A quick and innovative pipeline for producing chondrocyte-homing peptide-modified extracellular vesicles by three-dimensional dynamic culture of hADSCs spheroids to modulate the fate of remaining ear chondrocytes in the M1 macrophage-infiltrated microenvironment

(2024) 22:300 Chen et al. Journal of Nanobiotechnology https://doi.org/10.1186/s12951-024-02567-5 Journal of Nanobiotechnology Open Access RESEARCH A quick and innovative pipeline for producing chondrocyte‑homing peptide‑modified extracellular vesicles by three‑dimensional dynamic culture of hADSCs spheroids to modulate the fate of remaining ear chondrocytes in the M1 macrophage‑infiltrated microenvironment Jianguo Chen1, Enchong Zhang2*, Yingying Wan3*, Tianyu Huang1, Yuchen Wang1 and Haiyue Jiang1* Abstract Background Extracellular vesicles (EVs) derived from human adipose-derived mesenchymal stem cells (hADSCs) have shown great therapeutic potential in plastic and reconstructive surgery. However, the limited production and functional molecule loading of EVs hinder their clinical translation. Traditional two-dimensional culture of hADSCs results in stemness loss and cellular senescence, which is unfavorable for the production and functional molecule loading of EVs. Recent advances in regenerative medicine advocate for the use of three-dimensional culture of hADSCs to produce EVs, as it more accurately simulates their physiological state. Moreover, the successful application of EVs in tissue engineering relies on the targeted delivery of EVs to cells within biomaterial scaffolds. Methods and Results The hADSCs spheroids and hADSCs gelatin methacrylate (GelMA) microspheres are utilized to produce three-dimensional cultured EVs, corresponding to hADSCs spheroids-EVs and hADSCs microspheres-EVs respectively. hADSCs spheroids-EVs demonstrate excellent production and functional molecule loading compared with hADSCs microspheres-EVs. The upregulation of eight miRNAs (i.e. hsa-miR-486-5p, hsa-miR-423-5p, hsa-miR-92a-3p, hsa-miR122-5p, hsa-miR-223-3p, hsa-miR-320a, hsa-miR-126-3p, and hsa-miR-25-3p) and the downregulation of hsa-miR-146b-5p within hADSCs spheroids-EVs show the potential of improving the fate of remaining ear chondrocytes and promoting cartilage formation probably through integrated regulatory mechanisms. Additionally, a quick and innovative pipeline is developed for isolating chondrocyte homing peptide-modified EVs (CHP-EVs) from three-dimensional dynamic cultures of hADSCs spheroids. CHP-EVs are produced by genetically fusing a CHP at the N-terminus of the exosomal surface protein LAMP2B. The CHP + LAMP2B-transfected hADSCs spheroids were cultured with wave motion to promote the secretion *Correspondence: Enchong Zhang Yingying Wan Haiyue Jiang Full list of author information is available at the end of the article © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chen et al. Journal of Nanobiotechnology (2024) 22:300 Page 2 of 26 of CHP-EVs. A harvesting method is used to enable the time-dependent collection of CHP-EVs. The pipeline is easy to set up and quick to use for the isolation of CHP-EVs. Compared with nontagged EVs, CHP-EVs penetrate the biomaterial scaffolds and specifically deliver the therapeutic miRNAs to the remaining ear chondrocytes. Functionally, CHP-EVs show a major effect on promoting cell proliferation, reducing cell apoptosis and enhancing cartilage formation in remaining ear chondrocytes in the M1 macrophage-infiltrated microenvironment. Conclusions In summary, an innovative pipeline is developed to obtain CHP-EVs from three-dimensional dynamic culture of hADSCs spheroids. This pipeline can be customized to increase EVs production and functional molecule loading, which meets the requirements for regulating remaining ear chondrocyte fate in the M1 macrophage-infiltrated microenvironment. Keywords Adipose-derived mesenchymal stem cells, Chondrocyte homing peptide, Extracellular vesicles, M1 macrophage, Remaining ear chondrocytes Graphical Abstract Introduction Microtia has a significant impact on both the aesthetic appearance and psychological well-being of affected children. The current standard method for ear reconstruction in microtia involves autologous rib cartilage ear framework transplantation. However, this procedure is time-consuming, taking around 3 months to complete, and is costly, posing challenges for the child’s education and placing financial strain on families and society. Moreover, harvesting rib cartilage can lead to donor site damage and chest wall deformities. The intricate process of sculpting the ear framework requires a high level of expertise, making it difficult to train new doctors in this technique. Fortunately, recent advancements in materials science, three-dimensional printing, and tissue engineering technology have opened up new possibilities for creating tissue-engineered ear frameworks for clinical applications. Tissue-engineered ears offer great hope for children with microtia. Since 2018, our ear reconstruction center achieved a significant milestone by successfully conducting a clinical trial using tissue-engineered ears for external ear reconstruction, making this the first international achievement in this field [1]. However, the tissue-engineered ears implanted in the body exhibited varying levels of deformation and collapse in later stages due to inadequate synthesis of mature cartilage matrix by remaining ear chondrocytes. Therefore, it is crucial to investigate the reasons for the inadequate synthesis of tissue-engineered cartilage matrix and explore potential intervention strategies. Macrophage can be classified into two types: tissueresident macrophage and macrophage derived from circulating monocytes, which plays crucial roles in Chen et al. Journal of Nanobiotechnology (2024) 22:300 maintaining tissue balance and promoting tissue regeneration after injury. Macrophage can differentiate into at least two subgroups with distinct immune functions based on various stimuli. The M1 subgroup, known as classically activated macrophage, mainly contributes to inflammatory responses, while the M2 subgroup, known as alternatively activated macrophage, is primarily involv (...truncated)