Evaluation of silk biomaterials in combination with extracellular matrix coatings for bladder tissue engineering with primary and pluripotent cells

• Published in: PloS one Vol. 8; no. 2; p. e56237
• Main Authors: Franck, Debra, Gil, Eun Seok, Adam, Rosalyn M, Kaplan, David L, Chung, Yeun Goo, Estrada, Jr, Carlos R, Mauney, Joshua R
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 07-02-2013; Public Library of Science (PLoS)
• Subjects: Actin; Agriculture; Analysis; Animals; Attachment; Biocompatible Materials - pharmacology; Biological products; Biology; Biomaterials; Biomedical materials; Bladder; Cell adhesion; Cell differentiation; Cell Line; Cell lines; Coatings; Collagen; Cytology; Differentiation (biology); Embryo cells; Embryonic stem cells; Embryonic Stem Cells - cytology; Embryonic Stem Cells - drug effects; Embryos; Engineering; Evaluation; Extracellular matrix; Extracellular Matrix Proteins - pharmacology; Fibronectin; Fibronectins; Formulations; Gel spinning; Gene expression; Humans; Induced Pluripotent Stem Cells - cytology; Induced Pluripotent Stem Cells - drug effects; Laminates; Materials Science; Medicine; Mice; mRNA; Muscle contraction; Muscle proteins; Muscles; Myocytes, Smooth Muscle - cytology; Myocytes, Smooth Muscle - drug effects; Pluripotency; Polymerase chain reaction; Real time; Retinoic acid; Rodents; Scaffolds; Silk; Silk - chemistry; Smooth muscle; Stem cell transplantation; Stem cells; Studies; Tissue engineering; Tissue Engineering - methods; Tissue Scaffolds; Transforming growth factor-b1; Transforming growth factors; Urinary bladder; Urinary Bladder - cytology; Urothelium - cytology; Urothelium - drug effects; United States > US; Massachusetts
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0056237

Silk-based biomaterials in combination with extracellular matrix (ECM) coatings were assessed as templates for cell-seeded bladder tissue engineering approaches. Two structurally diverse groups of silk scaffolds were produced by a gel spinning process and consisted of either smooth, compact multi-laminates (Group 1) or rough, porous lamellar-like sheets (Group 2). Scaffolds alone or coated with collagen types I or IV or fibronectin were assessed independently for their ability to support attachment, proliferation, and differentiation of primary cell lines including human bladder smooth muscle cells (SMC) and urothelial cells as well as pluripotent cell populations, such as murine embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells. AlamarBlue evaluations revealed that fibronectin-coated Group 2 scaffolds promoted the highest degree of primary SMC and urothelial cell attachment in comparison to uncoated Group 2 controls and all Group 1 scaffold variants. Real time RT-PCR and immunohistochemical (IHC) analyses demonstrated that both fibronectin-coated silk groups were permissive for SMC contractile differentiation as determined by significant upregulation of α-actin and SM22α mRNA and protein expression levels following TGFβ1 stimulation. Prominent expression of epithelial differentiation markers, cytokeratins, was observed in urothelial cells cultured on both control and fibronectin-coated groups following IHC analysis. Evaluation of silk matrices for ESC and iPS cell attachment by alamarBlue showed that fibronectin-coated Group 2 scaffolds promoted the highest levels in comparison to all other scaffold formulations. In addition, real time RT-PCR and IHC analyses showed that fibronectin-coated Group 2 scaffolds facilitated ESC and iPS cell differentiation toward both urothelial and smooth muscle lineages in response to all trans retinoic acid as assessed by induction of uroplakin and contractile gene and protein expression. These results demonstrate that silk scaffolds support primary and pluripotent cell responses pertinent to bladder tissue engineering and that scaffold morphology and fibronectin coatings influence these processes.