Microfibrous Cotton-Wool-Like 3D Scaffold ... - ACS Publications

Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX-XXX

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Nano-/Microfibrous Cotton-Wool-Like 3D Scaffold with Core−Shell Architecture by Emulsion Electrospinning for Skin Tissue Regeneration Pallabi Pal,† Pavan Kumar Srivas,† Prabhash Dadhich,† Bodhisatwa Das,† Dhrubajyoti Maulik,‡ and Santanu Dhara*,† †

Biomaterials and Tissue Engineering Laboratory, School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, Kharagpur 721302, India ‡ Department of Surgery, Bankura Sammilani Medical College, Bankura, India S Supporting Information *

ABSTRACT: Electrospun nanofibrous scaffold has long been studied as skin substitutes for their structural resemblance to the dermal extracellular matrix. However, packed fibrous architecture with small pore size restricts cellular infiltration into nanofibrous mat. In this article, we report highly porous, nano-/microfibrous 3D structure using polycaprolactone-chitosan emulsion and its application in skin regeneration. Under the influence of electric field, the emulsion containing encapsulated charged chitosan droplets enhances charge of the spinning solution and residual charge in the core of the deposited fiber, thereby creating core− shell, cotton-like fluffy structure with average pore size 62 μm, fiber diameter ∼1.62 μm, contact angle of 72° and 80% water uptake capacity of the scaffold. Further, differential stirring period of the specific emulsion developed compact nanofibrous membrane with nanometer ranged pore size emphasizing the role played by emulsion droplet size and the charge carried thereafter. Presence of nanofibers with high-interconnected porosity promoted efficient cellular infiltration and proliferation from initial days of cell seeding. The scaffold supported extracellular matrix protein expression and stratified epithelialization in vitro. Effective integration and attachment of scaffold with margins of a full-thickness excision wound created in a rat model with accelerated healing within 3 weeks proved the efficiency of the scaffold as skin substitute. Additionally, gradual and prolong release of acidic chitosan from the core section benefitted wound healing by lowering the pH of wound environment. Simple technique with inexpensive raw materials endorsed the scaffold as a promising off-the-shelf matrix for skin tissue regeneration. KEYWORDS: RT-PCR, human fibroblast-keratinocyte coculture, porous scaffold, rat wound model infiltration.7−9 However, sacrificial polymer removal resulted in uncontrolled and irregular pore distribution. Additionally, sonic waves would loosen individual nanofibers resulting in perforated electrospun mats, eventually destabilizing the fiber integrity with compromised mechanical strength.10 Lee et al. and Nam et al. reported salt leaching method for creating porous 3D nanofiber scaffolds11,12 However, with this technique also the scaffold thus produced had irregular internal pore structure, which might collapse during the post-treatment process.13 Biopolymers have been explored the most for developing skin substitute as they are biocompatible, nontoxic and often resembling ECM cues. Chitosan, a natural polymer, in addition to having structural resemblance with glycosaminoglycan, an

1. INTRODUCTION Nanofibrous scaffold is favorable as skin substitute due to its morphological and architectural resemblance to the dermal extracellular matrix (ECM); promoting better cell attachment owing to its high surface area to volume ratio.1,2 Oxygen permeability, wound effluence release and inhibiting pathogenic microorganism infiltration are the added advantages of nanofibrous matrix.3 Faster cellular migration and proliferation are facilitated by scaffolds with porous and interconnected networks, enhancing vascularization and mechanical stability of the implant by facilitating mechanical interlocking between invading tissues and scaffold.4,5 However, the electrospun nanofiber mesh, usually exhibits packed fibrous structure with submicron pore size restricting cellular infiltration.6 For preparation of electrospun mat with larger pore sizes, “sacrificial” polymer like poly(ethylene oxide), gelatin and salt leaching had been introduced by several researchers who created porous nanofibrous scaffold with increased cellular © XXXX American Chemical Society

Received: September 14, 2017 Accepted: October 16, 2017 Published: October 16, 2017 A

DOI: 10.1021/acsbiomaterials.7b00681 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Figure 1. (A, C, E, G) SEM morphology, distribution of fiber diameter, and morphological appearance under TEM of as-spun nano-/microfibrous scaffolds prepared using different proportions of PCL−chitosan mix: 1:2 PCL−chitosan (P1C2), 1:1 PCL−chitosan (P1C1), 2:1 PCL−chitosan (P2C1), 3:1 PCL−chitosan (P3C1). (B, D, F, H) As-spun nano-/microfibrous scaffolds after heat treatment at 60 °C.

microfibers without using complex coaxial nozzle syringe as done by conventional coelectrospinning and also eliminated the need for a common solvent for natural and synthetic polymer.19 It has been employed to fabricate drug and growth factor encapsulated fibers for faster tissue regeneration. To the best of our knowledge, potential of PCL−chitosan core−shell fluffy skin regenerating scaffold through emulsion electrospinning has not been explored. We investigated the architectural and compositional essentialities of the scaffold and its effect in vitro and in vivo on rat full-thickness wound healing.

