Mesenchymal stem cell adhesion but not plasticity is affected by high substrate stiffness

Abstract

The acknowledged ability of synthetic materials to induce cell-specific responses regardless of biological supplies provides tissue engineers with the opportunity to find the appropriate materials and conditions to prepare tissue-targeted scaffolds. Stem and mature cells have been shown to acquire distinct morphologies in vitro and to modify their phenotype when grown on synthetic materials with tunable mechanical properties. The stiffness of the substrate used for cell culture is likely to provide cells with mechanical cues mimicking given physiological or pathological conditions, thus affecting the biological properties of cells. The sensitivity of cells to substrate composition and mechanical properties resides in multiprotein complexes called focal adhesions, whose dynamic modification leads to cytoskeleton remodeling and changes in gene expression. In this study, the remodeling of focal adhesions in human mesenchymal stem cells in response to substrate stiffness was followed in the first phases of cell–matrix interaction, using poly-ε-caprolactone planar films with similar chemical composition and different elasticity. As compared to mature dermal fibroblasts, mesenchymal stem cells showed a specific response to substrate stiffness, in terms of adhesion, as a result of differential focal adhesion assembly, while their multipotency as a bulk was not significantly affected by matrix compliance. Given the sensitivity of stem cells to matrix mechanics, the mechanobiology of such cells requires further investigations before preparing tissue-specific scaffolds.     

Substrate stiffness affects skeletal myoblast differentiation in vitro

To maximize the therapeutic efficacy of cardiac muscle constructs produced by stem cells and tissue engineering protocols, suitable scaffolds should be designed to recapitulate all the characteristics of native muscle and mimic the microenvironment encountered by cells in vivo. Moreover, so not to interfere with cardiac contractility, the scaffold should be deformable enough to withstand muscle contraction. Recently, it was suggested that the mechanical properties of scaffolds can interfere with stem/progenitor cell functions, and thus careful consideration is required when choosing polymers for targeted applications. In this study, cross-linked poly-ε-caprolactone membranes having similar chemical composition and controlled stiffness in a supra-physiological range were challenged with two sources of myoblasts to evaluate the suitability of substrates with different stiffness for cell adhesion, proliferation and differentiation. Furthermore, muscle-specific and non-related feeder layers were prepared on stiff surfaces to reveal the contribution of biological and mechanical cues to skeletal muscle progenitor differentiation. We demonstrated that substrate stiffness does affect myogenic differentiation, meaning that softer substrates can promote differentiation and that a muscle-specific feeder layer can improve the degree of maturation in skeletal muscle stem cells.     

Extracellular-matrix tethering regulates stem-cell fate

To investigate how substrate properties influence stem-cell fate, we cultured single human epidermal stem cells on polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) hydrogel surfaces, 0.1?kPa-2.3?MPa in stiffness, with a covalently attached collagen coating. Cell spreading and differentiation were unaffected by polydimethylsiloxane stiffness. However, cells on polyacrylamide of low elastic modulus (0.5?kPa) could not form stable focal adhesions and differentiated as a result of decreased activation of the extracellular-signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling pathway. The differentiation of human mesenchymal stem cells was also unaffected by PDMS stiffness but regulated by the elastic modulus of PAAm. Dextran penetration measurements indicated that polyacrylamide substrates of low elastic modulus were more porous than stiff substrates, suggesting that the collagen anchoring points would be further apart. We then changed collagen crosslink concentration and used hydrogel-nanoparticle substrates to vary anchoring distance at constant substrate stiffness. Lower collagen anchoring density resulted in increased differentiation. We conclude that stem cells exert a mechanical force on collagen fibres and gauge the feedback to make cell-fate decisions.     

