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Werner E G Muller - One of the best experts on this subject based on the ideXlab platform.
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polyphosphate as a donor of high energy phosphate for the synthesis of adp and atp
Journal of Cell Science, 2017Co-Authors: Werner E G Muller, Qingling Feng, Heinz C Schroder, Meik Neufurth, Shunfeng Wang, Maria Kokkinopoulou, Xiaohong WangAbstract:Here, we studied the potential role of inorganic polyphosphate (polyP) as an energy source for ADP and ATP formation in the extracellular space. In Saos-2 Cells, we show that matrix vesicles are released into the extracellular space after incubation with polyP. These vesicles contain both alkaline phosphatase (ALP) and adenylate kinase (AK) activities (mediated by ALPL and AK1 enzymes). Both enzymes translocate to the cell membrane in response to polyP. To distinguish the process(es) of AMP and ADP formation during ALP hydrolysis from the ATP generated via the AK reaction, inhibition studies with the AK inhibitor A(5')P5(5')A were performed. We found that ADP formation in the extracellular space occurs after enzymatic ATP synthesis. After exposure to polyP, a significant increase of the ADP level was observed, which is likely to be been catalyzed by ALP. This increase is not due to an intensified ATP release via exocytosis. The ATP level in the extracellular space of Saos-2 Cells is strongly increased in response to polyP, very likely mediated by the AK. We propose that the ALP and AK enzymes are involved in the extracellular ADP and ATP synthesis.
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effect of bioglass on growth and biomineralization of saos 2 Cells in hydrogel after 3d cell bioprinting
PLOS ONE, 2014Co-Authors: Xiaohong Wang, Barbel Diehlseifert, Qingling Feng, Heinz C Schroder, Meik Neufurth, Emad Tolba, Werner E G MullerAbstract:We investigated the effect of bioglass (bioactive glass) on growth and mineralization of bone-related Saos-2 Cells, encapsulated into a printable and biodegradable alginate/gelatine hydrogel. The hydrogel was supplemented either with polyphosphate (polyP), administered as polyP•Ca2+-complex, or silica, or as biosilica that had been enzymatically prepared from ortho-silicate by silicatein. These hydrogels, together with Saos-2 Cells, were bioprinted to computer-designed scaffolds. The results revealed that bioglass (nano)particles, with a size of 55 nm and a molar ratio of SiO2∶CaO∶P2O5 of 55∶40∶5, did not affect the growth of the encapsulated Cells. If silica, biosilica, or polyP•Ca2+-complex is co-added to the cell-containing alginate/gelatin hydrogel the growth behavior of the Cells is not changed. Addition of 5 mg/ml of bioglass particles to this hydrogel significantly enhanced the potency of the entrapped Saos-2 Cells to mineralize. If compared with the extent of the Cells to form mineral deposits in the absence of bioglass, the Cells exposed to bioglass together with 100 µmoles/L polyP•Ca2+-complex increased their mineralization activity from 2.1- to 3.9-fold, or with 50 µmoles/L silica from 1.8- to 2.9-fold, or with 50 µmoles/L biosilica from 2.7- to 4.8-fold or with the two components together (100 µmoles/L polyP•Ca2+-complex and 50 µmoles/L biosilica) from 4.1- to 6.8-fold. Element analysis by EDX spectrometry of the mineral nodules formed by Saos-2 revealed an accumulation of O, P, Ca and C, indicating that the mineral deposits contain, besides Ca-phosphate also Ca-carbonate. The results show that bioglass added to alginate/gelatin hydrogel increases the proliferation and mineralization of bioprinted Saos-2 Cells. We conclude that the development of cell-containing scaffolds consisting of a bioprintable, solid and cell-compatible inner matrix surrounded by a printable hard and flexible outer matrix containing bioglass, provide a suitable strategy for the fabrication of morphogenetically active and biodegradable implants.
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engineering a morphogenetically active hydrogel for bioprinting of bioartificial tissue derived from human osteoblast like saos 2 Cells
Biomaterials, 2014Co-Authors: Meik Neufurth, Barbel Diehlseifert, Qingling Feng, Heinz C Schroder, Xiaohong Wang, Thomas Ziebart, Renate Steffen, Shunfeng Wang, Werner E G MullerAbstract:Abstract Sodium alginate hydrogel, stabilized with gelatin, is a suitable, biologically inert matrix that can be used for encapsulating and 3D bioprinting of bone-related Saos-2 Cells. However, the Cells, embedded in this matrix, remain in a non-proliferating state. Here we show that addition of an overlay onto the bioprinted alginate/gelatine/Saos-2 cell scaffold, consisting of agarose and the calcium salt of polyphosphate [polyP·Ca 2+ -complex], resulted in a marked increase in cell proliferation . In the presence of 100 μ m polyP·Ca2+ -complex, the Cells proliferate with a generation time of approximately 47–55 h. In addition, the hardness of the alginate/gelatin hydrogel substantially increases in the presence of the polymer. The reduced Young's modulus for the alginate/gelatin hydrogel is approximately 13–14 kPa, and this value drops to approximately 0.5 kPa after incubation of the cell containing scaffolds for 5 d. In the presence of 100 μ m polyP·Ca2+-complex, the reduced Young's modulus increases to about 22 kPa. The hardness of the polyP·Ca2+ -complex containing hydrogel remains essentially constant if Cells are absent in the matrix, but it drops to 3.2 kPa after a 5 d incubation period in the presence of Saos-2 Cells, indicating that polyP·Ca 2+-complex becomes metabolized, degraded, by the Cells. The alginate/gelatine-agarose system with polyP·Ca2+ -complex cause a significant increase in the mineralization of the Cells. SEM analyses revealed that the morphology of the mineral nodules formed on the surface of the Cells embedded in the alginate/gelatin hydrogel do not significantly differ from the nodules on Cells growing in monolayer cultures . The newly developed technique, using Cells encapsulated into an alginate/gelatin hydrogel and a secondary layer containing the morphogenetically active, growth promoting polymer polyP·Ca 2+ -complex opens new possibilities for the application of 3D bioprinting in bone tissue engineering .
