C:\Users\JURNAL FKUSAKTI\Docume 135 *Postgraduate Biomedical Program, Faculty of Medicine, Universitas Islam Sultan Agung, Semarang **Department of Postgraduate Biomedical Science, Faculty of Medicine, Universitas Islam Sultan Agung, Semarang ***Stem Cell and Cancer Research Laboratory (SCCR), Faculty of Medicine, Univeritas Islam Sultan Agung, Semarang Correspondence : Dr. dr. Agung Putra, M. Si. Med. Candi Prambanan Utara Raya Kav. 1135-1136 Kelurahan Kalipancur, Manyaran, Kecamatan Ngaliyan, Semarang Phone: +62816 4251 646 Email: dr.agungptr@gmail.com ORCID ID: orcid.org/0000-0002- 9822-3119 Date of first submission, April 27, 2018 Date of final revised submission, July 23, 2018 Date of acceptance, July 24, 2018 This open access article is distributed under a Creative Commons Attribution- Non Commercial-Share Alike 4.0 International License ABSTRACT UNIVERSA MEDICINA Tumor necrosis factor-α-activated mesenchymal stem cells accelerate wound healing through vascular endothelial growth factor regulation in rats Aditya Nugraha* and Agung Putra**,*** BACKGROUND Wounds are areas of physical or thermal damage of the epithelial layer of skin or mucosa. The wound healing process consists of hemostasis, inflammation, proliferation, and remodeling. Mesenchymal stem cells (MSCs) play a role in wound healing by suppressing potent pro- inflammatory molecules, such as tumor necrosis factor-α (TNF-α), leading to macrophage polarization from the pro-inflammatory type to the pro- regeneration type characterized by increasing vascular endothelial growth factor (VEGF) production. MSCs are able to increase VEGF level in-vivo correlated with collagen synthesis. The objective of this study was to assess the role of TNF-α-activated MSCs on VEGF in rat wounds. METHODS An experimental animal study with post-test only control group design was performed involving 24 Wistar rats. The rats were randomized into four groups consisting of one control (K) and three treatment groups (P) (activated MSCs at doses of 3x105, 6x105, and 12x105 cells, respectively). The measurement of VEGF levels was done using ELISA assay while the collagen analysis was performed by light microscopy. One way ANOVA and Post Hoc LSD were used to analyze the data. RESULTS The results showed a significant increase in VEGF levels (p <0.05) on day 3 and then a significant decrease on day 5 along with a significant increase in the amount of collagen on day 7 (p<0.05). CONCLUSION This study demonstrated that TNF-α-activated MSCs were able to regulate VEGF levels and collagen synthesis in wound healing in rats. The molecular mechanism by which TNF-α-activated MSCs stimulate cutaneous wound healing should be clarified further. Keywords: Mesenchymal stem cells, tumor necrosis factor-α, vascular endothelial growth factor, collagen, rats DOI: http://dx.doi.org/10.18051/UnivMed.2018.v37.135-142 ORIGINAL ARTICLE pISSN: 1907-3062 / eISSN: 2407-2230 May-August, 2018 Vol.37- No.2 Cite this article as: Nugraha A, Putra A. Tumor necrosis factor--activated mes- enchymal stem cells accelerate wound healing through vascular endothelial growth factor regulation in rats. Univ Med 2018;37:135-42. doi: 10.18051/ UnivMed.2018.v37.135-142 136 Nugraha, Putra Activated MSCs regulate VEGF & collagen INTRODUCTION Wounds are defined as areas of physical or thermal damage in the cutaneous or mucosal epithelium that can be categorized as acute and chronic. (1) The wound healing process consists of the phases of hemostasis, inflammation, proliferation, and remodeling.(2) The inflammatory phase is initiated by neutrophil migration to the wound site as a result of stimulation by various chemoattractant molecules, such as tumor necrosis factor-α (TNF-α) or interleukin-1 (IL- 1). The proliferative phase is marked by an increase in a number of growth factors, such as vascular endothelial growth factor (VEGF).(3,4) The increase of these molecules may stimulate fibroblast proliferation that results in increased collagen synthesis, in addition to angiogenesis.