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Development of a Basement Membrane Substitute Incorporated Into an Electrospun Scaffold for 3D Skin Tissue Engineering
yesA major challenge in the production of 3D tissue engineered skin is the recreation of the basement
membrane region to promote secure attachment and yet segregation of keratinocytes from
the dermal substitute impregnated with fibroblasts. We have previously shown that simple electrospun
scaffolds provide fibres on which the cells attach, proliferate, and self-sort into epithelium and
dermis. In a development of this in this study tri-layered scaffolds were then electrospun from poly
L-lactic acid and poly hydroxybutyrate-co-hydroxyvalerate. In these a central layer of the scaffolds
comprising nano-porous/nano-fibrous poly hydroxybutyrate-co-hydroxyvalerate fibres was interwoven
into the bulk micro-porous poly L-lactic acid microfibers to mimic the basement membrane.
Keratinocytes and fibroblasts seeded onto these scaffolds and cultured for 2 weeks showed that
neither cell type was able to cross the central nano-porous barrier (shown by SEM, and fluorescence
monitoring with CellTrackerâą) while the micro-fibrous poly L-lactic acid provided a scaffold
on which keratinocytes could create an epithelium and fibroblasts could create a dermal substitute
depositing collagen. Although cells did not penetrate this barrier the interaction of cells was still
evident-essential for epithelial development
Functional characterisation of the amyotrophic lateral sclerosis risk locus GPX3/TNIP1
Background
Amyotrophic lateral sclerosis (ALS) is a complex, late-onset, neurodegenerative disease with a genetic contribution to disease liability. Genome-wide association studies (GWAS) have identified ten risk loci to date, including the TNIP1/GPX3 locus on chromosome five. Given association analysis data alone cannot determine the most plausible risk gene for this locus, we undertook a comprehensive suite of in silico, in vivo and in vitro studies to address this.
Methods
The Functional Mapping and Annotation (FUMA) pipeline and five tools (conditional and joint analysis (GCTA-COJO), Stratified Linkage Disequilibrium Score Regression (S-LDSC), Polygenic Priority Scoring (PoPS), Summary-based Mendelian Randomisation (SMR-HEIDI) and transcriptome-wide association study (TWAS) analyses) were used to perform bioinformatic integration of GWAS data (Ncases = 20,806, Ncontrols = 59,804) with âomics reference datasets including the blood (eQTLgen consortium N = 31,684) and brain (N = 2581). This was followed up by specific expression studies in ALS case-control cohorts (microarray Ntotal = 942, protein Ntotal = 300) and gene knockdown (KD) studies of human neuronal iPSC cells and zebrafish-morpholinos (MO).
Results
SMR analyses implicated both TNIP1 and GPX3 (p < 1.15 Ă 10â6), but there was no simple SNP/expression relationship. Integrating multiple datasets using PoPS supported GPX3 but not TNIP1. In vivo expression analyses from blood in ALS cases identified that lower GPX3 expression correlated with a more progressed disease (ALS functional rating score, p = 5.5 Ă 10â3, adjusted R2 = 0.042, Beffect = 27.4 ± 13.3âng/ml/ALSFRS unit) with microarray and protein data suggesting lower expression with risk allele (recessive model p = 0.06, p = 0.02 respectively). Validation in vivo indicated gpx3 KD caused significant motor deficits in zebrafish-MO (mean difference vs. control ± 95% CI, vs. control, swim distance = 112 ± 28âmm, time = 1.29 ± 0.59 s, speed = 32.0 ± 2.53âmm/s, respectively, p for all <â0.0001), which were rescued with gpx3 expression, with no phenotype identified with tnip1 KD or gpx3 overexpression.
Conclusions
These results support GPX3 as a lead ALS risk gene in this locus, with more data needed to confirm/reject a role for TNIP1. This has implications for understanding disease mechanisms (GPX3 acts in the same pathway as SOD1, a well-established ALS-associated gene) and identifying new therapeutic approaches. Few previous examples of in-depth investigations of risk loci in ALS exist and a similar approach could be applied to investigate future expected GWAS findings