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Introduction: Cleft lip and cleft palate are congenital disorders resulting from abnormal facial development. Current treatments require multiple surgeries, which have risks of scar formation and facial deformities. Recently, fetal treatments utilizing “scarless healing” have gained attention, as early intervention shows potential to suppress scarring. In the field of regenerative medicine, mesenchymal stem cell therapies using cell sheets have advanced, by which promotion of tissue repair is expected. However, researches for fetal treatment using small animal models of cleft lip are challenging due to the high fetal mortality caused by surgical invasiveness. Although organ culture methods may offer an alternative approach, no organ culture system for fetal cleft lip research has been reported. Methods: In this study, a cleft lip was surgically created on the upper left side lip of E15.5 mouse fetuses. These fetuses were cultured for four days using an organ culture system. Histological evaluation was performed to evaluate cell density, tissue morphology, and epithelialization. Additionally, adipose-derived stem cell (ADSC) sheets were transplanted two days after cleft lip creation to evaluate their effect on tissue repair. Results: The histological analysis showed that cell density and tissue morphology were stably maintained in the four-day culture period. Epithelialization of the incision site was observed two days after surgery, confirming the completion of cleft formation. In the ADSC-transplanted group, epithelialization of the cleft site was observed, which indicates that the stem cell sheets contributed to tissue repair. Conclusion: This research demonstrates the successful development of a cleft lip organ culture model and highlights the potential of ADSC sheets in promoting tissue repair. These findings provide a foundation for future regenerative medicine strategies in fetal cleft lip therapy.

Background: Cell therapy is a useful treatment method for wide spectrum of diseases which utilizes the immunosuppressive and regenerative abilities of administered cells. It is essential to build a transport system of tissues from which cells are harvested, because various external factors, such as temperature, time, air pressure, and vibration affect the cell functions isolated from body tissues. In particular, temperature is a critical factor which determines the viability of the cells and organs. In this study, we investigated the optimal temperature during the transportation of lipoaspirates from which adipose -derived stem cells (ASCs) were isolated. Method: Lipoaspirates obtained by liposuctions (lipomatic or vaser method) were transported in four different temperature zones (4, 20, 32, and 37 °C) in a transport container which is electrically controlled to maintain a constant temperature during transport. Stromal vascular fractions (SVFs) were harvested from the lipoaspirate, and the cell number, viability and proliferation rate and the yield of ASCs were examined. In addition, the metabolic state of the cells was examined. Results: ASCs from lipoaspirates transported at high temperature significantly decreased cell viability, while those at low temperature maintained high cell viability and showed good cell proliferation. In addition, transportation of lipoaspirates at low temperature resulted in a high level of NAD+/NADH, coenzymes involved in intracellular metabolism, and a low level of lactate in lipoaspirate suppressed the glycolytic system of intracellular metabolism, in ASCs. Conclusion: The lipoaspirate transported at 4 °C exhibited best results regarding live cell number, viability and cell proliferation in our experiments. This study offers a direction to build a transport system that connects laboratories and hospitals and achieve a beneficial therapy for patients.

Transforming growth factor β (TGFβ) induces epithelial–mesenchymal transition (EMT), which correlates with stemness and invasiveness. Mesenchymal–epithelial transition (MET) is induced by TGFβ withdrawal and correlates with metastatic colonization. Whether TGFβ promotes stemness and invasiveness simultaneously via EMT remains unclear. We established a breast cancer cell model expressing red fluorescent protein (RFP) under the E‐cadherin promoter. In 2D cultures, TGFβ induced EMT, generating RFPlow cells with a mesenchymal transcriptome, and regained RFP, with an epithelial transcriptome, after MET induced by TGFβ withdrawal. RFPlow cells generated robust mammospheres, with epithelio‐mesenchymal cell surface features. Mammospheres that were forced to adhere generated migratory cells, devoid of RFP, a phenotype which was inhibited by a TGFβ receptor kinase inhibitor. Further stimulation of RFPlow mammospheres with TGFβ suppressed the generation of motile cells, but enhanced mammosphere growth. Accordingly, mammary fat‐pad‐transplanted mammospheres, in the absence of exogenous TGFβ treatment, established lung metastases with evident MET (RFPhigh cells). In contrast, TGFβ‐treated mammospheres revealed high tumour‐initiating capacity, but limited metastatic potential. Thus, the biological context of partial EMT and MET allows TGFβ to differentiate between pro‐stemness and pro‐invasive phenotypes.

