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Covalent functionalization of bioceramics with adhesive peptides
No full textopenIl successo di un costrutto ingegnerizzato si basa sulla solidità strutturale, biocompatibilità e bioattività dello scaffold.
In questa tesi verrà riassunta la mia esperienza presso il laboratorio della Professoressa Dettin Monica a seguito della Dottoressa Zamuner Annj, durante la quale è stata prodotta della ardistonite arricchita con stronzio e magnesio, è stata funzionalizzata covalentemente con la sequenza peptidica D2HVP, sempre sintetizzata in laboratorio tramite sintesi su fase solida, e sono stati eseguiti dei test per verificare l’adesione tra il peptide ed il bioceramico.The successfulness of a engineered construct is based on structural integrity, biocompatibility and bioactivity of the scaffold.
This abstract will summarise my experience at Professor Dettin Monica's laboratory following PhD student Zamuner Annj in which some Mg/Sr-doped Hardystonite was sinterized, covalently functionalized using D2HVP peptide, also synthesized using Solid Phase Peptide Synthesis during this experience, and was later submitted to tests to prove the adhesion between bioceramic and peptide
Functionalized polymer-derived bioceramics for bone tissue engineering
Full text linkBioceramic foams, typically containing Ca or Ca-Mg silicates, as wollastonite (CaSiO3) and diopside (CaMgSi2O6), can be obtained from the thermal treatment of preceramic polymers (silicone resins) containing micro- and nano-sized filler powders. This innovative process is advantageous because of its simplicity, the limited processing temperature and the microstructural homogeneity. These foams can simulate the natural porous internal structure of human bones, and both physical and mechanical characteristics can be modulated by varying the parameters of bioceramic formation process, consequently can be considered as promising scaffolds for bone tissue engineering. The silicate scaffolds were functionalized to add a biochemical component miming organic structure of bone, thus improving the surface-to-cells interactions, complying with the new approach of “biochemical functionalization”. The nonapeptide (HVP) from the h-Vitronectin protein (sequence 352-360) was covalently and selectively anchored on wollastonite-diopside (W/D) foams’ surfaces. In addition, a dimeric analogue (2HVP) was designed in order to increase ionic interactions with cellular GAGs and two HVP retro-inverted sequences (DHVP, retro-inverted peptide of HVP, and D2HVP, retro-inverted peptide of 2HVP) were synthesized to increase the stability toward proteolytic degradation under physiological conditions. A first screening, through in vitro bioassays showed that D2HVP gave the best results in terms of h-Osteoblasts (HOB) adhesion, proliferation, gene expression without inducing cytotoxicity when anchored to W/D foams. In particular, HOB proliferation showed a 15-folds increase for D2HVP functionalized samples with respect to the silanized foams at 6 days. A next-generation scaffold in addition to the ability to “communicate” with the surrounding cells, has to improve angiogenesis by promoting the formation of an interconnected and stable vascular network, because angiogenesis is fundamental for implant’s survival and long term integration. In order to reach this goal, we combined the proved bioactivity of D2HVP functionalized polymer-derived silicate foams with the several properties of self-assembling peptides (SAPs). Preliminary in vitro screenings were carried out to understand how different cell types (i.e. macrophages, osteoblasts and HUVECs), involved in the processes of inflammatory response, osseointegration and angiogenesis, respond to scaffolds produced by SAPs. The results of these preliminary assays showed that EAK is able to induce the formation of longer HUVEC’s tubule structures, and to promote the highest increase in HOB proliferation and YAP1 expression. In vivo assays confirmed the osteoconductive and osteoinductive properties of D2HVP peptide. All W/D scaffolds showed external and internal cell colonization with formation of new blood vessels (neoangiogenesis). Only W/D foams functionalized with D2HVP were able to stimulate the process of ectopic mineralization. In vivo results showed that EAK enriched with the pro-angiogenic sequence TIM (from Tß4) grafted to the SAP itself, have a synergistic effect in promoting mineralization. Finally, we proposed a different covalent and selective functionalization methodology that, requiring only mild