Culture surface protein coatings affect the barrier properties and calcium signalling of hESC-RPE.

Human pluripotent stem cell-derived retinal pigment epithelium (RPE) transplantation is currently under evaluation as treatment for macular degeneration. For therapeutic applications, cryostorage during cell production is typically needed with potential consequences to cell functionality. We have previously shown that the culture substrate affects human embryonic stem cell-derived RPE (hESC-RPE) properties in fresh cultures. Here, we aimed to further identify the role of RPE basement membrane proteins type IV collagen (Col-IV), laminin (LN), and nidogen-1 in the maturation and functionality of hESC-RPE after cryopreservation. In addition to cell attachment and morphology, transepithelial electrical resistance, expression of key RPE proteins, phagocytosis capacity and Ca2+ signalling were analysed. After cryostorage, attachment of hESC-RPE on culture surfaces coated with Col-IV alone was poor. Combining Col-IV and LN with or without nidogen-1 significantly improved cell attachment and barrier properties of the epithelium. Furthermore, functional homogeneity of the hESC-RPE monolayer was enhanced in the presence of nidogen-1. Our results suggest that the choice of coating proteins for the cell culture may have implications to the functional properties of these cells after cryostorage cell banking.

Cell Type-Specific Adhesion and Migration on Laser-Structured Opaque Surfaces.

Cytocompatibility is essential for implant approval. However, initial in vitro screenings mainly include the quantity of adherent immortalized cells and cytotoxicity. Other vital parameters, such as cell migration and an in-depth understanding of the interaction between native tissue cells and implant surfaces, are rarely considered. We investigated different laser-fabricated spike structures using primary and immortalized cell lines of fibroblasts and osteoblasts and included quantification of the cell area, aspect ratio, and focal adhesions. Furthermore, we examined the three-dimensional cell interactions with spike topographies and developed a tailored migration assay for long-term monitoring on opaque materials. While fibroblasts and osteoblasts on small spikes retained their normal morphology, cells on medium and large spikes sank into the structures, affecting the composition of the cytoskeleton and thereby changing cell shape. Up to 14 days, migration appeared stronger on small spikes, probably as a consequence of adequate focal adhesion formation and an intact cytoskeleton, whereas human primary cells revealed differences in comparison to immortalized cell lines. The use of primary cells, analysis of the cell-implant structure interaction as well as cell migration might strengthen the evaluation of cytocompatibility and thereby improve the validity regarding the putative in vivo performance of implant material.

Correlation between ECM guidance and actin polymerization on osteogenic differentiation of human adipose-derived stem cells.

The correlation between extracellular matrix (ECM) components, cell shape, and stem cell guidance can shed light in understanding and mimicking the functionality of stem cell niches for various applications. This interplay on osteogenic guidance of human adipose-derived stem cells (hASCs) was focus of this study. Proliferation and osteogenic markers like alkaline phosphatase activity and calcium mineralization were slightly increased by the ECM components laminin (LA), collagen I (COL), and fibronectin (FIB); with control medium no differentiation occurred. ECM guided differentiation was rather dependent on osterix than on Runx2 pathway. FIB significantly enhanced cell elongation even in presence of actin polymerization blockers cytochalasin D (CytoD) and ROCK inhibitor Y-27632, which generally caused more rounded cells. Except for the COL surface, both inhibitors increased the extent of osterix, while the Runx2 pathway was more sensitive to the culture condition. Both inhibitors did not affect hASC proliferation. CytoD enabled osteogenic differentiation independently from the ECM, while it was rather blocked via Y-27632 treatment; on FIB the general highest extent of differentiation occurred. Taken together, the ECM effect on hASCs occurs indirectly and selectively via a dominant role of FIB: it sustains osteogenic differentiation in case of a tension-dependent control of actin polymerization.

Water-soluble photopolymerizable chitosan hydrogels for biofabrication via two-photon polymerization.

