Latent Oxidative Polymerization of Catecholamines as Potential Cross

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Latent Oxidative Polymerization of Catecholamines as Potential Crosslinkers for Biocompatible and Multifunctional Biopolymer Scaffolds Chetna Dhand, Veluchamy Amutha Barathi, Seow Theng Ong, Mayandi Venkatesh, Sriram Harini, Neeraj Dwivedi, Eunice Tze Leng Goh, Muruganantham Nandhakumar, Jayarama Reddy Venugopal, Silvia Marrero Diaz, Mobashar Hussain Urf Turabe Fazil, Xian Jun Loh, Liu Shou Ping, Roger W. Beuerman, Navin Kumar Verma, Seeram Ramakrishna, and Rajamani Lakshminarayanan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12544 • Publication Date (Web): 01 Nov 2016 Downloaded from http://pubs.acs.org on November 3, 2016

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Latent Oxidative Polymerization of Catecholamines as Potential Crosslinkers for Biocompatible and Multifunctional Biopolymer Scaffolds Chetna Dhand1, Veluchamy Amutha Barathi1,2,3, Seow Theng Ong4, Mayandi Venkatesh1, Sriram Harini1, Neeraj Dwivedi5, Eunice Tze Leng Goh1, Muruganantham Nandhakumar1, Jayarama Reddy Venugopal6, Silvia Marrero Diaz1, Mobashar Hussain Urf Turabe Fazil4, Xian Jun Loh7, Liu Shou Ping1,2, Roger W Beuerman1,2, Navin Kumar Verma1,4,*, Seeram Ramakrishna6,8,*, and Rajamani Lakshminarayanan1,2,* Affiliations 1

Anti-Infectives Research Group, Singapore Eye Research Institute, The Academia, 20 College

Road, Discovery Tower, Singapore 169856 2

Ophthalmology and Visual Sciences Academic Clinical Program, Duke-NUS Graduate Medical

School, Singapore 169857 3

Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of

Singapore, Singapore 119077 4

Lee Kong Chian School of Medicine, Nanyang Technological University, Experimental

Medicine Building, 59 Nanyang Drive, Singapore 636921 5

Department of Electrical and Computer Engineering, National University of Singapore, 3

Engineering Drive 3, Singapore 117583 6

Center for Nanofibers and Nanotechnology and Department of Mechanical Engineering,

National University of Singapore, Singapore 117576 7

Institute of Materials Research and Engineering, Agency for Science, Technology and Research

(A *STAR), Singapore 117602

1 ACS Paragon Plus Environment

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8

Guangdong-Hongkong-Macau Institute of CNS Regeneration (GHMICR), Jinan University,

Guangzhou, China 510632 *Correspondence should be addressed to Rajamani Lakshminarayanan ([email protected]), Navin Kumar Verma ([email protected]) and Seeram Ramakrishna ([email protected]).



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Abstract Electrospinning of naturally occurring biopolymers for biological applications requires postspinning crosslinking for endurance in protease-rich microenvironments and prevention of rapid dissolution. The most commonly used crosslinkers often generate cytotoxic byproducts, necessitate high concentrations or time-consuming procedures. Herein, we report the addition of “safe” catecholamine crosslinkers to collagen or gelatin dope solutions followed by electrospinning yielded junction-containing nanofibrous mats. Subsequent in situ oxidative polymerization of the catecholamines increased the density of soldered junctions and maintained the porous nanofiber architecture. This protocol imparted photoluminescence to the biopolymers, a smooth non-cytotoxic coating, and good mechanical/structural stability in aqueous solutions. The utility of our approach was demonstrated by the preparation of durable antimicrobial wound dressings and mineralized osteoconductive scaffolds via peptide antibiotics and calcium chloride (CaCl2) incorporation into the dope solutions. The mineralized composite mats consists of amorphous calcium carbonate that enhanced the osteoblasts cell proliferation, differentiation and expression of important osteogenic marker proteins. In proof-of-concept experiments, antibioticloaded mats displayed superior antimicrobial properties relative to silver (Ag)-based dressings, and accelerated wound healing in a porcine deep dermal burn injury model. Keywords: junction nanofibers, amorphous calcium carbonate, catecholamines, wound dressings, bone composites


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1. INTRODUCTION In recent years, electrospinning has received much scientific and commercial attention among the various methods available for generating nanofibers, because of its scalability, versatility, flexibility and ease of fiber production1,2. Electrospinning has the unique ability to morphologically transform almost any type of polymer or supramolecular assembly into a nanofibrous scaffolds3-7. The high aspect ratio and large surface area of electrospun fibers are advantageous for numerous biomedical applications including tissue engineering, controlled drug release and regenerative medicine. Moreover, nanofibers are particularly useful as implant materials and wound dressings when functionalized with “add-on” molecules8,9. Nevertheless, electrospun nanofibers prepared from biopolymers such as collagen or gelatin lack adequate mechanical stability and show a high degree of swelling in aqueous environments, limiting their long-term biomedical utility10. The aqueous stability and durability of electrospun biopolymers can be improved post-spinning through crosslinking by various physical means (e.g., dehydrothermal treatment or ultraviolet (UV) irradiation) or chemical tactics (e.g., crosslinking with bifunctional molecules). Crosslinking effectively increases nanofiber shape retention and integrity, and also aids in ease of scaffolds handling and manipulation during use in biomedical applications. While the available chemical crosslinkers provide adequate durability, a number of practical limitations are associated with the post-spinning crosslinking treatment of nanofibrous biopolymers.

For

example,

commonly

used

chemical

crosslinkers

(formaldehyde,

glutaraldehyde, glyceraldehyde, EDC/NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/Nhydroxysuccinimide) and genipin) either shows concentration-dependent cytotoxicity or require adverse pH/temperature conditions, use of organic solvents and long reaction times for efficient



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crosslinking11-14. Meanwhile, physical crosslinking techniques compromise the stability of collagen and other biological molecules due to thermal degradation. Therefore, alternative strategies are required to overcome the limitations of conventional crosslinking methods, thereby expanding the potential applications of electrospun biopolymers. Dopamine (DA) and norepinephrine (NE) belong to a group of naturally occurring catecholamines that readily form adherent material-independent polycatecholamine coatings on various substrates (e.g., synthetic polymers, metals, metal oxides, ceramics, and semiconductors) through pH adjustment to 8.515,16. Previous studies demonstrated that polycatecholamine coatings can be generated via enzymatic17 or oxidant-catalyzed reactions under neutral or weakly acidic conditions18, by UV irradiation19, by electrochemical20 or thermal treatment21, and also by addition of metal ions22 or catecholic polymers23. Polycatecholamine coating offers a generic way to functionalize substrates with biomacromolecules, minerals, noble metals, and/or living cells, thus expanding the repertoire of biomaterial substrates with diverse functional properties24. We recently demonstrated the in vivo efficacy of an antimicrobial peptide-functionalized polydopamine (pDA)-coated titanium implant in an alkali burn injury model of the rabbit cornea25. The pDA-coated implant provided complete protection to the injured cornea against Staphylococcus aureus infection without inducing an unwanted inflammatory response for more than 9 days, underscoring the durability and biocompatibility of the pDA coating in an in vivo environment. Despite these promising results, the use of polycatecholamine/pDA coatings on electrospun nanofibers has heretofore been limited to synthetic polymers possessing inherent aqueous stability, because the coating protocol involves exposure of substrates to aqueous conditions and an alkaline pH26,27.


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Here, we developed a simple methodology to prepare multifunctional polycatecholaminecrosslinked electrospun nanofibrous mats from water-labile biopolymers, gelatin and collagen. The polycatecholamines showed preferential crosslinking at nanofiber junctions, improved the photoluminescence properties of the electrospun mats, and formed a uniform coating on the fiber surface without affecting mat porosity. In the proof-of-concept experiments, crosslinked nanofiber mats were employed for the preparation of durable antimicrobial wound dressings and osteoconductive scaffolds. In vitro and animal model studies confirmed that the glycopeptide antibiotic loaded mats exhibited superior antimicrobial activity and wound healing properties when compared with commercial silver (Ag)-based dressings. Furthermore, osteoconductive scaffolds supported osteoblast growth in vitro. The overall strategy and results of this study are summarized in Scheme 1.