important constituent of native ECM, is nontoxic, biocompatible, biodegradable, and antimicrobial.14 Participation of chitosan molecule in various stages of wound healing cascade is overwhelming. It enhances adhesion and aggregation of platelets with rapid red blood cell mobilization to the injured site; aids in vasoconstriction; promotes formation of granulation tissue by inducing proliferation of dermal fibroblasts, decrease scar tissue formation, and faster reepithelialization.15,16 However, difficulty to electrospin chitosan alone primarily due to solvent limitation, and high viscosities at low concentrations, limits its use.17 Polycaprolactone (PCL) is an FDA approved, biocompatible, biodegradable polymer with favorable mechanical properties and electrospinnability. Although, high hydrophobicity and slow degradability restrict its application in skin regenerating scaffolds.18 Encapsulation of chitosan within PCL in the form of core−shell fiber would address these limitations as chitosan electrospun fibers could be easily fabricated. Further, cross-linking of the scaffolds can be avoided, which frequently leads to compaction, loss of porosity, eventually slowing degradation rate. Hydrophobicity and degradability of PCL will also be optimum for skin regeneration owing to its thin shell layer. In this study, we demonstrate differential stirring period for PCL−chitosan emulsion and subsequent electrospinning resulted in nano-/microfibrous cotton-wool-like fluffy structure and nanofibrous membrane. Emulsion electrospinning evolved as an alternative technique to develop core−shell nano/

2. MATERIALS AND METHODS 2.1. Preparation of the Electrospinning Emulsion. Emulsion electrospinning was performed using PCL (Mn 70−90 kDa; SigmaAldrich) and chitosan (MW 700 kDa; > 90% deacetylated, Marine Chemicals, India) blends. PCL (10 wt %) was prepared in chloroform−methanol (3:1) while chitosan solution (4 wt %) was prepared in acetic acid (90%). Different ratios of PCL−chitosan blends were prepared by mixing 1:1 (P1C1), 2:1 (P2C1), 3:1 (P3C1), and 1:2 (P1C2) volume ratios for 5 min to obtain an emulsion. Additionally, P2C1 emulsion was stirred for 12 h. 2.2. Fabrication of Nanofibers via Electrospinning. For electrospinning, 5 mL syringe was loaded with the emulsions fitted with a 26 gauge blunt needle tip. A syringe pump (KD Scientific, Switzerland) was used to maintain fluid flow at a rate of 3 μL/min, and DC power supply (30 kV, Glass Mann, Japan) of 22 kV was applied between the aluminum plate collector and needle kept 10 cm apart. All experiments were carried out at room temperature. Electrospinning was also carried out for 12 h stirred P2C1 emulsion and 10 wt % PCL. B


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ACS Biomaterials Science & Engineering Details of physicochemical characterization, in vitro assessment and in vivo application of the developed scaffold are detailed in the Supporting Information. 2.3. TGF-β1 Quantification by ELISA. Enzyme-linked immunosorbent assay (ELISA) was used to quantify TGF-β1 cytokine following the manufacturer’s protocol (Thermo Fisher Scientific). Briefly, scaffolds were seeded with fibroblast and keratinocyte cells individually, and after 3 and 7 days of growth, culture media was collected. Further, culture media was also collected after 3 and 7 days from scaffold with coculture of fibroblast and keratinocyte growing at air−liquid interface. Protein values were calculated in ng relating to the standard curve. 2.4. Statistical Analysis. All the experiments were carried out in triplicates and means were analyzed using the t-test and expressed as a mean ± SD. Differences were considered to be significant at p < 0.05.

increased which is clearly evident in Figure 1. However, distinct distribution of PCL and chitosan was not apparent in the obtained fiber morphology. The melting point of PCL is 60 °C while chitosan remains unaffected even until 150 °C. Thus, heat treatment of electrospun mats containing varied PCL−chitosan proportions could have significant influence on fiber morphology owing to compositional differences in core and shell. The samples microstructure after heat treatment at 60 °C revealed differential morphology as shown in Figure 1. The morphology of P1C2 fibers did not alter considerably as compared to asspun sample and accumulation of polymer droplet was also observed (Figure 1B). In case of P1C1 sample, fibers still retained their morphology with evidence of wetting the interfiber space by second phase (Figure 1D). Distinct alteration in fiber structure was noticeable in P2C1 and P3C1, where, P2C1 had very fine fibers with accumulation of large polymer droplets and significant filling of interfiber space (Figure 1F). Notably, in P3C1, the sample had fibrous remnants covered by molten phase leading to distinct shrinkage of scaffold (Figure 1H). The divergent morphology of nanofibers is associated with melting and differential drainage of PCL after heat treatment at 60 °C and is possible with core− shell structure containing PCL in the shell and chitosan in the core. TEM image showed increase in shell thickness with higher PCL proportion in the emulsion. The samples with higher PCL content had significant melting and filling of the interfiber space whereas the samples with relatively less PCL content had accumulation of droplets with thinning of fiber morphology. To further validate the distribution of chitosan and PCL in the core−shell structure, EDS was carried out. As PCL dissolves in chloroform while chitosan remains unaffected, significant morphological and compositional differences were noted in chloroform treated scaffold (Figure 2). The native sample and the scaffold part that dissolved in chloroform evidenced presence of only carbon and oxygen. The chloroform treated scaffold remnant although retained their fibrous morphology, had accumulation of polymer droplets and the fibrous surface had composition of carbon, nitrogen and oxygen. PCL is constituted of only carbon and oxygen while chitosan has carbon, nitrogen and oxygen. The EDS data is in accordance with our previous result confirming presence of PCL on shell of native sample while chloroform had dissolved PCL revealing the fibrous chitosan structures present in core. Therefore, owing to uniform bead free nanofiber morphology of P2C1 with thin PCL shell (∼20 nm thickness) on chitosan core (∼41 nm), it was chosen for further work. 3.2. Microscopic Observation of P2C1 Emulsion. The microscopic study of P2C1 emulsion at different time intervals is shown in Figure 3A. Emulsions are usually opaque with properties distinct from their constituents. Formation of emulsion droplets could be seen instantly after mixing for 5 min which after 2 h, started to coalesce and by 6 h, it started to separate out into individual layers and eventually complete separation took place after 12 h. Upon segregation, PCL layer would still be spinnable, whereas chitosan layer would form beaded morphology. In the case of emulsion with increasing chitosan amount, spinability was dependent on stability of individual chitosan droplets within PCL as dispersing medium (major phase), but as the chitosan amount increased as in P3C1, P2C1, and P1C1, the droplets began merging at higher rate. This phenomenon was more pronounced under static condition associated with accelerated rate of phase separation.

