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The straightfoward creation of an unreported glutathione-stabilised iron(III) complex is disclosed. In contrast to previous reports, glutathione was shown to coordinate and stabilise iron directly under physiological conditions in the absence of additional sulfur containing molecules, such as sodium sulfide. The complex was extensively characterised; the molecular geometry was determined as two inequivalent octahedra, approximately 2/3 of which are slightly distorted towards more tetrahedral in character, with the remaining 1/3 more regularly octahedral. The dispersion of the iron(III)–glutathione complex in aqueous solution yielded particles of 255 ± 4 nm in diameter that enhanced the growth and proliferation of L929 fibroblast cells over 7 days, and inhibited the activity of matrix metalloproteinase-13. Consequently, the unprecedented glutathione-stabilised iron(III) complex disclosed has potential use as a simple-to-prepare growth factor for inclusion within cell culture media, and is an excellent candidate as a therapeutic for the treatment of metalloproteinase-13-associated diseases. [ABSTRACT FROM AUTHOR]

Polymer-based hydrogels have been widely applied for chronic wound therapeutics, due to their well-acclaimed wound exudate management capability. At the same time, there is still an unmet clinical need for simple wound diagnostic tools to assist clinical decision-making at the point of care and deliver on the vision of patient-personalised wound management. To explore this challenge, we present a one-step synthetic strategy to realise a redox-responsive, hyaluronic acid (HA)-based hydrogel that is sensitive to wound environment-related variations in glutathione (GSH) concentration. By selecting aminoethyl disulfide (AED) as a GSH-sensitive crosslinker and considering GSH concentration variations in active and non-self-healing wounds, we investigated the impact of GSH-induced AED cleavage on hydrogel dimensions, aiming to build GSH-size relationships for potential point-of-care wound diagnosis. The hydrogel was also found to be non-cytotoxic and aided L929 fibroblast growth and proliferation over seven days in vitro. Such a material offers a very low-cost tool for the visual detection of a target analyte that varies dependent on the status of the cells and tissues (wound detection), and may be further exploited as an implant for fibroblast growth and tissue regeneration (wound repair). [ABSTRACT FROM AUTHOR]

Medical devices with matrix metalloproteinase (MMP)-modulating functionality are highly desirable to restore tissue homeostasis in critical inflammation states, such as chronic wounds, rotator cuff tears and cancer. The introduction of MMP-modulating functionality in such devices is typically achieved via loading of either rapidly diffusing chelating factors, e.g. EDTA, or MMP-cleavable substrates, raising issues in terms of non-controllable pharmacokinetics and enzymatic degradability, respectively. Aiming to accomplish inherent, long-term, device-induced MMP regulation, this study investigated the synthesis of a hydroxamic acid (HA)–methacrylated collagen conjugate as the building block of a soluble factor-free MMP-modulating hydrogel network with controlled enzymatic degradability. This was realised via a two-step synthetic route: (i) type I collagen was functionalised with photonetwork-inducing methacrylic anhydride (MA) adducts in the presence of triethylamine (TEA); (ii) this methacrylated product was activated with a water-soluble carbodiimide prior to reaction with hydroxylamine, resulting in MMP-chelating HA functions. Nearly-quantitative methacrylation of collagen amines was observed via 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay; this was key to avoiding intramolecular crosslinking side reactions during the carbodiimide-mediated activation of collagen carboxyl groups. The molar content of HA adducts was indirectly quantified via the conversion of remaining carboxyl functions into ethylenediamine (EDA), so that 12–16 mol% HA was revealed in the conjugate by both TNBS and Ninhydrin assays. Resulting UV-cured, HA-bearing collagen hydrogels proved to induce up to ∼13 and ∼32 RFU% activity reduction of MMP-9 and MMP-3, respectively, following 4-day incubation in vitro, whilst displaying an averaged mass loss in the range of 8–21 wt%. Dichroic and electrophoretic patterns of native type I collagen could still be observed following the introduction of HA adducts, suggesting preserved triple helix architecture and chemical sequence in respective HA–methacrylated collagen conjugate. No hydrogel-induced toxic response was observed following the 4-day culture of G292 cells, whilst a lower compression modulus and gel content were measured in HA-bearing compared to methacrylated hydrogels, likely related to HA radical scavenging activity. The novel synthetic strategies described in this work provide a new insight into the systematic chemical manipulation of collagen materials aiming at the design of biomimetic, inflammation-responsive medical devices. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Self-assembling peptides (SAPs) have been shown to offer great promise in therapeutics and have the ability to undergo self-assembly and form ordered nanostructures. However SAP gels are often associated with inherent weak and transient mechanical properties and incorporation of them into polymeric matrices is a route to enhance their mechanical stability. The aim of this work was to incorporate P11-8 peptide (CH3COQQRFOWOFEQQNH2) within poly(ε-caprolactone) (PCL) fibrous webs via one-step electrospinning, aiming to establish the underlying relationships between spinning process, molecular peptide conformation, and material internal architecture. Electrospinning of PCL solutions (6% w/w) in hexafluoro-2-propanol (HFIP) containing up to 40 mg mL−1 P11-8 resulted in the formation of fibres in both nano- (10–100 nm) and submicron range (100–700 nm), in contrast to PCL only webs, which displayed a predominantly submicron fibre distribution. FTIR and CD spectroscopy on both PCL/peptide solutions and resulting electrospun webs revealed monomeric and β-sheet secondary conformation, respectively, suggesting the occurrence of peptide self-assembly during electrospinning due to solvent evaporation. The peptide concentration (0 → 40 mg mL−1) was found to primarily affect the internal structure of the fabric at the nano-scale, whilst water as well as cell culture medium contact angles were dramatically decreased. Nearly no cytotoxic response (＞90% cell viability) was observed when L929 mouse fibroblasts were cultured in contact with electrospun peptide loaded samples. This novel nanofibrous architecture may be the basis for an interesting material platform for e.g. hard tissue repair, in light of the presence of the self-assembled P11-8 in the PCL fibrous structure. [ABSTRACT FROM AUTHOR]

