Assessment of Smooth Muscle Cell Differentiation and Cell-laden Biomaterials Properties for Urethral Reconstruction
Author
Pitsa, Aikaterini Chara
Term
4. term
Publication year
2019
Submitted on
2019-05-30
Pages
39
Abstract
Urethrastriktur er en hyppig tilstand hos mænd, og nuværende behandlinger for lange strikturer (>2 cm) er utilstrækkelige. Denne masteropgave undersøger, om glatte muskelceller (SMC’er) kan differentieres mod en kontraktil fænotype egnet til rekonstruktion af urinrøret, og sammenligner to celleholdige biomaterialer (CELLINK og Lifeink® 200) til 3D-bioprintning af vævslignende konstrukter. Differentiering blev forsøgt med et heparinholdigt medium og vurderet ved RT-qPCR, immunfluorescens og karbachol-induceret kontraktion. 3D-bioprintede konstrukter blev fremstillet med begge bioinks, og celleadfærd blev vurderet med Live/Dead-farvning, Alamar Blue, elastin- og hæmatoxylinfarvning samt RT-qPCR. Under de afprøvede betingelser viste analyserne ikke evidens for SMC-differentiering. Blandt bioinkene var Lifeink® 200 mere velegnet til fremstilling af celleholdige bioprintede konstrukter og peger dermed på et lovende biomateriale til rekonstruktion af urinrøret. Fremtidige studier bør afsøge andre mediatorer og opløselige faktorer for at fremme SMC-differentiering.
Urethral stricture is a common condition in men, and current treatments for long strictures (>2 cm) are inadequate. This master’s thesis evaluates whether smooth muscle cells (SMCs) can be driven toward a contractile phenotype suitable for urethral reconstruction and compares two SMC-laden biomaterials (CELLINK and Lifeink® 200) for 3D bioprinting of tissue-like constructs. Differentiation was attempted using a heparin-containing medium and assessed by RT-qPCR, immunofluorescence, and a carbachol-induced contraction assay. Cell-laden 3D constructs were fabricated with both bioinks, and cell behavior was evaluated by Live/Dead staining, Alamar Blue, elastin and hematoxylin staining, and RT-qPCR. Under the tested conditions, the assays did not demonstrate SMC differentiation. Among the bioinks, Lifeink® 200 was more suitable for fabricating cell-laden bioprinted constructs, indicating a promising biomaterial candidate for urethral reconstruction. Future work should explore alternative mediators and soluble factors to promote SMC differentiation.
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