Surface Modification of Eggshell Membrane with Electrospun Chitosan

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Surface Modification of Eggshell Membrane with electrospun Chitosan/ Polycaprolactone Nanofibers for Enhanced Dermal Wound Healing Preetam Guha Ray, Pallabi Pal, Pavan Kumar Srivas, Piyali Basak, Somenath Roy, and Santanu Dhara ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00169 • Publication Date (Web): 31 Aug 2018 Downloaded from http://pubs.acs.org on August 31, 2018

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Graphical Abstract 335x191mm (150 x 150 DPI)

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Surface Modification of Eggshell Membrane with electrospun Chitosan/ Polycaprolactone Nanofibers for Enhanced Dermal Wound Healing Preetam Guha Ray1,2, Pallabi Pal1, Pavan Kumar Srivas1, Piyali Basak2, Somenath Roy3, Santanu Dhara1* 1

Biomaterials and Tissue Engineering Laboratory, School of Medical Science and Technology (SMST), Indian Institute of Technology Kharagpur, Kharagpur 721302, India 2

School of Bioscience and Engineering, Jadavpur University, Kolkata-700032, India

3

Central Glass and Ceramic Research Institute, Khurja Center, Khurja-203131

*corresponding author Dr. Santanu Dhara E-mail: [email protected]

Abstract Eggshell membrane (ESM), a naturally occurring microfibrous biopolymer network comprising collagen I, V, X, GAGs and other significant proteins, is responsible for guided tissue regeneration. The extraction methodology of ESM and surface topography of the microfibers impede its extensive usage in skin tissue engineering. Herein we deploy a unique route of ESM surface modification utilizing chitosan/polycaprolactone (CS/PCL) nanofibers to fabricate a bilayered scaffold for wound healing application. Microstructural and surface topographic analysis of the construct confirms bilayered structure of the composite with smooth nanofibers of CS/PCL decorated on ESM. The two layers were corsslinked by carbodiimide chemistry as confirmed by XPS and FTIR analysis. Cytocompatibility of the scaffolds was evaluated with Human dermal fibroblast (HDF) cells culture study. The biomimetic architecture and composition of modified ESM facilitated extensive cell adhesion, migration and proliferation while an impeded cell 1 ACS Paragon Plus Environment

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adhesion was observed on the natural tissue. Moreover, owing to the presence of ESM, the scaffolds adhered naturally to the wound bed while implanted on full-thickness wound in rat model. Further, nanofiber modified ESM group showed extensive host cell migration and proliferation thus leading to faster re-epithelization and dermal regeneration with high collagen deposition in comparison to natural ESM. The above in vitro and in vivo results substantiate the effect of nanofiber functionalization on ESM surface thus making the bilayered construct a potential dermal substitute. Keywords: Eggshell Membrane, Chitosan/Polycaprolactone Nanofibers, Surface Modification, Human Dermal Fibroblast Cell adhesion, Full-Thickness Wound Healing.

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1. Introduction: Successful regeneration of wound involves complex biochemical phenomenon which include interaction between dermal and epidermal cells in order to form the extracellular matrix and promote angiogenesis, regulated through a cascade of growth factors and cytokines.1-2 In case of chronic wounds or excessive skin loss, immediate dressing of the wound bed is required for protecting it from exogenous microbial invasion.3 Apart from acting as an antimicrobial agent, an ideal wound dressing material should aid in removal of exudates while keeping the fluids and wound environment intact thus maintaining the aesthetic outlook of the wound bed.4-5 To fabricate an ideal scaffold, it is of paramount importance to select a polymer and fabrication methodology which can generate a scaffold with suitable surface topography that can facilitate cell adhesion, proliferation and cross-talk between cell lines to regenerate the extracellular matrix.6-10 Further, it was also reported that scaffolds possessing nano/micro fibers possess better wound healing characteristics owing to their ability to mimic the extracellular matrix (ECM).11-13 Eggshell Membrane (ESM) is a thin and naturally occurring protein-based fibrous tissue that lies in between the mineralized eggshell (ES) and egg yolk to provide protection against bacterial invasion and shows close resemblance with ECM constituents. The thickness of the membrane is approximately 50-70 μm and microstructural architecture of the ESM has entangled microfibers complemented by high porosity.14 The ESM microfibers are primarily composed of proteins (80-85%) and amongst which approximately 10% are collagen (Type I, V, X) and rest 7075% belong to other proteins and glycoproteins.15-18 Each fiber consists of collagen-rich core and glycoprotein-rich cortex.19 Presence of keratin in ESM fibers was also reported.20 The ability of ESM to mimic the ECM along with its unique chemical composition makes it a potential biomaterial for tissue engineering applications. Additionally, it is easily available, biocompatible, 3 ACS Paragon Plus Environment

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contaminant free and green material that can be easily functionalized or modified according to the need.21 The above physical and chemical properties of ESM facilitate its application in guided tissue regeneration.22 Maeda & Sasaki for the first time reported the usage of natural eggshell membrane as a biological dressing material in treatment of burn wounds.23 Following which Yang et al. successfully implemented raw ESM as a biological dressing material in split thickness skin grafting but some of the patients suffered from hypertrophic scar.24 Later, Fujita et al. immobilized hydrolyzed eggshell membrane with a biopolymer for studying attachment and proliferation of human dermal fibroblast.25 The hydrolyzed ESM immobilized biopolymer displayed good cell adhesion and proliferation but nothing was mentioned about the antibacterial activity or role of naturally occurring proteins in ESM. In an approach to enhance solubility, biodegradability, and processing of ESM into tissue engineering scaffolds or dressing material, soluble eggshell membrane protein (SEP) was extracted from ESM. Further SEP was combined with various biocompatible polymers like poly(ℇ-caprolactone) (PCL)26, poly (lactic-co-glycolic acid) (PLGA)27, poly(D,L-lactic acid) (PDLLA)28 and poly (ethylene oxide) (PEO)29 to facilitate the electrospinning of protein based nanofibers. Though SEP based nanofibers showed good cell adhesion and proliferation but the nanofibers essentially lack antibacterial activity with loss of proteins, glycosaminoglycans (GAGs), Hyaluronic acids as compared to natural ESM21. Moreover, the natural ESM and SEP based nanofibers displayed similar biodegradation rate when exposed to trypsin30. Though native ESM or hydrolyzed ESM has been successfully used in treating wounds but they had their own disadvantage. Various methods were adopted to modify the naturally occurring ESM but none of them met expectations due to lack of clarity regarding maintaining the proteinaceous structure or their antibacterial activity.