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 hours 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The straightfoward creation of an unreported glutathione-stabilised iron(iii) complex is disclosed. In contrast to previous reports, glutathione was shown to coordinate and stabilise iron directly under physiological conditions in the absence of additional sulfur containing molecules, such as sodium sulfide. The complex was extensively characterised; the molecular geometry was determined as two inequivalent octahedra, approximately 2/3 of which are slightly distorted towards more tetrahedral in character, with the remaining 1/3 more regularly octahedral. The dispersion of the iron(iii)-glutathione complex in aqueous solution yielded particles of 255 ± 4 nm in diameter that enhanced the growth and proliferation of L929 fibroblast cells over 7 days, and inhibited the activity of matrix metalloproteinase-13. Consequently, the unprecedented glutathione-stabilised iron(iii) complex disclosed has potential use as a simple-to-prepare growth factor for inclusion within cell culture media, and is an excellent candidate as a therapeutic for the treatment of metalloproteinase-13-associated diseases.

Medical devices with matrix metalloproteinase (MMP)-modulating functionality are highly desirable to restore tissue homeostasis in critical inflammation states, such as chronic wounds, rotator cuff tears and cancer. The introduction of MMP-modulating functionality in such devices is typically achieved via loading of either rapidly diffusing chelating factors, e.g. EDTA, or MMP-cleavable substrates, raising issues in terms of non-controllable pharmacokinetics and enzymatic degradability, respectively. Aiming to accomplish inherent, long-term, device-induced MMP regulation, this study investigated the synthesis of a hydroxamic acid (HA)-methacrylated collagen conjugate as the building block of a soluble factor-free MMP-modulating hydrogel network with controlled enzymatic degradability. This was realised via a two-step synthetic route: (i) type I collagen was functionalised with photonetwork-inducing methacrylic anhydride (MA) adducts in the presence of triethylamine (TEA); (ii) this methacrylated product was activated with a water-soluble carbodiimide prior to reaction with hydroxylamine, resulting in MMP-chelating HA functions. Nearly-quantitative methacrylation of collagen amines was observed via 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay; this was key to avoiding intramolecular crosslinking side reactions during the carbodiimide-mediated activation of collagen carboxyl groups. The molar content of HA adducts was indirectly quantified via the conversion of remaining carboxyl functions into ethylenediamine (EDA), so that 12-16 mol% HA was revealed in the conjugate by both TNBS and Ninhydrin assays. Resulting UV-cured, HA-bearing collagen hydrogels proved to induce up to ∼13 and ∼32 RFU% activity reduction of MMP-9 and MMP-3, respectively, following 4-day incubation in vitro, whilst displaying an averaged mass loss in the range of 8-21 wt%. Dichroic and electrophoretic patterns of native type I collagen could still be observed following the introduction of HA adducts, suggesting preserved triple helix architecture and chemical sequence in respective HA-methacrylated collagen conjugate. No hydrogel-induced toxic response was observed following the 4-day culture of G292 cells, whilst a lower compression modulus and gel content were measured in HA-bearing compared to methacrylated hydrogels, likely related to HA radical scavenging activity. The novel synthetic strategies described in this work provide a new insight into the systematic chemical manipulation of collagen materials aiming at the design of biomimetic, inflammation-responsive medical devices.

Self-assembling peptides (SAPs) have been shown to offer great promise in therapeutics and have the ability to undergo self-assembly and form ordered nanostructures. However SAP gels are often associated with inherent weak and transient mechanical properties and incorporation of them into polymeric matrices is a route to enhance their mechanical stability. The aim of this work was to incorporate P 11 -8 peptide (CH 3 COQQRFOWOFEQQNH 2 ) within poly(ε-caprolactone) (PCL) fibrous webs via one-step electrospinning, aiming to establish the underlying relationships between spinning process, molecular peptide conformation, and material internal architecture. Electrospinning of PCL solutions (6% w/w) in hexafluoro-2-propanol (HFIP) containing up to 40 mg mL -1 P 11 -8 resulted in the formation of fibres in both nano- (10-100 nm) and submicron range (100-700 nm), in contrast to PCL only webs, which displayed a predominantly submicron fibre distribution. FTIR and CD spectroscopy on both PCL/peptide solutions and resulting electrospun webs revealed monomeric and β-sheet secondary conformation, respectively, suggesting the occurrence of peptide self-assembly during electrospinning due to solvent evaporation. The peptide concentration (0 → 40 mg mL -1 ) was found to primarily affect the internal structure of the fabric at the nano-scale, whilst water as well as cell culture medium contact angles were dramatically decreased. Nearly no cytotoxic response (>90% cell viability) was observed when L929 mouse fibroblasts were cultured in contact with electrospun peptide loaded samples. This novel nanofibrous architecture may be the basis for an interesting material platform for e.g. hard tissue repair, in light of the presence of the self-assembled P 11 -8 in the PCL fibrous structure.