Tenogenic differentiation of human MSCs induced by the topography of electrochemically aligned collagen threads

Topographical cues from the extracellular microenvironment can influence cellular activity including proliferation and differentiation. Information on the effects of material topography on tenogenic differentiation of human mesenchymal stem cells (human MSCs) is limited. A methodology using the principles of isoelectric focusing has previously been developed in our laboratory to synthesize electrochemically aligned collagen (ELAC) threads that mimics the packing density, alignment and strength of collagen dense connective tissues. In the current study, human MSCs were cultured on ELAC and randomly oriented collagen threads and the effect of collagen orientation on cell morphology, proliferation and tenogenic differentiation was investigated. The results indicate that higher rates of proliferation were observed on randomly oriented collagen threads compared to ELAC threads. On the other hand, tendon specific markers such as scleraxis and tenomodulin, were significantly increased on ELAC threads compared to randomly oriented collagen threads. Additionally, osteocalcin, a specific marker of bone differentiation was suppressed on ELAC threads. Previous studies have reported that BMP-12 is a key growth factor to induce tenogenic differentiation of MSCs. To evaluate the synergistic effect of BMP-12 and collagen orientation, human MSCs were cultured on ELAC threads in culture medium supplemented with and without BMP-12. The results revealed that BMP-12 did not have an additional effect on the tenogenic differentiation of human MSCs on ELAC threads. Together, these results suggest that ELAC induces tenogenic differentiation of human MSCs by presenting an aligned and dense collagen substrate, akin to the tendon itself. In conclusion, ELAC has a significant potential to be used as a tendon replacement and in the development of an osteotendinous construct towards the regeneration of bone-tendon interfaces.     

Electrospinning of nano/micro scale poly(L-lactic acid) aligned fibers and their potential in neural tissue engineering

Efficacy of aligned poly(l-lactic acid) (PLLA) nano/micro fibrous scaffolds for neural tissue engineering is described and their performance with random PLLA scaffolds is compared as well in this study. Perfectly aligned PLLA fibrous scaffolds were fabricated by an electrospinning technique under optimum condition and the diameter of the electrospun fibers can easily be tailored by adjusting the concentration of polymer solution. As the structure of PLLA scaffold was intended for neural tissue engineering, its suitability was evaluated in vitro using neural stem cells (NSCs) as a model cell line. Cell morphology, differentiation and neurite outgrowth were studied by various microscopic techniques. The results show that the direction of NSC elongation and its neurite outgrowth is parallel to the direction of PLLA fibers for aligned scaffolds. No significant changes were observed on the cell orientation with respect to the fiber diameters. However, the rate of NSC differentiation was higher for PLLA nanofibers than that of micro fibers and it was independent of the fiber alignment. Based on the experimental results, the aligned nanofibrous PLLA scaffold could be used as a potential cell carrier in neural tissue engineering.     

Morphology,proliferation, alignment,and new collagen synthesis of mesenchymal stem cells on a microgrooved collagen membrane

The topographic cues of the extracellular matrix may have significant effects upon cellular behavior, such as adhesion, spreading, migration, proliferation, differentiation, and in particular, morphology and orientation. In this study, we examined the effects of microgrooved collagen membrane (MCM) on mesenchymal stem cell (MSC) behavior. The MCM (9 μm in periodicity, and 1–2 μm in depth) was fabricated on an untreated (nonpolar) and smooth polystyrene substrate, based on the absorption and self-assembly properties of collagen on the polystyrene substrate. Methyl thiazolyl tetrazolium assay revealed that cell proliferation on the MCM was enhanced compared with the smooth collagen membrane at day 2. Qualitative observation of MSC behavior using confocal laser scanning microscopy and scanning electron microscopy showed that MSCs grew with a highly elongated morphology and were aligned strictly along the direction of the microgrooves. Additionally, scanning electron microscopy revealed the oriented cells produced a collagenous matrix on the MCM that had a preferential orientation, whereas the collagenous matrix produced by randomly oriented MSCs on the smooth collagen membrane was disorganized. Future studies should investigate the fabrication of oriented topographical substrates, based on the natural biomaterial collagen, to guide cell alignment and oriented growth along definite directions. These substrates may help produce aligned collagenous matrices that could have good potential for the production of tissue substitutes.     

