The Efficacy of Mesenchymal Stem Cells loaded in Acacia Gum Hydrogel and Acacia Gum Hydrogel on Skin Wounds Healing in Rats

Cutaneous wound healing processes start at the beginning of the trauma leading to impairment of physical and functional tissue continuity. The present work aimed to clarify the efficacy of stem cells loaded in acacia gum hydrogel and acacia gum hydrogel on skin wound healing in rats. Thirty-six adult male rats (Wister rats). The weight of the rats was estimated between 150-160 grams and the animals were divided into three groups (n=12). In Group A (control group), the surgical skin wound was established and treated with normal saline only. Group B: surgical skin wound was made and covered with Acacia gum hydrogel. Group C: surgical skin wound was made and covered with mesenchymal stem cells loaded in Acacia gum hydrogel. Examination of wound healing on days 3, 7, 14 and 21 for gross and microscopic pathological changes. The gross pathological evaluation showed no significant difference between all groups but the histopathological result showed the Acacia gum improved wound recovery better than the control group, although the wound was not completely closed on day 21 in Acacia gum and control groups contrary to the stem cells treated group, which showed complete wound closure. The best ever histopathological result of wounds healing in mesenchymal stem cells loaded in acacia gum hydrogel treated groups. Abstract


Introduction
Skin is a complex structure designed to protect organism from exogenous environments and thus wound lead to damage of numerous structures, cellular layers and lineages, like the dermis, epidermal keratinocyte layer, hair follicles, sweat glands, fibroblasts, extracellular matrix, nerves, blood vessels and lymphatic vessels (1). Wound healing is complex regeneration processes involves three crucial stages (inflammatory reaction, proliferative and maturation phases), and also involves the interaction between may important components (e.g. cells, intercellular matrix ,signaling factors) (2, 3).
Acacia gum (Gum Arabic) is a branched-chain, complex mixture of glycoproteins and polysaccharides contains some minerals and salts (4). Acacia gum has the capability to absorb fluids and maintain wound hydration to speed up the wound healing process (5).
Bone marrow mesenchymal stromal cells (BM-MSCs) are most encouraging tools of wound healing. BM-MSCs are multipotential stem cells that can differentiate to give rise different types of cells, growth factors and cytokines for wound healing (6). It has the ability to move to the area of the injury and enhance wound healing (7).
The present study aims to evaluate and compare between BM-MSCs loaded in acacia gum hydrogel and acacia gum hydrogel in wound healing.

Preparation of Acacia gum hydrogel
Preparation of Acacia gum hydrogel according of technique by (8) with some modifications. The Acacia gum was grinded into a powder then digested by adding 20 mg of Acacia gum to 1 ml solution of 1 M of HCL acid and deionized water, then the solution was constant stirring for 24 h at room temperature for digestion of Acacia gum. Afterward pH was raised to 8 using NaOH .Added of phosphate buffered saline to lowering a pH-value of 7.4 (isotonic solution). The hydrogel was stored as neutralized samples with a pH 7.4 at 4 C°.

Isolation and culture of BM-MSCs
Six weeks old, adult male Wistar rats were sacrificed with overdose anesthesia (at 5 times the anesthetic dose) of ketamine and xylazine mixture according to animal euthanasia guidelines on the American Veterinary Medical Association (AVMA). Sterilization the animal by putting the whole body of animals in 70% alcohol. The femur and tibia were aseptically removed from body, and then loaded into sterile petri dish. Petri dish was placed under laminar flow hood, then the bones washed with phosphate buffer saline (PBS) and the cancellous bone was isolated from the femur and tibia by injection the cavity of the bone marrow with complete media to flushing bone marrow cells. The mixture plated in culture flask (T-25) and supplanted with 10 ml of Dulbecco's Modified Eagle's Medium (Sigma, USA). Examined under inverted microscope. The flask was labeled by cell type and date and then incubated in incubator (37 °C and 5% CO2) and the medium was changed every 3-4 days (after 4 days in first time) until cultures were reached 70% confluent (usually 5-14 days). For subculturing, BM-MSCs were detached with trypsin/EDTA, centrifuged, divided, and cultured.

Loading BM-MSCs in Acacia gum hydrogel
BM-MSCs at concentration of 3×10 6 were loaded in 0.5 ml Acacia gum hydrogel, and then incubated 6 h in a 5% CO2 humidified incubator at 37 °C.

Experimental Animal
Thirty-six rats were equally split into three groups The animals housed separately in laboratory cages and had free access to food (standard rodent pellet diet) and freely water access. Acclimatization the animals one-week before starting the experiment.
The animals were prepared for aseptic surgery and full thickness of circular skin circular incision of 1.5 cm in diameter was made in halfway between the imaginary line connecting the animal's shoulders according to (9). The group A, the wound defect was created by forceps and small size scissor and treated with normal saline and left without any treatment as group A. The group B, the wound was created and covered with 0.5 ml of acacia gum hydrogel. The group C, the wound was created and covered with 0.5 ml of BM-MSCs loaded in acacia gum hydrogel.

