White, Amanda B. MD; Keller, Patrick W. BS; Acevedo, Jesus F. BS; Word, R. Ann MD; Wai, Clifford Y. MD
Mechanical disruption of the anal sphincter complex during vaginal delivery is thought to be a major cause of anal incontinence in women.1–3 Perineal laceration, especially when it involves the anal sphincter, is an important risk factor for postpartum fecal incontinence.4 Sphincter defects are associated with flatal incontinence in 43% and 25% in women 3 and 6 months postpartum, respectively.5,6 These studies, together with others, indicate that direct injury of the anal sphincter musculature is associated with anal incontinence.
Previously, we demonstrated that significant impairment of contractile function occurs in rats 3 days after external anal sphincter transection.7 Ultimately, all parameters of anal sphincter physiology recovered by 3 months. Thus, even under ideal experimental conditions, the external anal sphincter requires time to heal. Although prevention of injury is of primary importance, when damage occurs, it is logical to focus on optimizing repair and providing an ideal environment for healing in the immediate period after injury.
The external anal sphincter is composed mainly of striated muscle. One of the unique properties of striated muscle is its ability to undergo spontaneous repair after injury. Wound healing in striated muscle involves activation, proliferation, and differentiation of special myogenic stem cells called satellite cells. Although these satellite cells are quiescent under normal conditions, they become activated in response to injury and play a major role in the regenerative phase of wound healing.8,9
The success of wound healing depends on having an adequate source of satellite cells and an appropriate physiologic environment. Regeneration or replacement of dysfunctional anal sphincter muscle through stem cell therapy and tissue engineering is a promising approach to wound healing and enhancing the physiologic function and morphology of a damaged anal sphincter. Although stem cell therapy has been shown to be beneficial in the treatment of urinary incontinence secondary to a deficient urethral sphincter10,11 and injured detrusor muscle,12 its effects on anal sphincter have not been defined. Thus, the objective of this study was to estimate the temporal effect of adjunctive myogenic stem cells on external anal sphincter neurophysiology after sphincter transection with and without repair.
MATERIALS AND METHODS
All procedures were approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center. After anesthesia with ketamine (40 mg/kg), acepromazine (0.2 mg/kg), and xylazine (10 mg/kg), for each of three time points (7, 21, or 90 days), young (2–3 months) virginal female Sprague-Dawley rats (200–300 g) were randomly assigned to one of four treatment groups using a random number table: anal sphincter transection with or without repair, and injection at the transection site with either phosphate-buffered saline alone (control) or stem cells in phosphate-buffered saline (stem cells) (Fig. 1).
For the anal sphincter transection with repair group, a 7-mm incision was made through the anal sphincter complex. This incision extended beyond the superior margins of the external anal sphincter, which is approximately 3–4 mm in longitudinal length. The rectal mucosa was reapproximated with single interrupted stitches (1 mm apart) of 5–0 braided polyglactin suture (Vicryl; Ethicon, Inc, Piscataway, NJ). A second layer of single interrupted stitches (1 mm apart) of the same suture and caliber was used as a reinforcing layer. The external anal sphincter was reapproximated with two single interrupted stitches of 5–0 braided polyglactin suture. For the anal sphincter transection without repair group, the same incision was created as described above but was not repaired. Procedures were performed by the same investigators to ensure standardization: treatment injections were performed by the lead investigator (A.B.W.) and surgical procedures by the senior investigator (C.Y.W.), who has had extensive experience with anal sphincter transection with repair in this animal model.
For animals randomly assigned to injection with phosphate-buffered saline, the ends of the external anal sphincter complex were identified microscopically before repair. Each end of the transection was injected with 20 microliters of phosphate-buffered saline using a 20-microliter Hamilton syringe (Hamilton, Reno, NV) and then repaired as detailed above.
