All human tissues are mainly made by fibrous networks (collagens and elastin) deeply interpenetrated by an amorphous polysaccharide matrix of proteoglycans and non-collagenous glycoproteins, forming an insoluble solid permeated by a ionic fluid, which is the extracellular matrix (ECM)1, in which there is also a cellular component which supplies nutrients for all the tissue components. The macroscopic properties of the tissue are determined by the peculiar composition and assemblage of the fibrillary components and of the matrix. The hierarchical structure of the tissues at a dimensional level [molecular scale (1-100 nm), ultramolecular scale (0.1-100 μm) and tissue scale (0.1-100 mm)] acquire a significant role in the determination of the physical and physiological features. The ECM, as a consequence of the specialization of the tissue, is able to meet expectations for the specific functions required, such as the mechanical resistance in the tendons and ligaments, the extreme hardness through calcification in bones and teeth, the glomerular filtration rate, the adherence to basal membranes, etc., so to be responsible for the mechanical support functions of elasticity, cellular anchoring, the determination of cell orientation and the exchange of molecules and fluids in human tissues. The different functions, thanks to their specific roles, allow us to distinguish the tissues at a micro and macro level, so to divide them easily in two categories: soft tissues (muscles, heart, nerves, tendons, ligaments, cartilage, skin, crystalline lens, etc.) and hard tissues (mainly bones and teeth). On the basis of the nature and the structure of the ECM, hydrogels seemed to be the best choice for the simulation of the chemical-physical features. Hydrogels are renowned for their hydrophilic reticulum, which inflate in water or in biological fluids, absorbing a high percentage of liquids, although remains insoluble. Moreover, they are excellent biomimetic materials for the capability of modulating their structural, morphologic and mechanical features on the basis of the specific need, by varying their composition and their grade of reticulation (such as for hydrogels with a polyvinyl alcohol basis2-4). Therefore, hydrogels were ideal candidates for tissue engineering and for regenerative medicine. The aim of the PhD research was the synthesis and characterization of bio composite materials with a hydrogel matrix to be used as tissue replacement. During this study, some of the most invalidating and high cost articular pathologies have been taken into consideration; among these:  Osteoarthritis (OA) of the knee cartilage, degenerative illness, which leads to the friction between the bones, with consequent atherosclerosis formations, eburnation of the bones, bone cists, synovitis, effusion and swelling of the knee and in large scale stiffness and pain which compromise the regular walking functions5.  The degeneration of the intervertebral disc (IDD), featured by a clear drop in cellular liveliness, a reduction of the water content and the consequent nourishment supply, a gradual substitution of the core with a fibrocartilage tissue and the formation of cracks and fractures in the external fibroses annul, which may lead to a structural fail of the IVD, causing also serious spine disorders, another invalidating syndrome6.  Meniscus injuries subject to wear, caused by demanding work, or by trauma or stress, especially for athletes. This last case is very common and it is characterized by pain and a period of impediment, with short or medium term prognosis or with the total block of the articulation. As a result, surgery includes a permanent change in the amount of load and stress on the articular cartilage, which often causes a premature onset of OA7. In order to face inconveniences produced by OA, a biomimetic material was projected and synthesized, so to use it as a substitute of the tibia cartilage. This material was the PVA hydrogel, reticulated chemically through STMP (both materials are notoriously atoxic and used as food additives)3,8. To determine the best conditions, five hydrogel samples with different molar ratios PVA:STMP (1:0.1; 1:0.25; 1:0.5; 1:1; 1:2)were synthetized and valued. While monitoring the effect of the variation of the parameters such as, pH (11, 12, 13), the concentration of the reticulated solution (10%/20% p/v), the reaction time (24, 48, 72 o 96 ore) and the different modalities of solution agitation (mechanical and magnetic), the desiccation methodology (through Lyophilisation (_L) or through heater (_E) 60°C) evaluating the results on the base of the reaction feedback, the solidity and continuity of the materials’ structure.The synthetized materials were later chemically characterized through an IR spectrometry, mass spectrometry of secondary ions (ToF-SIMS), colorimetric test and element analysis (AE), in order to determine the quality and quantity of the reticulum. From a physical point of view, the materials were characterizedthanks to the thermogravimetric analysis(TGA) and the evaluation of the water content (WC), it was possible to value if the entrance of the reticulum changed the thermic stability of the PVA matrix and, moreover, the inflatement capability of