Exosome-microneedle patches accelerate oral ulcer healing by remodeling macrophage-epithelial crosstalk via TSP‑1/CD47/NF‑κB signaling

Abstract

Oral ulcers are among the most common inflammatory lesions of the oral mucosa and often cause severe pain. However, current treatments are limited by short mucosal retention, suboptimal therapeutic efficacy, and steroid-associated side effects, underscoring the need for more effective and durable therapeutic strategies. Here, we developed exosome-loaded microneedle patches (Exo-MNPs) by incorporating mesenchymal stem cell-derived exosomes (MSC-exo) into gelatin methacryloyl (GelMA)/polyvinylpyrrolidone (PVP) microneedles and evaluated their potential to accelerate oral ulcer healing. Exo-MNPs were systematically characterized in terms of morphology, mechanical strength, and in vitro bioactivity. In rat ulcer models, Exo-MNPs markedly accelerated wound closure, promoted epithelial regeneration, reduced inflammation, and increased collagen deposition. Multi-omics profiling using single-cell RNA sequencing (scRNA-seq) and proteomics revealed that Exo-MNPs act through the TSP-1/CD47/NF-κB axis to reprogram macrophage phenotypes and promote regenerative epithelial subpopulations via intercellular crosstalk. These findings demonstrate that Exo-MNPs represent a promising localized bioactive therapy for oral ulcers and illuminate key immune–epithelial mechanisms underlying their therapeutic effects.

Keywords

Exosomes / Microneedle patches / Oral ulcers / Macrophage–epithelial crosstalk / TSP-1

References

[1] S.O. Akintoye, M.S. Greenberg, Recurrent aphthous stomatitis, Dent. Clin. North Am. 58 (2014) 281, https://doi.org/10.1016/j.cden.2013.12.002.

[2] S. Krisdapong, A. Sheiham, G. Tsakos, Impacts of recurrent aphthous stomatitis on quality of life of 12- and 15-year-old thai children, Qual. Life Res 21 (2012) 71, https://doi.org/10.1007/s11136-011-9925-4.

[3] C.T. Lee, T.J. Galloway, Pathogenesis and amelioration of radiation-induced oral mucositis, Curr. Treat. Options Oncol. 23 (2022) 311, https://doi.org/10.1007/s11864-022-00959-z.

[4] N. Lewkowicz, P. Lewkowicz, M. Banasik, A. Kurnatowska, H. Tchórzewski, Predominance of type 1 cytokines and decreased number of cd4(+)cd25(+high) t regulatory cells in peripheral blood of patients with recurrent aphthous ulcerations, Immunol. Lett. 99 (2005) 57 https://pubmed.ncbi.nlm.nih.gov/15894112.

[5] Y. Bi, G. Liu, R. Yang, Th17 cell induction and immune regulatory effects, J. Cell Physiol. 211 (2007) 273 https://pubmed.ncbi.nlm.nih.gov/17311299.

[6] N. Lewkowicz, P. Lewkowicz, K. Dzitko, B. Kur, M. Tarkowski, A. Kurnatowska, H. Tchórzewski, Dysfunction of cd4+cd25high t regulatory cells in patients with recurrent aphthous stomatitis, J. Oral. Pathol. Med 37 (2008) 454, https://doi.org/10.1111/j.1600-0714.2008.00661.x.

[7] M.R. Bazrafshani, A.H. Hajeer, W.E.R. Ollier, M.H. Thornhill, Il-1b and il-6 gene polymorphisms encode significant risk for the development of recurrent aphthous stomatitis (ras), Genes Immun. 3 (2002) 302 https://pubmed.ncbi.nlm.nih.gov/12140751.

[8] R.C. Borra, P.M. Andrade, I.D.C.G. Silva, A. Morgun, L.L.M. Weckx, A.S. Smirnova, M. Franco, The th1/th2 immune-type response of the recurrent aphthous ulceration analyzed by cdna microarray, J. Oral. Pathol. Med 33 (2004) 140 https://pubmed.ncbi.nlm.nih.gov/15128055.

[9] E.A. Georgakopoulou, C. Scully, Systemic use of non-biologic corticosteroids in orofacial diseases, Oral. Dis. 20 (2014) 127, https://doi.org/10.1111/odi.12132.

[10] Z. Shen, J. Wang, Q. Huang, Y. Shi, Z. Wei, X. Zhang, Y. Qiu, M. Zhang, Y. Wang, W. Qin, S. Huang, Y. Huang, X. Liu, K. Xia, X. Zhang, Z. Lin, Genetic modification to induce cxcr2 overexpression in mesenchymal stem cells enhances treatment benefits in radiation-induced oral mucositis, Cell death & Dis. 9 (2018) 229, https://doi.org/10.1038/s41419-018-0310-x.

