$â¡Corresponding Author Dr. Santanu Dhara

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Oleoyl Chitosan based Nanofiber Mats Impregnated with Amniotic Membrane Derived Stem Cells for Accelerated Full-Thickness Excisional Wound Healing Sayanti Datta, Arun Prabhu Rameshbabu, Kamakshi Bankoti, Priti Prasanna Maity, Dipankar Das, Sagar Pal, Sabyasachi Roy, Ramkrishna Sen, and Santanu Dhara ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00189 • Publication Date (Web): 26 Jun 2017 Downloaded from http://pubs.acs.org on July 2, 2017

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ACS Biomaterials Science & Engineering

Oleoyl Chitosan based Nanofiber Mats Impregnated with Amniotic Membrane Derived Stem Cells for Accelerated Full-Thickness Excisional Wound Healing

Sayanti Datta$, Arun Prabhu Rameshbabu$, Kamakshi Bankoti$, Priti Prasanna Maity$, Dipankar Das*, Sagar Pal*, Sabyasachi Roy¥, Ramkrishna Sen†, Santanu Dhara$‡

$

Biomaterials and Tissue Engineering Laboratory

School of Medical Science and Technology Indian Institute of Technology Kharagpur Kharagpur–721302, India †

Department of Biotechnology

Indian Institute of Technology Kharagpur Kharagpur–721302, India

*Department of Applied Chemistry Indian School of Mines Dhanbad–826004, India ¥

Department of Gynaecology,

Midnapore Medical College, Paschim Medinipur – 721101, India $‡

Corresponding Author

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

ACS Paragon Plus Environment


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ABSTRACT Wound healing management is a major challenge for critical full-thickness skin wounds. Development of nanofibrous scaffolds with tunable wettability, degradation and biocompatibility are highly desirable. Herein, we demonstrated synthesis of oleoyl chitosan (OC) by grafting mono-unsaturated fatty acid residue, C18 oleoyl chain, to the backbone of chitosan molecule and blending with gelatin to form the nanofiber mats. The physicochemical properties of the nanofiber mats revealed mechanical strength, moderate surface wettability, and suitable degradation rate. The nanofibrous mats showed excellent in vitro cytocompatibility with human amniotic membrane-derived stem cells (HAMSCs) in terms of enhanced adhesion and proliferation owing to biomimetic nano-architecture and chemical cues. Furthermore, the fabricated nanofiber was implanted with and without pre-seeded HAMSCs in the full-thickness wound to evaluate the skin wound healing efficacy in a rat model. Histological and immunohistochemical studies were conducted to evaluate the plausible changes of tissue architecture and expression of molecular markers involved in wound healing process. Both acellular and HAMSCs incorporated cellular nanofibers promoted wound contraction remarkably with superior skin tissue regeneration in terms of enhanced collagen synthesis, reepithelialization and initiation of epithelial cells stratification compared to control group.

KEYWORDS: oleoyl chitosan; electrospinning; nanofiber mat; stem cells; wound healing


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1. INTRODUCTION Skin is one of the largest organs of the body that executes numerous functions such as defense against pathogens, protection from mechanical injury, regulates body temperature as well as maintains fluid balance.1 Any injury in the skin should be quickly and efficiently restored as it serves as a protective barrier against the outside environment.2 Skin injury can occur from lacerations, burns, skin loss due to infection, skin cancer, pressure and diabetic ulcers. Skin regenerates spontaneously when the injury is subjected to epidermal layer (superficially or partially). It involves migration of epithelial cells i.e. keratinocytes from the periphery to the center of the wound bed as well as the formation of the newly stratified epidermis. However, the healing is impaired in case of severe full-thickness wounds. Re-epithelialization, stratification of the newly formed epidermis and angiogenesis are essential features for full-thickness wound healing. The healing of such cutaneous wounds involves complicated procedures like grafting (auto, allo or xenografting).3,

4

It may also face the problem of donor site morbidity, graft

rejection, probability of disease transmission and scar tissue formation.5,

6

Therefore, tissue

engineered graft is an emerging alternative to overcome these therapeutic dilemmas towards skin regeneration.7, 8 The important criteria of ideal tissue engineered graft is of readily adherent to the wound site, non-immunogenic, ability to mimic the native skin structure, sufficiently porous (template for cell infiltration and permit cell proliferation prior to regeneration), protect wounds from fluid loss, prevent infection, controlled degradation with suitable mechanical property.9,

10

Though

various forms of support matrices like film11, 12, hydrogel 13 are useful in skin tissue engineering, nanofiber mats are promising due to their structural similarity with natural ECM.14-16 Additionally, very high surface to volume ratio, tunable porosity, moderate mechanical strength,



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flexibility in surface functionality, cell-cell and cell-matrix interaction foster these nanofiber mats as aunique scaffold for full-thickness wound healing.17 Different cell-based products or bioengineered grafts combined with cells or other growth factors would be beneficial for the treatment of dermal wound healing and different nanofiber based matrices combined with cells/other factors used for wound healing applications.18-25 However, the role of human amniotic membrane-derived stem cells (HAMSCs) in wound healing is still not well studied and notably placental tissue derived stem cells secrete many growth factors as well as ECM in promoting wound repair.26,

