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Cited by 111Tissue Engineering and Regenerative MedicineMesenchymal stem cell researchMuscle Physiology and Disorders

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Mesenchymal stem cells and extracellular matrix scaffold promote muscle regeneration by synergistically regulating macrophage polarization toward the M2 phenotype

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Qiu, Xinyu, Liu, Shiyu, Zhang, Hao, Zhu, Bin, Su, Yuting, Zheng, Chenxi, Tian, Rong, Wang, Miao, Kuang, Huijuan, Zhao, Xinyi, Jin, Yan·2018·Stem Cell Research & Therapy
DOI 10.1186/s13287-018-0821-5PMC PMC5883419
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Abstract

BACKGROUND: Skeletal muscle plays an important role in the body's physiology but there are still no effective treatments for volumetric muscle loss (VML) resulting from severe traumatic injury or tumor excision. Recent studies show that a tissue engineering strategy using a compound containing mesenchymal stem cells (MSCs) and decellularized extracellular matrix (ECM) scaffold generates significant regenerative effects on VML injury, but the underlying mechanisms are not fully understood. METHODS: The characteristics of human umbilical cord MSCs, including multiplication capacity and multidifferentiation ability, were determined. We constructed a compound containing MSCs and decellularized ECM scaffold which was used for tissue regeneration in a VML model. RESULTS: We found that MSCs and decellularized ECM scaffold generated synergistic effects on promoting skeletal muscle tissue regeneration. Interestingly, both MSCs and decellularized ECM scaffold could promote macrophage polarization toward the M2 phenotype and suppress macrophage polarization toward the M1 phenotype, which is widely regarded as an important promoting factor in tissue regeneration. More importantly, MSCs and decellularized ECM scaffold generate synergistic promoting effects on macrophage polarization toward the M2 phenotype, not just an additive effect. CONCLUSIONS: Our findings uncover a previously unknown mechanism that MSCs and decellularized ECM scaffold promote tissue regeneration via collaboratively regulating macrophage polarization.

