Mechanical Cues Regulating Proangiogenic Potential of Human Mesenchymal Stem Cells through YAP‐Mediated Mechanosensing

Stem cells secrete trophic factors that induce angiogenesis. These soluble factors are promising candidates for stem cell–based therapies, especially for cardiovascular diseases. Mechanical stimuli and biophysical factors presented in the stem cell microenvironment play important roles in guiding their behaviors. However, the complex interplay and precise role of these cues in directing pro‐angiogenic signaling remain unclear. Here, a platform is designed using gelatin methacryloyl hydrogels with tunable rigidity and a dynamic mechanical compression bioreactor to evaluate the influence of matrix rigidity and mechanical stimuli on the secretion of pro‐angiogenic factors from human mesenchymal stem cells (hMSCs). Cells cultured in matrices mimicking mechanical elasticity of bone tissues in vivo show elevated secretion of vascular endothelial growth factor (VEGF), one of representative signaling proteins promoting angiogenesis, as well as increased vascularization of human umbilical vein endothelial cells (HUVECs) with a supplement of conditioned media from hMSCs cultured across different conditions. When hMSCs are cultured in matrices stimulated with a range of cyclic compressions, increased VEGF secretion is observed with increasing mechanical strains, which is also in line with the enhanced tubulogenesis of HUVECs. Moreover, it is demonstrated that matrix stiffness and cyclic compression modulate secretion of pro‐angiogenic molecules from hMSCs through yes‐associated protein activity.     

A 3D biodegradable protein based matrix for cartilage tissue engineering and stem cell differentiation to cartilage

A protein based 3D porous scaffold is fabricated by blending gelatin and albumin. The biomimetic biodegradable gelatin, promoted good cell adhesion and its hydrophilic nature enabled absorption of culture media. Albumin is proposed to serve as a nontoxic foaming agent and also helped to attain a hydrophobic-hydrophilic balance. The hydrophobic-hydrophilic balance and appropriate crosslinking of the scaffold avoided extensive swelling, as well as retained the stability of scaffold in culture medium for long period. The scaffold is found to be highly porous with open interconnected pores. The adequate swelling and mechanical property of the scaffold helped to withstand the loads imparted by the cells during in vitro culture. The scaffold served as a nontoxic material to monolayer of fibroblast cells and is found to be cell compatible. The suitability of scaffold for chondrocyte culture and stem cell differentiation to chondrocytes is further explored in this work. The scaffold provided appropriate environment for chondrocyte culture, resulting in deposition of cartilage specific matrix molecules that completely masked the pores of the porous scaffold. The scaffold promoted the proliferation and differentiation of mesenchymal stem cells to chondrocytes in presence of growth factors. The transforming growth factor, TGFbeta3 promoted better chondrogenic differentiation than its isoform TGFbeta1 in this scaffold.     

Emulsion electrospun nanofibers as substrates for cardiomyogenic differentiation of mesenchymal stem cells

The potential of cardiomyogenic differentiation of human mesenchymal stem cells (hMSCs) on emulsion electrospun scaffold containing poly(l-lactic acid)-co-poly-(ε-caprolactone), gelatin and vascular endothelial growth factor (PLCL/GV) was investigated in this study. The characterizations of the scaffold were carried out using scanning electron microscope (SEM), transmission electron microscope, water contact angle and porometer. The proliferation of hMSCs showed that 73.4 % higher cell proliferation on PLCL/GV scaffolds than that on PLCL scaffold after 20 days of cell culture. Results of 5-chloromethylfluorescein diacetate staining and SEM morphology analysis indicated that hMSCs differentiated on PLCL/GV scaffolds showed irregular morphology of cardiomyocyte phenotype compared to the typical long and thin hMSC phenotype. Immunostaining results showed the expression of alpha actinin and myosin heavy chain. Our studies identified emulsion electrospinning as a method for fabrication of core–shell fibers suitable for the differentiation of stem cells to cardiac cells, with potential application in cardiac regeneration.     

