Masticatory dysfunction in patients with diabetic neuropathy: A cross-sectional study.

INTRODUCTION: Chewing well is essential for successful diet therapy and control of blood glucose level in patients with diabetes. In addition, long-term hyperglycemia is a risk factor for microvascular complications, which are the main cause of morbidity and mortality in these patients. Hence, it is plausible that masticatory disorder may be relevant to diabetic microvascular complications which is caused by long-term hyperglycemia. The aim of this study was to investigate whether masticatory disorders are relevant to diabetic microvascular complications. METHODS: This cross-sectional study included 172 patients with type 2 diabetes who underwent educational hospitalization in the Department of Endocrinology and Diabetic Medicine, Hiroshima University Hospital, from April 2016 to March 2020. Masticatory efficiency was determined quantitatively by using the GLUCO SENSOR GS-Ⅱ. Multivariable linear regression models were constructed to examine which factors were related to masticatory efficiency. Statistical significance was defined as a two-sided p value of < 0.05. RESULTS: According to the bivariable analysis, masticatory efficiency was significantly correlated with duration of diabetes (p = 0. 049), number of remaining teeth (p < 0.0001), the number of moving teeth (p = 0.007) and condition of diabetic neuropathy (p < 0.0001). Moreover, the number of remaining teeth (p < 0.0001) and diabetic neuropathy (p = 0.007) remained significantly correlated with masticatory efficiency in the multivariable analysis. CONCLUSIONS: For the first time, we demonstrated that patients with type 2 diabetes who developed diabetic neuropathy had significantly reduced masticatory efficiency. Effective mastication is an important factor in successful diet therapy for diabetes. To prevent the progression of diabetic complications, especially in patients with diabetic neuropathy, it may be necessary to combine individualized therapies from dentists and nutritionists with consideration for the level of masticatory dysfunction.

Regulation of sphingolipid biosynthesis in the endoplasmic reticulum via signals from the plasma membrane in budding yeast.

Saccharomyces cerevisiae LIP1 encodes a regulatory subunit that forms a complex with the ceramide synthase catalytic subunits, Lag1/Lac1, which is localized on the membrane of endoplasmic reticulum. To understand the underlying regulatory mechanism of sphingolipid biosynthesis, we generated strains upon replacing the chromosomal LIP1 promoter with a Tet-off promoter, which enables the expression in Dox-dependent manner. The lip1-1 strain, obtained through the promoter substitution, exhibits severe growth inhibition and remarkable decrease in sphingolipid synthesis in the presence of Dox. Using this strain, we investigated the effect of a decrease in ceramide synthesis on TOR complex 2 (TORC2)-Ypk1 signaling, which senses the complex sphingolipid level at the plasma membrane and promotes sphingolipid biosynthesis. In lip1-1 cells, Ypk1 was activated via both upstream kinases, TORC2 and yeast PDK1 homologues, Pkh1/2, thereby inducing hyperphosphorylation of Lag1, but not of another Ypk1-substrate, Orm1, which is a known negative regulator of the first step of sphingolipid metabolism, in the presence of Dox. Therefore, our data suggest that the metabolic enzyme activities at each step of the sphingolipid biosynthetic pathway are controlled through a fine regulatory mechanism.

Cox2-mediated PGE2 production via p38/JNK-c-fos signaling inhibits cell apoptosis in 3D floating culture clumps of mesenchymal stem cell/extracellular matrix complexes.

Mesenchymal stem cells (MSCs), a class of adult stem cells, have attracted scientific and medical attention due to their self-renewing properties, multipotency, and trophic factor production. Although MSCs were originally studied on classical two-dimensional (2D) plastic plates, extensive scientific efforts have developed three-dimensional (3D) MSC culture systems, including MSCs spheroids and organoids that can mimic physical conditions. Moreover, we have recently developed 3D culture clumps of MSCs/extracellular matrix (ECM) complexes (C-MSCs) for novel bone regenerative cell therapy. Of note, even though it is widely accepted that cell detachment from the culture plate causes cell apoptosis, so called anoikis, these 3D MSCs constructs can be maintained in floating culture conditions. Currently, it is unclear why 3D floating-cultured MSCs constructs can escape from anoikis. To answer this question, the present study explored trophic factor production in 3D floating-cultured C-MSCs that play a cytoprotective role against anoikis and clarified the underlying molecular mechanism in vitro. Compared with cells cultured on 2D plastic plates, PGE2 production mediated by COX2 was significantly increased, and its inhibition drastically induced cell apoptosis in 3D floating-cultured C-MSCs. In the process of C-MSCs preparation, detachment of the cell sheet from culture plate activated the p38/JNK-c-Fos signaling pathway. Moreover, blockage of this signaling by chemical inhibitors abrogated COX2/PGE2 expressions and induced severe apoptosis. These results demonstrated that cell detachment facilitates cytoprotective COX2-mediated PGE2 synthesis via p38/JNK-c-Fos signaling, revealing a possible mechanism that allows resistance against anoikis in floating-cultured 3D MSCs constructs.

