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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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: This study aimed to determine the regulatory mechanism of bone marrow-derived mesenchymal stem cell (BM-MSC) differentiation mediated by humoral factors derived from human periodontal ligament (HPL) cells and human gingival fibroblasts (HGFs). We analyzed histone deacetylase (HDAC) expression and activity involved in BM-MSC differentiation and determined their regulatory effects in co-cultures of BM-MSCs with HPL cells or HGFs., Background: BM-MSCs can differentiate into various cell types and can, thus, be used in periodontal regenerative therapy. However, the mechanism underlying their differentiation remains unclear. Transplanted BM-MSCs are affected by periodontal cells via direct contact or secretion of humoral factors. Therefore, their activity is regulated by humoral factors derived from HPL cells or HGFs., Methods: BM-MSCs were indirectly co-cultured with HPL cells or HGFs under osteogenic or growth conditions and then analyzed for osteogenesis, HDAC1 and HDAC2 expression and activity, and histone H3 acetylation. BM-MSCs were treated with trichostatin A, or their HDAC1 or HDAC2 expression was silenced or overexpressed during osteogenesis. Subsequently, they were evaluated for osteogenesis or the effects of HDAC activity., Results: BM-MSCs co-cultured with HPL cells or HGFs showed suppressed osteogenesis, HDAC1 and HDAC2 expression, and HDAC phosphorylation; however, histone H3 acetylation was enhanced. Trichostatin A treatment remarkably suppressed osteogenesis, decreasing HDAC expression and enhancing histone H3 acetylation. HDAC1 and HDAC2 silencing negatively regulated osteogenesis in BM-MSCs to the same extent as that achieved by indirect co-culture with HPL cells or HGFs. Conversely, their overexpression positively regulated osteogenesis in BM-MSCs., Conclusion: The suppressive effects of HPL cells and HGFs on BM-MSC osteogenesis were regulated by HDAC expression and histone H3 acetylation to a greater extent than that mediated by HDAC activity. Therefore, regulation of HDAC expression has prospects in clinical applications for effective periodontal regeneration, mainly, bone regeneration., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2022 John Wiley & Sons Ltd.)

Background: Cytokines play key roles in stimulating periodontal regeneration; however, their exact mechanisms of action remain unclear. Mesenchymal stem cells (MSCs) are multipotent cells that have self-renewal abilities and can differentiate into periodontal tissues such as bone, cementum, and periodontal ligaments following transplantation, like periodontal progenitor cells. Here, we used MSCs to identify the regulatory genes induced by periodontal regenerative cytokines., Methods: Human MSCs (hMSCs) were cultured under conditions of periodontal regenerative cytokine stimulation or silencing of undifferentiated hMSC transcription factors. To characterize the changes associated with periodontal regenerative cytokine-regulated microRNAs (miRNAs), miRNA, and mRNA expression was evaluated using miRNA arrays and quantitative real-time polymerase chain reaction, respectively. One of the identified miRNAs, miR-628-5p, was then overexpressed or suppressed in hMSCs during osteogenesis; the effect of these changes on osteogenesis was investigated., Results: Cytokine-stimulated MSCs showed characteristic miRNA profiles and mRNA levels of undifferentiated hMSC transcription factors ETV1, SOX11, and GATA6. Next, we silenced these transcription factors in MSCs and examined the miRNA profiles. The levels of miR-628-5p were decreased upon all cytokine treatments and were increased upon silencing of ETV1, SOX11, and GATA6. Overexpression of miR-628-5p suppressed osteogenesis; however, its inhibition enhanced OPN, ALP, OC, BMP2, and RUNX2 mRNA levels, and bone matrix mineralization, but not OSX mRNA or ALP activity., Conclusions: miR-628-5p negatively regulates MSC stemness during periodontal regeneration. Periodontal regenerative cytokines act as miR-628-5p suppressors to support periodontal regeneration. Thus, selection of effective cytokines for different MSCs, based on miRNA profiling, is important for advancing regenerative therapies., (© 2021 American Academy of Periodontology.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction: Mesenchymal stem cells (MSCs) can differentiate into various types of cells and can thus be used for periodontal regenerative therapy. However, the mechanism of differentiation is still unclear. Transplanted MSCs are, via their transcription factors or microRNAs (miRNAs), affected by periodontal cells with direct contact or secretion of humoral factors. Therefore, transplanted MSCs are regulated by humoral factors from human gingival fibroblasts (HGF). Moreover, insulin-like growth factor (IGF)-1 is secreted from HGF and regulates periodontal regeneration. To clarify the regulatory mechanism for MSC differentiation by humoral factors from HGF, we identified key genes, specifically miRNAs, involved in this process, and determined their function in MSC differentiation., Materials and Methods: Mesenchymal stem cells were indirectly co-cultured with HGF in osteogenic or growth conditions and then evaluated for osteogenesis, undifferentiated MSC markers, and characteristic miRNAs. MSCs had their miRNA expression levels adjusted or were challenged with IGF-1 during osteogenesis, or both of which were performed, and then, MSCs were evaluated for osteogenesis or undifferentiated MSC markers., Results: Mesenchymal stem cells co-cultured with HGF showed suppression of osteogenesis and characteristic expression of ETV1, an undifferentiated MSC marker, as well as miR-101-3p. Over-expression of miR-101-3p regulated osteogenesis and ETV1 expression as well as indirect co-culture with HGF. IGF-1 induced miR-101-3p and ETV1 expression. However, IGF-1 did not suppress osteogenesis., Conclusions: Humoral factors from HGF suppressed osteogenesis in MSCs. The effect was regulated by miRNAs and undifferentiated MSC markers. miR-101-3p and ETV1 were the key factors and were regulated by IGF-1.

