NanoBioscaffolds as Wound Healing Biomaterials in Animals

Bio-based renewable materials are considered safer than synthetic materials. In recent days, research is focused on the development of biomaterials from naturally-derived polymers such as collagen, keratin and chitosan. They were considered healthy for skin because of their hydrophilic nature, biocompatibility, wound-healing property, biodegradability and antibacterial property (Xu et al., 2014) and they can be used to enrich human mesenchymal stem cells for clinical applications (Hartrianti et al., 2015). Among them, the extracellular matrix (ECM)-based scaffolds have an established place as medical devices in regenerative medicine. The forestomach matrix from a sheep have been shown to have low immunogenicity and is cleared by the US Food and Drug Administration (USFDA) for dermal indications and hence widely adopted in clinical practice (Lun et al., 2010). The physical, chemical and particularly the mechanical properties of collagen implants need to be adjusted according to the device requirements. Therefore, the decellularized products are gaining biomedical importance and market space due to their ease of standardized production, constant availability for grafting and mechanical or biochemical superiority against therapies/ modalities/ items, yielding clinical results ahead of the ones with autografts (Parmaksiz et al., 2016).
       
Wound healing is an inherent physiological response that helps repair cellular and anatomic continuity of a tissue. Wound care is a significant financial and social burden on both the patient and the medical system at large. Scaffold-associated bacterial infections are one of the most serious complications in animal and human orthopedic operations leading to worse clinical outcomes and ultimately increased financial costs. Recently, eco-friendly synthesized silver nanoparticles (SNPs) have attracted considerable interest in biomedical applications because of their ease of preparation, good biocompatibility, relatively large surface area and antimicrobial activity (Makarov et al., 2014). In the present study, preparation and characterization of bioscaffolds impregnated with silver nanoparticles were undertaken and were evaluated for its wound healing activity in wound model.

The research work was carried out at Veterinary College, KVAFSU, Shivamogga, Karnataka during the period from November-2015 to May-2017.
 
Preparation and characterization of decellularized extracellular matrix (dECM)
 
Fresh forestomach samples (n=6) of sheep (Ovis aries) were collected from a local abattoir in chilled (4°C) sterile phosphate buffered saline (PBS, pH 7.4) containing 0.1% amikacin, conditioned by freeze-thawing method and were decellularized using 0.5% bile. The decellularized and intact forestomach samples were processed for the evaluation of dECM for residual cellular material by routine hematoxylineosin (H&E) and 4, 6-diamidino-2-phenylindole (DAPI) (Luna, 2017). The DNA from both native and decellularized forestomach was quantified using spectrophotometer (Thermo Scientific NanoDrop 2000®, USA). The native and dECM tissues were analysed for morphology by scanning electron microscopy (Model: JOEL-JSM 5600). Fourier transform infrared (FTIR) spectroscopy was performed (Gasior-Glogowska et al., 2010) and FTIR spectra of the dECM in triplicates were measured with Nicolet 5700 FTIR spectrometer in the range of 500-4000 cm^-1. The tensile strength of the rectangular specimens of the dECM (6.0´1.0 cm) in wet state was tested with a trigger load of 100N load cell using Universal Testing Machine (M/s Instron, USA Model-3345) as per the standard procedures.
 
Preparation and impregnation of green silver nanoparticles (SNPs) on to the decellularized extracellular matrix
 
The ecofriendly SNPs were prepared as previously reported by Kahrilas et al., (2014). Briefly, an aqueous solution of 1 mM AgNO₃ was added into 175 ml of aqueous citrus fruit peel extract. The particle size and zeta potential of the resulting SNPs were determined by dynamic light scattering technique using Malvern Zetasizer (ZEN 3600; Malvern Instruments, Malvern, UK). The dECM was dip coated with SNPs (0.01 µg/ml) in keratin-chitosan (1%) solution and incubated at 37°C for 24 h by modified method of solvent casting technique (Shaik et al., 2013) and were characterized by X-Ray diffraction to confirm the nature of crystal structure formed. The dECM of biobased origin prepared in the study (Bioscaffold) is termed ‘NanoBioscaffod’ after coating with SNPs.
 
