Hypoxic mesenchymal stem cell‐conditioned medium accelerates wound healing by regulating IL‐10 and TGF‐β levels in a full‐thickness‐wound rat model

https://doi.org/10.22146/ijbiotech.63914

Adi Muradi Muhar(1), Faizal Mukharim(2), Dedy Hermansyah(3), Agung Putra(4*), Nurul Hidayah(5), Nur Dina Amalina(6), Iffan Alif(7)

(1) Department of Surgical, Medical Faculty, Universitas Sumatera Utara (USU), Medan 20155, Indonesia
(2) Graduate student of Medical Faculty, Sultan Agung Islamic University (UNISSULA), Semarang 50112, Indonesia
(3) Department of Surgical, Medical Faculty, Universitas Sumatera Utara (USU), Medan 20155, Indonesia
(4) Stem Cell and Cancer Research (SCCR), Medical Faculty, Sultan Agung Islamic University (UNISSULA), Semarang 50112, Indonesia; Department of Pathology, Medical Faculty, Sultan Agung Islamic University (UNISSULA), Semarang 50112, Indonesia; Department of Postgraduate Biomedical Science, Medical Faculty, Sultan Agung Islamic University (UNISSULA), Semarang 50112, Indonesia
(5) Stem Cell and Cancer Research (SCCR), Medical Faculty, Sultan Agung Islamic University (UNISSULA), Semarang 50112, Indonesia
(6) Stem Cell and Cancer Research (SCCR), Medical Faculty, Sultan Agung Islamic University (UNISSULA), Semarang 50112, Indonesia; Pharmacy Study Program, Faculty of Mathematics and Natural Sciences, Universitas Negeri Semarang, Semarang 50229, Indonesia
(7) Stem Cell and Cancer Research (SCCR), Medical Faculty, Sultan Agung Islamic University (UNISSULA), Semarang 50112, Indonesia
(*) Corresponding Author

Abstract


Full‐thickness wound healing is a complex process requiring a well‐orchestrated mechanism of various factors, including cytokines, particularly interleukin (IL)‐10 and transforming growth factor (TGF)‐β. IL‐10 and TGF‐β act as robust anti‐inflammatory cytokines in accelerating the wound healing process by regulating myofibroblasts. Hypoxic mesenchymal stem cell‐conditioned medium (hypMSC‐CM) containing cytokines potentially contribute to accelerate wound repair without scarring through the paracrine mechanism. This study aims to observe the role of hypMSC‐CM in controlling TGF‐β and IL‐10 levels to accelerate full‐thickness wound repair and regeneration. A total of 24 male Wistar rats were used in this study. Six healthy rats as a sham group and 18 rats were created as full‐thickness‐wound animal models using a 6 mm punch biopsy. The animals were randomly assigned into three groups (n = 6) consisting of two treatment groups treated with hypMSC‐CM at a low dose (200 µL hypMSC‐CM with 2 g water‐based gel added) and a high dose (400 µL hypMSC‐CM with 2 g water‐based gel added) and a control group (2 g water‐based gel only). The IL‐10 and TGF‐β levels were examined by ELISA. The results showed a significant increase in IL‐10 levels on day 3 after hypMSC‐CM treatment, followed by a decrease in platelet‐derived growth factor (PDGF) levels on days 6 and 9. In line with this finding, the TGF‐β levels also increased significantly on day 3 and then linearly decreased on days 6 and 9. HypMSC‐CM administra‐ tion may thus promote wound healing acceleration by controlling IL‐10 and TGF‐β levels in a full‐thickness‐wound rat model.


Keywords


fibroblast; hypMSC‐CM; IL‐10; TGF‐β; wound healing

Full Text:

PDF


References

Ahangar P, Mills SJ, Cowin AJ. 2020. Mesenchymal stem cell secretome as an emerging cell­free alternative for improving wound repair. Int. J. Mol. Sci. 21(19):1– 15. doi:10.3390/ijms21197038.

