Sphincter contractility after muscle-derived stem cells autograft into the cryoinjured anal sphincters of rats

Sung-Bum Kang, Haet Nim Lee, Ji Young Lee, Jun-Seok Park, Hye Seung Lee, Ji Youl Lee, Sung-Bum Kang, Haet Nim Lee, Ji Young Lee, Jun-Seok Park, Hye Seung Lee, Ji Youl Lee

Abstract

Purpose: This study was designed to determine whether the injection of muscle-derived stem cells into the anal sphincter can improve functional properties in a fecal incontinence rat model.

Methods: Cryoinjured rats were utilized as a fecal incontinence model. The gastrocnemius muscles of normal three-week-old female Sprague-Dawley rats were used for the purification of the muscle-derived stem cells. The experimental group was divided into three subgroups: normal control; cryoinjured; and muscle-derived stem cells (3 x 10(6) cells) injection group of cryoinjured rats. All groups were subsequently employed in contractility experiments using muscle strips from the anal sphincter, one week after preparation.

Results: Contractility in the cryoinjured group was significantly lower than in the control after treatment with acetylcholine and KCl. In the muscle-derived stem cells injection group, contraction amplitude was higher than in the cryoinjured group but not significantly (20.5 +/- 21.3 vs. 17.3 +/- 3.4 g per gram tissue, with acetylcholine (10(-4) mol/l); 31 +/- 14.2 vs. 18.4 +/- 7.9 g per gram tissue, with KCl (10(-4) mol/l)). PKH-26-labeled transplanted cells were detected in all of the grafted sphincters. Differentiated muscle masses stained positively for alpha smooth muscle actin and myosin heavy chain at the muscle-derived stem cells injection sites.

Conclusions: This is the first study reporting that autologous muscle-derived stem cell grafts may be a tool for improving anal sphincter function.

Figures

FIGURE 1
FIGURE 1
Representative contractility recording of muscle strips treated with acetylcholine. a. Normal control with acetylcholine. b. Cryoinjured group with acetylcholine. c. Muscle-derived stem cell injection group with acetylcholine.
FIGURE 2
FIGURE 2
Hematoxylin-eosin staining. a. Normal histology of the anal sphincter in normal control rats. Internal smooth muscle (arrowhead) and external skeletal muscle (arrow) (magnification, ×10). b. Damaged muscle fibers with cytoplasmic vacuolization and focal interstitial inflammatory cell infiltration in the cryoinjured group (magnification, ×400). c. Regenerating muscle fibers in the variable orientation with enlarged nuclei at the muscle-derived stem cell injection site (magnification, ×400).
FIGURE 3
FIGURE 3
Muscle formation in the cryoinjured anal sphincter following muscle-derived stem cell injection. a-c. Normal control group shows a thick smooth muscle layer encircled by a layer of outer striated muscle fibers. d-f. Cryoinjured group shows weakened smooth muscle and skeletal muscle layer. G. Muscle-derived stem cell injection group shows the variable orientation of the new muscle fiber. h. Differentiated smooth muscle (yellow) shown in colocalization of injected muscle-derived stem cell injection (PKH-26-staining, red) and smooth muscle via the immunostaining of alpha smooth muscle actin (green). i. Differentiated skeletal muscle (yellow) shown in the colocalization of injected muscle-derived stem cell injection (PKH-26-staining, red) and skeletal muscle via immunostaining of myosin heavy-chain (green). Hematoxylin-eosin staining (a, d, g), immunostaining of alpha smooth muscle actin (b, e, h), and myosin heavy-chain (c, f, i) (magnification, ×100).
FIGURE 4
FIGURE 4
CD3 staining. a. Scattered CD3-positive T cells (brown color) in anal mucosa of normal rat. b. CD3 staining was not observed after MDSC injection (magnification, ×200).

