Overexpression of miR-192 in Fibroblasts accelerates wound healing in diabetic rats

Overexpression of miR-192 in Fibroblasts accelerates wound healing in diabetic rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Overexpression of miR-192 in Fibroblasts accelerates wound healing in diabetic rats Forouzan Karam, Mahtab Sayadi, Saeedeh Dadi, Gholamreza Anani Sarab This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5290142/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Diabetic Foot Ulcer (DFU) is a severe diabetic complication.Transplantation of skin substitutes, stem cells, and Platelet-Rich Plasma (PRP) treatments are promising tools to promote ulcer healing in diabetes. An important aspect of the remodelling phase of wound healing is collagen deposition. miR-192 increases the expression of COL1A2 by specifically targeting Smad-interacting protein 1 (SIP1). This study was designed to investigate the impact of combined treatment with platelet-rich plasma and fibroblast cells expressing miR-192 on the healing process of wounds using an experimental diabetic animal model. Methods: After transfection of HDF cells and induction of increased miR-192 expression, relative changes in COL1A2 gene expression were determined by the RT-PCR method. Rats were randomly divided into 6 groups: non-diabetic control group, diabetic control, backbone, PRP, miR-192, and PRP+miR-192 groups. Diabetes was induced in male Wistar rats of all treated groups except non-diabetic control through a 21-day high-fat diet and an intraperitoneal injection of 40 mg/kg streptozotocin. A 10mm skin biopsy punch was used to create two full-thickness wounds on the dorsal part of the upper body in all six groups of animals. Hematoxylin-Eosin and Mason's trichrome staining were used to evaluate the wounds and analyze histological changes. Results: The overexpression of miR-192 in HDF cells resulted in a significant increase in COL1A2 gene expression, which was 15.77-fold higher than the control group. PRP and pLenti-III-miR-192-GFP-expressing cells significantly increased wound closure rates, granulation tissue area, and collagen fiber density in rats, according to a histological examination. Conclusion: The combined use of PRP and HDFs expressing pLenti-III-miR-192-GFP speeds up the healing of wounds by increasing collagen expression, demonstrating the efficacy of this approach in improving wound healing results. Cell & Tissue Engineering Hematology Pathology Diabetes Diabetic Foot Ulcer Fibroblast miR-192 Platelet-Rich Plasma wound healing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Background Diabetes mellitus (DM) is a widespread health condition impacting approximately 9.3% of the global population [ 1 ]. Diabetic foot ulcer (DFU) is a prevalent chronic complication of diabetes with significant medical, economic, and social impacts. research suggests that 15–25% of diabetic individuals are at risk of developing foot ulcers during their lifetime [ 2 ]. The stages of wound healing include homeostasis, which occurs immediately after wound formation, inflammation, which occurs from 10–15 minutes to 3 days after injury, proliferation, which occurs from 4 to 21 days after injury, and regeneration, which occurs from 21 days to a year [ 3 , 4 ]. Fibroblasts secrete growth factors, collagen, and other elements of the Extracellular Matrix (ECM), which are vital for wound healing. They release Platelet-Derived Growth Factor (PDGF), Fibroblast Growth Factors (FGF), and Transforming Growth Factor (TGF) to promote cell division, activity, or differentiation. [ 5 – 8 ]. Platelet-Rich Plasma (PRP) is considered a natural growth factor that is safe to use in enhancing the healing rate of wounds. It is particularly effective in treating chronic wounds associated with diabetes that require prompt repair to prevent infections. One clinical application is the use of PRP gel for chronic ulcers[ 9 – 11 ]. PRP is a product of blood plasma with a high platelet concentration that contains numerous growth factors and cytokines, such as platelet-derived growth factor (PDGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), and insulin-like growth factor[ 12 ]. More importantly, PRP can enhance the proliferation and migration of dermal fibroblasts, indicating that it may synergistically heal chronic wounds[ 13 ]. MicroRNAs (miRNAs) are small RNA molecules that are approximately 19 to 22 nucleotides in length. They regulate gene expression at the post-transcriptional level by binding to the untranslated 3' regions (UTR) of target messenger RNA (mRNA) molecules[ 14 ]. Multiple miRNAs have been identified in skin tissue, and they are believed to participate in various biological processes, including the regulation of wound healing [ 15 – 17 ]. The expression of miR-29a has been found to have a direct impact on collagen expression, whereas miR-192, miR-29b, and miR-29c are significantly upregulated during this process [ 18 – 21 ]. miR-192 promotes the expression of COL1A1 by targeting Smad-Interacting Protein 1 (SIP1) [ 22 ]. While current treatments for wound care have made significant progress, the replacement of lost skin remains a major challenge in the field of regenerative medicine. Despite several available treatment methods for wound healing, a treatment approach that accelerates the healing process would be highly valuable[ 23 ]. To our knowledge, relevant research on the combination of fibroblasts with overexpression of miR-192 and PRP for the treatment of wound healing is very limited. This research investigated the impact of co-treatment with fibroblasts expressing miR-192 and Platelet-Rich Plasma in the healing of wounds in a diabetic rat model. Materials and Methods Cell lines and culture Primary human dermal fibroblasts (obtained from the laboratory of Dr. Mohsen Khorashadizadeh) were cultured in DMEM (BIO-IDEA, Tehran, Iran) with 10% heat-inactivated Fetal Bovine Serum (FBS), 1% penicillin and streptomycin and incubated at 37°C in a 95% humidified incubator with 5% CO2. Plasmids construct and extraction The pLenti-III-pre-miR192-GFP(Green Fluorescent Protein) expression vector construct and pLenti-III-Backbone-GFP (mock) were purchased from ABM Inc (Applied Biological Materials, Richmond, BC, Canada). E.Coli Stbl4 strain harboring the vectors was cultured in Luria-Bertani (LB) broth medium with 50 µg/mL kanamycin. The plasmid was extracted with the Karmania Pars Gene plasmid extraction kit (Karmania Pars Gene, Kerman, Iran). Transient transfection For each well, 2×10^5 cells were seeded in a 6-well cell culture plate. Separately, 3 µg of plasmids and 4 µl of PEI in 100 µl of DMEM medium were dissolved in two 1.5 ml Microtubes, the mixture was added dropwise to the cells, 6 hours later medium was replaced with fresh medium. Confirmation of GFP expression by flow cytometry and Fluorescence microscope Forty-eight hours after the transfection, the expression of the labeled GFP protein in the cells was verified by trypsinizing the cells and analyzing them using a flow cytometry system. The cells were first diluted in PBS buffer at 3×10 4 cells per tube and homogenized using a vortex. The samples were then analyzed using the FL1 channel of the flow cytometry (CYFLOW CUBE 8, Sysmex, Germany). Additionally, to evaluate the expression of GFP, fluorescence microscopy (Olympus BX41TF, Japan) analysis was performed on transfected human dermal fibroblasts. RNA extraction Forty-eight hours post-transfection, total RNA was extracted from HDF cells using RNX-plus reagent (Sinaclon, Tehran, Iran) according to the manufacturer protocol. The extracted RNAs were then quantified using Nanodrop spectrophotometry (BioTek Epoch microplate spectrophotometer, USA) by measuring the absorbance at 260/280 and 260/230 nm wavelengths, Samples were kept at -80ᵒ C until use. cDNA synthesis and stem-loop qPCR of miR-192 Stem-loop primers and the miR-192-5P specific primer used was 5′- CTCTGACTTATGAATTGAC-3′ (forward) and reverse primers were designed to synthesize the cDNA of miR-192 according to the BON Stem High Sensitivity microRNA 1st Strand cDNA Synthesis kit protocol (Stem Cell Technology Research Center, Tehran, Iran) for qPCR, RT-qPCR was performed using StepOne™ Real-Time PCR System (StepOne™ Real-Time PCR System, USA) under the following conditions: 95°C for 2 minutes, and 60°C for 30 Seconds for up to 40 cycles. U6 primer used was 5́- AAGGATGACACGCAAAT-3́ (forward) and was utilized as an internal control to normalize the RNA input. The relative expression of miR-192 was evaluated using the ∆∆CT method, the experiment was performed in triplicate (n = 3). RT-qPCR for Collagen 1A2 gene expression The extracted RNA was reverse transcribed into cDNA using the Easy cDNA Synthesis Parstous Kit from Parstous, Mashhad, Iran. The qRT- PCR reactions were carried out using RealQ plus 2x Master Mix Green (AMPLIQON, Denmark) The experiment was conducted on a Real-time PCR System (StepOne™ Real-Time PCR System, USA) under the following conditions: 95°C for 2 minutes, and 61°C for 30 Seconds for up to 40 cycles. The primers used to target the COL1A2 gene were 5′GAGGGCAACAGCAGGTTCACTTA-3′ (forward) and 5′-TCAGCACCACCGATGTCCAA-3′ (reverse) (Table 1 ), GAPDH was used as an internal control gene. The ∆∆CT method was used for calculation. (n = 3) Table 1 Primers used in this study. Oligo Name Sequence 5́-3́ BC EC GC% MW (Da) *TM (℃) OD 260nm Product length Oligo ID/ metabion international AG Forward Primer COL1A2 TCT CTA CTG GCG AAA CCT GTA 21 197,0 47.62 6.381 59 3,5 98 220823B003H05 1/2 Reverse Primer COL1A2 TCC TAG CCA GAC GTG TTT CTT 21 189,0 47.62 6.363 59 4,3 98 220823B003A06 2/2 PRP Preparation PRP was prepared from 5 blood donors. Blood with sodium citrate anticoagulant (50 ml) was prepared and centrifuged at 1200 rpm for 10 minutes, the supernatant was separated and re-centrifuged at 3000 rpm for 10 minutes, the platelet precipitation was resuspended in the plasma. Experimental animals and study design 8-10-week-old male wistar rats were obtained from the Pasteur Institute of Iran (Tehran, Iran) for this study. Animals were kept in standard cages with four rats per cage. the rats were kept at a controlled room temperature of 22 ± 2°C and 60 ± 5% humidity. Animals were exposed to a 12-hour light-dark cycle and standard food and water. Rats weighing about 180–200 g were randomly divided into six groups with eight rats in each group: Group 1; wound model in non-diabetic rats, group 2; wound model in diabetic rats, group 3; wound model in diabetic rats treated with 100 µl of PRP, group 4; wound model in diabetic rats treated with 6×10 4 HDF expressing pLenti-III-Backbone-GFP suspended in 100 µl of DMEM medium, group 5; wound model in diabetic rats treated with 6×10 4 HDF expressing pLenti-III-miR-192-GFP suspended in 100 µl of DMEM medium, group 6; wound model in diabetic rats + 100 µl of PRP + 6×10 4 HDF expressing pLenti-III-miR-192-GFP. Induction of type 2 diabetes in rats The experiments were performed following institutional guidelines for animal care and approved by the local ethics committee (IR.BUMS.REC.1400.413). Type 2 diabetes was induced in rats using the fat-fed streptozotocin ( ZellBio GmbH, Berlin, Germany) (STZ) model mouse protocol. The rats were placed on a high-fat diet for three weeks, providing 60% of their caloric value as fat[ 24 ], on day 22, all rats were fasted for 8 h before STZ injection, and fasting blood glucose was measured, 40 mg of STZ was weighed, transferred to a 1.5 ml microcentrifuge tube and covered with aluminum foil, Immediately before injection, Citrate buffer was prepared, STZ was dissolved in 50 mM sodium citrate buffer at pH 4.5 to a final concentration of 40 mg/ml, administered within 5 minutes after dissolution, using a 1 ml syringe and 23-G needle, STZ was injected intraperitoneally (i.p.) into the test group at a dose of 