Isolation, identification, and differentiation analysis of rabbit BMSCs
Isolation of BMSCs
Eighteen 6-month-old New Zealand white rabbits (bought from Yunde Biotechnology Co., Ltd.) weighing 3.0–3.5 kg had been intravenously anesthetized. The surgical space was shaved and disinfected earlier than performing bone marrow aspiration to gather 3 mL of iliac bone marrow utilizing a syringe preloaded with 2 mL of heparin. The collected marrow was transferred to a ten mL centrifuge tube, combined with 3 mL of phosphate-buffered saline (PBS, Gibco, USA), and centrifuged at 1000 rpm for 10 min. The supernatant and fats layer had been discarded, and the cells had been washed twice with 3 mL PBS below the identical centrifugation situations.
The extracted cells had been resuspended in low-glucose Dulbecco’s Modified Eagle Medium (LG-DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin–streptomycin. They had been then seeded at a density of 1.4 × 104 cells/mL in tradition flasks and incubated at 37 °C with 5% CO2. The tradition medium was changed each 2–3 days. When cells reached 80–85% confluency, they had been digested with trypsin and passaged at a 1:2 ratio. The identical technique was utilized for subsequent passages. The expansion of main (P0) BMSCs was noticed from days 3–12 below a light-weight microscope, and the morphology of second by way of sixth-passaged cells (P2–P6) was evaluated utilizing phalloidin staining (No. C2201S, Beyotime, China) below a confocal microscope.
Identification of BMSCs
The floor markers CD29 and CD44 (MSC-related), in addition to CD34 and CD45 (hematopoietic-related), had been analyzed by circulate cytometry and immunofluorescence staining in passaged BMSCs (P0, P1, P3, P5, and P7).
For circulate cytometry, BMSCs at 80% confluency had been digested with 1 mL of 0.25% trypsin and neutralized with 4 mL of LG-DMEM containing 10% FBS. The cell suspension was pipetted to make sure uniformity, and the density was adjusted to 2 × 105 cells/mL. A 1 mL aliquot was transferred to fifteen mL centrifuge tubes, centrifuged at 1200 rpm for five min, and the supernatant discarded. The BMSCs had been washed with 1 mL of 5% bovine serum albumin (BSA, Gibco, USA) answer, centrifuged once more, and resuspended in 100 µL of 5% BSA. The suspension was divided into 5 teams and incubated in a single day at 4 °C with the corresponding antibodies (Desk S1, Supplemental Info): anti-CD29, anti-CD44, anti-CD34, anti-CD45, and a clean management. The next day, the cells had been washed with 1 mL of 5% BSA, centrifuged, and resuspended in 500 µL of 0.1 mol/L PBS. Evaluation was carried out utilizing a Cytomics FC500 (Beckman Coulter, Miami, FL, USA) or Accuri C6 Plus (BD Biosciences, San Jose, CA, USA) and Csampler software program (n = 5).
For immunofluorescence staining, cell slides had been ready by coating glass slides with poly-L-lysine for 120 min, adopted by air-drying and sterilization below ultraviolet (UV) gentle for 1 h. Slides had been positioned into tradition plate wells to facilitate cell attachment. Cells had been fastened with 4% paraformaldehyde for 10 min, washed thrice with PBS, and subjected to antigen retrieval in alkaline urea buffer at 95 °C for 10 min. After cooling, cells had been permeabilized with 0.1% Triton X-100 for 10 min, washed with PBS, and blocked. Major antibodies for CD29, CD44, CD34, and CD45 (Desk S2, Supplemental Info) had been incubated in a single day at 4 °C. After washing with PBST, secondary antibodies (Desk S2, Supplemental Info) had been utilized, adopted by nuclear staining with 4′,6-diamidino-2-phenylindole dilactate (DAPI, No. HY-D1738, MedChemExpress, USA). The slides had been mounted with anti-fade reagent and noticed below a confocal microscope. The relative expression ranges of CD29, CD44, CD34, and CD45 at numerous passages had been quantified utilizing ImageJ software program.