(1– 3) Several studies report that mesenchymal stem cells (MSCs) play an important role in wound healing, particularly in the inflammatory phase, because of their immunoregulator y characteristics, so that they are able to control the inflammatory process.(5) Mesenchymal stem cells are adult stem cells with the characteristics of adhering to plastic culture dishes, having various surface markers, such as CD73(+), CD90(+), CD105(+), CD45(- ), CD34(-), CD14(-) or CD11 (-), CD79(-) or CD19 (-) and HLA-DR(-),(6) and being able to differentiate into various cells, such as neurons and adipocytes.(6,7) Mesenchymal stem cells also play a role in immunoregulation of inflammation, by suppressing the release of pro-inflammatory molecules from various inflammatory cells, so accelerating the transition from the inflammatory phase into the regenerative phase.(1,8) Several potent pro-inflammatory cytokines, particularly TNF-α, play a n important role in MSCs activation.(9,10) Activated MSCs release various molecules, such as prostaglandin E 2 (PGE 2 ) that will bind to their EP4 and EP2 receptors on macrophages, so effecting the polarization of macrophages from the inflammatory into the regenerative type.(11) This is marked by the release of the anti-inflammatory interleukin-10 (IL-10).(12) Specifically, MSCs activation occurs through binding of TNF-α molecules to MSC receptors that impact on the activation of the nuclear factor kappa-B (NF-κB) pathway, so that several anti-inflammatory and proliferative molecules are expressed.(11) Theoretically, the TNF-α-activated MSCs are able to express va rious pro-r egenera tive growth factors, particularly VEGF, platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF), that can activate fibroblasts and impact on collagen synthesis.(9) Previous studies have reported that MSCs activated by the pro-inflammatory cytokine interferon gamma (IFN-γ) are able to increase wound regeneration and tensile strength that is correlated with the amount of collagen.(13) On the other hand, MSCs that are activated in vitro by TNF-α, lipopolysaccharide (LPS) and hypoxia, are able to increase the production of growth factors, particularly VEGF,(14) but the role of TNF-α- activated MSCs on VEGF concentrations and their relationship with collagen are as yet unknown. The aim of the present study was to evaluate the effect of MSCs that are activated by TNF-α at a dose of 10 ng/ml on VEGF and collagen concentrations in rats with incised wounds. METHODS Research design This study used a post-test only control group design and was conducted in the Stem Cell and Cancer Research Laboratory, Faculty of Medicine, Universitas Islam Sultan Agung (Unissula), Semarang, from 2 April to 25 August 2017. Animals The subjects of this study were 4 to 6-month old Wistar rats weighing 250-300 grams, that were certified healthy by the Agricultural and Fishery Service (Dinas Pertanian dan Perikanan) of Salatiga City under no. 524.3/0211/421. The sample size calculation was according to the 137 Federer formula for determining the required sample size: (n-1) x (t-1) 15. The sample used in this study comprised 6 animals per group with an estimated drop-out rate of 10%, so that the total sample size was 24 rats (n=6 rats/group). The rats were randomly divided into 4 groups (3 intervention groups P1-P3 and 1 control group K). The inclusion criteria were: purebred rats aged 4 to 6 months, weighing 250-300 grams, and without apparent anatomical defects. The exclusion criteria were: not active during the 7- day adaptation period and/or having apparent anatomical defects. The drop-out criterion was d yi ng i n th e c our se of t he e xper imen ta l interventions. The incised rats were injected with different doses of MSCs. The control group was injected with phosphate buffered saline (PBS). Groups P1, P2, and P3 received subcutaneous injections of activated MSCs at doses of 3x 105, 6 x 105 and 12 x 105 cells, respectively. The interventions were applied once daily in the morning. Surgical procedure Paravertebral full-thickness dorsal skin incisions were made aseptically with a No. 15 scalpel (B. Braun Aesculap AG, Tuttlingen, Germany) under general anesthesia in the longitudinal axis at least 1 cm apart a nd perpendicular to the skin cleavage lines. As a result, minimal strain was applied to the skin, allowing for direct visual control of the depth of the incision. For each incision, the dorsal muscles were the limits at which to stop.