Electrospun poly(vinylidene fluoride-trifluoroethylene)/zinc oxide nanocomposite tissue engineering scaffolds with enhanced cell adhesion and blood vessel formation

Piezoelectric materials that generate electrical signals in response to mechanical strain can be used in tissue engineering to stimulate cell proliferation.Poly (vinylidene fluoride-trifluoroethylene) (P(VDF-TrFE)),a piezoelectric polymer,is widely used in biomaterial applications.We hypothesized that incorporation of zinc oxide (ZnO) nanoparticles into the P(VDF-TrFE) matrix could promote adhesion,migration,and proliferation of cells,as well as blood vessel formation (angiogenesis).In this study,we fabricated and comprehensively characterized a novel electrospun P(VDF-TrFE)/ZnO nanocomposite tissue engineering scaffold.We analyzed the morphological features of the polymeric matrix by scanning electron microscopy,and utilized Fourier transform infrared spectroscopy,X-ray diffraction,and differential scanning calorimetry to examine changes in the crystalline phases of the copolymer due to addition of the nanoparticles.We detected no or minimal adverse effects of the biomaterials with regard to blood compatibility in vitro,biocompatibility,and cytotoxicity,indicating that P(VDF-TrFE)/ZnO nanocomposite scaffolds are suitable for tissue engineering applications.Interestingly,human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells cultured on the nanocomposite scaffolds exhibited higher cell viability,adhesion,and proliferation compared to cells cultured on tissue culture plates or neat P(VDF-TrFE) scaffolds.Nanocomposite scaffolds implanted into rats with or without hMSCs did not elicit immunological responses,as assessed by macroscopic analysis and histology.Importantly,nanocomposite scaffolds promoted angiogenesis,which was increased in scaffolds pre-seeded with hMSCs.Overall,our results highlight the potential of these novel P(VDF-TrFE)/ZnO nanocomposites for use in tissue engineering,due to their biocompatibility and ability to promote cell adhesion and angiogenesis.     

Substrate stiffness affects skeletal myoblast differentiation in vitro

Abstract

To maximize the therapeutic efficacy of cardiac muscle constructs produced by stem cells and tissue engineering protocols, suitable scaffolds should be designed to recapitulate all the characteristics of native muscle and mimic the microenvironment encountered by cells in vivo. Moreover, so not to interfere with cardiac contractility, the scaffold should be deformable enough to withstand muscle contraction. Recently, it was suggested that the mechanical properties of scaffolds can interfere with stem/progenitor cell functions, and thus careful consideration is required when choosing polymers for targeted applications. In this study, cross-linked poly-ε-caprolactone membranes having similar chemical composition and controlled stiffness in a supra-physiological range were challenged with two sources of myoblasts to evaluate the suitability of substrates with different stiffness for cell adhesion, proliferation and differentiation. Furthermore, muscle-specific and non-related feeder layers were prepared on stiff surfaces to reveal the contribution of biological and mechanical cues to skeletal muscle progenitor differentiation. We demonstrated that substrate stiffness does affect myogenic differentiation, meaning that softer substrates can promote differentiation and that a muscle-specific feeder layer can improve the degree of maturation in skeletal muscle stem cells.     

三维打印羟基磷灰石晶须增强复合骨修复支架

利用三维打印技术成功制备羟基磷灰石晶须(HAPw)增强的聚己内酯(PCL)复合骨修复支架。通过改变三维打印的挤出速度和挤出气压, 使不同含量HAPw均能在PCL基材中一致排列并均匀分布。PCL支架的机械强度随HAPw含量增加显著提高, 添加33wt%HAPw使PCL支架强度提升了高达3倍。此外, HAPw使PCL支架表面与水的接触角从近100º降低至约50º, 有效改善了细胞表面粘附。经过体外人类骨髓间充质干细胞(hBMSC)在支架上的培养实验, 发现添加HAPw的复合支架具有更好的生物相容性, 能够有效促进hBMSC的增殖生长, 且HAPw-PCL复合支架上细胞具有更高的碱性磷酸酶(ALP)活性和OCN、RUNX2等相关成骨基因表达, 显示出hBMSCs向成骨方向更好的分化及成骨活性。     

Design of novel three-phase PCL/TZ–HA biomaterials for use in bone regeneration applications

