Manufacture and characterisation of EmDerm—novel hierarchically structured bio-active scaffolds for tissue regeneration

Manufacture and characterisation of EmDerm—novel hierarchically structured bio-active scaffolds... There are significant challenges for using emulsion templating as a method of manufacturing macro-porous protein scaffolds. Issues include protein denaturation by adsorption at hydrophobic interfaces, emulsion instability, oil droplet and surfactant removal after protein gelation, and compatible cross-linking methods. We investigated an oil-in-water macro-emulsion stabilised with a surfactant blend, as a template for manufacturing protein-based nano-structured bio-intelligent scaffolds (EmDerm) with tuneable micro-scale porosity for tissue regeneration. Prototype EmDerm scaffolds were made using either collagen, through thermal gelation, fibrin, through enzymatic coagulation or collagen-fibrin composite. Pore size was controlled via surfactant-to-oil phase ratio. Scaffolds were crosslink-stabilised with EDC/NHS for varying durations. Scaffold micro-architecture and porosity were characterised with SEM, and mechanical properties by tensiometry. Hydrolytic and proteolytic degradation profiles were quantified by mass decrease over time. Human dermal fibroblasts, endothelial cells and bone marrow derived mesenchymal stem cells were used to investigate cytotoxicity and cell proliferation within each scaffold. EmDerm scaffolds showed nano-scale based hierarchical structures, with mean pore diameters ranging from 40–100 microns. The Young’s modulus range was 1.1–2.9 MPa, and ultimate tensile strength was 4–16 MPa. Degradation rate was related to cross-linking duration. Each EmDerm scaffold supported excellent cell ingress and proliferation compared to the reference materials Integra™ and Matriderm™. Emulsion templating is a novel rapid method of fabricating nano-structured fibrous protein scaffolds with micro-scale pore dimensions. These scaffolds hold promising clinical potential for regeneration of the dermis and other soft tissues, e.g., for burns or chronic wound therapies. 1 Introduction One method for achieving controlled porosity in protein hydrogels is controlled freezing and lyophilisation, in which Hierarchical interconnected porous architecture and nano- pores are formed by material exclusion from the ice crystal scale structure are fundamental requirements of three- porogen. This typically results in dense lamellar structured dimensional protein-based bio-active scaffolds, which are material, largely devoid of nano-scale structure. This is essential for the functions of cell conductivity, nutrient shown by many of the current scaffolds, notably acellular perfusion, angiogenesis and vasculogenic differentiation collagen scaffolds [4]. This method is still in widespread [1–3]. However, there is a need for effective, controllable, use despite the long processing times required to make such scalable methods of producing such nanostructured scaffolds [5]. Cellularisation and vascularisation into such scaffolds. materials is relatively slow [6]. Foam formation by aeration is another methodology [7–10], but also has some limita- tions, due to a large exposed air interface for protein denaturation, intrinsic bubble instability and foam drainage * Julian F. Dye Julian.dye@eng.ox.ac.uk during gelation and cross-linking, which cause difficulty in achieving a biologically acceptable degree of homogeneity. Tissue Engineering Group, Institute of Biomedical Engineering, Much recent attention has been focussed on the powerful Department of Engineering Science, University of Oxford, and highly controllable bottom-up manufacture methodol- ORCRB, Roosevelt Drive, Headington, Oxford OX3 7DQ, UK ogies [3, 11]. Electrospinning is an established method of Department of Plastic Surgery, Oxford University Hospitals NHS forming micro and nano-scale fibre meshes, but is not Foundation Trust, West Wing, John Radcliffe Hospital, Headington, Oxford OX9 9DU, UK amenable to manufacturing structures at the thickness and 1234567890();,: 1234567890();,: 79 Page 2 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:79 consistency for the commercial scale-up required for mar- issues required to establish a feasible manufacture process. keting three-dimensional scaffolds. While 3D-printing and A biocompatible emulsion has been established in our lab rapid-prototyping are emerging technologies which are able using non-ionic surfactants to control emulsion droplet size, to create soft scaffold structures, the concept of building prevent denaturation of proteins in the aqueous phase of macroscale structure from a nano-scaled filament at the emulsion, and allow successful formation of protein scaf- scale required for commercial manufacture, remains chal- folds. Almost all commercially available skin substitutes are lenging. Therefore, new methods of controllable rapid and made of collagen or collagen composites [20] and fibrin is versatile manufacture of nano-structured regenerative bio- also widely used as an extracellular matrix [21]. Therefore, materials would represent a significant advance in health- we have used these proteins as scaffold materials, to eval- care technology. uate the influence of protein type on biocompatibility of Emulsion templating, a form of ‘polymerisation of high emulsion templated dermal scaffolds (EmDerm). Their internal phase emulsion’ systems (polyHIPEs), is a well- efficacy in supporting cell proliferation and migration are established method of manufacturing nano-porous polymer compared to commercially the available scaffolds Matri- TM TM membranes e.g., for water purification films, catalytic derm and Integra . panels, controlled release storage and tissue regeneration [12]. Its potential benefits as a method of manufacturing porous scaffolds for tissue engineering are the ease of 2 Methods control of porosity by controlling emulsion droplets size, relatively rapidity structure formation in bulk, and cost- 2.1 Materials effectiveness. Its use with biological polymers has so far been limited to polysaccharides [13] and the denatured Reagents were obtained from Sigma Aldrich, Poole, UK polypeptide, gelatin [14] and not with native proteins such unless otherwise stated. Proteins used for scaffolds were as collagen and fibrin. type I rat tail collagen 5 mg/ml in acetic acid (First Link, There are several major limitations to emulsion tem- Wolverhampton, UK), bovine fibrin and bovine thrombin. plating of protein structures: protein denaturation (by 2-(N-morpholino) ethanesulfonic acid (MES) biochemical adsorption at hydrophobic interfaces and by surfactant grade (Fisher Scientific, Loughborough, UK) and sodium agents), emulsion instability, complexity of introducing chloride were used for the buffer. Decane, Tween 20 and stabilisation methods, and elution of the oil-phase and Span 20 were used for emulsions. 1-ethyl-3-(-3-dimethyla- surfactant [15]. Proteins typically adsorb to and denature at minopropyl) carbodiimide hydrochloride (EDC) and sulfo- oil-water interfaces [16–18] so the creation of very large N-hydroxysuccinimide (NHS), pure ethanol and iso- surface area to volume ratio interfaces (in the order of propanol were used for cross-linking and oil elution. The 500 cm /ml), and short diffusion distances (in the range excipients used were polyvinyl alcohol (PVA) (99% 1–10 μm) necessarily presents a system that will favour any hydrolysed, MW 89,000 to 98,000), polyethylene glycol denaturation process. Moreover, the use of surfactants (PEG) (MW 6000), Pluronic F-68 (P68) and mannitol (M). needed to form a stable emulsion will also be likely to 0.25% Trypsin-EDTA solution for cell culture was used for adsorb onto and denature proteins [19]. Additionally, any stability testing and the Cell Counting Kit (CCK-8) assay process of emulsion ripening or maturation, will counteract was used to measure cell proliferation. the desired stability of the emulsion droplets as a template. Further issues of particular consideration for protein scaf- 2.2 Manufacturing of scaffolds folds concern post-formation stabilisation, for example by a compatible chemical cross-linking method, and elution of Macro-emulsion mixtures comprising of decane, Span the oil phase without disruption of the formed scaffold. For 20/Tween 20 at a calculated HLB of 13 and an aqueous example, phase separation involves release of significant buffer (25 mM MES, 150 mM NaCl pH 7.4) were mixed in energy, which is capable of disrupting the formed material a 60 ml syringe with the tip removed, for 15 seconds at as the oil phase separates. All these considerations deter- 1000 rpm using a high-speed mixer (Ceframo BDC6015, mine the feasibility and resultant biocompatibility of the Ontario, Can). Emulsions with 0.1, 0.3, 