Parameters in three-dimensional osteospheroids of telomerized human mesenchymal (stromal) stem cells grown on osteoconductive scaffolds that predict in vivo bone-forming potential

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

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.
OriginalsprogEngelsk
TidsskriftTissue Engineering. Part A. Tissue Engineering
Vol/bind16
Udgave nummer7
Sider (fra-til)2331-42
Antal sider12
ISSN1937-3341
DOI
StatusUdgivet - 1. jul. 2010

Fingeraftryk

Stem cells
Mesenchymal Stromal Cells
Scaffolds
Bone
Collagen
Osteogenesis
Biglycan
Telomerase
Osteocalcin
Collagen Type I
Osteoblasts
Birefringence
Hydroxyapatite
Cell culture
Osteonectin
Phosphates
Decorin
Proteins
Elastin
Durapatite

Citer dette

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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.",
keywords = "Adult, Animals, Biological Markers, Birefringence, Bone Regeneration, Calcium Phosphates, Cell Aggregation, Cell Culture Techniques, Cell Differentiation, Cell Proliferation, Durapatite, Extracellular Matrix, Female, Gene Expression Regulation, Humans, Male, Mesenchymal Stem Cells, Mice, Mice, SCID, Osteoblasts, Osteogenesis, Spheroids, Cellular, Stromal Cells, Telomere, Tissue Scaffolds",
author = "Burns, {Jorge S} and Hansen, {Pernille Lund} and Larsen, {Kenneth H} and Schr{\o}der, {Henrik Daa} and Moustapha Kassem",
year = "2010",
month = "7",
day = "1",
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journal = "Tissue Engineering. Part A",
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TY - JOUR

T1 - Parameters in three-dimensional osteospheroids of telomerized human mesenchymal (stromal) stem cells grown on osteoconductive scaffolds that predict in vivo bone-forming potential

AU - Burns, Jorge S

AU - Hansen, Pernille Lund

AU - Larsen, Kenneth H

AU - Schrøder, Henrik Daa

AU - Kassem, Moustapha

PY - 2010/7/1

Y1 - 2010/7/1

N2 - 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.

AB - 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.

KW - Adult

KW - Animals

KW - Biological Markers

KW - Birefringence

KW - Bone Regeneration

KW - Calcium Phosphates

KW - Cell Aggregation

KW - Cell Culture Techniques

KW - Cell Differentiation

KW - Cell Proliferation

KW - Durapatite

KW - Extracellular Matrix

KW - Female

KW - Gene Expression Regulation

KW - Humans

KW - Male

KW - Mesenchymal Stem Cells

KW - Mice

KW - Mice, SCID

KW - Osteoblasts

KW - Osteogenesis

KW - Spheroids, Cellular

KW - Stromal Cells

KW - Telomere

KW - Tissue Scaffolds

U2 - 10.1089/ten.TEA.2009.0735

DO - 10.1089/ten.TEA.2009.0735

M3 - Journal article

C2 - 20196644

VL - 16

SP - 2331

EP - 2342

JO - Tissue Engineering. Part A

JF - Tissue Engineering. Part A

SN - 1937-3341

IS - 7

ER -