A novel nanoparticle-enhanced photoacoustic stimulus for bone tissue engineering

In this study, we introduce a novel nanoparticle-enhanced biophysical stimulus based on the photoacoustic (PA) effect. We demonstrate that the PA effect differentiates bone marrow-derived marrow stromal cells (MSCs) grown on poly(lactic-co-glycolic acid) (PLGA) polymer films toward osteoblasts. We further show that the osteodifferentiation of the MSCs due to PA stimulation is significantly enhanced by the presence of single-walled carbon nanotubes (SWCNTs) in the polymer. MSCs, without the osteogenic culture supplements (0.01 M β-glycerophosphate, 50 mg/L ascorbic acid, 10^-8 M dexamethasone), were seeded onto plain glass slides, glass slides coated with PLGA, or glass slides coated with SWCNT-PLGA films and photoacoustically stimulated by a 527 nm Nd:YLF pulse laser, with a 200 ns pulse duration, and 10 Hz pulse frequency for 10 min a day for 15 consecutive days. The study had four control groups; three baseline controls similar to the three experimental groups but without PA stimulation, and one positive control where MSCs were grown on glass slides without PA stimulation but with osteogenic culture supplements. The osteogenic differentiation of all the groups was evaluated using quantitative assays (alkaline phosphatase, calcium, osteopontin) and qualitative staining (alizarin red). After 15 days, the PA stimulated groups showed up to a 350% increase in calcium content when compared with the non-PA stimulated positive control. Further, within the PA stimulated group, the PLGA-SWCNT group had 130% higher calcium values than the PLGA film without SWCNTs. These results were further corroborated by the analysis of osteopontin secretion, alkaline phosphatase expression, and qualitative alizarin red staining of extracellular matrix calcification. The results indicate that PA stimulation holds promise for bone tissue engineering and that the nanomaterials which enhance the PA effect should allow the development of biophysical rather than biochemical strategies to induce osteoinductive properties into tissue engineering scaffolds.