Quantification of extracellular matrix components in samples stored with the SCM protocol.

<p>Quantification of extracellular matrix components in samples stored with the SCM protocol.</p

Composition and mechanical properties of decellularised scaffolds after storage.

<p>(A) Masson’s Trichrome staining demonstrated a progressive loss of architecture in 4°C-treated scaffolds. (B) Elastin staining showed maintenance of this protein in SCM scaffolds. Elastin was progressively lost in 4°C scaffolds. (C) Alcian Blue staining showed glycosaminoglycan maintenance in both storage methods. (D) Samples stored for 6 months with SCM maintained comparable ultimate tensile stress with 2 week-stored scaffolds despite a decreased Young's modulus with no impact on the maximum stress that the material could withstand. While 4°C samples at 2 weeks showed similar values to SCM, prolonged 4°C storage had a profound impact on the scaffold with a reduction of both values. *p<0.05 (bar = 100μm). SCM = slow cooling medium, 4°C = 4°C in PBS.</p

Functional analysis of decellularised scaffolds.

<p>(A,B) Synchrotron analysis after 2 DET cycles confirmed extracellular matrix preservation in the lamina propria, submucosa and muscularis. (C) An intact basement membrane was detected across the entire scaffold segment. (D) Blood vessels were seen converging towards the scaffold in a spoked wheel manner after 10 days from placement on the CAM as confirmed by blinded quantification of the vessels compared to the negative control (**p< 0.01). (E) The maximum tensile stress at which the samples broke and the elasticity modulus remained comparable to fresh after decellularisation. (F) Characteristic stress-strain curves showing that by increasing the number of DET cycles the tensile stress at which the samples break remains the same as seen in E. DET = Detergent-Enzymatic Treatment, CAM = Chicken chorioallantoic membrane assay.</p

Decellularisation efficiency and scaffolds characterization.

<p>(A) Efficient cell removal after 2 DET cycles was evident by macroscopic appearance, H&E staining and DNA quantification. (B) Immunohistochemistry for extracellular matrix composition. Masson’s Trichrome and Picrosirius Red staining demonstrated collagen preservation in both submucosa and among muscle fibers (inset). Elastin Van Gieson staining showed elastic fibers in the submucosa, around blood vessels and surrounding muscle fascicles both in the fresh and DET tissues. Muscular elastin staining was reduced after 3 DET cycles (inset). Alcian Blue staining indicated glycosaminoglycan preservation (bar = 100μm). (C) Extracellular component quantification demonstrated a gradual decrease in collagen after the first and second DET cycle. Elastin decreased after 3 DET cycles. glycosaminoglycan were partially reduced by the first DET cycle. DET = Detergent-Enzymatic Treatment, MT = Masson’s Trichrome, PR = Picrosirius Red, EVG = Elastin Van Gieson, AB = Alcian Blue. *p<0.05; **p<0.01.</p

Macro- and microscopic appearance of stored decellularised scaffolds at 6 months.

<p>(A) Macroscopic appearances varied in the two protocols. (B) Scanning electron microscopy analysis. While SCM scaffolds demonstrated a good preservation of all oesophageal layers, in 4°C samples extracellular matrix was falling apart with signs of degradation. SCM = slow cooling medium, 4°C = 4°C in PBS.</p