Nonwoven Reinforced Photocurable Poly(glycerol sebacate)-Based Hydrogels.

Implantable hydrogels should ideally possess mechanical properties matched to the surrounding tissues to enable adequate mechanical function while regeneration occurs. This can be challenging, especially when degradable systems with a high water content and hydrolysable chemical bonds are required in anatomical sites under constant mechanical stimulation, e.g., a foot ulcer cavity. In these circumstances, the design of hydrogel composites is a promising strategy for providing controlled structural features and macroscopic properties over time. To explore this strategy, the synthesis of a new photocurable elastomeric polymer, poly(glycerol-co-sebacic acid-co-lactic acid-co-polyethylene glycol) acrylate (PGSLPA), is investigated, along with its processing into UV-cured hydrogels, electrospun nonwovens and fibre-reinforced variants, without the need for a high temperature curing step or the use of hazardous solvents. The mechanical properties of bioresorbable PGSLPA hydrogels were studied with and without electrospun nonwoven reinforcement and with varied layered configurations, aiming to determine the effects of the microstructure on the bulk compressive strength and elasticity. The nonwoven reinforced PGSLPA hydrogels exhibited a 60% increase in compressive strength and an 80% increase in elastic moduli compared to the fibre-free PGSLPA samples. The mechanical properties of the fibre-reinforced hydrogels could also be modulated by altering the layering arrangement of the nonwoven and hydrogel phase. The nanofibre-reinforced PGSLPA hydrogels also exhibited good elastic recovery, as evidenced by the hysteresis in compression fatigue stress-strain evaluations showing a return to the original dimensions.

A collagen-based theranostic wound dressing with visual, long-lasting infection detection capability.

Continuous wound monitoring is one strategy to minimise infection severity and inform prompt variations in therapeutic care following infection diagnosis. However, integration of this functionality in therapeutic wound dressings is still challenging. We hypothesised that a theranostic dressing could be realised by integrating a collagen-based wound contact layer with previously demonstrated wound healing capability, and a halochromic dye, i.e. bromothymol blue (BTB), undergoing colour change following infection-associated pH changes (pH: 5-6 ➔ >7). Two different BTB integration strategies, i.e. electrospinning and drop-casting, were pursued to introduce long-lasting visual infection detection capability through retention of BTB within the dressing. Both systems had an average BTB loading efficiency of 99 wt% and displayed a colour change within 1 min of contact with simulated wound fluid. Drop-cast samples retained up to 85 wt% of BTB after 96 h in a near-infected wound environment, in contrast to the fibre-bearing prototypes, which released over 80 wt% of BTB over the same time period. An increase in collagen denaturation temperature (DSC) and red shifts (ATR-FTIR) suggest the formation of secondary interactions between the collagen-based hydrogel and the BTB, which are attributed to count for the long-lasting dye confinement and durable dressing colour change. Given the high L929 fibroblast viability in drop-cast sample extracts (92 %, 7 days), the presented multiscale design is simple, cell- and regulatory-friendly, and compliant with industrial scale-up. This design, therefore, offers a new platform for the development of theranostic dressings enabling accelerated wound healing and prompt infection diagnosis.

A Biomimetic Nonwoven-Reinforced Hydrogel for Spinal Cord Injury Repair.