Clumps of Mesenchymal Stem Cell/Extracellular Matrix Complexes Generated with Xeno-Free Conditions Facilitate Bone Regeneration via Direct and Indirect Osteogenesis.

Three-dimensional clumps of mesenchymal stem cell (MSC)/extracellular matrix (ECM) complexes (C-MSCs) consist of cells and self-produced ECM. We demonstrated previously that C-MSCs can be transplanted into bone defect regions with no artificial scaffold to induce bone regeneration. To apply C-MSCs in a clinical setting as a reliable bone regenerative therapy, the present study aimed to generate C-MSCs in xeno-free/serum-free conditions that can exert successful bone regenerative properties and to monitor interactions between grafted cells and host cells during bone healing processes. Human bone marrow-derived MSCs were cultured in xeno-free/serum-free medium. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and then torn off. The sheet was rolled to make a round clump of cells. Then, C-MSCs were transplanted into an immunodeficient mouse calvarial defect model. Transplantation of C-MSCs induced bone regeneration in a time-dependent manner. Immunofluorescence staining showed that both donor human cells and host mice cells contributed to bone reconstruction. Decellularized C-MSCs implantation failed to induce bone regeneration, even though the host mice cells can infiltrate into the defect area. These findings suggested that C-MSCs generated in xeno-free/serum-free conditions can induce bone regeneration via direct and indirect osteogenesis.

Type I collagen deposition via osteoinduction ameliorates YAP/TAZ activity in 3D floating culture clumps of mesenchymal stem cell/extracellular matrix complexes.

BACKGROUND: Three-dimensional (3D) floating culture clumps of mesenchymal stem cell (MSC)/extracellular matrix (ECM) complexes (C-MSCs) consist of cells and self-produced ECM. Previous studies have demonstrated that C-MSCs can be transplanted into bony lesions without an artificial scaffold to induce bone regeneration. Moreover, osteoinductive medium (OIM)-treated C-MSCs (OIM-C-MSCs) have shown rapid and increased new bone formation in vivo. To apply OIM-C-MSCs for novel bone regenerative cell therapy, their cellular properties at the molecular level must be elucidated. The transcriptional co-activators yes-associated protein/transcriptional co-activator with PDZ-binding motif (YAP/TAZ) have been recognized as key players in the mechanotransduction cascade, controlling cell lineage commitment in MSCs. It is plausible that 3D C-MSCs/OIM-C-MSCs cultured in floating conditions could provide distinct microenvironments compared to conventional 2D culture systems and thereby induce unique mechanotransduction cascades. Therefore, this study investigated the YAP/TAZ activity in 3D-cultured C-MSCs/OIM-C-MSCs in floating conditions. METHODS: Human bone marrow-derived MSCs were cultured in growth medium supplemented with ascorbic acid. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and were then torn off. The sheet was rolled to make round clumps of cells. Then, YAP/TAZ activity, filamentous actin (F-actin) integrity, collagen type I (COL1) production, and the differentiation potency in 3D floating culture C-MSCs/OIM-C-MSCs were analyzed. RESULTS: C-MSCs cultured in floating conditions lost their actin cytoskeleton to downregulate YAP/TAZ activity, which directed cells to undergo adipogenesis/chondrogenesis. OIM treatment induced abundant COL1 deposition, which facilitated Intß1-dependent actin fiber formation and YAP/TAZ activity to elevate the expression levels of osteogenic master transcriptional factor runt-related transcription factor 2 (RUNX2) mRNA in C-MSCs. Importantly, elevation of YAP/TAZ activity via OIM was associated with COL1 deposition and F-actin integrity, suggesting a positive feedback loop in OIM-C-MSCs. CONCLUSION: These findings suggest that OIM-C-MSCs, which form a unique microenvironment that maintains high YAP/TAZ activity, can serve as better candidates for bone regenerative cell therapy than C-MSCs.

