INTRODUZIONE. L'osso è una struttura rigida e dinamica che protegge diversi organi del corpo. Sebbene la riparazione delle fratture ossee di solito riporti l’osso danneggiato al suo stato pre-lesione, circa il 10% delle fratture non guarisce correttamente. Infatti, il processo di rigenerazione potrebbe fallire nel caso di resezioni ossee estese o di pseudoartrosi. Inoltre, la riabilitazione facciale, sia funzionale che estetica, ha acquisito nel tempo una maggiore importanza nella chirurgia maxillo-facciale. Le cellule staminali di diversa origine tissutale e distinti nuovi biomateriali osteoinduttivi sono impiegati per migliorare la riparazione ossea. Nella pratica clinica, le cellule staminali mesenchimali umane derivate da tessuto adiposo (hASCs) e da midollo osseo (hBMSCs) sono probabilmente quelle più utilizzate. In vitro, i cocktails impiegati per indurre il differenziamento osteogenico contengono almeno 10 nM di desametasone (dex). Il dex, però, induce anche un differenziamento adipocitario off-target nelle hBMSCs indotte al differenziamento osteogenico. Dex induce l'osteogenesi inibendo l'espressione di SRY-Box Transcription Factor 9 (SOX9) e non stimolando l'espressione di RUNX Family Transcription Factor 2 (RUNX2). Inoltre, dex stimola anche l'adipogenesi inducendo l'espressione di peroxisome proliferator-activated receptor gamma (PPARG). Tuttavia, ulteriori studi sono necessari per identificare quali mediatori portano alla down-regolazione di SOX9: PPARG è un candidato promettente. OBIETTIVI. Inizialmente, sono state valutate le proprietà di citocompatibilità e osteoinduttività di due biomateriali compositi, costituiti da idrossiapatite (HA)/collagene (Avitene), impiegati in chirurgia maxillo-facciale, ovvero Pro Osteon 200/Avitene e Bio-Oss/Avitene, utilizzando le hASCs come modello cellulare in vitro. Le loro caratteristiche sono state valutate fino a 40 giorni. Pazienti sottoposti ad aumento zigomatico utilizzando le suddette protesi, per malocclusione maxillo-mandibolare e/o asimmetria, sono stati valutati per la neoformazione ossea. Poiché l'infiammazione è un fattore importante da considerare per lo sviluppo di terapie efficaci a base di biomateriali, la risposta immunitaria delle hASCs cresciute su entrambi i biomateriali in esame è stata valutata dopo 21 giorni. In una seconda fase, è stato valutato un innovativo biomateriale HA-derivato ottenuto attraverso un nuovo processo di trasformazione “biomorfica”, in grado di trasformare pezzi di legno direttamente in biomateriali, preservando la struttura multi-scala originaria, senza adottare alcun processo di sinterizzazione. Nell'ultima parte di questo progetto, sono stati condotti esperimenti incentrati sui meccanismi dell'osteogenesi indotta da dex in hBMSCs utilizzando un inibitore di PPARG (T0070907). RISULTATI E CONCLUSIONE. I risultati ottenuti hanno dimostrato che: i) i biomateriali Pro Osteon 200/Avitene e Bio-Oss/Avitene sono idonei per l'uso in chirurgia maxillo-facciale, ii) il biomateriale biomorfico HA-derivato ha la capacità di modulare vari geni osteogenici e geni correlati al differenziamento degli osteoclasti nelle hASCs, indicando che l'uso di biomateriali con queste caratteristiche chimico-fisiche aprono a nuove prospettive di sviluppo di nuovi dispositivi con prestazioni superiori e iii) dex induce il differenziamento osteogenico delle hBMSCs migliorando il rapporto RUNX2/SOX9. Dex, inoltre, induce l'espressione del gene PPARG ed inibisce l'espressione di SOX9, come previsto. L'inibitore di PPARG, T0070907, sembra influenzare negativamente sia la sua attività di trans-attivazione che di trans-repressione. Sono necessarie ulteriori indagini per capire il motivo della down-regolazione di SOX9 in presenza di dex e T0070907. Ulteriori indagini potrebbero essere eseguite utilizzando un siRNAs per bloccare efficacemente l'espressione genica di PPARG, analizzando anche l'espressione di PPARG e SOX9 a livello proteico.

