Multiscale Mechanobiology in Tissue Engineering

The increased need to accelerate the healing process of critical size defects in the bone led to the study of optimal combination of cells, materials and external stimuli to obtain fully differentiated tissue to the injured site. Bioreactors play a crucial role in the control over the development of functional tissue allowing control over the surrounding chemical and mechanical environment. This chapter aims to review bioreactor systems currently available for monitoring mesenchymal stem cells (MSCs) behaviour under mechanical stimuli and to give an insight of their effect on cellular commitment. Shear stress, mechanical strain and pulsed electromagnetic field bioreactors are presented, and the effect of multiple conditions under varying parameters such as amplitude, frequency or duration of the stimuli on bone progenitor cells differentiation is considered and extensively discussed with particular focus on osteogenic and chondrogenic commitment.

Tissue grafts obtained from tissue engineering techniques can be developed with the application of cells in a scaffold within a bioreactor. In this chapter we present a multiscale method to simulate a bioreactor design that can adapt to the personalized tissue sought. It includes personalization of the bioreactor design but also personalization of the in vitro conditions. As the research area is going further and the computational possibilities as well, tools must be developed to design patient’s cell-specific pair of scaffold and bioreactor, as a virtual physiological human cell tool.

Thanks to a parametric geometry and a computational fluid dynamics model, we are able to design bioreactor chambers relying on the nearest boundary conditions in the bones to apply it to the bone substitute where cells have been seeded. First of all, considering an existing bioreactor chamber, we can design an optimized scaffold knowing the boundary conditions that the bioreactor chamber will impose. On the other hand, knowing the scaffold geometry used, a bioreactor chamber will be designed to reach appropriate environmental conditions at the cell scale.

It allowed testing two different bioreactor geometries showing no major interest within the simulation, but regarding the experimental process, the bubble traps presence is compulsory to avoid cell death. On the other hand, two scaffold geometries were tested highlighting a major difference regarding the local fluid flow within the scaffold pores and therefore on the cell development. Moreover, experimental analyses are required to correctly compare the simulation and improve the strength of the optimization process.

Three-dimensional (3D) scaffolds are increasingly employed as support for studies on cellular activities. They are widely shown to enhance cell survival and are a promising approach to be employed to mimic the in vivo conditions due to their controlled architecture. Moreover, 3D stiff structures fabricated by additive manufacturing are able to bear mechanical stimuli finding a role in the investigation of the effect of mechanical forces on cell proliferation and commitment. With this purpose, we propose a combination of a 3D polycaprolactone (PCL) scaffold and collagen soft gel as support for studying the response of mesenchymal stem cells following mechanical compression. This chapter focuses on the characterization of 3D Insert^® PCL scaffolds behaviour under mechanical compression. After defining mechanical properties and variability due to boundary effects, the focus moves on the development of a new composite scaffold made of a stiff PCL structure acting as support for cell activities and able to bear mechanical compression while embedding a soft collagen gel matrix responsible to provide an environment enhancing cellular activities as well as to transmit the stress resulting from the mechanical stimulation from the stiff matrix to the seeded cells. Finally, the last section focuses on the effect of low mechanical strain applied on seeded scaffolds and how the cellular response varies to bursts of compression applied at different time points.

Rapid prototyping is a powerful manufacturing technique to fabricate tissue engineering scaffolds as it provides control over scaffold architecture while processing biomaterials. For the successful integration of tissue engineering scaffolds in clinical applications, the manufacturing process needs to the meet good manufacturing practice standards delivering quality and reproducibility. Unexpected variations in the scaffold microstructure due to limitations in the fabrication process could lead to undesired mechanical stimuli at the cell level. Thus, if cell activity is affected, tissue growth will be perturbed. In this chapter, an in silico protocol to analyse fabricated scaffolds is presented and used to evaluate a commercial regular porous scaffold from 3D Biotek. The actual μCT-based morphology of five fabricated samples was analysed and then integrated into computational fluid dynamics simulations to analyse the local fluid flow conditions. The fabricated samples present variations in the internal microstructured and in the local fluid dynamics compared to the CAD scaffold. In addition, the five samples show intersample variability as well as internal variability from pore to pore. It is demonstrated that geometrical imperfections can deviate scaffold performance from the intended purpose. In this chapter, it is shown that in silico methods can be part of standard inspection protocols for tissue engineering applications.