含光诱导片段定向滑动多肽水凝胶诱导胚胎干细胞的外胚层分化（英文）

由细胞内行为或外源力引起的干细胞龛中存在的机械信号对干细胞的自我恢复和分化等基本功能具有重要影响.然而,关于具有分子机械运动产生的内在机械信号的人工细胞外基质鲜有报道.在此,我们报道了含光诱导片段定向滑动的机械动态水凝胶的合成及其作为人工细胞外基质在调节胚胎干细胞(ESC)分化中的功能.通过引入光笼蔽的半胱氨酸残基调控亲疏水交替多肽的自组装制备机械动态水凝胶.光笼蔽多肽组装体在光照射下转化为热力学非平衡的非笼蔽多肽双分子层,其进一步发生热力学有利的疏水性塌陷转变诱导的片段定向滑动.在机械动态水凝胶上培养鼠胚胎干细胞,该片段定向滑动诱导干细胞向外胚层谱系定向分化.进一步揭示了机械动态水凝胶促进机械转导蛋白YAP进入细胞核,表明其用于ESCs定向分化的潜在机械转导机制.细胞定向分化结果表明了机械动态水凝胶作为潜在的生物材料,有望用于疾病治疗和组织再生.     

Coupling of gelatin to inner surfaces of pore walls in spongy alginate-based scaffolds facilitates the adhesion,growth and differentiation of human bone marrow mesenchymal stromal cells

We have developed a novel wide-pore scaffold for cell 3D culturing, based on the technology of freeze-drying of Ca-alginate and gelatin. Two different preparation methodologies were compared: (i) freeze-drying of Na-alginate + gelatin mixed solution followed by the incubation of dried polymer in saturated ethanolic solution of CaCl₂; (ii) freeze-drying of the Na-alginate solution followed by the chemical “activation” of polysaccharide core with divinylsulfone with subsequent gelatin covalent attachment to the inner surfaces of pore walls. The scaffolds produced using the first approach did not provide adhesion and proliferation of human bone marrow mesenchymal stromal cells (MSCs). Conversely, the second approach allowed to obtain scaffolds with a high adherence ability for the cells. When cultured within the latter type of scaffold, MSCs proliferated and were able to differentiate into adipogenic, osteogenic and chondrogenic cell lineages, in response to specific induction stimuli. The results indicate that Ca-alginate wide-pore scaffolds with covalently attached gelatin could be useful for stem cell-based bone, cartilage and adipose tissue engineering.     

Stiff gelatin hydrogels can be photo-chemically synthesized from low viscous gelatin solutions using molecularly functionalized gelatin with a high degree of methacrylation

Gelatin is a very promising matrix material for in vitro cell culture and tissue engineering, e.g. due to its native RGD content. For the generation of medical soft tissue implants chemical modification of gelatin improves the mechanical properties of gelatin hydrogels and the viscous behavior of gelatin solutions for liquid handling. We present a systematic study on the influence of high degrees of methacrylation on the properties of gelatin solutions and photo-chemically crosslinked hydrogels. Changes from shear thinning to shear thickening behavior of gelatin solutions were observed depending on mass fraction and degree of methacrylation. Degrees of swelling of crosslinked hydrogels ranged from 194 to 770?% and storage moduli G′ from 368 to 5?kPa, comparable to various natural tissues including several types of cartilage. Crosslinked gels proofed to be cytocompatible according to extract testings based on DIN ISO 10933-5 and in contact with porcine chondrocytes.     

Mechanical properties and in vitro behavior of nanofiber-hydrogel composites for tissue engineering applications

Hydrogel-based biomaterial systems have great potential for tissue reconstruction by serving as temporary scaffolds and cell delivery vehicles for tissue engineering (TE). Hydrogels have poor mechanical properties and their rapid degradation limits the development and application of hydrogels in TE. In this study, nanofiber reinforced composite hydrogels were fabricated by incorporating electrospun poly(ε-caprolactone) (PCL)/gelatin 'blend' or 'coaxial' nanofibers into gelatin hydrogels. The morphological, mechanical, swelling and biodegradation properties of the nanocomposite hydrogels were evaluated and the results indicated that the moduli and compressive strengths of the nanofiber reinforced hydrogels were remarkably higher than those of pure gelatin hydrogels. By increasing the amount of incorporated nanofibers into the hydrogel, the Young's modulus of the composite hydrogels increased from 3.29 ± 1.02 kPa to 20.30 ± 1.79 kPa, while the strain at break decreased from 66.0 ± 1.1% to 52.0 ± 3.0%. Compared to composite hydrogels with coaxial nanofibers, those with blend nanofibers showed higher compressive strength and strain at break, but with lower modulus and energy dissipation properties. Biocompatibility evaluations of the nanofiber reinforced hydrogels were carried out using bone marrow mesenchymal stem cells (BM-MSCs) by cell proliferation assay and immunostaining analysis. The nanocomposite hydrogel with 25 mg ml(-1) PCL/gelatin 'blend' nanofibers (PGB25) was found to enhance cell proliferation, indicating that the 'nanocomposite hydrogels' might provide the necessary mechanical support and could be promising cell delivery systems for tissue regeneration.     