27

Also, HAMSCs were revealed to have better advantages over the bone

marrow-derived stem cells (BMSCs) because of its ease of availability, low risk of immunological rejection, high proliferative/differentiation potential which may promote accelerated wound closure, re-epithelialization as well as dermal tissue regeneration.28 Microbial infection prevention is also a vital challenge during cutaneous wound repair. Nanofiber with its nanotopography could prevent the invasion of microorganism and henceforth there is less chance of getting any infection during healing.29 Gelatin is widely used in various applications such as wound dressing30, drug delivery systems but it has some demerits like weak mechanical property, faster degradation and can be easily damaged by bacteria. Oleoyl chitosan (OC) is the modified form of chitosan consisting mono-unsaturated fatty acid i.e. oleic acid chain in its backbone. Research findings have shown that topical administration of non-essential fatty acid exhibited better cutaneous wound healing in comparison to other fatty acids by modulating the inflammation and thus speeding up the mending response in vivo, respectively.31, 32 Though OC nanoparticles have been studied in the literature for their antibacterial activity or oleic acid has been used for wound repair, yet nanofiber mats incorporated with OC as biomaterial is still unexplored. In this work, novel OC nanofibrous mats are prepared by blending with gelatin and


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studied for full-thickness dermal wound healing. The antimicrobial property of OC could be beneficial for preventing microbial invasion in the wound region. Additionally, OC based nanofiber may impart hydrophobicity to the scaffold surface that could be advantageous for cell migration as well as stratification of the newly formed epidermis.33 Therefore, composite gelatin/OC nanofiber may assist organized collagen deposition in the dermal region and thus could prevent scar tissue formation. The gelatin/OC nanofiber mats were investigated for physicochemical characterization and biological evaluation in vitro. Further, in vivo wound healing study was carried out with acellular scaffold (gelatin/OC nanofiber without HAMSCs), cellular scaffold (HAMSCs cultivated nanofiber for 5 days) by implantation to the full-thickness skin wound at the dorsal area of rats and compared with TegadermTM (a commercially available dressing for wound coverage) for ensuring its ability towards accelerated wound healing as well as skin tissue regeneration. 2. EXPERIMENTAL SECTION 2.1. Synthesis of OC OC was prepared by a method described by Liu et al..34 Briefly, chitosan (MW, 710 kDa; >90% deacetylated; Marine Chemicals, India) containing glucosamine moieties were charged with methane sulphonic acid (LobaChemie, India) to block the amine group by forming an ionic complex. Approximately, 1.5M methane sulphonic acid was mixed per mole of glucosamine unit and stirred for 30 minutes in an ice-bath. Oleoyl chloride (Sigma-Aldrich, Mw = 300.89 g/mol, ≥ 89%) was then added dropwise to this mixture. The reaction was continued for 5 h and allowed to stand overnight at 0 °C. The reaction mixture was precipitated by acetone and neutralized with ammonia water. The precipitate was filtered and purified by repeatedly washing with excess methanol and ethanol to remove any unreacted acid or oleoyl chloride. Finally, the synthesized



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derivative was obtained by vacuum drying at room temperature and stored in a desiccator until used. NMR study Synthesized dried sample (OC) was dissolved in dimethylsulfoxide-D6 (DMSO-D6) (Merck Millipore, Mw = 84.17 g/mol) at a concentration of 30 mg/ml and run through proton (1HNMR) and carbon (13CNMR) nuclear magnetic resonance. The spectra were recorded on an AVANCE DAX-600 (Bruker, Sweden) 600 MHz NMR spectrometer at room temperature under a static magnetic field 600 MHz. The degree of substitution (DS) can be calculated from 1H NMR. DS was determined by computing the integration of the methyl protons signals of the oleoyl compoundand the protons of the polysaccharide unit (excluding protons of hydroxyl and amine groups which were not measured by this method). 2.2. Electrospinning of gelatin/OC nanofibers In this study, gelatin (Type B gelatin from porcine bone, Loba Chemie, India) electrospinning was performed according to Gu et al..35 Particularly, gelatin/OC in different weight ratios (100:0, 90:10, 80:20, 75:25) were dissolved in 90% glacial acetic acid (Merck, India) and blend was kept overnight for uniform mixing. The prepared electrospinning solutions were filtered through filter mesh (100 µm) to remove undissolved particles and to avoid interrupted spinning. Freshly prepared electrospinning solutions were used for physicochemical characterization and the blends 90:10, 80:20, 75:25 were labeled as GOC1, GOC2, and GOC3, respectively. Physicochemical properties of different blends were determined by various parameters. Conductivity and pH of different blends were measured by conductivity/pH meter (Eutech, India) at 25° C. Bohlin CVO (Malvern, UK) rheometer was used for solution viscosity measurement at parallel plate geometry (20 mm diameter) with maintaining a gap of 100 µm at


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25° C. The shear rate was varied between 0.1 and 1000 s-1. The dynamic surface tension of those solutions was measured by tensiometer using aplatinum plate (DCAT 100, Data physics GmBH, Germany). For electrospinning, the polymer solution was placed in a plastic syringe fitted with a blunt end needle (22 G). The syringe was kept horizontally on the syringe pump (KD Scientific, USA) for controlling the solution flow rate from the needle tip. The solution was electrospun at a flow rate of 2 µL/ min with an applied high voltage of 20-22 kV. A distance of 15-18 cm was kept between the needle tip and the horizontal collector covered with aluminum foil. The optimization was carried out for obtaining samples with a distinct appearance of nanofibers by adjusting voltage parameter. Gelatin was electrospinnable35 at 18 kV, but nanofiber mats from gelatin/OC blend were prepared at a higher voltage range i.e. 20-22 kV. All the experiments were performed at 25 °C. Fabricated nanofiber mats were not stable in aqueous medium thus crosslinking

