Giuseppe Tronci

An entropy–elastic gelatin-based hydrogel system

Gelatin is a non-immunogenic and degradable biopolymer, which is widely applied in the biomedical field e.g. for drug capsules or as absorbable hemostats. However, gelatin materials present limited and hardly reproducible mechanical properties especially in aqueous systems, particularly caused by the uncontrollable partial renaturation of collagen-like triple helices. Therefore, mechanically demanding applications for gelatin-based materials, such as vascular patches, i.e. hydrogel films that seal large incisions in vessel walls, and for induced autoregeneration, are basically excluded if this challenge is not addressed. Through the synthesis of a defined chemical network of gelatin with hexamethylene diisocyanate (HDI) in DMSO, the self-organization of gelatin chains could be hindered and amorphous gelatin films were successfully prepared having Youngs moduli of 60–530 kPa. Transferring the crosslinking reaction with HDI and, alternatively, ethyl lysine diisocyanate (LDI), to water as reaction medium allowed the tailoring of swelling behaviour and mechanical properties by variation of crosslinker content while suppressing the formation of helices. The hydrogels had Youngs moduli of 70–740 kPa, compressive moduli of 16–48 kPa, and degrees of swelling of 300–800 vol%. Test reactions investigated by ESI mass spectrometry allowed the identification and quantification of reaction products of the crosslinking reaction. The HDI crosslinked networks were stabilized by direct covalent crosslinks (ca. 10 mol%), supported by grafting (50 mol%) and blending of hydrophobic oligomeric chains. For the LDI-based networks, less crosslinked (3 mol%) and grafted species (5 mol%) and much higher amounts of oligomers were observed. The adjustable hydrogel system enables the application of gelatin-based materials in physiological environments.

One Step Creation of Multifunctional 3D Architectured Hydrogels Inducing Bone Regeneration

Structured hydrogels showing form stability and elastic properties individually tailorable on different length scales are accessible in a one-step process. They support cell adhesion and differentiation and display growing pore size during degradation. In vivo experiments demonstrate their efficacy in biomaterial-induced bone regeneration, not requiring addition of cells or growth factors.

Controlled Change of Mechanical Properties during Hydrolytic Degradation of Polyester Urethane Networks

Polyester urethane networks are versatile polymer systems as it is possible to tailor their mechanical properties and their hydrolytic degradation profile. For biomedical applications, the biodegradability as well as the thermomechanical properties of the polymer networks during the course of degradation is of importance. Therefore, we investigated the change of thermomechanical properties of networks based on star-shaped precursors of rac-dilactide and diglycolide, e-caprolactone, or p-dioxanone, respectively, during hydrolytic degradation. Degradation rate and mechanical properties of the polymer networks were tailored by crosslink density, comonomers, and by changing the glass transition temperature. Most importantly, the degradation of the networks led to a controlled, step-by-step change of the mechanical properties of the networks.

Tunable drug-loading capability of chitosan hydrogels with varied network architectures

Advanced bioactive systems with defined macroscopic properties and spatio-temporal sequestration of extracellular biomacromolecules are highly desirable for next generation therapeutics. Here, chitosan (CT) hydrogels were prepared with neutral or negatively charged cross-linkers in order to promote selective electrostatic complexation with charged drugs. CT was functionalized with varied dicarboxylic acids, such as tartaric acid, poly(ethylene glycol) bis(carboxymethyl) ether, 1,4-phenylenediacetic acid and 5-sulfoisophthalic acid monosodium salt (PhS), whereby PhS was hypothesized to act as a simple mimetic of heparin. Attenuated total reflectance Fourier transform infrared spectroscopy showed the presence of CO amide I, N-H amide II and CO ester bands, providing evidence of covalent network formation. The cross-linker content was reversely quantified by proton nuclear magnetic resonance on partially degraded network oligomers, so that 18 mol.% PhS was exemplarily determined. Swellability (SR: 299 ± 65-1054 ± 121 wt.%), compressibility (E: 2.1 ± 0.9-9.2 ± 2.3 kPa), material morphology and drug-loading capability were successfully adjusted based on the selected network architecture. Here, hydrogel incubation with model drugs of varied electrostatic charge, i.e. allura red (AR, doubly negatively charged), methyl orange (MO, negatively charged) or methylene blue (MB, positively charged), resulted in direct hydrogel-dye electrostatic complexation. Importantly, the cationic compound, MB, showed different incorporation behaviours, depending on the electrostatic character of the selected cross-linker. In light of this tunable drug-loading capability, these CT hydrogels would be highly attractive as drug reservoirs towards e.g. the fabrication of tissue models in vitro.

