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• W85830812 abstract "Bone, a specialized connective tissue, consists of cells and mineralized extracellular matrix. The main cell types of bone tissue are: the osteoblasts, the osteocytes and the osteoclasts. Osteoblasts produce extracellular matrix, osteoclasts are responsible of its resorption, hence bone physiology is a delicate balance between synthesis of new bone and resorption of the old one. Osteoporosis is a disease in which catabolic activity of osteoclasts overtakes anabolic activity of osteoblasts leading to increased bone resorption and progressive bone fragility.Primary osteoporosis is a common disease among post-menopausal female population. Pathologies as diabetes mellitus, hyperparathyroidism and long-term treatment with glucocorticoids cause secondary osteoporosis. Glucocorticoid-induced osteoporosis is the most common type of secondary osteoporosis. Glucocorticoid treatment is a well known method to induce osteoporosis in animal models, hence it could be an example of “translational model” in which injured bone could be repopulated by stem cells or progenitors in clinical trials. The aim of this thesis is to investigate whether preosteoblasts could repopulate injured bone in an animal model treated with glucocorticoids. Preosteoblasts have been isolated from newborn calvariae of GFP mice. In these cells, expression of the osteogenic marker Runx2 has been assessed by Real time PCR, while osteogenic potential has been analysed by cytochemistry assays to detect alkaline phosphatase and mineralized bone nodules (Alizarin Red and Von Kossa staining).To realize the in vivo model, C57BL/6 three months aged mice have been divided into three groups [group I (n=4): mice not treated with drug and not infused with cells, group II (n=4): mice treated with drug and not infused with cells, group III (n=4): mice treated with drug and infused with cells]. Drug (methylprednisolone) has been administered for one month with a dose of 75 mg/Kg/week. In mice of group III, 5 x 105 GFP preosteoblasts, previously expanded in vitro, have been infused with injection into the tail vein. Mice have been sacrificed, tibial and femoral bones have been harvested, processed and analysed by istomorphometry and immunoistochemistry. Expression of Runx2, osteonectin (SPARC) and alkaline phosphatase (ALP) in these tissues has been detected with Real time PCR. In vitro preosteoblasts produce alkaline phosphatase during early time in culture with normal medium, while the level decreases in differentiating conditions with medium containing ascorbic acid and β-glycerophosphate. Preosteoblasts maintained in differentiation medium for 30 days are positive to Alizarin and Von Kossa staining, hence they are able to produce mineralized extracellular matrix that is a feature of functional mature osteoblasts. Runx2 expression increases during differentiating conditions; in cells maintained in differentiation medium for 30 days there is an increase of 50% compared to cells maintained in normal medium (p<0.05).In mice of group III an increased level of parameters concerning osteoid has been detected (O.Th, OS/BS, OV/BV) and an increased number of active osteoblasts (during synthetic activity) has been observed compared to group II. Between these two groups, significant variations of bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp) have not been detected. Microarchitecture parameters (Nd.N/TV, Nd/Tm) have not been affected. Similar results have been obtained from inderect microarchitecture parameters as Marrow Star Volume and Fractal Dimension.Real time PCR analysis revealed a reduction in osteogenic gene expression in group II compared to group I (ALP: -50%, p<0.01; Runx2: -56.75%, p<0.01; SPARC: -44.5%, p<0.05).In group III there is a recovery of expression of osteogenic markers (ALP: +40%, p<0.05; Runx2: +66.28%, p<0.001; SPARC: +55%; p<0.01) compared to group II. Immunoistochemistry is under investigation.In our glucocorticoid-induced osteoporosis model we sacrificed mice only one week after infusion of cells, this preparatory investigation shows that our model induces the engraftment of preosteoblasts in injured bone. However, a longer time, at least of 1-2 months, is needed to investigate if preosteoblasts are able not only to graft onto host tissue, but also to proliferate in vivo and to differentiate in full mature and functional osteoblasts." @default.
• W85830812 created "2016-06-24" @default.
• W85830812 creator A5020418022 @default.
• W85830812 date "2011-01-27" @default.
• W85830812 modified "2023-09-27" @default.
• W85830812 title "Trapianto di cellule staminali in un modello animale di danno osseo da glucocorticoidi" @default.
• W85830812 hasPublicationYear "2011" @default.
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