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Cited by 232Extracellular vesicles in diseaseBone Tissue Engineering MaterialsMesenchymal stem cell research

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Progenitor cell-derived exosomes endowed with VEGF plasmids enhance osteogenic induction and vascular remodeling in large segmental bone defects

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Zha, Yao, Li, Yawu, Lin, Tianyi, Chen, Jia, Zhang, Shengmin, Wang, Jianglin·2021·Theranostics
DOI 10.7150/thno.50741PMC PMC7681080
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Abstract

In our current work, we designed specifically engineered exosomes based on the requirements of vascularized bone repair in segmental bone defects. This work simultaneously illuminates the potential of functional exosomes in acellular tissue engineering.

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Figure 1 General idea of engineered exosome enhanced therapies on osteogenesis and angiogenesis. (A) 3D-printed porous PCL scaffolds were modified with 1,6-hexanediamine to generate the amino group on PCL scaffolds that were subsequently modified with the exosomal anchor peptide CP05. (B) Engineered exosomes were fabricated by encapsulating the VEGF plasmid DNA into ATDC5-derived exosomes. The well-designed bone scaffolds were constructed by combining the engineered exosomes with the CP05 modified 3D-printed scaffolds, and eventually implanted into a rat radial defect model to promote osteogenesis (C) and angiogenesis (D).
Figure 1 General idea of engineered exosome enhanced therapies on osteogenesis and angiogenesis. (A) 3D-printed porous PCL scaffolds were modified with 1,6-hexanediamine to generate the amino group on PCL scaffolds that were subsequently modified with the exosomal anchor peptide CP05. (B) Engineered exosomes were fabricated by encapsulating the VEGF plasmid DNA into ATDC5-derived exosomes. The well-designed bone scaffolds were constructed by combining the engineered exosomes with the CP05 modified 3D-printed scaffolds, and eventually implanted into a rat radial defect model to promote osteogenesis (C) and angiogenesis (D).CC BY · PubMed Central
Figure 2 Characterization of ATDC5-derived exosomes. (A) TEM images of ATDC5-derived exosomes (EXOs) presented a spherical morphology with bi-layered membrane structure. (B) Two EXOs protein markers CD63 and TSG101 were verified by western blot. (C) NTA analysis showed that the size of nanoscale EXOs was about 114.2 nm (n = 3). (D) Zeta potential distribution of EXOs was about -32.0 mV by DLS (n = 3). (E) The grafting efficiency of CP05 to EXOs was as high as 90.96% based on the analysis of flow cytometry.
Figure 2 Characterization of ATDC5-derived exosomes. (A) TEM images of ATDC5-derived exosomes (EXOs) presented a spherical morphology with bi-layered membrane structure. (B) Two EXOs protein markers CD63 and TSG101 were verified by western blot. (C) NTA analysis showed that the size of nanoscale EXOs was about 114.2 nm (n = 3). (D) Zeta potential distribution of EXOs was about -32.0 mV by DLS (n = 3). (E) The grafting efficiency of CP05 to EXOs was as high as 90.96% based on the analysis of flow cytometry.CC BY · PubMed Central
Figure 3 Expression and angiogenetic effect of VEGF. (A) The engineered exosomes (EXOs-VEGF) was successfully transfected into rBMSCs after culturing for 24 h and 48 h, and the positive gene expression of VEGF plasmid (pEGFP-kozVEGF165) was elevated with increased culture time. The nuclei of rBMSCs were stained with DAPI (blue), the cytoskeletons of rBMSCs were stained with ActinRed (red). (B) Tube formation assays with HUVECs was performed using the serum-free supernatant of rBMSCs with pure exosome (EXOs) and EXOs-VEGF for 1 h and 3 h. The results further demonstrated that the EXOs-VEGF extremely promoted tube formation of HUVECs in vitro. The cytoskeletons of HUVECs were stained with FITC (green). (C, D) Both qRT-PCR and ELISA clearly confirmed that the relative expression of VEGF in EXOs-VEGF was ultra-higher than that in pure EXOs (Independent-sample t-tests; **, p < 0.01; ***, p < 0.001 compared to EXOs group) (n = 4).
Figure 3 Expression and angiogenetic effect of VEGF. (A) The engineered exosomes (EXOs-VEGF) was successfully transfected into rBMSCs after culturing for 24 h and 48 h, and the positive gene expression of VEGF plasmid (pEGFP-kozVEGF165) was elevated with increased culture time. The nuclei of rBMSCs were stained with DAPI (blue), the cytoskeletons of rBMSCs were stained with ActinRed (red). (B) Tube formation assays with HUVECs was performed using the serum-free supernatant of rBMSCs with pure exosome (EXOs) and EXOs-VEGF for 1 h and 3 h. The results further demonstrated that the EXOs-VEGF extremely promoted tube formation of HUVECs in vitro. The cytoskeletons of HUVECs were stained with FITC (green). (C, D) Both qRT-PCR and ELISA clearly confirmed that the relative expression of VEGF in EXOs-VEGF was ultra-higher than that in pure EXOs (Independent-sample t-tests; **, p < 0.01; ***, p < 0.001 compared to EXOs group) (n = 4).CC BY · PubMed Central
Figure 4 Modification of 3D-printed PCL scaffolds. (A) 3D-printed PCL scaffolds exhibited a highly intercommunicating porous morphology by SEM observation (Left image for the top view and Right image for the side view). (B) XPS analysis verified that the amino group (-NH2) was successfully coated onto the surface of 3D-printed PCL scaffold. (C) The amino groups between PCL and PCL-NH2 scaffolds were comparably analyzed by ninhydrin coloration. (D) The graft efficiency of the anchor peptide CP05 was obviously higher in PCL NH2+ than that in PCL NH2-. The CP05 was conjugated with Alexa Fluor 488 to present green fluorescence.