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biosilica loaded poly ϵ caprolactone nanofibers mats provide a morphogenetically active surface scaffold for the growth and mineralization of the osteoclast related saos 2 Cells
Biotechnology Journal, 2014Co-Authors: Werner E G Muller, Thorben Link, Barbel Diehlseifert, Heinz C Schroder, Emad Tolba, Xiaohong WangAbstract:Bioprinting/3D cell printing procedures for the preparation of scaffolds/implants have the potential to revolutionize regenerative medicine. Besides biocompatibility and biodegradability, the hardness of the scaffold material is of critical importance to allow sufficient mechanical protection and, to the same extent, allow migration, cell–cell, and cell–substrate contact formation of the matrix-embedded Cells. In the present study, we present a strategy to encase a bioprinted, cell-containing, and soft scaffold with an electrospun mat. The electrospun poly(e-caprolactone) (PCL) nanofibers mats, containing tetraethyl orthosilicate (TEOS), were subsequently incubated with silicatein. Silicatein synthesizes polymeric biosilica by polycondensation of ortho-silicate that is formed from prehydrolyzed TEOS. Biosilica provides a morphogenetically active matrix for the growth and mineralization of osteoblast-related Saos-2 Cells in vitro. Analysis of the microstructure of the 300–700 nm thick PCL/TEOS nanofibers, incubated with silicatein and prehydrolyzed TEOS, displayed biosilica deposits on the mats formed by the nanofibers. We conclude and propose that electrospun PCL nanofibers mats, coated with biosilica, may represent a morphogenetically active and protective cover for bioprinted cell/tissue-like units with a suitable mechanical stability, even if the Cells are embedded in a softer matrix.
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induction of carbonic anhydrase in saos 2 Cells exposed to bicarbonate and consequences for calcium phosphate crystal formation
Biomaterials, 2013Co-Authors: Werner E G Muller, Vladislav A Grebenjuk, Heinz C Schroder, Hiroshi Ushijima, Ute Schlossmacher, Xiaohong WangAbstract:Ca-phosphate/hydroxyapatite crystals constitute the mineralic matrix of vertebrate bones, while Ca-carbonate dominates the inorganic matrix of otoliths. In addition, Ca-carbonate has been identified in lower percentage in apatite crystals. By using the human osteogenic Saos-2 Cells it could be shown that after exposure of the Cells to Ca-bicarbonate in vitro, at concentrations between 1 and 10 mm, a significant increase of Ca-deposit formation results. The crystallite nodules formed on the surfaces of Saos-2 Cells become denser and larger in the presence of bicarbonate if simultaneously added together with the mineralization activation cocktail (β-glycerophosphate/ascorbic acid/dexamethasone). In parallel, with the increase of Ca-deposit formation, the expression of the carbonic anhydrase-II (CA-II) gene becomes upregulated. This effect, measured on transcriptional level is also substantiated by immunohistological studies. The stimulatory effect of bicarbonate on Ca-deposit formation is prevented if the carbonic anhydrase inhibitor acetazolamide is added to the cultures. Mapping the surface of the Ca-deposit producing Saos-2 Cells by scanning electron microscopy coupled with energy-dispersive X-ray analysis revealed an accumulation of the signals for the element carbon and, as expected, also for phosphorus. Finally, it is shown that ortho-phosphate and hydrolysis products of polyphosphate inhibit CA-II activity, suggesting a feedback regulatory system between the CA-driven Ca-carbonate deposition and a subsequent inactivation of this process by ortho-phosphate. Based on the presented data we suggest that Ca-carbonate deposits act as bioseeds for a downstream Ca-phosphate deposition process. We propose that activators for CA, especially for CA-II, might be beneficial for the treatment of bone deficiency diseases.
Xiaohong Wang - One of the best experts on this subject based on the ideXlab platform.
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polyphosphate as a donor of high energy phosphate for the synthesis of adp and atp
Journal of Cell Science, 2017Co-Authors: Werner E G Muller, Qingling Feng, Heinz C Schroder, Meik Neufurth, Shunfeng Wang, Maria Kokkinopoulou, Xiaohong WangAbstract:Here, we studied the potential role of inorganic polyphosphate (polyP) as an energy source for ADP and ATP formation in the extracellular space. In Saos-2 Cells, we show that matrix vesicles are released into the extracellular space after incubation with polyP. These vesicles contain both alkaline phosphatase (ALP) and adenylate kinase (AK) activities (mediated by ALPL and AK1 enzymes). Both enzymes translocate to the cell membrane in response to polyP. To distinguish the process(es) of AMP and ADP formation during ALP hydrolysis from the ATP generated via the AK reaction, inhibition studies with the AK inhibitor A(5')P5(5')A were performed. We found that ADP formation in the extracellular space occurs after enzymatic ATP synthesis. After exposure to polyP, a significant increase of the ADP level was observed, which is likely to be been catalyzed by ALP. This increase is not due to an intensified ATP release via exocytosis. The ATP level in the extracellular space of Saos-2 Cells is strongly increased in response to polyP, very likely mediated by the AK. We propose that the ALP and AK enzymes are involved in the extracellular ADP and ATP synthesis.