INTRODUCTION. Bone is a rigid and highly dynamic structure that protects several organs of the body. Although bone fracture repair usually restores the injured skeletal organ to its pre-injury state, approximately 10% of fractures do not heal properly. Indeed, the bone regeneration process could fail in extensive bone resections or atrophic non-union. Moreover, face rehabilitation, both functional and aesthetic, has grown in importance in maxillofacial surgery. In tissue engineering, stem cells and new osteoconductive and osteoinductive biomaterials are employed to improve bone repair. Human mesenchymal stem cells derived from adipose tissue (hASCs) and bone marrow (hBMSCs) are probably the most common stem cells used in clinical practice. In vitro, osteogenic differentiation cocktails typically contain at least 10 nM of dexamethasone (dex). Recent studies provide evidence for an off-target adipocytic differentiation in standard osteogenic cultures of hBMSCs induced by dex. Dex induces osteogenesis by inhibiting SRY-Box Transcription Factor 9 (SOX9) gene expression and not by up-regulating the expression of RUNX Family Transcription Factor 2 (RUNX2). Moreover, dex also stimulates adipogenesis by inducing the expression of peroxisome proliferator-activated receptor gamma (PPARG), the adipogenic transcription factor. It is not clear which factor mediates SOX9 downregulation: PPARG is one promising candidate. AIMS. At first, the present project aims to evaluate the cytocompatibility and osteoinductivity proprieties of different scaffolds, which can improve bone regeneration, using hASCs as in vitro cellular model. The characteristics of two Hydroxylapatite (HA)/Collagen composite scaffolds employed in maxillofacial surgery, i.e. Pro Osteon 200/Avitene and Bio-Oss/Avitene, have been evaluated up to 40 days. Patients operated for maxillomandibular malocclusion and/or asymmetry, or for aesthetic reasons, who underwent malar augmentation with porous HA/Collagen prostheses, were evaluated for the new bone formation. Moreover, because inflammation is an important factor that should be considered to develop successful biomaterial-based therapeutics, the immune response of hASCs grown on both Pro Osteon 200/Avitene and Bio-Oss/Avitene scaffolds has been evaluated after 21 days. In the second part, it has been evaluated an innovative HA-derived biomaterial obtained through a new procedure of “biomorphic” transformation, which can directly transform wood pieces into large HA scaffolds, preserving the original multiscale structure through a heterogeneous reaction under supercritical conditions, directly in the 3D state, without adopting any sintering process. In the last part of this project, experiments focused on the mechanisms of dex-induced osteogenesis of hBMSCs have been conducted using T0070907 PPARG inhibitor. RESULTS AND CONCLUSION. My experimental data showed that: i) Pro Osteon 200/Avitene and Bio-Oss/Avitene are suitable materials to be used in maxillofacial surgery, ii) the biomorphic HA has the ability to modulate various osteogenic genes and genes related to osteoclast differentiation in hASCs, indicating that the use of scaffold associating biomimetic composition and nanostructure, enabling higher surface activity, is a promising route towards the development of new bio-devices with superior performance in the regeneration of bone tissue and iii) dex induces hBMSCs osteogenic differentiation enhancing RUNX2/SOX9 ratio. Dex induces PPARG gene expression and inhibits SOX9 expression, as expected. Moreover, the PPARG inhibitor T0070907 seems to inhibit both transactivation and transrepression activities of PPARG. Further investigations are needed to understand SOX9 down-regulation in the presence of dex and T0070907. Indeed, new analyses with siRNAs, which effectively knock down PPARG gene expression, together with PPARG and SOX9 protein expression experiments, may elucidate this issue.