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.

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 Intβ1-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.

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.

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 × 10 5 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.

LL37, an antimicrobial peptide, exhibits multiple bio-functions in various types of cells, including migration, cytokine production, apoptosis, and angiogenesis. Neovascularization and the subsequent recruitment of stem cells are essential for tissue engineering therapy, including bone regeneration. We hypothesized that LL37 can facilitate successful bone regeneration. To prove this hypothesis, the present study tested the effects of LL37 on bone formation in a rat calvarial bone defect model. Synthesized LL37 markedly induced newly formed bone. Interestingly, morphologically fibroblastic cells were observed in animals treated with LL37 on day 7, the early stage of tissue regeneration, which were positive for STRO-1, a marker of mesenchymal stem cells (MSCs), and accumulated in the bone defect area where cells positive for CD34, a marker of endothelial cells, were also localized. In addition, LL37 stimulated tube formation by endothelial cells and the proliferation of MSCs in vitro. These findings demonstrated for the first time that LL37 can regulate angiogenesis and the recruitment of stem cells to promote bone regeneration., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We recently reported that forced expression of basic helix-loop-helix transcription factor Dec1 accelerated chondrogenic differentiation of mesenchymal stem cells (MSC) in pellet cultures (Shen, M., Yoshida, E., Yan, W., Kawamoto, T., Suardita, K., Koyano, Y., Fujimoto, K., Noshiro, M., Kato, Y., 2002. Basic helix-loop-helix protein DEC1 promotes chondrocyte differentiation at the early and terminal stages. J. Biol. Chem. 277, 50112-50120). Since MSC have multilineage differentiation potential, we investigated the roles of Dec1 in osteogenic and adipogenic differentiation of human bone marrow-derived MSC. After osteogenic induction of MSC in medium containing dexamethasone, beta-glycerophosphate, and ascorbic acid, Dec1 expression gradually increased from day 5 to day 14, while expression levels of Dec1 mRNA markedly decreased on days 3 and 7 after adipogenic induction. Infection with adenovirus expressing Dec1 raised mRNA levels of several bone characteristic molecules such as osteopontin, PTH receptor, and alkaline phosphatase, even in the absence of the osteogenic induction medium, although it had little effect on Runx2 expression or calcification. In the osteogenic induction medium, Dec1 overexpression enhanced the expression of osteopontin and alkaline phosphatase and induced matrix calcification. Knockdown of Dec1 with siRNA suppressed the expression of osteoblastic phenotype by the induced MSC. Using MSC cultures, we also confirmed that forced expression of Dec1 suppressed adipogenic differentiation. These findings suggest that Dec1 modulates osteogenic differentiation of MSC by inducing the expression of several, but not all, bone-related genes.

