To Repair a Broken Heart: Stem Cells in Ischemic Heart Disease.

Despite improvements in contemporary medical and surgical therapies, cardiovascular disease (CVD) remains a significant cause of worldwide morbidity and mortality; more specifically, ischemic heart disease (IHD) may affect individuals as young as 20 years old. Typically managed with guideline-directed medical therapy, interventional or surgical methods, the incurred cardiomyocyte loss is not always completely reversible; however, recent research into various stem cell (SC) populations has highlighted their potential for the treatment and perhaps regeneration of injured cardiac tissue, either directly through cellular replacement or indirectly through local paracrine effects. Different stem cell (SC) types have been employed in studies of infarcted myocardium, both in animal models of myocardial infarction (MI) as well as in clinical studies of MI patients, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), Muse cells, multipotent stem cells such as bone marrow-derived cells, mesenchymal stem cells (MSCs) and cardiac stem and progenitor cells (CSC/CPCs). These have been delivered as is, in the form of cell therapies, or have been used to generate tissue-engineered (TE) constructs with variable results. In this text, we sought to perform a narrative review of experimental and clinical studies employing various stem cells (SC) for the treatment of infarcted myocardium within the last two decades, with an emphasis on therapies administered through thoracic incision or through percutaneous coronary interventions (PCI), to elucidate possible mechanisms of action and therapeutic effects of such cell therapies when employed in a surgical or interventional manner.

Reversible Switching CuII /CuI Single Sites Catalyze High-rate and Selective CO2 Photoreduction.

Herein, we first design a model of reversible redox-switching metal-organic framework single-unit-cell sheets, where the abundant metal single sites benefit for highly selective CO2 reduction, while the reversible redox-switching metal sites can effectively activate CO2 molecules. Taking the synthetic Cu-MOF single-unit-cell sheets as an example, synchrotron-radiation quasi in situ X-ray photoelectron spectra unravel the reversible switching CuII /CuI single sites initially accept photoexcited electrons and then donate them to CO2 molecules, which favors the rate-liming activation into CO2 δ- , verified by in situ FTIR spectra and Gibbs free energy calculations. As an outcome, Cu-MOF single-unit-cell sheets achieve near 100 % selectivity for CO2 photoreduction to CO with a high rate of 860 µmol g-1 h-1 without any sacrifice reagent or photosensitizer, where both the activity and selectivity outperform previously reported photocatalysts evaluated under similar conditions.

A morphological study of adipose-derived stem cell sheets created with temperature-responsive culture dishes using scanning electron microscopy.

Adipose-derived stem cell (ADSC) sheets have potential to be effective in various therapies. In this study, we first demonstrated that a cell sheet composed of human ADSCs could be created using a new temperature-responsive culture dish from the DIC Corporation. The dish can cause detachment of adherent cells due to temperature changes, but a few morphological analyses have evaluated the presence or absence of damage on the detached surface of cell sheet. To characterize our ADSC sheet, we tried to observe the surface of ADSC sheets with scanning electron microscope (SEM) using the ionic liquid, which enables the rapid preparation of samples. No damage was found on the surface of the ADSC sheets on the side that had been in contact with the surface of the culture dishes. In addition, when the transcriptomes of the harvested cell sheets were compared with those of monolayer cultures, no up-regulation of cell death related genes were detected. These results propose that the detachment from temperature-responsive culture dish causes no serious damage on the prepared ADSC sheet. It is also suggested that the SEM with ionic liquids is a useful and rapid method for the analysis of ADSC sheets for therapy.

Biobanked human foreskin epithelial cell sheets reduce inflammation and promote wound healing in a nude mouse model.

