The evaluation of the effects of Wharton gel mesenchymal stem cells with traditional medicine in the treatment of skin lesions caused by burns
The evaluation of the effects of Wharton gel mesenchymal stem cells with traditional medicine in the treatment of skin lesions caused by burns
hediyeh mohamedi,1,*Hamideh Abnoos,2
1. , Mashhad, Khorasan Razavi, Om al banin state model high school 2. Mashhad Khorasan Razavi biology teacher
Introduction: Burns are one of the most devastating injuries and major concerns of the World Health Organization (1, 2). It is the fourth most common injury after car accidents, falls and interpersonal violence. More than 300,000 people die from burns each year. Millions of people suffer from disabilities and social, psychological and economic disabilities caused by burns (4). Burns are injuries to the flesh or skin caused by heat, electricity, chemicals, friction, or radiation and can be associated with emotional and psychological disorders. In full-thickness burns or third-degree burns, all layers of the skin are damaged and the treatments required depend on the severity of the burn (5). It is also known as one of the main causes of skin tissue damage, which in addition to increasing vulnerability to the entry of foreign factors, causes dehydration and natural shape of the skin (7). In general, the burn repair process includes a set of cellular, molecular and tissue processes (8). In fact, wound healing is a four-step overlapping process involving the stages of coagulation, inflammation, fibroplasia, and tissue change. In the coagulation phase, diapause begins as a result of vasoconstriction and the release of catecholamines. In the inflammatory phase, the wound is occupied by phagocytic cells. In the fibroplasia phase, fibroblasts that have migrated to the wound synthesize and secrete different types of collagen, resulting in increased wound strength, and angiogenesis occurs at this stage. In the stage of changing the tissue arrangement, the tissue returns to its natural state (3). According to these studies, it seems that the use of new methods such as cell therapy as a non-invasive approach can be a good alternative to old methods of treatment. Cell therapy is an interesting and effective strategy in the treatment of diseases and includes the replacement of stem cells or tissue made from stem cells for various diseases and injuries. So far, various stem cells have been used in the treatment of diseases through various researches. Gone (6). Vinegar is an aqueous solution of acetic acid and other chemicals that may contain flavorings. Acetic acid is usually 5 to 20% of the volume of vinegar. Acetic acid is usually produced by the fermentation of ethanol or sugar by acetic acid bacteria. Natural vinegar has less tartaric acid and lemon essence. Ordinary vinegar, in addition to removing natural antibiotics, contains biological amino acids after fermentation, while synthetic vinegar does not contain this important substance. Natural vinegar regulates the body's metabolism because it secretes saliva secretion. Increases saliva secretion. According to biochemistry, it has a large amount of protein (albumin) that better digest and digest food. Mixing vinegar with saliva also protects teeth from decay. Vinegar is a body stimulant and is considered an antipyretic for some medications, such as atropine. Vinegar has long been considered a carcinogen, but is now considered to be partly a carcinogen. In a salt diet (salt loss and not eating it), it is good to eat vinegar because vinegar removes the salt in fruits. It stimulates and increases and excretes the body through sweating. The same goes for the meat diet, and it is not only suitable for people with liver disease. Vinegar is very useful for preventing obesity and weight loss as well as in anti-diabetic diet (33). It should be noted that white vinegar contains acetic acid (a component of aspirin), which can help relieve pain, itching and inflammation caused by burns, and as a disinfectant closes open pores of the skin. Prevents infection and inflammation at the site of the defect. (30). A stem cell is a cell with a high ability to divide that has not yet divided. Cells from mitotic division make more stem cells and can differentiate into other types of cells, and some, such as nerve cells, may lose the ability to divide during the process of differentiation. In mammals, there are two broad types of stem cells: embryonic stem cells and adult stem cells. In adult organisms, stem cells act as a repair system for the body and regenerate adult tissues. Today, the use of these cells to repair damaged tissues is expanding. Stem cells are divided into all (full), multipotent, multipotent and monopotent (26) based on their ability to make different tissues (26). The rate of angiogenesis and the development of blood