Towards a bioengineered uterus: bioactive sheep uterus scaffolds are effectively recellularized by enzymatic preconditioning
Uterine factor infertility was considered incurable until recently when they reported the first successful live birth after uterus transplantation. However, risky donor surgery and immunosuppressive therapy are factors that may be avoided with bioengineering. For example, transplanted recellularized constructs derived from decellularized tissue restored fertility in rodent models and mandate translational studies. In this study, they decellularized whole sheep uterus with three different protocols using 0.5% sodium dodecyl sulfate, 2% sodium deoxycholate (SDC) or 2% SDC, and 1% Triton X-100. Scaffolds were then assessed for bioactivity using the dorsal root ganglion and chorioallantoic membrane assays, and they found that all the uterus scaffolds exhibited growth factor activity that promoted neurogenesis and angiogenesis. Extensive recellularization optimization was conducted using multipotent sheep fetal stem cells and they report results from the following three in vitro conditions; (a) standard cell culturing conditions, (b) constructs cultured in transwells, and (c) scaffolds preconditioned with matrix metalloproteinase 2 and 9. The recellularization efficiency was improved short-term when transwells were used compared with standard culturing conditions. However, the recellularization efficiency in scaffolds preconditioned with matrix metalloproteinases was 200–300% better than the other strategies evaluated herein, independent of decellularization protocol. Hence, a major recellularization hurdle has been overcome with the improved recellularization strategies and in vitro platforms described herein. These results are an important milestone and should facilitate the production of large bioengineered grafts suitable for future in vivo applications in the sheep, which is an essential step before considering these principles in a clinical setting.
Hematoxylin and eosin-stained sections from normal and decellularized uterus showed that the extracellular matrix was well-preserved and that all hematoxylin positive nuclei had been removed by the different decellularization protocols (A–D; scale bar = 100 μm). Scanning electron microscopy pictures obtained at ×3000 magnification (E–H; scale bar = 8 μm) showed that protocol (P) 1 generated scaffolds resulted in an organized porous structure (F) while P2 and P3 derived scaffolds resulted in a more compact arrangement with less organized fiber structure. Higher magnification (×12,000; I–L; scale bar = 2 μm) showed that collagen bundles remained intact in all three scaffold types. The dorsal root ganglion (DRG) assay and the chorioallantioc membrane (CAM) assay showed that all scaffolds were functionally bioactive (M–V). The DRGs regenerated axons in wells coated with collagen that was given nerve growth factor supplement in the culture medium (positive control; M), but not when this growth factor was omitted (negative control, N). Scaffolds derived from the three different decellularization protocols all stimulated axonal regeneration (O–Q; scale bar = 500 µm).
Infertility, caused by an absent or dysfunctional uterus was considered incurable until their group succeeded with the world’s first uterus transplantation that resulted in live birth. This procedure has now been repeated at multiple centers resulting in more reported births. Uterus transplantation is still considered an experimental treatment procedure with protocols continuously being evaluated, i.e., organ preservation protocols10, positioning of the vascular anastomoses, and the use of robotic-assisted surgery to reduce recovery time and blood loss. So far, almost all successful uterus transplantation cases have used live donors. However, the risks entailed by live donor surgery and the negative side-effects caused by the required immunosuppressive treatment in the allograft recipient opens up discussions for an alternative donor source.
A Russell-Movat pentachrome stains collagen and reticular fibers (yellow), nuclei and elastic fibers (black), sulfated glycosaminoglycans (blue), muscle tissue (red), and fibrin intense red. Stained from scaffolds revealed that the matrix metalloproteinase 2 and 9 (MMPs) treatment resulted in a more porous scaffold and that elastic fibers became more distinct. Scale bars = 100 µm. B Each decellularization method resulted in scaffolds with very small amounts of remaining donor DNA, and that the MMPs were able to reduce this even more in protocol (P) 2 derived scaffolds. C The total protein content and D the hydroxyproline content were unaffected by the MMPs treatment while E the collagen fiber thickness was significantly reduced. Graphs show box plot with each sample value, median ± IQR, and range.