(15) Isolation of MSCs The umbilical cord of a female rat at 19 days of pregnancy was washed in PBS. The umbilical blood vessels were removed, then the umbilicus was cut into lengths of 2-5 mm using a sterile knife and the sections distributed evenly on a 66-mm culture dish. The medium used was Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO) mixed with 10% PBS, 100 IU/ml penicillin/streptomycin (GIBCO, Invitrogen), then incubated at 37oC with 5% CO 2 . The medium was refreshed once in 3 days.(16) Characteristics of the MSC phenotype The expression of the MSCs isolated from the umbilical cord of the female rat was evaluated by immunocytochemistry using MSC-positive markers. After the MSCs were 60-80% confluent, they were harvested and grown on coverslips, then fixed with 4% paraformaldehyde in 90% ethanol for 15 minutes at 4oC. The cells were incubated with CD73 and CD105 primary antibody as MSC-positive markers (1:100; BD Pharmingen, San Diego, CA, USA) for 60 minutes at room temperature, washed in PBS for 10 minutes then secondary antibody (1:2500) was added, the cells left to stand for 15 minutes at room temperature, then counterstained with diaminobenzene (DAB) (Santa Cruz Biotech) and observed under the microscope.(16) In-vitro differentiation The mesenchymal stem cells were grown in culture dishes at densities of 5x103 and 1x104 cells/well, to which was added osteogenic induction medium containing 10 mmol/L β- glycerophos phate, 10 -7 mol/L/ 0.1 µM dexamethasone, 50µmol/L ascorbate-2-phosphate (Sigma-Aldrich, Louis St, MO) and 10 % fetal bovine serum (FBS) in DMEM. Osteogenic differentiation was observed by Alizarin Red staining after 21 days of induction to find calcium deposits. The cells were rinsed in PBS and fixed with cold 70% ethanol (v/v) for 1 hour at room temperature, then rinsed three times with twice- distilled water. A volume of 1 ml 2% Alizarin Red solution (w/v) (pH 4.1-4.3) was added and the cells incubated for 30 minutes at room temperature, then rinsed four times in twice- distilled water.(17) TNF-α induction of MSCs MSCs (1x104 cells/well) were supplemented with 10 ng/ml recombinant TNF-α (BioLegend, San Diego, CA) on a 24-well plate using alphaMEM medium (Sigma-Aldrich, Louis St, Univ Med Vol. 37 No.2 138 Nugraha, Putra Activated MSCs regulate VEGF & collagen A B C MO), then incubated for 24 hours at 37o with 5% CO 2 . Rat VEGF ELISA assay The serum VEGF assay by means of an ELISA kit (Fine test, China) was performed at room temperatur e according to the manufacturer’s instructions. The ELISA plate was coated with capture antibody and incubated overnight at 4oC. The wells were washed, then blocked for 1 hour, after which they were incubated with rat VEGF standard solution and the intervention and control rat sera for 2 hours, then diluted to 1:1 00 so that the VEGF concentrations could be determined on the standard curves. After washing, the wells were incubated with detection antibody for one hour, then washed again several times. The wells were incubated with Avidin-HRP for 30 minutes, washed thoroughly, and incubated with substrate solution for 15 minutes, which was followed by the addition of the stop solution. The results were analyzed at a wavelength of 450 Å using a microplate reader. The VEGF concentrations (pg/ ml) were measured on days 3 and 5 of the experiment. Preparation of collagen slides Skin tissue was collected and placed in 10% neutral-buffered formalin then embedded in paraffin or Histogel (Thermo Scientific, Watham, MA, USA). Subsequently the tissue paraffin block was cut on a microtome to a thickness of 5-10 µm then stained with Masson Trichrome and observed under the microscope. The collagen density was calculated from the area of the collagenous tissue formed on each slide (%). Statistical analysis The amount of collagen and the VEGF concentration were presented descriptively. Data processing was performed using SPSS 