In clinical trials, new scaffolds for regeneration after spinal cord injury (SCI) should reflect the importance of a mechanically optimised, hydrated environment. Composite scaffolds of nonwovens, self-assembling peptides (SAPs) and hydrogels offer the ability to mimic native spinal cord tissue, promote aligned tissue regeneration and tailor mechanical properties. This work studies the effects of an aligned electrospun nonwoven of P11-8-enriched poly(ε-caprolactone) (PCL) fibres, integrated with a photo-crosslinked hydrogel of glycidylmethacrylated collagen (collagen-GMA), on neurite extension. Mechanical properties of collagen-GMA hydrogel in compression and shear were recorded, along with cell viability. Collagen-GMA hydrogels showed J-shaped stress-strain curves in compression, mimicking native spinal cord tissue. For hydrogels prepared with a 0.8-1.1 wt.% collagen-GMA concentration, strain at break values were 68 ± 1-81 ± 1% (±SE); maximum stress values were 128 ± 9-311 ± 18 kPa (±SE); and maximum force values were 1.0 ± 0.1-2.5 ± 0.1 N (±SE). These values closely mimicked the compression values for feline and porcine tissue in the literature, especially those for 0.8 wt.%. Complex shear modulus values fell in the range 345-2588 Pa, with the lower modulus hydrogels in the range optimal for neural cell survival and growth. Collagen-GMA hydrogel provided an environment for homogenous and three-dimensional cell encapsulation, and high cell viability of 84 ± 2%. Combination of the aligned PCL/P11-8 electrospun nonwoven and collagen-GMA hydrogel retained fibre alignment and pore structure, respectively, and promoted aligned neurite extension of PC12 cells. Thus, it is possible to conclude that scaffolds with mechanical properties that both closely mimic native spinal cord tissue and are optimal for neural cells can be produced, which also promote aligned tissue regeneration when the benefits of hydrogels and electrospun nonwovens are combined.

A long-lasting guided bone regeneration membrane from sequentially functionalised photoactive atelocollagen.

The fast degradation of collagen-based membranes in the biological environment remains a critical challenge, resulting in underperforming Guided Bone Regeneration (GBR) therapy leading to compromised clinical results. Photoactive atelocollagen (AC) systems functionalised with ethylenically unsaturated monomers, such as 4-vinylbenzyl chloride (4VBC), have been shown to generate mechanically competent materials for wound healing, inflammation control and drug delivery, whereby control of the molecular architecture of the AC network is key. Building on this platform, the sequential functionalisation with 4VBC and methacrylic anhydride (MA) was hypothesised to generate UV-cured AC hydrogels with reduced swelling ratio, increased proteolytic stability and barrier functionality for GBR therapy. The sequentially functionalised atelocollagen precursor (SAP) was characterised via TNBS and ninhydrin colourimetric assays, circular dichroism and UV-curing rheometry, which confirmed nearly complete consumption of collagen's primary amino groups, preserved triple helices and fast (< 180 s) gelation kinetics, respectively. Hydrogel's swelling ratio and compression modulus were adjusted depending on the aqueous environment used for UV-curing, whilst the sequential functionalisation of AC successfully generated hydrogels with superior proteolytic stability in vitro compared to both 4VBC-functionalised control and the commercial dental membrane Bio-Gide®. These in vitro results were confirmed in vivo via both subcutaneous implantation and a proof-of-concept study in a GBR calvarial model, indicating integrity of the hydrogel and barrier defect, as well as tissue formation following 1-month implantation in rats. STATEMENT OF SIGNIFICANCE: Collagen-based membranes remain a key component in Guided Bone Regeneration (GBR) therapy, but their properties, e.g. proteolytic stability and soft tissue barrier functionality, are still far from optimal. This is largely attributed to the complex molecular configuration of collagen, which makes chemical accessibility and structure-function relations challenging. Here, we fabricated a UV-cured hydrogel network of atelocollagen, whereby triple helices were sequentially functionalised with two distinct ethylenically unsaturated monomers. The effects of the sequential functionalisation and UV-curing on the macroscopic properties, degradation behaviour and GBR capability were investigated in vitro and in vivo. The results highlight the key role of the sequential functionalisation and provide important insights for the design of future, longer-lasting resorbable membranes for GBR therapy.

Characterisation of factors contributing to the performance of nonwoven fibrous matrices as substrates for adenovirus vectored vaccine stabilisation.

Adenovirus vectors offer a platform technology for vaccine development. The value of the platform has been proven during the COVID-19 pandemic. Although good stability at 2-8 °C is an advantage of the platform, non-cold-chain distribution would have substantial advantages, in particular in low-income countries. We have previously reported a novel, potentially less expensive thermostabilisation approach using a combination of simple sugars and glass micro-fibrous matrix, achieving excellent recovery of adenovirus-vectored vaccines after storage at temperatures as high as 45 °C. This matrix is, however, prone to fragmentation and so not suitable for clinical translation. Here, we report an investigation of alternative fibrous matrices which might be suitable for clinical use. A number of commercially-available matrices permitted good protein recovery, quality of sugar glass and moisture content of the dried product but did not achieve the thermostabilisation performance of the original glass fibre matrix. We therefore further investigated physical and chemical characteristics of the glass fibre matrix and its components, finding that the polyvinyl alcohol present in the glass fibre matrix assists vaccine stability. This finding enabled us to identify a potentially biocompatible matrix with encouraging performance. We discuss remaining challenges for transfer of the technology into clinical use, including reliability of process performance.