References

    1. Macmillan AK, Merrie AE, Marshall RJ, Parry BR. The prevalence of fecal incontinence in community-dwelling adults: a systematic review of the literature. Dis Colon Rectum. 2004;47:1341–1349. doi: 10.1007/s10350-004-0593-0.
    1. Shafik A. Polytetrafluoroethylene injection for the treatment of partial fecal incontinence. Int Surg. 1993;78:159–161.
    1. Kumar D, Benson MJ, Bland JE. Glutaraldehyde cross-linked collagen in the treatment of faecal incontinence. Br J Surg. 1998;85:978–979. doi: 10.1046/j.1365-2168.1998.00751.x.
    1. Shafik A. Perianal injection of autologous fat for treatment of sphincteric incontinence. Dis Colon Rectum. 1995;38:583–587. doi: 10.1007/BF02054115.
    1. Malouf AJ, Vaizey CJ, Norton CS, Kamm MA. Internal anal sphincter augmentation for fecal incontinence using injectable silicone biomaterial. Dis Colon Rectum. 2001;44:595–600. doi: 10.1007/BF02234337.
    1. Chan MK, Tjandra JJ. Injectable silicone biomaterial (PTQ) to treat fecal incontinence after hemorrhoidectomy. Dis Colon Rectum. 2006;49:433–439. doi: 10.1007/s10350-005-0307-2.
    1. Tjandra JJ, Lim JF, Hiscock R, Rajendra P. Injectable silicone biomaterial for fecal incontinence caused by internal anal sphincter dysfunction is effective. Dis Colon Rectum. 2004;47:2138–2146. doi: 10.1007/s10350-004-0760-3.
    1. Kenefick NJ, Vaizey CJ, Malouf AJ, Norton CS, Marshall M, Kamm MA. Injectable silicone biomaterial for faecal incontinence due to internal anal sphincter dysfunction. Gut. 2002;51:225–228. doi: 10.1136/gut.51.2.225.
    1. Davis K, Kumar D, Poloniecki J. Preliminary evaluation of an injectable anal sphincter bulking agent (Durasphere) in the management of faecal incontinence. Aliment Pharmacol Ther. 2003;18:237–243. doi: 10.1046/j.1365-2036.2003.01668.x.
    1. Pannek J, Brands FH, Senge T. Particle migration after transurethral injection of carbon coated beads for stress urinary incontinence. J Urol. 2001;166:1350–1353. doi: 10.1016/S0022-5347(05)65767-9.
    1. Emans PJ, Saralidze K, Knetsch ML, Gijbels MJ, Kuijer R, Koole LH. Development of new injectable bulking agents: biocompatibility of radiopaque polymeric microspheres studied in a mouse model. J Biomed Mater Res A. 2005;73:430–436.
    1. Kuang S, Kuroda K, Le Grand F, Rudnicki MA. Asymmetric self-renewal and commitment of satellite stem cells in muscle. Cell. 2007;129:999–1010. doi: 10.1016/j.cell.2007.03.044.
    1. Qu-Petersen Z, Deasy B, Jankowski R, et al. Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. J Cell Biol. 2002;157:851–864. doi: 10.1083/jcb.200108150.
    1. Lee JY, Paik SY, Yuk SH, Lee JH, Ghil SH, Lee SS. Long term effects of muscle-derived stem cells on leak point pressure and closing pressure in rats with transected pudendal nerves. Mol Cells. 2004;18:309–313.
    1. Cannon TW, Lee JY, Somogyi G, et al. Improved sphincter contractility after allogenic muscle-derived progenitor cell injection into the denervated rat urethra. Urology. 2003;62:958–963. doi: 10.1016/S0090-4295(03)00679-4.
    1. Yiou R, Yoo JJ, Atala A. Restoration of functional motor units in a rat model of sphincter injury by muscle precursor cell autografts. Transplantation. 2003;76:1053–1060. doi: 10.1097/01.TP.0000090396.71097.C2.
    1. Kwon D, Kim Y, Pruchnic R, et al. Periurethral cellular injection: comparison of muscle-derived progenitor cells and fibroblasts with regard to efficacy and tissue contractility in an animal model of stress urinary incontinence. Urology. 2006;68:449–454. doi: 10.1016/j.urology.2006.03.040.
