Juan Mora-Macías

Background: Bone tissue engineering has emerged as a promising technique for treating bone defects in large bones. Recent methods have enabled scaffold designs based on predefined microstructures or mechanical behavior patterns, including porosity-graded scaffolds adaptable to heterogeneous load states. However, there is no consensus on the optimal scaffold design strategy, which is sometimes chosen based on the intact bone or results from computational or in vivo experiments. Objective: This work proposes the design of graded-porosity triply periodic minimal surface (TPMS) scaffolds that mimic the mechanical environment within a bone transport callus at the peak of bone tissue production, according to in vivo load measurements. Methods: Finite element models based on computational tomography scans were used to define the strain field of the callus at the peak of bone tissue production. The developed scaffold models were evaluated through finite element simulation. Results: The callus simulations reported that the period in which maximum woven bone tissue production was achieved corresponds to the period of maximum axial strain. The graded-porosity scaffolds simulated demonstrated their ability to replicate this strain field along the callus. The microstructural parameters and strain environment of the proposed graded-porosity scaffolds were consistent with finding from studies assessing the influence of different microstructural parameters or strain conditions on bone ingrown within scaffolds. Conclusions: The proposed approach—designing graded-porosity scaffolds based on the callus strain field at the peak of bone tissue production—proved to be appropriate and may help improve future clinical applications.

El tejido óseo inmaduro generado durante procesos de regeneración ósea como fracturas simples o distracción osteogénica, es inicialmente un tejido heterogéneo que evoluciona mediante remodelación por la actividad de osteoblastos y osteoclastos, influido por la tensión mecánica a la que está sometido. Este tejido óseo inmaduro alcanza al final del proceso de regeneración unas propiedades mecánicas y microestructurales similares a las del hueso cortical. La caracterización del mismo a diferentes escalas es crucial para desarrollar modelos micromecánicos que optimicen los parámetros mecánicos de los que depende el proceso, controlando así la regeneración y previniendo las no uniones. La distracción osteogénica consiste en la separación gradual de dos segmentos óseos mediante el alargamiento del callo. Tiene gran importancia clínica por su aplicación en la corrección de defectos o alargamiento de extremidades para la rectificación de asimetrías óseas. Este estudio examina la evolución temporal del tejido óseo inmaduro en 6 muestras de callo de distracción en el metatarso de oveja. Se analizó el módulo elástico mediante nanoindentación, la microarquitectura trabecular vía microCT y la composición mediante análisis de cenizas y espectroscopia Raman. A escala macroscópica, el tejido óseo inmaduro del callo de distracción mostró un aumento considerable de la fracción de tejido óseo, debido principalmente al ensanchamiento de las trabéculas. Las ratios mineral/matriz medidos mediante espectroscopia Raman aumentaron hasta alcanzar niveles propios del hueso cortical durante la regeneración. Sin embargo, el módulo elástico local se mantuvo por debajo de los niveles en hueso cortical. Durante la consolidación del callo, el tejido óseo inmaduro experimentó cambios en la fracción de cenizas y en los porcentajes de calcio y fósforo. Correlacionando estos datos, se identificaron seis expresiones estadísticamente significativas para predecir el módulo elástico del tejido óseo inmaduro, a nivel local y aparente, en función de la fracción de ceniza, el porcentaje en volumen de tejido óseo y la composición química basada en espectroscopia Raman. Se concluye que la microarquitectura del tejido óseo inmaduro del callo de distracción desempeña un papel más significativo que su composición química en la determinación del módulo elástico aparente del tejido. Se demostró que los parámetros Raman proporcionan correlaciones más significativas con el módulo elástico a nivel microscópico que el contenido de mineral obtenido del análisis de cenizas.

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.

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.

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.

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.

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.

The woven bone created during the healing of bone regeneration processes is characterized as being extremely inhomogeneous and having a variable stiffness that increases with time. Therefore, it is important to study how the mechanical properties of woven bone are dependent on its microarchitecture and especially on its porosity and mineral content. The porosity and the x-ray greyscale of specimens taken from bone transport studies in sheep were assessed by means of ex vivo imaging. Our study demonstrates that the porosity of the woven bone in the distraction area diminishes during the healing process from 73.3% 35 days after surgery to 31.9% 525 days after surgery. In addition, the woven bone’s porosity is negatively correlated with its Young’s modulus. The x-ray greyscale, was measured as an indicator of the level of mineralization of the woven bone. Greyscale index has been demonstrated to be inversely proportional to porosity and to increase to up to 60–80% of the level in cortical bone. The results of this study may contribute to the development of micromechanical models of woven bone and improvements in in silico modelling.

Multiscale analysis has become an attractive technique to predict the behaviour of materials whose microstructure strongly changes spatially or among samples, with that microstructure controlling the local constitutive behaviour. This is the case, for example, of most biological tissues—such as bone. Multiscale approaches not only allow, not only to better characterise the local behaviour, but also to predict the field-variable distributions (e.g., strains, stresses) at both scales (macro and micro) simultaneously. However, multiscale analysis usually lacks sufficient experimental feedback to demonstrate its validity. In this paper an experimental and numerical micromechanics analysis is developed with application to cortical bone. Displacement and strain fields are obtained across the microstructure by means of digital image correlation (DIC). The other mechanical variables are computed following the micromechanics theory. Special emphasis is given to the differences found in the different field variables between the micro- and macro-structures, which points out the need for this multiscale approach in cortical bone tissue. The obtained results are used to establish the basis of a multiscale methodology with application to the analysis of bone tissue mechanics at different spatial scales.