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Synthesis and Fabrication of Collagen Coated Ostholamide Electrospun Nanofiber Scaffold for Wound Healing Subramani Kandhasamy, Sathiamurthi Perumal, Balaraman Madhan, Narayanan Umamaheswari, Javaid Ahmad Banday, Paramasivan Thirumalai Perumal, and Vichangal Pridiuldi Santhanakrishnan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16488 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 22, 2017

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ACS Applied Materials & Interfaces

Synthesis and Fabrication of Collagen Coated Ostholamide Electrospun Nanofiber Scaffold for Wound Healing Subramani Kandhasamy†, Sathiamurthi Perumal#, Balaraman Madhan#, Narayanan Umamaheswari†, Javid Ahmad Banday×, Paramasivan Thirumalai Perumal†, Vichangal Pridiuldi Santhanakrishnan*

†

Organic Chemistry Division, CSIR-Central Leather Research Institute, Chennai 600 020, Tamil Nadu, India # AcSIR, CHORD, CSIR-Central Leather Research Institute, Chennai 600 020, Tamil Nadu, India × Department of Chemistry, National Institute of Technology, Srinagar, India *Department of Plant Biotechnology, Centre for Plant Molecular Biology & Biotechnology Tamil Nadu Agricultural University, Coimbatore, India ABSTRACT: A novel scaffold for effective wound healing treatment was developed utilizing natural product bearing collagen based biocompatible electro-spun nanofibres. Initially, ostholamide (OSA) was synthesized from osthole (a natural coumarin), characterized by 1H, 13C, DEPT-135 NMR, ESI-MS and FT-IR spectroscopy analysis. OSA was incorporated into Polyhydroxybutyrate (PHB) and Gelatin (GEL), which serve as templates for electro-spun nanofibers. The coating of OSA-PHB-GEL nanofibers with collagen resulted in PHB-GEL-OSA-COL nanofibrous scaffold which mimics extra cellular matrix and serves as an effective biomaterial for tissue engineering applications, especially for wound healing. PHB-GEL-OSA-COL, along with PHB-GEL-OSA and collagen film (COLF), were characterized in vitro and in vivo to determine its efficacy. The developed PHB-GEL-OSA-COL nanofibers posed an impressive mechanical stability, an essential requirement for wound healing. The presence of OSA had contributed to antimicrobial efficacy. These scaffolds exhibited efficient antibacterial activity against common wound pathogens, Pseudomonas aeruginosa (P.aeruginosa) and Staphylococcus aureus (S.aureus). The zones of inhibition were observed to be 14±22 mm and 10±2 mm, respectively. It was observed that nanofibrous scaffold had the ability to release OSA in a controlled manner, and hence, OSA would be present at the site of application and exhibit bioactivity in a sustained manner. PHB-GEL-OSA-COL nanofiber was determined to be stable against enzymatic degradation, which is the most important parameter for promoting proliferation of cells contributing to repair and remodeling of tissues during wound healing applications. As hypothesized, PHB-GEL-OSA-COL was observed to imbibe excellent cytocompatibility, which was determined using NIH 3T3 fibroblast cell proliferation studies. PHB-GEL-OSA1 ACS Paragon Plus Environment


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COL exhibited excellent would healing efficacy which was confirmed using full thickness excision wound model in Wistar rats. The rats treated with PHB-GEL-OSA-COL nanofibrous scaffold displayed enhanced healing when compared to untreated control. Both, in vitro and in vivo analysis of PHB-GEL-OSA-COL presents a strong case of therapeutic biomaterial suiting wound repair and regeneration. KEYWORDS: Ostholamide, Collagen, Wound healing, Fibroblast, Electro-spun nanofibers