Figure 4 Modification of 3D-printed PCL scaffolds. (A) 3D-printed PCL scaffolds exhibited a highly intercommunicating porous morphology by SEM observation (Left image for the top view and Right image for the side view). (B) XPS analysis verified that the amino group (-NH2) was successfully coated onto the surface of 3D-printed PCL scaffold. (C) The amino groups between PCL and PCL-NH2 scaffolds were comparably analyzed by ninhydrin coloration. (D) The graft efficiency of the anchor peptide CP05 was obviously higher in PCL NH2+ than that in PCL NH2-. The CP05 was conjugated with Alexa Fluor 488 to present green fluorescence.CC BY · PubMed Central
Figure 5 In vitro interaction between exosome-mediated bone scaffolds and rBMSCs. (A) Schematic representation on the construction of the engineered exosome-mediated bone scaffolds via the linker of CP05 on combining nanoscale exosomes with macroscale bone scaffolds. (B) The graft efficiency of engineered exosomes connected onto 3D-printed bone scaffolds was significantly elevated with the help of the anchor peptide CP05. (C) Cell proliferation showed that there was no difference on biocompatibility before and after the modification with CP05. (one-way ANOVA followed by Tukey's post hoc test; NS, no significance compared to control group) (n = 4). (D) 3D-printed scaffolds were able to well support cell adhesion based on the analysis of confocal Z-stacks image and SEM image. (E) EXOs were internalized into 3D-printed scaffolds based on cell uptake. The nuclei of rBMSCs were stained with DAPI (blue), the cytoskeletons of rBMSCs were stained with FITC (green), and the EXOs were stained with DiI (red).
Figure 5 In vitro interaction between exosome-mediated bone scaffolds and rBMSCs. (A) Schematic representation on the construction of the engineered exosome-mediated bone scaffolds via the linker of CP05 on combining nanoscale exosomes with macroscale bone scaffolds. (B) The graft efficiency of engineered exosomes connected onto 3D-printed bone scaffolds was significantly elevated with the help of the anchor peptide CP05. (C) Cell proliferation showed that there was no difference on biocompatibility before and after the modification with CP05. (one-way ANOVA followed by Tukey's post hoc test; NS, no significance compared to control group) (n = 4). (D) 3D-printed scaffolds were able to well support cell adhesion based on the analysis of confocal Z-stacks image and SEM image. (E) EXOs were internalized into 3D-printed scaffolds based on cell uptake. The nuclei of rBMSCs were stained with DAPI (blue), the cytoskeletons of rBMSCs were stained with FITC (green), and the EXOs were stained with DiI (red).CC BY · PubMed Central
Figure 6 ATDC5-derived exosomes promoted osteogenic differentiation of rBMSCs. (A) Alizarin red staining showed EXOs could stimulate the formation of mineralization extracellular matrix of rBMSCs. (B) The osteogenic marker OCN was positively stained with Immunofluorescence staining, and cell nuclei and cytoskeletons were stained with DAPI (blue) and FITC-phalloidin (green), respectively. (C) QRT-PCR analysis of ALP, Col1a1, Runx2 and OCN further confirmed that both EXOs and EXOs-VEGF were able to promote osteogenic differentiation of rBMSCs and there was no significant difference between EXOs and EXOs-VEGF (one-way ANOVA followed by Tukey's post hoc test; *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to blank group) (n = 3).
Figure 6 ATDC5-derived exosomes promoted osteogenic differentiation of rBMSCs. (A) Alizarin red staining showed EXOs could stimulate the formation of mineralization extracellular matrix of rBMSCs. (B) The osteogenic marker OCN was positively stained with Immunofluorescence staining, and cell nuclei and cytoskeletons were stained with DAPI (blue) and FITC-phalloidin (green), respectively. (C) QRT-PCR analysis of ALP, Col1a1, Runx2 and OCN further confirmed that both EXOs and EXOs-VEGF were able to promote osteogenic differentiation of rBMSCs and there was no significant difference between EXOs and EXOs-VEGF (one-way ANOVA followed by Tukey's post hoc test; *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to blank group) (n = 3).CC BY · PubMed Central
Figure 7 Micro-CT and histological assessment of rat radial defect repair in vivo. (A) 3D reconstructed micro-CT images showed the experimental group of PCL-CP05~EXOs-VEGF exhibited the best repair and induced to form a bulk of new bone at 12 weeks after implantation. (B) Quantitative comparison of BV, BV/TV and Tb.Th presented the consistent results (one-way ANOVA followed by Tukey's post hoc test; NS, no significance; *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to blank group) (n = 5). (C) HE staining and (D) Masson staining clearly showed the formation of new bone in the experimental group of PCL-CP05~EXOs-VEGF at the endpoint 12 weeks. Right panels presented the enlargement views of the green rectangles in the left panels. All of these results solidly demonstrated that the experimental group could significantly stimulate the new bone forming. (E) Immunofluorescence staining of the angiogenic marker CD31 exhibited a positive staining at 12 weeks after implantation.
Figure 7 Micro-CT and histological assessment of rat radial defect repair in vivo. (A) 3D reconstructed micro-CT images showed the experimental group of PCL-CP05~EXOs-VEGF exhibited the best repair and induced to form a bulk of new bone at 12 weeks after implantation. (B) Quantitative comparison of BV, BV/TV and Tb.Th presented the consistent results (one-way ANOVA followed by Tukey's post hoc test; NS, no significance; *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to blank group) (n = 5). (C) HE staining and (D) Masson staining clearly showed the formation of new bone in the experimental group of PCL-CP05~EXOs-VEGF at the endpoint 12 weeks. Right panels presented the enlargement views of the green rectangles in the left panels. All of these results solidly demonstrated that the experimental group could significantly stimulate the new bone forming. (E) Immunofluorescence staining of the angiogenic marker CD31 exhibited a positive staining at 12 weeks after implantation.CC BY · PubMed Central