40 mg/kg of body weight (1.0 ml/kg). An equal volume of citrate buffer with pH 4.5 was injected i.p. into the control animals. For the diabetic groups, the rats received high-fat food, whereas the control group had a typical diet. Ten days following STZ delivery, blood glucose levels were assessed using the Infopia EasyGluco (Autocoding blood glucose metre EasyGlucoTM, South Korea) in a tail vein blood sample. Blood glucose levels exceeding 15 mmol/L (270 mg/dL) indicated that STZ-treated rats had successfully induced type 2 diabetes[ 24 ]. Experimental Induced Wounds and treatments Rats were anesthetized intraperitoneally using ketamine (Alphasan, Woerden, Holland) and xylazine (KELA.N.V, Belgie), and then the dorsal aspect of the upper part of the body was shaved and disinfected with 70% alcohol. A 10 mm skin biopsy punch was induced to create full-thickness wounds under aseptic conditions, treatments were performed in each group according to the previously mentioned protocol, in the form of subcutaneous injections in 5 points according to the Fig. 1 . Animals were sacrificed on the 3rd, 7th, 14th and 21st days after wounding. Macroscopic Observation Wounds were measured using a digital caliper and photographed on the day of wounding and subsequently on alternate days until healing was complete. Changes in wound areas were calculated at each time point to monitor the rate of wound contraction, the percentage reduction in wound size was calculated using the following equation[ 25 ]: % wound healing = \(\:\:\frac{\text{W}\text{o}\text{u}\text{n}\text{d}\:\text{a}\text{r}\text{e}\text{a}\:\text{d}\text{a}\text{y}\:0-\:\text{w}\text{o}\text{u}\text{n}\text{d}\:\text{a}\text{r}\text{e}\text{a}\:\text{d}\text{a}\text{y}\text{s}\:(3/7\:/14/\:21)\times\:100}{\text{w}\text{o}\text{u}\text{n}\text{d}\:\text{a}\text{r}\text{e}\text{a}\:\text{d}\text{a}\text{y}\:0}\) Haematoxylin and Eosin and Masson’s Trichrome Staining The wound area was removed using a sharp sterile scalpel. Then the cut tissue was fixed in a 10% formalin solution to preserve its structure and prevent destruction. The tissue was dehydrated through a series of alcohol solutions with increasing concentration. The tissue was cleared using xylene, tissue infiltration and embedding were performed using paraffin wax or other embedding materials to support the tissue during sectioning, then the histological sections were cut using a microtome and placed on glass slides, slides were stained with hematoxylin, eosin, and Masson's trichrome to observe collagen deposition and other histological features. An optical microscope (Microscope camera ODC series, England) was used to observe and photograph the slides, and all histological examinations were performed by two pathologists blindly. Statistical analysis In this study, Fiji/ImageJ 2.9.0 software was used for quantitative analysis of the density of collagen fibers, the number of fibroblast cells, and the amount of granulation during the different stages of wound healing. The data obtained from the Fiji/ImageJ software was analyzed and the results were presented using graphs and charts drawn with GraphPad Prism 9 software. The statistical significance of the data was determined using the two-way ANOVA and t-student test, and a significance level of p value less than 0.05 was considered in all experiments. Results Expression of GFP-tag and observation of green color as confirmation for transfection of expression plasmids in human Dermal fibroblast cells The transfection of HDF cells with pLenti-III plasmids was successful with a 20–25% transaction rate as estimated after 48 hours of transfection. The confirmation of the transfection was done using a fluorescence microscope, and the results were presented in Fig. 2 . The expression level of miR-192 was analyzed by flow cytometry using the FL1 channel compared to fresh and untransfected cells, the results of this analysis were presented in Fig. 3 of the study. Expression of miR-192 in transfected fibroblast cells increased COL1A2 gene expression The results of this analysis showed a significant increase (3-fold) in miR-192 expression in the fibroblast cells transfected with the miR-192 expressing plasmid compared to the cells transfected with the backbone expressing plasmid after 48 hours, as shown in Fig. 4 , this confirmed the successful transfection of the cells and the overexpression of miR-192 in the transfected cells. The study used RT-qPCR to test if miR-192 increased in fibroblast cells would change COL1A2 expression, Results showed a 15.77-fold increase in COL1A2 expression in transfected cells compared to the control group after 48 hours (Fig. 5 (, suggests miR-192 positively regulates COL1A2 expression and a potential mechanism for its effects on wound healing. Simultaneous treatment with HDF cells expressing miR-192 and PRP caused more wound closure than other groups. On the 3rd day, a significant difference was seen among the group receiving HDFs expressing pLenti-III-Backbone-GFP and the group treated with HDFs expressing pLenti-III-miR-192-GFP (P < 0 /0001) .There was a significant difference between the group that received PRP and HDFs expressing pLenti-III-miR-192-GFP and other treatment groups P < 0.0001 and more wound closure have occurred (Fig. 6 ). On the seventh day after wound formation, the wounds were examined macroscopically, and measurements were taken and recorded with a digital calliper. On the seventh day, the wound healing process accelerated in all intervention groups compared to the third day, and the difference between the groups became more obvious. According to the order of the groups in Fig. 7 , the average percentage of wound closure in various intervention groups versus the control groups is as follows: 68/33, 60/67, 66/83, 83/00, 91/53, and 89/03 percent. A significant difference of P = 0.0001 was seen in the healthy control group and the miR-192-expressing, PRP-group, and combined treatment groups, and these groups healed wounds quicker than the healthy control group. On the seventh day, the diabetic control group that did not receive treatment experienced a delay in the healing of the wound compared to the other groups. The difference between the rates of wound closure in the treated groups compared to the diabetic control group was almost completely different in the treated groups. Most of the wound area is closed, especially in the fifth and sixth groups, where it ranges from 80 to 90%, with diabetic wounds being close to 60%. In comparison to the group that only received fibroblast cells expressing pLenti-III-miR-192-GFP, more wounds closed in the combination therapy group (P = 0.0001). (Fig. 7 ) On the fourteenth day, there was a significant difference between the HDF groups expressing pLenti-III-Backbone-GFP and pLenti-III-miR-192-GFP plasmids (P < 0.0001), and more wound closure was observed in the HDF group expressing pLenti-III-miR-192-GFP. Between the PRP-treated group and the HDF-expressed group, there was a smaller difference (P < 0.05). The group that received the combination therapy with the groups treated with PRP and fibroblasts experiencing pLenti-III-miR-192-GFP alone is significant, with P < 0.0001 and NS (Not significant) respectively. On the 21st day after wound formation, healing occurred in almost all intervention groups, but the diabetic group without treatment has the lowest average percentage of wound closure (88.48%) compared to other groups, the group that simultaneously received PRP and fibroblast cells expressing pLenti-III-miR-192-GFP showed significant differences with each group that received the mentioned treatments alone, the group that was treated with combined treatment, the rate of wound closure and scars left from healing was lower than in other groups. (Fig. 8 ). The macroscopic views of the wound on different days are shown in Fig. 9 . The number of fibroblast cells in the group that simultaneously received HDF cells expressing miR-192 and PRP was higher than in the other groups. On the 14th and 21st days following the development of a wound, the average number of fibroblast cells was counted in the dermis of the wound area in various intervention groups. Figure 10 shows that the group that received the combined treatment had more fibroblast cells than the other groups. The fact that there was a noticeable difference between this group and the groups which received each treatment separately could mean that the injected fibroblast cells in the wound area were functional and alive. The density of collagen fibers in the group treated with PRP and HDF cells expressing miR-192 increased compared to other groups The density of collagen fibers in the group treated with PRP and HDF cells expressing miR-192 increased compared to other groups the group that got the combination therapy and HDF expressing pLenti-III-miR-192-GFP had a higher collagen fiber density on day 14 than the other groups, and there was a significant difference between these two groups and the PRP-treated group. A notable difference was observed between the groups that got HDF containing pLenti-III-miR-192-GFP plasmid and the backbone group on the 21st day following injury, additionally compared to each of the treatment groups separately, the combined treatment group's collagen fiber density increased, however, this rise was not statistically significant (Fig. 11 ). Figure 12 shows Masson-trichrome staining of the wound area in different groups on the 14th day after wound induction. Combined treatment with PRP and HDF cells expressing miR-192 increased the area of granulation tissue and wound healing . On the seventh day, the group treated at the same time with PRP and HDF expressing pLenti-III-miR-192-GFP, the average area of the granulation tissue is 502188 µm square and more than other groups, and there is a critical difference between this group and the two groups treated with PRP and HDF containing pLenti-III-miR-192-GFP expression plasmids alone was seen P < 0.0001. On the fourteenth day, a noteworthy distinction was observed between the group that received the combined treatment and the group that was treated with a PRP P < 0.05. But there was no difference between the combined group and the group that had fibroblasts containing pLenti-III-miR-192-GFP. There was no noteworthy difference between the PRP group and the group treated with HDF expressing pLenti-III-miR-192-GFP. But in all the groups that received treatment, compared to the healthy and diabetic control groups, there was a critical distinction, and the area of the granulation tissue was greater within the intervention groups than within the control groups. (Figure 13) . The area of granulation tissue can be seen in hematoxylin-eosin staining in Figure 14 . Discussion Present-day medications for wound care are not completely successful and chronic wounds present a challenge within regenerative medicine. Gene therapy and genetic engineering are new approaches that appear to have helpful potential for treating persistent wounds. Bioactive particles such as DNA, mRNA, siRNA, and miRNA may offer incredible clinical advantages, especially in regulating complex genetic systems and cellular signaling cascades related to skin repair. Changed expression of miRNA has appeared to play a central part in adjusting protein expression, and miRNA-based interventions may offer a wide run of targets that can be directed by a single miRNA [ 23 , 26 ]. In some studies, human skin fibroblast cells have been proposed as a capable helpful instrument for skin repair in wounds whose healing is delayed [ 27 – 29 ]. The present study used a genetic engineering approach to create more efficient fibroblast cells with increased expression of miR-192 and COL1A2. These modified cells were used in combination with Platelet-Rich Plasma (PRP) for wound healing in an experimental