Evaluation of NanoBioscaffods for wound healing activity
 
For in vitro evaluation for cell adhesion, the human dermal fibroblast (HDF) cells were used to culture on the surface of NanoBioscaffolds to assess the biocompatibility of scaffolds. The in vitro antimicrobial activity of the discs prepared from NanoBioscaffolds was studied using Bacillus cereus and Escherichia coli bacterial cultures (10⁸ CFU/ml) by modified agar well diffusion method and the diameter of the zone of inhibition was measured.
       
In vivo evaluation for wound healing activity was carried out as per the guidelines of the Organization for Economic Co-operation and Development (OECD). The ethical approval was obtained by the Institutional Animal Ethical Committee (IAEC-No RP41-1617) at WeInnovate Biosolutions Pvt. Ltd., Pune. The treatment protocol included two experimental groups of Wistar rats (150-180 g) each containing eight animals. Group I served as control (without any treatment) and was covered with paraffin film. Animals in the group II were treated with NanoBioscaffolds for the period of 21 days. Briefly, full thickness wounds of 8 mm diameter were created (Auddy et al., 2013). Wound tissue biopsies from both control and treated groups were processed for Histopathology on 7^th, 14^th and 21^st day of the experiment by routine haematoxylin and eosin staining (Luna, 2017). The keratinocyte proliferation factor (ki67) was detected using immunohistochemical staining. The catalase activity of the tissue was analyzed and expressed in micromoles (µM) of hydrogen peroxide (H₂O₂) separated at intervals one minute with one gram of wet cells.

In the present study, we have prepared the dECM from forestomach by using bile in place of synthetic detergents. Commonly, natural detergent based decellularization techniques are preferable to other methods, as synthetic detergents may deteriorate the scaffold integrity and intactness by decreasing the tensile strength and overall quality (Parmaksiz et al., 2016).
       
Morphological study of the dECM revealed visible differences in the surface topology of the luminal (rough) and abluminal (smooth) surfaces of the matrix. The current protocol removed the majority of cells of the forestomach tissue without altering the normal morphology of dECM as revealed by the histological analysis (Fig 1). Even the quantity of DNA was well below the general index for cell residues within dECM. Thus, the present decellularization process using bile (0.5%) was efficient making dECM appreciably biocompatible and non-immunogenic (Fig 1). The use of bile retained the normal morphology of the dECM which in turn enhanced the quality, efficacy and mechanical strength of dECM. The indicators of the effectiveness of decellularization process as set by earlier studies (Nagata et al., 2010) are; lack of visible nuclear material in tissue sections stained with DAPI or H&E and DNA lesser than 50 ng per mg ECM dry weight which directly correlated to adverse host reactions (Zheng et al., 2005).
 

       
The SEM analysis of dECM revealed the luminal surface showed more contoured with various pore sizes, while the abluminal surface more smoother (Fig 2). The findings of the present study indicated that the bimodal nature of the scaffold described by Ward et al., (2014) was found to be important in terms of its interactions with different cell types in a healing process, to encourage epithelial regeneration on the dense luminal surface and fibroblast invasion on the less dense abluminal surface when used for tissue regeneration.
 

       
The results of the FTIR analysis confirmed the intactness of the dECM, wherein Amide I band (1600-1700 cm^-1) appeared due to C=O bond stretching vibration. Amide II band appeared due to N-H bending vibration which was incapable of fully resolving the protein secondary conformation. The findings of the FTIR corroborated with the earlier report (Gasior-Glogowska et al., 2010). The mechanical properties were crucial when designing a scaffold for use in tissue engineering. The tensile strength of the dECM was 13.93±2.61 MPa in the present study, which was comparatively higher than that of decellularized collagen matrix (10.15±1.81 MPa) in which synthetic detergents were used. 
       
Any desirable bioactive molecule including certain antimicrobial agents can be incorporated into the biopolymer and it does not change the intrinsic biological properties of the ECM scaffold (Ward et al., 2014). The dECM was impregnated with 1% keratin-chitosan dissolved in 0.5% BMImCl solvent to increase the functionalization of Bioscaffolds. These hybrid scaffolds are considered superior as they have good porosity and homogenous pore distribution compared to those prepared from the individual biopolymers (Balaji et al., 2012).
 
