Tissue-engineered conduit using urine-derived stem cells seeded bacterial cellulose polymer in urinary reconstruction and diversion

Abstract

The objective of this study was to generate bacterial cellulose (BC) scaffolds seeded with human urine-derived stem cells (USC) to form a tissue-engineered conduit for use in urinary diversion. Microporous BC scaffolds were synthesized and USC were induced to differentiate into urothelial and smooth muscle cells (SMC). Induced USC (10⁶ cells/cm²) were seeded onto BC under static and 3D dynamic (10 or 40 RPM) conditions and cultured for 2 weeks. The urothelial cells and SMC derived from USC formed multilayers on the BC scaffold surface, and some cells infiltrated into the scaffold. The urothelium derived from USC differentiation expressed urothelial markers (uroplakin Ia and AE1/AE3) and the SMC expressed SMC markers (α-smooth muscle actin and desmin). In addition, USC/BC scaffold constructs were implanted into athymic mice, and the cells were tracked using immunohistochemical staining for human nuclear antigen. In vivo, the cells appeared to differentiate and express urothelial and SMC markers. In conclusion, porous BC scaffolds allow 3 dimensional growth of USC, leading to formation of a multilayered urothelium and cell–matrix infiltration. Thus, cell-seeded BC scaffolds hold promise for use in tissue-engineered urinary conduits for urinary reconstruction.

Introduction

Currently, bladder cancer is the second most common urologic malignancy in the United States, after prostate cancer. According to the American Cancer Society, almost 71,000 new cases of bladder cancer were diagnosed in 2009, and 14,000 patients with this type of cancer died [1]. The chance of developing bladder cancer is about 1 in 27 for men and 1 in 85 for women. In order to treat malignancies that have invaded the bladder muscle, surgical resection of the tumor, followed by the creation of a continent urinary reservoir using segments of the small or large intestine is often necessary. A number of surgical techniques have been used to perform urinary diversion, but the most common techniques are continent diversion and conduit diversion. In continent diversion, a continent stomal reservoir with a segment of intestine is created for catheterization, and this allows the patient to empty the reservoir via catheter as needed throughout the day. In conduit diversion a section of intestine, usually ileum is used to create a conduit that collects urine and allows it to drain through a stoma in the abdominal wall. The urine is then collected in a bag outside the body, and the bag must be emptied periodically. However, both continent diversion and conduit diversion can cause several potential complications, including hyperchloremic metabolic acidosis with hypokalemia, urinary tract infections, stone formation, malignancy, bowel obstruction, skin breakdown around the stoma, stenosis of the stoma, impaired renal function, or damage to the upper urinary tract resulting from urine reflux.

Tissue engineering technology may provide an alternative approach for building a functional urinary conduit to store urine for patients with bladder cancer who require total cystectomy. Various biodegradable scaffolds seeded with cells have been introduced for bladder reconstruction or urinary conduit procedures. Most of these biomaterials are biodegradable, including natural collagen materials i.e. bladder submucosa, (BSM) [2], small intestine submucosa (SIS) [3] or collagen type I matrix [4] and synthetic polymers such as polyglycolic acid (PGA), poly (lactic-co-glycolic acid) (PLGA)[5], [6] and biocarbon [7]. Most degradable biomaterials, promote cellular interaction and tissue development, and possess adequate mechanical and physical properties. However, natural collagen scaffolds cannot maintain a robust physical structure in an in vivo environment when used in total or subtotal bladder replacement, resulting in graft collapse, contraction, formation of fibrosis, and shrinkage of the new bladder, with resultant decreased bladder capacity [8]. A biomaterial with a retained hollow structure, anti-fibrosis properties, and a 3D porous microstructure for graft cell seeding would be highly desirable for creating a viable tissue-engineered urinary conduit.

Tissue-engineered urinary conduits and tissue-engineered bladders are similar in structure, but different in function. Both constructs require a hollow structure lined with urothelium on the lumen side. A new bladder or conduit covered with urothelial tissue can prevent stone, calcification, or scar formation caused by the naked muscle layer or biomaterial being directly exposed to urine [9], [10]. However, tissue-engineered urinary conduits are mainly used to store urine and they are emptied via catheterization in continent diversion or a channel in conduit diversion. Unlike engineered bladder tissue, an engineered conduit does not need to contract to empty itself, and thus the development of a thick muscular wall is not required. Furthermore, tissue-engineered bladders must be constructed using a degradable biomaterial to allow the necessary muscle contractility to develop, but this type of contractility is not required for the development of functional tissue-engineered conduits. Although either resorbable or non-resorbable materials can be used in tissue-engineered urinary conduits, a non-resorbable bacterial cellulose material can be an attractive alternative.

Bacterial cellulose is a natural biopolymer and consists of a network of nanofibrils which possess high mechanical strength [11]. This material is highly hydrophilic, non-cytotoxic, and stable within a wide range of temperatures and pH levels. Although bacterial cellulose polymer is non-degradable, it is an FDA-approved material used clinically in wound dressing [12], and it has been evaluated to repair cartilage, provide drug delivery, and replace dura mater or small-caliber blood vessels [13], [14], [15], [16]. Thus, this biomaterial might be a good candidate as scaffold for fabricating a urinary conduit.

Another critical factor in generating tissue-engineered urinary conduits for patients with bladder cancer is the cell source. Traditionally, cells obtained from bladder biopsies are a main cell source for cell-based tissue engineering in urinary tract reconstruction. However, healthy cells might not be available in patients with bladder cancer or chronic inflammation. Cancer-free urothelial cells or stem cells from the upper urinary tract provide an alternative cell source. Our recent study demonstrated that urine-derived cells can be collected from the upper urinary tract. These cells possess stem cell features, including self-renewal and multipotential differentiation [unpublished data]. Urine-derived stem cells from the upper urinary tract can be differentiated into urothelial and smooth muscle cells. It would be a simple and low-cost approach to harvest cells from patients who already have a nephrostomy tube in place.

The goal of this study was to produce a tissue-engineered urinary conduit structure with 3D urothelial mucosa using autologous urine-derived stem cells, cultured within 3D porous bacterial cellulose under dynamic culture conditions. This cell-based tissue-engineered conduit may be useful for patients with end-stage bladder diseases who need bladder reconstruction.