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. 2020 Sep 22;18(1):361.
doi: 10.1186/s12967-020-02525-3.

Synergic effects of decellularized bone matrix, hydroxyapatite, and extracellular vesicles on repairing of the rabbit mandibular bone defect model

Asrin Emami  1 , Tahereh Talaei-Khozani  1 , Saeid Tavanafar  2 , Nehleh Zareifard  1 , Negar Azarpira  3 , Zahra Vojdani  4
Affiliations

Synergic effects of decellularized bone matrix, hydroxyapatite, and extracellular vesicles on repairing of the rabbit mandibular bone defect model

Asrin Emami et al. J Transl Med. .

Abstract

Background: Extracellular vesicles (ECV) and bone extracellular matrix (ECM) have beneficial effects on the treatment of some pathological conditions. The purpose of this study was to find the synergic effects of decellularized bone (DB) ECM and ECVs on the repair of rabbit.

Methods: The quality of decellularized sheep bones was confirmed by H&E, Hoechst, DNA quantification, immunohistochemistry, histochemical staining, and scanning electron microscopy (SEM). Osteoblast-derived ECVs were evaluated by internalization test, Transmission electron microscopy, Dynamic light scattering, and flow cytometry for CD9, CD63, CD81 markers. The hydrogel containing DB and hydroxyapatite (HA) with or without ECVs was evaluated for osteoblast functions and bone repair both in vitro and in vivo.

Results: The data indicated ECM preservation after decellularization as well as cell depletion. In vitro assessments revealed that mineralization and alkaline phosphatase activity did not improve after treatment of MG63 cells by ECVs, while in vivo morphomatrical estimations showed synergic effects of ECVs and DB + HA hydrogels on increasing the number of bone-specific cells and vessel and bone area compared to the control, DB + HA and ECV-treated groups.

Conclusions: The DB enriched with ECVs can be an ideal scaffold for bone tissue engineering and may provide a suitable niche for bone cell migration and differentiation.

Keywords: Decellularized bone; Extracellular vesicle; Hydroxyapatite; Tissue engineering.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
a Comparison of the HA extracted from the bovine bone and commercial HA. It indicates that most of the extracted inorganic components are HA. b Electron micrograph of extracted HA nanoparticles.
Fig. 2
Fig. 2
The sections of decellularized scaffolds (a, c) and intact bone (b, d) stained with Hoechst and H&E. Both staining confirm cell and nuclei depletion. Also, the graph (e) compares DNA content of decellularized scaffold and the intact bone. Scanning electron microphotographs revealed scaffold porosity, ultrastructure preservation and orientation of the collagen fibers of decellularized scaffold (f) and intact bone (g). * Significant difference with control group (P < 0.05)
Fig. 3
Fig. 3
Histochemical Staining shows partially preservation of the ECM components after decellularization. Alcian blue (pH 2.5), aldehyde fuchsin and PAS stain acidic GAG and neutral carbohydrates preserved in ECM of decellularized and intact bone, and trichrome Masson stains collagen fibers
Fig. 4
Fig. 4
Immunohistochemistry shows preservation of collagen type I, fibronectin, laminin and osteopoitin in ECM of decellulsrized bone as compared to intact bone
Fig. 5
Fig. 5
Transmission electron micrograph of the EVCs. a Shows the ultrastructure of the EVCs. Due to different sizes, EVC population is heterogeneous and contains exosome and microvesicles. Internalization potential of the extracellular vesicles exposed to osteoblasts (b). The red particle in the osteoblast indicates internalized ECVs. Also, flow cytometry of a representative sample of ECVs (c) shows that they were positive for CD63, CD9 and CD81
Fig. 6
Fig. 6
MG63 cell line showed similar attachment property to both decellularized scaffold and polystyrene culture dish (a). The graph compares viability of the cell on decellularized scaffold (b). It was revealed that the scaffolds were not toxic and protected cell proliferation, but the presence of ECVs had no influence on cell viability. Calcium deposition by alizarin red S staining (c) and ALP activity (d) increased as the time progress; however, the presence of ECVs did not affect on mineralization
Fig. 7
Fig. 7
Comparing the gross and radiological examinations show that the repair accelerated in the defects treated with DB + HA + ECVs on 2 weeks after surgery. Arrows show the borders of the defects. DB decellularized bone, HA hydroxyapatite, ECVs extracellular vesicles
Fig. 8
Fig. 8
Comparing the gross and radiological examinations show that the repair in defects treated with DB + HA + ECVs was better than all other groups after 8 weeks. DB decellularized bone, HA hydroxyapatite, ECVs extracellular vesicles
Fig. 9
Fig. 9
Low magnification of the interface between the host bone and implant (arrows) after 2 weeks. Small square shows higher magnification of this region. (*) shows the implant that replaced with newly formed repairing tissue. It contains small islands of bone spicules
Fig. 10
Fig. 10
Morphometrical estimation of the bone area, fibrous connective tissue area, adipose tissue area and vessel area after 2 and 8 weeks. DB decellularized bone, HA hydroxyapatite, ECVs extracellular vesicles
Fig. 11
Fig. 11
Morphometrical estimation of the number of osteocytes, osteoblasts and osteoclasts after 2 and 8 weeks recovery. DB decellularized bone, HA hydroxyapatite, ECVs extracellular vesicles
Fig. 12
Fig. 12
Histological sections of repairing defects treated with various scaffolds after 2 weeks recovery. The control defect (A, a) and the defects treated with DB + HA + ECVs (B, b), DB + HA (C, c) and ECVs (D, d) after 2 (above) and 8 (below) weeks. DB decellularized bone, HA hydroxyapatite, ECVs extracellular vesicles
Fig. 13
Fig. 13
The remnant of the scaffolds (*) in the defect after 2 (a) and 8 (b) weeks
See this image and copyright information in PMC

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