A miniaturized bioreactor system for the evaluation of cell interaction with designed substrates in perfusion culture

In tissue engineering, the chemical and topographical cues within three-dimensional (3D) scaffolds are normally tested using static cell cultures but applied directly to tissue cultures in perfusion bioreactors. As human cells are very sensitive to the changes of culture environment, it is essential to evaluate the performance of any chemical, and topographical cues in a perfused environment before they are applied to tissue engineering. Thus the aim of this research was to bridge the gap between static and perfusion cultures by addressing the effect of perfusion on cell cultures within 3D scaffolds. For this we developed a scale down bioreactor system, which allows to evaluate the effectiveness of various chemical and topographical cues incorporated into our previously developed tubular ε-polycaprolactone scaffold under perfused conditions. Investigation of two exemplary cell types (fibroblasts and cortical astrocytes) using the miniaturized bioreactor indicated that: (1) quick and firm cell adhesion in 3D scaffold was critical for cell survival in perfusion culture compared with static culture, thus cell seeding procedures for static cultures might not be applicable. Therefore it was necessary to re-evaluate cell attachment on different surfaces under perfused conditions before a 3D scaffold was applied for tissue cultures, (2) continuous medium perfusion adversely influenced cell spread and survival, which could be balanced by intermittent perfusion, (3) micro-grooves still maintained its influences on cell alignment under perfused conditions, while medium perfusion demonstrated additional influence on fibroblast alignment but not on astrocyte alignment on grooved substrates. This research demonstrated that the mini-bioreactor system is crucial for the development of functional scaffolds with suitable chemical and topographical cues by bridging the gap between static culture and perfusion culture

Osteogenic lineage restriction by osteoprogenitors cultured on nanometric grooved surfaces – the role of focal adhesion maturation

The differentiation of progenitor cells is dependent on more than biochemical signalling. Topographical cues in natural bone extracellular matrix guide cellular differentiation through the formation of focal adhesions, contact guidance, cytoskeletal rearrangement and ultimately gene expression. Osteoarthritis and a number of bone disorders present as growing challenges for our society. Hence, there is a need for next generation implantable devices to substitute for, or guide, bone repair in vivo. Cellular responses to nanometric topographical cues need to be better understood in vitro in order to ensure the effective and efficient integration and performance of these orthopaedic devices. In this study, the FDA approved plastic polycaprolactone, was embossed with nanometric grooves and the response of primary and immortalised osteoprogenitor cells observed. Nanometric groove dimensions were 240 nm or 540 nm deep and 12.5 μm wide. Cells cultured on test surfaces followed contact guidance along the length of groove edges, elongated along their major axis and showed nuclear distortion, they formed more focal complexes and a lower proportions of mature adhesions relative to planar controls. Down-regulation of the osteoblast marker genes RUNX2 and BMPR2 in primary and immortalised cells was observed on grooved substrates. Down-regulation appeared to directly correlate with focal adhesion maturation, indicating the involvement of ERK 1/2 negative feedback pathways following integrin mediated FAK activation

A miniaturized bioreactor system for the evaluation of cell interaction with designed substrates in perfusion culture

In tissue engineering, the chemical and topographical cues within three-dimensional (3D) scaffolds are normally tested using static cell cultures but applied directly to tissue cultures in perfusion bioreactors. As human cells are very sensitive to the changes of culture environment, it is essential to evaluate the performance of any chemical, and topographical cues in a perfused environment before they are applied to tissue engineering. Thus the aim of this research was to bridge the gap between static and perfusion cultures by addressing the effect of perfusion on cell cultures within 3D scaffolds. For this we developed a scale down bioreactor system, which allows to evaluate the effectiveness of various chemical and topographical cues incorporated into our previously developed tubular ε-polycaprolactone scaffold under perfused conditions. Investigation of two exemplary cell types (fibroblasts and cortical astrocytes) using the miniaturized bioreactor indicated that: (1) quick and firm cell adhesion in 3D scaffold was critical for cell survival in perfusion culture compared with static culture, thus cell seeding procedures for static cultures might not be applicable. Therefore it was necessary to re-evaluate cell attachment on different surfaces under perfused conditions before a 3D scaffold was applied for tissue cultures, (2) continuous medium perfusion adversely influenced cell spread and survival, which could be balanced by intermittent perfusion, (3) micro-grooves still maintained its influences on cell alignment under perfused conditions, while medium perfusion demonstrated additional influence on fibroblast alignment but not on astrocyte alignment on grooved substrates. This research demonstrated that the mini-bioreactor system is crucial for the development of functional scaffolds with suitable chemical and topographical cues by bridging the gap between static culture and perfusion culture

Self-folding nano- and micropatterned hydrogel tissue engineering scaffolds by single step photolithographic process

Current progress in tissue engineering is focused on the creation of environments in which cultures of relevant cells can adhere, grow and form functional tissue. We propose a method for controlled chemical and topographical cues through surface patterning of self-folding hydrogel films. This provides a conversion of 2D patterning techniques into a viable method of manufacturing a 3D scaffold. While similar bilayers have previously been demonstrated, here we present a faster and high throughput process for fabricating self-folding hydrogel devices incorporating controllable surface nanotopographies by serial hot embossing of sacrificial layers and photolithography

