Marrow Mesenchymal Stem Cell Sheets Research Articles

Cell sheet-based in vitro bone defect model for long term evaluation of bone repair materials

Development of tissue-engineered in vitro human bone defect models for evaluation of bone repair materials (BRMs) is a promising approach for addressing both translational and ethical concerns regarding animal models. In this study, human bone marrow mesenchymal stem cell sheets were stacked to form a periosteum like tissue. HE staining showed a cell-dense, multilayered structure. BRMs were implanted in the defect area of the three-dimensional (3D) model. The CCK-8 test demonstrated that the 3D model was stronger in resisting the cytotoxicity of three kinds of commercial BRMs than the 2D culture model, which was consistent with in vivo results. After 28 d implantation in the 3D model, western blot and RT-qPCR showed that three materials induced increased expressions of RUNX2, OSX, OCN, OPN, while Materials B and C seemed to have stronger osteoinductivity than A. In vivo experiments also confirmed the osteoinductivity of the BRMs after 28 and 182 d implantation. Alizarin red staining proved that the mineralized nodules of Materials B and C were more than that of A. The differences of osteogenic properties among three BMRs might be attributed to calcium ion release. This cell sheet-based bone tissue model can resist cytotoxicity of BRMs, demonstrating the priority of long-term evaluation of osteoinductivity of BRMs. Further, the osteoinduction results of the 3D model corresponded to that of in vivo experiments, suggesting this model may have a potential to be used as a novel tool for rapid, accurate evaluation of BRMs, and thus shorten their research and development process.

The Role of Macrophage Phenotype in the Vascularization of Prevascularized Human Bone Marrow Mesenchymal Stem Cell Sheets.

With the development of tissue engineering and regenerative medicine, prevascularized marrow mesenchymal stem cell (BMSC) sheets have been regarded as a promising method for tissue regeneration. Furthermore, the inflammatory response is one of the main regulators of vascularization and the restoration of engineered tissue function; among them, macrophages and cytokines produced by them are considered to be the decisive factors of the downstream outcomes. This study investigated the effect of macrophages on the formation of microvascular-like structures of human umbilical vein endothelial cells (HUVECs) in BMSC sheets. First, a human monocytic leukaemia cell line (THP-1 cells) was differentiated into derived macrophages (M0) with phorbol 12-myristate 13-acetate (PMA) and further activated into proinflammatory macrophages (M1 macrophages) with IFN-γ and LPS or anti-inflammatory macrophages (M2 macrophages) with IL-4. Then, HUVECs and prevascularized sheets were treated with conditioned media (CM) from different macrophages, and the impact of macrophage phenotypes on vascularized network formation in prevascularized cell sheets was examined by HE staining, CD31 immunofluorescence staining and ELISA. Our study showed that macrophages may guide the arrangement of endothelial cells through a paracrine pathway. Cell sheets that were cultured in the conditioned media from M2 macrophages were thinner than those cultured in other media. At various time points, the levels of TNF-α and VEGF in prevascularized sheets cultured with CM(M1) was higher than that in sheets cultured with other media; however, the levels of PDGF-BB in prevascularized sheets cultured with CM(M2) was higher than that in sheets cultured with other media. These findings suggest that the paracrine effect of macrophages can influence the formation of microvascular networks in prevascularized sheets by regulating the arrangement of cells, the thickness of the cell sheet and the secretion of cytokines related to angiogenesis. Macrophages with different phenotypes have unique effects on prevascularized sheets.

Sutureless transplantation of in vivo priming human mesenchymal stem cell sheet promotes the therapeutic potential for cardiac repair

The heart, contrary to its small size, vigorously pumps oxygen and nutrients to our entire body indeterminably; and thus, its dysfunction could be devastating. Until now, there ave been several major obstacles to applying a cardiac patch for the treatment for myocardial infarction, including poor integration and low engraftment rates, due to the highly-curved surface of the heart and its dynamic nature. Here, we demonstrate a novel way for a comprehensive cardiac repair achieved by the sutureless transplantation of a highly integrable in vivo priming bone marrow mesenchymal stem cell (BMSC) sheet based on the utilization of a highly aligned thermoresponsive nanofiber membrane. Moreover, we developed a BMSC sheet specialized for vascular regeneration through ‘in-vivo priming’ using human umbilical vein endothelial cells. A prolonged secretion of multiple angiogenic cytokines, such as vascular endothelial growth factor, angiopoietin-1, insulin-like growth factor-1, which was observed in vitro from the specialized BMSC sheet seemed to lead a significant improvement in the cardiac function, including intrinsic contractibility and remodeling. In this study, we provide strong evidence that in vivo priming of a human BMSC sheet develops the therapeutic potential for cardiac repair.

