Data Stewardship Maturity Scoreboard and Rating Diagram Generator - PC version

A template to generate data stewardship maturity scoreboard and rating diagram with the assessment ratings for an individual dataset

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

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

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

Optimization of Liver Decellularization Maintains Extracellular Matrix Micro-Architecture and Composition Predisposing to Effective Cell Seeding

<div><p>Hepatic tissue engineering using decellularized scaffolds is a potential therapeutic alternative to conventional transplantation. However, scaffolds are usually obtained using decellularization protocols that destroy the extracellular matrix (ECM) and hamper clinical translation. We aim to develop a decellularization technique that reliably maintains hepatic microarchitecture and ECM components. Isolated rat livers were decellularized by detergent-enzymatic technique with (EDTA-DET) or without EDTA (DET). Histology, DNA quantification and proteomics confirmed decellularization with further DNA reduction with the addition of EDTA. Quantification, histology, immunostaining, and proteomics demonstrated preservation of extracellular matrix components in both scaffolds with a higher amount of collagen and glycosaminoglycans in the EDTA-DET scaffold. Scanning electron microscopy and X-ray phase contrast imaging showed microarchitecture preservation, with EDTA-DET scaffolds more tightly packed. DET scaffold seeding with a hepatocellular cell line demonstrated complete repopulation in 14 days, with cells proliferating at that time. Decellularization using DET preserves microarchitecture and extracellular matrix components whilst allowing for cell growth for up to 14 days. Addition of EDTA creates a denser, more compact matrix. Transplantation of the scaffolds and scaling up of the methodology are the next steps for successful hepatic tissue engineering.</p></div

Immunostaining demonstrates the preservation of ECM proteins in the decellularized scaffolds.

<p>(A) Collagen I staining in fresh tissue was positive as fine strands in the parenchymal space as well as around the blood vessels (asterisk). This was preserved following decellularization with a strong signal from vascular structures both in DET and EDTA-DET scaffolds. (B, C) Collagen III and collagen IV staining demonstrated a similar distribution and preservation in fresh tissue and decellularized scaffolds. Weak parenchymal staining was observed, and a strong signal surrounding vascular and biliary structures. EDTA-DET scaffolds demonstrated slightly increased staining in the parenchymal space when compared to DET scaffolds. (D) Fibronectin showed strong staining around the main blood vessels in fresh tissue with a more distributed signal pattern in decellularized scaffolds. (E) Laminin showed strong staining around the main blood vessels in fresh tissue with a more distributed signal pattern in decellularized scaffolds; scale bar: 100 μm.</p

Decellularization of the rat liver is achieved following one cycle of DET and EDTA-DET treatments.

<p>(A) Timeframe and infusion of solutions for the DET and EDTA-DET protocols. (B) Macroscopic appearance of the liver scaffolds showed no difference between the DET and EDTA-DET scaffolds. Following dH₂O addition, the livers became blanched, with SDC and DNase addition resulting in the livers becoming transparent. (C) H&E staining demonstrated absence of cells in sections from DET and EDTA-DET scaffolds. (D) DNA quantification reduced DNA in DET and EDTA-DET scaffolds compared with fresh tissue (p<0.001). (E-F) Histograms showing the signal intensities, associated to relevant nuclear proteins (E) and cytoplasmic proteins (F); ‡: p<0.001, compared to fresh tissue, #: p<0.001, compared to DET scaffold, scale bar in macroscopic images: 2cm, scale bar on histology: 100μm.</p

Addition of EDTA to the protocol makes the matrix more compact in the parenchyma and vasculature.

<p>(A-C) SEM confirmed acellularity in the scaffolds, demonstrating composition by a three-dimensional network of connective tissue fibers arranged in a honeycomb-like manner. The structure of the portal triads was identified clearly at low magnification (asterisk). (C) Scaffolds prepared with EDTA-DET were more tightly packed compared to the DET scaffolds, as the porous structure appeared to be compressed. (D) Quantification of the hepatocyte pockets in the EDTA-DET scaffold showed a reduction to approximately 50% of the size in the DET scaffolds. (E, F) Synchrotron-based x-ray phase contrast imaging corroborated these results, demonstrating a denser scaffold in scaffolds pre-treated with EDTA. (G) Infusion of trypan blue dye via the portal vein showed maintenance of the vascular network architecture. There was no dye leakage through the walls to the surrounding tissue; the dye followed the regular pathway of flow to the IVC. Macroscopically, the DET scaffolds were seen to possess a denser vascular network with the dye diffusing through the vessels more readily. (H) Dye intensity within the DET scaffolds followed an S-shaped curve, reaching a maximum point at approximately 35 seconds. Dye intensity within the EDTA-DET scaffold increased in a less steep S-shaped manner with the exponential part lasting 45 seconds instead of 15. Maximum intensity was reached at 75 seconds. (I) These results were paralleled by the quantification of surface area of dye distribution with the point of maximal surface area coverage having a difference of 40 seconds between the two scaffolds; #: p<0.001, compared to DET scaffold, scale bar: 1cm.</p