Cell Isolation and Expansion
The microtia tissue was obtained from patients (aged 5–17 years) who underwent ear reconstructive surgery at the Department of Plastic Surgery, Wuhan Union Hospital. Informed consent was signed with the donor's family before surgery, and the protocol was approved by the Ethics Committee of Huazhong University of Science and Technology.
The abandoned microtia tissue was immediately immersed in sterile normal saline containing 100 U mL-1 penicillin and 100 mg mL-1 streptomycin. The specimen was carefully dissected to strip the fibrous tissue and perichondrium, and then fragmented into 1 mm3. Chondrocytes were isolated with 0.25% trypsin (including 0.02% ethylene diamine tetraacetic acid (EDTA), Beyotime, China) for 0.5h and 0.15% type II collagenase (Sigma-Aldrich, USA) for 8h at 37℃. The obtained digestive liquid was filtered by a single-cell filter (100 mesh) and collected in a 50mL centrifuge tube. Centrifugation (1500rpm, 5min) was performed to discard the supernatant 3 times. Cell precipitates were suspended in complete medium [High glucose Dulbecco's modified eagle medium (H-DMEM, Hyclone, USA), 10% fetal bovine serum (FBS, Hyclone, USA), 100U mL-1 penicillin (Sigma-Aldrich, USA), 0.1mg mL-1 streptomycin (Sigma-Aldrich, USA) and 20ng mL-1 Fibroblast Growth Factor-basic (bFGF, Peprotech,USA)]. Chondrocytes were then inoculated in the 10cm cell culture dish (Corning, USA) for culture and expansion.
Immunofluorescence and Induced Differentiation of Microtia Chondrocytes
For immunofluorescence, the expression of CD90 and CD105 was detected using mouse anti-human CD90 and CD105 monoclonal antibodies (1:100, Abcam, UK), respectively, followed by rabbit anti-mouse immunoglobulin G (Abcam, UK). The expression of COL2, Aggrecan and CD73 was detected using rabbit anti-human COL2, aggrecan and CD73 monoclonal antibodies (1:100, Abcam, UK), respectively, followed by goat anti-rabbit immunoglobulin G (Abcam, UK). 4′,6-Diamidino-2-phenylindole (DAPI, Sigma-Aldrich, USA) was used for nuclear staining.
The osteogenic and adipogenic ability of microtia chondrocytes was detected by adipogenic and osteogenic induction solution. Osteogenic differentiation was induced with osteogenic differentiation medium [low-glucose Dulbecco's modified eagle medium (L-DMEM, Hyclone, USA), 10% fetal bovine serum (FBS), 100U mL-1 penicillin, 0.1mg mL-1 streptomycin ,10mM β-glycerol phosphate, 50mM vitamin C (Sigma-Aldrich, USA) and 0.1mM dexamethasone (Sigma-Aldrich, USA)] for 14 days. Microtia chondrocytes were fixed with 4% paraformaldehyde for 30-60min, and 0.1% (pH = 8.0) Alizarin Red S staining (Solarbio, China) solution was added to incubate for 60min. Adipogenic differentiation was induced with adipogenic differentiation medium [H-DMEM,10%FBS, 100U mL-1 penicillin, 0.1mg mL-1 streptomycin, 5mg mL-1 insulin (Sigma-Aldrich, USA), 0.5mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, USA), 200mM indomethacin (Sigma-Aldrich, USA) and 1mM dexamethasone] for 21 days. Microtia chondrocytes were fixed with 4% paraformaldehyde for 30-60min, and the Oil Red O staining solution kit (Solarbio, China) was used for staining.
Preparation of Cartilage Acellular Matrix (CAM)
Porcine ears were collected from the slaughter house and used with approval. The ears were carefully dissected to strip the fibrous tissue and perichondrium, and then fragmented into small pieces. The cartilage pieces were first freeze-dried for 12h, then placed in liquid nitrogen for 5min and frozen ground with a grinder (60Hz, 60s) to obtain cartilage microparticles. The microparticles were treated with 0.25% trypsin (containing 0.02%EDTA) in a constant temperature shaker (37℃, 150r min-1) for 24h, in which the new trypsin was changed every 4h. Trypsinized cartilage tissues were washed with hypertonic buffer solution (1.5mol L-1 NaCl dissolved in 50mM Tris-HCl, pH = 7.6, Sinopharm, China) for 5–8 times until the cartilage serous fluid turned white, and treated with nuclease solution [50U mL-1 DNase (Sigma-Aldrich, USA), 1U mL-1 RNaseA (Sigma-Aldrich, USA), dissolved in 10mM Tris-HCl (Beyotime, China), pH = 7.5] in the constant temperature shaker (37℃, 80r min-1) for 20h. To remove the enzymes, the tissues were washed with the 10mM Tris-HCl [containing 10U mL-1 aprotinin (Sigma-Aldrich, USA), pH = 8.0] for 20h and 1% TritonX-100/PBS (Beyotime, China) solution for 24h. The decellularized cartilage tissues were washed with PBS for 72h with changing the solution every 12h. Then the CAM tissues were frozen ground again (60Hz,60s) and sifted through the 50 mesh sieves to get CAM microparticles. To get different particle sizes, CAM microparticles were sifted through the 50,100,150 mesh sieves and stored at -20℃.