BACKGROUND: Human epithelial cell sheets (ECSs) are used to clinically treat epithelial conditions such as burns, corneal blindness, middle ear cholesteatoma and vitiligo. As a widely used material in clinic, there is little information on the biobanking of ECSs and its repair effect after storage. RESULTS: Two methods for biobanking foreskin ECSs were compared in a short term (7 days): 4-degree storage and programmed cryopreservation. Cell sheet integrity, viability, apoptosis, immunogenicity, mechanical properties and function were evaluated. In vivo, ECSs were directly transplanted to skin defect models and histological examination was performed at 1 week postoperatively. We successfully extracted human foreskin-derived primary epithelial cells and fabricated them into ECSs. Compared with 4-degree storage, programmed cryopreservation preserved the ECS structural integrity, enhanced the mechanical properties, decreased HLA-I expression, and increased cell viability and survival. An increased proportion of melanocytes with proliferative capacity remained in the cryopreserved sheets, and the undifferentiated epithelial cells were comparable to those of the fresh sheets. In vivo, cryopreserved ECSs could reduce inflammatory cell infiltration and promote connective tissue remodeling, epithelial cell proliferation and vascular regeneration. CONCLUSIONS: Programmed cryopreservation of ECSs was superior and more feasible than 4-degree storage and the cryopreserved ECSs achieved satisfying skin wound healing in vivo. We anticipate that the off-the-shelf ECSs could be quickly used, such as, to repair human epithelial defect in future.

Recent progress in induced pluripotent stem cell-derived 3D cultures for cardiac regeneration.

Cardiovascular diseases are the leading cause of death in the world due to the high incidence of the diseases coupled with the limited therapeutic options. In recent years, advances in regenerative medicine have emerged as a promising treatment. Differentiation of induced pluripotent stem cells (iPSCs) into cardiac cells and emerging technologies allowing arrangement of cells into complex 3D tissue-like structures open new frontiers for transplantation and engraftment of these tissue patches onto the damaged heart. Despite the cells integrating and presenting initial neovascularization, the functional and electric properties of these patches are still not comparable with those of the host cardiac tissue. Future research optimizing maturation and integration of the iPSC-derived cardiomyocytes is paramount for cardiac cell therapy to attain clinical use. Herein, we will review the state of the art and the different approaches to constructing these 3D transplantable structures.

Enhance Mandibular Symphyseal Surface Bone Growth with Autologous Mesenchymal Stem Cell Sheets: An Animal Study.

INTRODUCTION: The size and shape of the chin strongly influence facial profile and harmony. The current correction of chin deficiency mostly relies on genioplasty surgery involving osteotomy. To avoid osteotomy, one possible alternative is to enhance bone growth at the mental protuberance area with cell sheet transplantation. This study was undertaken to evaluate the efficacy of this approach in a pig model. MATERIALS AND METHODS: Five 4-month-old pigs were included for mandibular bone marrow aspiration and MSC isolation. Triple-layer MSC sheets were then fabricated and utilized using culture-expanded MSCs. Four weeks after bone marrow aspiration, subperiosteal pockets were created on the labial symphyseal surface, followed by transplantation of autogenous MSC sheets to one randomly chosen side with the other side (control) receiving no transplantation. Six weeks after the surgery, the pigs were euthanized and the specimens from both sides were collected for computed tomography (CT) and histological and immunohistochemical analysis. Measurements between the experimental and control sides were compared using paired t tests. RESULTS: MSC sheet fabrication and transplantation were reliably conducted. The labial cortical bone thickness increased significantly with MSC sheet transplantation by an average of 2 mm (p = 0.0001). The average measurements of mineral apposition rate and cell proliferation at the cell sheet side tended to be higher than the control side although the differences did not reach statistical significance (p = 0.1-0.2). Tissue mineral density measurements from CT images and bone volume fraction (BV/TV) measurements from histologic images were identical between the two sides (p > 0.5). CONCLUSION: These data provide a proof of concept that autologous MSC sheets may be transplanted to the subperiosteal region of the mandibular symphysis to stimulate local surface bone growth. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Mesenchymal stem cell sheets: a new cell-based strategy for bone repair and regeneration.

Mesenchymal stem cells (MSCs), a class of adult stem cells, are considered a promising source for bone regeneration. Although combining MSCs with biomaterial scaffolds offers an interesting clinical strategy for bone tissue engineering, the presence of the scaffolds could induce an undesirable effect on cell-cell interactions. Moreover, before the application of scaffold materials in bone tissue reconstruction, cells must be manipulated with proteolytic enzymes, such as trypsin or dispase that degrade extracellular matrix (ECM) molecules and cell surface proteins, which can result in the cell damage and loss of cellular activity. Therefore, the development of alternative strategies for bone regeneration is required to solve these problems. Recently, a novel tissue engineering technology named 'cell sheet' has been efficaciously utilized in the regeneration of bone, corneal, cardiac, tracheal and periodontal ligament-like tissues. The cell sheet is a layer of cells, which contains intact ECM and cell surface proteins such as growth factor receptors, ion channels and cell-to-cell junction proteins. MSC sheets can be easily fabricated by layering the recovered cell sheets without any scaffolds or complicated manipulation. This review summarizes the current state of the literature regarding the use of MSCs to produce cell sheets and assesses their applicability in bone tissue regeneration and repair.