model of diabetic rats. The results showed that increasing the expression of miR-192 in fibroblast cells resulted in increased expression of collagen, and the use of these modified cells locally in the wound area showed beneficial effects in accelerating the healing process of chronic wounds in the experimental model. Moreover, the combination treatment of PRP and fibroblast cells expressing miR-192 was found to be effective in promoting wound healing, suggesting a potential therapeutic application for this approach in the treatment of chronic wounds. Li et al. [ 30 ] showed that in hypertrophic scars, increasing the expression of miR-192 by direct suppression of SIP1 protein within the TGF-/Smad2-3 signaling pathway enhances the expression of type 1 and 3 collagen and α-SMA . According to Fang et al. [ 31 ] a cytoplasmic protein named SIP1 inhibits the phosphorylation of Smad2/3 and prevents the nuclear Smad from binding to the collagen gene promoter within the TGF-β/Smad2-3 signaling pathway and finally decreased the expression of collagen protein. In our study, HDF cells transfected with pLenti-III-miR-192-GFP expression plasmids demonstrate significantly increased in the level of COL1A2 expression compared to the group of HDF cells with pLenti-III-Backbone-GFP expression plasmids. Increasing the content of collagen peptides in skin fibroblast cells in laboratory conditions increases the proliferation of fibroblast cells and the content of fibroblast-derived cell matrix. [ 30 ], also the use of peptides derived from human COL1A2 in laboratory conditions leads to an increase in collagen levels and cell migration and the amount of elastin in skin fibroblast cells [ 31 ]. In the present study, the results showed that the use of combined treatment of PRP cells and HDF cells expressing miR-192 supported the wound healing process and faster wound closure compared to both PRP and HDF cells expressing miR-192 alone, in addition, the group treated with fibroblasts expressing miR-192 showed a more critical increase in wound closure compared to the backbone group which may be due to the increased expression of miR-192 and increment in collagen expression in HDF cells. consistent with our study, Zabihi et al. [ 32 ] examined the impact of injecting fibroblast cells on the healing of diabetic wounds in male rats, the speed of wound healing in conjunction with the thickness and elasticity of the skin increased altogether within the group treated with fibroblast cells, and it seems that fibroblast cells can accelerate healing by expanding the thickness and elasticity of the skin. Mohammadipour et al. [ 33 ] found that platelet gel treatment significantly improved wound healing in rats by increasing collagen formation and inflammatory cell infiltration. Immunohistochemistry revealed higher vascular growth factor expression in the treatment group compared to the control group, in the current study, we showed that in the group that received the combined treatment of PRP and fibroblast cells containing miR-192, the amount of collagen and granulation tissue formation on days 7 and 14 after wound formation compared to the groups of PRP and fibroblasts expressing backbone was increased. Chuncharoni et al.[ 28 ] investigated the effect of platelet gel on skin wound healing in desert rats and used human platelets to heal skin wounds in rats, in their study, microscopic evaluation showed that on the 3rd and 7th days after wound formation, within the treatment group, the increase in the migration of epithelial cells and the arrangement of epithelial tissue at the wound site were well seen, the results of the present study are also consistent with the above findings, as our study used human platelets for diabetic skin wound repair. The present study is consistent with the findings of the above study, in our study, human platelets were used to repair diabetic skin wounds, and microscopic evaluations on days 7, 14, and 21 showed an increase in the movement of epithelial cells and the arrangement of the epithelium in the group treated with PRP cells and Fibroblast expressed miR-192 compared to control groups. Shoaibi et al. [ 34 ] investigated the effect of Platelet-Rich Plasma on the proliferation and migration of human skin fibroblasts. They showed that PRP stimulates the proliferation and migration of skin fibroblasts and also increases the expression of procollagen 1 alpha , elastin , MMP1 and MMP2 in skin fibroblasts, thus, it can help accelerate healing. In the present study, the density of collagen fibers in the group treated with PRP and fibroblast cells expressing pLenti-III-miR-192-GFP was more than other groups on the 14th and 21st day after wounding, which indicates the effect of simultaneous use of PRP and fibroblast cells expressing pLenti-III-miR-192-GFP in the wound healing process. According to the data of the present study, the combination of PRP and fibroblasts expressing miR-192 was more effective than either alone and improved the wound healing process and its related characteristics, such as: wound closure rate, granulation tissue and the formation of epithelial tissue and angiogenesis and collagen fiber synthesis. In connection with our study Ni et al. [ 35 ] Combined PRP and adipose-derived stem cells (ADSC) to heal wounds in a mouse model, according to their study's findings, PRP and adipose-derived stem cells combined resulted in a better rate of wound healing than PRP and adipose-derived stem cells used alone, additionally the PRP + ADSC group had higher expression levels of p-STAT3 , VEGF , and SDF-1 than the other groups, moreover, combination therapy significantly increased the proliferation of endothelial cells. Conclusion The percentage of wound closure, the number of fibroblast cells, the area of granulation tissue, the area of epithelial tissue, and the collagen fibers could all be significantly increased by using fibroblasts expressing miR-192, which increased the expression of collagen, additionally, this treatment was able to boost angiogenesis in the area of the diabetic wound, which aided in hastening the healing process in wound diabetic model Abbreviations PRP Platelet-Rich Plasma DFU Diabetic Foot Ulcer COL1A2 Collagen type I alpha 2 chain SIP1 Smad-Interacting Protein 1 HDF Human Dermal Fibroblasts GFP Green Fluorescent Protein DM Diabetes Mellitus ECM Extracellular Matrix PDGF Platelet-Derived Growth Factor FGF Fibroblast Growth Factor TGF Transforming Growth Factor STZ Streptozotocin Declarations Availability of data and materials The datasets used and analyzed in the current study are available from the corresponding author upon reasonable request. Acknowledgments The authors sincerely thank Dr. Mohsen Khorashadizadeh and Dr. Mahdieh Rajabi Moghadam and the cooperation of all participants for carrying out this project in the university. Funding The research leading to these results received funding from the Hematology department of Birjand University of Medical Sciences under Grant Agreement No IR.BUMS.REC.1400.413. Author information Authors and Affiliations Department of Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran. Forouzan Karam,Saeedeh Dadi, Mahtab Sayadi and Gholamreza Anani Sarab Contributions FK, MS and SD performed and analyzed most of the experiments and were significant contributors on writing the manuscript. All authors read and approved the final manuscript. Corresponding author Correspondence to Gholamreza Anani Sarab Ethics Declarations Ethics approval and consent to participate The ethics governing the use and conduct of experiments on animals were strictly observed, and the experimental protocol was approved by the Birjand University of Medical Sciences committee on Medical Research ethics (IR.BUMS.REC.1400.413). Consent for publication Not applicable. Competing interest All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript, and all authors confirm its accuracy. References Saeedi P et al (2019) Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the International Diabetes Federation Diabetes Atlas. Diabetes research and clinical practice , 157: p. 107843 Raghav A et al (2018) Financial burden of diabetic foot ulcers to world: a progressive topic to discuss always. Therapeutic Adv Endocrinol metabolism 9(1):29–31 Li J, Chen J, Kirsner R (2007) Pathophysiology of acute wound healing. Clin Dermatol 25(1):9–18 Reinke J, Sorg Н (2012) Wound Repair and Regeneration. Eur Surg Res 49(1):35–43 Mahmoodi M (2018) Studying the effect of cord blood platelet rich plasma (PRP) on expression of miRNA21 and miRNA 146a in fibroblast cells. J Cell Sci Therapy Bainbridge P (2013) Wound healing and the role of fibroblasts. J Wound Care 22(8):407–411 Addis R et al (2020) Fibroblast proliferation and migration in wound healing by phytochemicals: Evidence for a novel synergic outcome. Int J Med Sci 17(8):1030–1042 desJardins-Park HE, Foster DS, Longaker MT Fibroblasts and wound healing: an update. 2018, Future Medicine . pp. 491–495 Anitua E et al (2004) Autologous platelets as a source of proteins for healing and tissue regeneration. Thromb Haemost 91(01):4–15 Green DM, Klink B (1998) Platelet gel as an intraoperatively procured platelet-based alternative to fibrin glue. Plast Reconstr Surg 101(4):1161–1162 O'Neill E et al (2001) Autologous platelet-rich plasma isolated using the Haemonetics Cell Saver 5 and Haemonetics MCS + for the preparation of platelet gel. Vox Sang 81(3):172–175 Aydin O et al (2018) Platelet-Rich Plasma May Offer a New Hope in Suppressed Wound Healing When Compared to Mesenchymal Stem Cells. J Clin Med , 7(6): p.143 Cho EB et al (2019) Effect of platelet-rich plasma on proliferation and migration in human dermal fibroblasts. J Cosmet Dermatol 18(4):1105–1112 Filipowicz W, Bhattacharyya SN, Sonenberg N (2008) Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9(2):102–114 Yi R et al (2006) Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs. Nat Genet 38(3):356–362 Yi R et al (2008) A skin microRNA promotes differentiation by repressing ‘stemness’. Nature 452(7184):225–229 Sonkoly E et al (2007) MicroRNAs: novel regulators involved in the pathogenesis of psoriasis? PLoS ONE 2(7):e610 Ciechomska M et al (2014) MiR-29a reduces TIMP-1 production by dermal fibroblasts via targeting TGF-β activated kinase 1 binding protein 1, implications for systemic sclerosis. PLoS ONE 9(12):e115596 Li Z et al (2009) Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. J Biol Chem 284(23):15676–15684 Van Rooij E et al (2008) Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis. Proceedings of the National Academy of Sciences , 105(35): pp. 13027–13032 Wang B et al (2010) E-cadherin expression is regulated by miR-192/215 by a mechanism that is independent of the profibrotic effects of transforming growth factor-β. Diabetes 59(7):1794–1802 Kato M et al (2007) MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-β-induced collagen expression via inhibition of E-box repressors. Proceedings of the National Academy of Sciences , 104(9): pp. 3432–3437 Padmakumari RG, Sherly CD, Ramesan RM (2022) Therapeutic delivery of nucleic acids for skin wound healing. Ther Deliv 13(6):339–358 Furman BL (2021) Streptozotocin-induced diabetic models in mice and rats. Curr protocols 1(4):e78 Chuncharunee A et al (2019) Invalid freeze-dried platelet gel promotes wound healing. Saudi Pharm J 27(1):33–40 Diener C, Keller A, Meese E (2022) Emerging concepts of miRNA therapeutics: from cells to clinic. Trends Genet 38(6):613–626 Cialdai F, Risaliti C, Monici M (2022) Role of fibroblasts in wound healing and tissue remodeling on Earth and in space. Front Bioeng Biotechnol 10:958381 Gharbia FZ et al (2023) Adult skin fibroblast state change in murine wound healing. Sci Rep 13(1):886 Talbott HE et al (2022) Wound healing, fibroblast heterogeneity, and fibrosis. Cell Stem Cell 29(8):1161–1180 Li Y et al (2017) MicroRNA-192 regulates hypertrophic scar fibrosis by targeting SIP1. J Mol Histol 48(5):357–366 Fang X et al (2019) Smad interacting protein 1 influences transforming growth factor-β 1/Smad signaling in extracellular matrix protein production and hypertrophic scar formation. J Mol Histol 50:503–514 Zabihi A, Mahmoodi M, Soltani S (2022) Effect of Fibroblast Cells Injection on Healing of Diabetic Wounds and Improvement of Skin Elasticity and Thickness in Male Rats. Res Med 46(3):105–114 Mohammadipour M et al (2019) Evaluation of human platelet gel effect on production process and expression level of VEGF in experimental skin wounds in rat. Sci J Iran Blood Transfus Organ 16(2):103–115 Cho EB et al (2019) Effect of platelet-rich plasma on proliferation and migration in human dermal fibroblasts. J Cosmet Dermatol 18(4):1105–1112 Ni X et al (2021) Adipose-derived stem cells combined with platelet-rich plasma enhance wound healing in a rat model of full-thickness skin defects. Stem Cell Res Ther 12:1–11 Additional Declarations The authors declare no competing interests. Supplementary Files GA.