3. RESULTS AND DISCUSSION 3.1. Nanofiber Fabrication. SEM micrographs of Figure 1 revealed the morphology of electrospun samples using different emulsions made by stirring for 5 min (see also Table 1). Table 1. SEM Morphology of Electrospun Samples Prepared Using Different Ratios of Chitosan in Acetic Acid with/ without PCL in Chloroform−Methanola sample

volume ratio of PCL:chitosan in emulsion

P0C1

0:1

P1C2

1:2

P1C1

1:1

P2C1

2:1

P3C1 P1C0

3:1 1:1

morphological appearance

average fiber diameter (μm)

nonelectrospinnable with bead bead on string with very thin fiber fiber with insignificant bead smooth fiber with uniform diameter uniform thick fiber uniform thin fiber

beaded 0.170 ± 0.06 1.22 ± 0.62 1.62 ± 1.26 3.77 ± 1.85 0.413 ± 0.19

a

Chitosan, 4 wt % solution in 90% acetic acid; PCL, 10 wt % solution in chloroform−methanol (3:1).

Chitosan solution in 90% acetic acid was nonelectrospinnable, while PCL in chloroform/methanol solution was electrospinnable. Incorporation of chitosan in PCL (P3C1) resulted in smooth fibers (average fiber diameter 3.77 μm and pore diameter 65 μm) due to higher percentage of PCL in the mix (Figure 1G). Smooth, uniform and bead free fiber (average fiber diameter 1.62 μm and pore diameter 62 μm) was still achievable by increasing chitosan amount as in P2C1 (Figure 1E). Further increase in chitosan amount yielded fibers (average fiber diameter 1.22 μm and pore diameter 60 μm) with sparsely distributed beads (P1C1) (Figure 1C); although fiber diameter decreased, significant reduction in pore diameter was not evident. However, with further enhancement of chitosan concentration as in P1C2, the resultant samples had extensive bead-on-string morphology of thin diameter and reduced pore diameter (average fiber diameter 170 nm and pore diameter 6.5 μm) (Figure 1A). It is noteworthy that surface tension and viscosity of different emulsions did not vary significantly. The compositions with higher amount of PCL yielded fibers of desired morphology although all emulsions had similar surface tension (30.13 ± 0.03 mN/m). Interestingly, TEM images revealed that emulsion electrospinning produced nanofibers with core−shell structure. As the chitosan amount increased in the mix (P1C2), the core thickness increased while with increase in PCL amount (P3C1), the shell thickness C


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Figure 2. SEM and EDS spectrum of native and chloroform treated nano-/microfibrous 2:1 PCL−chitosan scaffold.

Figure 3. (A) Microscopic observation of PCL, chitosan (Chn), and emulsion mix as in composition P2C1 at different time points under static conditions for 5 min and 12 h. (B) Structural evaluation of sample prepared using P2C1 emulsion through side view optical imaging of as-spun sample and SEM: cotton-wool-like 3D nano-/microfibrous scaffold by 5 min stirring and compact nanofibrous membrane by 12 h stirring.