the hydrogel. Then, the material was characterized morphologically, through scanning electron microscope (SEM) and differential scanning calorimeter (DSC)to analyse respectively the micro and the meso-structure. Finally, a rheological and mechanical characterization was made, thanks to a rheological analysis and a dynamo mechanical analysis(DMA) to determine if the viscoelastic properties of the materials are comparable to those of the tibia cartilage tissue. In conclusion, degradation and cytotoxic tests were made, following international procedures (ISO/FDIS 10993-5 e 10993-13 Biological evaluation of medical device), to evaluate the chemicalstability and the cytocompatibility of the synthetizedmaterials. From the analysis made on the sample PVA-H 0.5_E,it is possible to identify similar characteristics to the cartilage of the tibial plafond, in terms of hydration, stability, mechanical properties, and cytotoxicity and, therefore, it may be used in the realization of biomedical implants for the osteoarthritis therapy9,10. In order to face one of the most serious spinal disorders, the herniation of the intervertebral disc, in which a leak of material of the nucleus pulposus (NP) touches the nervous structures of the spine and causes intense pain, it was possible to propose a PVA hydrogel, synthetized chemically through STMP and enriched by PVP. The PVP is a highly hydrophilic polymer, which interacts through hydrogen bonds with the PVA chains11, and has allowed to decrease the degree of reticulation, producing a matrix with similar mechanical properties to the NP, and, at the same time, it has increased the water content of the samples, reaching the measures of the tissue that is being replaced.To determine the best conditions, three hydrogels were synthetized with the same molar ratioPVA:STMP (1:0.5), but with growing contents of PVP % p/v (1%, 2.5% e 5% p/v). The materials were later characterized through a chemical analysis, by IR spectrometry and colorimetric tests to determine the quality and quantity of the reticulum and to appraise if the introduction of PVP produced disturbing effects in the hydrogel’s matrix. More tests were made, by a physical viewpoint, thanks to thermogravimetric analysis(TGA) and evaluation of the water content(WC) to estimate the inflatement capacities of the hydrogels.Morphological tests, through differential scanning calorimeter (DSC) were made to determine the mesostructure. Rheological analyses (AR) were made to determine if the viscoelastic properties of the materials were comparable with those of the spinal nucleus pulposus, and finally cytotoxic tests were made, following international procedures (ISO/FDIS 10993-5 e 10993-13 Biological evaluation of medical device), to evaluate the chemical stability and the cytocompatibility of the synthetized materials. In conclusion, from the results obtained in this study, the best material which mimes best the NP features is the 5.0% sample, with hydration and mechanical properties similar to the NP. Therefore, the 5.0% sample may be considered an excellent possibility for the realization of biomedical implants for the therapy of spinal degenerations12. The research of biomimetic materials for the therapy of meniscal injuries was made by proposing two different samples of PVA based hydrogels for the substitution of regeneration of the meniscus, depending on the type of lesion: For those cases in which the patient had undergone Meniscectomy for a serious fracture, a PVA hydrogel reticulum obtained through STMP with ratioPVA:STMP 1:0.25, after three reticulations with the aim of incrementing the mechanical properties, so to reach similar features to those of the meniscus (approximately120 kPa) was proposed as a possible substitute13.For those cases in which the meniscal lesions were of a minor entity, a cellular scaffold was proposed. This scaffold was made by a hydrogel mix of PVA and Xanthan Gum, reticulated simultaneously through STMP for the regeneration of the meniscal tissue14. The research of the best substitute involved the synthesis of three samples with different content % p/v di XG-PVA (60-40; 30-70; 15-85).Once synthetized, both typologies of materials were cross-examined by a chemical, physical and mechanical characterization. Moreover, the materials undergone citotoxicological tests in vitro to evaluate the suitability for temporary or permanent meniscal substitutes. In the case of long-term substitutes, the sample 3R PVA-H 0.25 may be considered as a potential biomimetic substitute of the meniscus. To what may concern the stress relaxation tests, it is capable of instant dissipation of the deformation performed, by touching a decrease percentage > than 95% after 1 second, similarly to the human cartilage tissue15and with the same G value13.In the case of celluar scaffolds, the sample with improved skils such as biomimetic material is XG15.X15posses good properties in terms of hydration, mechanical properties apt for cellular colonization.Therefore, the sample XG15 is a potential candidate for the realization of biomedical implants for the therapy of meniscal lesions and it is qualified for the following evaluation fase of citotoxicology in vitro, in accordance with the law ISO/FDIS 10993-5.