Fabrication of three-dimensional (3D) hydrogel microenvironments with predefined geometry and porosity can facilitate important requirements in tissue engineering and regenerative medicine. Chitosan (CH) is well known as a biocompatible hydrogel with prospective biological properties for biomedical aims. So far, microstructuring of this soft material presents a great limitation for its application as functional supporting material for guided tissue formation. Enabling photopolymerization, chemically modified CH can be applied for the biofabrication of reproducible 3D scaffolds using rapid prototyping techniques like two-photon polymerization (2PP) or others. The application of this technique allows precise serial fabrication of computer-designed microstructure geometries by scanning a femtosecond laser beam within a photosensitive material. This work explores a new synthesis of water-soluble photosensitive chitosan and the fabrication of well-defined microstructures from the generated materials. To modulate the mechanical and biochemical properties of the material, CH was combined and cross-linked with synthetic poly(ethylene glycol) diacrylate. For a biological adaption to the in vivo situation, CH was covalently crosslinked with a photosensitive modified vascular endothelial growth factor (VEGF). Performed in vitro studies reveal that modified CH is biocompatible. VEGF enhances CH bioactivity. Furthermore, a 3D CH scaffold can be successfully seeded with cells. Therefore, the established CH holds great promise for future applications in tissue engineering.

Osteogenic differentiation of human mesenchymal stem cells in 3-D Zr-Si organic-inorganic scaffolds produced by two-photon polymerization technique.

Two-photon polymerization (2PP) is applied for the fabrication of 3-D Zr-Si scaffolds for bone tissue engineering. Zr-Si scaffolds with 150, 200, and 250 µm pore sizes are seeded with human bone marrow stem cells (hBMSCs) and human adipose tissue derived stem cells (hASCs) and cultured in osteoinductive and control media for three weeks. Osteogenic differentiation of hASCs and hBMSCs and formation of bone matrix is comparatively analyzed via alkaline phosphatase activity (ALP), calcium quantification, osteocalcin staining and scanning electron microscopy (SEM). It is observed that the 150 µm pore size Zr-Si scaffolds support the strongest matrix mineralization, as confirmed by calcium deposition. Analysis of ALP activity, osteocalcin staining and SEM observations of matrix mineralization reveal that mesenchymal stem cells cultured on 3-D scaffolds without osteogenic stimulation spontaneously differentiate towards osteogenic lineage. Nanoindentation measurements show that aging of the 2PP-produced Zr-Si scaffolds in aqueous or alcohol media results in an increase in the scaffold Young's modulus and hardness. Moreover, accelerated formation of bone matrix by hASCs is noted, when cultured on the scaffolds with lower Young's moduli and hardness values (non aged scaffolds) compared to the cells cultured on scaffolds with higher Young's modulus and hardness values (aged scaffolds). Presented results support the potential application of Zr-Si scaffolds for autologous bone tissue engineering.

Impact of laser-structured biomaterial interfaces on guided cell responses.

To achieve a perfect integration of biomaterials into the body, tissue formation in contact with the interface has to be controlled. In this connection, a selective cell control is required: fibrotic encapsulation has to be inhibited, while tissue guidance has to be stimulated. As conventional biomaterials do not fulfil this specification, functionalization of the biointerface is under development to mimic the natural environment of the cells. One approach focuses on the fabrication of defined surface topographies. Thereby, ultrashort pulse laser ablation is very beneficial, owing to a large variety of fabricated structures, reduced heat-affected zones, high precision and reproducibility. We demonstrate that nanostructures in platinum and microstructures in silicon selectively control cell behaviour: inhibiting fibroblasts, while stimulating neuronal attachment and differentiation. However, the control of fibroblasts strongly correlates with the created size dimensions of the surface structures. These findings suggest favourable biomaterial interfaces for electronic devices. The mechanisms which are responsible for selective cell control are poorly understood. To give an insight, cell behaviour in dependence of biomaterial interfaces is discussed-including basic research on the role of the extracellular matrix. This knowledge is essential to understand such specific cell responses and to optimize biomaterial interfaces for future biomedical applications.

Hyaluronic acid based materials for scaffolding via two-photon polymerization.

Hydrogels are able to mimic the basic three-dimensional (3D) biological, chemical, and mechanical properties of native tissues. Since hyaluronic acid (HA) is a chief component of human extracellular matrix (ECM), it represents an extremely attractive starting material for the fabrication of scaffolds for tissue engineering. Due to poor mechanical properties of hydrogels, structure fabrication of this material class remains a major challenge. Two-photon polymerization (2PP) is a promising technique for biomedical applications, which allows the fabrication of complex 3D microstructures by moving the laser focus in the volume of a photosensitive material. Chemical modification of hyaluronan allows application of the 2PP technique to this natural material and, thus, precise fabrication of 3D hydrogel constructs. To create materials with tailor-made mechanochemical properties, HA was combined and covalently cross-linked with poly(ethylene glycol) diacrylate (PEGDA) in situ. 2PP was applied for the fabrication of well elaborated 3D HA and HA-PEGDA microstructures. For enhanced biological adaption, HA was functionalized with human epidermal growth factor.

