Real-Time Wireless Platform for In Vivo Monitoring of Bone Regeneration.

For the monitoring of bone regeneration processes, the instrumentation of the fixation is an increasingly common technique to indirectly measure the evolution of bone formation instead of ex vivo measurements or traditional in vivo techniques, such as X-ray or visual review. A versatile instrumented external fixator capable of adapting to multiple bone regeneration processes was designed, as well as a wireless acquisition system for the data collection. The design and implementation of the overall architecture of such a system is described in this work, including the hardware, firmware, and mechanical components. The measurements are conditioned and subsequently sent to a PC via wireless communication to be in vivo displayed and analyzed using a developed real-time monitoring application. Moreover, a model for the in vivo estimation of the bone callus stiffness from collected data was defined. This model was validated in vitro using elastic springs, reporting promising results with respect to previous equipment, with average errors and uncertainties below 6.7% and 14.04%. The devices were also validated in vivo performing a bone lengthening treatment on a sheep metatarsus. The resulting system allowed the in vivo mechanical characterization of the bone callus during experimentation, providing a low-cost, simple, and highly reliable solution.

Quantitative atlas of collagen hydrogels reveals mesenchymal cancer cell traction adaptation to the matrix nanoarchitecture.

Collagen-based hydrogels are commonly used in mechanobiology to mimic the extracellular matrix. A quantitative analysis of the influence of collagen concentration and properties on the structure and mechanics of the hydrogels is essential for tailored design adjustments for specific in vitro conditions. We combined focused ion beam scanning electron microscopy and rheology to provide a detailed quantitative atlas of the mechanical and nanoscale three-dimensional structural alterations that occur when manipulating different hydrogel's physicochemistry. Moreover, we study the effects of such alterations on the phenotype of breast cancer cells and their mechanical interactions with the extracellular matrix. Regardless of the microenvironment's pore size, porosity or mechanical properties, cancer cells are able to reach a stable mesenchymal-like morphology. Additionally, employing 3D traction force microscopy, a positive correlation between cellular tractions and ECM mechanics is observed up to a critical threshold, beyond which tractions plateau. This suggests that cancer cells in a stable mesenchymal state calibrate their mechanical interactions with the ECM to keep their migration and invasiveness capacities unaltered. STATEMENT OF SIGNIFICANCE: The paper presents a thorough study on the mechanical microenvironment in breast cancer cells during their interaction with collagen based hydrogels of different compositions. The hydrogels' microstructure were obtained using state-of-the-art 3D microscopy, namely focused ion beam-scanning electron microscope (FIB-SEM). FIB-SEM was originally applied in this work to reconstruct complex fibered collagen microstructures within the nanometer range, to obtain key microarchitectural parameters. The mechanical microenvironment of cells was recovered using Traction Force Microscopy (TFM). The obtained results suggest that cells calibrate tractions such that they depend on mechanical, microstructural and physicochemical characteristics of the hydrogels, hence revealing a steric hindrance. We hypothesize that cancer cells studied in this paper tune their mechanical state to keep their migration and invasiveness capacities unaltered.

Mechanics Predicts Effective Critical-Size Bone Regeneration Using 3D-Printed Bioceramic Scaffolds.

BACKGROUND: 3D-printed bioceramic scaffolds have gained popularity due to their controlled microarchitecture and their proven biocompatibility. However, their high brittleness makes their surgical implementation complex for weight-bearing bone treatments. Thus, they would require difficult-to-instrument rigid internal fixations that limit a rigorous evaluation of the regeneration progress through the analysis of mechanic-structural parameters. METHODS: We investigated the compatibility of flexible fixations with fragile ceramic implants, and if mechanical monitoring techniques are applicable to bone tissue engineering applications. Tissue engineering experiments were performed on 8 ovine metatarsi. A 15 mm bone segment was directly replaced with a hydroxyapatite scaffold and stabilized by an instrumented Ilizarov-type external fixator. Several in vivo monitoring techniques were employed to assess the mechanical and structural progress of the tissue. RESULTS: The applied surgical protocol succeeded in combining external fixators and subject-specific bioceramic scaffolds without causing fatal fractures of the implant due to stress concentrator. The bearing capacity of the treated limb was initially altered, quantifying a 28-56% reduction of the ground reaction force, which gradually normalized during the consolidation phase. A faster recovery was reported in the bearing capacity, stiffening and bone mineral density of the callus. It acquired a predominant mechanical role over the fixator in the distribution of internal forces after one post-surgical month. CONCLUSION: The bioceramic scaffold significantly accelerated in vivo the bone formation compared to other traditional alternatives in the literature (e.g., distraction osteogenesis). In addition, the implemented assessment techniques allowed an accurate quantitative evaluation of the bone regeneration through mechanical and imaging parameters.