Differentiation potential analysis of passaged BMSCs
The differentiation potential of BMSCs was assessed by way of osteogenic and chondrogenic induction. Passaged BMSCs (P0, P1, P3, P5, and P7) at 70% confluency had been cultured in osteogenic medium (LG-DMEM supplemented with 10% FBS, 50 µM α-ascorbate-2-phosphate, 10 mM β-glycerophosphate, and 0.1 µM dexamethasone) or chondrogenic medium (LG-DMEM supplemented with 1% antibiotic–antimycotic, 1 × 10–3 M ascorbate-2-phosphate, 100 × 10–9 M dexamethasone, 40 µg/mL proline, insulin–transferrin–sodium selenite premix, 6.25 µg/mL insulin, 6.25 µg/mL transferrin, 6.25 µg/mL sodium selenite, 1.25 µg/mL BSA, 5.35 µg/mL linoleic acid, 100 ng/mL development differentiation issue 5, and 10 ng/mL remodeling development issue beta-3). Half of the medium was changed each 2–3 days.
Twin-lineage differentiation of BMSCs (P0–P7) was carried out utilizing: (i) Alizarin Crimson S staining to judge osteogenic potential and (ii) Alcian Blue staining to evaluate chondrogenic potential.
For Alizarin Crimson S staining (No. C0138, Beyotime, China), passaged BMSCs had been incubated with the staining answer for 30 min at 37 °C, rinsed with distilled water, and imaged below a light-weight microscope. For Alcian Blue staining (No. C0153S, Beyotime, China), BMSC slides had been ready, fastened, and stained with Alcian Blue answer for 30 min. After rinsing below working water and washing with PBS, slides had been dehydrated, mounted, and imaged. The depth of staining was quantified utilizing ImageJ software program.
Growth of cartilage organoids utilizing P3 BMSCs
Preparation of organoid mildew
A 25,000 DPI photo-plotting printer (Koenen, Germany) was employed to manufacture polydimethylsiloxane (PDMS) grasp molds (Sylgard 184, Dow Corning, USA), containing cylindrical arrays with diameters of 200 µm, depths of 150 µm, and inter-cylinder spacings of 100 µm. Subsequently, 3% (w/v) agarose (Invitrogen, China) was poured into the molds. Upon solidification, agarose replicas, roughly 1.8 cm2 in space, had been rigorously punched out and positioned into 24-well plates. The PDMS mildew and agarose gel duplicate had been photographed and documented. Following this, the agarose gel replicas had been freeze-dried and analyzed utilizing Scanning Electron Microscopy (SEM, FEI Examine F50, USA). Every effectively was then full of 1 mL of PBS and sterilized below UV gentle for 30 min. Every agarose gel duplicate contained roughly 2000 microwells, which served as inserts for high-throughput BMSC spheroid tradition.
Growth of cartilage organoids
P3 BMSCs, expanded in LG-DMEM containing 10% FBS, had been used for cartilage organoid improvement. The BMSCs had been harvested and seeded into every effectively of a 24-well plate (pre-loaded with agarose microwells) at a density of 1 × 106 cells per effectively. After self-aggregation, roughly 500 BMSCs had been current per microwell. The BMSC spheroids had been induced utilizing the beforehand described chondrogenic medium, with half of the medium changed each 2–3 days.
After 7 days, the BMSC spheroids had been transferred to 96-well plates for additional cartilage organoid improvement over an extra 21 days. This organoid preparation technique was outlined as 3D tradition. As a management, P3 BMSCs had been cultured on flat tradition dishes with the identical chondrogenic medium, known as 2D tradition. Pictures had been taken at completely different time factors all through the 28-day tradition interval utilizing a light-weight microscope.
To evaluate BMSC proliferation below 2D and 3D situations on days 1, 3, 7, and 14, reside/useless staining (Invitrogen, USA) was carried out to research cell survival, and a cell counting kit-8 (CCK-8, No. C0041, Beyotime, China) was used to measure cell viability [31]. Phalloidin staining (No. C2203S, Beyotime, China) was utilized to visualise the cell cytoskeleton. Chondrogenic differentiation was evaluated by immunofluorescence staining for aggrecan, Sox9, and COL II in BMSCs derived from each 3D organoid and 2D cultures (Desk S2, Supplemental Info). Quantitative evaluation of DNA, COL II, and glycosaminoglycan (GAG) content material was carried out as beforehand described [32], with outcomes expressed as COL II/DNA and GAG/DNA ratios.
Chondrogenic analysis of cartilage organoids
To verify the profitable improvement of cartilage organoids, hematoxylin and eosin (H&E, No. C0105S, Beyotime, China) staining was carried out. Briefly, organoids had been fastened, dehydrated, and sectioned. After rehydration by way of graded ethanol, the sections had been stained with hematoxylin and eosin, adopted by mounting for structural statement and imaging. Safranin O/Quick Inexperienced (Saf-O/FG) staining (No. C0621S, Beyotime, China) was additionally carried out. Sections had been stained with Quick Inexperienced for five min, rinsed with faucet water, differentiated in acetic acid for 30 s, washed, and stained with Safranin O for 8 min. After speedy dehydration with absolute ethanol, the sections had been mounted and analyzed.
