Biology subjects

Pelling, A.

Publications and source records attributed to Pelling, A..

11 recordsLinked to original sources

bioRxiv2023

Plant Cellulose as a Substrate for 3D Neural Stem Cell Culture

Neural stem cell (NSC) based therapies are at the forefront of regenerative medicine strategies for various neural defects and injuries such as stroke, traumatic brain injury and spinal cord injury. For several clinical applications, NSC therapies require biocompatible scaffolds to support cell survival and to direct differentiation. Here, we investigate decellularized plant tissue as a novel scaffold for three-dimensional (3D) in vitro culture of NSCs. Plant cellulose scaffolds were shown to support attachment and proliferation of adult rat hippocampal neural stem cells (NSCs). Further, NSCs differentiated on the cellulose scaffold had significant increases in their expression of neuron-specific beta-III tubulin and glial fibrillary acidic protein compared to 2D culture on a polystyrene plate, indicating that the scaffold may enhance differentiation of NSCs towards astrocytic and neuronal lineages. Our findings suggest that plant-derived cellulose scaffolds have the potential to be used in neural tissue engineering and can be harnessed to direct differentiation of NSCs.

bioengineering↗

bioRxiv2021

Biomechanical study of cellulose scaffolds for bone tissue engineering in vivo and in vitro.

Plant-derived cellulose biomaterials have recently been utilized in several tissue engineering applications. These naturally-derived cellulose scaffolds have been shown to be highly biocompatible in vivo, possess structural features of relevance to several tissues, and support mammalian cell invasion and proliferation. Recent work utilizing decellularized apple hypanthium tissue has shown that it possesses a pore size similar to trabecular bone and can successfully host osteogenic differentiation. In the present study, we further examined the potential of apple-derived cellulose scaffolds for bone tissue engineering (BTE) and analyzed their mechanical properties in vitro and in vivo. MC3T3-E1 pre-osteoblasts were seeded in cellulose scaffolds. Following chemically-induced osteogenic differentiation, scaffolds were evaluated for mineralization and for their mechanical properties. Alkaline phosphatase and Alizarin Red staining confirmed the osteogenic potential of the scaffolds. Histological analysis of the constructs revealed cell invasion and mineralization throughout the constructs. Furthermore, scanning electron microscopy demonstrated the presence of mineral aggregates on the scaffolds after culture in differentiation medium, and energy-dispersive spectroscopy confirmed the presence of phosphate and calcium. However, although the Youngs modulus significantly increased after cell differentiation, it remained lower than that of healthy bone tissue. Interestingly, mechanical assessment of acellular scaffolds implanted in rat calvaria defects for 8 weeks revealed that the force required to push out the scaffolds from the surrounding bone was similar to that of native calvarial bone. In addition, cell infiltration and extracellular matrix deposition were visible within the implanted scaffolds. Overall, our results confirm that plant-derived cellulose is a promising candidate for BTE applications. However, the discrepancy in mechanical properties between the mineralized scaffolds and healthy bone tissue may limit their use to low load-bearing applications. Further structural re-engineering and optimization to improve the mechanical properties may be required for load-bearing applications.

bioengineering↗

bioRxiv2020

Homemade Bread: Repurposing an Ancient Technology for Low Cost in vitro Tissue Engineering

