Collagen-silica bio-composite enriched with Cynodon dactylon extract for tissue repair and regeneration

Accepted Manuscript Collagen-silica bio-composite enriched with Cynodon dactylon extract for tissue repair and regeneration

Ramesh Kannan Perumal, Arun Gopinath, Thangam Ramar, Sathiamurthi Perumal, Dinesh Masilamani, Satiesh Kumar Ramadass, Madhan Balaraman PII: DOI: Reference:

S0928-4931(17)33354-4 doi:10.1016/j.msec.2018.06.050 MSC 8689

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

19 August 2017 17 May 2018 25 June 2018

Please cite this article as: Ramesh Kannan Perumal, Arun Gopinath, Thangam Ramar, Sathiamurthi Perumal, Dinesh Masilamani, Satiesh Kumar Ramadass, Madhan Balaraman , Collagen-silica bio-composite enriched with Cynodon dactylon extract for tissue repair and regeneration. Msc (2018), doi:10.1016/j.msec.2018.06.050

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ACCEPTED MANUSCRIPT Collagen-Silica Bio-composite enriched with Cynodon dactylon extract for Tissue Repair and Regeneration Ramesh Kannan Perumal, Arun Gopinath, Thangam Ramar, Sathiamurthi Perumal, Dinesh Masilamani, Satiesh Kumar Ramadass, Madhan Balaraman*

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CSIR - Central Leather Research Institute, Adyar, Chennai – 600020, Tamil Nadu, India

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Corresponding Author:

*Dr. B. Madhan, Principal Scientist CSIR- Central Leather Research Institute Adyar, Chennai 600020, India Tel: +91 44 24437169, Fax: +91 44 24911589 E-mail:[email protected], [email protected]

ACCEPTED MANUSCRIPT Abstract Development of biomaterials for tissue engineering applications is of great interest to meet the demand of different clinical requirements. The wound heal dressing biomaterials should necessarily contain well-defined therapeutic components and desirable physical,

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chemical and biological properties to support optimal delivery of therapeutics at the site of

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the wound. In this study, we developed collagen-silica wound heal scaffold incorporated

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with the extract of Cynodon dactylon, characterized and evaluated for its wound heal

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potential in vitro and in vivo against collagen (Col) and Collagen-silica (CS) scaffolds that served as controls. The prepared Collagen-Silica-Cynodon extract (CSCE) scaffold exhibits

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porous morphology with preferable biophysical, chemical, mechanical and mass transfer

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properties besides its controlled biodegradation at the wound site. Stability of CSCE was found to be better than that of native collagen due to intermolecular interactions between

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collagen and constituents of C. dactylon as confirmed by FTIR analysis. Notably, in vitro

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biocompatibility assay using DAPI and Rhodamine 123 staining demonstrated that the proliferation of NIH3T3 fibroblast cells was better for CSCE when compared to the Col and

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CS scaffolds. In vivo wound healing experiments with full-thickness excision wounds in

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wistar rat model demonstrated that the wounds treated with CSCE showed accelerated healing with enhanced collagen deposition when compared to wounds treated with Col and CS scaffolds, and these studies substantiated the efficacy of CSCE scaffold for treating wounds. Keywords: Wound healing; Biomaterial; Collagen; Cynodon dactylon; Cell proliferation.

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1. Introduction Wound healing is a multifaceted skin regenerative process comprising the events of suppression of inflammatory reactions, migration, and proliferation of connective tissue

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cells such as fibroblasts, collagen deposition, and remodeling of extracellular matrix (ECM)

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[1]. Effective wound dressings should induce conversion of cells from senescence to active

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state with an expression of optimal levels of collagen proteins and components of ECM but

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inhibit processes facilitating further deterioration to achieve regeneration of healthy tissue. The therapeutic biomaterials are expected to have large surface area with porosity, and

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mass transfer features for enabling effective wound cover, aeration, exudate removal and

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delivery of therapeutics, to facilitate proliferation of cells and controlled biodegradation for replacement with neo-tissues besides exhibiting low toxicity and high biocompatibility [2-

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5].

