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Modificazioni strutturali e del cell-signalling osteocitario nella rigenerazione ossea in risposta al trattamento con Teriparatide
No full textLo scopo della presente tesi di dottorato è stato quello di analizzare gli aspetti strutturali ossei le modificazioni del cell-signalling osteocitario durante la riparazione di lesioni scheletriche indotte (in vivo) e in un modello cellulare immortalizzato (in vitro) a seguito del trattamento con Teriparatide (farmaco osteo-protettore).
Il Teriparatide è il frammento attivo (1-34) dell’ormone paratiroideo umano endogeno (PTH). A dosaggi fisiologici il PTH induce ipercalcemia: in parte questo effetto è realizzato aumentando l’assorbimento intestinale di calcio ed incrementando il suo riassorbimento tubulare a livello renale; tuttavia, in condizioni non fisiologiche, l’ipercalcemia si realizza in modo consistente incrementando il riassorbimento osseo. E’ stato dimostrato che dosaggi cronicamente elevati di PTH comportano una riduzione della massa ossea mentre un'esposizione intermittente al PTH attiva gli osteoblasti più di quanto non agisca sugli osteoclasti. In particolare, è stato dimostrato che l’esposizione intermittente al PTH produce un netto aumento della densità minerale ossea, contrariamente al marcato riassorbimento osseo che si verifica a seguito di un’esposizione continua allo stesso ormone.
Gran parte degli studi strutturali hanno focalizzato la loro attenzione sull’effetto quantitativo del Teriparatide nella neo-deposizione di osso, senza indagare la qualità del tessuto neoformato; inoltre, da un punto di vista molecolare, la maggior parte delle ricerche finora condotte hanno prediletto lo studio dell’attività degli osteoblasti nel processo di riparazione ossea, tralasciando il ruolo fondamentale che gli osteociti giocano mediante la modulazione del loro cell-signalling. Gli osteociti sono infatti gli attori principali della comunicazione tra le cellule ossee, in particolare con gli osteoblasti con cui sono in diretta connessione tramite prolungamenti citoplasmatici e giunzioni specializzate (di tipo gap).
Durante la rigenerazione ossea vengono ripercorse le tappe tipiche dell’ossificazione membranosa; dopo la preliminare formazione di tessuto fibroso e la successiva proliferazione vascolare si susseguono due diversi tipi di osteogenesi: dapprima l’Osteogenesi Statica che, in base a fattori induttivi di origine vascolare, produce in fretta un preliminare trabecolato osseo e successivamente l’Osteogenesi Dinamica in cui gli osteoblasti, guidati dagli osteociti, producono un osso meccanicamente più valido. Allo stato attuale non risultano indagini focalizzate sulle relazioni funzionali tra osteociti e osteoblasti durante il processo di riparazione dell’osso con/senza farmaci.
Al fine di approfondire questo aspetto, il mio lavoro di dottorato si è concentrato inizialmente sull’analisi strutturale qualitativa della riparazione di lesioni sperimentalmente indotte in diafisi di femore di ratto e successivamente sull’analisi del cell-signalling osteocitario in vitro, entrambe in risposta al trattamento con Teriparatide.
I risultati della sperimentazione nel modello animale hanno mostrato che la somministrazione intermittente di Teriparatide accelera la comparsa dell’Osteogenesi Dinamica nel produrre un osso meccanicamente più valido. I dati di biologia molecolare su un modello cellulare immortalizzato osteocitario (MLO-Y4) trattato con il farmaco hanno mostrato (mediante analisi Microarray), a tempi differenti di trattamento, una diversa espressione dei geni coinvolti nel signaling osteocitario rispetto alla condizione di controllo. Il conseguimento di tali risultati ci ha indotto ad un approfondimento della tematica tramite l’analisi proteomica e l’utilizzo di una nuova linea cellulare ossea (IDG-SW3) in grado di andare incontro in vitro a completo differenziamento osteocitario partendo dallo stadio di pre-osteoblasta.The purpose of this thesis is to analyze the structural bone tissue modifications and the alteration of osteocyte cell-signaling during the repair of experimentally induced bone lesions (in vivo) and in an immortalized cell model (in vitro) under the treatment of the osteo-protective drug Teriparatide. Teriparatide is the active fragment (1-34) of the endogenous human parathyroid hormone (PTH). The physiological effect of PTH is to induce hypercalcemia: this effect is partially achieved by increasing both the calcium intestinal absorption and the renal tubular reabsorption; however, under non-physiological conditions the hypercalcemia is mainly resulted by the increase of bone resorption. Many studies showed that chronic administration of high doses of PTH results in decreased bone mass while intermittent exposure to PTH activates osteoblast bone deposition. In particular, it has been shown that intermittent exposure to PTH increases bone density by stimulating osteogenesis; whereas continued exposure to the same hormone causes strong bone reabsorption. Most of the structural studies published so far about the effect of Teriparatide focused their attention on the amount of newly-deposed bone without investigating the quality of the newly-formed bone tissue; moreover most of the papers enhance the attention to the osteoblast’s cellular and molecular involvement during the process of bone repair, ignoring the pivotal role that the osteocytes play in the bone environment by means of the modulation of their signaling. In many previous published papers it has been shown that osteocytes are the main actors of the communication among bone cells, in particular they are in close contacts with osteoblasts through cytoplasmic extensions and specialized gap-junctions, indicative of their guide role. Bone regeneration retraces the sequence of events occurring in membranous ossification: after a preliminary formation of fibrous tissue and the successive vascular proliferation, two different types of osteogenesis follow: firstly the process of Static Osteogenesis (SO), which produces preliminary bony trabeculae with woven texture, under factors/cytokines of vascular origin, and after that the process of Dynamic Osteogenesis (DO) starts, in which osteoblasts, driven by osteocytes, produce a more ordered (mainly lamellar) and more mechanical valid bone. At the present state of knowledge there are no scientific references focused on the functional relationships among osteocytes and osteoblasts during the bone repair process.
