Investigation of the Synergist Effect Extract of Licorice and Elaeagnus Angustifolia on the Chondrogenesis of Human Adipose-derived Stem Cells on Fibrin Scaffold

Ali Rajabi Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Majid Pourentezarizarch (  m.pourentezari@gmail.com ) Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Maryam Yadegari Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Morteza Anvari Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Abulghasem Abbasi Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Hengameh Dourtaj Department of Tissue Engineering and Applied Cell Science, Shiraz University of Applied Medical Science and Technologies, Shiraz, Iran Fatemeh Zakizade Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Romina Kardan Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Masoumeh Mozzafari Kalbibaki Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran


Introduction
Renovation of cartilage tissue damage is a signi cant problem in orthopedics, but more recently, tissue engineering techniques can be an acceptable solution to untangle this example of a problem. (1)The purpose of tissue engineering is to create options that enable the recovery, maintenance, and function of damaged tissue. In tissue engineering, cells are taken from a person, and after culturing and increasing their number, they are planted on scaffolding and new cells are formed in a short time. (2) Scaffolding plays an essential role in tissue regeneration and repair. (3)Scaffold is a three-dimensional structure used to guide the cell to grow and is an alternative to the extracellular matrix. (1)Cells penetrate the scaffolds and begin to grow, differentiate, multiply, and migrate against the physical and chemical signals of their environment, and if environmental conditions are favorable, they secrete an extracellular matrix. (3) The ideal scaffold should have good porosity for food distribution and good mechanical stability. It also increases cell adhesion and cellular signaling so that cells maintain their natural phenotype. (3)Fibrin, an irresoluble protein that is constructed in reply to bloodshed and is the main ingredient of the blood clot. it is organized from brinogen, a solvable protein that is created by the liver and found in blood plasma. (4) Fibrin is a natural biopolymer with many interesting characteristics. It is biocompatible, easy to process, and well adapted to different polymerization conditions. (5) Licorice (Lic L) is one of the medicinal plants in Iran that is exported in signi cant quantities annually.
The main ingredient of this species is a combination of a triterpenoid saponin called glycyrrhizinic acid or glycyrrhizin, which is used in the pharmaceutical and food industries and has a sweetness of 30 to 50 times that of sucrose. (6) Licorice root contains various compounds such as various sugars, avonoids, sterols, amino acids, gums and starches, essential oils, and saponins. (7) The compounds of glycyrrhizin plant have anti-in ammatory and antioxidant properties that are used to treat stomach ulcers, rheumatoid arthritis, respiratory diseases, etc. (8) Elaeagnus Angustifolia fruit(of E. angustifolia L) contains signi cant amounts of avonoids, terpenoids, phytosterols, carvacrol, glucose, fructose, phenolic acid. In traditional Iranian medicine,(9) elm fruit has been introduced as an analgesic agent in patients with arthritis. (10) Recent pharmacological studies have also shown that this plant has antibacterial, antioxidant, and anti-in ammatory effects.(11) Different tissues in the body are made up of cells that can produce cells like themselves or adjacent cells, including cells of the heart and nervous system. There are cells in the body that have special capacities to differentiate and become specialized cells. These cells have two distinct characteristics: one is that they can become specialized cells in the body, and second, they can form their stem cells and those around them and remain unchanged during large divisions and long periods. (12) ADSC also retains its differentiation potential in cells of its origin and is usually characterized by high immunity and modulatory effects. Abundance, accessibility, ease of stem cell isolation as well as differentiation and proliferation of these cells have made this tissue known as an important source for obtaining stem cells. (13) Therefore, the International Federation of Fat Treatment (IFATS) adopted the name ADSC for greater uniformity. (14) Methods The method of separating the stem cells of fat and tissue passage(DMEM high glucose (Gibco) Penicillin-streptomycin , Insulin transferrin-selenious sodium 50 µg/ml Ascorbate-2 phosphate (Sigma) Dexamethaoson (Sigma) ,Linoleic acid (Sigma) . collagenase(sigma) Adipose tissue was removed from the patient's abdomen using liposuction and surgery and placed in a sterile container containing 1% antibiotics and culture medium at 4 ° C to prevent cell destruction. Using a razor, the extra blood vessel and connective tissue were removed from the fat and then 0.5 mg per ml of collagenase was added per gram of fat. And incubated for 45 to 60 minutes at 37 ° C to separate stem cells from adipose tissue. Centrifugation was performed for 15 minutes at 2200 RPM. The cells deposit at the end of the Falcon tube, and in the Falcon tube, there are three stages: a layer of adipose tissue and the middle phase of the environment and enzyme, and the third stage is the stem cell. The asks contain DMEM. , Penicillin and streptomycin. The head of the cow embryo was transferred. It was placed in an incubator with a pressure of 5% carbon dioxide and a humidity of 95%. After 48 hours, the cells that were free and did not adhere to the end of the ask were removed by changing the culture medium. ( gure 1). (15) 1) Cut the fat into small pieces. 2) Add 0.5 mg of collagenase 2 per gram of fat. 3) Place in the incubator for 60 to 90 minutes. 4) Add the same amount of FBS. 5) for 15 minutes at 2200 RPM in a centrifuge. 6) Hold the third phase and remove the rest of the contents of the Falcon tube. 7) Add 6 ml of culture medium. 8)Transfer the cells to the ask. 9) Place in the incubator.