Tutti i tessuti presenti nel corpo umano sono prevalentemente costituiti da network fibrosi (collagene ed elastina) fortemente interpenetrati con una matrice polisaccaridica amorfa di proteoglicani e glicoproteine non collagenose, formando un solido insolubile permeato da un fluido ionico, la cosiddetta matrice extracellulare (ECM)1, all'interno della quale è inoltre presente una componente cellulare, con funzione di apporto nutrienti a tutti i componenti tissutali. Le proprietà macroscopiche del tessuto vengono determinate dalla particolare composizione e dall’assemblamento dei componenti fibrillari e della matrice. La struttura gerarchica dei tessuti a livello dimensionale [scala molecolare (1-100 nm), scala ultramolecolare (0.1-100 μm) e scala tissutale (0.1-100 mm)] assume un ruolo significativo nel determinarne le proprietà fisiche e fisiologiche. La ECM, in seguito ad una specializzazione del tessuto, riesce ad adempiere alle particolari funzioni richieste, come, ad esempio, la resistenza meccanica nei tendini e legamenti, l’estrema durezza tramite calcificazione nelle ossa e nei denti, la filtrazione nel glomerulo renale, l’adesione nelle membrane basali ecc., rendendosi responsabile di tutte le funzioni di supporto meccanico, elasticità, ancoraggio cellulare, determinazione dell’orientazione cellulare e scambio di molecole e fluidi dei tessuti umani. Le differenti funzioni, grazie ai loro specifici ruoli, rendono questi tessuti estremamente distinguibili a livello micro e macroscopici, permettendo di suddividerli facilmente in due tipologie: tessuti soft o molli (muscoli, cuore, nervi, tendini, legamenti, cartilagini, pelle, cristallino, ecc.) e tessuti hard (prevalentemente ossa e denti). Sulla base della natura e struttura della ECM, la scelta dei materiali per la simulazione delle caratteristiche chimico-fisiche della matrice extracellulare è ricaduta sugli idrogel, polimeri idrofili reticolati che in acqua o in fluidi biologici rigonfiano assorbendo elevate percentuali di liquido, pur rimanendo insolubili. Gli idrogeli sono degli ottimi materiali biomimetici grazie alla possibilità di modulare le loro proprietà strutturali, morfologiche e meccaniche sulla base dell’applicazione richiesta, semplicemente variando la loro composizione e il loro grado di reticolazione (come ad esempio gli idrogeli a base di polivinil alcool2-4). Essi rappresentano pertanto dei candidati ideali per l’ingegneria tissutale e per la medicina rigenerativa. L’attività del progetto di ricerca ha avuto come oggetto principale la sintesi e caratterizzazione di materiali biocompositi a matrice idrogelica da utilizzare come sostituti tissutali. Durante il lavoro svolto sono state identificate alcune tra le patologie articolari maggiormente invalidanti e onerose, da un punto di vista socio-economico, tra cui:  L'osteoartrosi (OA) del ginocchio, patologia di tipo degenerativo, caratterizzata dall’assottigliamento della cartilagine, che conduce, nei casi più avanzati, allo sfregamento delle unità ossee con conseguente formazione di osteosclerosi, eburneazione ossea, cisti ossee, sinoviti, versamento e tumefazione del ginocchio e, in larga scala, rigidità e dolore articolare che compromettono le normali funzioni di deambulazione5.  La degenerazione del disco intervertebrale (IDD) (caratterizzata da una netta diminuzione della vitalità cellulare, una riduzione del contenuto d’acqua del tessuto e conseguente apporto di nutrienti, una graduale sostituzione del nucleo con tessuto fibrocartilagineo e la formazione di fessure e fratture nella porzione esterna dell'anulo fibroso) che può condurre al fallimento strutturale dell'IVD comportando disordini spinali più gravi tra i quali l'erniazione del disco intervertebrale, altra patologia estremamente invalidante6.  