Scaffolds for whole organ tissue engineering: Construction and in vitro evaluation of a seamless, spherical and hollow collagen bladder construct with appendices

Abstract

The field of regenerative medicine has developed promising techniques to improve current neobladder strategies used for radical cystectomies or congenital anomalies. Scaffolds made from molecularly defined biomaterials are instrumental in the regeneration of tissues, but are generally confined to small flat patches and do not comprise the whole organ. We have developed a simple, one-step casting method to produce a seamless large hollow collagen-based scaffold, mimicking the shape of the whole bladder, and with integrated anastomotic sites for ureters and urethra. The hollow bladder scaffold is highly standardized, with uniform wall thickness and a unidirectional pore structure to facilitate cell infiltration in vivo. Human and porcine bladder urothelial and smooth muscle cells were able to attach to the scaffold and maintained their phenotype in vitro. The closed luminal side and the porous outside of the scaffold facilitated the formation of an urothelial lining and infiltration of smooth muscle cells, respectively. The cells aligned according to the provided scaffold template. The technology used is highly adjustable (shape, size, materials) and may be used as a starting point for research to an off-the-shelf medical device suitable for neobladders.

Statement of Significance

In this study, we describe the development of a simple, one-step casting method to produce a seamless large hollow collagen-based scaffold mimicking the shape of the whole bladder with integrated anastomotic sites for ureters and urethra. The hollow bladder scaffold is highly standardized with uniform wall thickness and a unidirectional pore structure to facilitate cell infiltration in vivo. The closed luminal surface and the porous exterior of the scaffold facilitated the formation of a urothelial lining and infiltration of smooth muscle cells, respectively. The applied technology is highly adjustable (shape, size, materials) and can be the starting point for research to an off-the-shelf medical device suitable for neobladders.

Introduction

In case of muscle-invasive and refractory superficial bladder cancer and end stage (congenital) bladder disease, the current clinical standard is radical cystectomy in combination with urinary diversion [1], [2]. The method of diversion depends on, amongst others, the nature of the defect, and the patient’s needs and wishes. Orthotopic bladder reconstruction is increasingly applied for urinary tract reconstruction [3]. However, current methods rely on autologous tissues that are harvested from the gastrointestinal tract. This can lead to severe complications including anastomotic leakages, enteric fistulae, bowel obstruction, prolonged episodes of ileus, life-threatening infections, nutritional mal-absorption, and/or intestinal failure [1], [4].

New techniques and materials generated in the field of regenerative medicine may provide useful alternatives. Regenerative medicine (RM) aims to regenerate tissues and organs by creating biological equivalents through the supplementation of scaffolding materials, bioactive components, cells or a combination thereof [5]. Within the field of RM different attempts have been made to reconstruct the bladder in both animal and human studies [6]. In 2006, a promising avenue for RM in producing a neobladder was published by Atala et al. where a collagen/polyglycolic acid composite was used which was sutured together into a partial bladder/cup shape and seeded with urothelial and smooth muscle cells [7]. Initial clinical results were promising, but a recent related phase II clinical trial demonstrated that an autologous cell cultured scaffold composed of synthetic polymers did not improve bladder compliance and was associated with serious adverse events that surpassed the acceptable safety standard [8]. In addition, the complicated and expensive nature of the procedure may not be feasible in most clinical centers [9], [10], [11]. Alternatively, a well-structured molecularly defined acellular scaffold resembling the whole bladder may be an option, using the body as a bioreactor. Previously, flat acellular collagen scaffolds have been used for bladder augmentation in patients with exstrophy-epispadias complex and were found to be completely lined with urothelial cells after implantation [12]. A tubular acellular collagen-based urostomy implanted in a pig model showed good results with respect to the re-urothelialization of the construct using the body as a bioreactor [13]. Flat scaffolds can be manually shaped into a sphere to create a bladder-like construct using sutures and a silicon breast prosthesis, as was shown by Baumert et al., who also pre-seeded the scaffold with urothelial and smooth muscle cells, and wrapped the construct in omentum for further cell differentiation in vivo [14]. Omental wrapping of tubular acellular collagen scaffolds resulted in good vascularization and tissue integration of the scaffold [15]. Bladder shaped acellular scaffolds for in vivo cellularization may be an option for urinary diversions and neobladder reconstructions and would be in line with statements from recent proceedings from the “2nd international consultation on bladder cancer: urinary diversion”, which indicated that widespread acceptance and success of a new technique is based on its simplicity [16]. For this, new methods in construct design are necessary. In this study, we have focused on the design of a novel, simple, standardized and adjustable process to produce resorbable seamless hollow scaffolds that mimic the size and shape of a human bladder and include appendices for anastomosis of the ureters and urethra. Cytocompatibility of bladder scaffolds, cellular influx and cell alignment were investigated using histology, immunohistochemistry, quantitative polymerase chain reaction, scanning electron microscopy and transmission electron microscopy.