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Collagen-based biomaterials remain as a treatment of choice for wound therapy as they

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possess the chemotactic ability to activate fibroblasts cells and thereby encourage the deposition and organization of newly formed collagen to create an environment that

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fosters healing. Also, they have other preferable properties such as smooth adherence to the wound bed, the absorbance of wound exudates, preserving the moist environment, shielding against mechanical harm and preventing secondary bacterial infections, facilitating fibroblast growth and minimizing matrix metalloproteinase (MMP) activities, biocompatibility, and safety [6, 7]. It is reported that the functional aspects of collagen biomaterials were modified or improved by several strategies [8, 9]. We have earlier reported collagen and silica based collagen biomaterials for soft tissue engineering

ACCEPTED MANUSCRIPT applications, in which the ratio of collagen and silica were optimized to yield scaffolds with desired physical properties such as porosity, water uptake, and mechanical strength as well as biocompatibility and wound heal potentials [10]. Though there are reports on collagen scaffolds incorporated with growth factors [11], or antibiotics[12], they suffer limitations

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due to the high cost of growth factors making them less affordable and the possibility of the

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emergence of antibiotic-resistant strains if antibiotics are used irrationally to prevent

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infections at the wound site [13]. Besides, the use of antibiotics in biomaterials does not

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significantly support wound healing due to cytotoxicity to cells.

Phytoconstituents or extracts from medicinal plants offer an excellent opportunity to

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compliment the preparation of biotherapeutic materials by means of strengthening their

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regenerative potentials such as angiogenesis, control of infection as well as inflammation with desirable features of high biocompatibility, safety, and affordability [14]. Cynodon

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dactylon L. (also known as Bermuda grass), belonging to the Poaceae family, is a perennial

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grass native to the warm temperate and tropical regions and it has many medicinal properties including antimicrobial, antiviral, anti-inflammatory [15], free radical

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scavenging potential and wound healing activity and these activities are attributed to

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phytochemicals such as hexadecanoic acid, apigenin, luteolin, quercetin etc. [16-18]. The use of C. dactylon, especially in reducing the inflammation as well as infection, angiogenesis and the associated wound healing benefits, has been widely reported in various traditional medicine systems [18, 19]. In this study, we attempt to develop Collagen-Silica-Cynodon extract (CSCE) scaffold comprising the collagen and phytoconstituents of C. dactylon for exhibiting synergistic and systemic effects on wound healing. The developed CSCE composite scaffolds were

ACCEPTED MANUSCRIPT characterized for biophysical, mechanical, biocompatibility and wound healing properties against Collagen and Collagen-Silica (CS) scaffolds in vitro and in vivo. 2. Materials and Methods

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2.1. Materials, cells, and animals

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Collagenase Type 1A, 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI), Rhodamine

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123 and reagents for MTT (3-(4, 5-dimethyl-thiazol-2-yl)- 2,5-di-phenyl-tetrazolium bromide) assay were purchased from Sigma-Aldrich (Bangalore, India). Tetraethoxysilane

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(TEOS) was purchased from Alfa Aesar (Heysham, UK). All other chemicals used in this

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study were of analytical grade. Ultra-pure water from a Siemens water purification system was used in all experiments.

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Forty-eight Wistar rats (female, six weeks old, 200 – 220 g) from Laboratory Animal

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Medicine (LAM) unit of Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Chennai were used in this study. The ambient temperature and relative humidity were

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maintained constant in the animal facility. Rats were acclimatized for one week before the

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experiments. Food and water were available ad libitum.

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2.2. Preparation of Cynodon dactylon extracts The extraction method, yield and chemical composition of cynodon plant have been reported elsewhere[18, 20]. The Cynodon dactylon plant was collected from Tiruvallur district of Tamil Nadu, India. The plant was washed with distilled water, shade dried and powdered with the help of domestic mixer. 10 g of the fine powder was soaked in 100 mL of 80% aqueous ethanol and stirred for 5 h and filtered using cheese cloth followed by centrifugation at 10,000 rpm for 15 min. After centrifugation, the ethanol was removed by

ACCEPTED MANUSCRIPT using rotary evaporator under reduced pressure at 35 °C, and the dried extract was placed at 4 °C until further use. The yield of the extract was about 8% of the crude powder (4 g of extract from 50 g of crude powder). The dried extract was dissolved in ethanol in a known concentration and used for further studies.

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2.3. Collagen extraction and scaffold preparation

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Acid solubilized collagen was isolated from bovine Achilles tendon according to the method of Tanaka et al. (1988) [21]. The collagen obtained was lyophilized, and redissolved in 0.05

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M acetic acid at a final concentration of 4 mg/mL and stored at 4 °C. This solution was continuously stirred using Ultra-Turrax IKA T25 Homogenizer at 15,000 rpm to generate

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uniform foam. During homogenization, a drop of Triton X-100 was added as a frothing

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agent. The collagen foam was poured immediately into a 90 mm sterile Petri dish and

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frozen at -80 °C overnight followed by freeze drying for 30 h continuously at temperature of -45 °C and pressure of 1 Pascal (Lark freeze dryer, perquin classic plus). The completely

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dried scaffolds were stored at 4 °C in airtight plastic containers and denoted as collagen

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sponge (Col).