In this article, we intend to develop a novel nano/micro biocomposite matrix by crosslinking electrospun nanofibers of chitosan (CS) and poly(ℇ-caprolactone) (PCL) to chemically modified ESM. To the best of our knowledge, this is the first report to modify natural ESM with polymeric nanofibers to form a single bilayered matrix, without disturbing its naturally occurring morphology and chemical compositions. The arrangement of nanofibers limits the pores thereby maintaining a controllable pore size distribution in order to reduce the water vapor transmission rate (WVTR) and also create a microenvironment for better cell adhesion and proliferation. It was also observed that inclusion of PCL in the matrix not only redeems the highly hydrophilic nature of the whole matrix owing to ESM and CS but also increases the tensile strength of the scaffold. Owing to the presence of chitosan, the nanobiocomposite displayed better biocompatibility and antibacterial activity in comparison to the native ESM. The ability of ESM to naturally integrate to the wound bed facilitated integration of the novel nanobiocomposite to the wound bed. Moreover, the presence of CS/PCL leads to faster wound closure, re-epithelization and reconstitution of collagen matrix in comparison to the natural ESM. 2. Materials and methods 2.1. Extraction of ESM The chemical modification of ESM using acetic acid was performed as described in our previous report31. In brief, egg yolk was first separated and eggshell containing the membrane was thoroughly washed in de-ionized water (DI) following which it was exposed to 5% acetic acid for 20 mins. Post incubation, eggshell with the membrane was rinsed with copious amount of DI water to remove any trace of unbound acid moiety from the surface of membrane to avoid further contamination. The extracted air dried membranes, named as ESM AA, was further stored in desiccator for later use for comparison.


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2.2. Surface Modification of ESM via Electrospinning The surface modification of ESM AA was carried out using nanofibers of CS and PCL. The fabrication of CS/PCL nanofibers were inspired from a previously illustrated report by Shalumon et.al.32 Briefly, 1g of CS (MW 700 kDa; > 90% deacetylated, Marine Chemicals, India) and 8g of PCL (Mn 70−90 kDa; Sigma-Aldrich) were dissolved individually in 100 ml solvent mixture containing formic acid/ acetone (70:30) to form 1% CS and 8% PCL solution respectively. The above solutions of CS and PCL were blended in 1:2 ratios and mixed for 2h before use. The asprepared blend of CS/PCL was homogenized and poured carefully into a 5 ml syringe with a 26gauge blunt needle tip. ESM AA samples were cut into appropriate shapes and affixed onto an aluminium foil which is used to cover the grounded stationary collector placed at a distance of 8 cm from the tip of the needle. The syringe containing polymer solution was mounted on a syringe pump (KD Scientific, Switzerland) and the flow rate was maintained at 0.5 ml/h. Electrospinning was performed under a constant voltage supply of 22 KV (30 KV, Glass Mann, Japan). The electrospinning of CS/PCL over as-affixed ESM AA was continued only for ~ 5-10 mins in order to get a thin layer of the nonwoven nanofibrous sheet on the chemically modified natural tissue. The as-obtained uncrosslinked bilayered matrice was then dried at room temperature for 1h. The samples were then crosslinked using 1:1 solution of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (Sigma) and 0.05 M N-Hydroxy succinimide (NHS) (Sigma), respectively, in DI water for 2h. The crosslinking procedure was followed by inactivation of the active groups using 1M di-sodium hydrogen phosphate (Na2HPO4) (Merck) solution in DI water for 2h, followed by rinsing with DI water for 4 times. Samples were further air dried on teflon sheets at room temperature to obtain a CS/PCL nanofiber modified bilayered scaffold of ESM AA, hereafter termed as CP-ESM. Further, morphological and biochemical evaluations of the as



obtained matrice were carried out to affirm crosslinking of surface modified bilayered membrane, which may eventually facilitate cell growth and wound healing. Details of experimental methodology adopted for microstructural evaluation, surface topography, biochemical analysis, physico-mechanical characterization of the fabricated scaffold are detailed in the Supporting Information. 2.3. In vitro Cytocompatibility Assessment with Human Dermal Fibroblast 2.3.1. Isolation of Human Dermal Fibroblast cells The isolation of human dermal fibroblast (HDF) cells were carried out using circumcised foreskin of human sample (age: 0 – 5 yrs) following a previously reported procedure12. The collection of Human tissue and isolation of HDFs were carried out under the approval from institutional ethical committee (IEC), Indian Institute of Technology, Kharagpur (Ref no. IIT/SRIC/AR/2012). Precisely, the foreskin samples were washed in 1x phosphate buffer saline (PBS) followed by overnight incubation in Dispase II solution (Sigma) for separating the dermis from epidermis. The collagenous dermis was treated with collagenase I (Gibco) followed by neutralization of the enzyme and collection of the fibroblast cells by centrifugation. Further, fibroblast cells were cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic (100 units/ml penicillin and 100 mg/ml amphotericin B). The isolated HDF cells of passage 3 to 7 were used in cytocompatibility studies. 2.3.2. Biocompatibility of HDF cells Adhesion, morphology and proliferation of HDF cells on control and CP-ESM scaffolds were evaluated by field emission scanning electron microscopy (FESEM), DAPI (4',6-diamidino2-phenylindole) staining and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Prior to cell seeding the samples were cut into appropriate dimensions and


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sterilized using 70% ethanol (Merck) for 20 mins on each side under UV irradiation. The sterilized samples were thoroughly washed in PBS followed by overnight incubation in DMEM medium. The HDF cells were seeded at a cell density of 5x104 cells/ sample and incubated at 37 ºC, 5% CO2 and relative humidity of 85%. The cell seeded scaffolds were incubated for 1, 3 and 7 days and post incubation the samples were retrieved to perform the above mentioned assays in order to determine their biocompatibility with HDF cells. At first the cell loaded samples at different time points were fixed with 4% paraformaldehyde in PBS followed by dehydration using serially diluted ethanol (50 – 100%). The as-fixed samples were examined using FESEM analysis to study cell adhesion and proliferation. Further the above fixed HDF cell loaded scaffolds were also stained with Rhodamine–phalloidin (R415, ThermoFisher Scientific) and DAPI (ThermoFisher Scientific) according to manufacturer’s protocol to study the migration of HDF cells within the scaffold. Image acquisition was performed using Axio Observer Z1 microscope (Carl Zeiss, Germany). The cytotoxicity of the above scaffolds was determined by studying the metabolic activity of seeded HDF cells using MTT assay. The cell seeded scaffolds were retrieved at every time point followed by addition of MTT (0.5 mg/ml in PBS) (Sigma) solution to each sample and incubating the same at 37 ºC for 4 h. Post incubation with MTT, the so formed formazan crystals were dissolved using dimethyl sulphoxide (DMSO, Sigma-Aldrich) and the absorbance for the same was measured at 595 nm using a microplate reader (iMark™, BIORAD). Tissue culture plate containing only cells was considered as control whereas wells without cells were considered as blank. Percent cell viability for each sample was calculated using the following equation: % Cell Viability = OD595[Sample] – OD595[Blank]/ OD595[Control] – OD595[Blank] X 100



