MI192 induced epigenetic reprogramming enhances the therapeutic efficacy of human bone marrows stromal cells for bone regeneration

Barroso

Brunet

et al. 2022

IJMS

Self Cite

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.

Section: Discussionsupporting

confidence: 61%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Barroso

Brunet

et al. 2022

IJMS

Self Cite

“…For example, Lee et al showed that HDAC activity is reduced during osteogenesis, which resulted in hyperacetylation induced gene activation ( Lee et al, 2006 ). As such, researchers have harnessed epigenetic modifying compounds including HDAC inhibitors (HDACis) to accelerate osteogenic differentiation both in vitro and in vivo ( Huynh et al, 2016 ; Man et al, 2021c ). Moreover, several studies have demonstrated the role of biomaterials substrates in augmenting key epigenetic functions via the modification of the cells actin cytoskeleton which transmits forces to the nuclear protein lamin A/C, ultimately resulting in enhanced transcriptional activation ( Li et al, 2011 ; Downing et al, 2013 ).…”

Section: Discussionmentioning

confidence: 99%

Development of a Bone-Mimetic 3D Printed Ti6Al4V Scaffold to Enhance Osteoblast-Derived Extracellular Vesicles’ Therapeutic Efficacy for Bone Regeneration

Front. Bioeng. Biotechnol.

Brunet

Louth

et al. 2021

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Extracellular Vesicles (EVs) are considered promising nanoscale therapeutics for bone regeneration. To date, EVs are typically procured from cells on 2D tissue culture plastic, an artificial environment that limits cell growth and does not replicate in situ biochemical or biophysical conditions. This study investigated the potential of 3D printed titanium scaffolds coated with hydroxyapatite to promote the therapeutic efficacy of osteoblast-derived EVs. Ti6Al4V titanium scaffolds with different pore sizes (500 and 1000 µm) and shapes (square and triangle) were fabricated by selective laser melting. A bone-mimetic nano-needle hydroxyapatite (nnHA) coating was then applied. EVs were procured from scaffold-cultured osteoblasts over 2 weeks and vesicle concentration was determined using the CD63 ELISA. Osteogenic differentiation of human bone marrow stromal cells (hBMSCs) following treatment with primed EVs was evaluated by assessing alkaline phosphatase activity, collagen production and calcium deposition. Triangle pore scaffolds significantly increased osteoblast mineralisation (1.5-fold) when compared to square architectures (P ≤ 0.001). Interestingly, EV yield was also significantly enhanced on these higher permeability structures (P ≤ 0.001), in particular (2.2-fold) for the larger pore structures (1000 µm). Furthermore osteoblast-derived EVs isolated from triangular pore scaffolds significantly increased hBMSCs mineralisation when compared to EVs acquired from square pore scaffolds (1.7-fold) and 2D culture (2.2-fold) (P ≤ 0.001). Coating with nnHA significantly improved osteoblast mineralisation (>2.6-fold) and EV production (4.5-fold) when compared to uncoated scaffolds (P ≤ 0.001). Together, these findings demonstrate the potential of harnessing bone-mimetic culture platforms to enhance the production of pro-regenerative EVs as an acellular tool for bone repair.

“…Poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) block copolymers were fabricated as previously described [ 33 ]. Briefly, PEGT/PBT scaffolds (300 g/mL, 55:45 wt%) were fabricated via fused deposition modelling in a layer-by-layer process using a 3D BioPlotter (EnvisionTec, Gladbeck, Germany) with a 1 mm fibre spacing.…”

Section: Methodsmentioning

confidence: 99%

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

Alcala

Mekhileri

et al. 2022

JFB

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Epigenetic approaches using the histone deacetylase 2 and 3 inhibitor-MI192 have been reported to accelerate stem cells to form mineralised tissues. Gelatine methacryloyl (GelMA) hydrogels provide a favourable microenvironment to facilitate cell delivery and support tissue formation. However, their application for bone repair is limited due to their low mechanical strength. This study aimed to investigate a GelMA hydrogel reinforced with a 3D printed scaffold to support MI192-induced human bone marrow stromal cells (hBMSCs) for bone formation. Cell culture: The GelMA (5 wt%) hydrogel supported the proliferation of MI192-pre-treated hBMSCs. MI192-pre-treated hBMSCs within the GelMA in osteogenic culture significantly increased alkaline phosphatase activity (p ≤ 0.001) compared to control. Histology: The MI192-pre-treated group enhanced osteoblast-related extracellular matrix deposition and mineralisation (p ≤ 0.001) compared to control. Mechanical testing: GelMA hydrogels reinforced with 3D printed poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) scaffolds exhibited a 1000-fold increase in the compressive modulus compared to the GelMA alone. MI192-pre-treated hBMSCs within the GelMA–PEGT/PBT constructs significantly enhanced extracellular matrix collagen production and mineralisation compared to control (p ≤ 0.001). These findings demonstrate that the GelMA–PEGT/PBT construct provides enhanced mechanical strength and facilitates the delivery of epigenetically-activated MSCs for bone augmentation strategies.