In vivo diabetic wound healing potential of nanobiocomposites containing bamboo cellulose nanocrystals impregnated with silver nanoparticles

Accepted Manuscript Title: In vivo diabetic wound healing potential of nanobiocomposites containing bamboo cellulose nanocrystals impregnated with silver nanoparticles Authors: Rubbel Singla, Sourabh Soni, Vikram Patial, Pankaj Markand Kulurkar, Avnesh Kumari, Mahesh S., Yogendra S. Padwad, Sudesh Kumar Yadav PII: DOI: Reference:

S0141-8130(17)31200-X http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.06.109 BIOMAC 7789

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

3-4-2017 5-6-2017 27-6-2017

Please cite this article as: Rubbel Singla, Sourabh Soni, Vikram Patial, Pankaj Markand Kulurkar, Avnesh Kumari, Mahesh S., Yogendra S.Padwad, Sudesh Kumar Yadav, In vivo diabetic wound healing potential of nanobiocomposites containing bamboo cellulose nanocrystals impregnated with silver nanoparticles, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.06.109 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

In vivo diabetic wound healing potential of nanobiocomposites containing bamboo cellulose nanocrystals impregnated with silver nanoparticles Rubbel Singlaa,c,‡, Sourabh Sonib,c,‡, Vikram Patialb,c, Pankaj Markand Kulurkarb, Avnesh Kumaria,c, Mahesh S.b, Yogendra S. Padwadb,c,*, Sudesh Kumar Yadava,c,d,*

‡

a

Equal contribution

Nanobiology Laboratory, Biotechnology Division, CSIR- Institute of Himalayan Bioresource

Technology, Palampur-176061 (H.P.), India b

Pharmacology and Toxicology Laboratory, Food and Nutraceuticals Division, CSIR- Institute of

Himalayan Bioresource Technology, Palampur (H.P.)-176061, India c

Academy of Scientific & Innovative Research (AcSIR), CSIR-IHBT, Palampur

d

Center of Innovative and Applied Bioprocessing (CIAB), Knowledge City, Sector-81, Mohali-

140306, India

Corresponding authors: *Email: [email protected]; [email protected] (SK Yadav) Email: [email protected] (YS Padwad)

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Graphical Abstract

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Highlights 

Nanobiocomposites (NCs) ointments contain bamboo cellulose nanocrystals and AgNPs.

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Reports the use of plant cellulose nanocrystals for diabetic wound healing.

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Wounds of diabetic mice treated with NCs showed full recovery within 18 days.

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NCs improved re-epithelialization and collagen deposition in the wounded skin.

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NCs applied tend to regulate the expression of growth factors for better healing.

Abstract In diabetes, hyperglycemic state immensely hinders the wound healing. Here, nanobiocomposites (NCs) developed by impregnation of in situ prepared silver nanoparticles in the matrix of bamboo cellulose nanocrystals were investigated for their ability to hasten the progress of healing events in streptozotocin induced diabetic mice model. Wounds treated with topically applied NCs (hydrogels) showed full recovery (98-100%) within 18 days post wounding in contrast to the various control groups where incomplete healing (88-92%) was noticed. Biochemical estimations documented a marked decrease in the levels of proinflammatory cytokines IL-6 and TNF-α leading to decreased inflammation in NCs treated mice. Significantly increased expression of collagen and growth factors (FGF, PDGF, VEGF) upon NCs treatment resulted in improved re-epithelialization, vasculogenesis and collagen deposition as compared to control groups. Hence, developed nanobiocomposites showcased potential to serve as highly effective and biocompatible wound dressings for diabetic patients.

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Keywords: Nanobiocomposites; Bamboo cellulose nanocrystals; Diabetic wound healing; Streptozotocin induced diabetes; Immunohistochemistry

1. Introduction Diabetes, a highly prevalent chronic disease leads to higher risk of delayed wound healing and has become a serious issue in public health care system. With ever increasing population prone to wound healing complications, a strong need is felt to develop strategies for both its prevention and treatment. Dermatological complications affect 15% of diabetic people and are the most recurrent reason of hospitalization that may lead to organ amputation in-spite of improved standards of wound care [1]. In diabetics, the orderly sequence of cellular and molecular events of healing cascade including homeostasis, inflammation, angiogenesis and neo-epithelialization is not followed that paves the way for delayed wound repair [2]. Imbalance between production and degradation of collagen along with higher concentration of proteases and lower levels of certain cytokines/growth factors causes diabetic wounds to stall in the inflammatory phase [3]. In diabetes, high levels of blood glucose (hyperglycemia) results from defects in secretion or function of insulin which ultimately produce reactive oxygen species that cause oxidative stress resulting in immune system dysfunction, neuropathy, cellular damage and poor neo-vascularization [4]. People suffering from diabetes are highly vulnerable to wound infections and inflammation due to high levels of pro-inflammatory cytokines such as TNF-α and IL-6 [5]. The elevated levels of these cytokines are linked to delayed healing causing lesser migration and proliferation of fibroblasts and keratinocytes, reduced accumulation of collagen and deferred reepithelialization [6]. Decoding the complex process of diabetic wound healing and designing a 4