Le lesioni meniscali da usura prodotte da lavori usuranti o di tipo traumatico in seguito a stress sportivo, che rappresentano una tipologia di infortunio molto comune, caratterizzata da dolore e invalità temporanea, che costringe il lavoratore o l'atleta a brevi-medi periodi di prognosi nei casi più lievi, e al blocco dell'articolazione nei casi più gravi. Tramite chirurgia è possibile asportare la parte di menisco danneggiata ripristinando la funzionalità dell'articolazione, tuttavia l'asportazione chirurgica comporta una modificazione permanete nell'applicazione dei carichi sulla cartilagine articolare e provoca spesso un'insorgenza precoce dell'OA7. Per far fronte agli inconvenienti prodotti dall'OA, patologia altamente invalidante, è stato progettato e sintetizzato un materiale biomimetico che potesse essere utilizzato come sostituto della cartilagine tibiale, scegliendo come materiale sostitutivo un idrogel di polivinil alcool (PVA), reticolato per via chimica mediante trisodio trimetafosfato (STMP) (entrambi materiali notoriamente atossici e impiegati come additivi alimentari)3,8. Al fine di realizzare un materiale biomimetico in grado di mimare al meglio le proprietà della cartilagine,e quindi scegliere le condizioni migliori di sintesi, sono stati sintetizzati e valutati cinque campioni di idrogel con differenti rapporti molari PVA:STMP (1:0.1; 1:0.25; 1:0.5; 1:1; 1:2), ed è stato monitorato l’effetto della variazione di parametri quali pH (11, 12, 13), concentrazione della soluzione di agente reticolante (10%/20% p/v), tempi di reazione (24, 48, 72 o 96 ore) e modalità di agitazione della soluzione (meccanica/magnetica), nonché metodologia di essiccamento [tramite liofilizzazione (_L) o tramite riscaldamento in stufa a 60°C (_E)],valutandoli in funzione della resa di reazione, solidità e continuità della struttura dei materiali. I materiali sintetizzati sono stati in seguito caratterizzati da un punto di vista chimico [mediante Spettrometria Infrarossa (IR), Spettrometria di Massa di Ioni Secondari (ToF-SIMS), Analisi Colorimetria e Analisi Elementare (AE)] per determinare qualitativamente e quantitativamente l'avvenuta reticolazione; da un punto di vista fisico [mediante Analisi Termogravimetrica (TGA) e valutazione del Contenuto d'acqua (WC)] per valutare l’effetto della reticolazione sulla stabilità termica nella matrice di PVA e per valutare la capacità di rigonfiamento degli idrogel; da un punto di vista morfologico [tramite Microscopia Elettronica a Scansione (SEM) e Calorimetria Differenziale a Scansione (DSC)] per valutare rispettivamente la micro e la mesostruttura delle matrici polimeriche; da un punto di vista reologico e meccanico [mediante Analisi Reologica (AR) e Dinamomeccanica (DMA)] per determinare se le proprietà viscoelastiche dei materiali sono confrontabili con quelle del tessuto cartilagineo del piatto tibiale. Infine, sono stati effettuati test degradativi e citotossicologici secondo le norme internazionali vigenti (ISO/FDIS 10993-5 e 10993-13 Biological evaluation of medical device) per valutare la stabilità chimica e la citotossicità dei materiali sintetizzati. Dalle analisi condotte il campione PVA-H 0.5_E mostra caratteristiche similari a quelle della cartilagine del piatto tibiale in termini di idratazione, stabilità, proprietà meccaniche e citotossicità e può essere potenzialmente impiegato nella realizzazione di impianti biomedici volti alla terapia dell’osteoartrosi9,10. Per far fronte ad uno dei disordini spinali più gravi, l'erniazione del disco intervertebrale, dove una fuoriuscita di materiale normalmente contenuto nel disco intervertebrale (nucleo polposo (NP)) entra in contatto con le strutture nervose contenute nel canale spinale, provocando intenso dolore, è stato proposto un idrogel di PVA, sintetizzato per via chimica mediante reticolazione tramite STMP e arricchito con PVP. Il PVP è un polimero altamente idrofilo che, interagendo mediante legami ad idrogeno con le catene di