Comparative Study of Effect of Silver Nitrate Gel and Differentiated Dermal Precursors (BMSCs) on Third Degree Burn in Wistar Rats

Skin is an ectodermal derivative that maintains internal homeostasis of the body. Any damage to the skin like burn injury internal homeostasis is lost, resulting in delayed healing. The aim is to study the histoarchitecture comparative effect of silver nitrate gel, and BMSCs (DDP) on third-degree burns in Wistar rats. A burn wound of size 2.5 cm (length) x 2.5 cm (breadth) x 6 mm (depth) was created using a preheated metal plate on (cid:977)lanks of Wistar rat. Every burn wound was treated with silver nitrate gel (commercially avail-able as silverex), bone marrow differentiated dermal precursors, and moni-tored for 1, 7, 14, 21 days until wound healing. Wound surface area was measured and compared among groups with histological and gross observations. The healing time was faster in bone marrow differentiated dermal precursors (DDP) group compared to control. Prolonged silver nitrate gel usage heals burn wound with no infection, but silver toxicity was noted. Wound contraction is slower but steady using bone marrow differentiated dermal precursors (DDP) cell when compared to the group treated with silver nitrate gel. The data from this study help use to use bone marrow differentiated dermal precursors (DDP) cells as an alternate and effective way to treat burn wounds.


INTRODUCTION
Burns are acquired tissue injuries when the skin is subjected to chemicals, radiation, hot liquids, hot solids, lames, etc. (Bosman, 2019). WHO states, burn injuries results in mortality and morbidity (Barillo et al., 2019). Depending on severity of burns and thickness involved with epidermis, dermis, and hypodermis of skin, burns are divided into irst degree -burn lesion restricted to epidermis of skin, second degree -burn involving epidermis and super icial dermis, third-degreeburns comprising dermis and subcutaneous layer of skin (Tredget et al., 2017). An autologous skin graft is used as a golden standard in the treatment of burn injuries (Varkey et al., 2019). Wound healing initiates the process of in lammation, odema, hypertrophy of burn site, leaving behind scar tissues (Chiang et al., 2016). Silver cream on topical wound application is a bene icial antibacterial agent (Branski et al., 2018) but maybe cytotoxic to target cells that it comes in contact. A stem cell has the remarkable potential to grow into many differentiated body cell types during early life and growth. They act as an internal repair system in many tissues, dividing essentially devoid of the limit to replenish other cells as long as person or animal live. Once the stem cell divides, every new cell remains as stem cell or become another cell type with a particular function such as cartilage, bone, muscle, nerve, and tooth. This differentiation potential of stem cells has enumerable therapeutic applications so that it can be bene ited by common people in trauma burns and old age patients. This study compares the effect of silver nitrate gel and DDP (BMSCs) cells on thirddegree burn in Wistar rats.

Animals
The test facility is registered with the Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment, Forest and Climate Change, Animal Welfare Division, Govt. of India (No.1182/PO/Rc/S/08/CPCSEA). The IAEC approval number of this study is MTR/IAEC/PRU/013-15. Animals were housed individually with stainless steel grill cages and provided with standard rodent pellet feed and daily UV puri ied RO iltered water ad libitum. Sterilized clean paddy husk was taken as the bedding material. Room Temperature and relative humidity were maintained. The test room facility was provided with 12 hrs light and 12 hrs of dark conditions throughout the experiment.

Third-degree Burn Model
Rats were anesthethetized using a combination of 10 mg/kg xylazine and 80 mg/kg ketamine intraperitoneally with a total average volume of 1.5 ml/animal. Third-degree Burn wound model was made on a shaved area by keeping a heated metal plate of 300ºC for 10 min on the lank of each rat with dimension 2.5 cm (length) x 2.5 cm (breadth) x 6 mm (depth).

Procedures
The third-degree burn Model rats (18nos) were classi ied into three random groups containing 6 ani-mals each. Group 1 -injured/ negative control, Group 2 treated with silver nitrate gel once daily for 21 days, Group 3 was treated once intralesionally, with 1 ml dermal precursors (DDP) from centre to periphery of the wound. No oral or parenteral antibiotics were directed to Wistar rats. Animals were weighed and observed for gross and histopathology observations.

Gross observation
Erythema, edema, and clinical signs was grossly observed under the burn model. The wound surface area was measured by radial planimetry (Foltynski et al., 2015). Wound contraction was observed every day by tracing the wound margins on acetate ilm.
Wound contraction percentage is calculated using formula, initial wound area-speci ic day wound area / initial wound area x 100 (Demilew et al., 2018).

Histopathology
At the end of the experiment, the third degree burn treated rats were given an overdose of Co2 anaesthesia. The treated area of wounded skin samples was taken from each group on day 7, 14, and 21. The Skin tissue measuring 5-6 µm thickness was collected and stored in neutral buffered formalin (10%) for the Histopathological process. Tissue, once embedded with wax, were subjected to microtomy and stained using hematoxylin and eosin dye for histological examination.

