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Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells.

Müller P, Bulnheim U, Diener A, Lüthen F, Teller M, Klinkenberg ED, Neumann HG, Nebe B, Liebold A, Steinhoff G, Rychly J - J. Cell. Mol. Med. (2008 Jan-Feb)

Bottom Line: Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell-extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin.In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium.An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Laboratory, Medical Faculty, University of Rostock, Rostock, Germany.

ABSTRACT
Although studies in vivo revealed promising results in bone regeneration after implantation of scaffolds together with osteogenic progenitor cells, basic questions remain how material surfaces control the biology of mesenchymal stem cells (MSC). We used human MSC derived from bone marrow and studied the osteogenic differentiation on calcium phosphate surfaces. In osteogenic differentiation medium MSC differentiated to osteoblasts on hydroxyapatite and BONITmatrix, a degradable xerogel composite, within 14 days. Cells revealed a higher alkaline phosphatase (ALP) activity and increased RNA expression of collagen I and osteocalcin using real-time RTPCR compared with cells on tissue culture plastic. To test whether material surface characteristics alone are able to stimulate osteogenic differentiation, MSC were cultured on the materials in expansion medium without soluble additives for osteogenic differentiation. Indeed, cells on calcium phosphate without osteogenic differentiation additives developed to osteoblasts as shown by increased ALP activity and expression of osteogenic genes, which was not the case on tissue culture plastic. Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell-extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin. On BONITmatrix, an increased mobility of focal adhesions was observed compared with cells on tissue culture plastic. In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium. An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation.

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(a) Fluorescence images of ALP activity in cells on tissue culture plastic (TC), hydroxyapatite (HA) and BONITmatrix® after mesenchymal stem cells were cultured in EM on different substrates for 14 days. Note that there was no visible ALP activity in cells on TC. (b) Quantitative analyses of ALP activity in cells on TC, HA and BM. The highest ALP activity was measured in cells on HA.
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fig04: (a) Fluorescence images of ALP activity in cells on tissue culture plastic (TC), hydroxyapatite (HA) and BONITmatrix® after mesenchymal stem cells were cultured in EM on different substrates for 14 days. Note that there was no visible ALP activity in cells on TC. (b) Quantitative analyses of ALP activity in cells on TC, HA and BM. The highest ALP activity was measured in cells on HA.

Mentions: Because on calcium phosphate a significant stimulation of osteoblastic differentiation was observed which exceeded the stimulation by the TCP surface, we next set out to test whether MSC on a calcium phosphate surface in EM without osteogenic differentiation additives are able to differentiate to osteoblasts. Measurements of ALP activity revealed that indeed MSC differentiated to osteoblasts on calcium phosphate substrates in the absence of differentiation additives in the medium (Fig. 4a and b). To confirm this result, we also tested the cell line MG-63 which is often used as a model for osteoblasts. Both in ODM and in the absence of osteogenic differentiation additives, MG-63 cells revealed ALP activity only on calcium phosphate surfaces but not on tissue culture plastic (Fig. 5). Gene expression analyses performed with MSC confirmed the osteogenic differentiation on calcium phosphate without differentiation additives in the medium (Fig. 6). In pure EM, RNA expression of osteogenic genes including Runx2 was detected after MSC were cultured for 3 or 14 days on BONITmatrix®. However, concerning the extent of gene expression when cells were cultured on the calcium phosphate substrate, with the exception of Runx2 we found a higher gene expression in ODM containing soluble differentiation factors compared with EM (Figs. 3 and 6). Concerning the time course of gene expression, both in ODM and EM we observed a similar expression profile. ALP and collagen I were more expressed on day 3, whereas bone sialoprotein and osteocalcin were highly expressed on day 14 (Figs. 3 and 6). Experiments with cells of the MG-63 cell line confirmed the results that a calcium phosphate surface induces gene expression without osteogenic differentiation additives. Both in ODM and in EM, MG-63 cells revealed an increased RNA expression of ALP both after 3 and 7 days on BONITmatrix® compared with cells on tissue culture plastic (Fig. 7).


Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells.