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effect of bioglass on growth and biomineralization of saos 2 Cells in hydrogel after 3d cell bioprinting
PLOS ONE, 2014Co-Authors: Xiaohong Wang, Barbel Diehlseifert, Qingling Feng, Heinz C Schroder, Meik Neufurth, Emad Tolba, Werner E G MullerAbstract:We investigated the effect of bioglass (bioactive glass) on growth and mineralization of bone-related Saos-2 Cells, encapsulated into a printable and biodegradable alginate/gelatine hydrogel. The hydrogel was supplemented either with polyphosphate (polyP), administered as polyP•Ca2+-complex, or silica, or as biosilica that had been enzymatically prepared from ortho-silicate by silicatein. These hydrogels, together with Saos-2 Cells, were bioprinted to computer-designed scaffolds. The results revealed that bioglass (nano)particles, with a size of 55 nm and a molar ratio of SiO2∶CaO∶P2O5 of 55∶40∶5, did not affect the growth of the encapsulated Cells. If silica, biosilica, or polyP•Ca2+-complex is co-added to the cell-containing alginate/gelatin hydrogel the growth behavior of the Cells is not changed. Addition of 5 mg/ml of bioglass particles to this hydrogel significantly enhanced the potency of the entrapped Saos-2 Cells to mineralize. If compared with the extent of the Cells to form mineral deposits in the absence of bioglass, the Cells exposed to bioglass together with 100 µmoles/L polyP•Ca2+-complex increased their mineralization activity from 2.1- to 3.9-fold, or with 50 µmoles/L silica from 1.8- to 2.9-fold, or with 50 µmoles/L biosilica from 2.7- to 4.8-fold or with the two components together (100 µmoles/L polyP•Ca2+-complex and 50 µmoles/L biosilica) from 4.1- to 6.8-fold. Element analysis by EDX spectrometry of the mineral nodules formed by Saos-2 revealed an accumulation of O, P, Ca and C, indicating that the mineral deposits contain, besides Ca-phosphate also Ca-carbonate. The results show that bioglass added to alginate/gelatin hydrogel increases the proliferation and mineralization of bioprinted Saos-2 Cells. We conclude that the development of cell-containing scaffolds consisting of a bioprintable, solid and cell-compatible inner matrix surrounded by a printable hard and flexible outer matrix containing bioglass, provide a suitable strategy for the fabrication of morphogenetically active and biodegradable implants.
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engineering a morphogenetically active hydrogel for bioprinting of bioartificial tissue derived from human osteoblast like saos 2 Cells
Biomaterials, 2014Co-Authors: Meik Neufurth, Barbel Diehlseifert, Qingling Feng, Heinz C Schroder, Xiaohong Wang, Thomas Ziebart, Renate Steffen, Shunfeng Wang, Werner E G MullerAbstract:Abstract Sodium alginate hydrogel, stabilized with gelatin, is a suitable, biologically inert matrix that can be used for encapsulating and 3D bioprinting of bone-related Saos-2 Cells. However, the Cells, embedded in this matrix, remain in a non-proliferating state. Here we show that addition of an overlay onto the bioprinted alginate/gelatine/Saos-2 cell scaffold, consisting of agarose and the calcium salt of polyphosphate [polyP·Ca 2+ -complex], resulted in a marked increase in cell proliferation . In the presence of 100 μ m polyP·Ca2+ -complex, the Cells proliferate with a generation time of approximately 47–55 h. In addition, the hardness of the alginate/gelatin hydrogel substantially increases in the presence of the polymer. The reduced Young's modulus for the alginate/gelatin hydrogel is approximately 13–14 kPa, and this value drops to approximately 0.5 kPa after incubation of the cell containing scaffolds for 5 d. In the presence of 100 μ m polyP·Ca2+-complex, the reduced Young's modulus increases to about 22 kPa. The hardness of the polyP·Ca2+ -complex containing hydrogel remains essentially constant if Cells are absent in the matrix, but it drops to 3.2 kPa after a 5 d incubation period in the presence of Saos-2 Cells, indicating that polyP·Ca 2+-complex becomes metabolized, degraded, by the Cells. The alginate/gelatine-agarose system with polyP·Ca2+ -complex cause a significant increase in the mineralization of the Cells. SEM analyses revealed that the morphology of the mineral nodules formed on the surface of the Cells embedded in the alginate/gelatin hydrogel do not significantly differ from the nodules on Cells growing in monolayer cultures . The newly developed technique, using Cells encapsulated into an alginate/gelatin hydrogel and a secondary layer containing the morphogenetically active, growth promoting polymer polyP·Ca 2+ -complex opens new possibilities for the application of 3D bioprinting in bone tissue engineering .