vessels is actually one of the most effective factors in wound healing. It also prevents the burn wound from deepening. If an agent such as a drug can accelerate the formation of blood vessels through biochemical and pharmacological mechanisms and be able to alter tissue circulation in the burn area, it will repair (9). The use of stem cells in tissue regeneration programs due to The ease of replication and isolation in vitro and their potential ability to advance and accelerate the healing process of tissue damage through the production of multiple growth factors have received much attention (12). Today, umbilical cord Wharton jelly stem cells have been considered as a suitable cellular source. The reason for using these cells is easy access, low cost, lack of invasive methods in separating tissue from the donor, the ability to differentiate into different cells and abundant sources of these cells (10). The umbilical cord consists of two arteries and a umbilical vein, both of which are located in a special mucosal connective tissue called the Wharton jelly, which is covered by the amniotic epithelium. The umbilical cord of hospital waste is considered and access to these cells is a non-invasive method. In addition, access to umbilical cord mesenchymal stem cells is not cumbersome and has no moral problem. Umbilical cord mesenchymal stem cells have a separate capacity for self-renewal. , While retaining their plurality and high differentiation ability, although some of these differentiation abilities are known in part (11, 13). Umbilical cord mesenchymal stem cells have received a great deal of attention due to their immunomodulatory properties. Today, mesenchymal stem cells have been proposed as a suitable source for medical resuscitation and immunotherapy (14). In 2019, Nazempour et al. Used human umbilical cord Wharton jelly stem cells to treat a burn pattern in rats. (16). The amniotic membrane is the innermost layer adjacent to the fetal amniotic fluid, which today is mainly excreted in the country as a biological waste. It has established itself in restorative medicine with numerous companies around the world processing and mass-supplying this biomaterial. (28) Combining traditional Iranian medicine with modern therapies can be a very effective idea for treating various diseases. It should be noted that reports from the World Health Organization confirm that more than 80% of the world's population still uses traditional medicine and herbs to treat a variety of diseases. Today, in some parts of Iran, a combination of animal oil and coriander gum is used to treat burns. Pistoia Atlantic is one of the wild pistachio species in Iran that is grown by car in many parts of the country, especially in the Zagros region (15). In traditional Iranian medicine, this plant and its gum are used in many cases, including in the treatment of stomach problems, infections, eczema, asthma, and as an anti-inflammatory, anti-viral and anti-bacterial. Coriander gum contains compounds such as fatty acid compounds, transbernol and pinene, which have been studied in relation to their biological activities, especially antimicrobial (18, 17). Sheep animal oil is also rich in unsaturated fatty acids, which are traditionally used in some parts of Iran to repair burns (19). Researchers at Iran University of Medical Sciences studied the effect of animal oil on the healing of second-degree burn wounds in rats (37). It should be noted that potatoes are the first treatment in the list of traditional and home remedies for burns. Raw has soothing properties and prevents skin irritation. In this way, it can prevent the formation of unpleasant blisters. And mixed water, which is marketed as vanilla extract, which generally contains 35% alcohol. Vanilla extract may also contain sugar, corn syrup or propylene glycol. The most famous use of this plant in Iran is in confectionery and ice cream. The Tutankhamun Indians were the early makers of vanilla. Vanilla beans contain 1-2% vanillin (4-hydroxy-3-methoxybenzaldehyde). Most of the vanilla flavor is due to the presence of vanillin in it. Vanillic acid, p-hydroxybenzoic aldehyde, tannins, polyphenols, free amino acids and resin are other components of this plant (34, 35) Vanilla is the most expensive spice after saffron. Of course, vanilla extract or essential oil can be used for minor burns (30)
Methods: In one study, acrylic acid at different concentrations was bonded to the silicone film at different concentrations after activating the surface of the silicon film by two-step plasma method. Chitosan and gelatin were then fixed in different percentages on the samples. Surface properties were evaluated by Fourier transform infrared spectroscopy and contact angle measurement. Finally, adhesion, dispersion and number of L929 cells on the samples were studied (9).