[11] J.R.J. Chew, S.J. Chuah, K.Y.W. Teo, S. Zhang, R.C. Lai, J.H. Fu, L.P. Lim, S.K. Lim, W.S. Toh, Mesenchymal stem cell exosomes enhance periodontal ligament cell functions and promote periodontal regeneration, Acta Biomater. 89 (2019) 252, https://doi.org/10.1016/j.actbio.2019.03.021.

[12] C. Zhou, B. Zhang, Y. Yang, Q. Jiang, T. Li, J. Gong, H. Tang, Q. Zhang, Stem cell-derived exosomes: Emerging therapeutic opportunities for wound healing, Stem Cell Res. & Ther. 14 (2023) 107, https://doi.org/10.1186/s13287-023-03345-0.

[13] R. Kalluri, V.S. LeBleu, The biology, function, and biomedical applications of exosomes, Science 367 (2020), https://doi.org/10.1126/science.aau6977.

[14] C. Wang, M. Wang, T. Xu, X. Zhang, C. Lin, W. Gao, H. Xu, B. Lei, C. Mao, Engineering bioactive self-healing antibacterial exosomes hydrogel for promoting chronic diabetic wound healing and complete skin regeneration, Theranostics 9 (2019) 65, https://doi.org/10.7150/thno.29766.

[15] X. Geng, Y. Qi, X. Liu, Y. Shi, H. Li, L. Zhao, A multifunctional antibacterial and self-healing hydrogel laden with bone marrow mesenchymal stem cell-derived exosomes for accelerating diabetic wound healing, Biomater. Adv. 133 (2022) 112613, https://doi.org/10.1016/j.msec.2021.112613.

[16] S. Sjöqvist, T. Ishikawa, D. Shimura, Y. Kasai, A. Imafuku, S. Bou-Ghannam, T. Iwata, N. Kanai, Exosomes derived from clinical-grade oral mucosal epithelial cell sheets promote wound healing, J. Extracell. vesicles 8 (2019) 1565264, https://doi.org/10.1080/20013078.2019.1565264.

[17] Z. Shen, S. Kuang, Y. Zhang, M. Yang, W. Qin, X. Shi, Z. Lin, Chitosan hydrogel incorporated with dental pulp stem cell-derived exosomes alleviates periodontitis in mice via a macrophage-dependent mechanism, Bioact. Mater. 5 (2020) 1113, https://doi.org/10.1016/j.bioactmat.2020.07.002.

[18] Y. Zhang, J. Chen, H. Fu, S. Kuang, F. He, M. Zhang, Z. Shen, W. Qin, Z. Lin, S. Huang, Exosomes derived from 3d-cultured mscs improve therapeutic effects in periodontitis and experimental colitis and restore the th17 cell/treg balance in inflamed periodontium, Int J. Oral. Sci. 13 (2021) 43, https://doi.org/10.1038/s41368-021-00150-4.

[19] I. Suharyani, A. Fouad Abdelwahab Mohammed, M. Muchtaridi, N. Wathoni, M. Abdassah, Evolution of drug delivery systems for recurrent aphthous stomatitis, Drug Des. Dev. Ther. 15 (2021) 4071, https://doi.org/10.2147/dddt.S328371.

[20] X. Qu, X. Guo, T. Zhu, Z. Zhang, W. Wang, Y. Hao, Microneedle patches containing mesoporous polydopamine nanoparticles loaded with triamcinolone acetonide for the treatment of oral mucositis, 11 (2023) 2023, https://doi.org/10.3389/fbioe.2023.1203709.

[21] W. Ge, Y. Gao, L. He, Y. Zeng, J. Liu, Y. Yu, X. Xie, R.-a Xu, Combination therapy using multifunctional dissolvable hyaluronic acid microneedles for oral ulcers, Int. J. Biol. Macromol. 251 (2023) 126333, https://doi.org/10.1016/j.ijbiomac.2023.126333.

[22] Y. Meng, X.J. Li, Y. Li, T.Y. Zhang, D. Liu, Y.Q. Wu, F.F. Hou, L. Ye, C.J. Wu, X.D. Feng, X.J. Ju, L. Jiang, Novel double-layer dissolving microneedles for transmucosal sequential delivery of multiple drugs in the treatment of oral mucosa diseases, ACS Appl. Mater. & Interfaces 15 (2023) 13892, https://doi.org/10.1021/acsami.2c19913.

[23] L. Long, D. Ji, C. Hu, L. Yang, S. Tang, Y. Wang, Microneedles for in situ tissue regeneration, Mater. Today Bio 19 (2023) 100579, https://doi.org/10.1016/j.mtbio.2023.100579.