Topographical cues regulate the crosstalk between MSCs and macrophages

Implantation of scaffolds may elicit a host foreign body response triggered by monocyte/macrophage lineage cells. Growing evidence suggests that topographical cues of scaffolds play an important role in MSC functionality. In this work, we examined whether surface topographical features can regulate paracrine interactions that MSCs establish with macrophages. Three-dimensional (3D) topography sensing drives MSCs into a spatial arrangement that stimulates the production of the anti-inflammatory proteins PGE(2) and TSG-6. Compared to two-dimensional (2D) settings, 3D arrangement of MSCs co-cultured with macrophages leads to an important decrease in the secretion of soluble factors related with inflammation and chemotaxis including IL-6 and MCP-1. Attenuation of MCP-1 secretion in 3D co-cultures correlates with a decrease in the accumulation of its mRNA levels in MSCs and macrophages. Using neutralizing antibodies, we identified that the interplay between PGE2, IL-6, TSG-6 and MCP-1 in the co-cultures is strongly influenced by the micro-architecture that supports MSCs. Local inflammatory milieu provided by 3D-arranged MSCs in co-cultures induces a decrease in monocyte migration as compared to-monolayer tells. This effect is partially mediated by reduced levels of IL-6 and MCP-1, proteins that up-regulate each other's secretion. Our findings highlight the importance of topographical cues in the soluble factor-guided communication between MSCs and macrophages

Surface Roughness Gradients Reveal Topography‐Specific Mechanosensitive Responses in Human Mesenchymal Stem Cells

The topographic features of an implant, which mechanically regulate cell behaviors and functions, are critical for the clinical success in tissue regeneration. How cells sense and respond to the topographical cues, e.g., interfacial roughness, is yet to be fully understood and even debatable. Here, the mechanotransduction and fate determination of human mesenchymal stem cells (MSCs) on surface roughness gradients are systematically studied. The broad range of topographical scales and high‐throughput imaging is achieved based on a catecholic polyglycerol coating fabricated by a one‐step‐tilted dip‐coating approach. It is revealed that the adhesion of MSCs is biphasically regulated by interfacial roughness. The cell mechanotransduction is investigated from focal adhesion to transcriptional activity, which explains that cellular response to interfacial roughness undergoes a direct force‐dependent mechanism. Moreover, the optimized roughness for promoting cell fate specification is explored

Controlling the morphology and outgrowth of nerve and neuroglial cells: The effect of surface topography

Unlike other tissue types, like epithelial tissue, which consist of cells with a much more homogeneous structure and function, the nervous tissue spans in a complex multilayer environment whose topographical features display a large spectrum of morphologies and size scales. Traditional cell cultures, which are based on two-dimensional cell-adhesive culture dishes or coverslips, are lacking topographical cues and mainly simulate the biochemical microenvironment of the cells. With the emergence of micro- and nano-fabrication techniques new types of cell culture platforms are developed, where the effect of various topographical cues on cellular morphology, proliferation and differentiation, can be studied. Different approaches (regarding the material, fabrication technique, topographical charactertistics, etc.) have been implemented. The present review paper aims at reviewing the existing body of literature on the use of artificial micro- and nano-topographical features to control neuronal morphology, outgrowth and neural network topology. The cell responses from phenomenology to investigation of the underlying mechanisms- on the different topographies, including both deterministic and random ones, are summarized

Micro- and nanoengineering approaches to control stem cell-biomaterial interactions.

As our population ages, there is a greater need for a suitable supply of engineered tissues to address a range of debilitating ailments. Stem cell based therapies are envisioned to meet this emerging need. Despite significant progress in controlling stem cell differentiation, it is still difficult to engineer human tissue constructs for transplantation. Recent advances in micro- and nanofabrication techniques have enabled the design of more biomimetic biomaterials that may be used to direct the fate of stem cells. These biomaterials could have a significant impact on the next generation of stem cell based therapies. Here, we highlight the recent progress made by micro- and nanoengineering techniques in the biomaterials field in the context of directing stem cell differentiation. Particular attention is given to the effect of surface topography, chemistry, mechanics and micro- and nanopatterns on the differentiation of embryonic, mesenchymal and neural stem cells

Enhanced differentiation of retinal progenitor cells using microfabricated topographical cues

Due to the retina’s inability to replace photoreceptors lost during retinal degeneration, significant interest has been placed in methods to implant replacement cells. Polymer scaffolds are increasingly being studied as vehicles for cellular delivery to degenerated retinas. Previously, we fabricated poly(methyl methacrylate) thin film scaffolds that increased survival and integration of implanted retinal progenitor cells (RPCs). Additionally, these scaffolds minimized the trauma and cellular response associated with implantation of foreign bodies into mouse eyes. Here, we demonstrate that biodegradable polycaprolactone (PCL) thin film scaffolds can be fabricated with integrated microtopography. Microfabricated topography in a PCL thin film enhanced the attachment and organization of RPCs compared to unstructured surfaces. Using real-time quantitative polymerase chain reaction we also observed that attachment to microtopography induced cellular differentiation. RPCs grown on PCL thin films exhibited an increase in gene expression for the photoreceptor markers recoverin and rhodopsin, an increase in the glial and Müller cell marker GFAP, and a decrease in SOX2 gene expression (a marker for undifferentiated progenitor cells) compared to cells grown on unmodified tissue culture polystyrene (TCPS)

Two Distinct Filopodia Populations at the Growth Cone Allow to Sense Nanotopographical Extracellular Matrix Cues to Guide Neurite Outgrowth

The process of neurite outgrowth is the initial step in producing the neuronal processes that wire the brain. Current models about neurite outgrowth have been derived from classic two-dimensional (2D) cell culture systems, which do not recapitulate the topographical cues that are present in the extracellular matrix (ECM) in vivo. Here, we explore how ECM nanotopography influences neurite outgrowth