Statistics
The wound half-lives were compared using the Kruskal-Wallis one-way ANOVA and by Scheffe's technique for multiple comparisons. All analyses were accomplished with the help of a SPSS 15 on a personal computer. Using the simple linear regression analysis, absolute area measurements showed that the initial wound area created no remarkable difference to half-life calculations.

RESULTS AND DISCUSSION
There was no mortality/morbidity for all animals were observed throughout the experimental day. The bodyweight of the individual animal was recorded during randomization, anaesthesia, and necropsy. There was no substantial difference in the weight gain between all treatment and control groups. None of the animals showed erythema, edema, and were observed individually for any clinical signs/toxicity signs once daily throughout the experimental days. The burnt wound rats have been depicted in Figure 1.

Figure 1: Effect of silver nitrate and DDP in burnt wound healing rats
Healing measurement of an individual animal was measured and recorded before application on day 1 of treatment followed by day 7, 14 and 21 (Figure 2). A better wound healing pattern with wound closure was observed in treated rats with 21 days of treatment in all the group. The wound contraction and reduction in healing on different days. There was a remarkable reduction in the size of a wound from 7 th day in treated rats and also, the closure rate was much faster in group 3 (DDP cells), in contrast to group 2 (silver nitrate gel) and group 1(control group) in the later days. Figure 3 depicts an extensive loss of epidermis with large scab consisting of necrotic cellular debris in ibrin coagulum bordered with numerous dege erated and intact neutrophils, increased mast cells, and histiocytes with moderate ibroblasts in the hypodermis (Figure 3 A). Extensive necrosis of epidermis with fatty changes di-organization of collagen and loss of adnexa in the dermis ; large scab with necrotic debris a d ibrin coagulum, numerous neutrophils and bacterial clumps distorting the muscle and adipose tissue in the hypodermis; increased ibroblasts, neovessels, mast cells and histiocytes in the hypodermis (Figure 3 B). Extensive loss of epidermis with a scab, increased ibroblasts, neovessels in the dermis with immature collagenisation, and a moderate number of neutrophils, mast cells, and histiocytes (Figure 3 C). Complete loss of epidermis and dermis and replacement with abundant scab tissue; numerous degenerated and intact neutrophils ; mild ibroblastic activity, increased mast cells, histiocytes, and neovascularization in the hypodermis (Figure 3 D).