Alagesan S, Brady J, Byrnes D, Fandiño J, Masterson C, McCarthy S, Laffey J, O’Toole D. 2022. Enhancement strategies for mesenchymal stem cells and related therapies. Stem Cell Res. Ther. 13(1):1–13. doi:10.1186/s13287­022­02747­w.

Batsali AK, Pontikoglou C, Koutroulakis D, Pavlaki KI, Damianaki A, Mavroudi I, Alpantaki K, Kouvidi E, Kontakis G, Papadaki HA. 2017. Differential expression of cell cycle and WNT pathway­related genes accounts for differences in the growth and differentiation potential of Wharton’s jelly and bone marrowderived mesenchymal stem cells. Stem Cell Res. Ther. 8(1):1–18. doi:10.1186/s13287­017­0555­9.

Beegle J, Lakatos K, Kalomoiris S, Stewart H, Isseroff RR, Nolta JA, Fierro FA. 2015. Hypoxic preconditioning of mesenchymal stromal cells induces metabolic changes, enhances survival, and promotes cell retention in vivo. Stem Cells 33(6):1818–1828. doi:10.1002/stem.1976.

Castrén E, Sillat T, Oja S, Noro A, Laitinen A, Konttinen YT, Lehenkari P, Hukkanen M, Korhonen M. 2015. Osteogenic differentiation of mesenchymal stromal cells in two­dimensional and three­dimensional cultures without animal serum. Stem Cell Res. Ther. 6(1):1–13. doi:10.1186/s13287­015­0162­6.

Darlan DM, Munir D, Putra A, Alif I, Amalina ND, Jusuf NK, Putra IB. 2022. Revealing the decrease of indoleamine 2,3­dioxygenase as a major constituent for B cells survival post­mesenchymal stem cells co­cultured with peripheral blood mononuclear cell (PBMC) of systemic lupus erythematosus (SLE) patients. Med. Glas. 19(1). doi:10.17392/1414­21.

Dominici M, Le Blanc K, Mueller I, Slaper­Cortenbach I, Marini FC, Krause DS, Deans RJ, Keating A, Prockop DJ, Horwitz EM. 2006. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 8(4):315–317. doi:10.1080/14653240600855905.

Drawina P, Putra A, Nasihun T, Prajoko YW, Dirja BT, Amalina ND. 2022. Increased serial levels of plateletderived growth factor using hypoxic mesenchymal stem cell­conditioned medium to promote closure acceleration in a full­thickness wound. Indones. J. Biotechnol. 27(1):36. doi:10.22146/ijbiotech.64021.

El Agha E, Kramann R, Schneider RK, Li X, Seeger W, Humphreys BD, Bellusci S. 2017. Mesenchymal Stem Cells in Fibrotic Disease. Cell Stem Cell 21(2):166–177. doi:10.1016/j.stem.2017.07.011.

Hamra NF, Putra A, Tjipta A, Amalina ND, Nasihun T. 2021. Hypoxia mesenchymal stem cells accelerate wound closure improvement by controlling α­smooth muscle actin expression in the full­thickness animal model. Open Access Maced. J. Med. Sci. 9:35–41. doi:10.3889/oamjms.2021.5537.

He X, Dong Z, Cao Y, Wang H, Liu S, Liao L, Jin Y, Yuan L, Li B, Bolontrade MF. 2019. MSC­Derived Exosome Promotes M2 Polarization and Enhances Cutaneous Wound Healing. Stem Cells Int. 2019:1–16. doi:10.1155/2019/7132708.

Ho CH, Lan CW, Liao CY, Hung SC, Li HY, Sung YJ. 2018. Mesenchymal stem cells and their conditioned medium can enhance the repair of uterine defects in a rat model. J. Chinese Med. Assoc. 81(3):268–276. doi:10.1016/j.jcma.2017.03.013.