Stober process involves sol-gel transmission assisted by the formation of silica from TEOS,

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which undergoes hydrolysis in the presence of acids and bases [22]. Acid catalyzed TEOS was added to the collagen solution, and the pH was adjusted to 6.0 using 0.32 M ammonium hydroxide to prepare collagen: silica (1:0.75 ratios) composite scaffold (CS) and other steps were followed as in the case of collagen sponge preparation. For the preparation of CSCE, 1.5 mL of Cynodon dactylon extract (CE) was added dropwise to the

ACCEPTED MANUSCRIPT above collagen and silica solution. Further steps were followed as mentioned above for the preparation of the scaffold. 2.4. Characterization of the scaffold Surface morphology of the prepared collagen and collagen composite scaffolds were

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studied using scanning electron microscopy (SEM, JEOL-JFC 6360), operated at an

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accelerating voltage of 5 kV. Then, the pore diameters of the SEM pictures of the scaffolds were analyzed using ImageJ (National Institute of Health, USA). The presence of functional

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groups in the collagen (Col), collagen-silica (CS) and CSCE scaffolds was analyzed using an FTIR Spectrometer (Jasco, Japan) in the range of 500 – 4000 cm-1. Thermal stability of the

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prepared scaffolds was investigated using Differential Scanning Calorimetry (DSC Q200

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Model, TA Instruments Inc., USA). DSC experiments were performed at temperatures

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ranging from 20 °C to 300 °C, at a controlled heating rate of 10 °C per min. In vitro enzymatic degradation studies of pure collagen and collagen, composite materials

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were performed with collagenase, and the release of hydroxyproline in the medium

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through degradation of collagen was analyzed according to the method of Woessner, 1961 [23]. Collagenase treatment was carried out in 0.04 M CaCl2 solution buffered at pH 7.2

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with 0.05 M Tris-HCl at 37 °C. The ratio of collagen and collagenase was maintained at 50:1 (w/w). The sampling was performed at desired time intervals (24 and 48 h). After incubation, the sample was immediately centrifuged at 2000 rpm at 4 °C for 10 min. An aliquot of the supernatant was hydrolyzed with 6 M HCl at 120 °C for 12 h. Degree of collagen degradation of collagen and collagen composite materials were assessed

ACCEPTED MANUSCRIPT spectrophotometrically through the measurement of hydroxyproline released in the supernatant. The Ethylene oxide (EO) sterilization was carried out at an external facility. For sterilization process, the scaffolds were placed in 35mm petri dishes and packaged in self-

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sealing sterilization pouches. EO sterilization was performed by exposure to an EO

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atmosphere (concentration of 321g/m3), at a relative humidity of 65%, for 8h at 50 °C. The

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scaffolds were then aerated with warm airflow (45 °C) at atmospheric pressure for 48 h to

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remove toxic EO.

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2.5. Porosity, Water Uptake, and Mechanical Properties

Scaffold porosity was measured using the liquid displacement technique [24]. Dimensions

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of the sponges were measured using Vernier caliper, and the volume (V) was calculated.

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The pre-weighed scaffold (Wi) was immersed in a known volume of n-hexane in a graduated measuring cylinder for 48 h until it was saturated. The scaffold was removed,

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and the weight of the wet sponge was noted as (Wf), and the following equation was used

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to calculate the porosity of the scaffold:

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𝑃𝑜𝑟𝑜𝑠𝑖𝑡𝑦 (%) =

(𝑊𝑓 − 𝑊𝑖) × 100 (𝜌ℎ𝑒𝑥𝑎𝑛𝑒 × 𝑉)

where ρhexane is the density of hexane. The water uptake ability of the scaffold was studied to understand the diffusion of medium and nutrients into the scaffold. Pre-weighed scaffolds were immersed in phosphate buffered saline (PBS) at 37 °C for 2 h. The excess PBS solution is removed by gently blotting

ACCEPTED MANUSCRIPT the scaffolds on a filter paper and weighed. The water uptake ratio of the scaffolds was calculated using the formula: 𝑊𝑎𝑡𝑒𝑟 𝑈𝑝𝑡𝑎𝑘𝑒 (%) =

(𝑊𝑒𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 − 𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 ) × 100 ( 𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 )

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The mechanical properties of the scaffolds were tested using an Instron texture analyzer at

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room temperature (25 °C). Briefly, freeze- dried samples of 5 mm thickness were cut into 5

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cm x 1 cm size. The gauge length between the two grips was set at 15 mm, and the speed of

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testing was set at 5 mm/min. The values of tensile strength and elongation at break were determined and were expressed as the mean ± standard deviation (n=3).