reactions’ conditions and aqueous solutions can be easily transferred to all types of polymer-derived bioglass (e.g. Mg/Sr doped hardystonite), for improving their bioactivity.Le schiume bioceramiche, tipicamente contenenti silicati di Ca o Ca-Mg, come wollastonite (CaSiO3) e diopside (CaMgSi2O6), possono essere ottenute dal trattamento termico di polimeri preceramici (resine siliconiche) contenenti fillers di dimensioni micro- e nanometriche. Questo processo innovativo è vantaggioso per la sua semplicità, per la limitata temperatura di lavorazione e l'omogeneità microstrutturale. Queste schiume simulano la naturale struttura porosa interna dell’osso trabecolare e, sia le caratteristiche fisiche che meccaniche possono essere modulate semplicemente variando i parametri iniziali del processo di produzione. Di conseguenza queste schiume possono essere considerate come scaffolds promettenti per l'ingegneria tessutale dell’osso. Gli scaffolds porosi sono stati funzionalizzati per aggiungere una componente biochimica che imiti la struttura organica dell'osso, migliorando così le interazioni superficie-cellula, attraverso l’innovativo approccio della "funzionalizzazione biochimica". Il nonapeptide (HVP) della Vitronettina (sequenza 352-360) è stato fissato in modo covalente e selettivo sulla superficie di schiume di wollastonite-diopside (W/D). Inoltre, un analogo dimerico (2HVP) è stato progettato per aumentare le interazioni ioniche con i GAG cellulari, e due sequenze retro-inverse del peptide HVP (DHVP, peptide retro-inverso di HVP e D2HVP, peptide retro-inverso di 2HVP) sono state sintetizzate per aumentare la stabilità verso la degradazione proteolitica osservata in condizioni fisiologiche. Un primo screening, attraverso prove biologiche in vitro, ha dimostrato che la sequenza D2HVP ha dato i migliori risultati in termini di adesione, proliferazione ed espressione genica di osteoblasti umani (HOB) senza indurre citotossicità quando ancorato a schiume W/D. In particolare, la proliferazione di HOB a 6 giorni su campioni funzionalizzati con D2HVP ha subito un incremento di 15 volte rispetto alle schiume silanizzate.
Inoltre, uno scaffold di nuova generazione in addizione alla capacità di "comunicare" con le cellule circostanti, deve possedere l’abilità di promuovere l'angiogenesi attraverso la formazione di una rete vascolare interconnessa e stabile, fondamentale per la sopravvivenza dell'impianto e la sua integrazione a lungo termine. Per raggiungere questo obiettivo, si è voluto sommare le proprietà dei peptidi auto-assemblanti (SAP) alla comprovata bioattività delle schiume da precursori polimerici funzionalizzate con D2HVP. Sono stati effettuati screening preliminari in vitro per comprendere come differenti tipi cellulari (i.e. macrofagi, osteoblasti e HUVEC), coinvolti nei processi di risposta infiammatoria, osteointegrazione e angiogenesi, rispondano a diversi scaffolds di SAP. I risultati di questi saggi preliminari hanno dimostrato che il peptide auto-aggregante EAK è in grado di indurre la formazione di strutture tubulari più estese di HUVEC e di aumentare la proliferazione di HOB e l'espressione di YAP1. I saggi in vivo hanno confermato le proprietà osteoconduttive e osteoinduttive del peptide D2HVP. Tutti gli scaffolds di W/D sono stati colonizzati in vivo sia sulla superficie esterna che interna, con formazione di nuovi vasi sanguigni (neoangiogenesi). Solo le schiume di W/D funzionalizzate con D2HVP sono state in grado di stimolare il processo di mineralizzazione ectopica. I risultati in vivo hanno dimostrato che EAK arricchito con la sequenza pro-angiogenica TIM (da Tß4) legata covalentemente al SAP stesso, ha un effetto sinergico nel promuovere la mineralizzazione. Infine, è stata proposta una diversa metodologia di funzionalizzazione covalente e selettiva che, richiedendo solo blande condizioni di reazione e soluzioni acquose, può essere facilmente trasferita a tutti i tipi di bioceramici derivati da precursori polimerici (e.g. Hardystonite arricchita con Mg/Sr), per migliorarne la bioattività
Self-seeding heart valve design: self-assembling peptide hydrogel as filler for decellularized pericardium
No full textInsertion of a flexible spacer increases the yield of site-specific bioconjugation through N-terminal transamination
No full textSelf-seeding heart valve design: Self-Assembling Peptide hydrogel as filler for decellularized pericardium (RENASCAT)
No full textSelf-assembling peptide hydrogels immobilized on silicon surfaces
