Calreticulin, an endoplasmic reticulum (ER)-resident protein, is a calcium buffering chaperone. In this study, with an optimized differentiation protocol from mouse R1 ES cells, we demonstrate a novel role of calreticulin in osteogenic commitment and differentiation. To enhance the efficacy of the method, we manipulated cell density and examined the addition of retinoic acid, dexamethasone and peroxisome proliferator-activated receptor γ. The regimen consisting of seeding 250 cells per embryoid body, with the addition of RA (from day 3 to 5) and Dex (from day 10 to 21) gave the most efficacious output. Using this optimized protocol, we investigated the potential involvement of calreticulin in osteogenesis. Calreticulin knock-out cells displayed impaired osteogenesis compared to wild-type cells. In particular, the nuclear translocation of the runt-domain related transcription factor 2 and Osterix, were impaired in the absence of calreticulin. The stimulatory effect of calreticulin on osteogenesis was mediated by its calcium buffering function.

Calreticulin, an endoplasmic reticulum (ER)-resident protein, is a calcium buffering chaperone. In this study, with an optimized differentiation protocol from mouse R1 ES cells, we demonstrate a novel role of calreticulin in osteogenic commitment and differentiation. To enhance the efficacy of the method, we manipulated cell density and examined the addition of retinoic acid, dexamethasone and peroxisome proliferator-activated receptor γ. The regimen consisting of seeding 250 cells per embryoid body, with the addition of RA (from day 3 to 5) and Dex (from day 10 to 21) gave the most efficacious output. Using this optimized protocol, we investigated the potential involvement of calreticulin in osteogenesis. Calreticulin knock-out cells displayed impaired osteogenesis compared to wild-type cells. In particular, the nuclear translocation of the runt-domain related transcription factor 2 and Osterix, were impaired in the absence of calreticulin. The stimulatory effect of calreticulin on osteogenesis was mediated by its calcium buffering function.

Biochemical and mechanical properties of the extracellular matrix (ECM) are known to independently influence cell function. Given the complexity of cellular responses, I hypothesized that the integration of multiple matrix factors as opposed to their individual contribution is key to understanding and controlling cell function. The objective of this thesis was to systematically investigate matrix biochemical and mechanical regulation of multipotent stromal cell (MSC) osteogenesis. First, I demonstrated that substrate stiffness-dependent MSC spreading, proliferation and osteogenic response were differentially regulated by matrix protein type (collagen I vs. fibronectin) and concentration. Second, I developed and characterized a matrix microarray platform that enabled the efficient screening of multiple matrix-derived cues (substrate stiffness, ECM type and density). I implemented the matrix microarray platform together with parametric regression models to elucidate novel matrix interactions in directing mouse MSC osteogenic and adipogenic differentiation. Third, I extended the screening study to examine matrix-dependent human MSC osteogenesis. Non-parametric regression models were used to provide a nuanced description of the multi-factorial matrix regulation in MSC osteogenesis. The response surfaces revealed a biphasic relationship between osteogenesis and substrate stiffness, with the exact location and magnitude of the optimum contingent on matrix composition. Guided by the screening results and perturbation to key cytoskeletal regulators, I identified a novel pathway involving Cdc42 in matrix mechanical and biochemical regulation of MSC osteogenesis. Surprisingly, Cdc42 mediated stiffness-dependent MSC osteogenesis independent of ROCK, suggestive of a contractility-independent mechanism in matrix rigidity signal transduction. In summary, the integration of cell-based arrays and statistical modeling has enabled the systematic investigation of complex cell-matrix interactions. This generalizable approach is readily adaptable to other cellular contexts, complementing hypothesis-driven strategies to facilitate non-intuitive mechanistic discovery. Moreover, the improved understanding of matrix-dependent MSC function also has practical relevance to the development of biomaterials for tissue engineering applications.

No description available.

