Sara Checa

A Mechanobiological Model for Tissue Differentiation that Includes Angiogenesis: A Lattice-Based Modeling Approach

Mechanobiological models have previously been used to predict the time course of the tissue differentiation process, with the local mechanical environment as the regulator of cell activity. However, since the supply of oxygen and nutrients to cells is also a regulator of cell differentiation and oxygen diffusion is limited to few hundred micrometers from capillaries, the morphology of the new vascular network may also play a critical role in the process. In this paper, a computational model for tissue differentiation based on the local mechanical environment and the local vascularity is presented. A regular lattice is used to simulate cell activity (migration, proliferation, differentiation, apoptosis, and angiogenesis). The algorithm for capillary network formation includes mechanoregulation of vessel growth. A simulation of tissue differentiation in a bone/implant gap under shear was performed. The model predicts capillary networks similar to those found in experimental studies and heterogeneous patterns of tissue differentiation, which are influenced by the morphology of the capillary network. Higher mechanical loads caused slower vascular development and delayed bone tissue formations.

Effect of cell seeding and mechanical loading on vascularization and tissue formation inside a scaffold: A mechano-biological model using a lattice approach to simulate cell activity

Achieving successful vascularization remains one of the main problems in bone tissue engineering. After scaffold implantation, the growth of capillaries into the porous construct may be too slow to provide adequate nutrients to the cells in the scaffold interior and this inhibits tissue formation in the scaffold core. Often, prior to implantation, a controlled cell culture environment is used to stimulate cell proliferation and, once in place, the mechanical environment acting on the tissue construct is determined by the loading conditions at the implantation site. To what extent do cell seeding conditions and the construct loading environment have an effect on scaffold vascularization and tissue growth? In this study, a mechano-biological model for tissue differentiation and blood vessel growth was used to determine the influence of cell seeding on vascular network development and tissue growth inside a regular-structured bone scaffold under different loading conditions. It is predicted that increasing the number of cells seeded homogeneously reduces the rate of vascularization and the maximum penetration of the vascular network, which in turn reduces bone tissue formation. The seeding of cells in the periphery of the scaffold was predicted to be beneficial for vascularization and therefore for bone growth; however, tissue formation occurred more slowly during the first weeks after implantation compared to homogeneous seeding. Low levels of mechanical loading stimulated bone formation while high levels of loading inhibited bone formation and capillary growth. This study demonstrates the feasibility of computational design approaches for bone tissue engineering.

Diminished response to in vivo mechanical loading in trabecular and not cortical bone in adulthood of female C57Bl/6 mice coincides with a reduction in deformation to load☆

Bone loss occurs during adulthood in both women and men and affects trabecular bone more than cortical bone. The mechanism responsible for trabecular bone loss during adulthood remains unexplained, but may be due at least in part to a reduced mechanoresponsiveness. We hypothesized that trabecular and cortical bone would respond anabolically to loading and that the bone response to mechanical loading would be reduced and the onset delayed in adult compared to postpubescent mice. We evaluated the longitudinal adaptive response of trabecular and cortical bone in postpubescent, young (10 week old) and adult (26 week old) female C57Bl/6J mice to axial tibial compression using in vivo microCT (days 0, 5, 10, and 15) and dynamic histomorphometry (day 15). Loading elicited an anabolic response in both trabecular and cortical bone in young and adult mice. As hypothesized, trabecular bone in adult mice exhibited a reduced and delayed response to loading compared to the young mice, apparent in trabecular bone volume fraction and architecture after 10 days. No difference in mechanoresponsiveness of the cortical bone was observed between young and adult mice. Finite element analysis showed that load-induced strain was reduced with age. Our results suggest that trabecular bone loss that occurs in adulthood may in part be due to a reduced mechanoresponsiveness in this tissue and/or a reduction in the induced tissue deformation which occurs during habitual loading. Therapeutic approaches that address the mechanoresponsiveness of the bone tissue may be a promising and alternate strategy to maintain trabecular bone mass during aging.

Simulation of angiogenesis and cell differentiation in a CaP scaffold subjected to compressive strains using a lattice modeling approach.