An attractive organ option which would overcome some of these limitations is a developed bioengineered uterus from an appropriate scaffold together with the patient’s own cells. Extracellular matrix (ECM) derived scaffolds proved successful in several bioengineering studies on female reproductive tissues. Recently, ECM-derived uterus scaffolds were created by decellularization for the mouse, the rat, the rabbit, the pig, and more recently, the sheep. Furthermore, a series of successful studies on rodents from multiple independent groups suggest that decellularized uterine tissue patches can be grafted to repair a full-thickness uterine wall injury and restore fertility. These successful results mandate a translational evaluation using larger animal models.They therefore developed decellularization protocols for the generation of sheep uterus scaffolds as the first step in this process. The sheep is by many considered the most suitable preclinical large animal model for uterine studies because of its physiological and anatomical resemblance to the human uterus, and the sheep model played a significant part in the lead up to the successful human uterus transplantation cases.
Immunohistochemistry stained sections from decellularized sheep uterus tissue before and after matrix metalloproteinase 2 and 9 (MMPs) preconditioning, including native uterus tissue for comparison. P protocol, Col. collagen. Scale bars = 100 µm.
However, a major obstacle using decellularized tissue for bioengineering applications is the limited ability to recellularize the constructs and achieve an appropriate cell density which seems to play an important role in the treatment outcome. For example, recellularized constructs seem to better stimulate tissue regeneration, recruit host cells and prevent rapid scaffold degeneration compared to scaffolds without cells, in particular for larger grafts.
The chorioallantioc membrane (CAM) bioactivity assay confirmed a significant increase in angiogenesis after the inoculation of the three scaffold types before the matrix metalloproteinase 2 and 9 (MMPs) preconditioning compared with the inert alginate reference point that indicate normal chick fetal angiogenesis.The bioactivity from the scaffolds stimulated a doubling of blood vessel formation, while there was a modest (non-significant) reduction in angiogenic effects after the MMPs treatment. Graphs show box plot with each sample value, median ± IQR, and range. P protocol. Significant levels; *p < 0.05; **p < 0.01.
Heterogeneous sheep fetal stem cells (SF-SCs) were isolated from a 6–8-week-old sheep fetus (A). These cells were expanded in vitro and their pluripotency was demonstrated after their differentiation into a chondrogenic lineage (alcian blue positive clusters; B), an osteogenic lineage positive for dentin matrix acidic phosphoprotein 1 (DMP1) and receptor activator of nuclear factor κ B (RANK; C), and a myogenic lineage positive for smooth muscle cell actin (SMA) and myoblast determination protein 1 (MyoD1; D). Scale bars; 5 cm (A), and 100 μm (B–D).
Representative hematoxylin and eosin-stained sections of recellularized constructs from each culturing condition after 14 days in vitro (A–C; scale bar = 100 μm). Cells were mainly located around the injection site when standard culturing conditions (RCSC) were used and there was limited migration (A). However, the different recellularization strategies (transwells, RCTW) and matrix metalloproteinase enzyme preconditioning in combination with TW (RCMMP+TW) clearly improved the cell density and the cellular distribution throughout the scaffolding structure (B, C). Surface cell density was visualized by scanning electron microscope and showed a dense coverage, independent of strategy used (D–F; inserts in C and F show the scaffold preconditioned with MMPs without cells; DCMMP; scale bar = 20 μm). Sheep fetal stem cells kept their pluripotency during recellularization independent of scaffold type, evident by the continuation of CD166, estrogen receptor-β (ER-β), vimentin (VIM), α-smooth muscle actin (SMA), and Ki67 (proliferation) expression (G–I; scale bars=100μm). When the recellularization efficiency was quantified and compared between groups and recellularization strategy, it became obvious that preconditioning with MMPs and using transwells were advantageous already after 3 days in vitro (J–L), but a substantial improvement was seen after 14 days (M–O) in protocol 1 (P1), protocol 2 (P2), and protocols 3 (P3), respectively. Graphs show box plot with each sample value, median ± IQR, and range. Significant levels; *p < 0.05; **p < 0.01; ***p < 0.001, and ****p < 0.0001.
They herein studied three different types of sheep uterus scaffolds created by decellularization and assessed their ability to stimulate growth and angiogenesis. They further describe novel strategies how to significantly increase the recellularization efficiency. This study is therefore an important in vitro milestone toward sheep uterus bioengineering in vivo1.
Padma, A.M., Carrière, L., Krokström Karlsson, F. et al. Towards a bioengineered uterus: bioactive sheep uterus scaffolds are effectively recellularized by enzymatic preconditioning. npj Regen Med6, 26 (2021). https://doi.org/10.1038/s41536-021-00136-0
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