Laser microstructured biodegradable scaffolds.

The two-photon polymerization technique (2PP) uses non-linear absorption of femtosecond laser pulses to selectively polymerize photosensitive materials. 2PP has the ability to fabricate structures with a resolution from tens of micrometers down to hundreds of nanometers. Three-dimensional microstructuring by the 2PP technique provides many interesting possibilities for biomedical applications. This microstructuring technique is suitable with many biocompatible polymeric materials, such as polyethylene glycol, polylactic acid, polycaprolactone, gelatin, zirconium-based hybrids, and others. The process of fabrication does not require clean room conditions and does not use hazard chemicals or high temperatures. The most beneficial property of 2PP is that it is capable of producing especially complex three-dimensional (3-D) structures, including devices with overhangs, without using any supportive structure. The flexibility in controlling geometries and feature sizes and the possibility to fabricate structures without the addition of new material layers makes this technique particularly appealing for fabrication of 3-D scaffolds for tissue engineering.

The selective role of ECM components on cell adhesion, morphology, proliferation and communication in vitro.

Cell binding to the extracellular matrix (ECM) is essential for cell and tissue functions. In this context, each tissue consists of a unique ECM composition, which may be responsible for tissue-specific cell responses. Due to the complexity of ECM-cell interactions-which depend on the interplay of inside-out and outside-in signaling cascades, cell and tissue specificity of ECM-guidance is poorly understood. In this paper, we investigate the role of different ECM components like laminin, fibronectin, and collagen type I with respect to the essential cell behaviour patterns: attachment dynamics such as adhesion kinetic and force, formation of focal adhesion complexes, morphology, proliferation, and intercellular communication. A detailed in vitro comparison of fibroblasts, endothelial cells, osteoblasts, smooth muscle cells, and chondrocytes reveals significant differences in their cell responses to the ECM: cell behaviour follows a cell specific ligand priority ranking, which was independent of the cell type origin. Fibroblasts responded best to fibronectin, chondrocytes best to collagen I, the other cell types best to laminin. This knowledge is essential for optimization of tissue-biomaterial interfaces in all tissue engineering applications and gives insight into tissue-specific cell guidance.

Topography and coating of platinum improve the electrochemical properties and neuronal guidance.

To improve neuronal-electrode interfaces, we analyzed the influence of surface topographies combined with coating on the electrochemistry of platinum and neuronal differentiation of PC-12 cells. Surface structuring on nanoscale was realized by femtosecond laser ablation. Additional coating with laminin (LA), collagen type I (COL) or poly-d-lysine (PDL) did not change the produced topography. We further demonstrated that impedance could be improved in all cases. The pre-requisites of differentiation - viability and attachment - were fulfilled on the topography. Cell attachment of non-differentiated and differentiated cells and their formation of focal adhesion complexes were even enhanced compared to unstructured platinum. However, without the nerve growth factor (NGF) no cellular outgrowth and differentiation were possible. The topography enabled cell elongation and reduced the amount of rounded cells, but less effective than coating. Differentiation was either comparable or increased on the structures when compared with unstructured coatings. For instance, microtubule associated protein (MAP2) was detected most on the topography alone. But a combination of surface structuring and coating had the strongest impact on differentiation: the usage of COL provoked best cell elongation and beta III tubulin expression, PDL best synaptophysin. LA-coating had no noteworthy effect. These findings point out that innovative electronic devices like cochlear implants include two aspects: (a) nanotopography to improve the transmission of electrical signals and neuronal attachment; and (b) an additional coating to stimulate neuronal differentiation.

Two-photon polymerization microstructuring in regenerative medicine.

Two-photon polymerization has developed as a powerful tool for making micro- and nanoscale structures for regenerative medicine applications. This review discusses micro- and nanoscale aspects of tissue engineering, which are followed by a brief description of the two-photon polymerization process and how it has been used thus far in tissue engineering and other regenerative medicine applications. Lastly, potential future applications of two-photon polymerization in regenerative medicine are presented. This review provides a comprehensive summary of the uses of two-photon polymerization thus far in regenerative medicine and a look into how this technique will be used in the future.