Gait analysis: An effective tool to mechanically monitor the bone regeneration of critical-sized defects in tissue engineering applications.

INTRODUCTION: Tissue engineering has emerged as an innovative approach to treat critical-size bone defects using biocompatible scaffolds, thus avoiding complex distraction surgeries or limited stock grafts. Continuous regeneration monitoring is essential in critical-size cases due to the frequent appearance of non-unions. This work evaluates the potential clinical use of gait analysis for the mechanical assessment of a tissue engineering regeneration as an alternative to the traditional and hardly conclusive manual or radiological follow-up. MATERIALS AND METHODS: The 15-mm metatarsal fragment of eight female merino sheep was surgically replaced by a bioceramic scaffold stabilized with an external fixator. Gait tests were performed weekly by making the sheep walk on an instrumented gangway. The evolution of different kinematic and dynamic parameters was analyzed for all the animal's limbs, as well as asymmetries between limbs. Finally, potential correlation in the recovery of the gait parameters was evaluated through the linear regression models. RESULTS: After surgery, the operated limb has an altered way of carrying body weight while walking. Its loading capacity was significantly reduced as the stance phases were shorter and less impulsive. The non-operated limbs compensated for this mobility deficit. All parameters were normalizing during the consolidation phase while the bone callus was simultaneously mineralizing. The results also showed high levels of asymmetry between the operated limb and its contralateral, which exceeded 150% when analyzing the impulse after surgery. Gait recovery significantly correlated between symmetrical limbs. CONCLUSIONS: Gait analysis was presented as an effective, low-cost tool capable of mechanically predicting the regeneration of critical-size defects treated by tissue engineering, as comparing regeneration processes or novel scaffolds. Despite the progressive normalization as the callus mineralized, the bearing capacity reduction and the asymmetry of the operated limb were more significant than in other orthopedic alternatives.

Evolution of relaxation properties of callus tissue during bone transport.

Callus tissue exhibits a viscoelastic behavior that has a strong influence on the distribution of stresses and their evolution with time and, thus, it can affect tissue differentiation during distraction procedures. For this reason, a deep knowledge of that viscoelastic behavior can be very useful to improve current protocols of bone distraction and bone transport. Monitoring stress relaxation of the callus during distraction osteogenesis allows characterizing its viscoelastic behavior. Different procedures have been used in the literature to fit the response of a given viscoelastic model to the force relaxation curve. However, these procedures do not ensure the uniqueness of that fit, which is of the utmost importance for statistical purposes. This work uses a fitting procedure already validated for other tissues that ensures that uniqueness. Very importantly too, the procedure presented here allows obtaining more information from the stress relaxation tests, distinguishing relaxation in different time scales, which provides a deeper insight into the viscoelastic behavior and its evolution over time. As it was observed in the results, relaxation is faster at the first days after osteotomy and becomes slower and more gradual with time. This fact can be directly linked to the temporal evolution of the callus composition (water, organic phase, and mineral content) and also to the progression of tissue differentiation, with a prevalence of hard tissues as time passes.

Time-Dependent Collagen Fibered Structure in the Early Distraction Callus: Imaging Characterization and Mathematical Modeling.

Collagen is a ubiquitous protein present in regenerating bone tissues that experiences multiple biological phenomena during distraction osteogenesis until the deposition of phosphate crystals. This work combines fluorescence techniques and mathematical modeling to shed light on the mechano-structural processes behind the maturation and accommodation-to-mineralization of the callus tissue. Ovine metatarsal bone calluses were analyzed through confocal images at different stages of the early distraction osteogenesis process, quantifying the fiber orientation distribution and mean intensity as fiber density measure. Likewise, a mathematical model based on the experimental data was defined to micromechanically characterize the apparent stiffening of the tissue within the distracted callus. A reorganization of the fibers around the distraction axis and increased fiber density were found as the bone fragments were gradually separated. Given the degree of significance between the mathematical model and previous in vivo data, reorganization, densification, and bundle maturation phenomena seem to explain the apparent mechanical maturation observed in the tissue theoretically.