Abstract Purpose Many points concerning the structure of osseointegration and the surrounding jaw bone remain unclear, and its optimal histological form has yet to be identified. The aim of this study was to clarify the structural characteristics of peri-implant jaw bone on the micro- and nano-scales by quantitatively evaluating bone quality. Methods Five samples of human mandibular bone containing dental implants and one dentate sample that had been in place for some years while the donors were still alive were collected. Bulk staining was performed, and 100-μm-thick polished specimens were prepared. The osteon distributions in peri-implant bone and mandibular cortical bone were measured, after which alignment analysis of biological apatite (BAp) crystallites and anisotropy analysis of collagen fiber orientation using second-harmonic generation imaging were carried out. Results Osteons in the vicinity of the implant body ran parallel to it. In the cortical bone at the base of the mandible, however, most osteons were oriented mesiodistally. The preferential alignment of BAp crystallites was generally consistent with osteon orientation. The orientation of collagen fibers in peri-implant jaw bone resembled the concentric rings seen in normal cortical bone, but there were also fibers that ran orthogonally across these concentric fibers. Conclusions These results suggest that the mechanical strain imposed by implants causes the growth of cortical bone-like bone in areas that would normally consist of cancellous bone around the implants, and that its structural characteristics are optimized for the load environment of the peri-implant jaw bone.

Introduction: In cartilage regenerative medicine, transplanted chondrocytes contain a mixture of populations, that complicates the regeneration of uniform cartilage tissue. Our group previously reported that chondrocytes with higher chondrogenic ability could be enriched by selection of rapidly growing cells. In this study, the detailed properties of rapidly growing chondrocytes were examined and compared to slowly growing cells. Methods: Human auricular chondrocytes were fluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE) and analyzed using flow cytometry, focusing on division rates as indicated by fluorescence intensity and cell morphology according to the forward scatter and side scatter. Rapid and slow growing cell groups were harvested on days 2 and 4 after CFSE labeling, and their ability to produce cartilage matrix in vitro was examined. To compare the chondrogenic ability in vivo, the cells were seeded on poly-l-lactic acid scaffolds and transplanted into nude mice. Gene expression differences between the rapid and slow cell groups were investigated by microarray analysis. Results: On day 2 after CFSE labeling, the rapidly growing cell group showed the highest proliferation rate. The results of pellet culture showed that the rapid cell group produced more glycosaminoglycans per cell than the slow cell group. The amount of glycosaminoglycan production was highest in the rapid cell group on day 2 after CFSE labeling, indicating high chondrogenic ability. Furthermore, microarray, gene ontology, and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed upregulation of genes that promote cell division such as origin recognition complex subunit 1 and downregulation of genes that inhibit cell division such as cyclin dependent kinase inhibitor 1A. Besides cell cycle-related genes, chondrocyte-related genes such as serpin family B member 2, clusterin, bone morphogenetic protein 2, and matrix metalloproteinase 3 were downregulated, while fibroblast growth factor 5 which is involved in stem cell maintenance, and coiled-coil and C2 domain containing 2A, which is required for cilia formation, were upregulated. Conclusion: The results showed that the rapid cell group proliferated well and had more undifferentiated properties, suggesting a higher stemness. The present findings provide a basis for the use of the rapid cell group in cartilage regeneration.

Abstract Regenerative cartilage prepared from cultured chondrocytes is generally immature in vitro and matures after transplantation. Although many factors, including host cells and humoral factors, have been shown to affect cartilage maturation in vivo, the requirement of direct cell–cell contact between host and donor cells remains to be verified. In this study, we examined the host cells that promote cartilage maturation via cell–cell contact. Based on analysis of the transplanted chondrocytes, we examined the contribution of endothelial cells and macrophages. Using a semiclosed device that is permeable to tissue fluids while blocking host cells, we selectively transplanted chondrocytes and HUVECs or untreated/M1-polarized/M2-polarized RAW264.7 cells. As a result, untreated RAW264.7 cells induced cartilage regeneration. Furthermore, an in vitro coculture assay indicated communication between chondrocytes and RAW264.7 cells mediated by RNA, suggesting the involvement of extracellular vesicles in this process. These findings provide insights for establishing a method of in vitro cartilage regeneration.

ABSTRACT Bone nodule formation by differentiating osteoblasts is considered an in vitro model that mimics bone modeling. However, the details of osteoblast behavior and matrix production during bone nodule formation are poorly understood. Here, we present a spatiotemporal analysis system for evaluating osteoblast morphology and matrix production during bone modeling in vitro via two‐photon microscopy. Using this system, a change in osteoblast morphology from cuboidal to flat was observed during the formation of mineralized nodules, and this change was quantified. Areas with high bone formation were densely populated with cuboidal osteoblasts, which were characterized by blebs, protruding structures on their cell membranes. Cuboidal osteoblasts with blebs were highly mobile, and osteoblast blebs exhibited a polar distribution. Furthermore, mimicking romosozumab treatment, when differentiated flattened osteoblasts were stimulated with BIO, a GSK3β inhibitor, they were reactivated to acquire a cuboidal morphology with blebs on their membranes and produced more matrix than nonstimulated cells. Our analysis system is a powerful tool for evaluating the cell morphology and function of osteoblasts during bone modeling. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