Scheme 1. Overall strategy for the preparation of durable antimicrobial wound dressings and osteoconducive scaffolds and a summary of the results. Catecholamines are mixed with biopolymer dope solution and electrospun. The as-electrospun mats are exposed to ammonium carbonate [(NH4)2CO3] in a closed desiccator for 24 h. Sublimation of (NH4)CO3 generates gaseous ammonia and CO2, which creates an alkaline atmosphere and triggers the oxidative 6 ACS Paragon Plus Environment


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polymerization of catecholamines. At the aqueous interface, CO2 readily dissolves and generates bicarbonate (HCO3¯) ions, which in turn deprotonate (due to alkaline atmosphere) to form carbonate (CO3═) ions. This facile chemistry is used to develop durable wound dressings or osteoconductive composites by incorporating peptide antibiotics or CaCl2 in the dope solution, respectively. The dotted box summarizes the salient features of the crosslinking method. 2. MATERIALS AND METHODS 2.1. Materials. Gelatin from porcine skin (Type A), trifluoroacetic acid, 1,1,1,3,3,3-hexafluoro2-propanol (HFIP), 2,2,2-trifluoroethanol (TFE), norepinephrine hydrochloride (NE), dopamine hydrochloride (DA), α-methyl norepinephrine (≥95%; AM), vancomycin hydrochloride hydrate (Vanco), caspofungin diacetate (Caspo), CaCl2, (NH4)2CO3, nutrient mixture F-12 (HAM), Dulbecco’s Modified Eagle’s Medium (DMEM), Hoechst nuclear stain solution, FluoromountTM aqueous mounting medium, alkaline phosphatase (ALP), hexamethyldisilazane, anti-goat TRITC and FITC-conjugated anti α-tubulin antibodies were obtained from Sigma-Aldrich, Singapore. Alexa Fluor 647 Phalloidin and Alexa Fluor 633 anti-mouse antibody were procured from Molecular Probe® (Thermo Fisher Scientific, Singapore). Collagen from calf skin (Type A) was obtained from Cosmo Bio, Japan.Primary human dermal fibroblasts (hDFs) and human fetal osteoblast cells (hFOB) were purchased from American Type Culture Collection (ATCC, VA, USA). Gibco™ fetal bovine serum (FBS), trypsin-EDTA and CellTracker™ Green CMFDA (5chloromethylfluorescein diacetate) were from Thermo Fisher Scientific. CellTiter 96® AQueous One solution was procured from Promega (Singapore). Antibodies against Bone Morphogenetic Protein 2 (BMP2), osteopontin and osteocalcin were purchased from Santa Cruz Biotechnology. 2.2. Electrospinning of Gelatin (Gel) and Collagen (Coll) Nanofibers. For electrospinning, first homogenous solution of gelatin (10%, Type A, Porcine skin) and collagen (8%, Type A, Calf skin) was prepared in TFE and HFIP, respectively, with 12 h continuous stirring at room temperature. The obtained biopolymer solutions were then loaded into the syringe (Becton Dickinson, BD, NJ, USA) and high voltages (Gamma High Voltage Research Inc., Ormond 7 ACS Paragon Plus Environment

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Beach, FL, USA) of 11.5 KV and 13 KV were applied for electrospun gelatin (ES_Gel) and collagen (ES_Coll) nanofiber mats, respectively, to draw the fibers onto the collector plate. The distance between the spinneret (27G needle) and the receiver was 12 cm/13 cm for collecting electrospun nanofibers of Gel/Coll and the flow rate was maintained at 0.8 mL h-1 using syringe pump (KD Scientific Inc., Holliston, MA, USA). The nanofibers were collected on different substrates including aluminum foil, coverslips (CS) and TEM grids for analysis by various characterization methods. All the electrospinning experiments were executed at room temperature and whole set up was maintained at ~25% relative humidity. 2.3. Electrospinning of Catecholamine (DA, NE and AM) Loaded Mats. To prepare the catecholamine loaded mats, DA, NE or AM was added at the concentration of 2% (w/w of Gel) and 10% (w/w of Coll) in gelatin and collagen dope solutions, respectively. The electrospinning parameters remained same as discussed above. 2.4. Electrospinning of Antibiotic Loaded Gelatin Mats for Wound Dressing Applications. For the preparation of antibiotics loaded mats, vancomycin or caspofungin (0.5% w/w of Gel) was added to Gel-DA dope solution. Since the addition of vancomycin caused substantial solution turbidity, electrospinning was carried out in 80% TFE at 14 KV. 2.5. Electrospinning of Calcium and Catecholamine Incorporated Collagen Scaffolds. For designing collagen based bone like composite scaffolds, dope solution composed of 8% collagen (w/v of 90% HFIP in water), 10% DA (w/w of Coll) and Ca2+ ions (20 mM) was used for electrospinning at DC voltage of 17 KV, as previously reported28. Photographs of various dope solutions used for the preparation of different gelatin and collagen based mats are shown in Figure S1. Fabrication of vancomycin loaded mat is presented



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in supporting video 1 (Initial electrospinning of Vanco_Gel_pDA nanofibers over aluminum foil) and supporting video 2 (Formation of Vanco_Gel_pDA mat after 3 h of electrospinning). 2.6. Ammonium Carbonate mediated Catecholamine-Based Crosslinking. For latent oxidative polymerization, the catecholamine loaded nanofibrous mats with/without antibiotics were exposed to powdered ammonium carbonate (~5 g) in a sealed desiccator for 24 h. For pDA coating by conventional method, pristine gelatin fiber mats was soaked for 5 h in 2% DA (w/v) solution in Tris-HCl buffer (10 mM, pH = 8.5) and then vacuum dried. Table 1 shows abbreviations used for various mats, composition of dope solutions and the spinning conditions. Table 1. Details of the electrospun gelatin and collagen mats prepared under various conditions and their acronym used in this article. Sample Abbreviation ES_Gel Gel_DA Gel_pDA Gel_NE Gel_pNE Gel_AM Gel_pAM Vanco_Gel_pDA

Caspo_Gel_pDA

ES_Coll

Details Pristine gelatin mats Dopamine (DA) incorporated gelatin mats Gel_DA mats after exposure to (NH4)2CO3 for 24 h Norepinephrine (NE) incorporated gelatin mats Gel_NE mats after exposure to (NH4)2CO3 for 24 h α-Methyl Norepinephrine (AM) incorporated gelatin mats Gel_AM mats after exposure to (NH4)2CO3 for 24 h Gel_DA mats containing vancomycin (0.5% w/w of gelatin) after exposure to (NH4)2CO3 for 24 h Gel_DA mats containing caspofungin (0.5% w/w of gelatin) after exposure to (NH4)2CO3 for 24 h Pristine Collagen Mats

Coll_DA

Dopamine incorporated collagen mats

Coll_pDA

Coll_DA mats after exposure to (NH4)2CO3 for 24 h Coll_DA mats containing 20 mM of Ca2+ ions

Coll_DA_Ca2+ Coll_pDA_Ca2+

Dope Solution Composition 10% Gelatin (w/v, in TFE) 10% Gelatin + 2% DA (w/w of gelatin) 10% Gelatin + 2% DA (w/w of gelatin) 10% Gelatin + 2% NE (w/w of gelatin) 10% Gelatin + 2% NE (w/w of gelatin) 10% Gelatin + 2% AM (w/w of gelatin) 10% Gelatin + 2% AM (w/w of gelatin) 10% Gelatin + 2% DA and 0.5% vancomycin (w/w of gelatin)

