Osteoblast and osteoclast differentiation in an in vitro three-dimensional model of bone.
There is increasing interest in developing new in vitro tissue models using typical tissue engineering approaches. This study was designed to (1) develop a novel three-dimensional (3D) in vitro model of bone by seeding murine primary osteoblasts and osteoclast precursors on a resorbable porous ceramic scaffold based on silicon-stabilized tricalcium phosphate (Skelite), and (2) investigate bone cell interactions in a 3D environment mimicking an in vivo condition and compare it to traditional two-dimensional (2D) cultures. Murine primary osteoblasts from C57Bl6/J mice and osteoclast precursors from C57Bl/6-Tg(ACTB-EGFP)1Osb/J mice were co-cultured on 3D Skelite scaffolds and on standard plastic culture dishes. The differentiation of these cells in both culture conditions was compared by histology (hematoxylin-eosin staining and polarized light analysis), immunohistochemistry (collagen type I), and gene expression analysis by real-time PCR for Runt-related transcription factor 2, osterix, osteocalcin, cathepsin K, and tartrate resistant acid phosphatase. To analyze and compare bone turnover in 3D and 2D co-cultures, we evaluated the modulation of RANKL and OPG mRNA expression. We observed an enhancement of osteoblast differentiation in the 3D mineralized environment that in turn promoted earlier osteoclast differentiation. In this paper, we also report that the increased osteoblast differentiation in the 3D model led to a deposition of extracellular matrix that faithfully reflected the morphology of bone tissue.
Tortelli
Federico
F
Dipartimento di Oncologia, Biologia e Genetica, Istituto Nazionale per la Ricerca sul Cancro, Università di Genova, Genova, Italy.
Pujic
Natalija
N
Liu
Yi
Y
Laroche
Norbert
N
Vico
Laurence
L
Cancedda
Ranieri
R
D000818:Animals; D001857:Bone Matrix / Q000166:cytology / Q000378:metabolism; D001842:Bone and Bones / Q000166:cytology* / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D005109:Extracellular Matrix / Q000378:metabolism; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology* / Q000378:metabolism; D010012:Osteogenesis; D053244:Osteoprotegerin / Q000235:genetics / Q000378:metabolism; D053245:RANK Ligand / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D054457:Tissue Scaffolds
D053244:Osteoprotegerin; D053245:RANK Ligand; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins