Oral condition at admission predicts functional outcomes and hospital-acquired pneumonia development among acute ischemic stroke patients.

INTRODUCTION: Oral care is crucial for the prevention of cardiovascular events and pneumonia. However, few studies have evaluated the associations between multidimensional assessments of oral status or functional outcomes and hospital-acquired pneumonia (HAP). METHODS: Consecutive patients with acute ischemic stroke (AIS) were retrospectively analyzed. We evaluated the modified oral assessment grade (mOAG) and investigated its association with a modified Rankin scale (mRS) score of 0‒2 (good stroke outcome) and HAP. The mOAG was developed to evaluate 8 categories (lip, tongue, coated tongue, saliva, mucosa, gingiva, preservation, and gargling) on a 4-point scale ranging from 0 to 3. We analyzed the effectiveness of the mOAG score for predicting stroke outcome or HAP using receiver operating characteristic (ROC) curve analysis. RESULTS: In total, 247 patients with AIS were analyzed. The area under the ROC curve of the mOAG for predicting poor outcomes was 0.821 (cutoff value: 7), and that for HAP incidence was 0.783 (cutoff value: 8). mOAG (a one-point increase) was associated with poor stroke outcome (odds ratio [OR] 1.31, 95% confidence interval [CI] 1.17‒1.48, P < 0.001) and HAP (OR 1.21, 95% CI 1.07‒1.38, P = 0.003) after adjusting for baseline clinical characteristics, including age and stroke severity. CONCLUSIONS: Lower mOAG scores at admission were independently associated with good outcomes and a decreased incidence of HAP. Comprehensive oral assessments are essential for acute stroke patients in clinical settings.

Periodontitis and postoperative inflammation in gastric cancer patients: Propensity score analysis.

OBJECTIVE: The objective of this study was to clarify the association between preoperative periodontitis and postoperative systemic inflammation in patients with gastric cancer. SUBJECTS AND METHODS: This retrospective cohort study analyzed data from 140 gastric cancer patients who underwent surgery at Hiroshima University Hospital between May 2019 and May 2022. Periodontal inflamed surface area (PISA) scores were determined to assess periodontitis severity using modified Nesse's methods. Propensity score matching was used to compare patients with high and low PISA scores (> or < the median PISA score of 92.4, respectively). Propensity scores were calculated using a logistic regression model, based on 17 clinical parameters: age, sex, smoking, alcohol consumption, hypertension, diabetes, dyslipidemia, cardiovascular disease, stroke, clinical stage, surgical procedure, surgical approach, neoadjuvant chemotherapy, surgery duration, blood loss during surgery, remaining teeth, and denture use. RESULTS: Thirty-seven patients were propensity-score-matched. Participants with high PISA scores had a higher incidence of surgical site infection (10.5%) than those with low PISA scores (5.3%). Moreover, participants with high PISA scores had significantly higher C-reactive protein levels on postoperative days 1 than those with low PISA scores. CONCLUSION: Preoperative periodontitis may determine the level of postoperative systemic inflammation in patients with gastric cancer.

Mechanosignaling YAP/TAZ-TEAD Axis Regulates the Immunomodulatory Properties of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have gained significant attention in cell therapies due to their multipotency and immunomodulatory capacities. The transcriptional co-activators YAP/TAZ, central to the mechanotransduction system in MSCs, dominantly direct MSCs lineage commitment. However, their role in immunomodulation remains elusive. Accordingly, this present study aimed to investigate the role of mechanotransducer YAP/TAZ and their binding target transcriptional factor, TEAD, in the immunomodulatory capacities of human bone marrow-derived MSCs. Reducing YAP/TAZ activity by altering the matrix stiffness, disrupting the F-actin integrity with chemical inhibitors, or using siRNAs increased the expression of immunomodulatory genes, such as TSG-6 and IDO, upon TNF-α stimulation. Similarly, transfection of TEAD siRNA also increased the immunomodulatory capacities in MSCs. RNA-seq analysis and inhibition assays demonstrated that the immunomodulatory capacities caused by YAP/TAZ-TEAD axis disruption were due to the NF-κB signaling pathway activation. Then, we also evaluated the in vivo anti-inflammatory efficacy of MSCs in a dextran sulfate sodium (DSS)-induced mice colitis model. The administration of human MSCs transfected with TEAD siRNA, which exhibited enhanced immunomodulatory properties in vitro, significantly ameliorated inflammatory bowel disease symptoms, such as body weight loss and acute colon inflammation, in the DSS-induced mice colitis model. Our findings underscore the mechanosignaling YAP/TAZ-TEAD axis as a regulator of MSCs immunomodulation. Targeting these signaling pathways could herald promising MSCs-based therapies for immune disorders.