PVA11, ha permesso di diminuirne il grado di reticolazione, producendo matrici con proprietà meccaniche più simili al NP, e allo stesso tempo di incrementare il contenuto acquoso dei campioni fino ai valori del tessuto da sostituire. Per determinare le migliori condizioni di sintesi, sono stati sintetizzati tre idrogel con uguale rapporto molare PVA:STMP(1:0.5), ma con crescenti contenuti % p/v di PVP (1%, 2.5% e 5% p/v). I materiali sintetizzati sono stati in seguito caratterizzati da un punto di vista chimico (mediante spettrometria infrarossa (IR) e analisi colorimetria) per determinare qualitativamente e quantitativamente l'avvenuta reticolazione e per valutare se l'introduzione del PVP producesse o meno effetti di disturbo all'interno della matrice dell'idrogel. Da un punto di vista fisico (mediante analisi termo gravimetrica (TGA) e valutazione del contenuto d'acqua (WC)) per valutare l’effetto della reticolazione sulla stabilità termica della matrice di PVA e per valutare la capacità di rigonfiamento degli idrogel. Da un punto di vista morfologico (tramite calorimetria a scansione differenziale (DSC)) per valutare la mesostruttura delle matrici sintetizzate. Da un punto di vista reologico (mediante analisi reologica (AR)) per determinare se le proprietà viscoelastiche dei materiali sono confrontabili con quelle del nucleo polposo spinale. Infine, sono stati effettuati test citotossicologici e di proliferazione cellulare secondo le norme internazionali vigenti (ISO/FDIS 10993-5 Biological evaluation of medical device) per valutarne la citotossicità e la citocompatibilità dei materiali sintetizzati. Dai risultati ottenuti il materiale che meglio mima le caratteristiche del NP è il campione 5.0%, con proprietà di idratazione e meccaniche in linea con quelle del NP. Pertanto, il campione 5.0% può essere considerato un valido candidato per la realizzazione di impianti biomedici per la cura delle degenerazioni spinali12. Il lavoro svolto alla ricerca di materiali biomimetici per la cura delle lesioni meniscali è stato affrontato proponendo due tipologie di idrogel differenti, a base di PVA, per la sostituzione/rigenerazione del menisco a seconda della tipologia di lesione subita. Per casi in cui il paziente sia stato sottoposto a menischectomia totale in seguito ad una grave frattura del menisco, è stato proposto un idrogel di PVA, reticolato mediante STMP con un rapporto PVA:STMP 1:0.25, sottoposto a tre reticolazioni consecutive allo scopo di incrementarne le proprietà meccaniche fino al raggiungimento di valori paragonabili a quelli del menisco (c.a. 120 kPa). Tale idrogel è quindi inteso come sostituto permanente13. Per i casi in cui le lesioni del menisco siano di minor entità, è stato proposto uno scaffold cellulare, costituito da un idrogel misto a base di PVA e Gomma-Xantano (XG), reticolati contemporaneamente mediante STMP, come sostituto temporaneo in grado di indurre e favorire la rigenerazione del tessuto meniscale. La XG è stata scelta poichè in grado di favorire e supportare l'adesione, la proliferazione e la differenziazione cellulare14. La ricerca del miglior sostituto ha coinvolto la sintesi di tre campioni con differenti contenuti % p/v di XG-PVA (60-40; 30-70; 15-85). Una volta sintetizzate, entrambe le tipologie di materiali sono state sottoposte a caratterizzazione chimica, fisica e meccanica. Inoltre, i materiali sono stati sottoposti a test di citotossicità in vitro per valutarne l’idoneità come sostituti meniscali temporanei e/o permanenti. Nel caso dei sostituti a lungo termine il campione 3R PVA-H 0.25 può essere considerato come un potenziale sostituto biomimetico del menisco. Da un punto di vista di rilassamento degli sforzi, esso è, infatti, in grado di dissipare istantaneamente la deformazione applicata, toccando mediamente una riduzione percentuale > dell’95% dopo 1 secondo allo stesso modo del tessuto cartilagineo umano15 e possiede valori di G' paragonabili a quelli del menisco13. Mentre nel caso degli scaffold cellulari il materiale che si comporta maggiormente come materiale biomimetico è il campione XG15 con caratteristiche, in termini di idratazione e proprietà meccaniche, idonee alla colonizzazione cellulare. Pertanto, il campione XG15 risulta un potenziale candidato per la realizzazione di impianti biomedici per la cura delle lesioni meniscali e viene abilitato alla successiva fase di valutazione della citotossicità in vitro secondo le norme ISO/FDIS 10993-5.