Triple-helical collagen hydrogels via covalent aromatic functionalisation with 1,3-phenylenediacetic acid

Chemical crosslinking of collagen is a general strategy to reproduce macroscale tissue properties in physiological environment. However, simultaneous control of protein conformation, material properties and biofunctionality is highly challenging with current synthetic strategies. Consequently, the potentially-diverse clinical applications of collagen-based biomaterials cannot be fully realised. In order to establish defined biomacromolecular systems for mineralised tissue applications, type I collagen was functionalised with 1,3-Phenylenediacetic acid (Ph) and investigated at the molecular, macroscopic and functional levels. Preserved triple helix conformation was observed in obtained covalent networks via ATR-FTIR (AIII/A1450 ~ 1) and WAXS, while network crosslinking degree (C: 87-99 mol.-%) could be adjusted based on specific reaction conditions. Decreased swelling ratio (SR: 823-1285 wt.-%) and increased thermo-mechanical (Td : 80-88 °C; E: 28-35 kPa; σmax : 6-8 kPa; εb : 53-58 %) properties were observed compared to state-of-the-art carbodiimide (EDC)-crosslinked collagen controls, likely related to the intermolecular covalent incorporation of the aromatic segment. Ph-crosslinked hydrogels displayed nearly intact material integrity and only a slight mass decrease (MR : 5-11 wt. %) following 1-week incubation in either PBS or simulated body fluid (SBF), in contrast to EDC-crosslinked collagen (MR : 33-58 wt. %). Furthermore, FTIR, SEM and EDS revealed deposition of a calcium-phosphate phase on SBF-retrieved samples, whereby an increased calcium phosphate ratio (Ca/P: 0.84-1.41) was observed in hydrogels with higher Ph content. 72-hour material extracts were well tolerated by L929 mouse fibroblasts, whereby cell confluence and metabolic activity (MTS assay) were comparable to those of cells cultured in cell culture medium (positive control). In light of their controlled structure-function properties, these biocompatible collagen hydrogels represent attractive material systems for potential mineralised tissue applications.

Multi-scale mechanical characterization of highly swollen photo-activated collagen hydrogels

Biological hydrogels have been increasingly sought after as wound dressings or scaffolds for regenerative medicine, owing to their inherent biofunctionality in biological environments. Especially in moist wound healing, the ideal material should absorb large amounts of wound exudate while remaining mechanically competent in situ. Despite their large hydration, however, current biological hydrogels still leave much to be desired in terms of mechanical properties in physiological conditions. To address this challenge, a multi-scale approach is presented for the synthetic design of cyto-compatible collagen hydrogels with tunable mechanical properties (from the nano- up to the macro-scale), uniquely high swelling ratios and retained (more than 70%) triple helical features. Type I collagen was covalently functionalized with three different monomers, i.e. 4-vinylbenzyl chloride, glycidyl methacrylate and methacrylic anhydride, respectively. Backbone rigidity, hydrogen-bonding capability and degree of functionalization (F: 16 ± 12–91 ± 7 mol%) of introduced moieties governed the structure–property relationships in resulting collagen networks, so that the swelling ratio (SR: 707 ± 51–1996 ± 182 wt%), bulk compressive modulus (Ec: 30 ± 7–168 ± 40 kPa) and atomic force microscopy elastic modulus (EAFM: 16 ± 2–387 ± 66 kPa) were readily adjusted. Because of their remarkably high swelling and mechanical properties, these tunable collagen hydrogels may be further exploited for the design of advanced dressings for chronic wound care.

Photo-active collagen systems with controlled triple helix architecture

The design of photo-active collagen systems is presented as a basis for establishing biomimetic materials with varied network architecture and programmable macroscopic properties. Following in-house isolation of type I collagen, reaction with vinyl-bearing compounds of varied backbone rigidity, i.e. 4-vinylbenzyl chloride (4VBC) and glycidyl methacrylate (GMA), was carried out. TNBS colorimetric assay, 1H-NMR and ATR-FTIR confirmed covalent and tunable functionalization of collagen lysines. Depending on the type and extent of functionalization, controlled stability and thermal denaturation of triple helices were observed via circular dichroism (CD), whereby the hydrogen-bonding capability of introduced moieties was shown to play a major role. Full gel formation was observed following photo-activation of functionalized collagen solutions. The presence of a covalent network only slightly affected collagen triple helix conformation (as observed by WAXS and ATR-FTIR), confirming the structural organization of functionalized collagen precursors. Photo-activated hydrogels demonstrated an increased denaturation temperature (DSC) with respect to native collagen, suggesting that the formation of the covalent network successfully stabilized collagen triple helices. Moreover, biocompatibility and mechanical competence of obtained hydrogels were successfully demonstrated under physiologically-relevant conditions. These results demonstrate that this novel synthetic approach enabled the formation of biocompatible collagen systems with defined network architecture and programmable macroscopic properties, which can only partially be obtained with current synthetic methods.