Preparation of NanoBioscaffods from dECM
 
The eco-friendly silver nanoparticles (SNPs) in liquid form were used to impregnate dECM. The appearance of the brownish colour of the solution used, indicated the formation of SNPs in the reaction mixture during the preparation of eco-friendly SNPs using citrus fruit peel extract. The colour change of the reaction mixture was due to the excitation of surface Plasmon Vibration in the SNPs (Kahrilas et al., 2014). Rich source of citric acid and ascorbic acid in the citrus fruits may possibly be responsible for the reduction of silver ions and efficient stabilization of SNPs. 
         
For the economic and efficient use of SNPs synthesized in the present study, the NanoBioscaffolds were prepared by dip coating the bioscaffolds with SNPs. The mean particle size of the chitosan impregnated SNPs were found to be 327.5±15.95 nm with a zeta potential value of 29.6±0.145 mV. This high value of zeta potential indicates the colloidal stability. The XRD patterns of NanoBioscaffolds indicated that the pure chitosan showed weak reflection at 2q of 20.96 and strong reflection at 2q of 30.06 which matches well with earlier reported values (Kong et al., 2005). The peaks for 022 and 104 plane of the silver in the samples showed that the main composition of NanoBioscaffolds was consisted of keratin, chitosan and SNPs. Hence, no other peaks present as impurities were found in the XRD report (Fig 3).
 

 
Evaluation of wound healing property of NanoBioscaffolds
 
The observations suggested that the NanoBioscaffolds increased human dermal fibroblasts adhesiveness to both luminal and abluminal surfaces and preferentially on to the abluminal surface. The In vitro cell adhesive property of the scaffolds could be due to the presence of cell adhesion sequence, RGD (Arg-Gly-Asp) and LDV (Leu-Asp-Val). The matrix served as a reservoir for many extracellular signaling molecules that control cell growth in the early stages of tissue wound healing assembly.
       
In vitro antibacterial activity of NanoBioscaffolds was evidenced by 8 mm and 6 mm of zone of inhibition exhibited against Bacillus cereus and Escherichia coli growth respectively. This may be due to release of SNPs from scaffolds and penetrate through the cell membrane to kill microorganisms instantly by blocking their respiratory enzymes (Hwang et al., 2012). Recently, the safety and efficacy of collagen-impregnated SNPs encapsulated in collagen hydrogels were shown in primary human skin fibroblasts and keratinocytes; while antimicrobial properties were shown against S. aureus, Staphylococcus epidermidis, E. coli and P. aeruginosa (Alarcon et al., 2015). 
       
The treated group showed increased healing by early wound contraction on the 14^th day of the treatment, when compared to the control group. The histomorphological examination of treated group showed a large amount of compact granulation tissue, a small number of mononuclear inflammatory cells, restoration of adnexa and extensive fibrosis (Fig 4b) as observed by Michel and Fredrickson (1990). The immunohistochemical analyses revealed the increased number of proliferative cells positive for Ki67 at wound site, treated with NanoBioscaffold which were corroborative with the earlier reports. In the present study, more catalase activity was observed in the treated group, in turn, less oxidized lipid when compared to the control group. Similar observations were made by Kurahashi and Fujii, (2015) wherein they opined that the reactive oxygen species (ROS) played a vital role in wound healing and antioxidative enzymes present abundantly in skin, notably catalase, played major roles in the detoxification of ROS, thus suggesting that NanoBioscaffold promotes wound healing in the rats.
 

The decellularized extracellular matrix samples were prepared using 0.5%bile which showed successful removal of cells as well as the intact preservation of structural and functional components and characterized by morphological, mechanical and biological methods. They were functionalized to form ‘NanoBioscaffolds’ by coating with SNPs. The NanoBioScaffolds were having favorable role in wound healing activity in wistar rats.

The fully biobased dECM (NanoBioscaffolds) can be suitable alternative scaffold for wound healing applications in human and veterinary therapeutics.