To characterize mesenchymal stem cells (MSC), we compared gene expression profiles in human bone marrow MSC (11 lines) and human fibroblasts (4 lines) by RT-PCR and real time PCR. Messenger RNA levels of MHC-DR-alpha, MHC-DR-beta, MHC-DR-associated protein CD74, tissue factor pathway inhibitor-2, and neuroserpin were much higher in MSC than in fibroblasts, even in the presence of large interindividual variations. Those of adrenomedullin, apolipoprotein D, C-type lectin superfamily member-2, collagen type XV alpha1, CUG triplet repeat RNA-binding protein, matrix metalloproteinase-1, protein tyrosine kinase-7, and Sam68-like phosphotyrosine protein/T-STAR were lower in MSC than in fibroblasts. FACS analysis showed that cell surface expression of MHC-DR was also higher in MSC than in fibroblasts. MHC-DR expression decreased after osteogenic differentiation, whereas the expression of adrenomedullin-a potent stimulator of osteoblast activity-along with collagen XV alpha1 and apolipoprotein D increased after osteogenic differentiation. The marker genes identified in this study should be useful for characterization of MSC both in basic and clinical studies.

Human gingival epithelial cells (HGECs) function as a mechanical barrier against invasion by pathogenic organisms through epithelial cell–cell junction complexes, which are complex components of integrin. Integrins play an important role in the protective functions of HGECs. Human periodontal ligament (HPL) cells regulate periodontal homeostasis. However, periodontitis results in the loss of HPL cells. Therefore, as replenishment, HPL cells or mesenchymal stem cells (MSCs) can be transplanted. Herein, HPL cells and MSCs were used to elucidate the regulatory mechanisms of HGECs, assuming periodontal tissue homeostasis. Human gingival fibroblasts (HGFs), HGECs, HPL cells, and MSCs were cultured, and the conditioned medium was collected. With or without silencing periostin mRNA, HGECs were cultured under normal conditions or in a conditioned medium. Integrin and periostin mRNA expression was determined using real-time polymerase chain reaction. Integrin protein expression was analyzed using flow cytometry, and periostin protein expression was determined via western blotting. The conditioned medium affected integrin expression in HGECs. Higher expression of periostin was observed in MSCs and HPL cells than in HGFs. The conditioned medium that contained periostin protein regulated integrin expression in HGECs. After silencing periostin in MSCs and HPL cells, periostin protein was not detected in the conditioned medium, and integrin expression in HGECs remained unaffected. Integrins in HGECs are regulated by periostin secreted from HPL cells and MSCs. This result suggests that periostin maintains gingival cell adhesion and regulates bacterial invasion/infection. Therefore, the functional regulation of periostin-secreting cells is important in preventing periodontitis. • Integrins on HGEC are regulated by humoral factor from HPL cells and MSCs. • Periostin is included in humoral factors from HPL cells and MSCs. • Periostin maintains gingival cell adhesion via the regulation of integrins. • Periostin can regulate bacterial invasion. • Regulation of periostin-secreting cells is important to prevent periodontitis. [ABSTRACT FROM AUTHOR]

