Ethical statement
Human articular cartilage and bone marrow samples were obtained with prior informed and written consent in accordance with the Declaration of Helsinki and clearance from the Ethics Committee Biomedical Health Research, Gleneagles Global Hospitals (File No: SPF/2021/000126).
Materials
The cell culture media High glucose Dulbecco’s Modified Eagle Medium (H-DMEM,1 g/L D-glucose, 11885-084 500 mL), foetal bovine serum, One Shot™ Format, US origin (FBS, A3160401 50 mL) and collagenase type II powder (1 mg/mL solution, 17101-015) were obtained from Gibco, Life Technologies Corp. HiSepTMLSM 1077 (LS001-500 mL) was purchased from HiMedia. L-ascorbic acid (50 µg/mL, A4403), dexamethasone (1 mM, D1756), transforming growth factor beta-2 (TGFβ-2, 50 µg/mL, T2815), basic fibroblast growth factor (bFGF, 10 ng/ml, F3685), sodium alginate powder (2 gm/100 mL, W201502), and chitosan (2%, cat no. 419419-50G) were obtained from Sigma Aldrich. All antibodies were purchased from Novus Biologicals (USA) and used at the recommended dilutions. Antibiotics (gentamycin, 50 IU/ml; amphotericin B, 2.5 µg/ml; penicillin–streptomycin, 100 IU/ml) were purchased from Thermo Fisher Scientific. A 70 µm cell strainer was obtained from Corning® (431751). The forward and reverse primers used were obtained from Eurofins. The remaining chemicals were purchased from Sigma–Aldrich Chemical Co. (USA).
Methodology
Human bone marrow (2 mL) and articular cartilage explants (1–2 mm) were collected from patients (n = 10) aged 23–70 years as part of a sports injury and knee replacement surgical procedure under aseptic conditions.
Bioink preparation and sterilisation
Cartilage explants were subjected to mechanical and enzymatic treatment with 0.25% collagenase type II and disrupted further with 2.5% sodium dodecyl sulphate (SDS) for 16 h to obtain cell-free ECM (herein represented as bioink) and achieve efficient decellularisation27. The bioink was rinsed with 1 × phosphate-buffered saline (PBS) to remove SDS and further treated with 0.1% Triton X-100 for 5 min to remove nucleic acids. The bioink was further rinsed with 1 × PBS to remove Triton X-10038. Before cell seeding, the bioink was sterilized in 1 × PBS solution containing antibiotics (penicillin/streptomycin (500 U/ml) and amphotericin B (0.5 μg/ml)) for 24 h at room temperature to prevent contamination39. The sections were then rinsed and stored in 1 × PBS until further use to construct biomimetic tissues.
Isolation and culture of native chondrocytes
Human articular cartilage explants were collected in H-DMEM and thoroughly rinsed with PBS containing antibiotics to remove undesirable contaminants. Tissue explants were minced using a sterile surgical blade (No. 21), subjected to mechanical and enzymatic digestion with collagenase type II supplemented with 2.5% FBS in H-DMEM supplemented with antibiotics, and incubated for 16 h at 37 °C on an orbital shaker. After incubation, the enzymatic reaction was stopped with 10% FBS, and the cells were strained through a 70 µm cell strainer to separate the chondrocytes from the ECM. The cell suspension was centrifuged at 1800 rpm for 5 min to obtain a cell pellet. The supernatant was discarded, and the cell pellet was rinsed with 1 × PBS by spinning at 1800 rpm for 5 min. The chondrocytes obtained as cell pellet was suspended in H-DMEM consisting of 10% FBS, L-ascorbic acid, dexamethasone, TGFβ-2, and antibiotics40 (hereafter referred to as chondrogenic media).
Isolation and culture of human BMSCs via the density gradient method and differentiation into the chondrogenic lineage
Human bone marrow tissue was collected in an anticoagulant, citrate dextrose (ACD) solution. The tissue was diluted in normal saline at a 1:1 ratio, layered on HiSep solution (1:3 ratio of HiSep to diluted tissue), and subjected to density gradient centrifugation at 1500 rpm for 30 min. The creamy white to buff-coloured layer was collected and centrifuged at 1500 rpm for 5 min. The resulting pellet was rinsed with sterile distilled water to avoid undesirable hematopoietic cell populations and centrifuged at 1500 rpm for 5 min. The cell pellet was suspended in H-DMEM containing 10% FBS, antibiotics and bFGF (BMSCs culture media) and strained through a 70 µm cell strainer to obtain a single-cell suspension41. An initial seeding density of 1 × 106 cells/mL was incubated at 37 °C in 5% CO2 till 70% confluency was achieved and were subjected to differentiation in chondrogenic media.