of

the

nanofibers

was

performed

in

90%

ethanolic

1-Ethyl-3-(3-

dimethylaminopropyl) carbodiimide (EDC, 40mM) and N-Hydroxysuccinimide (NHS, 10mM) solution at pH 5.5. Henceforth, nanofiber mats were washed with 0.1 M disodium phosphate buffer (Na2HPO4) to eliminate the residual carbodiimide groups and other byproducts.Further, the mats washed with distilled water, air dried and stored in desiccators. 2.3. Physicochemical characterization Ninhydrin assay for crosslinking density Fabricated nanofiber mats were crosslinked in ethanolic EDC/NHS (pH 5.5) solution and crosslinking density was evaluated by ninhydrin assay. 36 Details of ninhydrin assay are given as section S1 in Supporting Information. FTIR and XRD The details are provided in Supporting Information, sectionS2.



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Thermal behavior Details of Differential scanning calorimetry (DSC) and Thermogravimetric analysis (TGA) are given in section S3 in Supporting Information. Scanning electron microscopy Sample preparation for SEM analysis are given in Supporting Information, section S4. Mechanical testing Mechanical testing of nanofibers are presented in Supporting Information, section S5. 2.4. Interfacial studies Contact angle, Swelling study,and Biodegradation studyare provided in Supporting Information, section S6, S7 and S8 respectively. Hemocompatibility assay This characterization was evaluated according to the procedure described in the Supporting Information, section S9. 2.5. Cell culture studies Proliferation efficacy of HAMSCs on nanofibrous mats HAMSCs were isolated as described in our previous work

37

and cultured in Dulbecco’s

modified eagle’s media containing low glucose (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% antibiotic-antimycotic solution (Gibco) and maintained in humidified environment (37 °C, 5% CO2). Nanofibers were cut into 1 × 1 cm2 and sterilized by 70% ethanol and thoroughly washed with phosphate buffer saline (PBS) followed by overnight incubation in DMEM. HAMSCs at passage 3 were seeded on those nanofibers at a density of 1 × 104 cells in 24 well plates and cultured for 1, 3 and 5 days in triplicate. Viable cell proliferation on nanofibrous scaffolds was assessed by 3-(4, 5- dimethylthiazol-2-yl)-2, 5 diphenyl


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tetrazolium bromide (MTT) assay. At 1, 3, 5 days, the media was discarded and washed with PBS followed by addition of MTT solution and incubated at 37 °C for 4 h. At the end of the incubation period, dimethyl sulfoxide was added to the wells to dissolve insoluble formazan crystals. The culture plates were swirled for 30 min until the purple color became uniform. The absorbance was recorded at 590 nm on a microplate reader (RMS, Chandigarh, India). Cell proliferation activity was further analyzed by DNA quantification kit (DNA Quantification Kit (DNAQF), Sigma) as per the manufacturer protocol. Fluorescence imaging of cultured cells on nanofiber mats are described in Supporting Information, section S10. 2.6. In vivo wound healing The wound healing efficacy of the nanofibers mats was performed in full-thickness wounds using a rat model, and the procedure is described in section S11 of Supporting Information. 2.7. Statistical analysis Statistical analysis was performed using GraphPad Prism software (version 5.02, La Jolla, CA, USA) by one-way ANOVA. The experiments were evaluated in triplicates and expressed as a mean ± standard deviation. Significance was determined at p < 0.05. 3. RESULTS & DISCUSSION 3.1. Synthesis and chemical identification of OC by NMR Chitosan was chemically modified into oleoyl chitosan via condensation reaction forming esterification of –CH2OH group in sugar moiety with oleoyl chloride by releasing HCl as a byproduct. Oleoyl group was grafted at the C-6 position of chitosan whereas –NH2 at the C-2



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position was free. The final product obtained at the end of the reaction was pale yellow in color, and the yield of that product is ~80%. The chemical identification of synthesized product was ascertained by performing 1H NMR and

13

C NMR. The chemical shift values between δ = 3.299-3.999 were because of the

protons (Figure 1a) of the polysaccharide backbone (H2-H6). δ values 4.430, 4.721 and 5.051, represented –OH groups, –NH2 groups and anomeric protons (H1), respectively. Whereas, peaks at δ = 5.354, 2.796 and 2.394 are due to the H14/H15, H7, H13/H16 protons, respectively. The peaks at δ = 1.606, 1.450, 1.233 and 0.847 for the presence of H8, H22, H9-H12/H17-21 and H23 (-CH3) protons, respectively. Thus, appearance of the new peaks from H7-H23 confirmed the success of OC compound synthesis.38

13

CNMR spectrum of the synthesized

product (Figure 1b) showed chemical shift values were as follows: 13C NMR δ 100.4 (anomeric C1), 56.7 (C2), 65.2 (C6), 72.9 (C3), 75.4 (C5), 80.8 (C4), and 130.3 (C15/C16), respectively. Chemical shift value δ = 175.1 (C7) established the presence of carbonyl group of the oleoyl moiety; thus further demonstrates successful substitution of oleoyl group in the native chitosan molecule. Here we considered the base value of integration for –NH2 protons (2.00) of the chitosan and calculated all the integration. It has been observed that the DS is 42%.39, 40 3.2. Nanofiber formation through electrospinning In the present study, gelatin, OC and their blends dissolved in 90% acetic acid were evaluated for their fiber forming capability. Different parameters that are crucial for electrospinning solution were evaluated and have been tabulated in Table 1 in Supplementary Information, section S12. Gelatin and blends prepared from gelatin and OC were electrospinnable whereas pristine OC was not electrospinnable in 90% acetic acid. For successful electrospinning, optimum distance was kept 18 cm while maintaining flow rate at 2 µl/ min. The


