Horizon of exosome-mediated bone tissue regeneration: The all-rounder role in biomaterial engineering

Abstract

Bone injury repair has always been a tricky problem in clinic, the recent emergence of bone tissue engineering provides a new direction for the repair of bone injury. However, some bone tissue processes fail to achieve satisfactory results mainly due to insufficient vascularization or cellular immune rejection. Exosomes with the ability of vesicle-mediated intercellular signal transmission have gained worldwide attention and can achieve cell-free therapy. Exosomes are small vesicles that are secreted by cells, which contain genetic material, lipids, proteins and other substances. It has been found to play the function of material exchange between cells. It is widely used in bone tissue engineering to achieve cell-free therapy because it not only does not produce some immune rejection like cells, but also can play a cell-like function. Exosomes from different sources can bind to scaffolds in various ways and affect osteoblast, angioblast, and macrophage polarization in vivo to promote bone regeneration. This article reviews the recent research progress of exosome-loaded tissue engineering, focusing on the mechanism of exosomes from different sources and the application of exosome-loaded scaffolds in promoting bone regeneration. Finally, the existing deficiencies and challenges, future development directions and prospects are summarized.

Keywords: Biocompatibility; Biomaterials; Bone tissue engineering; Exosomes; Osteoinduction.

Graphical abstract

Fig. 1

Exosomes and bone regeneration. The main components of exosomes and the role of exosomes in repairing bone and cartilage damage. (mRNA: messenger RNA, miRNA: microRNA, lncRNA: long noncoding RNA, SM: sphingomyelin, PA: phosphatidic acid).

Fig. 2

Bone tissue engineering materials. The materials commonly used in bone tissue engineering can be generally divided into metal materials, organic materials, and inorganic materials. Different materials have their own characteristics and shortcomings. (TJR: total joint replacement, TCP: tricalcium phosphate, HAP: hydroxyapatite, BAG: bioactive glass, PLGA: poly(lactic-co-glycolic acid), PGA: polyglycolic acid, PCL: polycaprolactone, PLA: polylactic acid).

Fig. 3

Application of exosome-loaded inorganic materials in bone tissue engineering (A) Autophagic activity of hBMSCs stimulated by experimental group or control group. Representative confocal microscopic images of hBMSCs stimulated with experimental group or control group encapsulated nanotubes for 1, 3 and 7 days under osteogenic medium. (B) hBMSCs were incubated with experimental group encapsulated-nanotubes or control group encapsulated nanotubes under osteogenic differentiation for 4 days. The conditioned medium collected was measured using proteome profiling human XL cytokine array (Reproduced with permission of Ref. [85]). (C) Three-dimensional reconstruction and sagittal images showed different reparative effects of HA, exosomes derived from MSCs (MSC-Exos), and DMOG-stimulated MSCs (DMOG-MSC-Exos), and new blood vessels in calvarial defects are shown in three-dimensional reconstruction images (Reproduced with permission of Ref. [89]).

Fig. 4

Application of exosome-loaded MBG in bone tissue engineering (A) An overview of the study. (B):(i) Principal component analysis (PCA) of miRNAs of MSC-exos from different sources and conditions. (ii) Heatmaps of the differentially expressed miRNAs of BMSC-derived exosomes before and after osteoinductive treatment. (iii) Heatmaps of the differentially expressed exosomal miRNAs of exosomes under different interventions. (C) Histological evaluation of the bone sections 12 weeks post-implantation stained with VG (undecalcified), Masson's trichrome and HE (decalcified). (M: material; nb: new bone.) (Reproduced with permission of Ref. [90]).

Fig. 5

Application of exosome-loaded ECM in bone tissue engineering (A) Schematic illustration of the whole study. (B) (i): Micro-MRI findings. The red arrowheads show the locations of the defects. (ii): Micro-CT findings, the red rectangles show the subchondral bone at the defect sites. (C) (iii): Macroscopic results for new cartilage. The red circles show the locations of the defects. Scale bar ​= ​5 ​mm. (iv): ICRS cartilage repair macroscopic score. (∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.005, ∗∗∗∗P ​< ​0.001). (D) (v): Macroscopic view of the ACECM scaffold. (vi) SEM results of the cross-section of the ACECM scaffold. (vii): SEM results for a longitudinal section of the ACECM scaffold. (viii): SEM results for BMSCs planted on the ACECM scaffold for 4 days (the red arrowhead indicates the clustered cells). (ix–xi) Confocal laser observation of live/dead-stained BMSCs planted on the ACECM scaffold for 1, 4, and 7 days (green indicates live cells, and red indicates dead cells) (Reproduced with permission of Ref. [91]).

Fig. 6

Application of exosome-loaded liposomes and PCL scaffolds in bone tissue engineering (A) Schematic illustration of exosome-guided miRNA blocking. (B) (i) Biophotonic images of the organ distribution 4 ​h after intravenous injection of PBS, Cy5, Cy5 labeled liposomes and Cy5-labeled hybrid NPs, with various exosome-liposome ratios. (ii) Representative fluorescence microscopic images of the femur 1, 2, and 4 ​h after injection of Cy5-labeled CXCR4+ hybrid NPs (Reproduced with permission of Ref. [92]). (C) General idea of engineered exosome enhanced therapies on osteogenesis and angiogenesis. (iii) 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. (iv) 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 (v) and angiogenesis (vi). (D) Modification of 3D-printed PCL scaffolds. (vii) 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). (viii) 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 (Reproduced with permission of Ref. [93]).