No full textThe hydrogels of self-assembling ionic complementary peptides have collected in the scientific community increasing consensus as mimetics of the extracellular matrix that can offer 3D supports for cell growth or be vehicles for the delivery of stem cells or drugs. Such scaffolds have also been proposed as bone substitutes for small defects as they promote beneficial effects on human osteoblasts. In this context, our research deals with the introduction of a layer of self-assembling peptides on a silicon surface by covalent anchoring and subsequent physisorption. In this work, we present a spectroscopic investigation of the proposed bioactive scaffolds, carried out by surface-sensitive spectroscopic techniques such as XPS (X-ray photoelectron spectroscopy) and RAIRS (Reflection Absorption Infrared Spectroscopy) and by state-of-the-art synchrotron radiation methodologies such as angle dependent NEXAFS (Near Edge X-ray Absorption Fine Structure). XPS studies confirmed the change in the surface composition in agreement with the proposed enrichments, and led to assess the self-assembling peptide chemical stability. NEXAFS spectra, collected in angular dependent mode at the N K-edge, allowed to investigate the self-assembling behavior of the macromolecules, as well as to determine their molecular orientation on the substrate. Furthermore, Infrared Spectroscopy measurements demonstrated that the peptide maintains its secondary structure (β-sheet anti-parallel) after deposition on the silicon surface. The complementary information acquired by means of XPS, NEXAFS and RAIRS lead to hypothesize a “layer-by-layer” arrangement of the immobilized peptides, giving rise to an ordered 3D nanostructure
Peptide hydrogel decorated with growth factors and self-assembling adhesive sequences: covalent grafting on TiO2 surfaces investigated by SR-XPS, Angular Dependent NEXAFS and IR.
No full textSelective Grafting of Protease-Resistant Adhesive Peptides on Titanium Surfaces
Full text linkIn orthopedic, dental, and maxillofacial fields, joint prostheses, plates, and screws are widely used in the treatment of problems related to bone tissue. However, the use of these prosthetic systems is not free from complications: the fibrotic encapsulation of endosseous implants often prevents optimal integration of the prostheses with the surrounding bone. To overcome these issues, biomimetic titanium implants have been developed where synthetic peptides have been selectively grafted on titanium surfaces via Schiff base formation. We used the retro-inverted sequence (DHVPX) from [351–359] human Vitronectin and its dimer (D2HVP). Both protease-resistant peptides showed increased human osteoblast adhesion and proliferation, an augmented number of focal adhesions, and cellular spreading with respect to the control. D2HVP-grafted samples significantly enhance Secreted Phosphoprotein 1, Integrin Binding Sialoprotein, and Vitronectin gene expression vs. control. An estimation of peptide surface density was determined by Two-photon microscopy analysis on a silanized glass model surface labeled with a fluorescent analog
Nanocrystalline Cellulose from the Solitary Tunicate Phallusia Mammillata, a Valuable Nanocellulose Precursor
Full text linkAscidians are ubiquitous filter-feeding marine invertebrates, the only animals producing cellulose in their outer tunic, in the form of a composite structure of cellulose microfibrils linked to a protein matrix. This study reports the successful processing of Phallusia mammillata, a species prevalent in the Northern Adriatic Sea, into high-quality nanocellulose (NC). An optimized extraction method followed by chemical modification is applied to produce suitable precursors for cellulose-based products. Structural, compositional, and thermal characterization demonstrated the high degree of crystallinity of the extracted cellulose, which is further processed into both cationic and anionic forms by direct reaction of nanofibril suspensions. High-aspect-ratio nanofibrillated cellulose (NFC) with a fibrils diameter of 10 ± 2 nm is directly obtained from purified tunics. Anionic nanocrystals and cationic nanofibrils with a mean diameter of 8 ± 2 nm are obtained from pristine nanofibrillated cellulose suspensions. To the best of the authors’ knowledge, this is the first report on the extraction and processing of cellulose from Phallusia mammillata, opening new possibilities toward high-grade cellulose-based materials exploiting an abundant local resource
Spectroscopic characterization of Vitronectin-derived peptides enhancing cell adhesion on prosthetic devices
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