D


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Figure 4. Characterization of 3D nano-/microfibrous 2:1 PCL−chitosan porous scaffold (NF): (A) FTIR/ATR spectra of PCL, chitosan powder, and NF. (B) Swelling behavior examined for 48 h. (C) pH change due to acetic acid leaching monitored for 72 h. (D) Contact angle. (E) In vitro biodegradation observed for 2 months. (F) Tensile strength.

layer of the fiber than that of the inner layer and the viscosity difference directed chitosan to settle into the fiber interior.21 Further, the ionic chitosan emulsion droplets created islands of static charges. During spinning NF, in the presence of electric field the encapsulated chitosan imparted increased surface charge density on the fiber, causing radical charge repulsion, eventually splitting the ejected primary jet into multiple jets prior bending instability by a process called splaying, as illustrated by Reneker et al.22 The charged nanofibers although maintained distance due to electrostatic repulsions, the impact of electric field potential influenced them to move toward collector without collapsing. Further, residual charge in the core of the deposited fiber failed to dissipate immediately, resulting in maintenance of electrostatic repulsion between fibers even after deposition. Eventually, vaporization of solvents and solidification of fibers prompted the structure to grow as a stable self-assembled fluffy cone-shaped 3D scaffold. Further, owing to larger chitosan droplets the resultant fibers were thicker in diameter, thereby enhancing the overall pore diameter. During electrospinning NFM, owing to smaller size of emulsion droplet, the ejected fibers had lesser diameter and the charge density imparted on the fiber surface could easily dissipate. The fibers failed to maintain strong electrostatic repulsion between themselves resulting in their closer deposition and forming nanofibrous membrane. NF with its cotton-wool-like fluffy 3D architecture is promising to befit the 3D wound bed morphology. Hence, characterization of NF was pursued further to evaluate its potential as dermal ECM resembling matrix.

Interestingly, stirring P2C1 emulsion for 12 h intricately mixed the constituents resulting in emulsion with comparatively smaller droplet size (Figure 3A), which was stable until 6 h. It has been reported that emulsion droplet size plays a critical role in stability of an emulsion. A study by Miyagawa et al. showed that smaller droplets required larger kinetic energy to coalesce and is considered more stable than larger droplet.20 Therefore, it can be postulated that smaller droplet size might had imparted the 12 h emulsion more stability until 6 h, preventing coalescing of droplets as compared to 5 min stirring. 3.3. Morphological Evaluation of P2C1 Electrospun Structure. The antomical appearance of electrospun sample of P2C1 emulsion after 5 min mixing and 12 h stirring is shown in Figure 3B. NF scaffold had a cotton-wool-like, highly porous structure with loose fibers on top and height of ∼1.3 cm and pore size 62 ± 25 μm whereas NFM had a nanofibrous membranous architecture with average diameter 274.15 ± 121 nm and pore size 1.16 ± 0.715 μm. Further, depending on the need, varied thickness of NF could be generated, which is otherwise tough with conventional electrospinning. Owing to presence of PCL shell on chitosan, cross-linking step could be excluded while maintaining intact structure. Electrospinnable PCL worked as a carrier and spinning aid in formation of nanofibers of nonelectrospinnable chitosan. Under electric field, the emulsion droplet moved from the periphery to the central region, undergoing stretching into elliptical shape, and finally coalescing and encapsulating within PCL to form core−shell structure. Also, the chloroform−methanol mixture evaporated faster, causing an increase in viscosity on the outer E


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Figure 5. SEM micrograph of primary fibroblasts grown on 3D nano-/microfibrous scaffold at (A) 1 day (arrows indicate cellular attachment), (B) 3 days, (C) 7 days, and (D) 14 days and respective rhodamine cytoplasmic staining (red) and DAPI nuclear staining (blue) images at (E) 1 day, (F) 3 days, (G) 7 days, (H) 14 days. (I) Cross-sectional view of H&E stained fibroblast cell grown on NF for 14 days (arrows indicate cell attachment on fibers). (J) MTT assay of 3D nano-/microfibrous scaffold (NF) and tissue culture plate (control) for human fibroblast cells on different days. (K) Protein adsorption assay with PBS-FBS solution for 3D nano-/microfibrous scaffold. (L) Tensile strength of fibroblast seeded air-dried 3D nano-/ microfibrous scaffold on days 7 and 14.

3.4. NF Characterization. FTIR spectrum of chitosan in Figure 4A shows the peaks for amide A at 3446 cm−1 (N−H stretching), amide B at 2925 cm−1 (asymmetrical stretch of CH2), amide I at ∼1660 cm−1 (C = O stretching), amide II at ∼1593 cm−1 (N−H in-plane deformation) and amide III at ∼1381 cm−1. Few more significant bands were also observed at 1424 cm−1 (−CH2 wagging), at 1154 cm−1 (C−O−C bending) and at 897 cm−1 for presence of saccharide unit.23 The characteristic bands of PCL were exhibited at 2940 cm−1 (asymmetric CH2 stretching), at 2860 cm−1 (symmetric CH2 stretching), at 1728 cm−1 (carbonyl stretching), 1240 cm−1 (asymmetric COC stretching) and 1180 cm−1 (OC−O stretching).24 As done in ATR mode, NF displayed respective bands evidencing presence of both PCL and chitosan with 3295 cm−1 and 1663 cm-1 for chitosan and 2940 cm−1, 1728 and 1240 cm−1 for PCL. Scaffold with high water uptake capacity could effectively absorb moisture from exudating wound bed, promoting nutrient perfusion and maintaining scaffold morphology. It has been reported that moist scaffold promotes epidermal cell migration, fastening wound re-epithelialization twice as compared to dry wounds.25 Figure 4B shows NF to exhibit rapid swelling of ∼80% within 30 min of incubation and achieving equilibrium. Surface exposure of PCL on NF might have limited water uptake, however, presence of larger pores and high-interconnected porosity allowed efficient water uptake. It is reported that pH of wound environment changes from alkaline to neutral and eventually to acidic state with progression of healing. pH of wound also influences oxygen

