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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.

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Related in: MedlinePlus

Relative mRNA expression of osteoblast/ osteocyte markers.MSCs were cultured in unmodified (no ALP), ALP-modified (ALP) alginate beads or on traditional culture plates (2D). Samples were cultured in either growth medium (GM) or osteogenic medium (OM) for 21 days post encapsulation. RUNX2 (A), Osterix (SP7) (B), COL1A1 (C), and sclerostin (SOST) (E) mRNA expression are relative to mRNA expression in cells cultured on traditional culture plates in osteogenic medium for 7 days. Osteocalcin (BGLAP) (D) and DMP1 (F) mRNA expressions are relative to mRNA expression in cells in unmodified alginate beads cultured in growth medium for 7 days post encapsulation. ND = not detected.
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pone.0120374.g005: Relative mRNA expression of osteoblast/ osteocyte markers.MSCs were cultured in unmodified (no ALP), ALP-modified (ALP) alginate beads or on traditional culture plates (2D). Samples were cultured in either growth medium (GM) or osteogenic medium (OM) for 21 days post encapsulation. RUNX2 (A), Osterix (SP7) (B), COL1A1 (C), and sclerostin (SOST) (E) mRNA expression are relative to mRNA expression in cells cultured on traditional culture plates in osteogenic medium for 7 days. Osteocalcin (BGLAP) (D) and DMP1 (F) mRNA expressions are relative to mRNA expression in cells in unmodified alginate beads cultured in growth medium for 7 days post encapsulation. ND = not detected.

Mentions: To characterize the differentiation stage of the encapsulated cells, mRNA expression of markers for osteoblast differentiation were examined at day 7, 14 and 21 post encapsulation. As a reference the gene expression levels in MSC cultured on tissue culture plates in osteogenic media for 7, 14 and 21 days were also quantified. RUNX2 is considered an early osteoblastic gene [27], and we found RUNX2 to be expressed in all samples (Fig. 5A). RUNX2 mRNA was significantly higher in ALP-modified beads cultured in GM compared with unmodified beads cultured in GM (p ≤ 0.001, all time points, Tukey’s multiple comparison test). However, there was no significant difference in RUNX2 expression in the different bead types cultured in OM. The expression of the transcription factor osterix (SP7) increases during osteoblast maturation [28]. We found that SP7 was induced in cells cultured in traditional 2D in osteogenic medium; still, SP7 expression was higher in cells cultured in OM in the two bead types compared with cells cultured in 2D (p ≤ 0.001, both bead types, all time points, Tukey’s multiple comparison test). In comparison, SP7 expression in cells cultured in beads in GM was low, suggesting that a 3D environment by itself is not sufficient to promote the differentiation of fully mature osteoblasts (Fig. 5B). Collagen type I (COL1A1) is the major matrix protein produced by osteoblasts. Strikingly, cells cultured in osteogenic medium in both ALP-modified and non-modified alginate beads expressed higher levels of collagen type I than cells cultured in the same medium in traditional 2D, suggesting that a 3D environment might promote collagen type I expression (Tukey’s multiple comparison test p≤ 0.001 for both bead types) (Fig. 5C). At day 14, COL1A1 was higher in unmodified beads compared with ALP-modified beads for cells cultured in OM (p ≤ 0.001, Tukey’s multiple comparison test) but in cells cultured in growth media COL1A1 mRNA expression was similar in the two bead types. Similarly, gene expression levels of osteocalcin (bone gamma-carboxyglutamic acid-containing protein (BGLAP)) a secreted protein produced by mature osteoblasts, were higher in cells cultured in osteogenic medium in 3D compared with cells cultured in osteogenic medium in traditional 2D culture, in which we could not detect BGLAP mRNA (Fig. 5D). There was no difference in BGLAP mRNA expression between cells cultured in GM in the two bead types; however, BGLAP was significantly higher in ALP-modified beads cultured for 21 days in OM (p ≤ 0.001, Tukey’s multiple comparison test).