23.0 for Windows. The test of normality used the Shapiro- Wilk test and the test of homogeneity used Levene’s statistical test. Subsequently a parametric difference test was performed followed by ANOVA and LSD post-hoc test. Ethical clearance All research activities were in accordance to and approved by the Commission on Test Animal Ethics (Komisi Etik Hewan Uji), Faculty of Medicine, Universitas Islam Sultan Agung, Semarang, under No. 204/VI/2017/Komisi Bioetik. RESULTS Characteristics of MSCs The characteristics of the MSCs isolated by in-vitro dish culture until the cell population was at 80% confluence were as follows. The cells were spindle-shaped, resembling fibroblasts (Figure 1a). The confluent MSCs that were tested using an immunocytochemical technique showed positive expression of various protein surface markers, particularly CD105(+) (Figure 1b) and CD73(+) (Figure 1c), as indicated by the development of a brown color in the population of MSCs. Tests for differentiating the MSCs were Figure 1. Characteristics and differentiation of cultured mesenchymal stem cells Legend: (a) Umbilical cord candidate MSCs from in-vitro culture (b) CD105 expression, (c) CD73 expression. MSCs are brownish in color (d) Osteogenic differentiation with Alizarin Red staining 139 Univ Med Vol. 37 No.2 performed by administration of osteogenic medium and showed that MSCs were capable of differentiating into osteogenic cells, as indicated in the cell population by a red color from Alizarin Red staining (Figure 1d). VEGF concentration and collagen density The mean VEGF concentration increased on day 3, then decreased on day 5. The VEGF concentration on day 3 showed a significant increase (p<0.05) (Table 1) with the following mean values: K = 29.17 ±11.67 pg/mL; P1 = 87.38 ±17.38 pg/mL; P2 = 112.99 ±19.91 pg/mL; P 3= 98.52 ± 43.88 pg/mL. However, on day 5 a non significant decreased was found (p>0.05) (Table 1). The mean collagen density on day 7 differed significantly between the control group and the intervention groups (p<0.05). Mean collagen density was highest in group P3 (2.83 ± 0.80) and lowest in the control group, group P1, and group P2 (1.33 ± 0.51, 1.50 ± 0.54, and 2.50 ± 0.54, respectively) (Table 1). These results were also supported by the microscopic picture of collagen preparations with Masson Trichrome staining. The collagen was colored blue and filled the field of view (Figure 2). The results of the post-hoc analysis showed that mean VEGF concentration on days 3 and 5 differed significantly between the control group and groups P1, P2 and P3 (p<0.05). However, mean VEGF concentration on days 3 and 5 showed no significant difference between groups P1, P2 and P3 (Table 2). Mean collagen density on day 7 differed significantly between the control group and groups P1 and P2, between groups P1 and P3, and between groups P2 and P3. DISCUSSION Our study results showed that serum VEGF concentration increased significantly on day 3 in rat wounds treated with TNF-α activated MSCs, giving the impression that MSCs are able to accelerate the transition from the inflammatory phase into the proliferative phase through the polarization of type M1 proinflammatory Day Intervention group (mean±SD) p value Control (n=6) P1 (n=6) P2 (n=6) P3 (n=6) VEGF on day 3 (pg/mL) 29.17 ± 11.67 87.38 ± 17.38 112.99 ± 19.91 98.52 ± 43.88 0.001 VEGF on day 5 (pg/mL) 67.45 ± 53.65 5.09 ± 3.42 22.05 ± 8.23 15.93 ± 34.49 0.012 Collagen on day 7 (%) 1.33 ± 0.51 1.50 ± 0.54 2.50 ± 0.54 2.83 ± 0.80 0.000 Table 1. Mean VEGF concentration on days 3 and 5 and collagen density on day 7 *Significant at p<0.05; Note: P1: activated MSCs at dose of 3x 105 cells; P2: activated MSCs at dose of 6 x 105 cells; P3: activated MSCs at dose of 12 x 105 cells Figure 2. Microscopic preparations showing collagen density, with Masson trichrome staining. Collagen is bluish in color. Magnification 100x Legend : Panels A,B, C and D represent controls, P1, P2 and P3, respectively 140 Nugraha, Putra Activated MSCs regulate VEGF & collagen ma cr o pha ge s i n to typ e M 2 pr o l if e r a ti ve macrophages.