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biosilica loaded poly ϵ caprolactone nanofibers mats provide a morphogenetically active surface scaffold for the growth and mineralization of the osteoclast related saos 2 Cells
Biotechnology Journal, 2014Co-Authors: Werner E G Muller, Thorben Link, Barbel Diehlseifert, Heinz C Schroder, Emad Tolba, Xiaohong WangAbstract:Bioprinting/3D cell printing procedures for the preparation of scaffolds/implants have the potential to revolutionize regenerative medicine. Besides biocompatibility and biodegradability, the hardness of the scaffold material is of critical importance to allow sufficient mechanical protection and, to the same extent, allow migration, cell–cell, and cell–substrate contact formation of the matrix-embedded Cells. In the present study, we present a strategy to encase a bioprinted, cell-containing, and soft scaffold with an electrospun mat. The electrospun poly(e-caprolactone) (PCL) nanofibers mats, containing tetraethyl orthosilicate (TEOS), were subsequently incubated with silicatein. Silicatein synthesizes polymeric biosilica by polycondensation of ortho-silicate that is formed from prehydrolyzed TEOS. Biosilica provides a morphogenetically active matrix for the growth and mineralization of osteoblast-related Saos-2 Cells in vitro. Analysis of the microstructure of the 300–700 nm thick PCL/TEOS nanofibers, incubated with silicatein and prehydrolyzed TEOS, displayed biosilica deposits on the mats formed by the nanofibers. We conclude and propose that electrospun PCL nanofibers mats, coated with biosilica, may represent a morphogenetically active and protective cover for bioprinted cell/tissue-like units with a suitable mechanical stability, even if the Cells are embedded in a softer matrix.
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isoquercitrin and polyphosphate co enhance mineralization of human osteoblast like saos 2 Cells via separate activation of two runx2 cofactors aft6 and ets1
Biochemical Pharmacology, 2014Co-Authors: Xiaohong Wang, Qingling Feng, Barbel Diehlseifert, Heinz C Schroder, Vladislav A GrebenjukAbstract:Abstract Isoquercitrin, a dietary phytoestrogen, is a potential stimulator of bone mineralization used for prophylaxis of osteoporotic disorders. Here we studied the combined effects of isoquercitrin, a cell membrane permeable 3- O -glucoside of quercetin, and polyphosphate [polyP], a naturally occurring inorganic polymer inducing bone formation, on mineralization of human osteoblast-like Saos-2 Cells. Both compounds isoquercitrin and polyP induce at non-toxic concentrations the mineralization process of Saos-2 Cells. Co-incubation experiments revealed that isoquercitrin (at 0.1 and 0.3 μM), if given simultaneously with polyP (as Ca 2+ salt; at 3, 10, 30 and 100 μM) amplifies the mineralization-enhancing effect of the inorganic polymer. The biomineralization process induced by isoquercitrin and polyP is based on two different modes of action. After incubation of the Cells with isoquercitrin or polyP the expression of the Runt-related transcription factor 2 [RUNX2] is significantly upregulated. In addition, isoquercitrin causes a strong increase of the steady-state-levels of the two co-activators of RUNX2 , the activating transcription factor 6 [ATF6] and the Ets oncogene homolog 1 [Ets1]. The activating effect of isoquercitrin occurs via a signal transduction pathway involving ATF6, and by that, is independent from the induction cascade initiated by polyP. This conclusion is supported by the finding that isoquercitrin upregulates the expression of the gene encoding for osteocalcin , while polyP strongly increases the expression of the Ets1 gene and of the alkaline phosphatase . We show that the two compounds, polyP and isoquercitrin, have a co-enhancing effect on bone mineral formation and in turn might be of potential therapeutic value for prevention/treatment of osteoporosis.
Heinz C Schroder - One of the best experts on this subject based on the ideXlab platform.
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polyphosphate as a donor of high energy phosphate for the synthesis of adp and atp
Journal of Cell Science, 2017Co-Authors: Werner E G Muller, Qingling Feng, Heinz C Schroder, Meik Neufurth, Shunfeng Wang, Maria Kokkinopoulou, Xiaohong WangAbstract:Here, we studied the potential role of inorganic polyphosphate (polyP) as an energy source for ADP and ATP formation in the extracellular space. In Saos-2 Cells, we show that matrix vesicles are released into the extracellular space after incubation with polyP. These vesicles contain both alkaline phosphatase (ALP) and adenylate kinase (AK) activities (mediated by ALPL and AK1 enzymes). Both enzymes translocate to the cell membrane in response to polyP. To distinguish the process(es) of AMP and ADP formation during ALP hydrolysis from the ATP generated via the AK reaction, inhibition studies with the AK inhibitor A(5')P5(5')A were performed. We found that ADP formation in the extracellular space occurs after enzymatic ATP synthesis. After exposure to polyP, a significant increase of the ADP level was observed, which is likely to be been catalyzed by ALP. This increase is not due to an intensified ATP release via exocytosis. The ATP level in the extracellular space of Saos-2 Cells is strongly increased in response to polyP, very likely mediated by the AK. We propose that the ALP and AK enzymes are involved in the extracellular ADP and ATP synthesis.
