Extraction of Ceffe
The experiment received approval from the Ethics Committee of Hainan Affiliated Hospital, Hainan Medical University. Ceffe was extracted from fresh adipose tissue using a previously established method [25]. The conventional liposuction technique was employed to extract abdominal fat from six healthy female New Zealand White Rabbits (2 months old, weighing approximately 2.5 kg, Shanghai Yunde Biotechnology Co., Ltd, China). The freshly extracted adipose tissue underwent a process to remove excess blood cells, followed by centrifugation at 1200 rpm for 3 min. This step discarded the upper lipid and lower liquid layers, retaining the middle fat layer, which was then mechanically emulsified. The emulsified fat was frozen at -80 °C and subsequently thawed at 37 °C. After thawing, it underwent further centrifugation at 1200 g for 5 min at 4 °C. Post-centrifugation, the third liquid layer (Ceffe) was extracted, passed through a 0.22 μm filter to eliminate bacteria and cell debris, and stored at -20 °C for later use.
3D Printing of PLGA and Ceffe/PLGA scaffolds
1 g of PLGA crystals (Sigma, USA) was dissolved in 10 mL of dichloromethane-acetone organic solvent (8 ml dichloromethane and 2 ml acetone, Sigma, USA). The solution underwent vortex shaking three times for 15 s each, followed by centrifugation at 1000 rpm for 30 s to form a 10% w/v PLGA solution. Additionally, 5 g of polyvinyl alcohol dissolved in 50 mL phosphate buffer saline (PBS, Sigma, USA) solution. The PLGA solution was mixed with the polyvinyl alcohol solution, supplemented with 1 mg of Ceffe (designated as Ceffe/PLGA group) or without Ceffe (designated as PLGA group). The mixture was placed in a thermostatic stirrer and stirred overnight. Subsequently, the mixture underwent shock emulsification through an ultrasonic breaker for 5 min and stirring in a magnetic stirrer for 3 h. Finally, the mixture was centrifuged at 1000 rpm for 10 min to remove the organic solvent, resulting in the formation of Ceffe/PLGA and PLGA colloidal solutions.
Customized brackets, nipple-shaped in design, were created using computer-aided design through Magics software (Materialise Corporation). The scaffold design incorporated a porous structure (300 μm pore size for micropores) to facilitate nutrient diffusion and surrounding tissue growth. The Ceffe/PLGA and PLGA colloidal solutions were then 3D printed on a 3D printer (GeSim BioScaffolder, Germany) to obtain nipple-shaped Ceffe/PLGA and PLGA scaffolds. 3D printing parameter settings: nozzle temperature 25℃; platform cooling temperature 3℃; printing speed 2 mm/s; line spacing 0.5 mm. The scaffold specifications are 10 mm in diameter at the base and 5 mm in height.
Characterizations of PLGA and Ceffe/PLGA scaffolds
Morphological and pore size observation
The macromorphology of PLGA and Ceffe/PLGA scaffolds was observed using a digital camera (Nikon, Japan). Additionally, the micromorphology of the two scaffolds was assessed via scanning electron microscopy (SEM, S3400, Hitachi, Japan) at an acceleration voltage of 10 kV for microstructural inspection. ImageJ software was utilized for further analysis of the average pore size based on the SEM images.
Porosity determination
Porosity of PLGA and Ceffe/PLGA scaffolds was determined using a liquid replacement method. The original volume of ethanol was denoted as V1, the volume of the scaffold after 5 min of immersion in ethanol as V2, and the residual volume after removing the scaffold as V3. The porosity of scaffold was calculated using the formula: (V1-V3)/(V2-V3).
Shape maintenance
After immersion in PBS for 4 weeks, PLGA and Ceffe/PLGA scaffolds were photographed immediately upon removal from wells for area determination. The projected area of the scaffolds was assessed by ImageJ based on the images.
Mechanical strength
Mechanical strength of the scaffolds was determined using a mechanical testing machine. PLGA and Ceffe/PLGA scaffolds underwent continuous planar unconfined strain at a rate of 1 mm/min until 80% of maximal deformation was achieved. Young’s modulus was calculated according to the stress-strain curve.
Degradation rate
Dry PLGA and Ceffe/PLGA scaffolds were initially weighed as W1 and then immersed in sterile PBS (pH = 7.4) at 37 °C with continuous shaking. Scaffolds were retrieved at predetermined times (1, 2, 3, 4, 5, 6, 8, 10, 12 weeks), lyophilized, and weighed as W2. The degradation rate was calculated using the formula: W2/W1 × 100%.
Release kinetics of Ceffe from Ceffe/PLGA scaffold
The in vitro release kinetics of Ceffe from Ceffe/PLGA scaffolds were investigated by immersing the scaffolds in deionized water at 37 °C. The release medium was extracted at specific time intervals, replaced with an equal volume of deionized water. Ceffe concentration in the collected solution was determined by a BCA kit [2] at predetermined times (1, 2, 3, 4, 5, 6, 8, 10, 12 weeks), and Ceffe release percentage was calculated.
Isolation and culture of chondrocytes
All animal procedures received approval from the Ethics Committee of Hainan Affiliated Hospital, Hainan Medical University. Auricular cartilage was harvested from the aforementioned New Zealand White Rabbits, immersed in an antibiotic solution for 30 min, minced into approximately 0.5–2.0 mm³ pieces, and digested with 0.15% type II collagenase (Sigma, USA) in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) for 8 h at 37 °C to isolate chondrocytes. The isolated chondrocytes were cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% double antibody (Gibco, USA) at 37 °C in 5% CO2. Chondrocytes were collected at the second passage for use.
