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 1-2mm3 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. Copyright 2010, Mary Ann Liebert, Inc.
Parameters in Three-Dimensional Osteospheroids of Telomerized Human Mesenchymal (Stromal) Stem Cells Grown on Osteoconductive Scaffolds That Predict In Vivo Bone-Forming Potential
BURNS JSCo-primo
;
2010
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 1-2mm3 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. Copyright 2010, Mary Ann Liebert, Inc.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
