BMSCs were flushed from femurs and tibiae of 3 months old WT, AMBNHET and AMBNΔ5-6 littermates. The cell suspensions were filtered through a 40 μm cell strainer (Becton-Dickinson, Franklin Lakes, NJ) to obtain single cells. Nucleated cells were plated in 35 mm dishes at a density of 0.5×105/well in complete MesenCult medium (STEMCELL Technologies, Vancouver, Canada) with duplicated cultures. For the Colony Forming Unit-fibroblast numbers (CFU-F) assay, cell cultures were fixed and stained with Giemsa. For the CFU-osteoblast numbers (CFU-Ob) assay, BMSCs were cultured in growth medium for 14 days, followed by osteogenic medium (Lonza) for 7 days, and then analyzed for mineralized matrix formation by alizarin red staining. We determined the colony-forming efficiency by quantifying the number of colonies per 105 marrow cells. For chondrogenesis in standard pellet culture, 2×105 BMSCs were pelleted by centrifugation and cultured in chondrogenic medium (Lonza) for 18 days. The cultured pellets were then processed for histochemical analysis as previously described (48 (link)).
Osteogenic medium
Manufactured by Lonza
Sourced in United States
Osteogenic medium is a specialized cell culture medium designed to support the growth and differentiation of osteogenic (bone-forming) cells. Its core function is to provide the necessary nutrients, growth factors, and signaling cues to promote the development of bone-like tissues in vitro.
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Lab products found in correlation
Chondrogenic medium, by Lonza (2 mentions)
Osteoblasts, by Lonza (2 mentions)
Osteoblast growth medium, by Cambrex (2 mentions)
Human primary osteoblasts, by Lonza (2 mentions)
Mesencult medium, by STEMCELL (1 mentions)
40 μm cell strainer, by BD (1 mentions)
Pannoramic viewer software, by 3DHISTECH (1 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
Evos xl core microscope, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
4000b dmi, by Leica (1 mentions)
Alcian blue, by Merck Group (1 mentions)
Alizarin red s, by Merck Group (1 mentions)
Adipogenic induction medium, by Lonza (1 mentions)
Adipogenic maintenance medium, by Lonza (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Alp assay kit, by Beyotime (1 mentions)
9 protocols using osteogenic medium
1
Isolation and Characterization of Bone Marrow Stromal Cells
2
Histological Analysis of Calcium Deposition
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Achilles tendons were fixed in 2% (w/v) PFA in PBS for 20 minutes, wax processed. 5–6 µm sections were de-waxed before staining. Calcium deposition in primary human tenocytes was induced by culturing cells in Osteogenic Medium (Lonza) according to the manufacturer's protocol. After 14 days, cells were fixed with 2% (w/v) PFA in PBS for 20 minutes. Fixed cells or tissue sections were washed with distilled water and stained with 2% (w/v) Alizarin Red S, pH 4.2 for 10 min. Washed plates or sections were air-dried and imaged using a Pannoramic slide scanner and Pannoramic Viewer software (3DHISTECH).
3
Integrin-Specific Hydrogel Osteogenesis
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hMSCs were seeded at 5 × 106 cells/mL within integrin-specific hydrogels and cultured in osteogenic medium (Lonza). After 9 days of culture in induction medium, hMSCs were lysed and assayed for alkaline phosphatase activity (ALP) by incubating with 4-methylumbelliferyl phosphate disodium salt (MUP) substrate60 (link). Hydrogels were incubated in 1 mg/mL collagenase type I (ThermoFisher) at 37 °C until fully degraded. Cells were resuspended in 50 mM Tris HCl (pH 7.4) and lysed by sonication and freeze-thaw cycles. Samples and ALP standards were loaded into a 96-well plate, then incubated with 60 μg/mL MUP substrate at 37 °C for 1 h and read at 360 nm excitation/465 nm emission. Enzymatic activity was standardized using purified calf intestinal ALP at known dilutions and normalized to total protein. For mineralization studies, hMSC-laden hydrogels were fed every 3–4 days with hMSC growth media. On day 14, gels were fixed with 10% neutral buffered formalin, stained with 2% Alizarin red solution for 30 min, and washed repeatedly with DI water until dye stopped leaching out of the gel. Gels were imaged using an Evos XL Core microscope (ThermoFisher). Mean Alizarin red intensity was calculated using ImageJ software.
4
Epigenetic Changes in Osteogenic Differentiation
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In order to investigate epigenetic changes at basal levels and upon osteogenic differentiation, two PDLCs classified as l-PDLCs and h-PDLCs according to the above description were plated at 8.7 × 105 cells per 100 mm dishes either in Dulbecco’s Modified Eagle Medium (DMEM), containing 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 mg/mL) (Gibco, Carlsbad, CA, USA) (DMEM group) or in osteogenic medium (OM) (Lonza, Walkersville, MD, USA) supplemented as above (OM group), with media change every 3 days.
5
Multilineage Differentiation of MSCs
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Cells from cryopreserved stocks were grown to confluence in 12-well plates and subjected to osteogenic and chondrogenic differentiation using the appropriate induction media (Lonza osteogenic medium Cat# PT-4120, and chondrogenic Cat# PT-3003) supplemented with 10 ng/mL TGFβ3 Cat# PT-4124 under conditions described by the manufacturer.