0.5 and 0.7% sur- scaffold produced. factant concentration were prepared. To manufacture scaf- The underlying hypothesis we investigate is that a sur- folds from each protein type, each of the emulsions was factant hydrophilic boundary layer that prevents protein added to each protein solution designated as follows: col- adsorption and denaturation at the emulsion interface will lagen (Col), fibrinogen plus thrombin (Fbn) or a mixture of enable protein-based three-dimensional hydrogels to be collagen and fibrin solution at a 1:1 volume ratio (ColFbn). formed into porous scaffold structures with minimal dena- The gelling solution of type-I collagen was used at turation. We investigate this by addressing each of the 4.5 mg/ml, fibrinogen was prepared at 20 mg/ml and Journal of Materials Science: Materials in Medicine (2018) 29:79 Page 3 of 12 79 thrombin at 10 units/ml in in 25 mM MES/150 mM NaCl 2.6 Cell culture (pH 7.4) buffer. Each emulsion-scaffold was left to gel at 37 °C for 30 min and then cross-linked with EDC:NHS Human dermal fibroblasts derived from neonatal foreskin (21.9:8.68 mM, 5:2 molar ratio) in 80% ethanol [22]. The (HDF) were obtained from Invitrogen, UK. They were scaffolds were then washed in 80% isopropanol, then deio- cultured in standard Dulbecco’s Modified Eagle Medium nized water three times for 15 minutes. At this point scaffolds (DMEM) media (Gibco, ThermoFisher Scientific, UK) with were further incubated with a 1% w/v excipient solution (e.g. 20% foetal bovine serum and 1% penicillin/streptomycin. P68, PVA, PEG or M). Collagen scaffolds were freeze-dried Only cells from Passage 2 to 9 were used in the experiment. at −30 °C and fibrin scaffolds were freeze-dried at −40 °C. Human dermal microvascular endothelial cells derived from The possible effects of the different excipients used, on neonatal foreskin that have been hTERT immortalized functional parameters and scaffold morphology, were (HDE), were obtained from ATCC, UK. They were cultured compared using scaffolds made at large pore size with 0.1% using Vascular Cell Basal Medium with Microvascular surfactant concentration. The effect of surfactant con- Endothelial Cell Growth Kit-VEGF supplement containing: centration was compared between scaffolds made using F68 rhVEGF (5 ng/ml), rhEGF (5 ng/ml), rhFGF basic (5 ng/ml), as the excipient. rhIGF-1 (15 ng/ml), L-glutamine (10 mM), heparin sulfate (0.75 Units/ml), hydrocortisone (1 µg/ml), ascorbic acid 2.3 Pore size of scaffolds (50 µg/ml) and foetal bovine serum (5%). Human bone marrow mesenchymal stem cells transfected with GFP Scaffolds were cut using a scalpel so that the cross-section (MCS) were obtained courtesy of Dr James Li (Hong Kong of each scaffold was lying horizontally on the carbon tape University) and cultured in DMEM media. mounted on the aluminium stubs for scanning electron microscopy (SEM) imaging. Three images were taken per 2.7 Cytocompatibility of scaffolds scaffold and the diameters of 20 random pores were aver- aged out to calculate estimated mean pore size of each Scaffolds were cultured with each cell type to investigate scaffold. Scaffold morphology (e.g. fibrous or smooth) was cytocompatibility. Each scaffold was cut into 6 mm discs also noted. using a punch biopsy and washed with PBS three times before incubating overnight in media. On the following day, 2.4 Mechanical properties of scaffold each scaffold was seeded with 5000 cells/well and left overnight to allow cells to adhere. On the next day, the Scaffolds that were cross-linked for an hour, with different seeded scaffolds were transferred into a new well plate and excipients and surfactant concentrations, were tested for allowed to incubate for up to 14 days. Media was changed mechanical properties (Instron 5582 UTM). Each scaffold on alternate days. On days 3, 7 and 14, cell growth in each was cut into strips measuring 2 mm × 40 mm × T where T is scaffold was measured with the CCK-8 assay (2-(2-meth- the variable thickness of each scaffold. Each end of the oxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- strips were anchored into the jaws of the Deben tensile 2H-tetrazolium, monosodium salt (WST-8) plus 1-methoxy testing machine and stretched until the scaffold broke into phenazine methosulfate (PMS) reagent (WST8/PMS)) two pieces. Young’s modulus and ultimate tensile strength (Sigma, UK). Briefly, 10 μl of WST8/PMS reagent was (UTS) were calculated for each scaffold using stress-strain added to 100 μl of fresh media in each well and incubated curves obtained. Each test was performed in triplicate. for 4h at 37°C. 50 μl of the resulting solution was pipetted into a new plate for colorimetric assay of the reduced for- 2.5 Degradation profile of scaffold mazan product at 450 nm. Each test was done in triplicate. On day 14, the scaffolds were fixed in normal buffered Scaffolds were cut into 6 mm discs using a punch biopsy formalin for microscopy. and incubated in PBS for 7, 14, 21 28 and 35 days. The scaffolds were freeze-dried after each time point and dry 2.8 Wide-field imaging of seeded scaffolds mass of each scaffold was recorded. We introduced an enzymatic proteolysis assay to compare the degradation Each of the seeded scaffolds was stained using fluorescent profiles of each scaffold, over a relatively short period. antibodies for microscopy. Scaffolds seeded with human Scaffold discs were incubated in 0.25% (1×) Trypsin dermal fibroblasts were stained with 1:6 phalloidin (Alexa solution in PBS, which was changed daily for up to 7 days. Fluor 488, Thermo Fisher) and scaffolds seeded with Individual discs were freeze-dried at the following time human dermal endothelial cells were stained with 1:250 points: Days 1, 3, 5 and 7 and dry mass of each scaffold was mouse anti-human CD31 (Dako) and 1:1000 rabbit anti- measured. Each test was performed in triplicate. human vWF (Dako). Secondary staining was done using 79 Page 4 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:79 Fig. 1 Structure of EmDerm scaffolds formed with 0.1% surfactant surfactant concentration, from 0.1 to 0.7%, in the mixed phase tem- from: Collagen (a), Collagen-Fibrin (b) or Fibrin (c). SEM micro- plating system on the distribution of pore diameters (mean ± SD) of graphs of representative fields of scaffolds show meso-scale structure each type of EmDerm scaffold, collagen (d), collagen-fibrin (e) and and pore size (1000×) and nano-scale structure (2000×). The effect of fibrin scaffolds (f) Journal of Materials Science: Materials in Medicine (2018) 29:79 Page 5 of 12 79 Table 1 Mechanical strength characterisation of scaffolds (mean ± goat anti-mouse (Alexa Fluor 488, Thermo Fisher) and SD, n = 3) goat anti-rabbit (Alexa Fluor 568, Thermo Fischer). Scaf- Scaffold Young’s modulus (MPa) Ultimate tensile strength folds seeded with mesenchymal stem cells were not (MPa) stained, since they expressed GFP. Z-stacks were taken to visualize cell migration through the scaffold by wide-field Collagen scaffolds imaging at 20× magnification. Images were deconvoluted 0.1C 1.95 ± 0.49 8.28 ± 1.62 using AutoQuant X3 (Media Cybernetics) and visualised 0.3C 1.45 ± 0.76 7.87 ± 1.45 using Bitplane (Imaris software, Version 9.1). 0.5C 1.71 ± 0.21 7.99 ± 1.02 0.7C 2.14 ± 0.34 8.85 ± 0.97 2.9 Statistical analysis C-M 1.27 ± 0.48 9.65 ± 2.81 C-P68 1.25 ± 0.74 8.26 ± 1.78 Data between different scaffolds and time-points were ana- C-PEG 1.70 ± 0.51 8.87 ± 1.24 lysed using one-way analysis of variance (ANOVA) with Collagen-Fibrin scaffolds Tukey’s post-hoc multiple comparisons tests to evaluate 0.1CF 2.03 ± 0.81 12.82 ± 3.45 statistical significance. All statistical analysis was performed 0.3CF 2.43 ± 0.45 14.07 ± 2.67 using GraphPad Prism Version 4.0 for Windows. A p-value 0.5CF 2.55 ± 0.98 15.15 ± 3.92 of less than 0.05 is considered to be statistically significant. 