2.4. In vivo Analysis of Full-thickness Wound Healing The studies performed in this work pertaining to full-thickness wound repair on animal models using the above scaffolds were ethically approved by the ethical committee of Indian Institute of Technology Kharagpur. A total of 27 male Wistar rats (Rattus norvegicus) with an average weight of ~ 250 gm (per rat) were used for the study using the above fabricated scaffolds. The rats were maintained under controlled temperature of 22 ± 2 °C, relative humidity of 50 ± 10% and following a strict and regular photoperiod of 12 h (from 06:00 h to 18:00 h) in a pathogen free atmospheric condition. The in vivo experiments were carried out to analyze the impact of surface modification of ESM AA with CS/PCL nanofibers on full-thickness wound regeneration as compared to bare ESM AA. In order to perform the experiments, six weeks old Wistar rats were divided into 3 groups (n=3) including ESM AA, CP-ESM and open wound. Further the rats were voluntarily anesthetized with an intraperitoneal injection of ketamine hydrochloride (90 mg/kg) and xylazine hydrochloride (10 mg/kg) followed by shaving the dorso-lateral area with an electrical clipper. For each animal a 4 cm2 square shaped wound was created on the dorso-lateral region and the depth of the wound was kept until the subcutaneous panniculus carnosus. Before implantation, the samples were sterilized by incubating them in 70% ethanol (Merck, India) for 20 mins under UV irradiation on each side, followed by repeated wash with 1x PBS. The rats were divided into 3 groups as mentioned above and each group consist of 9 rats so that the experiments at each time interval i.e., 7, 14 and 21 days can be carried out in triplicates. The scaffolds were implanted on wound bed and owing to the presence of ESM, it adhered and integrated naturally onto the wound bed23 without any external facilitation. In order to secure the wounded region, TegadermTM dressing was deployed to cover


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the wounds of all the groups and it was eventually removed post 2 days of incubation. Post incubation for 7, 14 & 21 days, the animals from each group was euthanized and the final length of the cured wound bed was recorded using a Canon EOS 750D digital camera from a fixed distance. The length of the cured wound bed at each time point was evaluated by Image J (NIH, USA) software and the rate of wound healing was calculated using the following the equation: Rate of wound closure (Rwc) = (AWi – AWn)/ AWi X 100 AWi represents the initial area of the wound whereas AWn corresponds to the wound area at the nth day, when the animal was euthanized. Post euthanization at the prescribed time points, the reconstituted tissue along with surrounding uninjured tissue was excised and fixed in 10% formalin – phosphate buffered solution and embedded in paraffin blocks for Hematoxylin & Eosin or Masson’s trichrome (MT) staining. Further, 4 µm thin sections were made using a rotary microtome (RM2135, Leica, Germany) and the sections were stained with Hematoxylin and Eosin reagents following the routine procedures. Further immunohistochemical analysis of microtome sections were performed using anti CD31 (Abcam, USA) following manufacture’s protocol for studying neovascularization and development of blood vessels in order to understand healing kinetics at different time points. The collagen content of the tissue was determined by staining the sections with Masson’s trichrome (MT) stain (Sigma – Aldrich) following manufacture’s protocol. 2.5 Statistical Analysis All data were analyzed with GraphPad Prism software (version 5.02, La Jolla, CA, 394 USA) by one-way ANOVA t-test. The level of significance between data were considered significant at p < 0.05. All experiments were carried out in triplicates and data were denoted as mean ± standard deviation (SD) for n=3 except mentioned separately.



3. Results & Discussion The present study showcases utilization of surface modified ESM based bilayer scaffold for accelerating full thickness dermal wound healing. Dilute acetic acid treatment facilitated extraction of ESM AA while maintaining its structural and functional integrity. Owing to surface roughness or acetic acid treatment, the extracted ESM AA impeded cell adhesion. In order to ameliorate surface roughness of ESM AA, it was decorated with electrospun nanofibers of CS/PCL. CS is a polycationic polysaccharide derived from chitin containing naturally occurring functional moieties like β-(1-4)-linked Ɗ-glucosamine and N-acetyl-Ɗ-glucosamine groups that could be explored for efficient crosslinking with other polymeric substrates. This natural biopolymer possesses desirable characteristics like biocompatibility, biodegradability, nontoxicity, non-immunogenic and antimicrobial activity which potentially reinforces in vitro and in vivo applications.33-34 However, electrospinning of CS alone into nanofibers faced several challenges. Also CS display spontaneous biodegradability in vitro owing to its low mechanical stability and hydrophilicity. Therefore, CS was blended with a synthetic polymer like PCL to render smooth, reproducible and controllable nanofibers through electrospinning. PCL is an FDA approved hydrophobic aliphatic polyester having advantages including biocompatibility, high mechanical stability and processability.35 As reported previously, CS/PCL nanofibrous scaffold facilitated cell adhesion, growth and proliferation for HDF cells while acting as an efficient matrix for wound/burn tissue healing. 12 3.1. Microstructural Evaluation In order to explicitly evaluate and study the matrices, FESEM was performed on both scaffold surface and vertical-cross-sections. FESEM micrographs of ESM AA surface (Figure 1a) 11 ACS Paragon Plus Environment

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(a)

(b)

(c)

(d)

(e)

(f)

(g)

(i)

(h)

Figure 1. Microstructural evaluation of ESM AA and CP-ESM samples. The FESEM micrographs of (a) ESM AA (d) CP-ESM samples clearly depicts the micro/nano architecture of the as prepared samples. 3D AFM micrographs demonstrated the surface roughness of (b) ESM AA and (e) CP-ESM samples and also the effect of nanofiber functionalization on the same. Cross-sectional FESEM micrographs clearly illustrated the single layer and bilayered morphology of (c) ESM AA and (f) CP-ESM samples, respectively, along. Fiber diameter distribution of (g) ESM AA (h) CP-ESM and porosity (i) of both the scaffolds were also depicted.

depicted a highly intertwined network of intact microfibers with diameters ranging from 0.5 – 3.5 µm and the thickness of the sample varied between 50-60 µm (Figure 1c). The average inter-fiber distance in ESM AA samples was measured to be between 10-25 µm. Large inter-fiber distance led to cell penetration and high porosity of 94% for ESM AA. High porosity may lead to elevated WVTR of scaffold thus causing dryness in wound environment. Further, it was observed that there was no distortion in the fibrous architecture but the fiber surface displayed rough morphology (Figure 1b) which retarded cell adhesion. The surface roughness was further studied using AFM. 12 ACS Paragon Plus Environment


