Mineralization was induced on xerogel–HOB® constructs by the addition of Osteoblast Mineralization Medium (Promocell, Heidelberg, Germany) for at least 21 days. HOB® cultured with Osteoblast Growth Medium (Promocell, Heidelberg, Germany) was used as negative control, and cells grown with Osteoblast Mineralization Medium (Promocell, Heidelberg, Germany) were used as positive mineralization control. Media were changed three times per week, and cell cultures were incubated at 37 °C and 5% CO2.
Osteoblast mineralization medium
Manufactured by PromoCell
Sourced in Germany
Osteoblast mineralization medium is a specialized cell culture medium designed to support the differentiation and mineralization of osteoblasts, which are bone-forming cells. The medium provides the necessary nutrients and supplements to facilitate the process of bone matrix deposition and mineralization by osteoblasts.
Automatically generated - may contain errors
Lab products found in correlation
Osteoblast growth medium, by PromoCell (3 mentions)
Collagenase type ia, by Merck Group (2 mentions)
α mem medium, by PAN Biotech (2 mentions)
Alizarin red stain, by Merck Group (2 mentions)
Dispase 2, by Roche (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Alizarin red s, by ScienCell (1 mentions)
Collagenase, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg microbeads, by Miltenyi Biotec (1 mentions)
Dispase 2, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
β glycerol phosphate, by Merck Group (1 mentions)
Tetraethyl orthosilicate, by Thermo Fisher Scientific (1 mentions)
Absolute ethanol, by ITW Reagents (1 mentions)
Hob human osteoblasts, by PromoCell (1 mentions)
Osteoblast growing medium, by PromoCell (1 mentions)
Alizarin red staining solution, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
8 protocols using osteoblast mineralization medium
1
Mineralization of Xerogel-HOB Constructs
2
Culturing Mouse Fibroblasts and Human Osteoblasts
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The mouse fibroblast cell line L-929 was purchased from Sigma-Aldrich (St. Louis, Missouri, USA) and was cultured in MEM (Minimum Essential Medium) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Inc., Waltham, MA, USA), penicillin/streptomycin (100 U/mL each) and L-glutamine in a final concentration of 4 mM. The human osteoblasts were obtained from PromoCell (Heidelberg, Germany) and were grown in osteoblast growth medium (PromoCell, Heidelberg, Germany) without additional supplements. Cells were passaged when they reached about 80% confluence. Mineralization was performed by incubation with osteoblast mineralization medium (PromoCell, Heidelberg, Germany).
3
Quantification of Osteoblast Mineralization
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Cells were shifted to Osteoblast Mineralization Medium (PromoCell, Heidelberg, Germany) for 14 or 21 days, with medium change every 3 days to induce calcification. VSMC were washed three times with PBS and calcium deposition was visualized and quantified by Alizarin Red S (ARS staining quantification assay, Sciencell Research Laboratories, San Diego, CA, USA) as recommended by the manufacturer. Briefly, cells were fixed in 4% formaldehyde for 15 min. at room temperature and washed three times with distilled H2O. One ml of 40 mM ARS was added per well and incubated at room temperature for 30 min. With shaking. After washing of the cells five times with distilled H2O, images were taken using a microscope. Plates were stored at − 20 °C prior to dye extraction. For quantification, 800 μl of 10% acetic acid was added to each well of the 6-well plate for 30 min. Cells were collected using a cell scraper and transferred to a microcentrifuge tube. After vortexing for 30 s, samples were heated at 85 °C for 10 min. Samples were incubated on ice for 5 min and centrifuged at 20000 g for 15 min. Supernatants were transferred to a new tube and 200 μl of 10% ammonium hydroxide was added. Absorbance of the samples was read at 405 nm with a plate reader and plotted against an ARS standard curve to determine the concentration.
4
Isolation and Expansion of Primary Osteoblasts
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Primary osteoblast precursor cells were isolated from the calvarial bone of newborn C57BL/6 mice (1- to 2-day-old) through enzymatic digestion with α-MEM containing 0.1% collagenase (Life technologies) and 0.2% dispase II (Life Technologies). The isolated osteoblast precursor cells were promoted with osteogenic α-MEM medium with 10% FBS, 1% penicillin–streptomycin, 5 mM β-glycerol phosphate (Sigma), 0.1 mg ml−1 ascorbic acid (Sigma) and 10 nM dexamethasone (Sigma) for 9 days and culture medium was replaced every 2–3 days36 (link). The human primary osteoblasts (human OBs) were purchased from PromoCell. The human OB cells were cultured and promoted with osteoblast growth medium and osteoblast mineralization medium (PromoCell), respectively. The purified osteoblasts (ALP+ cells) were isolated by MACS using anti-ALP antibody (Abcam, Rabbit IgG, Ab108337) in combination with anti-Rabbit IgG MicroBeads (Miltenyi Biotec).
