title: Fluorescence microscopy imaging of bone for automated histomorphometry.
abstract: We have developed a computer-based method for the automated quantification of bone tissue in histological sections of decalcified specimens. Bone tissue was generated by ectopic implantation of ceramic-based carriers loaded with human bone marrow stromal cells (BMSCs). The method is based on the acquisition of multimodal images, in order to identify and measure the area covered by bone tissue (using fluorescent light) and the total area of tissue (using transmitted light), thereby excluding the regions corresponding to nonresorbed scaffold. The amount of bone as a percentage of the total area of interest (bone/area) and of the newly formed tissue (bone/tissue) is automatically derived. The computer-based results correlated closely with those obtained by manual identification of bone and tissue areas in the same histological fields (R(2) = 0.997; p < 0.0005), with errors dependent on the magnification used but always lower than 9.4%. The method was used to compare the bone/tissue and bone/area percentages in samples of engineered bone based on human BMSCs expanded in the presence of different biochemical factors and loaded onto different scaffolds. The technique thus represents a valuable tool to quantify reproducibly, accurately, and easily bone formation in a variety of tissue-engineering studies.
authors.affiliation: Dipartimento di Informatica, Sistemistica e Telematica (DIST), Università di Genova, Largo Rosanna Benzi n. 10, 16132 Genoa, Italy.
authors.forename: Ivan
authors.initials: I
authors.lastname: Martin
authors.forename: Maddalena
authors.initials: M
authors.lastname: Mastrogiacomo
authors.forename: Gianluca
authors.initials: G
authors.lastname: De Leo
authors.forename: Anita
authors.initials: A
authors.lastname: Muraglia
authors.forename: Francesco
authors.initials: F
authors.lastname: Beltrame
authors.forename: Ranieri
authors.initials: R
authors.lastname: Cancedda
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Quarto
mesh_terms: D001854:Bone Marrow Cells / Q000166:cytology; D016026:Bone Marrow Transplantation; D001842:Bone and Bones / Q000166:cytology*; D017886:Durapatite / Q000627:therapeutic use; D006801:Humans; D008856:Microscopy, Fluorescence; D023822:Tissue Engineering
chemical_list: D017886:Durapatite