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2.6. Cell compatibility assays

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The biocompatibility of synthesized scaffold was evaluated in vitro using MTT cell viability

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assay. Mouse embryonic fibroblasts (NIH 3T3 cells) were used as a reference cell line for this evaluation. The Ethylene oxide sterilized collagen (Col), CS and CSCE scaffolds (1 cm2 ×

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2 mm) were placed in 12-well plates which contained cells at a density of 1.5 × 105

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cells/well in 1 mL DMEM medium containing 10 % FBS, and the antibiotics penicillin, and streptomycin (100 IU/mL). The cells were cultured for 24 h in a humidified incubator with

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5% CO2 at 37 °C. The culture medium was replaced with fresh medium every 24 h. After treating the cells with developed scaffolds for about 1, 3and5 days, 500 μL MTT stock solutions (5 mg/mL in PBS) was added to each well and incubated for 4 h. The MTT solution was completely removed carefully and 250 μL DMSO was added to each well to dissolve the formazan blue crystals. The absorbance of the solution was measured using a microplate reader at 590 nm. The cell viability is determined as described below:

ACCEPTED MANUSCRIPT 𝐶𝑒𝑙𝑙 𝑉𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 (%) =

(𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒) × 100 ( 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙)

Here, control refers to collagen scaffold (Col), and samples refer to the CS, CSCE scaffolds.

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2.7. Cell viability and DAPI/Rhodamine 123

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The Col, CS, and CSCE scaffolds were placed onto 6-well plates, and 3T3 fibroblast cell

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suspension was added to the well (1.0 × 105 cells/well). After being incubated for 1, 3 and

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5days, the cells were fixed with methanol (75 mL) and acetic acid (25 mL) (3:1) in PBS for 10 min. Following that, the cells were stained with DAPI, and Rhodamine 123 (Sigma-

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Aldrich, USA) and the stained cells were observed using an inverted fluorescence

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microscope (Floid Cell imaging system, Invitrogen).

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2.8. F-actin filaments staining

A period of 24 h after the composite scaffolds was seeded with cells, F-actin staining was

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carried out to observe the cell interaction. In brief, the cells were initially fixed with 5%

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paraformaldehyde at 37°C for 15 min and permeabilized in 0.1% Triton X-100 at 37°C for 5 min. After permeation, cells were blocked with 10% fetal bovine serum at room for

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temperature

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The

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further

incubated

with

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FluoresceinIsothiocyanate (FITC)-phalloidin at room temperature for one hour. Further, the cell-scaffold constructs were washed with PBS, counterstained with DAPI for 30 min at room temperature for staining the nuclei and visualized under fluorescence microscope. 2.9. In vivo wound healing study

ACCEPTED MANUSCRIPT In vivo wound healing study was performed in a full thickness excision wound model. Female Wistar Rats (body weight range 200−220 g) were used for the study. All experimental protocols were approved by Institutional Animal Ethical Committee (Central Leather Research Institute, Chennai, India, IEAC No. 05/2014(b)) and NIH guidelines were

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followed for proper use of animals for biomedical research. A total of 48 animals were

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divided into four groups, each consisting of 12 rats: group 1, CSCE treatment group; group

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2, CS treatment group; group 3, Collagen (Col) group; and group 4, untreated group

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(Control). Each group of 12 rats was further divided into 3 subgroups of 4 rats each, which were sacrificed at predetermined time points for analysis. Animals were anesthetized by

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intraperitoneal injection of ketamine, at a dose of 50 mg/kg. The dorsal hair of the rats was removed, and the skin was disinfected with 70% ethanol. Full-thickness open excision

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wounds of 2X2 cm were created using scalpel blade by excising the dorsal skin. In this

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study, we have used a square shaped silicone splint to avoid wound contraction. The

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wound was centered within the splint, which was fixed to the surrounding skin by suturing at corners to stabilize its position. The wound was photographed, and the initial wound

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area was traced using a transparent sheet. The scaffolds were applied on excised wounds

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and covered with absorbent gauze to hold the material in the wound area. On days 5, 10, and 15 of post-wounding (PW), four animals from each group were sacrificed by using CO2 chamber, wound area was traced, and regenerated skin was excised for histopathological investigation. The samples of excised regenerated skin were preserved by fixing them in 10% neutral buffered formalin and were examined by stained with hematoxylin and eosin (H&E) and Masson's trichrome staining methods.

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2.10 Statistical Analysis Data were expressed as mean ± SD. One-way ANOVA was performed to determine the statistical significance of the experiments. Differences were considered statistically

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significant at p < 0.05. Statistical analyses were performed using GraphPad Prism 5

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software. 3. Results and discussion

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3.1. Physical and Mechanical Characteristics of Scaffolds

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The management of acute and chronic wounds represents a significant burden regarding pain, impaired quality of life of patients and direct costs of healthcare services. The

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ultimate goal of wound management is to promote the development of healthy granulation

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tissue while minimizing the risk of inflammation, degradation of existing or regenerative

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tissue and opportunistic infection. Hence, in this study, an attempt has been made to prepare a wound healing composite material using collagen, silica and Cynodon plant

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extract (CE). The SEM images of the freeze-dried Col, CS, and CSCE composite scaffolds as

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shown in Fig. 1. We observed that the CSCE scaffold exhibited smooth surface with numerous evenly distributed pores of almost similar size when compared to the Col and CS scaffolds. The average pore diameters of the CS and CSCE scaffolds were found to be 364 and 327 µm, respectively. A slight reduction in the pore size of CSCE was observed against CS scaffold, and this was due to the presence of Cynodon extract in CSCE. We have earlier reported that the addition of silica to collagen improves the porosity of the collagen scaffold [10]. SEM images of CSCE scaffold reveals porous collagen fibrous mesh-like

ACCEPTED MANUSCRIPT appearance suggesting that the material can facilitate air exchange and oxygen diffusion at the wound site, easy absorption of wound exudates, cell attachment, proliferation and migration for tissue regeneration, and these properties are essential for wound heal dressing materials. The results were in agreement with porosity and water uptake analysis

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of prepared scaffolds (Table 1 and Fig. 2). The porosity of the composite materials was

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measured by liquid displacement method in hexane at room temperature (Table 1). CS and

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CSCE scaffolds showed higher porosity at 89% and 93% respectively, whereas native Col

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scaffold showed reduced porosity (81%). This result is in agreement with those reported earlier for CHCS composite scaffold [25]. The addition of extract in the CS scaffold did not

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reduce the porosity of the material, and this was due to the dispersion of phytochemicals evenly throughout the matrix. Water uptake capacity of the prepared scaffolds is examined

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to evaluate the ability of these dressing materials to absorb exudates from the wound.

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Water uptake capacity of the CS was better than CSCE and Col scaffolds (Fig. 2), and this

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was due to the incorporation of silica in the CS composite [10]. The water holding capacity is an essential parameter for wound heal scaffold for preserving the moist environment at

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the wound site to promote rehydration of necrotic tissue for autolytic debridement, cell

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migration, cell-cell interaction, proliferation and migration of the fibroblast cells as well as collagen synthesis [26]. The mechanical properties of Col, CS, and CSCE scaffolds were presented in Table 1. Tensile strength and elongation at break of CS scaffold were reduced when compared to native collagen sponge due to the introduction of silica in the matrix. However, it could be seen that these properties for CSCE scaffold were increased when compared to Col scaffold due to the addition of Cynodon extract in the composite material and the extract has viscous in nature. The results of the study are in agreement with the

ACCEPTED MANUSCRIPT previous report on CS hybrid scaffold [10]. The collagen-based dressing materials should have sufficient mechanical strength suitable for handling the materials as well as for the desired clinical applications. 3.2. FT-IR and DSC analysis

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FT-IR spectrums of scaffolds are presented in Fig. 3a. The FTIR spectrum of native collagen

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showed the characteristics peaks of amide I, amide II and amide III at 1650 cm-1 (C=O stretching), 1554 cm-1 (N-H bending) and 1242 cm-1(C-N stretching)

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respectively. The FTIR spectrum of CE showed a broad and intense absorption band around at 3350-3385 cm-1 correspond to the O-H stretching vibrations of polyphenols and

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phenolic acid. The CS composite showed a shift in OH stretching vibration to a lower wave

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number and broadening, which indicates the hydrogen bonding interaction between

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collagen and silica. The silica is expected to exhibit non-covalent interactions viz., electrostatic and hydrogen bond interaction with collagen [27]. However, CSCE scaffold

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showed slight peak shift in the amide absorption region due to the interaction between Col

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and CE. We employed the DSC analysis to understand the thermal stability of native and composite scaffolds, and the thermogram was presented in Fig. 3b. DSC is a most widely