release, protease activity, angiogenesis, bacterial toxicity and enhanced macrophage and fibroblast activity.26 As shown in Figure 4C, pH of NF incubated DMEM cell culture medium gradually decreased rendering the medium acidic. The entrapped acetic acid in the core of the fiber was unable to vaporize, on leaching out gradually lowered the pH. PCL nanofibers are inherently hydrophobic and water contact angle (WCA) of 134° ± 2° was reported by Chen et al.27 However, NF had distinctly reduced WCA of 72° (Figure 4D). The decrease in WCA might be attributed to high porosity and large interfiber distances, resulting in faster water percolation.28 In a biological environment, body fluid rich in water molecules interact with scaffold creating shell on the materials surface. For moderately hydrophobic surfaces, the established water shell has reduced entropy, but disrupting this layer with proteins enhances entropy favoring adsorption. However, in hydrophilic scaffolds, the water molecules establish hydrogen bonding with surface paving the way for competition between water molecules and protein, resulting in less protein adsorption by a phenomenon known as nonfouling.29 Further, Hasskarl et al. and Tamada et al. reported that fibroblast and keratinocyte cells prefer a moderate hydrophobic surface for better attachment and growth.30,31 Thus, the presence of PCL on fibers along with high porosity allowed NF to attain moderate WCA beneficial for protein adhesion. PCL is reported to have slower degradation rate.18 However, NF degraded ∼25% in 2 months (Figure 4E). Surface exposure of PCL limited initial degradation rate, which eventually started to wear out owing to thin layer, exposing chitosan of the core region. Chitosan upon unmasking leached out faster enabling F


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Figure 6. (A) ECM protein expression by fibroblast on NF after 7 and 14 days of culture on 3D nano-/microfibrous scaffold. (B) TGF-β1 cytokine secretion on 3D nano-/microfibrous scaffold by fibroblast, keratinocyte cells, and fibroblast-keratinocyte coculture at air−liquid interface. (C) RTPCR expression studies of fibroblast and keratinocyte cocultured on NF. (D) H&E of fibroblast and keratinocyte cocultured cell/NF construct (arrows indicate the keratinocyte cell and fibroblast cell).

to fiber structures in the interior of the scaffold confirmed cellular infiltration and is shown with arrow (Figure 5I). Rhodamine-DAPI staining further confirmed the above result. Scaffold architecture and composition promoted efficient cell attachment by day 1 with distinct cytoplasmic filamentous spreading (Figure 5E), which increased by day 3 (Figure 5F). Cells had covered most of the surface by day 7 (Figure 5G), further proliferation by day 14 (Figure 5H) was mainly due to intrusion of cells into inner core of NF samples. Increase in cell density was observed by presence of glowing blue cell nuclei. It may be noted that NF enabled the cells to maintain their elongated structure with defined cytoplasmic protrusions as observed in natural environment. MTT assay measured the metabolic activity of actively growing cells calorimetrically. NAD(P)H-dependent oxidoreductase enzymes of an actively growing cell reduce the tetrazolium dye to purple colored formazan crystals. Therefore, an increase in absorbance value signifies presence of rapidly dividing cells. Quantitatively NF displayed 0.08 ± 0.005, 0.28 ± 0.01, 0.55 ± 0.02 and 0.82 ± 0.03 absorbance on day 1, 3, 7, and 14, respectively, in comparison to 0.06 ± 0.003, 0.15 ± 0.006, 0.32 ± 0.009, and 0.2 ± 0.01 in control, respectively (Figure 5J). It is noteworthy that in scaffolds contact inhibition was not observed even after 14 days although full surface coverage was evident after 7 days and is attributed to their 3D structure. However, declination in cell growth was noticed in control group after 7 days. Protein adsorption to the scaffold surface increased on timedependent scale from 1 to 24 h, as shown in Figure 5K. Moderate hydrophobicity was suggestive of minimizing nonspecific protein adsorption, and thus gradually protein adsorption enhanced which would eventually lead to higher

rapid degradation of the scaffold. Core−shell structure of NF thus proved beneficial for enhancing degradation rate. It is assumed that presence of degrading enzymes and low pH would further promote degradation in vivo. Tensile strength of NF shown in Figure 4F illustrates the strain−strain plot. The ultimate tensile strength and elastic modulus of the scaffold were 0.866 ± 0.06 and 9.34 ± 0.94 MPa, respectively. High cohesive forces due to intrafiber contacts resulted in an initial steep rise in the graph. However, it is to be noted that the strength of NF owing to its high porosity and large interfiber distances, was lesser as compared to PCL or PCL−chitosan nanofibers as reported elsewhere.32,33 3.5. Biological Assessment of Prepared Scaffolds. Fibroblast cell morphology, attachment, migration and proliferation on NF after 1, 3, 7, and 14 days were observed under SEM microscope. Uniform cell spreading with distinct cellular projections and active cell migration through the scaffold were noted from day 1 (Figure 5A). Eventually, as the culture period increased, cell coverage, infiltration, and thickening of cellular lamellipodial projections increased as shown in Figure 5B, C, D (inset) for 3, 7, and 14 days, respectively. It may also be noted that by day 7, cells were observed on the unseeded surface of NF (data not shown). Presence of a thick cell covering on the fiber was evidenced on day 14 (Figure 5D). It can the presumed that high porosity and presence of biocompatible nano/microfibers promoted initial cell attachment and migration. Chitosan on exposure to the surface intensified cell growth and proliferation, resulting in formation of thick cell sheet on NF surface. To observe cellular migration and proliferation within the scaffold, NF samples were examined in cross-section. Presence and adherence of cells G