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effect of acetic acid concentration on physicochemical properties was thoroughly investigated. Higher acetic acid concentration facilitated declination in surface tension. All the solutions prepared for study using 90% acetic acid had a specific range of pH (~2), conductivity (0.3-0.5 mS/cm) and surface tension (31.56-33.90 mN/m) which may assist fiber formation. Lower surface tension and conductivity may facilitate Taylor cone formation, higher jet instability and thus fabricate submicron range fiber. Viscosity diminished with a decrease in gelatin concentration as gelatin was the prime composition in all blends whereas OC concentration did not affect much in viscosity changes and exhibited the non-Newtonian behavior of the resultant blends (Details are given in Supporting Information in section S12 along with viscosity graph Figure S1). Electrospun fibers can be formed only when there is sufficient chain entanglement amongst the polymers. For continuous fiber fabrication, total polymer concentration should be at least 2-2.5 fold higher than the entanglement concentration.41-43 The entanglement concentration (Ce) was calculated for all the blends, and the total polymer concentration was found to be much higher (~8-9 times) than that of entanglement concentration Ce; thus all blend solutions were spinnable (entanglement numbers are provided in Table 1 of Supporting Information, Section S12). Finally, the fabricated nanofibers were crosslinked by EDC/NHS. The possible mechanism of nanofiber formation with gelatin and covalent crosslinking reaction by EDC-NHS is shown in Figure 2 along with OC synthesis. 3.3. Physicochemical characterization Ninhydrin assay for crosslinking density Gelatin is a water-soluble polymer, and it should be crosslinked or immobilized in an aqueous medium for its application to serve as extracellular matrix analog. A number of free amine groups in gelatin and blend nanofiber mat was determined by ninhydrin assay.



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Crosslinking of the nanofiber was achieved by forming amide bond through amine and carboxyl group of gelatin and OC. The formation of blue pigment is qualitatively linked with the extent of crosslinking with EDC/NHS. In the presence of EDC/NHS, –NH2 group of OC reacts with – COOH group of gelatin and forms an amide bond. Additionally –NH2 group of gelatin may also chemically bonded with –COOH group of gelatin. Uncrosslinked nanofibers showed deep blue color as compared to the crosslinked nanofibers as it contains arelatively higher number of the free amine group. From the absorbance, the degree of crosslinking was measured to be 35% ± 2% for all crosslinked nanofiber mats.44 Scanning electron microscopy The SEM images of different electrospun mats (GE, GOC1, GOC2, and GOC3) revealed the morphology of the nanofiber in the range of 150-400 nm (Figure 3). It is reported in the literature that gelatin can be easily electrospinnable45 while electrospinnabality of chitosan is very poor.46 As OC was synthesized from chitosan and it contained the bulky fatty acid group, it was also not spinnable in 90% acetic acid. Through mixing with gelatin, improvement of the spinning ability of OC was observed. Moreover, gelatin was acting like acarrier for OC in different blends, and it can carry up to a certain amount of OC (GOC2). Thus, no significant phase segregation was observed under SEM, and resulting fibers were significantly homogeneous in morphology, but GOC3 nanofiber (Figure 3d) exhibited diffused nanofiber morphology along with some beads that may be due to low conductivity owing to the higher amount of OC in GOC3 composition as seen in Table 1 in Supplementary information section S12. Though GOC2 generates nanofiber of finer diameter (Figure 3c) as well as contains a higher amount of OC; it could provide an adequate number of functional groups for improved cell attachment while mimicking the native ECM morphology. The extent of functional groups


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increases with reducing fiber diameter, and it has been studied earlier that the cells respond best to their in vivo environment at nanometer regime (ECM features ranging from 50 nm to 500 nm); which is closely similar to the fabricated nanofibers.47 FTIR spectroscopic analysis FTIR spectra of OC and gelatin/OC blends are shown in Figure 4. OC exhibited many alterations i.e. peaks at 1463 and 779 cm-1 which indicated thepresence of methyl and methylene functionality of the long carbon chain of oleoyl moiety, respectively. Absorption at 2922 and 2860 cm-1 was associated with stretching vibration of saturated C–H bonds. O–oleoyl carbonyl group was indicated at 1551 cm-1. In region 3200-3500 cm-1, the absorptions related to stretching vibrations of O–H and N–H bond were decreased. A strong peak at 1736 cm-1 was observed in the modified chitosan mainly due to oleoyl carbonyl stretching vibration.48 Both gelatin and OC has common functional groups like –OH and –NH2; thus exhibited significant intensity difference in the amide I and amide II regions (Figure 4). The formation of a new complex in the blend influences reduction in the extent of hydrogen bonding associated with the stronger molecular interaction between carboxyl and amide functionality. 49 Peaks at 1545 cm-1 attributed to the amino characteristics peak i.e. –NH2 stretching of amide II and a strong absorption was detectable around 1665 cm-1 that corresponded to the stretching vibration of amide I (–CONH–). A small peak at 2933 cm-1 in gelatin/OC blend nanofiber mats indicated the saturated C–H bonds and a broad band in the region of 3310 cm-1 corresponds to hydrogen bonded –OH groups. XRD and Thermal behavior XRD diffraction patterns for native chitosan, its derivative and blend nanofibers are depicted in Figure S2a and S2b in Supporting Information section S13. DSC graph of chitosan