Cryopreserved clumps of mesenchymal stem cell/extracellular matrix complexes retain osteogenic capacity and induce bone regeneration.

BACKGROUND: Three-dimensional (3D) cultured clumps of mesenchymal stem cell (MSC)/extracellular matrix (ECM) complexes (C-MSCs) consist of cells and self-produced ECM. C-MSCs can regulate cellular functions in vitro and can be grafted into a defect site without an artificial scaffold to induce bone regeneration. Long-term cryopreservation of C-MSCs, which can enable them to serve as a ready-to-use cell preparation, may be helpful in developing beneficial cell therapy for bone regeneration. Therefore, the aim of this study was to investigate the effect of cryopreservation on C-MSCs. METHODS: MSCs isolated from rat femurs were cultured in growth medium supplemented with ascorbic acid. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and were then torn off. The sheet was rolled to make a round clumps of cells. The C-MSCs were cryopreserved in cryomedium including 10% dimethyl sulfoxide. RESULTS: Cryopreserved C-MSCs retained their 3D structure and did not exhibit a decrease in cell viability. In addition, stem cell marker expression levels and the osteogenic differentiation properties of C-MSCs were not reduced by cryopreservation. However, C-MSCs pretreated with collagenase before cryopreservation showed a lower level of type I collagen and could not retain their 3D structure, and their rates of cell death increased during cryopreservation. Both C-MSC and cryopreserved C-MSC transplantation into rat calvarial defects induced successful bone regeneration. CONCLUSION: These data indicate that cryopreservation does not reduce the biological properties of C-MSCs because of its abundant type I collagen. More specifically, cryopreserved C-MSCs could be applicable for novel bone regenerative therapies.

Xenotransplantation of interferon-gamma-pretreated clumps of a human mesenchymal stem cell/extracellular matrix complex induces mouse calvarial bone regeneration.

BACKGROUND: Three-dimensional cultured clumps of a mesenchymal stem cell (MSC)/extracellular matrix (ECM) complex (C-MSC) consists of cells and self-produced ECM. C-MSC can regulate the cellular function in vitro and induce successful bone regeneration using ECM as a cell scaffold. Potentiating the immunomodulatory capacity of C-MSCs, which can ameliorate the allo-specific immune response, may be helpful in developing beneficial "off-the-shelf" cell therapy for tissue regeneration. It is well reported that interferon (IFN)-γ stimulates the immunosuppressive properties of MSC via upregulation of the immunomodulatory enzyme IDO. Therefore, the aim of this study was to investigate the effect of IFN-γ on the immunomodulatory capacity of C-MSC in vitro and to test the bone regenerative activity of C-MSC or IFN-γ-pretreated C-MSC (C-MSCγ) xenografts in a mice calvarial defect model. METHODS: Human bone marrow-derived MSCs were seeded at a density of 2.0 × 105 cells/well into 24-well plates and cultured with growth medium supplemented with 50 µg/mL L-ascorbic acid for 4 days. To obtain C-MSC, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and were then torn off. The cellular sheet was rolled to make a round clump of cells. C-MSC was stimulated with IFN-γ and IDO expression, immunosuppressive capacity, and immunophenotype were evaluated in vitro. Moreover, C-MSC or C-MSCγ was xenotransplanted into immunocompetent or immunodeficient mice calvarial defect models without artificial scaffold, respectively. RESULTS: IFN-γ stimulated IDO expression in C-MSC. C-MSCγ, but not C-MSC, attenuated CD3/CD28-induced T cell proliferation and its suppressive effect was reversed by an IDO inhibitor. C-MSCγ showed upregulation of HLA-DR expression, but its co-stimulatory molecule, CD86, was not detected. Xenotransplantation of C-MSCγ into immunocompetent mice calvarial defect induced bone regeneration, whereas C-MSC xenograft failed and induced T cell infiltration in the grafted area. On the other hand, both C-MSC and C-MSCγ xenotransplantation into immunodeficient mice caused bone regeneration. CONCLUSIONS: Xenotransplantation of C-MSCγ, which exerts immunomodulatory properties via the upregulation of IDO activity in vitro, may attenuate xenoreactive host immune response, and thereby induce bone regeneration in mice. Accordingly, C-MSCγ may constitute a promising novel allograft cell therapy for bone regeneration.