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Figure 7. Full-thickness wound healing effect of a porous 3D nano-/microfibrous scaffold (NF) as compared to open wound and compact PCL nanofibrous scaffold in rat model. (A−O) Optical photographs showing the progression of healing of the wound treated with NF, PCL, and the open wound after 3, 7, 14, and 21 days. (P) Percentage of wound area reduction after 3, 7, 14, and 21 days for NF, PCL, and the open wound. Bars expressed the mean of standard deviation (n ≥ 3) for independent samples per group and ∗ values indicated p > 0.05 compared with the control. Scale bar: 0.5 cm.

expression of collagen III and fibronectin on NF was desirable, as reports suggested their marginal expression as compared to collagen I in wound healing. MMPs remodel the newly formed granulation tissue, increasing its expression from an early healing stage to a late maturation stage.39 A lower expression level of MMP than collagen I suggested slower ECM degradation than synthesis. GAPDH served as the housekeeping gene in the expression profile study. 3.8. TGF-β1 Quantification by ELISA. TGF-β1’s role in wound healing is noteworthy, for its involvement in expression of fibronectin, collagen I, collagen III, and VEGF, and fastening wound closure by stimulating fibroblast contraction. However, prolonged expression of TGF-β1 leads to scarring in the later stages owing to excess ECM deposition and thus needs critical balance.40 Figure 6B shows TGF-β1 expression variation in fibroblast and keratinocyte monocultured and cocultured scaffolds. Fibroblasts enhance while keratinocytes reduce TGF-β1 expression from the third to the seventh day. A decline in expression from the third to seventh day was noted for cocultured samples also. Poole et al. also reported reduction in TGF-β1 level in a skin substitute model with a fibroblast and keratinocyte coculture.41 A decrease in TGF-β1 level by implanting a chitosan sponge in a full thickness wound and diminished scarring was observed by Baxter et al.42 From these results, we hypothesize that, during early stages of healing, NF would promote fibroblasts to synthesize TGF-β1, which would act in an autocrine manner, recruiting more fibroblasts, increasing ECM deposition, and fastening healing. However, during later stages, proliferating keratinocytes and chitosan leaching from the fiber core would suppress TGF-β1 expression and reduce scarring. However, conclusive results would be obtained from in vivo studies. 3.9. Coculture of Fibroblast and Keratinocyte. Histological evaluation with H&E staining revealed the formation of an epithelial layer over the fibroblast-laden scaffold after 14 days at the air−liquid interface (Figure 6D). RT-PCR results further confirmed the epithelial layer formation with initiation of the stratification process (Figure 6C). Protein markers specific to different layers of stratified epithelium like keratin 14, for dividing basal keratinocytes; keratin 10, for

cell adhesion and their migration. NF with perceptible fiber architecture, composition and porosity displayed efficient cell attachment and proliferation in vitro, suggesting its potentiality as a dermal substitute promoting effective tissue integration. 3.6. Mechanical Testing of Cell Seeded Scaffold. Mechanical property of the cell grown scaffold is shown in Figure 5L. The ultimate tensile strength and elastic modulus of the scaffold after 7 day cultures were 1.07 ± 0.1 and 7.29 ± 1.5 MPa, respectively, which increased to 1.45 ± 0.5 and 10.5 ± 0.79 MPa, respectively, after 14 days. Owing to cytocompatibility, high porosity, and architecture, cells easily infiltrated the scaffold, synthesizing ECM material, occupying interfiber spaces, and strengthening the scaffold. With an increase in cell proliferation by 14 days, ECM deposition increased, enhancing the tensile property of the scaffold. A 3D scaffold thus allowed development of an ECM/material construct, where both types of fibers were in close association. However, a compact 2D nanofibrous mat fabricated by conventional electrospinning fails to provide sufficient interfiber spaces for efficient ECM synthesis between fibers.34−36 Normal human skin is reported to have an ultimate tensile strength of 5−30 MPa.37 Therefore, it can be assumed that a further increase in growth period would allow deposition of enough ECM to strengthen the scaffold, reaching tensile strength near that of original skin. 3.7. ECM Protein Expression by Fibroblast on Scaffold. ECM proteins like collagen I, collagen III, fibronectin, and MMPs play an essential role in wound healing. The ability of dermal fibroblasts to synthesize these ECM proteins on the scaffold would be an essential parameter to evaluate its effect in vivo. Figure 6A shows the relative expression of collagen I, collagen III, fibronectin, and MMP protein after days 7 and 14. Collagen I, being the major structural protein synthesized by the fibroblast, had relatively pronounced expression, which increased from 7 to 14 days. Collagen III, fibronectin, and MMP expression also increased from 7 to 14 days but in a lesser amount as compared to collagen I. During early wound healing stages, fibronectin and collagen III help in the formation of granulation tissue, providing a matrix for migration and growth of cells.38 Lesser H