The combination of a 3D-Printed porous Ti-6Al-4V alloy scaffold and stem cell sheet technology for the construction of biomimetic engineered bone at an ectopic site.

Cell sheet technology has been widely used in bone tissue engineering and regenerative medicine. However, controlling the shape and volume of large pieces of engineered bone tissue remains impossible without additional suitable scaffolds. Three-dimensional (3D) printed titanium (Ti) alloy scaffolds are mostly used as implant materials for repairing bone defects, but the unsatisfactory bioactivities of traditional Ti-based scaffolds severely limit their clinical applications. Herein, we hypothesize that the combination of bone marrow mesenchymal stem cell (BMSC) sheet technology and 3D porous Ti–6Al–4V (PT) alloy scaffolds could be used to fabricate biomimetic engineered bone. First, various concentrations of BMSCs were directly cocultured with PT scaffolds to obtain complexes of osteoblastic cell sheets and scaffolds. Then, as an experimental control, an osteoblastic BMSC sheet was prepared by continuous culturing under osteogenic conditions for 2 weeks without passaging and used to wrap the scaffolds. The BMSC sheet was composed of several layers of extracellular matrix (ECM) and a mass of BMSCs. The BMSCs exhibited excellent adherent, proliferative and osteogenic potential when cocultured with PT scaffolds, which may be attributed to the ability of the 3D microstructure of scaffolds to facilitate the biological behaviors of cells, as confirmed by the in vitro results. Moreover, the presence of BMSCs and ECM increased the angiogenic potential of PT scaffolds by the secretion of VEGF. Micro-CT and histological analysis confirmed the in vivo formation of biomimetic engineered bone when the complex of cocultured BMSCs and PT scaffolds and the scaffolds wrapped by prepared BMSC sheets were implanted subcutaneously into nude mice. Therefore, the combination of BMSC sheet technology and 3D-printed PT scaffolds could be used to construct customized biomimetic engineered bone, offering a novel and promising strategy for the precise repair of bone defects.

Exosomes derived from NGF-overexpressing bone marrow mesenchymal stem cell sheet promote spinal cord injury repair in a mouse model

Cell transplantation has been an appealing way to improve the recovery of motor, sensory, and autonomic functions following spinal cord injury (SCI). Herein, we sought to elucidate the function of bone marrow mesenchymal stem cells (BMSCs) sheet in the progression of SCI and its underlying mechanism. BMSCs were extracted from bone marrow of femur and tibia collected from C57BL/6 mice, and the BMSC sheet was prepared when cells grew to 100% confluence after approximately 14 days. Exosomes (Exos) derived from BMSCs were isolated and characterized. The expression of NGF in the isolated Exos and neural stem cells (NSCs) was quantified. NSCs were co-cultured with Exos derived from the BMSC sheet that was treated with overexpressed NGF (oe-NGF) (Exos-oe-NGF). NSC differentiation, axonal regeneration and locomotor function were detected in vitro and in vivo. The BMSC sheet was successfully prepared and exerted a promoting effect on NSC differentiation into neuronal cells and axonal regeneration after SCI by releasing Exos. Co-culture data showed that NGF was highly expressed in the BMSC sheet-loaded Exos and facilitated neuronal differentiation of NSCs and axonal regeneration. In vivo experimental results unveiled that transplantation of BMSC sheet-loaded Exos-oe-NGF into SCI mice displayed enhanced functional recovery. Collectively, Exo-oe-NGF loaded on the BMSC sheet can accelerate NSC differentiation, axonal regeneration and SCI repair, therefore offering us with a potential therapeutic target for treating SCI.