Perfusion systems can help to drive cells to scaffold substrate with regular distribution and high efficiency. However, it is difficult to predict experimentally which are the best bioreactor conditions for such outcomes. In this chapter, a CFD model is developed to predict the local fluid flow conditions inside the scaffold, and a discrete phase representing the cells is included to describe cell motion near the scaffold substrate. The computational model is developed in combination with particle tracking experiments performed inside a 3D scaffold in order to configure and validate the model with well-characterized experimental data. The study showed that the computational model can describe the velocity profiles inside the scaffold as well as cell path. It was found that cells mainly follow the fluid streamlines passing throughout all scaffold pores. High permeability in scaffolds is beneficial to distribute cells; however, it does not guarantee the deposition of cells on the scaffold substrate and cell adhesion. The presented CFD model can help to design and optimize perfusion systems to reach the desired cell distribution and density which will affect the final tissue properties.

Mechanical forces and 3D topological environment can be used to control differentiation of mesenchymal stem cells (MSCs). However, the effects of physical and mechanical cues of the microenvironment on MSC fate determination have not yet been fully understood. This study investigates and compares the effect of mechanical stimulations on soft cellular microspheres when subjected to dynamic fluid compression. Microspheres were produced by gelation of bovine collagen type I with concentrations of 2 mg/ml and 1000–2000 hES-MP cells per 5 μl droplet. A loading condition of 10% dynamic loading was applied by a BOSE BioDynamic bioreactor for 15 and 40 min/day for 5 and 10 days on the cell-seeded collagen microspheres. Cell viability and proliferation, alkaline phosphatase activity and mineralization were compared with controls. Monitoring alkaline phosphatase level reported a significant increase in the enzyme activity by day 14 in loaded samples of 40 min/day loading protocol compared with other experimental conditions. Mineralization was assessed by measuring calcium, phosphorous concentrations and intensity of H&E and alizarin red S staining and showed the highest mineral accumulation in the loaded samples on day 28 post encapsulation. This study indicated that loading of very low cell number seeded on soft natural scaffold can encourage osteogenesis of cells by enhancing both early stage bone marker and mineralization. Self-assembled cell/collagen microspheres present exceptional cell delivery model in bone healing/repair process and field of regenerative medicine.

Mesenchymal stem cells (MSCs) are widely implicated for their potential use as a cell source for tissue engineering of skeletal tissue in regenerative medicine and tissue engineering. Mechanical forces from the microenvironment have a significant influence on differentiation of MSCs, and the resulting mechanotransduction would provide crucial adjuncts to standard biochemical signalling pathways. Combining microfluidic systems with mechanical stimulation for osteogenesis represents both a scientific and technological innovation that would greatly impact the field of regenerative medicine. We demonstrate a microfluidic chamber design for mechanical stimulation of flexible cellular microspheres and possibly a high-throughput microfluidic system for parallel processing of stem cell aggregation. We also showed that collagen microspheres serve an efficient cell delivery device supporting cell viability and migration post encapsulation.

A microfluidic chamber was made of PDMS with a central compression channel (0.6–0.9 mm). Bovine collagen type I with concentrations of 2 mg/ml and 2450 human embryonic mesenchymal stem progenitors (hES-MPs)/2.5 μl droplet was produced through gelation.

The microspheres were passed through the constriction at a flow rate of 320 μl/min for three cycles/day for 3 days starting at day 3 post encapsulation. Also effect of fluid sheer stress on the osteogenic differentiation of hES-MPs collagen microspheres was investigated using shaker and rocker systems. Cell viability, proliferation and bone markers alteration were monitored before and after compression.

