Mesenchymal Stem Cell Sheets Research Articles

Rapid harvesting of stem cell sheets by thermoresponsive bulk poly(N-isopropylacrylamide) (PNIPAAm) nanotopography.

To date, cell sheet engineering-based technologies have actualized diverse scaffold-free bio-products to revitalize unintentionally damaged tissues/organs, including cardiomyopathy, corneal defects, and periodontal damage. Although substantial interest is now centered on the practical utilization of these bio-products for patients, the long harvest period of stem cells- or other primary cell-sheets has become a huge hurdle. Here, we dramatically reduce the total harvest period of a cell sheet (from cell layer formation to cell sheet detachment) composed of human bone marrow mesenchymal stem cells (hBMSCs) down to 2 d with the help of bulk thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) substrate nanotopography, which is not achievable via the previous grafting methods using PNIPAAm. We directly replicated an isotropic 400 nm-nanopore-array pattern on a bulk PNIPAAm substrate through UV polymerization of highly concentrated NIPAAm monomers, which was achieved using a remarkably increased Young's modulus of bulk PNIPAAm that was 1500 times higher than conventional PNIPAAm. The rapid harvesting of the hBMSC sheet on the bulk PNIPAAm substrate nanotopography was not only based on the accelerated formation and maturation of the hBMSC layer, but also the easy detachment of the hBMSC sheet induced by the abrupt change in the surface roughness of the substrate below the lower critical solution temperature (LCST) owing to the enlarged surface area of the substrate. Our findings may contribute to reverse presumptions about the limitations regarding the grafting methods for the cell sheet harvest and could broaden the practical utilization of cell sheets for patients in the near future.

Phenotypic traits of mesenchymal stem cell sheets fabricated by temperature-responsive cell culture plate: structural characteristics of MSC sheets

BackgroundIn most stem cell therapy strategies reported to date, stem cells are introduced to damaged tissue sites to repair and regenerate the original tissue structure and function. MSC therapeutic efficacies are inconsistent, largely attributed to transplanted MSC difficulties both in engrafting at tissue sites and in retaining their therapeutic functions from suspension formulations. MSC functional components, including cell adhesion and cell–cell junction proteins, and ECM that contribute to essential cellular therapeutic effects, are damaged or removed by proteolytic enzymes used in stem cell harvesting strategies from culture. To overcome these limitations, methods to harvest and transplant cells without disrupting critical stem cell functions are required. Cell sheet technology, exploiting temperature-responsive cell culture surfaces, permits cell harvest without cell protein damage. This study is focused on phenotypic traits of MSC sheets structurally and functionally to understand therapeutic benefits of cell sheets.Methods/resultsThis study verified cleaved cellular proteins (vinculin, fibronectin, laminin, integrin β-1, and connexin 43) and increased apoptotic cell death produced under standard trypsin harvesting treatment in a time-dependent manner. However, MSC sheets produced without trypsin using only temperature-controlled sheet harvest from culture plastic exhibited intact cellular structures. Also, MSCs harvested using enzymatic treatment (i.e., chemical disruption) showed higher pYAP expression compared to MSC sheets.ConclusionRetention of cellular structures such as ECM, cell–cell junctions, and cell–ECM junctions is correlated with human umbilical cord mesenchymal stem cell (hUC-MSC) survival after detachment from cell culture surfaces. Retaining these proteins intact in MSC cultures using cell sheet technology is proposed to enhance stem cell survival and their function in stem cell-based therapy.

The Effect of Mesenchymal Stem Cell Sheets on Early Healing of the Achilles Tendon in Rats.

