Abstract
The need for bioengineering bladder tissue is critical for children with congenital diseases such as spina bifida and those suffering from spinal cord injury. These children have a neurogenic bladder and suffer symptoms such as incontinence, frequency, urgency, and retention which contributes to the morbidity of infection, stones, or renal injury. Bladder augmentation is the standard for treating small, non-compliant, high pressure neurogenic bladders and easing the associated complications. Bladder augmentation is performed by making a sagittal incision along the bladder and using sections of intestines as a tissue substitute to increase the bladder capacity. Augmentation does not, however, restore full functionality, as the bladder remains neurogenic. Although this is the standard protocol, there are short-and-long-term complications with using intestinal sections for bladder augmentation. Presently, bladder tissue bioengineering has primarily focused on regenerating the muscle and epithelial lining of the bladder, but not vasculature. Although it was successfully shown that augmenting bladders using non-vascularized bioengineered tissue worked in small animal models, the results did not translate to larger animal models and humans. Large grafts without a blood supply suffer from ischemia, which explains the high rate of graft contraction and bladder perforation seen in human clinical trials. The rate of angiogenesis from the host bladder is insufficient to supply blood to the central portion of a large, non-vascularized graft. Osborn et al. (2015) previously showed that prevascularized decellularized rat bladder tissue was able to inosculate, or connect, with the host vessels to establish blood supply soon after bladder wall transplant. Therefore, it is our hypothesis that creating vascularized bioengineered bladder tissue will allow for inosculation of host and graft vessels upon transplant. The overall goal of this project was to bioengineer vascularized porcine bladder tissue for proof-of-concept studies in a small animal model, with the long-term goal of testing bioengineered bladder tissue in pigs, which is a clinically-relevant model with regards to bladder function and size. We conceptualized two methods for vascularizing decellularized pig bladder tissue of which the bladder architecture, including the blood vessel structures, is preserved; this will be referred to as porcine urinary bladder matrix (pUBM). Our first method was an in vitro endothelization of the pUBM, where cells were seeded directly within the vascular scaffold. Different endothelial cell types such as rat bladder endothelial cells (RBECs) and human umbilical vein endothelial cells (HUVECs) as well as rat mesenchymal stem cells (rMSCs) where used for seeding into the pUBMs. Endothelial cells naturally line blood vessels while MSCs recruit cytokines necessary for creating vessels, thus making them prime cell types for pUBM seeding. Our pUBM seeding results indicated that all RBECs, HUVECs and rMSCs were found attached to the pUBMs, however only HUVECs and rMSCs morphologically showed the attachment of healthy cells. pUBMs were also intravascularly seeded with HUVECs in a static perfusion culture for 15 days to assess whether they attached to the inner lining of the vessels. Our microscopy results indicated that blood vessels were lined with cells for 6 days, however cells began to lose adherence around day 9. Our second method was an in vivo implantation of pUBMs onto the rectus abdominis muscle to assess whether this host tissue could induce vascularization of the decellularized bladder tissue. Our hypothesis was that a selectively-permeable filter would direct the cellular vascularization process from the edges of the pUBM towards the center to generate longitudinally-oriented blood vessels which would likely be better for subsequent grafting purposes. Grafts were harvested at 1, 2, and 6 weeks after implantation. Our immunohistochemistry results indicated that after 1 week, grafts without a filter were cellularized throughout in random directions; whereas grafts with a filter were only cellularized directionally from the periphery of the graft in a gradient manner. Grafts with and without a filter at two weeks were thoroughly cellularized. Preliminary data from our two-week experiments showed that grafts with a semipermeable filter had longer vessels while grafts without a filter contained shorter vessels. Thus, blood vessels can be generated within decellularized bladder scaffolds by seeding with cells utilizing in vitro and in vivo methodologies.