Optimization of a decellularization protocol of porcine tracheas. Long-term effects of cryopreservation. A histological study

Milián, Lara; Sancho‐Tello, María; Roig-Soriano, Joan; Foschini, Giovanna; Martínez-Hernández, Néstor J.; Más-Estellés, Jorge; Ruiz‐Saurí, Amparo; Carda, Javier Zurriaga; Cardá, Carmen; Mata, Manuel

doi:10.1177/03913988211008912

Cited by 7 publications

(8 citation statements)

References 32 publications

(53 reference statements)

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“…It allots a different score from 0 to 4 depending on the findings observed per high‐power field (400x) 20 . Decellularization was assessed through DAPI staining, and also by determination of DNA content as previously reported 18 …”

Section: Methodsmentioning

confidence: 99%

“…20 Decellularization was assessed through DAPI staining, and also by determination of DNA content as previously reported. 18 The expression of CD31 as a marker of neovascularization was evaluated by immunohistochemistry, using specific mouse anti-human predicted for rabbit antibody (ab212712; Abcam; Cambridge, United Kingdom). Immunohistochemistry was carried out following standard procedures.…”

Section: Histological Study Of Tracheal Replacementsmentioning

confidence: 99%

“…Tracheas were decellularized following previously published protocol. 18 Briefly, tracheas were submerged in a solution of PBS (phosphate buffered saline) containing 2% SDS (sodium dodecyl sulfate: Sigma-Aldrich, Missouri, United States), 5% penicillin-streptomycin, and 5% amphotericin B and they were maintained under stirring for 5 weeks at room temperature (RT). Each week the decellularization solution was replaced carrying out 2-hour osmotic shock with distilled water.…”

Section: Tracheal Decellularizationmentioning

confidence: 99%

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Decellularized tracheal prelamination implant: A proposed bilateral double organ technique

et al. 2021

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In tracheal replacement transplantation, prelamination is a critical stage. Nowadays, the most widely used prelamination technique is the prethoracic fascia flap with lateral thoracic artery. We propose a flap based on the internal thoracic artery, which allows a relatively non‐aggressive double organ implant, and we have tested its efficacy in decellularized tracheas. Tracheas of albino New Zealand rabbits were decellularized following a protocol that uses detergents and cryogenization, sterilized with 1kGy gamma radiation, and tutorized with a stent. Bilateral pedicled flaps made of pectoral fascia and a muscular component were harvested through a longitudinal 3‐cm central thoracic incision, wrapping the tracheas with them in 16 rabbits, remaining them implanted for 2, 4, 8, and 12 weeks. The tracheas were then studied histologically using standard stainings plus immunohistochemistry (CD31). The models were adjusted with Bayesian statistics using ordinal regression; results as odds ratios and credibility intervals. All analysis were performed using R software. Acute inflammatory cell invasion was observed at 2 weeks, which almost disappeared at week 8 after implant. Only macrophages and giant cells increased between Weeks 8 and 12 (OR 10.487, CI [1.603‐97.327]). The cartilage maintained its structure, with slight signs of ischemia in a few cases. New CD31‐positive vessels were observed from Week 2 and increasing thereafter, reaching a maximum peak at Week 8. We propose a bilateral implant technique that is viable and effective as a prelamination option for two concurrent tracheas, achieving perfect vascularization and integration of the organ with hardly any inflammatory response in the medium or long term.

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Section: Methodsmentioning

confidence: 99%

Section: Histological Study Of Tracheal Replacementsmentioning

confidence: 99%

Section: Tracheal Decellularizationmentioning

confidence: 99%

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Decellularized tracheal prelamination implant: A proposed bilateral double organ technique

et al. 2021

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“…As mentioned previously, depending on the selected tissue, decellularization protocols can differ. In other MSK tissues, such as cartilage [ 56 , 57 , 58 , 59 , 60 , 61 ] and ligaments [ 43 , 62 , 63 , 64 , 65 , 66 , 67 ], similar protocols can be used with different outcomes. However, the results have been shown to change when applying the decellularization protocol with a different harvest site of the same tissue [ 56 ].…”

