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. 2021 May:126:199-210.
doi: 10.1016/j.actbio.2021.03.030. Epub 2021 Mar 16.

3D Encapsulation and tethering of functionally engineered extracellular vesicles to hydrogels

Chun-Chieh Huang  1 , Miya Kang  1 , Sajjad Shirazi  1 , Yu Lu  1 , Lyndon F Cooper  1 , Praveen Gajendrareddy  2 , Sriram Ravindran  3
Affiliations

3D Encapsulation and tethering of functionally engineered extracellular vesicles to hydrogels

Chun-Chieh Huang et al. Acta Biomater. 2021 May.

Abstract

Mesenchymal stem cell (MSC) derived extracellular vesicles (EVs) in their naïve and engineered forms have emerged as potential alternatives to stem cell therapy. While they have a defined therapeutic potential, the spatial and temporal control of their activity in vivo remains a challenge. The objective of this study was to devise a methodology to encapsulate EVs in 3D hydrogels for prolonged delivery. To achieve this, we have leveraged the MSC EV interactions with ECM proteins and their derivative peptides. Using osteoinductive functionally engineered EVs (FEEs) derived from MSCs, we show that FEEs bind to mimetic peptides from collagen (DGEA, GFPGER) and fibronectin (RGD). In in vitro experiments, photocrosslinkable alginate hydrogels containing RGD were able to encapsulate, tether and retain the FEEs over a period of 7 days while maintaining the structural integrity and osteoinductive functionality of the EVs. When employed in a calvarial defect model in vivo, alginate-RGD hydrogels containing the FEEs enhanced bone regeneration by a factor of 4 compared to controls lacking FEEs and by a factor of 2 compared to controls lacking the tethering peptide. These results show that EVs can be tethered to biomaterials to promote bone repair and the importance of prolonged delivery in vivo. Results also provide a prelude to the possible use of this technology for controlled delivery of EVs for other regenerative medicine applications. STATEMENT OF SIGNIFICANCE: The beneficial effects of human MSC (HMSC) therapy are attributable to paracrine effects of the HMSC derived EVs. While EV engineering has the potential to impact several fields of regenerative medicine, targeted delivery of the engineered EVs with spatial and temporal control is necessary to prevent off-target effects and enhance tissue specificity. Here, we have leveraged the interactions of MSC EVs with ECM proteins to develop a tethering system that can be utilized to prolong EV delivery in vivo while maintaining the structural and functional integrity of the EVs. Our work has provided a tunable platform for EV delivery that we envision can be formulated as an injectable material or a bulk hydrogel suitable for regenerative medicine applications.

Keywords: Alginate; Bone regeneration; Controlled release; Exosomes; Extracellular vesicles; Integrins.

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Conflict of interest statement