0.7CF 2.89 ± 0.72 16.47 ± 5.39 Where applicable, * denotes a p–value of <0.05, ** denotes CF-M 1.89 ± 0.58 12.55 ± 3.89 ap–value of <0.01 and *** denotes a p-value of <0.001. CF-P68 1.46 ± 0.33 13.25 ± 2.66 CF-PEG 1.74 ± 0.76 11.53 ± 1.96 Fibrin scaffolds 3 Results 0.1F 1.52 ± 0.38 4.98 ± 0.96 3.1 Scaffold structure and porosity 0.3F 1.16 ± 0.29 4.81 ± 0.72 0.5F 1.18 ± 0.41 4.77 ± 0.42 The use of decane oil-in-water (o/w) emulsions was suc- 0.7F 1.12 ± 0.29 4.62 ± 0.58 cessful in forming scaffolds from each type of scaffold F-M 1.26 ± 0.35 4.25 ± 0.63 protein, neutralised type I collagen acetic acid extract, F-P68 1.48 ± 0.32 4.59 ± 0.22 which gels spontaneously on warming from a 4 °C solution F-PEG 1.37 ± 0.21 5.13 ± 0.51 to 37 °C; and fibrinogen, enzymatically coagulated with F-PVA 2.01 ± 0.11 4.43 ± 0.49 thrombin 37°C. Pore interconnectivity of scaffolds was obtained by controlling the oil:aqueous phase ratio to ≥0.5. Importantly, the formed scaffolds demonstrate a nano-scale approximately 1–2 MPa and UTS of 7–9 MPa. Fbn scaf- fibrous architecture, in addition to the micro-scale pores. folds had a Young’s modulus of around 1–2 MPa and UTS The use of an excipient was needed to preserve this struc- of 4–5 MPa. Change in porosity of the scaffolds and exci- ture on lyophilisation and reduce shrinkage. Figure 1a-c pients used did not have a significant impact on mechanical shows representative SEM images of each scaffold type. properties of the scaffolds. Pore size of the scaffolds inversely correlated with the surfactant concentration used to create the emulsion (Fig. 3.3 Hydrolytic degradation of scaffolds 1d-f). Previous work showed there was an inverse rela- tionship between surfactant concentration and emulsion Scaffold degradation by hydrolysis was determined by droplet diameter (not shown). For scaffolds made from measuring residual dry weight of scaffolds soaked in PBS emulsion with 0.1% surfactant, a mean pore size of around for extended periods (Fig. 2a-c). Collagen scaffolds showed 100 μm was obtained, and this decreased to approximately a large drop in dry mass of 40–50% over the first week. This 40 μm with 0.7% surfactant. is largely due to the dissolution of excipients during the initial hydration of the scaffold. At the end of 5 weeks, all 3.2 Mechanical properties scaffolds retained about 40–60% of their dry mass. Although Col scaffold stability was achieved at the shortest The measurement of the tensile properties of each EmDerm cross-linking time (Fig. 2c), ColFbn and Fbn scaffolds scaffold type (Table 1) showed that the ColFbn scaffolds which were cross-linked for a longer duration degraded had the highest Young’s modulus and UTS, approximately slower compared to scaffolds which were cross-linked for 1–2 MPa and 12–16 MPa respectively. This is followed by 30 min (Fig. 2b-c), although this was not statistically sig- Col scaffolds, which had a Young’s modulus of nificant in this experiment. Notably, Fbn scaffolds which 79 Page 6 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:79 Fig. 2 Hydrolytic and enzymatic degradation by mass over a period of EDC/NHS for different durations, after incubation in PBS (up to time. EmDerm scaffolds formed from: Collagen (a), Collagen-Fibrin 5 weeks) or Trypsin (up to 7 days). Data show % reduction of dry (b) or Fibrin (c) with 0.1% surfactant emulsions, cross-linked with weight (mean ± SD, n = 3) were cross-linked for only 30 min degraded completely by 3.5 Cytocompatibility of scaffolds 5 weeks. The assessment of the biocompatibility of EmDerm scaf- 3.4 Enzymatic degradation of scaffold folds was investigated by measuring the proliferation three cell types central to skin reconstruction, HDF, HDE and Col and ColFbn scaffolds remained incompletely degraded MSC. Net proliferation of each cell type occurred over by Day 7 when incubated in trypsin solution (Fig. 2a, b). 14 days in each scaffold material (Figs. 3–5). Importantly, All Fbn scaffolds that were cross-linked for less than 2 h there was no significant effect of varying the excipient used, were completely degraded within 7 days: Those that were compared to the P68 excipient (Fig. 3). However, it is cross-linked for 0.5 h were completely degraded by day 3, notable that HDE and MSC proliferation in Col scaffold followed by those cross-linked for 1 h, on Day 5 and those made without an excipient (C-GEL) was significantly lower cross-linked for 1.5 h by day 7 (Fig. 2c). As with the than Col scaffolds with excipient (C-P68 and C-M) (Fig. hydrolytic degradation, the mass of each scaffold decreased 3a). Also, HDF showed lower proliferation in ColFbn significantly over the first day. Dissolution of excipients is scaffolds with PEG (CF-PEG, Fig. 3b) and F-M (Fig. 3c) likely to contribute to the initial drop of scaffold dry mass. scaffolds. Journal of Materials Science: Materials in Medicine (2018) 29:79 Page 7 of 12 79 Fig. 3 Proliferation of HDF, HDE & MSC in EmDerm scaffolds of described in the methods. Scaffolds were washed, equilibrated and collagen (a), Collagen-Fibrin (b) or Fibrin (c) with 0.1% surfactant, cultured with each cell type, and proliferation was measured by WST- prepared with different excipients (PVA, M, P68, PEG) or nil, as 8/PMS reduction on days 3, 7 and 14 (data are mean ± SD, n = 3) While proliferation was marked in scaffolds with mesenchymal cell proliferation, both effects being statisti- high porosity, there was a general trend for proliferation cally significant (Fig. 5). to be progressively lower with decreased porosity (Fig. 4). This effect was most pronounced for HDF in 3.7 Wide-field microscope imaging of seeded Col scaffolds at the lowest porosity, corresponding to scaffolds 0.5 and 0.7% surfactant mix in the emulsion template (Fig. 4a), and in ColFbn scaffolds at the lowest porosity Based on the Z-stacks obtained, each cell type was found to (Fig. 4b). infiltrate into the scaffold uniformly over the XY plane and Z plane (Fig. 6). Due to limitations in light penetration, cells 3.6 Comparison with commercial scaffolds were only imaged up to a depth of about 100 microns. In particular, cell infiltration into Col scaffolds was observed Significantly greater proliferation of each cell type occurred to a similar extent as for fibrin-containing scaffolds, sug- in each of the high porosity EmDerm scaffolds from 0.1% gestive of effect of the preserved fibre nanostructure in these surfactant than the commercial comparator scaffolds, scaffolds. Interestingly, the endothelial cells seemed to form Matriderm and Integra (Fig. 5). It is notable that the Fbn ring-like structures when seeded onto EmDerm scaffolds, scaffold 0.1F-P68 supported greatest HDE proliferation, most notably within ColFbn and Fbn (arrows in Fig. 6, while the Col scaffold 0.1C-P68 promoted the better HDE panels). It is also notable that HDE ingress into the 79 Page 8 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:79 Fig. 4 Effect of porosity on in EmDerm collagen (a), collagen-fibrin (b) and fibrin (c) scaffolds achieved by varying surfactant concentration from 0.1 to 0.7% in primary manufacture step on proliferation of cell types (HDF, HDE and MSC) as in Fig. 3 (mean ± SD, n = 3) EmDerm Col scaffolds, and although show less cytoskeletal linking (data not shown). The resultant EmDerm scaffolds spreading, demonstrate some association into annular have consistent mechanical properties, are stable in phy- structures. By contrast, the stromal cell types adopt an siological solution, have good biocompatibility and cell elongated spindle-like morphology when seeded on to conductivity, and support cell proliferation. Effective EmDerm scaffolds, more pronounced with fibroblasts than ingress and proliferation of the main cell types required for mesenchymal stem cells (Fig. 6). dermal reconstruction, HDF, HDE & MSC are shown. Collectively, this suggests that the EmDerm have favour- able characteristics as dermal templates for skin regenera- 4 Discussion tion and wound healing. The characterisation and basic properties of these o/w In this paper, we demonstrate that an o/w macro-emulsion emulsions have been determined in our laboratory. Struc- system can be used successfully as a template for fabricat- turally, there was no evidence of protein denaturation. ing three-dimensional scaffolds with hierarchical porosity When used with acidic extracted type I collagen, which whilst preserving the intrinsic nano-scale protein hydrogel exhibits spontaneous gelation in warming after pH neu- structure. The method enables complex hierarchical struc- tralisation, the emulsion does not prevent gelation, and the ture from the nano-scale to be achieved in a relatively rapid nanostructure fibrils indicate preservation of the intrinsic process. Elution of the oil phase by washing can be structure of the hydrogel. Furthermore, the successful use of achieved with several alcohol-based solvents, after cross- emulsion system for fibrin, demonstrates that fibrinogen/ Journal of Materials Science: Materials in Medicine (2018) 29:79 Page 9 of 12 79 Fig. 5 Comparison of the proliferation of HDF, HDE and MSC scaffolds Matriderm™ and Integra™. Cell proliferation was measured between the three different types of EmDerm scaffolds made with as in Fig. 3 (mean ± SD, n = 3) 0.1% surfactant and P68 excipient, and the commercial reference thrombin enzymatic coagulation is able to proceed in the be sufficient to maintain integrity of the scaffolds post- mixed phased system, and the resultant fibrin fibril forma- implantation. A shorter duration significantly decreased tion is indicative of the native structural self-assembly hydrolytic and proteolytic stability of fibrin-based scaffolds, behaviour of fibrinogen after thrombin cleavage of the although the greater stabilisation achieved