Innovative P(3HB)/carbon-based material composites for bone tissue regeneration: Biocompatibility, thermo-mechanical, and structural insights

Bone tissue engineering (BTE) aims to address the challenge of repairing critical size bone defects, but effective substitutes with suitable mechanical properties and bioactivity are still needed. Poly(3-hydroxybutyrate), P(3HB)is a sustainable polymer with promising potential but suffers from poor mechanical properties and thermal instability. In this study, P(3HB) was reinforced with various carbon-based materials (CBMs) to evaluate thermomechanical and structural properties as well as biological responses, in composites before and after aging. CBMs with P(3HB) interactions and their spatial distribution were examined using advanced imaging, including Atomic Force Microscopy (AFM), Secondary Electron Hyperspectral Imaging (SEHI), and Short-Wave Infrared (SWIR) analysis. Biological responses were assessed using various biocompatibility assays; cytotoxicity and osteogenicity with primary human osteoblasts (ECACC, 406-05a) and MG63 cells. Aged P(3HB)/inkjet composites showed a 140 % increase in Young's modulus (1.2 GPa), matching trabecular bone stiffness, with a 3 % lower processing temperature than neat P(3HB), enhancing suitability for 3D printing. SEHI revealed elevated OH (4.8 eV) and CO (5.7 eV) functional groups, resulting in increased surface hydrophilicity and promoted cellular responses. P(3HB)/inkjet demonstrated the highest cell attachment (267.5 ± 43.3 cells) and ALP activity (6.3 ± 0.7 nmol PNP/min), outperforming composites with Starbon (150.1 ± 38.3 cells, 6.1 ± 0.8 ALP) and activated carbon (103.4 ± 24.5 cells, 5.7 ± 0.5 ALP). All aged composites showed improved performance over their fresh counterparts. In contrast, TCP and neat P(3HB) exhibited the lowest levels of mineralization. 3D printing offers further potential for enhancing P(3HB)/inkjet composites through precise and bespoke scaffold design and clinical feasibility.