Exploring peptide‐functionalized alginate scaffolds for engineering cardiac tissue from human embryonic stem cell‐derived cardiomyocytes in serum‐free medium

Engineering human cardiac tissue is a promising solution for myocardial repair of injured hearts and for drug screening. Herein, we examined the capability of chemically defined alginate scaffolds to promote cardiac tissue regeneration from human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs) in serum‐free, chemically defined medium. The cells were single seeded or coseeded with human dermal fibroblasts (HFs) in macroporous scaffolds made from pristine alginate or alginate modified with arginine‐glycine‐aspartate (RGD) peptide and heparin‐binding peptide (HBP). Our results show that the addition of fibroblasts to the 3‐D culture is indispensable for the formation of functional cardiac tissues and that the presence of RGD/HBP attached to the alginate matrix further improves its functionality. The engineered tissue displayed the typical fiber morphology with massive striation. An increase in contraction amplitude and calcium transients with time, together with a decrease in excitation threshold, indicated advancement toward tissue maturation. Our results thus point to the importance of co‐cultivating fibroblasts with hESCs‐CMs in chemically defined peptide‐functionalized alginate scaffolds and culture medium for regenerating functional cardiac tissue in vitro.     

A topographically optimized substrate with well-ordered lattice micropatterns for enhancing the osteogenic differentiation of murine mesenchymal stem cells

Although recently a growing number of reports demonstrate that topography or geometry of the substrate also plays an important role in the fate of the stem cells, most of these studies are usually completed by a few distinct patterns such as simple lines, posts, etc. As a result, there is a lack of quantitative analysis of the relationship between topographical variation and the differentiation of stem cells. Here, the effectiveness of topography variation is studied systematically in several microengineered substrates on osteogenic differentiation. It is found that the effectiveness of the osteogenic differentiation has a peak around 3 μm in the interval length of micropatterns.     

The influence of patterned nanofiber meshes on human mesenchymal stem cell osteogenesis

A specially designed electroconductive collector enables the electrospinning of P-NFM composed of areas of parallel/uniaxially aligned fibers and areas of random/orthogonal nanofiber distribution. The biological relevance of P-NFM is demonstrated using hBMSCs as an autologous cell source. The structures induce cell orientation along the uniaxially aligned fibers, mainly during earlier culturing periods under basal and osteogenic differentiation conditions. The microtopography of the P-NFM also controls the deposition of mineralized extracellular matrix along the pre-defined fiber direction. Genotypic characterization confirms the successful differentiation into the osteogenic lineage.     

Fabrication of graphene‐silver/polyurethane nanofibrous scaffolds for cardiac tissue engineering

The graphene‐based nanocomposites are considered as great candidates for enhancing electrical and mechanical properties of nonconductive scaffolds in cardiac tissue engineering. In this study, reduced graphene oxide‐silver (rGO‐Ag) nanocomposites (1 and 2 wt%) were synthesized and incorporated into polyurethane (PU) nanofibers via electrospinning technique. Next, the human cardiac progenitor cells (hCPCs) were seed on these scaffolds for in vitro studies. The rGO‐Ag nanocomposites were studied by X‐ray diffraction (XRD), Raman spectroscopy, and transmission electron microscope (TEM). After incorporation of rGO‐Ag into PU nanofibers, the related characterizations were carried out including scanning electron microscope (SEM), TEM, water contact angle, and mechanical properties. Furthermore, PU and PU/nanocomposites scaffolds were used for in vitro studies, wherein hCPCs showed good cytocompatibility via 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay and considerable attachment on the scaffold using SEM studies. Real‐time polymerase chain reaction (PCR) and immunostaining studies confirmed the upregulation of cardiac specific genes including GATA‐4, T‐box 18 (TBX 18), cardiac troponin T (cTnT), and alpha‐myosin heavy chain (α‐MHC) in the PU/rGO‐Ag scaffolds in comparison with neat PU ones. Therefore, these nanofibrous rGO‐Ag–reinforced PU scaffolds can be considered as suitable candidates in cardiac tissue engineering.     

