The high and growing number of trauma or disease-related non-union fractures in an aging population has increased the clinical demand for tissue-engineered bone. Bone tissue is considered a conductive and piezoelectric material as a result of being mainly composed by collagen and hydroxyapatite [1]. Thus, electroactive materials (both electroconductive and piezoelectric) have received recent interest for applications in bone tissue engineering (BTE) strategies [2]. In addition, electrical stimulation (EStim) has been shown to enhance cell osteogenesis and bone regeneration in several research/pre-clinical studies both in vitro and in vivo [3]. Nevertheless, the biological mechanisms through which EStim promotes bone regeneration are still poorly understood, indicating that further research is needed to fully explore the potential of EStim in BTE strategies. Additive manufacturing (AM) strategies (e.g. Fused Deposition Modelling-FDM) have been widely employed in BTE due to their ability to produce structured scaffolds with a high control over their architecture, microporosity and mechanical properties in a reproducible and scalable manner [4].

In this work, we combined AM technologies with electrically conductive and piezoelectric materials and explore the use of biophysical stimulation (EStim or ultrasound (US)) to enhance the osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells (hBMSCs).

Firstly, electro-bioreactor devices were fabricated by FDM using medical-grade electrode materials (stainless steel and Ti6Al4V) to apply EStim to 2D cell cultures and 3D cell-seeded scaffolds. A commercially available US system was used for the mechanical stimulation of piezoelectric scaffolds. Finite Element Analysis (FEA) was performed to predict the magnitude and distribution of electrical fields within the EStim devices and along the conductive scaffolds. Here, we highlight three different studies combining AM-based 3D structured functional scaffolds and biophysical stimuli for BTE:

i) Different 3D printed conductive scaffolds (conductive poly lactic acid (cPLA) and titanium) were seeded with hBMSCs and cultured for 21 days under osteogenic medium conditions with and without EStim and their biological performance was evaluated in comparison to non-conductive scaffolds;

ii) 3D piezoelectric polyvinyldiene fluoride (PVDF) scaffolds were seeded with hBMSCs and cultured with and without US stimuli to assess their potential for BTE applications;

iii) Several PEDOT:PSS-based coating solutions were screened to obtain PEDOT:PSS-coated polycaprolactone (PCL) scaffolds, in which hBMSCs were cultured for 21 days under osteogenic induction with and without EStim [5]. The fabricated 3D PEDOT:PSS-coated scaffolds presented a high electroconductivity (11.3-20.1 S/cm), which values were stable for 21 days in saline solution. When cultured under EStim, the best performance coating condition scaffold (PEDOT:PSS/Gelatin-PCL) was shown to improve significantly the osteogenic differentiation of hBMSCs in vitro, in particular, their cell-secreted calcium deposition (mineralization), bone-specific protein expression and osteogenic marker genes upregulation (in comparison to all other experimental groups). Overall, our results showed a clear synergy between 3D scaffolds’ conductivity and EStim on the improvement of the osteogenic differentiation of hBMSCs, highlighting this strategy potential for BTE clinical applications.

References

[1] F. Barbosa, F.C. Ferreira, J.C. Silva; International Journal of Molecular Sciences, 2022, 23, 2907.

[2] T. Zheng et al.; Journal of Materials Chemistry B, 2020, 8, 10221-10256.

[3] X. Zhang et al.; Materials Today, 2023, 68, 177-203.

[4] C. Garot et al.; Advanced Functional Materials, 2021, 31, 2006967.

[5] J.C. Silva et al.; Journal of Materials Chemistry B, 2024.