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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.

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: We devised a strategy for the fabrication of an ‘anatomy-mimicking’ cylinder-type engineered trachea combined with cartilage engineering. The engineered BIOTUBEs are used to support the architecture of the body tissue, for long-segment trachea (>5 cm) with carinal reconstruction. The aim of the present study was to fabricate an anatomy-mimicking cylinder-type regenerative airway, and investigate its applicability in a rabbit model. Methods: Collagen sponge rings (diameter: 6 mm) were arranged on a silicon tube (diameter: 6 mm) at 2-mm intervals. Chondrocytes from the auricular cartilage were seeded onto collagen sponges immediately prior to implantation in an autologous manner. These constructs were embedded in dorsal subcutaneous pouches of rabbits. One month after implantation, the constructs were retrieved for histological examination. In addition, cervical tracheal sleeve resection was performed, and these engineered constructs were implanted into defective airways through end-to-end anastomosis. Results: One month after implantation, the engineered constructs exhibited similar rigidity and flexibility to those observed with the native trachea. Through histological examination, the constructs showed an anatomy-mimicking tracheal architecture. In addition, the engineered constructs could be anastomosed to the native trachea without air leakage. Conclusion: The present study provides the possibility of generating anatomy-mimicking cylinder-type airways, termed BIO-AIR-TUBEs, that engineer cartilage in an in-vivo culture system. This approach involves the use of BIOTUBEs formed via in-body tissue architecture technology. Therefore, the BIO-AIR-TUBE may be useful as the basic architecture of artificial airways. Keywords: Anatomy-mimicking airway, Cylinder-type airway reconstruction, BIOTUBE, Engineered cartilage, BIO-AIR-TUBE

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

While regulation of gene-enhancer interaction is intensively studied, its application remains limited. Here, we reconstituted arrays of CTCF-binding sites and devised a synthetic topological insulator with tetO for chromatin-engineering (STITCH). By coupling STITCH with tetR linked to the KRAB domain to induce heterochromatin and disable the insulation, we developed a drug-inducible system to control gene activation by enhancers. In human induced pluripotent stem cells, STITCH inserted between MYC and the enhancer down-regulated MYC. Progressive mutagenesis of STITCH led to a preferential escalation of the gene-enhancer interaction, corroborating the strong insulation ability of STITCH. STITCH also altered epigenetic states around MYC. Time-course analysis by drug induction uncovered deposition and removal of H3K27me3 repressive marks follows and reflects, but does not precede and determine, the expression change. Finally, STITCH inserted near NEUROG2 impaired the gene activation in differentiating neural progenitor cells. Thus, STITCH should be broadly useful for functional genetic studies.

Abstract Background Contact domains of chromatin serve as a fundamental unit to regulate action of enhancers for target genes. Looping between a pair of CCCTC-binding factor (CTCF)-binding sites in convergent orientations underlies the formation of contact domains, while those in divergent orientations establish domain boundaries. However, every CTCF site is not necessarily engaged in loop or boundary structures, leaving functions of CTCF in varied genomic contexts still elusive. The locus containing Tfap2c and Bmp7 encompasses two contact domains separated by a region between the two genes, termed transition zone (TZ), characterized by two arrays of CTCF sites in divergent configuration. In this study, we created deletion and inversion alleles of these and other regions across the locus and investigated how they impinge on the conformation. Results Deletion of the whole two CTCF arrays with the CRISPR/Cas9 system resulted in impairment of blocking of chromatin contacts by the TZ, as assessed by the circular chromatin conformation capture assay (4C-seq). Deletion and inversion of either of the two arrays similarly, but less pronouncedly, led to reduction in the blocking activity. Thus, the divergent configuration provides the TZ with the strong boundary activity. Uniquely, we show the TZ harbors a 50-kb region within one of the two arrays that contacts broadly with the both flanking intervals, regardless of the presence or orientation of the other CTCF array. Further, we show the boundary CTCF array has little impact on intra-domain folding; instead, locally associating CTCF sites greatly affect it. Conclusions Our results show that the TZ not only separates the two domains, but also bears a wide interval that shows isotropic behavior of chromatin folding, indicating a potentially complex nature of actual boundaries in the genome. We also show that CTCF-binding sites inside a domain greatly contribute to the intra-domain folding of chromatin. Thus, the study reveals diverse and context-dependent roles of CTCF in organizing chromatin conformation at different levels.

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

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.

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: 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.

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: 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.