Fig. 7

Surface characterization of engineered PLGA substrates (A) Scanning electron microscopy of PLGA scaffolds (PLGA), PLGA scaffolds coated with polydopamine coating (PLGA/pDA), and PLGA scaffolds coated with polydopamine and exosomes (PLGA/pDA-Exo). (B) Distribution of PKH-26 labeled exosomes on the PLGA-only scaffold (middle) and PLGA/pDA scaffold (right), with PKH-26-stained scaffold as control (left). (C) In vitro exosome release kinetics in saline from exosomes-loaded PLGA by physical absorption (PLGA/Exo) and PLGA/pDA-Exo scaffolds. (D) Exosomes increased bone formation in critical-sized mouse calvarial defects. Mice were treated with PLGA scaffolds (PLGA), PLGA scaffolds with polydopamine coating (PLGA/pDA), or PLGA scaffolds coated with polydopamine and exosomes (PLGA/pDA-Exo). (i) Micro-CT images of bone formation in each group after 6 weeks. (ii) Quantitative comparison of new bone volume among the different groups. ∗∗p ​< ​0.01 compared with groups without exosomes. Histological assessment of bone formation in each group: (iii) HE staining. (iv) Masson staining. The collagen in the bone matrix was stained blue-green. The purple inclusions indicated by the white arrows were the remaining PLGA material. HB, host bone. Immunohistochemical staining for the osteogenic markers (v) RUNX2 and (vi) osteocalcin (OCN). Dark-brown granules indicating positive staining are marked by red arrows. The black arrows marked the newly formed tissue and white arrows indicated the area where the remaining PLGA material was located (Reproduced with permission of Ref. [97]).

Fig. 8

Flow chart of encapsulation of exosomes by double emulsion technology and application of exosome-loaded PLGA scaffolds in bone tissue engineering (A) Development of a dual flow-focusing junction microfluidic device to facilitate exosome encapsulation. The device was designed to facilitate the formation of a water/oil/water double emulsion (i) via two flow focusing junctions (ii) where immiscible solvents contacted and form droplets which later become particles (iii). CAD drawings of the device were used to simulate fluid flow using COMSOL, demonstrating the formation of droplets at both junctions (iv, v). Droplet size was controlled by the relative flow rates of two immiscible solvents when they contacted at the flow focusing junction (vi). PDMS devices were interfaced with pressure-driven pumps and droplet formation was visualized under a light microscope (vii, viii). Representative images shown. Scale ​= ​100 ​μm (Reproduced with permission of Ref. [100]). (B) Characteristics of different samples. (ix) Illustration of surface modification of PEEK. Fe3+ acts as an ionic cross-linker that can interact with up to three 3,4-dihydroxy-l-phenylalanine (DOPA) catechol functionalities to promote TA cross-linking. BMSC-derived Exos were reversibly bound to TA-SPEEK via hydrogen bond formation between phosphate groups in Exos phospholipid and polyphenol groups in the TA molecule. (x) FE-SEM images of PEEK, SPEEK, TA-SPEEK and Exo-TA-SPEEK. Scale bar represents 500 ​nm. (C) In vitro RAW264.7 ​cells polarization. (xi) IBa-1 and Arg-1 immunofluorescent staining of RAW264.7 ​cells on each sample surface are shown three days following culture. IBa-1 was stained green, Arg-1 was stained red and nuclei was stained blue. Scale bar represents 50 ​μm. (xii) IBa-1 and iNOS immunofluorescent staining of RAW264.7 ​cells on each sample surface three days after culture. IBa-1 was stained green, iNOS was stained red and nuclei was stained blue. Scale bar represents 50 ​μm (Reproduced with permission of Ref. [99]).

Fig. 9

Exosome-loaded hydrogels for cartilage regeneration (A) Schematic illustration of the one-step operation system for facilitating osteochondral defect regeneration. (i) Stereolithography-based ECM/GelMA/exosome bioprinting and osteochondral defect implantation. (ii) Migration of chondrocytes to the defect regions. (iii) Controlled administration of exosomes by the 3D-printed scaffolds. (iv) Enhanced chondrocyte mitochondrial biogenesis by the scaffolds. (B) Cartilage ECM/GelMA bioink preparation. (v) Images of the hydrogel before and after cross-linking. (vi) SEM images of the hydrogel with 1–3 ​wt % ECM in different magnifications (scale bar ​= ​100 ​μm or 50 ​μm). (vii) 2D and 3D AFM images of the hydrogel with different ECM concentrations. (viii) μCT reconstruction imaging of repaired knees at 6 and 12 weeks after surgery in various groups. (C) Differentially expressed proteins in normal and OA chondrocytes. (ix) Heat map of 289 downregulated and 191 upregulated proteins in OA samples compared with normal samples. (x-xii) GO classification of differentially expressed proteins (Reproduced with permission of Ref. [108]).

Fig. 10

Exosome-loaded hydrogels for OA treatment (A) As indicated at day 21, the pellets with different treatments were stained with Safranin O, Alcian blue and Toluidine blue. Experiments were repeated at least three times, and representative results are shown. Error bars show standard deviation. (B) Schematic diagram of the approach for combining sEVs derived from SMSCs (SMSC-sEVs) and sleep-related circRNA3503 used in this study. (C) Schematic diagram of the mechanism of sEVs derived from SMSCs with circRNA3503 overexpression (circRNA3503-OE-sEVs). (D) Schematic diagram summarising the mechanism by which circRNA3503-OE-sEVs prevent OA progression (Reproduced with permission of Ref. [109]).