Bone marrow-derived mesenchymal stem cells (BMSCs) have potential to accelerate flexor tendon healing and allow for earlier rehabilitation. The ideal BMSC construct and delivery method to the repair site remains unknown. We investigated the efficacy of interposed scaffold-free BMSC sheets on early Achilles tendon healing in rats. BMSCs were isolated from the femora and tibias of male Sprague-Dawley rats aged 8-12 weeks and BMSC sheets were produced on temperature-responsive culture dishes. Ninety-five male Sprague-Dawley rats aged 8-12 weeks were utilized. A bilateral Achilles tendon repair model was created. One side was randomly selected, and the tendon was repaired with the interposed BMSC sheet (BMSC group). The other side was repaired without BMSCs (control group). The bilateral tendons were harvested at 5, 6, 7, 10, and 14 days postoperatively for biomechanical analysis, measurement of the gene expression level of tendon markers scleraxis and tenomodulin by real-time polymerase chain reaction, and histological evaluation. The BMSC group had significantly higher maximum load to failure and stiffness at 5 and 6 days compared with the control group. Moreover, the BMSC group showed significantly increased gene expression of scleraxis and/or tenomodulin at all timepoints. The cross sectional areas in the BMSC group were significantly larger at 5, 6, and 14 days. However, hematoxylin and eosin staining of the central part of the repair site revealed no significant differences at all timepoints. These results suggest that the increased biomechanical strength afforded by BMSC sheet implantation into tendon repair sites may allow for the earlier onset of rehabilitation and improved clinical outcomes in flexor tendon surgery. Impact Statement We investigated the efficacy of interposed bone marrow-derived mesenchymal stem cell (BMSC) sheets on early Achilles tendon healing in rats. The tendons repaired with BMSC sheets revealed significantly increased mechanical strength compared with the control repairs (without the BMSC sheet) at 5 and 6 days. These data reveal that BMSC sheet implantation into tendon repair sites may allow for earlier onset of rehabilitation and improved clinical outcomes in flexor tendon surgery.

Transplantation of adipose-derived mesenchymal stem cell sheets directly into the kidney suppresses the progression of renal injury in a diabetic nephropathy rat model.

Aims/IntroductionAdipose‐derived mesenchymal stem cell (ASC) transplantation is a promising therapy for diabetic nephropathy (DN). However, intravascular administration of ASCs is associated with low engraftment in target organs. Therefore, we considered applying the cell sheet technology to ASCs. In this study, ASC sheets were directly transplanted into the kidneys of a DN rat model, and therapeutic consequences were analyzed.Materials and MethodsAdipose‐derived mesenchymal stem cells were isolated from adipose tissues of 7‐week‐old enhanced green fluorescent protein rats, and ASC sheets were prepared using a temperature‐responsive culture dish. A DN rat model was established from 5‐week‐old Spontaneously Diabetic Torii fatty rats. Seven‐week‐old DN rats (n = 21) were assigned to one of the following groups: sham‐operated (n = 6); ASC suspension (6.0 × 106 cells/mL) administered intravenously (n = 7); six ASC sheets transplanted directly into the kidney (n = 8). The therapeutic effect of the cell sheets was determined based on urinary biomarker expression and histological analyses.ResultsThe ASC sheets survived under the kidney capsule of the DN rat model for 14 days after transplantation. Furthermore, albuminuria and urinary tumor necrosis factor‐α levels were significantly lower in the ASC sheets transplanted directly into the kidney group than in the sham‐operated and ASC suspension administered intravenously groups (P < 0.05). Histologically, the ASC sheets transplanted directly into the kidney group presented mild atrophy of the proximal tubule and maintained the renal tubular structure.ConclusionsTransplantation of ASC sheets directly into the kidney improved transplantation efficiency and suppressed renal injury progression. Therefore, the ASC sheet technology might be a promising novel treatment for DN.

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.

Characteristic differences of cell sheets composed of mesenchymal stem cells with different tissue origins.

IntroductionStem cell therapy with mesenchymal stem cells (MSCs) has been widely used in many clinical trials, and therapy with MSC sheets shows promise for patients. However, there are few reports characterizing MSC sheets. In the present study, the properties of MSC sheets derived from bone marrow, adipose tissue, and umbilical cord were evaluated.MethodsCell sheets were fabricated with MSCs from different tissue origins in temperature-responsive cell culture dishes with and without pre-coating of fetal bovine serum (FBS). MSC adhesion behavior in the culture dish was observed. Secretion of cytokines related to cell proliferation and immune regulation from MSC sheets was investigated by ELISA. The adhesion properties of the MSC sheets were investigated by time-lapse microscopy.ResultsDifferent cell adhesion and proliferation rates in temperature-responsive cell culture dishes were observed among the three types of MSCs. FBS pre-coating of the dishes enhanced cell attachment and proliferation in all cell types. Harvested cell sheets showed high attachment capacity to tissue culture polystyrene dish surfaces.ConclusionsMSC sheets can be fabricated from MSCs from different tissue origins using temperature-responsive cell culture dishes. The fabricated MSC sheets could be useful in cell transplantation therapies by choosing appropriate types of MSCs that secrete therapeutic cytokines for the targeted diseases.