The three dimensional AFM micrograph (Figure 1b) demonstrated presence of “pine” like structures on the surface of ESM AA fibers and the average surface roughness was measured to be ~ 0.477 rms. In contrast, FESEM (Figure 1d) and 3D AFM micrograph (Figure 1e) of CP-ESM surface presented a thin sheath of CS/PCL electrospun nanofibers possessing smooth texture and morphology thus lowering the average surface roughness to ~ 0.186 rms. Vertical cross-section (Figure 1f) confirmed amalgamation of CS/PCL nanofibrous layer (~ 15 µm) with ESM AA. Statistical analysis of fiber diameter for CP-ESM revealed that 39% of the nanofibers were in the range of 400 – 700 nm while another 27% was between 100 – 400 nm (Figure 1h). Moreover, nanofiber decoration drastically reduced average inter-fiber distance in CP-ESM to 3 – 9 µm thus limiting the porosity of scaffold to 85% (Figure 1i). This drop in inter-fiber distance had significant impact on controlling cell penetration and WVTR of CP-SEM. Immobilization of nanofibers not only reduced the pore size of the scaffold but also rendered a nano/micro architecture which closely mimic the extracellular matrix thus facilitating the application of CP-ESM in skin tissue regeneration. Further 3D surface profilometry (Figure 2) was deployed to evaluate the homogeneity of nanofiber functionalization and its effect on ameliorating the overall surface roughness of ESM AA. Figure 2a(ii) & 2b(ii) illustrated four parameters to analyze surface roughness for each sample, i.e., Ra (average surface roughness), Rp (maximum profile peak height), Rq (root mean square roughness), Rt (maximum height of peak) and Rv (maximum profile valley depth). It was observed that Ra decreased from 4.639 µm in ESM AA to 4.084 µm in CP-ESM post immobilization of nanofibers. CP-ESM also demonstrated even distribution of nanofibers on the surface thus resulting in drastic fall of Rp to 23.807 µm from 40.958 µm in ESM AA. Moreover, a sharp decrease of Rv from -35.4 µm to -25.4 µm post nanofiber modification supported the above results.


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The above results concurrently suggest symmetric arrangement of nanofibers thus decreasing the inter-fiber spaces. Further, the surface profile for CP-ESM [Figure 2b (i)] appears to be homogeneous and evenly distributed in comparison to an irregular and uneven distribution for ESM AA surface [Figure 2a (i)]. The above results corroborate with FESEM micrographs results thus ensuring homogeneous modification of ESM AA surface.

b(i)

a(i)

a(ii)

c

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Figure 2. Three-Dimensional Surface Profilometry Analysis of a (i) ESM AA and b (i) CP-ESM samples were performed and the corresponding parameters for a (ii) ESM AA and b (ii) CP-ESM were demonstrated. Figure 3c and 3d illustrated the results for surface contact angle measurements conducted on ESM AA and CP-ESM samples respectively.

Another important parameter in analyzing the surface properties is to study its interaction with liquids. Figure 2c & 2d clearly states that the surface of ESM AA is highly hydrophilic with an average contact angle of 36.3º±3. This property of the natural tissue was justified by the presence of naturally occurring glycosaminoglycans (GAGs)21 and negatively charged carboxyl groups (COO-)31. Though, presence of GAGs makes the membrane act as a shock absorber but it also leads to high hydrophilicity which is inappropriate for protein adsorption. In comparison, CPESM showed moderate hydrophobic behavior with an average contact angle of 94º±3 due to the



inclusion of PCL in nanofibers. Therefore, integration of CS/PCL nanofibers not only increased hydrophobicity of the matrix but also provided a homogenous distribution of the fibrous matrix along with tailored pore size and porosity as a whole. 3.2. Chemical and Biochemical Composition of the Fibrous Matrices The chemical and biochemical composition of matrices were evaluated by deploying spectroscopic and staining techniques. Attenuated Total Reflectance - Fourier transform infrared (ATR – FTIR) and X-Ray Photoelectron Spectroscopic (XPS) studies were conducted to substantiate efficient crosslinking between the layers in order to affirm the stability of the bilayered matrix whereas staining with Sirius Red confirm the presence of intact collagenous structure. Figure S1 represents the FTIR spectra for chitosan, PCL, CS/PCL, ESM AA and CP-ESM samples. FTIR analysis for all samples is narrated in the supplementary information. Although FTIR analysis signify successful immobilization of CS/PCL nanofibers on surface of ESM AA but less was understood regarding the NHS/EDC crosslinking. Moreover, the covalent interaction between amine (–NH2) groups of chitosan and carboxyl (–COO-) groups of ESM AA was also not clear. Additionally, very limited footprint of ESM AA was obtained in the IR spectrum of CPESM. The surface characteristics and interactions of the fibers were further investigated using XPS studies. The CP-ESM samples were first plasma cleaned prior to XPS analysis in order to expose the interface, while all other samples were analyzed without pretreatment. Figure. 3 depicts the deconvoluted XPS spectra for C1s, N1s and O1s elemental composition of all the samples. As illustrated in Figure. 3(a), the C1s spectrum of chitosan clearly states its footprint at 284.4 eV corresponding to C-C and C-H bonds. The C1s peak at 285.7 eV and N1s peak at 399.35 eV (Figure. 3c) attributed to C-NH2 bonds thus affirming the presence of amine functional group on


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1 2 3 4 5 6 (a) (c) (b) 7 8 9 10 11 12 13 14 15 16 (e) 17 (d) 18 19 20 21 22 23 24 25 26 27 28 (g) (h) (f) 29 30 31 32 33 34 35 36 37 38 (i) (j) (k) 39 40 41 42 43 44 45 46 47 48 49 Figure 3. The deconvoluted XPS spectra for C1s, N1s and O1s elemental composition of Cs, PCL, ESM AA, CP-ESM, (a-c) XPS spectra 50 for C1s, O1s and N1s of Cs (d-e) XPS spectra for C1s and O1s of PCL (f-h) XPS spectra for C1s, O1s and N1s of ESM AA (i-k) XPS spectra 51 for C1s, O1s and N1s of CP-ESM samples. 52 53 54 55 56 57 58 16 59 ACS Paragon Plus Environment 60


chitosan. Finally, the presence of C-OH group was confirmed at 286.8 eV for chitosan. A relatively small peak for O=C-N bond was also seen at 400.14 eV due to presence of residual chitin in deacetylated Cs. Additionally the deconvoluted peaks of O1s spectrum at 532.33 and 533.07 eV confirmed the presence of C-OH and C-O-C groups of chitosan, respectively (Figure. 3b). In case of PCl, Figure. 3(d) demonstrated three characteristic binding energy peaks at 284.5, 285.45, 288.5 eV which were assigned to C-C or C-H, C-O-C and O-C=O chemical groups, respectively. The O1s spectrum for PCL further substantiated the presence of C=O, C-O-C or O-C=O at 531.7 and 533.07 eV, respectively (Figure. 3e). PCL doesn’t contain nitrogen thus justifying the absence of N1s spectrum. Now, ESM AA sample was evaluated through XPS to substantiate acetic acid functionalization and also confirm the intactness of the naturally occurring chemical groups of ESM. The deconvoluted C1s peak at 286.18 eV (Figure. 3f) and O1s peak at 533.1 eV (Figure. 3g) signifies the presence of –COO- groups. Further the O1s peak at 531.6 eV (Figure. 3g) also represents the C=O groups thus reinstating successful acetic acid functionalization of ESM AA. Also the above C1s peak at 286.18 eV and the N1s peak at 400.30 eV (Figure. 3h) confirms the naturally occurring amide linkages (O=C-N groups) while the C1s and N1s peaks at 285.22 and 399.66 eV, respectively confirms the presence of –C-NH- groups from collagen. The above amine and amide linkages elucidate the intact proteinaceous structure of ESM AA. Further strong footprints of GAGs were confirmed from C1s and O1s binding energies at 286.18 and 532.25 eV, respectively corresponding to –OH groups. Also, O1s peak at 533.1 eV certainly represents C-O-C bridging in GAGs chemical structure. Finally, C1s peaks at 284.6 and 285.22 eV represents the saturated (CC) and unsaturated (C=C) hydrocarbon backbone of ESM AA samples.


