TOWARDS UTERUS TISSUE ENGINEERING; SHEEP UTERUS DECELLULARIZATION PRODUCES BIOACTIVE SCAFFOLDS THAT CAN EFFICIENTLY BE RECELLULARIZED

Abstract

Introduction: Uterus transplantation (UTx) was introduced as a treatment option for uterus-related infertility which affects about 1 in 500 women of fertile age. After pre-clinical studies, our center initiated the first clinical trial of UTx which lead to the world’s first born baby after such procedure and proved that uterus-related infertility can be cured. Nevertheless, the risks involved in live-donor surgery and negative side-effects caused by immunosuppression after engraftment opens up discussion for other donor sources. For instance, novel bioengineering techniques may be an option to overcome these restraints which includes the development of a biologically-derived scaffold populated with the patient’s own cells to replace a donor organ in a UTx setting. Promising results were shown using decellularized scaffolds in rodent models. We herein report a translational approach of these ideas to a large animal model, and provide protocols for scaffold generation of the sheep uterus which is of a similar size to a human uterus, and show how to efficiently recellularize it. Materials and Methods: Decellularization was performed using vascular perfusion. Solutions were based on 0.5% sodium dodecyl sulfate, or 2% sodium deoxycholate, or with a sequential perfusion of 2% sodium deoxycholate and 1% Triton X-100. The scaffolds were examined by histology, extracellular matrix (ECM) quantification and mechanical properties. The scaffold bioactivity was analyzed using the dorsal root ganglion- (DRG) and chorioallantoic membrane (CAM) assays. Recellularization efficacy was quantified after standard cell culturing conditions, or when transwell culturing plates were used with- or without scaffold pre-conditioning with matrix metalloproteinases (MMP:s) -2 and -9. Results: Decellularization was confirmed and electron microscopy showed that each protocol resulted in a porous ECM-rich scaffold. Mechanical studies confirmed no loss of strength. The DRG- and CAM-assays confirmed that the scaffolds had growth promoting qualities. Compared to standard culturing methods, the recellularization efficiency could be improved by 200%-300% (depending on scaffold-type) when scaffolds were pre-conditioned with MMP:s and cultured in transwell plates for up to 14 days in vitro. Conclusion: Uteri decellularized with sodium deoxycholate seemed favorable based on ECM quantifications. However, all scaffolds showed bioactivity and an ability to support stem cell growth. These cells continued to express markers for proliferating stem cells. Recellularization using MMP:s to pre-condition the scaffolds and transwells during culture was advantageous, independent of scaffold type. These bioengineering strategies are therefore valuable for future in vivo transplantation studies on the sheep animal model to evaluate novel treatments for uterine-related infertility. Additionally, these successful de- and recellularization schemes may be effective when bioengineering other organs of similar size.