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.

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.

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.

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.

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.

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.

Comparison of methods for assigning the material properties of the distraction callus in computational models.

In silico models of distraction osteogenesis and fracture healing usually assume constant mechanical properties for the new bone tissue generated. In addition, these models do not always account for the porosity of the woven bone and its evolution. In this study, finite element analyses based on computed tomography (CT) are used to predict the stiffness of the callus until 69 weeks after surgery using 15 CT images obtained at different stages of an experiment on bone transport, technique in which distraction osteogenesis is used to correct bone defects. Three different approaches were used to assign the mechanical properties to the new bone tissue. First, constant mechanical properties of the hard callus tissue and no porosity were assumed. Nevertheless, this approach did not show good correlations. Second, random variations in the elastic modulus and porosity of the woven bone were taken from previous experimental studies. Finally, the elastic properties of each element were assigned depending on gray scale in CT images. The numerically predicted callus stiffness was compared with previous in vivo measurements. It was concluded firstly that assignment depending on gray scale is the method that provides the best results and secondly that the method that considers a random distribution of porosity and elastic modulus of the callus is also suitable to predict the callus stiffness from 15 weeks after surgery. This finding provides a method for assigning the material properties of the distraction callus, which does not require CT images and may contribute to improve current in silico models.

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.

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 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.

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.