In embryonic development of long bones, a cartilaginous anläge develops into bone tissue The development from embryonic cartilage to mature bone passes many stages like chondrocyte proliferation, chondrocyte hypertrophy, mineralization of the cartilage matrix, penetration of blood vessels in the tissue, resorption of mineralized cartilage, and bone formation These processes continue in the growth plates, where longitudinal growth takes place At matunty, the longitudinal growth stops and the growth plates close Mechanically, two types of bone are present in adult long bones, cortical bone, which is dense and compact, and trabecular bone, which is porous, owing to its structure of rods and plates.
The purpose of this thesis was to study the influence of mechanical loading on different processes in the development from embryonic to mature bone If for any reason one of these processes is disturbed, a compromised bone development and function may result Proper understanding of the regulatory mechanisms in bone development is important for prevention and treatment of musculoskeletal developmental deformities and osteoporosis, and also for bone regeneration during fracture healing and implant fixation, and for tissue engineering.
The first stage in bone development that was studied was chondrocyte hypertrophy In chapter three the hypothesis that hypertrophic chondrocytes pressunze the extracellular matrix and subsequently initiate or stimulate the mineralization process was discussed It was shown that during cell hypertrophy cell volume increased relatively to matrix volume, which is a precondition to support the theory that cell hypertrophy pressunzes the matrix In addition, increase of chondrocyte volume was accompanied by increase in mineralization rate, which is a precondition for the theory that larger cell volumes result in increased mineralization rates through the increased pressures However, we only showed that the hypothesis is feasible Additional research is required to test whether the hypothesis can be confirmed.
Cartilage mineralization was the next stage in bone development that was studied In chapter two, we showed that the stiffness for embryonic rudiments during mineralization increases by two orders of magnitude within one day Hence, within a penod of hours, the chondrocytes in the calcifying cartilage experience a significant change in mechanical environment that will lead to decreased cell deformation and stress shielding Since the transition is so sudden and gigantic, it can be seen as a process of 'catastrophic' proportion for the cells.
Indications were found that mechanical loads stimulate the mineralization process In chapter four, the mineralization process in the in vitro expenments, in which embryonic mouse metatarsals were externally loaded with an intermittent hydrostatic pressure, were evaluated. A poroelastic finite element analysis (FEA) was performed to calculate the local distributions of distortional strain and fluid pressure at the mineralization front in the metatarsal during intermittent hydrostatic loading. Distortional strains appeared to be insignificant under these loading conditions. Hydrostatic pressure was the only candidate left to have stimulated the mineralization process. We then hypothesized that the pressure may have created the physical environment enhancing the mineralization process, in which diffusion of ions may have contributed. In chapter five the influence of muscular forces on the local mineralization process in embryonic metatarsals of the mouse was studied. The mineralization front in vivo, in the presence of muscular forces, was found to be nearly straight, whereas in vitro it acquired a more convex shape. Using poroelastic FEA, local distributions of distortional strain and fluid pressure at the mineralization front during muscular loading showed that the role of hydrostatic pressure was unclear. It might have stimulated the mineralization front as a whole, but we could not prove nor exclude this possibility. The most likely candidate to explain the difference between in vivo and in vitro mineralization geometry was distortional strain, resulting from muscle contractions.
The last stage during development that was studied was trabecular bone. In chapter six, we questioned how trabecular bone density and architecture would change during growth. Morphological and mechanical parameters of three-dimensional (3D) trabecular bone samples from the vertebra and proximal tibia of pigs were studied using micro-computer tomography (μ€Τ) and micro FEA. The results showed that both bone volume fraction and stiffness increased rapidly in the initial growth phase, whereas the morphological anisotropy started to increase at a later stage. In addition, the anisotropy reached its highest value much later in the development than bone volume fraction did. We concluded that density is adapted to external load from the early phase of growth, whereas the trabecular architecture is adapted later in the development.
The results of chapter six let us hypothesize that during growth, the relatively older trabecular architecture in the epiphysis is better adapted to mechanical load than the younger architecture in the metaphysis, in which the trabeculae are gradually renewed due to the presence of a growth plate. As a first step to test this hypothesis, the 3D morphology of the trabecular structure in the metaphysis and epiphysis of growing pigs was characterized in chapter seven, using μ€Τ. The results showed that the trabecular architecture in the epiphysis had higher bone volume fraction, thicker trabeculae, and higher bone surface density compared to the structure in the metaphysis. In addition, its structure was plate-like, whereas the structure in the metaphysis was more rod-like. The anisotropy was somewhat higher in the epiphysis compared to the metaphysis, but not significantly so. However, the standard deviations of anisotropy were very significantly different. The trabecular structure of the epiphysis showed a low variety in anisotropy, which reflects its better structural organization compared to that in the metaphysis, for which the degree of anisotropy was much more variable. This sustains the hypothesis that the trabecular structure in the epiphysis reflects a more mature stage in growth and mechanical adaptation as compared to metaphyseal bone.
In chapter eight, we discussed the study in general and concluded that it is realistic that bone development is influenced by mechanical load. The research described in this thesis combined cell biological and biomechanical techniques, meant to gain more insight in the process of bone development and its relation to mechanical forces. For most of the developmental stages we suggested research directions to further study the process. Eventually, this could lead to a complete understanding of bone development, so that attempts could be made to better prevent and treat congenital deformities, osteoporosis, and osteoarthritis, and to make fracture healing more efficient.