Marrow Mesenchymal Stem Cell Sheets Research Articles

Light-Controlled BMSC Sheet-Implant Complexes with Improved Osteogenesis via an LRP5/β-Catenin/Runx2 Regulatory Loop.

The combination of bone marrow mesenchymal stem cell (BMSC) sheets and titanium implants (BMSC sheet-implant complexes) can accelerate osseointegration. However, methods of fabricating BMSC sheet-implant complexes are quite limited, and the survival of BMSC sheet-implant complexes is one of the key barriers. Here, we show that a light-controlled fabricating system can generate less injured BMSC sheet-implant complexes with improved viability and osteogenesis and that noninvasive monitoring of the viability of BMSC sheet-implant complexes using a lentiviral delivery system is feasible. Enhanced green fluorescent protein- and luciferase-expressing BMSC sheets were used to track the viability of BMSC sheet-implant complexes in vivo. The experiments of micro-computed tomography analysis and hard tissue slices were performed to evaluate the osteogenic ability of BMSC sheet-implant complexes in vivo. The results showed that BMSC sheet-implant complexes survived for almost 1 month after implantation. Notably, BMSC sheet-implant complexes fabricated by the light-controlled fabricating system had upregulating expression levels of low-density lipoprotein-receptor-related protein 5 (LRP5), β-catenin, and runt-related transcription factor 2 (Runx2) compared to the complexes fabricated by mechanical scraping. Furthermore, we found that Runx2 directly bound to the rat LRP5 promoter and the LRP5/β-catenin/Runx2 regulatory loop contributed to the enhancement of the osseointegrating potentials. In this study, we successfully fabricated BMSC sheet-implant complexes with improved viability and osteogenesis and established a feasible, noninvasive, and continuous method for tracking BMSC sheet-implant complexes in vivo. Our findings lay the foundation for the application of BMSC sheet-implant complexes in vivo and open new avenues for engineered BMSC sheet-implant complexes.

Laminin-521 Promotes Rat Bone Marrow Mesenchymal Stem Cell Sheet Formation on Light-Induced Cell Sheet Technology.

Rat bone marrow mesenchymal stem cell sheets (rBMSC sheets) are attractive for cell-based tissue engineering. However, methods of culturing rBMSC sheets are critically limited. In order to obtain intact rBMSC sheets, a light-induced cell sheet method was used in this study. TiO2 nanodot films were coated with (TL) or without (TN) laminin-521. We investigated the effects of laminin-521 on rBMSCs during cell sheet culturing. The fabricated rBMSC sheets were subsequently assessed to study cell sheet viability, reattachment ability, cell sheet thickness, collagen type I deposition, and multilineage potential. The results showed that laminin-521 could promote the formation of rBMSC sheets with good viability under hyperconfluent conditions. Cell sheet thickness increased from an initial 26.7 ± 1.5 μm (day 5) up to 47.7 ± 3.0 μm (day 10). Moreover, rBMSC sheets maintained their potential of osteogenic, adipogenic, and chondrogenic differentiation. This study provides a new strategy to obtain rBMSC sheets using light-induced cell sheet technology.

Improved osseointegrating functionality of cell sheets on anatase TiO2 nanoparticle surfaces

Bone marrow mesenchymal stem cell sheets (BMSC sheets) have been reported as a powerful tool for bioengineering applications in accelerating osseointegration.

Improving the osteogenesis of human bone marrow mesenchymal stem cell sheets by microRNA-21-loaded chitosan/hyaluronic acid nanoparticles via reverse transfection.

Cell sheet engineering has emerged as a novel approach to effectively deliver seeding cells for tissue regeneration, and developing human bone marrow mesenchymal stem cell (hBMMSC) sheets with high osteogenic ability is a constant requirement from clinics for faster and higher-quality bone formation. In this work, we fabricated biocompatible and safe chitosan (CS)/hyaluronic acid (HA) nanoparticles (NPs) to deliver microRNA-21 (miR-21), which has been proved to accelerate osteogenesis in hBMMSCs; then, the CS/HA/miR-21 NPs were cross-linked onto the surfaces of culture plates with 0.2% gel solution to fabricate miR-21-functionalized culture plates for reverse transfection. hBMMSC sheets were induced continuously for 14 days using a vitamin C-rich method on the miR-21-functionalized culture plates. For the characterization of CS/HA/miR-21 NPs, the particle size, zeta potential, surface morphology, and gel retardation were sequentially investigated. Then, the biological effects of hBMMSC sheets on the miR-21-functionalized culture plates were evaluated. The assay results demonstrated that the hBMMSC sheets could be successfully induced via the novel reverse transfection approach, and miR-21 delivery significantly enhanced the in vitro osteogenic differentiation of hBMMSC sheets in terms of upregulating calcification-related gene expression and enhancing alkaline phosphatase production, collagen secretion, and mineralized nodule formation. The enhanced osteogenic activity of hBMMSC sheets might promisingly lead to more rapid and more robust bone regeneration for clinical use.