This doctoral work focused at first on the morphological analysis of qualitative repair of lesions experimentally induced in diaphysis of rats femurs and then on the study of the osteocyte cell-signaling, both in response to the treatment with Teriparatide in order to deepen the knowledge of morphological and molecular events that occur in bone regeneration, with particular attention to the osteocyte signaling. The results obtained showed that intermittent administration of Teriparatide in the animal model anticipates the beginning of Dynamic Osteogenesis, which is characterized by the production of a more resistant bone. The molecular biology data were performed on an osteocyte immortalized cellular model (MLO-Y4) treated with the drug at different time points. Microarray analysis showed different expression of genes involved in osteocyte signaling compared with the control condition. These results lead us to perform new and more focused analysis of proteomics and to use a new bone cell line (IDG-SW3). This cell line is capable of going through a complete differentiation from pre-osteoblast to late osteocyte, allowing us to better understand the signaling modifications that may occur during the treatment with Teriparatide
Mineral and Skeletal Homeostasis Influence the Manner of Bone Loss in Metabolic Osteoporosis due to Calcium-Deprived Diet in Different Sites of Rat Vertebra and Femur
Full text linkRats fed calcium-deprived diet develop osteoporosis due to enhanced bone resorption, secondary to parathyroid overactivity
resulting from nutritional hypocalcemia.Therefore, rats provide a good experimental animal model for studying bone modelling
alterations during biochemical osteoporosis.Three-month-old Sprague-Dawley male rats were divided into 4 groups: (1) baseline,
(2) normal diet for 4 weeks, (3) calcium-deprived diet for 4 weeks, and (4) calcium-deprived diet for 4 weeks and concomitant
administration of PTH (1-34) 40 g/Kg/day. Histomorphometrical analyses were made on cortical and trabecular bone of lumbar
vertebral body as well as of mid-diaphysis and distal metaphysis of femur. In all rats fed calcium-deprived diet, despite the reduction
of trabecular number (due to themaintenance of mineral homeostasis), an intense activity of bone deposition occurs on the surface
of the few remaining trabeculae (in answering to mechanical stresses and, consequently, to maintain the skeletal homeostasis).