1. INTRODUCTION Natural product extracts contain secondary metabolites, which serve as precious medicinal sources for developing new drug1 for tissue engineering in biomedical application2,3. Natural products containing integrative bioactive ingredients play a pivotal role in enhancing their therapeutic value4. It has been formulated as nanofibers in the field of wound healing5. Nanofiber scaffolds have been one of the indispensable bio-materials used in biomedical applications6. Electro-spun nanofibers have an exclusive ability to deliver the drug in a controlled and sustained manner due to their high surface area, which go hand in hand with wound healing applications7-9 and they exhibit versatile biomedical properties due to the ease of fabrication with different bioactive molecules10. The nanofibrous scaffold fabricated with collagen11 in the presence of natural bioactive compounds, bearing different biopolymers, have been well established for tissue engineering applications12. Collagen is a protein of prime importance which provides structural rigidity and integrity to connective tissue of mammals. Collagen is effectively used for tissue engineering applications13,14 due to its excellent biocompatibility15, low antigenicity16 and noninflammatory17-19 characteristics. The nanofibrous scaffold based on collagen, mimics extracellular matrix (ECM) and exhibits good cell permeability to facilitate the repairing and regeneration of damaged tissues. Collagen coated nanofibrous scaffolds, in addition to cell attachment, possess good oxygen permeability, high surface to volume ratio and biocompatibility, thereby facilitating regeneration in wound healing applications20. Skin wound healing is a complex process due to the risk of bacterial infections21, 22, which is a major hindrance to easy recuperation and complicates rejuvenating the injured skin23. Haemostasis, inflammation, proliferation and remodeling are the consecutive phases of wound healing. Most of the synthetic wound dressing materials do not have a say due to the lower therapeutic values and short term drug effects at the wounded region24-27. Therefore, the design and development of suitable and effective biocompatible materials pose grave challenges in biomedical applications. 2 ACS Paragon Plus Environment

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Especially, they have to accelerate the elimination of excess exudates and toxic components, maintain essential humidity at the wound dressing interface28. In recent days, the naturally occurring anti-pathogenic compound combined nanofibrous scaffold have efficiently reduced the danger of infections29 and enhanced re-epithelialization. Osthole, (7-methoxy-8-(3methyl-2-butenyl) coumarin), a coumarin derivative is an important moiety of medicinal plants and herbs and was extracted from roots of the plant Prangos pabularia. Osthole derivatives have been widely applauded, recently due to its pharmacological applications, as it is a very promising essential natural compound for novel biomaterial production. Simultaneously, osthole exhibited versatile bioactivity viz., antimicrobial30,31, antiinflammatory32, vasorelaxant33, antioxidant34, neuroprotective35-37 and anti-cancerous38. Due to the nature of the bioactivities of osthole, it would be a prospective drug that will prevent infection, reduce inflammation or swelling of injury and reduction in high pressure of the blood vessels around the wounded area. Presence of osthole in the biomaterial platform could exhibit excellent therapeutic values in enhancing rapid wound healing. In addition, Poly (3hydroxybutyric acid) (PHB), a hydrophobic polymer39 has been selected for this study as a hydrophobic biomaterial. PHB has found excellent applications in the field of tissue engineering due to its biodegradable and biocompatible properties40,41. Gelatin (GEL) is an important biopolymer with versatile innovative properties42, such as non-antigenicity, good biocompatibility43, plasticity and superior adhesiveness44-46. These characteristics make it widely useful in the field of pharmaceutical and biomedical applications. Therefore, the collagen efficacy of biomaterial alone did not show any progressive development in wound healing process, but the combination of naturally occurring bioactive molecule with collagen, in the presence of biopolymer functionalized electro-spun nanofibrous scaffold have been developed for enhanced antibacterial and enhanced wound healing activity47. In our previous work, we have described the synthesis and biological evaluation of a novel 2-(methylamino)3-nitro-4-(4-oxo-4 H-chromen-3-yl) pyrano[3,2-c]chromen-5(4H)-one (CCN). The designed product was then applied for generating collagen based scaffold in tissue engineering which showed excellent compatibility and proliferation of NIH 3T3 fibroblast cells48. A closer literature survey in recent past, portrays the wide application of combinations of electro-spun coumarin in controlling cell morphology/ orientation49, gas sensing50, light emitting, photovoltaic devices, optical filters and waveguides51 and as green fluorescence dye for selfhealing agent52. In present study, we have carefully chosen coumarin based natural product, osthole, (7-methoxy-8-(3-methyl-2-butenyl) coumarin) as the starting material, and synthetically modified it to ostholamide (OSA), a bioactive compound, fabricated it with 3 ACS Paragon Plus Environment