A commercially available H9c2 myoblast cell line (American Type and Culture Company, Manassas, VA) was acquired for the preparation of myogenic cells. This cell line has been shown to differentiate into myotubes and adult striated muscle in vitro and in vivo13,14 and derived from Sprague-Dawley rats. Cells were cultured in Dulbecco's Modified Eagle Medium with 10% fetal bovine serum, 25 mM (4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid), and 1% antibiotic-antimycotic solution. To maintain the myoblastic component of the population, the cells were subcultured frequently to maintain preconfluence at all times. Immediately before injection, preconfluent cells were rinsed with phosphate-buffered saline and harvested by trypsin (0.25% vol/vol)-ethylene diamine tetra-acetic acid (1 mM). Cells (4×106) were suspended in phosphate-buffered saline (50 microleters). Animals randomly assinged to injection with myogenic stem cells underwent microscopic identification of the transected external anal sphincter complex followed by injection of each side with 1.6×106 cells (20 microliters) using a 20-microliter Hamilton syringe. A total of 3.2×106 cells were injected into the external anal sphincter complex of each rat.
Rats were killed with pentobarbital (50 mg/kg intraperitoneally) 7, 21, or 90 days (Fig. 1) after anal sphincter transection with or without repair plus either phosphate-buffered saline or stem cell injection at the transection site. The anal sphincter complex was dissected and removed by making a circumferential incision approximately 5 mm from the external anal orifice. Gentle traction of the perianal skin was used to facilitate sharp dissection of the anal complex from the connective tissue in the ischiorectal fossa laterally and the vagina anteriorly. The anal complex was removed intact after transecting the rectum 1.5–2 cm cephalad from the external anal orifice. Thereafter, a microscope was used to dissect the anal complex free of perianal fat and most of the perianal skin. To preserve integrity of the external anal sphincter, however, perianal tissue that was immediately adjacent to the sphincter was not teased away. The striated muscle of the external anal sphincter was identified and the lower rectum transected 1–2 mm above the sphincter. Immediately after dissection, tissues were mounted as a ring between two stainless steel wires in water-jacketed baths for assessment of contractile function as previously described.15 Within 30–60 minutes of mounting the tissue, muscles were stretched to optimal length for force development (ie. 1.2–1.8-g resting tension), and optimal resting tension was maintained throughout the course of the experiment. Tissue dissection and experimental protocols were performed in physiological salt solution of the following composition: NaCl (120.5 mM), KCl (4.8 mM), MgSO4 (1.2 mM), NaH2orally4 (1.2 mM), NaHCO3 (20.4 mM), CaCl2 (1.6 mM), D-glucose (10 mM), and pyruvate (1 mM), pH of 7.4. The solution was gassed with 95% oxygen and 5% carbon dioxide.
For electrical stimulation, platinum wire electrodes were mounted parallel to the suspended tissues. Electrodes were connected to an isolated pulse stimulator and a Grass stimulator (Grass Model S48; Grass Instrument Co., Quincy, MA) through a current-boosting amplifier (Bipolar Power Supply Amplifier, Model 6826A; Agilent Technologies, Santa Clara, CA). Stimulation of each tissue was also controlled by driver and signal conditioning/amplifier units (Grass DC driver amplifiers 7DAE or 7DAF, Grass low-level DC Amplifiers 7PIF). Analog force signals were captured with a multichannel analog-to-digital computer interface (National Instruments PCI-6032E, Austin, TX). Data were acquired at 100 Hz through the computerized interface. Data were collected before, during, and after initiation of field stimulation. Stimulation patterns were controlled by a computer program coupled to the voltage stimulator source using a computer interface (National Instruments PC-DIO-24 Digital I/O Board controlling a specially designed circuit). A central computerized controller delivered precise stimulation duration and frequencies. Voltage and pulse duration (in milliseconds) were determined manually through settings on the stimulator unit. Voltage settings, duration, and frequency were varied to obtain force-frequency and force-voltage response curves. Peak force production (defined as the maximum force produced during a contraction) was determined.
To determine twitch tension, tetanic force generation, and fatigue, experiments were conducted at 30°C. Twitch tension was determined after stimulation with 1 square 0.4-msec pulse of 50 V. For tetanic force generation, the muscle was allowed to recover and then force-frequency curves were determined by stimulation at 10–120 Hz, 50 V, 0.4-msec pulse duration, for 300 msec. After each frequency, muscles were stimulated with maximal stimulation of 50 V, 150 Hz. Muscles were allowed to recover for a minimum of 120 sec before the next increase in frequency. Forces obtained at each frequency were compared with forces obtained with maximal stimulation immediately thereafter. The ratio of single twitch tension to maximal tetanic tension was determined for each sphincter. Fatigue was determined by maximally stimulating the muscle for 30 sec at 50 V, 150 Hz. Force generation at 30 sec was expressed relative to initial maximal force generation. Maximal responses to electrical field stimulation were then determined at 37°C.