Moreover, immunofluorescence staining for COL II and aggrecan (Desk S2, Supplemental Info) was carried out on organoid sections. Following rehydration, antigen retrieval, antibody incubation, and DAPI staining had been carried out as described in Sect. “Identification of BMSCs“. The slides had been mounted, and pictures had been acquired for evaluation. Lastly, COL II and GAG content material had been measured at completely different time factors to additional consider chondrogenic protein expression.
In vitro differentiation analysis of cartilage organoids in avascular and vascular microenvironments
In vitro incubation of cartilage organoids in avascular and vascular microenvironments
Cartilage organoids, after 28 days of chondrogenic tradition, had been subjected to both a vascular (with vascular endothelial development issue, VEGF) or avascular (with out VEGF) medium for additional chondrogenic and hypertrophic analysis. Organoids had been divided into three teams (n = 5): (−)VEGF, (+)VEGF, and Management. The (−)VEGF group was induced utilizing the beforehand described chondrogenic medium, whereas the (+)VEGF group was handled with the identical medium supplemented with VEGF (5 ng/mL, No. GMP-VE5H23, ACROBiosystems, USA). The Management group consisted of cartilage organoids collected after 28 days of chondrogenic tradition, previous to additional induction (with out VEGF therapy). Half of the medium was changed each 2–3 days. After 4 weeks of induction, cartilage organoids from all teams had been collected for evaluation.
Hypertrophic differentiation of cartilage organoids with VEGF therapy
For quantitative real-time polymerase chain response (qPCR) evaluation, complete mRNA was extracted and purified from the induced cartilage organoids utilizing Trizol reagent (Thermo Fisher Scientific, USA) [33]. The amount and high quality of the mRNA had been decided by measuring absorbance at 260 nm with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). cDNA was synthesized from 1 µg of mRNA utilizing the Maxime RT PreMix package (Intron Biotechnology, Seoul, Korea) in a Bio-Rad Thermocycler (Bio-Rad Laboratories, Hercules, CA, USA). For amplification, a ten µL response system was ready with 5 µL SYBR Inexperienced PCR Premix (Roche, Switzerland), 2 µL RNA-free water, 2 µL primers, and 1 µL cDNA. The combination was positioned in a qPCR system and subjected to the next thermal biking situations: 95 °C for 10 min (denaturation); 40 cycles of 95 °C for 15 s (annealing) and 60 °C for 1 min (extension); adopted by a melting curve from 60 °C to 95 °C, with 0.5 °C increments each 5 s. Primer sequences are listed in Desk S3 (Supplemental Info). The relative fold modifications in gene expression for the (+)VEGF and (−)VEGF teams had been normalized to the Management group.
To additional assess hypertrophic differentiation in VEGF-treated cartilage organoids, western blotting was carried out [34]. Cartilage organoids had been lysed on ice for 30 min utilizing RIPA buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 100 µg/mL PMSF, and a protease inhibitor cocktail). The lysates had been clarified by centrifugation at 12,000 rpm for 20 min at 4 °C, and protein concentrations had been measured utilizing a BCA assay. Equal quantities of protein (20 µg) had been loaded onto 10% SDS-PAGE gels for electrophoresis and transferred to PVDF membranes. The membranes had been blocked with 5% non-fat milk for 1 h at room temperature and incubated in a single day at 4 °C with main antibodies (Desk S2, Supplemental Info). HRP-conjugated secondary antibodies (Desk S2, Supplemental Info) had been incubated for two h at room temperature. Enhanced chemiluminescence detection was used to visualise the blots, and quantitative evaluation was carried out utilizing ImageJ software program to judge goal protein expression ranges.
To validate differentiation outcomes, immunofluorescence staining was carried out to research the expression of chondrogenic proteins (COL II and GAG) and hypertrophic proteins (Matrix metalloproteinase-13 (Mmp13) and collagen sort X (COL X)). The staining process adopted the protocol described in Sect. “Identification of BMSCs“. Quantitative evaluation of fluorescence depth was carried out to evaluate differentiation.
