Indiana University-Purdue University Indianapolis (IUPUI) / The effect of weightlessness on bone and osteoblast precursor cells has previously been studied. A marked decrease in bone formation, an increase in less differentiated committed osteogenic cells (A+A'), and a decrease in preosteoblast cells (C+D) was noted. To date, the effect of hypergravity (2g) on osteoblast histogenesis has not been studied in vivo. In vitro studies using nonphysiologic high levels of gravity (20,40g) have shown an increased proliferation of cloned osteoblast-like cells. The purpose of the present study was to evaluate the effect of hypergravity (2g) on osteoblast histogenesis in the rat periodontal ligament (PDL), on the width of the mesial PDL, and on the percentage of forming bone surface on the mesial side of the tooth. Twenty male Wistar rats (SPF: Harlan Sprague Dawley) were randomly assigned to the centrifuge (experimental) or to the stationary (control) group. The experimental group was centrifuged for 14 days at 2g and the stationary group was housed in identical cages in the centrifuge room. The PDL of the mesial and distal surface of the mesial root of the first maxillary molar was analyzed microscopically 100 μm above and below the midroot area. Nuclear volume morphometry was used to classify periodontal ligament cells as: L cells (<40 μm3), A+A' cells (40-79 μm3), B cells (80-119 μm3), C cells (120-169 μm3), and D cells (>170 μm3). The percent of forming bone surface on the mesial side and the width of the PDL were also measured. A 2x2 factorial ANOVA with repeat measures revealed a significant (p < 0.05) decrease in the C cell population and a nearly significant (p < 0.06) increase in the A+A' cell population in the centrifuge group. Comparing bone surfaces, the forming surface had a significant (p < 0.01) increase in the C and D cell populations, a significant (p < 0.01) decrease in the L and A+A' cell populations, and a significant (p < 0.05) decrease in the B cell population. The stationary group weighed significantly (p < 0.01) more than the centrifuge group post-experiment. And an unpaired t-test revealed a nearly significant (p < 0.06) increase in the percent forming bone surface on the mesial side of the maxillary first molar and no significant difference in mesial PDL width. The results showed that the centrifuge group had a trend toward a block in preosteoblast formation. This is similar to that seen with hypogravity. However, it cannot be concluded at this time if this is a direct gravitational effect or related to other factors such as physiological response to stress. Physical stress has been suggested as a potential mechanism for the observed decrease in weight seen in centrifuged animal, while the PDL width does not seem to be affected by gravitational forces, and thus may not be a sensitive marker to osteoblast differentiation inhibition. Finally, the reason for the increase in forming bone surface in the centrifuged group is unclear.

Bibliography: leaves 184-220.

Periodontitis is a chronic inflammatory infection caused by the overgrowth of bacteria harbored in tooth-retained plaque. It is estimated to affect 50% of American adults over 30, with an increased incidence of up to 70% for those over 65. The disease is characterized by the destruction of the periodontal tissues, including the periodontal ligament (PDL), root cementum, and alveolar bone. As the PDL provides tooth anchorage by connecting the root cementum to the alveolar bone, damage to this tissue results in a loss of integration with the surrounding bone and cementum, eventually leading to complete tooth detachment. This is the primary reason for tooth extractions and/or loss. Current treatments for periodontitis fail to achieve consistent PDL regeneration and integration of soft and hard tissues, thus alternative approaches are needed to improve long term outcomes. This thesis focuses on the development of a biomimetic, fiber-based, polymer composite scaffold that will enable the regeneration and integration of the hard and soft tissues comprising the periodontium, while also controlling residual infection at the wound site. This work is guided by the hypothesis that a multi-phased scaffold optimized in structure and composition to promote tissue regeneration and integration, as well as control the presence of pathogenic organisms, will augment integrative periodontal healing. The first aim of this thesis investigated scaffold design parameters for ligament regeneration, exploring polymer chemistry, fiber alignment, and antibiotic dose for the support of PDL cell growth and matrix biosynthesis. In addition, the efficacy of antibiotic-containing scaffolds in controlling the growth of periodontal pathogens was evaluated. With the overarching goal of supporting hard tissue integration, aim two optimized scaffold fiber diameter, mineral composition and dose, as well as method of mineral incorporation in order to promote PDL cell viability, growth, differentiation, and mineralized matrix deposition. In the third aim of this thesis a composite scaffold was fabricated, combining the optimized elements from the previous two aims into a multi-phased system that is mimetic of the native periodontal structure. The composite scaffold was then evaluated for tissue healing as well as for integrative potential with native tissue in a tooth-in-bone explant model. Collectively, the results of this thesis demonstrates that a scaffold with optimal structure and composition for PDL growth and integration supports enhanced periodontal healing as assessed through functional evaluation and tissue biosynthesis. In summary, the studies in this thesis led to the development of a novel, anti-infective, multi-phased scaffold which promotes integrative periodontal ligament healing. The broader implications of this work, which includes the elucidation of cell-biomaterial interactions and the implementation of complex scaffold design strategies, can be extended toward the integrative and functional repair of other composite tissue systems.