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used technique for measuring of denaturation temperature (Td), which is a measure of cross-linking density [28]. The denaturation temperature of the Col scaffold was found to be 74°C, which corresponds to the destabilization or denaturation of the triple helical structure of the collagen. However, the composite scaffold CS (91°C) and CSCE (110°C) showed remarkable improvement of the denaturation temperature. The improved denaturation temperature of the composite scaffold was due to the possible bonding

ACCEPTED MANUSCRIPT interactions between collagen and silica, and with the plant extract in the case of CSCE composite scaffold. 3.3. In vitro enzymatic degradation study The in vitro degradation behavior of Col, CS, and CSCE scaffolds upon collagenase treatment

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were investigated by monitoring the release of hydroxyproline in the solution at different

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time periods. The CSCE scaffold showed slower degradation when compared to native collagen (Fig. 4). About 44% and 52% of degradation were observed in the CSCE and CS

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scaffold respectively, whereas the native collagen degraded rapidly to 62% at 24 h. After 48 h, the pure collagen scaffold showed 96% of degradation at ambient temperature

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conditions whereas the CSCE scaffold showed 63% of degradation indicating that the

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addition of TEOS and CE has made them resistant to collagenolytic activity. Enzymatic

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stability of CSCE was enhanced significantly due to the presence of phytochemical constituents especially flavonoids in the CE. It is reported that flavonoids act as potential

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collagenase or MMP inhibitors and thus protect collagen proteins against the degradative

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activities of these enzymes [29, 30]. The addition of such substances in the scaffold would result in the overall increase in the measurable collagen concentration in healing tissue.

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The active secretion and destruction of collagen proteins are natural events in wound healing. In acute wounds, the secretion of MMPs during the early phase followed by secretion of MMP inhibitors to facilitate skin regeneration. However, the MMP levels are abnormally elevated in the chronic wound with down-regulation of expression of MMP inhibitors, i.e. tissue inhibitors of matrix metalloproteinases (TIMPs) [31]. The elevated ratio of MMPs to TIMPs leads to excessive extracellular matrix degradation as several MMPs have collagen hydrolytic properties. Besides anti-inflammatory and antimicrobial

ACCEPTED MANUSCRIPT effects, it was reported that luteolin and apigenin, the major phytochemicals of CE, exhibited inhibitory activity against a range of MMPs and collagenases [32-34]. The other predominant flavonoids of the plant C. dactylon such as quercetin and catechin interact and stabilize the collagen against proteolytic degradation besides stimulating collagen

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synthesis [29, 30, 35, 36]. Since the CSCE of our study contains the Cynodon extract having

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the above phytoconstituents; the scaffold has the potential to accelerate healing of both

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acute and chronic wounds through the mechanisms mentioned above. Hence, it is

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suggested that the use of the CSCE scaffold is more advantageous than the CS and conventional Col scaffolds.

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3.4. Biocompatibility and cell viability Analysis

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The biocompatibility of developed scaffolds using proliferation rate of the cells was

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assessed using NIH 3T3 cells by MTT assay. It was observed that the NIH3T3 cells continued to proliferate on the surface of scaffolds, suggesting that the scaffolds were non-

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toxic to fibroblast cells. The results showed that the rate of cell proliferation was

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significantly higher in CS and CSCE scaffolds when compared to Col scaffold (Fig. 5). Both collagen and composite materials showed a time-dependent increase in cell proliferation at

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day 1, 3 and 5 of post seeding. The rate of cell proliferation was observed to be almost similar in all groups on 6 h, and day 1 of post seeding. However, it was observed that there were significant changes in the proliferation rate on day 3 and 5 among the scaffolds and there was a steady increase in the number of cells in CSCE group following the day 1, when compared to CS and Col materials (Fig. 5a). These results suggested that the porous and hydrophilic nature of CSCE scaffold has protective and proliferative effects on fibroblasts in addition to the chemotactic effects of collagen such as cell adhesion, cell differentiation, and

ACCEPTED MANUSCRIPT tissue regeneration. It is reported that the extract of C. dactylon has antibacterial activity [37]. Hence, the CSCE scaffold might prevent infection during the process of wound healing. Further, DAPI and Rh-123 staining were performed to detect metabolically active live cells grown on the prepared scaffolds at different time intervals such as day 1, 3 and 5. DAPI and

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Rh-123 respectively form fluorescent complexes with nuclear material and active

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mitochondrial membranes of proliferative cells [38]. As seen in fluorescence microscopic

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images of Fig. 5(b-d), the number of live cells were significantly higher in CSCE scaffold

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exposed group than controls. The live cells on the CSCE scaffold appeared to be well attached, networked and spread across the surface when compared to other groups as