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Figure 8. H&E staining images of rat full-thickness cutaneous wounds treated with a porous 3D nano-/microfibrous scaffold, nanofibrous PCL sheet, and control, after 3 and 7 days. S, scab; WM, wound margin; ET, epithelial tongue; E, epithelial layer; D, dermis; F, fibroblast; W, wound; GT, granulation tissue; SG, sebaceous gland; HF, hair follicle; and BV, blood vessel. Scale bar of A2, C2, and E2 is 0.5 mm.

suprabasal, postmitotic cells; involucrin, for terminal differentiation of keratinocytes, and E-Cadherin, noted for intercellular tight junction organization and keratinocyte stratification were found to express. Further, collagen IV and laminin 5, basement membrane proteins, were also detected, signifying development of a stable basement membrane. This result suggests that owing to its biofunctionality, NF would efficiently integrate within in vivo systems, supporting ECM materials synthesis and promoting stratified epithelial layer development. 3.10. Wound Size Measurements. The wound healing potential of NF was evaluated in a rat full-thickness wound model and compared with a nanofibrous PCL scaffold and open wound. To illustrate the difference between a conventional nanofibrous scaffold and cotton-wool-like fluffy scaffold, a PCL nanofibrous scaffold of an average fiber diameter of 500 nm and a pore diameter of 2.5 μm was used as a control. No infection was evident in the operative area of the rats throughout the experimental period. A decrease in wound area in a time-dependent manner was noted in all the groups as shown in Figure 7A−O with the percentage of wound closure

(Figure 7P). Notable reduction in wound area was not evident by day 3. The open wound was red and had a fresh wound-like appearance. Close interaction and bonding between NF with the adjacent tissue were evident (Figure 7B inset). NF porosity, architecture, and composition might have allowed efficient cell migration, resulting in NF/tissue attachment. Although PCL integrated with the wound bed, close interaction was not evident. By 7 days, significant reduction in wound area was prominent in the NF group (46 ± 2.34%) as compared to the open wound (21 ± 1.92%) and PCL group (22 ± 2.5%). The NF group followed a controlled healing pattern from the periphery to the central region from all sides, whereas control groups displayed irregular healing. Additionally, the NF structure was not prominently visible after 7 days, suggesting cellular migration from surrounding tissues. However, the presence of a PCL scaffold on the wound site could distinctly be seen even on the 14th day. Notable wound area diminution was observed by day 14 in the NF group (79 ± 2.9%) in comparison to the open wound (47.7 ± 3.2%) and PCL group (50.62 ± 1.4%). By day 21, the NF treated wound had significant healing with well-grown hair covering most areas, I


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Figure 9. H&E staining images of rat full-thickness cutaneous wounds treated by a porous 3D nano-/microfibrous scaffold, nanofibrous PCL sheet, and control, after 14 and 21 days. S, scab; WM, wound margin; E, epithelial layer; D, dermis; F, fibroblast; GT, granulation tissue; SG, sebaceous gland; HF, hair follicle; and BV, blood vessel. Scale bar of G3, I3, and K3 is 0.5 mm.

while the open wound group (75 ± 2.7%) and PCL (81.8 ± 1.2%) had healed partially. PCL and the open wound followed similar kinetics of healing, emphasizing the role played by porosity and chitosan in fastening healing with the NF scaffold. 3.11. Histological Examination. 3.11.1. Re-epithelialization. The re-epithelialization processes of the control, PCL, and scaffold implanted wound were analyzed by histological sections shown in Figures 8 and 9. During the third day postwounding, wound margins were distinctly visible with blood vessels and host cellular infiltration leading to the formation of a provisional matrix. NF and PCL laid on the subcutaneous tissues with its structure intact (Figure 8A1, C1). Epithelial tongue migration beneath the scab was noticed by the seventh day (Figure 8B1, D1, F1). Highly porous architecture and biocompatibility promoted NF to integrate with the newly formed granulation tissue matrix and hence could not be distinctly distinguished (Figure 8B2). Migration of the fibroblast toward the wound site and infiltrating the scaffold was more evident in the scaffold group (Figure 8B3) than the

control group (Figure 8F3) and PCL group (Figure 8D3). Limited porosity in the PCL nanofibrous scaffold resulted in less cellular invasion, and the sample was thus superficially present on wound bed (Figure 8D2). Initiation of epithelialization was evident by p63 expression in the migrating epithelial tongue.43 However, the number of basal epithelial cells was more in the NF group than in the PCL and control (Figure 11A, G, M). By the 14th day, the wound area had reduced, and stratified neoepithelium could be seen in the healed regions of the NF treated group (Figure 9A1). Further, the scaffold became a part of the tissue due to extensive cellular infiltration and hence could not be identified separately (Figure 9A2). However, the control displayed a larger unhealed area (Figure 9C2, E2), and the healed region was merely covered with a thin epidermal layer (Figure 9C1, E1). The PCL scaffold could still be seen on the wound surface in accordance with our previous data (Figure 7). Void spaces were more distinct in the control group as compared to the NF group. Maturation of granulation tissue promoted merging of the epithelial tongue and dermal J


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Figure 10. MT staining images of rat full-thickness cutaneous wounds treated by porous 3D nano-/microfibrous scaffold, nanofibrous PCL sheet, and control, after 3, 7, 14, and 21 days. S, scab; E, epithelial layer; D, dermis; GT, granulation tissue; and BV, blood vessel.

