Peng Yuan Wang

Modulation of alignment, elongation and contraction of cardiomyocytes through a combination of nanotopography and rigidity of substrates

The topographic and mechanical characteristics of engineered tissue constructs, simulating native tissues, should benefit tissue engineering. Previous studies reported that surface topography and substrate rigidity provide biomechanical cues to modulate cellular responses such as alignment, migration and differentiation. To fully address this issue, the present study aimed to examine the influence of nanogrooved substrates with different stiffnesses on the responses of rat cardiomyocytes. Nanogrooved substrates (450nm in groove/ridge width; 100 or 350nm in depth) made of polystyrene and polyurethane were prepared by imprinting from polydimethylsiloxane molds. The morphology and orientation of cardiomyocytes attached to the substrates were found to be influenced mainly by the nanogrooved structures, while the contractile function of the cells was regulated by the coupled effect of surface topography and substrate stiffness. The distribution of intracellular structural proteins such as vinculin and F-actin showed that the surface topography and substrate stiffness regulated the organization of the actin cytoskeleton and focal adhesion complexes, and consequently the contractile behavior of the cardiomyocytes. The beating rates of the cultured cardiomyocytes were dependent on both the surface topography and the substrate stiffness. The study provides insights into the interaction between cardiomyocytes and biomaterials, and benefits cardiac tissue engineering.

Screening of rat mesenchymal stem cell behaviour on polydimethylsiloxane stiffness gradients

Substrate stiffness is emerging as an effective tool for the regulation of cell behaviours such as locomotion, proliferation and differentiation. In order to explore the potential application of this biophysical tool, material platforms displaying lateral and continuously graded stiffness are advantageous since they allow the systematic exploration of adherent cell response to substrate stiffness and the tuning of the material to elicit the desired cell behaviour. Here, we demonstrate a simple approach towards the fabrication of polydimethylsiloxane (PDMS) stiffness gradients (with an indentation modulus of 190 kPa-3.1 MPa across a 12 mm distance) by means of a temperature gradient during curing. We then apply these stiffness gradients to the screening of osteogenic differentiation in rat mesenchymal stem cells (rMSCs). Our proof-of-principle results show that mineralization of rMSCs is strongly dependent on the PDMS substrate stiffness, but is also influenced by the display of extracellular matrix proteins preadsorbed on the gradients. This screening capability holds tremendous potential for the design of improved implant materials and tissue engineering scaffolds.

Modulation of alignment and differentiation of skeletal myoblasts by submicron ridges/grooves surface structure

Alignment and fusion of myoblasts into parallel arrays of multinucleated myotubes are critical in skeletal muscle tissue engineering. It is well known that contact guidance by grooves/ridges structures induces myoblasts to align and to migrate along the anisotropic direction. In this study, two series of grooved substrata with different widths (450 and 900 nm) and different depths (100, 350, and 550 nm) were studied on their effects on myoblast adhesion, proliferation, and differentiation into myotubes. We found that C2C12 cells were aligned and elongated along the direction of grooves. Groove depth was more influential on cellular morphology, proliferation, and differentiation than groove width. While cell proliferation was retarded on the grooved surfaces especially on the substrate with 900/550 nm (width/depth), differentiation was also enhanced on the patterned surfaces compared to the flat control. Our results demonstrated the potential of grooved substrata with submicron scale in skeletal muscle tissue engineering. Biotechnol. Bioeng. 2010;106: 285–294.

The roles of RGD and grooved topography in the adhesion, morphology, and differentiation of C2C12 skeletal myoblasts

Both chemical and topographic cues are crucial for the development of skeletal muscle. In this study, the relative roles of both signals in regard to cell adhesion, morphology, and differentiation of C2C12 skeletal myoblasts were investigated. Grooved polystyrene substrates containing grooves with approximately 900 nm in width with 600 nm ridge spans and 665 nm in depth were conjugated with the cell adhesion peptide arginine–glycine–aspartic acid (RGD). RGD conjugation significantly enhanced the adhesion, growth and differentiation of C2C12 cells. On the other hand, anisotropic topography primarily directed the direction and alignment of myoblasts and myotubes. The results in this study provide information regarding the relative roles of chemical and topographic cues in musculoskeletal myogenesis, and are of interest to applications in muscle tissue engineering. Biotechnol. Bioeng. 2012; 109:2104–2115.

Grooved PLGA films incorporated with RGD/YIGSR peptides for potential application on skeletal muscle tissue engineering.