The design of bioactive scaffold materials able to guide cellular processes involved in new-tissue genesis is key determinant in bone tissue engineering. The aim of this study was the design and characterization of novel multi-phase biomaterials to be processed for the fabrication of 3D porous scaffolds able to provide a temporary biocompatible substrate for mesenchymal stem cells (MSCs) adhesion, proliferation and osteogenic differentiation. The biomaterials were prepared by blending poly(ε-caprolactone) (PCL) with thermoplastic zein (TZ), a thermoplastic material obtained by de novo thermoplasticization of zein. Furthermore, to bioactivate the scaffolds, microparticles of osteoconductive hydroxyapatite (HA) were dispersed within the organic phases. Results demonstrated that materials and formulations strongly affected the micro-structural properties and hydrophilicity of the scaffolds and, therefore, had a pivotal role in guiding cell/scaffold interaction. In particular, if compared to neat PCL, PCL–HA composite and PCL/TZ blend, the three-phase PCL/TZ–HA showed improved MSCs adhesion, proliferation and osteogenic differentiation capability, thus demonstrating potential for bone regeneration.     

Mechanochemical Adhesion and Plasticity in Multifiber Hydrogel Networks

The extracellular matrix (ECM) has force-responsive (i.e., mechanochemical) properties that enable adaptation to mechanical loading through changes in fibrous network structure and interfiber bonding. Imparting such properties into synthetic fibrous materials will allow reinforcement under mechanical load, the potential for material self-adhesion, and the general mimicking of ECM. Multifiber hydrogel networks are developed through the electrospinning of multiple fibrous hydrogel populations, where fibers contain complementary chemical moieties (e.g., aldehyde and hydrazide groups) that form covalent bonds within minutes when brought into contact under mechanical load. These fiber interactions lead to microscale anisotropy, as well as increased material stiffness and plastic deformation. Macroscale structures (e.g., tubes and layered scaffolds) are fabricated from these materials through interfiber bonding and adhesion when placed into contact while maintaining a microscale fibrous architecture. The design principles for engineering plasticity described can be applied to numerous material systems to introduce unique properties, from textiles to biomedical applications.     

Focal complex maturation and bridging on 200 nm vitronectin but not fibronectin patches reveal different mechanisms of focal adhesion formation

The effects of protein type and pattern size on cell adhesion, spreading, and focal adhesion development are studied. Fibronectin and vitronectin patterns from 0.1 to 3 μm produced by colloidal lithography reveal important differences in how cells adhere to and bridge focal adhesions across protein nanopatterns versus micropatterns. Vinculin and zyxin in focal adhesions but not integrins are seen to bridge ligand gaps. Differences in protein mechanical properties are implicated as important factors in focal adhesion development.     

Patterning the mechanical properties of hydrogen silsesquioxane films using electron beam irradiation for application in mechano cell guidance

Hydrogen silsesquioxane (HSQ) is a material with the potential for studying the effect of surface stiffness on stem cell differentiation. Here, the effects of electron beam dose on the topography and the mechanical properties of HSQ obtained with or without trimethylamine (TMA) development are characterised by atomic force microscopy imaging and indentation. A correlation between the surface stiffness (uniform across the sample) and electron beam exposure is observed. Surface roughness of HSQ samples developed in TMA decreases exponentially with increasing electron beam exposure. Surface coating with plasma polymerised allylamine (ppAAm) leads to an overall decrease in stiffness values. However, the increase in surface stiffness with increasing electron beam exposure is still evident. The ppAAm coating is shown to facilitate human mesenchymal stem cell adhesion.     