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The deconvoluted C1s, N1s and O1s spectrum of CP-ESM was inspected to sought for fibrous interactions between nanofibers and microfibrous at the interface. It was speculated that NHS/ EDC activation of the –COO- groups of ESM-AA would lead to covalent bonding with free –NH2 groups of chitosan. The intensity of N1s peak at 400.26 eV (Figure. 3k) increased abruptly indicating a surge in amide linkages (O=C-N) in CP-ESM scaffolds. Additionally, it was seen that the C1s peak for C-N bond has a significant shift from 285.22 eV in ESM AA to 284.8 eV in CPESM (Figure. 3i). Also there was a sharp increase in peak intensity of C1s at 284.8 eV for CPESM samples as compared to the natural tissue thus indicating presence of more C-N bonds through newly formed amide linkages. Further there was a sharp decrease in peak area or intensity of C1s at 286.18 eV and O1s at 533.04 eV (Figure. 3j) for CP-ESM samples representing a significant decrease in carboxyl groups. The above results clearly states that the free carboxyl groups on ESM surface were covalently bonded to free amine groups of chitosan through amide linkages. Additionally, the footprint of PCL from nanofibers and GAGs from ESM AA was also spotted at C1s 285.7 eV (Figure. 3i) due to presence of C-O-C and C-OH groups, respectively. The presence of GAGs was also substantiated by O1s peak at 532.2 eV while the existence of collagen was confirmed by N1s peak at 399.61 eV in CP-ESM samples. Also, the presence of saturated (C-C) and unsaturated (C=C) hydrocarbon backbone of ESM in CP-ESM was ratified by C1s peaks at 284.3 and 284.8 eV, respectively. The immobilization of CS/PCL nanofibers on ESM AA microfibers by NHS/EDC crosslinking was thus proved and also justified the stability of bilayered construct. It was also observed that the surface of ESM AA reported an overall zeta potential of 5.31±1.21 mV which substantiated presence of COO- groups on the surface (Figure S1b). Owing to its cationic property, CS may also share ionic bonding with ESM AA surface. The surface



modified CP-ESM scaffolds reported a cumulative zeta potential of +2.41±1.1 mV thus neutralizing the negative potential of ESM AA with polycationic chitosan. The above results concluded presence of ionic and covalent interaction between the two layers thus adding to stability of the bilayered complex. Further, Sirius red staining of ESM AA and CP-ESM scaffolds confirm the presence of collagenous protein microfibers in both the matrices (Figure S2). The presence of reddish pink microfibers in both the scaffolds confirms the integrity of collagen from natural ESM which further facilitated proliferation of HDF cells. 3.3. Scaffold Characteristics An ideal wound healing material is expected to absorb wound exudates, maintain an adequately hydrated wound and absorb nutrient from the surrounding fluids in order to facilitate wound healing. The water retention capacity (WRC) of a scaffold determines its efficiency to absorb water and transfer the nutrient to the wound bed. The natural tissue showed above 80% water retention capacity within 15 mins of incubation and by the end of 1h its capacity increased to 110%. Owing to hydrophilic nature of ESM AA, it showed high water retention capacity. In comparison, CP-ESM samples demonstrated moderately high water uptake of upto 40% in the first half an hour and raised to 75% in 1h. The effective crosslinking between the layers and presence of PCL made CP-ESM samples hydrophobic thus restricting water uptake. However, inclusion of ESM and chitosan facilitated water retention upto 85% in 24h, which is adequate for maintaining proper wettability of scaffolds.36-37 Additionally, moderate hydrophobicity of the surface allows it to maintain its dimensional stability when exposed to wet environment for long periods. The wound bed requires adequate hydration for optimal wound regeneration but excessive fluid retention may lead to maceration of surrounding wound tissue and bacterial invasion.


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Moreover, high transmission rate may also lead to dryness of wound bed. The WVTR is a crucial factor in determining the fluid retention capacity of the wound bed. As illustrated in Figure 4(b), ESM AA samples possess a high WVTR in the range of 3132 g m-2 per 24 h. In case of CP-ESM samples, the WVTR was in the range of 2427 g m-2 per 24 h (Figure 4b) which was similar to commercially available grafts and normal healthy skin.37-38 Further in a study conducted by Xu et.al,39 it was reported that 2028 g m-2 per 24 h was the ideal WVTR required for optimal cell proliferation of fibroblast and epidermal cells. A high WVTR of 3132 g m-2 per 24 h for ESM AA may lead to dryness of wound tissue thus leading to scar formation which has been a reported phenomenon for the natural tissue.24 In contrast, CP-ESM displayed a WVTR of 2427 g m-2 per

(a)

(b)

(d)

(e)

(c)

(f)

Figure 4. (a) Water Retention Capacity (%) of ESM AA & CP-ESM scaffolds when exposed to SBF at 37ºC for varying time interval (b) WVTR of ESM AA and CP-ESM scaffolds when exposed to 35% RH at 37ºC for 24h (c) Biodegradation of ESM AA and CPESM matrices when incubated with lysozyme for varying period of time. (d) Protein adsorption capacity of ESM AA and CP-ESM samples when incubated in 5% FBS/PBS protein solution and its evaluation using BCA test at regular time intervals. Mechanical properties of the scaffolds were measured in wet conditions and the plots for (e) Elastic Modulus and (f) Tensile Strength illustrates the same.



24 h which is ideal for maintaining an optimal microenvironment for enhanced wound regeneration. Another important parameter in designing a wound dressing material is to determine its rate of biodegradation. An enzymatic degradation of both the scaffolds were carried out for 30 days at 37 ºC. It was observed that for ESM AA, only 37% of the initial weight was remaining after 15 days of incubation whereas CP-ESM had 42%. The complete degradation of AA samples took 27 days whereas CP-ESM samples degraded in little more than 30 days. The treatment of ESM with dilute acetic acid widened the pore size and also increased the fiber diameter as compared to the natural ESM31 which led to faster degradation rate of the microfibers. The surface of eggshell membrane was modified by a very thin layer (approx. 8-15 micron thick) of CS/PCL nanofibers. The nanofibers were fabricated from a blend of CS and PCL and in presence of lysozyme, chitosan in nanofibers degrades relatively faster which eventually facilitate faster dissolution of remnant PCL nanofibers. However, compact nano-micro fibrous structure of CPESM and presence of PCL delayed the degradation process but the result was appropriate to the required application. The Bicinchoninic Acid (BCA) assay was deployed to study protein adsorption of ESM AA and CP-ESM samples.40 In brief it was demonstrated that amino acids like cystine, tyrosine, tryptophan, and even the peptide bonds can reduce Cu2+ to Cu1+. The Cu1+ ions in turn form a purple-blue color complex when exposed to BCA in alkaline environment. The intensity of the purple-blue color complex is directly proportional to adsorbed protein. As depicted in Figure 4d, CP-ESM samples showed less protein adsorption in comparison of ESM AA for the first hour due less water uptake owing to hydrophobic nature of PCL. Post 3h of incubation, the protein adsorption started increasing for CP-ESM samples followed drastic increase after 6h of incubation.