Biochemical and mechanical properties of the extracellular matrix (ECM) are known to independently influence cell function. Given the complexity of cellular responses, I hypothesized that the integration of multiple matrix factors as opposed to their individual contribution is key to understanding and controlling cell function. The objective of this thesis was to systematically investigate matrix biochemical and mechanical regulation of multipotent stromal cell (MSC) osteogenesis. First, I demonstrated that substrate stiffness-dependent MSC spreading, proliferation and osteogenic response were differentially regulated by matrix protein type (collagen I vs. fibronectin) and concentration. Second, I developed and characterized a matrix microarray platform that enabled the efficient screening of multiple matrix-derived cues (substrate stiffness, ECM type and density). I implemented the matrix microarray platform together with parametric regression models to elucidate novel matrix interactions in directing mouse MSC osteogenic and adipogenic differentiation. Third, I extended the screening study to examine matrix-dependent human MSC osteogenesis. Non-parametric regression models were used to provide a nuanced description of the multi-factorial matrix regulation in MSC osteogenesis. The response surfaces revealed a biphasic relationship between osteogenesis and substrate stiffness, with the exact location and magnitude of the optimum contingent on matrix composition. Guided by the screening results and perturbation to key cytoskeletal regulators, I identified a novel pathway involving Cdc42 in matrix mechanical and biochemical regulation of MSC osteogenesis. Surprisingly, Cdc42 mediated stiffness-dependent MSC osteogenesis independent of ROCK, suggestive of a contractility-independent mechanism in matrix rigidity signal transduction. In summary, the integration of cell-based arrays and statistical modeling has enabled the systematic investigation of complex cell-matrix interactions. This generalizable approach is readily adaptable to other cellular contexts, complementing hypothesis-driven strategies to facilitate non-intuitive mechanistic discovery. Moreover, the improved understanding of matrix-dependent MSC function also has practical relevance to the development of biomaterials for tissue engineering applications.

Distraction osteogenesis is a recently developed option for surgical correction of the craniofacial discrepancy but there are few studies that look at the long-term effect of the relapse phenomenon. This study was conducted to look at the bone and muscle response at different consolidation and remodelling periods. Thirty ten-week-old Merino lambs were subjected to this current study. The surgically created defect was performed on the experimental side (right) by superficial masseter myectomy and a condylectomy. The lambs showed a midline shift to the affected side three months later. Marker screws were placed on both sides of the mandible to examine the adjustment of the vertical and horizontal dimension of the mandible. In this present study, the left side was used as the control. The affected mandible was surgically corrected using a vertical ramus distraction osteogenesis protocol. The latency period was 7 days where the device was inactive. Distraction was then performed at 1 mm per day until the distracted gap was 10 mm on a radiograph. Initially, the sheep were divided into 8 groups but later the groups were refined into 6 groups for statistical analysis; Group 1: Immediately post distraction Group 2: Consolidation 2 months and remodelling 1 month Group 3: Consolidation 3 months Group 4: Consolidation 3 months and remodelling 1 month Group 5: Consolidation 3 months and remodelling 2 months Group 6: Consolidation 4 months A ‘hemifacial microsomia like’ defect was successfully created and it was then corrected using vertical ramus distraction. Cephalometric examination showed that the vertical height was significantly increased after the distraction was completed. Examination of the bone and muscle was performed to look at the adjustment of bone structure in relation to relapse. The bone investigation was conducted using radiological analysis, histological analysis and direct measurement of the vertical screws at pre-distraction and at sacrifice as well as histomorphometric analysis. The radiological examination was conducted using cephalometric analysis of the distance between marker screws on both experimental and control sides. Bone histology was investigated on the middle and posterior position of the experimental side. The histomorphometric analysis was conducted using Quantiment analysis software. The muscle responses and adaptation were investigated by measuring: the weight of the masseter and medial pterygoid muscles; length between 6 different points (4 landmarks); cross section and thickness of masseter muscles by ultrasound and by histopathological examination of both masseter and medial pterygoid muscles on the experimental and the control sides. The distracted bone showed a completion of maturation after a three month consolidation period. Bone formation was shown to continue after longer consolidation periods. A two-month consolidation period was insufficient time for the bone to consolidate and stable, as there was evidenced of relapse during this period. There was no increased in muscle mass after distraction. The distraction altered the length of anterior and posterior planes and the size of cross sectional area and thickness of origin and middle level of masseter muscles. There were no changes in the length of the middle and oblique planes and the cross sectional area of the insertion of the masseter muscle. The distracted muscle adapted well after a longer consolidation and remodelling period but was sensitive to any surgical procedure such as device removal. The adjustment was observed to continue within the first month after surgical removal of device. The weight, length of planes, cross section and thickness was temporarily reduced on the experimental side but continued to improve and stabled during the second month after removal of the device. The first month after device removal also showed that histopathological activity was increased after both 2 and 3 month consolidation periods. Importantly, muscle histopathology was back to almost normal activity after the second month of device removal, following a 3 month consolidation period. This study showed that it was possible to create a ‘hemifacial microsomia like’ defect in very young lambs. The defect was then successfully corrected by a vertical distraction osteogenesis procedure. It was also shown that the sufficient time for the device to be fixed in place (consolidation period) was 3 months or longer as the bone and muscle was stable after that period of time. Results from this investigation have important implications to the management of similar conditions in humans. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1346836 / Thesis (Ph.D.) - University of Adelaide, School of Dentistry, 2008.

