Kazuto Hoshi

Cartilage is considered to be immune privileged in general. Clinically, live cells are removed from subcutaneously transplanted allogeneic cartilage mainly for preservation and for infection control. However, because maintaining cartilage feature requires live chondrocyte, it would be beneficial to subcutaneously transplant cartilage with live chondrocyte even if it was allogeneic. We harvested femoral head from 3-week-old male C57BL/6 mice, subcutaneously transplanted to 6-week-old male mice, BALB/c, BALB/c nu/nu , or C57BL/6-Tg (enhanced green fluorescent protein [EGFP] under the control of the CMV-IE enhancer, chicken beta-actin promoter, rabbit beta-globin genomic DNA [CAG promoter]), as allogeneic, allogeneic immunodeficient control, or syngeneic transplantation. We also transplanted cartilaginous particles from human induced pluripotent stem cells derived from human leukocyte antigen homozygous donor to 6-week-old male mice either BALB/c and BALB/c nu/nu as xenogeneic or xenogeneic immunodeficient control. The transplantation periods were 1, 2, 3, 4, 8, 12, and 24 weeks. As the result, we did not observe exposure of the transplant or apparent macroscopic inflammatory in all samples. Histological analysis suggested that the femoral head showed focal ossification and thinning in syngeneic transplantation. In allogeneic transplantation, slight invasion of CD3 (+) T cell and the denaturation of the cartilage were observed, suggesting immune reaction against allogeneic cartilage. In xenogeneic transplantation, slight invasion of CD3 (+) cell and CD4 (+) cell and the structure of the perichondrium-like tissue got unclear, suggesting slight immune reaction against xenogeneic cartilage. Our findings suggest that we should carefully investigate for appropriate procedure to control immune reaction against allogeneic cartilage with live chondrocyte and to maintain its cartilage feature for long time.

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare autosomal dominant disorder characterized by congenital skeletal malformation and progressive heterotopic ossification. In the oral and maxillofacial region, deformity of the temporoman-dibular joint is a common feature of FOP, as well as restricted mouth opening derived from heterotopic ossification in the masticatory muscles. Since surgical procedures are generally not recommended because of the risk of flare-ups and increased heterotopic ossification, reports of tooth extractions and their outcomes in patients with FOP are limited. The present article reports the long-term oral outcomes of three Japanese patients with FOP, in whom the teeth were deliberately extracted to avoid the risk of oral inflammation causing further heterotopic ossification. The extractions were conducted under local or general anesthesia, and healing of sockets was nonproblematic with the formation of new bone. Undesirable events, including progression of heterotopic ossification in the oral and maxillo-facial region and further restriction of mouth opening, were not apparent. The extractions also alleviated the existing inflammation, contributing to maintaining their oral hygiene. These cases suggest that deliberate planning and judicious surgery could induce favorable healing after tooth extractions in patients with FOP, leading to long-term stability of their oral health status.

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.

Microtia is a congenital malformation of the auricle. The conventional therapy for microtia is reconstruction of the auricle by using the patient's own costal cartilage. Because it is invasive to harvest costal cartilages, less invasive ways for auricular reconstruction need to be established. Recent reports have indicated a new method for the production of cartilaginous particles from human induced pluripotent stem cells. To adopt this method to create an auricular-shaped regenerative cartilage, a novel scaffold with the property of a three-dimensional shape memory was created. A scaffold with a three-dimensional shape of auricular frames composed of a helix and an antihelix, which was designed to mimic an auricular framework carved from autologous costal cartilage and transplanted in auricular reconstruction, was prepared, filled with cartilaginous particles, and subcutaneously transplanted in nude rats. The auricular-shaped regenerative cartilage maintained the given shape and cartilage features in vivo for 1 year. Our findings suggest a novel approach for auricular reconstruction.

Background: Vulvar Paget’s disease (VPD) is a rare malignant disorder originating in the external genitalia. It occasionally invades into urethral or vaginal mucosa of female, making surgical treatment more complicating. In case of urethral invasion of Paget’s cells, systematic mapping biopsy of urethral mucosa is the standard of care to determine the range of surgical resection. Resection of urethral mucosa and simple skin grafting often result in urethral stricture after surgery, which severely deteriorates patient’s quality of life. Case presentation: We applied a new technique of advancement urethral meatoplasty using buccal mucosa, in two Japanese cases of VPD with urethral invasion. After broad resection of vulvar skin together with the urethral mucosa, buccal mucosa was implanted between advanced urethral mucosa and skin graft. In both cases, we could prevent urethral stricture one year and two years after surgery, respectively. Conclusion: This technique prevented urethral stricture after surgery and could be a useful technique as part of urethroplasty for VPD.

