BioSphincters to treat Fecal Incontinence in Nonhuman Primates

Abstract

Loss of anorectal resting pressure due to internal anal sphincter (IAS) dysfunctionality causes uncontrolled fecal soiling and leads to passive fecal incontinence (FI). The study is focused on immediate and long-term safety and potential efficacy of bioengineered IAS BioSphincters to treat passive FI in a clinically relevant large animal model of passive FI. Passive FI was successfully developed in Non-Human Primates (NHPs) model. The implantation of autologous intrinsically innervated functional constructs resolved the fecal soiling, restored the resting pressure and Recto Anal Inhibitory Reflex (RAIR) within 1-month. These results were sustained with time, and efficacy was preserved up to 12-months. The histological studies validated manometric results with the regeneration of a well-organized neuro-muscular population in IAS. The control groups (non-treated and sham) remained affected by poor anal hygiene, lower resting pressure, and reduced RAIR throughout the study. The pathological assessment of implants, blood, and the vital organs confirmed biocompatibility without any adverse effect after implantation. This regenerative approach of implanting intrinsically innervated IAS BioSphincters has the potential to offer a better quality of life to the patients suffering from FI.

Figure 1

Development of passive FI following isolation of Cells from biopsies: (A) NHPs (n = 10) displayed a reduction in basal tone immediately after the hemi-sphincterectomy. (B) RAIR was reduced by 41% after sphincterectomy (n = 10); Fecal hygiene in NHP (C) before and (D) after hemi-sphincterectomy. *Values indicated p < 0.05 with Baseline; the horizontal bars exhibited mean and standard error.

Figure 2

Bioengineering and characterization of IAS BioSphincters: (A) Bioengineered IAS BioSphincter. (B) Viability assay (n = 3); (C) Proliferation Assay (n = 3); (D) mRNA expression of different proteins at day 1 and 10; Physiological functional analysis exhibited; (E) generation of basal tone (black) following rapid contraction response to treatment with 60 mM potassium chloride (orange), which was maintained for 6 min and returned to baseline; (F) Rapid contractile response to 40 µM acetylcholine (black) treatment which partially diminished on pre-treatment of TTX (40 µM) in red; (G) Electrical field stimulation caused relaxation (blue) for 20 min, which was partially improved on pre-treatment of nNOS inhibitor (green) and completely attenuated on pre-treatment of TTX (40 µM); (H) summarized expression of physiological response exhibited by all the BioSphincters (n = 6) prior to implantation. This graph expressed the mean and standard error (n = 6) independent samples per group (p > 0.05); Line graphs show representative tracings to portray the trend of each treatment. Scale bar 5 mm in image A.

Figure 3

Post-implantation in-vivo efficacy of IAS BioSphincters: Fecal hygiene of (A) non-treated NHPs displayed fecal soiling (yellow arrows) and staining of perineum; (B) after implantation, fecal soiling resolved within one month and perineum was clean; (C) sham group was unaffected and displayed sustained fecal output (yellow arrow); (D and G) IAS hemi-sphincterectomy (n = 10) resulted in a significant reduction in anal basal pressure and RAIR compared to baseline, which was sustained to 1, 3, 6 and 12 months (n = 2); (E and H) The reduction in anal basal pressure and RAIR compared to baseline was restored within 1 month of implantation (n = 6), further improved and preserved for next 12 months (n = 6); (F and I) The anal basal pressure and RAIR were remained low and unaffected in the sham group (n = 2) throughout the study. The horizontal bars exhibited mean and standard error.

Figure 4

Post-implantation histological analysis of IAS BioSphincters: The H&E stained surgical site section of NHP in both (A) non-treated and (B) sham group displayed irregular and discontinued arrangement of cells and extracellular matrix, but (C) 12-months post-implantation sections displayed regular arrangment of cells; MT stained sections of (D) non-treated group and (E) sham group confirmed random distribution of muscle cells (red) in collagen (blue), (F) 12-months post-implantation sections exhibited highly aligned and uniformly distributed muscle cells (red) in collagen matrix (blue); The immunostaining with caldesmon further confirmed the lack of muscle cells in both (G) non-treated group and (H) sham group, whereas (I) treated group displayed well-arranged muscle network; A poor neuronal network was appeared on immunostaining with βlll Tubulin on (J) non-treated group and (K) sham group compared to dense and innervated neuronal network in (L) treated group. Scale bar 500 µm (in A,B,E,F,I,J) and 100 µm (in C,D,G,H,K,L).