Computed tomography images are used for three-dimensional planning in orthognathic surgery. This facilitates the actual surgery by simulating the surgical scenario. We performed a computer-assisted virtual orthognathic surgical procedure using optically scanned three-dimensional (3D) data and real computed tomography data on a personal computer. It helped maxillary bone movement and positioning and the titanium plate temporary fixation and positioning. This simulated the surgical procedure, which made the procedure easy, and we could perform precise actual surgery and could forecast the postsurgery outcome. This simulation method promises great potential in orthognathic surgery to help surgeons plan and perform operative procedures more precisely.

Pluripotent stem cells are a promising tool for mechanistic studies of tissue development, drug screening, and cell-based therapies. Here, we report an effective and mass-producing strategy for the stepwise differentiation of mouse embryonic stem cells (mESCs) and mouse and human induced pluripotent stem cells (miPSCs and hiPSCs, respectively) into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenyl)pyrido[4′,3′:4,5]thieno[2,3-b]pyridine-2-carboxamide [TH]) under serum-free and feeder-free conditions. The strategy, which consists of mesoderm induction, osteoblast induction, and osteoblast maturation phases, significantly induced expressions of osteoblast-related genes and proteins in mESCs, miPSCs, and hiPSCs. In addition, when mESCs defective in runt-related transcription factor 2 (Runx2), a master regulator of osteogenesis, were cultured by the strategy, they molecularly recapitulated osteoblast phenotypes of Runx2 null mice. The present strategy will be a platform for biological and pathological studies of osteoblast development, screening of bone-augmentation drugs, and skeletal regeneration.

Odontogenic infection in immunocompromised patients tends to extend systemically beyond the oral cavity. Our case report presents a patient with sepsis due to a Streptococcus constellatus (S. constellatus) odontogenic infection in a 64-year-old-immunocompromised woman with Cogan’s syndrome. She had been suffering from chronic mandibular osteomyelitis which was thought to have been caused by dental caries and/or chronic periodontitis with furcation involvement of the left mandibular first molar. We suspect that the acute symptoms of the chronic osteomyelitis due to S. constellatus led to the systemic infection. This infection could be accelerated by the use of a corticosteroid and an alendronate. This is the first report which represents the potential association between odontogenic infection and Cogan’s syndrome.

In this study, we developed a new method to stimulate osteogenic differentiation in tissue-nonspecific alkaline phosphatase (TNAP)-positive cells liberated from human induced pluripotent stem cells (hiPSCs)-derived embryoid bodies (EBs) with 14 days long TGF-β/IGF-1/FGF-2 treatment. TNAP is a marker protein of osteolineage cells. We analyzed and isolated TNAP-positive and E-cadherin-negative nonepithelial cells by fluorescence-activated cell sorting. Treating the cells with a combination of transforming growth factor (TGF)-β, insulin-like growth factor (IGF)-1, and fibroblast growth factor (FGF)-2 for 14 days greatly enhanced TNAP expression and maximized expression frequency up to 77.3%. The isolated cells expressed high levels of osterix, which is an exclusive osteogenic marker. Culturing these TNAP-positive cells in osteoblast differentiation medium (OBM) led to the expression of runt-related transcription factor 2, type I collagen, bone sialoprotein, and osteocalcin (OCN). These cells responded to treatment with activated vitamin D3 by upregulating OCN. Furthermore, in OBM they were capable of generating many mineralized nodules with strong expression of receptor activator of NF-kappaB ligand and sclerostin (SOST). Real-time RT-PCR showed a significant increase in the expression of osteocyte marker genes, including SOST, neuropeptide Y, and reelin. Scanning electron microscopy showed dendritic morphology. Examination of semi-thin toluidine blue-stained sections showed many interconnected dendrites. Thus, TNAP-positive cells cultured in OBM may eventually become terminally differentiated osteocyte-like cells. In conclusion, treating hiPSCs-derived cells with a combination of TGF-β, IGF-1, and FGF-2 generated TNAP-positive cells at high frequency. These TNAP-positive cells had a high osteogenic potential and could terminally differentiate into osteocyte-like cells. The method described here may reveal new pathways of osteogenesis and provide a novel tool for regenerative medicine and drug development.

Hedgehog (Hh) signaling plays important roles in various development processes. This signaling is necessary for osteoblast formation during endochondral ossification. In contrast to the established roles of Hh signaling in embryonic bone formation, evidence of its roles in adult bone homeostasis is not complete. Here we report the involvement of Gli1, a transcriptional activator induced by Hh signaling activation, in postnatal bone homeostasis under physiological and pathological conditions. Skeletal analyses of Gli1+/- adult mice revealed that Gli1 haploinsufficiency caused decreased bone mass with reduced bone formation and accelerated bone resorption, suggesting an uncoupling of bone metabolism. Hh-mediated osteoblast differentiation was largely impaired in cultures of Gli1+/- precursors, and the impairment was rescued by Gli1 expression via adenoviral transduction. In addition, Gli1+/- precursors showed premature differentiation into osteocytes and increased ability to support osteoclastogenesis. When we compared fracture healing between wild-type and Gli1+/- adult mice, we found that the Gli1+/- mice exhibited impaired fracture healing with insufficient soft callus formation. These data suggest that Gli1, acting downstream of Hh signaling, contributes to adult bone metabolism, in which this molecule not only promotes osteoblast differentiation but also represses osteoblast maturation toward osteocytes to maintain normal bone homeostasis.