Bone marrow mesenchymal stem cell sheets with high expression of hBD3 and CTGF promote periodontal regeneration

The multi-bacterial environment of the oral cavity makes it hard for periodontal regeneration. As a class of antimicrobial peptide, beta defensin has been found to show broad-spectrum antibacterial ability. In addition, connective tissue growth factor (CTGF) is demonstrated to play a great role in multi-physiological events such as angiogenesis, wound healing and, more importantly, fibrogenesis. In this study, human β defensin 3 (hBD3) and CTGF were co-transfected into bone marrow derived mesenchymal stem cells (BMSCs) for preparing cell sheets. The transfection efficiency was detected through fluorescence of eGFP and western blot assay. Our results showed that the hBD3 and CTGF proteins were highly and stably expressed in the BMSCs after transfection. The results of RT-PCR and induced differentiation indicated that hBD3 promoted osteogenic differentiation of BMSCs, while CTGF significantly increased fibrogenic differentiation even in the presence of hBD3. The BMSCs acquired stronger capacity in terms of promoting M2 polarization of RAW 264.7 macrophages fulfilled by the transfection and secretion of hBD3 and CTGF. To further evaluate the periodontal remodeling performance of cell sheets, a coralline hydroxyapatite (CHA)-chitosan based hydrogel-human tooth system was designed to simulate the natural periodontal environment. The results showed that dense extracellular matrix, oriented fiber arrangement, and abundant collagen deposition appeared in the area of BMSCs sheets after subcutaneous transplantation. Altogether, our data showed that the lentivirus transfected BMSCs sheets had a promising application prospect for periodontal repair.

Effect of Bone Marrow Mesenchymal Stem Cell Sheets on Skin Capillary Parameters in a diabetic wound model: A Novel Preliminary Study

Background:Treatment with BMMSCs has anti-inflammatory, tissue regenerative, angiogenic, and immune-stimulating effects. When using as sheets or accumulate, BMMSCs causes the development of neoangiogenesis in damaged skin tissue. Diabetes, a metabolic disorder, can negatively affect many physiological functions, including the process of skin injury repair. This adverse impact may increase the risk of skin surgery. RSF is commonly used in reconstructive surgery. The terminal part of the RSF is often affected by necrosis because of impaired blood flow, which is exacerbated in diabetes. This study investigated the effect of stem cells, applied as accumulated or cell sheets, along with RSF surgery on skin capillaries in STZ-induced diabetic rats. Methods:Thirty male Wistar rats were divided into three groups (n = 10): diabetes-RSF control, diabetes-RSF local applied stem cells (loc-BMMSCs), diabetes-RSF applied stem cells as accumulated or cell sheets (ac-BMMSCs). Two weeks after the STZ injection, RSF surgery and stem cell therapy (6 × 109) were carried out (day zero). Furthermore, stereological methods were used to investigate the capillary patterns among the groups. Anti-CD31/PCAM1 immunohistochemistry was also used for further confirmation of changes in capillary parameters. Results:The results demonstrated that capillaries were protected by MSC sheets in the flap tissue, and the thickness of the epidermal layer was improved, indicationg the possible beneficial effects of MSC sheets on diabetic wound treatment. Conclusion:Stem cells, as ac-BMMSCs, may decrease the levels of wound healing complications in diabetes and can be considered as a cell therapy option in such conditions.

Engineering of axially vascularized bone tissue using natural coral scaffold and osteogenic bone marrow mesenchymal stem cell sheets

IntroductionBlood supply remains one of the obstacles to large bone tissue engineering. This study aimed to generate vascularized bone tissue by inducing axial vascularization into a construct combining natural coral scaffold and a bone marrow mesenchymal stem cells (BMSCs) sheet. Material and methodsIsolated BMSCs were cultured to form an osteogenic cell sheet using a continuous culture method. Natural coral scaffolds were prepared into customized shape with a cylinder of 20 mm length, 8 mm in outer diameter and 5 mm in inner diameter. Then, the freed superficial inferior epigastric vessel of rabbits was first wrapped with a cell sheet, and then inserted into the central passage of the scaffold, after being wrapped with another cell sheet, the complexes were implanted subcutaneously into a rabbit groin area. In contrast, the sheet-scaffold construct that implanted into groin subcutaneous area of the other side of the same rabbit with the distal end of the blood vessel was ligated, which was considered as control. New bone and vascularization formation were evaluated at 12 weeks postoperatively. ResultsThe volume of new bone formation and amount of capillary infiltration in the vascular circulation group were significantly greater than that in the vascular ligation group, which suggested that insertion of axial vessels could significantly promote angiogenesis and osteogenesis of the tissue-engineered bone. ConclusionsThese findings indicate that inserting an arteriovenous bundle into the constructs of mesenchymal stem cell sheet and coral has great potential for clinical applications to repair large bone defects.