Combination of Mesenchymal Stromal Cells and Cardiac Stem Cells in a Multilayer Cell Construct Promotes Activation of Notch Signaling and Initiation of Endothelial Differentiation.

We showed the possibility of generating combined tissue-engineered cell consisting of layers of rat cardiac stem cells and mesenchymal stromal cells from the adipose tissue. Cell-cell interaction within the cell sheet promoted proliferation of cardiac stem cells, expression of endothelial differentiation marker ETS1, and Notch signaling activation. The obtained results provide new insights into possible mechanisms of stimulation of endogenous regeneration processes after myocardial damage and demonstrate potential of cell-based cardiomyoplasty with the use of these combined cell sheets.

Treatment of Cutaneous Ulcers with Multilayered Mixed Sheets of Autologous Fibroblasts and Peripheral Blood Mononuclear Cells.

BACKGROUND/AIMS: We have developed a mixed-cell sheet consisting of autologous fibroblasts and peripheral blood mononuclear cells with a high potency for angiogenesis and wound healing against refractory cutaneous ulcers in mouse and rabbit models. To increase the effectiveness of the mixed sheet, we developed a multilayered mixed sheet. METHODS: We assessed the therapeutic effects of multilayered sheets on cutaneous ulcers in mice. Growth factors and chemokines were assessed by enzyme-linked immunosorbent assay. Angiogenesis and fibroblast migration were measured by using tube formation and migration assays. Wound healing rate of cutaneous ulcers was evaluated in mice with diabetes mellitus. RESULTS: The concentration of secreted vascular endothelial growth factor, hepatocyte growth factor, transforming growth factor, C-X-C motif chemokine ligand (CXCL)-1, and CXCL-2 in multilayered sheets was much higher than that in single-layered mixed-cell sheets (single-layered sheets) and multilayered sheets of fibroblasts alone (fibroblast sheets). The supernatant in multilayered sheets enhanced angiogenic potency and fibroblast migration compared with single-layered and fibroblast sheets in an in vitro experiment. The wound healing rate in the multilayered sheet-treated group was higher compared with the no-treatment group (control) at the early stage of healing. Moreover, both vessel lumen area and microvessel density in tissues treated with multilayered sheets were significantly increased compared with tissues in the control group. CONCLUSION: Multilayered sheets promoted wound healing and microvascular angiogenesis in the skin by supplying growth factors and cytokines. Accordingly, our data suggest that multilayered sheets may be a promising therapeutic material for refractory cutaneous ulcers.

Culture-expanded mesenchymal stem cell sheets enhance extraction-site alveolar bone growth: An animal study.

BACKGROUND AND OBJECTIVES: Impaired bone formation of the buccal alveolar plate after tooth extraction during adolescence increases the difficulty of future implant restoration. This study was undertaken to assess the feasibility and efficacy of transplanting autogenous scaffold-free culture-expanded mesenchymal stem cell (MSC) sheets to the buccal alveolar bone surface to stimulate local bone growth. MATERIAL AND METHODS: Mandibular bone marrow was aspirated from 3-month-old pigs (n = 5), from which MSCs were isolated and culture expanded. Triple-layer MSC sheets were then fabricated using temperature-responsive tissue culture plates. One month after bone marrow aspirations, the same pigs underwent bilateral extraction of mandibular primary molars, immediately followed by transplantation of 3 autogenous triple-layer MSC sheets on to the subperiosteal buccal alveolar surface of 1 randomly chosen side. The contralateral side (control) underwent the same periosteal reflection surgery without receiving MSC sheet transplantation. Six weeks later, the animals were killed and specimens from both sides were immediately harvested for radiographic and histological analysis. Buccal alveolar bone thickness, tissue mineral density (TMD), mineral apposition and bone volume fraction (BV/TV) were quantified and compared between the MSC sheet and control sides using paired t-tests. RESULTS: Triple-layer MSC sheets were reliably fabricated and the majority of cells remained vital before transplantation. The thickness of buccal bone tended to increase with MSC sheet transplantation (P = .18), with 4 of 5 animals showing an average of 1.82 ± 0.73 mm thicker bone on the MSC sheet side than the control side. After being normalized by the TMD of intracortical bone, the TMD of surface cortical bone was 0.5-fold higher on the MSC sheet side than the control side (P < .05). Likewise, the BV/TV measurements of the buccal surface region were also 0.4-fold higher on the MSC sheet side than the control side (P < .05) after being normalized by measurements from the intracortical region. Mineral apposition measurements were not different between the 2 sides. CONCLUSION: Mandibular marrow-derived MSCs can be fabricated into cell sheets and autogenous transplantation of MSC sheets onto the subperiosteal buccal alveolar bone surface at the tooth-extraction site may increase local bone density.

