Abstract:The development of more and more new dermal substitutes requires a reliable and effective animal model to evaluate their safety and efficacy. In this study we constructed a novel animal model using co-transplantation of autologous epidermal sheets with dermal substitutes to repair full-thickness skin defects. Autologous epidermal sheets were obtained by digesting the basement membrane (BM) and dermal components from rat split-thickness skins in Dispase II solution (1.2 u/ml) at 4°C for 8, 10 and 12 h. H&E, imm… Show more
“…As shown only a few epithelium cells remained attached to the LP indicating the separation of the layers occurred in the basement membrane. The specificity of the enzyme [33, 34] allowed us to isolate the epithelium layer from the LP without undue force to reduce tearing. Technical variation between samples due to the dissection procedure was accounted for by biological and technical replication described in the methods.Fig.…”
BackgroundThe rumen wall plays a major role in efficient transfer of digested nutrients in the rumen to peripheral tissues through the portal venous system. Some of these substrates are metabolised in the epithelium during this process. To identify the specific proteins involved in these processes, we used proteomic technologies. Protein extracts were prepared from ventral rumen tissue of six sheep fed a fibrous diet at 1.5× maintenance energy requirements. Using a newly developed method, we were able to enzymatically isolate the epithelial cells from underlying tissue layers, thus allowing cytosol and membrane fractions to be independently analysed using liquid chromatography tandem mass spectrometry (LC MS/MS).ResultsUsing our procedure we identified 570 epithelial proteins in the Ovis aries sequence database. Subcellular locations were largely cytosolic (n = 221) and extracellular (n = 85). However, a quarter of the proteins identified were assigned to the plasma membrane or organelle membranes, some of which transport nutrients and metabolites. Of these 91 were transmembrane proteins (TMHMM), 27 had an N-terminal signal peptide (signalP) and TMHMM motif, 13 had a glycosylphosphatidylinositol (GPI) anchor and signalP sequence, 67 had beta (β) strands or 17 β strands and a transit peptide sequence, indicating the identified proteins were integral or peripheral membrane proteins. Subunits of the 5 protein complexes involved in mitochondrial cellular energy production were well represented. Structural proteins (15%), proteins involved in the metabolism of lipids and proteins (26%) and those with steroid or cytokine action were a feature of the proteome.ConclusionOur research has developed a procedure to isolate rumen epithelium proteins from the underlying tissue layers so that they may be profiled using proteomic technologies. The approach improves the number of proteins that can be profiled that are specific to the epithelium of the rumen wall. It provides new insights into the proteins of structural and nutritional importance in the rumen epithelium, that carry out nutrient transport and metabolism, cell growth and signalling.Electronic supplementary materialThe online version of this article (10.1186/s40104-019-0314-0) contains supplementary material, which is available to authorized users.
“…As shown only a few epithelium cells remained attached to the LP indicating the separation of the layers occurred in the basement membrane. The specificity of the enzyme [33, 34] allowed us to isolate the epithelium layer from the LP without undue force to reduce tearing. Technical variation between samples due to the dissection procedure was accounted for by biological and technical replication described in the methods.Fig.…”
BackgroundThe rumen wall plays a major role in efficient transfer of digested nutrients in the rumen to peripheral tissues through the portal venous system. Some of these substrates are metabolised in the epithelium during this process. To identify the specific proteins involved in these processes, we used proteomic technologies. Protein extracts were prepared from ventral rumen tissue of six sheep fed a fibrous diet at 1.5× maintenance energy requirements. Using a newly developed method, we were able to enzymatically isolate the epithelial cells from underlying tissue layers, thus allowing cytosol and membrane fractions to be independently analysed using liquid chromatography tandem mass spectrometry (LC MS/MS).ResultsUsing our procedure we identified 570 epithelial proteins in the Ovis aries sequence database. Subcellular locations were largely cytosolic (n = 221) and extracellular (n = 85). However, a quarter of the proteins identified were assigned to the plasma membrane or organelle membranes, some of which transport nutrients and metabolites. Of these 91 were transmembrane proteins (TMHMM), 27 had an N-terminal signal peptide (signalP) and TMHMM motif, 13 had a glycosylphosphatidylinositol (GPI) anchor and signalP sequence, 67 had beta (β) strands or 17 β strands and a transit peptide sequence, indicating the identified proteins were integral or peripheral membrane proteins. Subunits of the 5 protein complexes involved in mitochondrial cellular energy production were well represented. Structural proteins (15%), proteins involved in the metabolism of lipids and proteins (26%) and those with steroid or cytokine action were a feature of the proteome.ConclusionOur research has developed a procedure to isolate rumen epithelium proteins from the underlying tissue layers so that they may be profiled using proteomic technologies. The approach improves the number of proteins that can be profiled that are specific to the epithelium of the rumen wall. It provides new insights into the proteins of structural and nutritional importance in the rumen epithelium, that carry out nutrient transport and metabolism, cell growth and signalling.Electronic supplementary materialThe online version of this article (10.1186/s40104-019-0314-0) contains supplementary material, which is available to authorized users.
