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Fabrication of Hybrid Collagen Aerogels Reinforced with Wheat Grass Bio-Actives as Instructive Scaffolds for Collagen Turnover and Angiogenesis for Wound Healing Applications Dharunya Govindarajan, Natarajan Duraipandy, Kunnavakkam Vinjimur Srivatsan, Rachita Lakra, Purna Sai Korrapati, Ramasamy Jayavel, and Manikantan Syamala Kiran ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 03 May 2017 Downloaded from http://pubs.acs.org on May 4, 2017

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ACS Applied Materials & Interfaces

Fabrication of Hybrid Collagen Aerogels Reinforced with Wheat Grass Bio-Actives as Instructive Scaffolds for Collagen Turnover and Angiogenesis for Wound Healing Applications Dharunya Govindarajan, † Natarajan Duraipandy, †, # Kunnavakkam Vinjimur Srivatsan, † Rachita Lakra, † Purna Sai Korapatti, †, # Ramasamy Jayavel, ‡ and Manikantan Syamala Kiran†, #, * †

Biological Materials Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai

600020, Tamil Nadu, India #

Academy of Scientific and Innovative Research, CSIR-Central Leather Research Institute,

Adyar, Chennai 600020, Tamil Nadu, India ‡ Centre for Research, Anna University, Chennai 600025, Tamil Nadu, India

*Corresponding Author Email: [email protected] and [email protected]

Keywords: Aerogel, collagen, wheat grass, wound care, angiogenesis. ABSTRACT. The present study illustrates the progress of wheat grass bio-actives reinforced collagen based aerogel system as instructive scaffolds for collagen turn over and angiogenesis for wound healing applications. The reinforcement of wheat grass bio-actives in collagen

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resulted in design and development of aerogels with enhanced physico-chemical and biomechanical properties due to the intermolecular interaction between the active growth factors of wheat grass and collagen fibril. DSC analysis revealed an enhanced denaturation temperature when compared to native collagen aerogels. FTIR analysis confirmed that the reinforcement of bio-actives in the wheat grass did not affect the structural integrity of the collagen molecule. Additionally, the reinforced biomaterial with a systematic absorptive morphology resulted in a three-dimensional sponge-like aerogel exhibiting a potent highly oriented 3-dimesional structural assembly that showed increased water retention ability and substance permeability that would enable the passage of nutrients and gaseous components for cellular growth. Furthermore, the cumulative effect of the growth factors in wheat grass and the collagen molecule augments the angiogenic ability and collagen production of the aerogel by restoration of the damaged tissue thereby making them a potential 3-D wound dressing scaffolds. The results were confirmed by in vivo wound healing assays. This study delivers possibility for the progress of a biocompatible, biodegradable and non-adhesive nutraceutical reinforced collagen aerogel as an instructive scaffold with good anti-microbial properties for collagen turn over and angiogenic response for wound healing application. INTRODUCTION Wound healing and repair management is well regulated complex processes which are spatiotemporally controlled by several cellular synergistic events and any alterations in these processes leads to delayed wound healing.1-3 Minor wound heals physiologically without any requirement for its dressing material. However, in case of wounds where substantial amounts of tissue loss has happened, wound bed should be covered with suitable wound dressing materials for early repair and closure.4 Essentially, these dressing materials should have good anti-proteolytic


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activity, anti-microbial activity to prevent sepsis and capability for enhanced collagen turnover.57

Currently, collagen based materials are gaining importance due to its conserved nature and

being a part of extracellular matrix; it seems to be a stockpile for prominent signaling elements to actuate cell attachment and proliferation for tissue regeneration.8,9 But, collagen based materials are endowed with drawbacks such as they are highly prone to microbial attack, susceptibility to proteolytic degradation as well as adhesive nature of collagen makes it difficult for its use as dressing material.10 Hence, there is a pressing demand for the development of dressing materials with good 3-Dimensional architecture having excellent anti-microbial, good proteolytic stability and collagen turn over property.11 One of the strategies to make good dressing materials is to use bioactive molecules such as nutraceuticals to bring in good therapeutic value. The beneficial effect of the nutraceutical requires a platform in modern regenerative medicine to ensure its authentic effect of biocompatibility and facilitated bioactivity against ailments.12 In the current tissue regeneration perspective, moisturized porous hydrogel systems engineered with bio-molecules are being utilized among several other wound dressing scaffolds. Moreover, the surface wound exudates are absorbed by these scaffolds from infected site and promote a firm with-holding of cells for differentiation.13 Hydrogels, made of polymers have gained a greater attention in medicine as they possess high swelling property, functional drug delivering capacity, non-toxicity, biodegradability and biocompatibility.14,