Numerous synthetic and naturally-occurring biomaterials have been developed to provide such architectures to support the proliferation of mammalian cells in vitro and in vivo. Our group, and others, have shown that scaffolds derived from plants can be utilized for tissue engineering applications in biomedicine and in the burgeoning cultured meat industry. Such scaffolds are ideally straightforward and inexpensive to prepare, allowing researchers to take advantage of their intrinsic 3D microarchitectures. These efforts inspired us to continue to pursue the development of novel and unconventional biomaterials that are easily produced and high performing in vitro. With this in mind, few plant-derived materials are more ubiquitous than bread. Having observed the porosity of the crumb (i.e. the internal bulk) we sought to investigate whether it might support the proliferation of mammalian cells in vitro. Here, we develop and validate a yeast-free "soda bread" that maintains its mechanical stability over several weeks in culture conditions. Importantly, we also demonstrate that control over the mechanical stability of the scaffold can also be achieved with both chemical and enzymatic means. The scaffolding is a heterogeneous and complex structure of isolated and interconnected pores which allow for the proliferation of multiple cell types. We demonstrate here that mouse fibroblasts, myoblasts and pre-osteoblasts are able to proliferate up to four weeks in culture. Immunohistochemistry demonstrates that the fibroblasts are able to deposit their own fibronectin extracellular matrix and that mouse myoblasts are able to differentiate and fuse into myotubes. Although the pre-osteoblasts proliferated over the course of four weeks their ability to differentiate was inconclusive. Metabolic analyses of proliferation, cytotoxicity and oxidative stress reveal that cells remain highly viable and functional on these novel bread scaffolds. While the results presented in this proof-of-concept study create many new questions and opportunities, the results open up novel possibilities in the development of edible scaffolds that may be utilized in future food applications. Bread derived scaffolds represent a surprising alternative to synthetic or animal-derived scaffolds for addressing a diverse variety of tissue engineering challenges in food science. Future studies will delve deeper into investigating these how possibilities might take advantage of the immense breadth of knowledge about bread making and examine their applicability in the development of lab grown foods and broader applications in cellular agriculture.

bioengineering↗

bioRxiv2020

Engineered Tissue Interfaces for in vitro and in vivo Bioengineering

In regenerative medicine, the healing of the interfacial zone between tissues is a major challenge, yet approaches for studying the complex microenvironment of this interface remain lacking. Here, we study these complex living interfaces by manufacturing modular "blocks" of naturally porous decellularized plant-derived scaffolds with a computer numerical controlled mill. We demonstrate how each scaffold can be seeded with different cell types and easily assembled in a manner akin to LEGO bricks to create an engineered tissue interface (ETI). Cells migrate across the interface formed between an empty scaffold and a scaffold pre-seeded with cells. However, when both scaffolds contain cells, only a shallow cross-over zone of cell infiltration forms at the interface. As a proof-of-concept study, we use ETIs to investigate the interaction between lab grown bone and connective tissues. Consistent with the above, a cross-over zone of the two distinct cell types forms at the interface between scaffolds, otherwise the populations remain distinct. Finally, we demonstrate how ETIs are biocompatible in vivo, becoming vascularized and integrated into surrounding tissue after implantation. This work creates new tissue design avenues for understanding biological processes or the development of synthetic artificial tissues.

biophysics↗

bioRxiv2020

Decellularized Plant-Based Scaffolds for Guided Alignment of Myoblast Cells

Alignment and orientation of cells in vivo plays a crucial role in the functionality of tissue. A challenged faced by traditional cell culture approaches is that the majority of two-dimensional substrates fail to induce a controlled alignment of cells in vitro. To address this challenge, approaches utilizing mechanical stresses, exposure to electrical fields, structurally aligned biomaterials and/or textured microfabricated substrates, have been developed to control the organization of cells through microenvironmental stimuli. In the field of muscle tissue engineering it is often desirable to control the alignment and fusion of muscle precursor cells as it more closely resembles in vivo conditions. In this study, we utilize plant-derived cellulose biomaterials to control the in vitro alignment of C2C12 murine myoblasts. We hereby report that cells display a clear sensitivity to the highly aligned vascular bundle architectures found in decellularized celery (Apium graveolens). Conveniently, the xylem and phloem channels lie within the 10-100m diameter, which has been shown to be optimal diameter for myoblast alignment through contact guidance. Following 10 days in proliferation media, F-actin filaments were observed to be aligned parallel to the longitudinal axis of the vascular bundle. Subsequently, following 5 days in differentiation media, myoblast maintained an aligned morphology, which led to the formation of aligned myotubes. We therefore conclude that the microtopography of the vascular bundle guides muscle cell alignment. The results presented here highlight the potential of this plant-derived scaffold for in vitro studies of muscle myogenesis, where structural anisotropy is required to more closely resemble in vivo conditions.

biophysics↗

bioRxiv2020

Time dependencies in the dynamic mechanical behavior of 3D microtissue cell cultures

