Synergy between 3D-extruded electroconductive scaffolds and electrical stimulation to improve bone tissue engineering strategies.

In this work, we propose a simple, reliable, and versatile strategy to create 3D electroconductive scaffolds suitable for bone tissue engineering (TE) applications with electrical stimulation (ES). The proposed scaffolds are made of 3D-extruded poly(ε-caprolactone) (PCL), subjected to alkaline treatment, and of poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS), anchored to PCL with one of two different crosslinkers: (3-glycidyloxypropyl)trimethoxysilane (GOPS) and divinyl sulfone (DVS). Both cross-linkers allowed the formation of a homogenous and continuous coating of PEDOT:PSS to PCL. We show that these PEDOT:PSS coatings are electroconductive (11.3-20.1 S cm-1), stable (up to 21 days in saline solution), and allow the immobilization of gelatin (Gel) to further improve bioactivity. In vitro mineralization of the corresponding 3D conductive scaffolds was greatly enhanced (GOPS(NaOH)-Gel - 3.1 fold, DVS(NaOH)-Gel - 2.0 fold) and cell colonization and proliferation were the highest for the DVS(NaOH)-Gel scaffold. In silico modelling of ES application in DVS(NaOH)-Gel scaffolds indicates that the electrical field distribution is homogeneous, which reduces the probability of formation of faradaic products. Osteogenic differentiation of human bone marrow derived mesenchymal stem/stromal cells (hBM-MSCs) was performed under ES. Importantly, our results clearly demonstrated a synergistic effect of scaffold electroconductivity and ES on the enhancement of MSC osteogenic differentiation, particularly on cell-secreted calcium deposition and the upregulation of osteogenic gene markers such as COL I, OC and CACNA1C. These scaffolds hold promise for future clinical applications, including manufacturing of personalized bone TE grafts for transplantation with enhanced maturation/functionality or bioelectronic devices.

A Novel Approach for Design and Manufacturing of Curvature-Featuring Scaffolds for Osteochondral Repair.

Osteochondral (OC) defects affect both articular cartilage and the underlying subchondral bone. Due to limitations in the cartilage tissue's self-healing capabilities, OC defects exhibit a degenerative progression to which current therapies have not yet found a suitable long-term solution. Tissue engineering (TE) strategies aim to fabricate tissue substitutes that recreate natural tissue features to offer better alternatives to the existing inefficient treatments. Scaffold design is a key element in providing appropriate structures for tissue growth and maturation. This study presents a novel method for designing scaffolds with a mathematically defined curvature, based on the geometry of a sphere, to obtain TE constructs mimicking native OC tissue shape. The lower the designed radius, the more curved the scaffold obtained. The printability of the scaffolds using fused filament fabrication (FFF) was evaluated. For the case-study scaffold size (20.1 mm × 20.1 mm projected dimensions), a limit sphere radius of 17.064 mm was determined to ensure printability feasibility, as confirmed by scanning electron microscopy (SEM) and micro-computed tomography (µ-CT) analysis. The FFF method proved suitable to reproduce the curved designs, showing good shape fidelity and replicating the expected variation in porosity. Additionally, the mechanical behavior was evaluated experimentally and by numerical modelling. Experimentally, curved scaffolds showed strength comparable to conventional orthogonal scaffolds, and finite element analysis was used to identify the scaffold regions more susceptible to higher loads.

Extracellular matrix decorated polycaprolactone scaffolds for improved mesenchymal stem/stromal cell osteogenesis towards a patient-tailored bone tissue engineering approach.

The clinical demand for tissue-engineered bone is growing due to the increase of non-union fractures and delayed healing in an aging population. Herein, we present a method combining additive manufacturing (AM) techniques with cell-derived extracellular matrix (ECM) to generate structurally well-defined bioactive scaffolds for bone tissue engineering (BTE). In this work, highly porous three-dimensional polycaprolactone (PCL) scaffolds with desired size and architecture were fabricated by fused deposition modeling and subsequently decorated with human mesenchymal stem/stromal cell (MSC)-derived ECM produced in situ. The successful deposition of MSC-derived ECM onto PCL scaffolds (PCL-MSC ECM) was confirmed after decellularization using scanning electron microscopy, elemental analysis, and immunofluorescence. The presence of cell-derived ECM within the PCL scaffolds significantly enhanced MSC attachment and proliferation, with and without osteogenic supplementation. Additionally, under osteogenic induction, PCL-MSC ECM scaffolds promoted significantly higher calcium deposition and elevated relative expression of bone-specific genes, particularly the gene encoding osteopontin, when compared to pristine scaffolds. Overall, our results demonstrated the favorable effects of combining MSC-derived ECM and AM-based scaffolds on the osteogenic differentiation of MSC, resulting from a closer mimicry of the native bone niche. This strategy is highly promising for the development of novel personalized BTE approaches enabling the fabrication of patient defect-tailored scaffolds with enhanced biological performance and osteoinductive properties.

Extruded Bioreactor Perfusion Culture Supports the Chondrogenic Differentiation of Human Mesenchymal Stem/Stromal Cells in 3D Porous Poly(ɛ-Caprolactone) Scaffolds.

Novel bioengineering strategies for the ex vivo fabrication of native-like tissue-engineered cartilage are crucial for the translation of these approaches to clinically manage highly prevalent and debilitating joint diseases. Bioreactors that provide different biophysical stimuli have been used in tissue engineering approaches aimed at enhancing the quality of the cartilage tissue generated. However, such systems are often highly complex, expensive, and not very versatile. In the current study, a novel, cost-effective, and customizable perfusion bioreactor totally fabricated by additive manufacturing (AM) is proposed for the study of the effect of fluid flow on the chondrogenic differentiation of human bone-marrow mesenchymal stem/stromal cells (hBMSCs) in 3D porous poly(ɛ-caprolactone) (PCL) scaffolds. hBMSCs are first seeded and grown on PCL scaffolds and hBMSC-PCL constructs are then transferred to 3D-extruded bioreactors for continuous perfusion culture under chondrogenic inductive conditions. Perfused constructs show similar cell metabolic activity and significantly higher sulfated glycosaminoglycan production (≈1.8-fold) in comparison to their non-perfused counterparts. Importantly, perfusion bioreactor culture significantly promoted the expression of chondrogenic marker genes while downregulating hypertrophy. This work highlights the potential of customizable AM platforms for the development of novel personalized repair strategies and more reliable in vitro models with a wide range of applications.