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Osteogenic differentiation of human mesenchymal stem cells in mineralized alginate matrices.

Westhrin M, Xie M, Olderøy MØ, Sikorski P, Strand BL, Standal T - PLoS ONE (2015)

Bottom Line: Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes.In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture.Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.

View Article: PubMed Central - PubMed

Affiliation: Kristian Gerhard Jebsen Center for Myeloma Research, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway.

ABSTRACT
Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.

No MeSH data available.


Related in: MedlinePlus

Viability of hMSCs in alginate beads cultured in osteogenic medium.(A) Live/dead fluorescent staining of cells cultured in osteogenic medium was visualized using confocal microscopy (LSM 510 META FCS, Zeiss). Left images: Confocal cross sections through overlaid transmitted light of hMSCs in alginate beads; Right images: three dimensional reconstructions of cross sections through the beads. Live cells appear green, dead cells appear red. Scale bar 500 μm. (B) Metabolic activity of MSC in alginate beads cultured in growth medium (GM) or osteogenic medium (OM) at day 2, 7, 14 and 21 post encapsulation measured by Alamar Blue assay. Metabolic activity was significantly different in ALP modified beads cultured in OM compared with unmodified beads cultured in OM at day 14, p <0.001, day 17, p< = 0.01 and day 21, p< = 0.001, Sidak’s multiple comparison test. The difference in metabolic activity between unmodified and ALP-modified beads cultured in GM was not statistically significant. The data presented are mean values +/- SD, n = 3. ALP: Containing 0.25mg/mL ALP.
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pone.0120374.g003: Viability of hMSCs in alginate beads cultured in osteogenic medium.(A) Live/dead fluorescent staining of cells cultured in osteogenic medium was visualized using confocal microscopy (LSM 510 META FCS, Zeiss). Left images: Confocal cross sections through overlaid transmitted light of hMSCs in alginate beads; Right images: three dimensional reconstructions of cross sections through the beads. Live cells appear green, dead cells appear red. Scale bar 500 μm. (B) Metabolic activity of MSC in alginate beads cultured in growth medium (GM) or osteogenic medium (OM) at day 2, 7, 14 and 21 post encapsulation measured by Alamar Blue assay. Metabolic activity was significantly different in ALP modified beads cultured in OM compared with unmodified beads cultured in OM at day 14, p <0.001, day 17, p< = 0.01 and day 21, p< = 0.001, Sidak’s multiple comparison test. The difference in metabolic activity between unmodified and ALP-modified beads cultured in GM was not statistically significant. The data presented are mean values +/- SD, n = 3. ALP: Containing 0.25mg/mL ALP.

Mentions: Viability of the encapsulated cells was high after encapsulation and remained high with an estimated 90% viability for the duration of the study, as examined by visual inspection of live/dead staining by confocal microscopy (Fig. 3A). Moreover, no significant differences in viability were observed for the different sample groups (Fig. 3A and S1 Fig.). The metabolic activity measured by Alamar Blue assay increased over time for cells in both types of beads cultured in either OM and GM which further support that the cells survive in the beads, and might also suggest that the cells divide during the culture period (Fig. 3B). The metabolic activity appeared higher in cells cultured in growth medium compared with cells cultured in osteogenic medium, although the difference was only statistically significant at day 14 for cells cultured in unmodified beads (p≤ 0.05, Sidak’s multiple comparison test). The lower metabolic activity in cells cultured in OM might reflect a lower number of cells, as differentiated osteoblasts rarely divide compared with the more immature cells [23]. There was no significant difference in metabolic activity between cells cultured in GM in unmodified versus ALP-modified beads. However, at day 7, 14 and 21 after encapsulation, cells cultured in OM had a significantly higher metabolic activity in ALP-modified beads compared with cells in unmodified beads (p≤ 0.001, p≤ 0.01, p≤ 0.001, respectively, Sidak’s multiple comparison test). In conclusion, encapsulation of hMSC in mineralized and unmineralized alginate beads maintain high viability and metabolic activity.