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PHB-GEL as an electro-spun fiber and coated with collagen to enhance the antibacterial activity in wound dressing for fast healing. To the best of our knowledge, no work is reported in literature on studies of the influence of electro-spun ostholamide (OSA) nanofibrous scaffold for wound dressing. Herein, we report, for the first time, the use of a natural product (osthole) based derivative for wound healing application. 2. MATERIALS AND METHODS 2.1. Materials Poly(3-hydroxybutyric acid) (PHB), Gelatin (GEL) and 1,1,1,3,3,3-hexafluoro-2propanaol, MTT assay reagents and Calcein AM, were purchased from Sigma-Aldrich. NIH 3T3 fibroblast cell line was obtained from the National Centre for Cell Science (NCCS), Pune, India. Other chemicals and culture wares were purchased from Sigma-Aldrich, unless specified otherwise. Analytical TLC was performed on precoated sheets of silica gel G/UV254 of 0.2 mm thickness (Macherey-Nagel, Germany) using analytical grade solvents and visualized with iodine spray (10% (w/w) I2 in silica gel) or UV light. All the experiments were done in compliance with the committee for the purpose of Control and Supervision of Experiments on Animal (CPCSEA) guidelines and were performed after approval by the Institutional Animal Care and Use Committee (IACUC). 2.2. Plant Material Collection Prangos pabularia plant is commonly found in the stony slopes of Ladakh (Drass, J&K, India). The plant material was collected after acquiring proper permission from the Department of Forestry (J&K, India) and it was authenticated by Dr. Akhtar H. Malik, (University of Kashmir). The plant material (Prangos pabularia) has been deposited under Accession No. 33214 and Collection No. 1203-Javid, Kash. There are no specific issues about its extinction as it does not fall under the category of endangered species53. 2.3. Preparation of Extract (1) The collected root parts of the Prangos pabularia plant were isolated, chopped, oven dried at 40 ͦ C and was made into a powder. The resulting powdered root (2.0 kg) was extracted using DCM: Methanol (1:1) solvent system. The crude DCM: Methanol (1:1) extract of the root (30.0 g) was obtained by removal of the solvent under vacuum on a rotary evaporator.

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2.4. Isolation of the Chemical Constituents (Osthole (2)) The crude DCM: Methanol (1:1) extract of root material (30.0 g) was column chromatographed and elution was carried out using the solvent of increasing polarity. The procedure was repeated with the fractions collected, followed by re-crystallization technique which resulted in ~98% pure compound. The isolated compound was characterized with the help of various spectroscopy techniques. 2.5.

Synthesis

of

N-(4-(7-methoxy-2-oxo-2H-chromen-8-yl)-2-methylbutan-2-yl)

acrylamide (OSA) (3) A magnetically stirred solution of osthole (100 mg, 0.41 mmol) in benzeneacrylonitrile (10 ml, 1:1) was treated with 0.5 ml of Boron triflouride in ether (BF3.O (C2H5)2). The reaction was observed at regular intervals using TLC (40%, Ethyl acetate: Hexane). The starting material had disappeared after an hour. A saturated solution of sodium bicarbonate was used to quench the reaction mixture, then separated using ethyl acetate and H2O, dried with anhydrous sodium sulphate. Removal of excess solvent under reduced pressure resulted in the pure compound (104 mg, 80%). 2.6. Isolation of Type-I Collagen Type-I collagen used in this study was isolated from the tails of healthy male albino Wistar rats (6 months-old) and purified. Saline and distilled water maintained at 4°C was used in washing the isolated tendons to remove blood and its constituents. Type-I collagen was isolated by solubilising the tendons with 500 mM acetic acid. The isolated type-I collagen was then dialyzed extensively against 50 mM acetic acid to eliminate all traces of salts54. The resulting pure type-I collagen solution was transferred to Petri plates, deep freezed for a minimum of 4 h maintained at -40°C and then lyophilized. The essential collagen sample was thereby prepared by dissolving a suitable amount of lyophilized collagen in 50 mM acetic acid for further usages. 2.7. Preparation of PHB-GEL-OSA-COL Nanofibrous Scaffold Poly (3-hydroxybutyric acid) (0.5g) and gelatin (0.5g) was dissolved in 10 mL of 1,1,1,3,3,3-hexafluoro-2-propanaol with constant stirring for 10 hours to obtain a 4 wt% 5 ACS Paragon Plus Environment