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5290142","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":367735287,"identity":"97b4bc83-c5ee-4638-b6f0-db06a9c9928f","order_by":0,"name":"Forouzan Karam","email":"","orcid":"https://orcid.org/0000-0002-0460-9038","institution":"Birjand University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Forouzan","middleName":"","lastName":"Karam","suffix":""},{"id":367735288,"identity":"5aecc1ba-5106-4784-905b-a2580f7b9c94","order_by":1,"name":"Mahtab Sayadi","email":"","orcid":"https://orcid.org/0000-0003-3087-705X","institution":"Birjand University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mahtab","middleName":"","lastName":"Sayadi","suffix":""},{"id":367735289,"identity":"42c540e7-0042-43f6-9984-f0ee301cf63d","order_by":2,"name":"Saeedeh Dadi","email":"","orcid":"https://orcid.org/0009-0008-8878-7915","institution":"Birjand University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Saeedeh","middleName":"","lastName":"Dadi","suffix":""},{"id":367735290,"identity":"fa8d7b7d-add7-40bc-bb07-3f08edaf9feb","order_by":3,"name":"Gholamreza Anani Sarab","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYDACCTBpA2MQryWNdC2HSdAiH9387OGPmvOJ/bObDz5gqLGJJqjF8M4xc2OeY7cTZ9w5lmzAcCwtt4GglhkJZtIMbLcTG27kmEkwNhwmRkv6N8kf/84lzidai7wEUCVv24HEDURrMZDIKZPm7Us23ngjLdkggRi/yM9I3yb545ud7LwbyQcffKixIcKWAxDaEawygZBysC1QQ+2JUTwKRsEoGAUjFAAANlpBcp5BLjsAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-3844-3643","institution":"Birjand University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Gholamreza","middleName":"Anani","lastName":"Sarab","suffix":""}],"badges":[],"createdAt":"2024-10-18 14:37:49","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-5290142/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5290142/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":67139071,"identity":"a2bd7c61-0b1f-4116-a4f7-6f02b35f0850","added_by":"auto","created_at":"2024-10-21 14:17:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":34315,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic figure of cell and PRP injection site. Treatments were injected at the center of circular wounds, positions 12, 3, 6, and 9.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5290142/v1/3d712a4e42cdb57dbf3e77c3.png"},{"id":67138853,"identity":"7f2de748-f302-44c1-9c34-bf4be0bba03a","added_by":"auto","created_at":"2024-10-21 14:09:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":299491,"visible":true,"origin":"","legend":"\u003cp\u003eGFP expression in human skin fibroblast cells. Magnification ×40. (A) HDF cells expressing GFP with visible light (B) HDF cells expressing GFP with fluorescent light.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5290142/v1/e88419e99f6592943811a714.png"},{"id":67138851,"identity":"b23d00e4-3cf4-4756-b2e6-58de0cadf5ef","added_by":"auto","created_at":"2024-10-21 14:09:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":292475,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of HDF cells transfected with pLenti-Ⅲ-miR-192-GFP and pLenti-Ⅲ-Backbone-GFP versus the negative control group by flow cytometry. Figure (A) negative control group with fresh HDF (GFP: 0.2% positive). Figure (B)) HDF group expressing pLenti-Ⅲ-miR-192-GFP (GFP: 22% positive). Figure (C) HDF group expressing pLenti-Ⅲ-Backbone-GFP (GFP: 20% positive).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5290142/v1/94cb5a87cfe4bef3bf57f38b.png"},{"id":67138850,"identity":"ae557287-d816-4d33-b1c1-9cc3fe92e053","added_by":"auto","created_at":"2024-10-21 14:09:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":73200,"visible":true,"origin":"","legend":"\u003cp\u003eIncreased expression of miR-192 in human skin fibroblast cell line. 48 hours after transfection with miR-192 expressing plasmid and backbone expressing plasmid, the level of miR-192 in both groups of cells was measured by qRT-PCR. (P = 0.0078) **. (n=3)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5290142/v1/03c313df19223c124e07724d.png"},{"id":67139072,"identity":"1387e555-bf4a-4fb3-b036-98f4b995fdef","added_by":"auto","created_at":"2024-10-21 14:17:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":78541,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of \u003cem\u003eCOL1A2\u003c/em\u003e gene expression in cells transfected with miR-192 expressing plasmids against the control group transfected with Backbone expressing plasmids. **** (P \u0026nbsp;\u0026lt; 0.0001). (n=3)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5290142/v1/aba149bf1417a09477e2cbdd.png"},{"id":67138857,"identity":"e46de1c9-55d2-4244-a377-0861ff3c9006","added_by":"auto","created_at":"2024-10-21 14:09:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":157202,"visible":true,"origin":"","legend":"\u003cp\u003eComparing the rate of wound closure on the third day after surgery, between the intervention groups with each other. NS (Not significant) and * (P \u0026lt; 0.05) and ** (P \u0026lt; 0.01) and *** (P \u0026lt; 0.001) and **** (P \u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5290142/v1/d1d8ea548f3a81e7c95d26e5.png"},{"id":67138859,"identity":"a5bd5a41-f8de-4efc-ae9b-49ae515570c8","added_by":"auto","created_at":"2024-10-21 14:09:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eComparing the rate of wound closure on the seventh day after surgery, between the intervention groups with each other. NS (Not significant) and * (P \u0026lt; 0.05) and ** (P \u0026lt; 0.01) and *** (P \u0026lt; 0.001) and **** (P \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"placeholderimage.png","url":"https://assets-eu.researchsquare.com/files/rs-5290142/v1/2923c252badae13f697cc332.png"},{"id":67138855,"identity":"66433082-b10b-4a3a-b780-c6558fe0009f","added_by":"auto","created_at":"2024-10-21 14:09:59","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":548422,"visible":true,"origin":"","legend":"\u003cp\u003eComparing the rate of wound closure on days 14 and 21 after wound formation between the intervention groups. \u003cstrong\u003eA:\u003c/strong\u003e Comparing the rate of wound closure on day 14. \u003cstrong\u003eB:\u003c/strong\u003e Comparing the rate of wound closure on day 21. NS (Not significant) and * (P \u0026lt; 0.05) and ** (P \u0026lt; 0.01) and *** (P \u0026lt; 0.001) and **** (P \u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5290142/v1/b81f9fbb63f3c260497b89d8.png"},{"id":67139077,"identity":"f098291e-1656-468e-a0bf-3d5e2ef428d6","added_by":"auto","created_at":"2024-10-21 14:17:59","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":996205,"visible":true,"origin":"","legend":"\u003cp\u003eMacroscopic views of wounds on different days. The wound pictures of the rats’ dorsal skin of the Non-Diabetic control group, Diabetic control and intervention groups were taken on postinjury days 3, 7, 14, and 21. Scale bar = 1 cm.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5290142/v1/3fecbf3a2ded41a23022f04b.png"},{"id":67139074,"identity":"07c95575-7d2c-4d11-93cd-33d3a285f518","added_by":"auto","created_at":"2024-10-21 14:17:59","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":217360,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the average number of fibroblast cells on the fourteenth and twenty-first day after wounding between different intervention groups. NS (Not significant) and * (P \u0026lt; 0.05) and ** (P \u0026lt; 0.01) and *** (P \u0026lt; 0.001) and **** (P \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-5290142/v1/a39a76cfb1f95553bbff6fba.png"},{"id":67139076,"identity":"dd6053b8-f14d-42ca-8c64-a3383ef396ef","added_by":"auto","created_at":"2024-10-21 14:17:59","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":236074,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the average density of collagen fibers on the 14th and 21st days after wounding in different intervention groups. NS (Not significant) and * (P \u0026lt; 0.05) and ** (P \u0026lt; 0.01) and *** (P \u0026lt; 0.001) and **** (P \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-5290142/v1/e9c54b35bfa2ffa5a8c6e800.png"},{"id":67139075,"identity":"1d894047-7b44-42c3-b3ca-eb8158af7482","added_by":"auto","created_at":"2024-10-21 14:17:59","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":1064224,"visible":true,"origin":"","legend":"\u003cp\u003eA Masson-trichrome staining of wound area in different groups on day 14 after wound induction. Note: Black arrow: epidermis area. Red arrow: blue collagen fibers in the dermis. photographed at a magnification of 400x, scale bar = 25 μm\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-5290142/v1/c7278f8303c60eb9a863ca83.png"},{"id":67138864,"identity":"c5043222-a33d-4b6b-a6bb-a6f07f83e034","added_by":"auto","created_at":"2024-10-21 14:09:59","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":509261,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the area of granulation tissue on the seventh day and the fourteenth day after wound formation. \u003cstrong\u003eA:\u003c/strong\u003e Comparison of the area of granulation tissue on the seventh day after wound formation in different intervention groups. \u003cstrong\u003eB:\u003c/strong\u003e Comparison of the area of granulation tissue on the fourteenth day after wound formation in different intervention groups. NS (Not significant) and *(P \u0026lt; 0.05) and **(P \u0026lt; 0.01) and ***(P \u0026lt; 0.001) and ****P \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-5290142/v1/3f5f84b47655a46009047284.png"},{"id":67138861,"identity":"6d890666-4c5f-4c0c-8a37-e8f6455b441b","added_by":"auto","created_at":"2024-10-21 14:09:59","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":951425,"visible":true,"origin":"","legend":"\u003cp\u003eGranulation tissue area in hematoxylin-eosin staining. H\u0026amp;E staining demonstrated the collagen fiber\u003cbr\u003e\ndeposition and changes in capillaries in the granulation tissues photographed at a magnification of 40x, scale bar=25 μm.\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-5290142/v1/3ebed6aaf274b23c09f98f7b.png"},{"id":67140498,"identity":"7fbed77a-48fb-47a1-a447-eb78e5c5b84f","added_by":"auto","created_at":"2024-10-21 14:34:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6265217,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5290142/v1/9d081eb2-5424-476b-89c7-8542e2839ede.pdf"},{"id":67140207,"identity":"46642dac-ba9b-4d96-b24b-cef14ce1dd4c","added_by":"auto","created_at":"2024-10-21 14:25:59","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":228802,"visible":true,"origin":"","legend":"","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-5290142/v1/b7e60e2944ef29c9a066040b.