Electrospinning Parameters Voltage =11.5 KV SD = 12 cm Voltage =11.5 KV SD = 12 cm Voltage =11.5 KV SD = 12 cm Voltage =11.5 KV SD = 12 cm Voltage =11.5 KV SD = 12 cm Voltage =11.5 KV SD = 12 cm Voltage =11.5 KV SD = 12 cm Voltage =14 KV SD = 12 cm

10% Gelatin + 2% DA + 0.5% caspofungin (w/w of gelatin)

Voltage =14 KV SD = 12 cm

8% Collagen (w/v, in HFIP) 8% Collagen + 10% DA (w/w of collagen) in HFIP 8% Collagen + 10% DA

Voltage =13 KV SD = 13 cm Voltage =13 KV SD = 13 cm

8% Collagen + 10% DA + 20 mM of Ca2+ (w/v, in 990% HFIP) Coll_DA_Ca2+mats after 8% Collagen + 10% DA exposure to (NHACS for 24 h + 20Environment mM of Ca2+ ions 4)2CO 3 Paragon Plus

Voltage =13 KV SD = 13 cm Voltage =17 KV SD = 13 cm Voltage =17 KV SD = 13 cm

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2.7. Morphology and Surface Characterization of ES Mats. Morphological analysis of electrospun gelatin and collagen mats were performed using Field Emission Scanning Electron Microscope (FE-SEM) and High Resolution Transmission Electron Microscope (HR-TEM). FESEM images were collected using JEOL-JSM6701F FE-SEM and TEM micrographs were collected using JEOL JEM-3010 TEM on gold coated copper grids. VCA Optima Surface Analysis system (AST Products, Billerica, MA, USA) was used to determine the contact angle of the electrospun mats. FTIR spectra were recorded using Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectrometer (Thermo Electron, Waltham, MA, USA). Kratos AXIS UltraDLD X-ray photoelectron spectrometer (Kratos Analytical Ltd, UK) was used to determine the surface composition of the mats. XPS studies were carried out under ultrahigh vacuum conditions of ~10-9 Torr by applying monochromatic Al-Kα X-ray beam at 1486.71 eV. Mechanical studies were performed using Instron tensile tester (Instron, Singapore) with a load cell capacity of 10 N at room temperature and relative humidity of 80%. Rectangular specimens (3-5 per sample) of each fibre mats with dimensions 30 mm ×10 mm were cut and tested at 5 mm min-1 cross-head speed. Reversed-phase high-performance liquid chromatography (RP-HPLC) experiments were carried out on a Waters HPLC system equiped with C18 analytical column (Phenomenex, CA, USA). A 10 mg of pDA crosslinked mats prepared by Tris-HCL and ADM was sonicated in 1 mL of MilliQ water at 50 ˚C for 30-45 minutes until the solution became optically clear and then centrifuged at 10,000 rpm. The supernatant solution (100 µL) was injected into a C18 (Phenomenex) analytical column that was equilibrated with buffer A (1% trifluoroacetic acid in distilled water). The oxidative products were eluted using a linear gradient of buffer B (1% trifluoroacetic acid in acetonitrile) and monitored by UV absorption at 280 nm.



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Zeiss LSM800 confocal microscope was used to investigate the photoluminescent properties of the mats. A Plan-Apochromat 63×/1.40 Oil DIC objective (Carl Zeiss Microimaging Inc., NY, USA) was used to image the photoluminescence of crosslinked mats, using excitation lasers of 405 nm, 488 nm, 561 nm and 640 nm wavelengths. The confocal pinhole was set to 1 Airy unit for green channel and other channels were adjusted to the same optical thickness accordingly. Acquired images were processed using ZEN imaging software (Carl Zeiss). 2.8. Cytocompatibility Analysis of ES Mats. Primary human dermal fibroblasts (hDFs) were cultured in DMEM medium supplemented with 50 U mL-1 penicillin, 50 µg mL-1 streptomycin and 10% (v/v) FBS at 37 ºC in a humidified incubator with 5% CO2. Cells (1 × 105 cells well-1) were seeded onto the ES fibre mats prepared on coverslips in a 12-well plate (Nunc®) and allowed to grow for 24 h. Cells were fixed using 3% paraformaldehyde in phosphate buffered saline (PBS). Alexa Fluor 569 phalloidin (Molecular Probes®) and anti-α-tubulin-FITC were then used to fluorescently label the cells to visualize the cellular morphologies and with Hoechst to visualize the nuclei. Coverslips were mounted with FlouromountTM on clear glass slides before imaging using Zeiss LSM800 confocal microscope (40× oil immersion objective lens). At least 20 different microscopic fields were captured and analyzed for each samples. Cell viability was quantified using CellTiter 96® Aqueous One Solution reagent as per the manufacturer’s instruction. Briefly, upon completion of the treatment, cells growing on different scaffolds were incubated with 10% (v/v) MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium) reagent at 37 ºC for 2 h and absorbace (490 nm) was recorded using a microplate reader (Infinite M200 Pro, Tecan,


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Mannedorf, Switzerland). Relative cell viability was determined in comparision to the control. Each sample treatment was done in three independent triplicates. 2.9. Bioactivity Assessment of Calcium Enriched Bone Like Collagen Scaffolds. Human fetal osteoblasts (hFOB) cells were grown in DMEM/F12 medium (1:1) supplemented with 1% antibiotic mixture and 10% FBS in a 75 cm2 cell-culture flask. The hFOB cells were then incubated under humidified conditions at 37ºC containing 5% CO2 for 1 week and once every three days the culture medium was changed. Trypsin-EDTA was used to trypsinize the hFOB cells, which were then replated after counting using trypan blue staining via a hemocytometer. The 15 mm diameter coverslips were used to collect various nanofibrous scaffolds and were placed in 24-well plates carrying stainless steel rings over them to avoid detachment of the mats. The scaffolds were then UV sterilized for 1 h and washed thrice with 10 mM PBS (pH 7) for 15 min each to eliminate any remaining solvent and subsequently immersed in complete medium before seeding the cells. The hFOB cell seeding were done at a density of 10,000 cells well-1 on ES_Coll, Coll_pDA, Coll_DA_Ca2+, Coll_pDA_Ca2+ nanofibrous scaffolds with CS as the control. To visualize cell proliferation on various mats, we used CMFDA (cell penetrating dye), which is readily cleaved by intracellular esterases available in live cells and produces fluorescent calcein. After 9 days of cell growth in 24-well plates, complete medium was replaced with 20 µl of CMFDA dye in medium (25 µM) at 37°C for 2 h. CMFDA medium was then replaced with complete medium for overnight incubation. Confocal fluorescence images with z-sectioning were then obtained using 40× oil imersion objective lens using 405 nm and 488 nm laser excitations.



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To analyze the morphologies of the cells growing on different mat preparations, cells were fixed with 3% paraformaldehyde after 9 days. Fixed cells were then labelled with Hoechst and Far-Red fluorescently labeled cytoplasmic dye, phalloidin (Molecular Probes®). Confocal fluorescence images and z-stacks were obtained with 405 nm, 488 nm and 640 nm laser excitations and processed using ZEN imaging software (Carl Zeiss). To evaluate the osteoblast proliferation on various nanofibrous scaffolds, MTS assay was used after culturing the cells for a period of 3, 6 and 9 days. For this, cellular nanofibrous scaffolds were first washed with PBS, incubated with MTS reagent for 3 h in serum-free medium followed by absorbance reading at 490 nm (FLUOstar OPTIMA microplate reader, BMG LABTECH GmbH, Germany). To assess the bone-forming ability of various scaffolds, the ALP activity of the cells was recorded. The cell-scaffold constructs were first washed with PBS for 15 min and then ALP reagent was added. After 1 h, the reaction was stopped by 2N NaOH. This assay uses p-nitrophenyl phosphate (pNPP) as a colourless phosphatase organic ester substrate, which turns yellow when dephosphorylated, or in other words, when catalyzed by ALP released from bone cells it forms p-nitrophenol and phosphate. The yellow colored p-nitrophenol product was aliquoted into 96-well plate and reaction absorbance (405 nm) was recorded using a microplate reader. The estimated ALP activity was normalized with cell number (as a marker for bone formation) and reported. Morphological analysis of in vitro cultured hFOB cells were carried out after 3, 6 and 9 days of cell culture using FE-SEM. For morphological analysis, cells were first fixed in 3% glutaraldehyde on the scaffolds after removing the non-adherent cells. The cell scaffolds were then dehydrated using increasing concentrations of ethanol (30%, 50%, 75%, 90% and 100%) for 15 min each concentration and finally treated with hexamethyldisilazane (HMDS) and air-