In Vitro Comparison of 2D-Cell Culture and 3D-Cell Sheets of Scleraxis-Programmed Bone Marrow Derived Mesenchymal Stem Cells to Primary Tendon Stem/Progenitor Cells for Tendon Repair.

The poor and slow healing capacity of tendons requires novel strategies to speed up the tendon repair process. Hence, new and promising developments in tendon tissue engineering have become increasingly relevant. Previously, we have established a tendon progenitor cell line via ectopic expression of the tendon-related basic helix-loop-helix (bHLH) transcription factor Scleraxis (Scx) in human bone marrow mesenchymal stem cells (hMSC-Scx). The aim of this study was to directly compare the characteristics of hMSC-Scx cells to that of primary human tendon stem/progenitors cells (hTSPCs) via assessment of self-renewal and multipotency, gene marker expression profiling, in vitro wound healing assay and three-dimensional cell sheet formation. As expected, hTSPCs were more naive than hMSC-Scx cells because of higher clonogenicity, trilineage differentiation potential, and expression of stem cell markers, as well as higher mRNA levels of several gene factors associated with early tendon development. Interestingly, with regards to wound healing, both cell types demonstrate a comparable speed of scratch closure, as well as migratory velocity and distance in various migration experiments. In the three-dimensional cell sheet model, hMSC-Scx cells and hTSPCs form compact tendinous sheets as histological staining, and transmission electron microscopy shows spindle-shaped cells and collagen type I fibrils with similar average diameter size and distribution. Taken together, hTSPCs exceed hMSC-Scx cells in several characteristics, namely clonogenicity, multipotentiality, gene expression profile and rates of tendon-like sheet formation, whilst in three-dimensional cell sheets, both cell types have comparable in vitro healing potential and collagenous composition of their three-dimensional cell sheets, making both cell types a suitable cell source for tendon tissue engineering and healing.

Evaluation of Mesenchymal Stem Cell Sheets Overexpressing BMP-7 in Canine Critical-Sized Bone Defects.

The aim of this study was to investigate the in vitro osteogenic capacity of bone morphogenetic protein 7 (BMP-7) overexpressing adipose-derived (Ad-) mesenchymal stem cells (MSCs) sheets (BMP-7-CS). In addition, BMP-7-CS were transplanted into critical-sized bone defects and osteogenesis was assessed. BMP-7 gene expressing lentivirus particles were transduced into Ad-MSCs. BMP-7, at the mRNA and protein level, was up-regulated in BMP-7-MSCs compared to expression in Ad-MSCs. Osteogenic and vascular-related gene expressions were up-regulated in BMP-7-CS compared to Ad-MSCs and Ad-MSC sheets. In a segmental bone-defect model, newly formed bone and neovascularization were enhanced with BMP-7-CS, or with a combination of BMP-7-CS and demineralized bone matrix (DBM), compared to those in control groups. These results demonstrate that lentiviral-mediated gene transfer of BMP-7 into Ad-MSCs allows for stable BMP-7 production. BMP-7-CS displayed higher osteogenic capacity than Ad-MSCs and Ad-MSC sheets. In addition, BMP-7-CS combined with demineralized bone matrix (DBM) stimulated new bone and blood vessel formation in a canine critical-sized bone defect. The BMP-7-CS not only provides BMP-7 producing MSCs but also produce osteogenic and vascular trophic factors. Thus, BMP-7-CS and DBM have therapeutic potential for the treatment of critical-sized bone defects and could be used to further enhance clinical outcomes during bone-defect treatment.