Characterizing the time-dependent mechanical properties of cells is not only necessary to determine how they deform, but also to fully understand how external forces trigger biochemical-signaling cascades to govern their behavior. Presently mechanical properties are largely assessed by applying local shear or compressive forces on single cells in isolation grown on non-physiological 2D surfaces. In comparison, we developed the microfabricated vacuum actuated stretcher to measure tensile loading of 3D multicellular microtissue cultures. With this approach, we assessed here the time-dependent stress relaxation and recovery responses of microtissues, and quantified the spatial remodeling that follows step length changes. Unlike previous results, stress relaxation and recovery in microtissues measured over a range of step amplitudes and pharmacological treatments followed a stretched exponential behavior describing a broad distribution of inter-related timescales. Furthermore, despite a performed compendium of experiments, all responses led to a single linear relationship between the residual elasticity and degree of stress relaxation, suggesting that these mechanical properties are coupled through interactions between structural elements and the association of cells with their matrix. Lastly, although stress relaxation could be quantitatively and spatially linked to recovery, they differed greatly in their dynamics; while stress recovery behaved as a linear process, relaxation time constants changed with an inverse power law with step size. This assessment of microtissues offers insights into how the collective behavior of cells in a 3D collagen matrix generate the dynamic mechanical properties of tissues, which is necessary to understanding how cells deform and sense mechanical forces in the body.

biophysics↗

bioRxiv2020

Mechanical stretch sustains myofibroblast phenotype and function in microtissues through latent TGF-β1 activation

Fibrosis is a leading cause of death in developed countries that is characterized by a progressive deterioration of tissue mechanical behavior. Developing methods to study tissue mechanics and myofibroblast activation may lead to new targets for therapeutic treatments that are urgently needed. Microtissue arrays are a promising approach to conduct relatively high throughput research into fibrosis as they recapitulate key biomechanical aspects of the disease through a relevant 3D extracellular environment. In early work, our group developed a device called the MVAS-force to stretch microtissues while enabling simultaneous assessment of their dynamic mechanical behavior. Here we investigated TGF-{beta}1 induced fibroblast to myofibroblast differentiation in microtissue cultures using our MVAS-force device through assessing -SMA expression, contractility and stiffness. By doing so, we linked cell-level phenotypic changes to functional changes that characterize the clinical manifestation of fibrotic disease. As expected, TGF-{beta}1 treatment promoted a myofibroblastic phenotype and microtissues became stiffer and possessed increased contractility. Furthermore, these changes were partially reversible upon TGF-{beta}1 withdrawal. In contrast, however, long-term cyclic stretching maintained myofibroblast activation. Furthermore stretching had no effect compared static cultures when TGF-{beta}1 receptors were inhibited and stretching promoted myofibroblast differentiation when given latent TGF-{beta}1. Together these results suggest that external mechanical stretch may activate latent TGF-{beta}1 and might be a powerful stimulus for continued myofibroblast activation to progress fibrosis. Further exploration of this pathway with our approach may yield new insights into myofibroblast activation and more effective therapeutic treatments for fibrosis. Insight boxUsing a novel high-throughput approach, we quantified the effects of dynamic mechanical stretching on the phenotype and function of cells in 3D microtissue cultures during myofibroblast activation with TGF-{beta}1 treatment and subsequent withdrawal. Our findings show that mechanical stretch may activate endogenously produced latent TGF-{beta}1 to maintain the presence and activity of myofibroblasts after tissue injury. Importantly, through this feed forward mechanism, mechanical stretch might be a powerful stimulus that directs tissues away from recovery and towards the development of fibrosis.

cell biology↗

bioRxiv2019

Structural and mechanical remodeling of the cytoskeleton studied in 3D microtissues under acute dynamic stretch

When stretched, cells cultured on 2D substrates share a universal softening and fluidization response that arises from poorly understood remodeling of well-conserved cytoskeletal elements. It is known, however, that the structure and distribution of the cytoskeleton is profoundly influenced by the dimensionality of a cells environment. Therefore, in this study we aimed to determine whether cells cultured in a 3D matrix share this softening behavior and to link it to cytoskeletal remodeling. To achieve this, we developed a high-throughput approach to measure the dynamic mechanical properties of cells and allow for sub-cellular imaging within physiologically relevant 3D microtissues. We found that fibroblast, smooth muscle and skeletal muscle microtissues strain softened but did not fluidize, and upon loading cessation, they regained their initial mechanical properties. Furthermore, microtissue prestress decreased with the strain amplitude to maintain a constant mean tension. This adaptation under an auxotonic condition resulted in lengthening. A filamentous actin cytoskeleton was required, and responses were mirrored by changes to actin remodeling rates and visual evidence of stretch-induced actin depolymerization. Our new approach for assessing cell mechanics has linked behaviors seen in 2D cultures to a 3D matrix, and connected remodeling of the cytoskeleton to homeostatic mechanical regulation of tissues.

