Solid-phase silica-based extraction leads to underestimation of residual DNA in decellularized tissues.

Decellularization of animal tissues is a novel route to obtain biomaterials for use in tissue engineering and organ transplantation. Successful decellularization is required as animal DNA causes inflammatory reactions and contains endogenous retroviruses, which could be transmitted to the patient. One of the criteria for successful decellularization is digestion (fragmentation) and elimination (residual quantity) of DNA from the tissue. Quantification of DNA can be done in many ways, but it has recently been shown that silica-based solid-phase extraction methods often do not completely purify in particular small DNA fragments. In the context of decellularization, this means that the measured DNA amount is underestimated, which could compromise safety of the processed tissue for in-patient use. In this article, we review DNA quantification methods used by researchers and assess their influence on the reported DNA contents after decellularization. We find that underestimation of residual DNA amount after silica-based solid-phase extraction may be as large as a factor of ten. We therefore recommend a direct assessment of DNA amount in tissue lysate using dsDNA-specific binding dyes, such as Picogreen, due to their higher accuracy for small fragment detection as well as ease of use and widespread availability.

The loop of phenotype: Dynamic reciprocity links tenocyte morphology to tendon tissue homeostasis.

Cells and their surrounding extracellular matrix (ECM) are engaged in dynamic reciprocity to maintain tissue homeostasis: cells deposit ECM, which in turn presents the signals that define cell identity. This loop of phenotype is obvious for biochemical signals, such as collagens, which are produced by and presented to cells, but the role of biomechanical signals is also increasingly recognised. In addition, cell shape goes hand in hand with cell function and tissue homeostasis. Aberrant cell shape and ECM is seen in pathological conditions, and control of cell shape in micro-fabricated platforms disclose the causal relationship between cell shape and cell function, often mediated by mechanotransduction. In this manuscript, we discuss the loop of phenotype for tendon tissue homeostasis. We describe cell shape and ECM organization in normal and diseased tissue, how ECM composition influences tenocyte shape, and how that leads to the activation of signal transduction pathways and ECM deposition. We further describe the use of technologies to control cell shape to elucidate the link between cell shape and its phenotypical markers and focus on the causal role of cell shape in the loop of phenotype. STATEMENT OF SIGNIFICANCE: The dynamic reciprocity between cells and their surrounding extracellular matrix (ECM) influences biomechanical and biochemical properties of ECM as well as cell function through activation of signal transduction pathways that regulate gene and protein expression. We refer to this reciprocity as Loop of Phenotype and it has been studied and demonstrated extensively by using micro-fabricated platforms to manipulate cell shape and cell fate. In this manuscript, we discuss this concept in tendon tissue homeostasis by giving examples in healthy and pathological tenson tissue. Furthermore, we elaborate this by showing how biomaterials are used to feed this reciprocity to manipulate cell shape and function. Finally, we elucidate the link between cell shape and its phenotypical markers and focus on the activation of signal transduction pathways and ECM deposition.

Cells Dynamically Adapt to Surface Geometry by Remodeling Their Focal Adhesions and Actin Cytoskeleton.

Cells probe their environment and adapt their shape accordingly via the organization of focal adhesions and the actin cytoskeleton. In an earlier publication, we described the relationship between cell shape and physiology, for example, shape-induced differentiation, metabolism, and proliferation in mesenchymal stem cells and tenocytes. In this study, we investigated how these cells organize their adhesive machinery over time when exposed to microfabricated surfaces of different topographies and adhesive island geometries. We further examined the reciprocal interaction between stress fiber and focal adhesion formation by pharmacological perturbations. Our results confirm the current literature that spatial organization of adhesive sites determines the ability to form focal adhesions and stress fibers. Therefore, cells on roughened surfaces have smaller focal adhesion and fewer stress fibers. Our results further highlight the importance of integrin-mediated adhesion in the adaptive properties of cells and provide clear links to the development of bioactive materials.

Multiscale characterization of pathological bone tissue.

Bone is a complex natural material with a complex hierarchical multiscale organization, crucial to perform its functions. Ultrastructural analysis of bone is crucial for our understanding of cell to cell communication, the healthy or pathological composition of bone tissue, and its three-dimensional (3D) organization. A variety of techniques has been used to analyze bone tissue. This article describes a combined approach of optical, scanning electron, and transmission electron microscopy for the ultrastructural analysis of bone from the nanoscale to the macroscale, as illustrated by two pathological bone tissues. By following a top-down approach to investigate the multiscale organization of pathological bones, quantitative estimates were made in terms of calcium content, nearest neighbor distances of osteocytes, canaliculi diameter, ordering, and D-spacing of the collagen fibrils, and the orientation of intrafibrillar minerals which enable us to observe the fine structural details. We identify and discuss a series of two-dimensional (2D) and 3D imaging techniques that can be used to characterize bone tissue. By doing so we demonstrate that, while 2D imaging techniques provide comparable information from pathological bone tissues, significantly different structural details are observed upon analyzing the pathological bone tissues in 3D. Finally, particular attention is paid to sample preparation for and quantitative processing of data from electron microscopic analysis.

Self-agglomerated collagen patterns govern cell behaviour.