Section: Decellularization Protocols For Tendonsmentioning

confidence: 99%

Decellularized Extracellular Matrix-Based Bioinks for Tendon Regeneration in Three-Dimensional Bioprinting

Khalak

García‐Villén

Ruiz‐Alonso

et al. 2022

IJMS

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In the last few years, attempts to improve the regeneration of damaged tendons have been rising due to the growing demand. However, current treatments to restore the original performance of the tissue focus on the usage of grafts; although, actual grafts are deficient because they often cannot provide enough support for tissue regeneration, leading to additional complications. The beneficial effect of combining 3D bioprinting and dECM as a novel bioink biomaterial has recently been described. Tendon dECMs have been obtained by using either chemical, biological, or/and physical treatments. Although decellularization protocols are not yet standardized, recently, different protocols have been published. New therapeutic approaches embrace the use of dECM in bioinks for 3D bioprinting, as it has shown promising results in mimicking the composition and the structure of the tissue. However, major obstacles include the poor structural integrity and slow gelation properties of dECM bioinks. Moreover, printing parameters such as speed and temperature have to be optimized for each dECM bioink. Here, we show that dECM bioink for 3D bioprinting provides a promising approach for tendon regeneration for future clinical applications.

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“…Scar tissue formation with graft stenosis and obstruction, not adequate biomechanical characteristics, required stenting to avoid collapse, lifelong immunosuppressive therapies, and technical surgical challenges were included among the main critical issues [ 12 ]. Additionally, as supported by preclinical and early clinical evidence, incomplete or even absent re-epithelization of the graft was possibly encountered, responsible for impaired mucus clearance, recurrent infections, and consequent implant failure [ 13 , 14 ]. Despite the need for further investigation, two alternatives may be promising due to a specific anatomical origin and possibly reduced/absent immunogenicity, which also makes them suitable for cancer patients who cannot tolerate immunosuppressive therapies [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

Development and In Vitro/In Vivo Comparative Characterization of Cryopreserved and Decellularized Tracheal Grafts

Stocco

Barbon

Mammana

et al. 2023

Cells

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Tracheal reconstruction represents a challenge when primary anastomosis is not feasible. Within this scenario, the study aim was to develop a new pig-derived decellularized trachea (DecellT) to be compared with the cryopreserved counterpart (CryoT) for a close predictive analysis. Tracheal segments underwent decellularization by a physical + enzymatic + chemical method (12 cycles); in parallel, cryopreserved samples were also prepared. Once decellularized (histology/DNA quantification), the two groups were characterized for Alpha-Gal epitopes/structural proteins (immunohistochemistry/histology/biochemical assays/second harmonic generation microscopy)/ultrastructure (Scanning Electron Microscopy (SEM))/mechanical behaviour. Cytotoxicity absence was assessed in vitro (extract-test assay/direct seeding, HM1SV40 cell line) while biocompatibility was verified in BALB/c mice, followed by histological/immunohistochemical analyses and SEM (14 days). Decellularization effectively removed Alpha-Gal epitopes; cartilage histoarchitecture was retained in both groups, showing chondrocytes only in the CryoT. Cryopreservation maintained few respiratory epithelium sparse cilia, not detectable in DecellT. Focusing on ECM, preserved structural/ultrastructural organization and collagen content were observed in the cartilage of both; conversely, the GAGs were significantly reduced in DecellT, as confirmed by mechanical study results. No cytotoxicity was highlighted by CryoT/DecellT in vitro, as they were also corroborated by a biocompatibility assay. Despite some limitations (cells presence/GAGs reduction), CryoT/DecellT are both appealing options, which warrant further investigation in comparative in vivo studies.

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Optimization of a decellularization protocol of porcine tracheas. Long-term effects of cryopreservation. A histological study

Cited by 7 publications

References 32 publications

Decellularized tracheal prelamination implant: A proposed bilateral double organ technique

Decellularized tracheal prelamination implant: A proposed bilateral double organ technique

Decellularized Extracellular Matrix-Based Bioinks for Tendon Regeneration in Three-Dimensional Bioprinting

Development and In Vitro/In Vivo Comparative Characterization of Cryopreserved and Decellularized Tracheal Grafts

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