Alignment of myocytes or myotubes is critical for skeletal muscle tissue engineering. In this study, grooved PLGA films (800nm in width of ridge/groove and 600nm in depth) incorporated with RGD or YIGSR peptides were fabricated to evaluate its efficacy for skeletal muscle tissue engineering. The growth and differentiation of C2C12 myoblasts were enhanced by the presentation of RGD or YIGSR compared with the untreated PLGA control. On the other hand, cell morphology was guided by the grooved structure, i.e. alignment of myoblasts and myotubes with the direction of grooves. This study elucidates the effects of both surface biochemical and topographic cues on the proliferation and differentiation of C2C12 myoblasts on biodegradable polymer films. Combination of surface topography and peptide presentation has a great potential in designing scaffolds for skeletal muscle tissue engineering.

Electrochemistry-enabled fabrication of orthogonal nanotopography and surface chemistry gradients for high-throughput screening.

Gradient surfaces are emerging tools for investigating mammalian cell-surface interactions in high throughput. We demonstrate the electrochemical fabrication of an orthogonal gradient platform combining a porous silicon (pSi) pore size gradient with an orthogonal gradient of peptide ligand density. pSi gradients were fabricated via the anodic etching of a silicon wafer with pore sizes ranging from hundreds to tens of nanometers. A chemical gradient of ethyl-6-bromohexanoate was generated orthogonally to the pSi gradient via electrochemical attachment. Subsequent hydrolysis and activation of the chemical gradient allowed for the generation of a cyclic RGD gradient. Whilst mesenchymal stem cells (MSC) were shown to respond to both the topographical and chemical cues arising from the orthogonal gradient, the MSCs responded more strongly to changes in RGD density than to changes in pore size during short-term culture.

Dynamic compression modulates chondrocyte proliferation and matrix biosynthesis in chitosan/gelatin scaffolds

It is well-documented that dynamical compression stimulates biosynthesis of extracellular biomacromolecules in cartilage explant or in chondrocyte/hydrogel systems. The object of this study was to apply high-strain dynamic compression to cell-seeded elastic scaffolds for articular cartilage tissue engineering. Rabbit chondrocytes had been cultured in chitosan/gelatin scaffolds for 3 days before dynamic compression. The chondrocyte/scaffold constructs were subjected to short-term (3 or 9 h) or long-term (6 h/day for 3 weeks) cyclic compression with 40% strain and 0.1 Hz. The expression of type II collagen and aggrecan was upregulated after 3-h of compression when compared with the free-swelling samples. Furthermore, long-term culture under dynamic compression facilitated cellular proliferation and deposition of glycosaminoglycan. Our results suggest that high-strain dynamic compression combined with elastic scaffolds might benefit articular cartilage tissue engineering.

Modulation of human mesenchymal stem cell behavior on ordered tantalum nanotopographies fabricated using colloidal lithography and glancing angle deposition

Ordered surface nanostructures have attracted much attention in biotechnology and biomedical engineering because of their potential to modulate cell-surface interactions in a controllable manner. However, the ability to fabricate large area ordered nanostructures is limited because of high costs and low speed of fabrication. Here, we have fabricated ordered nanostructures with large surface areas (1.5 × 1.5 cm(2)) using a combination of facile techniques including colloidal self-assembly, colloidal lithography and glancing angle deposition (GLAD). Polystyrene (722 nm) colloids were self-assembled into a hexagonally close-packed (hcp) crystal array at the water-air interface, transferred on a biocompatible tantalum (Ta) surface and used as a mask to generate an ordered Ta pattern. The Ta was deposited by sputter coating through the crystal mask creating approximately 60-nm-high feature sizes. The feature size was further increased by approximately 200-nm-height respectively using GLAD, resulting in the fabrication of four different surfaces (FLAT, Ta60, GLAD100, and GLAD200). Cell adhesion, proliferation, and osteogenic differentiation of primary human adipose-derived stem cells (hADSCs) were studied on these ordered nanostructures for up to 2 weeks. Our results suggested that cell spreading, focal adhesion formation, and filopodia extension of hADSCs were inhibited on the GLAD surfaces, while the growth rate was similar between each surface. Immunostaining for type I collagen (COL1) and osteocalcin (OC) showed that there was higher osteogenic components deposited on the GLAD surfaces compared to the Ta60 and FLAT surfaces after 1 week of osteogenic culture. After 2 weeks of osteogenic culture, alkaline phosphatase (ALP) activity and the amount of calcium was higher on the GLAD surfaces. In addition, osteoblast-like cells were confluent on Ta60 and FLAT surfaces, whereas the GLAD surfaces were not fully covered suggesting that the cell-cell interactions are stronger than cell-substrate interactions on GLAD surfaces. Visible extracellular matrix deposits decorated the porous surface can be found on the GLAD surfaces. Depth profiling of surface components using a new Ar cluster source and X-ray photoelectron spectroscopy (XPS) showed that deposited extracellular matrix on GLAD surfaces is rich in nitrogen. The fabricated ordered surface nanotopographies have potential to be applied in diverse fields, and demonstrate that the behavior of human stem cells can be directed on these ordered nanotopographies, providing new knowledge for applications in biomaterials and tissue engineering.