Tenomodulin (Tnmd) is a type II transmembrane protein characteristically expressed in dense connective tissues such as tendons and ligaments. Its expression in the periodontal ligament (PDL) has also been demonstrated, though the timing and function remain unclear. We investigated the expression of Tnmd during murine tooth eruption and explored its biological functions in vitro. Tnmd expression was related to the time of eruption when occlusal force was transferred to the teeth and surrounding tissues. Tnmd overexpression enhanced cell adhesion in NIH3T3 and human PDL cells. In addition, Tnmd-knockout fibroblasts showed decreased cell adhesion. In the extracellular portions of Tnmd, the BRICHOS domain or CS region was found to be responsible for Tnmd-mediated enhancement of cell adhesion. These results suggest that Tnmd acts on the maturation or maintenance of the PDL by positively regulating cell adhesion via its BRICHOS domain.

NF-κB signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-κB have been reported in mouse and human ES cells, and the role of NF-κB in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-κB signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-κB activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-κB activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-κB signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-κB signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.

To overcome the weak points of the present cartilage regenerative medicine, we applied a porous scaffold for the production of tissue-engineered cartilage with a greater firmness and a 3D structure. We combined the porous scaffolds with atelocollagen to retain the cells within the porous body. We conducted canine autologous chondrocyte transplants using biodegradable poly-l-lactic acid (PLLA) or poly-dl-lactic- co -glycolic acid (PLGA) polymer scaffolds, and morphologically and biochemically evaluated the time course changes of the transplants. The histological findings showed that the tissue-engineered constructs using PLLA contained abundant cartilage 1, 2, and 6 months after transplantation. However, the PLGA constructs did not possess cartilage and could not maintain their shapes. Biochemical measurement of the proteoglycan and type II collagen also supported the superiority of PLLA. The biodegradation of PLGA progressed much faster than that of PLLA, and the PLGA had almost disappeared by 2 months. The degraded products of PLGA may evoke a more severe tissue reaction at this early stage of transplantation than PLLA. The PLLA scaffolds were suitable for cartilage tissue engineering under immunocompetent conditions, because of the retarded degradation properties and the decrease in the severe tissue reactions during the early stage of transplantation.

Chondrocyte preparation with the safety and efficiency is the first step in cartilage regenerative medicine. To prepare a chondrocyte proliferation medium that does not contain fetal bovine serum (FBS) and that provides more than a 1000-fold increase in cell numbers within approximately 1 month, we attempted to use the medium containing 5% human serum (HS), but it exerted no more than twofold increase in 2 weeks. To compensate for the limited proliferation ability in HS, we investigated the combinational effects of 12 factors [i.e., fibroblast growth factor(FGF)-2, insulin-like growth factor(IGF)-I, insulin, bone morphogenetic protein-2, parathyroid hormone, growth hormone, dexamethasone, 1α25-dihydroxy vitamin D 3 , L-3,3′,5′-triodothyronine, interleukine-1 receptor antagonist, 17β-estradiol, and testosterone] on the proliferation of human auricular chondrocytes by analysis of variance in fractional factorial design. As a result, FGF-2, dexamethasone, insulin, and IGF-I possessed promotional effects on proliferation, while the combination of FGF-2 with insulin or IGF-I synergistically enhanced the proliferation. Actually, the chondrocytes increased 7.5-fold in number in 2 weeks in a medium containing 5% HS with 10 ng/ml FGF-2, while the cell number synergistically gained a 10–12-fold increase with 5 μg/ml insulin or 100 ng/ml IGF-I in the same period. The proliferation effects were more enhanced at a concentration of 100 ng/ml for FGF-2, and especially for the combination of 100 ng/ml FGF-2 and 5 μg/ml insulin (approximately 16-fold within 2 weeks). In the long-term culture with repeated passaging, this combination provided more than 10,000-fold within 8 weeks (i.e., passage 4). Thus, we concluded that such a combination of FGF-2 with insulin or IGF-I may be useful for promotion of auricular chondrocyte proliferation in a clinical application for cartilage regeneration.