Abstract
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.
Highlights
Surgical management of a variety of bladder disorders including neurogenic bladder, posterior urethral valves, and bladder exstrophy frequently requires augmentation cystoplasty in order to prevent renal damage and incontinence from increased urinary storage and voiding pressures [1]
We investigated two groups of silk scaffold configurations as potential templates for the development of cell-seeded constructs for bladder tissue engineering
Comparisons between these structurally diverse scaffold groups allowed for an interrogation of how silk biomaterial morphology affects primary and pluripotent cell responses
Summary
Surgical management of a variety of bladder disorders including neurogenic bladder, posterior urethral valves, and bladder exstrophy frequently requires augmentation cystoplasty in order to prevent renal damage and incontinence from increased urinary storage and voiding pressures [1]. Previous studies in bladder tissue engineering have investigated both natural and synthetic scaffolds either alone or seeded with autologous bladder cell populations as alternative strategies for defect repair or neobladder reconstruction [3] Conventional scaffolds such as acellular bladder matrix (ABM), poly-glycolic acid (PGA), and small intestinal submucosa (SIS) have been reported to support bladder tissue regeneration in various animal models [4,5,6] and in some cases short-term clinical trials [7,8]. We reported that manipulation of silk matrix properties can influence the extent of in vivo scaffold degradation and host tissue integration [17] These studies reveal that the silk gel spinning process represents a tunable system for understanding the ability of specific biomaterial characteristics to support reconstitution of lower urinary tract defects
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