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Mesenchymal stem cell (MSC) condensation contributes to membrane ossification by enhancing their osteodifferentiation. We investigated bone regeneration in rats using the human bone marrow-derived MSC-spheroids prepared by rotation culture, without synthetic or exogenous biomaterials. Bilateral calvarial defects (8 mm) were created in nude male rats; the left-sided defects were implanted with MSC-spheroids, β-tricalcium phosphate (β-TCP) granules, or β-TCP granules + MSC-spheroids, while the right-sided defects served as internal controls. Micro-computed tomography and immunohistochemical staining for osteocalcin/osteopontin indicated formation of new, full-thickness bones at the implantation sites, but not at the control sites in the MSC-spheroid group. Raman spectroscopy revealed similarity in the spectral properties of the repaired bone and native calvarial bone. Mechanical performance of the bones in the MSC-implanted group was good (50 and 60 % those of native bones, respectively). All tests showed poor bone regeneration in the β-TCP and β-TCP + MSC-spheroid groups. Thus, significant bone regeneration was achieved with MSC-spheroid implantation into bone defects, justifying further investigation.

Bone grafts currently used for the treatment of large bone defect or asymmetry in oral and maxillofacial region include autologous, allogeneic, and artificial bones. Although artificial bone is free from the concerns of donor site morbidity, limitation of volume, disease transmission, and ethical issues, it lacks osteogenic and osteoinductive activities. In addition, molding of the artificial bone is an issue especially when it is used for the augmentation of bone as onlay grafts. To solve this problem, additive manufacturing techniques have been applied to fabricate bones which have outer shapes conformed to patients' bones. We developed a custom-made artificial bone called a computed tomography (CT)-bone. Efficacy of CT-bone was proven in a clinical research and clinical trial, showing good manipulability, stability, and patient satisfaction. However, low replacement rate of artificial bones by endogenous bones remain an unsolved issue. Loading of cells and growth factors will improve the bone replacement by inducing osteogenic and osteoinductive activities. In addition, the three-dimensional bioprinting technique will facilitate bone regeneration by placing cells and biological substances into appropriate sites.

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.

Background This study evaluated the use of an augmented reality navigation system that provides a markerless registration system using stereo vision in oral and maxillofacial surgery. Method A feasibility study was performed on a subject, wherein a stereo camera was used for tracking and markerless registration. The computed tomography data obtained from the volunteer was used to create an integral videography image and a 3-dimensional rapid prototype model of the jaw. The overlay of the subject’s anatomic site and its 3D-IV image were displayed in real space using a 3D-AR display. Extraction of characteristic points and teeth matching were done using parallax images from two stereo cameras for patient-image registration. Results Accurate registration of the volunteer’s anatomy with IV stereoscopic images via image matching was done using the fully automated markerless system, which recognized the incisal edges of the teeth and captured information pertaining to their position with an average target registration error of < 1 mm. These 3D-CT images were then displayed in real space with high accuracy using AR. Even when the viewing position was changed, the 3D images could be observed as if they were floating in real space without using special glasses. Conclusion Teeth were successfully used for registration via 3D image (contour) matching. This system, without using references or fiducial markers, displayed 3D-CT images in real space with high accuracy. The system provided real-time markerless registration and 3D image matching via stereo vision, which, combined with AR, could have significant clinical applications.

Traditional tissue-engineered cartilage applied in clinical practice consists of cell suspensions or gel-form materials for which it is difficult to maintain their shapes. Although biodegradable polymer scaffolds are used for shape retention, deformation after transplantation can occur. Here, we showed that periostin (PN), which is abundantly expressed in fibrous tissues, contributes to the maturation and shape retention of tissue-engineered cartilage through conformational changes in collagen molecules. The tissue-engineered cartilage transplanted in an environment lacking PN exhibited irregular shapes, while transplants originating from chondrocytes lacking PN showed limited regeneration. In the in vitro assay, PN added to the culture medium of chondrocytes failed to show any effects, while the 3D culture embedded within the collagen gel premixed with PN (10 μg/mL) enhanced chondrogenesis. The PN-mediated collagen structure enhanced the mechanical strength of the surrounding fibrous tissues and activated chondrocyte extracellular signaling by interstitial fibrous tissues.

Hypercalcemia and hyperparathyroidism in patients receiving maintenance hemodialysis (MHD) can cause the progression of cardiovascular diseases (CVD) and mineral bone disorders (MBD). The KDIGO recommends the dialysates with a calcium (Ca) concentration of 1.25–1.5 mmol/L for MHD treatments, but the optimal concentration remains controversial. Here, we conducted a systematic review and a meta-analysis of seven randomized controlled trials examining a total of 622 patients to investigate the optimal concentration for MHD for 6 months or longer. The dialysates with a low Ca concentration (1.125 or 1.25 mmol/L) significantly lowered the serum Ca and raised the intact parathyroid hormone levels by 0.52 mg/dL (95% confidence interval, 0.20–0.85) and 39.59 pg/mL (14.80–64.38), respectively, compared with a high Ca concentration (1.50 or 1.75 mmol/L). Three studies showed that a low concentration was preferred for lowering arterial calcifications or atherosclerosis in different arteries, but one study showed that coronary arterial calcifications increased with a low concentration. Two studies showed contradictory outcomes in terms of MBD. Our meta-analysis showed that a dialysate with a low Ca concentration lowered the serum Ca levels in patients receiving long-term MHD, but further studies are needed to determine the optimal Ca concentration in terms of CVD and MBD.

Ideally, artificial bones should be dimensionally compatible with deformities, and be biodegradable and osteoconductive; however, there are no artificial bones developed to date that satisfy these requirements. We fabricated novel custom-made artificial bones from α-tricalcium phosphate powder using an inkjet printer and implanted them in ten patients with maxillofacial deformities. The artificial bones had dimensional compatibility in all the patients. The operation time was reduced due to minimal need for size adjustment and fixing manipulation. The postsurgical computed tomography analysis detected partial union between the artificial bones and host bone tissues. There were no serious adverse reactions. These findings provide support for further clinical studies of the inkjet-printed custom-made artificial bones.

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.

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.

PurposeThis study aimed to investigate whether intra-tracheal administration of basic fibroblast growth factor (b-FGF) promotes the growth of tracheal cartilage.MethodsTrachea of 4-week old mice were intubated and 2.5 μg b-FGF administered (Group 4) for periods from 1 to 5 days. Cervical tracheal outer diameter and tracheal ring length were compared in Group 1 (no intervention), Group 2 (tracheal intubation), Group 3 (intra-tracheal administration of distilled water) and Group 4, at 8 weeks of age. Outer diameter and tracheal ring length in Group 4 were also compared with that in Group 1 at 12 and 16 weeks of age.ResultsAt 8 weeks of age, tracheal ring length with b-FGF administration for more than 4 days in Group 4 was significantly increased over that following 1-day administration. At 8 weeks of age, mean outer diameter and the mean tracheal ring length in Group 4 were significantly greater than in the other groups. Mean outer diameter and mean tracheal ring length were significantly greater in Group 4 than in Group 1 at 12 and 16 weeks of age.ConclusionThis study has shown that intra-tracheal administration of b-FGF enlarges the tracheal lumen.