Gastroenterology
Volume 137, Issue 1 , Pages 53-61, July 2009

Bioengineered Internal Anal Sphincter Derived From Isolated Human Internal Anal Sphincter Smooth Muscle Cells

  • Sita Somara

      Affiliations

    • Department of Pediatrics-Gastroenterology University of Michigan, Ann Arbor, Michigan
    • ,
  • Robert R. Gilmont

      Affiliations

    • Department of Pediatrics-Gastroenterology University of Michigan, Ann Arbor, Michigan
    • ,
  • Robert G. Dennis

      Affiliations

    • Department of Biomedical Engineering, University of North Carolina, Chapel Hill, North Carolina
    • ,
  • Khalil N. Bitar

      Affiliations

    • Department of Pediatrics-Gastroenterology University of Michigan, Ann Arbor, Michigan
    • Corresponding Author InformationReprint requests Address requests for reprints to: Khalil N. Bitar, PhD, AGAF, University of Michigan Medical School, 1150 W. Medical Center Dr., MSRB I, Room A520, Ann Arbor, Michigan 48109-0658. fax: (734) 647-9703

Received 20 August 2008; accepted 17 March 2009. published online 31 March 2009.

Background & Aims

The internal anal sphincter (IAS) is a specialized circular smooth muscle that maintains rectoanal continence. In vitro models are needed to study the pathophysiology of human IAS disorders. We bioengineered sphincteric rings from human IAS smooth muscle cells (SMC) and investigated their response to cholinergic stimulation as well as investigated whether protein kinase C (PKC) and Rho kinase signaling pathways remain functional.

Methods

3-Dimensional bioengineered ring (3DBR) model of the human IAS was constructed from isolated human IAS SMC obtained from surgery. Contractile properties and force generation in response to acetylcholine, PKC inhibitor calphostin-C, Rho/ROCK inhibitor Y-27632, permeable Rho/ROCK inhibitor c3-exoenzyme, and PKC activator PdBU was measured.

Results

The human IAS 3DBR has the essential characteristics of physiologically functional IAS; it generated a spontaneous myogenic basal tone, and the constructs were able to relax in response to relaxants and contract in response to contractile agents. The constructs generated dose-dependent force in response to acetylcholine. Basal tone was significantly reduced by calphostin-C but not with Y-27632. Acetylcholine-induced force generation was also significantly reduced by calphostin-C but not with Y-27632. PdBU generated force that was equal in magnitude to acetylcholine. Thus, calphostin-C inhibited PdBU-induced force generation, whereas Y-27632 and c3 exoenzyme did not.

Conclusions

These data indicate that basal tone and acetylcholine-induced force generation depend on signaling through the PKC pathway in human IAS; PKC-mediated force generation is independent of the Rho/ROCK pathway. This human IAS 3DBR model can be used to study the pathophysiology associated with IAS malfunctions.

Abbreviations used in this paper: 3DBR, 3-dimensional bioengineered ring, IAS, internal anal sphincter, PKC, protein kinase C, SMC, smooth muscle cells

 

 Conflicts of interest The authors disclose no conflicts.

 Funding Supported by National Institutes of Health Grant NIDDK R01-DK071614.

PII: S0016-5085(09)00464-8

doi:10.1053/j.gastro.2009.03.036

Gastroenterology
Volume 137, Issue 1 , Pages 53-61, July 2009