Simvastatin inhibits sphingosylphosphorylcholine-induced differentiation of human mesenchymal stem cells into smooth muscle cells

Sphingosylphosphorylcholine (SPC) induces differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) into smooth muscle-like cells expressing α-smooth muscle actin (α-SMA) via transforming growth factor-β1/Smad2- and RhoA/Rho kinase-dependent mechanisms. 3-Hydroxy-3-methylglutaryl- coenzyme A reductase inhibitors (statins) have been known to have beneficial effects in the treatment of cardiovascular diseases. In the present study, we examined the effects of simvastatin on the SPC-induced α-SMA expression and Smad2 phosphorylation in hASCs. Simvastatin inhibited the SPC-induced α-SMA expression and sustained phosphorylation of Smad2 in hASCs. SPC treatment caused RhoA activation via a simvastatin-sensitive mechanism. The SPC-induced α-SMA expression and Smad2 phosphorylation were abrogated by pretreatment of the cells with the Rho kinase inhibitor Y27632 or overexpression of a dominant negative RhoA mutant. Furthermore, SPC induced secretion of TGF-β1 and pretreatment with either Y27632 or simvastatin inhibited the SPC-induced TGF-β1 secretion. These results suggest that simvastatin inhibits SPC-induced differentiation of hASCs into smooth muscle cells by attenuating the RhoA/Rho kinase-dependent activation of autocrine TGF-β1/Smad2 signaling pathway.     

组织工程治疗大鼠失神经肌萎缩的实验研究

探讨用骨髓基质细胞构建组织工程神经修复坐骨神经损伤的方法使失神经骨骼肌重获神经再支配的可行性.用80只Wistar大鼠随机分为4组,每组20只.除对照组(A组)外,其他组切断右侧坐骨神经5mm建立腓肠肌失神经实验模型,硅胶管桥接神经两断端.B组将BMSCs ECM凝胶(约1×106/mL)植入硅胶管内;C组硅胶管内植入同样稀释后的ECM凝胶;D组硅胶管内注满生理盐水.术后观察功能恢复及肌肉萎缩情况.14周进行电生理检查、再生轴突染色及肌肉形态学的检查.检测失神经腓肠肌是否重新获得再生轴突的再支配.结果表明:骨髓基质细胞组术后14周可检测到新生轴突,其再生的轴突与靶肌肉已经建立神经突触连接.肌肉萎缩情况及电生理指标明显优于术后其他各组.组织工程人工神经修复坐骨神经断伤能够使远端失神经骨骼肌重新获得神经再支配.     

Electrospun electroactive nanofibers of gelatin‐oligoaniline/Poly (vinyl alcohol) templates for architecting of cardiac tissue with on‐demand drug release

In this study, grafted gelatin with oligoaniline (GelOA) was synthesized and then mixed with Poly (vinyl alcohol) (PVA). Several scaffolds with different ratio of PVA/GelOA were electrospun to fabricate electroactive scaffolds. GelOA was characterized using Fourier‐transform infrared spectroscopy (FTIR); moreover, nanofiber properties were evaluated by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and scanning electron microscope (SEM) analyses. Nanofibers diameter was decreased with aniline oligomer increment form 300 to 150 nm because of the hydrophobic nature of the aniline oligomer. Aniline oligomer electroactivity was studied using cyclic voltammetry, which exhibited two redox peaks at 0.4 and 0.6. Moreover, aniline oligomer enhancement resulted in melting point increasing from 220°C to 230°C because of the crystallinity increment. To assess the biocompatibility of nanofibers, cell viability and cell adhesion were tracked using mesenchymal stem cell (MSCs). It was revealed that the presence of aniline oligomer leads to enhancing the conductivity, thermal properties and lowering the degradation rate and drug release. Among of different scaffolds, sample with high content of GelOA shows better behavior in physical and biological properties. Accumulative drug releases under applied electrical field at 40 minutes showed that the drug release for stimulated condition is about 33% more than the unapplied electrical field one.     

