Integration of In-Line Light-Mediated Cell Activation in Extrusion-Based Bioprinting: A New Biofabrication Strategy for Bone Tissue Engineering

Abstract

Controlled activation of cells through photobiomodulation can influence osteogenic outcomes. However, factors such as skin penetration depth limit its clinical relevance. In this study, we present a workflow integrating broadband light stimulation directly into the bioprinting process by equipping a custom, low-cost extrusion bioprinter with a self-made, high-voltage spark-gap stimulation unit, enabling in-line stimulation of cells embedded in hydrogel. Optimal duration of broadband light exposure was screened with SaOS-2 cells in 2D for 5, 10, or 15 min, and cultured under osteogenic and control conditions. In 3D, SaOS-2 or adipose-derived stromal cells (ASCs) were bioprinted in an alginate-gelatin bioink under 10 minutes of pulsatile light stimulation. Cellular responses were assessed after 21 days by cell yield, viability, metabolic activity, apoptosis, DNA damage and osteogenic differentiation through immunofluorescence, histology and qPCR. Additionally, vascularisation capacity was assessed in light-stimulated HUVEC/ASC co-cultures within bioprinted constructs. In 2D, light stimulation did not negatively affect cell yield, viability, metabolic activity, cytotoxicity and DNA damage across all exposure times. 3D cultures showed a 2.7-fold and 2.4-fold increase in calcium and hydroxyapatite, respectively, elevated osteocalcin/collagen type I protein expression and gene expression of ALPL, BGLAP, SPP-1, COL1A1, COL10A1 and no increase in DNA damage after 21 days. In the HUVEC/ASC co-culture, light stimulation promoted endothelial elongation (1.25-fold), network formation (2-fold) and perivascular ASC organisation. This work establishes a proof-of-concept methodology for in-line, broadband light cell activation during bioprinting as a strategy to enhance osteogenic differentiation and vascularisation potential.