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concentration of homogeneous polymer solution.1:1 ratio of this blended PHB and GEL solution was taken and mixed with 0.08 g per 2 mL of ostholamide (OSA). A 24 G needle was used to electro-spun the PHB-GEL-OSA solution, connected to the positive terminal of the high voltage DC power supply (ZEONICS, INDIA) before which a mat was prepared with the use of 100 ×100 mm2 sized plate which is covered inside with aluminium foil suitable for sample preparation. The homogeneous polymer solution was discharged into the plate at a rate of 1.5mL/h using a computer controlled syringe pump under an applied electric potential of 1.5 kV cm-1. The prepared PHB-GEL and PHB-GEL-OSA (100 ×100 mm2 size each) nanofibrous scaffold was kept over a grounded aluminium substrate around a rotary drum positioned at 12 cm in a direction perpendicular to the needle. Collagen film (COLF) was prepared by pouring 25 mL of 4 mg/mL collagen solution in 50 mM acetic acid into a 100 ×100 mm2 sized plate. PHB-GEL-COL and PHB-GEL-OSA-COL scaffolds were obtained by coating the electro-spun PHB-GEL and PHB-GEL-OSA scaffolds with the freshly prepared collagen solution and were dried at room temperature. The collagen coated scaffolds were stored at ambient temperature until further usage55. 2.8. Fourier Transform Infrared Spectra (FTIR) FTIR measurements were performed using ABB 3000 spectrometer with Grams as the operating software to determine the functional groups present in the prepared OSA, COLF, GEL, PHB, PHB-GEL-OSA and PHB-GEL-OSA-COL samples. The spectra were recorded in the frequency range of 4000-600 cm−1.

2.9. NMR and ESI-MS Measurements Commercially available solvents and reagents were used for instrumental analysis without further purification. Melting points were determined in centigrade scale in one end open capillary on Buchi-570 melting point apparatus and are uncorrected. 1H NMR,

13

C

NMR and DEPT-135 spectra were recorded using CDCl3 as a solvent with tetramethylsilane (TMS, δ=0.00) as an internal standard on a Bruker spectrometer at 400 and 100 MHz, respectively. NMR chemical shifts (δ) is expressed in parts per million (ppm). Spin multiplicities are denoted as s (singlet), d (doublet), t (triplet) and m (multiplet). Coupling constants (J) are given in hertz. Electrospray ionization (ESI) technique was used to record mass spectra and the corresponding [M++1] mass peaks were observed.


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2.10. Scanning Electron Microscopy (SEM) The scaffolds (10 x 10 mm2) were carefully mounted on adhesive tape placed over brass studs and gold coated using an ion coater (Emitech brand) in a vacuum condition. This improves the conductivity of the prepared samples. The surface morphology of the prepared scaffolds was examined using F E I Quanta FEG 200-HRSEM by which the secondary electrons were detected. It operates at an accelerating voltage of 10.00kV. Diameters of fibers of different scaffolds prepared were measured using UTHSCA image tool software. 2.11. In Vitro Enzymatic Degradation Study The in vitro enzymatic degradation study helps to predict the stability of prepared material at a wound site. The biological stability and the degradation rate of the material was determined by exposing a small square-shaped nanofibrous scaffold (10 × 10 mm2) to collagenase enzyme (Sigma #C0773). Before conducting the study, the prepared scaffolds were pre-weighed and air dried overnight at room temperature in a laminar hood. COLF, PHB-GEL-COL and PHB-GEL-OSA-COL scaffold were exposed to the collagenase enzyme (100 units per mL) with mild shaking at 37°C (pH 7.4) and samples were withdrawn at different time intervals, left to air dry for 24h in a laminar hood to remove moisture content from the samples. The percentage weight loss and end mass of the scaffold were calculated. Phosphate buffer solution (PBS, pH 7.4) was used for the preparation of enzyme solutions. The in vitro enzymatic degradation of prepared nanofibrous scaffolds were determined by the gravimetric method through the weight loss. All the study was carried out in triplicates.

2.12. In Vitro Swelling Study This primary principle of this study is to correlate the size of full thickness with the wound exudates absorption capacity of the prepared scaffolds. For this study, the scaffold was cut into a square piece (10 × 10 mm2 size) and immersed into phosphate buffer solution (PBS, pH 7.4) at room temperature. Once the equilibrium was reached, the samples were removed from the buffer solution. For measuring the in vitro swelling behaviour of the nanofibrous scaffold, the surface wetness was removed using filter paper and was hung for 1



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min to get rid of excessive moisture. The following equation was used to calculate the equilibrium-swelling ratio56. Swelling (%) = × 100

-----1

Where Wi and Wf is the initial and final weight of the scaffold, respectively.

