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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

Alkaline phosphatase activity in cells cultured in ALP-modified or unmodified beads for 9 days as indicated.p values indicate statistical significant differences by Sidak’s multiple comparison test.
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pone.0120374.g006: Alkaline phosphatase activity in cells cultured in ALP-modified or unmodified beads for 9 days as indicated.p values indicate statistical significant differences by Sidak’s multiple comparison test.

Mentions: Sclerostin (SOST) is a marker specific for osteocytes/late osteoblasts and is induced after BGLAP [29]. Indeed, in cells cultured in OM in both bead types SOST mRNA was highly expressed (Fig. 4E), many hundred-fold increased compared with cells induced to differentiate on plastic. After 21 days, SOST mRNA was significantly higher in unmodified alginate beads compared with ALP-beads (p ≤ 0.001, Tukey’s multiple comparison test). Moreover, the mRNA expression of dentin matrix protein 1 (DMP1), an extracellular matrix protein expressed by osteocytes, was highly expressed in encapsulated cells cultured in OM, and was also expressed by cells in beads cultured in growth medium (Fig. 5F). After 21 days, DMP1 was higher in ALP-modified beads compared with unmodified beads (p ≤ 0.05, Tukey’s multiple comparison test), whereas there was no difference in DMP1 expression in cells cultured in GM. Cells cultured in osteogenic conditions on plastic did not express DMP1. To further verify that the cells differentiated in osteogenic direction, we measured alkaline phosphatase (ALP) activity by an enzymatic assay. Indeed, alkaline phosphatase activity increased in cells cultured in OM compared with GM in both ALP-modified and unmodified beads (p ≤0.01, Sidak’s multiple comparison test) (Fig. 6). ALP-activity also appeared higher in unmodified beads compared with modified beads cultured in OM (p ≤ 0.05, Sidak’s multiple comparison test), but not in GM.


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)

Alkaline phosphatase activity in cells cultured in ALP-modified or unmodified beads for 9 days as indicated.p values indicate statistical significant differences by Sidak’s multiple comparison test.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120374.g006: Alkaline phosphatase activity in cells cultured in ALP-modified or unmodified beads for 9 days as indicated.p values indicate statistical significant differences by Sidak’s multiple comparison test.
Mentions: Sclerostin (SOST) is a marker specific for osteocytes/late osteoblasts and is induced after BGLAP [29]. Indeed, in cells cultured in OM in both bead types SOST mRNA was highly expressed (Fig. 4E), many hundred-fold increased compared with cells induced to differentiate on plastic. After 21 days, SOST mRNA was significantly higher in unmodified alginate beads compared with ALP-beads (p ≤ 0.001, Tukey’s multiple comparison test). Moreover, the mRNA expression of dentin matrix protein 1 (DMP1), an extracellular matrix protein expressed by osteocytes, was highly expressed in encapsulated cells cultured in OM, and was also expressed by cells in beads cultured in growth medium (Fig. 5F). After 21 days, DMP1 was higher in ALP-modified beads compared with unmodified beads (p ≤ 0.05, Tukey’s multiple comparison test), whereas there was no difference in DMP1 expression in cells cultured in GM. Cells cultured in osteogenic conditions on plastic did not express DMP1. To further verify that the cells differentiated in osteogenic direction, we measured alkaline phosphatase (ALP) activity by an enzymatic assay. Indeed, alkaline phosphatase activity increased in cells cultured in OM compared with GM in both ALP-modified and unmodified beads (p ≤0.01, Sidak’s multiple comparison test) (Fig. 6). ALP-activity also appeared higher in unmodified beads compared with modified beads cultured in OM (p ≤ 0.05, Sidak’s multiple comparison test), but not in GM.

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