The objective of this study is to clarify the effect of restoring the lowered masticatory muscle functional pressure and correcting bilateral differences in masticatory muscle functional pressure on jawbone growth during growth and development with a quantitative evaluation of the changes in the micro/nanostructural characteristics of entheses. Male Wistar rats aged 4 weeks were divided into an experimental group injected with a botulinum toxin serotype A (BoNT/A) formulation to reduce muscle function (BTX group) and a control group (CTRL group). They were euthanised after 6, 8, 10, 12, and 16 weeks after measuring the difference between the midline of the upper and lower incisors. The mandibles were harvested for histological examination, second harmonic generation imaging, and the quantitative evaluation of biological apatite (BAp) crystal alignment. The midline difference decreased with age in weeks. In rats from 6 weeks after BoNT/A administration to 12 weeks after administration, the collagen fibre bundle diameter was significantly smaller in the BTX group; the difference between the two groups decreased with increasing age. BAp crystal alignment was significantly different on the x-axis and the y-axis on the BTX group from 6 weeks after BoNT/A administration to 10 weeks after administration. Asymmetry of mandibular bone formation caused by load imbalance during growth could be corrected by the adjustment of the function of the masseter muscle on either side.

Abstract Cartilage tissues have poor self-repairing abilities. Regenerative medicine can be applied to recover cartilage tissue damage in the oral and maxillofacial regions. However, hitherto it has not been possible to predict the maturity of the tissue construction after transplantation or to prepare mature cartilage tissues before transplantation that can meet clinical needs. Macrophages play an important role in cartilage tissue regeneration, although the exact mechanisms remain unknown. In this study, we established and verified an in vitro experimental system for the direct co-culture of cell pellets prepared from mouse auricular chondrocytes and macrophages polarized into four phenotypes (M1-like, M1, M2-like, and M2). We demonstrate that cartilage pellets co-cultured with M1-like promoted collagen type 2 and aggrecan production and induced the most significant increase in chondrogenesis. Furthermore, M1-like shifted to M2 on day 7 of co-culture, suggesting that the cartilage pellet supplied factors that changed the polarization of M1-like. Our findings suggest that cartilage regenerative medicine will be most effective if the maturation of cartilage tissues is induced in vitro by co-culture with M1-like before transplantation.

Abstract Although multiple studies have investigated the mesenchymal stem and progenitor cells (MSCs) that give rise to mature bone marrow, high heterogeneity in their morphologies and properties causes difficulties in molecular separation of their distinct populations. In this study, by taking advantage of the resolution of the single cell transcriptome, we analyzed Sca-1 and PDGFR-α fraction in the mouse bone marrow tissue. The single cell transcriptome enabled us to further classify the population into seven populations according to their gene expression profiles. We then separately obtained the seven populations based on candidate marker genes, and specified their gene expression properties and epigenetic landscape by ATAC-seq. Our findings will enable to elucidate the stem cell niche signal in the bone marrow microenvironment, reconstitute bone marrow in vitro, and shed light on the potentially important role of identified subpopulation in various clinical applications to the treatment of bone- and bone marrow-related diseases.

Abstract Background Due to its low capacity for self-repair, articular cartilage is highly susceptible to damage and deterioration, which leads to the development of degenerative joint diseases such as osteoarthritis (OA). Menstrual blood-derived mesenchymal stem/stromal cells (MenSCs) are much less characterized, as compared to bone marrow mesenchymal stem/stromal cells (BMMSCs). However, MenSCs seem an attractive alternative to classical BMMSCs due to ease of access and broader differentiation capacity. The aim of this study was to evaluate chondrogenic differentiation potential of MenSCs and BMMSCs stimulated with transforming growth factor β (TGF-β3) and activin A. Methods MenSCs (n = 6) and BMMSCs (n = 5) were isolated from different healthy donors. Expression of cell surface markers CD90, CD73, CD105, CD44, CD45, CD14, CD36, CD55, CD54, CD63, CD106, CD34, CD10, and Notch1 was analyzed by flow cytometry. Cell proliferation capacity was determined using CCK-8 proliferation kit and cell migration ability was evaluated by scratch assay. Adipogenic differentiation capacity was evaluated according to Oil-Red staining and osteogenic differentiation according to Alizarin Red staining. Chondrogenic differentiation (activin A and TGF-β3 stimulation) was investigated in vitro and in vivo (subcutaneous scaffolds in nude BALB/c mice) by expression of chondrogenic genes (collagen type II, aggrecan), GAG assay and histologically. Activin A protein production was evaluated by ELISA during chondrogenic differentiation in monolayer culture. Results MenSCs exhibited a higher proliferation rate, as compared to BMMSCs, and a different expression profile of several cell surface markers. Activin A stimulated collagen type II gene expression and glycosaminoglycan synthesis in TGF-β3 treated MenSCs but not in BMMSCs, both in vitro and in vivo, although the effects of TGF-β3 alone were more pronounced in BMMSCs in vitro. Conclusion These data suggest that activin A exerts differential effects on the induction of chondrogenic differentiation in MenSCs vs. BMMSCs, which implies that different mechanisms of chondrogenic regulation are activated in these cells. Following further optimization of differentiation protocols and the choice of growth factors, potentially including activin A, MenSCs may turn out to be a promising population of stem cells for the development of cell-based therapies with the capacity to stimulate cartilage repair and regeneration in OA and related osteoarticular disorders.

