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

Abstract

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.

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

“…A growing body of evidence has demonstrated the potential of harnessing EVs as novel acellular tools to promote bone regeneration [ 24 , 26 , 32 ]. However, issues associated with the therapeutic potency of vesicles has hindered their clinical potential.…”

“…For example, 3D printed titanium scaffolds exhibiting a triangular pore shape accelerated osteoblast mineralisation when compared to square pore constructs. Osteoblast-derived EVs acquired from triangle pore scaffolds significantly increased hBMSCs osteogenic differentiation and mineralisation when compared to EVs acquired from other scaffold designs [ 26 ]. Therefore, in this study it is likely the cells within the LAP-containing gels release EVs with enhanced osteoinductive efficacy, promoting mineralisation in an autocrine/paracrine manner, however, this would require further investigation.…”

“…Additionally, we previously reported that the culture platform influences the transcriptional permissiveness of cells. Triangular pore titanium scaffolds substantially enhanced osteoblast histone acetylation, resulting in increased mineralisation [ 26 ]. Thus, GelMA-LAP 3D microenvironment likely altered the epigenetic landscape of encapsulated hBMSCs, priming them with enhanced differentiation capacity when compared to 2D cultured cells.…”

“…Hence, there have been extensive investigations into harnessing these naturally-derived nanoparticles as an acellular approach to stimulate bone repair, overcoming the numerous translational hurdles associated with cell-based therapies. Several studies have reported the considerable utility of EVs in stimulating osteogenesis [ 24 , 25 , 26 ]. Although great potential has been shown, there has been intensive research into EV engineering strategies to further enhance the therapeutic efficacy of these nano-sized vesicles for bone augmentation strategies [ 15 ].…”

The Impact of the Extracellular Matrix Environment on Sost Expression by the MLO-Y4 Osteocyte Cell Line

“…Whilst Lin determined that this response was not simply due to leached Ca and Pi ions, other studies have attributed the osteoinductive effect to them [ 66 ]. Lin’s work adds to increasing research suggesting HA can act as a focal point for growth factors or potential extracellular vesicles (EVs) [ 67 ], forming an osteoinductive niche [ 65 ]. We can infer that as a stem cell and osteoblast supporting material, HA would also serve to facilitate and support the activity of further differentiated osteocytes.…”