PBS + SDS 2% + penicillin-streptomycin 5% + amphotericin B 5% for 5 weeks under continual stirring
(1) HE: evaluating the decellularization process showed minimal chondrocyte debris in cartilage, and DNA quantification did not detect values >50 ng (almost 100% cell removal compared to fresh trachea) (2) Biomechanical test: using the tensile test and the radial compression test showed reduced biomechanical properties vs. native trachea
Group A: control group Group B: SDS decellularized only Group C: −80°C storage for 3 months only Group D: −80°C storage for 3 months and then decellularized Group E: after decellularization and then put into −80°C storage Decellularization solution: SDS 3%
(1) HE: group C still retained lots of nuclei, and groups B, D, and E have removed tracheal columnar epithelium cells and mucosal and submucosal, but ECM matrix still existed (2) MT: groups B, D, and E have removed epithelial layer, and all groups have enough ECM matrix (3) DAPI: compared with the control group, the experimental group (groups B, C, D, and E) did not significantly affect the cell nucleus in cartilage tissue (4) Safranin-O: there was no significant difference in the staining of the tracheal cartilage in the cryopreservation group compared with the control group (5) DNA quantification: group E (33.66 ng/mg), D (53.16), B (110.33), C (306.83), A (421 ng/mg) (6) Biomechanical test: decreased compressive strength in group E; tensile strength for all groups still good (within 50% tensile displacement, no fracture was found and the shape was preserved)
Lyophilization and DEM combination Group 1: LYP Group 2: LYP + DNase (150 U/mL) + MgSO4 (50 mmol) + LYP Group 3: LYP + deoxycholic acid + DNase + MgSO4 + LYP Group 4: LYP + deoxycholic acid + Triton X-100 + DNase + MgSO4 + LYP Group 5: LYP + SDS+1% (w/v) Triton X-100 + LYP Group 6: LYP + SDS + PBS + DNase + LYP Group 7: control: untreated trachea
(1) DNA concentration: <50 ng/mg (2) GAG: the cartilage matrix significantly decreased after decellularization and significantly different except group 3 (3) HE: group 1 with most cells retained, same with 4 and 6 (4) SEM: no cellular components on luminal surface of groups 2 and 6 and preserved cartilage matrix in group 6 (5) Biomechanical test: no different with the normal trachea but rigidities groups 1, 3, and 5 were higher
Decell A method: (i) Antibiotic rinse (ii) Perfused 50 ml/min: 30 min 0.0035% Triton X; 10 min 1M NaCl, 30 min PBS, 1.5 h 0.1% SDC, 30 min PBS, 1 hr 15 IU/ml benzonase in buffer, 30 min PBS, 10 min 0.5% Triton X, 4 × 20 min PBS Decell B method: (i) Antibiotic rinse (ii) 25x cycles perfused 50 ml/min: 4 h 4% SDC, 3 × 5 min diH2O, 3 h 2000 kU DNase-l in 1M NaCl, 2 × 5 min diH2O (iii) Store in PBS at 4C between cycles
(1) Cellular removal method B is harsher; complete cellular removal but DNA quantification in both reaches the goals but method A is better since it removes epithelial and stromal cells, while leaving the cartilage intact, where B removes all including chondrocytes (2) Matrix ECM and collagen are not well preserved in both methods (3) Mechanical structure: stiffer in both groups (4) Decell A produced a stiffer tissue, probably due to intact engaged collagen I in the setting of reduced collagen III. In contrast, Decell B resulted in a tissue with severely compromised ECM structure, including damage to collagen I fibers
Group 1: cryopreservation Group 2: tracheal decell using physical + enzymatic + chemical (i): Freezing and lyophilization for 12 h (ii): dH2O + 0.05% trypsin +0.02% EDTA enzymatic solution (iii): Washing w 2% Tergitol detergent solution + 0.8% NH3OH in dH2O (iv) Washing w dH2O repeated for 12 cycles and then frozen at −20°C and lyophilized