suitable dressing has been considered a challenging task for researchers. Traditionally used wound dressings like gauze, cotton wool and paraffin cause drying out of wounds leading to trauma on removal of dressing. In line with Winter’s findings, moist wounds are known to heal rapidly [7]. Moist dressings showcase antimicrobial trait, hence, act as highly desirable option for chronic wound management [8]. Most of the modern dressings were synthesized keeping this fact in mind. The occlusive dressings that came into practice, being impermeable to wound exudates and gaseous exchange did not offer much help leading to further development of porous dressings for effective drainage of wound exudates. Still issues regarding microbial infection and wound dehydration persisted [9]. Current clinical wound dressings such as hydrogels, alginates, collagen, gelatin and scaffolds used individually have disadvantages such as wound maceration, uncontrollable degradation and excessive hydrophilicity causing difficulty in their interactions with cutaneous proteins [10]. The usage of dressings depends upon the patient’s condition, wound type and microenvironment. Therefore, it is quite important to choose the correct dressing. Therefore, future research needs to develop intelligent dressings having the combined advantages of both occlusive as well as porous dressings. The recent surfacing of nanotechnology has provided a new therapeutic approach for the use of silver nanoparticles (AgNPs) in wound healing [11]. Nano-silver mixed with other natural/synthetic polymers have been widely used as topical dressings for the treatment of infections in acute as well as diabetic wounds [12,13]. Silver has well known anti-bacterial and anti-inflammatory properties, but the use of excessive amounts of

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this metal can be toxic [11]. Therefore, it necessitates the determination of an optimum and safe concentration of silver to be used in dressings for effective healing. In order to design an effective wound dressing, earlier we successfully developed inexpensive, natural polymer based nanobiocomposites (NCs) by impregnating low concentration of AgNPs (0.05±0.01 wt%) synthesized through greener route to plant cellulose nanocrystals (CNCs) isolated from the leaves of two bamboo species possessing anti-microbial and acute wound healing efficacy [13]. Although the process of diabetic and acute healing varies immensely in terms of altered healing events as well as major changes in cytokine profiles and levels of certain growth factors, it would be of great value if the same material fulfils the requirements of an ideal dressing (exudates absorption, gaseous exchange, anti-microbial nature, biocompatibility, etc.) for both. Diabetic wounds generally show delayed healing and take around 1-2 week more than the acute wounds to heal (~15 days). Thus, keeping this fact in mind, the present study deals with efficacy evaluation of NCs hydrogels on cutaneous wounds of streptozotocin (STZ) induced diabetic mice. Histopathological, immuno-histochemical and biochemical analysis has portrayed an increase in collagen and growth factors, decrease in proinflammatory cytokines in treated diabetic mice promoting faster healing and recreating normal skin strength and texture possibly due to synergistic action of plant CNCs and AgNPs. The topical application of developed NCs has led to accelerated wound healing in diabetic mice with wound closure in about 18 days, suggesting the role of NCs as potential candidates for biomedical applications. 2. Experimental 2.1. Materials

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Streptozotocin was purchased from SRL, India; chemicals for haematoxylin and eosin (H&E) staining from HiMedia, India; and chemicals for Masson’s Trichome (M&T) staining were supplied by CDH Pvt. Ltd., India. All the primary antibodies were procured from Biorbyt, U.S.A. and kit (ImmPRESS Excel staining) for immuno-histochemistry was obtained from Vector Laboratories, U.S.A. All the ELISA kits used for biochemical assays were provided by RayBiotech, U.S.A. 2.2. Synthesis of nanobiocomposites Nanobiocomposites (NCs) consisting of a mixture of cellulose nanocrystals (CNCs) and silver nanoparticles (AgNPs) were prepared following our already published method [13]. Briefly, CNCs were isolated from leaves of two bamboo species namely, Dendrocalamus hamiltonii (DH-CNCs) and Bambusa bambos (BB-CNCs) by a combination of chemo-mechanical approach. For preparation of CNC-Ag hydrogels, an in situ method was adopted, where 100 mg of lyophilized CNCs isolated from both the bamboo species were suspended each in 10 mL of 1 mM AgNO3 solution. The mixture was sonicated using ultra-probe sonicator for 2 min. Then, Syzygium cumini leaf extract (10% v/v) was added drop wise to the sonicated suspension and stirred at constant speed at room temperature for 6 h. Here, S. cumini leaf extract was used as a biological reducing agent to reduce AgNO3 solution to AgNPs. After 6 h, the suspension was centrifuged at 10,000 rpm for 10 min and washed repeatedly by centrifugation with distilled water. The pellet obtained was abbreviated as DH-CNC-Ag and BB-CNC-Ag hydrogels, respectively. Detailed characterization of these formulations has been described in our recent publication [13]. Further, DH-CNC-Ag and BB-CNC-Ag hydrogels were mixed with Vaseline as inert base (1:1) for topical application on the wounded skin of diabetic mice and termed as NCs.