Genomic evaluation of cartilage organoids in avascular and vascular microenvironments
RNA sequencing was carried out on cartilage organoids handled with or with out VEGF utilizing commonplace sequencing procedures supplied by Cloud-seq Firm, China [35]. A gene was thought of expressed in a pattern if its expression worth was ≥ 1. Differentially expressed genes had been recognized with a fold change of ≥ 4 and a p-value of ≤ 0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses had been carried out utilizing DAVID and REVIGO (https://david.ncifcrf.gov; http://revigo.irb.hr/). Goal gene screening was carried out utilizing the GeneCards database (https://www.genecards.org/). For every group, three organic replicates had been collected, and their RNA was extracted, sequenced, and analyzed.
In vivo cartilage and bone regeneration of Organoid/GelMA complexes in avascular and vascular microenvironments
Preparation of double-layer organoid/GelMA complexes with or with out axitinib
The gelatin methacryloyl (GelMA, No. LBH020001, Lab of Well being, China) precursor answer was ready by dissolving GelMA (pre-loaded with the blue gentle initiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate) in PBS to a last focus of 0.15 g/mL. To organize the organoid/GelMA complexes, induced cartilage organoids had been suspended within the GelMA precursor answer at a 1:1 quantity ratio. To create an avascular microenvironment, the anti-angiogenic drug Axitinib (No. HY-10065, MedChemExpress, USA) was added to the organoid/GelMA complicated at a last focus of 0.40 nM, designated because the (+)Axitinib layer answer. The vascular group didn’t embody Axitinib and was designated because the (−)Axitinib layer answer. These two options had been transferred to cylindrical molds with a diameter of 4 mm and top of 1.5 mm, and solidified after 15 s of publicity to Blu-ray gentle (wavelength 405 nm), leading to two sorts of cylinders. These cylinders had been then stacked to type a double-layer cylinder with an total diameter of 4 mm and a top of three mm, yielding three experimental teams: (+)Axitinib/(+)Axitinib, (−)Axitinib/(−)Axitinib, and (+)Axitinib/(−)Axitinib.
Cartilage and bone regeneration of organoid/GelMA complexes in avascular and vascular microenvironments in a nude mouse mannequin
Six-week-old feminine nude mice (n = 5) had been bought from Shanghai SLAC Laboratory Animal Co., Ltd. The double-layer organoid/GelMA complexes from every group had been subcutaneously implanted into the dorsal area of the mice. After 4 weeks, the mice had been euthanized below deep anesthesia, and the regenerated tissues had been harvested for evaluation.
Micro-computed tomography (Micro-CT) was used to judge the osseous traits of the double-layer organoid/GelMA complexes after subcutaneous implantation. The regenerated tissues had been fastened in 4% paraformaldehyde, then scanned at 40 kV, 600 µA, and a decision of 8.75 µm utilizing the Skyscan 1176 Micro-CT system (Bruker, Germany). The photographs had been reconstructed with a dynamic vary of 0–0.16, a beam hardening setting of 20, and a hoop artifact correction of 10. 3D reconstructions had been generated utilizing CTVox software program (Bruker, Germany). To quantify osseous options, the area of curiosity (ROI) was outlined utilizing CTAnalyser software program (Bruker, Germany), and key parameters, together with trabecular quantity (Tb.N) and trabecular thickness (Tb.Th), had been calculated inside the ROI (n = 3). These analyses supplied quantitative metrics for evaluating the osseous ECM deposition of the organoid/GelMA complexes.
The harvested tissues had been sectioned for histological evaluation, together with H&E and Saf-O/FG staining. Briefly, tissues had been fastened in 4% paraformaldehyde for twenty-four h, decalcified in 10% ethylenediamine tetraacetic acid (Chelatocal, Nationwide Diagnostics, Atlanta, GA, USA) for 4 weeks, then dehydrated, embedded, and sectioned. H&E and Saf-O/FG staining had been carried out to evaluate the chondrogenic and osteogenic potential of the regenerated tissues. Quantification of COL II and GAG content material was carried out as described in Sect. “Growth of cartilage organoids“.
To judge the impact of the vascular microenvironment on cartilage and bone regeneration, CD31 immunofluorescence staining was carried out on tissue sections. Semi-quantitative evaluation of CD31 expression adopted the protocol outlined in Sect. “Growth of cartilage organoids“, utilizing antibodies listed in Desk S2 (Supplemental Info).
Gradient heterogeneous osteochondral regeneration of organoid/GelMA cylinder in a rabbit OCD mannequin
Preparation of intact organoid/GelMA cylinder
To organize an intact organoid/GelMA cylinder, the GelMA precursor answer, with cartilage organoids added, was transferred right into a cylindrical mildew (4 mm diameter, 3 mm top). The combination was totally solidified after 30 s of publicity to Blu-ray gentle (wavelength 405 nm). A pure GelMA cylinder of the identical measurement because the organoid/GelMA cylinder was additionally ready as a management.