(1) DAPI and DNA quantification: show reduced number of cell and DNA content (2) HE: reduction in CryoT, no cells were detected in Decell T (3) Alcian blue staining: both group still has retained cytoplasmic granules and GAG, less intense in Decell T; however, 56.4% GAG in group B were maintained (4) Masson’s trichrome: all collagen remained intact in all (5) SEM: some cells were still recognizable in CryoT; complete removal of epithelium in decellT, both displayed organized collagen fiber
Tracheae were incubated in a detergent solution containing 0.25% Triton X-100 (Biofroxx, Einhausen, Germany) and 0.25% sodium deoxycholate (Sigma, California, USA) at 37°C for 24 h under vacuum. The scaffolds were then washed in sterile distilled water three times for 30 min and incubated for another 24 h in sterile distilled water at 4°C. Following the wash step, the scaffolds were subjected to enzymatic digestion with 2 kU/mL DNAse (Sigma, California, USA) and 4 U/mL RNAse (Biofroxx, Einhausen, Germany) in 1 M NaCl at 37°C at different times (8 h (VAD 8 h group), 16 h (VAD 16 h group), and 24 h (VAD 24 h group)) as experimental groups. Finally, the decellularized tracheal segments were stored in 4°C PBS containing 1% antibiotic and antimycotic solution
(1) HE: 24 h group showed the least cell nuclei (2) MT: all groups show similar structural integrity (3) Alcian blue: all groups show reduced GAG expression
(1) Hypotonic 10 mM tris buffer containing a serine protease inhibitor (5% phenylmethylsulfonylfluoride in ethanol, 0.35 mL/L), a metalloprotease inhibitor (5 mM ethylenediaminetetraacetic acid (EDTA)), and 5 mL/L penicillin/streptomycin solution (10,000 U/mL/10,000 mg/mL) for 36 h at room temperature (2) High saline (1.5 M potassium chloride), 50 mM tris buffer containing 1% Triton X-100 (octyl phenoxy polyethoxyethanol), as well as protease inhibitors and antibiotics for 48 h at room temperature (3) Rinsed with Hanks’ physiological buffer and then submerged for 5 h at 37°C in Hanks’ buffer containing 90 U/mL deoxyribonuclease (type II from bovine pancreas) and 85 mg/mL ribonuclease (type III A from bovine pancreas). In the fourth stage of the decellularization process, the three protocols differ. Samples were immersed in a 1% solution of either (i) sodium dodecyl sulfate (lauryl sulfate, SDS) identified as Triton-SDS, (ii) Triton X-100 identified as Triton-Triton, or (iii) tributyl phosphate (TnBP) identified as Triton-TnBP. This second wash was carried out with 50 mM Trizma’s base and antibiotics for 48 h at room temperature. Samples were subsequently rinsed with distilled water and then in a second modification of the process, immersed in a 50 mM tris buffer adjusted to pH 9.0 with antibiotics. Specimens remained in the pH 9.0 wash for 24 h at room temperature to remove residual surfactant before being washed in phosphate buffered saline (PBS) containing antibiotics for 24 h
(1) Overall, the DNA content was reduced by an average of 97.14% (fresh tissue mean DNA, 1857.59 ± 98.70 ng/mg and decellularized tissue mean DNA 53.21 ± 17.42 ng/mg; ) (2) The GAG content (n = 4 per run; n = 12 per group) was not significantly affected by the decellularization process (fresh tissue mean GAG 24.96 ± 1.89 μg/mg and decellularized tissue mean GAG 24.17 ± 2.01 μg/mg; ) (3) H&E revealed that cell nuclei in the perichondrium and the peripheral regions of the ECM were absent in the decellularized tissue and the modulus (fresh tissue mean 13.6 ± 1.8 MPa and decellularized tissue mean 17.3 ± 3.5 MPa; ) and ultimate tensile strength (fresh tissue mean 2.5 ± 0.6 MPa and decellularized tissue mean 3.0 ± 0.4 MPa; ) did not significantly change after decellularization
In brief, the samples were placed into cryovials that were immersed into liquid nitrogen for 15 minutes, and samples were then thawed in a water bath at 37°C for 30 minutes. This cycle was repeated 5 times.
