A biomimetic 3D printed gradient tri-layered scaffold stimulating osteochondral differentiation of mesenchymal stem cells for mandibular condyle regeneration

Abstract Objectives This study aimed to fabricate and assess the physical, mechanical, and in vitro biological properties of 3D printed single-layered scaffolds that simulate cartilage, calcified cartilage, and subchondral bone tissue. As well as a tri-layered osteochondral scaffold with a gradient composition and porosity. Methods Six groups were fabricated using a masked stereolithography-based 3D printer. The cartilage group consisted of polylactic acid (PLA)/ 2% hyaluronic acid (HA) with the smallest pores and percentage porosity. The calcified cartilage group comprised PLA/ 2% HA/10% nano-hydroxyapatite (nHAp). Subchondral bone scaffold was formed of PLA/ 25% nHAp with the largest pore size and % porosity. The three scaffold designs were assembled to create the tri-layered group. There were two control groups. A total of 683 scaffolds were 3D printed. Tensile strength, compressive strength, moduli, and interfacial adhesion strength were evaluated at 24 h and 30 days. The biodegradation % of different scaffolds was assessed after 7, 14, and 28 days. Cell viability was assessed through cell counting, cell adhesion, and H & E staining. Cell differentiation was assessed using fast green/safranin O, alizarin red staining, and immunofluorescence after coculturing with rat bone marrow mesenchymal stem at 7, 14, and 21 days. Data were statistically analyzed at p ≤ 0.05. Results The tensile strengths and moduli of the PLA/2%HA and PLA/25% nHAp scaffolds resembled those of normal cartilage and trabecular bone, respectively. The tri-layered scaffold exhibited strong interfacial adhesion strength and demonstrated 13.68% weight loss after 30 days. The tri-layered scaffold exhibited a stiffness gradient, supporting biological responses consistent with chondrogenic differentiation on the more flexible side of the scaffold and osteogenic differentiation on the stiffer side. It demonstrated evidence of both chondrogenic and osteogenic differentiation. Conclusions Incorporation of HA and nHAp, as well as variations in porosity, significantly impacted scaffolds' properties. Clinical relevance These findings suggested the potential of the fabricated scaffolds for medical and maxillofacial regenerative applications, particularly for repairing cartilage, bone, and osteochondral defects.