Efficacy of Low-Level Laser Therapy for Oral Mucositis in Hematologic Patients Undergoing Transplantation: A Single-Arm Prospective Study.

Oral mucositis significantly affects the quality of life in hematologic cancer patients undergoing hematopoietic stem cell transplantation. Despite global evidence supporting the efficacy of low-level laser therapy (LLLT) for mucositis prevention, its clinical adoption in Japan is limited. This study aimed to fill this gap by evaluating the safety and efficacy of LLLT in a Japanese patient population. In a single-group, non-blinded, exploratory trial, we compared 21 LLLT-treated patients against a historical control of 96 patients. The primary endpoint was the incidence of Grade ≥ 2 mucositis, based on NCI-CTCAE ver. 4.0. The LLLT group showed a significantly lower incidence of Grade ≥ 2 mucositis (23.8%) compared to the control group (64.6%) (p = 0.0006). Furthermore, Grade ≥ 2 mucositis correlated with increased oral dryness and longer hospital stays. Our study confirms the efficacy of LLLT in reducing the onset of severe oral mucositis among Japanese hematologic cancer patients, advocating for its clinical introduction as a preventive measure in Japan.

A Cartilaginous Construct with Bone Collar Exerts Bone-Regenerative Property Via Rapid Endochondral Ossification.

Three-dimensional clumps of mesenchymal stem cells (MSCs)/extracellular matrix (ECM) complexes (C-MSCs) can be implanted into tissue defects with no artificial scaffolds. In addition, the cellular properties and characteristics of the ECM in C-MSCs can be regulated in vitro. Most bone formation in the developmental and healing process is due to endochondral ossification, which occurs after bone collar formation surrounding cartilage derived from MSCs. Thus, to develop a rapid and reliable bone-regenerative cell therapy, the present study aimed to generate cartilaginous tissue covered with a mineralized bone collar-like structure from human C-MSCs by combining chondrogenic and osteogenic induction. Human bone marrow-derived MSCs were cultured in xeno-free/serum-free (XF) growth medium. Confluent cells that formed cellular sheets were detached from the culture plate using a micropipette tip. The floating cellular sheet contracted to round clumps of cells (C-MSCs). C-MSCs were maintained in XF-chondro-inductive medium (CIM) and XF-osteo-inductive medium (OIM). The biological and bone-regenerative properties of the generated cellular constructs were assessed in vitro and in vivo. C-MSCs cultured in CIM/OIM formed cartilaginous tissue covered with a mineralized matrix layer, whereas CIM treatment alone induced cartilage with no mineralization. Transplantation of the cartilaginous tissue covered with a mineralized matrix induced more rapid bone reconstruction via endochondral ossification in the severe combined immunodeficiency mouse calvarial defect model than that of cartilage generated using only CIM. These results highlight the potential of C-MSC culture in combination with CIM/OIM to generate cartilage covered with a bone collar-like structure, which can be applied for novel bone-regenerative cell therapy.

Clumps of mesenchymal stem cells/extracellular matrix complexes directly reconstruct the functional periodontal tissue in a rat periodontal defect model.

Periodontitis is an inflammatory disease characterized by tooth-supporting periodontal tissue destruction, including the cementum, periodontal ligament, and alveolar bone. To regenerate the damaged periodontal tissue, mesenchymal stem cells (MSCs) have attracted much scientific and medical attention. Recently, we generated clumps of MSCs/extracellular matrix (ECM) complexes (C-MSCs), which consist of cells and self-produced ECM. C-MSCs can be transplanted into lesion areas without artificial scaffold to induce tissue regeneration. To develop reliable scaffold-free periodontal tissue regenerative cell therapy by C-MSCs, this study investigated the periodontal tissue regenerative capacity of C-MSCs and the behavior of the transplanted cells. Rat bone marrow-derived MSCs were isolated from rat femur. Confluent cells were scratched using a micropipette tip and then torn off. The sheet was rolled to make a three-dimensional round clump of cells, C-MSCs. Then, ten C-MSCs were grafted into a rat periodontal fenestration defect model. To trace the grafted cells in the defect, PKH26-labeled cells were also employed. Micro-CT and histological analyses demonstrated that transplantation of C-MSCs induced successful periodontal tissue regeneration in the rat periodontal defect model. Interestingly, the majority of the cells in the reconstructed tissue, including cementum, periodontal ligaments, and alveolar bone, were PKH26 positive donor cells, suggesting the direct tissue formation by MSCs. This study demonstrates a promising scaffold-free MSCs transplantation strategy for periodontal disease using C-MSCs and offers the significance of multipotency of MSCs to induce successful periodontal tissue regeneration.

