Silk derived formulations for accelerated wound healing in diabetic mice

Background The present study aimed to prepare effective silk derived formulations in combination with plant extract (Aloe vera gel) to speed up the wound healing process in diabetic mice. Methods Diabetes was induced in albino mice by using alloxan monohydrate. After successful induction of diabetes in mice, excision wounds were created via biopsy puncture (6 mm). Wound healing effect of silk sericin (5%) and silk fibroin (5%) individually and in combination with 5% Aloe vera gel was evaluated by determining the percent wound contraction, healing time and histological analysis. Results The results indicated that the best biocompatible silk combination was of 5% silk fibroin and 5% Aloe vera gel in which wounds were healed in 13 days with wound contraction: 98.33 ± 0.80%. In contrast, the wound of the control group (polyfax) healed in 19 day shaving 98.5 ± 0.67% contraction. Histological analysis revealed that the wounds which were treated with silk formulations exhibited an increased growth of blood vessels, collagen fibers, and much reduced inflammation. Conclusion It can be concluded that a combination of Bombyx mori silk and Aloe vera gel is a natural biomaterial that can be utilized in wound dressings and to prepare more innovative silk based formulations for speedy recovery of chronic wounds.

109 glucose solution (10%) after receiving an injection of alloxan monohydrate to prevent them from 110 sudden hypoglycemic state (Vanitha et al., 2013;Bouzghaya et al., 2020). After 24 hrs of 111 induction of diabetes, blood samples were collected by pricking the tail tip of the mice. Blood 112 glucose level was measured with an electronic glucometer (On-call extra blood glucose meter 113 and test strips). Animals with a blood glucose level of ≥250 mg/dl were considered diabetic and 114 were selected for further experimentation (Chen et al., 2015). Mice were given free access to 115 food and water during the study and they were kept in standard plastic cages at room temperature 116 in the Animal House facility. Blood glucose levels of all albino mice were recorded before 117 starting of the experiment. Only those mice that have normal blood glucose levels were used for 118 further study and those having high blood glucose levels were excluded from the study (Dra et 119 al., 2019). 120 Creation of skin excision wound in mice 121 Mice were randomly divided into six groups with each group consisting of 6male mice. 122 Animals were anesthetized intraperitoneally with ketamine (100 mg/kg) and Xylazine (10 123 mg/kg) in saline before wound induction. The dorsal fur of mice was shaved completely by using 124 an electrical hand shaver. Two full thickness excision wounds were created on the dorsum of 125 each mouse by using a 6mm biopsy punch device. These surgical interventions were carried out 126 under sterile conditions. The total surgical time was 15-20 minutes for each mouse. All animals 127 received their respective treatments once a day from post wounding day till complete healing. 128 Body weight, skin color and skin irritation were observed and recorded daily.
129 Extraction of sericin from cocoons 130 Silk cocoons of B. mori (silkworm) were kindly supplied by the Sericulture section of 132 extraction, 5g of silk cocoon pieces were immersed in 100 ml of distilled water and autoclaved at 133 121 °C and 15 lb per square inch pressure for 1hr. After 1hr the sericin solution was allowed to 134 cool at room temperature and then filtered through a filter paper. The filtration process removed 135 impurities from the sericin solution. The filtered sericin solution was subjected to lyophilizer 136 (freeze drying) at -82 °C for 72 hrs to obtain sericin powder (Martínez et al., 2017). Extraction 137 procedures of sericin and fibroin were carried out separately by utilizing fresh silk cocoons each 138 time.
139 Extraction of fibroin 140 Degumming: Silk cocoons synthesized by B. mori silkworms were soaked in warm water 141 to loosen the threads. Silk threads from several cocoons were then unwound to obtain silk fibers.
194 Application of gel formulations on wounds 195 The diabetic mice were subjected to their respective treatments till complete wound 196 healing. The formulations were applied evenly on the wound surface daily.
197 Percent wound contraction 198 After wound creation, the wound margins were traced at 2 days interval on transparent 199 graph paper. Measurements were continued until the complete (98-99%) wound restoration.

203
The rate of healing as percentage contraction was calculated using the formula: For statistical analysis, the normality of the data was assessed using Shapiro-Wilk's test. 219 One-way ANOVA was conductedout to compare percent wound contractionin control and 220 treatment groups, followed by Tukey's post-hoc test using SPSS software (version 20). All data 221 were expressed as the mean ± SEM.

RESULTS
223 TEM analysis of silk fibroin and silk sericin 224 The transmission electron micrographs (TEM) showed 240-300 nm sized silk fibers of sonicated 225 samples (Figure 1). TEM micrographs of silk sericin at 2 µm scale bar are shown in Figure 1.
226 Results of TEM indicated that the size of the of silk sericin particles is approximately 102.5 nm.

GC-MS analysis of Aloe vera gel
228 A total of seventeen compounds were detected in Aloe vera gel by GC-MS analysis (Table 1). 229 Five major compounds (2,4:3,5:6,7-Tri-O-benzylidene-1-deoxy-d-gluco-d-gulo-heptitol, 230 stannane bis diphenyl, isopropyl myristate, 9-Octadecenoic acid and 10-Octadecenoic 231 acid)identified in Aloe vera gel. Their molecular formula, molecular weight (MW), retention 232 time (RT) and peak area (%) are presented in Table 2.Detail of major and minor compounds 233 (Table 1 and Table 2) detected through GC-MS analysis of Aloe vera gel will be helpful in future 234 wound healing studies and they may be utilized individually or in combinations for preparing 235 more effective gel formulations to treat chronic wounds.
236 Assessment of wound contraction 237 Healing area of wounds in treatment and control groups at day 11 is presented in figure 2 238 and at different days as percent wound contraction in Figure 3.  (Figure 3). However, the group C1 (positive control; polyfax) differs non-253 significantly from T1 (5% sericin) (P>0.05 ANOVA) and T2 (5% sericin and 5% Aloe vera) 254 (P>0.05 ANOVA). In the present study, the potential of silk-based formulations to accelerate the wound 274 healing process in diabetic mice was investigated. The results of this study indicated that silk 275 sericin and fibroin when blended with Aloe vera gel quicken the healing process without causing 276 any allergic reactions.