Different responses were detected in different sites of cortical bone, depending on their main function in answering mineral or
skeletal homeostasis. This study represents the starting point for work-in-progress researches, with the aim of defining in detail
timing and manners of evolution and recovery of biochemical osteoporosis
PTH(1-34) effects on repairing experimentally drilled holes in rat femur: novel aspects – qualitative vs. quantitative improvement of osteogenesis
Full text linkThe timetable of effects on bone repair of the active fraction-parathyroid hormone, PTH(1-34), was analytically investigated from the morphometric viewpoint in 3-month-old male Sprague-Dawley rats, whose femurs were drilled at mid-diaphyseal level (transcortical holes). The animals were divided into groups with/without PTH(1-34) administration, and sacrificed at different times (10, 28, 45 days after surgery). The observations reported here need to be framed in the context of our previous investigations regarding bone histogenesis (Ferretti et al. Anat Embryol. 2002; 206: 21–29) in which we demonstrated the occurrence of two successive bone-forming processes during both skeletal organogenesis and bone repair, i.e. static and dynamic osteogenesis: the former (due to stationary osteoblasts, haphazardly grouped in cords) producing preliminary bad quality trabecular bone, the latter (due to typical polarized osteoblasts organized in ordered movable laminae) producing mechanically valid bone tissue. The primary function of static osteogenesis is to provide a rigid scaffold containing osteocytes (i.e. mechano-sensors) for osteoblast laminae acting in dynamic osteogenesis. In the present work, histomorphometric analysis revealed that, already 10 days after drilling, despite the holes being temporarily filled by the same amount of newly formed trabecular bone by static osteogenesis independently of the treatment, the extent of the surface of movable osteoblast-laminae (covering the trabecular surface) was statistically higher in animals submitted to PTH(1-34) administration than in control ones; this datum strongly suggests the effect of PTH(1-34) alone in anticipating the occurrence of dynamic osteogenesis involved in the production of good quality bone (with more ordered collagen texture) more suitable for loading. This study could be crucial in further translational clinical research in humans for defining the best therapeutic strategies to be applied in recovering severe skeletal lesions, particularly as regards the time of PTH(1-34) administration
Bone texture modifications during bone regeneration and osteocyte cell-signaling changes in response to treatment with Teriparatide
No full textBone texture modifications during bone regeneration and osteocyte cell-signaling changes in response to treatment with Teriparatid
Up-regulation of the chemo-attractive receptor ChemR23 and occurrence of apoptosis in human chondrocytes isolated from fractured calcaneal osteochondral fragments
No full textTo study the expression level of a panel of pro/anti-apoptotic factors and inflammation-related receptors in
chondral fragments from patients undergoing surgical treatment for intra-articular calcaneal fractures, cartilage
fragments were retrieved from calcaneal fractures of 20 patients subjected to surgical treatment. Primary
cultures were performed using chondral fragments from fractured and control patients. Chondrocyte cultures
from each patient of the fractured and control groups were subjected to immunofluorescence staining and
quantitatively analyzed under confocal microscopy. Proteins extracted from the cultured chondrocytes taken
from the fractured and control groups were processed for Western blot experiments and densitometric analysis.
The percentage of apoptotic cells was determined using the cleaved PARP-1 antibody. The proportion of
labelled cells was 35% for fractured specimens, compared with 7% for control samples. Quantification of
caspase-3 active and Bcl-2 proteins in chondrocyte cultures showed a significant increase of the apoptotic
process in fractured specimens compared with control ones. Fractured chondrocytes were positively stained for
ChemR23 with statistically significant differences with respect to control samples. Densitometric evaluation of
the immunoreactive bands confirmed these observations. Human articular chondrocytes obtained from patients
with intra-articular calcaneal fractures express higher levels of pivotal pro-apoptotic factors, and of the chemoattractive
receptor ChemR23, compared with control cultures. On the basis of these observations, the authors
hypothesize that consistent prolonged chondrocyte death, associated with the persistence of high levels of proinflammatory
factors, could enhance the deterioration of cartilage tissue with consequent development of
post-traumatic arthritis following intra-articular bone fracture
COMPARATIVE MORPHOLOGICAL STUDY OF BONE REGENERATION IN DIFFERENT RABBIT CRANIAL OSTEOMOMIES: TRADITIONAL VERSUS NEW GENERATION OSTEOTOMES
No full textCOMPARATIVE MORPHOLOGICAL STUDY OF BONE REGENERATION IN DIFFERENT RABBIT CRANIAL OSTEOMOMIES: TRADITIONAL VERSUS NEW GENERATION OSTEOTOME
Effects of PTH(1-34) during fracture healing after experimental bone drilling in rat femur: novel aspects
Full text linkThe study concerns the role of PTH(1-34) during bone lesion repair. 3-month-old
male Sprague-Dawley rats, in which trans-cortical holes were drilled at femur middiaphysis,
were divided in groups with/without Teriparatide administration (40g/
Kg/day), and sacrificed at different times (10, 28, 45 days). In 2002 (1) we demonstrated
the occurrence of two successive bone forming processes during both skeletal
organogenesis and bone repair, i.e. static (SO) and dynamic (DO) osteogenesis: the
former (due to stationary osteoblasts, haphazardly grouped in cords) producing preliminary
bad quality trabecular bone, the latter (due to typical polarized osteoblasts
organized in ordered movable laminae) producing mechanically valid bone tissue.