Hierarchically Assembled Type I Collagen Fibres as Biomimetic Building Blocks of Biomedical Membranes.

Wet spinning is an established fibre manufacturing route to realise collagen fibres with preserved triple helix architecture and cell acceptability for applications in biomedical membranes. However, resulting fibres still need to be chemically modified post-spinning to ensure material integrity in physiological media, with inherent risks of alteration of fibre morphology and with limited opportunities to induce fibrillogenesis following collagen fixation in the crosslinked state. To overcome this challenge, we hypothesised that a photoactive type I collagen precursor bearing either single or multiple monomers could be employed to accomplish hierarchically assembled fibres with improved processability, macroscopic properties and nanoscale organisation via sequential wet spinning and UV-curing. In-house-extracted type I rat tail collagen functionalised with both 4-vinylbenzyl chloride (4VBC) and methacrylate residues generated a full hydrogel network following solubilisation in a photoactive aqueous solution and UV exposure, whereby ~85 wt.% of material was retained following 75-day hydrolytic incubation. Wide-angle X-ray diffraction confirmed the presence of typical collagen patterns, whilst an averaged compression modulus and swelling ratio of more than 290 kPa and 1500 wt.% was recorded in the UV-cured hydrogel networks. Photoactive type I collagen precursors were subsequently wet spun into fibres, displaying the typical dichroic features of collagen and regular fibre morphology. Varying tensile modulus (E = 5 ± 1 - 11 ± 4 MPa) and swelling ratio (SR = 1880 ± 200 - 3350 ± 500 wt.%) were measured following post-spinning UV curing and equilibration with phosphate-buffered saline (PBS). Most importantly, 72-h incubation of the wet spun fibres in PBS successfully induced renaturation of collagen-like fibrils, which were fixed following UV-induced network formation. The whole process proved to be well tolerated by cells, as indicated by a spread-like cell morphology following a 48-h culture of L929 mouse fibroblasts on the extracts of UV-cured fibres.

An injectable, self-healing and MMP-inhibiting hyaluronic acid gel via iron coordination.

Regulating the activity of matrix metalloproteinases (MMPs) is a potential strategy for osteoarthritis (OA) therapy, although delivering this effect in a spatially and temporally localised fashion remains a challenge. Here, we report an injectable and self-healing hydrogel enabling factor-free MMP regulation and biomechanical competence in situ. The hydrogel is realised within 1 min upon room temperature coordination between hyaluronic acid (HA) and a cell-friendly iron-glutathione complex in aqueous environment. The resultant gel displayed up to 300% in shear strain and tolerance towards ATDC 5 chondrocytes, in line with the elasticity and biocompatibility requirements for connective tissue application. Significantly enhanced inhibition of MMP-13 activity was achieved after 12 h in vitro, compared with a commercial HA injection (OSTENIL® PLUS). Noteworthy, 24-hour incubation of a clinical synovial fluid sample collected from a late-stage OA patient with the reported hydrogel was still shown to downregulate synovial fluid MMP activity (100.0 ± 17.6% ➔ 81.0 ± 7.5%), with at least comparable extent to the case of the OSTENIL® PLUS-treated SF group (100.0 ± 17.6% ➔ 92.3 ± 27.3%). These results therefore open up new possibilities in the use of HA as both mechanically-competent hydrogel as well as a mediator of MMP regulation for OA therapy.

Hydrolytic Degradability, Cell Tolerance and On-Demand Antibacterial Effect of Electrospun Photodynamically Active Fibres.

