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Isolation of osteogenic progenitors from human amniotic fluid using a single step culture protocol.

Antonucci I, Iezzi I, Morizio E, Mastrangelo F, Pantalone A, Mattioli-Belmonte M, Gigante A, Salini V, Calabrese G, Tetè S, Palka G, Stuppia L - BMC Biotechnol. (2009)

Bottom Line: Stem cells isolated from amniotic fluid are known to be able to differentiate into different cells types, being thus considered as a potential tool for cellular therapy of different human diseases.Electron microscopy analysis evidenced the best cell growth on this latter surface.The described protocol provides an efficient and time-saving tool for the production of osteogenic cells from amniotic fluid that in the future could be used in oral osteointegrated implantology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Sciences, G, d'Annunzio University, Chieti-Pescara, Italy. antonucciivana@libero.it

ABSTRACT

Background: Stem cells isolated from amniotic fluid are known to be able to differentiate into different cells types, being thus considered as a potential tool for cellular therapy of different human diseases. In the present study, we report a novel single step protocol for the osteoblastic differentiation of human amniotic fluid cells.

Results: The described protocol is able to provide osteoblastic cells producing nodules of calcium mineralization within 18 days from withdrawal of amniotic fluid samples. These cells display a complete expression of osteogenic markers (COL1, ONC, OPN, OCN, OPG, BSP, Runx2) within 30 days from withdrawal. In order to test the ability of these cells to proliferate on surfaces commonly used in oral osteointegrated implantology, we carried out cultures onto different test disks, namely smooth copper, machined titanium and Sandblasted and Acid Etching titanium (SLA titanium). Electron microscopy analysis evidenced the best cell growth on this latter surface.

Conclusion: The described protocol provides an efficient and time-saving tool for the production of osteogenic cells from amniotic fluid that in the future could be used in oral osteointegrated implantology.

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Related in: MedlinePlus

RT-PCR analysis of AFMSCs at day 20 of culture (protocol 1).
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Figure 3: RT-PCR analysis of AFMSCs at day 20 of culture (protocol 1).

Mentions: In Protocol 1, seven days after the initiation of the primary culture, fibroblast-like cells appeared both isolated and as colonies in the culture flask (Figure 2a). After 20–22 days of culture, at 70–80% confluence (Figure 2b), cells were treated with trypsin and EDTA and collected. RT-PCR analysis, carried out on RNA extracted from the cells at this stage, evidenced the presence of genes previously reported as expressed in AFMSCs [26], namely SDF1, CXCR4, Oct-4, SCF, GATA-4, Vim, FGF-5, Pax-6, NCAM, AFP, BMP-2 (Figure 3). Cells collected at day 20–22 were transferred and cultured in the osteogenic medium. After 18 days of culture in osteogenic medium (day 40 from withdrawal), the cells showed 70–75% confluence, and the presence of aggregates or nodules of calcium mineralization was appreciable. The number and size of these aggregates increased in the following days. Cells directly cultured in osteogenic medium (Protocol 2) reached 70–75% confluence after 18 days from withdrawal, and became over confluent in the following days (Figure 2c). In the following days the appearance of the first aggregates of calcium mineralization was observed (Figure 2). Alizarin Red staining confirmed the presence of biomineralization (Fig. 2e). An increase in the number and size of aggregates during the time was observed also in these cultures (Figure 2f). Cell count carried out on 5 cultures performed with protocol 2 at day 30 from withdrawal demonstrated the presence of cell number ranging from 8,9 × 106 9,7 × 106 cells.


Isolation of osteogenic progenitors from human amniotic fluid using a single step culture protocol.

Antonucci I, Iezzi I, Morizio E, Mastrangelo F, Pantalone A, Mattioli-Belmonte M, Gigante A, Salini V, Calabrese G, Tetè S, Palka G, Stuppia L - BMC Biotechnol. (2009)

RT-PCR analysis of AFMSCs at day 20 of culture (protocol 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654889&req=5

Figure 3: RT-PCR analysis of AFMSCs at day 20 of culture (protocol 1).
Mentions: In Protocol 1, seven days after the initiation of the primary culture, fibroblast-like cells appeared both isolated and as colonies in the culture flask (Figure 2a). After 20–22 days of culture, at 70–80% confluence (Figure 2b), cells were treated with trypsin and EDTA and collected. RT-PCR analysis, carried out on RNA extracted from the cells at this stage, evidenced the presence of genes previously reported as expressed in AFMSCs [26], namely SDF1, CXCR4, Oct-4, SCF, GATA-4, Vim, FGF-5, Pax-6, NCAM, AFP, BMP-2 (Figure 3). Cells collected at day 20–22 were transferred and cultured in the osteogenic medium. After 18 days of culture in osteogenic medium (day 40 from withdrawal), the cells showed 70–75% confluence, and the presence of aggregates or nodules of calcium mineralization was appreciable. The number and size of these aggregates increased in the following days. Cells directly cultured in osteogenic medium (Protocol 2) reached 70–75% confluence after 18 days from withdrawal, and became over confluent in the following days (Figure 2c). In the following days the appearance of the first aggregates of calcium mineralization was observed (Figure 2). Alizarin Red staining confirmed the presence of biomineralization (Fig. 2e). An increase in the number and size of aggregates during the time was observed also in these cultures (Figure 2f). Cell count carried out on 5 cultures performed with protocol 2 at day 30 from withdrawal demonstrated the presence of cell number ranging from 8,9 × 106 9,7 × 106 cells.

Bottom Line: Stem cells isolated from amniotic fluid are known to be able to differentiate into different cells types, being thus considered as a potential tool for cellular therapy of different human diseases.Electron microscopy analysis evidenced the best cell growth on this latter surface.The described protocol provides an efficient and time-saving tool for the production of osteogenic cells from amniotic fluid that in the future could be used in oral osteointegrated implantology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Sciences, G, d'Annunzio University, Chieti-Pescara, Italy. antonucciivana@libero.it

ABSTRACT

Background: Stem cells isolated from amniotic fluid are known to be able to differentiate into different cells types, being thus considered as a potential tool for cellular therapy of different human diseases. In the present study, we report a novel single step protocol for the osteoblastic differentiation of human amniotic fluid cells.

Results: The described protocol is able to provide osteoblastic cells producing nodules of calcium mineralization within 18 days from withdrawal of amniotic fluid samples. These cells display a complete expression of osteogenic markers (COL1, ONC, OPN, OCN, OPG, BSP, Runx2) within 30 days from withdrawal. In order to test the ability of these cells to proliferate on surfaces commonly used in oral osteointegrated implantology, we carried out cultures onto different test disks, namely smooth copper, machined titanium and Sandblasted and Acid Etching titanium (SLA titanium). Electron microscopy analysis evidenced the best cell growth on this latter surface.

Conclusion: The described protocol provides an efficient and time-saving tool for the production of osteogenic cells from amniotic fluid that in the future could be used in oral osteointegrated implantology.

Show MeSH
Related in: MedlinePlus