Enhanced yield and proteomic profile of osteoblast-derived extracellular vesicles from 3D MEW mPCL scaffolds

Biomaterials could influence the production and composition of cell derived extracellular vesicles (EVs), including osteoblast-derived EVs (OB-EVs), which are essential for cell-to-cell communication and hold potential for bone regeneration. Despite their promise, methods for enhancing OB-EVs yields, especially from 3D highly porous microfibrous polymeric scaffolds, remain limited. In this study, we cultured mouse osteoblasts cell line MC3T3-E1 on 3D melt electrowritten (MEW) medical grade polycaprolactone (mPCL) scaffolds and 2D tissue culture plates (TCPs) to compare EV yield, subtypes (small EVs, microvesicles, apoptotic bodies), and proteome profile using liquid chromatography coupled with Tandem mass spectrometry (LC/MS-MS). Our results revealed that OB cultured on MEW mPCL scaffolds significantly increased small EVs yield, with increased particles of small EVs and reduced apoptotic bodies. Notably, two 30 × 30 mm, 0.8 mm-thick MEW mPCL scaffolds (5.07 × 10⁸ sEVs per scaffold) produced the same sEVs yield comparable to that of a T175 TCP flask (9.37 × 10⁸ sEVs per flask). The LC-MS/MS results showed that MEW mPCL sEVs were enriched for 34 proteins associated with tight junction, cell adhesion, gap junction, proteasome, apoptosis and complement pathways. Key proteins such as tubulin superfamily members, myosin heavy chain 9, ezrin, complement 3, CD9, Decorin, and Biglycan were identified, all potentially contributing to tissue repair and regeneration. These findings suggest that 3D MEW mPCL scaffolds not only enhanced OB-sEVs production but also enriched sEVs-protein profiles, particularly those involved in cell-cell junctions and phagosome secretion, suggesting their strong potential in bone tissue engineering.