Scaffold-Free Bone Regeneration Through Collaboration Between Type IV Collagen and FBXL14

Background: The periosteum and periosteum-derived cells have attracted considerable attention for their potential use in clinical applications for treating bone defects. Bovine periosteum-derived cells have been investigated because of their capability for scaffold-free bone regeneration. Previous mass spectrometry (MS) and immunohistochemistry studies have shown the presence of F-box/leucine-rich repeat protein 14 (FBXL14) in bovine periosteum and periosteum-derived cells. Recently, studies using ESI-Q-Orbitrap MS suggested the presence of type IV collagen in the periosteum. The aim of the present study was to clarify the relationship between type IV collagen and FBXL14 in the formation of periosteum-derived cells. Methods: Bovine periosteum-derived cells were obtained from Japanese Black Cattle's legs in Medium 199 with ascorbic acid and 10% fetal bovine serum. Immunohistochemistry for type IV collagen and FBXL14 was performed using bovine bone with periosteum and periosteum alone for explant culture. Results: Both type IV collagen and FBXL14 were expressed in Volkmann's canals and the Haversian canals in bone and periosteum. After 5 weeks, type IV collagen and FBXL14 surrounded crystals containing osteocalcin and had formed periosteum-derived cells. Von Kossa staining and immunostaining of osteocalcin revealed that the crystals contained calcified substances and osteocalcin. Conclusions: Clinically, understanding osteocalcin-interacting proteins will help promote bone regeneration. Interactions between type IV collagen and FBXL14 may contribute to scaffold-free bone regeneration.