Vaginal childbirth inflicts substantial trauma to the pelvic floor and is the leading cause of pelvic floor disorders (PFDs), which often manifest later in life [1]. This affects 25% of women, highlighting the profound consequences of maternal childbirth injury on pelvic health [2]. The increasing demand for preventative treatment approaches for pelvic organ prolapse (POP) subsequent to birth injury has led to the exploration of novel biologically active scaffolds such as Aloe Vera-Alginate hydrogel (AV-ALG-Hyd) functionalized xenogeneic SUSD2+ human Endometrial Mesenchymal Stem Cells (eMSC) [3]. Thus, this pre-clinical study is the first to explore the therapeutic potential of AV-ALG-Hyd with and without eMSC in promoting healing post-birth injury, in a primiparous ovine model. It is hypothesized that this therapeutic injectable hydrogel may mitigate birth-related tissue injury by restoring extracellular matrix (ECM) homeostasis through targeted collagen realignment, elastin preservation, and smooth muscle regeneration.

Primiparous ewes underwent simulated birth injury via Bakri© balloon catheter. Four groups were evaluated: (1) uninjured controls, (2) AV-ALG-Hyd alone, (3) AV-ALG-Hyd with SUSD2+ eMSCs, and (4) sham injury. Explants were analysed at 30 and 90 days for POP-Q measurements (modified for ovine model), eMSC retention, histology (haematoxylin and eosin, masson’s trichrome, elastin), immunohistochemistry (α-SMA, CD45) (Figure 1). Stretch injury and architectural restoration was assessed quantitatively through extent   of epithelial detachment and stromal voids on masson’s trichome stain, and qualitatively using α-SMA to assess smooth muscle fibre alignment and integrity. Uniaxial tensiometry was used to assess tissue strength and deformation following exposure to repetitive cyclical loading (3 cycles, 70% stretch, wet condition), followed by monotonic tensile loading to mechanical failure . Statistical significance (p<0.05) was determined using ANOVA with Tukey post-hoc correction. Ethics approval was granted by Monash University (MMCA-2018).

Birth injury disrupted collagen, smooth muscle cells, and elastin in vaginal tissue, however AV-ALG + eMSC hydrogels reversed these changes. At 30 days post-injury, the hydrogel + eMSC group exhibited a 2.1-fold increase in α-SMA+ smooth muscle content compared to untreated injury (p<0.01), surpassing both sham and hydrogel-only groups (p=0.03). By 90 days, this group achieved complete restoration of tightly packed smooth muscle bundles, with α-SMA expression exceeding sham controls (p=0.004). Hydrogel injection alone and combined with eMSC reversed injury-induced architectural disruption within 30 days, showing reversal of epithelial detachment and disruptions in vaginal stroma, and complete restoration of histomorphometric parameters to uninjured levels by 90 days.  eMSC-based therapy promoted angiogenesis without foreign body response.  Untreated injury exhibited aberrant elastin accumulation (↑40% vs. baseline, p=0.01), whereas hydrogel + eMSC treatment normalized elastin content to parity-matched levels at 30d (↓25% vs. untreated, p=0.007) and 90d  (↓35%, p<0.001). CD45+ leukocyte density increased 3.8-fold in hydrogel + eMSC groups (p=0.0003), suggesting amplified immune-mediated remodelling. Biomechanical testing revealed a 2.3-fold improvement in maximum load tolerance (p<0.001) and a 180% increase in elastic modulus (p=0.004) at 90d, demonstrating restored tissue functionality.

AV-ALG hydrogels combined with eMSCs restored pelvic floor biomechanics by reversing birth injury-induced smooth muscle and stromal tissue loss, whilst mitigating elastin dysregulation and amplifying the immune response.  This proposes a multimodal mechanism of action for this hydrogel that addresses both structural degradation and functional impairment, offering a transformative approach to postpartum pelvic floor regeneration.

AV-ALG-Hyd with xenogeneic SUSD2+ eMSC shows promise for reversing birth injury structural malformations implicit in POP pathogenesis, thereby improving tissue healing after injury and serving as the world’s first preventative therapy for POP.

1.	Nygaard, I., Barber, M.D., Burgio, K.L., et al. (2008). "Prevalence of symptomatic pelvic floor disorders in US women." JAMA, 300(11), 1311-1316.
2.	Barber, M.D., et al. (2009). "Epidemiology and outcomes assessment of pelvic organ prolapse." International Journal of Gynecology & Obstetrics, 104(Suppl 1), S3-S7.
3.	Darzi S, Paul K, Leitan S, Werkmeister JA, Mukherjee S. Immunobiology and Application of Aloe Vera-Based Scaffolds in Tissue Engineering. Int J Mol Sci. 2021 Feb 8;22(4):1708. doi: 10.3390/ijms22041708. PMID: 33567756; PMCID: PMC7915752.