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However, the

collagen hydrogel faces several drawbacks due to its reduced mechanical stability. The ideal wound dressing materials are supposed to be designed in such a way that they should have higher bio-stability and mechanical stability.16 The strategy for the development of such wound dressing materials involve the usage of cross-linkers that cross-links collagen fibrils imparting them with good biomechanical stability. Currently, nutraceuticals are gaining importance as cross-linkers of


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collagen due to their therapeutic property as well as active functional groups that promote crosslinking of collagen.17-19 In this direction, various nutraceuticals have been tested as cross-linkers of collagen namely; curcumin, plumbagin, furfural, shikkimic acid. Wheat grass (Triticum aestivum) extract has been currently used as a health drink due to the presence of several bioactive constituents in it. It has been reported to have potent anti-inflammatory, anti-cancerous, anti-bacterial, anti-fungal and anti-oxidant effect.20-23 The significant amounts of mineral nutrients, chlorophyll, vitamin E, vitamin C, anti-anemic factors like vitamin B12, iron, folic acid, pyridoxine, amino acids and enzymes in wheat grass are known for their therapeutic value against various diseased conditions.24, 25 The presence of above mentioned supplements in wheat grass with active growth factors have also been reported to promote adequate re-epithelialization of cells in the infected site and favors reduction in scarring after the exudates get absorbed.26-29 However, there are no documented reports on the use of wheat grass as cross-linkers of collagen for wound healing applications. Hence, in this manuscript, the effect of novel wheat grass reinforced collagen aerogel was assessed as a moisture-free wound dressing material and the results showed good anti-bacterial activity, cell proliferation, collagen turnover required for wound healing with pro-angiogenic action. EXPERIMENTAL SECTION Materials. Analytical grade chemicals were used throughout the study. Food grade wheat grass powder was purchased from organic stores. Cell culture chemicals were purchased from Sigma Aldrich, USA. Extraction of collagen from Goat Tendon. Collagen from goat tendons was isolated by salt precipitation method.30 The tendons were cut into small pieces and washed for five times with distilled water followed by the treatment with diethyl ether: chloroform to remove lipids. The


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tendons were then immersed in 0.5 M acetic acid overnight at 4ºC for swelling. The mixture was then ground well and centrifuged at 14000 rpm for 30 minutes to obtain the supernatant. The supernatant was salt precipitated by slow adding 5% NaCl and centrifuged to collect the precipitated collagen which was re-dissolved in 0.5M acetic acid to obtain thick crude collagen. Finally, pure collagen solution was obtained by removing excess salts using dialysis against 0.05M acetic acid. The collagen solution was then lyophilized and stored at -40ºC. Preparation of wheat grass extract. 5 g of crude wheat grass powder was mixed with 100 ml of 1X Phosphate Buffer Saline and boiled to half the volume for about 20 minutes to obtain a concentrated extract. The solution was centrifuged at 5000 rpm for 15 minutes to collect the supernatant followed by filtering the extract using a 0.22 µm syringe filter. Further, three different concentrations of wheat grass extracts (1% w/v, 2% w/v and 3% w/v) were prepared from the concentrated mixture for the fabrication of wheat grass-collagen based aerogels. Characterization of wheat grass reinforced collagen aerogels. Scanning electron microscopic analysis. The surface morphology of native collagen and wheat grass (1%, 2% and 3%) reinforced collagen aerogels were analyzed using VEGA TESCAN 3 scanning electron microscope (SEM). About 1 mg of lyophilized native collagen and wheat grass reinforced collagen aerogels were mounted on to the stub and coated with gold by sputtering for 2-3 min. The sputtered aerogels were then scanned using a high resolution electron beam under vacuum. Atomic Force Microscopic analysis. The surface topography of native collagen and wheat grass reinforced collagen samples were analyzed using NTEGRA PRIMA atomic force microscope. All the samples were carefully dropped onto the sterile glass slides of 1cm x 1cm and kept for drying at room temperature for 24 h. The AFM outfitted with silicon nitride