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Metadata

Authors
Zha, Y., Li, Y., Lin, T., Chen, J., Zhang, S., & Wang, J.
Affiliations
Huazhong University of Science and Technology (CN)
Corresponding author
Zhang, Shengmin
ORCID
Venue
Theranostics
Publisher
Ivyspring International Publisher
Published
2021
Volume
11
Issue
1
Pages
397-409
ISSN
1838-7640 (ISSN-L)
DOI
10.7150/thno.50741
PMC
PMC7681080
OpenAlex
W3095185855
Cited by
232
References
47
Citations by year
2026: 25 · 2025: 55 · 2024: 55 · 2023: 48 · 2022: 39 · 2021: 10
FWCI
7.86
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Theranostics (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • National Natural Science Foundation of China (2017YFC1103900, 31670968, 91939111, SZSM201812055, 31800805, 81601610)
  • Sanming Project of Medicine in Shenzhen (SZSM201812055)
  • National Key Research and Development Program of China (2018YFC1105700, 2017YFC1103900)
  • Fundamental Research Funds for the Central Universities (2019kfyRCPY103)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Physical Sciences › Engineering › Biomedical Engineering › Bone Tissue Engineering Materials
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Mesenchymal stem cell research
Keywords
exosomes, progenitor cells, vascular endothelial growth factor, osteogenic induction, vascular remodeling, segmental bone defect, gene delivery
MeSH terms
Animals, Bone and Bones, Bone Regeneration, Cell Adhesion, Cell Line, Male, Materials Testing, Osteogenesis, Plasmids, Polyesters, Radius, Stem Cells, Tissue Engineering, Vascular Endothelial Growth Factor A, Cell Proliferation, Mice, Rats, Tissue Scaffolds, X-Ray Microtomography, Exosomes, Vascular Remodeling, Printing, Three-Dimensional

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