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2.3. Animals for diabetic wound healing experiments Healthy swiss albino mice at 6-8 weeks of age (27-35 g) procured from our institutional in-house animal facility (CPCSEA Registration no. 1381/ac/10/CPCSEA, DT: 27/10/2010) were used for this study. All the experimental procedures related to mice were performed in accordance with the guidelines set up by CPCSEA for the care and use of laboratory animals. All the protocols were priorly approved by the Institutional Animal Ethics Committee (IAEC approval no. IHBT6MAR2015). Animals were housed in separate cages and were kept in a pathogen free room at a constant temperature (25°C) and relative humidity (60-70%) with alternate 12 h light-dark cycles. Mice were fed a diet of commercial pellets and water was provided ad libitum. 2.4. Streptozotocin (STZ) induced diabetic mice model Before STZ injection for diabetes induction, all the animals were kept for overnight fasting. An intra-peritoneal injection of moderate dose of STZ (80 mg/kg body weight dissolved in freshly prepared ice-cold 0.1 M citrate buffer at pH = 4.5) was given to mice. Immediately after STZ injection, sucrose water (10%) was provided to mice to prevent sudden hypoglycaemic shock. For measurement of glucose at regular intervals, blood was drawn from ventral tail vein of mice by venipuncture and examined using a glucometer (Bayer Contour TS Blood Glucose Meter). After 72 h of first injection, a second STZ injection (80 mg/kg body weight) was given to mice following the same procedure. STZ injected mice were supervised regularly for any undesirable symptoms or mortality, blood glucose and insulin levels, and decrease in body weight. Insulin levels were measured in the serum of STZ injected mice at specific time intervals throughout the study period using ELISA kits following manufacturer’s protocol. Mice with glucose level >200 mg/dL were taken as diabetic. All the animals used in this study developed hyperglycemia according to the mentioned inclusion criteria. The protocol optimized for diabetes induction 8

using STZ at this dosage was found to be highly effective for stable induction of high glucose levels (>200 mg/dL) in swiss albino mice with minimum/no mortality. Mice having stable high blood glucose levels were housed individually for 1 week before commencing the wounding experiment. 2.5. Excision wound creation on diabetic mice skin and macroscopic examination The wound healing potential of developed NCs hydrogels was evaluated in diabetic swiss albino mice model. The mice were anaesthetized using an intra-peritoneal injection of a mixture of xylazine and ketamine (90 mg/kg and 5 mg/kg body weight, respectively). The dorsal surface of mice skin was shaved and sterilized with 5% povidone/iodine solution. Circular full thickness cutaneous wound of diameter 8 mm was created on the dorsal surface of mice using biopsy punch. The animals were then categorized randomly into 9 experimental groups (9 mice/group) according to the materials applied on the wounds for treatment (Table 1). The wounds were treated topically with each of the NCs formulation daily (50 mg/day/wound) over a period of 18 days. The amount of NCs used was enough to cover the wound bed completely. Blood glucose levels of each mice were measured at three time intervals (day 3, 10 and 18 post wound) and found to be >200 mg/dL consistently. The wound site of every animal was photographed and the wound diameter was measured at these time points using vernier calliper to calculate wound closure area (%) according to the formula:

 Initial area of wound  n th day area of wound    100 Wound closure (%)   Initial area of wound  

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(1)

2.6. Histopathology of diabetic mice wounded skin tissues Three mice from each group were euthanized on day 3, 10 and 18 and wounded skin along with marginal healthy skin was excised for downstream histopathological and immuno-histochemical studies to analyse the healing events occurring in the wounds of diabetic mice. The skin tissue sections were fixed in 10% neutral buffered formalin for histopathology. Once fixed, the tissues were dehydrated, cleared and then embedded in paraffin. Thin sections (4 μm) obtained using ultra-microtome were taken onto a slide and further processed for haematoxylin and eosin (H&E) staining. Briefly, the sections were deparaffinized, dehydrated and stained with haematoxylin. Counterstaining was done using eosin and the slides were examined for wound healing sequence of events under bright field microscope. The sections were also stained using Masson’s Trichome (M&T) procedure to analyse the extent of collagen formation during the healing period [13]. 2.7. Immuno-histochemistry (IHC) of wounded skin tissue sections of diabetic mice For IHC, paraffin embedded wounded skin tissue sections (4 μm) of diabetic mice were mounted on poly-L-lysine coated glass slides, deparaffinized and hydrated. Antigen retrieval was performed by sodium citrate buffer treatment to tissue sections. BLOXALL blocking solution (ImmPRESS excel staining kit) was used for quenching of endogenous peroxidases and blocking of exposed sites was done by incubating the tissue sections with 2.5% normal horse serum. Sections were then incubated with a specific primary antibody at a particular dilution. Repeated washes using phosphate buffer saline (PBS) were given to eliminate unbound primary antibody before incubation with horseradish peroxidase (HRP)-conjugated secondary antibody. The 10

sections were washed twice and incubated with DAB (3,3’-Diaminobenzidine) substrate [14]. Observations were made under bright field microscope in order to check the expression (%) and activation status of platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FGF), collagen I and collagen III for all the sections using ImageJ software (NIH, U.S.A.). 2.8. Estimation of hydroxyproline, IL-6, TGF-β and TNF-α levels in wounded skin tissue homogenate Wounded skin tissue excised from each animal at timelines (3, 10 and 18 days post wound) was also processed for biochemical analysis. The excised skin tissue (100 mg) was homogenized in 1 mL of 0.1 M PBS (pH= 7.4) using a homogenizer (IKA Laboratory, China). The skin homogenates were centrifuged at 10, 000 rpm for 10 min at 4 °C and the collected supernatants were stored at -80 °C until analyzed. The homogenates were estimated for hydroxyproline, IL-6, TGF-β and TNF-α contents using specific ELISA kits following manufacturer’s protocol. The absorbance was noted at a particular wavelength to calculate the amount of these factors at specified time intervals to study the effect of NCs treatment as compared to control groups on the wound healing events. 2.9. Statistical analysis For all the animal experiments, comparison and data analysis between control and NCs treated groups for quantification of wound closure area (%), IHC analysis and biochemical estimations was done with JMP software (SAS Institute). The results are presented as mean ± standard deviation (n=3 mice/group at each time interval). The significance of difference was assessed with one way ANOVA; statistical significance level was set at p < 0.05. 11