5
Osteoblast Mineralization from neonatal mice
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For osteoblast cultures mesenchymal cells were isolated from the calvariae of 3–6 days old neonatal hTNF-α tg mice. In brief, calvariae were digested in α-MEM medium (PAN Biotech, Aidenbach, Germany) with 0.1% collagenase type IA (Sigma-Aldrich) and 0.2% dispase II (Roche, Basle, Switzerland) at 37°C on a shaker for 5 × 10 min. Fractions 2–5 were collected and cells were cultured and expanded until P2 to P3. At subconfluency state, cells were plated at 104/cells/cm2. Mineralization assays were carried out in 12-well plate by changing the medium to osteoblast mineralization medium (PromoCell, Heidelberg, Germany) at 100% confluency and cells were irradiated 24 h after the first medium change. Mineralization media was used according to the manufacturer’s recommendation and formation of bone nodules was evaluated at d21 using Alizarin red stain (Millipore, Darmstadt, Germany). Total wells were scanned and images were then analyzed using ImageJ software (Version 1.46r).
6
Osteogenic Biomaterial Synthesis and Evaluation
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Tetraethylorthosilicate (TEOS, 99%) and chloride acid (HCl) (37%) were obtained from Alfa Aesar (Haverhill, MA, USA). Gelatin (type A porcine, gel strength 300) and 3-glycidoxypropyltrimethoxysilane (GPTMS, >98%) were purchased for Sigma Aldrich (St. Louis, MO, USA). Absolute ethanol (99.5%) was purchased from Panreac and acetid acid (Reagent Grade) was purchased from Scharlau (Barcelona, Spain). HOB® human osteoblasts, fetal calf serum, osteoblast growing medium and osteoblast mineralization medium were purchased from Promocell (Heidelberg, Germany). Paraformaldehyde, PBS, Triton FITC conjugate were all purchased from Sigma, (St. Louis, MI, USA) and Vectashield® (Vector Laboratories, Burlingame, CA, USA). Alizarin red staining solution was purchased from Fisher Scientific, (Waltham, MA, USA).
7
Osteoblast Differentiation from Neonatal Mouse Calvariae
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For osteoblast cultures mesenchymal cells were isolated from the calvariae of 3–6 days old neonatal mice. In brief, calvariae were digested in α-MEM medium (PAN Biotech, Aidenbach, Germany) supplemented with 0.1% collagenase type IA (Sigma-Aldrich) and 0.2% dispase II (Roche, Basel, Switzerland) at 37 °C on a shaker for 5 × 10 min. Fractions 2–5 were collected and cells were cultured and expanded until P2 to P3. At subconfluency state, cells were plated at 1 × 104 cells/cm2. Mineralization assays were carried out in 12-well plates by changing the medium to osteoblast mineralization medium (PromoCell, Heidelberg, Germany) at 100% confluency and cells were irradiated 24 h after the first medium change. Mineralization media was used according to the manufacturer’s recommendation and formation of bone nodules was evaluated at day 21 using Alizarin red stain (Millipore, Darmstadt, Germany). For analyses, total wells were scanned and images then were analyzed using ImageJ software (Version 1.46r).
8
Alizarin Red S Staining for Osteoblast Mineralization
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UMR 106 cells were harvested and seeded in six-well plates at a density of 1×10 5 cells per well. At 80% confluency, the growth medium (DMEM) was replaced by Osteoblast Mineralization Medium (PromoCell, Heidelberg, Germany) and cells were cultured for 5 days. The negative control received the standard growth medium (DMEM). Samples were taken at 0, 12, 24, 72 and 120 hours and centrifugated for 5min at 1000 rpm (Hettich Universal, RF30).
For the detection of Ca deposits, the staining solution was prepared dissolving Alizarin Red S (Carl Roth GmbH + Co. KG, Karlsruhe) in distilled water and pH was adjusted to 4.2. After washing the cells with PBS (Sigma Aldrich, St. Louis, Missouri, USA), the cellular monolayer was fixed with 10% neutral buffered formalin (Sigma Aldrich, St. Louis, Missouri, USA) for 30 min. Next, the fixed solution was removed and the cells were washed with distilled water. Finally, the staining solution was added and remained for 45min, before washing the cells again with distilled water.
For the detection of Ca deposits, the staining solution was prepared dissolving Alizarin Red S (Carl Roth GmbH + Co. KG, Karlsruhe) in distilled water and pH was adjusted to 4.2. After washing the cells with PBS (Sigma Aldrich, St. Louis, Missouri, USA), the cellular monolayer was fixed with 10% neutral buffered formalin (Sigma Aldrich, St. Louis, Missouri, USA) for 30 min. Next, the fixed solution was removed and the cells were washed with distilled water. Finally, the staining solution was added and remained for 45min, before washing the cells again with distilled water.
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