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indicated by staining of intact mitochondria and nuclei of the cells. Thus, the composite

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materials supported cell growth on the surfaces of composite materials, and the differences in cell distribution and viability among the groups were distinctly studied using these

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organelles-specific stains (Fig. 5b, c, and d). After 3 days of cell culture, the number of

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fibroblast cells attached to CS and CSCE was much higher than those of control (Fig. 5c). After 5 days of culture, cells completely covered the surface of CSCE scaffold whereas the

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cells in CS and Col scaffold groups did not show complete cover-up of the surface

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suggesting that the Cynodon extract significantly enhanced the proliferation of cells on the surface of CSCE scaffold (Fig. 5d). The porous structure of CSCE scaffold not only helped cell migration but also facilitated the exchange of oxygen and nutrients to cells, and it is essential for the construction of ECM and vascularization during the healing of wound [39].

ACCEPTED MANUSCRIPT 3.5. F-actin filaments FITC-phalloidin and DAPI staining were carried out to study the interaction of fibroblasts with composite scaffolds. Phalloidin is a bicyclic heptapeptide commonly used in imaging applications to selectively label filamentous actin in fixed cells [40]. Actin-tracker green

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and nucleus-tracker blue were used for F-actin microfilaments and cell nucleus

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respectively. Staining of cells of 24 h culture showed the distribution of F-actin filaments

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(stained green) in the cytoplasm under a fluorescence microscope (Fig. 6). We observed

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from the images that the density and morphology of fibroblasts were better in CSCE than CS and collagen scaffold groups. The cells supported by CSCE group revealed the

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distribution of a regular array of F-actin filaments stretched along the cell membranes, a

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characteristic cytoskeletal feature of metabolically active fibroblasts of connective tissue, for facilitating cell-cell interaction and tissue regeneration. These results substantiated that

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CSCE scaffolds were not only biocompatible but also could potentially promote adhesion,

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proliferation, and migration of cells for effective regeneration of ECM.

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3.6. In vivo Wound Healing

The wound healing activity of Col, CS and CSCE scaffolds were assessed in rats by full

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thickness excision wound model. The CSCE scaffold showed accelerated wound healing from day 5 onwards when compared to CS and Col scaffolds (Fig. 7). On day 10 of post wounding, it was observed that the CSCE scaffold treated wound showed healing progress comparable to rest of the groups. The extent of reduction of wound size for CSCE and CS groups on day 10 was 74% and 63% respectively, whereas this was 56% and 48% for collagen and control groups, respectively. However, on the 15th day of post wounding (PW),

ACCEPTED MANUSCRIPT the rate of re-epithelization (wound closure) was observed as 59%, 75%, 87% and 94% for control, Col, CS, and CSCE respectively. Complete healing was observed on the 17th day of PW for CSCE groups, whereas it was 19 and 22 days for CS and Col treatment, respectively. Enhanced wound healing by CSCE could be attributed to anti-inflammatory, antimicrobial,

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cell growth stimulating and tissue regenerative properties of CE and collagen constituents

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[15, 37]. Histological examinations were carried out on newly formed, healthy and healed

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tissues to analyze the collagen deposition, neovascularization, and re-epithelization. H&E

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stained skin sections of control and collagen composite scaffold treatments at different days intervals are presented in Fig. 8. It was noteworthy that control group, CS, and Col

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scaffolds exhibited some inflammatory cells after 5 days of PW. However, CSCE scaffold group showed some fibroblasts cells along with few neutrophils and macrophages

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indicating the progress of healing. The control group showed moderate inflammation after

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10 days whereas the absence of inflammatory cells and a few fibroblasts were observed in

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other groups. On day 15, the appearance of numerous fibroblasts accompanied by organized collagen deposition, epithelization and neovascularization were observed in all

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groups, and more particularly, these events were more pronounced in CSCE than other

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treatment groups. According to the report of Soraya et al. (2015) phytochemicals present in the aqueous extract of C. dactylon exhibited angiogenesis effect while reducing inflammation, which was advantageous properties for wound healing [19]. Saroja et al. reported that the aqueous extract of C. dactylon accelerated the activity of wound healing in an animal model with some basic physical parameters [41]. Collagen synthesis and deposition were the essential characteristics in tissue regeneration, and it is critical for the structure and function of healthy skin. Fig. 9 depicts the Masson’s

ACCEPTED MANUSCRIPT trichrome staining of the regenerated skin at different time intervals. On the day 5, the test groups exhibited the little amount of collagen deposition when compared to control. On the day 10, a large number of uniformly arranged thick collagen bundles as well as tissue granulation appeared in the regenerating wound area treated with CSCE scaffold when

