Reducing Inert Materials for Optimal Cell-Cell and Cell-Matrix Interactions within Microphysiological Systems.

In the pursuit of achieving a more realistic in vitro simulation of human biological tissues, microfluidics has emerged as a promising technology. Organ-on-a-chip (OoC) devices, a product of this technology, contain miniature tissues within microfluidic chips, aiming to closely mimic the in vivo environment. However, a notable drawback is the presence of inert material between compartments, hindering complete contact between biological tissues. Current membranes, often made of PDMS or plastic materials, prevent full interaction between cell types and nutrients. Furthermore, their non-physiological mechanical properties and composition may induce unexpected cell responses. Therefore, it is essential to minimize the contact area between cells and the inert materials while simultaneously maximizing the direct contact between cells and matrices in different compartments. The main objective of this work is to minimize inert materials within the microfluidic chip while preserving proper cellular distribution. Two microfluidic devices were designed, each with a specific focus on maximizing direct cell-matrix or cell-cell interactions. The first chip, designed to increase direct cell-cell interactions, incorporates a nylon mesh with regular pores of 150 microns. The second chip minimizes interference from inert materials, thereby aiming to increase direct cell-matrix contact. It features an inert membrane with optimized macropores of 1 mm of diameter for collagen hydrogel deposition. Biological validation of both devices has been conducted through the implementation of cell migration and cell-to-cell interaction assays, as well as the development of epithelia, from isolated cells or spheroids. This endeavor contributes to the advancement of microfluidic technology, aimed at enhancing the precision and biological relevance of in vitro simulations in pursuit of more biomimetic models.

The Role of Mechanical Properties and Structure of Type I Collagen Hydrogels on Colorectal Cancer Cell Migration.

Mechanical interactions between cells and their microenvironment play an important role in determining cell fate, which is particularly relevant in metastasis, a process where cells invade tissue matrices with different mechanical properties. In vitro, type I collagen hydrogels have been commonly used for modeling the microenvironment due to its ubiquity in the human body. In this work, the combined influence of the stiffness of these hydrogels and their ultrastructure on the migration patterns of HCT-116 and HT-29 spheroids are analyzed. For this, six different types of pure type I collagen hydrogels by changing the collagen concentration and the gelation temperature are prepared. The stiffness of each sample is measured and its ultrastructure is characterized. Cell migration studies are then performed by seeding the spheroids in three different spatial conditions. It is shown that changes in the aforementioned parameters lead to differences in the mechanical stiffness of the matrices as well as the ultrastructure. These differences, in turn, lead to distinct cell migration patterns of HCT-116 and HT-29 spheroids in either of the spatial conditions tested. Based on these results, it is concluded that the stiffness and the ultrastructural organization of the matrix can actively modulate cell migration behavior in colorectal cancer spheroids.

A mechanobiological model for tumor spheroid evolution with application to glioblastoma: A continuum multiphysics approach.

BACKGROUND: Spheroids are in vitro quasi-spherical structures of cell aggregates, eventually cultured within a hydrogel matrix, that are used, among other applications, as a technological platform to investigate tumor formation and evolution. Several interesting features can be replicated using this methodology, such as cell communication mechanisms, the effect of gradients of nutrients, or the creation of realistic 3D biological structures. The main objective of this work is to link the spheroid evolution with the mechanical activity of cells, coupled with nutrient consumption and the subsequent cell dynamics. METHOD: We propose a continuum mechanobiological model which accounts for the most relevant phenomena that take place in tumor spheroid evolution under in vitro suspension, namely, nutrient diffusion in the spheroid, kinetics of cellular growth and death, and mechanical interactions among the cells. The model is qualitatively validated, after calibration of the model parameters, versus in vitro experiments of spheroids of different glioblastoma cell lines. RESULTS: Our model is able to explain in a novel way quite different setups, such as spheroid growth (up to six times the initial configuration for U-87 MG cell line) or shrinking (almost half of the initial configuration for U-251 MG cell line); as the result of the mechanical interplay of cells driven by cellular evolution. CONCLUSIONS: Glioblastoma tumor spheroid evolution is driven by mechanical interactions of the cell aggregate and the dynamical evolution of the cell population. All this information can be used to further investigate mechanistic effects in the evolution of tumors and their role in cancer disease.