for collagen fibrinopeptides. scaffolds indicates greater reactivity to the EDC/NHS Scaffold porosity is a principle physical determinant of reagent. functionality of scaffolds [4, 23, 24]. Control of pore size Interestingly, there was a relationship between cell pro- was achieved by varying the emulsion droplet size through liferation and porosity, although the surfactant concentra- surfactant concentration in the primary manufacture step, at tion used in the scaffolds’ primary manufacture step is also a constant ratio of the aqueous protein phase to the tem- a variable. The use of concentrations over 0.5% in collagen plating oil phase. A direct relationship was found between scaffolds, and 0.7% in collagen-fibrin scaffolds, was asso- the surfactant concentration in the primary manufacture ciated with marked inhibition of HDF proliferation, possi- step, which determines the mixed phase template droplet bly suggesting some persistence of surfactant residue during diameter, and resultant lyophilised scaffold pore diameter the scaffold processing. However, the other cell types were by SEM. This applied over the range of mean pore dia- affected to a lesser extent, which suggests that if surfactant meters from around 40 to 100 μm. This suggests that the residues persist, the level is close to the threshold to cause porosity EmDerm scaffolds can be readily tuned according cytotoxicity. Additionally, the droplet diameters of emul- to needs. A mean pore size between 80 and 100 microns as sions with higher surfactant concentration are much smaller a suitable target is consistent with optimal migration and less ideal for promoting vascularisation. This observa- through scaffolds with this pore size range [25]. tion may therefore be useful in establishing a threshold for The mechanical properties of EmDerm scaffolds suggest the safe concentration of the current surfactant in the current that they can be strong enough to be handled physically and manufacture process, and confirms the need to evaluate do not tear easily. This is imperative as it allows the scaf- cytotoxicity. Possibly a more stringent wash process may be folds to be positioned and sutured during surgery. With Col required should the higher concentrations be needed. scaffolds there was an inverse relationship between the Despite this consideration, the overwhelming evidence of mechanical strength (Young’s modulus and UTS) and this work is that the EmDerm scaffolds are essentially porosity, which suggests that some differences in fibril cytocompatible. organisation. With Fbn and ColFbn scaffolds, the pore size Another aim of this method of manufacture is to create did not have a marked effect on these parameters scaffolds with nano-structured architecture, which current Post-implantation stability of scaffolds is an important commercial scaffolds lack. The nano-fibrous architecture aspect of their function, and resorption of tissue regenera- can provide cells with oriented cell adhesion signals and tion scaffolds allows for new extracellular matrix deposition promote migration of cells, as there is an aligned fibre [25]. Here, we introduced an assay of accelerated proteo- matrix for cells to move along [26]. In this regard, the effect lytic degradation in order to compare the kinetic profiles of on excipients on preserving the collagen nanostructure of degradation over a relatively short assay period. The cross- EmDerm scaffolds is notable. By contrast, scaffolds without linking duration had a direct effect on scaffold stability, excipients did not have nano-fibrous walls, rather they were demonstrating that this can be controlled. Based on our smooth and featureless when imaged under SEM. These findings, an incubation time of 1 h for cross-linking should findings suggest firstly that collagen is able to undergo 79 Page 10 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:79 Fig. 6 Morphology of HDF, HDE and MSC in the three different types seeded scaffolds, formation of annular structures is apparent (arrows). of EmDerm scaffolds made with 0.1% surfactant and P68 excipient, Scale bars = 50 μm collagen (a), collagen-fibrin (b) and fibrin (c) scaffolds. In HDE- structural reorganisation under freeze-drying involving of interconnected porosity [6, 28]. Thus, the present results fusion of the fibrillar hydrogel structure into microscale suggest that creating a nano-structured form of collagen can lamellae; secondly that excipient interaction with the pro- significantly increase the cell migration into the scaffold. tein matrix is a stabilising factor that prevents nano- This could translate to accelerated integration of collagen structure fusion. scaffolds in wound healing settings. The results of the biocompatibility studies with cells This paper demonstrates that the emulsion templating relevant to dermal reconstruction are particularly sig- technique is suitable for creating of a range of nano- nificant. A multi-parameter approach has recently been structured functional EmDerm scaffolds. The difference in validated that correlates in vitro cell responses to the clinical effect of each scaffold material on cell proliferation and properties of dermal biomaterials [27]. The generally morphology suggests that composition may have some cell greater cell proliferation and migration in the nano- type specific functional benefits. In particular, the greater structured EmDerm scaffold may be due to increase in proliferation of endothelial cells in collagen-fibrin and fibrin surface area available for cells to adhere to and migrate on, scaffolds than collagen scaffolds is associated with the as well as greater permeability to nutrients and oxygen formation of annular nascent vascular structures. This throughout the scaffold [25]. EmDerm scaffolds demon- behaviour, associated with the endothelial interaction with strate excellent cell penetrance, or conductivity, for different organised fibrin structures, is consistent with other angio- cell types. The present results contrast with previous studies genic properties of fibrin [21, 29–31]. However, the which have shown that collagen as a scaffold material in the migratory and morphological response of endothelial cells TM TM commercial scaffolds Integra and Matriderm , and also to the nano-structured collagen scaffolds suggests that the TM in decellularised dermal products Alloderm , Xeno- nano-structure increases its angiogenic potential. Further TM TM derm and Permacol and does not support complete cell examination of the cellular morphological and cell- conductivity, even though the structures have a high degree communication responses will extend our understanding Journal of Materials Science: Materials in Medicine (2018) 29:79 Page 11 of 12 79 of the role of scaffold nanostructure in influencing cellular 7. Barbetta A, Gumiero A, Pecci R, Bedini R, Dentini M. Gas-in- liquid foam templating as a method for the production of highly behaviour [27] and particularly angiogenesis and porous scaffolds. 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Production and character- Science, Technology and Research (A*STAR) and the Chinese ization of multicomponent films based on polysaccharides, gelatin Regenerative Medicine Institute (CRMI) for funding this project. and lipids: Effect of surfactants addition. International Congress on Engineering and Food; 2011 22-26/5/2011; Cosmosware, Compliance with ethical standards Athens, Greece: Elsevier Procedia. 15. Dalgleish DG. Adsorption of protein and the stability of emul- Conflict of interest The authors declare that they have no conflict of sions. Trends Food Sci Technol. 1997;8:1–6. interest. 16. Chen J, Dickinson E, Iveson G. Interfacial interactions, competi- tive adsorption and emulsion stability. Food Struct. 1993;12:1. Open Access This article is distributed under the terms of the Creative 17. Dickinson E. Proteins at interfaces and in emulsions stability, Commons Attribution 4.0 International License (http://crea rheology and interactions. 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Manufacture and characterisation of EmDerm—novel hierarchically structured bio-active scaffolds for tissue regeneration

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Springer Journals
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Copyright © 2018 by The Author(s)
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Materials Science; Biomaterials; Biomedical Engineering; Regenerative Medicine/Tissue Engineering; Polymer Sciences; Ceramics, Glass, Composites, Natural Materials; Surfaces and Interfaces, Thin Films
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0957-4530
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1573-4838
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10.1007/s10856-018-6060-6