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compared to other groups. Furthermore, the formation of epidermis was complete, and the

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newly formed epidermis was connected with the dermis. The bioactive compounds present

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in C. dactylon include phenolic acid, flavonoids and triterpenes support wound healing

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activity through anti-oxidative activity and inhibition of collagenases. 4. Conclusion

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In this paper, we have presented a novel collagen composite material incorporated with

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Cynodon dactylon extract having wound healing potential. The prepared CSCE scaffold

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showed superior porous structure, water uptake, mechanical properties and biostability than native collagen. In vitro studies revealed that the scaffold promoted cell-cell

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interaction, cell adhesion and proliferation of fibroblasts making the scaffold most suitable

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substrate for skin tissue engineering. In vivo studies revealed that CSCE scaffolds stimulated collagen deposition and enhanced the rate of wound healing. These results

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substantiated the tissue regeneration property of CSCE scaffold which could serve as an ideal and alternative biomaterial for the treatment of chronic wounds. Acknowledgments The authors are grateful to the Council of Scientific and Industrial Research (CSIR) for the providing financial support under the M2D XII plan project (CSC 0134). Author RKP thank UGC for Postdoctoral Research Fellowship.

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Biomedical Applications of

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Multiscale fibrous scaffolds in regenerative medicine,

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Table

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Table 1 Porosity and mechanical properties of Col, CS and CSCE scaffolds. Experiments are

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performed in triplicates, the *p < 0.05, considered to be statistically significant with control group (Col)

Scaffold

Col

Porosity (%)

81±1.62

Mechanical Properties Tensile Strength (Mpa)

Elongation at Break (%)

0.88 ± 0.05

14.3 ± 0.56

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0.53 ± 0.04*

10.8 ± 0.43*

CSCE

93±1.86*

0.92 ± 0.03*

15.9 ± 0.93*

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CS

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Figure Legends

Fig. 1 Photographic and SEM morphology of Col, CS and CSCE scaffolds at magnifications of

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40X. Pore size measurement of scaffold using ImageJ software.

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Fig. 2 In vitro water uptake ability of Col, CS and CSCE scaffolds. Data are expressed as

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mean ± SD (n = 5).

Fig. 3 (a) FTIR spectra of Collagen, CS, and Cynodon extract and CSCE scaffolds; (b) DSC thermogram of Col, CS and CSCE scaffolds Fig. 4 In vitro enzymatic degradation of Col, CS and CSCE scaffolds at different time intervals. A significant difference (**p < 0.01) in collagen degradation was measured between CS, CSCE, and collagen group.

ACCEPTED MANUSCRIPT Fig. 5 (a). In vitro biocompatibility of scaffolds by MTT assay. Values are expressed as mean ± SD and the level of significance is expressed as *and ** corresponding to p < 0.05 and p < 0.01, respectively compared with the corresponding control group. Fluorescence microscopy images (400X magnifications) of DAPI and Rh-123 stained 3T3 mouse

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fibroblasts cells on developed scaffolds at different time intervals (b) Day 1, (c) Day 3 and

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indicates active mitochondrial membranes of cells.

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(d) Day 5. DAPI staining (blue color) shows nucleus and Rh-123 staining (green color)

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Fig. 6 Fluorescence images of the cytoskeleton and nuclei of NIH3T3 fibroblasts cultured on Col, CS and CSCE scaffolds for 24 h. Actin microfilaments (green) and cell nuclei (blue)

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were visualized by FITC-phalloidin and DAPI respectively (The measurement of scale is 50

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µm).

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Fig. 7 Photographs of the Col, CS, CSCE and control (untreated) wounds. Each wound shown here is representative of four rats per group on the given day.

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Fig. 8 Hematoxylin and Eosin stained sections of the regenerated skins of control and

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experimental animal groups on 5th, 10th and 15th days. Arrow (Yellow in colour) indicates the fibroblasts cells of granulated tissues. The magnification is 20X with 50 µm scale bar.

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Fig. 9 Masson’s trichrome staining of skin tissues treated with Col, CS, and CSCE and an untreated control group of animals. The magnification is 20X with 50 µm scale bar.

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Graphical Abstract

ACCEPTED MANUSCRIPT Highlights Collagen based biomaterial have many application in the field tissue engineering

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The developed CSCE scaffolds support the proliferation of fibroblasts cells

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The stability of CSCE was better than native collagen scaffold

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CSCE scaffold has the potential for use of wound healing applications

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