Extracellular matrix derived from allogenic decellularized bone marrow mesenchymal stem cell sheets for the reconstruction of osteochondral defects in rabbits

Bioactive scaffolds from synthetical polymers or decellularized cartilage matrices have been widely used in osteochondral regeneration. However, the risks of potential immunological reactions and the inevitable donor morbidity of these scaffolds have limited their practical applications. To address these issues, a biological extracellular matrix (ECM) scaffold derived from allogenic decellularized bone marrow mesenchymal stem cell (BMSC) sheets was established for osteochondral reconstruction. BMSCs were induced to form cell sheets. Three different concentrations of sodium dodecyl sulfate (SDS), namely, 0.5%, 1%, and 3%, were used to decellularize these BMSC sheets to prepare the ECM. Histological and microstructural observations were performed in vitro and then the ECM scaffolds were implanted into osteochondral defects in rabbits to evaluate the repair effect in vivo. Treatment with 0.5% SDS not only efficiently removed BMSCs but also successfully preserved the original structure and bioactive components of the ECM When compared with the 1% and 3% SDS groups, histological observations substantiated the superior repair effect of osteochondral defects, including the simultaneous regeneration of well-vascularized subchondral bone and avascular articular cartilage integrated with native tissues in the 0.5% SDS group. Moreover, RT-PCR indicated that ECM scaffolds could promote the osteogenic differentiation potential of BMSCs under osteogenic conditions while increasing the chondrogenic differentiation potential of BMSCs under chondrogenic conditions. Allogenic BMSC sheets decellularized with 0.5% SDS treatment increased the recruitment of BMSCs and significantly improved the regeneration of osteochondral defects in rabbits, thus providing a prospective approach for both articular cartilage and subchondral bone reconstruction with cell-free transplantation.

Novel pre-vascularized tissue-engineered dermis based on stem cell sheet technique used for dermis-defect healing.

Insufficient donor dermis and the shortage of three-dimensional vascular networks are the main limitations in the tissue-engineered dermis (TED). To solve these problems, we initially constructed pre-vascularized bone marrow mesenchymal stem cell sheet (PBMCS) and pre-vascularized fibroblasts cell sheet (PFCS) by cell sheet technology, and then superimposed or folded them together to construct a pre-vascularized TED (PTED), aiming to mimic the real dermis structure. The constructed PTED was implanted in nude mice dorsal dermis-defect wound and the wound-healing effect was quantified at Days 1, 7 and 14 via the methods of histochemistry and immunohistochemistry. The results showed that PTED could rapidly promote the wound closure, especially at Day 14, and the wound-healing rate of three-layer PTED could reach 97.2% (P < 0.01), which was faster than the blank control group (89.1%), PBMCS (92.4%), PFCS (93.8%) and six-layer PTED (92.3%). In addition, the vessel density in the PTED group was higher than the other groups on the 14th day. Taken together, it is proved that the PTED, especially three-layer PTED, is more conducive to the full-thickness dermis-defect repair and the construction of the three-dimensional vascular networks, indicating its potential application in dermis-defect repair.

An effective light activated TiO2 nanodot platform for gene delivery within cell sheets to enhance osseointegration

Cell sheets play a vital role in gene therapies. Nonetheless, gene delivery platforms based on cell sheet technology are restricted to small genes. Here, we produced a rapid and efficient light activated gene delivery nanodot platform for large genes. Due to its large gene sequence, low-density lipoprotein receptor-related protein 5 (LRP5) is rarely used for gene therapy in vivo. Our study successfully constructed a nanodot platform using light and adenoviruses to harvest LRP5-overexpressing bone marrow mesenchymal stem cell (BMSC) sheets with a nearly 2-fold increase in efficiency and a 40% reduction in time compared with a traditional gene delivery platform. Overexpression of LRP5 upregulated the expression levels of alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP2), and runt-related transcription factor-2 (Runx2). Further, in vivo results suggested that LRP5 enhanced BMSC sheet-mediated osseointegration. This light activated gene delivery nanodot platform may serve as a potentially powerful tool for introducing various genes and for increasing the scope of application for cell sheet-based gene therapies.