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effect of bioglass on growth and biomineralization of saos 2 Cells in hydrogel after 3d cell bioprinting
PLOS ONE, 2014Co-Authors: Xiaohong Wang, Barbel Diehlseifert, Qingling Feng, Heinz C Schroder, Meik Neufurth, Emad Tolba, Werner E G MullerAbstract:We investigated the effect of bioglass (bioactive glass) on growth and mineralization of bone-related Saos-2 Cells, encapsulated into a printable and biodegradable alginate/gelatine hydrogel. The hydrogel was supplemented either with polyphosphate (polyP), administered as polyP•Ca2+-complex, or silica, or as biosilica that had been enzymatically prepared from ortho-silicate by silicatein. These hydrogels, together with Saos-2 Cells, were bioprinted to computer-designed scaffolds. The results revealed that bioglass (nano)particles, with a size of 55 nm and a molar ratio of SiO2∶CaO∶P2O5 of 55∶40∶5, did not affect the growth of the encapsulated Cells. If silica, biosilica, or polyP•Ca2+-complex is co-added to the cell-containing alginate/gelatin hydrogel the growth behavior of the Cells is not changed. Addition of 5 mg/ml of bioglass particles to this hydrogel significantly enhanced the potency of the entrapped Saos-2 Cells to mineralize. If compared with the extent of the Cells to form mineral deposits in the absence of bioglass, the Cells exposed to bioglass together with 100 µmoles/L polyP•Ca2+-complex increased their mineralization activity from 2.1- to 3.9-fold, or with 50 µmoles/L silica from 1.8- to 2.9-fold, or with 50 µmoles/L biosilica from 2.7- to 4.8-fold or with the two components together (100 µmoles/L polyP•Ca2+-complex and 50 µmoles/L biosilica) from 4.1- to 6.8-fold. Element analysis by EDX spectrometry of the mineral nodules formed by Saos-2 revealed an accumulation of O, P, Ca and C, indicating that the mineral deposits contain, besides Ca-phosphate also Ca-carbonate. The results show that bioglass added to alginate/gelatin hydrogel increases the proliferation and mineralization of bioprinted Saos-2 Cells. We conclude that the development of cell-containing scaffolds consisting of a bioprintable, solid and cell-compatible inner matrix surrounded by a printable hard and flexible outer matrix containing bioglass, provide a suitable strategy for the fabrication of morphogenetically active and biodegradable implants.
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engineering a morphogenetically active hydrogel for bioprinting of bioartificial tissue derived from human osteoblast like saos 2 Cells
Biomaterials, 2014Co-Authors: Meik Neufurth, Barbel Diehlseifert, Qingling Feng, Heinz C Schroder, Xiaohong Wang, Thomas Ziebart, Renate Steffen, Shunfeng Wang, Werner E G MullerAbstract:Abstract Sodium alginate hydrogel, stabilized with gelatin, is a suitable, biologically inert matrix that can be used for encapsulating and 3D bioprinting of bone-related Saos-2 Cells. However, the Cells, embedded in this matrix, remain in a non-proliferating state. Here we show that addition of an overlay onto the bioprinted alginate/gelatine/Saos-2 cell scaffold, consisting of agarose and the calcium salt of polyphosphate [polyP·Ca 2+ -complex], resulted in a marked increase in cell proliferation . In the presence of 100 μ m polyP·Ca2+ -complex, the Cells proliferate with a generation time of approximately 47–55 h. In addition, the hardness of the alginate/gelatin hydrogel substantially increases in the presence of the polymer. The reduced Young's modulus for the alginate/gelatin hydrogel is approximately 13–14 kPa, and this value drops to approximately 0.5 kPa after incubation of the cell containing scaffolds for 5 d. In the presence of 100 μ m polyP·Ca2+-complex, the reduced Young's modulus increases to about 22 kPa. The hardness of the polyP·Ca2+ -complex containing hydrogel remains essentially constant if Cells are absent in the matrix, but it drops to 3.2 kPa after a 5 d incubation period in the presence of Saos-2 Cells, indicating that polyP·Ca 2+-complex becomes metabolized, degraded, by the Cells. The alginate/gelatine-agarose system with polyP·Ca2+ -complex cause a significant increase in the mineralization of the Cells. SEM analyses revealed that the morphology of the mineral nodules formed on the surface of the Cells embedded in the alginate/gelatin hydrogel do not significantly differ from the nodules on Cells growing in monolayer cultures . The newly developed technique, using Cells encapsulated into an alginate/gelatin hydrogel and a secondary layer containing the morphogenetically active, growth promoting polymer polyP·Ca 2+ -complex opens new possibilities for the application of 3D bioprinting in bone tissue engineering .
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biosilica loaded poly ϵ caprolactone nanofibers mats provide a morphogenetically active surface scaffold for the growth and mineralization of the osteoclast related saos 2 Cells
Biotechnology Journal, 2014Co-Authors: Werner E G Muller, Thorben Link, Barbel Diehlseifert, Heinz C Schroder, Emad Tolba, Xiaohong WangAbstract:Bioprinting/3D cell printing procedures for the preparation of scaffolds/implants have the potential to revolutionize regenerative medicine. Besides biocompatibility and biodegradability, the hardness of the scaffold material is of critical importance to allow sufficient mechanical protection and, to the same extent, allow migration, cell–cell, and cell–substrate contact formation of the matrix-embedded Cells. In the present study, we present a strategy to encase a bioprinted, cell-containing, and soft scaffold with an electrospun mat. The electrospun poly(e-caprolactone) (PCL) nanofibers mats, containing tetraethyl orthosilicate (TEOS), were subsequently incubated with silicatein. Silicatein synthesizes polymeric biosilica by polycondensation of ortho-silicate that is formed from prehydrolyzed TEOS. Biosilica provides a morphogenetically active matrix for the growth and mineralization of osteoblast-related Saos-2 Cells in vitro. Analysis of the microstructure of the 300–700 nm thick PCL/TEOS nanofibers, incubated with silicatein and prehydrolyzed TEOS, displayed biosilica deposits on the mats formed by the nanofibers. We conclude and propose that electrospun PCL nanofibers mats, coated with biosilica, may represent a morphogenetically active and protective cover for bioprinted cell/tissue-like units with a suitable mechanical stability, even if the Cells are embedded in a softer matrix.
