Flow perfusion culture of human mesenchymal stem cells on coralline hydroxyapatite scaffolds with various pore sizes

Lea Bjerre, Cody Bünger, Anette Baatrup, Moustapha Kassem, Tina Mygind

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Bone grafts are widely used in orthopaedic reconstructive surgery, but harvesting of autologous grafts is limited due to donor site complications. Bone tissue engineering is a possible alternative source for substitutes, and to date, mainly small scaffold sizes have been evaluated. The aim of this study was to obtain a clinically relevant substitute size using a direct perfusion culture system. Human bone marrowderived mesenchymal stem cells were seeded on coralline hydroxyapatite scaffolds with 200 μm or 500 μm pores, and resulting constructs were cultured in a perfusion bioreactor or in static culture for up to 21 days and analysed for cell distribution and osteogenic differentiation using histological stainings, alkaline phosphatase activity assay, and real-time RT-PCR on bone markers. We found that the number of cells was higher during static culture at most time points and that the final number of cells was higher in 500 μm constructs as compared with 200 μm constructs. Alkaline phosphatase enzyme activity assays and real time RT-PCR on seven osteogenic markers showed that differentiation occurred primarily and earlier in statically cultured constructs with 200 μm pores compared with 500 μm ones. Adhesion and proliferation of the cells was seen on both scaffold sizes, but the vitality and morphology of cells changed unfavorably during perfusion culture. In contrast to previous studies using spinner flask that show increased cellularity and osteogenic properties of cells when cultured dynamically, the perfusion culture in our study did not enhance the osteogenic properties of cell/scaffold constructs. The statically cultured constructs showed increasing cell numbers and abundant osteogenic differentiation probably because of weak initial cell adhesion due to the surface morphology of scaffolds. Our conclusion is that the specific scaffold surface microstructure and culturing system flow dynamics has a great impact on cell distribution and proliferation and on osteogenic differentiation, and the data presented warrant careful selection of in vitro culture settings to meet the specific requirements of the scaffolds and cells, especially when natural biomaterials with varying morphology are used. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.
OriginalsprogEngelsk
TidsskriftJournal of Biomedical Materials Research. Part A
Vol/bind97A
Udgave nummer3
Sider (fra-til)251-63
Antal sider13
ISSN1549-3296
DOI
StatusUdgivet - 2011

Fingeraftryk

Stem cells
Hydroxyapatite
Cell culture
Scaffolds
Pore size
Bone
Phosphatases
Grafts
Alkaline Phosphatase
Assays
Differentiation Antigens
Cell adhesion
Orthopedics
Enzyme activity
Biocompatible Materials
Scaffolds (biology)
Bioreactors
Tissue engineering
Biomaterials
Surgery

Citer dette

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abstract = "Bone grafts are widely used in orthopaedic reconstructive surgery, but harvesting of autologous grafts is limited due to donor site complications. Bone tissue engineering is a possible alternative source for substitutes, and to date, mainly small scaffold sizes have been evaluated. The aim of this study was to obtain a clinically relevant substitute size using a direct perfusion culture system. Human bone marrowderived mesenchymal stem cells were seeded on coralline hydroxyapatite scaffolds with 200 μm or 500 μm pores, and resulting constructs were cultured in a perfusion bioreactor or in static culture for up to 21 days and analysed for cell distribution and osteogenic differentiation using histological stainings, alkaline phosphatase activity assay, and real-time RT-PCR on bone markers. We found that the number of cells was higher during static culture at most time points and that the final number of cells was higher in 500 μm constructs as compared with 200 μm constructs. Alkaline phosphatase enzyme activity assays and real time RT-PCR on seven osteogenic markers showed that differentiation occurred primarily and earlier in statically cultured constructs with 200 μm pores compared with 500 μm ones. Adhesion and proliferation of the cells was seen on both scaffold sizes, but the vitality and morphology of cells changed unfavorably during perfusion culture. In contrast to previous studies using spinner flask that show increased cellularity and osteogenic properties of cells when cultured dynamically, the perfusion culture in our study did not enhance the osteogenic properties of cell/scaffold constructs. The statically cultured constructs showed increasing cell numbers and abundant osteogenic differentiation probably because of weak initial cell adhesion due to the surface morphology of scaffolds. Our conclusion is that the specific scaffold surface microstructure and culturing system flow dynamics has a great impact on cell distribution and proliferation and on osteogenic differentiation, and the data presented warrant careful selection of in vitro culture settings to meet the specific requirements of the scaffolds and cells, especially when natural biomaterials with varying morphology are used. {\circledC} 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.",
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Flow perfusion culture of human mesenchymal stem cells on coralline hydroxyapatite scaffolds with various pore sizes. / Bjerre, Lea; Bünger, Cody; Baatrup, Anette; Kassem, Moustapha; Mygind, Tina.