Investigators were not blinded to the group assignment of each animal during neurophysiologic testing. Although this may have potentially biased the results, investigators had no knowledge of group assignment until after dissection and preparation of tissues for mounting into the apparatus.
Data were reported as force normalized to sphincter as opposed to weight or volume of tissue to avoid underestimation of true contractile forces generated. The surrounding tissues immediately adjacent to the sphincter complex, not removed to preserve sphincter integrity, have no contractile properties but contribute substantially to the weight thereby affecting the results if used for normalization. Statistical analysis was a three-factor analysis of variance with interaction. Subanalyses were performed as statistical contrasts to explore important aspects of the significant interactions. P<.05 was considered significant. The three-factor interaction was not significant but all two-factor interactions were significant (by days, P=.013; repair by days, P<.001; and repair by treatment, P=.011). Consequently no main effect P values are reported. Data from previous experiments were used to predict the force generating capacity of sphincters from control animals. We assumed that the maximal force-generating capacity of the external anal sphincter over time of control animals would not be different from previously studied sham animals and anticipated a 60% initial decrease of maximal force production that would return to baseline levels by 90 days. To examine differences in time profiles for the different experimental conditions (significance level of .05 and 80% power), a sample size of eight animals in each group for each of the time points was determined (significance level of .05 and 80% power, PROC GLMPOWER, SAS version 9.1; SAS Institute, Cary, NC). To allow for unequal cell sizes due to the nature of possible loss of animals, we targeted 10 animals per group (N=120 animals: 60 randomly assigned to repair, 60 randomly assigned to no repair after anal sphincter transection).
Maximal contractile responses of repaired (Fig. 2A) and unrepaired external anal sphincters (Fig. 2B) were evaluated at 7, 21, and 90 days after administration of phosphate-buffered saline or myogenic stems cells at the site of sphincter transection. During this time, mean (±standard deviation) animal weights increased 60±10 g with no differences in animal weight between treatment groups at any time point. Seven days after sphincter transection with repair, maximal contractile force was impaired significantly in sphincters injected with phosphate-buffered saline (Fig. 2A). Significant improvement was seen as early as 21 days after transection/repair and was maintained 90 days after treatment. Similarly, maximal field-stimulated responses of repaired sphincters treated with myogenic stem cells was significantly reduced at 7 days and was markedly improved by 90 days (Fig. 2A). Although both treatment groups showed significant recovery of maximal responses at 90 days, improvement was greater in sphincters receiving stems cells than in phosphate-buffered saline–administered controls (P=.001) (Fig. 2A). Interestingly, force generation 7 days after sphincter transection without repair was not as severely compromised as the repaired sphincter, suggesting that suture placement and sphincter manipulation may have an adverse effect on sphincter function at early time points. Nevertheless, as expected, sphincter function did not improve with time if unrepaired (Fig. 2B). Furthermore, injection of myogenic stem cells at the site of injury had no effect on contractile responses of the unrepaired sphincter at any time point compared with controls.
For anal sphincters with repair, twitch tension was dramatically reduced after 7 days in both control and stem cell–administered animals (Fig. 3A). Recovery of twitch tension occurred as early as 21 days after both phosphate-buffered saline and stem cells. Although the recovery of twitch tension in the phosphate-buffered saline group of animals leveled out 90 days after treatment, further improvement was evident in the stem cell group (Fig. 3A). At 90 days, twitch tension of sphincters from animals that had received stems cells was increased significantly compared with controls (P=.001).
A similar effect was observed in maximal tetanic force generation (Fig. 3C). Seven days after injury with repair, maximal tetanic force generation was significantly impaired in phosphate-buffered saline– and stem cell–treated animals (Fig. 3C). Although improved significantly at 90 days, tetanic force was again significantly increased in the stem cell group compared with controls (P=.001, Fig. 3C).