Resumo: O diagnóstico preciso e a elaboração de um plano de tratamento adequado podem constituir uma tarefa bastante complexa, especialmente nos traumatismos dentoalveolares, pois necessitam de uma abordagem multidisciplinar e conhecimento sobre o processo de reparo após o traumatismo. O objetivo do trabalho foi analisar o conhecimento dos cirurgiões dentistas sobre plano de tratamento das injúrias do ligamento periodontal após traumatismo dentoalveolar. Para tanto, a partir de um questionário, foram abordadas perguntas referentes ao perfil dos profissionais entrevistados e conduta frente às injúrias do ligamento periodontal (concussão, subluxação, luxação extrusiva, luxação lateral e luxação intrusiva) ocasionadas por traumatismo dentoalveolar. Seiscentos e noventa e três cirurgiões dentistas que participaram da 23ª Reunião Anual da SBPqO (2006) responderam o questionário e os dados obtidos foram submetidos à análise descritiva, enquanto o teste estatístico foi aplicado para demonstrar as freqüências e o nível de significância entre as variáveis (Teste qui-quadrado ou Teste Exato de Fisher). De acordo com os resultados obtidos, grandes dificuldades foram encontradas com relação ao plano de tratamento das luxações extrusiva, lateral e intrusiva. De maneira geral, a especialidade não influenciou na elaboração de planos adequados para as injúrias mais complexas. Foi possível concluir que os cirurgiões dentistas não apresentam conhecimento suficiente para tratar de maneira adequada as injúrias mais severas do ligamento periodontal após traumatismo dentoalveolar / Abstract: An accurate diagnosis and the establishment of an adequate treatment plan may constitute quite a complex task, particularly in cases of dentoalveolar trauma, which require a multidisciplinary approach and knowledge of the repair process in tooth injuries. The aim of this study was to analyze the dentists' knowledge of the treatment plan for periodontal ligament injuries after dentoalveolar trauma. For such purpose, a questionnaire was prepared with questions arguing about the profile of the interviewed professionals and their conduct facing periodontal ligament injuries (concussion, subluxation, extrusive luxation, lateral luxation and intrusive luxation) secondary to dentoalveolar trauma. Six hundred and ninety three dentists attending the 23rd Annual Meeting of the SBPqO (2006) filled out the questionnaire and the obtained data were subjected to descriptive analysis. Either chi-square test or Fisher's exact test was applied to determine the frequencies and the significance level among the variables. The results revealed great difficulties in establishing a treatment plan for extrusive, lateral and intrusive luxations. In general, the dental specialty of the participants did not influence the elaboration of adequate treatment plans for the most severe injuries. It could be concluded that dentists do not have sufficient knowledge to treat properly the most severe types of periodontal ligament injuries following a dentoalveolar trauma

Includes bibliographical references.

Bibliography: leaves 157-177.

Background and objectives: The most widely accepted tooth movement model is defined by the pressure-tension hypothesis. An orthodontic force applied to a tooth generates areas of compression and tension in the periodontal ligament (PDL), which are transmitted to the alveolar bone. Areas of tissue exposed to tensile strain undergo bone deposition, whereas areas of tissue exposed to compressive strain undergo bone resorption. We propose that human PDL cells in monolayer culture exposed to tensile mechanical strain would express multiple genes involved in osteogenesis. Materials and Methods: Human PDL cells were isolated and cultured from premolar teeth that were extracted for orthodontic reasons. These cells were plated on control and experimental Uniflex[TM] plates. Using a Flexercell FX4000 strain unit, PDL cells on experimental plates were exposed to a 12% uni-axial cyclic strain for 6 seconds out of every 90 seconds over a 24 hour period. RNA was extracted from the PDL cells at 6 hours, 12 hours and 24 hours. The differential expression of 78 genes implicated in osteoblast differentiation and bone metabolism was analysed using real-time reverse transcriptase polymerase chain reaction (RT-PCR) array technology. Results: Of the 78 genes tested, sixteen genes showed statistically significant (p<0.05) changes in expression in response to the mechanical strain regime. Eight genes were up-regulated (ALPL, BMP2, BMP6, COL2A1, ICAM1, PHEX, SOX9, and VEGFA) and eight genes were down-regulated (ANXA5, BMP4, COL11A1, COL3A1, EGF, ITGB1, MSX and SMAD1). Conclusions: This study has demonstrated that cultured human PDL cells express multiple osteogenic genes under tensile strain, which suggests that PDL cells may have a potential role in osseous remodeling during tooth movement. Key Words: Tooth movement, human PDL cells, tensile mechanical strain, osteogenic genes, real-time RT-PCR array, and Flexercell FX4000.

