Morphological, mechanical, and in-vitro bioactivity of gelatine/collagen/hydroxyapatite based scaffolds prepared by unidirectional freeze-casting

The fabrication of biomaterials to be used in segmental bone defects, mimicking the bone's organic-inorganic architecture and mechanical properties to induce osteogenesis, persists as a key challenge. The purpose of this study was to elucidate the effect of a lightweight, morphologically graded, and multiphase self-standing scaffold structure prepared from a combination of gelatine (Gel), collagen type 1 (Col) and/or hydroxyapatite (HAP) nanoparticles by a unidirectional freeze-casting process at different temperatures (−20, −40, −60 °C), followed by carbodiimide induced cross-linking, on their in-vitro mechanical stability and bioactive properties. In addition, the rheological study of differently formulated Gel solutions has been performed to determine the effect of Col and HAP content on their microstructural arrangement, which, together with the freezing kinetic, affects Gel/Col orientation and cross-linking, and, thus, the scaffold's mechanical strength and stability. A bone-like anisotropic, interconnected, and graded porosity (from 120 to a few μm) scaffold structure with up to 30% total porosity and ~61 μm average pores' diameter is obtained by using a higher Col content (Col: Gel = 2:5) and freezing temperature (−20 °C) while forming a few μm thick close-to-parallel lamellae, separated with a 10–100 μm space when prepared at −60 °C. Such a structure influenced in-vitro stability strongly (lower swelling without weight loss), being accompanied with a ~76% increase of compression strength (to 37 kPa) and ~67% decrease of elastic modulus (to 17 kPa) when prepared with HAP and incubated in HBSS for 7 days. On the other hand, a significant reduction of both strength (~78%, to 15 kPa) and elasticity (~95%, to 5 kPa) was noted for a scaffold prepared with HAP at −60 °C, being related to faster degradation and the formation of a highly opened structure on the bottom, required to stimulate the bone ingrowth, while a more closed network structure on the top to adhere with the surrounding soft tissue. None of the scaffolds induced cytotoxicity to human bone-derived osteoblasts, even after 19 days of incubation, but rather improved their viability while promoting cells' adhesions, proliferation, and differentiation, being supported with an increased alkaline phosphatase activity and rod-like CaP formation.