The multilineage differentiation potential of MSCs was assessed by histochemical staining and phase-contrast microscopy (Leica 4000b DMI, Leica Microsystems GmbH., Wetzlar, Germany). The degree of mineralization in osteogenic cultures was assessed by staining with 2% Alizarin Red S (Sigma-Aldrich, Saint Louis, MO, USA). Briefly, the cells were washed three times with phosphate buffered saline (PBS) pH 4.2 and fixed 20 min with 4% paraformaldehyde. The fixed cells were washed, stained, and washed again to remove excess stain. Similarly, the chondrogenic potential was evaluated by measuring the production of proteoglycans produced by chondrocytes after staining for 20 min with 1% Alcian blue (Sigma-Aldrich, Saint Louis, MO USA) (Figure S4 ).
The multilineage differentiation potential of MSCs was assessed by histochemical staining and phase-contrast microscopy (Leica 4000b DMI, Leica Microsystems GmbH., Wetzlar, Germany). The degree of mineralization in osteogenic cultures was assessed by staining with 2% Alizarin Red S (Sigma-Aldrich, Saint Louis, MO, USA). Briefly, the cells were washed three times with phosphate buffered saline (PBS) pH 4.2 and fixed 20 min with 4% paraformaldehyde. The fixed cells were washed, stained, and washed again to remove excess stain. Similarly, the chondrogenic potential was evaluated by measuring the production of proteoglycans produced by chondrocytes after staining for 20 min with 1% Alcian blue (Sigma-Aldrich, Saint Louis, MO USA) (
6
Multi-lineage Differentiation of SSEA-3+ Cells
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To investigate the multipotency of SSEA-3 positive cells in in vitro, osteogenic differentiation was performed using osteogenic medium (Lonza, Walkersville, MD, USA) consisting of dexamethasone, ascorbic acid, and β-glycerophosphate in a 6-well dish. In vitro adipogenic differentiation was also performed using adipogenic induction medium (Lonza) consisting of insulin, dexamethasone, indomethacin, and IBMX (3-isobutyl-methyl-xanthine) and adipogenic maintenance medium (Lonza) consisting of insulin in a 6-well dish. For in vitro chondrogenic differentiation, we utilized high-density three-dimensional micromass culture [21] (link), [22] (link), in which cells were trypsinized and resuspended at a density of 1 × 105 cells/10 μl. Ten microliter droplets were seeded in culture dishes and allowed to form cell aggregates and substratum at 37 °C for two and a half hours. Chondrogenic medium (Lonza), consisting of ITS + premix (6.25 μg/mL insulin, 6.25 μg/mL transferrin, 6.25 μg/mL selenous acid, 5.33 μg/mL linoleic acid, and 1.25 mg/mL bovine serum albumin), pyruvate (1 mmol/L), ascorbate 2-phosphate (0.17 mmol/L), proline (0.35 mmol/L), dexamethasone (0.1 μmol/L) and recombinant human TGF-β3 (10 ng/mL) was then carefully added around the cell aggregates. This Chondrogenic medium was replenished every three days.
7
Assessment of Osteoblast Differentiation
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Osteoblast differentiation ability was assessed in vitro by testing the alkaline phosphatase (ALP) activity of hBMSCs. The cells were seeded on the samples for 24 h, and then the medium was changed. Osteogenic medium (Lonza, USA) for inducing osteogenic differentiation contained 0.5% ascorbate, 0.5% dexamethasone, and 1% β-glycerophosphate. After 7 and 14 days of osteogenic induction on the scaffold, 500 µL lysate was added to the 48-well plates with the samples, and the cells were sonicated. Then, the lysate was centrifuged at 12,000 r/min for 5 min at 4 ℃. Absorbance values were measured at 405 nm using the ALP assay kit (Beyotime, China) and at 562 nm using the BCA protein assay kit (Beyotime, China). The ratio of the absorbance at 405 nm to that at 562 nm was used as the quantitative value of ALP.
8
Differentiation of Mesenchymal Stem Cells to Osteoblasts
9
Differentiation of Mesenchymal Stem Cells to Osteoblasts
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Human mesenchymal stem cells (MSC) and osteoblasts were obtained from Lonza Group Ltd (hereafter referred to as “Lonza”). Mesenchyme cells were grown and maintained in Mesenchymal Cell Growth Supplement (MCGS, PT-3001) with L-glutamine and containing 30 mg/ml gentamicin, 15 ug/ml amphotericin and 10% fetal bovine serum (FBS). Cells were cultured at 37 °C in the presence of 5% CO2 until ∼75% confluent. Mesenchyme cells were induced to differentiate to osteoblasts by culturing the cells in the presence of osteogenic medium from Lonza according to the manufacturer's protocol. Human primary osteoblasts were also obtained from Lonza. The primary osteoblasts were cultured in Clonetics Osteoblast Growth Medium especially formulated for the growth and expansion of human osteoblasts, and contained 10% FBS, ascorbic acid and gentamicin-amphotericin.
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