SpheroidJ: An Open-Source Set of Tools for Spheroid Segmentation.

BACKGROUND AND OBJECTIVES: Spheroids are the most widely used 3D models for studying the effects of different micro-environmental characteristics on tumour behaviour, and for testing different preclinical and clinical treatments. In order to speed up the study of spheroids, imaging methods that automatically segment and measure spheroids are instrumental; and, several approaches for automatic segmentation of spheroid images exist in the literature. However, those methods fail to generalise to a diversity of experimental conditions. The aim of this work is the development of a set of tools for spheroid segmentation that works in a diversity of settings. METHODS: In this work, we have tackled the spheroid segmentation task by first developing a generic segmentation algorithm that can be easily adapted to different scenarios. This generic algorithm has been employed to reduce the burden of annotating a dataset of images that, in turn, has been employed to train several deep learning architectures for semantic segmentation. Both our generic algorithm and the constructed deep learning models have been tested with several datasets of spheroid images where the spheroids were grown under several experimental conditions, and the images acquired using different equipment. RESULTS: The developed generic algorithm can be particularised to different scenarios; however, those particular algorithms fail to generalise to different conditions. By contrast, the best deep learning model, constructed using the HRNet-Seg architecture, generalises properly to a diversity of scenarios. In order to facilitate the dissemination and use of our algorithms and models, we present SpheroidJ, a set of open-source tools for spheroid segmentation. CONCLUSIONS: In this work, we have developed an algorithm and trained several models for spheroid segmentation that can be employed with images acquired under different conditions. Thanks to this work, the analysis of spheroids acquired under different conditions will be more reliable and comparable; and, the developed tools will help to advance our understanding of tumour behaviour.

Growth Plate Pathology in the Mucopolysaccharidosis Type VI Rat Model-An Experimental and Computational Approach.

BACKGROUND: Mucopolysaccharidoses (MPS) are a group of inherited metabolic diseases caused by impaired function or absence of lysosomal enzymes involved in degradation of glycosaminoglycans. Clinically, MPS are skeletal dysplasias, characterized by cartilage abnormalities and disturbances in the process of endochondral ossification. Histologic abnormalities of growth cartilage have been reported at advanced stages of the disease, but information regarding growth plate pathology progression either in humans or in animal models, as well as its pathophysiology, is limited. METHODS: Histological analyses of distal femur growth plates of wild type (WT) and mucopolysaccharidosis type VI (MPS VI) rats at different stages of development were performed, including quantitative data. Experimental findings were then analyzed in a theoretical scenario. RESULTS: Histological evaluation showed a progressive loss of histological architecture within the growth plate. Furthermore, in silico simulation suggest the abnormal cell distribution in the tissue may lead to alterations in biochemical gradients, which may be one of the factors contributing to the growth plate abnormalities observed, highlighting aspects that must be the focus of future experimental works. CONCLUSION: The results presented shed some light on the progression of growth plate alterations observed in MPS VI and evidence the potentiality of combined theoretical and experimental approaches to better understand pathological scenarios, which is a necessary step to improve the search for novel therapeutic approaches.

Biophysical Stimuli: A Review of Electrical and Mechanical Stimulation in Hyaline Cartilage.

OBJECTIVE: Hyaline cartilage degenerative pathologies induce morphologic and biomechanical changes resulting in cartilage tissue damage. In pursuit of therapeutic options, electrical and mechanical stimulation have been proposed for improving tissue engineering approaches for cartilage repair. The purpose of this review was to highlight the effect of electrical stimulation and mechanical stimuli in chondrocyte behavior. DESIGN: Different information sources and the MEDLINE database were systematically revised to summarize the different contributions for the past 40 years. RESULTS: It has been shown that electric stimulation may increase cell proliferation and stimulate the synthesis of molecules associated with the extracellular matrix of the articular cartilage, such as collagen type II, aggrecan and glycosaminoglycans, while mechanical loads trigger anabolic and catabolic responses in chondrocytes. CONCLUSION: The biophysical stimuli can increase cell proliferation and stimulate molecules associated with hyaline cartilage extracellular matrix maintenance.

Proximal femoral growth plate mechanical behavior: Comparison between different developmental stages.