Cell-biomaterial mechanical interaction in the framework of tissue engineering: insights, computational modeling and perspectives.

Tissue engineering is an emerging field of research which combines the use of cell-seeded biomaterials both in vitro and/or in vivo with the aim of promoting new tissue formation or regeneration. In this context, how cells colonize and interact with the biomaterial is critical in order to get a functional tissue engineering product. Cell-biomaterial interaction is referred to here as the phenomenon involved in adherent cells attachment to the biomaterial surface, and their related cell functions such as growth, differentiation, migration or apoptosis. This process is inherently complex in nature involving many physico-chemical events which take place at different scales ranging from molecular to cell body (organelle) levels. Moreover, it has been demonstrated that the mechanical environment at the cell-biomaterial location may play an important role in the subsequent cell function, which remains to be elucidated. In this paper, the state-of-the-art research in the physics and mechanics of cell-biomaterial interaction is reviewed with an emphasis on focal adhesions. The paper is focused on the different models developed at different scales available to simulate certain features of cell-biomaterial interaction. A proper understanding of cell-biomaterial interaction, as well as the development of predictive models in this sense, may add some light in tissue engineering and regenerative medicine fields.

Towards the Experimentally-Informed In Silico Nozzle Design Optimization for Extrusion-Based Bioprinting of Shear-Thinning Hydrogels.

Research in bioprinting is booming due to its potential in addressing several manufacturing challenges in regenerative medicine. However, there are still many hurdles to overcome to guarantee cell survival and good printability. For the 3D extrusion-based bioprinting, cell viability is amongst one of the lowest of all the bioprinting techniques and is strongly influenced by various factors including the shear stress in the print nozzle. The goal of this study is to quantify, by means of in silico modeling, the mechanical environment experienced by the bioink during the printing process. Two ubiquitous nozzle shapes, conical and blunted, were considered, as well as three common hydrogels with material properties spanning from almost Newtonian to highly shear-thinning materials following the power-law behavior: Alginate-Gelatin, Alginate and PF127. Comprehensive in silico testing of all combinations of nozzle geometry variations and hydrogels was achieved by combining a design of experiments approach (DoE) with a computational fluid dynamics (CFD) of the printing process, analyzed through a machine learning approach named Gaussian Process. Available experimental results were used to validate the CFD model and justify the use of shear stress as a surrogate for cell survival in this study. The lower and middle nozzle radius, lower nozzle length and the material properties, alone and combined, were identified as the major influencing factors affecting shear stress, and therefore cell viability, during printing. These results were successfully compared with those of reported experiments testing viability for different nozzle geometry parameters under constant flow rate or constant pressure. The in silico 3D bioprinting platform developed in this study offers the potential to assist and accelerate further development of 3D bioprinting.

Mechanobiology of Bone Consolidation During Distraction Osteogenesis: Bone Lengthening Vs. Bone Transport.

Bone lengthening and bone transport are regeneration processes that commonly rely on distraction osteogenesis, a widely accepted surgical procedure to deal with numerous bony pathologies. Despite the extensive study in the literature of the influence of biomechanical factors, a lack of knowledge about their mechanobiological differences prevents a clinical particularization. Bone lengthening treatments were performed on sheep metatarsus by reproducing the surgical and biomechanical protocol of previous bone transport experiments. Several in vivo monitoring techniques were employed to build an exhaustive comparison: gait analysis, radiographic and CT assessment, force measures through the fixation, or mechanical characterization of the new tissue. A significant initial loss of the bearing capacity, quantified by the ground reaction forces and the limb contact time with the ground, is suffered by the bone lengthening specimens. The potential effects of this anomaly on the musculoskeletal force distribution and the evolution of the bone callus elastic modulus over time are also analyzed. Imaging techniques also seem to reveal lower bone volume in the bone lengthening callus than in the bone transport one, but an equivalent mineralization rate. The simultaneous quantification of biological and mechanical parameters provides valuable information for the daily clinical routine and numerical tools development.