Photodynamically active fibres (PAFs) are a novel class of stimulus-sensitive systems capable of triggering antibiotic-free antibacterial effect on-demand when exposed to light. Despite their relevance in infection control, however, the broad clinical applicability of PAFs has not yet been fully realised due to the limited control in fibrous microstructure, cell tolerance and antibacterial activity in the physiologic environment. We addressed this challenge by creating semicrystalline electrospun fibres with varying content of poly[(l-lactide)-co-(glycolide)] (PLGA), poly(ε-caprolactone) (PCL) and methylene blue (MB), whereby the effect of polymer morphology, fibre composition and photosensitiser (PS) uptake on wet state fibre behaviour and functions was studied. The presence of crystalline domains and PS-polymer secondary interactions proved key to accomplishing long-lasting fibrous microstructure, controlled mass loss and controlled MB release profiles (37 °C, pH 7.4, 8 weeks). PAFs with equivalent PLGA:PCL weight ratio successfully promoted attachment and proliferation of L929 cells over a 7-day culture with and without light activation, while triggering up to 2.5 and 4 log reduction in E. coli and S. mutans viability, respectively. These results support the therapeutic applicability of PAFs for frequently encountered bacterial infections, opening up new opportunities in photodynamic fibrous systems with integrated wound healing and infection control capabilities.

The facile and additive-free synthesis of a cell-friendly iron(iii)-glutathione complex.

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.

Induced Periosteum-Mimicking Membrane with Cell Barrier and Multipotential Stromal Cell (MSC) Homing Functionalities.

The current management of critical size bone defects (CSBDs) remains challenging and requires multiple surgeries. To reduce the number of surgeries, wrapping a biodegradable fibrous membrane around the defect to contain the graft and carry biological stimulants for repair is highly desirable. Poly(ε-caprolactone) (PCL) can be utilised to realise nonwoven fibrous barrier-like structures through free surface electrospinning (FSE). Human periosteum and induced membrane (IM) samples informed the development of an FSE membrane to support platelet lysate (PL) absorption, multipotential stromal cells (MSC) growth, and the prevention of cell migration. Although thinner than IM, periosteum presented a more mature vascular system with a significantly larger blood vessel diameter. The electrospun membrane (PCL3%-E) exhibited randomly configured nanoscale fibres that were successfully customised to introduce pores of increased diameter, without compromising tensile properties. Additional to the PL absorption and release capabilities needed for MSC attraction and growth, PCL3%-E also provided a favourable surface for the proliferation and alignment of periosteum- and bone marrow derived-MSCs, whilst possessing a barrier function to cell migration. These results demonstrate the development of a promising biodegradable barrier membrane enabling PL release and MSC colonisation, two key functionalities needed for the in situ formation of a transitional periosteum-like structure, enabling movement towards single-surgery CSBD reconstruction.

Biomimetic peptide enriched nonwoven scaffolds promote calcium phosphate mineralisation.

Cell-free translational strategies are needed to accelerate the repair of mineralised tissues, particularly large bone defects, using minimally invasive approaches. Regenerative bone scaffolds should ideally mimic aspects of the tissue's ECM over multiple length scales and enable surgical handling and fixation during implantation in vivo. Leveraging the knowledge gained with bioactive self-assembling peptides (SAPs) and SAP-enriched electrospun fibres, we presented a cell free approach for promoting mineralisation via apatite deposition and crystal growth, in vitro, of SAP-enriched nonwoven scaffolds. The nonwoven scaffold was made by electrospinning poly(ε-caprolactone) (PCL) in the presence of either peptide P11-4 (Ac-QQRFEWEFEQQ-Am) or P11-8 (Ac QQRFOWOFEQQ-Am), in light of the polymer's fibre forming capability and its hydrolytic degradability as well as the well-known apatite nucleating capability of SAPs. The 11-residue family of peptides (P11-X) has the ability to self-assemble into ß-sheet ordered structures at the nano-scale and to generate hydrogels at the macroscopic scale, some of which are capable of promoting biomineralisation due to their apatite-nucleating capability. Both variants of SAP-enriched nonwoven used in this study were proven to be biocompatible with murine fibroblasts and supported nucleation and growth of apatite minerals in simulated body fluid (SBF) in vitro. The fibrous nonwoven provided a structurally robust scaffold, with the capability to control SAP release behaviour. Up to 75% of P11-4 and 45% of P11-8 were retained in the fibres after 7 day incubation in aqueous solution at pH 7.4. The encapsulation of SAP in a nonwoven system with apatite-forming as well as localised and long-term SAP delivery capabilities is appealing as a potential means of achieving cost-effective bone repair therapy for critical size defects.