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ESM AA samples didn’t show much increase in protein adsorption rate after 3h and was always trailing in compare to CP-ESM samples on the following time points. Owing to the hydrophilic surface of natural tissue, the adsorption of desired proteins was competitively inhibited by water molecules and unwanted proteins moieties. The hydrophobic nature of CP-ESM, due to the presence of PCL, restricted water adsorption on the surface thus leading to enhanced protein adsorption. Moreover, the CS/PCL nanofibers possess shorter diameters and it was observed that protein adsorption increases with decreasing diameter of fibers.40 Similarly, presence of hydrophilic microfibers in ESM AA inhibited the protein adsorption kinetics. ESM AA surface showed comparable protein adsorption but the reduction of Cu2+ to Cu1+ could be also due to naturally occurring proteins present in the ESM AA. Though it is not a protein content study but indirectly it corroborates to the above results that the proteinaceous structure of ESM AA was intact. The study of protein adsorption is an important phenomenon as it controls the cell growth and differentiation. The mechanical strength of a scaffold evaluates its sustainability during cellular morphogenesis. Moreover, fibrous architecture of scaffolds must possess adequate tensile strength in order to withstand any external forces at the wound bed during in vivo experiments. The tensile strength of ESM AA sample was measured to be 6.88 ± 0.81 MPa which was in compliance with previous reports.41 However, this value significantly increased ~ 1.5 times for CP-ESM sample and was measured to be 10.27 ± 0.9 MPa. The densely arranged CS/PCL nanofibers and decreasing porosity led to increase in tensile strength of the bilayered composite. Thus it was evident that surface modification of ESM with CS/PCL nanofibers improved tensile property of the scaffold. Further, it was noted that tensile strength of electrospun fibrous scaffold between 0.8 and 18.0 MPa was found to be sufficiently durable for dermal cell culture.42 The average of ultimate tensile



strength for human skin was reported to be 7.7 MPa.38 Also, the composite reported much higher tensile strength in comparison to previously reported scaffolds comprising of PCL/collagen (marine source) ~ 5 MPa,43 PCL/collagen (porcine source) ~ 2.02 ± 0.03 MPa,44 Cs/PCL/collagen ~ 1.92 ± 1.26 MPa45 or other combinations of CS/Col/PCL38. Additionally, the CP-ESM composite reported substantially high elastic modulus of 249 ± 9.47 MPa in comparison to 153.8 ± 7.24 MPa for ESM AA samples thus CP-ESM scaffolds were significantly elastic in comparison to ESM AA scaffolds. The above noted results clearly suggest that crosslinking between microfibers and nanofibers of CP-ESM led to increased tensile strength and elasticity of the bilayered scaffold making it durable for skin tissue engineering applications. CP-ESM revealed a well-defined bilayered matrix with interconnected structures which presented excellent scaffold characteristics and mechanical properties. The in vitro and in vivo activity of the bilayered composite was further evaluated to determine its cytocompatibility and wound regeneration kinetics. 3.4. Determination of in vitro biocompatibility and antibacterial activity The present study was intended to modify the surface properties of ESM with nanofibers in order to promote cell adhesion and proliferation of HDF cells. Figure 5 (a-c & g-i) illustrates the adhesion and proliferation characteristics of HDF cells when seeded onto ESM AA and CPESM matrices. Figure 5a demonstrated poor cell adhesion of HDF cells on the surface of microfibrous natural tissue and even the adhered cells showed deformity in their shape and size on day one. In contrast, the HDF cells were not only able to easily adhere on the surface of CP-ESM samples through their filopodial extensions but also maintained their elongated spindle morphology with well-defined cytoplasmic protrusions (Figure 5d) after day one. Moreover, it was


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1 2 3 4 5 6 (b) (c) (a) 7 8 9 10 11 12 13 14 (d) (e) (f) 15 16 17 18 19 20 21 22 23 24 (g) (h) (i) 25 26 27 28 29 30 31 32 33 (j) (k) (l) 34 35 36 37 38 39 40 41 42 (n) (o) (m) 43 0 44 45 46 47 48 49 50 51 52 53 54 55 Figure 5. Evaluation of in-vitro cytocompatibility. (a-l) FESEM Micrograph and DAPI stained Fluorescence Microscopy images of HDF cells 56 seeded on ESM AA and CP-ESM scaffolds post incubation at 37ºC for 1, 3 & 7 days. (m) Plot for MTT assay at varying time interval to check the 57 metabolic activity of cells when seeded onto ESM AA and CP-ESM samples respectively. (n) & (o) FESEM Micrograph of fractured scaffolds 58 24 deviation (n ≥ 3) of independent samples per group, double asterisks post 7 days of incubation with HDF cells at 37ºC. Error bars depicts standard 59 indicate P < 0.001. (Single arrow: Cellular lumping, double arrow: Cell migration into the scaffold) (Scale: 10 µm) ACS Paragon Plus Environment 60


observed that cells were able to communicate among each other without showing any deformity in their shape or size. On the 3rd day, cells formed a sheet like morphology on the bilayered scaffold (Figure 5h) depicting excellent cell migration and proliferation. Owing to large inter-fiber space in ESM AA sample, the cells showed good cell migration on 3rd day but proliferation was limited. The cells, instead of forming a layered structure, were piling up to form lumps as it was unable to adhere on the surface (Figure 5b). Also the cells portrayed regional migration keeping certain areas in the scaffold devoid of cells. The matrix of ESM AA showed good cell proliferation and migration after 7 days of incubation but the cells were still unable to adhere properly on the fiber surface which impeded formation of cell sheath. The overall negative potential of ESM AA scaffold due to presence of –COO- groups46 as explained before and “Pine” like morphology of ESM microfibers retarded HDF cell adhesion and proliferation. Due to above reasons, cells were unable to grow or adhere homogeneous followed by occurrence of cellular lumps at certain areas on ESM AA samples. On the other hand, HDF cells on 7th day were able to form cell sheath on the CP-ESM fibers with ECM deposition and superior interconnectivity through lamellipodial extension (Figure 5f). It was also noted that with time, microfibers of ESM degraded thus providing naturally occurring proteins for enriching the growth of HDF cells. The fluorescence microscopy results (Figure 5d-f & 5j-l) with rhodamine phalloidin - DAPI staining were concurrent with above observation and demonstrated similar activity of HDF cells on ESM AA and CP-ESM samples. An excellent surge of glowing blue cell nuclei with homogenous distribution was observed for CP-ESM as compared to ESM AA wherein the distribution of slowly increasing cell nuclei were concentrated at certain areas rather than spreading evenly across the matrice. FESEM micrograph of vertical cross-section of cell loaded ESM AA and CP-ESM fractured scaffolds (Figure 5n and 5o) clearly indicated excellent migration & proliferation of HDF cells. It was also