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cantilevers was used to analyze the thin surfaces of collagen samples using semi-contact mode and the spring constant of the probes used was 3.1-37.6 Nm-1. The roughness of specimen was calculated from data obtained from the equipment along with their topographical images. Differential Scanning Calorimetric analysis. Denaturation temperature of native and wheat grass reinforced collagen aerogels was studied using DSC PERKIN ELMER. In brief, 5-6 mg of the samples were taken individually and kept into the aluminum pans for sealing. These sealed pans containing the samples were then heated at a temperature of 50ºC-350ºC at a heating rate of 5ºC/min. Fourier Transform Infra-Red Spectroscopic analysis. FTIR spectrum of native collagen and wheat grass reinforced collagen aerogels were recorded under transmission mode using a SPECTRUM TWO PERKIN-ELMER SPECTROPHOTOMETER. The samples were ground with KBr in a ratio of 1:100 and pelleted using Atlas Manual 15T Hydraulic Press pellet maker. A scanning range from 1000 cm-1 to 4000 cm-1 was recorded with 7 scans per sample and resolution of 1 cm-1. TNBS assay. The cross-linking degree of wheat grass reinforced collagen aerogels was quantified using trinitrobenzenesulfonic acid (TNBS) assay by calculating the amount of free amino groups present in the collagen aerogels reinforced with three different concentrations of wheat grass and it was compared against control collagen aerogels (Blank). The native collagen and reinforced aerogels were treated with 500 µl of 4% NaHCO3 and 500 µl of 0.05% TNBS and the tubes were kept for incubation at 37°C. After two hours of incubation, 1.5 ml of 6 N HCl was added to the aerogels to cease the reaction. These reaction mixtures were then diluted with distilled water (2.5 ml) and the absorbance was measured at 345 nm.


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Water uptake study. Water uptake study was performed to determine the permeability of native collagen aerogels and wheat grass reinforced collagen aerogels. In brief, the samples were immersed in distilled water and incubated at room temperature for different time periods. At certain intervals (1 h, 3 h, 5h, 7h, 22h, 24h, 36h and 48h), aerogels were removed from water and the wet weight was measured. The swelling behavior of the aerogels was calculated using the following equation 1: Swelling (%) = [(Ws-Wd)/Wd] x100 Where, Wd - weight of dry scaffold; Ws - Weight of wet scaffold. Substance permeability assay. In vitro substance permeability assay was carried out to assess the porous nature of native collagen and wheat grass reinforced collagen aerogels. The permeating efficacy of the aerogels was determined by calculating the amount of dye (rhodamine B) passed through the samples at different intervals of time. The absorbance value of rhodamine B present in the medium was measured at 550 nm using a Bio-Rad Elisa Plate reader to confirm the permeable nature of aerogels. Mechanical properties. Mechanical properties such as tensile strength, elongation at break (%) and extension at maximum load (mm) of native collagen and wheat grass reinforced collagen aerogels were measured. The bandage like scaffolds were prepared with the dimensions of 5 cm x 1 cm x 0.5 cm whose ends was clipped with a gripper. The measurements were made using an Instron tensile tester instrument with a load of 1 N and the crosshead speed of 5mm/min at room temperature. Rheological measurements. Shear studies on collagen aerogels were performed on Brooke field R/S + Rheometer with coaxial measuring at different shear rates of 28ºC. The shear stress and


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shear viscosity of native collagen and wheat grass reinforced collagen samples were tested against shear rate. Native collagen was used as a control. Hemo-compatibility assay. Hemo-compatible behavior of native collagen and wheat grass reinforced collagen aerogels was tested using human heparinized blood.31 RBC pellet was separated from blood by centrifugation process. HEPES (5mM) buffer containing sodium chloride (150mM) was used for washing the red blood cells for at least three to four times. The aerogels were kept in the microfuge tubes containing 1 ml (107cells) suspension and incubated at 37ºC for 30 minutes. The absorbance values of supernatant were measured at 540 nm after centrifugation. Percentage of hemolysis was calculated from the equation: 2 mentioned below; Hemolysis (%) = [(Absorbance of sample-Absorbance of negative control)/(Absorbance of positive control-Absorbance of negative control)] x 100 Biomedical Applications of aerogels. Cell compatibility assay. Biocompatibility of control collagen and wheat grass reinforced collagen aerogels was studied by MTT assay. The aerogels of 6mm x 6mm size were placed in the tissue culture plates containing approximately 30,000 EA Hy926 (Endothelial cells) and incubated in a CO2 incubator. The culture medium was removed from the culture well after 24h and added with 0.5 mg/mL 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) salt dissolved in PBS. The culture plate was then incubated at 37ºC for four hours. The insoluble formazan crystals formed was solubilized in DMSO (dimethyl sulphoxide) and the optical density was measured at 570 nm. The cell viability was calculated from the following equation: 3, Cell viability (%) = [Optical density of aerogel/Optical density of control] * 100 Evaluation of collagen expression.