Mechanical stimuli are one of the factors that influence tissue differentiation. In the development of biomaterials for bone tissue engineering, mechanical stimuli and formation of a vascular network that transport oxygen to cells within the pores of the scaffolds are essential. Angiogenesis and cell differentiation have been simulated in scaffolds of regular porosity; however, the dynamics of differentiation can be different when the porosity is not uniform. The objective of this study was to investigate the effect of the mechanical stimuli and the capillary network formation on cell differentiation within a scaffold of irregular morphology. A porous scaffold of calcium phosphate based glass was used. The pores and the solid phase were discretized using micro computed tomography images. Cell activity was simulated within the interconnected pore domain of the scaffold using a lattice modeling approach. Compressive strains of 0.5 and 1% of total deformation were applied and two cases of mesenchymal stem cells initialization (in vitro seeding and in vivo) were simulated. Similar capillary networks were formed independently of the cell initialization mode and the magnitude of the mechanical strain applied. Most of vessels grew in the pores at the periphery of the scaffolds and were blocked by the walls of the scaffold. When 0.5% of strain was applied, 70% of the pore volume was affected by mechano-regulatory stimuli corresponding to bone formation; however, because of the lack of oxygen, only 40% of the volume was filled with osteoblasts. 40% of volume was filled with chondrocytes and 3% with fibroblasts. When the mechanical strain was increased to 1%, 11% of the pore volume was filled with osteoblasts, 59% with chondrocytes, and 8% with fibroblasts. This study has shown the dynamics of the correlation between mechanical load, angiogenesis and tissue differentiation within a scaffold with irregular morphology.

Corroboration of mechanobiological simulations of tissue differentiation in an in vivo bone chamber using a lattice-modeling approach.

It is well established that the mechanical environment modulates tissue differentiation, and a number of mechanoregulatory theories for describing the process have been proposed. In this study, simulations of an in vivo bone chamber experiment were performed that allowed direct comparison with experimental data. A mechanoregulation theory for mesenchymal stem cell differentiation based on a combination of fluid flow and shear strain (computed using finite element analysis) was implemented to predict tissue differentiation inside mechanically controlled bone chambers inserted into rat tibae. To simulate cell activity, a lattice approach with stochastic cell migration, proliferation, and selected differentiation was adopted; because of its stochastic nature, each run of the simulation gave a somewhat different result. Simulations predicted the load‐dependency of the tissue differentiation inside the chamber and a qualitative agreement with histological data; however, the full variability found between specimens in the experiment could not be predicted by the mechanoregulation algorithm. This result raises the question whether tissue differentiation predictions can be linked to genetic variability in animal populations.

Mineralizing surface is the main target of mechanical stimulation independent of age: 3D dynamic in vivo morphometry

Mechanical loading can increase cortical bone mass by shifting the balance between bone formation and resorption towards increased formation. With advancing age resorption outpaces formation resulting in a net loss in cortical bone mass. How cortical bone (re)modeling - especially resorption - responds to mechanical loading with aging remains unclear. In this study, we investigated age-related changes in the modulation of cortical bone formation and resorption sites by mechanical loading. Using in vivo microCT we determined the kinetics of three dimensional formation and resorption parameters. To analyze age-associated adaptation, the left tibiae of young, adult and elderly female C57BL/6 mice were cyclically loaded for 2weeks. Our data showed that in the nonloaded limbs, cortical bone loss with age is the result of an imbalance of resorption to formation thickness, while the surface of resorption is comparable to formation. Loading has a much stronger effect on formation than on resorption; more specifically this effect is due to an increase in formation surface with mechanical stimulation. This is the only effect of loading which is conserved into old age. The resorption thickness is independent of loading in all age groups. Using this novel image analysis technique, we were able for the first time to quantify age-related changes in cortical (re)modeling and the adaptive capacity to mechanics. Most likely a therapy against age-related bone loss combining physical exercise and pharmaceuticals is most efficient if they each act on different parameters of the (re)modeling process. Despite some differences in skeletal aging between mice and humans, our results would suggest that physical exercise in old individuals can positively influence only the formation side of (re) modeling.

Spatial-temporal mapping of bone structural and elastic properties in a sheep model following osteotomy.

The course of bone healing in animal models is conventionally monitored by morphologic approaches, which do not allow the determination of the material properties of the tissues involved. Mechanical characterization techniques are either dedicated to the macroscopic evaluation of the entire organ or to the microscopic evaluation of the tissue matrix. The latter provides insight to regionally specific alterations at the tissue level in the course of healing. In this study, quantitative scanning acoustic microscopy was used at 50 MHz to investigate microstructural and elastic alterations of mineralized callus and cortical tissue after transverse osteotomy in sheep tibiae. Analyses were performed after 2, 3, 6 and 9 weeks of consolidation with stabilization by either a rigid or a semi-rigid external fixator. Increased stiffness and decreased porosity were observed in the callus tissue over the course of the healing process, which was dependent on the fixator type. In the adjacent cortical tissue, stiffness decreased during the first 3 weeks. Cortical porosity increased over time but the time-dependence was different between the two fixator types. The changes of stiffness of cortical and callus tissues were measured with respect to the distance to the periosteal cortex-callus boundary. Stiffness of cortex and callus tissue smoothly decreased as a function of the distance from the inner cortical region. The data obtained in this study can help to understand the processes involved in tissue maturation during endogenous bone healing.