Colony Formation, Migratory, and Differentiation Characteristics of Multipotential Stromal Cells (MSCs) from "Clinically Accessible" Human Periosteum Compared to Donor-Matched Bone Marrow MSCs.

Periosteum is vital for fracture healing, as a highly vascular and multipotential stromal cell- (MSC-) rich tissue. During surgical bone reconstruction, small fragments of periosteum can be "clinically accessible," yet periosteum is currently not ultilised, unlike autologous bone marrow (BM) aspirate. This study is aimed at comparing human periosteum and donor-matched iliac crest BM MSC content and characterising MSCs in terms of colony formation, growth kinetics, phenotype, cell migration patterns, and trilineage differentiation capacity. "Clinically accessible" periosteum had an intact outer fibrous layer, containing CD271+ candidate MSCs located perivasculary; the inner cambium was rarely present. Following enzymatic release of cells, periosteum formed significantly smaller fibroblastic colonies compared to BM (6.1 mm2 vs. 15.5 mm2, n = 4, P = 0.0006). Periosteal colonies were more homogenous in size (range 2-30 mm2 vs. 2-54 mm2) and on average 2500-fold more frequent (2.0% vs. 0.0008%, n = 10, P = 0.004) relative to total viable cells. When expanded in vitro, similar growth rates up to passage 0 (P0) were seen (1.8 population doublings (PDs) per day (periosteum), 1.6 PDs per day (BM)); however, subsequently BM MSCs proliferated significantly slower by P4 (4.3 PDs per day (periosteum) vs. 9.3 PDs per day (BM), n = 9, P = 0.02). In early culture, periosteum cells were less migratory at slower speeds than BM cells. Both MSC types exhibited MSC phenotype and trilineage differentiation capacity; however, periosteum MSCs showed significantly lower (2.7-fold) adipogenic potential based on Nile red : DAPI ratios with reduced expression of adipogenesis-related transcripts PPAR-γ. Altogether, these data revealed that "clinically accessible" periosteal samples represent a consistently rich source of highly proliferative MSCs compared to donor-matched BM, which importantly show similar osteochondral capacity and lower adipogenic potential. Live cell tracking allowed determination of unique morphological and migration characteristics of periosteal MSCs that can be used for the development of novel bone graft substitutes to be preferentially repopulated by these cells.

A redox-responsive hyaluronic acid-based hydrogel for chronic wound management.

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).

Optimising proliferation and migration of mesenchymal stem cells using platelet products: A rational approach to bone regeneration.

This study investigates how mesenchymal stem cell's (MSCs) proliferation and migration abilities are influenced by various platelet products (PP). Donor-matched, clinical-, and control laboratory-standard PPs were generated and assessed based on their platelet and leukocyte concentrations. Bone marrow derived MSCs were exposed to these PP to quantify their effect on in vitro MSC proliferation and migration. An adapted colony forming unit fibroblast (CFU-F) assay was carried out on bone marrow aspirate using clinical-standard PP-loaded electrospun poly(ϵ-caprolactone) (PCL) membrane to mimic future clinical applications to contain bone defects. Clinical-standard PP had lower platelet (2.5 fold, p < 0.0001) and higher leukocyte (14.1 fold, p < 0.0001) concentrations compared to laboratory-standard PP. It induced suboptimal MSC proliferation compared to laboratory-standard PP and fetal calf serum (FCS). All PP induced significantly more MSC migration than FCS up to 24 h. The removal of leukocytes from PP had no effect on MSC proliferation or migration. The PP-loaded membranes successfully supported MSC colony formation. This study indicates that platelet concentrations in PP impact MSC proliferation more than the presence of leukocytes, whilst MSC migration in response to PP is not influenced by platelet or leukocyte numbers. Clinical-standard PP could be applied alongside manufactured membranes in the future treatment of bone reconstruction. © 2019 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:1329-1338, 2019.

