Assessment of Decellularized Scaffolds for Skeletal Muscle Engineering
Author
Krzyslak, Hubert
Term
4. term
Publication year
2022
Pages
42
Abstract
Traumatiske skeletmuskelsskader er hyppige og behandles i dag med løsninger, der ofte er utilstrækkelige. Decellulariserede biomaterialer og den ekstracellulære matrix (ECM) aflejret af humane adipose-afledte stamceller (hASC) er lovende alternativer, hvor fibrets orientering antages at være vigtig for muskelcellers modning. Dette projekt havde til formål at undersøge, om hASC-aflejret ECM på orienterede (alignerede) kontra tilfældigt orienterede elektrosppundne fibermåtter kan understøtte og styre differentiering af skeletmuskelceller. hASC blev dyrket til at aflejre ECM på alignerede eller tilfældige fibermåtter, hvorefter stilladserne blev decellulariseret. ECM-sammensætning blev vurderet ved farvebaseret kollagenmåling, immunfluorescens for kollagen I, VI og fibronectin samt qPCR for generne kollagen I, VI, fibronectin og decorin. C2C12-myoblaster blev efterfølgende dyrket på de decellulariserede stilladser, og differentiering og orientering blev vurderet ved farvninger; som pilot blev humane skeletmuskel-myoblaster (hSkMM) også testet. ECM fulgte underlagets retning på de alignerede stilladser, men både kollagenindhold og proteinforekomst (kollagen I, VI, fibronectin) var reduceret, og genudtryk var lavere dag 1 (vedvarende for kollagen I og decorin dag 3). C2C12-myotuber var mere orienterede på alignerede stilladser, men blev samtidig tyndere, kortere og færre på begge scaffoldtyper. Overordnet var C2C12-differentiering kun let forbedret med ECM ift. uden ECM, og alignerede ECM-stilladser gav ikke bedre effekt end ECM på flade underlag. Pilotforsøg med hSkMM viste derimod lovende vækst og differentiering på ECM og peger på en relevant retning for fremtidige studier.
Traumatic skeletal muscle injuries are common and current treatments often fall short. Decellularized biomaterials and extracellular matrix (ECM) deposited by human adipose-derived stem cells (hASC) are promising, with fiber alignment considered important for muscle cell maturation. This project aimed to test whether hASC-deposited ECM on aligned versus randomly oriented electrospun fibers supports and guides skeletal muscle cell differentiation. hASC were cultured to deposit ECM on aligned or random fibrous substrates, which were then decellularized. ECM composition was evaluated by a colorimetric collagen assay, immunofluorescence for collagen I, collagen VI and fibronectin, and qPCR for collagen I, collagen VI, fibronectin and decorin. C2C12 myoblasts were subsequently seeded on the decellularized scaffolds and their differentiation and alignment were assessed by staining; as a pilot, human skeletal muscle myoblasts (hSkMM) were also tested. ECM alignment mirrored the substrate on aligned scaffolds, but both collagen content and protein levels (collagen I, VI, fibronectin) were reduced, and gene expression decreased at day 1 (with collagen I and decorin remaining reduced at day 3). C2C12 myotubes aligned more on aligned scaffolds but became thinner, shorter and less abundant on both scaffold types. Overall, C2C12 differentiation was only slightly improved with ECM compared to without ECM, and aligned ECM-coated scaffolds did not outperform ECM on flat substrates. In contrast, the pilot with hSkMM showed encouraging growth and differentiation on ECM, suggesting a relevant direction for future studies.
[This summary has been generated with the help of AI directly from the project (PDF)]
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