In cartilage tissue engineering, research on materials for three-dimensional (3D) scaffold has attracted attention. Decellularized matrix can be one of the candidates for the scaffold material. In this study, decellularization of regenerated cartilage was carried out and its effectiveness as a scaffold material was examined. Three-dimensionally-cultured cartilage constructs in the differentiation medium containing IGF-1 produced more cartilage matrix than those in the proliferation medium. Detergent-enzymatic method (DEM) could decellularize 3D-cultured cartilage constructs only by 1 cycle without breaking down the structure of the constructs. In vitro, newly-seeded chondrocytes were infiltrated and engrafted into decellularized constructs in the proliferation medium, and newly formed fibers were observed around the surface where newly-seeded cells were attached. Recellularized constructs could mature similarly as those without decellularization in vivo. The decellularized 3D-cultured matrix from regenerative cartilage is expected to be used as a scaffold material in the future.

Introduction: Currently, various kinds of materials are used for the treatment of bone defects. In general, these materials have a problem of formativeness. The three -dimensional (3D) printing technique has been introduced to fabricate artificial bone with arbitrary shapes, but poor bone replacement is still problematic.Our group has created a β⁻tricalcium phosphate (β⁻TCP) scaffold by applying 3D printing technology. This scaffold has an arbitrary shape and an internal structure suitable for cell loading, growth, and colonization. The scaffold was coated with a recombinant collagen peptide (RCP) to promote bone replacement.As indicated by several studies, cells loaded to scaffolds promote bone regeneration, especially when they are induced osteoblastic differentiation before transplantation. In this study, culture duration for bone marrow cells was optimized before being loaded to this new scaffold material. Method: Bone marrow cells isolated from C57BL/6J mice were subjected to osteogenic culture for 4, 7, and 14 days. The differentiation status of the cells was examined by alkaline phosphatase staining, alizarin red staining, and real-time RT-PCR for differentiation markers. In addition, the flow of changes in the abundance of endothelial cells and monocytes was analyzed by flow cytometry according to the culture period of bone marrow cells.Next, cells at days 4, 7, and 14 of culture were placed on a β-TCP/RCP scaffold and implanted subcutaneously into the back of C57BL/6J mice. Grafts were harvested and evaluated histologically 8 weeks later. Finally, Cells cultured for 7 days were also transplanted subperiosteally in the skull of the mouse with scaffolds. Result: Alkaline phosphatase staining was most prominent at 7 days, and alizarin red staining was positive at 14 days. Real-time RT-PCR revealed that Runx2 and Alp peaked at 7 days, while expression of Col1a1 and Bglap was highest at 14 days. Flow cytometry indicated that endothelial cells increased from day 0 to day 7, while monocytes increased continuously from day 0 to day 14. When transplanted into mice, the scaffold with cells cultured for 7 days exhibited the most prominent osteogenesis. The scaffold, which was transplanted subperiosteally in the skull, retained its shape and was replaced with regenerated bone over a large area of the field of view. Conclusion: Osteoblasts before full maturation are most efficient for bone regeneration, and the pre-culture period suitable for cells to be loaded onto a β-TCP/RCP hybrid scaffold is approximately 7 days.This β-TCP/RCP hybrid scaffolds will also be useful for bone augmentation.

Introduction: Achilles tendinopathy is characterized by scar formation or ectopic ossification, both of which result in pain and worsened physical function in athletes and older people. Although cell therapy using adipose-derived stem cells (ASCs) has been shown to be effective for tendinopathy, the underlying mechanisms by which ASCs result in tendon healing in vivo have not yet been fully clarified. Methods: ASCs were obtained from the fat pads of EGFP transgenic mice by collagenase digestion. C57BL/6 mice were used in a collagenase-induced injury model. ASCs were transplanted into injury sites at 1 week after injury. Tendons were harvested at 9 days, 2 weeks, and 4 weeks after transplantation, and analyzed by histological examination and μCT scanning. Results: Histological analysis and μCT scanning revealed greater recovery of collagen fibers and suppression of ectopic ossification in the ASC-treated group than in the control group at 2 and 4 weeks after injury. Immunohistochemical analysis identified transplanted ASCs in the tendon core close to peritenon and connective tissue at 2 days and 1 week after transplantation, but not at 3 weeks. Furthermore, while the expression levels of IL-1β, GLUT1, and CA9 were significantly reduced in the ASC group compared to the control group at 9 days after injury, those of VEGF and the number of CD31 positive vessels were significantly increased. Conclusion: The efficacy of ASCs for tendon repair and the prevention of ectopic ossification in Achilles tendinopathy were demonstrated. Our data suggest that ASCs can modulate inflammation and induce neovascularization in the early stage of tendon injury.