Two-layer Electrospun System Enabling Wound Exudate Management and Visual Infection Response.

The spread of antimicrobial resistance calls for chronic wound management devices that can engage with the wound exudate and signal infection by prompt visual effects. Here, the manufacture of a two-layer fibrous device with independently-controlled exudate management capability and visual infection responsivity was investigated by sequential free surface electrospinning of poly(methyl methacrylate-co-methacrylic acid) (PMMA-co-MAA) and poly(acrylic acid) (PAA). By selecting wound pH as infection indicator, PMMA-co-MAA fibres were encapsulated with halochromic bromothymol blue (BTB) to trigger colour changes at infection-induced alkaline pH. Likewise, the exudate management capability was integrated via the synthesis of a thermally-crosslinked network in electrospun PAA layer. PMMA-co-MAA fibres revealed high BTB loading efficiency (>80 wt.%) and demonstrated prompt colour change and selective dye release at infected-like media (pH > 7). The synthesis of the thermally-crosslinked PAA network successfully enabled high water uptake (WU = 1291 ± 48 - 2369 ± 34 wt.%) and swelling index (SI = 272 ± 4 - 285 ± 3 a.%), in contrast to electrospun PAA controls. This dual device functionality was lost when the same building blocks were configured in a single-layer mesh of core-shell fibres, whereby significant BTB release (~70 wt.%) was measured even at acidic pH. This study therefore demonstrates how the fibrous configuration can be conveniently manipulated to trigger structure-induced functionalities critical to chronic wound management and monitoring.

Photodynamically Active Electrospun Fibers for Antibiotic-Free Infection Control.

Antimicrobial biomaterials are critical to aid in the regeneration of oral soft tissue and prevent or treat localized bacterial infections. With the rising trend in antibiotic resistance, there is a pressing clinical need for new antimicrobial chemistries and biomaterial design approaches enabling on-demand activation of antibiotic-free antimicrobial functionality following an infection that are environment-friendly, flexible and commercially viable. This study explores the feasibility of integrating a bioresorbable electrospun polymer scaffold with localized antimicrobial photodynamic therapy (aPDT) capability. To enable aPDT, we encapsulated a photosensitizer (PS) in polyester fibers in the PS inert state, so that the antibacterial function would be activated on-demand via a visible light source. Fibrous scaffolds were successfully electrospun from FDA-approved polyesters, either poly(ε-caprolactone (PCL) or poly[(rac-lactide)-co-glycolide] (PLGA), with encapsulated PS (either methylene blue (MB) or erythrosin B (ER)). These were prepared and characterized with regards to their loading efficiency (UV-vis spectroscopy), microarchitecture (SEM, porometry, and BET (Brunauer-Emmett-Teller) analysis), tensile properties, hydrolytic behavior (contact angle, dye release capability, degradability), and aPDT effect. The electrospun fibers achieved an ∼100 wt % loading efficiency of PS, which significantly increased their tensile modulus and reduced their average fiber diameter and pore size with respect to PS-free controls. In vitro, PS release varied between a burst release profile to limited release within 100 h, depending on the selected scaffold formulation, while PLGA scaffolds displayed significant macroscopic shrinkage and fiber merging, following incubation in phosphate buffered saline solution. Exposure of PS-encapsulated PCL fibers to visible light successfully led to at least a 1 log reduction in Escherichia coli viability after 60 min of light exposure, whereas PS-free electrospun controls did not inactive microbes. This study successfully demonstrates the significant potential of PS-encapsulated electrospun fibers as photodynamically active biomaterial for antibiotic-free infection control.

A UV-cured nanofibrous membrane of vinylbenzylated gelatin-poly(ɛ-caprolactone) dimethacrylate co-network by scalable free surface electrospinning.