Collagen is one of the most used biomaterials in tissue engineering applications. It is abundant in biological tissues, namely, constituting approximately 30% of all musculoskeletal tissues, through the presence in the extracellular matrix. Along the years, several works tried to characterize this biomaterial, as isolated fibres or as part of hydrogels, but the fragility of this material has been deferring its full characterization. This chapter presents an experimental and numerical characterization of a highly hydrated collagen hydrogel, with 0.20% of collagen concentration. The experimental data obtained through rheology experiments with three hydrogel samples was complemented with numerical simulation through finite element techniques. This framework was complemented with literature data, resulting on a set of biomechanical parameters that can describe the hydrogel behaviour, namely, a shear modulus of 0.023 kPa and a bulk modulus of 0.769 kPa, corresponding to a Poisson’s ratio of 0.485. The ultimate aim for this work is to contribute for the determination of the load transfer mechanisms through a collagen medium, i.e. to understand the contribution of collagen on the mechanical stimuli that affect cell behaviour on scaffold cell seeding.

Cells interact with their extracellular environment, from which they gather information that influences their behaviour. The cytoskeleton provides a bridge to transmit information between the extracellular and the intracellular environments. It has been suggested that the CSK components may have distinct mechanical roles in the cell and that they might form the structure that defines cell rigidity. One approach to studying the mechanosensing processes is to understand the mechanical properties of cells’ constitutive components individually. In this chapter we describe the development of a multi-structural 3D finite element model of a single-adherent cell to investigate the biophysical differences of the mechanical role of each cytoskeleton component. The model includes prestressed actin bundles and microtubule within the cytoplasm and nucleus, which are surrounded by the actin cortex.

With the multi-structural model, we predicted that actin cortex and microtubules were targeted to respond to compressive loads, while actin bundles and microtubules were major components in maintaining cell forces during stretching. Additionally, corroboration of the multi-structural model regarding its ability to identify the role of the CSK components was obtained by comparing the numerical predictions with AFM force measurements on U2OS-osteosarcoma cells exposed to different cytoskeleton-disrupting drugs. Overall, the multi-structural model not only illustrates that a combination of cytoskeletal structures with their own properties is necessary for a complete description of cellular mechanics but also clarifies the effects of cytoskeletal heterogeneity on the interpretation of force-deformation measurements.

The ability to predict the mechanical responses of different adherent cell types presents many opportunities to mechanobiology research to further identify changes from cell physiological conditions to disease. Using the multi-structural cell model presented in Chap 9, we aim to show how variation of the material properties of the intracellular components affects cell response after compression and shearing. A parametric study was performed to understand the key mechanical features from different cell types, focussing on variation of the mechanical properties of specific cytoskeleton components and prestress. Using the previously defined elastic formulation, we predicted that cell force is mainly affected by changes in cortex thickness, cortex Young’s modulus and rigidity of the cytoplasm. Changes in rigidity of actin bundles and number of microtubules influence cell response to shear loads, while a higher number of actin bundles aligned in the direction of applied compression increase cell response to compression.

Furthermore, we also performed a parametric study to evaluate the contribution of the viscoelastic properties of the cortex and cytoplasm under compressive loading, in which the mechanical behaviour of these components was represented by a power-law model. The time-dependent responses observed were remarkably similar to those reported for a variety of measurements with atomic force microscopy, suggesting this model is a consensus description of the fundamental principles defining cell mechanics. Additionally, we reported that viscoelastic properties of the cortex are essential to define the time-dependent response of the cell to compressive loads.

Scaffold biomaterials for tissue engineering can be produced in many different ways depending on the applications and the materials used. Most research into new biomaterials is based on an experimental trial-and-error approach that limits the possibility of making many variations to a single material and studying its interaction with its surroundings. Instead, computer simulation applied to tissue engineering can offer a more exhaustive approach to test and screen out biomaterials. In this chapter, current perspectives will be presented to indicate that more efforts need to be put into the development of such advanced studies, and a new workflow including the use of computer modelling for the development of new tissue engineering product is presented.