3. Results and Discussion 3.1. Synthesis of nanobiocomposites (NCs) The isolated CNCs (DH-CNCs and BB-CNCs) were used as a matrix for in situ preparation of NCs. Under this approach, Ag ions from AgNO3 solution were initially adsorbed onto CNCs surface by electrostatic or van der Waals forces and then Ag ions were reduced into AgNPs by S. cumini leaf extract (biological reducing agent) leading to impregnation of AgNPs on to CNCs matrix. During NCs formation, CNCs acted as a template and form 3-D sheet like structure for the adsorption of Ag ions via strong ion-dipole interactions of Ag+ with hydroxyl and carboxyl groups of CNCs. This has decreased the mobility of Ag+ and resulted in controlled size distribution and stabilization of AgNPs, thus preventing their aggregation. Likewise, hyaluronan fibers have been reported previously to act both as a template and stabilizer during AgNPs formation using AgNO3 as a precursor [15]. Average diameter and length of DH-CNCs was 18±0.5 nm and 272±52 nm, respectively. BB-CNCs were 20±1 nm in diameter and 385±97 nm in length. The impregnated spherical AgNPs were of diameter 16±3 and 22±7 nm, in case of DH-CNC-Ag and BB-CNC-Ag hydrogels, respectively [13]. These hydrogels are stable for a minimum of 1 year at room temperature (25±2˚C). These NCs hydrogels at 25, 50 and 100 mg/mL concentrations were found to be non-toxic (<40% cytotoxicity) against primary keratinocytes of mice after 24 and 48 h incubation as reported in our recent publication [13]. Similarly, another study has also documented that schizophyllan/silver nanoparticle composites formed from reduction of AgNO3 were non-toxic to keratinocytes and can be used for wound healing applications [16]. Cytotoxicity studies have confirmed the safe usage of NCs hydrogels developed in this study for wound healing purposes. 3.2. Establishment of the diabetic animal model 12

STZ, a widely known chemical for diabetes mellitus type 1 induction, elicits an inflammatory reaction upon injection into mice. STZ is toxic and causes β-cells destruction in pancreas leading to induction of the hyperglycemia and inhibition of insulin release [17]. The variations in body weight, insulin and glucose levels in each animal were monitored throughout the experimental time period. After STZ injection, a marked decrease in insulin levels and severe hyperglycemia were detectable throughout the experiment as compared to controls. After 1 week of STZ injection, the diabetic mice showed a visible decrease in body weight compared to non-diabetic controls. Additionally, the diabetic mice were observed to exhibit a substantial rise in blood glucose as compared to control animals and were randomly distributed into various groups after cutaneous injury. 3.3. NCs accelerated wound closure in diabetic mice Diabetic mice were subjected to NCs hydrogels and various controls for wound treatment and further monitored for macroscopic changes in wounded skin to evaluate in vivo performance of treatment groups by quantitative measurement of wound closure area (%). The wound sites of NCs treated groups exhibited morphology that was different at day 3, 10 and 18 post surgery (Fig. 1a) with greater degree of wound closure in contrast to untreated diabetic control (UC) and vehicle controls where a delay in wound closure was noticed (Fig. 1b). On day 3, no evidence of infection with slight wound closure was observed in NCs treated groups whereas wound sepsis with signs of pus was seen in control groups. The absorption of wound fluids by CNCs present in NCs might have assisted in increasing the release of AgNPs from NCs which has converted into active Ag+ form and accelerated the evading of bacteria around the wound milieu [18]. Similarly, polymeric hydrogels absorbing excessive wound exudates have been recently reported to enhance healing [19]. Statistical analysis by one way ANOVA exhibited that there was no 13

significant difference between wound closure area (%) in any of the groups on day 3 (p < 0.05). On day 10, large scab tissue covering the excision wound was observed in control groups while scab tissue formation was minimal in skin wounds treated with NCs. This could be due to the presence of AgNPs in hydrogels that might have helped to minimize scarring [18]. Likewise, alginate dressings have been reported to have good absorption capability, limiting wound exudates and minimizing bacterial contamination, due to strong hydrophilic gel formation. Some reports suggest that alginate inhibits keratinocytes migration and leads to wound dehydration thus minimizing their use in wound dressings [20]. On the other hand, CNCs present in NCs provides moist and clean environment to the exuding wounds by absorbing wound exudates [13]. Statistically on day 10, wounds treated with NCs hydrogels showed faster healing as compared to UC group (p < 0.05). Similarly, another polymer based chitosan/hyaluronan/nonwoven fabrics has also been reported to enhance the rate of healing in diabetic and non-diabetic mice [21]. On day 18, NCs treated skin healed almost 100% with growth of new hair follicles whereas in all control groups, there were still signs of skin injury indicating incomplete tissue repair. Statistical analysis showed that DH-CNC-Ag, BB-CNC-Ag and NDUC (non-diabetic untreated control) groups showed almost complete wound closure and also significantly (p < 0.05) accelerated the wound closure rate as compared to UC group (Fig. 1b). Rapid but nonsignificant (p ≥ 0.05) wound healing was observed in NCs hydrogel treated groups in comparison to positive as well as vehicle controls. Similar results were obtained in non-diabetic mice treated with DH-CNCs-Ag and BB-CNC-Ag as shown in our previous paper. However, the rate of complete healing was rapid in non-diabetics as compared to diabetic mice [13]. The enhanced rate of wound closure in NCs treated groups relative to vehicle controls suggested synergistic effect due to surface functional groups of CNCs and anti-bacterial AgNPs in NCs. 14

