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Derivation and characterization of porcine vocal fold extracellular matrix scaffold
Author(s) -
Wrona Emily A.,
Peng Robert,
Born Hayley,
Amin Milan R.,
Branski Ryan C.,
Freytes Donald O.
Publication year - 2016
Publication title -
the laryngoscope
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.181
H-Index - 148
eISSN - 1531-4995
pISSN - 0023-852X
DOI - 10.1002/lary.25640
Subject(s) - extracellular matrix , scaffold , fold (higher order function) , microbiology and biotechnology , chemistry , matrix (chemical analysis) , biophysics , biology , computer science , biomedical engineering , engineering , programming language , chromatography
Objectives/Hypothesis To optimize decellularization of porcine vocal folds (VF) and quantify human bone marrow‐derived mesenchymal stem cell (BM‐MSC) interactions with this matrix to provide a foundation for regenerative approaches to VF repair. Study Design and Methods Vocal folds were dissected from porcine larynges and three decellularization protocols were compared, each consisting of washes and mechanical agitations with different combinations of reagents. DNA content was analyzed via Quant‐iT Picogreen assay and hematoxylin and eosin staining. Bone marrow‐derived MSCs were then seeded onto the decellularized VF matrices. Morphology, metabolic activity, DNA content, and gene expression were assessed using LIVE/DEAD Cell Viability, alamarBlue Cell Viability Assay, Quant‐iT Picogreen assay, and quantitative polymerase chain reaction, respectively. Results The most successful decellularization protocol removed 95% DNA content within 1 day, compared to several days required for previously described protocols. Histology confirmed the retention of extracellular matrix (ECM) and its components, including glycosaminoglycans, collagen, and fibrin, while void of nuclear/cellular content. Decellularized scaffolds were then seeded with BM‐MSCs. Similar DNA quantities were observed after 24 hours of seeding within the VF‐ECM scaffold when compared to cells on tissue culture plastic (TCP). LIVE/DEAD staining of the seeded VF‐ECM confirmed excellent cell viability, and the metabolic activity of BM‐MSCs increased significantly on VF‐ECM compared to TCP. Endoglin gene expression decreased, suggestive of differentiation. Conclusion Porcine VFs can be efficiently decellularized within 5 hours using a combination of sodium deoxycholate and peracetic acid. Decellularized VF‐ECM supported attachment and growth of human BM‐MSCs, with evidence of differentiation. Level of Evidence N/A Laryngoscope , 126:928–935, 2016