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Cell culture. A density of 2,00,000 Mouse Fibroblasts (Swiss3T6) and Human Keratinocytes (HaCaT) were seeded in the tissue culture flasks containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 24 mM sodium bicarbonate and 25 mM HEPES, 20% Heatinactivated Fetal Bovine Serum and buffered at pH 7.4 for the cells to reach 70-80% confluence. The atmosphere of 5% CO2/95% air at 37°C was maintained. The cells were then subjected to serum starvation followed by the treatment with control collagen and three different concentrations of wheat grass reinforced (CC, WG1%, WG2%, WG3%) collagen aerogels to study the expression of collagen. After 24 hours of incubation, fibroblast and keratinocyte cells were harvested for total RNA isolation. RNA Isolation and PCR Analysis. To evaluate the collagen expression in fibroblasts and keratinocytes, the confluent cells in the culture flasks were homogenized using trizol reagent followed by phase separation using chloroform. Then the colorless upper aqueous phase was collected by centrifugation at 12,000 x g for 15 minutes at 4°C to obtain total RNA. Collagen primer pair includes forward primer-5’-GAGATGATGGGGAAGCTGGAAAAC-3’ and reverse primer-5’-GGCACCATCCAAACCACTGAAA-3’ (140 bp). The primer sequences were designed and custom synthesized by Priority Lifescience, India. Semi-Quantitative PCR was performed in an Eppendorf thermocycler, using MASTER CYCLER GRADIENT PCR. The isolated RNA (10 µl) was quantified by absorbance at 260 nm and used as template for reverse transcription followed by amplification. It was then size fractionated by 2% agarose gel electrophoresis. Anti-bacterial activity. Anti-microbial activity of native collagen and wheat grass reinforced collagen aerogels were determined using agar diffusion method. A fresh sample of native collagen and wheat grass reinforced collagen aerogels (6mm x 6mm) were placed in the agar


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plates containing E.coli (Gram negative) and B.subtilis (Gram positive) cultures. The plates were incubated at 37ºC overnight and the bactericidal effect was determined by measuring the inhibitory zones formed around the samples. Chorioallantoic membrane (CAM) assay. CAM assay was performed on fertilized white leghorn eggs to check the angiogenic activity and embryo development. On fifth day, the eggs were cleaned using alcohol and the shells were opened on the blunt end to create a window opening at the central region. Wheat grass reinforced collagen aerogels (6mm x 6mm) were placed on the CAM in a suitable position over the vasculature. These eggs were then sealed with a sterile parafilm and kept under incubation at 37ºC in a humid atmosphere. The seal was removed at certain time intervals and the development of micro vessel with prominent embryonic growth was photographed. Aortic ring assay. Aortic ring assay was performed to study the extent of endothelial sprouting in the aortas treated with different concentrations of wheat grass based collagen aerogels. Thoracic aortas excised from female wistar rat (130-150 g) were cut into small pieces of 0.1 cm long rings rinsed in DMEM medium. These rings were placed into the culture plates containing collagen aerogels with and without wheat grass of various concentrations. The plates containing the samples were kept under incubation with 95% air and 5% CO2 at 37ºC. The culture medium was replaced every day and the sprouting from aortic rings was observed under microscope at different time intervals. Wound Healing. The approval was obtained from IAEC- Institutional Animal Ethical Committee (CSIR-Central Leather Research Institute, Chennai) for the use of animals in biomedical research. The rules and regulations were authentically followed according to the proper guidelines of IAEC. The open excision wound healing assay was performed on female


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wistar rats weighing 130-150 g and the animals were maintained under standard habitual conditions for seven to nine days. Before starting the experiment, the rats were anaesthetized by ketamine/xylazine through intra-peritoneal route. The hair on the dorsal side of the skin was shaved and disinfected for the creation of open excision wounds. The size of wound (2cm x 2cm) was marked out and the wound regions on the dorsal side of the rats were photographed. The test groups were categorized by treating the wounded rats with wheat grass reinforced collagen aerogels; native collagen aerogels were used as control groups and the untreated controls was grouped separately. The open excision wounds were then treated with aerogel scaffolds and the wound dressing was made using sterile gauze. The wound was cleaned with sterile saline and the dressing was changed every third day. The wound size reduction was outlined and photographed at different periods of time. The percentage of wound reduction was calculated using Equation: 4; Cn= [(so-sn) /so]*100 Where, Cn represents the percentage of wound size reduction on days 3, 9, 12, 15 and 18, So represents initial wound size and Sn represents the wound size on day 3, 9, 12, 15 and 18 after treatment. Statistical analysis. The analysis throughout the study was carried out in triplicates. The final results were obtained from the average of three experiments. The statistical analysis was performed using SPSS 22 software. The P value

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