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Fig. 1 Decellularization of native heart tissue and biochemical analysis. a Representative optical images of native and decellularized heart tissue. b H&E staining images, Masson’s Trichrome staining images and SEM images of native and decellularized heart tissue. Scale bar, 100 μm. c Representative images of immunofluorescence staining for collagen I, fibronectin and laminin of native and decellularized heart tissue. Scale bar, 200 μm. d ECM components (COL and GAGs) and DNA contents of native and decellularized heart tissue. All experiments performed in triplicate. Data shown as mean ± SD. *P < 0.05; NS, not significant (P > 0.05). H&E hematoxylin and eosin, SEM scanning electron microscopy, H-dECM heart decellularized extracellular matrix, GAG glycosaminoglycan
Fig. 1 Decellularization of native heart tissue and biochemical analysis. a Representative optical images of native and decellularized heart tissue. b H&E staining images, Masson’s Trichrome staining images and SEM images of native and decellularized heart tissue. Scale bar, 100 μm. c Representative images of immunofluorescence staining for collagen I, fibronectin and laminin of native and decellularized heart tissue. Scale bar, 200 μm. d ECM components (COL and GAGs) and DNA contents of native and decellularized heart tissue. All experiments performed in triplicate. Data shown as mean ± SD. *P < 0.05; NS, not significant (P > 0.05). H&E hematoxylin and eosin, SEM scanning electron microscopy, H-dECM heart decellularized extracellular matrix, GAG glycosaminoglycanCC BY · PubMed Central
Fig. 2 Characterization of human umbilical cord mesenchymal stem cells (hUCMSCs). a Flow cytometric analysis of ex-vivo-expanded hUCMSCs revealed positive expression of CD29, CD44, CD90 and CD105, and negative expression of CD31, CD34 and CD45. b Representative percentage of cell markers for identification. c Representative proliferation of single clone of hUCMSCs. d hUCMSCs seeded in plates induced with osteogenic medium for 10 days. Activity of ALP detected by ALP staining. e hUCMSCs cultured in osteogenic inductive conditions for 28 days, mineralized nodules found by Alizarin Red staining. f Cultured hUCMSCs formed Oil Red O-positive lipid cluster following 14 days of adipogenic induction. g–j Cell aggregate (g control) positively expressed fibronectin (h), collagen I (i) and integrin-β1 (j). k Arrangement of cells in cell aggregate observed by SEM. l H&E staining showing cell aggregate was dense and contained plenty of cells. n = 6 per group. Scale bar, 100 μm. PE phycoerythrin, CFU-F fibroblastic colony-forming unit, ALP alkaline phosphatase, Con control, SEM scanning electron microscopy, H&E hematoxylin and eosin
Fig. 2 Characterization of human umbilical cord mesenchymal stem cells (hUCMSCs). a Flow cytometric analysis of ex-vivo-expanded hUCMSCs revealed positive expression of CD29, CD44, CD90 and CD105, and negative expression of CD31, CD34 and CD45. b Representative percentage of cell markers for identification. c Representative proliferation of single clone of hUCMSCs. d hUCMSCs seeded in plates induced with osteogenic medium for 10 days. Activity of ALP detected by ALP staining. e hUCMSCs cultured in osteogenic inductive conditions for 28 days, mineralized nodules found by Alizarin Red staining. f Cultured hUCMSCs formed Oil Red O-positive lipid cluster following 14 days of adipogenic induction. g–j Cell aggregate (g control) positively expressed fibronectin (h), collagen I (i) and integrin-β1 (j). k Arrangement of cells in cell aggregate observed by SEM. l H&E staining showing cell aggregate was dense and contained plenty of cells. n = 6 per group. Scale bar, 100 μm. PE phycoerythrin, CFU-F fibroblastic colony-forming unit, ALP alkaline phosphatase, Con control, SEM scanning electron microscopy, H&E hematoxylin and eosinCC BY · PubMed Central
Fig. 3 Gross morphology and functional evaluation of TA muscle after VML injury and repair. a Gross appearance of VML-injured muscle in four groups. b–d Representative central thickness of TA muscle (b) and widths of the distal (c) and proximal (d) thirds of the TA muscle in each group. ECM-treated group, MSC-treated group and compound-treated group can all partially recover central thickness and distal width of TA muscle. e, f Mechanical function evaluation by ES-induced contractions observed in each group presented at 1 month (e) and 2 months (f). g Mean body weight of four groups over the course of 8 weeks. No differences observed among all groups at each time point. n= 6 per group. Data shown as mean ± SD. *P < 0.05; **P < 0.01; NS, not significant (P > 0.05). PBS phosphate buffer saline, ECM extracellular matrix, MSC mesenchymal stem cell, TA tibialis anterior
Fig. 3 Gross morphology and functional evaluation of TA muscle after VML injury and repair. a Gross appearance of VML-injured muscle in four groups. b–d Representative central thickness of TA muscle (b) and widths of the distal (c) and proximal (d) thirds of the TA muscle in each group. ECM-treated group, MSC-treated group and compound-treated group can all partially recover central thickness and distal width of TA muscle. e, f Mechanical function evaluation by ES-induced contractions observed in each group presented at 1 month (e) and 2 months (f). g Mean body weight of four groups over the course of 8 weeks. No differences observed among all groups at each time point. n= 6 per group. Data shown as mean ± SD. *P < 0.05; **P < 0.01; NS, not significant (P > 0.05). PBS phosphate buffer saline, ECM extracellular matrix, MSC mesenchymal stem cell, TA tibialis anteriorCC BY · PubMed Central
Fig. 4 TA muscle histological study following repair in four groups after transplantation for 8 weeks. a TA muscles harvested 8 weeks after VML injury. Longitudinal and cross-sections from four groups stained with H&E (D–K) and Masson’s Trichrome (L–S). b, c Surface ratio of new muscle fibers (b) and collagen fibers (c) formed in each group analyzed by Image-Pro Plus 6.0. n = 6 per group. #Area of the defect. Dashed line represents separation between two areas. Scale bar, 200 μm. Data shown as mean ± SD. **P < 0.01; ***P < 0.001; NS, not significant (P > 0.05). PBS phosphate buffer saline, ECM extracellular matrix, MSC mesenchymal stem cell
Fig. 4 TA muscle histological study following repair in four groups after transplantation for 8 weeks. a TA muscles harvested 8 weeks after VML injury. Longitudinal and cross-sections from four groups stained with H&E (D–K) and Masson’s Trichrome (L–S). b, c Surface ratio of new muscle fibers (b) and collagen fibers (c) formed in each group analyzed by Image-Pro Plus 6.0. n = 6 per group. #Area of the defect. Dashed line represents separation between two areas. Scale bar, 200 μm. Data shown as mean ± SD. **P < 0.01; ***P < 0.001; NS, not significant (P > 0.05). PBS phosphate buffer saline, ECM extracellular matrix, MSC mesenchymal stem cellCC BY · PubMed Central
Fig. 5 Macrophage polarization toward M2 phenotype after transplantation for 2 weeks. a Only a small amount of CD206 (red)-positive macrophage appeared in the PBS-treated group, few in the ECM-treated group and MSC-treated group, and plenty of CD206-positive macrophages detected in the compound treated group. Nuclei stained with Hoechst 33342 (blue). b Field of view for CD206-positive macrophages in each group analyzed by Image-Pro Plus 6.0. c Plenty of iNOS (red)-positive macrophages showed in the PBS-treated group, meanwhile, only few can be detected in the compound-treated group. Nuclei were stained with Hoechst 33342 (blue). d Field of view for iNOS-positive macrophages in each group analyzed by Image-Pro Plus 6.0. n = 6 per group. #Area of defect. Dashed line represents separation between two areas. Scale bar, 200 μm. Data shown as mean ± SD. **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant (P > 0.05). PBS phosphate buffer saline, ECM extracellular matrix, MSC mesenchymal stem cell, iNOS inducible nitric oxide synthase
Fig. 5 Macrophage polarization toward M2 phenotype after transplantation for 2 weeks. a Only a small amount of CD206 (red)-positive macrophage appeared in the PBS-treated group, few in the ECM-treated group and MSC-treated group, and plenty of CD206-positive macrophages detected in the compound treated group. Nuclei stained with Hoechst 33342 (blue). b Field of view for CD206-positive macrophages in each group analyzed by Image-Pro Plus 6.0. c Plenty of iNOS (red)-positive macrophages showed in the PBS-treated group, meanwhile, only few can be detected in the compound-treated group. Nuclei were stained with Hoechst 33342 (blue). d Field of view for iNOS-positive macrophages in each group analyzed by Image-Pro Plus 6.0. n = 6 per group. #Area of defect. Dashed line represents separation between two areas. Scale bar, 200 μm. Data shown as mean ± SD. **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant (P > 0.05). PBS phosphate buffer saline, ECM extracellular matrix, MSC mesenchymal stem cell, iNOS inducible nitric oxide synthaseCC BY · PubMed Central
Fig. 6 Gene expression in inguinal lymph node after 2 weeks post operation. Gene expression measured in local (inguinal) lymph nodes at 2 weeks after transplantation to measure M1 phenotype-related (TNF-α, IL-1β, IL-6) (a–c) and M2 phenotype-related (IL-4, IL-10, IL-13) (d–f) cytokine changes dependent on ECM and MSCs application. n = 6 per group. Data shown as mean ± SD. **P < 0.01; ****P < 0.0001; NS, not significant (P > 0.05). IL, interleukin, PBS phosphate buffer saline, ECM extracellular matrix, MSC mesenchymal stem cell, TNF-α tumor necrosis factor alpha
Fig. 6 Gene expression in inguinal lymph node after 2 weeks post operation. Gene expression measured in local (inguinal) lymph nodes at 2 weeks after transplantation to measure M1 phenotype-related (TNF-α, IL-1β, IL-6) (a–c) and M2 phenotype-related (IL-4, IL-10, IL-13) (d–f) cytokine changes dependent on ECM and MSCs application. n = 6 per group. Data shown as mean ± SD. **P < 0.01; ****P < 0.0001; NS, not significant (P > 0.05). IL, interleukin, PBS phosphate buffer saline, ECM extracellular matrix, MSC mesenchymal stem cell, TNF-α tumor necrosis factor alphaCC BY · PubMed Central

