Isolation of Matrix-bound Nanovesicles from Porcine Vaginal Extracellular Matrix

Objectives: Polypropylene (PP) mesh is used in urogynecologic surgery to improve durability and reduce the invasiveness of surgical treatment of pelvic organ prolapse and stress urinary incontinence. However, PP mesh is associated with a strong pro-inflammatory and fibrotic response and the resulting high clinical complication rate precludes use for vaginal prolapse repair. Extracellular matrix (ECM) based therapies are promising potential therapeutics to improve the foreign body response after mesh implantation or vaginal insult. Indeed, ECMs constitute a resource of proteins, growth factors, and exosomes that encourage tissue regeneration, wound healing, and immune regulation. Heterologous extracellular matrix tissue sources from porcine small intestine submucosa (SIS) and porcine urinary bladder matrix (UBM) have been used to improve vaginal wound healing after implantation of PP mesh or after vaginal insults such as childbirth injury or disruption of pelvic support. However, this has resulted in only partial improvement in healing. This may be due to differences in ECM composition by tissue source.

Porcine vaginal ECM (vECM) has recently been developed as a homologous source, however, its regenerative and immunomodulatory properties have not yet been characterized. Matrix-bound vesicles (MBV) are important regenerative elements of the ECM, shown to produce the regenerative and anti-inflammatory properties associated with SIS and UBM ECM when isolated and administered alone. MBV contents have also been shown to vary based on tissue source. Here we aim to isolate MBVs from vECM to allow for characterization.

Methods: The porcine vagina was harvested from 6-month-old nulliparous pigs immediately after euthanasia. Decellularization was performed via serial washing in detergent and delipidating agents as previously described. vECM powder was solubilized by digesting with collagenase overnight with constant agitation. Nanoparticles were isolated in duplicate using sequential centrifugation at 500 g, 2500 g, and 10,000 g to separate and remove insoluble collagen fibrils and other non-soluble remnants followed by ultracentrifugation method at 100.000 g at 4°C for 2 h. The recovered pellet was resuspended with PBS. Nanoparticle size distribution was determined using Nanoparticle Tracking Analysis (NanoSight LM10, Malvern Panalytical) at a dilution of 1:100.

Results: Qiagen DNEasy DNA extraction followed by fluorescent Picogreen dsDNA assay demonstrated an average 75.8% reduction of dsDNA after decellularization. NTA analysis demonstrated recovery of nanoparticles with a concentration of 1.25e+10 +/- 1.44e+09 nanoparticles/ml. The mean size of particles was 95.8 +/- 6.8 nm and the most frequent particle size was 103.5 nm, consistent with previously described MBV size, which is between 30 nm and 150 nm.

Conclusions: Characterizing and isolating beneficial components of the ECM is crucial to determining vECM’s place in restoring tissue integrity and minimizing deleterious foreign body response secondary to PP mesh. Here nanoparticles were isolated from decellularized vECM and will be further characterized for surface markers and protein, cytokine, and miRNA content.