Effect of book-shaped acellular tendon scaffold with bone marrow mesenchymal stem cells sheets on bone–tendon interface healing

BackgroundTissue engineering has exhibited great effect on treatment for bone-tendon interface (BTI) injury. The aim of this study was to evaluate the effect of a book-shaped acellular tendon scaffold (ATS) with bone marrow mesenchymal stem cells sheets (MSCS) for BTI injury repair.MethodsATS was designed based on the shape of “book”, decellularization effect was evaluated by Hematoxylin and eosin (H&E), 4′, 6-diamidino-2-phenylindole (DAPI) and scanning electron microscopy (SEM), then bone marrow mesenchymal stem cells (MSCs) were cultured on ATS to assess the differentiation inductivity of ATS. A rabbit right partial patellotomy model was established, and MSCS seeded on ATS were implanted into the lesion site. The patella-patellar tendon (PPT) at 2, 4, 8 or 16 weeks post-operation were obtained for histological, biomechanical and immunofluorescence analysis.ResultsH&E, DAPI and SEM results confirmed the efficiency of decellularization of ATS, and their in vitro tenogenic and chondrogenic ability were successfully identified. In vivo results showed increased macrophage polarization toward the M2 phenotype, IL-10 expression, regenerated bone and fibrocartilage at the patella-patellar tendon interface of animals received MSCS modified ATS implantation. In addition, the level of tensile strength was also the highest in MSCS modified ATS implantation group.ConclusionThis study suggests that ATS combined with MSCS performed therapeutic effects on promoting the regeneration of cartilage layer and enhancing the healing quality of patella-patellar tendon interface.The translational potential of this articleThis study showed the good biocompatibility of the ATS, as well as the great efficacy of ATS with MSCS on tendon to bone healing. The results meant that the novel book-shaped ATS with MSCS may have a great potential for clinical application.

Structure and ingredient-based biomimetic scaffolds combining with autologous bone marrow-derived mesenchymal stem cell sheets for bone-tendon healing

Tendon attaches to bone across a robust fibrocartilaginous tissue termed the bone-tendon interface (BTI), commonly injured in the field of sports medicine and orthopedics with poor prognosis. So far, there is still a lack of effective clinical interventions to achieve functional healing post BTI injury. However, tissue-engineering may be a promising treatment strategy. In this study, a gradient book-type triphasic (bone-fibrocartilage-tendon) scaffold is fabricated based on the heterogeneous structure and ingredient of BTI. After decellularization, the scaffold exhibits no residual cells, while the characteristic extracellular matrix of the original bone, fibrocartilage and tendon is well preserved. Meanwhile, the bone, fibrocartilage and tendon regions of the acellular scaffold are superior in osteogenic, chondrogenic and tenogenic inducibility, respectively. Furthermore, autologous bone marrow mesenchymal stem cell (BMSC) sheets (CS) combined with the acellular scaffolds is transplanted into the lesion site of a rabbit BTI injury model to investigate the therapeutic effects. Our results show that the CS modified scaffold not only successfully achieves triple biomimetic of BTI in structure, ingredient and cell distribution, but also effectively accelerates bone-tendon (B-T) healing. In general, this work demonstrates book-type acellular triphasic scaffold combined with autologous BMSCs sheets is a promising graft for repairing BTI injury.

Bone regeneration capacities of alveolar bone mesenchymal stem cells sheet in rabbit calvarial bone defect

