published_or_final_version / abstract / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

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Tooth extraction is necessary when there is advanced decay, significance trauma or injury to the oral cavity, misalignment associated with crowded teeth or even, poor eruption of third molars. Typically, during extraction procedures, bone grafting material is inserted to minimize bone loss. Bone grafting has become a standard procedure in dentistry and an essential component in modern medicine. The practice started nearly 350 years ago when a Dutch doctor in 1668 performed the first and successful bone grafting operation. Currently, research studies are ongoing to enhance the success and viability of dental reconstruction. This thesis examines the effect of varying dental graft techniques and methodology on the stability and long-term effect on the jawbone. These techniques include five primary bone grafting methods, onlay, inlay, ridge expansion, distracted osteogenesis and guided bone regeneration (GBR) have been approved to enhance the outcome of dental implants. The review has presented the documented limitations and viability of each method. In this paper, the alternatives to bone grafting have also been elaborated. Based on the findings depicted in this research, the following recommendations will assist in achieving the best jawbone outcomes in long-term and short-term assessments. These recommendations are as follows, a proper evaluation of each patient to determine their health condition. The nature of the infection, injury, and trauma that led to the extraction of the tooth should be well documented. The selection of technique pertaining to dental restoration, functionality, and aesthetic needs are of primary factors considered. A proper clinical follow-up and monitoring of recovery process is an essential part that contributes to valid results. Finally, patient awareness is essential as well. The study found out that although some of the bone grafting techniques have shown a high survival rate, significant alveolar bone quality and quantity, and success of the implants, several reports that the use of bone grafts and implants is a practice that will still dominate dental surgery and attract more clinical assessments.

Bone is the most commonly replaced organ, with nearly 1 million grafting procedures performed annually in the United States. Inherent limitations associated with bone grafts, such as graft availability and donor site morbidity, leave room for alternative grafting solutions. Current mineralized tissue engineering approaches include the use of synthetic hydroxyapatite as cement or as nano- or micro-particles pre-incorporated into a tissue engineering scaffold prior to cell seeding or implantation. While promising results have been reported with such methods, these constructs are not biomimetic as they fail to replicate neither the size, distribution, nor density of mineral inherent in the native bone, leading to inferior mechanical properties and supra-physiologic levels of calcium phosphate that can disrupt healing, alter cell response and inhibit normal tissue homeostasis. To address these issues, inspiration is taken from the native biomineralization process which is often facilitated by matrix vesicles, a lipid-based nanocarrier within which calcium and phosphate ions are combined to form calcium phosphate mineral in hard tissues such as bone. Synthetic matrix vesicles (SMV) formulated from self-assembling liposomes have emerged as a promising model both for studying the biomineralization process as it relates to matrix vesicles and for use in regenerative medicine. The ideal SMV system is defined as follows: the mineral formed should match the native calcium phosphate in both structure and chemistry, the mineral must be stable in the physiological environment and can continue to grow in size when necessary and the matrix vesicles should also be able to work in conjunction with a scaffold tailored for bone tissue engineering. It is hypothesized that the formation of native bone-like calcium phosphate can be achieved with the controlled optimization of matrix vesicles in terms of fabrication parameters, ion transport, cell response and interactions with a gelatinous matrix. To this end, a liposome-based, biomimetic matrix vesicle system was designed to facilitate vesicle-mediated biomineralization for regeneration of calcified tissues. Synthetic matrix vesicles were fabricated from two different phospholipids, DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DMPC (1,2-bis(myristoyl)-sn-glycero-3-phosphocholine) and optimized in terms of membrane composition, alkaline phosphatase bioconjugation, and ion encapsulation. Calcium (Ca2+) and phosphate (Pi) ions were successfully encapsulated within the liposomes. Ion permeability across the bi-layer membrane, which is necessary for Ca2+ and Pi to combine within the SMV for mineralization, was found to increase with increasing DMPC composition, validated through ion release studies and diffusion modeling through Fick's 2nd Law. In addition, alkaline phosphatase (ALP), an enzyme which cleaves Pi from organic phosphate molecules for mineral formation with Ca2+, was successfully conjugated to the SMV membrane through the use of biotin-functionalized phospholipids and streptavidin-ALP. Human osteoblast-like cells were dosed with the optimized SMV and the effects of SMV type and dosage on mineralization response was evaluated. Mineralization potential of human osteoblast-like cells was found to decrease through exposure to Pi-encapsulated SMV similar to the response found for human osteoblast-like cells supplemented with beta-glycerophosphate (beta-GP), an organic phosphate source typically used in mineralization in vitro studies. Human osteoblast-like cells were also dosed with two different configurations of ALP SMV liposomes with ALP bound within (ALP-inside SMV) and liposomes with ALP bound to the membrane on the outside (ALP-outside SMV). ALP-outside SMV were ultimately selected for further study since the location of the ALP in the outside configuration more closely mimics the structure of native matrix vesicles. While mineral-like structures were observed in several types of SMV under cryo-electron microscopy, no bulk mineralization was observed by human osteoblast-like cells from SMV supplementation alone. This motivated a dosage study conducted with the Pi SMV which optimized the cell-to-liposome ratio and the concentration of Pi encapsulated. The optimized ALP-outside SMV and Pi SMV were individually combined with an electrospun gelatin nanofiber scaffold to further promote cell mineral deposition by acting as a biomimetic substrate for calcium phosphate nucleation. It was demonstrated that in the absence of growth factor stiumulation, culture of human osteoblast-like cells with SMV+beta-GP and Pi SMV resulted in mineral deposition on the gelatin nanofiber scaffold. Human mesenchymal stem cells (hMSC), a more clinically relevant cell type, were also cultured on the SMV-gelatin scaffold system. Mineralization potential was found to increase for hMSC cultured with ALP SMV, and the osteogenic marker osteocalcin was upregulated for cultures with Pi SMV. Dosage of hMSC with SMV+beta-GP and Pi SMV alone resulted in the formation of a mineralized matrix. In summary, this thesis focuses on the design of a biomimetic, liposome-based synthetic matrix vesicle system and elucidates the compositional and dosage parameters for the formation of calcified tissue by human osteoblast-like cells and MSCs. The synthetic matrix vesicle system developed in this thesis can be utilized for further investigation into the mechanisms of biomineralization, in addition to its potential for use in promoting cell-mediated regeneration of a variety of calcified tissues, including bone, teeth and mineralized soft-to-hard tissue interfaces.