Jiang D, Scharffetter­Kochanek K. 2020. Mesenchymal Stem Cells Adaptively Respond to Environmental Cues Thereby Improving Granulation Tissue Formation and Wound Healing. Front. Cell Dev. Biol. 8:1– 13. doi:10.3389/fcell.2020.00697.

Kucharzewski M, Rojczyk E, Wilemska­Kucharzewska K, Wilk R, Hudecki J, Los MJ. 2019. Novel trends in application of stem cells in skin wound healing. Eur. J. Pharmacol. 843:307–315. doi:10.1016/j.ejphar.2018.12.012.

Li M, Xu J, Shi T, Yu H, Bi J, Chen G. 2016. Epigallocatechin­3­gallate augments therapeutic effects of mesenchymal stem cells in skin wound healing. Clin. Exp. Pharmacol. Physiol. 43(11):1115– 1124. doi:10.1111/1440­1681.12652.

Lurier EB, Dalton D, Dampier W, Raman P, Nassiri S, Ferraro NM, Rajagopalan R, Sarmady M, Spiller KL. 2017. Transcriptome analysis of IL­10­ stimulated (M2c) macrophages by next­generation sequencing. Immunobiology 222(7):847–856. doi:10.1016/j.imbio.2017.02.006.

Menssen A, Häupl T, Sittinger M, Delorme B, Charbord P, Ringe J. 2011. Differential gene expression profiling of human bone marrow­derived mesenchymal stem cells during adipogenic development. BMC Genomics 12:461. doi:10.1186/1471­2164­12­461.

Mesquita I, Ferreira C, Barbosa AM, Ferreira CM, Moreira D, Carvalho A, Cunha C, Rodrigues F, Dinis­Oliveira RJ, Estaquier J, Castro AG, Torrado E, Silvestre R. 2018. The impact of IL­ 10 dynamic modulation on host immune response against visceral leishmaniasis. Cytokine 112:16–20. doi:10.1016/j.cyto.2018.07.001.

Muhar AM, Putra A, Warli SM, Munir D. 2019. Hypoxiamesenchymal stem cells inhibit intra­peritoneal adhesions formation by upregulation of the il­10 expression. Open Access Maced. J. Med. Sci. 7(23):3937– 3943. doi:10.3889/oamjms.2019.713.

Nakanishi K, Sato Y, Mizutani Y, Ito M, Hirakawa A, Higashi Y. 2017. Rat umbilical cord blood cells attenuate hypoxic­ischemic brain injury in neonatal rats. Sci. Rep. 7:1–14. doi:10.1038/srep44111.

Numakura S, Uozaki H, Kikuchi Y, Watabe S, Togashi A, Watanabe M. 2019. Mesenchymal stem cell marker expression in gastric cancer stroma. Anticancer Res. 39(1):387–393. doi:10.21873/anticanres.13124.

Ohashi CM, Caldeira FAM, Feitosa DJS, Valente AL, Dutra PRW, Miranda MdS, Santos SdSD, Brito MVH, Ohashi OM, Yasojima EY. 2016. Stem cells from adipose tissue improve the time of wound healing in rats. Acta Cir. Bras. 31(12):821–825. doi:10.1590/S0102­ 865020160120000007.

Putra A, Alif I, Hamra N, Santosa O, Kustiyah AR, Muhar AM, Lukman K. 2020a. Msc­released tgf­β regulate α­sma expression of myofibroblast during wound healing. J. Stem Cells Regen. Med. 16(2):73–79. doi:10.46582/jsrm.1602011.

Putra A, Antari AD, Kustiyah AR, Intan YSN, Sadyah NAC, Wirawan N, Astarina S, Zubir N, Munir D. 2018a. Mesenchymal stem cells accelerate liver regeneration in acute liver failure animal model. Biomed. Res. Ther. 5(11):2802–2810. doi:10.15419/bmrat.v5i11.498.

