Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jan 2;12(1):23259671231210304.
doi: 10.1177/23259671231210304. eCollection 2024 Jan.

Effect of Exosomes From Bone Marrow-Derived Mesenchymal Stromal Cells and Adipose-Derived Stromal Cells on Bone-Tendon Healing in a Murine Rotator Cuff Injury Model

Xiaoqian Tan  1   2   3 , Han Xiao  1   2   3 , An Yan  1   2   3 , Miao Li  1   2   3 , Linfeng Wang  4
Affiliations

Effect of Exosomes From Bone Marrow-Derived Mesenchymal Stromal Cells and Adipose-Derived Stromal Cells on Bone-Tendon Healing in a Murine Rotator Cuff Injury Model

Xiaoqian Tan et al. Orthop J Sports Med. .

Abstract

Background: Bone-tendon injury is characterized by poor self-healing. It is established that exosomes are favorable for tissue repair and regeneration. However, their effect on bone-tendon healing has not yet been determined.

Purpose: To compare the effectiveness of exosomes derived from adipose-derived mesenchymal stromal cells (ADSC-Exos) and bone marrow-derived mesenchymal stromal cells (BMSC-Exos) on bone-tendon interface healing in murine rotator cuff injury model and explore the underlying mechanisms thereof.

Study design: Controlled laboratory study.

Methods: A total of 63 male C57BL6 mice with rotator cuff injuries underwent surgery and were randomly assigned to a control group treated without exosomes (n = 21), an ADSC-Exos group (n = 21), or a BMSC-Exos group (n = 21). The mice were sacrificed 4 or 8 weeks after surgery, and tissues were collected for histologic examination and radiographic and biomechanical testing. For exosome tracing in vivo, mice were sacrificed 7 days after surgery. A series of functional assays (radiographic evaluation, proliferation assay, Alizarin Red staining, alkaline phosphatase staining and activity, Alcian blue staining, quantitative polymerase chain reaction analyses, and glycosaminoglycans quantification) were conducted to evaluate the effect of exosomes on the cellular behaviors of the BMSCs in vitro. A statistical analysis of multiple-group comparisons was performed by 1-way analysis of variance, followed by the Bonferroni post hoc test to assess the differences between the 2 groups.

Results: The ADSCs and BMSCs were positive for surface markers CD29 and CD90 and negative for surface markers CD34 and CD45 and could differentiate into osteoblasts, chondrocytes, and adipocytes. Exosomes showed a cup- or sphere-shaped morphology and were positive for CD63 and TGS101. Local injection of ADSC-Exos and BMSC-Exos could recruit BMSCs and promote osteogenesis, chondrogenesis, and bone-tendon healing. In vitro, ADSC-Exos and BMSC-Exos could significantly promote the proliferation, migration, osteogenic differentiation, and chondrogenic differentiation ability of BMSCs. In vivo, ADSC-Exos and BMSC-Exos significantly accelerated bone-tendon injury healing, with no significant statistical difference between them.

Conclusion: ADSC-Exos and BMSC-Exos exhibited similar therapeutic effects on bone-tendon healing in our murine animal model.

Clinical relevance: ADSC-Exos and BMSC-Exos may be used to develop a new cell-free therapy method for promoting rotator cuff injury repair.

Keywords: adipose-derived mesenchymal stromal cell; bone marrow–derived mesenchymal stromal cell; bone-tendon healing; exosomes; rotator cuff.

PubMed Disclaimer

Conflict of interest statement

One or more of the authors has declared the following potential conflict of interest or source of funding: Research support was received from the National Key Clinical Specialty Construction Project at Pediatric Surgery of Hunan Children's Hospital (XWYF [2022] No. 2), the Hunan Province Science and Technology Innovation Plan Project (2021SK50516), and the 440 Young Talents of 1233 program of Hunan Children Hospital. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto. Ethical approval for this study was obtained from the Animal Care and Use Committee of Hunan Children's Hospital (ref No: HCHDWLL-2022-16).