Reciprocity between cells and their surrounding extracellular matrix is one of the main drivers for cellular function and, in turn, matrix maintenance and remodelling. Unravelling how cells respond to their environment is key in understanding mechanisms of health and disease. In all these examples, matrix anisotropy is an important element, since it can alter the cell shape and fate. In this work, the objective is to develop and exploit easy-to-produce platforms that can be used to study the cellular response to natural proteins assembled into diverse topographical cues. We demonstrate a robust and simple approach to form collagen substrates with different topographies by evaporating droplets of a collagen solution. Upon evaporation of the collagen solution, a stain of collagen is left behind, composed of three regions with a distinct pattern: an isotropic region, a concentric ring pattern, and a radially oriented region. The formation and size of these regions can be controlled by the evaporation rate of the droplet and initial collagen concentration. The patterns form topographical cues inducing a pattern-specific cell (tenocyte) morphology, density, and proliferation. Rapid and cost-effective production of different self-agglomerated collagen topographies and their interfaces enables further study of the cell shape-phenotype relationship in vitro. Substrate topography and in analogy tissue architecture remains a cue that can and will be used to steer and understand cell function in vitro, which in turn can be applied in vivo, e.g. in optimizing tissue engineering applications.

Tendon-Derived Biomimetic Surface Topographies Induce Phenotypic Maintenance of Tenocytes In Vitro.

The tenocyte niche contains biochemical and biophysical signals that are needed for tendon homeostasis. The tenocyte phenotype is correlated with cell shape in vivo and in vitro, and shape-modifying cues are needed for tenocyte phenotypical maintenance. Indeed, cell shape changes from elongated to spread when cultured on a flat surface, and rat tenocytes lose the expression of phenotypical markers throughout five passages. We hypothesized that tendon gene expression can be preserved by culturing cells in the native tendon shape. To this end, we reproduced the tendon topographical landscape into tissue culture polystyrene, using imprinting technology. We confirmed that the imprints forced the cells into a more elongated shape, which correlated with the level of Scleraxis expression. When we cultured the tenocytes for 7 days on flat surfaces and tendon imprints, we observed a decline in tenogenic marker expression on flat but not on imprints. This research demonstrates that native tendon topography is an important factor contributing to the tenocyte phenotype. Tendon imprints therefore provide a powerful platform to explore the effect of instructive cues originating from native tendon topography on guiding cell shape, phenotype, and function of tendon-related cells.

Expanding Biomaterial Surface Topographical Design Space through Natural Surface Reproduction.

Surface topography is a tool to endow biomaterials with bioactive properties. However, the large number of possible designs makes it challenging to find the optimal surface structure to induce a specific cell response. The TopoChip platform is currently the largest collection of topographies with 2176 in silico designed microtopographies. Still, it is exploring only a small part of the design space due to design algorithm limitations and the surface engineering strategy. Inspired by the diversity of natural surfaces, it is assessed as to what extent the topographical design space and consequently the resulting cellular responses can be expanded using natural surfaces. To this end, 26 plant and insect surfaces are replicated in polystyrene and their surface properties are quantified using white light interferometry. Through machine-learning algorithms, it is demonstrated that natural surfaces extend the design space of the TopoChip, which coincides with distinct morphological and focal adhesion profiles in mesenchymal stem cells (MSCs) and Pseudomonas aeruginosa colonization. Furthermore, differentiation experiments reveal the strong potential of the holy lotus to improve osteogenesis in MSCs. In the future, the design algorithms will be trained with the results obtained by natural surface imprint experiments to explore the bioactive properties of novel surface topographies.

Identification of topographical architectures supporting the phenotype of rat tenocytes.

Tenocytes, the main cell type of the tendon, require mechanical stimuli for their proper function. When the tenocyte environment changes due to tissue damage or by transferring tenocytes from their native environment into cell culture, the signals from the tenocyte niche are lost, leading towards a decline of phenotypic markers. It is known that micro-topographies can influence cell fate by the physical cues they provide. To identify the optimal topography-induced biomechanical niche in vitro, we seeded tenocytes on the TopoChip, a micro-topographical screening platform, and measured expression of the tendon transcription factor Scleraxis. Through machine learning algorithms, we associated elevated Scleraxis levels with topological design parameters. Fabricating micro-topographies with optimal surface characteristics on larger surfaces allowed finding an improved expression of multiple tenogenic markers. However, long-term confluent culture conditions coincided with osteogenic marker expression and the loss of morphological characteristics. In contrast, passaging tenocytes which migrated from the tendon directly on the topography resulted in prolonged elongated morphology and elevated Scleraxis levels. This research provides new insights into how micro-topographies influence tenocyte cell fate, and supports the notion that micro-topographical design can be implemented in a new generation of tissue culture platforms for supporting the phenotype of tenocytes. STATEMENT OF SIGNIFICANCE: The challenge in controlling in vitro cell behavior lies in controlling the complex culture environment. Here, we present for the first time the use of micro-topographies as a biomechanical niche to support the phenotype of tenocytes. For this, we applied the TopoChip platform, a screening tool with 2176 unique micro-topographies for identifying feature characteristics associated with elevated Scleraxis expression, a tendon related marker. Large area fabrication of micro-topographies with favorable characteristics allowed us to find a beneficial influence on other tenogenic markers as well. Furthermore, passaging cells is more beneficial for Scleraxis marker expression and tenocyte morphology compared to confluent conditions. This study presents important insights for the understanding of tenocyte behavior in vitro, a necessary step towards tendon engineering.