    1. Lee JY, Cannon TW, Pruchnic R, Fraser MO, Huard J, Chancellor MB. The effects of periurethral muscle-derived stem cell injection on leak point pressure in a rat model of stress urinary incontinence. Int Urogynecol J Pelvic Floor Dysfunct. 2003;14:31–37. doi: 10.1007/s00192-002-1004-5.
    1. Yiou R, Dreyfus P, Chopin DK, Abbou CC, Lefaucheur JP. Muscle precursor cell autografting in a murine model of urethral sphincter injury. BJU Int. 2002;89:298–302. doi: 10.1046/j.1464-4096.2001.01618.x.
    1. Chermansky CJ, Tarin T, Kwon DD, et al. Intraurethral muscle-derived cell injections increase leak point pressure in a rat model of intrinsic sphincter deficiency. Urology. 2004;63:780–785. doi: 10.1016/j.urology.2003.10.035.
    1. Strasser H, Marksteiner R, Margreiter E, et al. Autologous myoblasts and fibroblasts versus collagen for treatment of stress urinary incontinence in women: a randomised controlled trial. Lancet. 2007;369:2179–2186. doi: 10.1016/S0140-6736(07)61014-9.
    1. Garcia-Olmo D, Garcia-Arranz M, Garcia LG, et al. Autologous stem cell transplantation for treatment of rectovaginal fistula in perianal Crohn’s disease: a new cell-based therapy. Int J Colorectal Dis. 2003;18:451–454. doi: 10.1007/s00384-003-0490-3.
    1. Garcia-Olmo D, Garcia-Arranz M, Herreros D, Pascual I, Peiro C, Rodriguez-Montes JA. A phase I clinical trial of the treatment of Crohn’s fistula by adipose mesenchymal stem cell transplantation. Dis Colon Rectum. 2005;48:1416–1423. doi: 10.1007/s10350-005-0052-6.
    1. Rando TA, Blau HM. Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J Cell Biol. 1994;125:1275–1287. doi: 10.1083/jcb.125.6.1275.
    1. Huard J, Yokoyama T, Pruchnic R, et al. Muscle-derived cell-mediated ex vivo gene therapy for urological dysfunction. Gene Ther. 2002;9:1617–1626. doi: 10.1038/sj.gt.3301816.
    1. Gulbins H, Pritisanac A, Anderson I, et al. Myoblasts for survive 16 weeks after intracardiac transfer and start differentiation. Thorac Cardiovasc Surg. 2003;51:295–300. doi: 10.1055/s-2003-45418.
    1. Hwang JH, Yuk SH, Lee JH, et al. Isolation of muscle derived stem cells from rat and its smooth muscle differentiation. Mol Cells. 2004;17:57–61.
    1. Qu Z, Balkir L, van Deutekom JC, Robbins PD, Pruchnic R, Huard J. Development of approaches to improve cell survival in myoblast transfer therapy. J Cell Biol. 1998;142:1257–1267. doi: 10.1083/jcb.142.5.1257.
    1. Beauchamp JR, Heslop L, Yu DS, et al. Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells. J Cell Biol. 2000;151:1221–1234. doi: 10.1083/jcb.151.6.1221.
    1. Yokoyama T, Huard J, Chancellor MB. Myoblast therapy for stress urinary incontinence and bladder dysfunction. World J Urol. 2000;18:56–61. doi: 10.1007/s003450050010.
    1. Hagège AA, Carrion C, Menasché P, et al. Viability and differentiation of autologous skeletal myoblast grafts in ischaemic cardiomyopathy. Lancet. 2003;361:491–492. doi: 10.1016/S0140-6736(03)12458-0.
    1. Alameddine HS, Louboutin JP, Dehaupas M, Sebille A, Fardeau M. Functional recovery induced by satellite cell grafts in irreversibly injured muscles. Cell Transplant. 1994;3:3–14.
    1. Guettier-Sigrist S, Coupin G, Warter JM, Poindron P. Cell types required to efficiently innervate human muscle cells in vitro. Exp Cell Res. 2000;259:204–212. doi: 10.1006/excr.2000.4968.
    1. Alessandri G, Pagano S, Bez A, et al. Isolation and culture of human muscle-derived stem cells able to differentiate into myogenic and neurogenic cell lineages. Lancet. 2004;364:1872–1883. doi: 10.1016/S0140-6736(04)17443-6.

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