For unrepaired sphincters, all measures of physiologic function (twitch tension and maximal tetanic contractile force generation in response to electrical field stimulation) were significantly impaired 7 days after sphincter transection (Fig. 3B and D). Unlike the repaired group in which all measures significantly improved with time, unrepaired sphincters did not show improvement of twitch tension (Fig. 3B) nor tetanic force generation (Fig. 3D) with administration of either phosphate-buffered saline or stem cells at 90 days.
To further define responses to electrical field stimulation, force-frequency and force-voltage response curves were determined in the external anal sphincters from all animals at each time point (Fig. 4). For animals that underwent sphincter transection with repair (Fig. 4A and B), frequency- and voltage-induced force generation for both the phosphate-buffered –and stem cell–treated groups at 90 days were statistically different from values 7 days after treatment, with the myogenic stem cell group demonstrating the greatest amount of improvement at 90 days (P<.001) compared with phosphate-buffered saline. Consistent with the findings for twitch tension, tetanic force generation, and maximal force in response to electrical field stimulation, unrepaired animals did not exhibit any difference between treatment groups (phosphate-buffered saline compared with stem cell), nor did they demonstrate any interval improvement from 7 to 90 days (Fig. 4B and D).
External anal sphincters were stimulated at 50V, 150 Hz for 30 sec, and fatigue was expressed as a percentage of force generation at 30 sec relative to initial maximal force generation (Fig. 5). Denervation injury of striated muscle is accompanied by increases in slow:fast twitch muscle fibers resulting in resistance to fatigue and increases in twitch:tetanic tension ratios. To determine if repaired or unrepaired transection of sphincters resulted in denervation, these parameters were quantified as a function of time after injury (Fig. 5, Table 1). Fatigability was not altered in repaired or unrepaired external anal sphincters with or without stem cells at any time point with the single exception of 7 days after repair (Fig. 5A). Repaired sphincters without myogenic stem cells were less fatigable, and the twitch:tetanic tension ratio was increased early after injury (7 days) but resolved with time (Fig. 5A, Table 1). Overall, these results suggest that although repair of the transected sphincter may be accompanied by transient denervation (likely due to suture-induced nerve injury and edema), the predominant phenotype 90 days after injury is not one of denervation. The external anal sphincter of the rat is composed of both slow- and fast-twitch fibers. In general, after denervation, the proportion of fast-twitch fibers increases in slow-twitch muscles.16 Fatigability of fast-twitch skeletal muscle is more pronounced and the twitch:tetany ratio is decreased.17 Denervated slow twitch muscles exhibit increased fatigability and increases the twitch:tetany ratio as the muscle adapts to denervation and transitions from the slow- to fast-twitch phenotype.16,17 With the twitch:tetany ratio unchanged, and fatigability not altered (except for transiently 7 days after repair), these findings suggest that the primary cause of the impaired function of the sphincter under these experimental conditions was not denervation but mechanical disruption. Further, re-innervation of a denervated muscle is not likely with a normal twitch:tetany ratio.18
As an additional control, limited neurophysiologic studies (twitch tension and response to maximal electrical field stimulation) were conducted in eight unoperated animals to determine baseline contractile properties. Maximal force generated in response to electrical field stimulation (6.65±1.15 g) and maximal twitch tension (0.94±0.25 g) in these control animals were not significantly different from phosphate-buffered saline–administered animals at 90 days. However, animals that were administered stem cells had superior function compared with these unoperated controls.
Consistent with previous work,19 findings from this study confirm that recovery of physiologic function of the transected and repaired external anal sphincter occurs with time. More importantly, these data demonstrate that a single administration of myogenic stem cells at the site and time of injury with concomitant repair provides additional benefit to various parameters of striated muscle physiology (twitch tension, maximal tetanic contraction, and maximal contractile force in response to electrical field stimulation).
