Decellularization method and tissue region impact the structural properties and composition of porcine vaginal extracellular matrix

Pelvic regenerative medicine lags behind other fields in availability of regenerative bioscaffolds derived from decellularized vagina, resulting in fewer platforms for investigating pelvic pathology in vitro and an absence of regenerative grafts mimicking unique vaginal properties. Utilizing nulliparous porcine vagina obtained from an abattoir, the goal of this study was to 1) evaluate the efficacy of different modalities of decellularization (freeze-thaw, detergent, and a combined hybrid method) on reducing cellularity, the primary outcome of decellularization, and 2) characterize in-depth the impact of these modalities on the structural properties and composition of the remaining matrix using mechanical and biochemical analyses. While all methods tested showed roughly equivalent performance in reducing cellularity, success diverged regarding preservation of matrix properties. Freeze-thaw decellularization reduced cellular components the least but preserved fiber architecture and gross mechanics. Detergent decellularization achieved moderate cellular component reduction and preserved glycosaminoglycan content but distorted fiber architecture. Overall, hybrid decellularization resulted in maximum antigenic cellular component reduction but poorly retained gross mechanics and glycosaminoglycan content. Additionally, heterogeneity in composition and mechanics between vaginal regions was observed between the proximal-distal orientations, mirroring observations in other animal models. Like other tissues, there is no one-size-fits-all approach to decellularizing the vagina, and selection of the decellularization method should be concordant with the most desired properties of the resultant matrix product. This study suggests that freeze-thaw methods are ideal for investigation of native biomechanical properties, detergent methods for generating raw materials for further manufacturing, and hybrid methods are best suited for generating whole-tissue acellular matrices. STATEMENT OF SIGNIFICANCE: Vaginal tissue engineering is understudied, hampering investigations into clinical regenerative potential and informed choice of decellularization method. We selected three decellularization methods and evaluated their efficacy and off-target effects on full thickness adult nulliparous porcine vagina isolated from six distinct vaginal regions. We found that while all methods similarly reduced cellular contents, each altered the resulting vaginal extracellular matrix structure and composition in a region-specific manner, which may have implications for generation of regenerative bioscaffolds or mechanistic studies of matrix mechanics. Data generated from this study will shape guidelines for vaginal decellularization based on scientific or clinical need, establish a link between changes in matrix properties and decellularization method, and further define regional complexity within the adult porcine vagina.