J Adv Prosthodont.  2014 Oct;6(5):379-386. 10.4047/jap.2014.6.5.379.

Optimization of growth inducing factors for colony forming and attachment of bone marrow-derived mesenchymal stem cells regarding bioengineering application

• Quan H
• Kim SK
• Heo SJ
• Koak JY
• Lee JH

• KMID: 2284738
• DOI: http://doi.org/10.4047/jap.2014.6.5.379

Abstract

PURPOSE
These days, mesenchymal stem cells (MSCs) have received worldwide attention because of their potentiality in tissue engineering for implant dentistry. The purpose of this study was to evaluate various growth inducing factors in media for improvement of acquisition of bone marrow mesenchymal stem cells (BMMSCs) and colony forming unit-fibroblast (CFU-F).
MATERIALS AND METHODS
The mouse BMMSCs were freshly obtained from female C3H mouse femur and tibia. The cells seeded at the density of 106/dish in media supplemented with different density of fetal bovine serum (FBS), 1alpha, 25-dihydroxyvitamin (VD3) and recombinant human epidermal growth factor (rhEGF). After 14 days, CFU-F assay was conducted to analyze the cell attachment and proliferation, and moreover for VD3, the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was additionally conducted.
RESULTS
The cell proliferation was increased with the increase of FBS concentration (P<.05). The cell proliferation was highest at the density of 20 ng/mL rhEGF compared with 0 ng/mL and 200 ng/mL rhEGF (P<.05). For VD3, although the colony number was increased with the increase of its concentration, the difference was not statistically significant (P>.05).
CONCLUSION
FBS played the main role in cell attachment and growth, and the growth factor like rhEGF played the additional effect. However, VD3 did not have much efficacy compare with the other two factors. Improvement of the conditions could be adopted to acquire more functional MSCs to apply into bony defect around implants easily.

Keyword

Bone marrow mesenchymal stem cells; Cell proliferation; Fetal bovine serum; 1alpha; 25-dihydroxyvitamin; Recombinant human epidermal growth factor

MeSH Terms

Animals
Bioengineering*
Bone Marrow
Cell Proliferation
Dentistry
Epidermal Growth Factor
Female
Femur
Humans
Mesenchymal Stromal Cells*
Mice
Mice, Inbred C3H
Tibia
Tissue Engineering
Epidermal Growth Factor

Figure

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  Fig. 1 Primary isolated mBMMSCs cultured in 0%, 1%, 5%, 10%, 20% FBS medium for 14 days to do colony forming unit (CFU) assay. (A-E) show the mBMMSCs cultured in 0%, 1%, 5%, 10%, 20% FBS medium after 14 days by microscope. Magnification ×100. And (a-e) is magnification ×200.

• 

  Fig. 2 The images show the colonies stained by toluidine blue after 14 days of primary cultured in 0%, 1%, 5%, 10%, 20% FBS medium.

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  Fig. 3 Primary isolated mBMMSCs cultured in 0 nM, 1 nM, 10 nM, 100 nM VD3 medium for 14 days to do colony forming unit (CFU) assay. (A-D) show the mBMMSCs cultured in 0 nM, 1 nM, 10 nM, 100 nM VD3 medium after 14 days by microscope. Magnification ×100. And (a-d) is magnification ×200.

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  Fig. 4 The images show the colonies stained by toluidine blue after 14 days of primary cultured in 0 nM, 1 nM, 10 nM, 100 nM VD3 medium.

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  Fig. 5 Primary isolated mBMMSCs cultured in 0 ng/mL, 20 ng/mL, 200 ng/mL EGF medium for 14 days to do colony forming unit (CFU) assay. (A-C) show the mBMMSCs cultured in 0 ng/mL, 20 ng/mL, 200 ng/mL EGF medium after 14 days by microscope. Magnification ×100. And (a-c) is magnification ×200.

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  Fig. 6 The images show the colonies stained by toluidine blue after 14 days of primary cultured in 0 ng/mL, 20 ng/mL, 200 ng/mL EGF medium.

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  Fig. 7 The results of colony forming unit assay for 0%, 1%, 5%, 10%, 20% FBS after 14 days of primary cultured. The data are expressed as the mean ± SD of triplicate determinations (*P<.05).

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  Fig. 8 The results of colony forming unit assay for 0 nM, 1 nM, 10 nM, 100 nM VD3 after 14 days of primary cultured. The data are expressed as the mean ± SD of triplicate determinations (P>.05).

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  Fig. 9 The results of colony forming unit assay for in 0 ng/mL, 20 ng/mL, 200 ng/mL EGF after 14 days of primary cultured. The data are expressed as the mean ± SD of triplicate determinations (*P<.05).

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  Fig. 10 Primary isolated mBMMSCs cultured in 0 nM, 1 nM, 10 nM, 100 nM VD3 medium for 14 days. The results of MTS assay for VD3 after 1 day, 4 days, 7 days, 10 days and 14 days of primary cultured. The data are expressed as the mean ± SD of triplicate determinations (P>.05).

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