Teeth are exposed to various stimuli, including bacterial, thermal, and physical stimuli. Therefore, immune cells present in the normal dental pulp and the immune response to these stimuli have been studied. However, the relationship between systemic inflammation, such as that induced by viral infection, and changes occurring in dental pulp is not well known. This study aimed to investigate the immunological and hematological responses to systemic inflammation in dental pulp. Poly(I:C), a toll-like receptor 3 agonist, was injected into mice every two days to simulate a systemic inflammatory state in which type I interferon (IFN–I) was produced. The untreated normal state was defined as a steady state, and the states of acute and chronic inflammation were defined according to the period of administration. Changes in the abundance and dynamics of hematopoietic and immune cells in dental pulp, bone marrow and peripheral blood were quantitatively investigated in the steady state and under conditions of inflammation induced by IFN-l. We found that dental pulp in the steady state contained only a few hematopoietic cells, but a greater variety of immune cells than previously reported. B cells were also found in the steady state. An increase in multipotent progenitor cell levels was observed in the dental pulp during both acute and chronic inflammation. The increased multipotent progenitor cells in the dental pulp during acute inflammation tended to differentiate into the myeloid lineage. On the other hand, there was an influx of B cells into the dental pulp during chronic inflammation. These results revealed that a unique immune response is induced in the dental pulp by systemic inflammation, which would lead to a significant change in the perspective of dentists on the utility of dental pulp in the management of systemic diseases.

Introduction: Cartilage regeneration have been attracted attentions because of the poor ability of cartilage tissues to regenerate. Three-dimensional (3D) culture of chondrocytes is considered to be advantageous for cartilage regeneration. Although it is plausible that maturation of the constructs before transplantation positively affects the chondrogenesis, matured constructs after cultures for longer periods do not necessarily result in effective cartilage regeneration. In this study, we compared different types of culture media including growth factors which are clinically available. We prepared differentiation medium containing insulin-like growth factor-1 (IGF-1), proliferation medium containing fibroblast growth factor-2 (FGF-2) and insulin, and combination of them, and compared their efficacies on chondrogenesis when used in 3D culture of engineered cartilage constructs. Methods: Cartilage constructs were fabricated by auricular chondrocytes and atelocollagen, and they were 3D-cultured with four types of media: control medium, differentiation medium, proliferation medium, and combination medium. After 3 weeks of culture, the constructs were analyzed for cell number, gene and protein expressions and mechanical properties. The constructs were also transplanted into nude mice. After 8 weeks, the degree of cartilage regeneration was evaluated. Constructs manufactured with canine auricular chondrocytes were subjected to autologous transplantation into beagles and examined for cartilage regeneration. Results: During 3D culture, remarkably high gene expression of type II collagen was detected in the construct cultured with the differentiation medium whereas cell apoptosis were suppressed in the proliferation medium. When transplanted into nude mice, the constructs 3D-cultured in the proliferation medium produced abundant cartilage matrices. In autologous implantation model, the construct cultured in the proliferation medium again showed better chondrogenesis than those in other media. Conclusions: The present study indicates that 3D culture with the proliferation medium maintains the cell viability to potentiate the subsequent cartilage regeneration. Here, we propose that not only differentiation but also high cell viability accompanied by proliferation factors should be taken into account to improve cartilage regeneration. Keywords: Cartilage regeneration, Three-dimensional culture, Culture medium

Introduction: Pluripotent stem cells have an advantage that they can proliferate without reduction of the quality, while they have risk of tumorigenesis. It is desirable that pluripotent stem cells can be utilized safely with minimal effort in cartilage regenerative medicine. To accomplish this, we examined the potential usefulness of induced pluripotent stem cells (iPS cells) after minimal treatment via cell isolation and hydrogel embedding for cartilage regeneration using a large animal model. Methods: Porcine iPS-like cells were established from the CLAWN miniature pig. In vitro differentiation was examined for porcine iPS-like cells with minimal treatment. For the osteochondral replacement model, osteochondral defect was made in the quarters of the anteromedial sides of the proximal tibias in pigs. Porcine iPS-like cells and human iPS cells with minimal treatment were seeded on scaffold made of thermo-compression-bonded beta-TCP and poly-L-lactic acid and transplanted to the defect, and cartilage regeneration and tumorigenesis were evaluated. Results: The in vitro analysis indicated that the minimal treatment was sufficient to weaken the pluripotency of the porcine iPS-like cells, while chondrogenic differentiation did not occur in vitro. When porcine iPS-like cells were transplanted into osteochondral replacement model after minimal treatment in vitro, cartilage regeneration was observed without tumor formation. Additionally, fluorescent in situ hybridization (FISH) indicated that the chondrocytes in the regenerative cartilage originated from transplanted porcine iPS-like cells. Transplantation of human iPS cells also showed the regeneration of cartilage in miniature pigs under immunosuppressive treatment. Conclusion: Minimally-treated iPS cells will be a useful cell source for cartilage regenerative medicine. Keywords: iPS cells, Minimal treatment, Osteochondral replacement model, Cartilage regeneration

