The Impact of Different Ratios of Chondrocytes and Mesenchymal Stem Cells on Condrogenic Potential of Extracellular Vesicles Derived from Coculture of Chondrocytes and Mesenchymal Stem Cells

Hosseinzadeh, M; Hosseini, S; Baghaban Eslaminejad, M

doi:10.52547/JCT.13.4.268

The Impact of Different Ratios of Chondrocytes and Mesenchymal Stem Cells on Condrogenic Potential of Extracellular Vesicles Derived from Coculture of Chondrocytes and Mesenchymal Stem Cells

Document Type : Research - Scientific

Authors

M Hosseinzadeh

S Hosseini

M Baghaban Eslaminejad

Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

https://doi.org/10.52547/JCT.13.4.268

Abstract

Aim: Cartilage tissue has limited capacity for spontaneous repair and self-renew due to the absence of vascularization and progenitor cells, thus requiring a therapeutic approach to repair tissue damage. Recently, extracellular vesicles (EVs) have attracted attentions because of their major roles in cell communication and tissue repair by regulating cellular processes such as cell growth, differentiation, and proliferation. Therefore, it is necessary to isolate extracellular vesicles with chondrogenic potential from an appropriate cellular source for cartilage regeneration. This study aims to compare the chondrogenic ability of extracellular vesicles derived from cocultured -chondrocytes / mesenchymal stem cells (CHO / MSC) at ratios of 1/2 and 1/4.
Material and Methods: Towards this goal, chondrocytes and mesenchymal stem cells were isolated from rabbit articular cartilage and bone marrow, respectively. Mesenchymal phenotype of isolated MSCs were characterized based on their surface markers and by differentiation into mesenchymal lineages. Chondrocytes and mesenchymal stem cells were co-cultured with defined ratios, their conditioned media were collected and extracellular vesicles were extracted with an ultracentrifuge. Concentrations of extracellular vesicles were determined using BCA Protein Assay Kit, then EVs were characterized in terms of size, morphology, and expression of surface markers by dynamic light scattering (DLS), scanning electron microscope (SEM) and western blotting, respectively. Mesenchymal stem cells were treated with different concentrations of extracellular vesicles (50, 100, and 150 ug/ml) for 21 days. Quantitative real time-PCR (qRT-PCR) and histological analysis were subsequently performed to assess the quality of chondrogenic differentiation among experimental groups. The differentiation of MSCs to osteogenic and adipogenic lineages was demonstrated by Oil Red and Alizarin Red staining after 21 days.
Results: The results of flow cytometry showed that CD90 was expressed by 88.6% of the cell population as a positive marker and CD34 as a negative marker was only expressed in 5.48% of the cell population. SEM micrographs confirmed the spherical shape of CHO/ MSC-EV 1/2 and CHO/MSC-EV 1/4 and the mean particle size of isolated EVs were 51.66 ± 8.15 and 19.11 ± 6.03 nm, respectively. Special surface markers for extracellular vesicles containing CD9 and CD81 were expressed in both groups. At concentrations of 100 and 150 ug/ml of CHO/MSC-EV 1/2, higher cartilage - specific markers, including Col II were observed compared to CHO/MSC-EV 1/4. Similarly, safranin O and toluidine blue staining revealed the more deposition of aminoglycan and proteoglycan at concentrations of 100 and 150 ug/ml of CHO/ MSC-EV 1/2, compared to CHO/ MSC-EV 1/4. This study demonstrated the chondrogenesis potential of extracellular vesicles, especially in CHO /MSC-EV 1/2 and extracellular vesicles derived from co-culturing chondrocytes/MSCs improve matrix production and chondrogenesis.
Conclusion: Our research shows that chondrocyte-MSCs proximity is essential for the response as improved viability, chondrogenesis, and matrix formation in recipient MSCs. It is also proposed that co-cultured derived EVs with the ability to promote chondrogenesis have the potential to be utilized in cartilage regeneration. Due to their immunogenicity and their low tumorigenic potential, extracellular vesicles can be used to help repair cartilage tissue.