Thesis (Ph. D.)--School of Dentistry. University of Missouri--Kansas City, 2006. / "A dissertation in oral biology and cell biology and biophysics." Advisor: Lynda F. Bonewald. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Nov. 12, 2007. Includes bibliographical references (leaves 121-139). Online version of the print edition.

Σκοπός της διπλωματικής αυτής εργασίας είναι η μελέτη ρομποτικής διάταξης οστεογένεσης και πιο συγκεκριμένα η μελέτης της δυναμικής και ο έλεγχος αυτής. Πιο αναλυτικά, τα κεφάλαια που ακολουθούν πραγματεύονται τα ακόλουθα: θα παρουσιαστεί γενικά ο μηχανισμός της οστεογένεσης από ιατρικής πλευράς, καθώς επίσης και οι διάφορες διατάξεις με τις οποίες πραγματοποιείται η διατατική οστεογένεση. Στη συνέχεια θα παρουσιάσουμε τις κινηματικές και δυναμικές εξισώσεις της πλατφόρμας Stewart. Το τελευταίο μέρος αποτελεί και το πειραματικό μέρος της παρούσας εργασίας δηλαδή τον έλεγχο της συγκεκριμένης διάταξης μέσω του SimMechanics toolbox του πακέτου MatLab. Στο παράρτημα παρουσιάζονται τα αποτελέσματα των πειραμάτων με μορφή διαγραμμάτων. / Distraction osteogenesis and simulation of stewart platform on Simulink.