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clear that CP-ESM samples supported better cell proliferation as compared to ESM AA therefore favoring migration of more number of cells into the scaffold. The metabolic activity of HDF cells on above mentioned scaffolds were demonstrated by MTT assay. The active HDF cells were capable of transforming the tetrazolium salt to coloured formazan crystals. As demonstrated in Figure 5m, the CP-ESM samples displayed higher percent viability of HDF cells in comparison to ESM AA samples irrespective of the time interval. Post 7 days of incubation, the ESM AA samples elucidated close to 90% cell viability whereas CP-ESM possessed an excellent 140% cell viability. The above results summarized to the fact that surface modification of ESM AA with nanofibers of CS/PCL had a massive impact on increasing cell viability and cytocompatibility of the scaffolds. The inclusion of CS/PCL nanofibers acted as a passivating layer and reduced the surface roughness, thereby facilitated cell adhesion and proliferation. Also the inclusion of PCL increased hydrophobicity of the construct to a moderate level which encouraged protein adsorption required for cell adhesion and proliferation. The presence of CS/PCL also re-inforced cytocompatibility factor of the bilayered matrices. Though for ESM AA, the cells are directly seeded onto collagenous microfibers wherein the surface roughness, chemical composition and surface charge collagenous microfibers wherein the surface roughness, chemical composition and surface charge of microfibers impeded cell adhesion and proliferation. The anti-bacterial activity of ESM AA and CP-ESM samples were evaluated against S. aureus and E. coli bacterial cultures using disc diffusion or plate count method. Figure 6a revealed that ESM AA scaffold possessed a zone of inhibition of ~ 1.2 ± 0.12 cm2 and ~ 1.13 ± 0.11 cm2 for E. coli and S. aureus bacterial cultures respectively. In comparison, CP-ESM scaffolds demonstrated an increased zone of inhibition of 2.68 ± 0.21 cm2 and 2.19 ± 0.17 cm2 for E. coli and S. aureus bacterial cultures respectively. The plate count experiment also revealed that CP-



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Figure 6. Evaluation of Antibacterial Activity. (a) Zone of inhibition created by ESM AA & CP-ESM when incubated with E. coli and S. aureus respectively for 24 h at 37ºC under continuous shaking. (b)&(c) Determination of colony size when exposed to ESM AA or CP-ESM as compared to control (without treatment) for varying interval of time. Error bars depicts standard deviation (n = 3) of independent samples per group, double asterisks indicate P < 0.001

ESM scaffolds projected a colony size of 3 x 101 cfu/ml and 1 x 102 cfu/ml for E. coli and S. aureus bacterial cultures respectively post 6 h of incubation (Figure 6b & c). No colonies were observed in case of CP-ESM scaffolds post 12h of incubation in E. coli or S. aureus bacterial cultures. ESM AA on the other hand presented a colony size of 1x 104 and 2 x 104 cfu/ml for E. coli or S. aureus bacterial cultures respectively post 6h of contact time. The above results clearly suggest that ESM AA samples retained its antibacterial activity on both gram positive, S. aureus and gram negative, 27 ACS Paragon Plus Environment

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E. coli bacterial cultures owing to the presence of intact GAGs. Presence of polycationic CS reinforced the antibacterial activity of CP-ESM scaffolds by facilitating the adhesion of negatively charged bacterial cells47. Presence of a thin cell wall for E. coli cells made it more susceptible to bactericidal activity of ESM AA and CP-ESM as compared to S. aureus cells. Further, the CS containing nanofibers led to membrane perforation and leakage of cytosolic compounds including DNA and proteins finally leading to cell lysis and disintegration. 3.5. Evaluation of full-thickness wound regeneration 3.5.1 Wound Size Measurements Full-thickness wound on Wister rat was deployed to evaluate the effect of surface functionalization of ESM samples on wound healing efficacy. CP-ESM and ESM AA samples were implanted on the wound bed for 7, 14 and 21 days and the results were compared with open wound (control) (Figure 7). Significant reduction in wound size was observed for CP-ESM samples (50 ± 3.8%) post 7 days of implantation in comparison to ESM AA (25 ±2.6%) or control samples (15 ± 2%). Owing to presence of the natural tissue, the samples portrayed excellent integration to the wound bed without any external aid. Due to high WVTR for

Figure 7. Digital photographs representing the efficiency of wound healing when remain untreated, Control [A (i-iv)] and treated with ESM AA [B (i-iv)] or CP-ESM [C (i-iv)] scaffolds for 7, 14 and 21 days. (D) Plot suggested the wound closure rate and kinetics when treated with ESM AA and CP-ESM scaffolds for 7, 14 and 21 days. (E) and (F) are digital photographs which demonstrated the highly flexible nature of the developed material required during in vivo implantation. Error bars depicts standard deviation (n = 3) of independent samples per group, double asterisks indicate P < 0.001 and triple asterisks signify P < 0.05. (Scale: 1mm) asterisk values indicate p < 0.05 with TCP, and double asterisk values display p < 0.05 with CSCAP.



ESM AA samples and open wound, the wound surface became dry with time whereas the same for CP-ESM samples were adequately moist without any noticeable tissue maceration. Post 14 days of incubation, CP-ESM grafted wounds displayed remarkable wound closure rate of 90 ± 2% with hairs covering most of the healed regions in comparison to ESM AA (65±2.7%) and control samples (40±2.6%). After 21 days, CP-ESM transplanted wounds showed complete wound closure with full grown hairs covering the entire healed area whereas ESM AA samples still possessed unhealed region. The wound closure rate suggested that decoration of the natural tissue with nanofibers enhanced the healing phenomena and also avoided scar formation by keeping the wound adequately moist. 3.5.2 Histological Examination Re-epithelialization & Neo-vascularization: A scaffold acts as a biomimicking template for influx of host cells and supports the formation of a provisional matrix. Figure 8 & 10 depicts histological sections of treated and open wounds at different time intervals, which explains the reepithelialization process. On 7th day, it was observed that ESM AA and CP-ESM scaffolds have integrated excellently to the wound bed and bonded with the wound margin. ESM’s natural proteinaceous contents with microfibrous architecture stimulated fibroblast migration to scaffold implanted wound sites, but superior and homogenous fibroblast infiltration was observed for CPESM samples. However, open wound samples showed limited host cellular migration to the wound bed with poorly developed granular tissue in comparison to the treated samples. Post 14 days of incubation, as the wound area had decreased substantially (Figure. 7), formation of stratified neoepithelium was observed at the healed site of CP-ESM. Moreover, CP-ESM scaffold could not be recognized separately from the surrounding tissues post 14th day of incubation due to extensive


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Figure 8. Hematoxylin–eosin (H&E) staining images of the wister rat full-thickness wound treated with CP-ESM, ESM AA in comparison with control (open wound). S: scab, E: epithelial layer, D: dermis, GT: granulation tissue, BV: blood vessel, SG: sebaceous gland and HF: hair follicle. (Scale Bar: 100 µm)

cellular infiltration, migration and tissue integration. Moreover, it was apparent that the scaffold was in process of degradation as CP-ESM samples demonstrated close to ~ 60% degradation when 30 ACS Paragon Plus Environment