Highlights

Keywords

Extracellular vesicles

Mesenchymal stem cells

Chondrocyte

Chondrogenesis

مقدمه

بیماری‌های غضروف مفصلی یکی از اختلالات شایع به‫ویژه در میان قشر میانسال و مسن جامعه است. غضروف مفصلی به‫علت عدم وجود رگ خونی و تراکم پایین سلولی ظرفیت محدودی برای ترمیم خودبه‌خودی دارد، در نتیجه آسیب‌های جزئی می‌توانند به آسیب‌های پیشرونده مانند استئوآرتریت (OA) منجر شوند (1). روش‌های سنتی شامل دارو درمانی و جراحی برای درمان آسیب غضروف مفصلی به‫کار گرفته شده است اما هیچکدام در درمان کامل آسیب غضروفی موفق نبوده‌اند (2, 3). سلول‌درمانی یکی از روش‌های نوین در درمان آسیب‌های غضروفی است و سلول‌های بنیادی مزانشیمی (MSCs) براساس دارا بودن توانایی تمایز به غضروف و استخوان به‫عنوان یک منبع سلولی مناسب مورد توجه قرار گرفته‌اند (4-6). علی‌رغم امکان تکثیر MSCها در شرایط برون‌تنی استفاده از این سلول‌ها همچنان با چالش‌هایی از جمله از دست رفتن ظرفیت تمایز به غضروف در شرایط کشت و تکثیر، خطرتومورزایی و ایمنی‌زایی همراه است (7). در سال‌های اخیر استفاده از وزیکول‌های خارج سلولی که یکی از انواع ترشحات سلولی است به‫عنوان یک رویکرد درمانی عاری از سلول و بدون عوارض ناشی از سلول درمانی از جمله تومورزایی و ایمنی‌زایی مورد توجه قرار گرفته است. وزیکول‌های خارج سلولی، وزیکول‌هایی با غشای دو لایه‌ی لیپیدی هستند و از انواع سلول‌ها و همچنین از مایعات مختلف بدن مانند سرم، بزاق، مایع مغزی-نخاعی و مایع سینوویال استخراج میشوند (12-8). وزیکول‌های خارج سلولی با تنظیم فرایندهای سلولی از جمله رشد، تمایز و فراخوانی سلول‌‌ها، در فرایند ترمیم بافت‌های مختلف، نقش مهمی ایفا می‌نماید و نقش درمانی آن‌ها برای ترمیم بافت‌های مختلف شامل قلب، کلیه، کبد، ریه و پوست نیز مورد مطالعه قرار گرفته است (13-18).Wang و همکاران با استفاده از EV‌های مشتق از CPC‌ها (Chondrogenic Progenitor Cells) در مدل موشی دچار استئوآرتریت (OA) نشان دادند که این EV‌ها قادر به جلوگیری از پیشرفت و شدت OA در داخل بدن و همچنین افزایش تحریک در روند تکثیر کندروسیت‌ها در شرایط آزمایشگاهی هستند. همچنین نتایج qRT-PCR نشان داد که ژن‌های دخیل در فرایند ترمیم غضروف در موش‌های دریافت کننده‌ی EVهای مشتق از CPC‌ها افزایش بیان و ژن‌های دخیل در التهاب کاهش بیان داشتند (19). نتایج حاصل از مطالعه‌ای که توسط Zhang و همکاران در بررسی نقش وزیکول‌های خارج سلولی جدا شده از سلول‌های بنیادی مزانشیمی مشتق از جنین انسانی در ترمیم آسیب استئوکندرال انجام شد، نشان داد که وزیکول‌های خارج سلولی منجر به بهبود آسیب استئوکندرال از طریق تشکیل بافت شبه استخوان سابکندرال و بافت شبه غضروف هیالین شد (20). همچنین گروه Tao در سال ۲۰۱۷ اثر وزیکول‌های خارج سلولی مشتق از سلولهای MSC مایع سینوویال با افزایش بیان miRNA- 140 را در آسیب استئوکندرال بررسی کردند. نتایج این مطالعه نشان داد که استفاده از این وزیکول‌های خارج سلولی سبب افزایش تکثیر کندروسیت‌ها در شرایط برونتنی و جلوگیری از پیشرفت استئوآرتریت در شرایط درونتنی گردید (21). در مطالعه‌ای دیگر که سال ۲۰۱۸ توسط گروه Vonk

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