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. 2024 Sep;28(17):e70049.
doi: 10.1111/jcmm.70049.

Bone marrow stromal and anterior cruciate ligament remnant cell co-culture-derived extracellular vesicles promote cell activity in both cell types

Sung-Yen Lin  1   2   3   4   5 , Shun Cheng Wu  4   5   6 , Zi-Miao Liu  2 , Paul Pei-Hsi Chou  2   7 , Chunfeng Zhao  8 , Mei-Ling Ho  4   5 , Cheng-Chang Lu  2   4   5   7   9
Affiliations

Bone marrow stromal and anterior cruciate ligament remnant cell co-culture-derived extracellular vesicles promote cell activity in both cell types

Sung-Yen Lin et al. J Cell Mol Med. 2024 Sep.

Abstract

The significance of anterior cruciate ligament (ACL) remnants during reconstruction remains unclear. Co-culturing ACL remnant cells and bone marrow stromal cells (BMSCs) may reduce apoptosis and enhance hamstring tendon activity. This study investigated whether extracellular vesicles (EVs), which facilitate cell-cell interactions, act as the active components, improving graft maturation in this co-culture. The effects of EVs on cell viability, proliferation, migration and gene expression in the rabbit ACL remnant cells and BMSCs were assessed using control (BMSC-only culture), co-culture (ACL remnant cells and BMSCs, CM) and co-culture without EVs (CM ∆ EVs) media. EVs were isolated from control (BMSC-EV) and co-culture (CM-EV) media and characterized. CM significantly enhanced the proliferation, migration and expression of transforming growth factor (TGF-β)-, vascular endothelial growth factor (VEGF)-, collagen synthesis- and tenogenesis-related genes. However, CM-induced effects were reversed by the CM ∆ EVs treatment. CM-EV treatment exhibited higher potential to enhance proliferation, migration and gene expression in the ACL remnant cells and BMSCs than BMSC-EV and non-EV treatments. In conclusion, EVs, secreted under the coexistence of ACL remnant cells and BMSCs, primarily increase the cell viability, proliferation, migration and gene expression of collagen synthesis-, TGF-β-, VEGF- and tenogenesis-related genes in both cell types.

Keywords: anterior cruciate ligament; bone marrow stromal cells; extracellular vesicle; tenogenesis.

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Conflict of interest statement

Cheng‐Chang Lu and all authors, or any member of his or her immediate family has no funding or commercial associations (e.g. consultancies, stock ownership, equity interest, patent/licensing arrangements) that might pose a conflict of interest in connection with the submitted article. I agree and confirm that this statement is true.

Figures

FIGURE 1
FIGURE 1
Experimental design of the study. ACL, anterior cruciate ligament; BMSC, bone marrow stromal cell; CM, co‐culture medium; EV, extracellular vesicle.
FIGURE 2
FIGURE 2
Cell proliferation, migration and expression of collagen I and III‐, TGF‐β‐, VEGF‐ and tenogenic‐related genes in ACL remnant cells following treatment with BMSC‐only culture medium (control), ACL remnant cell/BMSC co‐culture medium (CM) and co‐culture medium without extracellular vesicles (CM ∆ EVs). Migration is evaluated via transwell and scratch migration assays. In the transwell assay, cells that traverse the transwell membrane are stained in purple with crystal violet, and their migration ratio is determined. In the scratch assay, results are presented as the percentage of initial scratch closure. *p < 0.05; **p < 0.01.
FIGURE 3
FIGURE 3
Cell proliferation, migration and expression of collagen I and III‐, TGF‐β‐, VEGF‐ and tenogenic‐related genes in BMSCs following treatment with BMSC‐only culture medium (control), ACL remnant cell/BMSC co‐culture medium (CM) and co‐culture medium without extracellular vesicles (CM ∆ EVs). Migration is evaluated via transwell and scratch migration assays. In the transwell assay, cells that traverse the transwell membrane are stained in purple with crystal violet, and their migration ratio is determined. In the scratch assay, results are presented as the percentage of initial scratch closure. *p < 0.05; **p < 0.01.
FIGURE 4
FIGURE 4
(A) Particle size distribution, expression levels of biomarkers and transmission electron microscopic images of EVs derived from BMSC‐only culture (BMSC‐EV) and ACL remnant cell/BMSC co‐culture medium (CM‐EV). (B) Immunofluorescence pictures show the CM‐EV uptake and internalization by the ACL remnant cells and BMSCs.
FIGURE 5
FIGURE 5
Cell viability, proliferation, migration and expression of collagen I and III‐, TGF‐β‐, VEGF‐ and tenogenic‐related genes in ACL remnant cells following treatment with EVs derived from BMSC‐only culture medium (BMSC‐EV), ACL remnant cell/BMSC co‐culture medium (CM‐EV) and non‐EV medium (control) group. Migration is evaluated via transwell and scratch migration assays. In the transwell assay, cells that traverse the transwell membrane are stained in purple with crystal violet, and their migration ratio is determined. In the scratch assay, results are presented as the percentage of initial scratch closure. *p < 0.05; **p < 0.01.
FIGURE 6
FIGURE 6
Cell viability, proliferation, migration and expression of collagen I and III‐, TGF‐β‐, VEGF‐ and tenogenic‐related genes in BMSCs following treatment with EV derived from BMSC‐only culture medium (BMSC‐EV), ACL remnant cell/BMSC co‐culture medium (CM‐EV) and non‐EV medium (control) groups. Migration is evaluated via transwell and scratch migration assays. In the transwell assay, cells that traverse the transwell membrane are stained in purple with crystal violet, and their migration ratio is determined. In the scratch assay, results are presented as the percentage of initial scratch closure. *p < 0.05; **p < 0.01.
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