Design of decorated self-assembling peptide hydrogels as architecture for mesenchymal stem cells

Hydrogels from self-assembling ionic complementary peptides have been receiving a lot of interest from the scientific community as mimetic of the extracellular matrix that can offer three-dimensional supports for cell growth or can become vehicles for the delivery of stem cells, drugs or bioactive proteins. In order to develop a 3D "architecture" for mesenchymal stem cells, we propose the introduction in the hydrogel of conjugates obtained by chemoselective ligation between a ionic-complementary self-assembling peptide (called EAK) and three different bioactive molecules: an adhesive sequence with 4 Glycine-Arginine-Glycine-Aspartic Acid-Serine-Proline (GRGDSP) motifs per chain, an adhesive peptide mapped on h-Vitronectin and the growth factor Insulin-like Growth Factor-1 (IGF-1). The mesenchymal stem cell adhesion assays showed a significant increase in adhesion and proliferation for the hydrogels decorated with each of the synthesized conjugates; moreover, such functionalized 3D hydrogels support cell spreading and elongation, validating the use of this class of self-assembly peptides-based material as very promising 3D model scaffolds for cell cultures, at variance of the less realistic 2D ones. Furthermore, small amplitude oscillatory shear tests showed that the presence of IGF-1-conjugate did not alter significantly the viscoelastic properties of the hydrogels even though differences were observed in the nanoscale structure of the scaffolds obtained by changing their composition, ranging from long, well-defined fibers for conjugates with adhesion sequences to the compact and dense film for the IGF-1-conjugate

Breast cancer cells grown on hyaluronic acid-based scaffolds as 3D in vitro model for electroporation

Nowadays, electroporation (EP) represents a promising method for the intracellular delivery of anticancer drugs. To setting up the process, the EP efficiency is usually evaluated by using cell suspension and adherent cell cultures that are not representative of the in vivo conditions. Indeed, cells are surrounded by extracellular matrix (ECM) whose composition and physical characteristics are different for each tissue. So, various three-dimensional (3D) in vitro models, such as spheroids and hydrogel-based cultures, have been proposed to mimic the tumour microenvironment. Herein, a 3D breast cancer in vitro model has been proposed. HCC1954 cells were seeded on crosslinked and lyophilized matrices composed of hyaluronic acid (HA) and ionic complementary self-assembling peptides (SAPs) already known to provide a fibrous structure mimicking collagen network. Herein, SAPs were functionalized with laminin derived IKVAV adhesion motif. Cultures were characterized by spheroids surrounded by ECM produced by cancer cells as demonstrated by collagen1a1 and laminin B1 transcripts. EP was carried out on both 2D and 3D cultures: a sequence of 8 voltage pulses at 5 kHz with different amplitude was applied using a plate electrode. Cell sensitivity to EP seemed to be modulated by the presence of ECM and the different cell organization. Indeed, cells cultured on HA-IKVAV were more sensitive than those treated in 2D and HA cultures, in terms of both cell membrane permeabilization and viability. Collectively, our results suggest that HA-IKVAV cultures may represent an interesting model for EP studies. Further studies will be needed to elucidate the influence of ECM composition on EP efficiency

Facile and selective covalent grafting of an RGD-peptide to electrospun scaffolds improves HUVEC adhesion

The development of a biomimetic surface able to promote endothelialization is fundamental in the search for blood vessel substitutes that prevent the formation of thrombi or hyperplasia. This study aims at investigating the effect of functionalization of poly-ε-caprolactone or poly(L-lactic acid-co-ɛ-caprolactone) electrospun scaffolds with a photoreactive adhesive peptide. The designed peptide sequence contains four Gly-Arg-Gly-Asp-Ser-Pro motifs per chain and a p-azido-Phe residue at each terminus. Different peptide densities on the scaffold surface were obtained by simply modifying the peptide concentration used in pretreatment of the scaffold before UV irradiation. Scaffolds of poly-ε-caprolactone embeddedwith adhesive peptideswere produced to assess the importance of peptide covalent grafting. Our results show that the scaffolds functionalized with photoreactive peptides enhance adhesion at 24h with a dosedependent effect and control the proliferation of human umbilical vein endothelial cells, whereas the inclusion of adhesive peptide in the electrospun matrices by embedding does not give satisfactory results

Design of Decorated Self-Assembling Peptide Hydrogels as Architecture for Mesenchymal Stem Cells

Hydrogels from self-assembling ionic complementary peptides have been receiving a lot of interest from the scientific community as mimetic of the extracellular matrix that can offer three-dimensional supports for cell growth or can become vehicles for the delivery of stem cells, drugs or bioactive proteins. In order to develop a 3D “architecture” for mesenchymal stem cells, we propose the introduction in the hydrogel of conjugates obtained by chemoselective ligation between a ionic-complementary self-assembling peptide (called EAK) and three different bioactive molecules: an adhesive sequence with 4 Glycine-Arginine-Glycine-Aspartic Acid-Serine-Proline (GRGDSP) motifs per chain, an adhesive peptide mapped on h-Vitronectin and the growth factor Insulin-like Growth Factor-1 (IGF-1). The mesenchymal stem cell adhesion assays showed a significant increase in adhesion and proliferation for the hydrogels decorated with each of the synthesized conjugates; moreover, such functionalized 3D hydrogels support cell spreading and elongation, validating the use of this class of self-assembly peptides-based material as very promising 3D model scaffolds for cell cultures, at variance of the less realistic 2D ones. Furthermore, small amplitude oscillatory shear tests showed that the presence of IGF-1-conjugate did not alter significantly the viscoelastic properties of the hydrogels even though differences were observed in the nanoscale structure of the scaffolds obtained by changing their composition, ranging from long, well-defined fibers for conjugates with adhesion sequences to the compact and dense film for the IGF-1-conjugate

Functionalized polymer-derived bioceramics for bone tissue engineering

Bioceramic 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

Progettazione di valvole cardiache "self-seeding": peptidi auto-aggreganti come "filler" per pericardio decellularizzato

Il lavoro si inserisce nel panorama attuale della progettazione di valvole cardiache "self-seeding". In particolare, si valuta l'utilizzo di nanofibre di peptidi auto-assemblanti quali "filler" per matrici di pericardio decellularizzate, verificandone, in primo luogo, la capacità di penetrazione all'interno della matrice stessa. In seguito si quantificano le variazioni delle proprietà fisiche del pericardio bovino e porcino dovute al trattamento con peptide auto-assemblante e non auto-assemblante. La finalità è di creare scaffolds per bioprotesi valvolari graditi alle cellule, in modo da consentirne un completo ripopolamento in vivo, apportando, così, notevoli miglioramenti nel campo della cardiochirurgia valvolare. Inoltre viene utilizzata la tecnica della chemoselective ligation per coniugare peptidi auto-assemblanti ad una sequenza adesiva ed a un fattore di crescit

MODELLI IN VITRO PER ELETTROPORAZIONE

La presente invenzione riguarda il campo della coltura cellulare tridimensionale e le matrici adatte alla coltura cellulare. La presente invenzione riguarda matrici solide reticolate, i procedimenti per la loro preparazione e il loro impiego nelle colture cellulare

Selective Grafting of Protease-Resistant Adhesive Peptides on Titanium Surfaces

In 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