Putra A, Ridwan FB, Putridewi AI, Kustiyah AR, Wirastuti K, Sadyah NAC, Rosdiana I, Munir D. 2018b. The role of tnf­α induced mscs on suppressive inflammation by increasing tgf­β and il­10. Open Access Maced. J. Med. Sci. 6(10):1779–1783. doi:10.3889/oamjms.2018.404.

Putra A, Rosdiana I, Darlan DM, Alif I, Hayuningtyas F, Wijaya I, Aryanti R, Makarim FR, Antari AD. 2020b. Intravenous Administration is the Best Route of Mesenchymal Stem Cells Migration in Improving Liver Function Enzyme of Acute Liver Failure. Folia Med. (Plovdiv). 62(1):52–58. doi:10.3897/folmed.62.e47712.

Rahmani F, Ziaee V, Assari R, Sadr M, Rezaei A, Sadr Z, Reza Raeeskarami S, Hassan Moradinejad M, Aghighi Y, Rezaei N. 2019. Interleukin 10 and transforming growth factor beta polymorphisms as risk factors for kawasaki disease: A case­control study and meta­analysis. Avicenna J. Med. Biotechnol. 11(4):325–333.

Restimulia L, Ilyas S, Munir D, Putra A, Madiadipoera T, Farhat F, Sembiring RJ, Ichwan M, Amalina ND. 2022. Rats’ umbilical­cord mesenchymal stem cells ameliorate mast cells and Hsp70 on ovalbumininduced allergic rhinitis rats. Med. Glas. 19(1). doi:10.17392/1421­21.

Rezapour­Lactoee A, Yeganeh H, Gharibi R, Milan PB. 2020. Enhanced healing of a full­thickness wound by a thermoresponsive dressing utilized for simultaneous transfer and protection of adipose­derived mesenchymal stem cells sheet. J. Mater. Sci. Mater. Med. 31(11):101. doi:10.1007/s10856­020­06433­2.

Sabry D, Mohamed A, Monir M, Ibrahim HA. 2019. The effect of mesenchymal stem cells derived microvesicles on the treatment of experimental CCL4 induced liver fibrosis in rats. Int. J. Stem Cells 12(3):400–409. doi:10.15283/IJSC18143.

Saheli M, Bayat M, Ganji R, Hendudari F, Kheirjou R, Pakzad M, Najar B, Piryaei A. 2020. Human mesenchymal stem cells­conditioned medium improves diabetic wound healing mainly through modulating fibroblast behaviors. Arch. Dermatol. Res. 312(5):325– 336. doi:10.1007/s00403­019­02016­6.

Sapudom J, Wu X, Chkolnikov M, Ansorge M, Anderegg U, Pompe T. 2017. Fibroblast fate regulation by time dependent TGF­β1 and IL­10 stimulation in biomimetic 3D matrices. Biomater. Sci. 5(9):1858– 1867. doi:10.1039/c7bm00286f.

Schreier C, Rothmiller S, Scherer MA, Rummel C, Steinritz D, Thiermann H, Schmidt A. 2018. Mobilization of human mesenchymal stem cells through different cytokines and growth factors after their immobilization by sulfur mustard. Toxicol. Lett. 293:105–111. doi:10.1016/j.toxlet.2018.02.011.

Shi J, Li J, Guan H, Cai W, Bai X, Fang X, Hu X, Wang Y, Wang H, Zheng Z, Su L, Hu D, Zhu X. 2014. Anti­fibrotic actions of interleukin­ 10 against hypertrophic scarring by activation of PI3K/AKT and STAT3 signaling pathways in scarforming fibroblasts. PLoS One 9(5):e98228. doi:10.1371/journal.pone.0098228.

Steen EH, Wang X, Balaji S, Butte MJ, Bollyky PL, Keswani SG. 2020. The Role of the Anti­Inflammatory Cytokine Interleukin­10 in Tissue Fibrosis. Adv. Wound Care 9(4):184–198. doi:10.1089/wound.2019.1032.