Mesenchymal stem cells sheets have been verified as a promising non-scaffold strategy for bone regeneration. Alveolar bone marrow mesenchymal stem cells, derived from neural crest, have the character of easily obtained and strong multi-differential potential. However, the bone regenerative features of alveolar bone marrow mesenchymal stem cells sheets in the craniofacial region remain unclear. The purpose of the present study was to compare the osteogenic differentiation and bone defect repairment characteristics of bone marrow mesenchymal stem cells sheets derived from alveolar bone (alveolar bone marrow mesenchymal stem cells) and iliac bone (Lon-bone marrow mesenchymal stem cells) in vitro and in vivo. Histology character, osteogenic differentiation, and osteogenic gene expression of human alveolar bone marrow mesenchymal stem cells and Lon-bone marrow mesenchymal stem cells were compared in vitro. The cell sheets were implanted in rabbit calvarial defects to evaluate tissue regeneration characteristics. Integrated bioinformatics analysis was used to reveal the specific gene and pathways expression profile of alveolar bone marrow mesenchymal stem cells. Our results showed that alveolar bone marrow mesenchymal stem cells had higher osteogenic differentiation than Lon-bone marrow mesenchymal stem cells. Although no obvious differences were found in the histological structure, fibronectin and integrin β1 expression between them, alveolar-bone marrow mesenchymal stem cells sheet exhibited higher mineral deposition and expression levels of osteogenic marker genes. After being transplanted in the rabbit calvarial defects area, the results showed that greater bone volume and trabecular thickness regeneration were found in bone marrow mesenchymal stem cells sheet group compared to Lon-bone marrow mesenchymal stem cells group at both 4 weeks and 8 weeks. Finally, datasets of bone marrow mesenchymal stem cells versus Lon-bone marrow mesenchymal stem cells, and periodontal ligament mesenchymal stem cells (another neural crest derived mesenchymal stem cells) versus umbilical cord mesenchymal stem cells were analyzed. Total 71 differential genes were identified by overlap between the 2 datasets. Homeobox genes, such as LHX8, MKX, PAX9, MSX, and HOX, were identified as the most significantly changed and would be potential specific genes in neural crest mesenchymal stem cells. In conclusion, the Al-bone marrow mesenchymal stem cells sheet-based tissue regeneration appears to be a promising strategy for craniofacial defect repair in future clinical applications.

Preparation and effect of lyophilized platelet-rich fibrin on the osteogenic potential of bone marrow mesenchymal stem cells in vitro and in vivo

ObjectivesThe goal of this study was to prepare lyophilized platelet-rich fibrin (L-PRF) and analyze the combined use of L-PRF and osteogenic bone marrow mesenchymal stem cell (BMSC) sheet fragments for bone tissue engineering via in vivo injection. MethodsFirst, fresh PRF (F-PRF) was lyophilized to prepare L-PRF, the characteristics of which were examined through gross morphological, and histological and microstructural observations. In addition, the kinetics of growth factor release from L-PRF and F-PRF were also determined by enzyme-linked immunosorbent assay (ELISA). Subsequently, after assessing the proliferation and osteogenic differentiation of BMSCs exposed to L-PRF or F-PRF in vitro, we subcutaneously injected BMSC sheet fragments with L-PRF or F-PRF into nude mice and assessed bone formation through microcomputed tomography and histological analyses. ResultsWe observed that L-PRF released growth factors that favored BMSC proliferation and osteogenic differentiation in vitro. The combined use of L-PRF and osteogenic BMSC sheet fragments enabled bone tissue regeneration in vivo, and no significant difference between the F-PRF and L-PRF groups was observed (P = 0.24). ConclusionsThe results of this study demonstrate that the combined use of L-PRF and osteogenic BMSC sheets may have potential in the fabrication of engineered bone.

Additively Manufactured Multiphasic Bone-Ligament-Bone Scaffold for Scapholunate Interosseous Ligament Reconstruction.

The scapholunate interosseous ligament (SLIL) is a frequently torn wrist ligament, and current surgical options for SLIL tears are suboptimal. This research aims to develop a novel multiphasic bone-ligament-bone scaffold (BLB) with a porous interface using 3D-printing and cell sheet technology for the reconstruction of the dorsal scapholunate interosseous ligament. The BLB comprises two bone compartments bridged by aligned polycaprolactone fibers mimicking the architecture of the native tissue. Mechanical testing of the BLBs shows their ability to withstand physiological forces. Combination of the BLB with human bone marrow mesenchymal stem cell sheet demonstrates that the harvesting did not compromise cell viability, while allowing homogeneous distribution in the ligament compartment. The BLBs are loaded with cell sheets and bone morphogenetic protein-2 in the ligament and bone compartment respectively prior to ectopic implantation into athymic rats. The histology demonstrates rapid tissue infiltration, high vascularization, and more importantly the maintenance of the compartmentalization as bone formation remains localized to the bone compartment despite the porous interface. The cells in the ligament compartment become preferentially aligned, and this proof-of-concept study demonstrates that the BLB can provide sufficient compartmentalization and fiber guiding properties necessary for the regeneration of the dorsal SLIL.