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and its derivative is shown in Figure S2c. Details of XRD and thermal are described in Supporting Information at section S13 and section S14 respectively. Mechanical testing The mechanical property of a tissue-engineered scaffold is a significant parameter as it offers an initial biomechanical profile for the cells before the formation of neo-tissue. Thus, it should be consistent with the anatomical site itself where it is to be implanted. Tensile stressstrain and % elongation curves of crosslinked gelatin/OC nanofibers in the dry state with different ratio are described in Table 1. It clearly demonstrates that GE nanofiber mats exhibited a moderate amount of tensile strength and minimum % elongation i.e. 10.75 MPa and 4.35%, respectively. Nanofibers fabricated from increasing amount of OC results in an increased % elongation and show comparable tensile strength. GOC1 nanofiber exhibited % elongation of 11.86% while GOC2 and GOC3 nanofiber showed 15.88% and 16.40% elongation, respectively. The tensile strength of fabricated nanofiber mats (GE, GOC1, GOC2, and GOC3) is comparable to the normal skin requirements, and it lies in the range of 4-15 MPa.50 Tensile strength of the sample increases after crosslinking as the structural integrity of electrospun fiber, thus showing improved mechanical properties.51 Less % elongation and high young’s modulus illustrated the weak physical properties and brittle nature of GE nanofibers whereas an increased % elongation and comparable tensile strength were observed for blend nanofibers (demonstrated in Table 1) that described flexible nature of the nanofiber mats that may satisfactorily perform as a good wound dressing material.52 3.4. Interfacial studies Contact angle


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Surface wettability of crosslinked nanofibers is shown in Table 1. It can be observed that contact angle altered with the addition of OC in the solution blend. Crosslinked GE nanofiber exhibited surface wettability of 36° whereas the gelatin/OC nanofiber surface became moderately hydrophilic (50° – 80°) with OC incorporation in the gelatin solution. Contact angles of different nanofibers were 50°(GOC1), 62°(GOC2), 80°(GOC3), respectively. The surface wettability of nanofiber mats could influence the healing rate as it is reported that moderate hydrophilic scaffold with a contact angle around 55°-70° (GOC2 = 62°) facilitates most efficient wound healing by enabling proper contact with the wound bed.53 Swelling study The most predominant criteria of wound healing involve not only attachment of the scaffold to the wound bed but also uptake of wound exudates, support new tissue formation and also transports nutrients in the scaffold during in vitro cell culture. The equilibrium swelling behavior of the nanofiber mats was measured in PBS buffer (pH 7.4) at 25° C (Figure 5a). GE nanofiber showed improved swelling with respect to gelatin/OC blend nanofibers i.e. 440%. Nanofiber composed of higher OC content (GOC3) had approx 380% swelling followed by % swelling of 400% (GOC2) and 420% (GOC1), respectively. Swelling behavior is directly related to the gelatin concentration54 (Figure 5a) in blend solution, and the swelling rate was highest for GE nanofiber, whereas GOC3 nanofiber had least percentage swelling. Biodegradation study In vitro enzymatic degradation of different nanofibrous mats was performed for 15 days by immersing in lysozyme solution. The degradation rate was very rapid in GE nanofiber mats and it showed complete enzymatic degradation only after 10 days while oleoyl groups containing nanofiber mats (GOC1, GOC2, and GOC3) sustained in enzymatic solution even after 15 days



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(Figure 5b). Percentage loss was directly proportional to the gelatin contained in nanofiber mat. It has been previously demonstrated that higher swelling percentage may affect the stability of the scaffold in the moist environment.55 Therefore, GE nanofiber revealed faster degradation while unsaturated fatty acid group containing OC improves the endurance of the blend nanofiber mats (GOC1, GOC2, and GOC3) as depicted in biodegradation study (Figure 5b). It may be attributed to strong intermolecular covalent bonding amongst gelatin/OC, leading to moderate hydrophilic nature of those composite nanofibers and hence relatively higher durability of nanofiber in the moist environment.56 Hemocompatibility assay Significant hemolytic activity is not desired for the scaffold to any tissue engineering application since it affects the cellular response when the scaffold is implanted in vivo. From hemocompatibility study, it was evident that the polymeric nanofibers with different compositions did not induce any hemorrhaging or antigenic response and thus insignificant hemolytic activity was observed (less than 5% hemolysis) which could be beneficial for tissue engineering application. In Table 1, the hemocompatible behavior of GE as well as blend nanofibers (GOC1, GOC2 and GOC3) is mentioned. Table 1. Mechanical Property, Contact angle and Hemolytic Activity of Different Nanofiber Mats Sample Elongation %

Tensile Strength (MPa)

Young’s Modulus (MPa)

Contact

Hemolysis %

angle(°)