Reversal of established liver fibrosis by IC-2-engineered mesenchymal stem cell sheets

Chronic hepatitis viral infection, alcoholic intoxication, and obesity cause liver fibrosis, which progresses to decompensated liver cirrhosis, a disease for which medical demands cannot be met. Since there are currently no approved anti-fibrotic therapies for established liver fibrosis, the development of novel modalities is required to improve patient prognosis. In this study, we clarified the anti-fibrotic effects of cell sheets produced from human bone marrow-derived mesenchymal stem cells (MSCs) incubated on a temperature-sensitive culture dish with the chemical compound IC-2. Orthotopic transplantation of IC-2-engineered MSC sheets (IC-2 sheets) remarkably reduced liver fibrosis induced by chronic CCl4 administration. Further, the marked production of fibrolytic enzymes such as matrix metalloproteinase (MMP)-1 and MMP-14, as well as thioredoxin, which suppresses hepatic stellate cell activation, was observed in IC-2 sheets. Moreover, the anti-fibrotic effect of IC-2 sheets was much better than that of MSC sheets. Finally, knockdown experiments revealed that MMP-14 was primarily responsible for the reduction of liver fibrosis. Here, we show that IC-2 sheets could be a promising therapeutic option for established liver fibrosis.

Allogeneic adipose-derived mesenchymal stem cell sheet that produces neurological improvement with angiogenesis and neurogenesis in a rat stroke model.

Stem cell therapy is a promising strategy for the treatment of severe cerebral ischemia. However, targeting sufficient grafted cells to the affected area remains challenging. Choosing an adequate transplantation method for the CNS appears crucial for this therapy to become a clinical reality. The authors used a scaffold-free cell sheet as a translational intervention. This method involves the use of cell sheet layers and allows the transplantation of a large number of cells, locally and noninvasively. The authors evaluated the effectiveness of allogeneic adipose tissue-derived mesenchymal stem cell sheets in a rat model of stroke. The animals, subjected to middle cerebral artery occlusion, were randomly divided in two groups: one in which a cell sheet was transplanted and the other in which a vehicle was used (n = 10/group). Over a period of 14 days after transplantation, the animals' behavior was evaluated, after which brain tissue samples were removed and fixed, and the extent of angiogenesis and infarct areas was evaluated histologically. Compared to the vehicle group, in the cell sheet group functional angiogenesis and neurogenesis were significantly increased, which resulted in behavioral improvement. Transplanted cells were identified within newly formed perivascular walls as pericytes, a proportion of which were functional. Newly formed blood vessels were found within the cell sheet that had anastomosed to the cerebral blood vessels in the host. The transplantation approach described here is expected to provide not only a paracrine effect but also a direct cell effect resulting in cell replacement that protects the damaged neurovascular unit. The behavioral improvement seen with this transplantation approach provides the basis for further research on cell sheet-based regenerative treatment as a translational treatment for patients with stroke.

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.

Effective stacking and transplantation of stem cell sheets using exogenous ROS-producing film for accelerated wound healing

Extensive skin loss caused by burns or diabetic ulcers may lead to major disability or even death. Therefore, cell-based therapies that enhance skin regeneration are clinically needed. Previous approaches have been applied the injections of cell suspensions and the implantation of biodegradable three-dimensional scaffolds seeded cells. However, these treatments have limits due to poor localization of the injected cells and insufficient delivery of oxygen and nutrients to cells. Recently, cell sheet-based tissue engineering has been developed to transplant cell sheets, which are cell-dense tissues without scaffolds. Because cell density is one of the important factors for improving the therapeutic effect of cell transplantation, transplanting layered cell sheet constructs can promote the recovery of tissue function and tissue regeneration compared with a single cell sheet. Thus, this study designed ROS-induced cell sheet stacking method with newly fabricated hematoporphyrin-incorporated polyketone film (Hp-PK film) to enhance cell sheet delivery efficiency and application in wound healing. We have demonstrated the therapeutic effect of a multi-layered mesenchymal stem cell sheets onto a full-thickness wound defect in nude mice. Consequentially, three-layered cell sheets transplanted and stacked by ROS-induced method promoted angiogenesis and skin regeneration at the wound site. Thus, our strategy based on Hp-PK film, which allows for easy stacking and transplantation of cell sheets, could be applied to enhance tissue regeneration. Statement of SignificanceWe herein report exogenous ROS-induced cell sheet stacking method with newly fabricated hematoporphyrin-incorporated polyketone film (Hp-PK film) to enhance cell sheet transplantation efficiency and application in wound healing. Although there are several ways to stack-up cell sheets, all of these methods have limitations in transplanting the cell sheet directly to the target site. The method is simple and takes a relatively short time compared to previously reported methods for stacking and transplanting cell sheets. Thus, our study will provide a scientific impact because the method of applying exogenous ROS generated from Hp-PK film on cell detachment can transplant the cell sheet through a process of putting a cell sheet-cultured film on the lesion, irradiating with light, and then removing only the film.

