Table 1 depicts the results of all the physical properties of the chitosan sheet. All used chitosan sheets were transparent and slightly yellowish in colour. The SEM images show the uniform thickness of the sheet and it also shows the uniform binding of the drug throughout the sheet. The thickness of the chitosan sheet was 40 - 45 μm.
Moisture content (%) and Swelling index
The moisture content of the chitosan sheet was observed to be
16.2 + 0.9. The swelling index of the chitosan sheet was 469.63 + 15. This shows the high efficiency of dressing to absorb wound exudates. The swelling rate depends on the extent of cross linking (cross-linking density), the porous structure of the material, and the forces that act between the solvent (water) and the material (chitosan).
Water vapour transmission rate (WVTR)
Water-vapour transmission rate is important for wound dressing, because it controls the wound fluid evaporation across the dressing. At the same time, it should not allow the accumulation of exudates in the wound area. The rate of water vapour permeability of chitosan sheets were measured at different levels and it shows a transmission rate of 180 + 10 done at 80% RH and at 35ºC.
Agar Diffusion Test
The activity of the released drug from the dressing shows a clear zone of inhibition of 22 mm controlling the growth of mixed cultures inoculated in the Mueller Hinton agar, whereas full microbial growth was seen around the chitosan sheet without drug, that is, the control. The clear zone of inhibition was maintained for more than 10 days in AMP incorporated chitosan sheet and shown in Figure 1.
FT-IR spectrum of chitosan sheets show the characteristic peaks of chitosan at 899.20 and 1158 cm-1 corresponding to saccharide structure. Strong absorption peaks at 1640.69 and 1324.02 cm-1 are characteristics of amide I and III peaks, respectively. The peak at 1425.18 cm-1 is assigned to the CH3 symmetrical deformation mode. The peak at 1040.30 cm-1 indicates the C-O stretching vibration in chitosan. Another broad peak at 3447 cm-1 is caused by amine N-H symmetrical vibration. Peak at 2950 cm-1 is the typical C-H stretch vibrations (Figure 2.).
Thermal studies of sheets
Thermogravimetric analyses (TGA) (Figure 3a.) revealed that the chitosan sheet showed stability upto 2000C. The second stage of weight loss started at 200ºC and continued upto 781ºC (with 31.85% remaining).
DSC thermogram (Figure 3b.) of chitosan showed an initial broad peak at around 980C which is due to loss of moisture on heating. This was followed by further decomposition of chitosan as a broad exotherm in sheet at around 279ºC.
The wide angle X-ray diffraction (WAXD) pattern of pure chitosan powder and chitosan sheet in Figure 4. showed the main diffraction peaks around 2q = 20º. But the peak intensity ratio of chitosan sheet was decreased when compared to peak intensity of pure chitosan powder, although the crystalline peaks still remain. This indicated the crystallinity of the chitosan has been decreased.
Rate of wound contraction
Visual inspection of the wound showed that all the animals had well formed granulation tissues by day 4. A visual proof of the healing pattern of the wound was photographed on 0, 4, 8, 12 and 16 days after wound creation and depicted in Figures 5A, 5B, 5C, 5D, 5E & 5F.
Wounds of group I rats (Negative control) were reduced to 27.42% of the original wound area on day 4 of wound creation. The experimental groups (G II – G VI) showed significant (p<0.05) wound reduction (40%, 36.3%, 40%, 32% and 42.25%, respectively) compared to the control animals (G I) on day 4 of wound creation with more prominent reduction in animals treated with AMP incorporated chitosan sheet. The same trend continued after 4, 12 and 16th day, indicating wound contraction was significant throughout the healing period. Animals treated with AMP incorporated chitosan showed faster rate of wound contraction (97.23%) on day 14, followed by AMP at higher concentration (96.71%) on day 15, then by AMP at lower concentration (94.0%) on day 16, and animals treated only with chitosan showed 92.98% healing on day 16 (Figure 6.). The period of epithelialization of control and experimental groups is presented in Table 2. In our study, the rate of wound contraction in treated rats was significantly higher. Furthermore, the period of epithelialization was shorter for the treated wounds. These results further support the effectiveness of AMP produced by B.amyloliquefaciens MBL27 for wound healing.
Swab was taken on the wounded site on 3rd, 5th, 7th, 14th and 21st day taking care that the entire site of the wound was covered, from the day of wound creation and the microbial count was estimated as colony forming units (CFU/ml). There was a significant (p<0.001) reduction in bacterial population from 104 cells to 101 cells on the 3rd and 8th day, respectively for AMP incorporated chitosan sheet. But for animals treated with chitosan sheet the reduction is from 104 to 102 cells only (Figure 7.). Similarly, significant (p<0.05) reduction in microbial count was noticed for other groups also when compared to control animals. The antimicrobial property of the AMP is responsible for the reduction of the microbial count.