In vitro mesenchymal stem cell osteogenic differentiation and immunomodulation, and bone regrowth in patients, are induced by innovative biomaterials

IAQUINTA, Maria Rosa
2022-06-14T00:00:00+02:00

Abstract

INTRODUCTION. Bone is a rigid and highly dynamic structure that protects several organs of the body. Although bone fracture repair usually restores the injured skeletal organ to its pre-injury state, approximately 10% of fractures do not heal properly. Indeed, the bone regeneration process could fail in extensive bone resections or atrophic non-union. Moreover, face rehabilitation, both functional and aesthetic, has grown in importance in maxillofacial surgery. In tissue engineering, stem cells and new osteoconductive and osteoinductive biomaterials are employed to improve bone repair. Human mesenchymal stem cells derived from adipose tissue (hASCs) and bone marrow (hBMSCs) are probably the most common stem cells used in clinical practice. In vitro, osteogenic differentiation cocktails typically contain at least 10 nM of dexamethasone (dex). Recent studies provide evidence for an off-target adipocytic differentiation in standard osteogenic cultures of hBMSCs induced by dex. Dex induces osteogenesis by inhibiting SRY-Box Transcription Factor 9 (SOX9) gene expression and not by up-regulating the expression of RUNX Family Transcription Factor 2 (RUNX2). Moreover, dex also stimulates adipogenesis by inducing the expression of peroxisome proliferator-activated receptor gamma (PPARG), the adipogenic transcription factor. It is not clear which factor mediates SOX9 downregulation: PPARG is one promising candidate. AIMS. At first, the present project aims to evaluate the cytocompatibility and osteoinductivity proprieties of different scaffolds, which can improve bone regeneration, using hASCs as in vitro cellular model. The characteristics of two Hydroxylapatite (HA)/Collagen composite scaffolds employed in maxillofacial surgery, i.e. Pro Osteon 200/Avitene and Bio-Oss/Avitene, have been evaluated up to 40 days. Patients operated for maxillomandibular malocclusion and/or asymmetry, or for aesthetic reasons, who underwent malar augmentation with porous HA/Collagen prostheses, were evaluated for the new bone formation. Moreover, because inflammation is an important factor that should be considered to develop successful biomaterial-based therapeutics, the immune response of hASCs grown on both Pro Osteon 200/Avitene and Bio-Oss/Avitene scaffolds has been evaluated after 21 days. In the second part, it has been evaluated an innovative HA-derived biomaterial obtained through a new procedure of “biomorphic” transformation, which can directly transform wood pieces into large HA scaffolds, preserving the original multiscale structure through a heterogeneous reaction under supercritical conditions, directly in the 3D state, without adopting any sintering process. In the last part of this project, experiments focused on the mechanisms of dex-induced osteogenesis of hBMSCs have been conducted using T0070907 PPARG inhibitor. RESULTS AND CONCLUSION. My experimental data showed that: i) Pro Osteon 200/Avitene and Bio-Oss/Avitene are suitable materials to be used in maxillofacial surgery, ii) the biomorphic HA has the ability to modulate various osteogenic genes and genes related to osteoclast differentiation in hASCs, indicating that the use of scaffold associating biomimetic composition and nanostructure, enabling higher surface activity, is a promising route towards the development of new bio-devices with superior performance in the regeneration of bone tissue and iii) dex induces hBMSCs osteogenic differentiation enhancing RUNX2/SOX9 ratio. Dex induces PPARG gene expression and inhibits SOX9 expression, as expected. Moreover, the PPARG inhibitor T0070907 seems to inhibit both transactivation and transrepression activities of PPARG. Further investigations are needed to understand SOX9 down-regulation in the presence of dex and T0070907. Indeed, new analyses with siRNAs, which effectively knock down PPARG gene expression, together with PPARG and SOX9 protein expression experiments, may elucidate this issue.
MARTINI, Fernanda
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11392/2489305
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