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Thesis (Ph.D.). / Written for the Dept. of Biology. Title from title page of PDF (viewed 2008/01/12). Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 182-200) Also available in print.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23058号 / 医博第4685号 / 新制||医||1048(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 浅野 雅秀, 教授 別所 和久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The synchronization of flight mechanics with respiration and echolocation call emission by bats, while economizing these behaviors, presumably puts compressive loads on the cartilaginous rings that hold open the respiratory tract. Previous work has shown that during postnatal development of Artibeus jamaicensis (Phyllostomidae), the onset of adult echolocation call emission rate coincides with calcification of the larynx, and the development of flight coincides with tracheal ring calcification. In the present study, I assessed the level of reinforcement of the respiratory system in 13 bat species representing six families that use stereotypical modes of echolocation (i.e. duty cycle % and intensity). Using computed tomography, the degree of mineralization or ossification of the tracheal rings, cricoid, thyroid and arytenoid cartilages were determined for non-echolocators, tongue clicking, low-duty cycle low-intensity, low-duty cycle high-intensity, and high-duty cycle high-intensity echolocating bats. While all bats had evidence of cervical tracheal ring mineralization, about half the species had evidence of thoracic tracheal ring calcification. Larger bats (Phyllostomus hastatus and Pterpodidae sp.) exhibited more extensive tracheal ring mineralization, suggesting an underlying cause independent of laryngeal echolocation. Within most of the laryngeally echolocating species, the degree of mineralization or ossification of the larynx was dependent on the mode of echolocation system used. Low-duty cycle low-intensity bats had extensively mineralized cricoids, and zero to very minor mineralization of the thyroids and arytenoids. Low-duty cycle high-intensity bats had extensively mineralized cricoids, and patches of thyroid and arytenoid mineralization. The high-duty cycle high-intensity rhinolophids and hipposiderid had extensively ossified cricoids, large patches of ossification on the thyroids, and heavily ossified arytenoids. The high-duty cycle high-intensity echolocator, Pteronotus parnellii, had mineralization patterns and laryngeal morphology very similar to the other low-duty cycle high-intensity mormoopid species, perhaps suggesting relatively recent evolution of high-duty cycle echolocation in P. parnellii compared with the Old World high-duty cycle echolocators (Rhinolophidae and Hipposideridae). All laryngeal echolocators exhibited mineralized or ossified lateral expansions of the cricoid for articulation with the inferior horn of the thyroid, these were most prominent in the high-duty cycle high-intensity rhinolophids and hipposiderid, and least prominent in the low-duty cycle low-intensity echolocators. The non-laryngeal echolocators had extensively ossified cricoid and thyroid cartilages, and no evidence of mineralization/ossification of the arytenoids or lateral expansions of the cricoid. While the non-echolocators had extensive ossification of the larynx, it was inconsistent with that seen in the laryngeal echolocators.

Among vertebrates, oviparous animals lay eggs with a calcified eggshell and eggs are laid in an external environment, while viviparous animals retain eggs in oviduct until they give birth to live young. Viviparity has evolved in the lineage of snakes and lizards (squamates) over 100 times, more than all other vertebrate groups. Embryos of oviparous squamates obtain calcium from both yolk and eggshell while their viviparous counterparts lack a calcified eggshell and must obtain their calcium solely from yolk, or from yolk plus placental transfer. During embryonic development, squamates rely on calcium to build the skeleton before hatching. The extent of skeletal ossification at hatching or birth varies considerably among vertebrates. This study aims to determine if skeletal development/ossification varies in association with reduced eggshell calcium in embryos of viviparous squamate species. We studied the amount of ossification and overall size of embryos and hatchlings from an oviparous and a viviparous population of the reproductively bimodal lizard Zootoca vivipara. Previous studies suggested that limb development is delayed, and that overall size is reduced in viviparous squamates. We tested the hypothesis that viviparous embryos and hatchlings are more skeletally immature and smaller in size than oviparous embryos and hatchlings in squamates. To achieve this, specimens from both populations, spanning multiple stages of embryonic development including hatchlings, were cleared & stained to reveal skeletal cartilage and bone. Lengths of total body, humerus, femur, skull and Meckel’s cartilage were measured from photographs of cleared & stained specimens taken with a Cannon EOS 70D camera on a Motic, Leica MZ9 Compound Microscope, with a measurement reference in each picture. Photos were calibrated to the measurement reference and total length measurements were obtained using iSolutionLite® software. In addition to total measurements, the lengths of ossified portions on the humerus and femur were also measured. Preliminary results revealed that total length of the skull and body are reduced in embryos and hatchlings of viviparous specimens. Total length of the limbs and of Meckel’s cartilage are not significantly different between populations. However, it appears that the amount of ossification in the limbs is reduced in oviparous specimens. These preliminary findings do not support our hypothesis and indicate that reduction in eggshell calcium in embryos of viviparous populations does not negatively impact ossification during development but does influence overall size.