Although the exact mechanisms by which myogenic stem cells affect recovery from injury remains unclear, it provides an attractive and feasible treatment adjunct for anal sphincter laceration. Some hypothesize that the ability of the stem cells to differentiate and form multinucleated myotubes facilitates recovery of striated muscle function after injury.20–22 Others suggest it is the recruitment, activation, and subsequent proliferation of once-quiescent myogenitor precursor cells that enhance the reparative process.23 Generally, injury in skeletal muscle results in macrophage activity and local angiogenesis. The initial muscle damage and mediators released trigger satellite cell activation and chemotactic migration to the site of injury. Cells fuse to form myoblasts, then into myotubes and, in turn, mature into muscle fibers. The response by skeletal muscle to injury can be classified into three interrelated phases: destruction, repair, and remodeling.24 Administration of myogenic stem cells is believed to play a role in the remodeling phase (regeneration and reorganization of muscle fibers).
Not surprisingly, unrepaired sphincters remained compromised even at 90 days after injury. However, it was interesting to note that unrepaired sphincters injected with stem cells also demonstrated no improvement of any parameter of sphincter function. These results suggest that administration of stem cells does not always result in recovery of function, especially in unrepaired sphincters. To optimize the effect of stem cells on the healing process, an intact innervation is required, as well as the cells' ability to fuse with myofibers from the transected sphincter muscle. The absence of an intact sphincter, in which the muscle fibers are separated by some distance, may impede fusion with existing myofibers. Additionally, the ends of the musculature may need to be in close proximity so that local release of growth factors can regulate proliferation and differentiation of myoblasts at the injured site.25,26 This ultimately would facilitate muscle regeneration and repair. It is important to recognize that repair of the external anal sphincter in this study was performed under optimal experimental conditions after a clean surgical incision. In practice, the injury is not precise and not completely clean, nor is the repair performed with the assistance of a microscope. The persistence of impaired physiologic function in unrepaired sphincters highlights the importance of recognition with subsequent repair, especially in the context of myogenic stem cell administration.
The transected and repaired external anal sphincter of both nonpregnant19 and pregnant27 rats recovers to baseline function by 90 days. It should be emphasized, however, that the sphincter of this experimental animal model is simply transected and repaired under optimal surgical conditions using a dissecting microscopic. In women, the sphincter is torn, stretched, and likely exposed to hypoxia, free radicals, and fecal contamination. Conditions for postpartum repair are almost always suboptimal. The experimental findings of this work indicate that administration of myogenic stem cells results in superior contractile function compared with phosphate-buffered saline–administered controls. As in our previous studies, contractile function of the external anal sphincter from phosphate-buffered saline controls at 90 days was virtually identical to that at baseline. We suggest that myogenic stem cells may not be needed to augment repair of a sharply-defined transected sphincter with optimal repair. Myogenic stem cells, however, may serve as a therapeutic adjunct for severe and complicated anal lacerations at the time of vaginal delivery.
Although this study focuses on the effects of administering myogenic stem cells after acute injury, the clinical utility may be applied to the chronically injured anal sphincter. Given the positive findings of this study, injection at other times and sites remote from injury would be the focus of subsequent studies. As scientists continue to explore the potential role for stem cell therapy in recovery of tissue from injury, our study indicates that in an animal model stem cells provide a useful adjunct to the surgical management of acute external anal sphincter injury and may have potential utility in recovery of a physiologically impaired sphincter.
In conclusion, this study demonstrates that administration of myogenic stem cells enhances contractile function of repaired external anal sphincters in an animal model after 90 days compared with repair alone. The unrepaired sphincter, however, did not benefit from the reparative potential of stem cells. The mechanisms by which myogenic stem cells improved function of the repaired sphincter are not known. Previous studies indicate that stem cells are incorporated into injured anal sphincter.28,29 Studies in other models of tissue injury indicate that stem cells improve organ function through secretion of antiinflammatory proteins.30 Our study did not address this issue but demonstrated functional improvement of the sphincter with stem cell injection. Studies are in progress to determine whether these results are unique to myogenic stem cells and if enhancement of the microenvironment through growth factor release, secretion of antiinflammatory molecules, and matrix reconstruction contributes to this phenomenon.
Taken together, findings from this study provide experimental evidence that myogenic stem cells may serve as positive adjunctive therapy in the treatment of anal sphincter injury. Myogenic stem cells improve both acute and long-term function of the external anal sphincter after mechanical injury. Perhaps equally significant, the results underscore the importance of recognition of anal sphincter defects and subsequent repair.
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