Nelli, N. (2017). “Sviluppo di materiali biocompositi per la realizzazione di sostituti tissutali per impianti a breve e lungo termine”.

“Sviluppo di materiali biocompositi per la realizzazione di sostituti tissutali per impianti a breve e lungo termine”

NELLI, NICOLA
2017-01-01

Abstract

All human tissues are mainly made by fibrous networks (collagens and elastin) deeply interpenetrated by an amorphous polysaccharide matrix of proteoglycans and non-collagenous glycoproteins, forming an insoluble solid permeated by a ionic fluid, which is the extracellular matrix (ECM)1, in which there is also a cellular component which supplies nutrients for all the tissue components. The macroscopic properties of the tissue are determined by the peculiar composition and assemblage of the fibrillary components and of the matrix. The hierarchical structure of the tissues at a dimensional level [molecular scale (1-100 nm), ultramolecular scale (0.1-100 μm) and tissue scale (0.1-100 mm)] acquire a significant role in the determination of the physical and physiological features. The ECM, as a consequence of the specialization of the tissue, is able to meet expectations for the specific functions required, such as the mechanical resistance in the tendons and ligaments, the extreme hardness through calcification in bones and teeth, the glomerular filtration rate, the adherence to basal membranes, etc., so to be responsible for the mechanical support functions of elasticity, cellular anchoring, the determination of cell orientation and the exchange of molecules and fluids in human tissues. The different functions, thanks to their specific roles, allow us to distinguish the tissues at a micro and macro level, so to divide them easily in two categories: soft tissues (muscles, heart, nerves, tendons, ligaments, cartilage, skin, crystalline lens, etc.) and hard tissues (mainly bones and teeth). On the basis of the nature and the structure of the ECM, hydrogels seemed to be the best choice for the simulation of the chemical-physical features. Hydrogels are renowned for their hydrophilic reticulum, which inflate in water or in biological fluids, absorbing a high percentage of liquids, although remains insoluble. Moreover, they are excellent biomimetic materials for the capability of modulating their structural, morphologic and mechanical features on the basis of the specific need, by varying their composition and their grade of reticulation (such as for hydrogels with a polyvinyl alcohol basis2-4). Therefore, hydrogels were ideal candidates for tissue engineering and for regenerative medicine. The aim of the PhD research was the synthesis and characterization of bio composite materials with a hydrogel matrix to be used as tissue replacement. During this study, some of the most invalidating and high cost articular pathologies have been taken into consideration; among these:  Osteoarthritis (OA) of the knee cartilage, degenerative illness, which leads to the friction between the bones, with consequent atherosclerosis formations, eburnation of the bones, bone cists, synovitis, effusion and swelling of the knee and in large scale stiffness and pain which compromise the regular walking functions5.  