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isoquercitrin and polyphosphate co enhance mineralization of human osteoblast like saos 2 Cells via separate activation of two runx2 cofactors aft6 and ets1
Biochemical Pharmacology, 2014Co-Authors: Xiaohong Wang, Qingling Feng, Barbel Diehlseifert, Heinz C Schroder, Vladislav A GrebenjukAbstract:Abstract Isoquercitrin, a dietary phytoestrogen, is a potential stimulator of bone mineralization used for prophylaxis of osteoporotic disorders. Here we studied the combined effects of isoquercitrin, a cell membrane permeable 3- O -glucoside of quercetin, and polyphosphate [polyP], a naturally occurring inorganic polymer inducing bone formation, on mineralization of human osteoblast-like Saos-2 Cells. Both compounds isoquercitrin and polyP induce at non-toxic concentrations the mineralization process of Saos-2 Cells. Co-incubation experiments revealed that isoquercitrin (at 0.1 and 0.3 μM), if given simultaneously with polyP (as Ca 2+ salt; at 3, 10, 30 and 100 μM) amplifies the mineralization-enhancing effect of the inorganic polymer. The biomineralization process induced by isoquercitrin and polyP is based on two different modes of action. After incubation of the Cells with isoquercitrin or polyP the expression of the Runt-related transcription factor 2 [RUNX2] is significantly upregulated. In addition, isoquercitrin causes a strong increase of the steady-state-levels of the two co-activators of RUNX2 , the activating transcription factor 6 [ATF6] and the Ets oncogene homolog 1 [Ets1]. The activating effect of isoquercitrin occurs via a signal transduction pathway involving ATF6, and by that, is independent from the induction cascade initiated by polyP. This conclusion is supported by the finding that isoquercitrin upregulates the expression of the gene encoding for osteocalcin , while polyP strongly increases the expression of the Ets1 gene and of the alkaline phosphatase . We show that the two compounds, polyP and isoquercitrin, have a co-enhancing effect on bone mineral formation and in turn might be of potential therapeutic value for prevention/treatment of osteoporosis.
Barbel Diehlseifert - One of the best experts on this subject based on the ideXlab platform.
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effect of bioglass on growth and biomineralization of saos 2 Cells in hydrogel after 3d cell bioprinting
PLOS ONE, 2014Co-Authors: Xiaohong Wang, Barbel Diehlseifert, Qingling Feng, Heinz C Schroder, Meik Neufurth, Emad Tolba, Werner E G MullerAbstract:We investigated the effect of bioglass (bioactive glass) on growth and mineralization of bone-related Saos-2 Cells, encapsulated into a printable and biodegradable alginate/gelatine hydrogel. The hydrogel was supplemented either with polyphosphate (polyP), administered as polyP•Ca2+-complex, or silica, or as biosilica that had been enzymatically prepared from ortho-silicate by silicatein. These hydrogels, together with Saos-2 Cells, were bioprinted to computer-designed scaffolds. The results revealed that bioglass (nano)particles, with a size of 55 nm and a molar ratio of SiO2∶CaO∶P2O5 of 55∶40∶5, did not affect the growth of the encapsulated Cells. If silica, biosilica, or polyP•Ca2+-complex is co-added to the cell-containing alginate/gelatin hydrogel the growth behavior of the Cells is not changed. Addition of 5 mg/ml of bioglass particles to this hydrogel significantly enhanced the potency of the entrapped Saos-2 Cells to mineralize. If compared with the extent of the Cells to form mineral deposits in the absence of bioglass, the Cells exposed to bioglass together with 100 µmoles/L polyP•Ca2+-complex increased their mineralization activity from 2.1- to 3.9-fold, or with 50 µmoles/L silica from 1.8- to 2.9-fold, or with 50 µmoles/L biosilica from 2.7- to 4.8-fold or with the two components together (100 µmoles/L polyP•Ca2+-complex and 50 µmoles/L biosilica) from 4.1- to 6.8-fold. Element analysis by EDX spectrometry of the mineral nodules formed by Saos-2 revealed an accumulation of O, P, Ca and C, indicating that the mineral deposits contain, besides Ca-phosphate also Ca-carbonate. The results show that bioglass added to alginate/gelatin hydrogel increases the proliferation and mineralization of bioprinted Saos-2 Cells. We conclude that the development of cell-containing scaffolds consisting of a bioprintable, solid and cell-compatible inner matrix surrounded by a printable hard and flexible outer matrix containing bioglass, provide a suitable strategy for the fabrication of morphogenetically active and biodegradable implants.