Surface topography enhances differentiation of mesenchymal stem cells towards osteogenic and adipogenic lineages

Surface topography impacts on cell growth and differentiation, but it is not trivial to generate defined surface structures and to assess the relevance of specific topographic parameters. In this study, we have systematically compared in vitro differentiation of mesenchymal stem cells (MSCs) on a variety of groove/ridge structures. Micro- and nano-patterns were generated in polyimide using reactive ion etching or multi beam laser interference, respectively. These structures affected cell spreading and orientation of human MSCs, which was also reflected in focal adhesions morphology and size. Time-lapse demonstrated directed migration parallel to the nano-patterns. Overall, surface patterns clearly enhanced differentiation of MSCs towards specific lineages: 15 μm ridges increased adipogenic differentiation whereas 2 μm ridges enhanced osteogenic differentiation. Notably, nano-patterns with a periodicity of 650 nm increased differentiation towards both osteogenic and adipogenic lineages. However, in absence of differentiation media surface structures did neither induce differentiation, nor lineage-specific gene expression changes. Furthermore, nanostructures did not affect the YAP/TAZ complex, which is activated by substrate stiffness. Our results provide further insight into how structuring of tailored biomaterials and implant interfaces – e.g. by multi beam laser interference in sub-micrometer scale – do not induce differentiation of MSCs per se, but support their directed differentiation.     

Shape-memory-actuated change in scaffold fiber alignment directs stem cell morphology

Tissue engineering scaffolds have traditionally been static physical structures poorly suited to mimicking the complex dynamic behavior of in vivo microenvironments. Here we present a thermoresponsive scaffold that can be programmed to change macroscopic shape and microscopic architecture during cell culture. The scaffold, which was prepared by electrospinning a shape memory polymer (SMP), was used to test the hypothesis that a shape-memory-actuated change in scaffold fiber alignment could be used to control the behavior of attached and viable cells. To test this hypothesis, we stretched an SMP scaffold of randomly oriented fibers and fixed the scaffold in a temporary but stable elongated shape in which fibers were aligned by the strain. Following seeding and culture of human adipose-derived stem cells on the strain-aligned scaffold, the scaffold was triggered to transition back to its initial shape and random fiber orientation via shape memory actuation using a cytocompatible temperature increase. We found that cells preferentially aligned along the fiber direction of the strain-aligned scaffold before shape memory actuation. After shape memory actuation, cells remained attached and viable but lost preferential alignment. These results demonstrate that shape-memory-actuated changes in scaffold fiber alignment can be achieved with attached and viable cells and can control cell morphological behavior. The incorporation of shape memory into cytocompatible scaffolds is anticipated to facilitate the development, delivery and functionality of tissue engineering scaffolds and the in vitro and in vivo study and application of mechanobiology.     

Tissue-to-cellular level deformation coupling in cell micro-integrated elastomeric scaffolds

In engineered tissues we are challenged to reproduce extracellular matrix and cellular deformation coupling that occurs within native tissues, which is a meso-micro scale phenomenon that profoundly affects tissue growth and remodeling. With our ability to electrospin polymer fiber scaffolds while simultaneously electrospraying viable cells, we are provided with a unique platform to investigate cellular deformations within a three dimensional elastomeric fibrous scaffold. Scaffold specimens micro-integrated with vascular smooth muscle cells were subjected to controlled biaxial stretch with 3D cellular deformations and local fiber microarchitecture simultaneously quantified. We demonstrated that the local fiber geometry followed an affine behavior, so that it could be predicted by macro-scaffold deformations. However, local cellular deformations depended non-linearly on changes in fiber microarchitecture and ceased at large strains where the scaffold fibers completely straightened. Thus, local scaffold microstructural changes induced by macro-level applied strain dominated cellular deformations, so that monotonic increases in scaffold strain do not necessitate similar levels of cellular deformation. This result has fundamental implications when attempting to elucidate the events of de-novo tissue development and remodeling in engineered tissues, which are thought to depend substantially on cellular deformations.     