2.13. Tensile Strength Measurement Tensile strength and elongation are the two important phenomena of the prepared nanofibrous scaffolds as they play a vital role in covering the irregular shapes of wounds. The tensile strength and load elongation measurement were performed by cutting all the scaffolds into dumb-bell shaped specimens (100 × 16 mm2). According to Vogel, a universal testing machine (INSTRON model 1405) was used to carry out load-elongation measurement at an extension rate of 5 mm/min57.

2.14. In Vitro Biocompatibility, Cell Adhesion and Proliferation Studies By using MTT assay, the cell viability of the nanofibrous scaffold has been measured. The fibroblast (NIH 3T3) cell line was grown on the COLF, PHB-GEL-COL and PHB-GELOSA-COL nanofibrous scaffold placed in a 24-well plates (Corning, NY) and kept in DMEM with 10 % fetal calf

serum along with the addition of penicillin (120 units per mL),

streptomycin B (75 mg mL-1) at 37°C at a density of 5×104 cells per mL, incubated over a period of time intervals 24 h, 72 h and day 5 in a humidified atmosphere containing 5 % CO2. Blank wells were cultured with cells and used as a control. Culture medium was replaced by serum-free

medium

comprising

of

10

mL

of

3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT), and incubated for four h at 37°C in a humidified atmosphere with 5 % CO2 After 24 h, 72 h and day 5. The medium was aspirated with 500 mL dimethyl sulfoxide (DMSO) per well to dissolve the Formazan needles with slow agitation for 10 min to yield a bluish purple solution. The absorbance was measured at 570 nm by using Universal Microplate Reader. The live/dead assay of NIH 3T3 fibroblast cell attachment and proliferation were quantified at regular time intervals (day 1, 3, 5), the medium was removed and washed with PBS. To this, Calcein AM solution (4 µM; 500 µL) was added and incubated for 30 min. The scaffold plates were washed with PBS and viewed under the blue filter containing fluorescence microscope58 (Euromex, Holland).


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2.15. Antimicrobial Activity of Osthole, Ostholamide (OSA), COLF and PHB-GELOSA-COL Nanofibrous Scaffold

2.15.1. Preparation of Seeded Plates The nutrient agar medium (50 mL) was prepared and was sterilized using an autoclave at 15 min with the pressure of 15 lbs per square inch at 121°C and cooled for about 30 min. The sterilized medium was then removed from the autoclave and the suitable organism was taken to the laminar airflow room which was previously sterilized using a U.V. lamp. A loopful of the organism from the solid or liquid culture (as the case may be) were taken and seeded on a nutrient agar medium in a conical flask. At around 50°C, the nutrient agar and the seeded organisms were mixed thoroughly. The seeded medium was shaken well and was transferred into previously sterilized Petri dishes and was left undisturbed. The medium settled within 15-20 min at room temperature.

2.15.2. Disc Diffusion Method Gram-positive

organisms

such

as

Staphylococcus

aureus

(ATCC

9144),

Staphylococcus epidermidis(ATCC 155), Bacillus subtilis (ATCC 6633), Bacillus cereus (ATCC 11778) and Micrococcus luteus and Gram–negative organisms such as Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 9027) and fungal pathogenic strains were used to study the antibacterial and antifungal studies of Osthole, Ostholamide(OSA), COLF and PHB-GEL-OSA-COL fibrous scaffolds. Standard drugs, Gentamycin (Std), Ciprofloxacin (Std) and Ketoconazole (Std) served as the control for both antibacterial and antifungal studies, respectively. The organisms were obtained from the Institute of Microbial Technology, Sector 39A, Chandigarh, India 160036. Disc diffusion method was employed using inoculums containing nutrient agar medium to perform antimicrobial tests. The dried plant extracts and ostholamide were dissolved in 5 mg/mL of DMF. To the sterile filter paper disc of 6 mm diameter, 5 µL of the plant extract and compound (250 µg/disc) was applied. Inoculated nutrient agar/sabouraud dextrose medium in the presence of solvent control was used to place the prepared discs and scaffold. For both bacteria and fungi, suitable reference antibiotic disc was applied. Gentamycin 10 µg/disc and ciprofloxacin 25 µg/disc served as positive control for bacteria and ketoconazole 25 µg/disc served as a positive control for fungi. DMF impregnated on the filter paper disc served as the solvent control. Incubation was carried out for 24 h at 37.5°C for bacterial Petri dishes and 9 ACS Paragon Plus Environment


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24-48 h at 37°C for fungal petri dishes, respectively. The zone of inhibition (mm) displayed the antimicrobial activity of osthole, ostholamide (OSA), COLF and PHB-GEL-OSA-COL fibrous scaffolds. All experiments were carried out in duplicate.