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exposed to lysozyme under in vitro experimental conditions. At the same time, ESM AA and control samples portrayed greater unhealed portions as compared to CP-ESM. Also, ESM AA sample was still visible at the wound site due to lesser cell migration while open wound possessed thin layer of neoepithelium at the healed sites. It was also observed that the development of skin appendages had already initiated in the healed regions of all samples and was predominantly visible in CP-ESM scaffold. Further, immunofluorescence histochemical analysis of CD31 was performed for both the scaffolds and control groups in order to demonstrate neo-vascularization

Figure 9. Anti CD31 staining images of wister rat full-thickness wound treated with CP-ESM, ESM AA and control (open wound) to demonstrate angiogenesis; Green signify expression of anti CD31 antibody and blue colour represents nucleus staining using DAPI. (Scale Bar: 100 µm)


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and development of blood vessels with incubation time. Post 7 days of treatment, an excellent surge in neo-microvessels were observed for CP-ESM samples as compared to ESM AA and control experiments (Figure 9). Efficient vascularization for CP-ESM treated group may ensure continues blood supply to wound site thus accelerating the regeneration process. After 14 days, significant reduction of neo-microvessels were observed for CP-ESM group thus ratifying superior healing kinetics. In contrast, presence of neo-microvessels in developing stage was apparent for ESM AA and control group indicating slower wound healing. As the healing progressed, microvessels were further replaced with matured blood vessels. After 21 days of incubation the CP-ESM scaffold grafted wound bed demonstrated complete wound closure with a stratified epithelium layer and mature dermal tissue comprising of completely developed skin appendages. At the same time ESM AA and control samples demonstrated incomplete healing in certain parts but the healed regions were observed to possess a stratified epidermal layer. Moreover, the skin appendages in the healed regions of ESM AA and control samples were still in the developing stages. The above results conclude to the fact that though the ESM AA and control samples possessed progressive wound healing kinetics but CP-ESM lead to faster wound closure and reepithelialization.

Dermal Regeneration: Collagen deposition, alignment and development of connective tissue defines various characteristic phases of a complete wound healing process. Figure 10 reveals the MT staining of as-obtained healed tissue from the animal model experiments at different time intervals to monitor the collagen deposition kinetics and development of connective tissue. It was observed that after 7 days of incubation, CP-ESM and ESM AA samples depicted good and uniform collagen deposition with effective migration of host cells to form the granulation tissue.



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Figure 10. Masson’s trichrome (MT) staining images of Wister rat full-thickness wound treated with CP-ESM, ESM AA in comparison with control (untreated). S: scab, E: epithelial layer, D: dermis, SG: sebaceous gland and HF: hair follicle. (Scale Bar: 100 µm)

In comparison, the control showed very sparse distribution of collagen along the wound tissue with 21 days of incubation in CP-ESM scaffolds, complete wound closure along with fully developed


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and aligned collagen bundles in the form of “angel curl shaped structures”, identical to mature a less organized granular tissue. Post 14 days of incubation, CP-ESM samples depicted predominantly mature granular tissue and more homogenous distribution of collagen bundles with developing skin appendages. However, ESM AA showed irregular distribution of collagen and control samples projected less collagen deposition with limited growth in the granular tissue. After human skin was observed. The healed construct witnessed a decrease in cellular structure and regression of vasculature which promoted dermal maturation. ESM AA group depicted significantly developed collagen bundles with well-connected skin appendages and possibility of complete wound closure but the progress was far slower than CP-ESM group. Excess collagen deposition for ESM AA group may also lead to scar tissue formation. The presence of irregular surface topography in ESM AA and lack of supporting platform for control group impeded host cell migration and thereby retarding the wound healing kinetics in these groups. The results from H&E and MT staining coherently suggested successful re-epithelialization and dermal regeneration in CP-ESM group post 21 days of incubation and were in accordance with wound closure rate results. Moreover, anti CD31 staining of sample groups demonstrated excellent neovascularization and development of blood vessels for CP-ESM samples in comparison to ESM AA and control groups. The study clearly showed the effect of surface functionalization of ESM on wound healing process wherein the naturally occurring collagens and GAGs were effectively utilized without impeding cell growth and proliferation. Moreover, the presence of chitosan promoted the release of interleukin by fibroblast which is required for its own migration and proliferation thus promoting wound closure by facilitating the formation of granular tissue and angiogenesis. Additionally, the presence of chitosan along with ESM enhanced the overall antibacterial activity of the scaffold thus maintaining a pathogen free wound bed.



4. Conclusion Herein, a unique route of surface modification for ESM AA was followed to develop a bilayered scaffold through electrospinning of CS/PCL nanofibers. Notably, the fabricated CPESM matrix has resemblance with natural ECM owing to its architecture and presence of collagens, GAGs and other glycoproteins, which enhances in vitro and in vivo cellular adhesion and wound healing efficacy. CP-ESM was crosslinked by Carbodiimide chemistry as confirmed by XPS and FTIR spectra to improve its structural integrity under hydrated condition. Moreover, physico-mechanical studies confirmed improvement in mechanical properties and protein adsorption facilitating superior cell attachment, proliferation and migration of HDF cells as compared to the ESM AA. Subcutaneous implantation of the bilayered scaffold in full-thickness wound model led to 90% wound closure in 14 days. Post three weeks, complete scarless wound healing with excellent re-epithelization and organized dermal regeneration was observed. In vivo results corroborate to the fact that surface modification of the natural tissue with CS/PCL nanofibers reinforces its wound healing properties. Moreover, easy processability, availability and low immunogenicity of ESM makes it a good source of collagen in comparison to its porcine or marine counterfeits. Thus it can be noted that the as fabricated novel bilayered construct, CP-ESM can be potentially used as a dermal substitute in future application. 5. Acknowledgement The authors of present work want to acknowledge fellowship support received from Council of Scientific and Industrial Research (CSIR) for Preetam Guha Ray (File No. 31/015(0134)/2017EMR-I) and Pallabi Pal. The authors are also thankful to Indian Institute of Technology, Kharagpur 35 ACS Paragon Plus Environment

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for providing financial support and research facilities. The authors also would like to acknowledge Miss. Shreya Biswas from School of Bioscience and Engineering of Jadavpur University, Kolkata for facilitating in antibacterial study and Mr. Nantu Dogra for helping in immunohistochemical staining experiments. The authors would also like to acknowledge Dr. Arun Achar from Medinipur Medical College for providing Human tissue samples (IEC/2014/4). 6. Supporting Information Available: Experimental procedure adopted for evaluating microstructure, surface topography, porosity, water retention ability of scaffolds, water vapour transmission rate (WVTR), in vitro biodegradation kinetics, ATR-FTIR, X-Ray Photoelectron Spectroscopy (XPS), tensile properties, protein adsorption and result for ATR-FTIR was also narrated. Figures of results related to ATR-FTIR, zeta potential and sirius red staining were also included.



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Surface Modification of Eggshell Membrane with Electrospun Chitosan

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