Orientador: Idelmo Rangel Garcia Junior / Banca: Francisley Avila Souza / Banca: Igor Mariotto Beneti / Banca: André Luis da Silva Fabris / Banca: Sônia Regina Panzarini Barioni / Resumo: A perda dentária promove uma severa e irreversível reabsorção do osso alveolar, especialmente na região posterior da maxila, dificultando a reabilitação com implantes ósseointegrados e para conseguir um aumento em altura e espessura do osso maxilar, pode ser necessário o uso de biomateriais como subtitutos ósseos. A proposta deste estudo foi avaliar o processo de ossificação com o uso do biomaterial Biogran® (Orthovita, Malvern, PA, EUA), à base de vidro bioativo, como uma opção de substituto ósseo em técnica de levantamento do assoalho do seio maxilar através de análises histológica e histométrica. Para tal, foram utilizados quinze coelhos Albinus machos brancos da raça Nova Zelandia pesando 3 a 4 Kg aproximadamente que foram divididos aleatoriamente em dois grupos segundo o material de enxerto do seio maxilar: Grupo osso autógeno e grupo vidro bioativo/Biogran®. Foi realizado o descolamento bilateral da membrana sinusal e realizado o enxerto com o Biogran® e osso autógeno respeitivamente e os períodos estudados foram de 7, 15 e 40 dias para a análise histológica e histométrica. A porcentagem de neoformação óssea foi maior no grupo osso autógeno aos 7 e 15 dias, entretanto, não houve diferença estatistica entre os grupos aos 40 dias (p>0,05). Foi possível concluir que o vidro bioativo/ Biogran® é capaz de conduzir o processo de formação óssea de forma satisfatória, apresentando resultados semelhantes ao enxerto de osso autógeno e tornando-o uma alternativa viável para aument... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The dental loss promotes a severe and irreversible reabsorption of the alveolar bone, especially in the posterior region of the maxilla, making it difficult to rehabilitate with osseointegrated implants and to achieve an increase in height and thickness of the maxillary bone, it may be necessary to use biomaterials as bone substitutes. The purpose of this study was to evaluate the ossification process using Biogran® (Orthovita, Malvern, PA, USA) bioactive glass based biomaterial as a bone substitute option in the technique of lifting the floor of the maxillary sinus through histological and histometric analyzes. For this purpose, fifteen white New Zealand Albinus male rabbits weighing approximately 3 to 4 kg were randomly divided into two groups according to the maxillary sinus graft material: Autogenous bone group and Bioactive glass/ Biogran® glass group. Bilateral detachment of the sinus membrane was performed and the graft was performed with Biogran® and autogenous bone respectively and the periods studied were 7, 15 and 40 days for histological and histometric analysis. The percentage of new bone formation was higher in the autogenous bone group at 7 and 15 days, however, there was no statistical difference between groups at 40 days (p> 0.05). It was possible to conclude that BioGran®/bioactive glass is capable of conducting the bone formation process satisfactorily, presenting results similar to the autogenous bone graft and making it a viable alternative to increase bone height in the maxillary posterior region / Doutor

Pericytes represent a population of potential mesenchymal stem cells (MSC) that reside within a perivascular niche until they are required in normal homeostasis and the response to injury. Their mesenchymal capacities for multipotent differentiation, immune modulation and release of trophic factors hold great promise for regenerative therapies. Pathological expression of these potentials has been described in disease states, while acute or chronic inflammation following injury can lead to the production of signalling molecules that ultimately drive these progenitors to a fibrotic fate. The aim of this work was to explore how fate decisions of pericytes are regulated by their niche (in the setting of osteogenesis), and in the response to acute and chronic injury (in the setting of fibrosis). It was hypothesized that interactions between pericytes and endothelial cells (EC) within their perivascular niche are responsible for regulating mesenchymal differentiation. The osteogenic, adipogenic and chondrogenic potential of pericytes following isolation from multiple human organs was confirmed. The interactions between pericytes and EC in 2D and 3D coculture and the production of basement membrane proteins in these settings were confirmed. The osteogenic differentiation of pericytes was accelerated by EC but no influence of EC on the adipogenic and chondrogenic differentiation of pericytes was detected. Furthermore, data indicated that the influence on pericyte osteogenic potential by EC may occur through wnt signaling. The activation of TGFβ (transforming growth factor beta) through αv integrins has been suggested as central mediator of fibrosis in multiple organs. We hypothesized that selective αv integrin deletions in PDGFRβ (platelet derived growth factor receptor beta) expressing pericytes identifies a targetable pathway regulating fibrosis in skeletal muscle. We report that PDGFRβ-Cre inactivates genes in murine skeletal muscle pericytes with high efficiency. Deletion of the αv integrin subunit in pericytes protected mice from chemical injury induced skeletal muscle fibrosis. Pharmacological blockade of αv integrins by a novel small molecule (CWHM 12) attenuated muscle fibrosis, even when administered after fibrosis was established.