The faster wound closure in NCs treated groups could be attributed to fast epidermal growth/skin regeneration due to the potential of CNCs to preserve moisture around wound environment, as the rate of epithelialization is hastened in moist wounds [22,23]. Similarly, wound healing effect of hyaluronan-silver nanocomposites nonwoven fabrics in diabetic and non-diabetic rats has explained the synergetic effects between antibacterial performance of AgNPs and the functional groups of hyaluronan [24]. Moist conditions permit the migration of epidermis through the exudate to the wound surface, keeping the dermis intact. The original wound site lies beneath the neo-epidermis, reducing scarring [7]. Larger availability of NCs at the wound area and also steady and sustained supply of AgNPs from the CNCs matrix leads to enhanced healing [25]. The concentration of Ag+ released from NCs was within the safety limits that didn’t cause any damage to normal skin cells [13]. The use of Vaseline as inert base provided good attachment to NCs hydrogels with wound surface to prevent being washed off by excessive exudates. Moreover, there was no need of a secondary dressing required to cover the hydrogel and facilitated better healing benefit. Furthermore, NCs wound dressings does not leave any residue at the wound site. 3.4. Histopathological examination of wounded skin tissues by H&E staining Diabetes has been known to disturb immune functions of the body and hamper wound healing, which is a critical factor for the survival of diabetic patients with wound injury [26]. H&E staining was carried out to visualize the effect of topically applied NCs hydrogels on the wounded skin of diabetic mice at specific time points (day 3, 10 and 18 post wound) to study the alterations in wound healing events like inflammation, proliferation, dermal remodeling and regeneration. Semi-quantitative analysis of H&E images performed by ImageJ software is presented in Table 2. On day 3, H&E images showed abundance of inflammatory cells in control 15

groups representing inflammatory chronicity whereas lesser degree of inflammation was seen in wounds treated with NCs hydrogel and AgNPs (Fig. 2). Control over inflammation is a critical step towards healing of impaired wounds where NCs dressings could play a greater role. Evidence of a few fibroblasts and blood vessels was also observed in NCs treated mice. Similar observations have been made in a study that demonstrated lesser inflammation in rats after application of hydro cellular foam dressings of hydrophilic polyurethane and polyethylene glycol [27]. The reason behind the decreased inflammation in NCs treated groups may be due to existence of large number of carboxylate moieties of CNCs that hold a higher concentration of Ag. AgNPs are believed to decrease the time for fibroblast’s invasion into wound tissue, and also possess anti-inflammatory properties [28]. Additionally, it has been previously postulated that the diabetic wounds have a prolonged inflammatory response due to enhanced proteolytic activity caused by bacterial infection. AgNPs are known protease inactivators that act to decrease inflammation and also reduce the time for granulation tissue formation [29]. Hence, both CNCs and AgNPs present in NCs act synergistically to reduce inflammation. On day 10, high persistence of inflammatory cells with only a few fibroblasts was found in UC, Vas and Bet groups (Table 2 and Fig. 2). Skin sections of NCs treated groups have shown thick granulation with negligible number of inflammatory cells (Fig. 2). NCs treated and NDUC groups showed increase in proliferation of epithelial tissue covering the wound area. Fibroblasts and compact collagen regeneration was also observed in NCs treated sections. In skin tissue sections of mice treated with vehicle controls, the process of dermal remodeling was slow as evidenced by partially formed thin layer of neo-epithelium. Similar healing properties have been reported by hyaluronan/silver nanocomposites [24]. Minimal improvement in healing process was noticed in control groups in terms of lower epithelialization along with lesser development 16

of collagen fibers. Fibroblasts differentiation into myofibroblasts was initiated in NCs treated groups leading to rapid healing due to the presence of AgNPs in NCs which are known to increase the angiogenetic effect [30]. On day 18 post injury, the wounds of all the vehicle and NCs treated groups have shown almost complete healing, with newly synthesized fibrous tissue fully covered by a neoepithelium. However, wounds treated with NCs hydrogels exhibited more stratum corneum than others. Similar results showing complete epithelialization were reported when nanofibrous membranes containing poly(lactic-co-glycolic acid) and metformin were applied to diabetic rats [31]. Abundance of well-arranged collagen bundles and newly formed blood vessels was seen in NCs treated wounds (Fig. 2). Conversion of fibroblasts into myofibroblasts and also the presence of a few hair follicles has given an indication of complete dermal modeling in NCs treated mice. The reason might be due to the presence of AgNPs in NCs that could have quicken the repair process by providing the required energy for contraction of wound area by conversion of fibroblast into myofibroblasts [11]. In Vas and UC group, thin collagen fibrils distributed in irregular fashion were accompanied by a thin granulation tissue covered with thin epithelium with a large epithelial gap. Nanofibrillar cellulose has been speculated to promote rapid epithelialization due to their tendency to absorb water in nano-structural pores that keep the wound site moist [23]. 3.5. Masson’s Trichome staining showed faster progression in collagen fibrils upon NCs application A special Masson’s Trichome (M&T) staining procedure was carried out to estimate the density of collagen fibrils as tissue repair progresses. At day 10 post wound, NCs were able to promote fibroblast migration, collagen deposition in dermis and wound closure more efficiently than 17