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Abstract

There are significant challenges for using emulsion templating as a method of manufacturing macro-porous protein scaffolds. Issues include protein denaturation by adsorption at hydrophobic interfaces, emulsion instability, oil droplet and surfactant removal after protein gelation, and compatible cross-linking methods. We investigated an oil-in-water macro-emulsion stabilised with a surfactant blend, as a template for manufacturing protein-based nano-structured bio-intelligent scaffolds (EmDerm) with tuneable micro-scale porosity for tissue regeneration. Prototype EmDerm scaffolds were made using either collagen, through thermal gelation, fibrin, through enzymatic coagulation or collagen-fibrin composite. Pore size was controlled via surfactant-to-oil phase ratio. Scaffolds were crosslink-stabilised with EDC/NHS for varying durations. Scaffold micro-architecture and porosity were characterised with SEM, and mechanical properties by tensiometry. Hydrolytic and proteolytic degradation profiles were quantified by mass decrease over time. Human dermal fibroblasts, endothelial cells and bone marrow derived mesenchymal stem cells were used to investigate cytotoxicity and cell proliferation within each scaffold. EmDerm scaffolds showed nano-scale based hierarchical structures, with mean pore diameters ranging from 40–100 microns. The Young’s modulus range was 1.1–2.9 MPa, and ultimate tensile strength was 4–16 MPa. Degradation rate was related to cross-linking duration. Each EmDerm scaffold supported excellent cell ingress and proliferation compared to the reference materials Integra™ and Matriderm™. Emulsion templating is a novel rapid method of fabricating nano-structured fibrous protein scaffolds with micro-scale pore dimensions. These scaffolds hold promising clinical potential for regeneration of the dermis and other soft tissues, e.g., for burns or chronic wound therapies. 1 Introduction One method for achieving controlled porosity in protein hydrogels is controlled freezing and lyophilisation, in which Hierarchical interconnected porous architecture and nano- pores are formed by material exclusion from the ice crystal scale structure are fundamental requirements of three- porogen. This typically results in dense lamellar structured dimensional protein-based bio-active scaffolds, which are material, largely devoid of nano-scale structure. This is essential for the functions of cell conductivity, nutrient shown by many of the current scaffolds, notably acellular perfusion, angiogenesis and vasculogenic differentiation collagen scaffolds [4]. This method is still in widespread [1–3]. However, there is a need for effective, controllable, use despite the long processing times required to make such scalable methods of producing such nanostructured scaffolds [5]. Cellularisation and vascularisation into such scaffolds. materials is relatively slow [6]. Foam formation by aeration is another methodology [7–10], but also has some limita- tions, due to a large exposed air interface for protein denaturation, intrinsic bubble instability and foam drainage * Julian F. Dye Julian.dye@eng.ox.ac.uk during gelation and cross-linking, which cause difficulty in achieving a biologically acceptable degree of homogeneity. Tissue Engineering Group, Institute of Biomedical Engineering, Much recent attention has been focussed on the powerful Department of Engineering Science, University of Oxford, and highly controllable bottom-up manufacture methodol- ORCRB, Roosevelt Drive, Headington, Oxford OX3 7DQ, UK ogies [3, 11]. Electrospinning is an established method of Department of Plastic Surgery, Oxford University Hospitals NHS forming micro and nano-scale fibre meshes, but is not Foundation Trust, West Wing, John Radcliffe Hospital, Headington, Oxford OX9 9DU, UK amenable to manufacturing structures at the thickness and 1234567890();,: 1234567890();,: 79 Page 2 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:79 consistency for the commercial scale-up required for mar- issues required to establish a feasible manufacture process. keting three-dimensional scaffolds. While 3D-printing and A biocompatible emulsion has been established in our lab rapid-prototyping are emerging technologies which are able using non-ionic surfactants to control emulsion droplet size, to create soft scaffold structures, the concept of building prevent denaturation of proteins in the aqueous phase of macroscale structure from a nano-scaled filament at the emulsion, and allow successful formation of protein scaf- scale required for commercial manufacture, remains chal- folds. Almost all commercially available skin substitutes are lenging. Therefore, new methods of controllable rapid and made of collagen or collagen composites [20] and fibrin is versatile manufacture of nano-structured regenerative bio- also widely used as an extracellular matrix [21]. Therefore, materials would represent a significant advance in health- we have used these proteins as scaffold materials, to eval- care technology. uate the influence of protein type on biocompatibility of Emulsion templating, a form of ‘polymerisation of high emulsion templated dermal scaffolds (EmDerm). Their internal phase emulsion’ systems (polyHIPEs), is a well- efficacy in supporting cell proliferation and migration are established method of manufacturing nano-porous polymer compared to commercially the available scaffolds Matri- TM TM membranes e.g., for water purification films, catalytic derm and Integra . panels, controlled release storage and tissue regeneration [12]. Its potential benefits as a method of manufacturing porous scaffolds for tissue engineering are the ease of 2 Methods control of porosity by controlling emulsion droplets size, relatively rapidity structure formation in bulk, and cost- 2.1 Materials effectiveness. Its use with biological polymers has so far been limited to polysaccharides [13] and the denatured Reagents were obtained from Sigma Aldrich, Poole, UK polypeptide, gelatin [14] and not with native proteins such unless otherwise stated. Proteins used for scaffolds were as collagen and fibrin. type I rat tail collagen 5 mg/ml in acetic acid (First Link, There are several major limitations to emulsion tem- Wolverhampton, UK), bovine fibrin and bovine thrombin. plating of protein structures: protein denaturation (by 2-(N-morpholino) ethanesulfonic acid (MES) biochemical adsorption at hydrophobic interfaces and by surfactant grade (Fisher Scientific, Loughborough, UK) and sodium agents), emulsion instability, complexity of introducing chloride were used for the buffer. Decane, Tween 20 and stabilisation methods, and elution of the oil-phase and Span 20 were used for emulsions. 1-ethyl-3-(-3-dimethyla- surfactant [15]. Proteins typically adsorb to and denature at minopropyl) carbodiimide hydrochloride (EDC) and sulfo- oil-water interfaces [16–18] so the creation of very large N-hydroxysuccinimide (NHS), pure ethanol and iso- surface area to volume ratio interfaces (in the order of propanol were used for cross-linking and oil elution. The 500 cm /ml), and short diffusion distances (in the range excipients used were polyvinyl alcohol (PVA) (99% 1–10 μm) necessarily presents a system that will favour any hydrolysed, MW 89,000 to 98,000), polyethylene glycol denaturation process. Moreover, the use of surfactants (PEG) (MW 6000), Pluronic F-68 (P68) and mannitol (M). needed to form a stable emulsion will also be likely to 0.25% Trypsin-EDTA solution for cell culture was used for adsorb onto and denature proteins [19]. Additionally, any stability testing and the Cell Counting Kit (CCK-8) assay process of emulsion ripening or maturation, will counteract was used to measure cell proliferation. the desired stability of the emulsion droplets as a template. Further issues of particular consideration for protein scaf- 2.2 Manufacturing of scaffolds folds concern post-formation stabilisation, for example by a compatible chemical cross-linking method, and elution of Macro-emulsion mixtures comprising of decane, Span the oil phase without disruption of the formed scaffold. For 20/Tween 20 at a calculated HLB of 13 and an aqueous example, phase separation involves release of significant buffer (25 mM MES, 150 mM NaCl pH 7.4) were mixed in energy, which is capable of disrupting the formed material a 60 ml syringe with the tip removed, for 15 seconds at as the oil phase separates. All these considerations deter- 1000 rpm using a high-speed mixer (Ceframo BDC6015, mine the feasibility and resultant biocompatibility of the Ontario, Can). Emulsions with 0.1, 0.3, 0.5 and 0.7% sur- scaffold produced. factant concentration were prepared. To manufacture scaf- The underlying hypothesis we investigate is that a sur- folds from each protein