Contributions of Bioactive Molecules in Stem Cell-Based Periodontal Regeneration.

Periodontal disease is a widespread disease, which without proper treatment, may lead to tooth loss in adults. Because stem cells from the inflammatory microenvironment created by periodontal disease exhibit impaired regeneration potential even under favorable conditions, it is difficult to obtain satisfactory therapeutic outcomes using traditional treatments, which only focus on the control of inflammation. Therefore, a new stem cell-based therapy known as cell aggregates/cell sheets technology has emerged. This approach provides sufficient numbers of stem cells with high viability for treating the defective site and offers new hope in the field of periodontal regeneration. However, it is not sufficient for regenerating periodontal tissues by delivering cell aggregates/cell sheets to the impaired microenvironment in order to suppress the function of resident cells. In the present review, we summarize some promising bioactive molecules that act as cellular signals, which recreate a favorable microenvironment for tissue regeneration, recruit endogenous cells into the defective site and enhance the viability of exogenous cells.

Customizable engineered blood vessels using 3D printed inserts.

Current techniques for tissue engineering blood vessels are not customizable for vascular size variation and vessel wall thickness. These critical parameters vary widely between the different arteries in the human body, and the ability to engineer vessels of varying sizes could increase capabilities for disease modeling and treatment options. We present an innovative method for producing customizable, tissue engineered, self-organizing vascular constructs by replicating a major structural component of blood vessels - the smooth muscle layer, or tunica media. We utilize a unique system combining 3D printed plate inserts to control construct size and shape, and cell sheets supported by a temporary fibrin hydrogel to encourage cellular self-organization into a tubular form resembling a natural artery. To form the vascular construct, 3D printed inserts are adhered to tissue culture plates, fibrin hydrogel is deposited around the inserts, and human aortic smooth muscle cells are then seeded atop the fibrin hydrogel. The gel, aided by the innate contractile properties of the smooth muscle cells, aggregates towards the center post insert, creating a tissue ring of smooth muscle cells. These rings are then stacked into the final tubular construct. Our methodology is robust, easily repeatable and allows for customization of cellular composition, vessel wall thickness, and length of the vessel construct merely by varying the size of the 3D printed inserts. This platform has potential for facilitating more accurate modeling of vascular pathology, serving as a drug discovery tool, or for vessel repair in disease treatment.

Assessment of cellular materials generated by co-cultured 'inflamed' and healthy periodontal ligament stem cells from patient-matched groups.

Recently, stem cells derived from the'inflamed' periodontal ligament (PDL) tissue of periodontally diseased teeth (I-PDLSCs) have been increasingly suggested as a more readily accessible source of cells for regenerative therapies than those derived from healthy PDL tissue (H-PDLSCs). However, substantial evidence indicates that I-PDLSCs exhibit impaired functionalities compared with H-PDLSCs. In this study, patient-matched I-PDLSCs and H-PDLSCs were co-cultured at various ratios. Cellular materials derived from these cultures were investigated regarding their osteogenic potential in vitro and capacity to form new bone following in vivo transplantation. While patient-matched I-PDLSCs and H-PDLSCs could co-exist in co-culture systems, the proportion of I-PDLSCs tended to increase during in vitro incubation. Compared with H-PDLSC monoculture, the presence of I-PDLSCs in the co-cultures appeared to enhance the overall cell proliferation. Although not completely rescued, the osteogenic and regenerative potentials of the cellular materials generated by co-cultured I-PDLSCs and H-PDLSCs were significantly improved compared with those derived from I-PDLSC monocultures. Notably, cells in co-cultures containing either 50% I-PDLSCs plus 50% H-PDLSCs or 25% I-PDLSCs plus 75% H-PDLSCs expressed osteogenesis-related proteins and genes at levels similar to those expressed in H-PDLSC monocultures (P>0.05). Irrespective of the percentage of I-PDLSCs, robust cellular materials were obtained from co-cultures with 50% or more H-PDLSCs, which exhibited equivalent potential to form new bone in vivo compared with sheets generated by H-PDLSC monocultures. These data suggest that the co-culture of I-PDLSCs with patient-matched H-PDLSCs is a practical and effective method for increasing the overall osteogenic and regenerative potentials of resultant cellular materials.