control groups of diabetic mice. The plausible reason could be nanoporous structure of CNCs which is beneficial for cell migration, growth and proliferation [8]. Similarly, bacterial celluloseZnO nanocomposites have also been reported to accelerate healing by increasing proliferation of fibroblasts and keratinocytes [32]. Indeed, by day 18, NCs treated groups have documented the re-establishment of a dense, well oriented layer of thick collagen fibers (Fig. 3). However, the extent of collagen synthesis was remarkable in NCs treated groups. While less developed and randomly organized collagen fibrils were observed in granulation tissue of all the control groups. Fully distinguished and symmetrically distributed collagen fibers under the neo-epithelium in hyaluronan-4 treated group has been reported previously [33]. These outcomes have indicated the complete and efficient wound healing of skin, demarcated by reconstitution of dermalepidermal junction, in NCs treated mice as compared to control groups. 3.6. Immunohistochemistry (IHC) documented the role of NCs in diabetic wound healing In the wound microenvironment, the growth factors such as platelet derived growth factor (PDGF), vascular epidermal growth factor (VEGF) and fibroblast growth factor (FGF) change the wound healing cascade through stimulatory or inhibitory effect on the wound microenvironment [34,35]. These growth factors are related to inflammation, proliferation, cellular differentiation, migration and matrix substances required for healing. Therefore, the changes in secretion or absence of such growth factors in diabetic wounds can potentially hinder the healing process. Many growth factors have been reported to be clearly involved in fibroblast activation and migration [36]. IHC images are presented in Fig. 4 and the % expression (n = 5 observation fields/ group) of various proteins as analyzed from IHC images is plotted (Fig. 5). Collagen, the most abundant connective tissue protein, is a vital component of dermis. The complex processes of collagen synthesis and degradation in wound repair continue at the 18

wound site extensively after injury. The equilibrium between collagen synthesis and degradation is tenuous in diabetes where collagen synthesis noticeably decreases, causing chronic connective tissue complications [37]. With the progression of wound maturity, collagen III get converted to collagen I to strengthen the wound [38]. As evident from IHC images, expression of collagen is lower in control groups as compared to NCs treated ones (Fig. 4). NCs treated groups have significantly (p < 0.05) higher collagen levels as compared to control groups suggesting their probable role in collagen enhancement (Fig. 5). Recenlty, another polysaccharide based chitosanPVP-nano silver oxide wound dressing has also been reported to increase collagen density in rat wounds [39]. Vascular endothelial growth factor (VEGF) plays an essential role in vascular network formation that is found to be deficient in diabetics after cutaneous injury [40]. VEGF induces wound repair by aiding in vascular permeability, thus permitting inflammatory cells to reach the injured site, and also enhances the proliferation and migration of the already present endothelial cells [41]. During the initial healing process, a significant (p < 0.05) rise in VEGF level was observed in non-diabetic mice that has been accredited in the past for improved reepithelialization at the wound site. Previous reports have also suggested that declining VEGF levels in diabetics could be due to their inability to accurately up-regulate VEGF expression [42]. NCs constituted of plant CNCs and AgNPs have been found to enhance VEGF production due to the angiogenetic effect of AgNPs, clearly evident from IHC images (Fig. 4). Statistically, NCs hydrogels have shown significant (p < 0.05) increase in VEGF levels as compared to control groups which has resulted in intensified angiogenesis and accelerated healing (Fig. 5). PDGF is known to be an effective mitogen and chemotactic agent which acts to increase wound vascularization. Levels of PDGF were observed to be statistically lower in control as 19

compared to NCs treated groups (p < 0.05) (Fig. 4 and 5). NCs could augment wound healing through PDGF expression by endothelial cells and platelets. Past research has shown the autocrine and mitogenic effects of PDGF on keratinocytes and has supported epidermal proliferation, stimulation of vessel maturation and stabilization of epidermal junction occurred during closure of wound [43]. Basic fibroblast growth factor (bFGF) is a potent mitogen that gives the initial stimulation to endothelial cell migration and proliferation for diabetic wound repair [41]. Absence of FGF leads to delayed healing in diabetic wounds. In this study, significantly higher levels of FGF (p < 0.05) were observed in NCs treated groups in comparison to controls, suggesting the role of NCs in accelerating wound repair process (Fig. 4 and 5). Due to water absorption capacity of CNCs present in NCs, the proteolytic activity might be elevated in a moist micro-environment which prompts stimulation and accumulation of certain growth factors like VEGF, TGF and FGF at wound site as was reviewed in case of microbial cellulose [44]. The decreased production of inflammatory cytokines and increased fibroblasts proliferation, angiogenesis and collagen deposition which is consistent with enhanced in vivo skin tissue repair in NCs treated mice. 3.7. Skin homogenate of NCs treated mice showed increased hydroxyproline and decreased IL-6 levels A non-proteinogenic amino acid, hydroxyproline is mainly restricted to collagen and is measured as a collagen content indicator. Collagen is crucial in reconstructing the injured or disrupted tissue, as it restores the mechanical strength, anatomical structure and function of repaired skin [43]. The collagen levels showed significant variations among control and NCs treated groups at day 10 but no significant changes were observed at day 18 (Fig. 6). The hydroxyproline content 20