Enhancing endothelial differentiation of human mesenchymal stem cells by culture on a nanofibrous polycaprolactone/(poly-glycerol sebacate)/gelatin scaffold

Cardiovascular diseases have always been one of the main causes of death worldwide and eventually one of the major medical concerns. Tissue engineering is promising strategies of treating cardiovascular, which can be an effective approach with the design of appropriate scaffold. In this study, to develop engineering basement membrane for endothelial differentiation with good cell attachment, we produced polycaprolactone (PCL)/poly (glycerol sebacate) (PGS)/gelatin nanofibrous scaffold via electrospinning. Attenuated total reflectance-Fourier transform infrared and the proton nuclear magnetic resonance results confirmed the chemical structure of synthesized PGS. Scanning electron microscope images of the electrospun scaffold revealed that the nanofibers are smooth, continues and uniform. Moreover, due to the presence of hydrophilic functional groups in the scaffold, the contact angle is in the appropriate range for cell adhesion especially endothelial cells. The elastic modulus and ultimate tensile stress of electrospun scaffold were calculated 1.32 ± 0.27 MPa and 1.23 ± 0.18 MPa respectively. Quantitative polymerase chain reaction was performed for evaluation of endothelial differentiation of mesenchymal stem cells cultured on standard plate and fibrous scaffold under chemical stimulation with growth factor. Specific endothelial gene expression results postulated that our modified scaffold could support and significantly promote endothelial differentiation of MSCs.     

Polypyrrole thin films formed by admicellar polymerization support the osteogenic differentiation of mesenchymal stem cells

The objective of this study was to evaluate the attachment, proliferation, and differentiation of rat mesenchymal stem cells (MSC) toward the osteoblastic phenotype seeded on polypyrrole (PPy) thin films made by admicellar polymerization. Three different concentrations of pyrrole (Py) monomer (20, 35, and 50 x 10(-3) M) were used with the PPy films deposited on tissue culture polystyrene dishes (TCP). Regular TCP dishes and PPy polymerized on TCP by chemical polymerization without surfactant using 5 x 10(-3) M Py, were used as controls. Rat MSC were seeded on these surfaces and cultured for up to 20 d in osteogenic media. Surface topography was characterized by atomic force microscopy, X-ray photoelectron spectroscopy, and static contact angle. Cell attachment, proliferation, alkaline phosphatase (ALP) activity, and calcium content were measured to evaluate the ability of MSC to adhere and differentiate on PPy-coated TCP. Increased monomer concentrations resulted in PPy films of increased thickness and surface roughness. PPy films generated by different monomer concentrations induced drastically different cellular events. A wide spectrum of cell attachment characteristics (from excellent cell attachment to the complete inability to adhere) were obtained by varying the monomer concentration from 20 m to 50 x 10(-3) M. In particular the 20 x 10(-3) M PPy thin films demonstrated superior induction of MSC osteogenicity, which was comparable to standard TCP dishes, unlike PPy films of similar thickness prepared by chemical polymerization without surfactant. Adhesion of mesenchymal stem cells on tissue culture plates (TCP) coated with polypyrrole thin films made by admicellar polymerization.     

Microtopography based on inverse opal structures regulates the behavior of bone marrow‐derived mesenchymal stem cells

Physical cues from the extracellular microenvironment play an important role in regulating cell behavior, such as adhesion, migration, and differentiation. Many studies have shown that different physical parameters (eg, stiffness and topography) could modulate the in vitro differentiation of mesenchymal stem cells (MSCs), which had multilineage differentiation potential and could be easily isolated from various tissues such as bone marrow, adipose tissue, and the umbilical cord. However, the underlying mechanism of the topographical influence on MSCs and the detailed cell‐substrate interaction remain unclear. Here, we present oriented elliptical inverse opal structures for regulating the morphology and alignment of bone marrow‐derived MSCs. The inverse opal structures were made through a convenient bottom‐up approach of self‐assembly, which is facile and cost effective. MSCs cultured on the oriented structures were highly aligned and extended highly oriented thick lamellipodia. Moreover, the oriented substrates cracked along the lateral boundary of the cells, suggesting that a strong cell‐substrate interaction was induced by the response of MSCs to the oriented topography. These features of the oriented elliptical topography indicated their promising value in stem cell research and tissue engineering.     