Effects of cartilage-derived morphogenetic protein 1 (CDMP1) transgenic mesenchymal stem cell sheets in repairing rabbit cartilage defects.

The aim of this study was to investigate the abilities of cartilage-derived morphogenetic protein 1 (CDMP1) transgenic cell sheets in repairing rabbit cartilage defects. Rabbit CDMP1 transgenic bone marrow mesenchymal stem cell (BMSC) sheets (CDMP1-BMSCs) were cultured on temperature-sensitive culture dishes, and CDMP1 expression and type II collagen protein in the cell sheets were detected. Tissue-engineered cell sheets were constructed and transplanted into defect rabbit thyroid cartilage, to investigate the expression of engineered cartilage collagen protein and proteoglycan (GAG). The experiment was divided into three groups; A) BMSC sheet, B) Ad-CMV-eGFP-transfected cell sheet, and C) Ad-CMV-hCDMP1-IRES-eGFP-transfected cell sheet. The expression of CDMP1 was detected in the transgenic cell sheets. The engineered cartilage exhibited positive immunohistochemical and Alcian blue staining. The expression levels of type II collagen protein and GAG in group A were positive, whereas those in group B and group C were negative (P < 0.05). The CDMP1-BMSC sheets had a good cartilage differentiation activity, and could effectively repair rabbit laryngeal cartilage defects.

Influence of nanotopography on periodontal ligament stem cell functions and cell sheet based periodontal regeneration.

Periodontal regeneration is an important part of regenerative medicine, with great clinical significance; however, the effects of nanotopography on the functions of periodontal ligament (PDL) stem cells (PDLSCs) and on PDLSC sheet based periodontal regeneration have never been explored. Titania nanotubes (NTs) layered on titanium (Ti) provide a good platform to study this. In the current study, the influence of NTs of different tube size on the functions of PDLSCs was observed. Afterward, an ectopic implantation model using a Ti/cell sheets/hydroxyapatite (HA) complex was applied to study the effect of the NTs on cell sheet based periodontal regeneration. The NTs were able to enhance the initial PDLSC adhesion and spread, as well as collagen secretion. With the Ti/cell sheets/HA complex model, it was demonstrated that the PDLSC sheets were capable of regenerating the PDL tissue, when combined with bone marrow mesenchymal stem cell (BMSC) sheets and HA, without the need for extra soluble chemical cues. Simultaneously, the NTs improved the periodontal regeneration result of the ectopically implanted Ti/cell sheets/HA complex, giving rise to functionally aligned collagen fiber bundles. Specifically, much denser collagen fibers, with abundant blood vessels as well as cementum-like tissue on the Ti surface, which well-resembled the structure of natural PDL, were observed in the NT5 and NT10 sample groups. Our study provides the first evidence that the nanotopographical cues obviously influence the functions of PDLSCs and improve the PDLSC sheet based periodontal regeneration size dependently, which provides new insight to the periodontal regeneration. The Ti/cell sheets/HA complex may constitute a good model to predict the effect of biomaterials on periodontal regeneration.

A novel experimental study on the fabrication and biological characteristics of canine bone marrow mesenchymal stem cells sheet using vitamin C.

The aim of this study was to fabricate canine bone marrow mesenchymal stem cell sheet through the use of vitamin C, to identify the biological characteristics of the resulting cell sheets, and to reveal the potential mechanism of cell-sheet promotion by vitamin C. This study used vitamin C to induce bone marrow mesenchymal stem cells to proliferate. The resulting cells secreted large amounts of collagen, thereby shortening the construction time of the cell-sheet layer. In addition to these aims, we identified biological microcharacteristics of the cell sheet through histological observation, transmission electron microscopy, real-time PCR detection, immunohistochemical detection, and osteogenesis-induction experiments on the cell sheet. We were able to stably and rapidly construct bone marrow mesenchymal stem cell sheet, effectively harvest it, and transfer the seed cells for tissue engineering. This study indicates that the use of vitamin C for harvesting mesenchymal stem cell sheets from bone marrow may provide an easy and practical approach for bone tissue regeneration.