Biomimetic wet-stable fibres via wet spinning and diacid-based crosslinking of collagen triple helices

One of the limitations of electrospun collagen as bone-like fibrous structure is the potential collagen triple helix denaturation in the fibre state and the corresponding inadequate wet stability even after crosslinking. Here, we have demonstrated the feasibility of accomplishing wet-stable fibres by wet spinning and diacid-based crosslinking of collagen triple helices, whereby fibre ability to act as bonemimicking mineralisation system has also been explored. Circular dichroism (CD) demonstrated nearly complete triple helix retention in resulting wet-spun fibres, and the corresponding chemically crosslinked fibres successfully preserved their fibrous morphology following 1-week incubation in phosphate buffer solution (PBS). The presented novel diacid-based crosslinking route imparted superior tensile modulus and strength to the resulting fibres indicating that covalent functionalization of distant collagen molecules is unlikely to be accomplished by current state-of-the-art carbodiimide-based crosslinking. To mimic the constituents of natural bone extra cellular matrix (ECM), the crosslinked fibres were coated with carbonated hydroxyapatite (CHA) through biomimetic precipitation, resulting in an attractive biomaterial for guided bone regeneration (GBR), e.g. in bony defects of the maxillofacial region.

Compositional and in vitro evaluation of nonwoven type I collagen /poly-DL-lactic acid scaffolds for bone regeneration

Poly-dl-lactic acid (PDLLA) was blended with type I collagen to attempt to overcome the instantaneous gelation of electrospun collagen scaffolds in biological environments. Scaffolds based on blends of type I collagen and PDLLA were investigated for material stability in cell culture conditions (37 °C; 5% CO2) in which post-electrospinning glutaraldehyde crosslinking was also applied. The resulting wet-stable webs were cultured with bone marrow stromal cells (HBMSC) for five weeks. Scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), Fourier transform infra-red spectroscopy (FTIR) and biochemical assays were used to characterise the scaffolds and the consequent cell-scaffold constructs. To investigate any electrospinning-induced denaturation of collagen, identical PDLLA/collagen and PDLLA/gelatine blends were electrospun and their potential to promote osteogenic differentiation investigated. PDLLA/collagen blends with w/w ratios of 40/60, 60/40 and 80/20 resulted in satisfactory wet stabilities in a humid environment, although chemical crosslinking was essential to ensure long term material cell culture. Scaffolds of PDLLA/collagen at a 60:40 weight ratio provided the greatest stability over a five-week culture period. The PDLLA/collagen scaffolds promoted greater cell proliferation and osteogenic differentiation compared to HMBSCs seeded on the corresponding PDLLA/gelatine scaffolds, suggesting that any electrospinning-induced collagen denaturation did not affect material biofunctionality within 5 weeks in vitro.

Protease-sensitive atelocollagen hydrogels promote healing in a diabetic wound model

The design of exudate-managing wound dressings is an established route to accelerated healing, although such design remains a challenge from material and manufacturing standpoints. Aiming towards the clinical translation of knowledge gained in vitro with highly-swollen rat tail collagen hydrogels, this study investigated the healing capability in a diabetic mouse wound model of telopeptide-free, protease-inhibiting collagen networks. 4-Vinylbenzylation and UV irradiation of type I atelocollagen (AC) led to hydrogel networks with chemical and macroscopic properties comparable to previous collagen analogues, attributable to similar lysine content and dichroic properties. After 4 days in vitro, hydrogels induced nearly 50 RFU% reduction in matrix metalloproteinase (MMP)-9 activity, whilst showing less than 20 wt% mass loss. After 20 days in vivo, dry networks promoted 99% closure of 10 × 10 mm full thickness wounds and accelerated neo-dermal tissue formation compared to Mepilex®. This collagen system can be equipped with multiple, customisable properties and functions key to personalised chronic wound care.