Figures

Figure 1.
Figure 1.
Identification of ADSCs and ADSC-Exos. (A) Identification of MSCs by optical morphology (scale bar: 100 μm), Alizarin Red staining (scale bar: 100 μm), Oil Red O staining (scale bar: 50 μm), and Alcian blue staining (scale bar: 200 μm). (B) Results of flow cytometry analysis. (C) Morphology of ADSC-Exos under a transmission electron microscope (scale bar: 200 nm). (D) Size distribution profile of AMSC-Exos. (E) Western blot analysis of exosome-specific CD63 and TSG101 proteins. ADSCs, adipose-derived mesenchymal stromal cells; MSCs, mesenchymal stromal cells.
Figure 2.
Figure 2.
(A) DiR-labeled MSC-Exos existed in the injury site for about 1 week. (B) Cell tracking showed exogenous BMSCs could be recruited into the healing area (Scale bar = 100 μm). BMSCs, bone marrow–derived mesenchymal stromal cells; Exos, exosomes; MSCs, mesenchymal stromal cells.
Figure 3.
Figure 3.
Histological evaluation of the bone-tendon interface. Representative (A) hematoxylin and eosin staining and (B) Toluidine Blue and Fast-Green staining of the bone-tendon interface at 4 and 8 weeks postoperatively in the control, ADSC-Exos, and BMSC-Exos groups (n = 3 per group). The ADSC-Exos and BMSC-Exos groups showed better healing results than the control group at postoperative 4 and 8 weeks (Scale bar: 50 μm). Dashed boxes represent the BTI; ADSCs, adipose-derived mesenchymal stromal cells; BMSCs, bone marrow–derived mesenchymal stromal cells; bone; Exos, exosomes; SB, subchondral; ST, supraspinatus tendon.
Figure 4.
Figure 4.
Radiographic evaluation and mechanical testing of BTI. (A) Representative Xradia tomography of the BTI at postoperative 4 and 8 weeks. The triangle indicated the BTI healing site (Scale bar: 1cm). (B) Comparison of BV/TV and Tb.Th around the injury site among 3 different groups (n = 6 per group). (C) Comparison of mechanical tests among the 3 groups (n = 6 per group). ADSCs, adipose-derived mesenchymal stromal cells; BMSCs, bone marrow–derived mesenchymal stromal cells; bone; BTI, bone-tendon interface; BV/TV, bone volume/tissue volume; Exos, exosomes; ns, not significant versus ADSC-Exos group; Tb.Th, trabecular thickness. *Statistically significant difference versus controls (P < 0.05).
Figure 5.
Figure 5.
ADSC-Exos and BMSC-Exos promoted the proliferation and migration of BMSCs in vitro. (A) Internalization of PKH26-labeled ADSC-Exos or BMSC-Exos by BMSCs. The red-labeled exosomes were around the nucleus (scale bar: 50 μm). (B) The proliferation rate of BMSCs receiving different treatments was tested by CCK-8 (n = 3 per group). (C) Representative picture of Transwell assay in BMSCs treated with ADSC-Exos, BMSC-Exos, or PBS (scale bar: 100 μm). (D) Quantitative analysis of migrated cells in (C); n = 3 per group. (E) Representative picture of the scratch wound in BMSCs treated with ADSC-Exos, BMSC-Exos, or PBS (scale bar: 250 μm). (F) Quantitative analysis of migration rates in (E); n = 3 per group. ADSCs, adipose-derived mesenchymal stromal cells; BMSCs, bone marrow–derived mesenchymal stromal cells; Exos, exosomes; ns, not significant versus the ADSC-Exos group; OD, optical density. *Statistically significant difference versus controls (P < .05).
Figure 6.
Figure 6.
ADSC-Exos and BMSC-Exos promoted osteogenic and chondrogenic differentiation of BMSCs in vitro. (A) Representative images of Alizarin Red staining (scale bar: 100μm); (B) quantitative analysis of Alizarin Red staining in (A); n = 3 per group; (C) Representative images of Alcian blue staining; (D) quantitative analysis of GAG content, normalized to DNA content (n = 3 per group). (E) The osteogenesis and chondrogenesis-related gene expression by BMSCs induced in osteogenic or chondrogenesis medium supplemented with ADSC-Exos, BMSC-Exos, or PBS. ADSCs, adipose-derived mesenchymal stromal cells; ALP, alkaline phosphatase; BMSCs, bone marrow–derived mesenchymal stromal cells; Col2A1, collagen type 2 alpha 1 chain; DNA, deoxyribonucleic acid; Exos, exosomes; GAG, glycosaminoglycan; ns, not significant versus the ADSC-Exos group; PBS, phosphate-buffered saline; RUNX2, runt-related transcription factor 2; SOX9, SRY-Box Transcription Factor 9. *Statistically significant difference versus controls (P < .05).
See this image and copyright information in PMC

References

    1. Baglio SR, Rooijers K, Koppers-Lalic D, et al. Human bone marrow- and adipose-mesenchymal stem cells secrete exosomes enriched in distinctive miRNA and tRNA species. Stem Cell Res Ther. 2015;6:127. - PMC - PubMed
    1. Basu J, Ludlow JW. Exosomes for repair, regeneration and rejuvenation. Expert Opin Biol Ther. 2016;16:489-506. - PubMed
    1. Benjamin M, Toumi H, Ralphs JR, Bydder G, Best TM, Milz S. Where tendons and ligaments meet bone: attachment sites (‘entheses’) in relation to exercise and/or mechanical load. J Anat. 2006;208:471-490. - PMC - PubMed
    1. Cavallo C, Cuomo C, Fantini S, et al. Comparison of alternative mesenchymal stem cell sources for cell banking and musculoskeletal advanced therapies. J Cell Biochem. 2011;112:1418-1430. - PubMed
    1. Chaubey S, Thueson S, Ponnalagu D, et al. Early gestational mesenchymal stem cell secretome attenuates experimental bronchopulmonary dysplasia in part via exosome-associated factor TSG-6. Stem Cell Res Ther.2018;9:173. - PMC - PubMed