Osteogenic differentiation of human mesenchymal stem cells in mineralized alginate matrices.

Westhrin M, Xie M, Olderøy MØ, Sikorski P, Strand BL, Standal T - PLoS ONE (2015)

Viability of hMSCs in alginate beads cultured in osteogenic medium.(A) Live/dead fluorescent staining of cells cultured in osteogenic medium was visualized using confocal microscopy (LSM 510 META FCS, Zeiss). Left images: Confocal cross sections through overlaid transmitted light of hMSCs in alginate beads; Right images: three dimensional reconstructions of cross sections through the beads. Live cells appear green, dead cells appear red. Scale bar 500 μm. (B) Metabolic activity of MSC in alginate beads cultured in growth medium (GM) or osteogenic medium (OM) at day 2, 7, 14 and 21 post encapsulation measured by Alamar Blue assay. Metabolic activity was significantly different in ALP modified beads cultured in OM compared with unmodified beads cultured in OM at day 14, p <0.001, day 17, p< = 0.01 and day 21, p< = 0.001, Sidak’s multiple comparison test. The difference in metabolic activity between unmodified and ALP-modified beads cultured in GM was not statistically significant. The data presented are mean values +/- SD, n = 3. ALP: Containing 0.25mg/mL ALP.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4358956&req=5

pone.0120374.g003: Viability of hMSCs in alginate beads cultured in osteogenic medium.(A) Live/dead fluorescent staining of cells cultured in osteogenic medium was visualized using confocal microscopy (LSM 510 META FCS, Zeiss). Left images: Confocal cross sections through overlaid transmitted light of hMSCs in alginate beads; Right images: three dimensional reconstructions of cross sections through the beads. Live cells appear green, dead cells appear red. Scale bar 500 μm. (B) Metabolic activity of MSC in alginate beads cultured in growth medium (GM) or osteogenic medium (OM) at day 2, 7, 14 and 21 post encapsulation measured by Alamar Blue assay. Metabolic activity was significantly different in ALP modified beads cultured in OM compared with unmodified beads cultured in OM at day 14, p <0.001, day 17, p< = 0.01 and day 21, p< = 0.001, Sidak’s multiple comparison test. The difference in metabolic activity between unmodified and ALP-modified beads cultured in GM was not statistically significant. The data presented are mean values +/- SD, n = 3. ALP: Containing 0.25mg/mL ALP.
Mentions: Viability of the encapsulated cells was high after encapsulation and remained high with an estimated 90% viability for the duration of the study, as examined by visual inspection of live/dead staining by confocal microscopy (Fig. 3A). Moreover, no significant differences in viability were observed for the different sample groups (Fig. 3A and S1 Fig.). The metabolic activity measured by Alamar Blue assay increased over time for cells in both types of beads cultured in either OM and GM which further support that the cells survive in the beads, and might also suggest that the cells divide during the culture period (Fig. 3B). The metabolic activity appeared higher in cells cultured in growth medium compared with cells cultured in osteogenic medium, although the difference was only statistically significant at day 14 for cells cultured in unmodified beads (p≤ 0.05, Sidak’s multiple comparison test). The lower metabolic activity in cells cultured in OM might reflect a lower number of cells, as differentiated osteoblasts rarely divide compared with the more immature cells [23]. There was no significant difference in metabolic activity between cells cultured in GM in unmodified versus ALP-modified beads. However, at day 7, 14 and 21 after encapsulation, cells cultured in OM had a significantly higher metabolic activity in ALP-modified beads compared with cells in unmodified beads (p≤ 0.001, p≤ 0.01, p≤ 0.001, respectively, Sidak’s multiple comparison test). In conclusion, encapsulation of hMSC in mineralized and unmineralized alginate beads maintain high viability and metabolic activity.

Bottom Line: Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes.In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture.Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.

View Article: PubMed Central - PubMed

Affiliation: Kristian Gerhard Jebsen Center for Myeloma Research, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway.

ABSTRACT
Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.

No MeSH data available.


Related in: MedlinePlus