GE

4.35 ± 1.21

10.75 ± 1.52

525 ±21.65

36

0.36 ±0.018

GOC1

11.86 ± 0.46

9.74 ± 0.32

405±63.23

50

0.35 ±0.02

GOC2

15.88 ± 0.89

9.85 ± 0.64

323 ±56.85

62

0.34 ±0.022

GOC3

16.40 ± 0.42

8.28 ± 1.68

212 ±74.98

80

0.33 ±0.02


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3.5. Cell culture studies Cell proliferation activity Cell proliferation and viability was performed by MTT assay and DNA quantification (Figure 6a and 6b). The results suggested that GE nanofibers as well as gelatin/OC blend nanofibers were cytocompatible and non-toxic in nature. MTT result was found statistically significant, and it was p < 0.05. Nanofibers with higher OC showed higher cell viability and enhanced proliferation rate as evident from DNA quantification assay. It is reported that the maximum adhesion of the cells observed on the surface with moderate hydrophilicity (water contact angle of 60°) compared to extremely hydrophilic/hydrophobic surface.57 Also, the morphology of the cells was fully spread when it was cultured on a surface with 55°-70° wettability. GOC2 nanofiber exhibited the contact angle of 62° whereas the contact angle for GE nanofiber was 36° as seen in Table 1. Therefore, GOC2 nanofiber showed higher cell viability as well as the proliferation compared to GE and other nanofibers (GOC1 and GOC3), which is evidenced by MTT and DNA quantification assay (Figure 6a and 6b). Based on all physicochemical characterization and in vitro cell culture studies, it was prominent that all the samples were biocompatible in nature but GOC2 exhibited finer nanofiber morphology, good mechanical property, suitable contact angle, low swelling rate along with controlled degradation under aqueous environment and thus may help to choose GOC2 nanofiber as appropriate tissue engineered graft. Cell adhesion and morphology Cell seeded nanofibrous mats (GE and GOC2) were stained by rhodamine-phalloidin (cytoskeleton staining (green)) and DAPI (nucleus staining (red)) to observe the proliferation of the HAMSCs onto the nanofibers. The fluorescent images are depicted in Figure 7a. Significant



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numbers of well spread cytoskeleton of HAMSCs were visible in GOC2 nanofibrous mats compared to GE nanofiber at day 1, 3, and 5 days. SEM images of cell cultured nanofiber mats also revealed a significant amount of cells along with good morphology after 5th day (Figure 7b). The reason behind this might be due to the change of surface wettability with the addition of OC in blend nanofibers. Also, the presence of biological cues along with CH3/NH2 or CH3/COOH groups in the GOC2 nanofiber with an optimum contact angle (62°) contributed the superior cell adhesion and proliferation.58 3.6. In vivo wound healing Xenotransplantation of mesenchymal stem cells for full-thickness wound healing is of paramount importance as it may reduce the donor site morbidity (during autotransplantation) by providing abundant supply and ease of use. Many kinds of literature describe the potential of mesenchymal stem cells in wound healing.59, 60 Though mesenchymal stem cells may promote improved wound healing without the need of any scaffold, the major problem is the short halflife of secreted proteins due to inactivation by protease inhibitors present in the wound site.61 It is also reported that nanofiber scaffold impregnated with mesenchymal stem cells promotes enhanced excisional full-thickness wound healing.62 In this context, we performed xenotransplantation of HAMSCs in an animal model to investigate the possibility of xenogenic transplantation in skin regeneration. To evaluate the potential and effectiveness of GOC2 in accelerating the healing of thefull-thickness wound, acellular and cellular scaffold (GOC2) were implanted in the dorsal area of the rat model and compared with control (without scaffold or cells). During the healing process, wound-healing rate (%) was higher in the acellular group and the cellular group as compared to the control group. Wound healing phase at different days (5, 10 and 15 days) was photographed (Figure 8a) whereas wound closure rate of the various groups


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was demonstrated in Figure 8b. Cellular group showed maximum (64%) closure rate rather than acellular (45%) and control group (15%) after 5 days of post wounding while after 10 days of wounding, % reduction of wound closure rate of control, acellular and cellular scaffold implanted group was 39%, 72%, and 87%, respectively. At day 15, the wound did not heal completely in control group with a wound closure rate of approx 50% while most of the wound area was covered with continuous epidermis in acellular nanofiber mat treated group and cellular group with closures of ~ 90% and ~ 100%. Hence control group illustrated incomplete epithelium whereas one small wound mark was visible in the acellular group and but wound remnant mark was disappeared in the cellular group after 15 days of post wounding. Histological findings Wound tissue sections of a different group with various time periods were stained with H & E, MT for histological analysis. H & E staining reveals the overall tissue structure at various time points after wounding and is shown in Figure 9. Acellular as well as cellular scaffold was well integrated into the host tissue within 5 days due to a large number of cell infiltration into the membranes without a sign of acute immunogenic response or tissue necrosis. At day 5, neovascularization was also detected in both acellular and cellular group compared to control group (Figure 9a, 9b and 9c). After 10 days of post wounding, control group exhibited increased number dermal fibroblasts population with respect to other groups (Figure 9d). However, at day 10, keratinocytes migrated to the wound site and started forming a new epithelial layer at the wound edges in both acellular and cellular group (Figure 9e and 9f). Moreover, H & E staining of the cellular group at 10th day (Figure 9f) illustrates horizontal reconstructed dermis along with thick epithelium layer; whereas acellular group did not yet form any proper epidermis layer (Figure 9e). After 15 days, control group disclosed the partial development of epithelium (Figure