[24] L.E.N. Ferreira, M. Franz-Montan, B. Benso, H.S. Gill, Microneedles for oral mucosal delivery - current trends and perspective on future directions, Expert Opin. Drug Deliv. 20 (2023) 1251, https://doi.org/10.1080/17425247.2023.2264189.

[25] W. Ge, Y. Gao, Y. Zeng, Y. Yu, X. Xie, L. Liu, Silk fibroin microneedles loaded with lipopolysaccharide-pretreated bone marrow mesenchymal stem cell-derived exosomes for oral ulcer treatment, ACS Appl. Mater. Interfaces 16 (2024) 37486, https://doi.org/10.1021/acsami.4c04804.

[26] J. Gan, X. Zhang, W. Ma, Y. Zhao, L. Sun, Antibacterial, adhesive, and msc exosomes encapsulated microneedles with spatio-temporal variation functions for diabetic wound healing, Nano Today 47 (2022) 101630, https://doi.org/10.1016/j.nantod.2022.101630.

[27] M. Han, H. Yang, X. Lu, Y. Li, Z. Liu, F. Li, Z. Shang, X. Wang, X. Li, J. Li, H. Liu, T. Xin, Three-dimensional-cultured msc-derived exosome-hydrogel hybrid microneedle array patch for spinal cord repair, Nano Lett. 22 (2022) 6391, https://doi.org/10.1021/acs.nanolett.2c02259.

[28] R. Naomi, H. Bahari, P.M. Ridzuan, F. Othman, Natural-based biomaterial for skin wound healing (gelatin vs. Collagen): Expert review, Polym. (Basel) 13 (2021), https://doi.org/10.3390/polym13142319.

[29] A.B. Bello, D. Kim, D. Kim, H. Park, S.-H. Lee, Engineering and functionalization of gelatin biomaterials: From cell culture to medical applications, Tissue Eng. Part B Rev. 26 (2020) 164, https://doi.org/10.1089/ten.TEB.2019.0256.

[30] K.N. Mangang, P. Thakran, J. Halder, K.S. Yadav, G. Ghosh, D. Pradhan, G. Rath, V.K. Rai, Pvp-microneedle array for drug delivery: Mechanical insight, biodegradation, and recent advances, J. Biomater. Sci. Polym. Ed. 34 (2023) 986, https://doi.org/10.1080/09205063.2022.2155778.

[31] H. Fu, J. Yang, Z. Shen, Y. Zhang, S. Kuang, L. Li, Z. Lin, X. Shi, Antibacterial, wet adhesive, and healing-promoting nanosheets for the treatment of oral ulcers, Biomater. Sci. 11 (2023) 3214, https://doi.org/10.1039/d2bm02063g.

[32] E.V. Stein, T.W. Miller, K. Ivins-O’Keefe, S. Kaur, D.D. Roberts, Secreted thrombospondin-1 regulates macrophage interleukin-1β production and activation through cd47, Sci. Rep. 6 (2016) 19684, https://doi.org/10.1038/srep19684.

[33] D.D. Roberts, J.S. Isenberg, Cd47 and thrombospondin-1 regulation of mitochondria, metabolism, and diabetes, Am. J. Physiol. Cell Physiol. 321 (2021) C201, https://doi.org/10.1152/ajpcell.00175.2021.

[34] E.V. Stein, T.W. Miller, K. Ivins-O′Keefe, S. Kaur, D.D. Roberts, Secreted thrombospondin-1 regulates macrophage interleukin-1β production and activation through cd47, Sci. Rep. 6 (2016) 19684, https://doi.org/10.1038/srep19684.

[35] F. Chen, Z. Zhao, X. Liu, H. Chen, L. An, Y. Wang, W. Xu, S. Guo, S. Jiang, G.-Q. Chen, Y. Sun, X. Zhang, A multifunctional microneedle patch loading exosomes and magnetic nanoparticles synergistically for treating oral mucosal lesions, Appl. Mater. Today 40 (2024) 102382, https://doi.org/10.1016/j.apmt.2024.102382.

[36] Y. Zeng, X. Xie, Y. Gao, W. Ge, B. Fang, C. Han, Antibacterial composite protein microneedle loaded with hypoxia-treated exosomes for oral ulcer healing, J. Drug Deliv. Sci. Technol. 99 (2024) 105952, https://doi.org/10.1016/j.jddst.2024.105952.

[37] Y. Li, D. Bi, Z. Hu, Y. Yang, Y. Liu, W.K. Leung, Hydrogel-forming microneedles with applications in oral diseases management, Mater. (Basel Switz.) 16 (2023), https://doi.org/10.3390/ma16134805.

[38] D. Baykara, T. Bedir, E. Ilhan, M.E. Mutlu, O. Gunduz, R. Narayan, C.B. Ustundag, Fabrication and optimization of 3d printed gelatin methacryloyl microneedle arrays based on vat photopolymerization, Front. Bioeng. Biotechnol. 11 (2023) 1157541, https://doi.org/10.3389/fbioe.2023.1157541.