I: Journal of Biomedical Materials Research. Part A, Bind 97A, Nr. 3, 2011, s. 251-63.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Flow perfusion culture of human mesenchymal stem cells on coralline hydroxyapatite scaffolds with various pore sizes

AU - Bjerre, Lea

AU - Bünger, Cody

AU - Baatrup, Anette

AU - Kassem, Moustapha

AU - Mygind, Tina

N1 - Copyright © 2011 Wiley Periodicals, Inc.

PY - 2011

Y1 - 2011

N2 - Bone grafts are widely used in orthopaedic reconstructive surgery, but harvesting of autologous grafts is limited due to donor site complications. Bone tissue engineering is a possible alternative source for substitutes, and to date, mainly small scaffold sizes have been evaluated. The aim of this study was to obtain a clinically relevant substitute size using a direct perfusion culture system. Human bone marrowderived mesenchymal stem cells were seeded on coralline hydroxyapatite scaffolds with 200 μm or 500 μm pores, and resulting constructs were cultured in a perfusion bioreactor or in static culture for up to 21 days and analysed for cell distribution and osteogenic differentiation using histological stainings, alkaline phosphatase activity assay, and real-time RT-PCR on bone markers. We found that the number of cells was higher during static culture at most time points and that the final number of cells was higher in 500 μm constructs as compared with 200 μm constructs. Alkaline phosphatase enzyme activity assays and real time RT-PCR on seven osteogenic markers showed that differentiation occurred primarily and earlier in statically cultured constructs with 200 μm pores compared with 500 μm ones. Adhesion and proliferation of the cells was seen on both scaffold sizes, but the vitality and morphology of cells changed unfavorably during perfusion culture. In contrast to previous studies using spinner flask that show increased cellularity and osteogenic properties of cells when cultured dynamically, the perfusion culture in our study did not enhance the osteogenic properties of cell/scaffold constructs. The statically cultured constructs showed increasing cell numbers and abundant osteogenic differentiation probably because of weak initial cell adhesion due to the surface morphology of scaffolds. Our conclusion is that the specific scaffold surface microstructure and culturing system flow dynamics has a great impact on cell distribution and proliferation and on osteogenic differentiation, and the data presented warrant careful selection of in vitro culture settings to meet the specific requirements of the scaffolds and cells, especially when natural biomaterials with varying morphology are used. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.

AB - Bone grafts are widely used in orthopaedic reconstructive surgery, but harvesting of autologous grafts is limited due to donor site complications. Bone tissue engineering is a possible alternative source for substitutes, and to date, mainly small scaffold sizes have been evaluated. The aim of this study was to obtain a clinically relevant substitute size using a direct perfusion culture system. Human bone marrowderived mesenchymal stem cells were seeded on coralline hydroxyapatite scaffolds with 200 μm or 500 μm pores, and resulting constructs were cultured in a perfusion bioreactor or in static culture for up to 21 days and analysed for cell distribution and osteogenic differentiation using histological stainings, alkaline phosphatase activity assay, and real-time RT-PCR on bone markers. We found that the number of cells was higher during static culture at most time points and that the final number of cells was higher in 500 μm constructs as compared with 200 μm constructs. Alkaline phosphatase enzyme activity assays and real time RT-PCR on seven osteogenic markers showed that differentiation occurred primarily and earlier in statically cultured constructs with 200 μm pores compared with 500 μm ones. Adhesion and proliferation of the cells was seen on both scaffold sizes, but the vitality and morphology of cells changed unfavorably during perfusion culture. In contrast to previous studies using spinner flask that show increased cellularity and osteogenic properties of cells when cultured dynamically, the perfusion culture in our study did not enhance the osteogenic properties of cell/scaffold constructs. The statically cultured constructs showed increasing cell numbers and abundant osteogenic differentiation probably because of weak initial cell adhesion due to the surface morphology of scaffolds. Our conclusion is that the specific scaffold surface microstructure and culturing system flow dynamics has a great impact on cell distribution and proliferation and on osteogenic differentiation, and the data presented warrant careful selection of in vitro culture settings to meet the specific requirements of the scaffolds and cells, especially when natural biomaterials with varying morphology are used. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.

U2 - 10.1002/jbm.a.33051

DO - 10.1002/jbm.a.33051

M3 - Journal article

C2 - 21442726

VL - 97A

SP - 251

EP - 263

JO - Journal of Biomedical Materials Research. Part A

JF - Journal of Biomedical Materials Research. Part A

SN - 1549-3296

IS - 3

ER -