Analysis of cell-seeded 3-dimensional bone constructs manufactured in vitro with hydroxyapatite granules obtained from red algae.
OBJECTIVE
Bone tissue engineering is a promising approach for the treatment of defective or lost bone in the maxillofacial region. Biocompatible and biodegradable scaffolds seeded with living cells are used to create functional tissue for load-bearing bone reconstruction. The aim of this study was to manufacture cell-seeded 3-dimensional bone constructs based on hydroxyapatite ceramic granule calcified from red algae and mesenchymal cambial-layer precursor cells. The ability of these cells to grow on hydroxyapatite ceramic was quantitatively investigated to evaluate 3-dimensional bone constructs for their potential use in bone tissue engineering.


METHODS
Mesenchymal cambial-layer precursor cells were isolated from mandibular periosteum biopsy samples of 3 patients. To manufacture 72 bone constructs, these cells and hydroxyapatite ceramic granules (C GRAFT/Algipore; Clinician's Preference LLC, Golden, CO) were cultivated under osteogenic differentiation conditions in a rotating wall vessel system. After 6 and 21 days, histologic examination and scanning electron microscopy were performed. The absolute DNA content, protein synthesis, and alkaline phosphatase activity were also quantified. The osteoblastic phenotype of the constructs was confirmed by the expression of bone-specific genes (osteocalcin, osteonectin, osteopontin, and core binding factor alpha1) using semiquantitative reverse transcription-polymerase chain reaction and Western blot analysis.


RESULTS
Cells within the constructs showed good viability, which was evidenced by an increase in DNA content over the culture period. The decrease in alkaline phosphatase-specific activity could be an indicator of the maturation of cells and the induction of mineralization. The osteoblastic phenotype of the constructs was demonstrated on protein and at the RNA level over the entire culture period.


CONCLUSIONS
We observed a positive effect of hydroxyapatite ceramic granules on mesenchymal cambial-layer precursor cell behavior in cell-seeded 3-dimensional bone constructs, indicating the potential applicability of C GRAFT/Algipore composites in bone tissue engineering.
Turhani
Dritan
D
Hospital of Cranio-Maxillofacial and Oral Surgery, Medical University of Vienna, Austria. dritan.turhani@meduniwien.ac.at
Watzinger
Elisabeth
E
Weissenböck
Martina
M
Cvikl
Barbara
B
Thurnher
Dietmar
D
Wittwer
Gert
G
Yerit
Kaan
K
Ewers
Rolf
R
D015153:Blotting, Western; D001861:Bone Regeneration; D018786:Bone Substitutes; D018929:Cell Culture Techniques; D017886:Durapatite / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008422:Materials Testing; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D015675:Osteocalcin / Q000378:metabolism; D015676:Osteonectin / Q000378:metabolism; D053495:Osteopontin; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D000461:Rhodophyta; D012795:Sialoglycoproteins / Q000378:metabolism; D023822:Tissue Engineering / Q000379:methods*
C483718:Algipore; D018786:Bone Substitutes; D015676:Osteonectin; D012795:Sialoglycoproteins; D015675:Osteocalcin; D053495:Osteopontin; D017886:Durapatite