We focus on two topics, multi-scale experimental characterization of structure and mechanical properties of bone as a function of age and regeneration of bone in long bone critical sized defects.

In the first part of this study, a multi-scale experimental work was carried out to characterize cortical bone as a heterogeneous material with hierarchical structure. We analyzed bone at several different length scales:​ nanoscale (1 nm -100 nm, apatite crystal and collagen fibril level), sub-microscale (1-10μm, single lamella level), microscale (10 -500μm, single osteon level), and mesoscale (1–10 cm, involving a random arrangement of osteons, lamellar bone and/or woven bone, representing cortical bone). Macroscale level represents a whole bone level which includes both cortical and trabecular bones. Samples prepared from swine femoral cortical bones from three age groups (6-month, 12-month and 42-month) were used to study the age-related changes. The mechanical properties of cortical bone at meso-scale were measured by tensile and compression testing and the modulus and hardness were measured at the single lamella level using nanoindentation. Scanning electron microscopy (SEM) and micro-computed tomography (micro-CT) were used to analyze the structural variations in bones from different age groups from sub-micro to meso-scale levels. The bone’s chemical composition and its spatial distribution were characterized by combining the ash content method, Duel Energy X-ray Absortionmetry (DEXA) and Fourier transform infrared microspectroscopy (FTIRM). These experimental results indicated significant age-related changes in both structure and chemical composition of cortical bone. Woven bone was dominant in 6-month samples, lamellar bone was a prevalent structure in organic ratio increased as bone matured. The superior bone structure and high mineralization level led to the increase in the elastic modulus. The increase of the tensile strength with age could be attributed to the decrease of the porosity and the increased fraction of tough and stiff microstructures. In addition to these measurements, the effect of sample geometry and shape as well as bone’s anisotropy on tensile properties were investigated.

In the second part, our work focused on strategies for healing critical size defects in long bones resulting from traumatic injuries or diseases. We have developed a small animal in vivo load bearing model to study the effect of a biocompatible artificial polymer scaffold on regeneration of long bone defects in adult African Clawed Frogs (Xenopus laevis) hind limbs. We first designed and fabricated scaffolds made of 1,6 hexanediol diacrylate (HDDA) using an innovative three dimensional microfabrication technology called Projection Micro-Stereolithography. Critical size defects were made in one bone of the dual skeletal element hind limb tarsus bone in adult Xenopus laevis frog. HDDA scaffolds were soaked with two growth factors:​ BMP4 and VEGF. Defects in control frogs were left empty, or were implanted with scaffolds lacking growth factors. The limbs were harvested at a series of time points ranging from 3 weeks to 6 months after implantation. We employed Micro-CT to assess the shape and density of the regenerated tarsus, and standard histology to evaluate tissue types and the anatomical relationships. In frogs treated with growth factors soaked scaffolds, five out of eight defects were completely filled with cartilage by 6 weeks. Blood vessels had invaded the cartilage, and bone was beginning to form in ossifying centers. By 3 months these processes were well advanced. In contrast, defects in control frogs showed formation of fibrous scar tissue and the negligible cartilage formation was observed in defects. Our studies demonstrate the feasibility of using scaffolds loaded with carefully selected growth factors to repair long bone defects over gaps of critical size by developmental regeneration.