The degeneration of the intervertebral disc (IDD), featured by a clear drop in cellular liveliness, a reduction of the water content and the consequent nourishment supply, a gradual substitution of the core with a fibrocartilage tissue and the formation of cracks and fractures in the external fibroses annul, which may lead to a structural fail of the IVD, causing also serious spine disorders, another invalidating syndrome6.  Meniscus injuries subject to wear, caused by demanding work, or by trauma or stress, especially for athletes. This last case is very common and it is characterized by pain and a period of impediment, with short or medium term prognosis or with the total block of the articulation. As a result, surgery includes a permanent change in the amount of load and stress on the articular cartilage, which often causes a premature onset of OA7. In order to face inconveniences produced by OA, a biomimetic material was projected and synthesized, so to use it as a substitute of the tibia cartilage. This material was the PVA hydrogel, reticulated chemically through STMP (both materials are notoriously atoxic and used as food additives)3,8. To determine the best conditions, five hydrogel samples with different molar ratios PVA:STMP (1:0.1; 1:0.25; 1:0.5; 1:1; 1:2)were synthetized and valued. While monitoring the effect of the variation of the parameters such as, pH (11, 12, 13), the concentration of the reticulated solution (10%/20% p/v), the reaction time (24, 48, 72 o 96 ore) and the different modalities of solution agitation (mechanical and magnetic), the desiccation methodology (through Lyophilisation (_L) or through heater (_E) 60°C) evaluating the results on the base of the reaction feedback, the solidity and continuity of the materials’ structure.The synthetized materials were later chemically characterized through an IR spectrometry, mass spectrometry of secondary ions (ToF-SIMS), colorimetric test and element analysis (AE), in order to determine the quality and quantity of the reticulum. From a physical point of view, the materials were characterizedthanks to the thermogravimetric analysis(TGA) and the evaluation of the water content (WC), it was possible to value if the entrance of the reticulum changed the thermic stability of the PVA matrix and, moreover, the inflatement capability of the hydrogel. Then, the material was characterized morphologically, through scanning electron microscope (SEM) and differential scanning calorimeter (DSC)to analyse respectively the micro and the meso-structure. Finally, a rheological and mechanical characterization was made, thanks to a rheological analysis and a dynamo mechanical analysis(DMA) to determine if the viscoelastic properties of the materials are comparable to those of the tibia cartilage tissue. In conclusion, degradation and cytotoxic tests were made, following international procedures (ISO/FDIS 10993-5 e 10993-13 Biological evaluation of medical device), to evaluate the chemicalstability and the cytocompatibility of the synthetizedmaterials. From the analysis made on the sample PVA-H 0.5_E,it is possible to identify similar characteristics to the cartilage of the tibial plafond, in terms of hydration, stability, mechanical properties, and cytotoxicity and, therefore, it may be used in the realization of biomedical implants for the osteoarthritis therapy9,10. In order to face one of the most serious spinal disorders, the herniation of the intervertebral disc, in which a leak of material of the nucleus pulposus (NP) touches the nervous structures of the spine and causes intense pain, it was possible to propose a PVA hydrogel, synthetized chemically through STMP and enriched by PVP. The PVP is a highly hydrophilic polymer, which interacts through hydrogen bonds with the PVA chains11, and has allowed to decrease the degree of reticulation, producing a matrix with similar mechanical properties to the NP, and, at the same time, it has increased the water content of