Aligned neurite outgrowth and directed cell migration in self-assembled monodomain gels

Regeneration of neural tissues will require regrowth of axons lost due to trauma or degeneration to reestablish neuronal connectivity. One approach toward this goal is to provide directional cues to neurons that can promote and guide neurite growth. Our laboratory previously reported the formation of aligned monodomain gels of peptide amphiphile (PA) nanofibers over macroscopic length scales. In this work, we modified these aligned scaffolds specifically to support neural cell growth and function. This was achieved by displaying extracellular matrix (ECM) derived bioactive peptide epitopes on the surface of aligned nanofibers of the monodomain gel. Presentation of IKVAV or RGDS epitopes enhanced the growth of neurites from neurons encapsulated in the scaffold, while the alignment guided these neurites along the direction of the nanofibers. After two weeks of culture in the scaffold, neurons displayed spontaneous electrical activity and established synaptic connections. Scaffolds encapsulating neural progenitor cells were formed in situ within the spinal cord and resulted in the growth of oriented processes in vivo. Moreover, dorsal root ganglion (DRG) cells demonstrated extensive migration inside the scaffold, with the direction of their movement guided by fiber orientation. The bioactive and macroscopically aligned scaffold investigated here and similar variants can potentially be tailored for use in neural tissue regeneration.     

Novel biodegradable three-dimensional macroporous scaffold using aligned electrospun nanofibrous yarns for bone tissue engineering

This study aimed to develop a practical three-dimensional (3D) macroporous scaffold from aligned electrospun nanofibrous yarns for bone tissue engineering. A novel 3D unwoven macroporous nanofibrous (MNF) scaffold was manufactured with electrospun poly(L-lactic acid) and polycaprolactone (w/w 9:1) nanofibers through sequential yarns manufacture and honeycombing process at 65°C. The efficacy of 3D MNF scaffold for bone formation were evaluated using human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) differentiation model and rabbit tibia bone defect model. In vitro, more cell proliferation and cell ingrowth were observed in 3D MNF scaffold. Moreover, calcium deposit was obviously detected in vitro differentiation of hESC-MSCs. In vivo, histology and X-ray showed that 3D MNF scaffold treated bone defect had fine 3D bony tissue formation around the scaffold as well as inside the scaffold at 3 weeks and 6 weeks. This study demonstrated that 3D MNF scaffold provides a structural support for hESC-MSCs growth and guides bone formation suggesting that this novel strategy successfully makes use of electrospun fibers for bone tissue engineering, which may help realize the clinical translation of electrospun nanofibers for regenerative medicine in future.     

Bone augmentation by bone marrow mesenchymal stem cells cultured in three-dimensional biodegradable polymer scaffolds

Poly-lactic-glycolic acid (PLGA) is a biocompatible as well as biodegradable polymer and used in various medical applications. In this study, we evaluated efficiency of the specially designed three-dimensional porous PLGA as a scaffold for bone augmentation. First, cell attachment/proliferation, differentiation, and mineralization of Fisher 344 rat marrow mesenchymal stem cells (MSCs) cultured on the PLGA scaffold were analyzed. Viable MSCs were impregnated into pore areas of the scaffold and a moderate increase of DNA contents was seen. High alkaline phosphatase, osteocalcin content, and calcium content of MSCs in PLGA scaffolds under osteogenic differentiation conditions were seen after 14 or 21 days of culture. Subsequently, we implanted the PLGA/MSCs composites on rat calvaria bone for 30 days. Newly formed bone was seen in only the composite PLGA/MSCs implantation group, which had been precultured under osteogenic condition. We also demonstrated that the newly formed bone originated from the donor composites. These results demonstrate that the three-dimensional PLGA scaffold can support osteogenic differentiation of MSCs, and the scaffold combined with osteogenic MSCs can be used for in vivo bone tissue augmentation.     

胶原-壳聚糖支架材料与间充质干细胞的组织相容性

背景：近年来一些研究发现胶原蛋白-壳聚糖复合支架材料可作为神经组织工程的支架材料,但相关细胞相容性研究较少。 目的：观察兔骨髓间充质干细胞在胶原蛋白-壳聚糖复合支架材料表面生长及分化情况。 方法：分离培养兔骨髓间充质干细胞,无血清培养液培养,流式细胞仪检查细胞表型;然后,将其接种到凝胶支架材料表面(实验组)及多聚赖氨酸包被的盖玻片表面(对照组),神经诱导培养基内培养,倒置相差显微镜观察干细胞的生长及分化情况。 结果与结论：细胞表型为CD29⁺、CD44⁺、CD166⁺。倒置相差显微镜观察：实验组中,接种的骨髓间充质干细胞生长良好,7 d后可见有突起神经细胞,细胞生长情况与对照组未见有明显差别。证实胶原蛋白-壳聚糖复合支架材料对骨髓间充质干细胞有良好细胞相容性。     