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engineering a morphogenetically active hydrogel for bioprinting of bioartificial tissue derived from human osteoblast like saos 2 Cells
Biomaterials, 2014Co-Authors: Meik Neufurth, Barbel Diehlseifert, Qingling Feng, Heinz C Schroder, Xiaohong Wang, Thomas Ziebart, Renate Steffen, Shunfeng Wang, Werner E G MullerAbstract:Abstract Sodium alginate hydrogel, stabilized with gelatin, is a suitable, biologically inert matrix that can be used for encapsulating and 3D bioprinting of bone-related Saos-2 Cells. However, the Cells, embedded in this matrix, remain in a non-proliferating state. Here we show that addition of an overlay onto the bioprinted alginate/gelatine/Saos-2 cell scaffold, consisting of agarose and the calcium salt of polyphosphate [polyP·Ca 2+ -complex], resulted in a marked increase in cell proliferation . In the presence of 100 μ m polyP·Ca2+ -complex, the Cells proliferate with a generation time of approximately 47–55 h. In addition, the hardness of the alginate/gelatin hydrogel substantially increases in the presence of the polymer. The reduced Young's modulus for the alginate/gelatin hydrogel is approximately 13–14 kPa, and this value drops to approximately 0.5 kPa after incubation of the cell containing scaffolds for 5 d. In the presence of 100 μ m polyP·Ca2+-complex, the reduced Young's modulus increases to about 22 kPa. The hardness of the polyP·Ca2+ -complex containing hydrogel remains essentially constant if Cells are absent in the matrix, but it drops to 3.2 kPa after a 5 d incubation period in the presence of Saos-2 Cells, indicating that polyP·Ca 2+-complex becomes metabolized, degraded, by the Cells. The alginate/gelatine-agarose system with polyP·Ca2+ -complex cause a significant increase in the mineralization of the Cells. SEM analyses revealed that the morphology of the mineral nodules formed on the surface of the Cells embedded in the alginate/gelatin hydrogel do not significantly differ from the nodules on Cells growing in monolayer cultures . The newly developed technique, using Cells encapsulated into an alginate/gelatin hydrogel and a secondary layer containing the morphogenetically active, growth promoting polymer polyP·Ca 2+ -complex opens new possibilities for the application of 3D bioprinting in bone tissue engineering .
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biosilica loaded poly ϵ caprolactone nanofibers mats provide a morphogenetically active surface scaffold for the growth and mineralization of the osteoclast related saos 2 Cells
Biotechnology Journal, 2014Co-Authors: Werner E G Muller, Thorben Link, Barbel Diehlseifert, Heinz C Schroder, Emad Tolba, Xiaohong WangAbstract:Bioprinting/3D cell printing procedures for the preparation of scaffolds/implants have the potential to revolutionize regenerative medicine. Besides biocompatibility and biodegradability, the hardness of the scaffold material is of critical importance to allow sufficient mechanical protection and, to the same extent, allow migration, cell–cell, and cell–substrate contact formation of the matrix-embedded Cells. In the present study, we present a strategy to encase a bioprinted, cell-containing, and soft scaffold with an electrospun mat. The electrospun poly(e-caprolactone) (PCL) nanofibers mats, containing tetraethyl orthosilicate (TEOS), were subsequently incubated with silicatein. Silicatein synthesizes polymeric biosilica by polycondensation of ortho-silicate that is formed from prehydrolyzed TEOS. Biosilica provides a morphogenetically active matrix for the growth and mineralization of osteoblast-related Saos-2 Cells in vitro. Analysis of the microstructure of the 300–700 nm thick PCL/TEOS nanofibers, incubated with silicatein and prehydrolyzed TEOS, displayed biosilica deposits on the mats formed by the nanofibers. We conclude and propose that electrospun PCL nanofibers mats, coated with biosilica, may represent a morphogenetically active and protective cover for bioprinted cell/tissue-like units with a suitable mechanical stability, even if the Cells are embedded in a softer matrix.
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isoquercitrin and polyphosphate co enhance mineralization of human osteoblast like saos 2 Cells via separate activation of two runx2 cofactors aft6 and ets1
Biochemical Pharmacology, 2014Co-Authors: Xiaohong Wang, Qingling Feng, Barbel Diehlseifert, Heinz C Schroder, Vladislav A GrebenjukAbstract:Abstract Isoquercitrin, a dietary phytoestrogen, is a potential stimulator of bone mineralization used for prophylaxis of osteoporotic disorders. Here we studied the combined effects of isoquercitrin, a cell membrane permeable 3- O -glucoside of quercetin, and polyphosphate [polyP], a naturally occurring inorganic polymer inducing bone formation, on mineralization of human osteoblast-like Saos-2 Cells. Both compounds isoquercitrin and polyP induce at non-toxic concentrations the mineralization process of Saos-2 Cells. Co-incubation experiments revealed that isoquercitrin (at 0.1 and 0.3 μM), if given simultaneously with polyP (as Ca 2+ salt; at 3, 10, 30 and 100 μM) amplifies the mineralization-enhancing effect of the inorganic polymer. The biomineralization process induced by isoquercitrin and polyP is based on two different modes of action. After incubation of the Cells with isoquercitrin or polyP the expression of the Runt-related transcription factor 2 [RUNX2] is significantly upregulated. In addition, isoquercitrin causes a strong increase of the steady-state-levels of the two co-activators of RUNX2 , the activating transcription factor 6 [ATF6] and the Ets oncogene homolog 1 [Ets1]. The activating effect of isoquercitrin occurs via a signal transduction pathway involving ATF6, and by that, is independent from the induction cascade initiated by polyP. This conclusion is supported by the finding that isoquercitrin upregulates the expression of the gene encoding for osteocalcin , while polyP strongly increases the expression of the Ets1 gene and of the alkaline phosphatase . We show that the two compounds, polyP and isoquercitrin, have a co-enhancing effect on bone mineral formation and in turn might be of potential therapeutic value for prevention/treatment of osteoporosis.