Effects of chondrogenic and osteogenic regulatory factors on composite constructs grown using human mesenchymal stem cells, silk scaffolds and bioreactors.

Human mesenchymal stem cells (hMSCs) isolated from bone marrow aspirates were cultured on silk scaffolds in rotating bioreactors for three weeks with either chondrogenic or osteogenic medium supplements to engineer cartilage- or bone-like tissue constructs. Osteochondral composites formed from these cartilage and bone constructs were cultured for an additional three weeks in culture medium that was supplemented with chondrogenic factors, supplemented with osteogenic factors or unsupplemented. Progression of cartilage and bone formation and the integration between the two regions were assessed by medical imaging (magnetic resonance imaging and micro-computerized tomography imaging), and by biochemical, histological and mechanical assays. During composite culture (three to six weeks), bone-like tissue formation progressed in all three media to a markedly larger extent than cartilage-like tissue formation. The integration of the constructs was most enhanced in composites cultured in chondrogenic medium. The results suggest that tissue composites with well-mineralized regions and substantially less developed cartilage regions can be generated in vitro by culturing hMSCs on silk scaffolds in bioreactors, that hMSCs have markedly higher capacity for producing engineered bone than engineered cartilage, and that chondrogenic factors play major roles at early stages of bone formation by hMSCs and in the integration of the two tissue constructs into a tissue composite.     

Spatial Micropatterning of Growth Factors in 3D Hydrogels for Location‐Specific Regulation of Cellular Behaviors

Growth factors are potent stimuli for regulating cell function in tissue engineering strategies, but spatially patterning their presentation in 3D in a facile manner using a single material is challenging. Micropatterning is an attractive tool to modulate the cellular microenvironment with various biochemical and physical cues and study their effects on stem cell behaviors. Implementing heparin's ability to immobilize growth factors, dual‐crosslinkable alginate hydrogels are micropatterned in 3D with photocrosslinkable heparin substrates with various geometries and micropattern sizes, and their capability to establish 3D micropatterns of growth factors within the hydrogels is confirmed. This 3D micropatterning method could be applied to various heparin binding growth factors, such as fibroblast growth factor‐2, vascular endothelial growth factor, transforming growth factor‐betas and bone morphogenetic proteins while retaining the hydrogel's natural degradability and cytocompability. Stem cells encapsulated within these micropatterned hydrogels have exhibited spatially localized growth and differentiation responses corresponding to various growth factor patterns, demonstrating the versatility of the approach in controlling stem cell behavior for tissue engineering and regenerative medicine applications.     

Labeling of mesenchymal stem cells by bioconjugated quantum dots

Long-term labeling of stem cells during self-replication and differentiation benefits investigations of development and tissue regeneration. We report the labeling of human mesenchymal stem cells (hMSCs) with RGD-conjugated quantum dots (QDs) during self-replication, and multilineage differentiations into osteogenic, chondrogenic, and adipogenic cells. QD-labeled hMSCs remained viable as unlabeled hMSCs from the same subpopulation. These findings suggest the use of bioconjugated QDs as an effective probe for long-term labeling of stem cells.     

Comparison of bone marrow cell growth on 2D and 3D alginate hydrogels

Calcium cross-linked sodium alginate hydrogels have several advantageous features making them potentially suitable as tissue engineering scaffolds and this material has been previously used in many biomedical applications. 3D cell culture systems are often very different from 2D petri dish type cultures. in this study the effect of alginate hydrogel architecture was investigated by comparing rat bone marrow cell proliferation and differentiation on calcium cross linked sodium alginate discs and 1mm internal diameter tubes. It was found that bone marrow cell proliferation was diminished as the concentration of alginate in the 2D hydrogel substrates increased, yet proliferation was extensive on tubular alginate constructs with high alginate contents. Alginate gel thickness was found to be an important parameter in determining cell behaviour and the different geometries did not generate significant alterations in BMC differentiation profiles.     

New perspectives on the roles of nanoscale surface topography in modulating intracellular signaling

The physical properties of biomaterials, such as elasticity, stiffness, and surface nanotopography, are mechanical cues that regulate a broad spectrum of cell behaviors, including migration, differentiation, proliferation, and reprogramming. Among them, nanoscale surface topography, i.e., nanotopography, defines the nanoscale shape and spatial arrangement of surface elements, which directly interact with the cell membranes and stimulate changes in the cell signaling pathways. In biological systems, the effects of nanotopography are often entangled with those of other mechanical and biochemical factors. Precise engineering of 2D nanopatterns and 3D nanostructures with well-defined features has provided a powerful means to study the cellular responses to specific topographic features. In this Review, we discuss efforts in the last three years to understand how nanotopography affects membrane receptor activation, curvature-induced cell signaling, and stem cell differentiation.     