In long bones the growth plate is a cartilaginous structure located between the epiphysis and the diaphysis. This structure regulates longitudinal growth and helps determine the structure of mature bone through the process of endochondral ossification. During human growth the femur's proximal growth plate experiences changes in its morphology that may be related to its mechanical environment. Thus, in order to test this hypothesis from a computational perspective, a finite element analysis on a proximal femur was performed on which we modeled different physeal geometries corresponding to the shapes acquired for this structure in a child between the ages of five to eleven. Results show augmented Von Mises stress values with increasing irregularities in physeal geometry, whereas displacement decreased with increased irregularities in the growth plate's morphology. Such observations suggest that growth plate's shape changes follows a possible mechanical adaptation on imposed loads to sustain a person's increasing body mass during growth.

Geometrical and mechanical factors that influence slipped capital femoral epiphysis: a finite element study.

Slipped capital femoral epiphysis (SCFE) is an orthopedic pathology in which damage of the growth plate leads to the anterosuperior displacement of the femoral body in respect to the femoral head. Despite being a widely studied disease, its etiology is still unknown. This study was carried out to determine the influence of the physeal-diaphysis angle, body mass, the presence of the perichondrial ring, the type of physical activity, and physeal thickness on SCFE. For this purpose, a finite element analysis of the hip joint and the femur-physis interface was carried out. With the computational model, the Von Mises stresses along the growth plate were calculated and subsequently analyzed statistically to find their correlation with the studied factors. It was found that body mass, the type of physical activity, and the presence of the perichondrial ring had more statistical relevance for the physeal stresses than the physeal-diaphysis angle and the physeal thickness. Thus, our work suggests that changes in growth plate inclination and thickness do not influence the etiology of SCFE.

Modelado por elementos finitos del deslizamiento epifisiario / Modélisation par éléments finis du déplacement épiphysaire / Finite element modeling of epiphyseal gliding

Objetivo: desarrollar un análisis por elementos finitos de la influencia del ángulo fisis-diáfisis, la masa corporal y la actividad física con el fin de observar su predominancia en la incidencia de deslizamiento epifisiario. Métodos: se elaboraron los modelos correspondientes a las combinaciones entre cada uno de los parámetros definidos (ángulo, masa y actividad física), generando 20 casos diferentes, y se evaluaron los esfuerzos presentes a lo largo de la placa de crecimiento. Resultados: se muestra un comportamiento uniforme y similar entre cada combinación, así como un aumento en las tensiones en la medida en que se incrementaba el valor de la carga y del ángulo. Conclusiones: el esfuerzo tiende a aumentar cuando se incrementa tanto el ángulo como la masa física, lo cual sugiere que estos dos factores podrían influir de manera decisiva en el origen del deslizamiento epifisiario(AU)

Objective: to develop a finite element analysis of the influence of physis-diaphysis angle, body mass and physical activity to observe its predominance in the incidence of epiphyseal gliding. Methods: models corresponding to the combinations among each of the defined parameters (angle, mass and physical activity) were developed, generating 20 different cases and efforts present through the growth plate were evaluated. Results: a similar and uniform behaviour between each of the combinations is shown as well as an increase in tension at the same time as the value of the load and angle increases. Conclusions: effort tends to increase when there is an increment in both the angle and the physical mass what suggests that these two factors could have a decisive influence on the origin of the epiphyseal gliding(AU)

But: en s'appuyant sur la technique des éléments finis, une analyse de l'influence de l'angle physe-diaphyse, la masse corporelle et l'activité physique a été réalisée afin d'observer cette influence sur l'incidence du déplacement épiphysaire. Méthodes: des modèles correspondant aux combinaisons entre chaque paramètre défini (angle, masse et activité physique), en résultant 20 cas différents, ont été élaborés, et les efforts présents tout au long de la plaque de croissance ont été évalués. Résultats: un comportement uniforme et similaire entre chaque combinaison est montré, ainsi qu'une élevée des tensions au fur et à mesure que la valeur de la charge et l'angle augmentaient. Conclusions: l'effort tend à augmenter lorsque l'angle et la masse physique s'accroissent, ce qui indique que ces deux facteurs pourraient influer certainement sur l'origine du déplacement épiphysaire(AU)