Histopathology
Epidermis and dermis intact; reduction in the size of scab and in lammatory cells; intense ibroblastic activity with collagenisation and neovascularization; a few scattered mast cells and histiocytes in the hypodermis (Figure 3 E). Loss of epidermis and scab formation; intense ibroblastic activity and neovascularization with numerous mast cells, histiocytes, and a few scattered neutrophils in the dermis; mild collagenisation (Figure 3 F).
Reduction in scab in hypodermis; increased neutrophils and ibroblastic activity with neovascularization ; mild collagenisation (Figure 3 G). Reduction in scab in hypodermis increased neutrophils and ibroblastic activity with neovascularization ; mild collagenisation (Figure 3 H). Loss of epidermis with scab; partial mild re-epithelization; increased ibroblastic activity, neovascularization, and moderate collagenisation; a few scattered neutrophils and mast cells (Figure 3 I).
The epidermis of thin skin is exposed to moderate sunburn or ultraviolet light. It causes capillary, venules in the papillary and sub papillary region to dilate by making it turn red -(erythema). In severe burns, the capillaries and venules allow plasma to leak through them, resulting in accumulation of luid between the epidermis and dermis, resulting in blisters. Under natural conditions, the epidermal cells surviving in the lower part of hair follicles regenerate over the blisters surface resulting in healing.
Acute wound luids have a positive effect of the wound. This could be due to increased stem cells (Krzyszczyk et al., 2018). If the burn injury involves the super icial part of the dermis, epithelial cells from a deep part of the hair follicle proliferate to grow along with the interface between living and the dead dermis. However, if the burn damage extends beyond the base of the hair follicle, regrowth of epidermis can occur only at the perimeter of the burns. Delayed healing in chronic wounds is due to stem cell de iciency (Ghieh et al., 2015).
Healing of chronic burn wounds occurs so slowly that a skin graft is necessary. Autologous Skin graft transplanted from the different sites of the body is used as a golden standard in treating chronic burn injuries (Gardien et al., 2016). Epidermis and supericial part of the dermis is transplanted. Skin grafts derive its vascularity and nutrients from tissue luid exudate, where the skin graft is placed, resulting in a healing area covered with new skin. Slow regrowth of epidermis occurs at the periphery of the burn site. Systemic transplantation of dermis derived multipotent cells has no superior effect in the healing of irradiated wounds (Francis et al., 2019). Mesenchymal stem cells were applied in addition to surgical laps (Linard et al., 2018). The bone marrow mesenchymal stem cells application reduces in lammation and healing time (Dehkordi et al., 2019). BMSCs were used as an arti icial dermal substitute to wound healing by increasing the rate of epithelisation and vascular density. After burns injury, healing of wound progress with haemostasis, in lammation, proliferation, and tissue remodelling.
In the present study, a third-degree burn model was created and was divided into group 1 -injured control, group 2 -treated with silver nitrate gel, and group 3-treated with bone marrow differentiated dermal precursors. Wound healing was noted on day 1, 7, 14, and 21 by gross and histological observations. Under gross observation, all animals were healthy, and none of them reported with erythema, edema, weight loss, morbidity, mortality, & clinical signs/toxicity signs.
Wound contracture was greater in group 2 treated with silver nitrate gel, followed by group 3 treated with bone marrow differentiated dermal precursors (DDP) when compared to the control group. This could be due to the daily application of silver nitrate gel. Whereas bone marrow differentiated dermal precursors (DDP) was injected once intralesionally from the periphery to central on thirddegree burn wound. Under histological observations, day 7: group 1 showed extensive loss of epidermis with large scab consisting of necrotic cellular debris in ibrin coagulum bordered with numerous degenerated and intact neutrophils when compared to group 2 & 3. Stem cells injected to rectangular thermal burns with unburned interspaces did not necrose after 7 days, thereby preventing the progression of burn injuries. Day 14: histological observations of group 1 shows epidermal necrosis with collagen disorganization and loss of adnexa in the dermis with bacterial clumps distorting the muscle and adipose tissue in the dermis. Group 2 shows a reduction in the size of scab and in lammatory cells. This could be due to the potent antimicrobial and anti-in lammatory properties of silver (Burdus , el et al., 2018).
Argyria, a perpetual disorder caused by the deposition of silver in the skin's micro-vessels in people who are exposed to chronic silver toxicity (Magh-  (Qi et al., 2014). Day 7: Group 3 showed a reduction in scab in the hypodermis, increased neutrophils, and ibroblastic activity with neovascularisation and mild collagenisation. Day 14: group 3 showed partial mild re-epithelisation; this could be due to the proliferation of stem cells to keratinocytes, thereby promoting re-epithelisation and wound healing (Yang et al., 2019). epithelial cells on wound margin move to seal the entire wound by process of "epiboly". Day 14 of group 3 showed increased ibroblastic activity. Bone marrow cells exhibit ibroblast-like morphology. Fibroblast moves to the injured target tissue to secrete collagen matrix. Collagen provides strength and stability to underlying tissue in order to retain structural and functional anatomy. There exists a homeostasis between normal and abnormal collagen deposition.
Group 3 showed mild to moderate collagenisation from day 7, 14, and 21 days. Fibroblast adhere to collagen and elastin ibre which they laid down. Bone marrow differentiated dermal precursors are a principal source of dermal ibroblast. Dermal ibroblast secretes type 1 collagen (Tracy et al., 2016). Fibroblast particularly active during wound repair, multiplying, and laying down a ibrous matrix which becomes invaded by numerous blood vessels (Caballé-Serrano et al., 2019). Neovascularisation was observed in group 3 & group 2 on 7, 14, and 21 st day. This is to attain Cell nourishment. The nutrients to the building block are transferred by the process of "diffusion". Mesenchymal stem cells injected in the burn-injured mouse model showed faster healing and denser collagen (Revilla et al., 2016). Mesenchymal stem cell medium is chemo attractant invitro, thereby recruiting speci ic cells like epidermal keratinocytes and dermal ibroblast to wound via Mesenchymal stem cell paracrine signalling. This mechanism of paracrine signalling favours angiogenesis and tissue remodelling such as ibroblast proliferation and epithelisation in all phases of wound repair (Lee et al., 2016). BMSCs differentiated into dermal cells when injected into the targeted dermal injury progresses wound healing by maintaining tissue homeostasis, in lammation, proliferation, and tissue remodelling. BMSCs differentiated dermal fastens healing time, so as to reject and overcome graft and minor surgeries.

CONCLUSIONS
Culturing DDP cells ex-vivo is a long process which needs a GMP environment. DDP cells are expensive and cannot be afford by common people. Bone marrow differentiated dermal precursors ful ill the DDP functions by accelerating wound closure with increased epithelisation, granulation tissue formation, and angiogenesis. The BMSCs differentiated dermal precursors (DDP) cells showed faster tissue healing than the control group. Continuous silver nitrate gel usage causes silver toxicity. Intralesional administration of DDP cells once from inside out of lesion showed steady wound healing when compared with control groups. However, healing is incomplete and required some more cells for it to be complete and replace tissue damage. Therapeutic administration of BMSCs differentiated dermal precursors (DDP) on third-degree burns will replace damaged skin and can be used as an alternate therapeutic option in treating dermal injuries with reduced treatment duration.