Improved efficacy of bio‐mineralization of human mesenchymal stem cells on modified PLLA nanofibers coated with bioactive materials via enhanced expression of integrin α2β1

Current therapeutic interventions in bone defects are mainly focused on finding the best bioactive materials for inducing bone regeneration via activating the related intracellular signaling pathways. Integrins are trans‐membrane receptors that facilitate cell‐extracellular matrix (ECM) interactions and activate signal transduction. To develop a suitable platform for supporting human bone marrow mesenchymal stem cells (hBM‐MSCs) differentiation into bone tissue, electrospun poly L‐lactide (PLLA) nanofiber scaffolds were coated with nano‐hydroxyapatite (PLLA/nHa group), gelatin nanoparticles (PLLA/Gel group), and nHa/Gel nanoparticles (PLLA/nHa/Gel group) and their impacts on cell proliferation, expression of osteoblastic biomarkers, and bone differentiation were examined and compared. MTT data showed that proliferation of hBM‐MSCs on PLLA/nHa/Gel scaffolds was significantly higher than other groups (P < .05). Alkaline phosphatase activity was also more increased in hBM‐MSCs cultured under osteogenic media on PLLA/nHa/Gel scaffolds compared to others. Gene expression evaluation confirmed up‐regulation of integrin α2β1 as well as the osteogenic genes BGLAP, COL1A1, and RUNX2. Following use of integrin α2β1 blocker antibody, the protein level of integrin α2β1 in cells seeded on PLLA/nHa/Gel scaffolds was decreased compared to control, which confirmed that most of the integrin receptors were bound to gelatin molecules on scaffolds and could activate the integrin α2β1/ERK axis. Collectively, PLLA/nHa/Gel scaffold is a suitable platform for hBM‐MSCs adhesion, proliferation, and osteogenic differentiation in less time via activating integrin α2β1/ERK axis, and thus it might be applicable in bone tissue engineering.     

Fabrication of random and aligned electrospun nanofibers containing graphene oxide for skeletal muscle cells scaffold

Graphene oxide (GO)‐based materials have been explored in biomedical applications as active engineered materials for diagnosis and therapy. Although a large number of studies have been carried out in the last years, aspects involving the orientation and elongation of cells on GO immobilized on polymeric nanofibers are still scarce. We investigated the interactions between skeletal muscle cells and GO immobilized on random and aligned electrospun nanofibers of poly(caprolactone) (PCL), a biocompatible and biodegradable polymer. Oxygen plasma was employed to modify the nanofiber polymer surface to enhance the interactions between the PCL fibers and GO. Scanning electron microscopy and confocal microscopy revealed the morphology and orientation of skeletal muscle cells (C2C12 cells) on random and aligned GO/PCL nanofibers. The approach employed here is useful to investigate the interaction of skeletal muscle cells with biocompatible polymer nanofibers modified with GO intended for cell scaffolds and tissue engineering.     

The potential use of mesenchymal stem cells in hematopoietic stem cell transplantation

In the last 10 years, mesenchymal stem cells (MSCs) have emerged as a therapeutic approach to regenerative medicine, cancer, autoimmune diseases, and many more due to their potential to differentiate into various tissues, to repair damaged tissues and organs, and also for their immunomodulatory properties. Findings in vitro and in vivo have demonstrated immune regulatory function of MSCs and have facilitated their application in clinical trials, such as those of autoimmune diseases and chronic inflammatory diseases. There has been an increasing interest in the role of MSCs in allogeneic hematopoietic stem cell transplantation (HSCT), including hematopoietic stem cell engraftment and the prevention and treatment of graft-versus-host disease (GVHD), and their therapeutic potential has been reported in numerous clinical trials. Although the safety of clinical application of MSCs is established, further modifications to improve their efficacy are required. In this review, we summarize advances in the potential use of MSCs in HSCT. In addition, we discuss their use in clinical trials of the treatment of GVHD following HSCT, the immunomodulatory capacity of MSCs, and their regenerative and therapeutic potential in the field of HSCT.     