Electrospun nanofibrous membranes of natural polymers, such as gelatin, are fundamental in the design of regenerative devices. Crosslinking of electrospun fibres from gelatin is required to prevent dissolution in water, to retain the original nanofibre morphology after immersion in water, and to improve the thermal and mechanical properties, although this is still challenging to accomplish in a controlled fashion. In this study, we have investigated the scalable manufacture and structural stability in aqueous environment of a UV-cured nanofibrous membrane fabricated by free surface electrospinning (FSES) of aqueous solutions containing vinylbenzylated gelatin and poly(ɛ-caprolactone) dimethacrylate (PCL-DMA). Vinylbenzylated gelatin was obtained via chemical functionalisation with photopolymerisable 4-vinylbenzyl chloride (4VBC) groups, so that the gelatin and PCL phase in electrospun fibres were integrated in a covalent UV-cured co-network at the molecular scale, rather than being simply physically mixed. Aqueous solutions of acetic acid (90 vol%) were employed at room temperature to dissolve gelatin-4VBC (G-4VBC) and PCL-DMA with two molar ratios between 4VBC and DMA functions, whilst viscosity, surface tension and electrical conductivity of resulting electrospinning solutions were characterised. Following successful FSES, electrospun nanofibrous samples were UV-cured using Irgacure I2959 as radical photo-initiator and 1-Heptanol as water-immiscible photo-initiator carrier, resulting in the formation of a water-insoluble, gelatin/PCL covalent co-network. Scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, differential scanning calorimetry (DSC), tensile test, as well as liquid contact angle and swelling measurements were carried out to explore the surface morphology, chemical composition, thermal and mechanical properties, wettability and water holding capacity of the nanofibrous membranes, respectively. UV-cured nanofibrous membranes did not dissolve in water and showed enhanced thermal and mechanical properties, with respect to as-spun samples, indicating the effectiveness of the photo-crosslinking reaction. In addition, UV-cured gelatin/PCL membranes displayed increased structural stability in water with respect to PCL-free samples and were highly tolerated by G292 osteosarcoma cells. These results therefore support the use of PCL-DMA as hydrophobic, biodegradable crosslinker and provide new insight on the scalable design of water-insoluble, mechanical-competent gelatin membranes for healthcare applications.

Monomer-Induced Customization of UV-Cured Atelocollagen Hydrogel Networks.

The covalent functionalization of type I atelocollagen with either 4-vinylbenzyl or methacrylamide residues is presented as a simple synthetic strategy to achieve customizable, cell-friendly UV-cured hydrogel networks with widespread clinical applicability. Molecular parameters, i.e., the type of monomer, degree of atelocollagen functionalization and UV-curing solution, have been systematically varied and their effect on gelation kinetics, swelling behavior, elastic properties, and enzymatic degradability investigated. UV-cured hydrogel networks deriving from atelocollagen precursors functionalized with equivalent molar content of 4-vinylbenzyl (F 4VBC = 18 ± 1 mol.%) and methacrylamide (F MA = 19 ± 2 mol.%) adducts proved to display remarkably-different swelling ratio (SR = 1963 ± 58-5202 ± 401 wt.%), storage modulus (G' = 17 ± 3-390 ± 99 Pa) and collagenase resistance (µ rel = 18 ± 5-56 ± 5 wt.%), similarly to the case of UV-cured hydrogel networks obtained with the same type of methacrylamide adduct, but varied degree of functionalization (F MA = 19 ± 2 - 88 ± 1 mol.%). UV-induced network formation of 4VBC-functionalized atelocollagen molecules yielded hydrogels with increased stiffness and enzymatic stability, attributed to the molecular rigidity of resulting aromatized crosslinking segment, whilst no toxic response was observed with osteosarcoma G292 cells. Although to a lesser extent, the pH of the UV-curing solution also proved to affect macroscopic hydrogel properties, likely due to the altered organization of atelocollagen molecules during network formation. By leveraging the knowledge gained with classic synthetic networks, this study highlights how the type of monomer can be conveniently exploited to realize customizable atelocollagen hydrogels for personalized medicine, whereby the structure-property relationships can be controlled to meet the requirements of unmet clinical applications.

A hydroxamic acid-methacrylated collagen conjugate for the modulation of inflammation-related MMP upregulation.

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.