Tissue engineered periodontal products.

Attainment of periodontal regeneration is a significant clinical goal in the management of advanced periodontal defects arising from periodontitis. Over the past 30 years numerous techniques and materials have been introduced and evaluated clinically and have included guided tissue regeneration, bone grafting materials, growth and other biological factors and gene therapy. With the exception of gene therapy, all have undergone evaluation in humans. All of the products have shown efficacy in promoting periodontal regeneration in animal models but the results in humans remain variable and equivocal concerning attaining complete biological regeneration of damaged periodontal structures. In the early 2000s, the concept of tissue engineering was proposed as a new paradigm for periodontal regeneration based on molecular and cell biology. At this time, tissue engineering was a new and emerging field. Now, 14 years later we revisit the concept of tissue engineering for the periodontium and assess how far we have come, where we are currently situated and what needs to be done in the future to make this concept a reality. In this review, we cover some of the precursor products, which led to our current position in periodontal tissue engineering. The basic concepts of tissue engineering with special emphasis on periodontal tissue engineering products is discussed including the use of mesenchymal stem cells in bioscaffolds and the emerging field of cell sheet technology. Finally, we look into the future to consider what CAD/CAM technology and nanotechnology will have to offer.

Addition of Adipose-Derived Stem Cells to Mesenchymal Stem Cell Sheets Improves Bone Formation at an Ectopic Site.

To determine the effect of adipose-derived stem cells (ADSCs) added to bone marrow-derived mesenchymal stem cell (MSC) sheets on bone formation at an ectopic site. We isolated MSCs and ADSCs from the same rabbits. We then prepared MSC sheets for implantation with or without ADSCs subcutaneously in the backs of severe combined immunodeficiency (SCID) mice. We assessed bone formation at eight weeks after implantation by micro-computed tomography and histological analysis. In osteogenic medium, MSCs grew to form multilayer sheets containing many calcium nodules. MSC sheets without ADSCs formed bone-like tissue; although neo-bone and cartilage-like tissues were sparse and unevenly distributed by eight weeks after implantation. In comparison, MSC sheets with ADSCs promoted better bone regeneration as evidenced by the greater density of bone, increased mineral deposition, obvious formation of blood vessels, large number of interconnected ossified trabeculae and woven bone structures, and greater bone volume/total volume within the composite constructs. Our results indicate that although sheets of only MSCs have the potential to form tissue engineered bone at an ectopic site, the addition of ADSCs can significantly increase the osteogenic potential of MSC sheets. Thus, the combination of MSC sheets with ADSCs may be regarded as a promising therapeutic strategy to stimulate bone regeneration.

ROCK inhibitor Y-27632 maintains the proliferation of confluent human mesenchymal stem cells.

BACKGROUND AND OBJECTIVE: The transplantation of cell sheets of mesenchymal stem cells (MSCs) is expected to be the next generation of periodontal regenerative therapy. An adequate method of multilayering MSCs has yet to be established. When cell sheets proliferate, they usually contract and detach from culture dishes and then the proliferation of cells in the contracted areas is arrested. ROCK-mediated contractile force causes cell contraction. Although multilayer formation medium (MFM) stimulated the proliferation of growth-arrested confluent MSCs, MSCs detached from the culture dish. Therefore, we investigated the effects of ROCK inhibitor Y-27632 on the proliferation and detachment of confluent MSCs, and examined the ability of cells to differentiate within the cell sheets. MATERIAL AND METHODS: Confluent MSCs were cultured in MFM containing transforming growth factor-ß1, ascorbic acid and fetal bovine serum either with or without Y-27632. Cell proliferation was examined by bromodeoxyuridine incorporation assays and total DNA measurement. Sheet contractions were examined by light microscopy and stereomicroscopy. Multilayer formations and focal adhesion assembly were observed with confocal microscopy. Characteristic of cells were examined by flow cytometric analysis. Osteoblast lineage differentiation was observed with alkaline phosphatase and alizarin red S staining. Adipocyte lineage differentiation was observed with oil red O staining. RESULTS: The addition of Y-27632 to MFM prevented the cell sheets from detaching and did not inhibit MSC growth. The cell numbers cultured with MFM/Y-27632 were significantly higher than that obtained with MFM-only on day 4. Cell sheets detached from the culture dish on day 4, and the number of bromodeoxyuridine-positive cells in the detached area decreased. Cells in the cell sheets had similar characteristics to primary MSCs, and differentiated into osteoblast and adipocyte lineages. CONCLUSION: Y-27632 both prevented the MSC sheets from detaching and maintained the multilayered proliferation of confluent MSCs by MFM, and then cells in the sheets had differentiation potency.

