Autologous regeneration of blood vessels in urinary bladder matrices provides early perfusion after transplant to the bladder
Stephanie L. Osborn, Ph.D., Leanna W. Mah, M.D., Erica V. Ely, B.S., Stefania Ana, Pharm.D., Christina Huynh, M.A., Naveena S. Ujagar, B.S., Serena C. Chan, B.S. expected 2022, Philip Hsiao, M.D., Jonathan C. Hu, M.D., Eric A. Kurzrock, M.D..
UC Davis School of Medicine, Sacramento, CA, USA.
Background. For patients with neuropathic bladder from spina bifida or spinal cord injury, the current surgery of using intestine for bladder augmentation causes high morbidity, as well as short- and long-term complications. In clinical trials, grafts have lacked existing vasculature and have ultimately failed due to dehiscence and contraction from ischemia. To address this critical issue of blood supply, we developed an in vivo staged implant strategy to create autologous, vascularized bioengineered bladder tissue with potential for clinical translation.
Methods. The staged implant technique was tested in two animal models: rat and pig. Pig bladders were used to create acellular urinary bladder matrices (UBMs), which were implanted on the rectus abdominus muscles to generate large cellular, vascularized grafts. Rectus-matured UBMs (RM-UBMs) were harvested at early (1-2 weeks) and late (1-3 months) time-points and evaluated for cellularity, vascularity, smooth muscle formation and blood vessel function by immunohistochemistry. Large RM-UBM or UBM were then transplanted to the bladders of six pigs after partial cystectomy and harvested for similar analyses at 2 weeks.
Results. In both animal models at early timepoints, rectus-matured UBMs (RM-UBMs) were highly cellularized and contained an abundance of CD31-positive blood vessels, which were shown to be functional by perfusion studies using India ink. Mean vessel density (MVD) remained stable at later timepoints and was comparable to native bladder wall. Muscle patterns within grafts showed smooth muscle formation over time, with no evidence of striated muscle ingrowth from the rectus. At early timepoints, smooth muscle actin (SMA) expression was associated primarily with vascular smooth muscle. At later timepoints, however, SMA expression was increased specifically within the detrusor area of the UBM, where the pattern began to morphologically mirror the smooth muscle layers of native bladder wall. In some grafts, the skeletonized blood vessels of the UBM were marked with colored India ink prior to implantation to determine the path of angiogenesis. Upon recovery of grafts, ink was found within the lumens of newly formed vessels, suggesting that angiogenesis occurs within the decellularized vessel spaces. To determine whether the pre-vascularized tissue would support early perfusion after transplant, large autologous RM-UBMs were harvested from the rectus muscle at 2 months and transplanted to the bladder after partial cystectomy. Large UBMs were transplanted as controls. At 2 weeks post-transplant, RM-UBMs maintained approximately 90% of their transplant size, while UBMs maintained only 20% of their size. Functional, ink-perfused blood vessels were found in the central portion of all RM-UBM grafts at 2 weeks, while UBM grafts were significantly deteriorated, contracted and lacked central cellularization and vascularization.
Conclusions. This study suggests that the rectus muscle is a viable space to bioengineer autologous bladder tissue. Graft regeneration recapitulates native bladder wall and smooth muscle and blood vessels develop within their specific compartments of the extracellular matrix. The blood supply of these autologous grafts is capable of inosculating with host vessels to quickly perfuse the graft and prevent contraction after transplant to the bladder, suggesting this staged implantation technique holds great potential for clinical translation.
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