In vitro biocompatibility
Cell proliferation assay
To assess the biocompatibility of the PLGA and Ceffe/PLGA scaffolds, chondrocytes were resuspended in DMEM containing 10% FBS, and the cell density was adjusted to 1.0 × 106 cells/mL before seeding onto PLGA and Ceffe/PLGA scaffolds. Cells were cultivated in vitro for 5 days in DMEM at 37 °C in 5% CO2. The viability of chondrocytes directly seeded on scaffolds on days 1, 3, and 5 was evaluated using a live/dead cell viability assay (Sigma, USA). Live/dead staining images were observed under a laser scanning confocal microscope (CLSM, Leica, Germany). Cell proliferation was measured using a cell counting kit-8 (CCK-8, Sigma, USA) according to the manufacturer’s instructions, with optical density (OD) measured at 450 nm.
Cell morphology
The morphology of chondrocytes cultured in vitro within PLGA and Ceffe/PLGA scaffolds was observed with CLSM. Samples collected on days 1, 3, and 5 were fixed in 4% paraformaldehyde for 30 min, washed three times with PBS, permeabilized with 0.1% Triton X-100 (Sigma, USA) for 30 min, and then incubated with Phalloidin-iFluor for 30 min. After further washing, the chondrocytes were stained with 4’,6-diamidino-2-phenylindole (DAPI, Sigma, USA) for 10 min.
Angiogenic evaluation
Extraction of scaffolds
PLGA and Ceffe/PLGA scaffolds were disinfected with a 75% ethanol solution for 60 min, washed twice with PBS, and then placed into DMEM. The solution was incubated for 24 h at 37 °C, collected, filtered with a 0.22 μm filter, and stored at 4 °C for long-term preservation.
Migration of human umbilical vein endothelial cells (HUVECs)
1.0 × 105 HUVECs, obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), were seeded in the upper chamber of transwell 24-well plates. PLGA and Ceffe/PLGA scaffold extracts were added to the lower chamber of the transwell. After 4 and 12 h, the upper chambers were removed, and the cells on the lower chamber were stained with 0.1% crystal violet. HUVEC counts per field were analyzed using ImageJ software.
Capillary formation assay
HUVECs were used for capillary formation assays. MatrigelTM was prepared overnight at 4°C, with 50 µL added per well of a chilled 96-well plate. HUVECs and extracts of PLGA and Ceffe/PLGA scaffolds were subsequently added (3 × 104 cells/well), followed by incubation for 4 and 12 h at 37 °C. Tube formation in both groups was observed via a light microscope, and quantification for tubes and branch points per field were measured using Image J software.
In vivo nipple-shaped cartilage regeneration
Preparation of chondrocyte-scaffold construct
PLGA and Ceffe/PLGA scaffolds were sterilized in a 75% ethanol solution overnight, followed by three washes with sterile saline solution. Chondrocytes at the 2nd passage, with a density of 1.0 × 108 cells/mL, were then separately packed into PLGA and Ceffe/PLGA scaffolds, forming chondrocyte-scaffold constructs.
Subcutaneous implantation of chondrocyte-scaffold construct
Six nude mice (4 weeks old, weighing approximately 200 g, Shanghai Yunde Biotechnology Co., Ltd, China) were anesthetized using 1% sodium pentobarbital. The backs were sterilized, and a skin incision was made, creating a pocket in the subcutaneous tissue. The constructs in both PLGA and Ceffe/PLGA groups were implanted into the pocket, the incision was closed, and the mice were incubated for 2 and 8 weeks. Samples were collected from the backs of nude mice for gross observation and sectioned for histological evaluation.
Histological evaluation
After gross observation using a digital camera, samples were fixed in 4% buffered formalin for 24 h, embedded in paraffin, and cut into 5 μm sections. One part of the sections was used for histological staining, and the other for multiple immunofluorescence (mHIC) staining. Sections were stained with hematoxylin & eosin (HE) and Safranin-O to evaluate the histological structure and cartilage-specific extracellular matrix (ECM) deposition in the regenerated tissue.
Biochemical analysis
Specimens from nude mice were digested in papain solution at 65 °C. The sulfated glycosaminoglycan (GAG) content of samples in PLGA and Ceffe/PLGA groups was quantified by Alcian Blue. The samples were hydrolyzed at 100 °C with HCl, and hydroxyproline content was determined using the Hydroxyproline Assay Kit. In addition, collagen type II (COL II) content was quantified using Enzyme-linked Immunosorbent Assay.
mHIC staining
The sections were fixed with 4% paraformaldehyde for 30 min, permeabilized in PBS containing 1% Triton X-100 for 10 min, followed by washing three times with PBS, and blocked with 10% goat serum in PBS for 30 min. Then, the sections were incubated with primary antibodies against CD31 (abcam, UK) or collagen type II (abmart, China) in the Superblock solution overnight at 4 °C. On the next day, sections were washed with PBS three times for 5 min each, followed by incubation with the Goat Anti-Rabbit IgG (abcam, UK) or Goat Anti-Rat IgG H&L (abcam, UK) for 2 h at room temperature. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) for 2 min. After washing, the tissue was observed under the CLSM in the darkroom. Quantification for CD31 and COL II intensities were determined based on the obtained mHIC images via ImageJ software.
Statistical analysis
All numerical data were presented as mean ± standard deviation. Differences between two groups were analyzed using Student’s t-test, and differences between multiple groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. All analyses were performed using SPSS 22.0 software (IBM SPSS, Chicago, IL, USA). Differences were considered statistically significant at P < 0.05.
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