Biochemical Characterization of CAM
To verify the extent of decellularization, the residual DNA and glycosaminoglycan (GAG) were measured and histological sections were analyzed with H&E staining and Masson staining. For DNA and GAG quantification, 20mg CAM and 20mg native cartilage tissue were weighed respectively. DNA was extracted according to instructions of Blood/Cell/Tissue Genomic DNA Extraction Kit (TianGen DP304, China) and measured using the Quant-iTTM PicoGreen dsDNA (ThermoFisher, USA). GAG content was measured with Blyscan™ Glycosaminoglycan Kit (Biocolor, UK). For histological evaluation, both native and decellularized tissues were fixed in 4% paraformaldehyde (Sinopharm, China). After dehydration, samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) and Masson’s trichrome (Solarbio, China). Three independent experiments were performed in triplicate for each group.
Particle Size Analysis
The test was carried out with the laser-assisted particle size analyzer (Malvern Mastersizer 2000, UK), and the dispersing solvent was distilled water. CAM microtissues to be measured were slowly dispersed into distilled water in the test container, and the test was started when the shading background intensity reached 12%. The refractive coefficients of CAM microtissues and distilled water were 1.50 and 1.33, respectively.
Fabrication of Microtissues
CAM microparticles were treated with 0.1% peracetic acid (Sinopharm, China) solution in combination with 4% ethanol (Sinopharm, China) for 4h for disinfection, and then washed 3 times with sterile PBS. 500mg CAM microparticles were added to 50ml phosphate buffer saline (PBS) to get 10mg mL-1 CAM suspension. 2% agarose solution was added to the 12-well plate in advance to cool to gel. The CAM suspension was blown and mixed, and then added to the 12-well plate. After CAM microparticles settled into the lower layer, the upper PBS was sucked with the 1mL syringe. Meanwhile, P3 microtia chondrocytes were digested and suspended into 1×106 cell mL-1 cell suspension, dropped on CAM with the inoculation density of 5×104 chondrocytes per 1mg CAM and incubated at 37℃ for 2h to allow for cell attachment. After that, the complete culture medium [H-DMEM, 10% FBS, 100U mL-1 penicillin, 0.1mg mL-1 streptomycin and 20ng mL-1 bFGF] was added for long-time culture.
Assessment of Chondrocytes Viability in Microtissues
For live/dead assay, calcein (Beyotime, China) and propidium iodide (PI, Sigma-Aldrich, USA) were used to stain living cells and dead cells, respectively. Before staining, calcein mother solution was diluted 1000 times with PBS, and 10mg PI was dissolved in 1mL acetone and diluted to 1ug/ mL with PBS. The samples were rinsed with PBS for 3 times in advance. The samples were incubated at 37℃ with calcein working solution for 30min and then replaced with culture medium for another 30min. After that, the medium was sucked out, and PI working solution was added and incubated for 5min. Finally, the samples were rinsed with PBS for 3 times and observed under the confocal laser microscope (Nikon, Japan).
Cell proliferation within microtissues was detected by Cell Counting Kit-8 (Beyotime, China). Briefly, standard curves were measured using P3 chondrocytes. After that, microtissues were detected by Cell Counting Kit-8 at day1, day3 and day5.
Preparation of Bioink
10% w/v GelMA (EFL-GM-90, EFL, China) solution was prepared and sterilized in advance, in which the lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, EFL-LAP, EFL, China) concentration was 0.25% w/v as photoinitiator, and 0.1% w/v tartrazine (Sinopharm, China) as photoabsorber for DLP bioprinting. In order to observe the effects of different CAM concentrations and particle sizes on the performance of bioink, 1%, 5% and 10% w/v concentrations and three sizes of CAM microparticles with 0-150µm, 150–300µm and > 300µm were selected for testing, and the suitable CAM concentration and particle size were chosen for the preparation of microtissue bioink.