Finite element study of scaffold architecture design and culture conditions for tissue engineering

Tissue engineering scaffolds provide temporary mechanical support for tissue regeneration and transfer global mechanical load to mechanical stimuli to cells through its architecture. In this study the interactions between scaffold pore morphology, mechanical stimuli developed at the cell microscopic level, and culture conditions applied at the macroscopic scale are studied on two regular scaffold structures. Gyroid and hexagonal scaffolds of 55% and 70% porosity were modeled in a finite element analysis and were submitted to an inlet fluid flow or compressive strain. A mechanoregulation theory based on scaffold shear strain and fluid shear stress was applied for determining the influence of each structures on the mechanical stimuli on initial conditions. Results indicate that the distribution of shear stress induced by fluid perfusion is very dependent on pore distribution within the scaffold. Gyroid architectures provide a better accessibility of the fluid than hexagonal structures. Based on the mechanoregulation theory, the differentiation process in these structures was more sensitive to inlet fluid flow than axial strain of the scaffold. This study provides a computational approach to determine the mechanical stimuli at the cellular level when cells are cultured in a bioreactor and to relate mechanical stimuli with cell differentiation.     

The growth and differentiation of mesenchymal stem and progenitor cells cultured on aligned collagen matrices

Cell-matrix interactions are paramount for the successful repair and regeneration of damaged and diseased tissue. Since many tissues have an anisotropic architecture, it has been proposed that aligned extracellular matrix (ECM) structures in particular could guide and support the differentiation of resident mesenchymal stem and progenitor cells (MSCs). We therefore created aligned collagen type I structures using a microfluidic set-up with the aim to assess their impact on MSC growth and differentiation. In addition, we refined our aligned collagen matrices by incorporating the glycosaminoglycan (GAG) heparin to demonstrate the versatility of the applied methodology to study multiple ECM components in a single system. Our reconstituted, aligned ECM structures maintained and allowed multilineage (osteogenic/adipogenic/chondrogenic) differentiation of MSCs. Most noticeable was the observation that during osteogenesis, aligned collagen substrates choreographed ordered matrix mineralization. Likewise, myotube assembly of C2C12 cells was profoundly influenced by aligned topographic features resulting in enhanced myotube organization and length. Our results shed light on the regulation of MSCs through directional ECM structures and demonstrate the versatility of these cell culture platforms for guiding the morphogenesis of tissue types with anisotropic structures.     

Osteogenic differentiation of rabbit mesenchymal stem cells in thermo-reversible hydrogel constructs containing hydroxyapatite and bone morphogenic protein-2 (BMP-2)

The aim of this study was to assess the efficacy of ectopic bone formation in a three-dimensional hybrid scaffold in combination with hydroxyapatite (HA) and poly(NiPAAm-co-AAc) as an injectable vehicle in the form of a supporting matrix for the osteogenic differentiation of rabbit mesenchymal stem cells (MSCs). Osteogenic differentiation of MSCs in the hybrid scaffold was greatly influenced by the addition of growth factors. When the osteoinduction activity of hybrid scaffold was studied following implantation into the back subcutis of nude mouse in terms of histological and biochemical examinations, significantly homogeneous bone formation was histologically observed throughout the hybrid scaffolds containing growth factor (BMP-2: bone morphogenic protein-2). The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high for the perfusion group compared with those in static culture group. We conclude that combination of MSC-seeded hybrid scaffold containing BMP-2 was a promising method by which to enhance in vitro osteogenic differentiation of MSC and in vivo ectopic bone formation.     