Müller P, Bulnheim U, Diener A, Lüthen F, Teller M, Klinkenberg ED, Neumann HG, Nebe B, Liebold A, Steinhoff G, Rychly J - J. Cell. Mol. Med. (2008 Jan-Feb)

(a) Fluorescence images of ALP activity in cells on tissue culture plastic (TC), hydroxyapatite (HA) and BONITmatrix® after mesenchymal stem cells were cultured in EM on different substrates for 14 days. Note that there was no visible ALP activity in cells on TC. (b) Quantitative analyses of ALP activity in cells on TC, HA and BM. The highest ALP activity was measured in cells on HA.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3823489&req=5

fig04: (a) Fluorescence images of ALP activity in cells on tissue culture plastic (TC), hydroxyapatite (HA) and BONITmatrix® after mesenchymal stem cells were cultured in EM on different substrates for 14 days. Note that there was no visible ALP activity in cells on TC. (b) Quantitative analyses of ALP activity in cells on TC, HA and BM. The highest ALP activity was measured in cells on HA.
Mentions: Because on calcium phosphate a significant stimulation of osteoblastic differentiation was observed which exceeded the stimulation by the TCP surface, we next set out to test whether MSC on a calcium phosphate surface in EM without osteogenic differentiation additives are able to differentiate to osteoblasts. Measurements of ALP activity revealed that indeed MSC differentiated to osteoblasts on calcium phosphate substrates in the absence of differentiation additives in the medium (Fig. 4a and b). To confirm this result, we also tested the cell line MG-63 which is often used as a model for osteoblasts. Both in ODM and in the absence of osteogenic differentiation additives, MG-63 cells revealed ALP activity only on calcium phosphate surfaces but not on tissue culture plastic (Fig. 5). Gene expression analyses performed with MSC confirmed the osteogenic differentiation on calcium phosphate without differentiation additives in the medium (Fig. 6). In pure EM, RNA expression of osteogenic genes including Runx2 was detected after MSC were cultured for 3 or 14 days on BONITmatrix®. However, concerning the extent of gene expression when cells were cultured on the calcium phosphate substrate, with the exception of Runx2 we found a higher gene expression in ODM containing soluble differentiation factors compared with EM (Figs. 3 and 6). Concerning the time course of gene expression, both in ODM and EM we observed a similar expression profile. ALP and collagen I were more expressed on day 3, whereas bone sialoprotein and osteocalcin were highly expressed on day 14 (Figs. 3 and 6). Experiments with cells of the MG-63 cell line confirmed the results that a calcium phosphate surface induces gene expression without osteogenic differentiation additives. Both in ODM and in EM, MG-63 cells revealed an increased RNA expression of ALP both after 3 and 7 days on BONITmatrix® compared with cells on tissue culture plastic (Fig. 7).

Bottom Line: Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell-extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin.In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium.An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Laboratory, Medical Faculty, University of Rostock, Rostock, Germany.

ABSTRACT
Although studies in vivo revealed promising results in bone regeneration after implantation of scaffolds together with osteogenic progenitor cells, basic questions remain how material surfaces control the biology of mesenchymal stem cells (MSC). We used human MSC derived from bone marrow and studied the osteogenic differentiation on calcium phosphate surfaces. In osteogenic differentiation medium MSC differentiated to osteoblasts on hydroxyapatite and BONITmatrix, a degradable xerogel composite, within 14 days. Cells revealed a higher alkaline phosphatase (ALP) activity and increased RNA expression of collagen I and osteocalcin using real-time RTPCR compared with cells on tissue culture plastic. To test whether material surface characteristics alone are able to stimulate osteogenic differentiation, MSC were cultured on the materials in expansion medium without soluble additives for osteogenic differentiation. Indeed, cells on calcium phosphate without osteogenic differentiation additives developed to osteoblasts as shown by increased ALP activity and expression of osteogenic genes, which was not the case on tissue culture plastic. Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell-extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin. On BONITmatrix, an increased mobility of focal adhesions was observed compared with cells on tissue culture plastic. In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium. An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation.

Show MeSH
Related in: MedlinePlus