the samples, reaching the measures of the tissue that is being replaced.To determine the best conditions, three hydrogels were synthetized with the same molar ratioPVA:STMP (1:0.5), but with growing contents of PVP % p/v (1%, 2.5% e 5% p/v). The materials were later characterized through a chemical analysis, by IR spectrometry and colorimetric tests to determine the quality and quantity of the reticulum and to appraise if the introduction of PVP produced disturbing effects in the hydrogel’s matrix. More tests were made, by a physical viewpoint, thanks to thermogravimetric analysis(TGA) and evaluation of the water content(WC) to estimate the inflatement capacities of the hydrogels.Morphological tests, through differential scanning calorimeter (DSC) were made to determine the mesostructure. Rheological analyses (AR) were made to determine if the viscoelastic properties of the materials were comparable with those of the spinal nucleus pulposus, and finally cytotoxic tests were made, following international procedures (ISO/FDIS 10993-5 e 10993-13 Biological evaluation of medical device), to evaluate the chemical stability and the cytocompatibility of the synthetized materials. In conclusion, from the results obtained in this study, the best material which mimes best the NP features is the 5.0% sample, with hydration and mechanical properties similar to the NP. Therefore, the 5.0% sample may be considered an excellent possibility for the realization of biomedical implants for the therapy of spinal degenerations12. The research of biomimetic materials for the therapy of meniscal injuries was made by proposing two different samples of PVA based hydrogels for the substitution of regeneration of the meniscus, depending on the type of lesion: For those cases in which the patient had undergone Meniscectomy for a serious fracture, a PVA hydrogel reticulum obtained through STMP with ratioPVA:STMP 1:0.25, after three reticulations with the aim of incrementing the mechanical properties, so to reach similar features to those of the meniscus (approximately120 kPa) was proposed as a possible substitute13.For those cases in which the meniscal lesions were of a minor entity, a cellular scaffold was proposed. This scaffold was made by a hydrogel mix of PVA and Xanthan Gum, reticulated simultaneously through STMP for the regeneration of the meniscal tissue14. The research of the best substitute involved the synthesis of three samples with different content % p/v di XG-PVA (60-40; 30-70; 15-85).Once synthetized, both typologies of materials were cross-examined by a chemical, physical and mechanical characterization. Moreover, the materials undergone citotoxicological tests in vitro to evaluate the suitability for temporary or permanent meniscal substitutes. In the case of long-term substitutes, the sample 3R PVA-H 0.25 may be considered as a potential biomimetic substitute of the meniscus. To what may concern the stress relaxation tests, it is capable of instant dissipation of the deformation performed, by touching a decrease percentage > than 95% after 1 second, similarly to the human cartilage tissue15and with the same G value13.In the case of celluar scaffolds, the sample with improved skils such as biomimetic material is XG15.X15posses good properties in terms of hydration, mechanical properties apt for cellular colonization.Therefore, the sample XG15 is a potential candidate for the realization of biomedical implants for the therapy of meniscal lesions and it is qualified for the following evaluation fase of citotoxicology in vitro, in accordance with the law ISO/FDIS 10993-5.
2017
Nelli, N. (2017). “Sviluppo di materiali biocompositi per la realizzazione di sostituti tissutali per impianti a breve e lungo termine”.
Nelli, Nicola
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11365/1011390
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