Shape-defined poly(lactic acid)-nanohydroxyapatite composite microparticles modulate osteogenic differentiation in 3D microtissues of human mesenchymal stromal cells

Microparticles have gained significant attention as promising injectable fillers for tissue defect repair, particularly in bone regeneration. While tailoring microparticle chemistry to guide osteogenic differentiation and bone regeneration has been extensively studied, the influence of microparticle shape remains less explored. We hypothesized that, similar to chemistry, microparticle shape can modulate the osteogenic differentiation of human mesenchymal stromal cells (hMSCs). To test this, we employed a micromolding method to fabricate shape-defined microparticles from poly(lactic acid) (PLA) or PLA-nanohydroxyapatite (nHA) composites with different aspect ratios and sizes, and then co-cultured them with hMSCs to self-assemble into 3D microtissues. Microtissues containing composite microparticles showed significantly higher alkaline phosphatase activity, with high-aspect-ratio and small-sized microparticles eliciting the strongest response. Both microparticle shape and composition regulate hMSC osteogenic differentiation according to gene expression analysis. In the absence of nHA, PLA microparticles with higher aspect ratios significantly increased the expression of osteogenesis-related genes, including IBSP , SPP1 , and MMP13 , whereas others showed minimal effects. Introducing nHA altered this trend, with small-sized microparticles inducing the highest SPP1 expression and osteopontin production at late time points. Small-sized microparticles further promoted the expression of vinculin and yes-associated protein. Furthermore, etching composite microparticles to expose nHA on their surface amplified this size-dependent effect, leading to enhanced expression of the late osteogenic marker, BGLAP, in hMSC microtissues containing small cube composite microparticles. Our findings establish microparticle shape, especially size and aspect ratio, as fundamental design parameters that synergize with microparticle composition to direct hMSCs toward osteogenic lineage, offering a promising strategy for engineering injectable fillers for bone regeneration. • Micromolding enabled fabrication of shape- and chemistry-tuned microparticles. • Rectangular polymeric microparticles induced the highest osteogenic gene expression. • Hydroxyapatite addition made small square microparticles most potent in osteogenesis. • Microparticle shape and chemistry synergistically drive osteogenesis in 3D tissues.