Perianal implantation of bioengineered human internal anal sphincter constructs intrinsically innervated with human neural progenitor cells

Abstract

Background: The internal anal sphincter (IAS) is a major contributing factor to pressure within the anal canal and is required for maintenance of rectoanal continence. IAS damage or weakening results in fecal incontinence. We have demonstrated that bioengineered, intrinsically innervated, human IAS tissue replacements possess key aspects of IAS physiology, such as the generation of spontaneous basal tone and contraction/relaxation in response to neurotransmitters. The objective of this study is to demonstrate the feasibility of implantation of bioengineered IAS constructs in the perianal region of athymic rats.

Methods: Human IAS tissue constructs were bioengineered from isolated human IAS circular smooth muscle cells and human enteric neuronal progenitor cells. After maturation of the bioengineered constructs in culture, they were implanted operatively into the perianal region of athymic rats. Platelet-derived growth factor was delivered to the implanted constructs through a microosmotic pump. Implanted constructs were retrieved from the animals 4 weeks postimplantation.

Results: Animals tolerated the implantation well, and there were no early postoperative complications. Normal stooling was observed during the implantation period. At harvest, implanted constructs were adherent to the perirectal rat tissue and appeared healthy and pink. Immunohistochemical analysis revealed neovascularization. Implanted smooth muscle cells maintained contractile phenotype. Bioengineered constructs responded in vitro in a tissue chamber to neuronally evoked relaxation in response to electrical field stimulation and vasoactive intestinal peptide, indicating the preservation of neuronal networks.

Conclusion: Our results indicate that bioengineered innervated IAS constructs can be used to augment IAS function in an animal model. This is a regenerative medicine based therapy for fecal incontinence that would directly address the dysfunction of the IAS muscle.

Figure 1

1A) A circumferential incision was made in the rat perineum, and a bioengineered innervated IAS construct was implanted over the rectum. B) A micro-osmotic pump delivering PDNF (52.8ng/day) was implanted subcutaneously, in a pocket created in the abdomen. C) The catheter from the micro-osmotic pump was directed towards the implanted bioengineered innervated human IAS construct. D) Rats recovered from anesthesia and stooling was observed within 30 minutes post-operatively.

Figure 2

Implanted constructs were retrieved between 28–32 days post operatively. (A) The surgical site had healed very well. (B) Circumferential dissection around the anal verge was performed, and the implanted constructs were identified by the non-absorbable suture placed around them (arrow). The implanted constructs were found integrated within the perirectal soft tissue. (C) Further exploration of the rat abdomen revealed normal proximal bowel without signs of obstruction.

Figure 3

(A) Cross-sections of implanted constructs were neovascularized and several blood vessels were seen embedded within the constructs. (B) Implanted human IAS smooth muscle cells stained positive with human specific HSP27, indicated by the red fluorescence. (C–D). Implanted smooth muscle cells within bioengineered constructs also stained positive for smooth muscle actin and smooth muscle caldesmon, indicating the maintenance of a contractile phenotype in the human smooth muscle.

Figure 4

Explanted bioengineered constructs were analyzed for force generation: (A) Treatment with relaxant peptide neurotransmitter VIP resulted in a relaxation of approximately 90% of basal tone. This relaxation was attenuated in the presence of neuronal blocker TTX, indicating neural integrity and survival of inhibitory motor neurons within the implanted construct. (B) Treatment with electrical field stimulation (EFS) resulted in neuronally evoked relaxation of the smooth muscle, which was attenuated completely in the presence of TTX.