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dried overnight in order to maintain normal cell morphology. Dried cellular constructs were sputter-coated with platinum and observed under FE-SEM at an accelerating voltage of 10 KV. After 11 days of cell culture, hFOB cultured on different nanofibrous scaffolds and CS were processed for osteogenic markers staining. For this, 3% paraformaldehyde solution was used to fix the cells followed by permeabilization using 0.3% Triton X-100. Staining of the cells was then performed using antibodies against bone specific markers, osteopontin (OPN), osteocalcin (OCN) and Bone Morphogenetic Protein 2 (BMP2) Confocal images were acquired using Zeiss LSM800 as described above. 2.10. In vivo Porcine Skin Model of Burn Injury. Burns were created under the anaesthetic conditions on the pig skin via direct contact with a hot water beaker preheated to 92 °C for 15 sec, following the protocol reported by Cuttle et al29. Eight second degree burns, up to dermis layer, were created on the thoracic ribs of each pig. Burns were created 4 on each side, i.e. four on the cranial end and the four on the caudal end, with 1 cm distance in-between. During burn creation, surgical drapes with absorbent pads were used around animal to avoid spillage and leakage from the burning procedure. In context to the circular burning device, we have used 100 ml beaker (with 4 cm diameter and ~12.57 cm2 surface area) filled with 50 ml sterile water maintained at 92 °C. The pressure to the burning device was induced using 500 ml schott duran bottle filled with 300 ml warm water (Temp ~45°C). The burning device was maintained in contact with the porcine skin for an optimized time of 15 sec to generate uniform burn wounds. The created wounds were photographed immediately to estimate the initial wound area for each burn. The wounds were cleaned to remove the burned epithelium debris. The test wound dressings including ES_Gel, Vanco_Gel_pDA and Aquacel® Ag were applied on the designated 14 ACS Paragon Plus Environment


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wounds, two wounds for each dressing type, to completely cover the wound area. Two wounds were left uncovered and were labelled as untreated control burn wounds. To prevent inter-wound cross contamination, Tegaderm films were used for covering the wound dressing area. Dressings were changed at the frequency of twice a week under anaesthetic conditions. Animal was first sedated using 40% ketamine/xylazine intramuscular dose to induce anaesthesia (1 mg kg-1 xylasine/13 mg kg-1 ketamine) and maintained with 1-2% isofluorane. To reduce the discomfort, buprenorphrine (0.01 mg kg-1) was applied intramuscularly before and 2 days after the burn wounds. At the time of dressing change, 0.05% chlorhexidine solution and cotton gauze was used to clean the wounds before placing fresh dressing material. The clinical description of the wound was then recorded after inspecting the wounds thoroughly. Wound photographs were taken for all the groups, using a Nikon D90 digital SLR camera. A template was employed to mark four dots on pig skin surface which were then ruled up with the focusing spots in the camera viewfinder to verify the camera was at a standard distance from the wound. A CyanMagenta-Yellow-Black (CMYK) color scale was kept alongside the wound to standardize the colors on different photographs with each other. The SigmaScan Pro 5 software was used to calculate the total wound area in square centimetre for different wound dressings. Statistical analysis. The data was reported as mean ± standard error of mean. One-way analysis of variance (ANOVA) was used for comparison of two groups and p-values ≥ 0.05 was taken as statistically insignificant. 3. RESULTS AND DISCUSSIONS 3.1. Preparation of Catecholamine-Crosslinked Electrospun Biopolymers by Latent Oxidation. Electrospun nanofiber mats were prepared using 10% (w/v) gelatin or 8% (w/v) collagen dope solutions in fluoro alcohols as described previously30,31. Morphologically, the


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electrospun gelatin and collagen (ES_Gel and ES_Coll) fibers appeared smooth and unbranched, with average diameters (ø) of 798 ± 115 and 900 ± 150 nm, respectively (Figure 1a,b). No obvious interactions were observed between individual pristine (un-crosslinked) gelatin or collagen nanofibers (Figure 1a,b and insets). Exposure of electrospun gelatin to an alkaline DA solution resulted in the complete loss of fibrous morphology and a rough coating deposited over the entire mat (Figure 1c). The high swellability of gelatin in aqueous solutions was probably responsible for the observed non-uniform coating. To overcome this problem, we added DA to the gelatin/collagen dope solution before spinning, which was then electrospun into nanofibers as before. Transmission electron microscopy (TEM) analysis revealed strong interactions between individual nanofibers in the DA-loaded, as-spun gelatin or collagen (Gel_DA or Coll_DA) mats at the junction points, which permitted the formation of soldered or welded junctions (Figure 1d,e). Rather than exposing the DA-loaded biopolymer mats to aqueous alkaline pH conditions, we instead exposed them to ammonium carbonate vapor in a closed desiccator for 24 h. This method, termed as the ammonium carbonate diffusion method (ADM), rapidly saturated the surrounding gas phase with ammonia (NH3) and carbon dioxide (CO2), followed by slow diffusion of NH3 and CO2 across the interface and a concomitant increase in the pH to > 8.532. The rise in pH then triggered the oxidative polymerization of DA, with no deleterious effects on nanofiber morphology. Interestingly, ADM increased the density of the soldered junctions in the as-spun Gel_DA and Coll_DA mats while maintaining their macroporous morphology (Figure 1f,g). In addition, the oxidative polymerization of DA caused nanofiber fusion along the fiber axis and the formation of branched structures, thus allowing numerous soldered junctions in a



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small volume. TEM images were indicative of a pDA coating along the nanofiber junctions, and the nanofibers displayed smooth surface features with no particulate matter (Figure 1f,g, insets). Determination of the root-mean square surface roughness by atomic force microscopy imaging further confirmed the formation of a smooth pDA coating across the entire surface of the gelatin nanofibers, unlike pristine ES_Gel nanofibers (Figure S2). These results are quite different from those obtained with pDA coating of electrospun fibers and nanoparticles by conventional Tris-HCl-mediated protocols, which yield rough particulate morphologies and nonhomogeneous coatings27,33,34. Similarly, the addition of other catecholamines (i.e., NE or α-methyl norepinephrine (AM)) to the gelatin dope solution and subsequent electrospinning promoted the formation of many soldered junctions in a small volume without affecting nanofiber morphology (Figure S3). Mats comprised of gelatin electrospun with NE or AM (Gel_NE or Gel_AM mats, respectively) showed all the hallmarks of crosslinked scaffolds, including a high density of soldered junctions, numerous inter-fiber fusion points and extensive branching (Figure S3a,b)35,36. In spite of limited solubility of NE/AM in TFE, the as-spun mats appeared smooth and did not display the presence of beads or “beads on string” morphology. ADM treatment of the NE/AM-loaded gelatin mats further increased the density of the soldered junctions (Figure S3c,d), as previously observed for ADM-treated, DA-loaded mats (Figure 1f,g). Like the Gel_NE and Gel_AM mats, NE- and AM-loaded electrospun collagen (Coll_NE and Coll_AM) mats clearly revealed the presence of soldered junctions in TEM images of the asspun biopolymers (Figure S4a,c, insets). Furthermore, incorporation of catecholamines into the dope solution markedly decreased the ø -values for both as-spun collagen and gelatin nanofibers (Figure 1h). ADM treatment of catecholamine-loaded electrospun collagen further increased the


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formation of soldered junctions and promoted inter-fiber adhesion (Figure S3c,d). Therefore, the latent oxidative polymerization of catecholamine-loaded electrospun scaffolds by ADM is an effective way of stabilizing water-labile, mechanically weak biopolymer nanofiber mats. Given that the entire reaction takes place in the solid phase, ADM can apparently retain the anisotropy and porosity of electrospun scaffolds in comparison with the conventional method.