Expansion
After the density of cultured cells in the ask reached above 80-90%, the whole-cell culture medium was drained and washed with 5 ml PBS to remove proteins and disturbing cell components. Then PBS was removed and 2 ml of trypsin / EDTA was added to it and placed in an incubator for 2-3 minutes. Using neo bar slide and trypan blue stain, we counted the number of cells and divided them in a ratio of 1: 3 to be used for the next passage. (16) differentiation of adipose stem cells into cartilage cells:DMEM(Contains 4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate. The cultured mesenchymal stem cells were isolated from the bottom of the culture ask using trypsin and cultured in a ready-made brin scaffold. Cells in certain groups containing compounds (the rst group of of Lic L and the second group of of E. angustifolia L and the third group of of E. angustifolia L and Lic L simultaneously and four control group(medium chondrogenic)) and DMEM with insulin and 1% FBS and penicillin and streptomycin ascorbate 2 phosphate store 50 micrograms per liter They were incubated for 14 days at 37 ° C and 5% carbon dioxide pressure and we change the culture medium every three days.
Fibrin scaffold design: The new plasma was prepared in coordination with the blood ward of Shahid Sadoughi Hospital in Yazd. Fresh plasma was mixed with calcium gluconate in a ratio of 5 to 3 and incubated at 37 ° C for 60 to 90 minutes. It was then centrifuged at 2200 rpm for 10 minutes. After doing this, the contents of the Falcon tube form two separate phases, the transparent thrombin layer at the top, which is used by us to make hydrogel scaffolds. (1) Extraction and Puri cation of RNA The use of RNA XPLUS is a common method in most laboratories in Iran. The basis of this method is based on guanidine thiocyanate and phenol-chloroform.
Turn on the centrifuge and place it at 4 ° C.2-Prepare microtube 2 containing 1000 λ RNA XPLUS.3-Prepare the razor and slide and put the ice bucket under the hood.4-Pour the cell and scaffold into RNX PLUS and mix for 15 seconds. Leave at room temperature for 5 minutes.5-Then vertex the microtube containing the homogenized cell and scaffold for 15 seconds and place it for 5 min at room temperature.6-Add chloroform with 200 λ with the yellow head sampler and make 15 s stylish by hand.7-Incubate on ice for 5min.8-Then place in a centrifuge at 12000 rpm at 4 ° C and Rpm for 15 min.9-150λ three to 50 λ with the yellow head sampler, remove from the upper phase and transfer to microtube 1.5.10-Add the same fuzzy volume that we took, 150 λ isopropanol, mix 5 s, and put 15 min on ice.11-Place for 15 minutes in a centrifuge at 12,000 rpm and 4 ° C. At this stage, we make 75% alcohol for To make 1 cc of 75% ethanol, we use 750 λ alcohol + 250 λ sterile distilled water and turn on the heater, and put it at 60 degrees Celsius.12-Discard all supernatants (total microtube content) and add 1 cc or λ 1000% 75% alcohol made in the previous step to the microtube and place in a centrifuge at 7500 rpm and 4 ° C for 8min.13-Remove the entire contents of the microtube and place it on sterile gas for 6 minutes.14-Add 35 λ DEPCwater.15-Put the heater at the set temperature for 10 minutes.16-We put 2 λ RNA in the spectrometry .

Results
Evaluation of cell viability using trypan blue staining Examination of cell viability revealed that all groups were signi cantly different from the control group, but the group in which and Lic L was used was not signi cantly different from other groups. ( Figure 3) Evaluation of the results of gene expression through RT-PCR technique: Examination of chondrocyte-speci c genes in different groups showed that after 14 days, the cells were in the path of chondrogenesis induction. Gene expression in different groups was compared with the control group.
Investigation expression of gene: Examination of gene expression, in general, showed that in the speci ed period, the presence of E. angustifolia L and Lic L affected gene expression (P-value ≤0.05). The expression level of SOX9 gene was signi cantly higher in the group that received the extracts at the same time than the other groups was 28 times higher than the control group Also, the extracts alone were effective in gene expression. Agrecan gene expression was 17.3 times higher in the group that used the combination of licorice extract and Elaeagnus angustifolia extract than in the control group. The expression level of type II collagen gene in a given period was signi cantly higher in the group that received Elaeagnus Angustifolia fruit extract and licorice than other groups. The expression of type X collagen gene in the combination group of licorice extract and Elaeagnus Angustifolia was 4.8 times higher than the control group. ( Figure 5)

Discussion
Hyaline cartilage tissue lacks any blood supply and therefore the associated lesions are almost di cult to repair; On the other hand, the treatment method that is commonly used to repair these lesions is not effective. From this, tissue engineering and restorative medicine have expanded as an alternative method in recovering lost tissues in the last decade.Mesenchymal stem cells are considered an ideal source for use in tissue engineering due to their ability to proliferate and differentiate into several cell lines. Since normal articular cartilage typically has low cell density and does not repair spontaneously, (17,18), su cient cells with high bioavailability must be transplanted in the area of cell lesions. ADSC is considered an ideal source due to its easy availability and the number of stem cells obtained each time adipose tissue is extracted. In this study, it was used as a cellular source to prepare cartilage tissue.One of the important factors and features in cartilage tissue engineering is the preparation of scaffolds that can mimic or resemble the extracellular matrix and connect well with the host tissue. (19,20) Studies have shown that such a density of cells that interact with each other should use three-dimensional culture media (scaffolds) through which to achieve a high density of cells in an environment. In addition, scaffolds are effective in increasing the biological function of cells and producing an extracellular matrix (129). So far, extensive studies have been used on the use of a wide range of biomaterials as scaffolds, each with its advantages and disadvantages. In general, there are two types of scaffolding: natural scaffolding such as brin, collagen, alginate, hyaluronic acid ... and synthetic scaffolding such as polylactide acid, polyethylene glycol, co-polymer polylactide glycolic acid.
Fibrin scaffolds can be used as effective scaffolds because of their ability to help cells differentiate and proliferate, but the main problem with these scaffolds is their poor stability. In the study, brin hydrogel was effective in cell survival, proliferation, and differentiation. Although brin gel has been introduced as a sponge, it can be used as a three-dimensional platform for cartilage repair. Hamidpour et. al(2017) intending to investigate Elm (ElaeagnusAngustifolia L.) from a variety of traditional drug programs to its new roles as active antioxidants, anti-in ammatory, anti-mutant, and analgesic, as well as In wound healing, the ndings of the study indicate that after the use of E. Angustifolia L. fruit extract in the wound area, the content of hydroxyproline, an important factor in collagen production, increased in the tissue. This is in line with our research that using licorice and elm extract simultaneously and separately, the expression of collagen genes has increased compared to the control group, which did not nd any effective substances. (21) SoheilaHamidpour and Ra Hamidpour et. al(2019) with the aim of chemistry, pharmacy, and medicine of Russian olive or ElaeagnusAngustifolia L, it has been determined that the compounds of sesame extract or olive Russian cause the production of proline and hydroxyproline, which are the main components of collagen, which improves wounds and anti-in ammatory effects. Which is observed simultaneously or separately with the results of real-time tests related to licorice and elm extracts.
According to the study, these extracts cause the expression of collagen genes. ( It has antioxidant properties and anti-in ammatory functions. The study found that glycyrrhizin reverses the degradation of aggregates and collagen II and improves their production. In summary, all results suggest that glycyrrhizin may be a potential treatment for OA. . According to our real-time test data, which shows the expression of collagen and if genes more than the control group, we can say that licorice can be an effective factor in the expression of cartilage genes.(29)

Conclusion
Finally, we can conclude that the simultaneous use of both extracts improves the expression of cartilagespeci c genes and, to a very small extent, promotes cells to hypertrophy, which we can also improve by changing the doses used.The viability of cells during concomitant use of the extract was higher than other groups, which can be concluded that the use of the extract with brin scaffold was effective on cell survival.      Quantitative Expression Status of the Agg, Sox 9, Coll2, Coll10 Genes Analyzed by Real-Time PCR Figure 5