Integration of mesenchymal stem cell sheet and bFGF-loaded fibrin gel in knitted PLGA scaffolds favorable for tendon repair.

Tendon injuries are common and require a long time to heal, and are particularly associated with some adverse problems such as adhesion and rupture. Herein, we aim to develop new bioactive scaffolds endowed with stem cell sheets and growth factors to enable cell migration and proliferation favorable for tendon regeneration in situ. An exogenous basic fibroblast growth factor (bFGF)-loaded fibrin gel was firstly incorporated into the porous network of knitted poly(lactide-co-glycolide) (PLGA) scaffolds and then sheets of mesenchymal stem cells (MSCs) were also integrated into the scaffolds. It was shown that the pores in the knitted PLGA scaffold were readily filled with a complex network of fibrin fiber gel and the fibrin fibers were beneficial for the controlled release of bFGF over a long time period. After transplantation in a critical-size Achilles tendon defect model (7 mm) in the rat right hindlimb, gross observation revealed no immunologic incompatibility or rejection derived from the scaffold systems. It was observed that the MSC sheets contributed directly to tendon regeneration, and exerted an environment-modifying effect on the injuries in situ, consistent with the beneficial effect of bFGF. It was interesting that the knitted PLGA-fibrin gel scaffolds loaded with MSC sheets and bFGF showed the highest expression of tendon-related gene markers and outstanding repair efficacy, including appreciable biomechanical strength and native-like histological microstructures. Therefore, the integration of MSC sheets and bFGF into PLGA/bFGF-fibrin gel scaffolds may stimulate the proliferation and tenogenic differentiation of MSCs in situ and synergistically enhance the injured tendon reconstruction.

Engineered tendon-fibrocartilage-bone composite and bone marrow-derived mesenchymal stem cell sheet augmentation promotes rotator cuff healing in a non-weight-bearing canine model

Reducing rotator cuff failure after repair remains a challenge due to suboptimal tendon-to-bone healing. In this study we report a novel biomaterial with engineered tendon-fibrocartilage-bone composite (TFBC) and bone marrow-derived mesenchymal stem cell sheet (BMSCS); this construct was tested for augmentation of rotator cuff repair using a canine non-weight-bearing (NWB) model. A total of 42 mixed-breed dogs were randomly allocated to 3 groups (n = 14 each). Unilateral infraspinatus tendon underwent suture repair only (control); augmentation with engineered TFBC alone (TFBC), or augmentation with engineered TFBC and BMSCS (TFBC + BMSCS). Histomorphometric analysis and biomechanical testing were performed at 6 weeks after surgery. The TFBC + BMSCS augmented repairs demonstrated superior histological scores, greater new fibrocartilage formation and collagen fiber organization at the tendon-bone interface compared with the controls. The ultimate failure load and ultimate stress were 286.80 ± 45.02 N and 4.50 ± 1.11 MPa for TFBC + BMSCS group, 163.20 ± 61.21 N and 2.60 ± 0.97 MPa for control group (TFBC + BMSCS vs control, P = 1.12E-04 and 0.003, respectively), 206.10 ± 60.99 N and 3.20 ± 1.31 MPa for TFBC group (TFBC + BMSCS vs TFBC, P = 0.009 and 0.045, respectively). In conclusion, application of an engineered TFBC and BMSCS can enhance rotator cuff healing in terms of anatomic structure, collagen organization and biomechanical strength in a canine NWB model. Combined TFBC and BMSCS augmentation is a promising strategy for rotator cuff tears and has a high potential impact on clinical practice.

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System.