Structural optimization of 3D-printed patient-specific ceramic scaffolds for in vivo bone regeneration in load-bearing defects.

Tissue engineering has recently gained popularity as an alternative to autografts to stimulate bone tissue regeneration through structures called scaffolds. Most of the in vivo experiments on long-bony defects use internally-stabilized generic scaffolds. Despite the wide variety of computational methods, a standardized protocol is required to optimize ceramic scaffolds for load-bearing bony defects stabilized with flexible fixations. An optimization problem was defined for applications to sheep metatarsus defects. It covers biological parameters (porosity, pore size, and the specific surface area) and mechanical constraints based on in vivo and in vitro results reported in the literature. The optimized parameters (59.30% of porosity, 5768.91 m-1 of specific surface area, and 360.80 µm of pore size) and the compressive strength of the selected structure were validated in vitro by means of tomographic images and compression tests of six 3D-printed samples. Divergences between the design and measured values of the optimized parameters, mainly due to manufacturing defects, are consistent with the previous studies. Using the mixed experimental-mathematical scaffold-design procedure described, they could be implanted in vivo with instrumented external fixators, therefore facilitating biomechanical monitoring of the regeneration process.

Mechanical Influence of Surrounding Soft Tissue on Bone Regeneration Processes: A Bone Lengthening Study.

Bone lengthening is a bone regeneration technique with multiple clinical applications. One of the most common complications of this treatment is the lack of adaptation of the surrounding soft tissue to their extension. A better understanding of the mechanobiology of the tissues involved in distraction osteogenesis would allow better control of the clinical cases. Bone lengthening treatments were performed in vivo in the metatarsus of Merino sheep, measuring the distraction forces by means of an instrumented fixator. The tissue relaxation after distraction was analyzed in this study. A viscoelastic model was also applied to distraction data to assess the mechanical behavior of the tissues during the distraction phase. Tissue relaxation is similar to other bone regeneration processes which do not imply surrounding soft tissue extension, e.g. bone transport. The effects of this tissue on distraction forces are limited to the first minutes of distraction and elongations above 4% of the original length with the protocol applied. Moreover, the surrounding soft tissue initially loses some of its viscoelasticity and subsequently suffers strain hardening from day 5 of distraction until the end of the distraction phase, day 15. Finally, anatomical changes were also evidenced in the elongated limb of our specimens.

Comparison of the volumetric composition of lamellar bone and the woven bone of calluses.

Woven tissue is mainly present in the bone callus, formed very rapidly either after a fracture or in distraction processes. This high formation speed is probably responsible for its disorganized microstructure and this, in turn, for its low stiffness. Nonetheless, the singular volumetric composition of this tissue may also play a key role in its mechanical properties. The volumetric composition of woven tissue extracted from the bone transport callus of sheep was investigated and compared with that of the lamellar tissue extracted from the cortical shell of the same bone. Significant differences were found in the mineral and water contents, but they can be due to the different ages of both tissues, which affects the mineral/water ratio. However, the content in organic phase remains more or less constant throughout the mineralization process and has proven to be a good variable to measure the different composition of both tissues, being that content significantly higher in woven tissue. This may be linked to the abnormally high concentration of osteocytes in this tissue, which is likely a consequence of the more abundant presence of osteoblasts secreting osteoid and burying other osteoblasts, which then differentiate into osteocytes. This would explain the high formation rate of woven tissue, useful to recover the short-term stability of the bone. Nonetheless, the more abundant presence of organic phase prevents the woven tissue from reaching a stiffness similar to that of lamellar tissue in the long term, when it is fully mineralized.

Histological study of the docking site after bone transport. Temporal evolution in a sheep model.

INTRODUCTION: Bone transport appears to be a solution for segmental bone defects; specifically, the "docking site" is where the transported segment meets the target segment at the end of the process. A lack of its consolidation is one of the major causes of failure for this technique. Many studies have been performed in order to enhance the consolidation of the docking site, but histological changes occurring in it remain unknown. The aim of this study was to determine microscopic changes present in this area, from distraction to remodeling, in order to clarify the best options to facilitate the success of this technique. MATERIALS AND METHODS: Ten adult sheep were submitted to bone transport using an Ilizarov external fixator. Histomorphometry and immunohistochemical studies were performed in the docking site to determine the main types of ossification, the evolutions of tissues and blood vessels and the distributions of collagen I and II. RESULTS: Ossification was mainly intramembranous with some areas of endochondral ossification. Fibrous tissue was predominant until 98 days after surgery. The area occupied by blood vessels increased until 50 days after surgery, when it decreased slowly until the end of the study. CONCLUSIONS: As far as the authors know, this is the first histological study performed in the docking site reporting the complete evolution of tissues until the end of remodeling, showing results contrary to those published by others authors. This could help to clarify information about its union and may be useful for future investigations about techniques for improving the consolidation of the docking site in humans.