was almost similar in most of the groups at day 10 and 18 post wound (Fig. 6). The possible reason for this could be that by day 18, most of the groups have entered a phase of dermal remodeling where equilibrium was achieved between the rate of collagen formation and degradation [45]. Histopathological and biochemical analysis have documented early collagen deposition in NCs treated wounds. Higher hydroxyproline levels at day 18 has indicated good healing as collagen has enhanced wound strength and was crucial for processes involved in early stages of healing of a wound. Findings of this study were consistent with past reports that demonstrated the correlation of presence of abundant collagen fibers with better wound healing [26]. The ultimate aim of wound healing is fast recovery accompanied with minimum scar formation. An altered inflammatory response and cytokine pattern in the wound microenvironment at different time points is a hallmark of delayed diabetic wound healing in vivo. The pro-inflammatory cytokine IL-6 is largely released by immune cells early during wound healing cascade and it acts as an effector for fibroblasts and keratinocytes thus enhancing epithelialization [46]. Changes in the level of IL-6 during NCs treatment at day 3, 10 and 18 are illustrated in Fig. 6. Significantly higher levels of IL-6 at day 3 were found in UC and Bet group, while its level was decreased at day 10 and 18 post wound in all groups. Significant reduction in IL-6 level was observed at day 10 and 18 in NCs treated groups as compared to control groups (Fig. 6). Thus, the reduction in inflammation due to declining IL-6 levels could be attributed to anti-inflammatory property of AgNPs present in NCs [47]. Hyper inflammation and wound infection are common in diabetic individuals due to higher levels of pro-inflammatory cytokines TNF-α and IL-6 [48]. Chronic wounds and delayed healing occur due to persistent inflammation [6]. Furthermore elevated levels of such cytokines 21

are common in diabetics and are often related with extracellular matrix (ECM) degradation [49]. In this study, TNF-α levels in diabetic wounds were observed at lower levels in NCs treated groups as compared to control groups at day 3, 10 and 18 post wound (Fig. 6). This might be due to the presence of AgNPs that are known to have an inhibitory effect on the release of proinflammatory cytokines [50]. TGF-β is released quite early during the healing process by platelets and is a driving force for cellular differentiation and chemotaxis of immune and inflammatory cells to the wounded region [51]. In addition to it, TGF-β is involved in synthesis of granulation tissue and accumulation of ECM proteins [52]. Furthermore, TGF-β is essential during the last phase of healing process, where it helps in the conversion of collagen III to collagen I causing tissue remodeling [53], and stimulates keratinocyte proliferation thus supporting wound epithelialization [54]. Indeed, defects in TGF-β signaling in diabetics contribute to delay in healing [55]. Therefore, TGF-β level was evaluated to ascertain its role in wound healing enhancement following NCs treatment. TGF-β level in NCs treated groups was decreased drastically at day 10 but regained normal levels by day 18 of post wound. Results suggested that NCs treatment has led to enhancement of TGF-β levels in wounded mice at later stage (Fig. 6). 4. Conclusions Nanobiocomposite (NCs) hydrogels prepared from bamboo cellulose nanocrystals impregnated with silver nanoparticles serve as effective dressing materials for healing wounds in streptozotocin induced diabetic mice model. The developed NCs hydrogel resulted in accelerated diabetic wound healing within a time span of 18 days, as compared to various control groups that took longer time to heal. NCs hasten the healing process by reducing inflammation along with 22

early proliferation, collagen formation and epithelialization through regulating the expression of certain pro-inflammatory cytokines and growth factors responsible for delaying diabetic wound healing. In a nutshell, these NCs have immense potential to be used as ideal wound dressing materials for efficient and faster healing in diabetic patients. 5. Acknowledgements Authors are highly grateful to the Director, CSIR-IHBT for providing research facilities and infrastructure. We would like to thank the Council of Scientific and Industrial Research (CSIR), Govt. of India for financial support in the form a research project (BSC-0112, MLP-0039 and MLP-0068). RS is thankful to UGC and SS to CSIR for providing senior research fellowships.

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Figure Captions Fig. 1 Digital camera photographs presenting the macroscopic examination of wounded skin at day 0, 3, 10 and 18 post wound showing the effect of different treatment groups in in vivo diabetic mice model (a); histogram showing the wound closure area (%) of each treated diabetic mice group at day 3, 10 and 18 post wounding. The measurements are presented as mean ± standard deviation, n= 3 mice/group at each time interval. Groups not connected by same alphabets represent statistically significant differences at p < 0.05 (b); and Magnified image of untreated diabetic mice and mice treated with DH-CNC-Ag at 18 day (c). Fig. 2 Bright field micrographs of Haematoxylin and Eosin staining of wounded skin tissue sections of diabetic mice determining the changes in wound healing events at day 3, 10 and 18 after wounding. Fig. 3 Bright field micrographs of Masson’s Trichome staining of wounded skin tissue sections of each group of diabetic mice at day 10 and 18 post wound where presence of blue color indicates the density of collagen fibrils. Fig. 4 Representative immuno-histochemistry (IHC) staining images of different proteins i.e. collagen I and collagen III (at day 18), FGF, PDGF and VEGF (at day 10 post wound) in the tissue sections of diabetic mice. Brown color represents positive immuno-staining and was observed in epidermal and dermal layers in an uneven distribution. Fig. 5 Histograms represent the level of protein expression (%) in the wound tissue sections of diabetic mice on day 18 (collagen 1 and 3) and day 10 (FGF, PDGF and VEGF) post wound. The