Dual‐micropillar‐based microfluidic platform for single embryonic stem cell‐derived neuronal differentiation

We developed the dual‐micropillar‐based microfluidic platform to direct embryonic stem (ES) cell fate. 4 × 4 dual‐micropillar‐based microfluidic platform consisted of 16 circular‐shaped outer micropillars and 8 saddle‐shaped inner micropillars in which single ES cells were cultured. We hypothesized that dual‐micropillar arrays would play an important role in controlling the shear stress and cell docking. Circular‐shaped outer micropillars minimized the shear stress, whereas saddle‐shaped inner micropillars allowed for docking of individual ES cells. We observed the effect of saddle‐shaped inner micropillars on cell docking in response to hydrodynamic resistance. We also demonstrated that ES cells cultured for 6 days within the dual‐micropillar‐based microfluidic platform differentiated into neural‐like cells. Therefore, this dual‐micropillar‐based microfluidic platform could be a potentially powerful method for screening of lineage commitments of single ES cells.     

The Development of Genipin‐Crosslinked Poly(caprolactone) (PCL)/Gelatin Nanofibers for Tissue Engineering Applications

Composite nanofibers of poly(caprolactone) (PCL) and gelatin crosslinked with genipin are prepared. The contact angles and mechanical properties of crosslinked PCL‐gelatin nanofibers decrease as the gelatin content increases. The proliferation of myoblasts is higher in the crosslinked PCL‐gelatin nanofibers than in the PCL nanofibers, and the formation of myotubes is only observed on the crosslinked PCL‐gelatin nanofibers. The expression level of myogenin, myosin heavy chain, and troponin T genes is increased as the gelatin content is increased. The results suggest that PCL‐gelatin nanofibers crosslinked with genipin can be used as a substrate to modulate proliferation and differentiation of myoblasts, presenting potential applications in muscle tissue engineering.

     

A novel silk/PES hybrid nanofibrous scaffold promotes the in vitro proliferation and differentiation of adipose‐derived mesenchymal stem cells into insulin producing cells

Using stem cells to replace the lost beta cells is a hopeful strategy in the treatment of diabetic patients. Furthermore, during stem cell culture and therapy, it is a need to use a substrate to act as a supportive matrix to mimic 3D in vivo microenvironment. Therefore, in this study, human adipose‐derived stem cells were used to differentiate into insulin‐producing cells (IPCs) on a silk/polyethersulfone (PES) scaffold. After exposing to the differentiation media, 2D and 3D (silk/PES) cultured cells were gradually aggregated and formed spherical shaped clusters. The viability of cells was comparable in both 3D and 2D culture. As the results of gene expression assay in both RNA and protein level showed, the differentiation efficiency was higher in 3D culture. Furthermore, ELISA revealed that the release of C‐peptide and insulin was higher in 3D than 2D culture. It seems that silk/PES nanofibrous hybrid scaffold could provide an appropriate matrix to mimic in vivo microenvironment and therefore increases the IPC differentiation potency of stem cells.     

多层面验证共培养微环境诱导法诱导骨髓间充质干细胞心肌样分化

为了多层面探讨共培养微环境诱导法定向诱导骨髓间充质干细胞(MSCs)心肌样分化的可行性,取第3代MSCs与原代心肌细胞(CMs)进行共培养。在显微镜下观察诱导1周后的MSCs形态学变化,用免疫荧光和实时荧光定量聚合酶链式反应(RT-PCR)分别检测诱导的MSCs中心肌肌钙蛋白I(cTnI)、α-肌动蛋白(α-actin)、Nkx-2.5和GATA-4的基因表达变化情况。采用超高效液相色谱-串联质谱(UPLC-MS/MS)分别检测诱导组和对照组的代谢产物。诱导1周后的MSCs形态呈心肌样改变,cTnI、α-actin、Nkx-2.5和GATA-4的基因表达均明显升高,正交偏最小二乘法判别分析(OPLS-DA)模型显示诱导的MSCs代谢物向CMs转变趋势明显。通过多元和单元统计分析筛选差异变量,根据一级质谱和二级质谱比对结果,最终确定12种差异代谢物。与未经诱导的MSCs相比,经诱导的MSCs与CMs中变化趋势相同的差异代谢物有7种,变化趋势不同的差异代谢物有5种。实验结果表明,无论从形态、基因、蛋白质还是代谢层面看,MSCs通过与CMs间接接触共培养后均发生了心肌样改变,但是与CMs仍存在差异。