Mesenchymal tissue is a multipotent tissue with the capability to differentiate into a variety of skeletal tissues including bone, cartilage, fibrocartilage, or fibrous tissue. Mechanical stresses play an important role in regulating this differentiation in a variety of contexts during skeletal development and regeneration. This dissertation presents four studies analyzing the effects of mechanical loading on mesenchymal tissue differentiation during 1) oblique pseudarthrosis formation, 2) mandibular distraction osteogenesis (one experimental and one computational study), and 3) soft skeletal tissue regeneration.

The first study examines the role of mechanical loading on tissue differentiation during the development of oblique pseudarthroses. It implements a previously developed mechanobiological tissue differentiation concept relating hydrostatic stress and maximum principal tensile strain to the formation of bone, cartilage, fibrous tissue, or fibrocartilage along with two-dimensional finite element analysis to predict locations of cartilage, fibrocartilage, and bone within a typical oblique fracture. Finite element analysis is used to determine regions of maximum principal tensile strain and hydrostatic stress. It is confirmed that locations of hydrostatic pressure corresponded to regions of cartilage formation, locations of tensile strain combined with hydrostatic pressure correspond to regions of fibrocartilage formation, and that locations of mild hydrostatic tension and low tensile strain correspond to bone formation. However, it is also shown that regions of excessive tensile strain correspond to locations of mesenchymal tissue failure and that regions of excessive hydrostatic pressure correspond to locations of periosteal bone resorption. Information from this study is used to both explain how pseudarthrosis formation might occur in oblique fractures as a result of mechanical loading in vivo and to update the previous mechanobiological tissue differentiation concept to include the limiting boundaries of pressure necrosis and tensile failure.

The second study is an experimental analysis of mesenchymal tissue differentiation during mandibular distraction osteogenesis. Both successful (bony union) and unsuccessful (fibrous union) protocols of mandibular distraction osteogenesis in a rat model are analyzed. The protocols for successful (gradual lengthening) and unsuccessful (acute lengthening) consolidation are obtained from a previously published analysis of rat mandibular distraction osteogenesis. Our focus in this study is to quantify the actual timedependent changes in the mechanical environment within the tissue regenerate occurring as a result of the distraction procedure during mandibular distraction osteogenesis. We perform mechanical tests on the tissue regenerate within the distraction gap of gradually lengthened specimens at 4 different time points:​ end latency, distraction day 2, distraction day 5, and distraction day 8. Results from these mechanical tests are used to:​ 1) determine if tissue failure occurs in vivo as a result of tissue distraction;​ and, 2) calculate the actual tensile forces, stresses, and strains occurring in the tissue regenerate as a result of the distraction protocol. Mechanical testing is followed by histological analyses of the specimens to obtain correlations between the distraction-induced mechanical loads and de novo skeletal tissue regeneration within the regenerate tissue. Histological data is further used to determine the amounts of new bone apposition within the distraction gap so that tensile strains occurring in the mesenchymal tissue during each distraction can be calculated. This study provides new information about the actual mechanical loads resulting from distraction osteogenesis and, further, correlates specific magnitudes of tensile strains occurring during distraction to de novo bone formation.

The third study expands upon the second by utilizing three-dimensional finite element analysis to obtain information about magnitudes and locations of hydrostatic stress and tensile strain within the multipotent mesenchymal tissue regenerate. We obtain computed tomography (CT) scans of each specimen at the specific time point of interest (end latency, distractions days 2, 5, or 8) and use the CT data to create finite element models of the true geometry of the tissue regenerate at each time point. Boundary condition information is obtained from the experimental analyses described in the second study above. Rnite element analyses are performed to determine hydrostatic stresses and maximum principal tensile strains within the tissue regenerate at each time point. Magnitudes of tensile strain are then compared to those determined empirically. Information from this study is used to determine the time-dependent changes in magnitudes and patterns of hydrostatic stress and tensile strain within the tissue regenerate and to correlate these changes to skeletal tissue regeneration throughout mandibular distraction osteogenesis.

The final study examines how the material properties of multipotent mesenchymal tissue change during the differentiation process associated with soft skeletal tissue regeneration. Using a fiber-reinforced poroelastic model of soft skeletal tissues, we track time-dependent material property adaptations in tensile elastic modulus (E), permeability (k), and aggregate modulus (H_A) during the differentiation of mesenchymal tissue into articular cartilage, fibrocartilage, or fibrous tissue. In this mathematical approach, intermittently imposed fluid pressure and tensile strain regulate proteoglycan synthesis and collagen fibrillogenesis, assembly, cross-linking, and alignment Cyclic fluid pressure causes an increase in proteoglycan synthesis, resulting in a decrease in k and increase in H_A due to the hydrophilic nature and large size of the aggregating proteoglycans. It further causes a slight increase in E due to the stiffness added by newly synthesized type II collagen. Tensile strain increases the density, size, alignment, and cross-linking of collagen fibers, thereby increasing E while also decreasing transverse k as a result of an increased flow path length. Implementing a computer algorithm based on these concepts, we simulate progressive changes in material properties for differentiating soft skeletal tissues.

The results of this thesis verify the importance of mechanobiological factors in multipotent mesenchymal tissue differentiation during skeletal tissue regeneration. The relationships examined in this thesis are essential to understanding the time-dependent changes that occur during differentiation as a result of both physiologic and artificially imposed mechanical loads at a site of regenerating tissue.