Endothelial progenitor cells improve the therapeutic effect of mesenchymal stem cell sheets on irradiated bone defect repair in a rat model

BackgroundThe reconstruction of bone defects is often impaired by radiotherapy since bone quality is compromised by radiation. This study aims to investigate the therapeutic efficacy of the composite cell sheets-bone marrow mesenchymal stem cell (BMSC) sheets cocultured with endothelial progenitor cells (EPCs)-in the healing of irradiated bone defects and the biological effects of EPCs on the osteogenic properties of BMSC sheets.MethodsBMSCs and EPCs were isolated from rat bone marrow. BMSCs were used to form cell sheets by the vitamin C inducing method. EPCs were seeded on BMSC sheets to make EPCs–BMSC sheets. Osteogenesis of EPCs–BMSC sheets and BMSC sheets were tested. In vitro osteogenesis tests included ALP, Alizarin Red S, Sirius Red staining, qRT-PCR and Western blot analysis after 3 and 7 days of osteogenic incubation. Subcutaneous osteogenesis was tested by H&E staining and immunohistochemical staining 8 weeks after transplantation. EPCs–BMSC sheets and BMSC sheets were used in the 3 mm defects of non-irradiated and irradiated rat tibias. Micro-CT and histological analysis were used to test the healing of bone defects 4 and 8 weeks after transplantation.ResultsEPCs–BMSC sheets showed enhanced osteogenic differentiation in vitro with increased expression of osteoblastic markers and osteogenesis related staining compared with BMSC sheets. In subcutaneous osteogenesis test, EPCs–BMSC sheets formed larger areas of new bone and blood vessels. The EPCs–BMSC group had the highest volume of newly formed bone in the defect area of irradiated tibias.ConclusionsEPCs improved the osteogenic differentiation of BMSC Sheets and enhanced the ectopic bone formation. EPCs–BMSC sheets promoted bone healing in irradiated rat tibias. EPCs–BMSC sheets are potentially useful in the reconstruction of bone defect after radiotherapy.

Semaphorin 3A-stimulated bone marrow mesenchymal stem cells sheets promotes osteogenesis of type 2 diabetic rat

Objective: To evaluate the effect of semaphorin 3A (Sema3A) pre-treated bone marrow mesenchymal stem cells (BMSC) sheets on new bone formation in type 2 diabetes mellitus rats. Methods: Type 2 diabetes mellitus (T2DM) were induced by injection of streptozotocin, and the BMSC were isolated, controlled, identified and induced into cell sheets. Fifteen T2DM rats were randomly divided into control, sheets and Sema3A-sheets group and the calvarial critical size defect (CSD) model of rats were established. The defect zone of rats from control group were implanted with bone powder. The defect zone of rats from sheets group were implanted with bone powder and BMSC sheets. The defect zone of rats from Sema3A-sheets group were implanted with bone powder and BMSC sheets pretreated with 1.0 mg/L Sema3A. After 8 weeks, the bone samples were harvested and analyzed by micro-CT scanning, HE staining for the evaluation of new bone formation, and the immunohistochemical analysis for the expression of osteogenesis-related proteins including type Ⅰ collagen (COL- Ⅰ ), bone morphogenetic protein-2 (BMP-2), and osteocalcin (OCN). Results: The BMSC were isolated and cultured, and oil red O and Alizarin red S staining proved the multi-potential differentiation. Eight weeks after the establishment of calvarial CSD model, Sema3A-sheet group showed the most abundant new bone formation (0.516±0.070), with increased bone volume fraction, namely bone volume/tissue volume (BV/TV) compared with sheets group (0.319±0.050) and control group (0.224±0.037) (P<0.05), and the sheets group showed increased BV/TV compared with control group (P<0.05). While trabecular thickness (Tb.Th) control group showed no difference in three groups (P>0.05). HE staining also confirmed that Sema3A-sheets group showed the most new bone formation. Sheet group (0.174±0.051) compared showed difference with control group (0.099±0.033) (P< 0.05), and Sema3A-sheet group (0.421±0.069) showed increased bone formation compared with sheet group and control group (P<0.05). Immunohistochemistry showed that BMSC sheet increased the expression of osteogenesis-related proteins including COL-Ⅰ, BMP-2 and OCN, while Sema3A pretreatment showed more obvious increase of the expression of COL-Ⅰ and OCN. Conclusions: The combined implantation of bone powder and Sema3A stimulated BMSC sheets significantly increased bone regeneration in vivo. Therefore, Sema3A pre-treated BMSC sheets transplantation provides a new strategy for restoring bone defect in T2DM.