For the preparation of microtissue bioink, the construction method of microtissues was mentioned above. After 5 days of culture in vitro, the amount of living cells on microtissues was measured, then the microtissues were gently mixed with sterilized 10% w/v GelMA solution. For the bioink as control, the same amount of chondrocytes (about 6.26x106 cells in 1mL GelMA solution) of the same generation were mixed with sterilized 10% w/v GelMA solution directly. All the above operations were carried out at room temperature as soon as possible, and protected from light. The above experiment was repeated for 5 times, and stable microtissue bioink was obtained each time. We then tried replacing GelMA with ethylene diacrylate (PEGDA, M.V. 1000, Aladdin, China) and repeated the above steps to observe the growth of microtissues in PEGDA.
Scanning Electron Microscopy (SEM)
The surface morphology was measured by Environmental Scanning Electron Microscope (Quanta 200, FEI, USA). All the samples were fixed with 2.5% v/v glutaraldehyde for 4h, gradient dehydrated by different levels of ethanol (30%, 50%, 70%, 90%, 100%), treated with n-butanol and then lyophilized. The dried samples were sputtered with gold under vacuum (20mA,150s) and observed.
Disc shaped test specimen of 8mm diameter and 3mm height were prepared using bioink with different concentrations and particle sizes of CAM. A 20g load cell was used for testing. Samples were compressed to a final strain of 30% at a rate of 0.01 mm s− 1. The compression modulus was calculated from the slope of the linear 2%-10% of the stress–strain curve. Three independent experiments were performed in triplicate for each group.
Rheological measurements were conducted on an Anton Paar MCT 302 rheometer (Anton Paar, Austria) at 37°C. To evaluate the shear thinning behavior, the bioink was placed between the parallel plate subjected to an increasing shear of 0.01 ~ 200 s− 1. The viscosity of the bioink before printing was evaluated by subjecting the bioink to a low shear of 1 s− 1 and held for 600s. Then the bioink was solidified into a gel sheet with 10mm diameter and a 1mm height, the storage and loss modulus were evaluated by time sweeps under fixed strain and frequency conditions (1% strain and 5 rad s− 1) for 300s.
The swelling ratio values were determined following the previous study procedure. Disc shaped test specimen of 8 mm diameter and 3 mm height were prepared using bioink with different concentrations and particle sizes of CAM. After photo-crosslinking with 405nm ultraviolet light, three samples from each condition were soaked in PBS at 37℃ for 12h and weighted as M1, then lyophilized for 12h and weighted as M2. The swelling ratio (q) was calculated using Equation q = M1/M2. Three independent experiments were performed in triplicate for each group.
Sedimentation Characteristics of Microtissue in GelMA
The sedimentation characteristics was monitored according to the previous study procedure. Briefly, three particle sizes of 0-150µm, 150–300µm and > 300µm CAM microparticles were added into DMEM to the concentration of 10% w/v, left to rest at 37℃ and monitored the sedimentation behavior with time. As for the bright field images at high magnification, the samples standing for 0h, 1h and 3h were solidified and formed at 4℃ respectively, and then placed horizontally under a stereomicroscope for photographing, so as to ensure that the distribution of particles inside the samples remained unchanged. Three samples were tested for each group, and the sedimentation distance was then measured and statistical analysis was done with one-way ANOVA test.
The 3D auricular model was desighed with UG 10.0 software and 3D bioprinting experiments were conducted using DLP printer (EFL-BP-8600, China). The bioink was prepared in advance and mixed by gently blowing, then added on the platform under sterile conditions. For GelMA or GelMA + chondrocytes bioinks, the printing parameters were set as: light intensity 14mW cm-2, exposure time 30s, and penetration depth 30µm. As for GelMA + CAM or GelMA + microtissues bioinks, the printing parameters were set as: light intensity 14mW cm-2, exposure time 35s, and penetration depth 30µm. After conducting the printing process, the constructs were soaked in H-DMEM for further culturing.
In Vitro Culture
The printed auricular constructs were placed in the complete culture medium [H-DMEM, 10% FBS, 100U mL-1 penicillin, 0.1mg mL-1 streptomycin and 20ng mL-1 bFGF] for culture, and the culture medium was changed every 3 days for continuous culture for 20 days. The cell growth was observed under confocal laser microscope at day1, day10 and day20.
To observe the growth of microtisues in different gel networks and to explore whether the construction method of microtissue bioink is universal, we switched GelMA to another gel. PEGDA is a common hydrogel. Different from GelMA, it has denser gel network and higher mechanical strength, but the biocompatibility needs to be improved. Here, 10% w/v PEGDA (M.V. 1000, Aladdin, China) was mixed with microtissues (10% w/v) for in vivo culture, and the cell growth was observed under confocal laser microscope at day1 and day10.