biophysics↗

bioRxiv2019

Mechanotransduction of strain regulates an invasive phenotype in newly transformed epithelial cells

Our organs and tissues are in constant motion, exposing epithelial cells to mechanical stretch. However, how these external forces impact cellular morphology, organization and dynamics in healthy and diseased tissues is still being elucidated. In several studies, we and others have shown how mechanical stresses and strains in the epithelium can modulate the dynamics and invasiveness of transformed cells. Carcinoma, the most common type of cancer, develops in the sheets of cells forming the epithelium and lining our organs and cavities. It usually begins with the transformation of a single cell via the activation of oncogenes such as Ras. Here, we show in a model system how mechanical stretch in epithelial sheets results in a more invasive phenotype in transformed cells. Cyclic strain prevents the formation of strong circumferential belts of actin in RasV12 cells and greatly promoting the formation of RasV12 protrusions, typical of a more invasive phenotype. We also show that RasV12 and wild type MDCK cells possess distinct sensitivity to strain. External forces remodel their actin cytoskeletons and adhesion complexes differently, resulting in a more invasive system dynamic. Our work demonstrates that the Rho-ROCK mechanotransduction pathway is involved in regulating a mechanically-induced switch to a more invasive phenotype. In a mechanically dynamic microenvironment, transformed cells exhibit drastically different cellular dynamics and movements when compared to static conditions. They grow larger invasive protrusions, potentially making them harder to be eliminated from healthy tissues. The insights gained in this study reveal the complex dynamics at play in healthy and transformed epithelial cells which are found in a mechanically active microenvironment.

biophysics↗

bioRxiv2017

Mechanical mismatch between Ras transformed and untransformed epithelial cells.

The organization of the actin cytoskeleton plays a key role in regulating cell mechanics. It is fundamentally altered during transformation, affecting how cells interact with their environment. We investigated mechanical properties of cells expressing constitutively active, oncogenic Ras (RasV12) in adherent and suspended states. To do this, we utilized atomic force microscopy and a microfluidic optical stretcher. We found that adherent cells stiffen and suspended cells soften with the expression of constitutively active Ras. The effect on adherent cells was reversed when contractility was inhibited with the ROCK inhibitor Y-27632, resulting in softer RasV12 cells. Our findings suggest that increased ROCK activity as a result of Ras has opposite effects on suspended and adhered cells. Our results also establish the importance of the activation of ROCK by Ras and its effect on cell mechanics.

biophysics↗

bioRxiv2017

A vacuum-actuated microtissue stretcher for long-term exposure to oscillatory strain within a 3D matrix

Although our understanding of cellular behavior in response to extracellular biological and mechanical stimuli has greatly advanced using conventional 2D cell culture methods, these techniques lack physiological relevance. We developed the microtissue vacuum-actuated stretcher (MVAS) to probe cellular behavior within a 3D multicellular environment composed of innate matrix protein, and in response to continuous uniaxial stretch. The MVAS consists of an array of fifty self-assembled microtissues bordered by vacuum chambers. When a vacuum is applied, the microtissues stretch in plane allowing live imaging. The MVAS is highly suitable for biomedical research and pharmaceutical discovery due to a high-throughput array format and scalable fabrication steps outlined in this paper. We validated our approach by characterizing the bulk microtissue strain, the microtissue strain field and single cell strain, and by assessing F-actin expression in response to chronic cyclic strain of 10%. The MVAS was shown to be capable of delivering reproducible dynamic bulk strain amplitudes up to 13% and the strain field had local maxima around each of the cantilevers. The strain at the single cell level was found to be 10.4% less than the microtissue axial strain due to cellular rotation. Chronic cyclic strain produced a 35% increase in F-actin expression consistent with previously observed cytoskeletal reinforcement in 2D cell culture. The MVAS may further our understanding of the reciprocity shared between cells and their environment, which is critical to meaningful biomedical research and successful therapeutic approaches.

bioengineering↗