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eOverexpression of miR-192 in Fibroblasts accelerates wound healing in diabetic rats\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eDiabetes mellitus (DM) is a widespread health condition impacting approximately 9.3% of the global population [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Diabetic foot ulcer (DFU) is a prevalent chronic complication of diabetes with significant medical, economic, and social impacts. research suggests that 15\u0026ndash;25% of diabetic individuals are at risk of developing foot ulcers during their lifetime [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe stages of wound healing include homeostasis, which occurs immediately after wound formation, inflammation, which occurs from 10\u0026ndash;15 minutes to 3 days after injury, proliferation, which occurs from 4 to 21 days after injury, and regeneration, which occurs from 21 days to a year [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFibroblasts secrete growth factors, collagen, and other elements of the Extracellular Matrix (ECM), which are vital for wound healing. They release Platelet-Derived Growth Factor (PDGF), Fibroblast Growth Factors (FGF), and Transforming Growth Factor (TGF) to promote cell division, activity, or differentiation. [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Platelet-Rich Plasma (PRP) is considered a natural growth factor that is safe to use in enhancing the healing rate of wounds. It is particularly effective in treating chronic wounds associated with diabetes that require prompt repair to prevent infections. One clinical application is the use of PRP gel for chronic ulcers[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. PRP is a product of blood plasma with a high platelet concentration that contains numerous growth factors and cytokines, such as platelet-derived growth factor (PDGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), and insulin-like growth factor[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. More importantly, PRP can enhance the proliferation and migration of dermal fibroblasts, indicating that it may synergistically heal chronic wounds[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMicroRNAs (miRNAs) are small RNA molecules that are approximately 19 to 22 nucleotides in length. They regulate gene expression at the post-transcriptional level by binding to the untranslated 3' regions (UTR) of target messenger RNA (mRNA) molecules[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Multiple miRNAs have been identified in skin tissue, and they are believed to participate in various biological processes, including the regulation of wound healing [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The expression of miR-29a has been found to have a direct impact on collagen expression, whereas miR-192, miR-29b, and miR-29c are significantly upregulated during this process [\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. miR-192 promotes the expression of \u003cem\u003eCOL1A1\u003c/em\u003e by targeting Smad-Interacting Protein 1 (SIP1) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. While current treatments for wound care have made significant progress, the replacement of lost skin remains a major challenge in the field of regenerative medicine. Despite several available treatment methods for wound healing, a treatment approach that accelerates the healing process would be highly valuable[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo our knowledge, relevant research on the combination of fibroblasts with overexpression of miR-192 and PRP for the treatment of wound healing is very limited. This research investigated the impact of co-treatment with fibroblasts expressing miR-192 and Platelet-Rich Plasma in the healing of wounds in a diabetic rat model.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and culture\u003c/h2\u003e \u003cp\u003ePrimary human dermal fibroblasts (obtained from the laboratory of Dr. Mohsen Khorashadizadeh) were cultured in DMEM (BIO-IDEA, Tehran, Iran) with 10% heat-inactivated Fetal Bovine Serum (FBS), 1% penicillin and streptomycin and incubated at 37\u0026deg;C in a 95% humidified incubator with 5% CO2.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlasmids construct and extraction\u003c/h3\u003e\n\u003cp\u003eThe pLenti-III-pre-miR192-GFP(Green Fluorescent Protein) expression vector construct and pLenti-III-Backbone-GFP (mock) were purchased from ABM Inc (Applied Biological Materials, Richmond, BC, Canada). E.Coli Stbl4 strain harboring the vectors was cultured in Luria-Bertani (LB) broth medium with 50 \u0026micro;g/mL kanamycin. The plasmid was extracted with the Karmania Pars Gene plasmid extraction kit (Karmania Pars Gene, Kerman, Iran).\u003c/p\u003e\n\u003ch3\u003eTransient transfection\u003c/h3\u003e\n\u003cp\u003eFor each well, 2\u0026times;10^5 cells were seeded in a 6-well cell culture plate. Separately, 3 \u0026micro;g of plasmids and 4 \u0026micro;l of PEI in 100 \u0026micro;l of DMEM medium were dissolved in two 1.5 ml Microtubes, the mixture was added dropwise to the cells, 6 hours later medium was replaced with fresh medium.\u003c/p\u003e\n\u003ch3\u003eConfirmation of GFP expression by flow cytometry and Fluorescence microscope\u003c/h3\u003e\n\u003cp\u003eForty-eight hours after the transfection, the expression of the labeled GFP protein in the cells was verified by trypsinizing the cells and analyzing them using a flow cytometry system. The cells were first diluted in PBS buffer at 3\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per tube and homogenized using a vortex. The samples were then analyzed using the FL1 channel of the flow cytometry (CYFLOW CUBE 8, Sysmex, Germany). Additionally, to evaluate the expression of GFP, fluorescence microscopy (Olympus BX41TF, Japan) analysis was performed on transfected human dermal fibroblasts.\u003c/p\u003e\n\u003ch3\u003eRNA extraction\u003c/h3\u003e\n\u003cp\u003eForty-eight hours post-transfection, total RNA was extracted from HDF cells using RNX-plus reagent (Sinaclon, Tehran, Iran) according to the manufacturer protocol. The extracted RNAs were then quantified using Nanodrop spectrophotometry (BioTek Epoch microplate spectrophotometer, USA) by measuring the absorbance at 260/280 and 260/230 nm wavelengths, Samples were kept at -80ᵒ C until use.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ecDNA synthesis and stem-loop qPCR of miR-192\u003c/h2\u003e \u003cp\u003eStem-loop primers and the miR-192-5P specific primer used was 5\u0026prime;- CTCTGACTTATGAATTGAC-3\u0026prime; (forward) and reverse primers were designed to synthesize the cDNA of miR-192 according to the BON Stem High Sensitivity microRNA 1st Strand cDNA Synthesis kit protocol (Stem Cell Technology Research Center, Tehran, Iran) for qPCR, RT-qPCR was performed using StepOne\u0026trade; Real-Time PCR System (StepOne\u0026trade; Real-Time PCR System, USA) under the following conditions: 95\u0026deg;C for 2 minutes, and 60\u0026deg;C for 30 Seconds for up to 40 cycles. U6 primer used was 5́- AAGGATGACACGCAAAT-3́ (forward) and was utilized as an internal control to normalize the RNA input. The relative expression of miR-192 was evaluated using the ∆∆CT method, the experiment was performed in triplicate (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRT-qPCR for Collagen 1A2 gene expression\u003c/h3\u003e\n\u003cp\u003eThe extracted RNA was reverse transcribed into cDNA using the Easy cDNA Synthesis Parstous Kit from Parstous, Mashhad, Iran. The qRT- PCR reactions were carried out using RealQ plus 2x Master Mix Green (AMPLIQON, Denmark) The experiment was conducted on a Real-time PCR System (StepOne\u0026trade; Real-Time PCR System, USA) under the following conditions: 95\u0026deg;C for 2 minutes, and 61\u0026deg;C for 30 Seconds for up to 40 cycles.\u003c/p\u003e \u003cp\u003eThe primers used to target the \u003cem\u003eCOL1A2\u003c/em\u003e gene were 5\u0026prime;GAGGGCAACAGCAGGTTCACTTA-3\u0026prime; (forward) and 5\u0026prime;-TCAGCACCACCGATGTCCAA-3\u0026prime; (reverse) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), \u003cem\u003eGAPDH\u003c/em\u003e was used as an internal control gene. The ∆∆CT method was used for calculation. (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers used in this study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOligo Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence 5́-3́\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGC%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMW (Da)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e*TM (℃)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eOD\u003c/p\u003e \u003cp\u003e260nm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eProduct length\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eOligo ID/ metabion international AG\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eForward Primer COL1A2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTCT CTA CTG GCG AAA CCT GTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e197,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e47.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.381\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e220823B003H05 1/2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReverse Primer COL1A2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTCC TAG CCA GAC GTG TTT CTT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e189,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e47.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.363\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4,3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e220823B003A06 2/2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003ePRP Preparation\u003c/h3\u003e\n\u003cp\u003ePRP was prepared from 5 blood donors. Blood with sodium citrate anticoagulant (50 ml) was prepared and centrifuged at 1200 rpm for 10 minutes, the supernatant was separated and re-centrifuged at 3000 rpm for 10 minutes, the platelet precipitation was resuspended in the plasma.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperimental animals and study design\u003c/b\u003e \u003c/p\u003e \u003cp\u003e8-10-week-old male wistar rats were obtained from the Pasteur Institute of Iran (Tehran, Iran) for this study. Animals were kept in standard cages with four rats per cage. the rats were kept at a controlled room temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5% humidity. Animals were exposed to a 12-hour light-dark cycle and standard food and water. Rats weighing about 180\u0026ndash;200 g were randomly divided into six groups with eight rats in each group:\u003c/p\u003e \u003cp\u003eGroup 1; wound model in non-diabetic rats, group 2; wound model in diabetic rats, group 3; wound model in diabetic rats treated with 100 \u0026micro;l of PRP, group 4; wound model in diabetic rats treated with 6\u0026times;10\u003csup\u003e4\u003c/sup\u003e HDF expressing pLenti-III-Backbone-GFP suspended in 100 \u0026micro;l of DMEM medium, group 5; wound model in diabetic rats treated with 6\u0026times;10\u003csup\u003e4\u003c/sup\u003e HDF expressing pLenti-III-miR-192-GFP suspended in 100 \u0026micro;l of DMEM medium, group 6; wound model in diabetic rats\u0026thinsp;+\u0026thinsp;100 \u0026micro;l of PRP\u0026thinsp;+\u0026thinsp;6\u0026times;10\u003csup\u003e4\u003c/sup\u003e HDF expressing pLenti-III-miR-192-GFP.