Restoration of damaged cartilage tissue has been deemed futile with current treatments. Although there have been many studies on cartilage regeneration thus far, there is no report that chondrocytes were completely re-differentiated in vitro. The clarification of cellular composition and matrix production during cartilage regeneration must be elucidated to fabricate viable mature cartilage in vitro. In order to achieve this aim, the chondrocytes cultured on coverslips were transplanted into the peritoneal cavities of mice. At different time points post-transplantation, the cartilage maturation progression and cells composing the regeneration were examined. Cartilage regeneration was confirmed by hematoxylin & eosin (HE) and toluidine blue staining. The maturation progression was carefully examined further by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). At the first and second week time points, various cell shapes were observed using SEM. Chronologically, by the third week, the number of fibers increased, suggesting the progression of extracellular matrix (ECM) maturation. Observation through TEM revealed the chondrocytes located in close proximity to various cells including macrophage-like cells. On the second week, infiltration of lymphocytes and capillary vessels were observed, and after the third week, the chondrocytes had matured and were abundantly releasing extracellular matrix. Chronological observation of transplanted chondrocytes by electron microscopy revealed maturation of chondrocytes and accumulation of matrix during the re-differentiation process. Emerging patterns of host-derived cells such as macrophage-like cells and subsequent appearance of lymphocytes-like cells and angiogenesis were documented, providing crucial context for the identification of the cells responsible for in vivo cartilage regeneration. Keywords: Chondrocytes, Macrophage, Lymphocyte, Endothelial cells, Peritoneal cavity, Electron microscopy

Remarkable advances have been made in cartilage regenerative medicine to cure congenital anomalies including microtia, tissue defects caused by craniofacial injuries, and geriatric diseases such as osteoarthritis. However, those procedures require a substantial quantity of chondrocytes for tissue engineering. Previous studies have required several passages to obtain sufficient cell numbers for three-dimensional and monolayer cultures. Thus, our objective was to improve the quantity of chondrocytes that can be obtained by examining an anti-fouling polyhydrophilic chemical called poly(2-hydroxyethyl methacrylate) (pHEMA). To determine the effectiveness of the chemical, pHEMA solution was applied via dip-coating to centrifuge tubes, serological pipettes, and pipette tips. The cell quantity obtained during standard cell culturing and passaging procedures was measured alongside non-coated materials as a control. A significant 2.2-fold increase of chondrocyte yield was observed after 2 passages when pHEMA was applied to the tubes compared to when non-coated tubes were utilized. The 3-dimensional chondrocyte pellets prepared from the respective cell populations and transplanted into nude mice were histologically and biochemically analyzed. No evidence of difference in matrix production for in vitro and in vivo cultures was found as well as similar proliferation rates and colony formation abilities. The use of pHEMA provides a powerful alternative method for expanding the quantity of chondrocytes harvested and handled during cell isolation and passaging to enhance cartilage tissue engineering.

Introduction: We have developed an implant-type tissue-engineered cartilage using a poly-l-lactide scaffold. In a clinical study, it was inserted into subcutaneous areas of nasal dorsum in three patients, to correct cleft lip–nose deformity. The aim of this study was to helping evaluation on the efficacy of the regenerative cartilage. Methods: 3D data of nasal shapes were compared between before and after surgery in computed tomography (CT) images. Morphological and qualitative changes of transplants in the body were also evaluated on MRI, for one year. Results: The 3D data from CT images showed effective augmentation (>2 mm) of nasal dorsum in almost whole length, observed on the medial line of faces. It was maintained by 1 year post-surgery in all patients, while affected curves of nasal dorsum was not detected throughout the observation period. In magnetic resonance imaging (MRI), the images of transplanted cartilage had been observed until 1 year post-surgery. Those images were seemingly not straight when viewed from the longitudinal plain, and may have shown gentle adaptation to the surrounding nasal bones and alar cartilage tissues. Conclusion: Those findings suggested the potential efficacy of this cartilage on improvement of cleft lip–nose deformity. A clinical trial is now being performed for industrialization.

Cartilage has a poor capacity for healing due to its avascular nature. Therefore, cartilage regenerative medicine including autologous chondrocyte implantation (ACI) could be a promising approach. Previous research has proposed various methods to enrich the cultured chondrocytes for ACI, yet it has been difficult to regenerate homogeneous native-like cartilage in vivo. The cell populations with an increased ability to produce cartilage matrix can show somatic stem cells-like characteristics. Stem cells, especially somatic stem cells are able to grow rapidly in vitro yet the growth rate is drastically reduced when placed in in vivo conditions [14]. Thus, in this study we investigated whether proliferation rate has an impact on in vivo regeneration of cartilage constructs by sorting human chondrocytes. The human chondrocytes were fluorescently labeled with CFSE and then cultured in vitro; once analyzed, the histogram showed a widening of fluorescence level, indicating that the cells with various division rates were included in the cell population. To compare the characteristics of the cell groups with different division rates, the chondrocytes were sorted into groups according to the fluorescence intensity (30 or 45 percent of cells plotted in the left and right sides of histogram). Then the cells of the rapid cell group and slow cell group were seeded into PLLA scaffolds respectively, and were transplanted into nude mice. Metachromatic regions stained with toluidine blue were larger in the rapid cell group compared to the slow cell group, indicating that the former had higher chondrogenic ability. We proposed a new method to enrich cell population with high matrix production, using proliferation rate alone.