2.16. Encapsulation Efficiency and In Vitro Drug Release Assay A small piece (20 × 20 mm2) of an electro-spun PHB-GEL-OSA-COL scaffold and OSA-COL film were selected to determine the drug (OSA) loading capacity, encapsulation efficiency and release of OSA from PHB-GEL-COL nanofibrous scaffold and collagen film (triplicates). Each piece was kept in a round bottom flask, covered with aluminium foil and dissolved in ethyl acetate. The dissolving process was carried out at room temperature and upon completion, the saturated homogeneous solution was taken and analyzed using UV. The percentage drug (OSA) loading and encapsulation efficiency were calculated by the following formula59. !"#$ % &' () % drug (OSA)loading = × 100 *"+*"+) &' () !"#$ % &' () % Encapsulation Efficiency = × 100 )"#$ #+) % &' ()

-----2 -----3

The Franz diffusion model was used to determine drug release study. In the upper chamber, a 20×20 mm2 sized fibrous scaffold and collagen film was placed. The lower chamber was filled with 17.5 mL of physiological synthetic serum electrolyte solution (pH 7.4 maintained at 37 °C, with constant stirring). A wet dialysis membrane placed over the aperture of Franz diffusion apparatus separates the two chambers. One mL of aliquots was withdrawn at various time intervals which was subsequently replaced by same volume of fresh medium. The release of osthole was determined spectrophotometrically. A calibration curve was taken by UV-Visible spectrophotometer at 235 nm for the determination of ostholamide drug concentration. The concentration of drug release was calculated60. A plot of cumulative in vitro release of drug from the nanofibrous scaffold Vs time was obtained. 2.17. In Vivo Studies of Full Thickness Excision Wound Healing Rat Model. In this study, Wistar rats weighing 180-200 g were used as experimental objects. The permission to carry out the animal study for biomedical research and protocols related to the experiments were approved by CSIR-Central Leather Research Institutional animal ethical committee, Chennai, India, IAEC No. 17/2015 (A) and in agreement with the NIH guidelines.


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The 48 animals used in this study were divided similarly into four groups, each group comprising of 12 rats. Group 1, control (sterile cotton gauze); group 2, collagen film (COLF); group 3, PHB-GEL-Collagen fibrous scaffold; group 4, PHB-GEL-OSA COL nanofibrous scaffold. A dosage of 60 mg/Kg of intraperitoneal injection of ketamine was used as an anaesthesia for the rats. For the evaluation of wound healing, the dorsal hairs of the rats were shaved and ethyl alcohol (70%) was used for disinfecting the skin. A scalpel blade was used to create full thickness excision open wounds of 2 cm2 area and excising of the dorsal skin was continued further. The wound region of the rats were photographed and the transparent sheet was used for tracing. The excised wounds were covered with the prepared scaffold and it was protected using an absorbent gauze for the applied material to be held in the wound area. After a period of 5, 10 and 15 days of wounding, cervical dislocation of four animals from each group was done to sacrifice them. The wound areas were identified and regenerated skins were exercised for histopathological investigation. The effect of percentage wound closure was measured using the formula. Cn = [(So-Sn)/So] ×100

---4

Where Cn is the reduction of wound size after 5, 10 and 15 days of wounding, So is the original wound area and Sn is a wound area on day 5, 10 and 15 days of post wounding. 10% neutral buffered formalin, stained with haematoxylin and eosin (H&E) was used for performing the histology of exercised regenerated skin. Determination of the histology and scoring was done by the pathologist in a treatment-blinded valuation manner. The observed scoring method was followed for determination of histological scoring for neovascularisation and inflammatory infiltrate.

2.18. Statistical Analysis The experiments were done in triplicate. Results are presented as mean ± S.D. (n = 3). ANOVA (analysis of variance) and student’s t-test was performed to determine the significant differences between the groups. The observed differences showed statistical significance when p

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