. Osteogenic differentiation was examined using quantitative polymerase chain reaction and immunofluorescence after confirmation by Alizarin red staining. We found that between 14 and 21 days, CHP-HA exhibited a well-organized matrix distribution, a high degree of cell proliferation, and a high level of Alizarin red staining in comparison to NanoHA. Expression of all the osteogenic markers examined was increased significantly relative to NanoHA at 14 days, but no significant differences in some osteogenic genes were found at 21 days. Immunofluorescence revealed stronger staining in the CHP-HA group. In conclusion, PDLSCs cultivated with this novel CHP-HA show enhanced cellular responses. We propose that CHP-HA may be a promising alternative biomaterial for periodontal tissue engineering.

The miniature pig is a useful large laboratory animal model. Various tissues and organs of miniature pigs are similar to those of humans in terms of developmental, anatomical, immunological, and physiological characteristics. The oral and maxillofacial region of miniature pigs is often used in preclinical studies of regenerative dentistry. However, there is limited information on the dentition and tooth structure of miniature pigs. The purpose of this study was to examine the time-course changes of dentition and tooth structure (especially the root) of the miniature pig mandibular cheek teeth through X-ray analyses using soft X-ray for two-dimensional observations and micro-CT for three-dimensional observations. The mandibles of male Clawn strain miniature pigs (2 weeks and 3, 5, 7, 9, 11, 14, 17, and 29 months of age) were used. X-ray analysis of the dentition of miniature pig cheek teeth showed that the eruption pattern of the miniature pig is diphyodont and that the replacement pattern is vertical. Previous definitions of deciduous and permanent teeth often varied and there has been no consensus on the number of teeth (dentition); however, we found that three molars are present in the deciduous dentition and that four premolars and three molars are present in the permanent dentition. Furthermore, we confirmed the number of tooth roots and root canals. We believe that these findings will be highly useful in future studies using miniature pig teeth., (Copyright © 2013 Wiley Periodicals, Inc.)

The periodontal ligament (PDL) contains various cell populations and plays a central role in the maintenance, repair, and regeneration of the periodontium, i.e., tooth-supporting structures. Because primary cells isolated from PDL tissue are heterogeneous, the establishment of an effective isolation method for cells of interest is desired. In the present study, two morphologically distinct cell types were identified in confluent primary cultures derived from rat PDL. To isolate these cell populations, a small piece of filter paper soaked with trypsin-EDTA was placed directly onto the target cell population, enabling the cells to detach from the culture dish. The filter papers were then transferred into fresh culture dishes to establish outgrowth cultures; these two steps constitute the "cell fishing" method. The "fished" cell types were propagated and subcultured for further analyses. In morphological evaluation, immunocytochemical analyses, and reverse transcription-polymerase chain reaction, the isolated cells exhibited a polygonal appearance or a mono- or multinucleated appearance, with a high cytoplasm-to-nucleus ratio, leading to their being characterized as epithelial or myogenic cell populations, respectively. Surprisingly, a notable proportion of the multinuclear cells in the primary and subsequent isolated cultures demonstrated dramatic, spontaneous contractions, a feature typical of skeletal muscle cells. Finally, the isolated cell populations maintained a normal karyotype with a diploid chromosomal number. These results demonstrated that physiological epithelial and skeletal muscle cells can be obtained from primary PDL cultures without artificial induction using growth factors or chemicals, and can be propagated as individual lineage-committed cell populations; the populations consisted of differentiated and progenitor cells that maintained chromosomal stability. This simple, classical culture procedure provides new insights into the biological properties of PDL cells, which are potentially important for the differentiation of tissue or somatic stem cells and for the development of future cell-based therapies for dental and muscular diseases., (Copyright © 2013 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

This article focuses on recent advances in the regeneration of periodontium using tissue engineering and examines new technologies that will lead to further advances in periodontal therapy. The various advantages and drawbacks of protein-based, cell-based, and genetic-engineering approaches are evaluated. The debate between those who aim to regenerate periodontal tissues and researchers who have focused on the reconstitution of structural elements of the teeth is examined. The isolation of human dental stem cells from deciduous and adult wisdom teeth might hold the key to allowing the replacement of teeth and the regeneration of supporting tissue. The combination of scientific research, following on from advances in other fields, with clinical research in dentistry could yield a solution to the debilitating and widespread problem of periodontitis.

Synthetic biomaterials have been developed and used for bone grafting. Here, we developed a biodegradable sponge composite for bone tissue engineering by combining beta-tricalcium phosphate (beta-TCP) and collagen. In addition, we sought to determine the optimal beta-TCP granules/collagen ratio by evaluating and bone formation in vivo. Porous beta-TCP granules were mixed with atelocollagen hydrochloride solution at various ratios--0.02, 0.05, 0.1, and 0.2 g/mL. The resultant mixtures were freeze-dried and subjected to dehydrothermal treatment in vacuo. The final composites obtained were designated beta-TCP/collagen sponge composites (beta-TCP/CS). Through compression testing, it was found that the stress values for beta-TCP/CS (0.2 g/mL) were higher than those of the other three composites over the whole strain range. Histological evaluation at four weeks after implantation revealed that the collagen sponge had degraded and newly formed bone was present on the surface of the beta-TCP granules. At 12 weeks, the beta-TCP granules were completely degraded and remodeling of the lamellar bone was observed.

The feasibility of an in situ tissue-engineering method employing cell-based therapy with autologous periodontal ligament-derived cells was investigated. Periodontal ligament cells were obtained from six beagle dogs. Periodontal fenestration defects (6 x 4 mm) were created bilaterally at a location 6 mm apical to the marginal alveolar crest in the maxillary canines. Alkaline phosphatase-positive periodontal ligament cells (3 x 10(5) cells) were seeded onto a collagen sponge scaffold just before implantation. One defect was filled with the cell-scaffold construct, and another was left empty as the control. All animals were killed 4 weeks after surgery, and specimens were evaluated histomorphometrically. All the histomorphometrical data were analyzed by three-way analysis of variance with the Bonferroni multiple comparisons test. Regeneration of apical tissue was faster than that of coronal and isolated tissues on the control side (apical > coronal > isolated; p < 0.0001). On the other hand, on the cell-seeded side, regeneration of the cementum was observed uniformly on the root surface. Our data suggest that the seeded cells induced cementum regeneration on the root surface, indicating the potential of in situ tissue engineering using autologous cells for the regeneration of periodontal tissues.

To regenerate periodontal tissues, a sandwich membrane composed of a collagen sponge scaffold and gelatin microspheres containing basic fibroblast growth factor (bFGF) in a controlled-release system was developed according to the new concept of "in situ tissue engineering." A three-walled alveolar bone defect (3 x 4 x 4 mm) was made bilaterally in edentulous regions created mesially to the canines in both the maxilla and mandible of nine beagle dogs. A sandwich membrane with or without bFGF (100 microg) was implanted in each defect (each group, n = 18). During weeks 1, 2, and 4, histologic evaluation and histometric analyses were performed on three dogs. Throughout the 4 weeks, vascularization and osteogenesis were active only in the bFGF-treated group (p < 0.01). New cementum was formed (2.4 +/- 0.9 mm) on the exposed root surface at 4 weeks, and functional recovery of the periodontal ligament was indicated in part by the perpendicular orientation of regenerated collagen fibers. In the control group, epithelial downgrowth and root resorption occurred and the defects were filled with connective tissue. Thus, our sandwich membrane induced successful regeneration of the periodontal tissues in a short period of time.