Implantation of organoid/GelMA cylinder in a rabbit OCD mannequin
A full-thickness OCD mannequin was created in grownup male New Zealand white rabbits. The rabbits had been sedated with Zoletil50 (0.35 mL/kg, Virbac, France), and preoperative antibiotics (10 mg/kg, Baytril, Bayer Korea) and analgesics (5 mg/kg, Tramadol HCl, HanAll Biopharma, Korea) had been administered subcutaneously to forestall issues. A lateral parapatellar strategy was used to show the patellar groove, with sequential incisions by way of the pores and skin, subcutaneous tissue, and joint capsule. The patella was dislocated medially to disclose the trochlear groove. A 4-mm diameter cylindrical defect, 3 mm in depth, was created within the femoral trochlea utilizing a high-speed drill (Colibri II, Depuy Synthes, Oberdorf, Switzerland).
The rabbits had been randomly divided into three teams: the empty group, the GelMA group, and the organoid/GelMA group (n = 6). The defect websites had been implanted with both a GelMA cylinder or an autologous organoid/GelMA cylinder, whereas the empty group acquired no implant. The joint capsule and subcutaneous tissue had been sutured with absorbable sutures, and the pores and skin was closed with nylon sutures. Postoperative care concerned 7 days of monitoring, with the surgical web site disinfected with chlorhexidine and antibiotics and analgesics administered subcutaneously. The rabbits had been housed for 12 weeks post-surgery. On the endpoint, the rabbits had been euthanized below deep anesthesia (5 MAC isoflurane) and intravenous KCl (150 mg/kg). The repaired knee joints had been harvested, photographed, and stuck in 4% paraformaldehyde for additional evaluation.
Analysis of gradient heterogeneous osteochondral tissue regeneration
Regenerated tissue was assessed utilizing the Worldwide Cartilage Restore Society (ICRS) scoring system (Desk S4, Supplemental Info). Closing scores had been decided by averaging the outcomes from 5 unbiased researchers (n = 5).
Micro-CT evaluation was carried out on the repaired joint tissue to acquire 2D and 3D photographs. Parameters had been set at 40 kV, 600 μA, and a decision of 8.75 μm. Tb.Th and bone quantity fraction (BV/TV) had been quantified inside the area of curiosity (ROI) utilizing CTAnalyser software program, as described in Sect. “Cartilage and bone regeneration of organoid/GelMA complexes in avascular and vascular microenvironments in a nude mouse mannequin“.
Histological analyses had been carried out to judge cartilage and subchondral bone regeneration within the knee joint. Tissue sections had been stained with H&E and Saf-O/FG. For toluidine blue staining (No. HY-D0220, MedChemExpress, USA), hydrated sections had been incubated with toluidine blue staining answer at room temperature for 30 min, adopted by differentiation in 1% hydrochloric acid-alcohol and additional rinsing. Sections had been then dehydrated and mounted for statement. Immunohistochemical staining for COL II was carried out to substantiate cartilage formation. After rehydration, antigen retrieval was carried out utilizing an alkaline urea buffer. Permeabilization was executed with 0.1% Triton X-100 in PBS, adopted by blocking. Sections had been incubated with main and secondary antibodies (Desk S2, Supplemental Info). DAB staining (No. P0203, Beyotime, China) was used for visualization, adopted by hematoxylin counterstaining and mounting. Outcomes had been imaged and semi-quantitatively analyzed.
Biosafety evaluation
To evaluate the biosafety of the organoid/GelMA cylinder, tissue samples from the guts, liver, spleen, lungs, and kidneys had been collected from handled rabbits for histological evaluation (n = 5). Stomach aortic blood was additionally collected for full blood depend and liver and kidney perform checks (n = 5) to judge systemic biosafety.
Statistical evaluation
Statistical evaluation was carried out utilizing SPSS software program (Model 22.0, SPSS Inc., USA), ImageJ software program (Model 1.8.0, Nationwide Institutes of Well being, USA), and GraphPad Prism (Model 9.0, GraphPad Software program Inc., USA). All knowledge are offered as imply ± commonplace deviation (SD) and had been derived from not less than three unbiased experiments. For 2-group comparisons, the Pupil’s t-test was used. For multi-group comparisons, one-way evaluation of variance (ANOVA) was carried out. A p-value of lower than 0.05 was thought of statistically important.
