Controlled Release of Epigenetically-Enhanced Extracellular Vesicles from a GelMA/Nanoclay Composite Hydrogel to Promote Bone Repair

Abstract

Extracellular vesicles (EVs) have garnered growing attention as promising acellular tools for bone repair. Although EVs’ potential for bone regeneration has been shown, issues associated with their therapeutic potency and short half-life in vivo hinders their clinical utility. Epigenetic reprogramming with the histone deacetylase inhibitor Trichostatin A (TSA) has been reported to promote the osteoinductive potency of osteoblast-derived EVs. Gelatin methacryloyl (GelMA) hydrogels functionalised with the synthetic nanoclay laponite (LAP) have been shown to effectively bind, stabilise, and improve the retention of bioactive factors. This study investigated the potential of utilising a GelMA-LAP hydrogel to improve local retention and control delivery of epigenetically enhanced osteoblast-derived EVs as a novel bone repair strategy. LAP was found to elicit a dose-dependent increase in GelMA compressive modulus and shear-thinning properties. Incorporation of the nanoclay was also found to enhance shape fidelity when 3D printed compared to LAP-free gels. Interestingly, GelMA hydrogels containing LAP displayed increased mineralisation capacity (1.41-fold) (p ≤ 0.01) over 14 days. EV release kinetics from these nanocomposite systems were also strongly influenced by LAP concentration with significantly more vesicles being released from GelMA constructs as detected by a CD63 ELISA (p ≤ 0.001). EVs derived from TSA-treated osteoblasts (TSA-EVs) enhanced proliferation (1.09-fold), migration (1.83-fold), histone acetylation (1.32-fold) and mineralisation (1.87-fold) of human bone marrow stromal cells (hBMSCs) when released from the GelMA-LAP hydrogel compared to the untreated EV gels (p ≤ 0.01). Importantly, the TSA-EV functionalised GelMA-LAP hydrogel significantly promoted encapsulated hBMSCs extracellular matrix collagen production (≥1.3-fold) and mineralisation (≥1.78-fold) in a dose-dependent manner compared to untreated EV constructs (p ≤ 0.001). Taken together, these findings demonstrate the potential of combining epigenetically enhanced osteoblast-derived EVs with a nanocomposite photocurable hydrogel to promote the therapeutic efficacy of acellular vesicle approaches for bone regeneration.

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

“…Importantly, we demonstrated that the EV-functionalised composite gels significantly enhanced hBMSCs osteogenic differentiation and mineralisation when compared to the EV-free groups, consistent with observations in the literature ( Man et al, 2021b ). Similar to the effects on proliferation, our findings showed an EV dose-dependent increase in mineralisation within the hydrogel, consistent with the concentration-dependent increase in hBMSCs mineralisation observed within a GelMA nanocomposite hydrogel ( Man et al, 2022 ). These findings further signify the efficacy of encapsulated EVs in stimulating hBMSCs osteogenesis within this hydrogel system.…”

“…At day 1, 3, 5, and 7, the receiving medium was collected, and replaced by an equal volume of fresh PBS. Previous studies have shown that EVs derived from mineralising osteoblasts were CD63 positive and as such we selected this marker for the release study ( Man et al, 2021b ; Man et al, 2022 ). The EV concentration in the collected medium was evaluated using the CD63 ExoELISA-ULTRA complete kit (System Biosciences, United States) following the manufacturer’s protocol.…”

GelMA Hydrogel Reinforced with 3D Printed PEGT/PBT Scaffolds for Supporting Epigenetically-Activated Human Bone Marrow Stromal Cells for Bone Repair

“…Hence, there is growing precedence for controlling the delivery of MSCs in situ to maximise their therapeutic efficacy. GelMA hydrogels have demonstrated their potential for different tissue engineering applications due to their plethora of desirable properties [ 34 , 38 , 39 ]. Hence, this study aimed to evaluate the capability of GelMA hydrogels reinforced with a 3D printed structural scaffold to support the MI192-induced mineralisation of hBMSCs.…”

“…The samples were centrifuged at 10,000× g for 10 min at 4 °C, and lysates were collected for ALPSA assay. ALPSA quantification was determined using the 4-nitrophenyl colorimetric phosphate liquid system (pNPP, Sigma-Aldrich, Gillingham, UK) as previously described [ 15 , 34 ]. ALPSA was determined by normalisation with the total DNA content of the same sample.…”

Printable Hydrogels Based on Alginate and Halloysite Nanotubes

“…Among the various counterparts used in the preparation strategy, nanoparticles such as nanoclays hold a certain importance [ 15 , 16 ]. Halloysite nanotubes (HNTs) are naturally occurring aluminosilicates possessing a hollow tubular shape in the nanometric scale and opposite charges between the external surface, which is negative, and the internal surface, which is positive [ 17 , 18 , 19 , 20 ].…”