A hydrophobically-modified alginate gel system: utility in the repair of articular cartilage defects

Alginate is a family of natural polysaccharides, widely used in industry and medicine for many applications, with its non toxic nature, gentle sol/gel transition procedure and low cost, alginate inferior biomechanical properties have limited its utility especially in tissue engineering. Additionally, ionically cross-linked alginate hydrogels generally lose most of their initial mechanical and swelling properties within a few hours in physiological solution. In order to overcome these limitations, the referenced alginate was treated by covalent fixation of octadecyl chains onto the polysaccharide backbone by esterification. In semi dilute solution, intermolecular hydrophobic interactions of long alkyl chains result in the formation of physical hydrogels, which can then be reinforced by the addition of calcium chloride. FTIR studies clearly showed the presence of ester bonds at 1612 and 1730?cm(-1) indicating that the alkyl groups are incorporated in the backbone of resulting polymer. The endothermic peak and exothermic peak present in the DSC thermogram of Alg-C18 had shifted to lower temperatures comparing to native alginate (from 106 to 83°C and from 250 to 245°C, respectively) due to the esterification reaction that leads to high hydrophobic nature of the modified sample. From rheological experiments, it can be inferred that the combination of both calcium bridges and intermolecular hydrophobic interaction in the treated alginate leads to enhanced gel strength accompanied by more stable structure in physiological solution comparing to native sodium alginate hydrogel. Finally, the modified alginate tended to have no toxic effects on mesenchymal stem cell culture, rather it supported MSC chondrogenic differentiation.     

“All‐in‐One” Gel System for Whole Procedure of Stem‐Cell Amplification and Tissue Engineering

Microsphere (MS)‐based systems provides great advantages for cell expansion and transplantation due to their high surface‐to‐volume ratio and biomimetic environment. However, a MS‐based system that includes cell attachment, proliferation, passage, harvest, cryopreservation, and tissue engineering together has not been realized yet. An “all‐in‐one” gel MS‐based system is established for human adipose‐derived mesenchymal stem cells (hADSCs), realizing real 3D culture with enhanced expansion efficiency and simplified serial cell culture operations, and construction of macrotissues with uniform cell distribution and specific function. A 3D digital light‐processing technology is developed to fabricate gel MSs in an effective way. The printed MSs present a suitable environment with rough surface architecture and the mechanical properties of soft tissues, leading to high cell viability, attachment, proliferation, activity, and differentiation potential. Further, convenient standard operation procedures, including cell passage, detachment, and cryopreservation, are established for cell culture on the gel MSs. Finally, hADSCs‐loaded gel MSs form macrotissues through a “bottom‐up” approach, which demonstrates the potential applications for tissue engineering. These findings exhibit the feasibility and beauty of “all‐in‐one” stem cell culture and tissue engineering system.     

Spatially controlled simultaneous patterning of multiple growth factors in three-dimensional hydrogels

Three-dimensional (3D) protein-patterned scaffolds provide a more biomimetic environment for cell culture than traditional two-dimensional surfaces, but simultaneous 3D protein patterning has proved difficult. We developed a method to spatially control the immobilization of different growth factors in distinct volumes in 3D hydrogels, and to specifically guide differentiation of stem/progenitor cells therein. Stem-cell differentiation factors sonic hedgehog (SHH) and ciliary neurotrophic factor (CNTF) were simultaneously immobilized using orthogonal physical binding pairs, barnase-barstar and streptavidin-biotin, respectively. Barnase and streptavidin were sequentially immobilized using two-photon chemistry for subsequent concurrent complexation with fusion proteins barstar-SHH and biotin-CNTF, resulting in bioactive 3D patterned hydrogels. The technique should be broadly applicable to the patterning of a wide range of proteins.     

Solvent‐Assisted Micromolding of Biohybrid Hydrogels to Maintain Human Hematopoietic Stem and Progenitor Cells Ex Vivo

Array‐format cell‐culture carriers providing tunable matrix cues are instrumental in current cell biology and bioengineering. A new solvent‐assisted demolding approach for the fabrication of microcavity arrays with very small feature sizes down to single‐cell level (3 µm) of very soft biohybrid glycosaminoglycan–poly(ethylene glycol) hydrogels (down to a shear modulus of 1 kPa) is reported. It is further shown that independent additional options of localized conjugation of adhesion ligand peptides, presentation of growth factors through complexation to gel‐based glycosaminoglycans, and secondary gel deposition for 3D cell embedding enable a versatile customization of the hydrogel microcavity arrays for cell culture studies. As a proof of concept, cell‐instructive hydrogel compartment arrays are used to analyze the response of human hematopoietic stem and progenitor cells to defined biomolecular and spatial cues.     