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9g) with incomplete wound contraction while wound covered with acellular scaffold and cellular scaffold showed almost complete wound contraction as well as newly formed epithelium (Figure 9h and 9i). Moreover, cellular group exhibited reconstructed dermis, complete reepithelialization as well as theformation of secondary features like hair follicle, nerves and sweat glands (Figure 9i). MT staining of a different group is shown in Figure 10. The presence of collagen, an essential component in the ECM, was detected by MT staining which exhibited an increased and defined formation of collagen layer in the wound covered with cellular nanofiber mat. In unwounded skin, MT stained sections show dense blue color which indicates collagen bundles arranged in basket weave pattern while newly formed collagen assembled in a lattice pattern.63 It was clearly observed that cellular scaffold inspired the initiation of forming basket weave type architecture (of collagen fibers) after 10 days (Figure 10f) whereas collagen was deposited randomly in control and acellular scaffold treated wound (Figure 10d and 10e). After 15 days, it is properly visible that wound wrapped with cellular scaffold possessed improved (epidermal as well as dermal tissue regeneration) potential compared to the acellular scaffold in terms of faster re-epithelialization and enhanced angiogenesis with generating some of the secondary structures (Figure 10h and 10i). Immunohistochemical analysis The tissue sections of control, acellular and cellular groups were subjected for different immunohistochemical staining to understand the phenomenon of complete wound healing as shown in Figure 11. Proliferation phase initiated with themigration of fibroblasts in the wounded region, their differentiation into contractile myofibroblasts (wound contraction) followed by their depletion (wound closure). Angiogenesis is another central feature that involves in granulation


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tissue formation, and that was immunohistochemically detected by CD 31 antibody (Figure 11a, 11b, and 11c). At day 5, finer blood vessels (neovascularization) were advancing as well as little bit ramifying into the granulation tissue in acellular (Figure 11b) and cellular (Figure 11c) nanofiber mats implanted group, which represented more blood supply in wound area thus may precede the healing phenomenon; whereas thicker blood vessels were visible in control group (Figure 11a). After 15 days, re-epithelialization and epidermal differentiation were determined by expression of suprabasal epithelial marker anti-CK10 (cytoplasm) and anti-p63 (nucleus). In the case of the control group, there was incomplete epithelium formed at the wound area (Figure 9g) hence anti-CK10 (Figure 11d) as well as anti-p63 antibodies (Figure 11g) were not expressed. Also, acellular scaffold treated group did not significantly express anti-CK10 (Figure 11e) and anti-p63 (Figure 11h) at the epithelium suprabasal layer after 15 days since the epithelialization and its stratification were not distinct as shown in H & E image (Figure 9h). However, in the case of acellular group, reconstituted tissue expressed anti-p63 (Figure 11i) and anti-CK10 (Figure 11f) antibodies distinctly that depicted the development of new stratified epithelial layer after 15 days post wounding (Figure 11f and 11i). Though collagen deposition in the dermal region was evident from MT staining, it was further confirmed by anti-COL III antibody. From the MT micrograph, it has been observed that after 15 days, cellular group (Figure 10i) expressed highest as well as matured basket weave structured collagen deposition in the dermal region compared to control (Figure 10g) and acellular group (Figure 10h). Immunohistochemical staining of thecellular group after 15 days (Figure 11l) by anti-COL-III antibody showed more collagen deposition as compared to acellular (Figure 11k) and control groups (Figure 11j).



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The possible explanations for cellular and acellular groups for exhibiting efficient wound healing compared to control group might be due to (a) GOC2 (both acellular and cellular scaffold) containing oleoyl group, could play a vital role in wound repair by inhibiting the generation of inflammatory mediators, raft formation and the fluidity of cell membrane;

64

(b)

neo-angiogenesis, being a foremost part of wound healing phenomenon, is promoted by high metabolic rate due to proliferating cells and low oxygen content in the damaged tissue;

65

(c)

ECM protein as well as some cytokines and growth factors are synthesized by HAMSCs that may govern influential role in wound remodeling by assisting the formation of continuous epithelium, better reconstructed dermis with improved secondary features like hair follicle, nerves and sweat glands within 15 days approximately.66 4. CONCLUSION In conclusion, we developed cellular nanofiber (GOC2) for ameliorating full-thickness wound healing in a rat model. In addition to the excellent biocompatibility, the nanofibrous membranes impart moderate hydrophilicity and thus facilitates the prolonged sustain period of the nanofiber mats in biological systems. In vivo full-thickness wound healing potential was evaluated by creating three groups: control, acellular and cellular scaffold. Both acellular and cellular group demonstrated enhanced wound healing as compared to control, but cellular GOC2 exhibited superior tissue regeneration in terms of re-epithelialization and enhanced collagen deposition. Therefore, we envision that these HAMSCs impregnated nanofibrous GOC2 will exhibit significant wound healing efficacy for the treatment of large skin defects or chronic wounds. Also, long oleoyl tail of OC can be used for sustained drug release study and this pristine nanofiber along with drugs may be applied to burn wound that not only act as a drug releasing matrix but also impede the bacterial infection during healing.


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ACKNOWLEDGEMENTS Fellowship from Ministry of Human Resource Development, Govt. of India, New Delhi is acknowledged for Sayanti Datta and Arun Prabhu Rameshbabu. Fellowship support from Department of Science and Technology (DST) Govt. of India, New Delhi for Kamakshi Bankoti and Indian Council of Medical Research (ICMR) for Priti Prasanna Maity is acknowledged. Financial aid from Department of Biotechnology (BT/TR/7818/MED/32/279/2013), Govt. of India, New Delhi is also acknowledged. ASSOCIATED CONTENT: Supporting Information: Ninhydrin assay, FTIR, XRD, thermal behavior, physicochemical characterization of electrospinning solution and nanofibers, interfacial studies, fluorescence imaging and in vivo experimentations.