“…Epidermis and dermis samples from abdominal skins were prepared by Dispase II (Sigma) treatment according to the method of Huang et al . 57 . Epidermis or dermis samples were cut into small pieces and used for the determination of GAGs and polyamine analysis ( Supplementary Table S1 ).…”
Proteoglycans (PGs), a family of glycosaminoglycan (GAG)-protein glycoconjugates, contribute to animal physiology through interactions between their glycan chains and growth factors, chemokines and adhesion molecules. However, it remains unclear how GAG structures are changed during the aging process. Here, we found that polyamine levels are correlated with the expression level of heparan sulfate (HS) in human skin. In cultured cell lines, the EXT1 and EXT2 enzymes, initiating HS biosynthesis, were stimulated at the translational level by polyamines. Interestingly, the initiation codon recognition by 43S preinitiation complex during EXT2 translation is suppressed by let-7b, a member of the let-7 microRNA family, through binding at the N-terminal amino acid coding sequence in EXT2 mRNA. Let-7b-mediated suppression of initiation codon depends on the length of 5′-UTR of EXT2 mRNA and its suppression is inhibited in the presence of polyamines. These findings provide new insights into the HS biosynthesis related to miRNA and polyamines.
“…Maximising wound coverage with available donor site involves thin, widely-meshed, or expanded (Meek-Wall technique) ( 24 ) skin autografts, resulting in poor functional and aesthetic outcomes. In addition, donor sites generated by split-thickness skin graft harvesting are extremely painful, may require opiate analgesia, limit mobilisation, and discourage compliance with physical therapy ( 25 ). Skin autografts, however, have properties that promote their continued widespread use for the closure of large, deep skin wounds (no rejection, vascular inosculation, high efficacy, long-term stability), but their correlation with increased morbidity, especially in the elderly, is a significant disadvantage, which motivates the search for alternatives ( 26 , 27 ).…”
Skin tissue bioengineering is an emerging field that brings together interdisciplinary teams to promote successful translation to clinical care. Extensive deep tissue injuries, such as large burns and other major skin loss conditions, are medical indications where bioengineered skin substitutes (that restore both dermal and epidermal tissues) are being studied as alternatives. These may not only reduce mortality but also lessen morbidity to improve quality of life and functional outcome compared with the current standards of care. A common objective of dermal-epidermal therapies is to reduce the time required to accomplish stable closure of wounds with minimal scar in patients with insufficient donor sites for autologous split-thickness skin grafts. However, no commercially-available product has yet fully satisfied this objective. Tissue engineered skin may include cells, biopolymer scaffolds and drugs, and requires regulatory review to demonstrate safety and efficacy. They must be scalable for manufacturing and distribution. The advancement of technology and the introduction of bioreactors and bio-printing for skin tissue engineering may facilitate clinical products' availability. This mini-review elucidates the reasons for the few available commercial skin substitutes. In addition, it provides insights into the challenges faced by surgeons and scientists to develop new therapies and deliver the results of translational research to improve patient care.
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