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Figure 1. SEM/TEM images of ES gelatin and ES collagen nanofibrous mats prepared under various electrospinning conditions. SEM micrographs for (a) As-spun gelatin and (b) collagen. Insets in (a) and (b) show TEM images indicating the lack of any interactions between individual gelatin/collagen fibres. The scale bar is 1 µm for inset (a) and 0.5 µm for (b). (c) Morphology of ES gelatin mats after exposure to 2% (w/v) DA prepared in Tris-HCl (pH 8.5). Note that pDA covered the entire mat with a rough coating. The inset in (c) is a TEM image showing the formation of a non-porous and rough coating of pDA by the Tris-HCl route. Scale bar = 1 µm. (d) ES gelatin and (e) ES collagen mats containing DA. TEM images in the insets indicate that DA promotes an interaction between the nanofibers at the junction. Inset scale bar is 0.5 µm for panel (d) and (e), respectively. Morphology of gelatin (f) and collagen (g) mats containing DA after (NH4)2CO3 exposure. Note the formation of extensive soldered junctions and branching after ADM. Insets show TEM images of smooth pDA coating at the junction (scale bar = 0.5 µm). Scale bar is 1 µm for all the SEM images. (h) Effect of catecholamines on the average diameter of as-spun gelatin and collagen mats. The final amount of DA used was 2% and 10% (w/w of the biopolymer) for gelatin and collagen mats, respectively. For SEM, electrospun fibers were collected on coverslips whereas for TEM analysis, fibers were collected on gold coated copper grids.


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3.2. Characterization of pDA-Crosslinked Electrospun Mats. A number of reports address the material-independent capacity of pDA coatings for surface modification, including that of electrospun gelatin nanofibers. We then characterized the ES_Gel mats crosslinked with pDA by ADM, hereafter termed Gel_pDA mats. To do this, we took advantage of XPS to ascertain the effect of ADM-generated pDA coatings on nanofiber surface properties. General survey scans of ES_Gel, Gel_DA, and Gel_pDA samples revealed the presence of carbon, nitrogen, and oxygen in the XPS spectra, consistent with the expected nanofiber compositions (Figure S5a). Deconvolution analyses of the core-level spectra of C 1s, O 1s and N 1s, was carried out to confirm the bonding status of carbon with carbon, nitrogen, and oxygen. The detailed corelevel spectra data for all samples are provided in Figure S5. Because the alkaline oxidative polymerization of DA generates heteroaromatic dopaminechrome, we further examined the N 1s core-level spectra of the samples. For all mats, the N 1s spectra could be fitted into three constituent peaks assigned to RNH2, R2NH and C=NR bonding (Figure 2a–c). The constituent peak values were estimated by an area ratio method (Figure 2d). An increase in C=NR bonding intensity was observed for Gel_DA mats relative to pristine gelatin mats. This probably resulted from the partial oxidation of DA molecules during electrospinning, which generates conjugated intermediates like dopaminechrome. Furthermore, a significant increase in C=NR bonding intensity for the Gel_pDA mats supported pDA structure formation after (NH4)2CO3 treatment. To confirm the presence of crosslinking between pDA and gelatin, we estimated the C=O bonding intensities in Gel_DA and Gel_pDA mats from the O 1s core-level spectra. As expected, a significant decrease in the C=O bonding intensity (p < 0.01) was observed for Gel_pDA versus Gel_DA mats (Figure S5b–e), thus establishing that pDA can crosslink gelatin nanofibers.



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The formation of pDA coating on the gelatin nanofibers was also assessed by ATR-FTIR analysis. Pristine gelatin mats displayed ATR-FTIR absorption peaks at 1240 cm-1, 1540 cm-1 and ~1640 cm-1 which were assigned to amide III (C-N stretching and N-H in-phase bending), amide II (N-H bending and stretching) and amide I (C=O stretching) bonds, respectively. A broad band located at 3300 cm-1 was attributed to the combination of amide A and B peaks due to N-H stretching37 (Figure S6a). For Gel_pDA, a significant broadening and increase in the intensity of a band traversing 3200–3500 cm-1 designated the presence of more hydroxyl functionalities resulting from formation of the pDA coating. Notably, all of the inherent spectroscopic features of gelatin were still observed in the Gel_pDA mats. Contrarily, the welldefined gelatin peaks were completely obscured in pDA-coated gelatin mats prepared by conventional methods37. Moreover, the indoline and indole structures at 1515 cm-1 and 1605 cm-1 observed after DA polymerization overlapped with the gelatin amide I and II peaks, leading to formation of intense broad bands. These observations indicate that conventional pDA coating methodologies disrupt the anisotropic properties of electrospun gelatin nanofibers. Given that the formation of a pDA coating introduces additional functional groups onto nanofibers, we examined the wettability of gelatin mats after ADM by measurement of dynamic water contact angles. The wetting dynamics were assessed by goniometric measurements of temporal changes in the water contact angle (Figure 2e). Initially a high contact angle was obtained for ES_Gel mats, which decreased rapidly to achieve a zero-degree contact angle within 50 s. For Gel_DA mats, the zero-degree contact angle was achieved more rapidly, within 25 s. However, the contact angle values decreased gradually for Gel_pDA mats, reaching a plateau of 64.7 ± 1.2°. These data suggest that DA polymerization enhanced the hydrophobicity of the pDA-coated gelatin surface relative to that of the pristine gelatin surface,


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rendering an observed final contact angle that was consistent with reported values for other pDA-coated substrates24,38. Because oxidative polymerization of DA generates heteroaromatic chromophores, we examined the fluorescence emission characteristics of the gelatin nanofiber mats. The ES_Gel mats displayed weak fluorescence when excited at a wavelength of 360 nm, whereas intensely blue-colored fluorescent nanofibers were visible in the Gel_pDA mats (Figure 2f,g)39. Fluorescence imaging further confirmed the uniform distribution of pDA on the nanofiber surfaces. A λ-scan of ES_Gel mats revealed a broad band spanning the 415–440 nm region, but no characteristic emission maximum was observed (Figure S6b). Contrarily, Gel_pDA mats displayed a characteristic emission maximum at ~457 nm, and the emission intensity increased with increasing concentrations of DA in the dope solution (Figure S6b). Next, we used RP-HPLC to investigate differences between the oxidative products formed by the ADM coating method and a conventional Tris-HCl-mediated coating method. The composition of the pDA coating facilitated by the Tris-HCl route is complex, and contains a mixture of oligomeric species40. The high aggregation tendency of the oligomers and the heterogeneity of the pDA coating are responsible for the surface roughness of Tris-HClgenerated products. Supernatants were collected from Gel_pDA mats and the pDA coating prepared by the Tris-HCl method and then compared via RP-HPLC (Figure 2h). The monomer peak of DA at ~6 min was barely visible in the Gel_pDA supernatant, and only a single peak at ~25 min was observed. This signifies a complete conversion of monomeric DA into polymeric DA, as well as the homogenous polymerization of DA41. However, several peaks were observed in the Tris-HCl pDA supernatant, in addition to a small amount of monomeric DA. These results imply that the



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homogenous product formed by ADM accounts for the uniform inter-fiber fusion, smooth surface coating, and photoluminescent properties of Gel_pDA nanofibers. The increased formation of nanofiber junctions and the subsequent generation of a pDA surface coating are expected to enhance mechanical fiber properties. Therefore, we assessed the tensile properties of the as-spun, pristine gelatin mats and the DA-doped gelatin mats before and after ADM. Figure 2i shows the typical stress-strain curves for ES_Gel, Gel_DA, and Gel_pDA samples. Analyses of the mechanical characteristics indicated an increase of approximately 2fold in ultimate tensile strength and work of failure for Gel_pDA mats versus pristine ES_Gel mats, where the increase is dependent on the amount of DA initially present in the dope solution (Table S1). Thus, we have developed a simple crosslinking protocol that can be carried out entirely in the solid-air interfaces without exposing the electrospun gelatin to organic solvents. As such, the approach allows nanofiber crosslinking at junction points and maintains the anisotropic properties. In contrast to conventional catecholamine coating methods, the current protocol produced a smooth pDA coating along the entire length of the fibers, but did not compromise the porous structure of the electrospun mats. The pDA coating also afforded a blue photoluminescence to the nanofibers and decreased their wettability.


