4.1. Fabrication of biotracheal construct
The experiments on human nasal septum-derived chondrocytes were performed in compliance with the Institutional Review Board of the Catholic Medical Center Clinical Research Coordinating Center (KC08TISS0341), and the written informed consents from the donors were obtained. The chondrocytes were isolated as previously described.[15] The chondrocytes (5⋅106 cells/mL )were then mixed with collagen gel (2.7% w/v; Ubiosis, Korea).
The PCL frame, as the basic structure of the biotracheal scaffold, was 3D-printed based on the previously designed bellows structure.[6] PCL pellets (molecular weight 43–50 kDa; Polyscience Inc., USA) were prepared and extruded via a 90 °C printing head using the in-house 3D-printing system. The bellows-type framework had following dimensions: length 13 mm; internal diameter 7 mm; pore size 200 µm; and wall thickness 400 µm. The chondrocyte-encapsulated collagen was 3D-printed in each groove of the bellows. Completed biotracheal grafts were cultured in vitro for 5 d before implantation.
4.2. Fabrication of tracheal construct and chitosan membrane
Electrospinning solutions for chitosan (High molecular weight: 310–375 kDa; Sigma-Aldrich, USA) and PCL (Mn 80,000; Sigma-Aldrich, USA) were prepared separately. Chitosan (12.5% w/v) was dissolved in a mixed solvent containing trifluoroacetic acid (TFA; 99%; Sigma-Aldrich, USA) and dichloromethane (DCM, > 99.8%; Sigma-Aldrich, USA) at a volume ratio of 7:3, followed by stirring at 50°C for 6 h. PCL pellets (at 7.5% w/w) were dissolved in 2,2,2-trifluoroethanol (TFE, 99%; Alfa Aesar, USA) and stirred at 25 °C (room temperature) for 10 h.
To fabricate CHIM, the chitosan solution and PCL solution were blended at a weight ratio of 1:3. Due to acidic hydrolysis and degradation of PCL caused by TFA, the blended solution should be prepared just before electrospinning; the blended solution retains its electrospinnability for up to 1 h after preparation.[16] The blended solution was loaded into a gastight syringe (Hamilton, USA) and ejected through a 21-gauge needle at a constant flow rate (1.5 mL/h) using a syringe pump (KDS200, KD Scientific, USA). Next, a high voltage (19 kV) was applied between the metal capillary and a metal collector using a power supply (HV30, NanoNC, Korea) at a vertical distance of 14 cm. During electrospinning for 20–30 min, a relative humidity of 50–60% and a temperature of 20–25°C were maintained. The thickness of the fabricated membranes was within 40–50 µm, as measured using a micrometer (Mitutoyo, Japan). PCLM was fabricated using the same electrospinning conditions, by loading the PCL solution into the gastight syringe.
4.3. Electrospun nanofiber membrane treatment for in vivo implantation
Before in vivo implantation, the CHIM need to be neutralized and sterilized.[17] The prepared CHIM was desiccated for 24 h to evaporate residual toxic solvent, then neutralized in ammonium hydroxide (14% by weight) (OCI Company, Korea) for 15 min and rinsed with deionized water three times to remove the remaining ammonium hydroxide. Then, the CHIM was then sterilized using 70% ethanol for 15 min and in fresh 70% ethanol for an additional 45 min, sequentially. The CHIM was rinsed with deionized water three times for three min each, followed by rinsing with phosphate-buffered saline (PBS, Hyclone, USA) three times for three min each. Subsequently, the CHIM was immersed in fresh PBS overnight at 4°C. Prior to in vivo implantation, the CHIM was transferred to fresh PBS.
4.4. Measurement of contact angle
To investigate the surface wettability of the electrospun nanofiber membranes, 10 µL of deionized water was placed on the membrane. The SmartDrop device (Femtobiomed, Korea) was used to measure the static contact angle of the sessile water droplet on the membrane surface at room temperature.
4.5. Quantification of fiber diameter
To examine the structure of the electrospun membranes, the membranes were dried, and sputter coated with Au–Pd at 10 mA for three min. SEM (FE-SEM SU6600, Hitachi, Japan) images of the membrane were obtained at an accelerating voltage of 15 kV. The membrane fiber diameter was quantified from the SEM images using ImageJ software (NIH, USA).
4.6. Mechanical properties
To investigate the mechanical properties, the electrospun membrane was trimmed into a dog-bone-shaped specimen using a laser cutter. The specimen, which was downsized based on the ASTM D638 standards, had a 6 mm gauge length, 4 mm width, and 50 µm thickness. Each end of the specimen was gripped in a clamp and its tensile properties was tested at a constant tensile speed of 10 mm min− 1, using a customized testing machine comprising a linear actuator and a load cell with a resolution of 0.01 gf.[18] The resultant force–distance curve was converted to a stress–strain curve for evaluating the elastic modulus, ultimate tensile strength, and resilience.
4.7. In vivo assessment
Six Sprague-Dawley male rats (approximately 300–450 g, approximately 8–9 months old) were used for the in vivo study. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at POSTECH and performed in strict accordance with the recommendations in the Guide for IACUC guidelines (IACUC permit No. POSTECH-2018-0031). In addition, the study was carried out in compliance with the ARRIVE guidelines. The rats were categorized into two groups (n = 5 each): the first group received a biotrachea scaffold only (control), while the second group a biotrachea scaffold surrounded by the CHIM. The rats were anesthetized using isoflurane (induced at 4% and maintained at 2%), shaved, and administered subcutaneous injection of buprenorphine (0.6 mg kg− 1). The animals were positioned in sternal recumbency for dorsal implantation. After scrubbing with betadine, dorsal side incisions (10 mm) were made over the posterior region. Twelve scaffolds were inserted subcutaneously on both sides.
4.8. Histological examination
Two weeks after implantation, the rats were sacrificed according to the euthanasia guidelines adapted from the Veterinary Medical Association Guidelines for the Euthanasia of Animals. Harvested samples were embedded in paraffin and slides were stained with hematoxylin and eosin and Alcian blue. The stained specimens were visualized using a microscope. Tracheal structure, chondrocyte density, and inflammatory cell infiltration were evaluated by a pathologist blinded to the treatment modalities. Chondrocyte density was quantified as the number of chondrocytes in three randomly selected fields per slide viewed at 10× magnification. The intensity of Alcian blue staining was measured using ImageJ software.
4.9. Distribution of pores on tracheal construct
To observe morphological changes in the 3D-printed scaffolds during degradation, the surface structures and pore sizes were observed using SEM (FE-SEM SU6600, Hitachi, Japan). Pore size was measured using ImageJ software.
4.10 Statistical analysis
All statistical data are expressed as mean ± standard deviation. Data were analyzed using two-way analysis of variance followed by post-hoc Tukey test. Results with p < 0.05 were considered statistically significant.
4.11 Study approval
All procedures involving human subjects were approved by the Institutional Review Board of the Catholic Medical Center Clinical Research Coordinating Center (KC08TISS0341) and conducted in accordance with relevant guidelines and regulations[6]. Investigations were conducted according to the principles expressed in the Declaration of Helsinki, and the written informed consents from the donors were obtained. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at POSTECH and performed in strict accordance with the recommendations in the Guide for IACUC guidelines and regulations (IACUC permit No. POSTECH-2018-0031).