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Artificial extracellular matrices with oversulfated glycosaminoglycan derivatives promote the differentiation of osteoblast-precursor cells and premature osteoblasts.

Hempel U, Preissler C, Vogel S, Möller S, Hintze V, Becher J, Schnabelrauch M, Rauner M, Hofbauer LC, Dieter P - Biomed Res Int (2014)

Bottom Line: Oversulfated GAG derivatives with a sulfate group at the C-6 position of the N-acetylglycosamine revealed the most pronounced proosteogenic effect as determined by tissue nonspecific alkaline phosphatase activity and calcium deposition.A subset of the aECM was further analysed with different primary osteoblasts and cell lines reflecting different maturation stages to test whether the effect of sulfated GAG derivatives depends on the maturation status of the cells.It was shown that the proosteogenic effect of aECM was most prominent in early osteoblasts.

View Article: PubMed Central - PubMed

Affiliation: Institute of Physiological Chemistry, Faculty of Medicine Carl Gustav Carus, TU Dresden, Fiedlerstraße 42, 01307 Dresden, Germany.

ABSTRACT
Sulfated glycosaminoglycans (GAG) are components of the bone marrow stem cell niche and to a minor extent of mature bone tissue with important functions in regulating stem cell lineage commitment and differentiation. We anticipated that artificial extracellular matrices (aECM) composed of collagen I and synthetically oversulfated GAG derivatives affect preferentially the differentiation of osteoblast-precursor cells and early osteoblasts. A set of gradually sulfated chondroitin sulfate and hyaluronan derivatives was used for the preparation of aECM. All these matrices were analysed with human bone marrow stromal cells to identify the most potent aECM and to determine the influence of the degree and position of sulfate groups and the kind of disaccharide units on the osteogenic differentiation. Oversulfated GAG derivatives with a sulfate group at the C-6 position of the N-acetylglycosamine revealed the most pronounced proosteogenic effect as determined by tissue nonspecific alkaline phosphatase activity and calcium deposition. A subset of the aECM was further analysed with different primary osteoblasts and cell lines reflecting different maturation stages to test whether the effect of sulfated GAG derivatives depends on the maturation status of the cells. It was shown that the proosteogenic effect of aECM was most prominent in early osteoblasts.

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Influence of medium supplements on TNAP activity of osteoblast-precursor cells, premature and mature osteoblasts. Various human (hBMSC, MG-63, SaOS-2) (a), rat (rCaOB, rBMSC) (b), and mouse cell (line)s (MC3T3-E1, MLO-Y4) (c) were plated in BM on TCPS. At day 4 after plating cells were cultured either in BM, OM, or OM/D. At day 11 after plating, TNAP activity was determined in cell lysates with p-nitrophenylphosphate as a substrate. The released p-nitrophenolate was measured photometrically at 405 nm. TNAP activity in mU/mL was normalized to protein concentration in mg/mL determined with Rotiquant assay. Significant differences were calculated by one-way ANOVA analysis and indicated with a (P < 0.05), b (P < 0.01) and c (P < 0.001), n = 4.
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fig3: Influence of medium supplements on TNAP activity of osteoblast-precursor cells, premature and mature osteoblasts. Various human (hBMSC, MG-63, SaOS-2) (a), rat (rCaOB, rBMSC) (b), and mouse cell (line)s (MC3T3-E1, MLO-Y4) (c) were plated in BM on TCPS. At day 4 after plating cells were cultured either in BM, OM, or OM/D. At day 11 after plating, TNAP activity was determined in cell lysates with p-nitrophenylphosphate as a substrate. The released p-nitrophenolate was measured photometrically at 405 nm. TNAP activity in mU/mL was normalized to protein concentration in mg/mL determined with Rotiquant assay. Significant differences were calculated by one-way ANOVA analysis and indicated with a (P < 0.05), b (P < 0.01) and c (P < 0.001), n = 4.

Mentions: Each experiment was performed with nonpooled cells from four different donors (in the case of primary cells: hBMSC, rCaOB, rBMSC) or four independent cell cultures (in the case of cell lines: MG-63, SaOS-2, MC3T3-E1, and MLO-Y4) each in triplicate. The results are presented as mean ± standard error of the mean (SEM). Statistical significance was analyzed with GraphPad Prism 5.04 software (Statcon, Witzenhausen, Germany) by one-way ANOVA (Figures 2 and 3) and two-way ANOVA analysis (Figure 4, Table 3) with Bonferroni's posttest.