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eInduction of type 2 diabetes in rats\u003c/h2\u003e \u003cp\u003e The experiments were performed following institutional guidelines for animal care and approved by the local ethics committee (IR.BUMS.REC.1400.413). Type 2 diabetes was induced in rats using the fat-fed streptozotocin \u003cb\u003e(\u003c/b\u003eZellBio GmbH, Berlin, Germany) (STZ) model mouse protocol. The rats were placed on a high-fat diet for three weeks, providing 60% of their caloric value as fat[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], on day 22, all rats were fasted for 8 h before STZ injection, and fasting blood glucose was measured, 40 mg of STZ was weighed, transferred to a 1.5 ml microcentrifuge tube and covered with aluminum foil, Immediately before injection, Citrate buffer was prepared, STZ was dissolved in 50 mM sodium citrate buffer at pH 4.5 to a final concentration of 40 mg/ml, administered within 5 minutes after dissolution, using a 1 ml syringe and 23-G needle, STZ was injected intraperitoneally (i.p.) into the test group at a dose of 40 mg/kg of body weight (1.0 ml/kg). An equal volume of citrate buffer with pH 4.5 was injected i.p. into the control animals. For the diabetic groups, the rats received high-fat food, whereas the control group had a typical diet. Ten days following STZ delivery, blood glucose levels were assessed using the Infopia EasyGluco (Autocoding blood glucose metre EasyGlucoTM, South Korea) in a tail vein blood sample. Blood glucose levels exceeding 15 mmol/L (270 mg/dL) indicated that STZ-treated rats had successfully induced type 2 diabetes[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Induced Wounds and treatments\u003c/h2\u003e \u003cp\u003eRats were anesthetized intraperitoneally using ketamine (Alphasan, Woerden, Holland) and xylazine (KELA.N.V, Belgie), and then the dorsal aspect of the upper part of the body was shaved and disinfected with 70% alcohol. A 10 mm skin biopsy punch was induced to create full-thickness wounds under aseptic conditions, treatments were performed in each group according to the previously mentioned protocol, in the form of subcutaneous injections in 5 points according to the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Animals were sacrificed on the 3rd, 7th, 14th and 21st days after wounding.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMacroscopic Observation\u003c/h2\u003e \u003cp\u003eWounds were measured using a digital caliper and photographed on the day of wounding and subsequently on alternate days until healing was complete. Changes in wound areas were calculated at each time point to monitor the rate of wound contraction, the percentage reduction in wound size was calculated using the following equation[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]:\u003c/p\u003e \u003cp\u003e% wound healing =\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\frac{\\text{W}\\text{o}\\text{u}\\text{n}\\text{d}\\:\\text{a}\\text{r}\\text{e}\\text{a}\\:\\text{d}\\text{a}\\text{y}\\:0-\\:\\text{w}\\text{o}\\text{u}\\text{n}\\text{d}\\:\\text{a}\\text{r}\\text{e}\\text{a}\\:\\text{d}\\text{a}\\text{y}\\text{s}\\:(3/7\\:/14/\\:21)\\times\\:100}{\\text{w}\\text{o}\\text{u}\\text{n}\\text{d}\\:\\text{a}\\text{r}\\text{e}\\text{a}\\:\\text{d}\\text{a}\\text{y}\\:0}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHaematoxylin and Eosin and Masson\u0026rsquo;s Trichrome Staining\u003c/h2\u003e \u003cp\u003eThe wound area was removed using a sharp sterile scalpel. Then the cut tissue was fixed in a 10% formalin solution to preserve its structure and prevent destruction. The tissue was dehydrated through a series of alcohol solutions with increasing concentration.\u003c/p\u003e \u003cp\u003eThe tissue was cleared using xylene, tissue infiltration and embedding were performed using paraffin wax or other embedding materials to support the tissue during sectioning, then the histological sections were cut using a microtome and placed on glass slides, slides were stained with hematoxylin, eosin, and Masson's trichrome to observe collagen deposition and other histological features. An optical microscope (Microscope camera ODC series, England) was used to observe and photograph the slides, and all histological examinations were performed by two pathologists blindly.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eIn this study, Fiji/ImageJ 2.9.0 software was used for quantitative analysis of the density of collagen fibers, the number of fibroblast cells, and the amount of granulation during the different stages of wound healing. The data obtained from the Fiji/ImageJ software was analyzed and the results were presented using graphs and charts drawn with GraphPad Prism 9 software. The statistical significance of the data was determined using the two-way ANOVA and t-student test, and a significance level of p value less than 0.05 was considered in all experiments.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eExpression of GFP-tag and observation of green color as confirmation for transfection of expression plasmids in human Dermal fibroblast cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe transfection of HDF cells with pLenti-III plasmids was successful with a 20\u0026ndash;25% transaction rate as estimated after 48 hours of transfection. The confirmation of the transfection was done using a fluorescence microscope, and the results were presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eThe expression level of miR-192 was analyzed by flow cytometry using the FL1 channel compared to fresh and untransfected cells, the results of this analysis were presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression of miR-192 in transfected fibroblast cells increased\u003c/strong\u003e \u003cstrong\u003eCOL1A2\u003c/strong\u003e \u003cstrong\u003egene expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of this analysis showed a significant increase (3-fold) in miR-192 expression in the fibroblast cells transfected with the miR-192 expressing plasmid compared to the cells transfected with the backbone expressing plasmid after 48 hours, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, this confirmed the successful transfection of the cells and the overexpression of miR-192 in the transfected cells.\u003c/p\u003e\n\u003cp\u003eThe study used RT-qPCR to test if miR-192 increased in fibroblast cells would change \u003cem\u003eCOL1A2\u003c/em\u003e expression, Results showed a 15.77-fold increase in \u003cem\u003eCOL1A2\u003c/em\u003e expression in transfected cells compared to the control group after 48 hours (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e(, suggests miR-192 positively regulates \u003cem\u003eCOL1A2\u003c/em\u003e expression and a potential mechanism for its effects on wound healing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSimultaneous treatment with HDF cells expressing miR-192 and PRP caused more wound closure than other groups.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn the 3rd day, a significant difference was seen among the group receiving HDFs expressing pLenti-III-Backbone-GFP and the group treated with HDFs expressing pLenti-III-miR-192-GFP (P\u0026thinsp;\u0026lt;\u0026thinsp;0 /0001) .There was a significant difference between the group that received PRP and HDFs expressing pLenti-III-miR-192-GFP and other treatment groups P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and more wound closure have occurred (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eOn the seventh day after wound formation, the wounds were examined macroscopically, and measurements were taken and recorded with a digital calliper. On the seventh day, the wound healing process accelerated in all intervention groups compared to the third day, and the difference between the groups became more obvious. According to the order of the groups in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, the average percentage of wound closure in various intervention groups versus the control groups is as follows: 68/33, 60/67, 66/83, 83/00, 91/53, and 89/03 percent.\u003c/p\u003e\n\u003cp\u003eA significant difference of P\u0026thinsp;=\u0026thinsp;0.0001 was seen in the healthy control group and the miR-192-expressing, PRP-group, and combined treatment groups, and these groups healed wounds quicker than the healthy control group. On the seventh day, the diabetic control group that did not receive treatment experienced a delay in the healing of the wound compared to the other groups. The difference between the rates of wound closure in the treated groups compared to the diabetic control group was almost completely different in the treated groups. Most of the wound area is closed, especially in the fifth and sixth groups, where it ranges from 80 to 90%, with diabetic wounds being close to 60%. In comparison to the group that only received fibroblast cells expressing pLenti-III-miR-192-GFP, more wounds closed in the combination therapy group (P\u0026thinsp;=\u0026thinsp;0.0001). (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e\n\u003cp\u003eOn the fourteenth day, there was a significant difference between the HDF groups expressing pLenti-III-Backbone-GFP and pLenti-III-miR-192-GFP plasmids (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and more wound closure was observed in the HDF group expressing pLenti-III-miR-192-GFP. Between the PRP-treated group and the HDF-expressed group, there was a smaller difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The group that received the combination therapy with the groups treated with PRP and fibroblasts experiencing pLenti-III-miR-192-GFP alone is significant, with P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and NS (Not significant) respectively.\u003c/p\u003e\n\u003cp\u003eOn the 21st day after wound formation, healing occurred in almost all intervention groups, but the diabetic group without treatment has the lowest average percentage of wound closure (88.48%) compared to other groups, the group that simultaneously received PRP and fibroblast cells expressing pLenti-III-miR-192-GFP showed significant differences with each group that received the mentioned treatments alone, the group that was treated with combined treatment, the rate of wound closure and scars left from healing was lower than in other groups. (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). The macroscopic views of the wound on different days are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe number of fibroblast cells in the group that simultaneously received HDF cells expressing miR-192 and PRP was higher than in the other groups.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn the 14th and 21st days following the development of a wound, the average number of fibroblast cells was counted in the dermis of the wound area in various intervention groups. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e shows that the group that received the combined treatment had more fibroblast cells than the other groups. The fact that there was a noticeable difference between this group and the groups which received each treatment separately could mean that the injected fibroblast cells in the wound area were functional and alive.