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Figure 2. Characterization of polydopamine crosslinked electrospun gelatin nanofibers prepared by ADM. N 1s core-level spectra of (a) ES_Gel, (b) Gel_DA, and (c) Gel_pDA mats. (d) >C=NR bonding ratio for the three mats determined from N 1s core-level spectra. (e) Dynamic contact angle measurements determined for the three ES mats. The inset images show photographs of droplets after 20 s. Fluorescence images of ES_Gel (f) and Gel_pDA (g) nanofibers showing the enhanced fluorescence properties after pDA formation. Scale bar = 20 µm. (h) RP-HPLC analysis of the composition after the formation of polydopamine by Tris-HCl and ADM protocols. For a comparison, the elution profile of pure dopamine is also shown. (i) Mechanical properties of ES gelatin mats showing enhanced tensile properties after pDA crosslinking. 3.3. Preparation and Characterization of Osteoconductive Collagen Scaffolds. As mentioned above, ADM supersaturates the surrounding atmosphere with NH3 and CO2. The later readily dissolves in water or aqueous interfaces to form carbonic acid. Due to the increased pH created by an ammoniacal atmosphere, carbonic acid deprotonates and generates carbonate ions, which



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can then be combined with Ca2+ ions to produce CaCO3 (Scheme 1). We hypothesized that the simultaneous crosslinking of DA and mineralization of calcium facilitated by ADM would enhance the mechanical and biological properties of electrospun collagen. To investigate this hypothesis, 20 mM calcium chloride (CaCl2) was mixed with a DAcontaining collagen dope solution prior to electrospinning. After initial optimization, electrospun mats with smooth nanofibrous morphology were successfully produced. The mats were termed Coll_DA_Ca2+ and Coll_pDA_Ca2+ scaffolds, for as-spun, DA-/Ca2+-containing mats and the same mats after ADM treatment, respectively. Interestingly, the scaffold color was markedly altered upon addition of CaCl2 to the dope solution (Figure 3a, inset). The inherent redox characteristics of DA and increased conductivity stemming from Ca2+ incorporation into the mats probably triggered partial electrochemical oxidation and the color change, leading to formation of a pDA structure42. After ADM, color of the mats became intense brown and the porosity of the Coll_pDA_Ca2+ mats was greatly decreased (Figure 3a,b). A considerable increase in fiberover-fiber adhesion and the number of nanofiber junctions was also observed for the Coll_pDA_Ca2+ mats, resulting in a dense nanofiber network with intermittent pores. HR-TEM micrographs revealed the dissemination of CaCO3 minerals along the length of the fiber axis as well as at the junctions (Figure S7a-d). The diffused ring patterns appeared in the selected area electron diffraction (SAED) confirmed the formation of amorphous CaCO3 (Figure S7e,f). Interestingly, confocal fluorescence image scans indicated intense blue and green fluorescence in the Coll_DA_Ca2+ and Coll_pDA_Ca2+, suggesting enhancement in the photoluminescence of ES collagen after mineralization (Figure S8). Uniaxial tensile testing revealed considerable changes in the mechanical parameters for collagen mats with different compositions (Figure 3c; Table S2). Pristine electrospun collagen


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(ES_Coll) mats displayed brittle-like characteristics, including high tensile strength, stiffness, and a low failure strain. A marked increase in the tensile properties was observed for Coll_DA_Ca2+ which displayed plastic deformation after peak stress, indicative of significant improvements in the elastic properties of the scaffolds and an increase in toughness. These results suggest that the partial oxidation of DA during electrospinning caused considerable increase in the tensile properties of ES_Coll. Tensile strength and Young’s modulus showed further enhancement for Coll_pDA_Ca2+ mats, and no necking was observed, signifying that simultaneous mineralization and pDA formation afforded maximum scaffold reinforcements. These findings imply that numerous soldered junctions and branched structures result from pDA crosslinking consolidate the nanofiber interfaces, whereas the formation of a mineral phase reinforces the interface to impart high strength and stiffness. Next, the potential of various nanofibrous mats to support the growth of human fetal osteoblasts (hFOB) was investigated by MTS-based cell proliferation and ALP activity assays at different day points (3, 6, and 9 days) of cell growth on various collagen scaffolds. Cells seeded on electrospun mats displayed significant increase in proliferation at 6 or 9 days of post seeding compared to TCP (Figure 3d). No apparent differences (p>0.05 at 3, 6 and 9 days p.s.) were observed between ES_Coll and Coll_pDA mats. However, a greater increase in metabolic activity was observed for cells seeded on Coll_DA_Ca2+ and Coll_pDA_Ca2+ mats in comparison to ES_Coll and Coll_pDA mats. Supplementary Table S3 details the statistical comparison on cell proliferation between various groups. The results confirmed that simultaneous crosslinking and mineralization enhanced hFOB proliferation. Among the various groups, Coll_pDA_Ca2+ enhanced the osteogenic differentiation of hFOB, as indicated by >1.5fold increase in ALP activity (an early indication of osteogenesis) after 9 days p.s. (Figure 3e).



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Altogether, these results demonstrate the enhancement in the osteoconductive potential of collagen after simultaneous pDA crosslinking and mineralization.

Figure 3. Evaluation of the osteoconductive properties of electrospun collagen-CaCO3 scaffolds. (a) SEM image of Coll_DA_Ca2+ with the inset showing a photograph revealing brown coloration indicative of the formation of pDA in the as-electrospun mats. (b) SEM micrograph of Coll_pDA_Ca2+ with the insets showing photographs (top-left) displaying dark brown coloration and a TEM image (top-right). Scale bar in the SEM and TEM images is equal to 5 µm and 1 µm, respectively. (c) Stress-strain curves for various electrospun collagen scaffolds. (d) MTS and (e) ALP assay results showing significant enhancement in osteoblast proliferation and differentiation in Coll_pDA_Ca2+ compared with the control coverslip (CS) and other electrospun collagen scaffolds. For clarity, significant values are shown between Coll_pDA and Coll_pDA_Ca2+ samples only. To gain insight into the depth of cell infiltration and visualize morphology of the cells seeded on various scaffolds, confocal images of hFOB cells labelled with Hoechst dye and Alexa Fluor 647-Phalloidin were captured (Figure 4a-d, left panel). Confocal z-stack images demonstrated cell penetration into various mat preparations at 9 days p.s. (Figure 4a-d, z-


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sections on the top of left panel images). By taking advantage of the photoluminescent properties of the scaffolds, we used confocal microscopy to confirm the integrity of the fiber mats at 9 days p.s. in the cell culture. The fluorescence images indicated presence of intact collagen fibers after masking the fluorescent signals from the cells, thus confirming the stability of the mats in the culture media (Figure 4a-d, second left panel). Enhancement in the hFOB cell populations and spreading was also observed after 9 days p.s. on various scaffolds as indicated by CellTracker Green CMFDA and nuclei (blue) staining (Figure 4a-d). Confocal z-sectioning further demonstrated the level of cell infiltration into various scaffolds as indicated by the z-stack thickness plots (Figure 4a-d, right panels). Zsection plots of the XY images indicated thicker cell coverage in Coll_DA_Ca2+ and Coll_pDA_Ca2+ than on ES_Coll and Coll_pDA mats (Figure 4a-d, right panels).