Pathological examinations A. Gross pathological study of wound healing
Gross pathological examinations included percentage ratio of wound contraction on day 3, 7, 14 and 21 after induced of wound. The percentage of wound healing was calculated according to (9) as calculating the distance and size of the wound by using numerical calipers as the following equation (9).

Microscopic evaluation of wound healing:
The histological evaluation done on day 3, 7, 14, and 21 of the surgery, the specimens of all the rats were collected from skin wound and washed with normal saline then the samples preserved in neutral buffered formalin (10%), processed and stained with hematoxylin and eosin stain (H&E). Five fields were examined and the lesions were examined for any under light microscopy (Olympus Japan) for pathological changes and the photo captured (Sony digital camera)

Statistical Analysis
The statistical analysis using analysis of variance of one-way ANOVA test (with Tukey test afterwards in SPSS statistical software (version 24). Probability at 0.05 level were used for statistical difference.

Results and Discussion
In present study, efforts have been made for providing promising tools in the wound regeneration depending on gross and histopathological evaluation of healing capability of BM-MSCs loaded in Acacia gum hydrogel and Acacia gum hydrogel on skin wound healing in rats. This study was the first study use the combined (BM-MSCs loaded in Acacia gum hydrogel).

Gross pathological study of wound healing
Experimental rats expressed good model in wound study as they tolerated surgery well and survived healthy during the study period. This result in consisted with other researchers that served the rats as a good model for skin wound regeneration study (2,3) In gross pathological study of wound healing and showed no difference in wound healing between all groups , these findings could be attributed to use small size wound in study. Gross pathological study and measurements of wounds at days 0, 3, 7, 14 and 21 post-wound presented in Figure 1 and Table 1. The wound appearance and calculations showed no difference in diameter at day 0 and days 3 in all groups. Meanwhile, at days 7 and 14 the contraction appeared highest in both treated groups compared to group A but these results are considered statistically non-significant. Complete wound closure in the group C at day 21 showed minimal scar formation when compared with other groups (A and B) which showed incomplete wound closure at day 21 although these results was considered statistically non-significant.
These results is in contrast with (10) that found the significant result in gross pathological appearance of mesenchymal stem cells treated groups. Furthermore, faster contraction of wound and complete close wound was mentioned in mesenchymal stem cells treated groups by (11,12). Karp and Teo (13) were also clarified the best wound healing results of local wound injection of mesenchymal stem cells.
Histopathological evaluation of healed wound area: Day 3 : The histology of the wound tissues on the days 3 after induced of wound showed same histological changes including infiltration inflammatory cells, granulation tissue formation with and blood vessels congestion and presence of angiogenesis and collagen distribution in all of experiment groups. Day 7: Group A and Group B at this time-showed sever inflammatory reaction meanwhile the group C showed less inflammatory cells and more collagen, fibroblast, and blood proliferating capillaries than the control group, Day 14: The histological study of group A showed necrosis, incomplete epithelialization of epidermis with high numbers of inflammatory cell, while group B and group C showed numerous of collagen distribution with increase of intensity and complete epithelia layer formation. Day 21: Group A showing presence of covering crust with filling of wound bed and presence of granulation tissue with epidermis disappearance of the skin growing and the surface of wound covering with epidermal regeneration and filling of wound bed with regenerated epidermis and increasing thickness of epidermis. Group C showed fully regeneration of the dermis and epidermis. The dermal regeneration with fully hair follicles formation and presence of regenerated sebaceous glands and absence of inflammatory cells.
Histopathological study of healed wound area show superior wound healing in stem cells group as dermal regeneration with fully hair follicles formation and presence of regenerated sebaceous glands and absence of inflammatory cells. Similar findings were seen is several studies that shown the efficiency of BM-MSCs for enhancing wound healing (10). The same advantages were obtained by (14) that he ensure the enhancement effect of mesenchymal stem cells in dermal regeneration in mice and regard good cells source for burns and wounds treatments. The uses Acacia gum to treat wound also done by (5,15) results demonstrate that topical application of Acacia gum hydrogel improvement the wound healing.

Conclusion
Mesenchymal Stem Cells loaded in Acacia gum hydrogel and Acacia gum hydrogel treated groups enhanced of wound healing in rats. the study reveal no significant difference in gross appearances but histopathological evaluation clearly showed that the BM-MSCs loaded in acacia gum hydrogel treated group superior than the control group or acacia gum hydrogel group.