Porous gelatin-chondroitin-hyaluronate tri-copolymer scaffold containing microspheres loaded with TGF-beta1 induces differentiation of mesenchymal stem cells in vivo for enhancing cartilage repair

The aim of the study was to produce a novel porous gelatin-chondroitin-hyaluronate scaffold in combination with a controlled release of transforming growth factor beta1 (TGF-beta1), which induced the differentiation of mesenchymal stem cells (MSCs) in vivo for enhancing cartilage repair. Gelatin microspheres loaded with TGF-beta1 (MS-TGFbeta1) showed a fast release at the initial phase (37.4%), and the ultimate accumulated release was 83.1% by day 18. The autologous MSCs seeded on MS-TGFbeta1/scaffold were implanted to repair full-thickness cartilage defects in rabbits as in vivo differentiation repair group, while MSCs differentiated in vitro were seeded on scaffold without MS-TGFbeta1 to repair the contra lateral cartilage defects (n = 30). Fifteen additional rabbits without treatment for defects were used as control. Histology observation showed that the in vivo differentiation repair group had better chondrocyte morphology, integration, continuous subchondral bone, and much thicker newly formed cartilage layer when compared to in vitro differentiation repair group 12 and 24 weeks, postoperatively. There was a significant difference in histological grading score between these two experimental groups, and both showed much better repair than that of the control. The present study implied that the novel scaffold with MS-TGFbeta1 might serve as a new way to induce the differentiation of MSCs in vivo to enhance the cartilage repair.     

Well-aligned chitosan-based ultrafine fibers committed teno-lineage differentiation of human induced pluripotent stem cells for Achilles tendon regeneration

Physical property of substrates such as stiffness and topography have been reported to induce mesenchymal stem cells differentiation into bone, muscle and neuron lineages. Human-induced pluripotent stem cells (hiPSCs) are a highly promising cell source for regenerative medicine. However, physical properties have not yet been reported to successfully induce pluripotent stem cells into specific lineages. This study aimed to develop a robust, stepwise topographic strategy to induce hiPSCs differentiate into teno-lineage. A novel spinning approach termed stable jet electrospinning (SJES), is utilized to fabricate continuous well-aligned ultrafine fibers (891 ± 71 nm), which mimic the native tendon's microstructure and mechanical properties. hiPSCs are first differentiated into MSCs on smooth plastic surface as confirmed by the differentiations into three mesenchymal lineages and expression of characteristic MSC surface markers through an EMT (Epithelial–Mesenchymal Transition) process. Subsequently, the hiPSC derived MSCs are seeded onto well-aligned fibers to differentiate into tenocyte-like cells through activating mechanic-signal pathway. The in situ tendon repair study further confirms that aligned fiber scaffold with hiPSC-MSCs had significant effect on improving the structural and mechanical properties of tendon injury repair. These findings indicate that the stepwise physical substrate change strategy can be adopted to induce hiPSCs differentiation for tendon tissue regeneration.     

Aligned hybrid silk scaffold for enhanced differentiation of mesenchymal stem cells into ligament fibroblasts

The concept of contact guidance utilizes the phenomenon of anchorage dependence of cells on the topography of seeded surfaces. It has been shown in previous studies that cells were guided to align along the topographical alignment of the seeding substrate and produced enhanced amounts of oriented extracellular matrix (ECM). In this study, we aimed to apply this concept to a three-dimensional full silk fibroin (SF) hybrid scaffold system, which comprised of knitted SF and aligned SF electrospun fibers (SFEFs), for ligament tissue engineering applications. Specifically, knitted SF, which contributed to the mechanical robustness of the system, was integrated with highly aligned SFEF mesh, which acted as the initial ECM to provide environmental cues for positive cellular response. Mesenchymal stem cells seeded on the aligned hybrid scaffolds were shown to be proliferative and aligned along the integrated aligned SFEF, forming oriented spindle-shaped morphology and produced an aligned ECM network. Expression and production of ligament-related proteins were also increased as compared to hybrid SF scaffolds with randomly arranged SFEFs, indicating differentiative cues for ligament fibroblasts present in the aligned hybrid SF scaffolds. Consequently, the tensile properties of cultured aligned constructs were significantly improved and superior to the counterpart with randomly arranged SFEF. These results thus show that the aligned hybrid scaffold system is promising for enhancing cell proliferation, differentiation, and function for ligament tissue engineering applications.     