31

measurements are presented as mean ± standard deviation, n= 5 observation fields/group. Groups not connected by same alphabets represent statistically significant differences at p < 0.05. Fig. 6 Biochemical ELISA assays quantify the levels of hydroxyproline (a); IL-6 (b); TGF-β (c) and TNF-α (d) in skin homogenate of all the mice groups at specific time points of post wound. The results are presented as mean ± standard deviation (n = 3 mice/group). Groups not connected by same alphabets represent statistical significant differences at p < 0.05.

32

a)

NDUC

UC

Vas

Bet

AgNPs

DH-CNCs

BB-CNCs DH-CNC-Ag BB-CNC-Ag

0 day

3 day

10 day

18 day

b)

c)

120 A

100

Wound closure area (%)

AAAAAA BBBBB A

B C D

80 60

B C DC D D

A BA ACBAA BDC B C D

B

NDUC

UC

UC (18 day) Enlarged

Vas Bet AgNPs DH-CNCs

40

BB-CNCs DH-CNC-Ag

20

BB-CNC-Ag 0 3 Day

10 Day

18 Day

Figure 1

33

DH-CNC-Ag (18 day) Enlarged

3 day

10 day

18 day

NDUC

UC

Vas

Bet

AgNPs

DH-CNCs

BB-CNCs

DH-CNC-Ag

BB-CNC-Ag

Figure 2

34

10 day

18 day

NDUC

UC

Bet

Vas

AgNPs

DH-CNCs

BB-CNCs

DH-CNC-Ag

BB-CNC-Ag

Figure 3

35

Collagen 1

Collagen 3

FGF

PDGF

NDUC

UC

Vas

Bet

AgNPs

DH-CNCs

BB-CNCs

DH-CNC-Ag

BB-CNC-Ag

Figure 4

36

VEGF

Figure 5

37

1200 1000

A A AA BA AA B B

A B

A B AA AA A A B B AB BB B C C B C C C C D D D D D

1600

UC

Vas 800

Bet

B

AgNPs

600

DH-CNCs 400

BB-CNCs

A

A

1400

NDUC

Concentration of IL-6 (pg/mL)

Concentration of hydroxyproline (ng/mL)

1400

1200

A

1000 800

B C

600

CB C

B C

B C D

B C

C D

C C D D

C D

400

DH-CNC-Ag

200

A B

BB CB C

A AB B C D

D

200

BB-CNC-Ag

0

D

AB BC B CD C C D D

0 10 Day

18 Day

3 Day

10 Day

18 Day

8000 450

C D

D

200

AAA B BBB CC CB C

A B BB B C CC C

A A B B C

A BA B B C C C C

C

150

Concentration of TNF-α (pg/mL)

Concentration of TGF-β (ng/L)

250

A A AA AB B B BC C C CD D C D

A B C

350

300

7000

A B

400

6000 5000

4000

A

A AA BB

B C B C

B C

3000

B BC C

AA A A BB B C C D

A B

B C D

C D

C D

2000

D

D

A

AA BB C

A B B BC C

B C C

100

1000

50 0

0 3 Day

10 Day

18 Day

3 Day

Figure 6

38

10 Day

18 Day

D

Table 1. Categorization of mice in different groups according to the materials applied topically for diabetic wound healing. Group no.

Types of material applied

No. of mice/group

1.

Non-diabetic untreated control (NDUC)

9

2.

Diabetic untreated control (UC)

9

3.

Vaseline treated (Vas) - Vehicle control

9

4.

Betadine (Bet) – Positive controls

9

5.

Silver nanoparticles (AgNPs) – Vehicle control

9

6.

DH-CNCs (Vehicle control)

9

7.

BB-CNCs (Vehicle control)

9

8.

DH-CNC-Ag hydrogel

9

9.

BB-CNC-Ag hydrogel

9

39

Table 2. Semi-quantitative analysis of H&E images of skin tissue sections of each treatment group of diabetic mice done by using ImageJ software. S. Treatment groups Degree of Extent of Epithelializati No. Inflammation Fibrous tissue on formation Day Day Day Day Day Day 3 10 10 18 10 18 1. Non-diabetic untreated control +++ + + ++ P C (NDUC) 2. Diabetic untreated control (UC) +++ ++ + + P P 3. Vaseline treated (Vas) +++ ++ + ++ P P Vehicle control 4. Betadine (Bet) – Positive ++ ++ + ++ P C controls 5. Silver nanoparticles (AgNPs) – +++ ++ + ++ P P Vehicle control 6. DH-CNCs (Vehicle control) ++ + + ++ P C 7. BB-CNCs (Vehicle control) ++ ++ + ++ P C 8. DH-CNC-Ag hydrogel + ++ +++ C C 9. BB-CNC-Ag hydrogel + + ++ +++ C C The symbols representing + = Few inflammatory cells, ++ = Moderate number of inflammatory cells, +++ = Large number of inflammatory cells, + = Mild presence of fibrous tissue, ++ = Moderate, +++ = Abundant, P= partial epithelialization, and C= complete epithelialization.

40