Featured Application: Through a comprehensive comparison between the widely-used MSC spheroids and the newly-developed C-MSCs, this study sheds light on the distinct biological properties of each 3D cell culture method, advancing our understanding and potentially paving the way for more effective and versatile applications in MSC-based cell therapy. Background: Cells typically function and behave within a three-dimensional (3D) environment. Mesenchymal stem cells (MSCs), known for their self-renewal, multi-lineage differentiation capabilities, and paracrine effects, have garnered significant medical interest. MSC spheroid culture is widely adopted to study the biological properties of MSCs in a 3D context. In contrast, we previously developed 3D clumps of MSC/ECM complexes termed C-MSCs. C-MSCs consisted of cells and self-produced ECM proteins, allowing grafting into tissue defects without any artificial scaffolds. This present study aimed to elucidate the fundamental biological distinctions between 3D MSC spheroids and C-MSCs. Methods: MSC spheroids and C-MSCs are generated from human bone-marrow-derived MSCs. The physical properties, histological structures, and gene expression patterns were compared in vitro. Results: Macroscopic and histological examinations revealed that, whereas MSC spheroids are dense cell clusters primarily formed through Cadherin-mediated cell–cell interactions, C-MSCs are cell aggregates anchored by the ECM component COL1, enabling them to form larger structures. Furthermore, transcriptome analysis showed that C-MSCs possess enhanced capacities to produce immunomodulatory and cytoprotective factors, a prominent biological characteristic of MSCs. Conclusion: Recognizing the distinct attributes of each cell aggregate offers insights into the potential evolution of 3D cell culture techniques and possible therapeutic implications. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: Periodontitis causes periodontal tissue destruction and results in physiological tooth dysfunction. Therefore, periodontal regeneration is ideal therapy for periodontitis. Mesenchymal stem cells (MSCs) are useful for periodontal regenerative therapy as they can differentiate into periodontal cells; however, the underlying regulatory mechanism is unclear. In this study, we attempted to identify regulatory genes involved in periodontal cell differentiation and clarify the differentiation mechanism for effective periodontal regenerative therapy. Background: The cementum and periodontal ligament play important roles in physiological tooth function. Therefore, cementum and periodontal ligament regeneration are critical for periodontal regenerative therapy. Mesenchymal stem cell transplantation can be a common periodontal regenerative therapy because these cells have multipotency and self‐renewal ability, which induces new cementum or periodontal ligament formation. Moreover, MSCs can differentiate into cementoblasts. Cementoblast‐ or periodontal ligament cell–specific proteins have been reported; however, it is unclear how these proteins are regulated. MicroRNA (miRNA) can also act as a key regulator of MSC function. Therefore, in this study, we identified regulatory genes involved in cementoblast or periodontal cell differentiation and commitment. Methods: Human MSCs (hMSCs), cementoblasts (HCEM), and periodontal ligament cells (HPL cells) were cultured, and mRNA or miRNA expression was evaluated. Additionally, cementoblast‐specific genes were overexpressed or suppressed in hMSCs and their expression levels were investigated. Results: HCEM and HPL cells expressed characteristic genes, of which we focused on ets variant 1 (ETV1), miR‐628‐5p, and miR‐383 because ETV1 is a differentiation‐related transcription factor, miR‐628‐5p was the second‐highest expressed gene in HCEM and lowest expressed gene in HPL cells, and miR‐383 was the highest expressed gene in HCEM. miR‐628‐5p and miR‐383 overexpression in hMSCs regulated ETV1 mRNA expression, and miR‐383 overexpression downregulated miR‐628‐5p expression. Moreover, miR‐383 suppression decreased miR‐383 expression and enhanced ETV1 mRNA expression, but miR‐383 suppression also decreased miR‐628‐5p. Furthermore, silencing of ETV1 expression in hMSCs regulated miR‐628‐5p and miR‐383 expression. Concerning periodontal cell commitment, miR‐628‐5p, miR‐383, and ETV1 regulated the expression of HCEM‐ or HPL cell–related genes by adjusting the expression of these miRNAs. Conclusion: HCEM and HPL cells show characteristic mRNA and miRNA profiles. In particular, these cells have specific miR‐383, miR‐628‐5p, and ETV1 expression patterns, and these genes interact with each other. Therefore, miR‐383, miR‐628‐5p, and ETV1 are key genes involved in cementogenesis or HPL cell differentiation. [ABSTRACT FROM AUTHOR]

Background: The purpose of this study is to evaluate whether (β-tricalcium phosphate (β-TCP) could be a promising modality to help augment alveolar bone in periodontal tissue regeneration by bone marrow mesenchymal stem cells (BMMSCs). Methods: Expanded BMMSCs and atelocollagen (Col) were mixed together (MSC/Col). A combination of β-TCP with MSC/Col was also prepared (MSC/Col/TCP). MSC/ Col/TCP or MSC/Col was transplanted into experimental periodontal Class III furcation defects that had been exposed to inflammation in beagle dogs. Periodontal tissue regeneration was evaluated by histologic and morphometric analyses at 4 and 8 weeks after transplantation. Results: MSC/Col and MSC/Col/TCP enhanced periodontal tissue regeneration compared to Col and TCP/Col according to hematoxylin and eosin staining. The percentage of new cementum length in the MSC/Col/TCP group was not significantly different from that in the MSC/Col group at 4 and 8 weeks. On the other hand, the percentage of new bone area in the MSC/Col/TCP group was much higher than that in the MSC/TCP group at 4 weeks. However, at 8 weeks, no significant difference in new bone area was found between the two groups. In the MSC/Col/TCP group, β-TCP was surrounded by newly formed bone. Multinucleated cells, which were positive for osteopontin and tartrate-resistant acid phosphatase, were present in the interconnected mac-ropores of β-TCP. Conclusion: These findings suggest that β-TCP is applicable as a scaffold for BMMSCs transplantation and helps augment alveolar bone without impairing regeneration of cementum. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]