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe density of collagen fibers in the group treated with PRP and HDF cells expressing miR-192 increased compared to other groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe density of collagen fibers in the group treated with PRP and HDF cells expressing miR-192 increased compared to other groups the group that got the combination therapy and HDF expressing pLenti-III-miR-192-GFP had a higher collagen fiber density on day 14 than the other groups, and there was a significant difference between these two groups and the PRP-treated group. A notable difference was observed between the groups that got HDF containing pLenti-III-miR-192-GFP plasmid and the backbone group on the 21st day following injury, additionally compared to each of the treatment groups separately, the combined treatment group's collagen fiber density increased, however, this rise was not statistically significant (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eFigure 12 shows Masson-trichrome staining of the wound area in different groups on the 14th day after wound induction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCombined treatment with PRP and HDF cells expressing miR-192 increased the area of granulation tissue and wound healing\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eOn the seventh day, the group treated at the same time with PRP and HDF expressing pLenti-III-miR-192-GFP, the average area of the granulation tissue is 502188 \u0026micro;m square and more than other groups, and there is a critical difference between this group and the two groups treated with PRP and HDF containing pLenti-III-miR-192-GFP expression plasmids alone was seen P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the fourteenth day, a noteworthy distinction was observed between the group that received the combined treatment and the group that was treated with a PRP P \u0026lt; 0.05. But there was no difference between the combined group and the group that had fibroblasts containing pLenti-III-miR-192-GFP. There was no noteworthy difference between the PRP group and the group treated with HDF expressing pLenti-III-miR-192-GFP. But in all the groups that received treatment, compared to the healthy and diabetic control groups, there was a critical distinction, and the area of the granulation tissue was greater within the intervention groups than within the control groups. (Figure 13)\u003cspan dir=\"RTL\"\u003e.\u0026nbsp;\u003c/span\u003eThe area of granulation tissue can be seen in hematoxylin-eosin staining in Figure 14\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePresent-day medications for wound care are not completely successful and chronic wounds present a challenge within regenerative medicine. Gene therapy and genetic engineering are new approaches that appear to have helpful potential for treating persistent wounds. Bioactive particles such as DNA, mRNA, siRNA, and miRNA may offer incredible clinical advantages, especially in regulating complex genetic systems and cellular signaling cascades related to skin repair. Changed expression of miRNA has appeared to play a central part in adjusting protein expression, and miRNA-based interventions may offer a wide run of targets that can be directed by a single miRNA [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In some studies, human skin fibroblast cells have been proposed as a capable helpful instrument for skin repair in wounds whose healing is delayed [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe present study used a genetic engineering approach to create more efficient fibroblast cells with increased expression of miR-192 and \u003cem\u003eCOL1A2.\u003c/em\u003e These modified cells were used in combination with Platelet-Rich Plasma (PRP) for wound healing in an experimental model of diabetic rats. The results showed that increasing the expression of miR-192 in fibroblast cells resulted in increased expression of collagen, and the use of these modified cells locally in the wound area showed beneficial effects in accelerating the healing process of chronic wounds in the experimental model. Moreover, the combination treatment of PRP and fibroblast cells expressing miR-192 was found to be effective in promoting wound healing, suggesting a potential therapeutic application for this approach in the treatment of chronic wounds.\u003c/p\u003e \u003cp\u003eLi et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] showed that in hypertrophic scars, increasing the expression of miR-192 by direct suppression of SIP1 protein within the TGF-/Smad2-3 signaling pathway enhances the expression of type 1 and 3 collagen and \u003cem\u003eα-SMA\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAccording to Fang et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] a cytoplasmic protein named SIP1 inhibits the phosphorylation of Smad2/3 and prevents the nuclear Smad from binding to the collagen gene promoter within the TGF-β/Smad2-3 signaling pathway and finally decreased the expression of collagen protein.\u003c/p\u003e \u003cp\u003eIn our study, HDF cells transfected with pLenti-III-miR-192-GFP expression plasmids demonstrate significantly increased in the level of \u003cem\u003eCOL1A2\u003c/em\u003e expression compared to the group of HDF cells with pLenti-III-Backbone-GFP expression plasmids.\u003c/p\u003e \u003cp\u003eIncreasing the content of collagen peptides in skin fibroblast cells in laboratory conditions increases the proliferation of fibroblast cells and the content of fibroblast-derived cell matrix. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], also the use of peptides derived from human \u003cem\u003eCOL1A2\u003c/em\u003e in laboratory conditions leads to an increase in collagen levels and cell migration and the amount of elastin in skin fibroblast cells [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In the present study, the results showed that the use of combined treatment of PRP cells and HDF cells expressing miR-192 supported the wound healing process and faster wound closure compared to both PRP and HDF cells expressing miR-192 alone, in addition, the group treated with fibroblasts expressing miR-192 showed a more critical increase in wound closure compared to the backbone group which may be due to the increased expression of miR-192 and increment in collagen expression in HDF cells. consistent with our study, Zabihi et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] examined the impact of injecting fibroblast cells on the healing of diabetic wounds in male rats, the speed of wound healing in conjunction with the thickness and elasticity of the skin increased altogether within the group treated with fibroblast cells, and it seems that fibroblast cells can accelerate healing by expanding the thickness and elasticity of the skin.\u003c/p\u003e \u003cp\u003eMohammadipour et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] found that platelet gel treatment significantly improved wound healing in rats by increasing collagen formation and inflammatory cell infiltration. Immunohistochemistry revealed higher vascular growth factor expression in the treatment group compared to the control group, in the current study, we showed that in the group that received the combined treatment of PRP and fibroblast cells containing miR-192, the amount of collagen and granulation tissue formation on days 7 and 14 after wound formation compared to the groups of PRP and fibroblasts expressing backbone was increased. Chuncharoni et al.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] investigated the effect of platelet gel on skin wound healing in desert rats and used human platelets to heal skin wounds in rats, in their study, microscopic evaluation showed that on the 3rd and 7th days after wound formation, within the treatment group, the increase in the migration of epithelial cells and the arrangement of epithelial tissue at the wound site were well seen, the results of the present study are also consistent with the above findings, as our study used human platelets for diabetic skin wound repair. The present study is consistent with the findings of the above study, in our study, human platelets were used to repair diabetic skin wounds, and microscopic evaluations on days 7, 14, and 21 showed an increase in the movement of epithelial cells and the arrangement of the epithelium in the group treated with PRP cells and Fibroblast expressed miR-192 compared to control groups. Shoaibi et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] investigated the effect of Platelet-Rich Plasma on the proliferation and migration of human skin fibroblasts. They showed that PRP stimulates the proliferation and migration of skin fibroblasts and also increases the expression of \u003cem\u003eprocollagen 1 alpha\u003c/em\u003e, \u003cem\u003eelastin\u003c/em\u003e, \u003cem\u003eMMP1 and MMP2\u003c/em\u003e in skin fibroblasts, thus, it can help accelerate healing. In the present study, the density of collagen fibers in the group treated with PRP and fibroblast cells expressing pLenti-III-miR-192-GFP was more than other groups on the 14th and 21st day after wounding, which indicates the effect of simultaneous use of PRP and fibroblast cells expressing pLenti-III-miR-192-GFP in the wound healing process. According to the data of the present study, the combination of PRP and fibroblasts expressing miR-192 was more effective than either alone and improved the wound healing process and its related characteristics, such as: wound closure rate, granulation tissue and the formation of epithelial tissue and angiogenesis and collagen fiber synthesis. In connection with our study Ni et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] Combined PRP and adipose-derived stem cells (ADSC) to heal wounds in a mouse model, according to their study's findings, PRP and adipose-derived stem cells combined resulted in a better rate of wound healing than PRP and adipose-derived stem cells used alone, additionally the PRP\u0026thinsp;+\u0026thinsp;ADSC group had higher expression levels of \u003cem\u003ep-STAT3\u003c/em\u003e, \u003cem\u003eVEGF\u003c/em\u003e, and \u003cem\u003eSDF-1\u003c/em\u003e than the other groups, moreover, combination therapy significantly increased the proliferation of endothelial cells.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe percentage of wound closure, the number of fibroblast cells, the area of granulation tissue, the area of epithelial tissue, and the collagen fibers could all be significantly increased by using fibroblasts expressing miR-192, which increased the expression of collagen, additionally, this treatment was able to boost angiogenesis in the area of the diabetic wound, which aided in hastening the healing process in wound diabetic model\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePRP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePlatelet-Rich Plasma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDFU\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDiabetic Foot Ulcer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCOL1A2\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCollagen type I alpha 2 chain\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSIP1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSmad-Interacting Protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHDF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman Dermal Fibroblasts\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGFP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGreen Fluorescent Protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDiabetes Mellitus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eECM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtracellular Matrix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePDGF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePlatelet-Derived Growth Factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eFGF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFibroblast Growth Factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTGF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransforming Growth Factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSTZ\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStreptozotocin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed in the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors sincerely thank \u003cstrong\u003eDr. Mohsen Khorashadizadeh\u003c/strong\u003e and \u003cstrong\u003eDr. Mahdieh Rajabi Moghadam\u003c/strong\u003e and the cooperation of all participants for carrying out this project in the university.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research leading to these results received funding from\u003cstrong\u003e\u0026nbsp;the Hematology department of Birjand University of Medical Sciences\u003c/strong\u003e under Grant Agreement No\u0026nbsp;IR.BUMS.REC.1400.413.