An in vitro model system utilizing de-mineralized periodontium tissues was developed for investigating the molecular controls involved in the spatial deposition of minerals on collagenous tissues. Preferential mineral deposition was observed when the de-mineralized tissue sections were incubated in solutions containing a supersaturation of calcium and phosphate ions with respect to hydroxyapatite (HA). Energy dispersive X-Ray (EDX) analysis demonstrated that these minerals are likely to be octacalcium phosphate (OCP) or dicalcium phosphate dihydrate (DCPD); further characterization with a secondary technique is required to draw a more definitive conclusion. The role of collagen fibrils in mineralization was tested by removing all the non-collagenous components from the tissue sections with proteolytic enzymes and exposing them to similar mineralization conditions as the control samples. A substantially less amount of minerals were formed in these samples; this correlates well with the hypothesis in the literature that collagen fibrils alone cannot initiate mineral formation.

An in vitro model system utilizing de-mineralized periodontium tissues was developed for investigating the molecular controls involved in the spatial deposition of minerals on collagenous tissues. Preferential mineral deposition was observed when the de-mineralized tissue sections were incubated in solutions containing a supersaturation of calcium and phosphate ions with respect to hydroxyapatite (HA). Energy dispersive X-Ray (EDX) analysis demonstrated that these minerals are likely to be octacalcium phosphate (OCP) or dicalcium phosphate dihydrate (DCPD); further characterization with a secondary technique is required to draw a more definitive conclusion. The role of collagen fibrils in mineralization was tested by removing all the non-collagenous components from the tissue sections with proteolytic enzymes and exposing them to similar mineralization conditions as the control samples. A substantially less amount of minerals were formed in these samples; this correlates well with the hypothesis in the literature that collagen fibrils alone cannot initiate mineral formation.

This study used vital bone markers to investigate mineralized tissue adaptation in the periodontium of rats after a hypothermic insult to their maxillary first molars. This hypothermic insult has been shown in previous studies to induce aseptic root resorption with variable effects on ankylosis. A secondary objective was to assess the pulpal changes that occurred concurrent with the changes in the periodontium. Four groups of 7, eight-week old male Sprague Dawley rats were assigned to be euthanased at the day 7, 14, 21 and 28 observation periods. At day 0, 4 groups of 6 rats were subject to a single 20 minute application of dry ice on their maxillary right first molar. The remaining 1 rat within each group did not receive the dry ice. All rats were given 2 sequential bone labels, calcein 5mg/kg and alizarin red 30mg/kg, administered intraperitoneally 8 days apart. The timing of the labels was such that all rats were euthanased 2 days after the last label. The rat maxillae were fixed in ethanol and embedded undecalcified in methylmethacrylate. Ten micrometre coronal sections were obtained through the furcation of the first molars with three of each group of ten consecutive sections being unstained, stained with von Kossa/ hematoxylin and eosin counterstain, or decalcified and stained with hematoxylin and eosin, respectively. Unstained sections were viewed under fluorescence, while transmitted light microscopy was used for the other sections. Mineral apposition rates along the bone, root and pulpal surfaces as well as periodontal ligament width were measured using histomorphometry. Semiquantitative measurements of the resorptive surfaces within the periodontium were also noted. Multivariate and negative binomial regression statistical analyses were used to identify influencing variables. A focal pattern of ankylosis was observed at days 14 and 21 in 3 rats and was not seen at day 28. In both the treated and control teeth, appositional activity was greatest along bone and least along the root surface. Mineral tissue apposition rates along the bone and root surface displayed an initial spike during day 14 but declined to levels of the control teeth by day 28. A longer time lag was observed with appositional activity in the pulp which also displayed a declining trend towards the control teeth values by day 28. Resorption levels along the root surface continued to remain significantly (p<0.0001) elevated. The significantly (p<0.0001) wider periodontal ligament width in the treated molars showed a declining trend towards that of the control teeth by day 28. There was a temporary disturbance of mineralized tissue adaptation on the bone and root surfaces with a recovery of cellular vitality within the periodontium and pulp and a trend towards homeostasis of the periodontal ligament width. The null hypotheses that a single prolonged thermal insult on a rat has no effect on mineralized tissue adaptation within the periodontium and pulp chamber and that the periodontal ligament width within the interradicular region does not change in response to thermal trauma induced by the present study were rejected. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1345386 / Thesis (D.Clin.Dent) - University of Adelaide, School of Dentistry, 2008