Adaptive Liquid Interfacially Assembled Protein Nanosheets for Guiding Mesenchymal Stem Cell Fate

There is a growing interest in the development of dynamic adaptive biomaterials for regulation of cellular functions. However, existing materials are limited to two-state switching of the presentation and removal of cell-adhesive bioactive motifs that cannot emulate the native extracellular matrix (ECM) in vivo with continuously adjustable characteristics. Here, tunable adaptive materials composed of a protein monolayer assembled at a liquid–liquid interface are demonstrated, which adapt dynamically to cell traction forces. An ultrastructure transition from protein monolayer to hierarchical fiber occurs through interfacial jamming. Elongated fibronectin fibers promote formation of elongated focal adhesion structures, increase focal adhesion kinase activation, and enhance neuronal differentiation of stem cells. Cell traction force results in spatial rearrangement of ECM proteins, which feeds back to alter stem cell fate. The reported biomimetic adaptive liquid interface enables dynamic control of stem cell behavior and has potential translational applications.     

Interactions of Fibroblasts with Different Morphologies Made of an Engineered Spider Silk Protein

Spider silk has been investigated for decades due to the intriguing mechanical and also biomedical properties of the silk fibers. Previously, it has been shown that recombinant silk proteins can also be processed into other morphologies. Here, we characterized scaffolds made of the recombinant spider silk protein eADF4(C16) concerning their surface interactions with fibroblasts. Studies of BALB/3T3 cells on hydrogels and films made of eADF4(C16) showed low cell adhesion without observable duplication. Electro‐spun non‐woven scaffolds made of eADF4(C16), however, enabled both their adhesion and proliferation. Since eADF4(C16) lacks specific motifs for cell attachment, fibroblasts cannot generate focal adhesions with the material's surface, and, therefore, other cell–interface interactions such as topographical anchorage or cell attachment mediated by adhesion of extracellular matrix proteins are discussed in this paper. On non‐woven meshes protrusion of filopodia and/or lamellipodia between individual fibers increase the surface contact area, which depends on the diameter of the fibers of the non‐woven meshes. In contrast, at flat (film) or microstructured surfaces (hydrogels) such interactions seem to be precluded.     

Adhesion formation of primary human osteoblasts and the functional response of mesenchymal stem cells to 330nm deep microgrooves.

The surface microtexture of an orthopaedic device can regulate cellular adhesion, a process fundamental in the initiation of osteoinduction and osteogenesis. Advances in fabrication techniques have evolved to include the field of surface modification; in particular, nanotechnology has allowed for the development of experimental nanoscale substrates for investigation into cell nanofeature interactions. Here primary human osteoblasts (HOBs) were cultured on ordered nanoscale groove/ridge arrays fabricated by photolithography. Grooves were 330nm deep and either 10, 25 or 100mum in width. Adhesion subtypes in HOBs were quantified by immunofluorescent microscopy and cell-substrate interactions were investigated via immunocytochemistry with scanning electron microscopy. To further investigate the effects of these substrates on cellular function, 1.7K gene microarray analysis was used to establish gene regulation profiles of mesenchymal stem cells cultured on these nanotopographies. Nanotopographies significantly affected the formation of focal complexes (FXs), focal adhesions (FAs) and supermature adhesions (SMAs). Planar control substrates induced widespread adhesion formation; 100mum wide groove/ridge arrays did not significantly affect adhesion formation yet induced upregulation of genes involved in skeletal development and increased osteospecific function; 25mum wide groove/ridge arrays were associated with a reduction in SMA and an increase in FX formation; and 10mum wide groove/ridge arrays significantly reduced osteoblast adhesion and induced an interplay of up- and downregulation of gene expression. This study indicates that groove/ridge topographies are important modulators of both cellular adhesion and osteospecific function and, critically, that groove/ridge width is important in determining cellular response.     

Small-diameter tissue engineered vascular graft made of electrospun PCL/lecithin blend