Preparation of high bioactivity multilayered bone-marrow mesenchymal stem cell sheets for myocardial infarction using a 3D-dynamic system

Cell sheet techniques offer a promising future for myocardial infarction (MI) therapy; however, insufficient nutrition supply remains the major limitation in maintaining stem cell bioactivity in vitro. In order to enhance cell sheet mechanical strength and bioactivity, a decellularized porcine pericardium (DPP) scaffold was prepared by the phospholipase A2 method, and aspartic acid was used as a spacer arm to improve the vascular endothelial growth factor crosslink efficiency on the DPP scaffold. Based on this scaffold, multilayered bone marrow mesenchymal stem cell sheets were rapidly constructed, using RAD16-I peptide hydrogel as a temporary 3D scaffold, and cell sheets were cultured in either the 3D-dynamic system (DCcs) or the traditional static condition (SCcs). The multilayered structure, stem cell bioactivity, and ultrastructure of DCcs and SCcs were assessed. The DCcs exhibited lower apoptosis, lower differentiation, and an improved paracrine effect after a 48 h culture in vitro compared to the SCcs. Four groups were set to evaluate the cell sheet effect in rat MI model: sham group, MI control group, DCcs group, and SCcs group. The DCcs group improved cardiac function and decreased the infarcted area compared to the MI control group, while no significant improvements were observed in the SCcs group. Improved cell survival, angiogenesis, and Sca-1+ cell and c-kit+ cell amounts were observed in the DCcs group. In conclusion, the DCcs maintained higher stem cell bioactivity by using the 3D-dynamic system to provide sufficient nutrition, and transplanting DCcs significantly improved the cardiac function and angiogenesis. Statement of SignificanceThis study provides an efficient method to prepare vascular endothelial growth factor covalent decellularized pericardium scaffold with aspartic acid, and a multilayered bone marrow mesenchymal stem cell (BMSC) sheet is constructed on it using a 3D-dynamic system. The dynamic nutrition supply showed a significant benefit on BMSC bioactivity in vitro, including decreasing cell apoptosis, reducing stem cell differentiation, and improving growth factor secretion. These favorable bioactivity improved BMSC survival, angiogenesis, and cardiac function of the infarcted myocardium. The study highlights the importance of dynamic nutrition supply on maintaining stem cell bioactivity within cell sheet, and it stresses the necessity and significance of setting a standard for assessing cell sheet products before transplantation in the future application.

Curcumin-mediated bone marrow mesenchymal stem cell sheets create a favorable immune microenvironment for adult full-thickness cutaneous wound healing

BackgroundAdult full-thickness cutaneous wound repair suffers from an imbalanced immune response, leading to nonfunctional reconstructed tissue and fibrosis. Although various treatments have been reported, the immune-mediated tissue regeneration driven by biomaterial offers an attractive regenerative strategy for damaged tissue repair.MethodsIn this research, we investigated a specific bone marrow-derived mesenchymal stem cell (BMSC) sheet that was induced by the Traditional Chinese Medicine curcumin (CS-C) and its immunomodulatory effects on wound repair. Comparisons were made with the BMSC sheet induced without curcumin (CS-N) and control (saline).ResultsIn vitro cultured BMSC sheets (CS-C) showed that curcumin promoted the proliferation of BMSCs and modified the features of produced extracellular matrix (ECM) secreted by BMSCs, especially the contents of ECM structural proteins such as fibronectin (FN) and collagen I and III, as well as the ratio of collagen III/I. Two-photon fluorescence (TPF) and second-harmonic generation (SHG) imaging of mouse implantation revealed superior engraftment of BMSCs, maintained for 35 days in the CS-C group. Most importantly, CS-C created a favorable immune microenvironment. The chemokine stromal cell-derived factor 1 (SDF1) was abundantly produced by CS-C, thus facilitating a mass migration of leukocytes from which significantly increased expression of signature TH1 cells (interferon gamma) and M1 macrophages (tumor necrosis factor alpha) genes were confirmed at 7 days post-operation. The number of TH1 cells and associated pro-inflammatory M1 macrophages subsequently decreased sharply after 14 days post-operation, suggesting a rapid type I immune regression. Furthermore, the CS-C group showed an increased trend towards M2 macrophage polarization in the early phase. CS-C led to an epidermal thickness and collagen deposition that was closer to that of normal skin.ConclusionsCurcumin has a good regulatory effect on BMSCs and this promising CS-C biomaterial creates a pro-regenerative immune microenvironment for cutaneous wound healing.