[39] S. Jacob, A.B. Nair, S.H.S. Boddu, B. Gorain, N. Sreeharsha, J. Shah, An updated overview of the emerging role of patch and film-based buccal delivery systems, Pharmaceutics 13 (2021), https://doi.org/10.3390/pharmaceutics13081206.

[40] J. Xu, H. Wang, K. Li, S. Pan, G. Guo, K. Liu, N. Wang, L. Xiang, Localized treatment of oral ulcers via responsive microneedle patch by enhancing mucosal penetration, Int. J. Pharm. 691 (2026) 126598, https://doi.org/10.1016/j.ijpharm.2026.126598.

[41] S. Kuang, J. Yang, Z. Shen, J. Xia, Z. Lin, Single-cell and spatial multi-omics analysis reveal that targeting jag1 in epithelial cells reduces periodontal inflammation and alveolar bone loss, Int. J. Mol. Sci. 25 (2024), https://doi.org/10.3390/ijms252413255.

[42] Y. Dai, Y. Chen, Targeting persistently activated inflammatory microenvironment to promote chronic wound healing, Front. Immunol. 16 (2025) 1708358, https://doi.org/10.3389/fimmu.2025.1708358.

[43] A.I. Toma, J.M. Fuller, N.J. Willett, S.L. Goudy, Oral wound healing models and emerging regenerative therapies, Transl. Res. J. Lab. Clin. Med. 236 (2021) 17, https://doi.org/10.1016/j.trsl.2021.06.003.

[44] N. Stadelmann, R.E. Horch, R. Schmid, D. Ostendorf, A. Peddi, T. Promny, A.M. Boos, A. Kengelbach-Weigand, Growth factors igf-1 and kgf and adipose-derived stem cells promote migration and viability of primary human keratinocytes in an in vitro wound model, Front. Med. 12 (2025) 1516116, https://doi.org/10.3389/fmed.2025.1516116.

[45] S.S. Natah, R. Häyrinen-Immonen, J. Hietanen, M. Malmström, Y.T. Konttinen, Immunolocalization of tumor necrosis factor-alpha expressing cells in recurrent aphthous ulcer lesions (rau), J. Oral. Pathol. Med 29 (2000) 19 https://pubmed.ncbi.nlm.nih.gov/10678712.

[46] M.R. Bazrafshani, A.H. Hajeer, W.E.R. Ollier, M.H. Thornhill, Recurrent aphthous stomatitis and gene polymorphisms for the inflammatory markers tnf-alpha, tnf-beta and the vitamin d receptor: No association detected, Oral. Dis. 8 (2002) 303 https://pubmed.ncbi.nlm.nih.gov/12477062.

[47] Q.S. Geng, R.J. Liu, Z.B. Shen, Q. Wei, Y.Y. Zheng, L.Q. Jia, L.H. Wang, L.F. Li, J. Li, W.H. Xue, Transcriptome sequencing and metabolome analysis reveal the mechanism of shuanghua baihe tablet in the treatment of oral mucositis, Chin. J. Nat. Med. 19 (2021) 930, https://doi.org/10.1016/s1875-5364(22)60150-x.

[48] J. Reckenbeil, D. Kraus, H. Stark, B. Rath-Deschner, A. Jäger, M. Wenghoefer, J. Winter, W. Götz, Insulin-like growth factor 1 (igf1) affects proliferation and differentiation and wound healing processes in an inflammatory environment with p38 controlling early osteoblast differentiation in periodontal ligament cells, Arch. Oral. Biol. 73 (2017) 142, https://doi.org/10.1016/j.archoralbio.2016.10.010.

[49] Z. Lopez-Dee, K. Pidcock, L.S. Gutierrez, Thrombospondin-1: Multiple paths to inflammation, Mediat. Inflamm. 2011 (2011) 296069, https://doi.org/10.1155/2011/296069.

[50] Y.Z. Lu, B. Nayer, S.K. Singh, Y.K. Alshoubaki, E. Yuan, A.J. Park, K. Maruyama, S. Akira, M.M. Martino, Cgrp sensory neurons promote tissue healing via neutrophils and macrophages, Nature 628 (2024) 604, https://doi.org/10.1038/s41586-024-07237-y.

[51] X. Chen, B. Yang, J. Tian, H. Hong, Y. Du, K. Li, X. Li, N. Wang, X. Yu, X. Wei, Dental follicle stem cells ameliorate lipopolysaccharide-induced inflammation by secreting tgf-β3 and tsp-1 to elicit macrophage m2 polarization, Cellular physiology biochemistry international journal experimental cellular physiology biochemistry pharmacology 51 (2018) 2290, https://doi.org/10.1159/000495873.