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)

Relative mRNA expression of osteoblast/ osteocyte markers.MSCs were cultured in unmodified (no ALP), ALP-modified (ALP) alginate beads or on traditional culture plates (2D). Samples were cultured in either growth medium (GM) or osteogenic medium (OM) for 21 days post encapsulation. RUNX2 (A), Osterix (SP7) (B), COL1A1 (C), and sclerostin (SOST) (E) mRNA expression are relative to mRNA expression in cells cultured on traditional culture plates in osteogenic medium for 7 days. Osteocalcin (BGLAP) (D) and DMP1 (F) mRNA expressions are relative to mRNA expression in cells in unmodified alginate beads cultured in growth medium for 7 days post encapsulation. ND = not detected.
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Related In: Results  -  Collection

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pone.0120374.g005: Relative mRNA expression of osteoblast/ osteocyte markers.MSCs were cultured in unmodified (no ALP), ALP-modified (ALP) alginate beads or on traditional culture plates (2D). Samples were cultured in either growth medium (GM) or osteogenic medium (OM) for 21 days post encapsulation. RUNX2 (A), Osterix (SP7) (B), COL1A1 (C), and sclerostin (SOST) (E) mRNA expression are relative to mRNA expression in cells cultured on traditional culture plates in osteogenic medium for 7 days. Osteocalcin (BGLAP) (D) and DMP1 (F) mRNA expressions are relative to mRNA expression in cells in unmodified alginate beads cultured in growth medium for 7 days post encapsulation. ND = not detected.
Mentions: To characterize the differentiation stage of the encapsulated cells, mRNA expression of markers for osteoblast differentiation were examined at day 7, 14 and 21 post encapsulation. As a reference the gene expression levels in MSC cultured on tissue culture plates in osteogenic media for 7, 14 and 21 days were also quantified. RUNX2 is considered an early osteoblastic gene [27], and we found RUNX2 to be expressed in all samples (Fig. 5A). RUNX2 mRNA was significantly higher in ALP-modified beads cultured in GM compared with unmodified beads cultured in GM (p ≤ 0.001, all time points, Tukey’s multiple comparison test). However, there was no significant difference in RUNX2 expression in the different bead types cultured in OM. The expression of the transcription factor osterix (SP7) increases during osteoblast maturation [28]. We found that SP7 was induced in cells cultured in traditional 2D in osteogenic medium; still, SP7 expression was higher in cells cultured in OM in the two bead types compared with cells cultured in 2D (p ≤ 0.001, both bead types, all time points, Tukey’s multiple comparison test). In comparison, SP7 expression in cells cultured in beads in GM was low, suggesting that a 3D environment by itself is not sufficient to promote the differentiation of fully mature osteoblasts (Fig. 5B). Collagen type I (COL1A1) is the major matrix protein produced by osteoblasts. Strikingly, cells cultured in osteogenic medium in both ALP-modified and non-modified alginate beads expressed higher levels of collagen type I than cells cultured in the same medium in traditional 2D, suggesting that a 3D environment might promote collagen type I expression (Tukey’s multiple comparison test p≤ 0.001 for both bead types) (Fig. 5C). At day 14, COL1A1 was higher in unmodified beads compared with ALP-modified beads for cells cultured in OM (p ≤ 0.001, Tukey’s multiple comparison test) but in cells cultured in growth media COL1A1 mRNA expression was similar in the two bead types. Similarly, gene expression levels of osteocalcin (bone gamma-carboxyglutamic acid-containing protein (BGLAP)) a secreted protein produced by mature osteoblasts, were higher in cells cultured in osteogenic medium in 3D compared with cells cultured in osteogenic medium in traditional 2D culture, in which we could not detect BGLAP mRNA (Fig. 5D). There was no difference in BGLAP mRNA expression between cells cultured in GM in the two bead types; however, BGLAP was significantly higher in ALP-modified beads cultured for 21 days in OM (p ≤ 0.001, Tukey’s multiple comparison test).

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