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Figure 4. Confocal microscopy of hFOB cells seeded on different collagen scaffolds at 9 days post seeding (a) ES_Coll, (b) Coll_pDA, (c) Coll_DA_Ca2+, (d) Coll_pDA_Ca2+. The left panel is the hFOB cells labelled with Alexa Fluor 647-Phalloidin and Hoechst. The second left panel is the confocal fluorescent images highlighting the presence of intact collagen fibers after immersion in culture media after masking the fluorescence signals from the cells. The third panel shows the XY scans of CMFDA stained hFOB cells. The plots next to the XY CMFDA images (right panel) are the z-scan sections showing the position and thickness of hFOB cell layers grown on various mats. Scale bar = 10 µm.


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Analysis of scanning electron microscopy images of osteoblasts grown on various substrates at 6 and 9 days are shown in Supplementary Figure S9. Cells seeded onto Coll_pDA_Ca2+ mats showed characteristic polygonal morphologies with extensive network formation over the nanofibrous scaffold, even at early stages of culture (3 days; Figure 5a). After 6 and 9 days, the cells retained their polygonal morphology and formed many confluent multilayer nodules of interconnected osteoblast cells (Figure 5b,c). This is an important feature of the in vitro differentiation/maturation of osteoblasts43. Immunofluorescence images confirmed the expression of important osteogenic proteins (osteocalcin, osteopontin, and bone morphogenetic protein) in cells seeded on various electrospun mats (Figure 5d–g). From the confocal images, we determined the mean fluorescence intensity (MFI) of the respective marker proteins from cells seeded on various electrospun mats (Figure S10). The results indicated a marked increase in the MFI values for Coll_DA_Ca2+ and Coll_pDA_Ca2+ mats. Therefore, simultaneous mineralization and pDA crosslinking of collagen nanofibers increased osteoblast proliferation, differentiation, maturation and expression of key osteogenic markers when compared to Coll_pDA mats. It is likely that the increase in matrix stiffness and calcium ions present in the mats may contribute to the enhancement of focal adhesion and cytoskeletal organization of hFOB44. Several studies investigating the influence of CaCO3 or hydroxy apatite minerals on the osteoblasts cell proliferation were previously reported45-50. The composite mats were prepared by adding a slurry of preformed minerals in to the dope solution. As a result, these mats require higher amounts of minerals (>20% w/w of the polymers) to achieve adequate mechanical strength and osteoconductive properties. However, this study indicated that mechanically tough



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collagen mats with photoluminescent and superior osteoconductive properties can be achieved at a much lower mineral:matrix ratio, using our methodology.

Figure 5. Osteoconductive properties of various nanofibrous collagen scaffolds. SEM micrographs of osteoblasts grown over Coll_pDA_Ca2+ scaffolds after (a) day 3, (b) day 6, and (c) day 9. Scale bar is 10 µm. Immunofluorescence images showing the expression of important osteogenic marker proteins after 11 days p.s. (d) coverslip; (e) ES_Coll; (f) Coll_pDA; (g) Coll_DA_Ca2+ and (h) Coll_pDA_Ca2+. Scale bar is 20 µm. 3.4. Preparation of Durable Antimicrobial Wound Dressings. Surface coating of electrospun nanofibers with pDA have been used to covalently link cell adhesion peptides, growth factors, bone matrix protein-2 and bone forming peptide-1 through imine functionalization or Michael addition reactions51-54. Having verified the generation of pDA crosslinking and the formation of osteoconductive scaffolds by ADM, we next evaluated the prospective utility of our approach in the preparation of durable wound dressings for biomedical applications. We used ES_Gel mats 31 ACS Paragon Plus Environment

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for this purpose owing to their numerous advantages such as good biocompatibility, biodegradability, absorbency, and cell adhesion properties, easy availability, and low cost. We first examined the cytotoxicity of the mats for primary human dermal fibroblasts (hDFs), which has been used extensively for evaluating skin compatibility of dressings and implants. Exposure of hDFs to ES_Gel or Gel_pDA mats for 24 h failed to alter cellular or nuclear morphology, cytoskeletal architecture, or cell adhesion properties (Figure 6a–c). MTS-based chromogenic assay further showed that pDA coating of the gelatin mats did not interfere with hDFs viability, confirming the biocompatibility of the crosslinked scaffold (Figure 6d). Therefore, the oxidative polymerization of DA-loaded electrospun gelatin nanofibers by ADM yields a benign, noncytotoxic pDA coating on the fibers.

Figure 6. Biocompatibility evaluation of Gel_pDA mats. Confocal images showing the morphology of primary human dermal fibroblasts (hDFs) seeded on (a) pristine ES_Gel, (b) Gel_DA, and (c) Gel_pDA. Scale bar = 20 µm. (d) MTS assay confirming the metabolic activity of hDFs seeded on various ES mats and coverslips (CS). We selected two US FDA approved lipopeptide antibiotics, vancomycin and caspofungin, to assess the antimicrobial efficacy of the pDA-coated electrospun nanofiber mats. Vancomycin 32 ACS Paragon Plus Environment


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is a mainstay therapeutic agent for invasive infection treatment against the “superbug”, methicillin-resistant S. aureus (MRSA)55, whereas caspofungin is approved for treatment of invasive fungal infections and for patients who are allergic to amphotericin B and itraconazole antifungal medications56. The concentration of the antibiotics was chosen to maintain the antimicrobial component at 50% lower than that maintained by the commercial Ag-based wound dressings, Aquacel® Ag and Melgisorb®. Incorporation of antibiotics at 0.5% (w/w) concentration into gelatin nanofibers did not alter fiber morphology before or after ADM (Figure 7a–d). Dynamic contact angle studies indicated that the contact angle decreased abruptly for Vanco_Gel_pDA mats to 0º within 30 seconds (Figure S11). For Caspo_Gel_pDA, the values decreased gradually and a plateau of 70.3±1.6º was observed after 60 seconds. These results confirm that incorporation of vancomycin decreased the wettability of the mats quite significantly compared to caspofungin. Mechanical studies indicated that both the antibiotics enhanced the tensile strength and stiffness while decreasing the elasticity (Table S1). To confirm the retention of antibiotic activity post-incorporation, a disc diffusion assay was performed following the Clinical and Laboratory Standard

Institute (CLSI) guidelines. Both

Vanco_Gel_pDA and Caspo_Gel_pDA mats displayed excellent zones of inhibition against pathogenic Gram-positive S. aureus, MRSA, and assorted C. albicans bacterial strains compared with Aquacel® Ag and Melgisorb® dressings (Figure 7e,f; Table S4), indicating that the crosslinking process did not impair antimicrobial properties. To determine whether adherent pDA crosslinking can maintain long-term antimicrobial activity, we investigated the leaching durability of the mats in accordance with the guidelines of the American Society for Testing and Materials for antimicrobial-coated medical devices57. Briefly, Vanco_Gel_pDA and Caspo_Gel_pDA mats were immersed in PBS (pH 7.0) with


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continuous shaking. After the indicated intervals, mats were taken out followed by their washing with water and assessment by the disc diffusion method. A complete retention of antimicrobial activity was observed in both types of antibiotic-loaded mats even after 20 days of immersion in PBS (Figure 7g,h). In particular, the vancomycin-loaded mas exhibited superior retention of anti-MRSA activity relative to that exhibited by the commercial Ag-based wound dressing, Aquacel® Ag (Figure 7g). Previous studies reported the occurrence of a ~80–90% weight loss in glutaraldehyde-crosslinked electrospun gelatin mats within < 5 h of incubation with PBS, indicative of leaching58. These results confirm the excellent aqueous stability and durability of the antibiotic-loaded mats to leaching conferred by pDA crosslinking. In addition, performance of the entire crosslinking process in the solid phase permitted a high encapsulation efficiency of the antibiotics as well.