type, each of the emulsions was factant hydrophilic boundary layer that prevents protein added to each protein solution designated as follows: col- adsorption and denaturation at the emulsion interface will lagen (Col), fibrinogen plus thrombin (Fbn) or a mixture of enable protein-based three-dimensional hydrogels to be collagen and fibrin solution at a 1:1 volume ratio (ColFbn). formed into porous scaffold structures with minimal dena- The gelling solution of type-I collagen was used at turation. We investigate this by addressing each of the 4.5 mg/ml, fibrinogen was prepared at 20 mg/ml and Journal of Materials Science: Materials in Medicine (2018) 29:79 Page 3 of 12 79 thrombin at 10 units/ml in in 25 mM MES/150 mM NaCl 2.6 Cell culture (pH 7.4) buffer. Each emulsion-scaffold was left to gel at 37 °C for 30 min and then cross-linked with EDC:NHS Human dermal fibroblasts derived from neonatal foreskin (21.9:8.68 mM, 5:2 molar ratio) in 80% ethanol [22]. The (HDF) were obtained from Invitrogen, UK. They were scaffolds were then washed in 80% isopropanol, then deio- cultured in standard Dulbecco’s Modified Eagle Medium nized water three times for 15 minutes. At this point scaffolds (DMEM) media (Gibco, ThermoFisher Scientific, UK) with were further incubated with a 1% w/v excipient solution (e.g. 20% foetal bovine serum and 1% penicillin/streptomycin. P68, PVA, PEG or M). Collagen scaffolds were freeze-dried Only cells from Passage 2 to 9 were used in the experiment. at −30 °C and fibrin scaffolds were freeze-dried at −40 °C. Human dermal microvascular endothelial cells derived from The possible effects of the different excipients used, on neonatal foreskin that have been hTERT immortalized functional parameters and scaffold morphology, were (HDE), were obtained from ATCC, UK. They were cultured compared using scaffolds made at large pore size with 0.1% using Vascular Cell Basal Medium with Microvascular surfactant concentration. The effect of surfactant con- Endothelial Cell Growth Kit-VEGF supplement containing: centration was compared between scaffolds made using F68 rhVEGF (5 ng/ml), rhEGF (5 ng/ml), rhFGF basic (5 ng/ml), as the excipient. rhIGF-1 (15 ng/ml), L-glutamine (10 mM), heparin sulfate (0.75 Units/ml), hydrocortisone (1 µg/ml), ascorbic acid 2.3 Pore size of scaffolds (50 µg/ml) and foetal bovine serum (5%). Human bone marrow mesenchymal stem cells transfected with GFP Scaffolds were cut using a scalpel so that the cross-section (MCS) were obtained courtesy of Dr James Li (Hong Kong of each scaffold was lying horizontally on the carbon tape University) and cultured in DMEM media. mounted on the aluminium stubs for scanning electron microscopy (SEM) imaging. Three images were taken per 2.7 Cytocompatibility of scaffolds scaffold and the diameters of 20 random pores were aver- aged out to calculate estimated mean pore size of each Scaffolds were cultured with each cell type to investigate scaffold. Scaffold morphology (e.g. fibrous or smooth) was cytocompatibility. Each scaffold was cut into 6 mm discs also noted. using a punch biopsy and washed with PBS three times before incubating overnight in media. On the following day, 2.4 Mechanical properties of scaffold each scaffold was seeded with 5000 cells/well and left overnight to allow cells to adhere. On the next day, the Scaffolds that were cross-linked for an hour, with different seeded scaffolds were transferred into a new well plate and excipients and surfactant concentrations, were tested for allowed to incubate for up to 14 days. Media was changed mechanical properties (Instron 5582 UTM). Each scaffold on alternate days. On days 3, 7 and 14, cell growth in each was cut into strips measuring 2 mm × 40 mm × T where T is scaffold was measured with the CCK-8 assay (2-(2-meth- the variable thickness of each scaffold. Each end of the oxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- strips were anchored into the jaws of the Deben tensile 2H-tetrazolium, monosodium salt (WST-8) plus 1-methoxy testing machine and stretched until the scaffold broke into phenazine methosulfate (PMS) reagent (WST8/PMS)) two pieces. Young’s modulus and ultimate tensile strength (Sigma, UK). Briefly, 10 μl of WST8/PMS reagent was (UTS) were calculated for each scaffold using stress-strain added to 100 μl of fresh media in each well and incubated curves obtained. Each test was performed in triplicate. for 4h at 37°C. 50 μl of the resulting solution was pipetted into a new plate for colorimetric assay of the reduced for- 2.5 Degradation profile of scaffold mazan product at 450 nm. Each test was done in triplicate. On day 14, the scaffolds were fixed in normal buffered Scaffolds were cut into 6 mm discs using a punch biopsy formalin for microscopy. and incubated in PBS for 7, 14, 21 28 and 35 days. The scaffolds were freeze-dried after each time point and dry 2.8 Wide-field imaging of seeded scaffolds mass of each scaffold was recorded. We introduced an enzymatic proteolysis assay to compare the degradation Each of the seeded scaffolds was stained using fluorescent profiles of each scaffold, over a relatively short period. antibodies for microscopy. Scaffolds seeded with human Scaffold discs were incubated in 0.25% (1×) Trypsin dermal fibroblasts were stained with 1:6 phalloidin (Alexa solution in PBS, which was changed daily for up to 7 days. Fluor 488, Thermo Fisher) and scaffolds seeded with Individual discs were freeze-dried at the following time human dermal endothelial cells were stained with 1:250 points: Days 1, 3, 5 and 7 and dry mass of each scaffold was mouse anti-human CD31 (Dako) and 1:1000 rabbit anti- measured. Each test was performed in triplicate. human vWF (Dako). Secondary staining was done using 79 Page 4 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:79 Fig. 1 Structure of EmDerm scaffolds formed with 0.1% surfactant surfactant concentration, from 0.1 to 0.7%, in the mixed phase tem- from: Collagen (a), Collagen-Fibrin (b) or Fibrin (c). SEM micro- plating system on the distribution of pore diameters (mean ± SD) of graphs of representative fields of scaffolds show meso-scale structure each type of EmDerm scaffold, collagen (d), collagen-fibrin (e) and and pore size (1000×) and nano-scale structure (2000×). The effect of fibrin scaffolds (f) Journal of Materials Science: Materials in Medicine (2018) 29:79 Page 5 of 12 79 Table 1 Mechanical strength characterisation of scaffolds (mean ± goat anti-mouse (Alexa Fluor 488, Thermo Fisher) and SD, n = 3) goat anti-rabbit (Alexa Fluor 568, Thermo Fischer). Scaf- Scaffold Young’s modulus (MPa) Ultimate tensile strength folds seeded with mesenchymal stem cells were not (MPa) stained, since they expressed GFP. Z-stacks were taken to visualize cell migration through the scaffold by wide-field Collagen scaffolds imaging at 20× magnification. Images were deconvoluted 0.1C 1.95 ± 0.49 8.28 ± 1.62 using AutoQuant X3 (Media Cybernetics) and visualised 0.3C 1.45 ± 0.76 7.87 ± 1.45 using Bitplane (Imaris software, Version 9.1). 0.5C 1.71 ± 0.21 7.99 ± 1.02 0.7C 2.14 ± 0.34 8.85 ± 0.97 2.9 Statistical analysis C-M 1.27 ± 0.48 9.65 ± 2.81 C-P68 1.25 ± 0.74 8.26 ± 1.78 Data between different scaffolds and time-points were ana- C-PEG 1.70 ± 0.51 8.87 ± 1.24 lysed using one-way analysis of variance (ANOVA) with Collagen-Fibrin scaffolds Tukey’s post-hoc multiple comparisons tests to evaluate 0.1CF 2.03 ± 0.81 12.82 ± 3.45 statistical significance. All statistical analysis was performed 0.3CF 2.43 ± 0.45 14.07 ± 2.67 using GraphPad Prism Version 4.0 for Windows. A p-value 0.5CF 2.55 ± 0.98 15.15 ± 3.92 of less than 0.05 is considered to be statistically significant. 