Extracellular Vesicles Derived from Neutrophils Accelerate Bone Regeneration by Promoting Osteogenic Differentiation of BMSCs.

The reconstruction of bone defects has been associated with severe challenges worldwide. Nowadays, bone marrow mesenchymal stem cell (BMSC)-based cell sheets have rendered this approach a promising way to facilitate osteogenic regeneration in vivo. Extracellular vesicles (EVs) play an essential role in intercellular communication and execution of various biological functions and are often employed as an ideal natural endogenous nanomedicine for restoring the structure and functions of damaged tissues. The perception of polymorphonuclear leukocytes (neutrophils, PMNs) as indiscriminate killer cells is gradually changing, with new evidence suggesting a role for these cells in tissue repair and regeneration, particularly in the context of bone healing. However, the role of EVs derived from PMNs (PMN-EVs) in bone regeneration remains largely unknown, with limited research being conducted on this aspect. In the current study, we investigated the effects of PMN-EVs on BMSCs and the underlying molecular mechanisms as well as the potential application of PMN-EVs in bone regeneration. Toward this end, BMSC-based cell sheets with integrated PMN-EVs (BS@PMN-EVs) were developed for bone defect regeneration. PMN-EVs were found to significantly enhance the proliferation and osteogenic differentiation of BMSCs in vitro. Furthermore, BS@PMN-EVs were found to significantly accelerate bone regeneration in vivo by enhancing the maturation of the newly formed bone in rat calvarial defects; this is likely attributable to the effect of PMN-EVs in promoting the expression of key osteogenic proteins such as SOD2 and GJA1 in BMSCs. In conclusion, our findings demonstrate the crucial role of PMN-EVs in promoting the osteogenic differentiation of BMSCs during bone regeneration. Furthermore, this study proposes a novel strategy for enhancing bone repair and regeneration via the integration of PMN-EVs with BMSC-based cell sheets.

Immunomodulatory Behavior of Tendon Magnetic Cell Sheets can be Modulated in Hypoxic Environments under Magnetic Stimulus.

Tissue environments play a crucial role in orchestrating cell behavior, guided by a complex interplay of various factors. Long lasting inflammatory signals compromise tendon homeostasis and promote tissue degeneration, while tissue oxygen levels affect local cells' responses with hypoxic environments influencing apoptosis, inflammatory mediators, and matrix production. Recent works have unveiled the therapeutic potential of pulsed electromagnetic field (PEMF) in modulating inflammatory signals expressed by human tendon cells (hTDCs), and in mitigating the hypoxia-induced effects on the regulation of inflammatory cytokines. Thus, we sought to investigate the role of hypoxic environments, namely, 1 and 2% oxygen tension, in the inflammatory profiles of magnetic cell sheets (magCSs) formed by magnetic nanoparticles internalized in contiguous hTDCs with intact cell-cell junctions and deposited matrix. We also aimed to explore the impact of PEMF over hypoxia-treated magCSs, including IL-1ß-primed-magCSs, with the objective of harnessing magnetic stimulation to guide abnormal inflammatory cell responses toward efficient treatments supporting tendon regenerative potential. Our findings revealed that low oxygen tensions amplified the expression of hypoxia-associated genes and of inflammatory markers in IL-1ß-primed-magCSs with an involvement of the NF-κB signaling pathway. Encouragingly, when PEMF was applied to IL-1ß-primed-magCSs under hypoxic conditions, it successfully modulated inflammatory cues by favoring IL-10 and IL-4, via the NF-κB pathway. These results signify the remarkable potential of PEMF in driving proregenerative strategies and opens up new approaches in tendon therapies, highlighting the transformative impact of immunomodulatory magnetic cell sheets.