In this study, natural lecithin was incorporated into cholesterol-poly(ε-caprolactone) (Chol-PCL) by solution blending in order to modify the performance of the hydrophobic and bio-inert PCL. The fibrous Chol-PCL/lecithin membranes were fabricated by electrospinning, and the surface morphology and properties were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, static water contact angle, and mechanical tensile testing. The blood compatibility of the scaffolds was evaluated by in vitro hemolysis assay. The cytocompatibility of the scaffolds was investigated by cell adhesion and proliferation using bone-marrow mesenchymal stem cells (MSCs). Subcutaneous implantation was also performed to evaluate the in vivo inflammatory reaction. The tubular tissue-engineered vascular graft (TEVG) was further constructed by rolling cell sheet comprising fibrous membrane and MSCs. Furthermore, endothelial cells (ECs) were seeded onto the lumen of the graft with the aim to form vascular endothelium. The preliminary results indicate that electrospun Chol-PCL/lecithin scaffolds show improved hemocompatibility and cytocompatibility compared with neat Chol-PCL, and combining the Chol-PCL/lecithin fibrous scaffold with MSCs and ECs with well controlled distribution is a promising strategy for constructing TEVGs.     

Powder Metallurgical Near‐Net‐Shape Fabrication of Porous NiTi Shape Memory Alloys for Use as Long‐Term Implants by the Combination of the Metal Injection Molding Process with the Space‐Holder Technique

A new method was developed for producing highly porous NiTi for use as an implant material. The combination of the space‐holder technique with the metal injection molding process allows a net‐shape fabrication of geometrically complex samples and the possibility of mass production for porous NiTi. Further, the porosity can be easily adjusted with respect to pore size, pore shape, and total porosity. The influence of the surface properties of powder metallurgical NiTi on the biocompatibility was first examined using human mesenchymal stem cells (hMSCs). It was found that pre‐alloyed NiTi powders with an average particle size smaller than 45 μm led to the surface properties most suitable for the adhesion and proliferation of hMSCs. For the production of highly porous NiTi, different space‐holder materials were investigated regarding low C‐ and O‐impurity contents and the reproducibility of the process. NaCl was the most promising space‐holder material compared to PMMA and saccharose and was used in subsequent studies. In these studies, the influence of the total porosity on the mechanical properties of NiTi is investigated in detail. As a result, bone‐like mechanical properties were achieved by the choice of Ni‐rich NiTi powder and a space‐holder content of 50 vol% with a particle size fraction of 355–500 μm. Pseudoelasticity of up to 6% was achieved in compression tests at 37 °C as well as a bone‐like loading stiffness of 6.5 GPa, a sufficient plateau stress σ₂₅ of 261 MPa and a value for σ₅₀ of 415 MPa. The first biological tests of the porous NiTi samples produced by this method showed promising results regarding proliferation and ingrowth of mesenchymal stem cells, also in the pores of the implant material.     

Novel 3D porous multi-phase composite scaffolds based on PCL,thermoplastic zein and ha prepared via supercritical CO2 foaming for bone regeneration

The aim of this study was the design of novel biodegradable porous scaffolds for bone tissue engineering (bTE) via supercritical CO₂ (scCO₂) foaming process. The porous scaffolds were prepared from a poly(ε-caprolactone)-thermoplastic zein multi-phase blend w/o interdispersed hydroxyapatite particles (HA) and the porous structure achieved via the scCO₂ foaming technology. The control of scaffolds porosity was obtained by modulating materials formulation and foaming temperature (T_F). The scaffolds were subjected to morphological, micro-structural and biodegradation analyses, as well as in vitro biocompatibility tests. Results demonstrated that both HA concentration and T_F significantly affected the micro-structural features of the scaffolds. In particular, scaffolds with porosity and pore size distribution, mechanical properties and biodegradability adequate for bTE were designed and produced by selecting a T_F equal to 100 °C for all the compositions used. The biocompatibility of these scaffolds was assessed in vitro by using osteoblast-like MG63 and human mesenchymal stem cells (hMSCs).     