Sun ZL, Feng Y, Zou ML, Zhao BH, Liu SY, Du Y, Yu S, Yang ML, Wu JJ, Yuan ZD, Lv GZ, Zhang JR, Yuan FL. 2020. Emerging role of IL­10 in hypertrophic scars. Front. Med. 7:1–8. doi:10.3389/fmed.2020.00438.

Sungkar T, Putra A, Lindarto D, Sembiring RJ. 2020. Intravenous Umbilical Cord­derived Mesenchymal Stem Cells Transplantation Regulates Hyaluronic Acid and Interleukin­10 Secretion Producing Lowgrade Liver Fibrosis in Experimental Rat. Med. Arch. (Sarajevo, Bosnia Herzegovina) 74(3):177– 182. doi:10.5455/medarh.2020.74.177­182.

Wu P, Zhang B, Shi H, Qian H, Xu W. 2018. MSCexosome: A novel cell­free therapy for cutaneous regeneration. Cytotherapy 20(3):291–301. doi:10.1016/j.jcyt.2017.11.002.

Xu J, Zanvit P, Hu L, Tseng PY, Liu N, Wang F, Liu O, Zhang D, Jin W, Guo N, Han Y, Yin J, Cain A, Hoon MA, Wang S, Chen WJ. 2020. The Cytokine TGF­β Induces Interleukin­31 Expression from Dermal Dendritic Cells to Activate Sensory Neurons and Stimulate Wound Itching. Immunity 53(2):371–383. doi:10.1016/j.immuni.2020.06.023.

Xu Y, Tang X, Yang M, Zhang S, Li S, Chen Y, Liu M, Guo Y, Lu M. 2019. Interleukin 10 genemodified bone marrow­derived dendritic cells attenuate liver fibrosis in mice by inducing regulatory T cells and inhibiting the TGF­β/Smad signaling pathway. Mediators Inflamm. 2019:1–15. doi:10.1155/2019/4652596.

Yew TL, Hung YT, Li HY, Chen HW, Chen LL, Tsai KS, Chiou SH, Chao KC, Huang TF, Chen HL, Hung SC. 2011. Enhancement of wound healing by human multipotent stromal cell conditioned medium: The paracrine factors and p38 MAPK activation. Cell Transplant. 20(5):693–706. doi:10.3727/096368910X550198.

Yustianingsih V, Sumarawati T, Putra A. 2019. Hypoxia enhances self­renewal properties and markers of mesenchymal stem cells. Universa Med. 38(3):164. doi:10.18051/univmed.2019.v38.164­171.

Zhao G, Liu F, Liu Z, Zuo K, Wang B, Zhang Y, Han X, Lian A, Wang Y, Liu M, Zou F, Li P, Liu X, Jin M, Liu JY. 2020. MSC­derived exosomes attenuate cell death through suppressing AIF nucleus translocation and enhance cutaneous wound healing. Stem Cell Res. Ther. 11(1):1–18. doi:10.1186/s13287­020­ 01616­8.

Zheng X, Ding Z, Cheng W, Lu Q, Kong X, Zhou X, Lu G, Kaplan DL. 2020. Microskin­Inspired Injectable MSC­Laden Hydrogels for Scarless Wound Healing with Hair Follicles. Adv. Healthc. Mater. 9(10):1–14. doi:10.1002/adhm.202000041.

