Development of an efficient perfusion-based protocol for whole-organ decellularization of the ovine uterus as a human-sized model and in vivo application of the bioscaffolds

Abstract

Purpose The main purpose of this investigation was to determine an efficient whole-organ decellularization protocol of a humansized uterus and evaluate the in vivo properties of the bioscaffold. Methods Twenty-four ovine uteri were included in this investigation and were decellularized by three different protocols (n 6). We performed histopathological and immunohistochemical evaluations, 4,6-diamidino-2-phenylindole (DAPI) staining, DNA quantification, MTT assay, scanning electron microscopy, biomechanical studies, and CT angiography to characterize the scaffolds. The optimized protocol was determined, and patches were grafted into the uterine horns of eight female Wistar rats. The grafts were extracted after 10 days; the opposite horns were harvested to be evaluated as controls.Results Protocol III (perfusion with 0.25% and 0.5% SDS solution and preservation in 10% formalin) was determined as the optimized method with efficient removal of the cellular components while preserving the extracellular matrix. Also, the bioscaffolds demonstrated native-like biomechanical, structural, and vascular properties. Histological and immunohistochemical evaluations of the harvested grafts confirmed the biocompatibility and recellularization potential of bioscaffolds. Also, the grafts demonstrated higher positive reaction for CD31 and Ki67 markers compared with the control samples which indicated eminent angiogenesis properties and proliferative capacity of the implanted tissues. Conclusions This investigation introduces an optimized protocol for whole-organ decellularization of the human-sized uterus with native-like characteristics and a prominent potential for regeneration and angiogenesis which could be employed in in vitro and in vivo studies. To the best of our knowledge, this is the first study to report biomechanical properties and angiographic evaluations of a large animal uterine scaffold.

Applications of Biomaterials in 3D Cell Culture and Contributions of 3D Cell Culture to Drug Development and Basic Biomedical Research

“…One of the ideal scaffolds is a decellularized matrix that provides natural geometric morphology, flexibility, and mechanical strength, which is difficult to mimic perfectly with synthetic scaffolds. Recently, various approaches have been introduced to fabricate decellularized scaffolds, including perfusion of the whole organ (recommended for dense organs/tissues), application of a pressure gradient (employed for hollow tissues), use of supercritical fluid (appropriate for long-standing storage of decellularized scaffolds), and immersion and agitation (suitable for thin tissues) [ 152 , 153 , 154 , 155 , 156 , 157 ]. During the decellularization process, the cells are eliminated to inhibit inflammatory reactions or immediate rejection after implantation.…”

Bioengineering of the Uterus

“…Decellularization and recellularization techniques for regeneration of the uterus have emerged since 2013 as shown in Table 2 [81][82][83][84][85][86][87][88][89][90][91][92][93][94][95][96][97][98].…”

“…It was repopulated with primary endometrial cells. Daryabari et al found that perfusion with SDS and preservation in formalin could be used for preparation of a decellularized ovine uterine scaffold that was capable of regenerating the uterus when grafted into the uteri of rats [ 96 ]. Yao et al decellularized whole rabbit uteri for xenografting to rat full thickness uterine walls [ 97 ].…”

Advances in the Application of Biomimetic Endometrium Interfaces for Uterine Bioengineering in Female Infertility

“…Campo et al (2017) employed decellularization and recellularization technique in the fabrication of a scaffold from porcine uterus and displayed the excellent vascular network in the ECM after recellularization by human side population stem cells. Similarly, Daryabari et al (2019) reported a wholeorgan perfusion decellularization method for production of scaffold from ovine uterus and implanted its segments into rats. The scaffold successfully retained the vascular structure after decellularization and started recellularization in the endometrium and myometrium after implantation, potentially due to homing of the circulating and local stem cells.…”