A Micro‐Ark for Cells: Highly Open Porous Polyhydroxyalkanoate Microspheres as Injectable Scaffolds for Tissue Regeneration

To avoid large open surgery using scaffold transplants, small‐sized cell carriers are employed to repair complexly shaped tissue defects. However, most cell carriers show poor cell adherences and viability. Therefore, polyhydroxyalkanoate (PHA), a natural biopolymer, is used to prepare highly open porous microspheres (OPMs) of 300–360 µm in diameter, combining the advantages of microspheres and scaffolds to serve as injectable carriers harboring proliferating stem cells. In addition to the convenient injection to a defected tissue, and in contrast to poor performances of OPMs made of polylactides (PLA OPMs) and traditional less porous hollow microspheres (PHA HMs), PHA OPMs present suitable surface pores of 10–60 µm and interconnected passages with an average size of 8.8 µm, leading to a high in vitro cell adhesion of 93.4%, continuous proliferation for 10 d and improved differentiation of human bone marrow mesenchymal stem cells (hMSCs). PHA OPMs also support stronger osteoblast‐regeneration compared with traditional PHA HMs, PLA OPMs, commercial hyaluronic acid hydrogels, and carrier‐free hMSCs in an ectopic bone‐formation mouse model. PHA OPMs protect cells against stresses during injection, allowing more living cells to proliferate and migrate to damaged tissues. They function like a micro‐Noah's Ark to safely transport cells to a defect tissue.     

Click chemistry approach for fabricating PVA/gelatin nanofibers for the differentiation of ADSCs to keratinocytes

Every year, millions of people suffer from dermal wounds caused by heat, fire, chemicals, electricity, ultraviolet radiation or disease. Tissue engineering and nanotechnology have enabled the engineering of nanostructured materials to meet the current challenges in skin treatments owing to such rising occurrences of accidental damages, skin diseases and defects. The abundance and accessibility of adipose derived stem cells (ADSCs) may prove to be novel cell therapeutics for skin regeneration. The nanofibrous PVA/gelatin/azide scaffolds were then fabricated by electrospinning using water as solvent and allowed to undergo click reaction. The scaffolds were characterized by SEM, contact angle and FTIR. The cell–scaffold interactions were analyzed by cell proliferation and the results observed that the rate of cell proliferation was significantly increased (P ≤ 0.05) on PVA/gelatin/azide scaffolds compared to PVA/gelatin nanofibers. In the present study, manipulating the biochemical cues by the addition of an induction medium, in combination with environmental and physical factors of the culture substrate by functionalizing with click moieties, we were able to drive ADSCs into epidermal lineage with the development of epidermis-like structures, was further confirmed by the expression of early and intermediate epidermal differentiation markers like keratin and filaggrin. This study not only provides an insight into the design of a site-specific niche-like microenvironment for stem cell lineage commitment, but also sheds light on the therapeutic application of an alternative cell source—ADSCs, for wound healing and skin tissue reconstitution.     

High Throughput Screening of Cell Mechanical Response Using a Stretchable 3D Cellular Microarray Platform

Cells in vivo are constantly subjected to multiple microenvironmental mechanical stimuli that regulate cell function. Although 2D cell responses to the mechanical stimulation have been established, these methods lack relevance as physiological cell microenvironments are in 3D. Moreover, the existing platforms developed for studying the cell responses to mechanical cues in 3D either offer low‐throughput, involve complex fabrication, or do not allow combinatorial analysis of multiple cues. Considering this, a stretchable high‐throughput (HT) 3D cell microarray platform is presented that can apply dynamic mechanical strain to cells encapsulated in arrayed 3D microgels. The platform uses inkjet‐bioprinting technique for printing cell‐laden gelatin methacrylate (GelMA) microgel array on an elastic composite substrate that is periodically stretched. The developed platform is highly biocompatible and transfers the applied strain from the stretched substrate to the cells. The HT analysis is conducted to analyze cell mechano‐responses throughout the printed microgel array. Also, the combinatorial analysis of distinct cell behaviors is conducted for different GelMA microenvironmental stiffnesses in addition to the dynamic stretch. Considering its throughput and flexibility, the developed platform can readily be scaled up to introduce a wide range of microenvironmental cues and to screen the cell responses in a HT way.