Dimethylmethylene blue

Cultures were harvested with selective papain digestion for aggregates. Cell proliferation was assessed with the Cell Proliferation reagent WST-1 [16 (link),44 (link)]. The DNA contents were determined with a fluorimetric assay by using Hoechst 33258 [16 (link),35 (link),41 (link)-44 (link)]. The proteoglycan contents were measured by binding to dimethylmethylene blue dye [16 (link),35 (link),41 (link)-44 (link)], and those for type I, type II, and type X collagen with ELISA [16 (link),41 (link)-44 (link)]. The ALP activities were analyzed with a colorimetric assay to measure the hydrolysis of p-nitrophenol by using a standard curve made of this reagent [16 (link)]. All measurements were performed by using a GENios spectrophotometer/fluorometer (Tecan, Crailsheim, Germany).

Isolation and proliferation of ADSCsWritten consent was obtained from all patients in an operation room. Subcutaneous adipose tissue (∼10 g) was obtained from 4 patients (30-50 years age), under sterile conditions and transferred to the lab. Afterward, it was digested with 0.075% collagenase type I (Sigma) at 37°C for 30 min. The enzyme was inactivated by DMEM LG (Sigma) containing 10% FBS (Invitrogen). Subsequently, the resultant solution was centrifuged (1200 rpm, 15 min) and cell pellet was cultured in 25 cm² flasks with DMEM LG, 10% FBS, 1% penicillin & streptomycin (Gibco) and incubated with 5% CO₂, 37°C. The medium was changed every 4 days. , Cells were detached with 0.05% trypsin/0.53 mM EDTA (Sigma) and passaged, when they reached 80% confluence. P3-p5 cells were seeded in an alginate scaffold.
Isolation& proliferation of articular chondrocytesAfter taking a written consent, a bit of articular cartilage was obtained by the knee arthroscopy from 4 patients (20-30 years age) whose joints did not show any signs of a degenerative arthritis. The specimens were taken from non-weight bearing areas and transferred within PBS (Sigma) to the lab. The specimens were then divided into 1×1 mm slices and digested with collagenase type II (Sigma) 350 u/ml in 37° for 4 hr. After inactivation with DMEM F12 (Gibco) with 10% FBS, the solution was centrifuged and cell pellets were cultured in 25 cm² flask with DMEM F12, 10% FBS, 1% penicillin and streptomycin. When cells reached 80% confluence, they were detached with trypsin/EDTA and passaged. P2-P4 cells were used for seeding in an alginate scaffold.
Encapsulation and culture procedure in alginateInitially, ADSCs (P3-P5) and articular chondrocytes (p2-p4) were separately resuspended in 1.5% alginate (Sigma) at 5×10⁶ cells/ml. The alginate/cell suspension was expressed through a 23-gauge needle into a 102 mM CaCl₂ solution (Merck). The alginate beads after 15 min were washed twice in 0.9% saline solution and once in DMEM-HG (Gibco), and finally 2 ml chondrogenic media was added to each well of 12-well. Chondrogenic culture media contained: DMEM-HG (High Glucose)(Gibco), penicillin & streptomycin 1% (Gibco), dexamethasone 10^-7M (Sigma), ascorbat-2-phosphate 50 µg/ml (Sigma), bovine serum albumin 1% (Sigma), linoleic acid 5µg/ml (Sigma), insulin-transferrin-selenium (ITS) 1% (Sigma),with and without adding transforming growth factor- β₃ (TGFβ₃₎ 10 ng /ml (Sigma)
The plates were incubated with 5% CO₂ at 37° and replaced every 4 days. Supernatant mediums on days 14 and 21 were frozen at -20° for ELISA.
ELISAAGC and type I collagen produced in supernatant media with and without TGFβ₃ on days 14 and 21 was measured by ELISA. For AGC the kit was from Invitrogen (Cat No KAP1461) and type I collagen was from MD bioproducts (Cat No M036007) according to their manufacturers’ protocols. Finally samples were read in ELISA reader (Hyperion, Microreader 4 plus) with 450 nm wave length.
Biochemical assaysDNA quantificationIn order to determinate DNA and GAG , first, alginate beads on days 14 and 21 digested for 18 h at 60° in papain solution 125µg/ml(Sigma) containing cistein10 mM (Sigma) in PBE buffer (Na₂Hpo₄ 100 mM, EDTA 10 mM, pH=6.5). For each 12 beads, one ml enzyme was used. The resultant solutions were used for determination of DNA and GAG.
The content of DNA (ng/ml) was measured using DNA Quantification Kit, Fluorescence Assay (Sigma, Cat. No. DNAQF), according to its manufacturer’s protocol. Briefly, resultant solution was read by dyed Hoechst 33258 and absorbance of each sample was read by spectrofluorometer (Perkin Elmer LS-3) at 360 nm in excitation and 460 nm wave length in emission. Afterward, the DNA content was calculated according to the thymic calf standard curve.
GAG AssayThe amount of GAG (µg/ml) was quantified using 1, 9-dimethylmethylene blue (DMMB) dye. Briefly, 100μl of resultant solution was mixed with 2400 μl of DMMB solution in a cuvette and then its absorbance in 525 nm wave length was read by spectrophotometer (Spectonic 70, Baucsh&lomb). The GAG content was calculated using standard curve chondroitin sulfate of bovine trachea.
Finally, the ratio of GAG/DNA in each sample was normalized. All experiments were performed twice.
Real-time PCRAt first, alginate beads on days 14 and 21 were washed with PBS and for digestion, they were placed in 1.5% 55 mM citrate sodium (Sharlau) and 0.15 mM NaCl (Merck). The resultant solution centrifuged for 10 min at 1200 rpm and the derived cell pellet was used for the extraction of RNA with RNeasy mini kit (Qiagen,Cat. No. 74101) with a little modification (36 (link)). For lysing cells, firstly a solution mixed of 990 µl Trizol (Invitrogen) and 10 µl of 2-mercaptoethanol (Sigma), was kept in room temperature (RT) for 5 min. Next, 200 µl chloroform was added and shacked vigorously for 15 second kept in RT for 2-3 min and then centrifuged for 15 min at 4° at 12000 g.
The supernatant aqueous phase was transported into a 1.5 ml microtube and the same volume of ethanol 70% was added and then mixed. The resultant solution transferred to columns in kit and the rest of instruction was carried out according to kit protocol in which to kit protocol in which RNase free DNase set (Qiagen) applied for elimination of possible DNA contamination. The extent of derived RNA was measured by spectrophotometer (Biophotometer, Eppendorf) at 260/280 nm wave length. Reverse transcription for cDNA, 100 ng RNA used by recruitment of RevertAid^TM First Strand cDNA Synthesis Kit (Fermentas,Cat. No. #K 1621) according to manufacturer’s protocol.
Relative quantification of the expression types II and X collagens was measured, using Maxima SYBR® Green/RoxqPCR master Mix 2X (Fermentas), with GAPDH primer as an internal control. The calculation was performed via comparative Ct (ΔΔ Ct). The reactions conducted in 20 µl with, 10 µl SYBR® Green, 7.5 µl H₂o, 0.25 µM forward and reverse primers and 1.5 µl cDNA as following planned by StepOne Plus Real Time PCR system (Applied Biosystem): primary denaturation in 95° for 10 min, denaturation in 95° for 15 sec, Annealing and Extension in 60° for 1 min –the whole process was done 40 cycles- and finally melt curve (increment 0.3 °C, 60°C→95°C) was depicted. All experiments were performed in triplicates for each specimen. The applied primers for Real-Time PCR were designed by AlleleID 7/6 software which indicated in Table 1.
Statistical testsKolmogorov-Simonov test was used for assessing the normal distribution of variables. ANOVA (one-way-analysis of variance) with LSD post hoc test used for comparison of ELISA, Real-Time PCR and GAG assay results in different groups.