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dual effect of inorganic polymeric phosphate polyphosphate on osteoblasts and osteoclasts in vitro
Journal of Tissue Engineering and Regenerative Medicine, 2012Co-Authors: Xiaohong Wang, Barbel Diehlseifert, Heinz C Schroder, Matthias Wiens, Klaus Kropf, Ute Schlossmacher, Werner E G MullerAbstract:Inorganic polymeric phosphate/polyphosphate (polyP) is a natural polymer existing in both pro- and eukaryotic systems. In the present study the effect of polyP as well as of polyP supplied in a stoichiometric ratio of 2 m polyP:1 m CaCl2 [polyP (Ca2+ complex)] on the osteoblast-like Saos-2 Cells and the osteoclast-like RAW 264.7 Cells was determined. Both polymers are non-toxic for these Cells up to a concentration of 100 µm. In contrast to polyP, polyP (Ca2+ complex) significantly induced hydroxyapatite formation at a concentration > 10 µm, as documented by alizarin red S staining and scanning electron microscopic (SEM) inspection. Furthermore, polyP (Ca2+ complex) triggered in Saos-2 Cells transcription of BMP2 (bone morphogenetic protein 2), a cytokine involved in maturation of hydroxyapatite-forming Cells. An additional activity of polyP (Ca2+ complex) is described by showing that this polymer impairs osteoclastogenesis. At concentrations > 10 µm polyP (Ca2+ complex) slows down the progression of RAW 264.7 Cells to functional osteoclasts, as measured by the expression of TRAP (tartrate-resistant acid phosphatase). Finally, it is shown that 10–100 µm polyP (Ca2+ complex) inhibited phosphorylation of IκBα by the respective kinase in RAW 264.7 Cells. We concluded that polyP (Ca2+ complex) displays a dual effect on bone metabolizing Cells. It promotes hydroxyapatite formation in Saos-2 Cells (osteoblasts) and impairs maturation of the osteoclast-related RAW 264.7 Cells. Copyright © 2012 John Wiley & Sons, Ltd.
Giovanni Magenes - One of the best experts on this subject based on the ideXlab platform.
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effects of electromagnetic stimulation on calcified matrix production by saos 2 Cells over a polyurethane porous scaffold
Tissue Engineering, 2006Co-Authors: Lorenzo Fassina, Livia Visai, Francesco Benazzo, Laura Benedetti, Alberto Calligaro, Maria Gabriella Cusella De Angelis, Aurora Farina, Valentina Maliardi, Giovanni MagenesAbstract:There is increasing interest in designing new biomaterials that could potentially be used in the form of scaffolds as bone substitutes. In this study we used a hydrophobic crosslinked polyurethane in a typical tissue-engineering approach, that is, the seeding and in vitro culturing of Cells using a porous scaffold. Using an electromagnetic bioreactor (magnetic field intensity, 2 mT; frequency, 75 Hz), we investigated the effect of the electromagnetic stimulation on Saos-2 human osteoblast proliferation and calcified matrix production. Cell proliferation was twice as high; expression of decorin, osteocalcin, osteopontin, type I collagen, and type III collagen was greater (1.3, 12.2, 12.1, 10.0, and 10.5 times as great, respectively); and calcium deposition was 5 times as great as under static conditions without electromagnetic stimulation. RT-PCR analysis revealed the electromagnetically upregulated transcription specific for decorin, fibronectin, osteocalcin, osteopontin, transforming growth factor-beta, type I collagen, and type III collagen. The immunolocalization of the extracellular matrix constituents showed their colocalization in the cell-rich areas. The bioreactor and the polyurethane foam were designed to obtain cell colonization and calcified matrix deposition. This cultured biomaterial could be used, in clinical applications, as an osteoinductive implant for bone repair.
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calcified matrix production by saos 2 Cells inside a polyurethane porous scaffold using a perfusion bioreactor
Tissue Engineering, 2005Co-Authors: Lorenzo Fassina, Livia Visai, L Asti, Francesco Benazzo, Pietro Speziale, M C Tanzi, Giovanni MagenesAbstract:The repair and regeneration of damaged or resected bone are problematic. Bone autografts show optimal skeletal incorporation, but often bring about complications. Hence, there is increasing interest in designing new biomaterials that could potentially be used in the form of scaffolds as bone substitutes. In this study we used a hydrophobic cross-linked polyurethane in a typical tissue-engineering approach, that is, the seeding and in vitro culturing of Cells within a porous scaffold. The polyurethane porous scaffold had an average pore diameter of 624 µm. Using a perfusion bioreactor, we investigated the effect of shear stress on Saos-2 human osteoblast proliferation and calcified matrix production. The physical, morphological, and compressive properties of the polyurethane foam were characterized. At a scaffold perfusion rate of 3 mL/min, in comparison with static conditions without perfusion, we observed 33% higher cell proliferation; higher secretion of osteopontin, osteocalcin, decorin, and type I col...