Inter-species investigation of the mechano-regulation of bone healing: Comparison of secondary bone healing in sheep and rat

Inter-species differences in regeneration exist in various levels. One aspect is the dynamics of bone regeneration and healing, e.g. small animals show a faster healing response when compared to large animals. Mechanical as well as biological factors are known to play a key role in the process. However, it remains so far unknown whether different animals follow at all comparable mechano-biological rules during tissue regeneration, and in particular during bone healing. In this study, we investigated whether differences observed in vivo in the dynamics of bone healing between rat and sheep are only due to differences in the animal size or whether these animals have a different mechano-biological response during the healing process. Histological sections from in vivo experiments were compared to in silico predictions of a mechano-biological computer model for the simulation of bone healing. Investigations showed that the healing processes in both animal models occur under significantly different levels of mechanical stimuli within the callus region, which could explain histological observations of early intramembranous ossification at the endosteal side. A species-specific adaptation of a mechano-biological model allowed a qualitative match of model predictions with histological observations. Specifically, when keeping cell activity processes at the same rate, the amount of tissue straining defining favorable mechanical conditions for the formation of bone had to be increased in the large animal model, with respect to the small animal, to achieve a qualitative agreement of model predictions with histological data. These findings illustrate that geometrical (size) differences alone cannot explain the distinctions seen in the histological appearance of secondary bone healing in sheep and rat. It can be stated that significant differences in the mechano-biological regulation of the healing process exist between these species. Future investigations should aim towards understanding whether these differences are due to differences in cell behavior, material properties of the newly formed tissues within the callus and/or differences in response to the mechanical environment.

Aging Leads to a Dysregulation in Mechanically Driven Bone Formation and Resorption

Physical activity is essential to maintain skeletal mass and structure, but its effect seems to diminish with age. To test the hypothesis that bone becomes less sensitive to mechanical strain with age, we used a combined in vivo/in silico approach. We investigated how maturation and aging influence the mechanical regulation of bone formation and resorption to 2 weeks of noninvasive in vivo controlled loading in mice. Using 3D in vivo morphometrical assessment of longitudinal microcomputed tomography images, we quantified sites in the mouse tibia where bone was deposited or resorbed in response to controlled in vivo loading. We compared the (re)modeling events (formation/resorption/quiescent) to the mechanical strains induced at these sites (predicted using finite element analysis). Mice of all age groups (young, adult, and elderly) responded to loading with increased formation and decreased resorption, preferentially at high strains. Low strains were associated with no anabolic response in adult and elderly mice, whereas young animals showed a strong response. Adult animals showed a clear separation between strain ranges where formation and resorption occurred but without an intermediate quiescent “lazy zone”. This strain threshold disappeared in elderly mice, as mechanically induced (re)modeling became dysregulated, apparent in an inability to inhibit resorption or initiate formation. Contrary to what is generally believed until now, aging does not shift the mechanical threshold required to initiate formation or resorption, but rather blurs its specificity. These data suggest that pharmaceutical strategies augmenting physical exercise should consider this dysfunction in the mechanical regulation of bone (re)modeling to more effectively combat age‐related bone loss.

The influence of age on adaptive bone formation and bone resorption

Bone is a tissue with enormous adaptive capacity, balancing resorption and formation processes. It is known that mechanical loading shifts this balance towards an increased formation, leading to enhanced bone mass and mechanical performance. What is not known is how this adaptive response to mechanical loading changes with age. Using dynamic micro-tomography, we show that structural adaptive changes of trabecular bone within the tibia of living mice subjected to two weeks of in vivo cyclic loading are altered by aging. Comparisons of 10, 26 and 78 weeks old animals reveal that the adaptive capacity diminishes. Strikingly, adaptation was asymmetric in that loading increases formation more than it reduces resorption. This asymmetry further shifts the (re)modeling balance towards a net bone loss with age. Loading results in a major increase in the surface area of mineralizing bone. Interestingly, the resorption thickness is independent of loading in trabecular bone in all age groups. This data suggests that during youth, mechanical stimulation induces the recruitment of bone modeling cells whereas in old age, only bone forming cells are affected. These findings provide mechanistic insights into the processes that guide skeletal aging in mice as well as in other mammals.