(18) This is in line with previous studies stating that MSCs activated by 50 ng/ml of TNF-α are able to increase VEGF expression significantly in 24 hours post-induction,(14) indicating that the MSCs are induced by the T NF-α i n f l a mma to r y s ign a l s t o s e c r e te proliferative molecules, such as VEGF, in addition to anti-inflammatory molecules.(19) Other studies have reported that in normal wound healing the serum VEGF concentration starts to increase on the fourth day and continues until the seventh day of the prolifer ative phase.(1,20) T his gives the impression that activated MSCs are more potent in suppressing inflammation in wounds and promote the activation of various pro-proliferative cells, so accelerating the wound healing process. On the other hand, our study also found that the VEGF concentration decreased significantly on day 5 (p<0.05). We surmise that this decrease in VEGF concentration on day 5 was due to the more rapid transition from the proliferative to the remodeling phase, as a result of the administration of TNF-α-activated MSCs. This agrees with previous studies stating that in the remodeling phase the VEGF concentration decreases significantly, marking the end of the proliferative phase.(21) A high VEGF concentration in the remodeling phase may cause an interference with wound healing that is marked by tissue fibrosis.(22) Our study also found a significantly increased collagen density in wounds receiving TNF-α-activated MSCs. The collagen density is shown by the microscopic picture of a bluish color in preparations with Masson’s trichrome staining. This collagen density correlates with the number of TNF-α-activated MSCs administered at each of the interventions. These results are supported by previous studies, in that MSC administration is able to accelerate wound regeneration by modulating collagen synthesis, in addition to inhibiting apoptosis.(23) We think that the increase in collagen density on day 7 is related to the ability of TNF-α-ac tivated MSCs for pa racrine expression of various growth factors, such as VEGF, so accelerating the transition from the inflammatory to the proliferative phase, in addition to differentiating into fibroblasts and activating endogenous stem cells to promote fibroblast formation and collagen synthesis. This agrees with previous studies stating that fibroblasts that express large amounts of VEGF may trigger collagen formation.(24) One limitation of the present study is that we did not determine the concentrations of PDGF and transforming growth factor (TGF) that are potent mediators of fibroblast activation in collagen synthesis. We also did not measure VEGF concentrations at baseline and at the end o f t h e i n te r ve nt i o ns t o d e t e rmi ne t he i r r e l a ti o ns hi p w it h t he in f l a mma t or y a n d remodeling phases. This study has the potential to become in the future one of the solutions to the problem of accelerating wound healing, particularly chronic wounds. Mean difference p value VEGF on day 3 Controls P1 P2 P3 P1 P2 P3 P2 P3 62.36 45.40 51.51 16.96 10.848 6.114 0.002* 0.018* 0.009* 0.339 0.587 0.727 VEGF on day 5 Controls P1 P2 P3 P1 P2 P3 P2 P3 57.21 82.82 68.35 25.61 11.13 14.47 0.003* 0.000* 0.001* 0.144 0.513 0.398 Collagen on day 7 Controls P1 P2 P3 P1 P2 P3 P2 P3 1.00 1.33 0.16 0.33 0.16 1.500 0.003* 0.000* 0.576 0.269 0.001* 0.000* Table 2. Post-hoc test for VEGF on days 3 and 5 and collagen on day 7 *Significant at p<0.05; Note : P1 : activated MSCs at dose of 3x 105 cells; P2 : activated MSCs at dose of 6 x 105 cells; P3 : activated MSCs at dose of 12 x 105 cells 141 Univ Med Vol. 37 No.2 CONCLUSION This study demonstrated that TNF-α- activated MSCs are able to regulate VEGF concentrations and collagen density in wound healing. The VEGF concentrations expressed by activated MSCs accelerate the transition from the inflammatory to the regenerative phase and trigger collagen synthesis in wound healing. CONFLICT OF INTEREST Competing interests: no relevant disclosures FINANCIAL DISCLOSURE The authors did not receive any specific grant from any public funding agency. 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