The Dimethylmethylene Blue (DMMB) assay was used to quantify total glycosaminoglycan (GAG) amount in each material at 7 days and 21 days of culture. A stock solution of dimethylmethylene blue (DMMB) was made by dissolving 32 mg of 1,9-DMMB in 20 mL of pure ethanol overnight on an orbital shaker at room temperature. This stock solution was added to a mixture of 1.5 L distilled water, 59 mL 1 M NaOH and 7 mL 98% formic acid and left to mix for 2 h. The pH of the dye was adjusted and verified to be pH 1.5 prior to use. The cell-laden biomaterial hemispheres of each bioink were lysed in Radioimmunoprecipitation Assay (RIPA) buffer (Thermo Fisher Scientific, MA, USA) to yield protein isolates for GAG quantification along with material only control samples. Isolates were diluted 1 in 50 with distilled water and 40 μL added to the wells of a 96 well plate in triplicate with 200 μL DMMB reagent per well. The plates were read immediately at 525 nm compared to a series of chondroitin standards ranging from 0 to 50 μg/mL (0.03–0.75 μg). The total content of glycosaminoglycan in each sample was acquired using the standard curve of chondroitin samples, corrected for sample volume and dilution.

hPCs were plated on a 24‐well plate (40000 cells per well). The next day, culture media was replaced with osmolarity adjusted media (either 330 or 400 mOsm) with or without TGFβ1 (10 ng mL⁻¹, R&D systems, 7754‐BH‐005). After 96 h of replacing the media, supernatant was collected, and glycosaminoglycan content was quantified using dimethylmethylene blue (DMMB) assay as described before.^[59 (link)
^] All measurements were performed in duplicate and repeated in three OA donors.