(1) Safranin-O staining evidenced a certain loss of GAG only in the regions immediately surrounding the pores but a preserved GAG intense matrix in the remaining regions of the LPTCs (2) DNA content of TCs after the decellularization process averaged 35 ± 12 ng/mg. Results showed that endotoxin concentrations were similarly low (less than 1.0 IE/ml) in the conditioned media and control medium
10 cycles of agitation for 48 h with 30 mL of a 2% sodium deoxycholate detergent and ethylenediaminetetraacetic acid (EDTA) 0.02% in an incubator at 36°C and 180 rpm, followed by 3 washings with phosphate-buffered solution (PBS) for 10 min. At the end of this process, the scaffolds were exposed to 30 mL of DNase solution 3 L of DNase +1 mL of 1.3 mM MgSO4 and 2 mM CaCl2 at 37°C for 12 h under constant agitation
(1) The DNA contents fell from 850 to +123 ng/mg of dry tissue to 20 +8 ng/mg of dry tissue, (2) Cytotoxicity, the cellular viability under the challenge with the homogenate was 42.7% (3) There is an increase in the maximum load and stress in the maximum load in the longitudinal axis, while there was a decrease in the transverse axis. The mechanical properties of the scaffold in PBS solution at 4°C did not show significant differences in the Young modulus, maximum load, and stress at the maximum load (4) There was no significant difference in the percentage of collagen in the trachea before and after decellularization
Group 1: Supercritical carbon dioxide processed at 37°C with 250 bar for 12 hours as a dynamic run (constant flow of CO2) Washed by 25% H2O2 for 60 min Washed by H2O Washed by a 1.25 M NaOH for 30 min Washed by H2O pH set at 7.4 ± 0.2 with 0.25 M NaH2PO4 buffer Group 2: Following the group 1 without NaOH wash Followed by lyophilization Group 3: Following group 2: Subsequently scCO2 sterilized with low dose peracetic acid (4.5 mL freshly prepared PAA with 1.25 mL H2O, 0.75 mL 25% H2O2 and 2.5 mL 2 M acetic acid) with 160 bar for 4 h at 35°C as a static run Control group: Treated with Triton X-100/sodium deoxycholate for 24 h under vacuum conditions followed by a 48 h wash step Freeze dried for 3 days under vacuum conditions
(1) HE: method 1 resulted in efficient chondrocyte nuclei removal compared to other methods (2) Gomori’s trichrome: methods 2 and 3 and DEM were comparable to native trachea. Methods 2 and 3 resulted in more incomplete washout of cellular remnants compared to method 1 (3) Fibronectin and laminin staining: Laminin and fibronectin were not observed in all groups (4) GAG: GAG was best preserved in method 3 (48.8 ± 5.5 μg/mg; native 41.7 ± 4.7 μg/mg), followed by method 2 (33.1 ± 11.0 μg/mg), and significantly reduced in DEM and method 1 (20.3 ± 1.9 μg/mg tissue; 5.7 ± 3.2 μg/mg) (5) SEM: basement membrane was disrupted due to method 3 while staying intact in other methods (6) Collagen was loss in method 2 and DEM (7) DNA quantification: the least DNA was found in DEM (15.2 ± 9.2 ng/mg), followed by methods 1, 2, and 3 (87.7 ± 59.7 ng/mg, 385.7 ± 116.8 ng/mg, 412.6 ± 193.8 ng/mg) (8) Mechanical properties: method 3 (10.48 ± 0.45 ng/mg) has the most similar properties to native (8.8 ± 0.8 ng/mg), followed by DEM, method 2, and 1 (4.7 ± 1.4 ng/mg; 4.2 ± 1.4 ng/mg; 1.3 ± 0.9 ng/mg)
DEM with adjusted DNase I dose: 2 kU/ml 4 kU/mL 6 kU/mL 8 kU/mL
(1) HE: 8 kU resulted in almost completely removal of cartilage nuclei (2) Masson trichrome: no significant change in each group compared to the natives (3) Alician blue: GAG n the matrix was defected more as DNase I concentration increase (4) DAPI: blue fluorescence got dimmer as the DNase I concentration increased, indicating less residual DNA (5) DNA quantitative: DNA content was significantly less as the DNase I concentration increased (6) IHC: IHC showed significantly reduced expression of MHC-I and MHC-II in all groups compared to native trachea (7) sGAG: all groups had significant loss of sGAG as DNase I concentration increased (8) Type II collagen: only 8 kU/mL DNase I has significant loss of collagen compared to native trachea (9) Biomechanical properties: anticompression properties got weaken as DNase I increased but not significant in group 2 kU/mL (10) SEM: 6 kU resulted in small gaps in the basal membrane, 8 kU resulted in larger crack in basal membrane