Introduction: Achondroplasia (ACH) is a congenital disease which causes dwarfism and many symptoms resulting from skeletal dysplasia. Because present therapeutic strategies are mainly surgical procedures as symptomatic treatments, development of a radical treatment is desired. Clarification of the ACH pathology is essential for creating a new remedy. However, there are many questions about the disease mechanisms that have not been answered. Methods: As a single base substitution of the FGFR3 gene had been proved to be the ACH causing genome mutation, our group established disease specific iPS cells by introducing the causative mutation of achondroplasia into human iPS cells by CRISPR/Cas9 based genome editing. These cells were differentiated towards chondrocytes, then the gene and protein expressions were examined by real time RT-PCR and Western blotting, respectively. Results: Based on the western blotting analysis, the FGFR3 protein and phosphorylated ERK were increased in the FGFR3 mutated iPS cells compared to the control cells, while the FGFR3 gene expression was suppressed in the FGFR3 mutated iPS cells. According to chondrogenic differentiation experiments, the IHH expression level was increased in the control cells as the differentiation progressed. On the other hand, up-regulation of the IHH gene expression was suppressed in the FGFR3 mutated iPS cells. Conclusions: These results suggested that chondrocyte maturation was impaired between the proliferative stage and prehypertrophic stage in the chondrocytes of ACH. The development of chemical compounds which affect the specific maturation stage of chondrocytes is expected to contribute to the ACH treatment, and FGFR3 genome-edited hiPSCs will be a valuable tool in such research studies.

Introduction: There are various types of cartilage, including the auricular and articular cartilages. These cartilages have different functions, and their matrix volume and density of chondrocytes may differ. Thus, different protocols may be required to digest different types of cartilage. Methods: In this study, we examined protocols for the digestion of articular and auricular cartilages and determined the optimal conditions for articular cartilage digestion. Results: Our histological findings showed that the articular cartilage has a larger matrix area and fewer cells than the auricular cartilage. In 1-mm2 areas of articular and auricular cartilages, the average numbers of cells were 44 and 380, respectively, and the average matrix areas were 0.94 and 0.77 mm2, respectively. The maximum numbers of viable cells (approximately 1 × 105 cells/0.1 g of tissue) were obtained after digestion in 0.15, 0.3, or 0.6% collagenase for 24 h, in 1.2% collagenase for 6 h, or in 2.4% collagenase for 4 h. In tissues incubated in 0.15 or 0.3% collagenase, the cell numbers were lower than 1 × 105, even at 24 h, possibly reflecting incomplete digestion of cartilage. No significant differences were observed in the results of apoptosis assays for all collagenase exposure times and concentrations. However, cell damage appeared to be greater when collagenase concentrations were high. When cells obtained after digestion with different concentrations of collagenase were seeded at a density of 3000 cells/cm2, they yielded the maximum cell numbers after 1 week. Conclusions: We recommend a 24-h incubation in 0.6% collagenase as the optimal condition for chondrocyte isolation from articular cartilage. Moreover, we found that the optimum cell-seeding density is approximately 3000 cells/cm2. Conditions determined in this study would maximize the yield of isolated articular chondrocytes and enable the generation of a large quantity of cultured cells.

Objective: To explore the involvement of bone marrow cells and angiogenesis in the pathogenesis of antiresorptive agent-related osteonecrosis of the jaw (ARONJ). Methods: We performed micro-computed tomography (CT) and histological analyses in an ARONJ mouse model generated using bisphosphonate (BP) and cyclophosphamide (CY). Results: Micro-CT analysis showed that BP and CY inhibited osteoneogenesis in the extraction socket. Histological analysis at 3 days after tooth extraction showed inhibition of vascular endothelial cell and mesenchymal stem cell mobilization into the extraction socket. When neovascularization of the extraction fossa was observed from as early as 1 day after extraction, it occurred predominantly in the area adjacent to the extraction fossa and close to the bone marrow cavity. In addition, the extraction fossa communicated with the adjacent bone marrow via the vasculature. Histological evaluation of the alveolar bone marrow around the extraction socket showed a decrease in bone marrow cells in the BP + CY group. Conclusion: Both inhibition of angiogenesis and suppression of bone marrow cell mobilization are involved in the pathogenesis of ARONJ. [ABSTRACT FROM AUTHOR]