But also a study to prove the effect of animal oil on burns: This study is an experimental type and was conducted in 2013 in the animal laboratory of Hazrat Fatemeh Hospital in Tehran. Thirty-six adult male Sprague-Dawley rats weighing approximately 250-300 g were selected and kept in accordance with the animal rights standard and the agenda of the Medical Ethics Committee on the use and care of laboratory animals at Iran University of Medical Sciences. Thus, they were kept in standard separate cages with a light cycle of 12 hours of darkness and 12 hours of light and a temperature of 22-24 ° C. During this time, the animals had sufficient access to water and food. 60 mg / kg), the hairs on the back of the rats were shaved with an electric shaver. Then, to create a deep second degree burn on the back of the rats, 2 cm 2 cm metal stamps placed in boiling water at 90 ° C for three minutes were placed in contact with the skin on the back of the animal for eight seconds. Rats were randomly divided into three groups. In the first group with a thick layer of ointment with a diameter of 2-3 mm, the second group with a layer of animal oil and the third group with Vaseline were treated daily. Every five days, the burn site was photographed near the ruler using a Nikon D300digital camera (Nikon Corporation, Tokyo, Japan) and a 60 mm macro lens with a magnification of 1:10 and a distance of 80 cm. After transferring the images to the computer, the size of the wound in each image was measured and morphologically calculated using ImageJ software, version 1.45 (National Institutes of Health, Bethesda, Maryland, USA). Pathology samples were taken from 30 of the restored area. Histopathological slides were examined using Hematoxylin and Eosin (H&E) stains to evaluate and determine the extent of epithelialization, collagen filament arrangement, acute and chronic inflammatory cells, vascularization, and local fibroplasia. At the end of the study (30 days), rats were exterminated as standard using a high dose of Nesdonal. The collected data were analyzed by SPSS software, version 16 (IBM SPSS, Armonk, NY, USA). The central index of mean and scattering of standard deviation was used to describe the quantitative data. Data distribution and their normality were measured. To analyze the data in the study groups, Post hoc analysis of variance, ANOVA and Kruskal-Wallis tests were used, p <0.05 was considered statistically significant (36).
Isolation and culture of mesenchymal stem cells from Wharton's umbilical cord jelly:
First, after obtaining and removing blood, the umbilical cord was immediately transferred to a cell culture laboratory in physiological serum containing antibiotic (penicillin / streptomycin 5%) and then washed with saline phosphate buffer (PBS) containing 1% amphotericin and 3% antibiotic. given. According to the methods mentioned in previous articles, cell isolation was performed (10), the umbilical cord was fragmented, blood vessels and fetal membranes were removed, and Wharton jelly was isolated and dissolved by tissue fragmentation. After isolation, the cells were transferred to a flask and cultured in an incubator at 37 ° C, 5% carbon dioxide and 95% moisture for several weeks until the third passage. The culture medium was changed every 3 days and the cells were examined daily under a microscope. The cells were then placed in a T25 flask (SPL, Korea) and in a DMEM culture medium (g I b co, USA) with 10% serum. FBS bovine embryos (GI bco, USA) and 1% strepsin antibiotic (GI b co, USA) were kept until the third passage, followed by filling the flask with a density of over 80% using terpsin / EDTA ( GI b co, USA, .25%) Passage and counted using a neobar slide and poured in equal numbers 160X1 in each microtype containing half (0.5) ml and subcutaneously using insulin syringe on the edges of fresh burn wounds Created injected (14)
Animal studies and modeling of burns:
In this study, which was performed on male Wistaran rats, 28 rats weighing 250-200 g were prepared from the animal house of Shahid Chamran University of Veterinary Medicine and after purchasing the animals, they were transferred to the animal house of the Faculty of Science and transferred to They were kept in standard conditions for a week to adapt to the new environment (access to water and food was unlimited and 12 hours of light and 12 hours of darkness). The boil was placed at 100 ° C and formed. First, the animals were anesthetized by intraperitoneal injection of a mixture of ketamine (80 mg / kg) and xylazine (10 mg / kg). The backs of the animals were shaved and disinfected with betadine. The hot stamp was placed on the body for 30 seconds. After induction of the burn model, rats were randomly divided into two groups of control and treatment. The rats of the control group were divided into three groups without using special treatment and the rats of the treatment group were divided into three groups: The second subcutaneous injection of stem cells around the burn area and the third group were treated with simultaneous cell injection and daily rubbing of the poultice. Animals were kept separately in standard cages with normal day and night conditions and unlimited access to water and food until the end of the treatment period (20, 21).
For microscopic studies, the animals underwent chloroform facilitation on day 30 and the skin of the scalpel-treated area was dissected in 2x2 dimensions. After preparation of tissue sections and general staining of hematoxylin-eosin and specific Trichrome-Mason imaging, light microscopy was performed for histological examinations using I mage J software. Results using SPSS software (Ver.16) and ANOVA and T uke y statistical tests were evaluated as Mean ± SEM). The tables were drawn in Word 2016 and the graphs were drawn in Excel 2016 software and differences with p <0.05 were considered significant.