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.

This study focused on the Kuchikara Taberu Balance Chart (KTBC) as a tool for swallowing function evaluation. To clarify the relationship between videoendoscopic (VE) examination of swallowing function and the KTBC, we compared median KTBC scores with and without laryngeal penetration identified by VE. Sixty-five patients with a mean age of 84.3 ± 7.9 years were examined at the Towada City Hospital. The patients were classified into groups based on laryngeal penetration, including 28 patients with and 37 patients without penetration. We found no significant differences in patient backgrounds. The median KTBC score (interquartile range) was 36.5 (31–44.5) in the group with laryngeal penetration and 42 (35–48.5) in the group without penetration, but the scores were not significantly different (level of statistical significance at α = 0.0036 determined by the Bonferroni correction method) when compared with the Mann–Whitney U test (36.5 vs. 42, z = -2.33, p = 0.020). The median respiratory condition (4 vs. 3, z = − 3.23; p < 0.0036), oral preparatory and propulsive phases (4 vs. 3, z = − 2.96; p < 0.0036), and position and endurance (3 vs. 1, z = − 3.25; p < 0.0036) scores were significantly lower in the group without laryngeal penetration. This study revealed a correlation between laryngeal penetration confirmed by VE and KTBC scores. Consequently, respiratory condition, oral preparatory and propulsive phases, and position and endurance may be useful as tools for the assessment of swallowing. In particular, we recommend adding respiratory status to dysphagia screening.

Purpose: There are no long‐term reports on morphologic changes of nasal deformity in patients with cleft lip and palate (CLP). This retrospective study was conducted to clarify changes in the nose morphology of patients with CLP.Materials and Methods: We investigated 47 patients in our department. The evaluation time was as follows: T1 (before or after attending elementary school), T2 (after puberty growth), and T3 (after rhinoplasty). We evaluated the dorsal nose deviation angle, the nasal tip angle, and the nasolabial angle with standardized facial photographs, and nasal septum with computed tomography (CT) images. Computer software ImageJ was used for measurements. For statistical analysis, the Mann‐Whitney U test was used (significance level; P < 0.05).Results: Analysis showed that in patients with unilateral cleft lip and palate (UCLP), twisted nose and reduction of the nasolabial angle became more prominent after puberty. In patients with bilateral cleft lip and palate (BCLP), the nasal tip was larger than in patients with UCLP and tended to be directed upward. The nasolabial angle in BCLP was more obtuse than the general mean value, but that in UCLP tended to be more acute. CT showed that in UCLP patients, the nasal septum was displaced toward the affected side, and the inferior nasal turbinate on the unaffected side was thickened. Nasal septum deviation was less severe in BCLP patients.Conclusions: UCLP and BCLP patients showed different nasal morphology at T1 and they grew differently from T1 to T2.

Instead of the silicone implants previously used for repair and reconstruction of the auricle and nose lost due to accidents and disease, a new treatment method using tissue-engineered cartilage has been attracting attention. The quality of cultured cells is important in this method because it affects treatment outcomes. However, a marker of chondrocytes, particularly auricular chondrocytes, has not yet been established. The objective of this study was to establish an optimal marker to evaluate the quality of cultured auricular chondrocytes as a cell source of regenerative cartilage tissue. Gene expression levels were comprehensively compared using the microarray method between human undifferentiated and dedifferentiated auricular chondrocytes to investigate a candidate quality control index with an expression level that is high in differentiated cells, but markedly decreases in dedifferentiated cells. We identified glial fibrillary acidic protein (GFAP) as a marker that decreased with serial passages in auricular chondrocytes. GFAP was not detected in articular chondrocytes, costal chondrocytes, or fibroblasts, which need to be distinguished from auricular chondrocytes in cell cultures. GFAP mRNA expression was observed in cultured auricular chondrocytes, and GFAP protein levels were also measured in the cell lysates and culture supernatants of these cells. However, GFAP levels detected from mRNA and protein in cell lysates were significantly decreased by increases in the incubation period. In contrast, the amount of protein in the cell supernatant was not affected by the incubation period. Furthermore, the protein level of GFAP in the supernatants of cultured cells correlated with the in vitro and in vivo production of the cartilage matrix by these cells. The productivity of the cartilage matrix in cultured auricular chondrocytes may be predicted by measuring GFAP protein levels in the culture supernatants of these cells. Thus, GFAP is regarded as a marker of the purity and properties of cultured auricular chondrocytes.