Repairing Larynx Cartilage Defects with Prefabricated PLAG Scaffold Compounded with BMSCs Cell Sheets Transfected by GDF5

Cell sheet technology is a new skill for cell harvesting and transferring in medicine tissue engineering,which makes the cultured cells separate from the dish surface to form a layer of complete lamellar structure containing extracellular matrix. It has circumvent these limitations,such as insufficient biological activity,low cell-attachment efficiency,which being in traditional tissue engineering. So a compound which was constituted with mesenchymal stem cell sheet transfected with GDF5 by adenovirus vector and PLGA scaffold prefabricated with GDF5 gene-transferred BMSCs was structured,explore the clinical application and make a preliminary assessment on the repairing effect. Isolated and cultured rabbit bone marrow mesenchymal stem cells in vitro,tansfected the passage 4 cells with exogenous recombinant growth differentiation factor 5 by adenovirus vector. Harvested the transgenic bone marrow mesenchymal stem cell sheet through temperature-responsive culture dish after culturing 7 ~ 14 days. Using real time PCR to detect the express of GDF5 in transgenic cell sheet. Preparing a prefabricated PLAG scaffold through injecting GDF5 gene-transferred BMSCs to PLGA scaffold,then co-culture the compound constituted with the cell sheet and prefabricated PLGA scaffold for 2 days. Transplant the compound to rabbit larynx cartilage defects and discuss the repair capacity after 4 or 8 weeks at the gross level as well as at the histological level. The experiment divided into three groups:(A)the compound constituted with the transgenic cell sheet and prefabricated PLGA scaffold;(B)prefabricated PLGA scaffold;(C) PLGA scaffold loaded BMSCs. The Results showed the whole transgenic cell sheet have been harvested successfully,real time PCR can detect the express of GDF5 in transgenosis cell sheets. The results of immunohistochemistry and Alcian Blue staining showed the expression of collagen II and GAG in group A and group B,furthermore,there was more collagen II and GAG in group A than that in group B with statistical difference( P 0. 05). Thus,the compound constituted with the transgenosis BMSCs cell sheet and PLGA scaffold prefabricated with transgenosis BMSCs has the more superior chondrogenic capacity than traditional PLGA scaffold loaded with cells,it can promote the repair of cartilage effectively.

The effect of the coumarin-like derivative osthole on the osteogenic properties of human periodontal ligament and jaw bone marrow mesenchymal stem cell sheets

Cell sheet engineering is a scaffold-free delivery concept that has been shown to improve mesenchymal stem cell-mediated regeneration of injured or pathologically damaged periodontal tissues in preclinical studies and several clinical trials. However, the best strategy for cell sheet production remains to be identified. The aim of this study was to investigate the biological effects of osthole, a coumarin-like derivative extracted from Chinese herbs, on the cell sheet formation and osteogenic properties of human periodontal ligament stem cells (PDLSCs) and jaw bone marrow mesenchymal stem cells (JBMMSCs). Patient-matched PDLSCs and JBMMSCs were isolated, and an appropriate concentration of osthole for cell culture was screened for both cell types in terms of cell proliferation and alkaline phosphatase (ALP) activity. Next, the best mode of osthole stimulation for inducing the formation of sheets by each cell type was selected by evaluating the amount of their extracellular matrix (ECM) protein production as well as osteogenic-related gene expression. Furthermore, both PDLSC and JBMMSC sheets obtained from each optimized technique were transplanted subcutaneously into nude mice to evaluate their capacity for ectopic bone regeneration. The results revealed that 10−5 m/L osthole significantly enhanced the proliferation of both PDLSCs and JBMMSCs (P < 0.05), although for JBMMSCs, there was no concentration-related change among the four established osthole groups (P > 0.05). In addition, 10−5 m/L osthole was the best concentration to promote the ALP activities of both cells (P < 0.01). Based on both the production of ECM proteins (collagen type I, integrin β1, and fibronectin) and the expression of osteogenic genes (ALP, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)), the provision of 10−5 m/L osthole throughout the entire culture stage (10 days) for PDLSCs or at the early stage (first 3 days) for JBMMSCs was the most effective osthole administration mode for cell sheet formation (P < 0.05). The results of in vivo transplantation showed that osthole-mediated PDLSC and JBMMSC sheets formed more new bone than those obtained without osthole intervention (P < 0.001). Our data suggest that a suitable concentration and mode of osthole stimulation may enhance ECM production and positively affect cell behavior in cell sheet engineering.

Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity

Cell sheet engineering has been developed as an alternative approach to improve mesenchymal stem cell-mediated tissue regeneration. In this study, we found that vitamin C (Vc) was capable of inducing telomerase activity in periodontal ligament stem cells (PDLSCs), leading to the up-regulated expression of extracellular matrix type I collagen, fibronectin, and integrin β1, stem cell markers Oct4, Sox2, and Nanog as well as osteogenic markers RUNX2, ALP, OCN. Under Vc treatment, PDLSCs can form cell sheet structures because of increased cell matrix production. Interestingly, PDLSC sheets demonstrated a significant improvement in tissue regeneration compared with untreated control dissociated PDLSCs and offered an effective treatment for periodontal defects in a swine model. In addition, bone marrow mesenchymal stem cell sheets and umbilical cord mesenchymal stem cell sheets were also well constructed using this method. The development of Vc-mediated mesenchymal stem cell sheets may provide an easy and practical approach for cell-based tissue regeneration.