Live/dead cell staining and immunofluorescence staining of in vitro culture constructs
For live/dead assay, calcein (Beyotime, China) and PI (Sigma-Aldrich, USA) were used to stain living cells and dead cells, respectively. Before staining, calcein mother solution was diluted 1000 times with PBS, and 10mg PI was dissolved in 1mL acetone and diluted to 1ug/ mL with PBS. The samples were rinsed with PBS for 3 times in advance. The samples were incubated at 37℃ with calcein working solution for 30min and then replaced with culture medium for another 30min. After that, the medium was sucked out, and PI working solution was added and incubated for 5min. Finally, the samples were rinsed with PBS 3 times for 5 minutes each and observed under the confocal laser microscope (Nikon, Japan). Z-axis scan length was 250µm and the experiment was performed in triplicate (n = 3), with the viability of each sample averaged over 3 pictures of randomly chosen positions inside the hydrogel.
For immunofluorescence staining, the expression of COL2 and Aggrecan was detected using rabbit anti-human COL2 and aggrecan monoclonal antibodies (1:200, Abcam, UK), respectively, followed by goat anti-rabbit immunoglobulin G (Abcam, UK).
For cocultivation experiments, 6-well transwell coculture plates (0.4µm pore size membrane, Corning, USA) were chosen. 100ul of chondrocytes were seeded in each lower well plate, at the density of 1 million cells mL-1. Subsequently, transwell chambers containing chondrocytes and microtissues were respectively inserted into the 6-well plates with equal amounts of 1x105 chondrocytes in each chamber, and then cocultured for 3 and 5 days. The experiment was repeated three times.
Gene Expression Analysis
For the microtia chondrocytes cultured in petri dish and microtissues, cells were collected by trypsin digestion. For the chondrocytes encapsulated in GelMA (EFL-GM-90, EFL, China), GelMA Lysis Buffer (EFL-GM-LS-001, EFL, China) was used to dissolve the gel and release the cells in advance. The total RNA was extracted using TRIzol (ThermoFisher, USA). RNA was reversed transcribed into cDNA and the real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) detection was performed following the manufacturer’s recommendation. Briefly, qRT-PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems) with the following cycling scheme: 1) Pre-denaturation (95℃, 10min), 2) Denaturation (95℃) + Annealing extension (60℃) was used as a cycle, repeated 40 times, 3) Determination of dissolution curve: (95℃, 15s) - (60℃, 60s) - (95℃, 15s). For the detection of mRNA expression, all primers were synthesized by Wuhan Biofavor Biotech. Primers used for qRT-PCR are presented in Table S1. Three independent experiments were performed in triplicate for each group.
Subcutaneous Nude Mice Implantation
All experiments involving animals were performed in accordance with the guidelines of the Ethics Committee of Huazhong University of Science and Technology. Male BALB/c-nu nude mice (7 weeks old, weighing 17 to 20g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China). To investigate the effects of CAM microtissues on cartilage regeneration in vivo, 40 mice were divided randomly into 4 groups (n = 10 mice per group). GelMA (CAM-, chondrocytes-), GelMA + chondrocytes (CAM-, chondrocytes+), GelMA + CAM (CAM+, chondrocytes-) and GelMA + microtissues (CAM+, chondrocytes+) three-dimensional (3D) printed auricular constructs were prepared under sterile conditions in advance and soaked in complete medium. Recipient mice were anesthetized by means of inhaled 3% isoflurane. A 10mm skin incision was made along the midline of the dorsal, two subcutaneous pockets are then bluntly stripped outwards. Auricular constructs were placed under the skin, and 5 − 0 cosmetic sutures were used to suture the skin incision intermittently, so as to keep the stent in its original position and avoid damaging the scaffold structure. After 6 and 12 weeks, five randomly selected animals in each treatment group were euthanized, and the grafts were harvested for the following analyses.
An incision along the dorsal midline was created and two subcutaneous pockets created lateral to the dorsal midline by blunt dissection.
The specimens were placed in the − 80℃ refrigerator immediately after harvested. 7µm frozen sections were prepared for all stainings of auricular constructs with cryostat microtome (ThermoFisher, USA). Samples were stained with hematoxylin and eosin (H&E) and Safranin O (Solarbio, China). For immunohistochemical evaluation, rabbit anti-human COL2 monoclonal antibodies (1:100, Abcam, UK) was used, followed by goat anti-rabbit immunoglobulin G (Abcam, UK).
For immunofluorescence staining, the expression of Lamin A/C was detected using rabbit anti-human LaminA + LaminC monoclonal antibodies (1:400, Abcam, UK), respectively, followed by goat anti-rabbit immunoglobulin G (Abcam, UK).
Quantitative results were analyzed with GraphPad Prism (v.7.0.0). One-way analysis of variance (ANOVA) and student’s t-test were applied to mean comparisons. All data in this study are expressed as the mean values ± standard deviation. Statistically significant differences were considered when p-values below 0.05 (p < 0.05).