P-788 Analysis of tissue regeneration after transplantation of bioengineered uterine grafts in a sheep model of uterine injury
Authors: De Miguel-Gómez L , Sehic E , Thorén E , Gudmundsdottir I , Rabe H , Oltean M , Akouri R , Brännström M , Hellström M 
Published: July 2024

Abstract

Study question
Can we regenerate a damaged uterus using decellularized (further repopulated with stem cells or not) uterine scaffolds in a sheep model?

Summary answer
Bioengineered uterine grafts can promote the regeneration of a damaged uterus, but this process is highly dependent on the basal immune status of the recipient.

What is known already
Uterus bioengineering aims to develop scaffolds to repair defective uterine tissue that may cause infertility. The remaining extracellular matrix after uterus decellularization has frequently been used as a scaffold material for preclinical treatment applications. So far, multiple in vivo studies using rodents have shown successful restoration of uterine tissue and even fertility when small grafts were used. Furthermore, this pro-regenerative effect is enhanced when repopulating the scaffolds with mesenchymal stem cells (MSCs). However, studies using larger animal models and larger scaffolds are still missing. Therefore, we investigated the therapeutic potential of transplanting these scaffolds in the sheep model.

Study design, size, duration
All experiments were performed in university facilities, including the sheep animal work. Uterine scaffold donor sheep (from a local abattoir) and recipient sheep (n = 10) were of the same mixed sheep breed (Swedish fin-ull and Dutch texel mixed breed) and age (8-12 months old). Recipient sheep were transplanted with uterine decellularized (either recellularized or not) grafts that were maintained for 6 weeks.

Participants/materials, setting, methods
We implemented our previously established decellularization protocol (2% sodium deoxycholate) for sheep uterine scaffold production, generating 3x2cm grafts; half of these were recellularized with sheep MSCs. Decellularized (DC; n = 7) and recellularized (RC; n = 8) grafts were transplanted as replacements for native uterine tissue. After six weeks, histological, immunohistochemistry (CD31, CD45), and gene expression (progesterone/estrogen receptors, HOXA10, VEGF, vWF, TNFA, IFNG) analysis were conducted. Simultaneously, we used cell sorting to monitor the systemic immune response over time.

Main results and the role of chance
Angiogenesis was increased in the RC group as more new blood vessels (CD31+ cells) were evidenced (P=0.0120). VEGF and vWF gene expression also indicated the presence of blood vessels in the grafts but without differences among the groups. Regarding the immune response, CD45 staining revealed leukocyte recruitment into both transplanted grafts, with an increased significance in the DC group (P=0.0071) compared to the RC (P=0.0181). Furthermore, the pro-inflammatory genes TNFA and IFNG expression remained invariable compared to the native tissue. Finally, hormone receptor gene levels were comparable to those in the native tissue, with HOXA10 presenting higher values in the RC group compared to the DC (P=0.0548). So, the RC group showed signs of better uterine regeneration, probably due to the MSC’s anti-inflammatory action. Still, differences were subtle between groups. So, we blindly analyzed the histology of all grafts and detected substantially but also poorly regenerated grafts in both DC and RC groups. The analysis of the systemic immune response likely answers this, as when segregating individuals in substantially and poorly regenerated grafts, there was a significant difference in the percentage of activated CD4+ T-cells, being higher (P&lt;0.0001) in those with poorly regenerated grafts already at day 0.

Limitations, reasons for caution
Due to the lack of available sheep antibodies, we did not study specific subtypes of leukocytes by immunohistochemistry as we did for the cell sorting analysis. Also, despite experienced group members performing the blinded histological analysis of the grafts to detect substantially/poorly regenerated grafts, this method requires refinement.

Wider implications of the findings
Basal immune system status should be considered before transplanting uterine grafts, as inflammation seems crucial in tissue regeneration.

Trial registration number
Not applicable