Modulation of cell attachment and collagen production of anterior cruciate ligament cells via submicron grooves/ridges structures with different cell affinity

This study aimed to investigate the effects of submicron‐grooved topography and surface cell affinity on the attachment, proliferation and collagen synthesis of anterior cruciate ligament (ACL) cells. Two grooved polystyrene (PS) surfaces (equal groove/ridge width of 800 nm) with a groove depth of 100 or 700 nm were fabricated and modified by oxygen plasma treatment, dopamine deposition and conjugation of RGD‐containing peptides to enhance cell affinity. The elongation and alignment of ACL cells was enhanced by grooved structures with increasing groove depths regardless of surface chemistry. On the other hand, cell spreading and proliferation mainly depended on surface chemistry, in accordance with surface cell affinity: O2 plasma < dopamine deposition < RGD conjugation. The synthesis of type I collagen was the highest by the ACL cells cultured on the 700 nm grooved surface conjugated with RGD peptides, indicating that both surface grooved topography and chemistry play a role in modulating collagen production of ACL cells. Furthermore, the type I collagen deposited on the 700 nm PS surface was aligned with grooves/ridges. Our results indicated that both ligand presentation and cell alignment are important in the physiological activities of ACL fibroblasts. Such information is critical for design of biomaterials for ACL tissue engineering. Biotechnol. Bioeng. 2013; 110: 327–337.

Modulation of human multipotent and pluripotent stem cells using surface nanotopographies and surface-immobilised bioactive signals: A review

The ability to control the interactions of stem cells with synthetic surfaces is proving to be effective and essential for the quality of passaged stem cells and ultimately the success of regenerative medicine. The stem cell niche is crucial for stem cell self-renewal and differentiation. Thus, mimicking the stem cell niche, and here in particular the extracellular matrix (ECM), in vitro is an important goal for the expansion of stem cells and their applications. Here, surface nanotopographies and surface-immobilised biosignals have been identified as major factors that control stem cell responses. The development of tailored surfaces having an optimum nanotopography and displaying suitable biosignals is proposed to be essential for future stem cell culture, cell therapy and regenerative medicine applications. While early research in the field has been restricted by the limited availability of micro- and nanofabrication techniques, new approaches involving the use of advanced fabrication and surface immobilisation methods are starting to emerge. In addition, new cell types such as induced pluripotent stem cells (iPSCs) have become available in the last decade, but have not been fully understood. This review summarises significant advances in the area and focuses on the approaches that are aimed at controlling the behavior of human stem cells including maintenance of their self-renewal ability and improvement of their lineage commitment using nanotopographies and biosignals. More specifically, we discuss developments in biointerface science that are an important driving force for new biomedical materials and advances in bioengineering aiming at improving stem cell culture protocols and 3D scaffolds for clinical applications. Cellular responses revolve around the interplay between the surface properties of the cell culture substrate and the biomolecular composition of the cell culture medium. Determination of the precise role played by each factor, as well as the synergistic effects amongst the factors, all of which influence stem cell responses is essential for future developments. This review provides an overview of the current state-of-the-art in the design of complex material surfaces aimed at being the next generation of tools tailored for applications in cell culture and regenerative medicine. STATEMENT OF SIGNIFICANCE This review focuses on the effect of surface nanotopographies and surface-bound biosignals on human stem cells. Recently, stem cell research attracts much attention especially the induced pluripotent stem cells (iPSCs) and direct lineage reprogramming. The fast advance of stem cell research benefits disease treatment and cell therapy. On the other hand, surface property of cell adhered materials has been demonstrated very important for in vitro cell culture and regenerative medicine. Modulation of cell behavior using surfaces is costeffective and more defined. Thus, we summarise the recent progress of modulation of human stem cells using surface science. We believe that this review will capture a broad audience interested in topographical and chemical patterning aimed at understanding complex cellular responses to biomaterials.