Pellet culture with bioink
Isolated native chondrocytes and BMSCs (6 × 106 cells/mL) along with bioink (~ 100 μL) were seeded in chondrogenic and BMSCs culture media, respectively, and grown as pellet cultures. The cells were pelleted every 3 days at 1800 rpm for 8 min and half replenished with fresh chondrogenic media to retain the growth factors and matrix deposited on the biomimetic tissue constructs. The culture was monitored for an increase in pellet size over three weeks. Simultaneously, a pellet culture without bioink was used as a control.
Preparation of alginate polymer beads with bioink
Sodium alginate powder (2%) was dissolved in distilled water by stirring on a magnetic stirrer, and the final pH was adjusted to 6.8–7.2. A calcium chloride (100 mM) solution was prepared in 1 × PBS and allowed to dissolve completely to obtain a homogenous solution. The sodium alginate and calcium chloride solutions were filter sterilised with a 0.2 µm syringe filter before alginate bead preparation. Chondrocytes and BMSCs (6 × 106 cells/mL) were suspended in chondrogenic media with sodium alginate solution supplemented with bioink (~ 100 μL), and using a 23-gauge needle, the final suspension was dropped as beads in cold calcium chloride solution. Calcium-alginate beads were incubated at 37 °C for 30 min for polymerisation and encapsulation. The beads were then washed with 1 × PBS and cultured in chondrogenic media42. Alginate beads were also prepared and cultured using a similar process without bioink as a control. The cultured alginate beads were paraffin-embedded and sectioned (2 µm thickness) using a microtome for histology and immunocytochemistry (ICC) characterisation.
In vitro assays
Cell viability using a flow cytometer
Chondrocytes and BMSCs-induced chondrocytes (1 × 106 cells/mL) from native and in vitro cultures were stained with 10 µl of 7-AAD cell viability dye, with unstained cells serving as a control. The cells were incubated for 20 min at 4 °C in the dark, and the percentage of live to dead cells among the unstained and stained cells was analysed using a flow cytometer (Becton Dickinson FACS Calibur™).
Proliferation assay
To bioink-laden chondrocytes and BMSCs-induced chondrocytes (6 × 106 cells/mL) in H-DMEM without FBS, 5 μM Cell Tracker™ Green CMFDA Dye (Cat. C2925; Invitrogen) was added. The cells were incubated at 37 °C for 30 min and rinsed with 1 × PBS43. The cultures were imaged for three weeks at 0, 7, 14, and 21 days using the CLSM software LAS X Office v1.4.5 (Leica Microsystems). The quantitative mean fluorescence values were plotted for biomimetic cultures with and without bioink, and proliferation patterns were analysed.
SEM characterisation
The SEM was performed to characterise the cellular attachment and aggregation of the obtained biomimetic tissue constructs without and with bioink. The samples were maintained in a freezer at − 20 °C for 24 h and were lyophilized in a freeze-dryer. The samples were fixed with 4% paraformaldehyde for 30 min at room temperature, followed by rinsing with 1 × PBS three times to remove the fixative. The samples were dehydrated through an ascending concentration of absolute ethanol, starting with 30%, 50%, 70%, 80%, 90%, and 100%, incubated for 30 min time interval and spin at 1200 rpm, 10 min, 4 °C at each step. After the final step of dehydration, another 100% ethanol was added and incubated at room temperature for 30 min. Then, the samples were allowed to dry under high vacuum for 2 h. The samples were mounted on aluminium metal stubs using double-sided adhesive tapes and coated with thin layer (300 angstrom) of ionic gold nanoparticles in sputter coating unit E-1010 (Hitachi Japan) under high vacuum44.The gold nanoparticle coating enabled the samples conductive to electrons for capturing images at × 8.0–10.0 k magnification, 15 kilovolts (kV), under high vacuum (10−7 torr) using SEM.
