The Combined Effect of Substrate Stiffness and Surface Topography on Chondrogenic Differentiation of Mesenchymal Stem Cells.

Stem cell differentiation is guided by contact with the physical microenvironment, influence by both topography and mechanical properties of the matrix. In this study, the combined effect of substratum nano-topography and mechanical stiffness in directing mesenchymal stem cell (MSC) chondrogenesis was investigated. Three polyesters of varying stiffness were thermally imprinted to create nano-grating or pillar patterns of the same dimension. The surface of the nano-patterned substrate was coated with chondroitin sulfate (CS) to provide an even surface chemistry, with cell-adhesive and chondro-inductive properties, across all polymeric substrates. The surface characteristic, mechanical modulus, and degradation of the CS-coated patterned polymeric substrates were analyzed. The cell morphology adopted on the nano-topographic surfaces were accounted by F-actin distribution, and correlated to the cell proliferation and chondrogenic differentiation outcomes. Results show that substratum stiffness and topographical cues affected MSC morphology and aggregation, and influenced the phenotypic development at the earlier stage of chondrogenic differentiation. Hyaline-like cartilage with middle/deep zone cartilage characteristics was generated on softer pillar surface, while on stiffer nano-pillar material MSCs showed potential to generate constituents of hyaline/fibro/hypertrophic cartilage. Fibro/superficial zone-like cartilage could be derived from nano-grating of softer stiffness, while stiffer nano-grating resulted in insignificant chondrogenesis. This study demonstrates the possibility of refining the phenotype of cartilage generated from MSCs by manipulating surface topography and material stiffness.

Substrate topography determines the fate of chondrogenesis from human mesenchymal stem cells resulting in specific cartilage phenotype formation.

To reproduce a complex and functional tissue, it is crucial to provide a biomimetic cellular microenvironment that not only incorporates biochemical cues, but also physical features including the nano-topographical patterning, for cell/matrix interaction. We developed spatially-controlled nano-topography in the form of nano-pillar, nano-hole and nano-grill on polycaprolactone surface via thermal nanoimprinting. The effects of chondroitin sulfate-coated nano-topographies on cell characteristics and chondrogenic differentiation of human mesenchymal stem cell (MSC) were investigated. Our results show that various nano-topographical patterns triggered changes in MSC morphology and cytoskeletal structure, affecting cell aggregation and differentiation. Compared to non-patterned surface, nano-pillar and nano-hole topography enhanced MSC chondrogenesis and facilitated hyaline cartilage formation. MSCs experienced delayed chondrogenesis on nano-grill topography and were induced to fibro/superficial zone cartilage formation. This study demonstrates the sensitivity of MSC differentiation to surface nano-topography and highlights the importance of incorporating topographical design in scaffolds for cartilage tissue engineering. From the clinical editor: These authors have developed spatially-controlled nano-topography in the form of nano-pillar, nano-hole and nano-grill on polycaprolactone surface via thermal nanoimprinting, and the effects of chondroitin sulfate-coated nano-topographies on cell characteristics and chondrogenic differentiation of human mesenchymal stem cells (MSC) were investigated. It has been concluded that MSC differentiation is sensitive to surface nano-topography, and certain nano-imprinted surfaces are more useful than others for cartilage tissue engineering.

Bioinspired ultrahigh water pinning nanostructures.

Rose petal mimetic surfaces with ultrahigh water pinning forces have been fabricated via nanoimprinting process onto three different polymer films. Water pinning forces ranging from 104 to 690 µN are obtained on free-standing polycarbonate films with imprinted nanostructures. Through a systematic variation of the surface structures, this study provides experimental evidence that an ultrahigh water pinning force can be achieved by combining two surface topographical designs: (1) conical- or parabolic-shaped nanoprotrusions and (2) isotropic and continuous nanoprotrusions. These design criteria ensure that a continuous solid-liquid contact line is achieved and provide a rule-of-thumb to engineer surfaces with tunable water pinning forces. The ultrahigh water pinning film is further demonstrated to mitigate the "coffee ring" effect, a phenomenon associated with nonuniform deposition from a drying solute-laden liquid droplet.

Microarray with micro- and nano-topographies enables identification of the optimal topography for directing the differentiation of primary murine neural progenitor cells.

During development and tissue repair, progenitor cells are guided by both biochemical and biophysical cues of their microenvironment, including topographical signals. The topographical cues have been shown to play an important role in controlling the fate of cells. Systematic investigation of topographical structures with different geometries and sizes under the identical experimental conditions on the same chip will enhance the understanding of the role of shape and size in cell-topography interactions. A simple customizable multi-architecture chip (MARC) array is therefore developed to incorporate, on a single chip, distinct topographies of various architectural complexities, including both isotropic and anisotropic features, in nano- to micrometer dimensions, with different aspect ratios and hierarchical structures. Polydimethylsiloxane (PDMS) replicas of MARC are used to investigate the influence of different geometries and sizes in neural differentiation of primary murine neural progenitor cells (mNPCs). Anisotropic gratings (2 µm gratings, 250 nm gratings) and isotropic 1 µm pillars significantly promote differentiation of mNPCs into neurons, as indicated by expression of ß-III-tubulin (59%, 58%, and 58%, respectively, compared to 30% on the control). In contrast, glial differentiation is enhanced on isotropic 2 µm holes and 1 µm pillars. These results illustrate that anisotropic topographies enhance neuronal differentiation while isotropic topographies enhance glial differentiation on the same chip under the same conditions. MARC enables simultaneous cost-effective investigation of multiple topographies, allowing efficient optimization of topographical and biochemical cues to modulate cell differentiation.

Early integrin binding to Arg-Gly-Asp peptide activates actin polymerization and contractile movement that stimulates outward translocation.

Integrin-mediated adhesions are critical for stem cell differentiation, cancer metastasis, and the immune response [Hynes RO (2009) Science 326:1216-1219]. However, the mechanisms of early adhesion formation remain unclear, especially the effects of lateral clustering of integrins and the role of the Src family kinases. Using mobile Arg-Gly-Asp (RGD) peptide ligands on lipid bilayers with nano-fabricated physical barriers [Salaita K, et al. (2010) Science 327:1380-1385], we observe surprising long-range lateral movements of ligated integrins during the process of cell spreading. Initially, RGD-activated integrin clusters stimulate actin polymerization that radiates from the clusters. Myosin II contraction of actin from adjacent clusters produces contractile pairs that move toward each other against barriers. Force generated by myosin II stimulates a Src kinase-dependent lamellipodial extension and outward movement of clusters. Subsequent retraction by myosin II causes inward movement of clusters. The final cell spread area increases with the density of periodic barriers. Early integrin clustering recruits adhesion proteins, talin, paxillin, and FAK, irrespective of force generation. However, recruitment of vinculin is only observed upon contraction. Thus, we suggest that integrin activation and early clustering are independent of lateral forces. Clustering activates Src-dependent actin polymerization from clusters. Myosin contraction of clusters to lines stimulates active spreading with outward forces from actin polymerization followed by a second wave of contraction. Many of these early mechanical steps are not evident in cells spreading on immobilized matrices perhaps because of the low forces involved. These observations can provide new targets to control integrin-dependent adhesion and motility.