Figure 8. shows the total protein content in the granulation tissues of control (G I) and experimental wounds (G II – G VI). The protein content had a statistically significant (p<0.05) increase in experimental groups (G II – G VI) and was maintained upto day 12 of wound creation indicating the synthesis of other extracellular matrix protein other than collagen in the granulation tissues by the infiltrating cells. Group III showed a significant (p<0.05) increase in protein level in the granulation tissues on 4th day by 43.19% compared to control animals (G I). Similar trend was observed on 12th day. Group IV showed a significant (p<0.05) increase in protein level in the granulation tissues on 4th day by 59.69% compared to control animals (G I). Similar trend was observed on 12th day by 62%. Group V showed a significant (p<0.05) increase from 4th day (30.89%) to 12th day (47.58%), respectively whereas Group VI showed a significant (p<0.001) increase from 4th day (69.63%) to 12th day (77.79%), respectively compared to controls (G I). The increase in the total protein content indicates active synthesis and deposition of matrix proteins in the granulation tissues.
The total collagen content of granulation tissues on various days is presented in Figure 9. A significant (p<0.05) increase in collagen content was observed in experimental rats (G II – G VI) compared to controls (G I), throughout the course of healing, which is an important constituent of extracellular matrix for healing. Group II animals showed a significant (p<0.05) increase in collagen content on day 4, 8, 12 and 16th day when compared to controls (G I). Similar observations were found in the cases of G III, G IV, G V and G VI animals. A significant (p<0.001) increase in collagen content from day 4 (143.45%) to 8 (106.09%), respectively was observed in G VI animals compared to control animals.
Increased fibroblasts proliferation and dense collagen deposition is observed during later stages of healing particularly in the remodelling phase. As a result there is an increase in the total collagen content in all the experimental groups undergoing proper healing.
The results of hexosamine content in granulation tissues of control and experimental wounds have been shown in Figure 10. Hexosamine content was significantly increased in all experimental groups (G II – G VI) compared to controls (G I) with G VI showing increased content than all other groups. G III animals showed a significant (p<0.05) increase in hexosamine content when compared to controls (G I) and it was 48.75%, 32.26%, 13.27% and 58.4% on 4, 8, 12 and 16th day, respectively. A significant (p<0.05) increase in hexosamine content was found in G IV animals when compared to controls (G I) and it was 90.82%, 70.92%, 31.41% and 78.73% on 4, 8, 12 and 16th day, respectively. A significant (p<0.001) increase was observed in G VI animals also when compared to controls (G I). In addition, among the groups, G III and G VI had significantly higher values (p<0.05) than the other experimental groups, followed by G V and G VI and then by G III and G IV.
Uronic acid content
Table 3 presents the uronic acid levels in the granulation tissues of the control and experimental wounds. The synthesis of ground substance uronic acid was increased upto day 12 post-wounding in the treated groups (G II – G VI), thereafter the levels decreased.
The increase in hexosamine and uronic acid contents in the granulation tissues could be attributed to the formation of glycosaminoglycans (one of the ECM proteins) for which these two ingredients forms the backbone. They are the first components of ECM to be synthesized during wound healing and form the template for collagen and elastin deposition. There was a significant (p<0.05) increase in the uronic acid levels in all experimental groups (G II – G VI) compared to controls (G I), with G VI showing higher values compared to other treatment groups. Among the groups, G IV and G VI showed a statistically significant (p<0.001) increase on 8, 12 and 16th day.
In G I (Negative control) epidermal layer is still not formed well. But infiltration was reduced and macrophages were also observed below the epidermal layer. In G II (Positive control) histological features of normal skin were clearly observed with beginning of remodelling of skin. A well formed epidermis was noticed along with hair follicles emerging from the epidermal layer. In G III (AMP at lower concentration) well formed epithelia were seen with dermal layer containing mature fibroblasts and collagen deposition. In G IV (AMP at higher concentration) epithelial proliferation with well formed collagen bundles was observed. Hair follicles were also found. In G V (Chitosan sheet) wounded area was covered with epithelium. Collagen deposition was also observed. In G VI (AMP incorporated Chitosan sheet) complete epithelialization was seen. Mature fibroblastic cells were seen in the dermal region with collagen deposition. Healing was complete with surface being covered by epithelial cells (Figure 11.).
Masson’s Trichrome staining was used to examined the extent of collagen deposition in the wounds. Figure 12. shows the histological sections of both control (G I) and experimental groups (G II – G VI) taken on 16th day. Masson’s trichrome staining stains collagen and yields a blue colour. The pattern of staining intensity corresponds to the relative quantity of collagen-fiber deposit, which reflects the process of synthesis and degradation and remodeling as well as the timing of the lesion. Experimental groups (G II – G VI) showed well formed epithelial layer with intense collagen deposition when compared to controls (G I). Well formed hair follicles along with sebaceous gland were also observed. Experimental groups (G II – G VI) also depict the compact and well aligned arrangement of collagen layers except for controls (G I) where only very slight collagen was observed. The bundles of collagen were also thicker in AMP incorporated chitosan sheet (G VI) treated group than controls (G I).