(1) HE: groups C and E had superior effect on chondrocyte removal, while B and D had lower effect (2) Collagen and GAGs: groups C and D preserved more compared to groups B and E (3) DNA: groups C, D, and E resulted in significant decrease of DNA content (4) Elastic: groups D and E preserved elastic fiber, resulting in a nonsignificant difference with the control group Experiment 2: (1) HE: group H removed the most cells (2) Collagen: group E lost the most collagen, while other groups had minimal effect on the collagen
Mix of physical-chemical process: (1) Freezing and thawing for 10 cycles (i) Frozen at −80°C for 24 hours (ii) Thawed for 40 min in 36°C water bath (iii) Washed with PBS 3x and exposed to 2% sodium deoxycholate (iv) Shaken in the incubator at 180 rpm, 36°C for a 48-hour cycle (2) DNA extractions (i) DNase solution for 12 hours (ii) Proteinase K for 12 hours (iii) Extraction with phenol chloroform
(1) HE: complete denudation of the epithelium and mixed glands over the cartilage: 20 ± 8 ng/mg (2) Marked by reduction of the DNA content (mean DNA content)
Multistep chemical process involving: (1) Solution A, pH 8 in room temperature for 12 hours (i) Trizma base (hypotonic solution) (ii) EDTA (metalloprotease inhibitor) (iii) PMSF (serine protease inhibitor) (iv) Pen/strep (2) Solution B, pH 8 in room temperature for 12 hours (i) Triton X-100 (nonionic surfactant) (ii) EDTA (iii) PMSF (iv) KCl (hypertonic solutions) (v) Trizma base (vi) Pen/strep (3) DNase and RNase, pH 7.35, 37°C for 5.5 hours (4) Solution C, pH 9 in room temperature for 12 hours (i) 70% ethanol (ii) Trizma base (iii) TBP (surfactant like solvent) (iv) Pen/strep (5) Solution pH 9 in room temperature for 22 hours (6) 1% paracetic acid in room temperature for 4 hours (7) Phosphate buffer saline in room temperature for 4 hours
(1) HE: cell nuclei in the perichondrium and the peripheral regions of the ECM were absent in the decellularized tissue (2) Masson’s trichrome: major histoarchitecture of the trachea remained unchanged following decellularization (3) Overall DNA count reduced by average of 97.14% (mean DNA content: 53.21 ± 17.42 ng/mg)
Chemical process using: (1) CHAPS solution (8 mM CHAPS, 1M NaCl, and 25 mM EDTA diluted at 1 × PBS; pH 8) for 22 hours at room temperature (2) PBS 1× washing for 30 min at 4°C (3) SDS solution (1.8 mM SDS, 1 M NaCl, and 25 mM EDTA diluted at 1× PBS; pH 8) for 22 hours at room temperature (4) PBS 1× washing for 30 min at 4°C (5) Place sample in α-MEM/40% FBS at 37 ̊C for 48 hours All steps were done under constant agitation and repeated 3 times
(1) HE: complete removal of nuclear and cellular materials after three cycles of decellularization compared to native samples (2) Masson’s trichrome: sulfated glycosaminoglycans, collagen, and elastin were preserved in decellularized tissue (3) DNA contents post decellularization was significantly lower (4.1 ng DNA/mg dry tissue) compared to native tissue (722.9 ng DNA/mg dry tissue)
Chemical process using: (1) 10 min with 1X DPBS (100 mL) (2) 8 min with 0.25% SDS (w/v) diH2O (40 mL) (3) 8 min with diH2O (4) 8 min with triton X-100 v/v in diH2O (40 mL) (5) 50 min with 1x DPBS (250 mL)
(1) HE: Luminal cells are absent (2) Masson’s trichrome: no difference found between the decellularized group and the control group (3) DNA quantification showed that the decellularized trachea’s DNA count is significantly lower compared to the control group (350 ng/mg)