Enhanced differentiation of mesenchymal stem cells co-cultured with ligament fibroblasts on gelatin/silk fibroin hybrid scaffold

The differentiation of mesenchymal stem cells (MSCs) towards fibroblasts is a crucial issue in ligament tissue engineering. This study aims to investigate the feasibility of using co-culture system to induce the differentiation of MSCs for constructing the tissue-engineered ligament in vitro. A kind of silk cable-reinforced gelatin/silk fibroin hybrid scaffold was used to provide three-dimensional (3-D) culture environments for MSCs. The 3-D co-culture system was set up by culturing MSCs/scaffold and ligament fibroblasts in the transwell insert and lower chamber, respectively. The regulatory effects of fibroblasts on MSCs were determined. After 2 weeks of co-culture the MSCs showed faster proliferation and higher DNA content compared with MSCs non-co-cultured. The MSCs were distributed uniformly throughout the scaffold and showed good viability. The collagen production also increased significantly with culture time. The MSCs in co-culture system were proved to differentiate into ligament fibroblasts by expressing ligament extra-cellular matrix (ECM)-specific genes including collagen I, collagen III, and tenascin-C in mRNA and protein level. The immunohistochemistry staining also confirmed the synthesis of key ligament ECM components. This study reveals that specific regulatory signals released from fibroblasts in 3-D co-culture system can enhance the differentiation of MSCs for ligament tissue engineering.     

Engineering cell matrix interactions in assembled polyelectrolyte fiber hydrogels for mesenchymal stem cell chondrogenesis

Cell–cell and cell–matrix interactions are important events in directing stem cell chondrogenesis, which can be promoted in matrix microenvironments presenting appropriate ligands. In this study, interfacial polyelectrolyte complexation (IPC) based hydrogels were employed, wherein the unique formation of submicron size fibers facilitated spatial orientation of ligands within such hydrogels. The influence of aligned, collagen type I (Col I) presentation in IPC hydrogel on chondrogenic differentiation of human mesenchymal stem cells (MSC) was investigated. Early morphological dynamics, onset of N-cadherin/β-catenin mediated chondrogenic induction and differentiation were compared between MSCs encapsulated in IPC-Col I and IPC-control (without Col I) hydrogels, and a conventional Col I hydrogel. MSCs in IPC-Col I hydrogel aligned and packed uniformly, resulting in enhanced cell–cell interactions and cellular condensation, facilitating superior chondrogenesis and the generation of mature hyaline neocartilage, with notable downregulation of fibrocartilaginous marker. Inhibition study using function blocking β1-integrin antibodies reversed the aforementioned outcomes, indicating the importance of coupling integrin mediated cell–matrix interactions and N-cadherin/β-catenin mediated downstream signaling events. This study demonstrated the significance of oriented ligand presentation for MSC chondrogenesis, and the importance of facilitating an orderly sequence of differentiation events, initiated by proximal interactions between MSCs and the extracellular matrix for robust neocartilage formation.     

Ectopic cartilage formation induced by mesenchymal stem cells on porous gelatin-chondroitin-hyaluronate scaffold containing microspheres loaded with TGF-beta1

The study aimed to produce a novel porous gelatin-chondroitin-hyaluronate scaffold in combination with a controlled release of TGF- beta1 and to evaluate its potentials in ectopic cartilage formation. The gelatin-chondroitin-hyaluronate scaffold was developed to mimic the natural extra cellular matrix of cartilage. Gelatin microspheres loaded with TGF- beta1 (MS-TGF beta1) showed a fast cytokine release at initial phase (37.4%) and the ultimate accumulated release was 83.1% by day 18. Then MS-TGF beta1 were incorporated into scaffold. The MSCs seeded on scaffold with or without MS-TGF beta1 were incubated in vitro or implanted subcutaneously in nude mice. In vitro study showed that, compared to the scaffold, the scaffold/MS-TGF beta1 significantly augmented the proliferation of MSCs and GAG synthesis. Three weeks postoperatively histology observation showed that in MSCs/scaffold/MS-TGF beta1 implantation group, cells of newly formed ectopic cartilage were located within typical lacunae and demonstrated morphological characteristics of chondrocytes. Six weeks later the ectopic cartilage grew more and islands of cartilage were observed. The matrix was extensively metachromatic by safranin-O/Fast green staining. Immunohistochemical staining also indicated ectopic cartilage was intensely stained for type II collagen. Instead, in the MSCs/scaffold implantation group, no cartilage-like tissue formed and matrix showed negative or weak positive staining. The percentage of positive staining area was significantly larger in MSCs/scaffold/MS-TGF beta1 group (p<0.05) at each time point. The results indicated that the novel gelatin-chondroitin-hyaluronate scaffold with MS-TGF beta1 could induce the chondral differentiation of MSCs to form cartilage and might serve as a new way to repair cartilage defects.