Histology and ICC characterisation
Histological sections were stained with H&E and alcian blue for morphological identification and accumulation of GAG, respectively. The sections for ICC, were fixed in a 4% paraformaldehyde solution for 5 min, washed 2× with 1 × PBS for 5 min, subjected to antigen retrieval with sodium citrate buffer for 20 min, heated at 95–100 °C, and allowed to cool at room temperature for 20 min. Further blocking and permeabilization were performed using 0.5% Triton X-100 and 5% FBS in 1 × PBS for 20 min at room temperature. The sections were incubated with primary antibodies in a humidifying chamber for 16 h at 4 °C. The primary antibodies used were against CD44 (chondrocyte-specific cellular marker), VCAM1 (differentiated chondrocyte-specific cellular marker), and the ECM-specific markers collagen type I, collagen type II, aggrecan, and laminin to characterise their expression. The primary antibodies were removed by rinsing three times with 1 × PBS for 5 min and incubated with secondary antibodies for 1 h at room temperature on an orbital shaker. Further washes were performed with 1 × PBS, and the sections were counterstained for 5 min with the nuclear stain 4′,6-diamidino-2-phenylindole (DAPI, 1 mg/mL concentration, further diluted to a 1:1000 working stock)45. The sections were rinsed with distilled water and mounted with glycerol for imaging using CLSM (Leica TCS SPE).
Quantitative PCR (qPCR) gene expression profile
The cultured biomimetic tissue constructs were subjected to RNA isolation using a Qiagen Miniprep Kit (cat. no. 74104), and the quality and purity were checked using a spectrophotometer (NanoDrop2000, Thermo Scientific). The isolated RNA template (2 µg) was subjected to cDNA conversion using 2 × reverse transcriptase (RT) master mix consisting of 10 × RT buffer, 25 × dNTP mixture (100 mM), 10 × RT hexamer random primers, Multiscribe™ RT enzyme, and nuclease-free water in a final reaction volume of 10 µL. The thermocycling conditions were optimized for the initial hold (25 °C, 10 min), RT enzyme activation step (48 °C, 30 min), and final enzyme inactivation step (95 °C, 5 min).
The converted cDNA was subjected to qPCR gene expression profiling using ECM-specific forward and reverse primers for collagen type I, collagen type II, aggrecan, and laminin, with normalization against the housekeeping gene GAPDH (Table 1). qPCR was performed using 5 µg of cDNA added to 2 × SYBR® Select master mix supplemented with the aforementioned forward and reverse primers (10 pmol/µL) in nuclease-free water for a final reaction volume of 20 µL. The PCR cycling and melting curve conditions were set to the initial activation step (95 °C, 5 min), followed by 45 cycles of two-step denaturation (95 °C, 15 s) and annealing/extension (60 °C, 60 s). The final run data were analysed with a threshold value set to 0.05, and gene expression levels and fold changes were calculated using cycle threshold (Ct) values and the 2−∆∆Ct method, respectively46.
Alginate–chitosan composite hydrogel preparation
The alginate–chitosan hydrogel was prepared with various concentrations of sodium alginate and chitosan solutions polymerised with calcium chloride and glutaraldehyde and mixed with and without bioink (Table 2). A 2% chitosan solution was prepared by dissolving chitosan in 1% aqueous glacial acetic acid and heating it in a water bath at 60 °C to obtain a homogenous solution. The sodium alginate solution along with the cell-laden bioink (~ 100 µL) in chondrogenic media was layered over the chitosan solution, followed by layering with a cross-linking solution (1 mL of glyceraldehyde solution mixed in an equal volume of calcium chloride solution) under gentle rotation on an orbital shaker for uniform mixing of the hydrogel. The hydrogel was incubated at 37 °C for polymerisation47,48.
Statistical analysis
The unpaired two-tailed type t-test statistical analysis was performed for all samples (n = 10) in triplicates using GraphPad Prism software. The mean Ct values were used to calculate standard deviation for each target gene under culture conditions without and with bioink. Samples exhibiting t-test with significant difference in p-value less than 0.05, were considered to be statistically significant, rejecting the null hypothesis.
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- Source: https://www.nature.com/articles/s41598-024-62656-1