An ECM-Mimetic Hydrogel to Promote the Therapeutic Efficacy of Osteoblast-Derived Extracellular Vesicles for Bone Regeneration

Abstract

The use of extracellular vesicles (EVs) is emerging as a promising acellular approach for bone regeneration, overcoming translational hurdles associated with cell-based therapies. Despite their potential, EVs short half-life following systemic administration hinders their therapeutic efficacy. EVs have been reported to bind to extracellular matrix (ECM) proteins and play an essential role in matrix mineralisation. Chitosan and collagen type I are naturally-derived pro-osteogenic biomaterials, which have been demonstrated to control EV release kinetics. Therefore, this study aimed to develop an injectable ECM-mimetic hydrogel capable of controlling the release of osteoblast-derived EVs to promote bone repair. Pure chitosan hydrogels significantly enhanced compressive modulus (2.48-fold) and osteogenic differentiation (3.07-fold), whilst reducing gelation times (2.09-fold) and proliferation (2.7-fold) compared to pure collagen gels (p ≤ 0.001). EV release was strongly associated with collagen concentration (R² > 0.94), where a significantly increased EV release profile was observed from chitosan containing gels using the CD63 ELISA (p ≤ 0.001). Hydrogel-released EVs enhanced human bone marrow stromal cells (hBMSCs) proliferation (1.12-fold), migration (2.55-fold), and mineralisation (3.25-fold) compared to untreated cells (p ≤ 0.001). Importantly, EV-functionalised chitosan-collagen composites significantly promoted hBMSCs extracellular matrix mineralisation when compared to the EV-free gels in a dose-dependent manner (p ≤ 0.001). Taken together, these findings demonstrate the development of a pro-osteogenic thermosensitive chitosan-collagen hydrogel capable of enhancing the therapeutic efficacy of osteoblast-derived EVs as a novel acellular tool for bone augmentation strategy.

Keywords: bone; controlled release; drug delivery; extracellular vesicle; hydrogel; tissue engineering.

FIGURE 1

Experimental outline detailing the development of a chitosan-collagen composite hydrogel for promoting EV therapeutic efficacy for site specific bone regeneration. 1) The influence of chitosan-collagen hydrogel content on material properties and osteogenic differentiation. 2) EV isolation, characterisation, and hydrogel-EV release kinetics. 3) The biological efficacy of hydrogel-released EVs on hBMSCs behaviour. 4) The influence of EV-functionalised hydrogel on encapsulated hBMSCs mineralisation. Created with BioRender.com.

FIGURE 2

Physiochemical properties of chitosan-collagen hydrogels. (A) pH of composite hydrogels before and after β-GP addition. (B) Gelation time of hydrogels at 37°C. (C) Compressive modulus. (D) Macroscopic image of hydrogels after gelation. Data are expressed as mean ± SD (n = 3). *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

FIGURE 3

The effects of chitosan-collagen composite hydrogel on proliferation and osteogenic differentiation. (A) Schematic representation of functional assessments. The influence of different hydrogel formulations on (B) proliferation, (C) ALP activity and (D,E) calcium deposition. Black staining indicates mineral nodules. Scale bar = 200 µm. Data are expressed as mean ± SD (n = 3). *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

FIGURE 4

Characterisation of isolated osteoblast-derived EVs and hydrogel-EV release kinetics. (A) TEM image of EVs. Scale bar = 50 nm. (B) Size distribution of isolated EV by NTA. Insert shows snapshot of particles during analysis. (C) Detection of tetraspanin-positive nanoparticles (CD81 and/or CD9) via interferometry after immuno-capture onto ExoView™ chip. (D) Quantification of EVs released from chitosan-collagen hydrogels assessed via CD63 positive ELISA. Data are expressed as mean ± SD (n = 3). *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

FIGURE 5

The biological efficacy of hydrogel-released EVs on hBMSCs behaviour. The influence on hydrogel-released EVs on hBMSCs (A) EV cell uptake. Scale bar = 20 μm, (B) proliferation, (C) migration, (D,F) collagen production, and (E,F) calcium deposition. Black staining indicates mineral nodules. Scale bar = 100 µm. Data are expressed as mean ± SD (n = 3). *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

FIGURE 6

The effect of EV-functionalised chitosan-collagen hydrogel on encapsulated hBMSCs osteogenic differentiation. (A) Schematic representation of EV-hydrogel groups (0 µg/ml = untreated, 50 µg/ml = EV-50, 100 µg/ml = EV-100). (B) Live/dead staining of encapsulated hBMSCs after 7 days of culture. Scale bar = 200 µm. (C) DNA content within EV-hydrogels after 7 days of culture. The influence on EV-hydrogels on hBMSCs (D) ALP activity, (E) collagen production, and (F,G) calcium deposition during osteogenic culture. Black staining indicates mineral nodules. Scale bar = 200 µm. Data are expressed as mean ± SD (n = 3). *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.