Co-aggregation of MSC/chondrocyte in a dynamic 3D culture elevates the therapeutic effect of secreted extracellular vesicles on osteoarthritis in a rat model

www.nature.com/scientificreports OPEN Co‑aggregation of MSC/ chondrocyte in a dynamic 3D culture elevates the therapeutic effect of secreted extracellular vesicles on osteoarthritis in a rat model Abazar Esmaeili1,2, Samaneh Hosseini1,3, Amir Kamali1, Maryam Hosseinzadeh1, Faezeh Shekari1 & Mohamadreza Baghaban Eslaminejad1,2* Extracellular vesicles (EVs) have therapeutic effects on osteoarthritis (OA). Some recent strategies could elevate EV’s therapeutic properties including cell aggregation, co-culture, and 3D culture. It seems that a combination of these strategies could augment EV production and therapeutic potential. The current study aims to evaluate the quantity of EV yield and the therapeutic effect of EVs harvested from rabbit mesenchymal stem cells (MSCs) aggregates, chondrocyte aggregates, and their co-aggregates in a dynamic 3D culture in a rat osteoarthritis model. MSC and chondrocytes were aggregated and co-aggregated by spinner flasks, and their conditioned medium was collected. EVs were isolated by size exclusion chromatography and characterized in terms of size, morphology and surface markers. The chondrogenic potential of the MSC-ag, Cho-ag and Co-ag EVs on MSC micromass differentiation in chondrogenic media were assessed by qRT-PCR, histological and immunohistochemical analysis. 50 μg of MSC-ag-EVs, Cho-ag-EVs and Co-ag-EVs was injected intraarticularly per knee of OA models established by monoiodoacetate in rats. After 8 weeks follow up, the knee joints were harvested and analyzed by radiographic, histological and immunohistochemical features. MSC/chondrocyte co-aggregation in comparison to MSC or chondrocyte aggregation could increase EV yield during dynamic 3D culture by spinner flasks. Although MSC-ag-, Cho-ag- and Co-agderived EVs could induce chondrogenesis similar to transforming growth factor-beta during in vitro study, Co-ag-EV could more effectively prevent OA progression than MSC-ag- and Cho-ag-EVs. Our study demonstrated that EVs harvested from the co-aggregation of MSCs and chondrocytes could be considered as a new therapeutic potential for OA treatment. Hyaline cartilage repair using various therapeutic approaches such as pharmaceutical drugs, surgical interventions and cell therapy, although hopeful, has not led to the desired o utcomes1. Recently, extracellular vesicle (EV) therapy for cartilage repair has been considered as a promising cell-free s trategy2. However, this approach still needs to be improved in terms of producing the quality and quantity of EVs to achieve a more therapeutic effect for the restoration of cartilage defects with natural properties. Researchers have initially tried to produce EVs with more effective therapeutic properties by selecting the proper cell s ource3. In most studies, the EVs of MSC (MSC-EVs), and in some cases chondrocyte (Cho-EVs) and chondrogenic progenitor cells have been considered for OA t reatment4,5. Thus far, some research groups have studied the effect of MSC-EVs on OA models with the use of different MSC sources including amniotic fluid stem c ells6, human embryonic M SCs7,8, bone marrow MSCs (BMSCs)9,10, infrapatellar fat pad-derived11 1 Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. 2Faculty of Sciences and Advanced Technologies in Biology, University of Science and Culture, Tehran, Iran. 3Department of Cell Engineering, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. *email: Scientific Reports | (2022) 12:19827 | https://doi.org/10.1038/s41598-022-22592-4 1 Vol.:(0123456789) www.nature.com/scientificreports/ and other cells. According to studies, BMSCs and articular chondrocytes (ACs) could be the appropriate cells to harvest EVs for OA t reatment2,12–14. They reported articular Cho-EVs could induce a chondrogenic effect on MSCs5,15. BMSCs exhibited more immunomodulatory activity than adipose tissue and Wharton’s jelly MSCs16. It has been shown that transplantation of spheroids formed from synovium-derived cells and chondrocytes was also effective in cartilage r epair17. Cell therapy studies have demonstrated that cellular architectures in the form of aggregation improves a range of biological properties of MSCs, including the ability of differentiation, cell survival and the secretion of therapeutic factors18. It seems that the study of the therapeutic effect of EVs secreted by MSCs and ACs aggregates for the treatment of OA could be a valuable strategy. Many studies have shown improvement in chondrogenic differentiation by co-culture of MSCs and chondrocytes19,20. Moreover, other research has proved that chondrocytes induce chondrogenesis of MSCs and, MSCs are effective in reshaping and stimulating the proliferation of chondrocytes21. It was suggested that these mutual effects could be through trophic factors and paracrine secretion, especially by E Vs22. MSC-EVs interceded cartilage repair by increasing chondrocyte proliferation, decreasing apoptosis, and controlling immune reactivity23. In contrast, Cho-EVs elevated the proliferation and chondrogenic differentiation of MSCs15. Diao et al. suggested that 3D co-culture of human MSCs and ACs is mutually advantageous in their therapeutic potential for OA t reatment24. It is demonstrated that co-culture of MSCs and ACs could reduce and suppress hypertrophy during chondrogenesis and enhance the functional properties of c artilage25–27. According to these studies, it is interesting to investigate the effect of co-aggregation of MSCs/ACs in terms of their EVs’ therapeutic effect for chondrogenic differentiation and treatment of OA. EVs derived from 3-dimensionally (3D) cultured MSCs provided better therapeutic outcome compared to 2D culture28. Recently, Rocha et al. have shown that 3D cellular architecture impacts the microRNA (miR) and protein cargo of E Vs29. 3D MSC cultures improved small interfering RNA (siRNA) transfer to recipient c ells30. In addition, dynamic 3D culture could increase EV yield30,31. It has been shown that the dynamic MSC 3D culture in an orbital-shake sharply enhanced EV y ield31. Haraszt et al. have demonstrated that microcarrier-based (3D) MSC cultures in spinner flasks improve EV yield and siRNA t ransfer30. Spinner flask culture in addition to inducing de-differentiation of redifferentiated chondrocytes32, could be effective for scale-up of MSC and AC aggregate culture by dynamic collision-based a ssembly33. Despite the proved impact of cells produced by dynamic 3D culture on therapeutic outcomes, there is less evidence on the quality of the EV cargo and the quantitative efficiencies of EVs harvested from the aggregates of MSCs and ACs and their co-aggregates by spinner flasks for OA treatment. In this study, we selected BMSCs and ACs as the most common EV cell sources with considering the prospect of clinical application potential (...truncated)