Model of the distraction callus tissue behavior during bone transport based in experiments in vivo.

Bone transport studies have measured the forces related to bone segment distraction (Brunner et al., 1994; Hyodo et al., 1996). However, no distraction force distribution between callus and docking-site was reported. Besides, most of these works have not provided continuous and long-term force relaxation measurements. The fit of the relaxation curves allows for modeling the mechanical behavior of the callus tissue during distraction osteogenesis, particularly in bone transport, where the resistance of the soft tissue and muscle is reduced compared with the bone lengthening. Bone transport experiments were carried out in sheep in which the distraction force was monitored continuously in vivo. The daily force relaxation curves were fitted, and two experimental models of the mechanical behavior of the callus tissue were obtained, assuming the total daily force relaxation or the accumulation of the residual forces. According to these models, the residual force 24h after each distraction step was a maximum of 71.6N, and the peak distraction force increased with the number of steps from 7-34N to 41-246N. The maximum residual force values that were predicted are much lower than those measured during bone lengthening in the literature. These results indirectly differentiate the influence of the surrounding soft tissues during bone transport compared with bone lengthening. Moreover, experimental measurements showed that distraction force through the docking-site was negligible with respect to distraction force through the callus. Experimental models of the callus tissue allow for an understanding of the mechanobiology of distraction osteogenesis and for predicting outcomes in its application processes.

Histological evolution of the regenerate during bone transport: an experimental study in sheep.

INTRODUCTION: Bone transport (BT) for segmentary bone defects is a well-known technique as it enables correction with new bone formation, which is similar to the previous bone. Despite the high number of experimental studies of distraction osteogenesis in bone lengthening, the types of ossification and histological changes that occur in the regenerate of the bone transport process remain controversial. OBJECTIVE: The aim of this study is to provide the complete evolution of tissues and the types of ossification in the regenerate during the different phases of bone formation after BT until the end of the remodelling period. METHODS: A histological study was performed using ten adult sheep that were submitted to BT. The types of ossification as well as the evolution of different tissues in the regenerate were determined using histomorphometry and inmunohistochemical studies. The evolution of trabeculae thickness, osteoblast and osteoclast densities, relationship between collagen types and changes in vascularization were also studied. RESULTS: Ossification was primarily intramembranous, with some focus of endochondral ossification in isolated animals. The cell counts showed a progression of cellular activity from the periphery to the centre, presenting the same progression as the growth of bone trabeculae, whose trabeculae thickness was quadrupled at the end of remodelling. Inmunohistochemical studies confirmed the prevalence of type I collagen and the ratio of the Type I/Type II collagen ratio was found to be 2.48. The percentages of the vascularized areas were proximally higher than distally in all animals, but distal zone obtained higher rates than the central region. CONCLUSIONS: Bone transport regenerate exhibits a centripetal ossification model and a mixed pattern with predominance of intramembranous over endochondral ossification. The data obtained resemble partially to those found in models of bone lengthening applied to large animals. This study provides a detailed structural characterization of the newly formed tissue, which may help to explain the development of the regenerate of bone transport in humans. It will also serve for future mechanobiological models that may aid research on the effect of loading or distractor stiffness in clinical results.

Biomechanical design of less invasive stabilization system femoral plates: computational evaluation of the fracture environment.