0.7CF 2.89 ± 0.72 16.47 ± 5.39 Where applicable, * denotes a p–value of <0.05, ** denotes CF-M 1.89 ± 0.58 12.55 ± 3.89 ap–value of <0.01 and *** denotes a p-value of <0.001. CF-P68 1.46 ± 0.33 13.25 ± 2.66 CF-PEG 1.74 ± 0.76 11.53 ± 1.96 Fibrin scaffolds 3 Results 0.1F 1.52 ± 0.38 4.98 ± 0.96 3.1 Scaffold structure and porosity 0.3F 1.16 ± 0.29 4.81 ± 0.72 0.5F 1.18 ± 0.41 4.77 ± 0.42 The use of decane oil-in-water (o/w) emulsions was suc- 0.7F 1.12 ± 0.29 4.62 ± 0.58 cessful in forming scaffolds from each type of scaffold F-M 1.26 ± 0.35 4.25 ± 0.63 protein, neutralised type I collagen acetic acid extract, F-P68 1.48 ± 0.32 4.59 ± 0.22 which gels spontaneously on warming from a 4 °C solution F-PEG 1.37 ± 0.21 5.13 ± 0.51 to 37 °C; and fibrinogen, enzymatically coagulated with F-PVA 2.01 ± 0.11 4.43 ± 0.49 thrombin 37°C. Pore interconnectivity of scaffolds was obtained by controlling the oil:aqueous phase ratio to ≥0.5. Importantly, the formed scaffolds demonstrate a nano-scale approximately 1–2 MPa and UTS of 7–9 MPa. Fbn scaf- fibrous architecture, in addition to the micro-scale pores. folds had a Young’s modulus of around 1–2 MPa and UTS The use of an excipient was needed to preserve this struc- of 4–5 MPa. Change in porosity of the scaffolds and exci- ture on lyophilisation and reduce shrinkage. Figure 1a-c pients used did not have a significant impact on mechanical shows representative SEM images of each scaffold type. properties of the scaffolds. Pore size of the scaffolds inversely correlated with the surfactant concentration used to create the emulsion (Fig. 3.3 Hydrolytic degradation of scaffolds 1d-f). Previous work showed there was an inverse rela- tionship between surfactant concentration and emulsion Scaffold degradation by hydrolysis was determined by droplet diameter (not shown). For scaffolds made from measuring residual dry weight of scaffolds soaked in PBS emulsion with 0.1% surfactant, a mean pore size of around for extended periods (Fig. 2a-c). Collagen scaffolds showed 100 μm was obtained, and this decreased to approximately a large drop in dry mass of 40–50% over the first week. This 40 μm with 0.7% surfactant. is largely due to the dissolution of excipients during the initial hydration of the scaffold. At the end of 5 weeks, all 3.2 Mechanical properties scaffolds retained about 40–60% of their dry mass. Although Col scaffold stability was achieved at the shortest The measurement of the tensile properties of each EmDerm cross-linking time (Fig. 2c), ColFbn and Fbn scaffolds scaffold type (Table 1) showed that the ColFbn scaffolds which were cross-linked for a longer duration degraded had the highest Young’s modulus and UTS, approximately slower compared to scaffolds which were cross-linked for 1–2 MPa and 12–16 MPa respectively. This is followed by 30 min (Fig. 2b-c), although this was not statistically sig- Col scaffolds, which had a Young’s modulus of nificant in this experiment. Notably, Fbn scaffolds which 79 Page 6 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:79 Fig. 2 Hydrolytic and enzymatic degradation by mass over a period of EDC/NHS for different durations, after incubation in PBS (up to time. EmDerm scaffolds formed from: Collagen (a), Collagen-Fibrin 5 weeks) or Trypsin (up to 7 days). Data show % reduction of dry (b) or Fibrin (c) with 0.1% surfactant emulsions, cross-linked with weight (mean ± SD, n = 3) were cross-linked for only 30 min degraded completely by 3.5 Cytocompatibility of scaffolds 5 weeks. The assessment of the biocompatibility of EmDerm scaf- 3.4 Enzymatic degradation of scaffold folds was investigated by measuring the proliferation three cell types central to skin reconstruction, HDF, HDE and Col and ColFbn scaffolds remained incompletely degraded MSC. Net proliferation of each cell type occurred over by Day 7 when incubated in trypsin solution (Fig. 2a, b). 14 days in each scaffold material (Figs. 3–5). Importantly, All Fbn scaffolds that were cross-linked for less than 2 h there was no significant effect of varying the excipient used, were completely degraded within 7 days: Those that were compared to the P68 excipient (Fig. 3). However, it is cross-linked for 0.5 h were completely degraded by day 3, notable that HDE and MSC proliferation in Col scaffold followed by those cross-linked for 1 h, on Day 5 and those made without an excipient (C-GEL) was significantly lower cross-linked for 1.5 h by day 7 (Fig. 2c). As with the than Col scaffolds with excipient (C-P68 and C-M) (Fig. hydrolytic degradation, the mass of each scaffold decreased 3a). Also, HDF showed lower proliferation in ColFbn significantly over the first day. Dissolution of excipients is scaffolds with PEG (CF-PEG, Fig. 3b) and F-M (Fig. 3c) likely to contribute to the initial drop of scaffold dry mass. scaffolds. Journal of Materials Science: Materials in Medicine (2018) 29:79 Page 7 of 12 79 Fig. 3 Proliferation of HDF, HDE & MSC in EmDerm scaffolds of described in the methods. Scaffolds were washed, equilibrated and collagen (a), Collagen-Fibrin (b) or Fibrin (c) with 0.1% surfactant, cultured with each cell type, and proliferation was measured by WST- prepared with different excipients (PVA, M, P68, PEG) or nil, as 8/PMS reduction on days 3, 7 and 14 (data are mean ± SD, n = 3) While proliferation was marked in scaffolds with mesenchymal cell proliferation, both effects being statisti- high porosity, there was a general trend for proliferation cally significant (Fig. 5). to be progressively lower with decreased porosity (Fig. 4). This effect was most pronounced for HDF in 3.7 Wide-field microscope imaging of seeded Col scaffolds at the lowest porosity, corresponding to scaffolds 0.5 and 0.7% surfactant mix in the emulsion template (Fig. 4a), and in ColFbn scaffolds at the lowest porosity Based on the Z-stacks obtained, each cell type was found to (Fig. 4b). infiltrate into the scaffold uniformly over the XY plane and Z plane (Fig. 6). Due to limitations in light penetration, cells 3.6 Comparison with commercial scaffolds were only imaged up to a depth of about 100 microns. In particular, cell infiltration into Col scaffolds was observed Significantly greater proliferation of each cell type occurred to a similar extent as for fibrin-containing scaffolds, sug- in each of the high porosity EmDerm scaffolds from 0.1% gestive of effect of the preserved fibre nanostructure in these surfactant than the commercial comparator scaffolds, scaffolds. Interestingly, the endothelial cells seemed to form Matriderm and Integra (Fig. 5). It is notable that the Fbn ring-like structures when seeded onto EmDerm scaffolds, scaffold 0.1F-P68 supported greatest HDE proliferation, most notably within ColFbn and Fbn (arrows in Fig. 6, while the Col scaffold 0.1C-P68 promoted the better HDE panels). It is also notable that HDE ingress into the 79 Page 8 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:79 Fig. 4 Effect of porosity on in EmDerm collagen (a), collagen-fibrin (b) and fibrin (c) scaffolds achieved by varying surfactant concentration from 0.1 to 0.7% in primary manufacture step on proliferation of cell types (HDF, HDE and MSC) as in Fig. 3 (mean ± SD, n = 3) EmDerm Col scaffolds, and although show less cytoskeletal linking (data not shown). The resultant EmDerm scaffolds spreading, demonstrate some association into annular have consistent mechanical properties, are stable in phy- structures. By contrast, the stromal cell types adopt an siological solution, have good biocompatibility and cell elongated spindle-like morphology when seeded on to conductivity, and support cell proliferation. Effective EmDerm scaffolds, more pronounced with fibroblasts than ingress and proliferation of the main cell types required for mesenchymal stem cells (Fig. 6). dermal reconstruction, HDF, HDE & MSC are shown. Collectively, this suggests that the EmDerm have favour- able characteristics as dermal templates for skin regenera- 4 Discussion tion and wound healing. The characterisation and basic properties of these o/w In this paper, we demonstrate that an o/w macro-emulsion emulsions have been determined in our laboratory. Struc- system can be used successfully as a template for fabricat- turally, there was no evidence of protein denaturation. ing three-dimensional scaffolds with hierarchical porosity When used with acidic extracted type I collagen, which whilst preserving the intrinsic nano-scale protein hydrogel exhibits spontaneous gelation in warming after pH neu- structure. The method enables complex hierarchical struc- tralisation, the emulsion does not prevent gelation, and the ture from the nano-scale to be achieved in a relatively rapid nanostructure fibrils indicate preservation of the intrinsic process. Elution of the oil phase by washing can be structure of the hydrogel. Furthermore, the successful use of achieved with several alcohol-based solvents, after cross- emulsion system for fibrin, demonstrates that fibrinogen/ Journal of Materials Science: Materials in Medicine (2018) 29:79 Page 9 of 12 79 Fig. 5 Comparison of the proliferation of HDF, HDE and MSC scaffolds Matriderm™ and Integra™. Cell proliferation was measured between the three different types of EmDerm scaffolds made with as in Fig. 3 (mean ± SD, n = 3) 0.1% surfactant and P68 excipient, and the commercial reference thrombin enzymatic coagulation is able to proceed in the be sufficient to maintain integrity of the scaffolds post- mixed phased system, and the resultant fibrin fibril forma- implantation. A shorter duration significantly decreased tion is indicative of the native structural self-assembly hydrolytic and proteolytic stability of fibrin-based scaffolds, behaviour of fibrinogen after thrombin cleavage of the although the greater stabilisation achieved for collagen fibrinopeptides. scaffolds indicates greater reactivity to the EDC/NHS Scaffold porosity is a principle physical determinant of reagent. functionality of scaffolds [4, 23, 24]. Control of pore size Interestingly, there was a relationship between cell pro- was achieved by varying the emulsion droplet size through liferation and porosity, although the surfactant concentra- surfactant concentration in the primary manufacture step, at tion used in the scaffolds’ primary manufacture step is also a constant ratio of the aqueous protein phase to the tem- a variable. The use of concentrations over 0.5% in collagen plating oil phase. A direct relationship was found between scaffolds, and 0.7% in collagen-fibrin scaffolds, was asso- the surfactant concentration in the primary manufacture ciated with marked inhibition of HDF proliferation, possi- step, which determines the mixed phase template droplet bly suggesting some persistence of surfactant residue during diameter, and resultant lyophilised scaffold pore diameter the scaffold processing. However, the other cell types were by SEM. This applied over the range of mean pore dia- affected to a lesser extent, which suggests that if surfactant meters from around 40 to 100 μm. This suggests that the residues persist, the level is close to the threshold to cause porosity EmDerm scaffolds can be readily tuned according cytotoxicity. Additionally, the droplet diameters of emul- to needs. A mean pore size between 80 and 100 microns as sions with higher surfactant concentration are much smaller a suitable target is consistent with optimal migration and less ideal for promoting vascularisation. This observa- through scaffolds with this pore size range [25]. tion may therefore be useful in establishing a threshold for The mechanical properties of EmDerm scaffolds suggest the safe concentration of the current surfactant in the current that they can be strong enough to be handled physically and manufacture process, and confirms the need to evaluate do not tear easily. This is imperative as it allows the scaf- cytotoxicity. Possibly a more stringent wash process may be folds to be positioned and sutured during surgery. With Col required should the higher concentrations be needed. scaffolds there was an inverse relationship between the Despite this consideration, the overwhelming evidence of mechanical strength (Young’s modulus and UTS) and this work is that the EmDerm scaffolds are essentially porosity, which suggests that some differences in fibril cytocompatible. organisation. With Fbn and ColFbn scaffolds, the pore size Another aim of this method of manufacture is to create did not have a marked effect on these parameters scaffolds with nano-structured architecture, which current Post-implantation stability of scaffolds is an important commercial scaffolds lack. The nano-fibrous architecture aspect of their function, and resorption of tissue regenera- can provide cells with oriented cell adhesion signals and tion scaffolds allows for new extracellular matrix deposition promote migration of cells, as there is an aligned fibre [25]. Here, we introduced an assay of accelerated proteo- matrix for cells to move along [26]. In this regard, the effect lytic degradation in order to compare the kinetic profiles of on excipients on preserving the collagen nanostructure of degradation over a relatively short assay period. The cross- EmDerm scaffolds is notable. By contrast, scaffolds without linking duration had a direct effect on scaffold stability, excipients did not have nano-fibrous walls, rather they were demonstrating that this can be controlled. Based on our smooth and featureless when imaged under SEM. These findings, an incubation time of 1 h for cross-linking should findings suggest firstly that collagen is able to undergo 79 Page 10 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:79 Fig. 6 Morphology of HDF, HDE and MSC in the three different types seeded scaffolds, formation of annular structures is apparent (arrows). of EmDerm scaffolds made with 0.1% surfactant and P68 excipient, Scale bars = 50 μm collagen (a), collagen-fibrin (b) and fibrin (c) scaffolds. In HDE- structural reorganisation under freeze-drying involving of interconnected porosity [6, 28]. Thus, the present results fusion of the fibrillar hydrogel structure into microscale suggest that creating a nano-structured form of collagen can lamellae; secondly that excipient interaction with the pro- significantly increase the cell migration into the scaffold. tein matrix is a stabilising factor that prevents nano- This could translate to accelerated integration of collagen structure fusion. scaffolds in wound healing settings. The results of the biocompatibility studies with cells This paper demonstrates that the emulsion templating relevant to dermal reconstruction are particularly sig- technique is suitable for creating of a range of nano- nificant. A multi-parameter approach has recently been structured functional EmDerm scaffolds. The difference in validated that correlates in vitro cell responses to the clinical effect of each scaffold material on cell proliferation and properties of dermal biomaterials [27]. The generally morphology suggests that composition may have some cell greater cell proliferation and migration in the nano- type specific functional benefits. In particular, the greater structured EmDerm scaffold may be due to increase in proliferation of endothelial cells in collagen-fibrin and fibrin surface area available for cells to adhere to and migrate on, scaffolds than collagen scaffolds is associated with the as well as greater permeability to nutrients and oxygen formation of annular nascent vascular structures. This throughout the scaffold [25]. EmDerm scaffolds demon- behaviour, associated with the endothelial interaction with strate excellent cell penetrance, or conductivity, for different organised fibrin structures, is consistent with other angio- cell types. The present results contrast with previous studies genic properties of fibrin [21, 29–31]. However, the which have shown that collagen as a scaffold material in the migratory and morphological response of endothelial cells TM TM commercial scaffolds Integra and Matriderm , and also to the nano-structured collagen scaffolds suggests that the TM in decellularised dermal products Alloderm , Xeno- nano-structure increases its angiogenic potential. Further TM TM derm and Permacol and does not support complete cell examination of the cellular morphological and cell- conductivity, even though the structures have a high degree communication responses will extend our understanding Journal of Materials Science: Materials in Medicine (2018) 29:79 Page 11 of 12 79 of the role of scaffold nanostructure in influencing cellular 7. Barbetta A, Gumiero A, Pecci R, Bedini R, Dentini M. Gas-in- liquid foam templating as a method for the production of highly behaviour [27] and particularly angiogenesis and porous scaffolds. Biomacromolecules. 2009;10:3188–92. https:// vasculogenesis. doi.org/10.1021/bm901051c. 8. Barbetta A, Rizzitelli G, Bedini R, Pecci R, Dentini M. Porous gelatin hydrogels by gas-in-liquid foam templating. Soft Matter. 2010;6. https://doi.org/10.1039/b920049e. 5 Conclusions 9. Schacht K, Vogt J, Scheibel T. Foams made of engineered recombinant spider silk proteins as 3D scaffolds for cell growth. We demonstrate the feasibility of using emulsion templating ACS Biomater Sci Eng. 2016;2:517–25. https://doi.org/10.1021/a as a novel method of fabricating micro-porous nano-fibrous csbiomaterials.5b00483. 10. Vrana NE, Builles N, Kocak H, Gulay P, Justin V, Malbouyres M, protein scaffolds which are unique and easily tuned et al. EDC/NHS cross-linked collagen foams as scaffolds for according to wound healing and tissue regeneration needs. artificial corneal stroma. J Biomater Sci Polym Ed. This is a versatile method of templating various protein 2007;18:1527–45. polymers, including collagen and fibrin, and is amenable to 11. Zhu N, Chen X. Biofabrication of tissue scaffolds. In: Pignatello R, editor. Advances in biomaterials science and biomedical clinical manufacture scaleup. These scaffolds also have applications. InTech: London, UK; 2013. pp. 315–28. https://doi. excellent cytocompatibility and are able to support various org/10.5772/54125. types of cell growth and have excellent potential as dermal 12. Cameron NR. High internal phase emulsion templating as a route or soft tissue substitutes. to well-defined porous polymers. Polymer. 2005;46:1439–49. 13. Franks GV, Moss B, Phelan D. Chitosan tissue scaffolds by emul- sion templating. J Biomater Sci Polym Edn. 2006;17:1439–50. Acknowledgements The authors would like to thank the Agency of 14. Hulda-Chambi CG, Grosso C, editors. 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Journal of Materials Science: Materials in MedicineSpringer Journals

Published: Jun 5, 2018

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