Direct Ink Writing of Osteoconductive Scaffolds using a Crosslinked Collagen-Hydroxyapatite Ink

Abstract Large bone defects present a major clinical challenge, exceeding the body’s natural regenerative capacity. In this study, we investigated the physicochemical and biological properties of a novel cross-linked collagen-hydroxyapatite (Coll-HA-XL) biomaterial ink designed for additive manufacturing of scaffolds for bone tissue engineering. The biomaterial ink was developed through three stages: initially as a hydrogel, then molded and freeze-dried into disk-shaped forms, and finally tailored into 3D-printed scaffolds subjected to subsequent freeze-drying. To optimize the ink, we systematically varied the hydroxyapatite (HA) proportions and the sequence of HA incorporation and cross-linking. The composite materials were then 3D-printed into scaffolds by a direct ink writing method, and were seeded with primary human osteoblasts (hOB). The introduction of HA and subsequent collagen cross-linking induced a significantly increased storage modulus and thermal stability of the material, when compared with the non-crosslinked, HA-containing controls. Biocompatibility of the materials was assessed by hOB cultures, and Coll-HA-XL induced higher alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) activity when compared to the non-crosslinked control. After four weeks of culture on 3D-printed Coll-HA-XL scaffolds, high ALP and LDH activities and osteocalcin (OCN) staining of hOB indicated robust osteoblastic differentiation. Our findings show that a crosslinked, collagen-based biomaterial ink supplemented with HA is suitable for direct ink writing of scaffolds tailored for bone tissue engineering.