Bone formation occurs through two ossification processes, intramembranous and endochondral. Intramembranous ossification is characterized by the direct differentiation of stem cells into osteoblasts, which then create bone. Endochondral ossification involves an intermediate step, as stem cells first differentiate into chondrocytes and produce a cartilage anlage. The chondrocytes mature into hypertrophic chondrocytes, which transform the cartilage anlage into bone. Bone tissue engineering has predominantly mimicked intramembranous ossification, creating osteoblast-based grafts through the direct differentiation of stem cells. Though successful in specific applications, greater adoption of osteoblast-based grafts has failed due to incomplete integration, limited regeneration, and poor mechanical maintenance. To overcome these obstacles, inspiration was drawn from native bone fracture repair, creating tissue engineered bone grafts replicating endochondral ossification. Hypertrophic chondrocytes, the key cell in endochondral ossification, were differentiated from mesenchymal stem cell sources by first generating chondrocytes and then instigating maturation to hypertrophic chondrocytes. Conditions influencing this differentiation were investigated, indicating the necessity of prolonged chondrogenic cultivation and elevated oxygen concentrations to ensure widespread hypertrophic maturation. Comparing the bone production performance of differentiated hypertrophic chondrocytes to differentiated osteoblasts revealed that hypertrophic chondrocytes deposit significantly greater volume of bone mineral at a higher density than osteoblasts, albeit in a more juvenile form. When implanted subcutaneously, the hypertrophic chondrocytes stimulated turnover of this juvenile template into compact-like bone, whereas osteoblasts proceeded with processes similar to bone remodeling, generating spongy-like bone. Implanting these tissue engineered constructs into an orthotopic, critical-sized femoral defect saw hypertrophic chondrocyte-based constructs integrate quickly with the femur and facilitate the creation of significantly more bone, resulting in a successful bridging of the defect. The success of hypertrophic chondrocyte-based grafts in overcoming the failures of tissue engineered bone grafts demonstrates the potential of endochondral ossification inspired bone strategies and prompts its further investigation towards clinical utilization.

The articular cartilage has a limited capacity to regenerate. Cartilage lesions often result in degeneration, leading to osteoarthritis. Current treatments are mostly palliative and reparative, and fail to restore cartilage function in the long term due to the replacement of hyaline cartilage with fibrocartilage. Although a stem-cell based approach towards regenerating the articular cartilage is attractive, cartilage generated from human mesenchymal stem cells (hMSCs) often lack the function, organization and stability of the native cartilage. Thus, there is a need to develop effective methods to engineer physiologic cartilage tissues from hMSCs in vitro and assess their outcomes in vivo. This dissertation focused on three coordinated aims: establish a simple in vivo model for studying the maturation of osteochondral tissues by showing that subcutaneous implantation in a mouse recapitulates native endochondral ossification (Aim 1), (ii) develop a robust method for engineering physiologic cartilage discs from self-assembling hMSCs (Aim 2), and (iii) improve the organization and stability of cartilage discs by implementing spatiotemporal control during induction in vitro (Aim 3). First, the usefulness of subcutaneous implantation in mice for studying the development and maintenance of osteochondral tissues in vivo was determined. By studying juvenile bovine osteochondral tissues, similarities in the profiles of endochondral ossification between the native and ectopic processes were observed. Next, the effects of extracellular matrix (ECM) coating and culture regimen on cartilage formation from self-assembling hMSCs were investigated. Membrane ECM coating and seeding density were important determinants of cartilage disc formation. Cartilage discs were functional and stratified, resembling the native articular cartilage. Comparing cartilage discs and pellets, compositional and organizational differences were identified in vitro and in vivo. Prolonged chondrogenic induction in vitro did not prevent, but expedited endochondral ossification of the discs in vivo. Finally, spatiotemporal regulation during induction of self-assembling hMSCs promoted the formation of functional, organized and stable hyaline cartilage discs. Selective induction regimens in dual compartment culture enabled the maintenance of hyaline cartilage and potentiated deep zone mineralization. Cartilage grown under spatiotemporal regulation retained zonal organization without loss of cartilage markers expression in vivo. Instead, cartilage discs grown under isotropic induction underwent extensive endochondral ossification. Together, the methods established in this dissertation for investigating cartilage maturation in vivo and directing hMSCs towards generating physiologic cartilage in vitro form a basis for guiding the development of new treatment modalities for osteochondral defects.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Boyan, Barbara; Committee Co-Chair: Schwartz, Zvi; Committee Member: Bonewald, Lynda; Committee Member: Jo, Hanjoong; Committee Member: Sambanis, Athanassios. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.