title: Parameters in three-dimensional osteospheroids of telomerized human mesenchymal (stromal) stem cells grown on osteoconductive scaffolds that predict in vivo bone-forming potential.
abstract: Osteoblastic differentiation of human mesenchymal stem cells (hMSC) in monolayer culture is artefactual, lacking an organized bone-like matrix. We present a highly reproducible microwell protocol generating three-dimensional ex vivo multicellular aggregates of telomerized hMSC (hMSC-telomerase reverse transcriptase (TERT)) with improved mimicry of in vivo tissue-engineered bone. In osteogenic induction medium the hMSC were transitioned with time-dependent specification toward the osteoblastic lineage characterized by production of alkaline phosphatase, type I collagen, osteonectin, and osteocalcin. Introducing a 1-2 mm(3) crystalline hydroxyapatite/beta-tricalcium phosphate scaffold generated osteospheroids with upregulated gene expression of transcription factors RUNX2/CBFA1, Msx-2, and Dlx-5. An organized lamellar bone-like collagen matrix, evident by birefringence of polarized light, was deposited in the scaffold concavities. Here, mature osteoblasts stained positively for differentiated osteoblast markers TAZ, biglycan, osteocalcin, and phospho-AKT. Quantification of collagen birefringence and relatively high expression of genes for matrix proteins, including type I collagen, biglycan, decorin, lumican, elastin, microfibrillar-associated proteins (MFAP2 and MFAP5), periostin, and tetranectin, in vitro correlated predictively with in vivo bone formation. The three-dimensional hMSC-TERT/hydroxyapatite-tricalcium phosphate osteospheroid cultures in osteogenic induction medium recapitulated many characteristics of in vivo bone formation, providing a highly reproducible and resourceful platform for improved in vitro modeling of osteogenesis and refinement of bone tissue engineering.
authors.affiliation: Laboratory for Molecular Endocrinology (KMEB), Department of Endocrinology and Metabolism, Odense University Hospital and Medical Biotechnology Center, Odense, Denmark.
authors.forename: Jorge S
authors.initials: JS
authors.lastname: Burns
authors.forename: Pernille L
authors.initials: PL
authors.lastname: Rasmussen
authors.forename: Kenneth H
authors.initials: KH
authors.lastname: Larsen
authors.forename: Henrik Daa
authors.initials: HD
authors.lastname: Schrøder
authors.forename: Moustapha
authors.initials: M
authors.lastname: Kassem
mesh_terms: D000328:Adult; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001718:Birefringence; D001861:Bone Regeneration / Q000187:drug effects / Q000502:physiology*; D002130:Calcium Phosphates / Q000494:pharmacology; D002449:Cell Aggregation / Q000187:drug effects; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D017886:Durapatite / Q000494:pharmacology; D005109:Extracellular Matrix / Q000187:drug effects / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects / Q000502:physiology*; D018874:Spheroids, Cellular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016615:Telomere / Q000378:metabolism*; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D015415:Biomarkers; D002130:Calcium Phosphates; D017886:Durapatite; C018392:tricalcium phosphate