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDepartment of Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eForouzan Karam,Saeedeh Dadi, Mahtab Sayadi and Gholamreza Anani Sarab\u003c/p\u003e\n\u003cp\u003eContributions\u003c/p\u003e\n\u003cp\u003eFK, MS and SD performed and analyzed most of the experiments and were significant contributors on writing the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eCorresponding author\u003c/p\u003e\n\u003cp\u003eCorrespondence to\u0026nbsp;Gholamreza Anani Sarab\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe ethics governing the use and conduct of experiments on animals were strictly observed, and the experimental protocol was approved by the\u0026nbsp;\u003cstrong\u003eBirjand University of Medical Sciences\u003c/strong\u003e committee on Medical Research ethics\u0026nbsp;(IR.BUMS.REC.1400.413).\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eCompeting interest\u003cbr\u003e\u0026nbsp;All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript, and all authors confirm its accuracy.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSaeedi P et al (2019) Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the International Diabetes Federation Diabetes Atlas. \u003cem\u003eDiabetes research and clinical practice\u003c/em\u003e, 157: p. 107843\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaghav A et al (2018) Financial burden of diabetic foot ulcers to world: a progressive topic to discuss always. Therapeutic Adv Endocrinol metabolism 9(1):29\u0026ndash;31\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Chen J, Kirsner R (2007) Pathophysiology of acute wound healing. Clin Dermatol 25(1):9\u0026ndash;18\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReinke J, Sorg Н (2012) Wound Repair and Regeneration. Eur Surg Res 49(1):35\u0026ndash;43\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahmoodi M (2018) Studying the effect of cord blood platelet rich plasma (PRP) on expression of miRNA21 and miRNA 146a in fibroblast cells. J Cell Sci Therapy\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBainbridge P (2013) Wound healing and the role of fibroblasts. J Wound Care 22(8):407\u0026ndash;411\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAddis R et al (2020) Fibroblast proliferation and migration in wound healing by phytochemicals: Evidence for a novel synergic outcome. Int J Med Sci 17(8):1030\u0026ndash;1042\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003edesJardins-Park HE, Foster DS, Longaker MT Fibroblasts and wound healing: an update. 2018, \u003cem\u003eFuture Medicine\u003c/em\u003e. pp. 491\u0026ndash;495\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnitua E et al (2004) Autologous platelets as a source of proteins for healing and tissue regeneration. Thromb Haemost 91(01):4\u0026ndash;15\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreen DM, Klink B (1998) Platelet gel as an intraoperatively procured platelet-based alternative to fibrin glue. Plast Reconstr Surg 101(4):1161\u0026ndash;1162\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO'Neill E et al (2001) Autologous platelet-rich plasma isolated using the Haemonetics Cell Saver 5 and Haemonetics MCS\u0026thinsp;+\u0026thinsp;for the preparation of platelet gel. Vox Sang 81(3):172\u0026ndash;175\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAydin O et al (2018) Platelet-Rich Plasma May Offer a New Hope in Suppressed Wound Healing When Compared to Mesenchymal Stem Cells. \u003cem\u003eJ Clin Med\u003c/em\u003e, 7(6): p.143\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCho EB et al (2019) Effect of platelet-rich plasma on proliferation and migration in human dermal fibroblasts. J Cosmet Dermatol 18(4):1105\u0026ndash;1112\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFilipowicz W, Bhattacharyya SN, Sonenberg N (2008) Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9(2):102\u0026ndash;114\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYi R et al (2006) Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs. Nat Genet 38(3):356\u0026ndash;362\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYi R et al (2008) A skin microRNA promotes differentiation by repressing \u0026lsquo;stemness\u0026rsquo;. Nature 452(7184):225\u0026ndash;229\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSonkoly E et al (2007) MicroRNAs: novel regulators involved in the pathogenesis of psoriasis? PLoS ONE 2(7):e610\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCiechomska M et al (2014) MiR-29a reduces TIMP-1 production by dermal fibroblasts via targeting TGF-β activated kinase 1 binding protein 1, implications for systemic sclerosis. PLoS ONE 9(12):e115596\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z et al (2009) Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. J Biol Chem 284(23):15676\u0026ndash;15684\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Rooij E et al (2008) Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e, 105(35): pp. 13027\u0026ndash;13032\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang B et al (2010) E-cadherin expression is regulated by miR-192/215 by a mechanism that is independent of the profibrotic effects of transforming growth factor-β. Diabetes 59(7):1794\u0026ndash;1802\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKato M et al (2007) MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-β-induced collagen expression via inhibition of E-box repressors. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e, 104(9): pp. 3432\u0026ndash;3437\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePadmakumari RG, Sherly CD, Ramesan RM (2022) Therapeutic delivery of nucleic acids for skin wound healing. Ther Deliv 13(6):339\u0026ndash;358\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFurman BL (2021) Streptozotocin-induced diabetic models in mice and rats. Curr protocols 1(4):e78\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChuncharunee A et al (2019) Invalid freeze-dried platelet gel promotes wound healing. Saudi Pharm J 27(1):33\u0026ndash;40\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiener C, Keller A, Meese E (2022) Emerging concepts of miRNA therapeutics: from cells to clinic. Trends Genet 38(6):613\u0026ndash;626\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCialdai F, Risaliti C, Monici M (2022) Role of fibroblasts in wound healing and tissue remodeling on Earth and in space. Front Bioeng Biotechnol 10:958381\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGharbia FZ et al (2023) Adult skin fibroblast state change in murine wound healing. Sci Rep 13(1):886\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalbott HE et al (2022) Wound healing, fibroblast heterogeneity, and fibrosis. Cell Stem Cell 29(8):1161\u0026ndash;1180\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y et al (2017) MicroRNA-192 regulates hypertrophic scar fibrosis by targeting SIP1. J Mol Histol 48(5):357\u0026ndash;366\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang X et al (2019) Smad interacting protein 1 influences transforming growth factor-β 1/Smad signaling in extracellular matrix protein production and hypertrophic scar formation. J Mol Histol 50:503\u0026ndash;514\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZabihi A, Mahmoodi M, Soltani S (2022) Effect of Fibroblast Cells Injection on Healing of Diabetic Wounds and Improvement of Skin Elasticity and Thickness in Male Rats. Res Med 46(3):105\u0026ndash;114\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohammadipour M et al (2019) Evaluation of human platelet gel effect on production process and expression level of VEGF in experimental skin wounds in rat. Sci J Iran Blood Transfus Organ 16(2):103\u0026ndash;115\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCho EB et al (2019) Effect of platelet-rich plasma on proliferation and migration in human dermal fibroblasts. J Cosmet Dermatol 18(4):1105\u0026ndash;1112\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNi X et al (2021) Adipose-derived stem cells combined with platelet-rich plasma enhance wound healing in a rat model of full-thickness skin defects. Stem Cell Res Ther 12:1\u0026ndash;11\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"b4c40d19-558d-4316-bf1e-c0b4d860e880","identifier":"10.13039/501100005117","name":"Birjand University of Medical Sciences","awardNumber":"IR.BUMS.REC.1400.413.","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Birjand University of Medical Sciences","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Diabetes, Diabetic Foot Ulcer, Fibroblast, miR-192, Platelet-Rich Plasma, wound healing","lastPublishedDoi":"10.21203/rs.3.rs-5290142/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5290142/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Diabetic Foot Ulcer (DFU) is a severe diabetic complication.Transplantation of skin substitutes, stem cells, and Platelet-Rich Plasma (PRP) treatments are promising tools to promote ulcer healing in diabetes. An important aspect of the remodelling phase of wound healing is collagen deposition. miR-192 increases the expression of \u003cem\u003eCOL1A2 \u003c/em\u003eby specifically targeting Smad-interacting protein 1 (SIP1). This study was designed to investigate the impact of combined treatment with platelet-rich plasma and fibroblast cells expressing miR-192 on the healing process of wounds using an experimental diabetic animal model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eAfter transfection of HDF cells and induction of increased miR-192 expression, relative changes in \u003cem\u003eCOL1A2\u003c/em\u003egene expression were determined by the RT-PCR method. Rats were randomly divided into 6 groups: non-diabetic control group, diabetic control, backbone, PRP, miR-192, and PRP+miR-192 groups. Diabetes was induced in male Wistar rats of all treated groups except non-diabetic control through a 21-day high-fat diet and an intraperitoneal injection of 40 mg/kg streptozotocin. A 10mm skin biopsy punch was used to create two full-thickness wounds on the dorsal part of the upper body in all six groups of animals. Hematoxylin-Eosin and Mason's trichrome staining were used to evaluate the wounds and analyze histological changes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe overexpression of miR-192 in HDF cells resulted in a significant increase in \u003cem\u003eCOL1A2\u003c/em\u003e gene expression, which was 15.77-fold higher than the control group. PRP and pLenti-III-miR-192-GFP-expressing cells significantly increased wound closure rates, granulation tissue area, and collagen fiber density in rats, according to a histological examination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThe combined use of PRP and HDFs expressing pLenti-III-miR-192-GFP speeds up the healing of wounds by increasing collagen expression, demonstrating the efficacy of this approach in improving wound healing results.\u003c/p\u003e","manuscriptTitle":"Overexpression of miR-192 in Fibroblasts accelerates wound healing in diabetic rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-21 14:09:54","doi":"10.21203/rs.3.rs-5290142/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fca47250-7e2c-4586-81cb-923b1af56174","owner":[],"postedDate":"October 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":39133781,"name":"Cell \u0026 Tissue Engineering"},{"id":39133782,"name":"Hematology"},{"id":39133783,"name":"Pathology"}],"tags":[],"updatedAt":"2024-10-21T14:09:54+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-21 14:09:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5290142","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5290142","identity":"rs-5290142","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}