Research has also been done on the use of the amniotic membrane. According to this research, the amniotic membrane of the fetus can be stored in certain conditions after freezing and drying to be used free of microbial contamination. With this measure, the amniotic membrane of the fetus can be stored for one to two years. (28) And normal saline (80 m / l). Placental blood samples were sent to the laboratory for HBS-AG, HIVAB and HCVAB tests. After separation from the chorion and purification, the amniotic membrane was placed in normal saline solution containing gentamicin (80 mg / l) and stored at 4 ° C. In case of negative test results, amniotic fluid was used for patients' biological dressing (29). In some articles, the treatment of burns with vinegar can be seen. First, a clean cotton pad or a piece of sterile gauze was placed in vinegar and placed directly on the burn site. In order to have a greater effect, they used a thin towel in a dilute vinegar solution as a soothing compress on the gas. [30] Or a sterile gauze was dipped in some of this essential oil and gently placed on the burned area of the skin to evaporate the essential oil to cool the burn and gradually heal. (30).
There are people who say that if we put flour on the burn for ten minutes, it will not even blister, but this method has not been recognized yet and many people consider this method dangerous. (31)In other studies, potatoes were also a very effective treatment for burns. In this method, raw potatoes were first cut into thin slices and then placed on the burn and sliced. They had to rub the potatoes on the burnt part, but they did it carefully so that no pressure was applied to the burn and they waited for the juice to be released all over the burnt part. (32)
Results: Evaluation of Wharton Jelly Mesenchymal Stem Cell Morphology:Morphology of Wharton jelly mesenchymal stem cells cultured using an inverted microscope was performed. 1))
Histomorphometric results:
Also, microscopic examination of samples with H&E staining on day 30 showed that the thickness of dermis and epidermis in treatment groups was significantly higher than the control group. Color changes in samples with Mason Trichrome staining showed that collagen synthesis in the samples of the treatment group started faster than the samples of the control group and the amount of collagen was higher. The intensity of the blue color of collagen, which indicates the amount of collagen formation, showed that the process of collagen formation in the treatment groups was clearly faster than the control group. The mean number of blood vessels and fibroblasts on day 40 after treatment showed a significant difference (P <0.05) in the treatment and control groups. The morphological and histological results in this study showed that among a sample of treatment after 30 days In group samples that received only cells, wound healing and closure occurred faster than in samples that received only poultices (Figures 2 and 3). There was no significant difference between the number of blood vessels and the thickness of the epidermis in these two groups, but the comparison of the number of fibroblasts, collagen formation and the thickness of the dermis in the cell receiving group was significantly higher than the group receiving the dressing. Studies also showed that the simultaneous treatment group (dressing + cell) was repaired much faster than the other two groups and quantitative data from histological studies showed a significant increase in fibroblast cell count, angiogenesis and collagen formation and increased dermis thickness in The treatment group was simultaneous compared to the other two groups (Tables 1 and 2)
Figure 1- Images obtained from morphological examination of human umbilical cord Wharton jelly mesenchymal stem cells
Figure 1 - Images obtained from the morphology of human umbilical cord Wharton jelly mesenchymal stem cells. Images were recorded using an inverted microscope at 20% magnification. The red star refers to the exit of mesenchymal cells from tissue fragments.
Figure 2. Morphological examination of dermis and epidermis of skin on day 30 using H&E staining using light microscope. A * and A. control sample, and B. sample treatment with poultice, C * and C. cell therapy sample, D * D. (cell therapy + poultice) Row A images are taken with a magnification of 4 ˟ and row B images are taken with a magnification of 40 (. In this figure, blue stars represent the epidermis and orange stars represent the dermis. And black arrows indicate blood vessels.
Figure 3 - Morphological study of collagen formation and density on day 30: Collagen density in the control group was significantly lower than the treatment groups. Collagen density was significantly higher in cell therapy + dressing group than in the two treatment groups. Collagen concentration was higher in the cell therapy group compared to the dressing group. A. control sample, B. poultice sample, C. cell therapy sample, D. cell therapy sample + poultice (images taken with 4˟ magnification). In this figure, blue stars represent the epidermis and orange stars represent the dermis.