Ahangar P, Mills SJ, Cowin AJ. 2020. Mesenchymal stem
cell secretome as an emerging cell­free alternative for
improving wound repair. Int. J. Mol. Sci. 21(19):1–
15. doi:10.3390/ijms21197038.
Alagesan S, Brady J, Byrnes D, Fandiño J, Masterson C,
McCarthy S, Laffey J, O’Toole D. 2022. Enhancement strategies for mesenchymal stem cells and related therapies. Stem Cell Res. Ther. 13(1):1–13.
doi:10.1186/s13287­022­02747­w.
Batsali AK, Pontikoglou C, Koutroulakis D, Pavlaki KI,
Damianaki A, Mavroudi I, Alpantaki K, Kouvidi E,
Kontakis G, Papadaki HA. 2017. Differential expression of cell cycle and WNT pathway­related genes accounts for differences in the growth and differentiation potential of Wharton’s jelly and bone marrowderived mesenchymal stem cells. Stem Cell Res.
Ther. 8(1):1–18. doi:10.1186/s13287­017­0555­9.
Beegle J, Lakatos K, Kalomoiris S, Stewart H, Isseroff
RR, Nolta JA, Fierro FA. 2015. Hypoxic preconditioning of mesenchymal stromal cells induces
metabolic changes, enhances survival, and promotes
cell retention in vivo. Stem Cells 33(6):1818–1828.
doi:10.1002/stem.1976.
Castrén E, Sillat T, Oja S, Noro A, Laitinen A, Konttinen
YT, Lehenkari P, Hukkanen M, Korhonen M. 2015.
Osteogenic differentiation of mesenchymal stromal
cells in two­dimensional and three­dimensional cultures without animal serum. Stem Cell Res. Ther.
6(1):1–13. doi:10.1186/s13287­015­0162­6.
Darlan DM, Munir D, Putra A, Alif I, Amalina ND, Jusuf
NK, Putra IB. 2022. Revealing the decrease of indoleamine 2,3­dioxygenase as a major constituent
for B cells survival post­mesenchymal stem cells
co­cultured with peripheral blood mononuclear cell
(PBMC) of systemic lupus erythematosus (SLE) patients. Med. Glas. 19(1). doi:10.17392/1414­21.
Dominici M, Le Blanc K, Mueller I, Slaper­Cortenbach
I, Marini FC, Krause DS, Deans RJ, Keating A,
Prockop DJ, Horwitz EM. 2006. Minimal criteria for defining multipotent mesenchymal stromal
cells. The International Society for Cellular Therapy position statement. Cytotherapy. 8(4):315–317.
doi:10.1080/14653240600855905.
Drawina P, Putra A, Nasihun T, Prajoko YW, Dirja BT,
Amalina ND. 2022. Increased serial levels of plateletderived growth factor using hypoxic mesenchymal
stem cell­conditioned medium to promote closure acceleration in a full­thickness wound. Indones. J.
Biotechnol. 27(1):36. doi:10.22146/ijbiotech.64021.
El Agha E, Kramann R, Schneider RK, Li X, Seeger
W, Humphreys BD, Bellusci S. 2017. Mesenchymal Stem Cells in Fibrotic Disease. Cell Stem Cell
21(2):166–177. doi:10.1016/j.stem.2017.07.011.
Hamra NF, Putra A, Tjipta A, Amalina ND, Nasihun T.
2021. Hypoxia mesenchymal stem cells accelerate
wound closure improvement by controlling α­smooth
muscle actin expression in the full­thickness animal
model. Open Access Maced. J. Med. Sci. 9:35–41.
doi:10.3889/oamjms.2021.5537.
He X, Dong Z, Cao Y, Wang H, Liu S, Liao L, Jin Y, Yuan
L, Li B, Bolontrade MF. 2019. MSC­Derived Exosome Promotes M2 Polarization and Enhances Cutaneous Wound Healing. Stem Cells Int. 2019:1–16.
doi:10.1155/2019/7132708.
Ho CH, Lan CW, Liao CY, Hung SC, Li HY, Sung YJ.
2018. Mesenchymal stem cells and their conditioned
medium can enhance the repair of uterine defects in



DOI: https://doi.org/10.22146/ijbiotech.63914

Article Metrics

Abstract views : 1458 | views : 1185

Refbacks

  • There are currently no refbacks.


Copyright (c) 2022 The Author(s)

Creative Commons License
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.