Artificial extracellular matrices with oversulfated glycosaminoglycan derivatives promote the differentiation of osteoblast-precursor cells and premature osteoblasts.

Hempel U, Preissler C, Vogel S, Möller S, Hintze V, Becher J, Schnabelrauch M, Rauner M, Hofbauer LC, Dieter P - Biomed Res Int (2014)

Influence of medium supplements on TNAP activity of osteoblast-precursor cells, premature and mature osteoblasts. Various human (hBMSC, MG-63, SaOS-2) (a), rat (rCaOB, rBMSC) (b), and mouse cell (line)s (MC3T3-E1, MLO-Y4) (c) were plated in BM on TCPS. At day 4 after plating cells were cultured either in BM, OM, or OM/D. At day 11 after plating, TNAP activity was determined in cell lysates with p-nitrophenylphosphate as a substrate. The released p-nitrophenolate was measured photometrically at 405 nm. TNAP activity in mU/mL was normalized to protein concentration in mg/mL determined with Rotiquant assay. Significant differences were calculated by one-way ANOVA analysis and indicated with a (P < 0.05), b (P < 0.01) and c (P < 0.001), n = 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4020545&req=5

fig3: Influence of medium supplements on TNAP activity of osteoblast-precursor cells, premature and mature osteoblasts. Various human (hBMSC, MG-63, SaOS-2) (a), rat (rCaOB, rBMSC) (b), and mouse cell (line)s (MC3T3-E1, MLO-Y4) (c) were plated in BM on TCPS. At day 4 after plating cells were cultured either in BM, OM, or OM/D. At day 11 after plating, TNAP activity was determined in cell lysates with p-nitrophenylphosphate as a substrate. The released p-nitrophenolate was measured photometrically at 405 nm. TNAP activity in mU/mL was normalized to protein concentration in mg/mL determined with Rotiquant assay. Significant differences were calculated by one-way ANOVA analysis and indicated with a (P < 0.05), b (P < 0.01) and c (P < 0.001), n = 4.
Mentions: Each experiment was performed with nonpooled cells from four different donors (in the case of primary cells: hBMSC, rCaOB, rBMSC) or four independent cell cultures (in the case of cell lines: MG-63, SaOS-2, MC3T3-E1, and MLO-Y4) each in triplicate. The results are presented as mean ± standard error of the mean (SEM). Statistical significance was analyzed with GraphPad Prism 5.04 software (Statcon, Witzenhausen, Germany) by one-way ANOVA (Figures 2 and 3) and two-way ANOVA analysis (Figure 4, Table 3) with Bonferroni's posttest.

Bottom Line: Oversulfated GAG derivatives with a sulfate group at the C-6 position of the N-acetylglycosamine revealed the most pronounced proosteogenic effect as determined by tissue nonspecific alkaline phosphatase activity and calcium deposition.A subset of the aECM was further analysed with different primary osteoblasts and cell lines reflecting different maturation stages to test whether the effect of sulfated GAG derivatives depends on the maturation status of the cells.It was shown that the proosteogenic effect of aECM was most prominent in early osteoblasts.

View Article: PubMed Central - PubMed

Affiliation: Institute of Physiological Chemistry, Faculty of Medicine Carl Gustav Carus, TU Dresden, Fiedlerstraße 42, 01307 Dresden, Germany.

ABSTRACT
Sulfated glycosaminoglycans (GAG) are components of the bone marrow stem cell niche and to a minor extent of mature bone tissue with important functions in regulating stem cell lineage commitment and differentiation. We anticipated that artificial extracellular matrices (aECM) composed of collagen I and synthetically oversulfated GAG derivatives affect preferentially the differentiation of osteoblast-precursor cells and early osteoblasts. A set of gradually sulfated chondroitin sulfate and hyaluronan derivatives was used for the preparation of aECM. All these matrices were analysed with human bone marrow stromal cells to identify the most potent aECM and to determine the influence of the degree and position of sulfate groups and the kind of disaccharide units on the osteogenic differentiation. Oversulfated GAG derivatives with a sulfate group at the C-6 position of the N-acetylglycosamine revealed the most pronounced proosteogenic effect as determined by tissue nonspecific alkaline phosphatase activity and calcium deposition. A subset of the aECM was further analysed with different primary osteoblasts and cell lines reflecting different maturation stages to test whether the effect of sulfated GAG derivatives depends on the maturation status of the cells. It was shown that the proosteogenic effect of aECM was most prominent in early osteoblasts.

Show MeSH
Related in: MedlinePlus