Background: Bone-grafting materials are required in orthopaedic surgery to treat bone defects. Bone formation assessment is required for the development of new strategies and approaches and for quality assurance and quality control of currently available materials. Approaches to the assessment of bone formation are yet to be systematically established, quantified and standardized. Aims: the overall aim of this study was to establish a set of comprehensive quantitative approaches for the assessment of bone formation and to evaluate the role of osteoblastic cells, growth factors, and scaffolds on this process. Materials & methods: both in vitro and in vivo parameters for osteoblast phenotype and bone formation were tested in osteosarcoma cell lines, Saos-2 and U2OS cells, mesenchymal cell line, C2C12 cells, primary adipose derived stromal cells (ADSCs), platelet rich plasma (PRP), and morselized bone grafts. The in vitro parameters used were measurement of alkaline phosphatase (ALP) activity, detection of bone nodules and biomineralization, and quantification of immunocytochemistry and conventional RT-PCR of osteoblast genotyping. In vivo parameters involved ectopic bone formation in nude mice and nude rats and a tibial defect model in nude rats. Histomorphometric and quantitative immunohistochemical analyses were also performed. Results: The in vitro characterization and ectopic bone formation capabiltity of Saos-2 and U2OS cells have been established. Saos-2 cell line, which presents many osteoblast genotype and phenotype, is a stable positive control for both in vitro and in vivo bone formation assessments. The measurement of ALP activity in both solid and liquid phases has been standardized. Both the genotype and phenotype of osteoblast lineage cells has been quantitatively assessed during the capability testing of ADSCs and PRP. Quantitative assessment of new bone formation and related protein markers in vivo has been successfully established through the testing of the biological properties of gamma irradiated morselized bone grafts. Conclusion: A comprehensive knowledge of the assessment of bone regeneration and formation in vitro and in vivo has been integrated and developed through years of study. A whole set of in vitro and in vivo approaches for the assessment of bone formation has been modified and standardized to best suit the different clinical applications. This thesis provides an outline of both in vitro and in vivo bone formation assessment and their clinical applications.

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Materials Science and Engineering." Includes bibliographical references.

Thesis (M.S.)--University of Michigan, 1986.

Thesis (M.S.)--University of Michigan, 1985.

Akademisk avhandling--Universitetet i Göteborg. / Extra t.p., with thesis statement, inserted. Bibliography: p. 88-95.

Thesis (M.S.)--University of Michigan, 1985.