Preparation of Hydroxyapatite-Aligned Collagen Sheets and Their Evaluation for Fibroblast Adhesion and Collagen Secretion

The structure of many native tissues consists of aligned collagen (Col) fibrils, some of which are further composited with dispersed hydroxyapatite (HAp) nanocrystals. Accurately mimicking this inherent structure is a promising approach to enhance scaffold biocompatibility in tissue engineering. In this study, biomimetic sheets composed of highly aligned Col fibrils were fabricated using a plastic compression and tension method, followed by the deposition of HAp nanocrystals on the surface via an alternate soaking method. The fabricated Col sheets exhibited high solid density, retained the native periodicity (D-band) of Col fibrils, and displayed plate-like HAp nanocrystals dispersed on their surface. In vitro experiments demonstrated that these sheets could regulate fibroblasts adhesion, inducing more elongated nuclei and oriented actin bundles on the aligned Col sheets. Analysis of focal adhesion assembly revealed greater cell focal adhesions on the aligned composite sheets compared to those with random Col fibril structures. Fibroblasts cultured on aligned Col with partly HAp-mineralized sheets exhibited the highest cell-extracellular matrix (ECM) protein secretion, highlighting that HAp incorporation and fibroblast alignment synergistically promote early ECM formation and wound healing. These results suggest that highly aligned Col fibrils with dispersed HAp nanocrystals, closely mimicking the microarchitecture of natural tissues, have significant potential to control cell adhesion and protein secretion for tissue engineering applications.