Objective: Scar formation is an inevitable outcome after craniofacial surgery in the congenital facial anomaly. Scarless healing is the ultimate treatment after the surgery. Therefore, we elucidate the mechanism underlying scarless healing during fetal development. Methods: A full-thickness back skin excision (1× 0.5 mm) was made at embryonic day 16.5 (E16.5) and 18.5 (E18.5) in fetal C57BL/6J mice and examined the histochemical and morphometrical findings of wound healing after 48 hours. Results: The wound made at E16.5 showed almost complete re-epithelialization with fine reticular dermal collagen fibers, but not at E18.5. The ratio of CK5 positive area was significantly higher in the wound of E16.5 operation than in the E18.5. The wounds made at E18.5 showed granulation tissue formation which will lead to subsequent scar formation. The collagen fibers tended to be thinner in wound than in normal skin, while the decrease in the number of fibers but the increase in the straightness of fibers were evident in the wound at E18.5. Conclusion: Transition point of scarless healing seemed between E16.5 and E18.5 in mice, which may imply that the potential of epithelial regeneration and matrix formation was changed, possibly due to alteration of cell constitution and decrease in stemness, at that time. [ABSTRACT FROM AUTHOR]

Arkadia, a positive regulator of Smad-dependent signalling via the transforming growth factor-β (TGF-β) family, is an E3 ubiquitin ligase that induces ubiquitylation and proteasome-dependent degradation of TGF-β suppressors such as Smad7, c-Ski and SnoN. In this study, we examined the effects of Arkadia on bone morphogenetic protein (BMP)-induced osteoblast differentiation. Knockdown of Arkadia reduced mineralization and expression of osteoblast differentiation markers. Furthermore, we showed that Smad6, a BMP-specific inhibitory Smad, is a target of Arkadia: wild-type (WT) Arkadia, but not the C937A (CA) mutant lacking E3 ubiquitin-ligase activity, induced ubiquitylation and proteasome-dependent degradation of Smad6. Accordingly, protein levels of Smad6, Smad7 and c-Ski were elevated in MEFs from Arkadia KO mice. Finally, expression of Arkadia attenuated blockade of BMP signalling by Smad6 in a transcriptional reporter assay. These results demonstrate that Smad6 is a novel target of Arkadia, and that Arkadia positively regulates BMP signalling via degradation of Smad6, Smad7 and c-Ski/SnoN. [ABSTRACT FROM AUTHOR]

Abstract  Idiopathic osteonecrosis of the femoral head (ION) is a devastating pathological condition of unknown etiology. In this study, we developed a simple murine model of osteonecrosis and investigated the underlying molecular mechanisms. In this model, the central portion of the tails of male C57BL/6 mice were tightly ligated to produce ischemic regions at sites distal to the ligatures. The occlusive ligatures were maintained for the indicated periods and then removed to induce reperfusion. The tails were histologically examined, and gene expression was analyzed by PCR array. The effect of p53 expression on osteocytes apoptosis was examined using preosteocytic MLO-A5 cells. In addition, the expression of p53 was analyzed in the femoral head samples obtained from hip osteoarthritis (OA) patients and ION patients. Caudal vertebrae distal to the ligatures (distal region) exhibited histological changes mimicking those observed in ION. Expression of p53 was increased in the distal region, and overexpression of p53 induced apoptosis in MLO-A5 cells. Treatment with a p53 inhibitor suppressed osteocyte apoptosis in the distal region. Strong p53 immunostaining was observed in osteocytes, vascular endothelial cells, and bone marrow cells in the femoral heads from ION patients but not from OA patients. Ischemia/reperfusion of the caudal vertebrae is a useful murine model of osteonecrosis, mimicking the histological changes found in ION. Using this model, we found the possible involvement of p53 in the osteocyte apoptosis observed in ION. Therapeutics targeting p53 might be a useful approach to ameliorating or even preventing osteonecrosis in ION patients. [ABSTRACT FROM AUTHOR]

Osteoclasts (OCs) undergo rapid apoptosis without trophic factors, such as macrophage colony-stimulating factor (M-CSF). Their apoptosis was associated with a rapid and sustained increase in the pro-apoptotic BH3-only Bcl-2 family member Bim. This was caused by the reduced ubiquitylation and proteasomal degradation of Bim that is mediated by c-Cbl. Although the number of OCs was increased in the skeletal tissues of bim-/- mice, the mice exhibited mild osteosclerosis due to reduced bone resorption. OCs differentiated from bone marrow cells of bim-/- animals showed a marked prolongation of survival in the absence of M-CSF, compared with bim+/+ OCs, but the bone-resorbing activity of bim-/- OCs was significantly reduced. Overexpression of a degradation-resistant lysine-free Bim mutant in bim-/- cells abrogated the anti-apoptotic effect of M-CSF, while wild-type Bim did not. These results demonstrate that ubiquitylation-dependent regulation of Bim levels is critical for controlling apoptosis and activation of OCs. [ABSTRACT FROM AUTHOR]