Xenotransplantation of cryopreserved human clumps of mesenchymal stem cells/extracellular matrix complexes pretreated with IFN-γ induces rat calvarial bone regeneration.

Introduction: Three-dimensional (3D) clumps of mesenchymal stem cells (MSCs)/extracellular matrix (ECM) complexes, composed with cells and self-produced intact ECM, can be grafted into defect areas without artificial scaffold to induce successful bone regeneration. Moreover, C-MSCs pretreated with IFN-γ (C-MSCsγ) increased the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) expression and thereby inhibited T cell activity. Xenotransplantation of human C-MSCsγ suppressed host T cell immune rejection and induced bone regeneration in mice. Besides, we have also reported that C-MSCs retain the 3D structure and bone regenerative property even after cryopreservation. To develop the "off-the-shelf" cell preparation for bone regenerative therapy that is promptly provided when needed, we investigated whether C-MSCsγ can retain the immunosuppressive and osteogenic properties after cryopreservation. Methods: Confluent human MSCs 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. The round cell clumps were incubated with a growth medium for 3 days, and then C-MSCs were obtained. To generate C-MSCsγ, after 2 days' culture, C-MSCs were stimulated with 50 ng/ml of IFN-γ. Both C-MSCs and C-MSCsγ were cryopreserved for 2 days and then thawed to obtain Cryo-C-MSCs and Cryo-C-MSCsγ, respectively. The biological properties of those cell clumps were assessed in vitro. In addition, to test whether human Cryo-C-MSCsγ attenuates immune rejection to induce bone regeneration, a xenograft study using a rat calvarial defect was performed. Results: Both IFN-γ pretreatment and cryopreservation process did not affect the 3D structure and cell viability in all human cell clumps. Interestingly, Cryo-C-MSCsγ showed significantly increased IDO mRNA expression equivalent to C-MSCsγ. More importantly, xenotransplantation of human C-MSCsγ and Cryo-C-MSCsγ induced rat calvarial bone regeneration by suppressing rat T cells infiltration and the grafted human cells reduction in the grafted area. Finally, there were no human donor cells in the newly formed bone, implying that the bone reconstruction by C-MSCsγ and Cryo-C-MSCsγ can be due to indirect host osteogenesis. Conclusion: These findings implied that Cryo-C-MSCsγ can be a promising bone regenerative allograft therapy that can be certainly and promptly supplied on demand.

Clumps of Mesenchymal Stem Cells/Extracellular Matrix Complexes Generated with Xeno-Free Chondro-Inductive Medium Induce Bone Regeneration via Endochondral Ossification.

Three-dimensional clumps of mesenchymal stem cells (MSCs)/extracellular matrix (ECM) complexes (C-MSCs) can be transplanted into tissue defect site with no artificial scaffold. Importantly, most bone formation in the developing process or fracture healing proceeds via endochondral ossification. Accordingly, this present study investigated whether C-MSCs generated with chondro-inductive medium (CIM) can induce successful bone regeneration and assessed its healing process. Human bone marrow-derived MSCs were cultured with xeno-free/serum-free (XF) growth 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. The cell clumps, i.e., C-MSCs, were maintained in XF-CIM. C-MSCs generated with XF-CIM showed enlarged round cells, cartilage matrix, and hypertrophic chondrocytes genes elevation in vitro. Transplantation of C-MSCs generated with XF-CIM induced successful bone regeneration in the SCID mouse calvaria defect model. Immunofluorescence staining for human-specific vimentin demonstrated that donor human and host mouse cells cooperatively contributed the bone formation. Besides, the replacement of the cartilage matrix into bone was observed in the early period. These findings suggested that cartilaginous C-MSCs generated with XF-CIM can induce bone regeneration via endochondral ossification.

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.