Editors
B. Singh
David A. Turner
Raphaël Lévy
Sandrine Willaime-Morawek
Rhiannon Grant, John Hallett, Stuart Forbes, David Hay, Anthony Callanan
PubMed: 31000735 DOI: 10.1038/s41598-019-42627-7
The authors report the preparation and application of electrospun scaffolds incorporating either a single protein (Laminin, collagen or fibronectin) or an extracellular matrix preparation from donated human livers (hlECM) in a poly-L-lactic acid matrix. The hlECM preparation is obtained by decellularization of a small piece of human liver tissue (by extensive washing with 0.25% SDS), followed by freeze drying and milling. The electrospun scaffolds are used to grow THLE-3 cells which are immortalised liver cells. The authors present data on the mechanical properties (Fig 1 and table 1), microstructure (Fig 5), the presence of the proteins on the scaffold (Fig 2), cell numbers and cell viability at 5, 10 and 16 days (Fig 3 and 4), presence of hepatic functional markers (Fig 6 and 7). The authors claim that there is a significant increase in young modulus of the scaffolds incorporating the hlECM, and that that same scaffold exerts an influence on production of albumin and expression of key hepatic genes which is not recapitulated by individual components. Whilst the general motivation of the article line of research is clearly explained (generating tissues for transplantation), the article does not make it easy for the reader to identify exactly what has been done (explained above), why and what are the limitations.
Figure 1 shows how incorporating different proteins (or the hlECM preparation) affects the Young modulus. The experiment is done on 6 individual 10 mm diameter scaffolds for each condition. The results are presented as the mean ± confidence interval. The conclusions the authors draw from these results are that the hlECM scaffold has “significant increase in Young modulus”. We think that the presentation of the data and the overuse of statistics are in fact hiding the main result from this experiment which is the very large scaffold to scaffold variability in mechanical properties. If the 95% confidence interval (with N=6) is ~±25% for a single condition, that indicates that the distribution of individual scaffold mechanical properties is very broad indeed. The authors report a 66% Young Modulus increase between the Polymer only and the hlECM condition but is this plausible? To answer this question, one needs to look at the protocol for preparing the hlECM (or proteins) incorporating fibres. The hlECM (or proteins) is dissolved at 25 microg/mL in 0.25 M acetic acid. The other component of the scaffold, the poly-L-Lactic acid, is dissolved at 22% w/v in HFIP. These two solutions are then mixed in 1:9 ratio and electrospun. From these numbers, one can conclude that there is ~100,000 times more poly-L-Lactic acid than hlECM (or proteins) in the electrospun fibres. Whilst it is possible that such a low percentage might have a biological effect, it is not plausible that a composition variation of the order of 0.00001 could lead to a 66% change in mechanical properties. Therefore the most likely explanation of the results is that it is a random variation due to the variability of electrospun scaffolds (see above) rather than anything related to composition.
Figure 2 is an immunofluorescence microscopy study of the different scaffolds. The authors claim that it demonstrates that the proteins they want to be there are there; i.e. the hBTC1 is positive for collagen 1, etc, and the hlECM condition has Collagen 1, Fibronectin and Laminin. They also claim that recognition by the primary antibodies indicates that the proteins have retained some bioactivity. Before discussing the figure, it is worth noting three shortcomings of the article: 1) there is no characterization of the extracellular matrix preparation so we know nothing about its composition before being incorporated into the scaffold (not even whether it contains Collagen 1, Fibronectin and Laminin); 2) the preparation is called hlECM but based on the protocol it is unclear why there would be more extracellular components than intracellular 3) the electrospinning process is not gentle and it is likely that the proteins are denatured even before getting electrospun given the HFIP /acetic acid solvent. None of these points are discussed in the article. Coming back to the figure, we first note that for single channel microscopy images, it is better practice to present images in gray scale as this is where human eyes are best at distinguishing contrast. Here however, the bigger problem is that the conditions to be compared are presented in different colours: green for the “positive” and red for the “negative”. It is very hard to find a rationale for that choice.
Figure 3 shows that: 1) cells grow, 2) they do grow on all tested scaffold. These are the main results. Statistical analysis is used here again to highlight a very small variation (a tiny increase with hlECM at day 5). Whether that small variation is real or not (i.e. whether it would be confirmed on a much larger scale experiment or not) is largely irrelevant. The effect, even if it is there is clearly of no biological importance. The authors say that reassuringly the same pattern is observed in the DNA concentration but that is not the case: at day 5 there is no difference between DNA per scaffold between the hlECM and polymer-only conditions.
We do not have any particular comments about Figures 4 and 5.
The last two data figures demonstrate the presence of hepatic functional markers at the gene level (Figure 6) and protein level (Figure7). Figure 6 quantifies the mRNA levels of key hepatic genes; Albumin, Cyp1A1, Cyp1A2, Cyp3A4, Collagen I and Collagen IV and Fibronectin. It demonstrates there is little difference between these genes across the different scaffolds at the mRNA level, indicating that the type of protein incorporated into the scaffold has no effect. The authors write Only the human liver hlECM scaffold maintains albumin expression in the expected pattern, increasing over time; as seen in primary hepatocytes and other liver cell lines [40] but their data show no significant difference between the different scaffolds. Incidentally, the choice of a bar chart is really confusing (these are fold differences on a log scale so the bar is really meaningless); with the standard deviation shown as an error bar, it makes it look like a box plot which it is not.
Figure 7 is a plot of serum albumin production over 24 hours, across the different scaffolds at 5, 10 and 16 days. The authors write hBTC1, polymer only and liver ECM all exhibit increasing production of albumin over time (Fig. 7), as expected in a healthy hepatocyte culture and correlating with gene expression patterns observed in Fig. 6. It is in fact particularly difficult to study the correlation between Fig 6 and 7 because Fig 6 is q-PCR presented as fold change compared to polymer only, whilst Fig 7 is absolute protein production. Fig 7 again suffers from statistics overuse. Many of these lines and stars are really not helpful in understanding the results. For example, the authors highlight on the figure (and in the text) a significant difference (**) between the hlECM condition at day 10 and the laminin scaffold (hRL521) at day 10, but this difference is due, not to the superior performance of the hlECM scaffold (smaller than polymer only), but to the unexplained zero value for albumin at day 10 for hRL521. This zero value is clearly an anomaly; it is smaller than the value at day 5 which given that the cells grow is absurd and it does not correlate with the mRNA results.
Overall, for the reasons above, we are not convinced that the results support the conclusions of this article.
Raphaël Lévy and Jonathan Temple
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