In brief, the primary function of SO is to provide a rigid scaffold, containing osteocytes
(i.e. mechano-sensors), to DO-osteoblastic laminae; therefore, in DO mechanical
factors can play a crucial role in transduction of mechanical stresses into biological
signals. In the present work, histomorphometric analysis showed that, already after
10 days from drilling, notwithstanding the holes are temporarily filled by the same
amount of newly-formed trabecular bone (produced by SO) independently from the
treatment, the number of movable osteoblast laminae (typical of DO), covering the
trabecular surface, is statistically higher in animals submitted to PTH(1-34) administration
than in the control ones; this suggests that the mere effect of Teriparatide is
to anticipate the occurrence of dynamic osteogenesis involved in the production of
good quality bone more suitable to loading. These findings are also supported by the
higher values of microhardness as well as the more ordered-fibered texture (observed
by polarized light) in treated animals with respect to control ones that strongly indicates
the qualitative (instead of quantitative) effect of PTH (1-34) in improving bone
healing. The present investigation could be of crucial importance in further translational
clinical research in humans to define the best therapeutic strategies in recovering
skeletal lesions, particularly in terms of time of administration of PTH(1-34)
Effect of PTH (1-34) on trabecular bone of rat vertebral body in induced-biochemical osteoporosis by calcium- deprived diet
Full text linkRats fed calcium-deprived diet were used as experimental model for studying bone modelling alterations during biochemical osteoporosis and recovery of bone loss. Such model is suitable to evaluate the possible effects exerted by PTH(1-34) in preventing as well as in recovering metabolic osteoporosis. Three-month-old Sprague Dawley male rats were divided in different groups: some fed normal diet or calcium-deprived diet with/without 40μg/Kg/day PTH(1-34), provided by Eli Lilly-USA, for 4 weeks and some with restoration of normal diet with/without PTH (1-34) for further 4 weeks. To evaluate the occurrence of osteogenesis during the first 4 weeks of the experimental period, rats received three labels of bone deposition at 1st, 20th and 27th day (and then were sacrificed); during the successive 4 weeks (in which those rats previously fed with calcium-deprived diet had restoration of normal diet), animals received three labels of bone deposition at 1st, 7th and 14th day. Histomorphometrical analyses were performed on cortical and trabecular bone taken from the central level of the 5th lumbar vertebral body, transversely sectioned. The results showed that differences among the groups were observed mainly in trabecular bone with respect to cortical one, thus underlining the different role of the two types of bone architecture in mineral and skeletal homeostasis. Concerning trabecular bone, the observations showed that administration of PTH (1-34) during calcium-deprived diet and/or during the restoration of normal diet induces higher deposition of trabecular bone with respect to that recorded in rats that never received PTH(1-34), neither during calcium-deprived diet nor during restoration of normal diet. Since increments of trabecular bone are detectable only after the period of diet restoration (but not before), the authors suggest that a chronic administration of PTH (1-34) is necessary to achieve appreciable results on bone mass recovery
Induced Biochemical osteoporosis: Effects of 1-month calcium–deprived diet on rat bone remodelling with/without contemporary administration of PTH(1-34)
No full textIt is known that rats fed calcium-deprived diet develop osteoporosis due to en-hanced bone resorption secondary to parathyroid overactivity resulting from nutritional hypocalcemia. Therefore, rats provide a good experimental animal model for studying bone remodelling alterations during biochemical osteoporosis. This preliminary study is performed in 3 month-old Sprague Dawley male rats, divided into 4 groups (5 rats each): 1) base line, 2) normal diet for 4 weeks, 3) calcium-deprived diet for 4 weeks; 4) calcium-deprived diet for 4 weeks plus contemporary administration of PTH(1-34) 40μg/kg/day. Three labels of osteogenesis were performed at 1st , 20th and 27th day of experimental period in order to evaluate bone formation during animal treatment. His-tomorphometrical analyses were performed on cortical bone of femoral diaphyses, as well as on trabecular bone of distal femoral metaphyses, both transversely sectioned. The preliminary results showed that at femur mid-diaphyseal level the diet induced a reduction of cortical bone area (even if not significant) with enlargement of the medul-lary canal due to endosteal resorption, while periosteal neo-deposition is similar in all groups and particularly abundant in those periosteal regions mainly devoted in answering the mechanical demands. PTH(1-34) treatment seems to reduce endosteal resorption only in those surfaces where periosteal mechanical loading are less consistent. Conversely, PTH(1-34) treatment doesn't seem to affect osteoblast activity. Moreover, in distal femoral metaphyses, diet induced osteoclast activity, with a decrease in the amount of trabecular bone volume, confirming that this architecture is mainly devoted in answering the metabolic demands. The novelty of the proposed model Is the contemporary administration of PTH(1-34) together with calcium deprived diet to evaluate induced-biochemical osteoporosis. This model seems a good starting point for successive studies in order to study bone alterations during unbalanced calcium metabolism frequently occurring in aging and to define time and manner of bone mass recovery
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