3D printed poly(3-hydroxybutyrate-co-3-hydroxyexanoate) scaffolds support chondrogenic potential of human primary chondrocytes during in vitro culture

Three-dimensional (3D) scaffolds are a critical component in guided-tissue regeneration strategies, particularly in cartilage engineering, by providing an adequate structural and physical environment for seeded cells to proliferate and eventually differentiate. Here we investigate the use of microbial poly(3-hydroxybutyrate-co-3-hydroxyexanoate) (PHBHHx) as polymeric inks for 3D printing of scaffolds and weigh their chondrogenic potential against commonly used poly(ε-caprolactone) (PCL). A set of processing parameters is first optimized for extrusion-based printing of porous PHBHHx and PCL scaffolds with well-defined architectures and without affecting the polymer's physicochemical properties. Mechanical testing results obtained under static compression confirm the fabrication of PHBHHx scaffolds with elastic modulus values comparable to those of human mature cartilage. LIVE/DEAD™ and AlamarBlue assays do not reveal any cytotoxic effect of PHBHHx scaffolds on primary human chondrocytes, which remain viable over 14 days of in vitro static culture. Additionally, real-time RT-qPCR analysis of key chondrogenic markers (i.e., SOX9, COL2A1 and ACAN) suggests chondrocytes retain their phenotype over the studied period, independently of the scaffolding material. Taken together, our results confirm the suitability of PHBHHx scaffolds to support the function of chondrocytes in vitro, opening new opportunities for their application in the field of cartilage tissue engineering.