B one regeneration is a well-orchestrated natural process of bone reconstruction during the continuous remodeling throughout adult life, fracture healing, or complex clinical conditions, including the skeletal reconstruction of bone defects created by infection, trauma, tumor resection, and skeletal abnormalities and discrepancies. In this biological problem, the role of engineering is essential for several aspects, including the control of the callus mechanical environment, predicting the complex mineralization phenomena, and designing materials and structures to optimize osteogenesis. In this line, the aim of this Ph.D. thesis is to find, through in vivo and ex vivo experiments in a fixed animal model and mechanical condition, biological and mechanical solutions to current challenging limitations. The following ones stand out:​ the current mechanical incompatibility between brittle implants and external fixations in load-bearing models, the insufficient knowledge of the mechanical changes suffered by the surrounding soft tissues (e.g., tendons, muscles, or skin) during an elongation process, and their biological limits, the lack of understanding of the mechanical environment that promotes mineralization, or the need of quantitative tools for better clinical control of the processes to avoid premature fractures.

Distraction osteogenesis and tissue engineering experiments were carried out on a total of 20 merino sheep (12 and 8 animals, respectively) through the cooperation of a multidisciplinary team composed of veterinarians, surgeons and animal anesthetists, and engineers. A flexible Ilizarov-type external fixator (593 N/mm) was initially implanted in the metatarsus of their ipsilateral hindlimb to stabilize the resulting bony fragments. The distraction group underwent an osteotomy in a middle cross-section of the bone that, after a latency period of 7 days, was elongated to a final length of 15 mm (distraction rate of 1 mm/day). By cons, the tissue engineering group had 15 mm of bone directly replaced with a porous subject-specific bioceramic scaffold biologically enhanced with cancellous bone autograft. Animals were slaughtered at different time-points of the consolidation phase, up to a year after surgery, to analyze ex vivo callus samples with several levels of maturation and ossification under different approaches.

Before starting the experiments, the design and in vitro validation of both external fixator and scaffold were required. A versatile instrumented external fixator was devised to control bone formation through mechanical parameters indirectly. By working with a low-cost and size-optimized real-time wireless acquisition system, they reported promising results in estimating callus forces and stiffening, average errors and uncertainties below 6.7% and 14.04%. The resulting system offers mechanobiological comparisons of the evolutions of different regeneration treatments (fracture healing, bone lengthening, bone transport, bone grafts and tissue engineering) under a fixed mechanical environment. Concerning the scaffold design, a standardized optimization problem was defined and applied to optimize their internal architecture by covering biological parameters (porosity, pore size, and specific surface area) and mechanical constraints to ensure its structural integrity in vivo. After manufacturing by robocasting, the optimized structure (59.30% of porosity, 5768.91 m⁻¹ of specific surface area, and 360.80 µm of pore size) and its compressive strength under the flexible fixation environment were tested and validated in vitro using tomographic images and compression tests.

During the bone lengthening distraction phase, higher viscoelastic forces with a similar relaxation rate were quantified compared to other distraction processes that do not involve limb elongation. After applying rheological models to the distraction raw data, the mechano-structural changes suffered by this bone callus and its surrounding soft tissues were estimated. The contribution of these adjacent tissues to the distraction forces was limited to the elongations above 4% of the original length when stain hardening and anatomical changes were evidenced. Likewise, the quantified elastic callus stiffening was mathematically modeled to elucidate the mechano-structural phenomena behind its maturation and its successful accommodation-to-mineralization. The mathematical model was based on ex vivo confocal imaging data applied to non-mineralized callus samples. The continuous collagen reorientation, densification and maturation seemed to control the problem.

In the consolidation stage, several in vivo monitoring techniques were employed to build an exhaustive comparison between distraction and tissue engineering processes:​ gait analysis of the ground reaction force and limb contact time with the ground, internal force distribution, bone callus stiffening, x-rays, and macro- and micro-computed tomographic imaging. All the parameters analyzed generally tended to healthy values in both regeneration groups but at different rates. In the short-term, the bone callus recovered its loading capacity, and it exponentially increased its apparent stiffness, being faster in the tissue engineering group due to its premature mechanical and structural contribution of the scaffold. The healthy callus mineral density and limb-bearing capacity were reached around 5-6 months after surgery. However, the recovery time of this last parameter seems to have been influenced by the walking conditions acquired by the animals due to pain and low confidence in the treated limb in the early stages. This makes it a poor parameter for clinical decision-making, especially in the distraction group with additional elongation complications. Finally, both the apparent geometry of the callus and its trabecular microstructure seemed to recover in the long remodeling phase (>1 year), although its growth stage at both scales maintained a certain degree of significance in its evolution.