Conclusion: The results of the present study showed that subcutaneous injection of Wharton jelly mesenchymal stem cells in the burn area accelerated the wound healing process and reduced the time required for complete wound healing. The exact mechanism of action of stem cells in wound healing is not fully understood, although according to studies so far, the effect of various drugs and cell therapy together has accelerated wound healing (17).
In general, the results of this study indicate that with the help of traditional medicine, a lot of valuable help has been given to the treatment of burns. For example, the substances mentioned above, such as vanilla essential oil, vinegar, potatoes, etc., although they help with burns, but the role of Wharton gel is more important. In the laboratory, the resulting cells were injected at the wound site and caused the wound to heal. In this study, the simultaneous use of coriander gum poultice and animal oil along with cell therapy had a significant effect on improving the healing process. It seems that the most important effect of coriander gum in improving the wound process can be due to its antimicrobial and anti-inflammatory properties. Also, although the mechanism of the effect of topical application of animal oil on wound healing is not clear, but it seems that the use of animal oils that contain large amounts of unsaturated fatty acids due to antimicrobial effects, reduce inflammation at the wound site and Acceleration of short-chain fat by providing essential fatty acids can increase cell proliferation in the skin and help form hypertrophic scars to repair skin damage and burns. In addition, scientific studies have reported that they inhibit the topical use of acids (22). Has been. In 2017, Bagheri et al. Used animal oil to treat a second-degree burn model in the burn model in Wistar rats and performed pathological and morphological studies on different days during the repair process. Contrary to expectations, however, their results showed that animal oil could not improve the fuel repair process, and they stated that this method did not improve the pathological monitoring criteria (24), although these differences could be due to differences in the type and quality of oil used. In the type of burn model creation and so on. The study also showed that concomitant use of coriander gum with animal oil compared with control improved type 2 burn wounds in rats, but their comparison with silver sulfadiazine ointment showed that the effect of the ointment was significantly greater than the simultaneous use of gum. Coriander has been used with animal oil (20) In another study, Zohour et al. Stated that animal oil can have a positive effect on the healing process of burn wounds in rabbits (23) Comparing the average thickness of dermis and epidermis in treatment groups The control group was significantly higher. Studies by Marchin et al. (2012) showed that the repair of the epidermis is faster and the dermis is slow, and it takes about 3-4 weeks for my body to fully heal (23). Collagen fibers in tissue sections related to the group As shown in the results, collagen fibers in tissue sections belong to groups The treatments are network-like and wavy. Although the presence of collagen was observed in the control group, but this accumulation of collagen was localized and heterogeneous and no wavy and network arrangement of collagen fibers was seen. Also, fibroblast cells are the most effective cells in wound healing. These cells are involved in the formation of healthy tissue by increasing the number of damaged areas and the formation of a matrix of collagen fibers (25). Tetamanti et al. (2004) used fibroblast counting to determine wound quality and connective tissue formation in leech wound healing (25). Introduced surgical wound healing. (7) Our overall conclusion from the above studies is that Fibroblast count was used as an indicator in the evaluation of healing tissue. In addition, if a factor can stimulate and intensify the formation of new blood vessels at the right time and cause blood flow to the area. It can be said that the healing process is successful and the wound is prevented from deepening. Angiogenesis is essential for wound nutrition and oxygen supply. The effect of mesenchymal cells, especially through the production of angiogenesis factors, is a reason for stimulating the formation of the largest number of vessels on day 30 in cell-treated groups. In general, the results of this study showed that the simultaneous use of new methods of treatment, ie cell therapy with traditional methods can be appropriate and effective in improving the healing process of burns. Of course, to prepare a poultice for gum and oil, these two substances must be completely combined and combined with one hand, and it is recommended that the oil be sterilized if possible. Of course, in the continuation of the researchers' research, biological dressing had no effect on preventing the occurrence of thrombophlebitis in patients, and certainly traditional medicine has a special role in the treatment of burns. Conveniently and in the shortest time and at the lowest cost using the above-mentioned ingredients such as vanilla (in the treatment of minor burns), potatoes, vinegar and animal oil, etc.
Deafness and appreciation to the good and patient teacher, Ms. abnoos, who provided the necessary instructions throughout the process.
Keywords: Cell Therapy, Burn, Stem Cells of the Wharton's Jelly, Coriander Tree Gum, Traditional Medicine