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Figure 7. Morphology of antibiotics loaded ES gelatin mats containing DA before and after ADM exposure. (a) and (c) contain vancomycin whereas (b) and (d) contain caspofungin as the 35 ACS Paragon Plus Environment

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antibiotics. The amount of antibiotics loaded was 0.5% (w/w of the polymer). Note that the fibre diameters in vancomycin-loaded mats is significantly decreased due to differences in solvent conditions. Scale bar = 1 µm. Photographs of disc diffusion assays showing the efficacy of Gel_pDA mats loaded with vancomycin (e) and caspofungin (f). Scale bar = 1 cm. Durability of vancomycin (g) and caspofungin (h) antibiotic-loaded Gel_pDA mats to leaching. Note that the antibiotic-loaded Gel_pDA mats displayed excellent durability when compared with the silverbased wound dressing Aquacel® Ag. 3.5. Wound Healing Efficacy of Vancomycin-Loaded, pDA-Crosslinked Mats in a Porcine Deep Dermal Burn Injury Model. Finally, we evaluated the wound healing properties of pristine ES_Gel and Vanco_Gel_pDA mats using a porcine deep dermal burn wound healing model. In terms of anatomical and physiological properties, porcine skin closely resembles human skin, and the wound healing properties of the pig are considered similar to those of the human59. Here, burns were created under anesthetic conditions by placing a hot water beaker preheated to 92°C for 15 sec on the dorsum of the animals (Figure 8a). Eight second degree burns (up to the dermal layer) were created on the thoracic ribs of each pig and distributed in 4 groups. Untreated control served as Group I whereas ES_Gel (Group II), Vanco_Gel_pDA (Group III) and Aquacel Ag (Group IV) served as treated groups. Vanco_Gel_pDA was soaked in PBS for 30 minutes for proper coverage over the injured site and ease of handling whereas ES_Gel and Aquacel® Ag were applied without any prior soaking in PBS (Figure 8b). The wounds were cleaned to remove the burned epithelium debris. Two wounds were left uncovered and were labelled as untreated control burn wounds. To prevent inter-wound cross contamination, Tegaderm film was used for covering the wound dressing area and protected with a white garment to avoid tearing of the dressings (Figure 8b,c). Aquacel® Ag-treated and untreated wounds served as positive and negative controls, respectively. Photographs of the wounds were taken with a Nikon D90 digital SLR camera, and the images were processed using SigmaScan Pro 5 software. For each wound at every time point, the wound size was measured and compared with that of untreated control wounds. 36 ACS Paragon Plus Environment


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Figure 8. Establishment of porcine burn injury model. (a) Digital photographs showing the induction of thermal injury. (b) Photograph showing the application of Vanco_Gel_pDA and Aquacel® Ag covered with Tegaderm wound dressings. (c) Photograph of white garment covering the wounds. Figure 9a–d shows the wound area at the starting day (day 0) and the ending day (day 46) of the wound healing process for all four wound groups (ES_Gel, Vanco_Gel_pDA, Aquacel® Ag, and untreated control). Photographs displaying the progression of wound healing events among the various groups are shown in Figure S12. The total wound size reduction was plotted as a percentage of the initial wound size (Figure 9e, f). An increased wound closure (89.6%) was observed for burn wounds treated with Vanco_Gel_pDA mats versus untreated control wounds (p ≤ 0.01) or wounds treated with pristine ES_Gel mats (p ≤ 0.05). Additionally, a faster wound healing rate was observed for wounds treated with Vanco_Gel_pDA mats than for the other three groups at 2 weeks after injury. Moreover, the final wound closure area displayed improvements compared with those afforded by the commercial Ag-based dressing. These results indicate that antibiotic-loaded, pDA-crosslinked electrospun nanofibrous mats promote rapid re-epithelialization of burn wounds, and establish the in vivo efficacy and biocompatibility of the scaffolds.


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Figure 9. In vivo wound healing efficacy of Vanco_Gel_pDA mats in a porcine burn injury model. Digital photographs showing the wound closure during the course of the treatments. (a) Untreated, (b) ES_Gel, (c) Vanco_Gel_pDA mats, and (d) Aquacel® Ag wound dressings. For clarity, only photographs of the wounds before and after treatment are shown. (e) Temporal changes in the wound closure area after various treatments during the entire course of the study. (f) Quantitative estimation of the wound closure area for treated and untreated wounds. Note the increased wound closure for Vanco_Gel_pDA mats when compared with pristine and silverbased dressings.



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4. Conclusions In this study, using the oxidative polymerization of catecholamines under alkaline conditions, we successfully developed protocols for the generation of durable antimicrobial or mineralized scaffolds for advanced wound dressings and bone tissue engineering, respectively. The protocol outlined in this investigation offers a simple strategy for nanofiber production that utilizes a polycatecholamine coating and relatively mild conditions for the preparation of electrospun collagen or gelatin scaffolds. Our strategy employs no organic solvents, toxic additives, or extreme temperatures, and is therefore readily applicable to manufacturing practices. Furthermore, our method confers additional benefits to the procured nanofibrous scaffolds, including excellent surface smoothness, desirable mechanical properties, the ability to anchor peptide antibiotics to the nanofiber surface for an extended period of time, inherent nanofiber photoluminescent characteristics, and both in vitro and in vivo biocompatibility. The polycatecholamine coating can also be mineralized to generate osteoconductive scaffolds. Accordingly, our approach opens a new direction of research utilizing catecholamines as crosslinkers for biomacromolecules, and expands the portfolio of technologies for the development of multifunctional nanostructures for biomedical applications. Associated content Supporting information available: Figure S1 to S12 comprising of pictures of all dope solution, Atomic force microscopy analysis, Morphology of the ES_Gel and ES_Coll nanofibers incorporated with NE and AM before and after ADM, XPS deconvolution analyses, ATR-FTIR spectra, Flourescence spectral scans, TEM micrographs, SAED pattern, Confocal fluorescence images, SEM images of the hFOB cells

cultured on different nanofibrous

collagen scaffolds, Plot showing the mean fluorescence intensity measured from the confocal


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images after immunostaining with respect to protein markers, Dynamic contact angle measurements for the antibiotic loaded mats and photographs of in vivo porcine burn injury model, Table S1-S4. Supporting Videos: Supporting Video 1 shows initial electrospinning of Van_Gel_pDA nanofibers over aluminium foil. Supporting Video 2 shows the formation of Van_Gel_pDA mat after 3 h of electrospinning. Acknowledgements This research was supported by the Translational and Clinical Research Flagship Program of Singapore National Research Foundation (NMRC/TCR/008-SERI/2013) and administered by National Medical Research Council of Singapore Ministry of Health. R. L thanks the funding support from Co-operative Basic Research Grant from Singapore National Medical Research Council (NMRC/CBRG/0048/2013) and SNEC Ophthalmic Technologies Incubator Program grant (R1181/83/2014); N.K.V. acknowledges LKC Medicine, Nanyang Technological University Singapore Start-Up Grants (L0412130 & L0412290) and the Ministry of Education Singapore AcRF-Tier I Grant (2014-T1-001-141).



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Under

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Conditions,

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ASTM

International,

West

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TOC GRAPHIC


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Photoluminescent Crosslinked Mats for Wound Healing 1. Vancomycin (NH ) CO 4 2 3 2. Caspofungin exposure

Biopolymers + Catecholamines

(NH4)2CO3 2NH3(g) + CO2(g) + H2O(g) NH3(g) + H2O NH4OH CO2(g) + H2O HCO3¯  CO3═ + Ca2+CaCO3(s)

+ + + + +

High Voltage

CaCl2

(NH4)2CO3 exposure

In-situ Mineralized Photoluminescent Osteoconductive Scaffold

ACS Paragon Plus Environment

Latent Oxidative Polymerization of Catecholamines as Potential Cross

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