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Metadata

Authors
Qiu, X., Liu, S., Zhang, H., Zhu, B., Su, Y., Zheng, C., Tian, R., Wang, M., Kuang, H., Zhao, X., & Jin, Y.
Affiliations
  • Air Force Medical University (CN)
  • Chinese People's Liberation Army (CN)
Corresponding author
  • Zhao, Xinyi
  • Jin, Yan
ORCID
Venue
Stem Cell Research & Therapy
Publisher
Springer Science and Business Media LLC
Published
2018-04-03
Volume
9
Issue
1
Pages
88-88
ISSN
1757-6512 (ISSN-L)
DOI
10.1186/s13287-018-0821-5
PMC
PMC5883419
OpenAlex
W2800380986
Cited by
111
References
64
Citations by year
2026: 5 · 2025: 12 · 2024: 13 · 2023: 7 · 2022: 17 · 2021: 17 · 2020: 21 · 2019: 13 · 2018: 6
FWCI
14.81
Type
journal article
Language
en
Open access
Licence
Locations
  • Stem Cell Research & Therapy (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Natural Science Foundation of China (31670995, 81470679)
  • National Key Research and Development Program of China (2016YFC1101400, 2016TFC1102903)
Topics
  • Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Mesenchymal stem cell research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
Keywords
mesenchymal stem cells, skeletal muscle regeneration, volumetric muscle loss, macrophage polarization, M2 macrophages, M1 macrophages, human umbilical cord mesenchymal stem cells, synergistic effect, tissue engineering, traumatic muscle injury
MeSH terms
Animals, Cell Differentiation, Cells, Cultured, Extracellular Matrix, Humans, Macrophages, Regeneration, Rats, Sprague-Dawley, Muscle, Skeletal, Mesenchymal Stem Cell Transplantation, Rats, Mesenchymal Stem Cells

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