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

HEART RATE VARIABILITY AND BLOOD PRESSURE IN PARKINSON'S DISEASE PATIENTS UNDER ANALOGUE MICROGRAVITY

Patients with compromised bone conditions still suffer the problem of deficient osseointegration during dental implant treatment. Developing mesenchymal stem cell (MSC) sheet functionalized titanium implant with proper inductive cue to promote osteogenesis and angiogenesis coupling shall be a good solution. In the present study, the antimiR-138 delivered MSC sheet is used to functionalize the Ti implant. The cell sheet can well integrate with the Ti implant to form the MSC sheet-implant complex (MSIC). The antimiR-138 delivered MSIC shows greatly improved osteogenesis and angiogenesis coupling both in vitro and in vivo. In vitro, the antimiR-138 delivered MSIC significantly promotes the expression of endogenous osteogenesis and angiogenesis related genes and proteins, alkaline phosphatase activity, extracellular matrix mineralization and collagen secretion compared to the antimiR-control and the nothing delivered control. The in vivo ectopic implantation assay uncovers the robust vascularized bone formation of the antimiR-138 delivered MSIC. The antimiR-138 delivered MSIC with promoted osteogenesis and angiogenesis coupling is anticipated to lead to rigid osseointegration in the compromised bone conditions.

Mesenchymal Stem Cell Sheets Exert Antistenotic Effects in a Rat Arterial Injury Model

Restenosis after catheter or surgical intervention substantially affects the prognosis of arterial occlusive disease. Mesenchymal stem cells (MSCs) may have antistenotic effects on injured arteries. MSC transplantation from the adventitial side of an artery is safer than endovascular transplantation but has not been extensively examined. In this study, a rat model of femoral artery injury was used to compare the antistenotic effects of transplanted cell sheets and transplanted cell suspensions. Rat adipose-derived stem cells (ASCs) were used as the source of MSCs. For both cell sheets and suspensions, 6 × 106 MSCs were transplanted on the day of arterial injury. MSC sheets attenuated neointimal hyperplasia more than MSC suspensions (intima-to-media ratio in hematoxylin/eosin-stained sections: 0.55 ± 0.13 vs. 1.14 ± 0.12; p < 0.05). Cell engraftment (assessed by immunohistochemistry or bioluminescence imaging of luciferase-expressing cells), arterial re-endothelialization (evaluated by immunohistochemical staining for rat endothelial cell antigen-1), and restriction of vascular smooth muscle cell proliferation in the neointima (double-staining of alpha-smooth muscle actin and phospho-histone H3) were greater when MSC sheets were applied than when MSC suspensions were used. In conclusion, MSC sheets exhibited better antistenotic and cell engraftment properties than MSC suspensions. MSC sheet transplantation from the adventitial side is a promising therapy for prevention of arterial restenosis.

Novel engineered tendon-fibrocartilage-bone composite with cyclic tension for rotator cuff repair.

Surgical repair of rotator cuff tears presents a significant clinical challenge with high failure rates and inferior functional outcomes. Graft augmentation improves repair outcomes; however, currently available grafting materials have limitations. Although cell-seeded decellularized tendon slices may facilitate cell infiltration, promote tendon incorporation, and preserve original mechanical strength, the unique fibrocartilage zone is yet to be successfully reestablished. In this study, we investigated the biological and mechanical properties of an engineered tendon-fibrocartilage-bone composite (TFBC) with cyclic tension (3% strain; 0.2Hz). Decellularized TFBCs seeded with bone marrow-derived mesenchymal stem cell (BMSCs) sheets and subjected to mechanical stimulation for up to 7days were characterised by histology, immunohistochemistry, scanning electron microscopy, mechanical testing, and transcriptional regulation. The decellularized TFBC maintained native enthesis structure and properties. Mechanically stimulated TFBC-BMSC constructs displayed increased cell migration after 7days of culture compared with static groups. The seeded cell sheet not only integrated well with tendon scaffold but also distributed homogeneously and aligned to the direction of stretch under dynamic culture. Developmental genes were regulated including scleraxis, which was significantly upregulated with mechanical stimulation. The Young's modulus of the cell-seeded constructs was significantly higher compared with the noncell-seeded controls. In conclusion, the results of this study reveal that the TFBC-BMSC composite provides an ideal multilayer construct for cell seeding and growth, with mechanical preconditioning further enhances cell penetration and differentiation. The BMSC cell sheet revitalised TFBC in conjunction with mechanical stimulation could serve as a novel and primed biological patch to improve rotator cuff repair.

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.