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1.
Figure 1.. Characterization of EVs:
A) Immunoblots of FEE and cell lysates for the presence of TSG101, HSP70, CD63 exosomal marker proteins as well as for the intracellular protein control tubulin. Note the absence of tubulin in the EV lysates. B) Representative NTA plot of FEEs indicating the size distribution of the isolated EVs. C) Representative TEM image of FEEs immunolabeled for CD63 (20 nm gold dots). The insert represents the boxed area. The arrows in the insert point to the EV membrane. D) Confocal micrograph showing a representative image of the uptake of the fluorescently labeled FEEs (green) by HMSCs fluorescently stained with tubulin antibody (red) and DAPI nuclear stain (blue).
Figure 2.
Figure 2.. Binding of FEEs to ECM proteins:
A) Confocal micrograph showing a representative image of the binding of fluorescently labeled FEEs (green) to the decellularized ECM of HMSCs immunostained for type I collagen (red). Arrow in merged image points to a representative area of colocalization. Scale bar represents 10μm. B) Graphical representation of dose-dependent and saturable binding of fluorescently labeled FEEs to type I collagen coated assay plates (data points represent mean +/− SD, n=6). C) Confocal micrograph showing a representative image of the binding of fluorescently labeled FEEs (green) to the decellularized ECM of HMSCs immunostained for fibronectin (red). Arrow in merged image points to a representative area of colocalization. Scale bar represents 10μm. D) Graphical representation of dose-dependent and saturable binding of fluorescently labeled FEEs to fibronectin coated assay plates (data points represent mean +/− SD, n=6).
Figure 3.
Figure 3.. Binding of FEEs to ECM derivative peptides (2D kinetics):
A) Graphical representation of dose-dependent binding of fluorescently labeled FEEs to RGD, DGEA and GFPGER peptide coated assay plates. B) Graphical representation of dose-dependent binding of FEEs (with or without pre-treatment with collagen mimetic peptides DGEA and GFPGER) to type I collagen. Note the reduction in binding to type I collagen when the FEEs are pre-treated with the peptides. C) Graphical representation of dose-dependent binding of FEEs (with or without pre-treatment with RGD peptide) to fibronectin. Note the reduction in binding to fibronectin when the FEEs are pre-treated with the RGD peptide. D) Graphical representation of the fluorescently labeled FEE release from type I collagen, fibronectin, RGD, DGEA, and GFPGER coated assay plates over time. In all the graphs, the blue lines represent fibronectin and/or derivative peptides and black lines represent type I collagen and/or derivative peptides. For all graphs, data points represent mean +/− SD, n=6, * represents statistical significance of the peptide treated group to untreated control group at all concentrations calculated using Tukey’s ad hoc test post ANOVA. E) Immunoblots of FEE lysates probed for the presence of ITGA5 and ITGAV proteins. Yellow arrow in the ITGAV blot points to the expected molecular weight of the protein.
Figure 4.
Figure 4.. Release and integrity of alginate hydrogel encapsulated FEEs in a 3D system using RGD as a tether:
A) Graphical representation of 3D encapsulated fluorescently labeled FEE release from 2% (blue lines) and 4% (black lines) alginate hydrogels +/− RGD peptide over time. Data points represent mean +/− SD, n=6. * represents statistical significance with respect to RGD vs no RGD containing hydrogel pairs and # represents statistical significance of the 4% A-RGD group compared to the 2% A-RGD group calculated using Tukey’s ad hoc test post ANOVA. B, C, D, E and F) Representative NTA plots of FEEs released from each of the hydrogel groups indicating EV size distribution before (control (B)) and after release from the 2% (C) and 4% (D) alginate hydrogels as well as the 2% and 4% alginate hydrogels containing the RGD peptide (E and F respectively). The inserts in the figure show average particle size and the poly dispersity index (PDI).
Figure 5.
Figure 5.. In vitro functionality of hydrogel encapsulated FEEs:
A) A schematic representation of the contactless experimental setup followed by confocal micrograph showing a representative image of the uptake of the fluorescently labeled FEEs (red) released from 4% A-RGD alginate hydrogels by HMSCs fluorescently stained with actin (green) and DAPI (blue) at 24 hours post the no contact experiment start time. Note the absence of EV presence in the cells indicating that minimal amounts of EVs were released from the hydrogels. The following graph depicts fold change in osteogenic gene expression at 3 and 5 days post the no contact experiment start time. No significant changes in expression levels were noted in comparison to control setup lacking EVs. The final graph shows fold change in alkaline phosphatase (ALP) activity in HMSCs 3 and 5 days the start of the contactless experiment. No significant change was observed with respect to the control group. B) A schematic representation of the contact experimental setup followed by 3D Confocal micrograph showing a representative image of the uptake of the fluorescently labeled BMP2 FEEs (red) by HMSCs fluorescently stained with actin (green) at 24 hours post the contact experiment start time. The following graph depicts fold change in osteogenic gene expression at 3 and 5 days post the contact experiment start time. The final graph shows fold change in ALP activity in HMSCs 3 and 5 days the start of the contact experiment. For all graphical figures, data points represent mean fold change +/− SD. * represents statistical significance with respect to the control group measured by student’s t-test.
Figure 6.
Figure 6.. In vivo functionality of the encapsulated FEEs:
A) Representative μCT images of 5 mm calvarial defects that were treated with alginate hydrogels (+/−FEEs) and alginate-RGD hydrogels +/− FEEs at 4-weeks and 8-weeks post wounding. Scale bar in all μCT images represents 5mm. B) Volumetric quantitation of the μCT data expressed as percentage bone volume regenerated to total void volume (n=6 defects per group and per time point). * represents statistical significance with respect to the alginate alone control group (no RGD, no FEEs) and # represents statistical significance with respect to the A-RGD alone (no FEEs) group. $ represents statistical significance for A+FEE versus A-RGD+FEE. * represents statistical significance for A-RGD+FEE 4 weeks versus A-RGD+FEE 8 weeks calculated using Tukey’s post hoc test post ANOVA.
Figure 7.
Figure 7.. Histological evaluation of calvarial defects:
Images are representative light microscopy images of H&E stained demineralized calvarial samples of defects treated with alginate hydrogels (+/−FEEs) and alginate-RGD hydrogels +/− FEEs at 4- and 8-weeks post wounding. The black arrows in the images point to regenerated bone tissue. The yellow arrows in the images point to alginate hydrogel. Scale bar represents 100μm in all images.
Figure 8.
Figure 8.. IHC of calvarial defects:
Images represent the expression levels of osteoinductive marker protein DMP1 in the calvarial sections from the demineralized calvarial samples of defects treated with alginate hydrogels (+/− FEEs) and alginate-RGD hydrogels +/− FEEs at 4- and 8-weeks post wounding. Scale bar represents 50μm in all images. In all images green represents DMP1 presence in the section and the nuclei are stained in blue.
Figure 9.
Figure 9.. IHC of calvarial defects:
Images represent the expression levels of osteoinductive marker protein BSP in the calvarial sections from the demineralized calvarial samples of defects treated with alginate hydrogels (+/− FEEs) and alginate-RGD hydrogels +/− FEEs at 4- and 8-weeks post wounding. Scale bar represents 50μm in all images. In all images red represents BSP presence in the section and the nuclei are stained in blue.
Figure 10.
Figure 10.. IHC of calvarial defects:
Images represent the expression levels of osteoinductive marker protein OCN in the calvarial sections from the demineralized calvarial samples of defects treated with alginate hydrogels (+/− FEEs) and alginate-RGD hydrogels +/− FEEs at 4- and 8-weeks post wounding. Scale bar represents 50μm in all images. In all images green represents OCN presence in the section and the nuclei are stained in blue.
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