Cell proliferation,viability, and in vitro differentiation of equine mesenchymal stem cells seeded on bacterial cellulose hydrogel scaffolds

The culture of multipotent mesenchymal stem cells on natural biopolymers holds great promise for treatments of connective tissue disorders such as osteoarthritis. The safety and performance of such therapies relies on the systematic in vitro evaluation of the developed stem cell-biomaterial constructs prior to in vivo implantation. This study evaluates bacterial cellulose (BC), a biocompatible natural polymer, as a scaffold for equine-derived bone marrow mesenchymal stem cells (EqMSCs) for application in bone and cartilage tissue engineering. An equine model was chosen due to similarities in size, load and types of joint injuries suffered by horses and humans. Lyophilized and critical point dried BC hydrogel scaffolds were characterized using scanning electron microscopy (SEM) to confirm nanostructure morphology which demonstrated that critical point drying induces fibre bundling unlike lyophilisation. EqMSCs positively expressed the undifferentiated pluripotent mesenchymal stem cell surface markers CD44 and CD90. The BC scaffolds were shown to be cytocompatible, supporting cellular adhesion and proliferation, and allowed for osteogenic and chondrogenic differentiation of EqMSCs. The cells seeded on the BC hydrogel were shown to be viable and metabolically active. These findings demonstrate that the combination of a BC hydrogel and EqMSCs are promising constructs for musculoskeletal tissue engineering applications.     

Reduced graphene oxide: osteogenic potential for bone tissue engineering

Collagen (Col) type I, as the major component of the bone extracellular matrix has been broadly studied for bone tissue engineering. However,inferior mechanical properties limit its usage for load bearing applications. In this research, freeze dried Col scaffolds are coated with graphene oxide (GO) through a covalent bond of the amine Col with the graphene carboxyl groups. The prepared scaffolds were then reduced using a chemical agent. Scanning electron microscopy exhibited a porous structure for the synthesized scaffolds with an approximate pore size of 100–220 ± 12 µm, which is in the suitable range for bone tissue engineering application. Reducing the GO coating improved the compressive modulus of the Col from 250 to 970 kPa. Apatite formation was also indicated by immersing the scaffolds in simulated body fluid after five days. The cytocompatibility of the scaffolds, using human bone marrow‐derived mesenchymal stem cells, was confirmed with MTT analysis. Alkaline phosphatase assay revealed that reducing the Col–GO scaffolds can effectively activate the differentiation of hBM‐MSCs into osteoblasts after 14 days, even without the addition of an osteogenic differentiation medium. The results of this study highlight that GO and its reduced form have considerable potential as bone substitutes for orthopaedic and dental applications.Inspec keywords: molecular biophysics, tissue engineering, biochemistry, cellular biophysics, graphene, biomedical materials, bone, proteins, scanning electron microscopy, porous materials, compressive strength, biomechanicsOther keywords: human bone marrow‐derived mesenchymal stem cells, reduced graphene oxide, bone extracellular matrix, inferior mechanical properties, load bearing applications, freeze‐dried Col scaffolds, amine Col groups, graphene carboxyl groups, bone tissue engineering, collagen type I, GO‐Col scaffolds, covalent bond, scanning electron microscopy, compressive modulus, apatite formation, cytocompatibility, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide analysis, alkaline phosphatase assay, osteogenic differentiation medium, dental applications, orthopaedic applications, porous structure, time 14.0 day, CO     

Adipose-derived stem cells could sense the nano-scale cues as myogenic-differentiating factors

Microenvironmental cues, such as surface topography and substrate stiffness, may affect stem cells adhesion, morphology, alignment, proliferation and differentiation. Adipose derived stem cells (ASCs) have attracted considerable interest in regenerative medicine due to their easy isolation, extensive in vitro expandability and ability to differentiate along a number of different tissue-specific lineages. The aim of this work was to investigate ASCs adhesion, alignment and differentiation into myogenic lineage on nanofibrous polymeric scaffolds with anisotropic topography. Nanostructured scaffolds with randomized or parallel fibers were fabricated by electrospinning using polycaprolactone (PCL) and the polycarbonate-urethane ChronoFlex AL 80A (CFAL). Cells expressed myosin (fast skeletal) and tropomyosin in all surface topographies 7 days after seeding but myotube formation was only observed on CFAL scaffolds and only few myotubes were formed on PCL scaffolds. The different cell behavior could be ascribed to two main parameters: fibers dimensions and fibers orientation of the substrates that could result in a better myotube formation on CFAL scaffolds.