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(62) Ma, K.; Liao, S.; He, L.; Lu, J.; Ramakrishna, S.; Chan, C. K. Effects of nanofiber/stem cell composite on wound healing in acute full-thickness skin wounds. Tissue Eng. Part A. 2011, 17 (9-10), 1413–1424. (63) Bonvallet, P. P.; Schultz, M. J.; Mitchell, E. H.; Bain, J. L.; Culpepper, B. K.; Thomas, S. J.; Bellis, S. L. Microporous dermal-mimetic electrospun scaffolds pre-seeded with fibroblasts promote tissue regeneration in full-thickness skin wounds. PLoS One 2015, 10 (3): e0122359. (64) Cardoso, C. R.; Favoreto, S. Jr.; Oliveira, L. L.; Vancim, J. O.; Barban, G. B.; Ferraz, D. B.; Silva, J. S. Oleic acid modulation of the immune response in wound healing: a new approach for skin repair. Immunobiology 2011, 216 (3), 409–415. (65) Wei, X.; Yang, X.; Han, Z. P.; Qu, F. F.; Shao, L.; Shi, Y. F. Mesenchymal stem cells: a new trend for cell therapy. Acta Pharmacol. Sin. 2013, 34 (6), 747–754. (66) Skardal, A.; Mack, D.; Kapetanovic, E.; Atala, A.; Jackson, J. D.; Yoo, J.; Soker, S. Bioprinted amniotic fluid-derived stem cells accelerate healing in large skin wounds. Stem Cells Transl. Med. 2012, 1 (11), 792–802.


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Oleoyl Chitosan based Nanofiber Mats Impregnated with Amniotic Membrane Derived Stem Cells for Accelerated Full-Thickness Excisional Wound Healing

Sayanti Datta$, Arun Prabhu Rameshbabu$, Kamakshi Bankoti$, Priti Prasanna Maity$, Dipankar Das*, Sagar Pal*, Sabyasachi Roy¥, Ramkrishna Sen†, Santanu Dhara$‡

Table of Contents Graphic



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Figure 1. Chemical identification of Oleoyl Chitosan (OC) by (a) 1H NMR and (b) 13C NMR. 188x279mm (300 x 300 DPI)


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Figure 2. Synthesis representation of (a) OC synthesis; (b) Mechanism of gelatin (GE) and OC interaction during electrospinning in acetic acid environment (forming ionically crosslinked nanofibers through COO− NH3 +,); (c) Crosslinking reactions of gelatin/OC nanofibers with NHS-EDC at pH 5.5 resulted in covalent amide linkages (covalently crosslinked nanofibers). 184x268mm (300 x 300 DPI)



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Figure 3. Morphology of GE and gelatin/OC blend nanofiber (GOC1, GOC2, GOC3) observed under scanning electron microscope (SEM) at lower (5 KX) and higher (15 KX) magnification. Scale bar 1 µm. 186x275mm (300 x 300 DPI)


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Figure 4. Demonstrates FTIR spectra of OC, GE and blend gelatin/OC nanofibers (GOC1, GOC2, GOC3). 139x153mm (300 x 300 DPI)



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Figure 5. (a) Equilibrium swelling percentage in PBS at different time intervals and (b) Degradation rate of GE and gelatin/OC blend nanofiber mats (GOC1, GOC2, GOC3). Y-error bars represent standard deviation. 54x23mm (300 x 300 DPI)


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Figure 6. (a) MTT assay and (b) DNA quantification study of GE and gelatin/OC blend (GOC1, GOC2, GOC3) nanofiber mats. Y-error bars represent standard deviation. 48x18mm (300 x 300 DPI)



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Figure 7. (a) Fluorescence imaging of GE and GOC2 blend nanofibers by Rhodamine (green)-DAPI (red) staining at day 1, day 3 and day 5, respectively. Scale bar 50 µm. (b) Cell attachment onto GE and GOC2 nanofiber mats after 5 days observed under scanning electron microscope (SEM). Scale bar 2 µm. 117x108mm (300 x 300 DPI)


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Figure 8. (a) Demonstrates the effects of wound healing by taking optical images and (b) wound closure rate of control, acellular and cellular group at different time phase. Y-error bars represent standard deviation. 45x16mm (300 x 300 DPI)



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Figure 9. Represents hematoxylin & eosin staining of control (a, d, g), acellular (b, e, h) and cellular (c, f, i) scaffolds after 5, 10 and 15 days post-wounding at 10 X magnification. Scale bar 100 µm. 15 days pictures were depicted at 20 X magnification for control, acellular and cellular (j, k, l) group, respectively. Scale bar 50 µm. 71x40mm (300 x 300 DPI)


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Figure 10. Represents Masson's trichome staining of control (a, d, g), acellular (b, e, h) and cellular (c, f, i) scaffolds after 5, 10 and 15 days post-wounding at 10 X magnification. Scale bar 100 µm. 15 days pictures were depicted at 20 X magnification for control, acellular and cellular (j, k, l) group, respectively. Scale bar 50 µm. 71x40mm (300 x 300 DPI)



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Figure 11. Immunohistochemical staining of anti-CD31 of different groups after 5 days post-wounding of control, acellular and cellular group (a, b, c), respectively. Photographs at 20X magnification (Scale bar 50 µm) and arrows represent blood vessel formation. Anti-CK10 (d, e, f), anti-p63 (g, h, i) and anti-COLIII (j, k, l) represents immunohistochemical staining for control, acellular and cellular group after 15 days postwounding. Anti-CK10, anti-p63 images were captured at 40X (Scale bar 25 µm), and anti-COL III was captured at 20X (Scale bar 50 µm) magnification. 81x51mm (300 x 300 DPI)


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$â¡Corresponding Author Dr. Santanu Dhara

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$â¡Corresponding Author Dr. Santanu Dhara