Less Invasive Stabilization System femoral plates are currently accepted as a suitable fixation technique for supra-intercondylar femoral fractures. However, general agreement does not exist regarding the optimum design of this fixator type. Therefore, the aim of this article is to reduce the intrinsic Less Invasive Stabilization System complications by clarifying, from a biomechanical point of view, how the number of screws, the screw connection type (unicortical or bicortical), or the structured position of the screws can influence the outcome of the fracture site. These studies include a specific finite element analysis that determines how several biomechanical variables, such as the movement at the fracture site, are influenced by the preconditions of bone healing. The results of this study show that the screw type affects the mechanical stabilization of the femur to a greater extent than the material type of the Less Invasive Stabilization System femoral plates. The most significant differences among all the analyzed configurations are observed in the shear interfragmentary strain between screw types. Values are approximately 50% higher with unicortical screws than with bicortical ones.

Hyperelastic remodeling in the intrauterine growth restricted (IUGR) carotid artery in the near-term fetus.

A constitutive model for a fiber reinforced hyperelastic material was applied to understand arterial fiber remodeling in a sheep model of Intrauterine Growth Restriction (IUGR). IUGR is associated altered hemodynamics characterized by increased resistance to blood flow in the placenta and elevated fetal arterial pressure and pulsatility. The constitutive model describes the collagen contribution to the mechanics within the arterial wall in both control and IUGR carotid artery through defining the material modulus and the orientation of the microstructure. A sheep model of placental insufficiency induced IUGR (PI-IUGR) was created by exposure of the pregnant ewe to elevated ambient temperatures. Experimental data was collected using pressure-diameter measurements to measure passive compliance in control and PI-IUGR carotid arteries. The constitutive model was optimized to fit the experimental data predicting the material parameters. Specifically, the collagen fiber predicted angle (γ) in the control artery was 49.9° from the circumferential axis while the PI-IUGR was 16.6° with a 23.5% increase in fiber orientation (κ). Quantitative assessment of collagen fiber orientation in secondary harmonic generation images confirmed the shift in orientation between the two groups. Together these suggest vascular remodeling of the ECM fiber orientation plays a major role in arterial stiffening in the PI-IUGR near-term fetal sheep.

Influence of high-frequency cyclical stimulation on the bone fracture-healing process: mathematical and experimental models.

Mechanical stimulation affects the evolution of healthy and fractured bone. However, the effect of applying cyclical mechanical stimuli on bone healing has not yet been fully clarified. The aim of the present study was to determine the influence of a high-frequency and low-magnitude cyclical displacement of the fractured fragments on the bone-healing process. This subject is studied experimentally and computationally for a sheep long bone. On the one hand, the mathematical computational study indicates that mechanical stimulation at high frequencies can stimulate and accelerate the process of chondrogenesis and endochondral ossification and consequently the bony union of the fracture. This is probably achieved by the interstitial fluid flow, which can move nutrients and waste from one place to another in the callus. This movement of fluid modifies the mechanical stimulus on the cells attached to the extracellular matrix. On the other hand, the experimental study was carried out using two sheep groups. In the first group, static fixators were implanted, while, in the second one, identical devices were used, but with an additional vibrator. This vibrator allowed a cyclic displacement with low magnitude and high frequency (LMHF) to be applied to the fractured zone every day; the frequency of stimulation was chosen from mechano-biological model predictions. Analysing the results obtained for the control and stimulated groups, we observed improvements in the bone-healing process in the stimulated group. Therefore, in this study, we show the potential of computer mechano-biological models to guide and define better mechanical conditions for experiments in order to improve bone fracture healing. In fact, both experimental and computational studies indicated improvements in the healing process in the LMHF mechanically stimulated fractures. In both studies, these improvements could be associated with the promotion of endochondral ossification and an increase in the rate of cell proliferation and tissue synthesis.

Growth mixture model of distraction osteogenesis: effect of pre-traction stresses.

In tensional studies of bone fragments during limb lengthening, it is usually assumed that the stress level in the gap tissue before each distraction step (pre-traction stress) is rather modest. However, during the process of distraction osteogenesis, a large interfragmentary gap is generated and these pre-traction stresses may be important. To date, to the authors' knowledge, no computational study has been developed to assess the effect of stress accumulation during limb lengthening. In this work, we present a macroscopic growth mixture formulation to investigate the influence of pre-traction stresses on the outcome of this clinical procedure. In particular, the model is applied to the simulation of the regeneration of tibial defects by means of distraction osteogenesis. The evolution of pre-traction forces, post-traction forces and peak forces is evaluated and compared with experimental data. The results show that the inclusion of pre-traction stresses in the model affects the evolution of the regeneration process and the corresponding reaction forces.