Application of the SPRI biosensor for the analysis of osteogenic differentiation under the influence of multilayered bioactive scaffolds.

• Grant number: 24/21693-9
• Support Opportunities: Scholarships in Brazil - Master
• Start date: September 01, 2025
• End date: February 28, 2026
• Field of knowledge: Interdisciplinary Subjects
• Principal Investigator: Luana Marotta Reis de Vasconcellos
• Grantee: Juliana dos Santos Lupp
• Host Institution: Instituto de Ciência e Tecnologia (ICT). Universidade Estadual Paulista (UNESP). Campus de São José dos Campos. São José dos Campos , SP, Brazil

Abstract

Mesenchymal cells (MCs) are widely used in regenerative therapies, particularly in bone regeneration, due to their pluripotent potential. In vitro studies are essential for understanding the stages of osteogenic differentiation, as they allow the identification of factors that influence each phase and contribute to advances in regenerative medicine. To promote this process, the use of bioactive scaffolds has been extensively explored, as they provide physical support, mimic the characteristics of bone tissue, and possess properties that stimulate cell adhesion, proliferation, and differentiation. However, conventional methods for assessing osteogenesis have limitations, including the requirement for cell lysis, high costs, and the inability to perform continuous and non-destructive analyses. In this context, imagebased surface plasmon resonance (SPRi) biosensors emerge as a promising alternative, as they enable real-time monitoring of protein expression while preserving cell viability. This study aims to apply the SPRi biosensor to analyze the osteogenic differentiation of mesenchymal cells under the influence of multilayered bioactive scaffolds composed of hydrogel and poly(lactic acid) (PLA) incorporated with 45B5 bioactive glass, through the identification and quantification of osteogenic proteins. The 45B5 bioactive glass was previously synthesized and characterized by X-ray diffraction (XRD). PLA nanofibers containing 5% (w/v) of 45B5 bioactive glass were then produced by electrospinning and coated with a sodium alginate hydrogel previously shaped by 3D printing. The scaffolds were characterized using scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR), and are currently undergoing energy-dispersive X-ray spectroscopy (EDS) and wettability analyses. For the in vitro analysis of osteogenesis, four groups were established: a positive control group with cells cultured directly on the well plate using osteogenic induction medium; a group with cells cultured on an inert titanium substrate using osteopermissive medium; an experimental group with cells cultured on the scaffold composed of hydrogel and PLA nanofibers incorporated with 45B5 bioactive glass using osteopermissive medium; and a basal control group with cells cultured directly on the well plate using basal medium. Mesenchymal cells will be isolated from the femurs of rats and seeded in 6-well plates at a density of 5×10¿ cells per well along with the respective experimental conditions. Supernatants will be collected on days 0, 4, 8, 12, 16, 20, 24, and 28. The levels of type I collagen (COL-1), alkaline phosphatase (ALP), and osteocalcin (OCN) will be quantified in these supernatants using both ELISA and SPRi biosensors. A direct comparison of the results obtained by both methods will allow assessment of the sensitivity and efficiency of the SPRi biosensor. Additionally, the same experimental groups will be analyzed in 24-well plates for ALP activity, mineralized nodule formation, and calcium quantification. The data obtained will be subjected to normality testing and statistical analysis, considering a significance level of p<0.05. It is expected that the SPRi biosensor will detect these osteogenic markers more rapidly and accurately than ELISA and other conventional techniques. (AU)