Figure 11. Immunostaining images of rat full-thickness cutaneous wounds treated by porous 3D nano-/microfibrous scaffold, nanofibrous PCL sheet, and control after 7 and 21 days.

tissue with the wound site. Skin appendages, like hair follicles and sebaceous glands, were observed in the healed regions of all the groups. By the 21st day, a healed stratified epithelial layer covering the mature dermal tissue was evidenced in the NF group (Figure 9B). Islands of unhealed regions were still evident in PCL and the open wound group on the 21st day (Figure 9D,F). However, the healed regions had a stratified epidermal layer. Immunostaining also confirmed these observations with p63 and keratin 10 expressions for stratified epithelial layer formation in the NF group (Figure 11C,D). The PCL treated and open wound also manifested progressive healing; however, expression of p63 and keratin 10 was significantly less as compared to NF (Figure 11I,J,O,P). 3.11.2. Dermal Regeneration. Figure 10 shows the kinetics of collagen deposition and connective tissue regeneration by MT staining at different time points. During the third day in the NF group, the presence of the scaffold acted as a template for cell attachment and matrix deposition, limiting the existence of

void spaces which were prominent in the control groups. Neocollagen deposition was observed as light blue staining beneath NF, PCL, and the scab in the control. By the seventh day, granulation tissue established richly in cells, but with poorly developed collagen bundles, as evident by light and diffuse blue staining. Both the NF and PCL groups had uniform collagen distribution (Figure 10B,F), but the control group had dispersed islands of collagen bundles (Figure 10J). Immunostaining for collagen I expression also evidenced uniform collagen I distribution in the NF group (Figure 11B) and faint, dispersed staining for the PCL and open wound (Figure 11H,N). On the 14th day, the granulation tissue was more mature, and a uniform deposition and distribution of collagen was noted in both healed and unhealed regions of the NF group, evidenced by dark blue staining with linearly arranged collagen bundles (Figure 10C). However, the PCL and open wound groups had a nonuniform, differential collagen matrix deposition pattern which stained light blue (Figure 10G,K). By K


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ACS Biomaterials Science & Engineering the 21st day in the NF group, the collagen bundles were “angel curl shaped structures,” characteristic of mature human skin, while the number of cellular structures decreased, suggesting a healed area.44 Regression of the vasculature was even noted, which allowed dermal maturation. The presence of islands of the unhealed region was still evident in control groups (Figure 10H,L). Collagen I and collagen III staining confirmed the maturation of dermal region (Figure 11E,F). Our result of profound expression of collagen I as compared to collagen III was in accordance with the report that granulation tissue expresses 40% type III collagen.45 Although, collagen I and collagen III expression was much less in PCL and the open wound (Figure 11K,L,Q,R). Healing progression and dermal maturation of the PCL-treated and open wounds followed a similar pattern, suggesting restricted contribution of the compact nanofibrous scaffold in wound healing. Faster maturation of granulation tissue in NF could be attributed to the role played by chitosan on exposure. Chitosan is reported to induce the fibroblast to release interleukin necessary for its migration and proliferation, enhancing the wound healing rate by elevating granulation tissue formation along with angiogenesis.16,46 Thus, it can be hypothesized that the core−shell structure played a dual role in quickening healing, where PCL being biocompatible allowed initial cell attachment and proliferation, and eventually chitosan on exposure played its part. It was also noted that the entrapped acetic acid in the core reduced the pH. In both acute and chronic wounds, the rate of healing declines under high alkaline pH. Therefore, it can be postulated that in vivo the leached acetic acid would lower the pH of the wound environment and prove beneficial for treating chronic wounds. Overall, our study showed that without external inclusion of growth factors or cytokines, NF alone encouraged neo-vascularization, stratified epithelialization, and dermal maturation with all appendages. Hence, NF has great potential to heal full thickness wounds and to be used as a dermal regenerating material.

of application also proved the applicability of NF as a dermal substitute.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00681. Experimental procedure for microscopic imaging, FTIR, contact angle, swelling study, mechanical properties, biodegradation kinetics, acetic acid release kinetics, cell culture studies, RT-PCR, and a full-thickness wound healing experiment (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Santanu Dhara: 0000-0001-8599-569X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Council of Scientific & Industrial Research (CSIR) and Indian Institute of Technology, Kharagpur for financial and research facilities. Financial support of DBT, Govt. of India (project code, CST; grant no., BT/ PR7818/MED/32/279/2013) is fully acknowledged. The authors are thankful to all group members of the Biomaterials and Tissue Engineering laboratory for their guidance and support.

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REFERENCES

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