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Obstetrics & Gynecology:
doi: 10.1097/AOG.0b013e3182397009
Original Research

Recovery of the Injured External Anal Sphincter After Injection of Local or Intravenous Mesenchymal Stem Cells

Pathi, Sujatha D. MD; Acevedo, Jesus F. BA; Keller, Patrick W. BA; Kishore, Annavarapu H. PhD; Miller, Rodney T. PhD; Wai, Clifford Y. MD; Word, R. Ann MD

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Author Information

From ProPath Laboratory, and the Division of Female Pelvic Medicine and Reconstructive Surgery, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas.

Supported by the AUGS Foundation and an Astellas research grant.

Presented at the 31st Annual Scientific Meeting of the American Urogynecologic Society, September 29–October 2, 2010, Long Beach, California.

Corresponding author: R. A. Word, Division of Urogynecology and Reconstructive Surgery, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX 75390-9032; e-mail: ruth.word@utsouthwestern.edu.

Financial Disclosure The authors did not report any potential conflicts of interest.

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Abstract

OBJECTIVES: To understand the endogenous process of wound healing after anal sphincter injury and to determine possible mechanisms by which mesenchymal stem cells (MSCs) exert their regenerative potential.

METHODS: Virginal female rats (n=204) underwent anal sphincter laceration and repair. Thereafter, animals were randomly assigned to control injection, injection with intravenous MSCs, or direct injection of MSCs into the injured sphincter. Twenty uninjured animals served as baseline controls. Sphincters were analyzed for contractile function and parameters of wound healing 24 hours, 48 hours, 7 days, and 21 days after injury.

RESULTS: Direct injection of MSCs into the injured anal sphincter resulted in improved contractile function 21 days after injury compared with controls. Although expression of both proinflammatory (cyclooxygenase-2 and interleukin-6) and anti-inflammatory (interleukin-10 and tumor necrosis factor-α–stimulated gene-6) genes were increased dramatically and transiently after injury, MSCs did not alter this response. In contrast, transforming growth factor (TFG)-β1 (an important mediator of matrix deposition by mesenchymal cells) and lysyl oxidase (an enzyme important for synthesis of collagen and elastin) expression increased dramatically at earlier time points in the direct MSC injection group compared with controls. Increased expression of TFG-β1 and lysyl oxidase in directly injected sphincters was associated with increased collagen deposition and engraftment of MSCs in the sphincter.

CONCLUSION: In this preclinical animal model, direct, but not intravenous, injection of MSCs into the injured anal sphincter at the time of repair resulted in improved contractile function of the sphincter after injury, increased matrix deposition in the external anal sphincter, and increased expression of TFG-β1 and lysyl oxidase in the acute phase after injury.

Anal sphincter laceration during child birth is a risk factor for postpartum anal incontinence. Anal sphincter lacerations have been reported to occur in from 0.5% to 18% of vaginal deliveries.1 Fecal incontinence affects 7–28% of women24 with obstetric anal sphincter injuries. Prospective studies have shown that within 5 years of sustaining an anal sphincter tear, 42% of women have bothersome flatal incontinence and 11% have fecal incontinence.2

Despite repair at time of injury, up to 41% of women with a third-degree or fourth-degree tear report anal incontinence at 9 months postpartum.3 It is therefore important to understand prevention and treatment of obstetric anal sphincter injuries. Currently, cell-based treatments are being explored as a potential adjunct to address the poor outcomes of primary sphincter repair (Zutshi M, Salcedo L, Penn M, Mayorga M, Hull TL, Damaser M. Mesenchymal stem cells improve functional outcome after anal sphincter injury in an animal model. 39th Annual Scientific Meeting of the International Continence Society, September 29–October 3, 2009, San Francisco, California, 2009).57 One goal of these treatments is to optimize wound healing and foster regeneration of specialized cells to preinjury functional status. Recently, we found that injection of myogenic stem cells at the time of anal sphincter repair in the rat model resulted in superior contractile function at 90 days compared with repair alone.6 Other studies have examined the use of mesenchymal stem cells (MSCs) injected at the time of sphincter repair and found similar improvements in sphincter function (Zutshi M et al. Mesenchymal stem cells improve functional outcome after anal sphincter injury in an animal model. 39th Annual Scientific Meeting of the International Continence Society, 2009).5 The mechanisms underlying this improved wound healing remain unknown.8

Mesenchymal stem cells have been used with good results in animal models examining osteogenesis imperfecta, myocardial infarction, spinal cord injury, stroke, and cartilage repair.911 In these studies, MSCs were delivered intravenously with the assumption that these cells homed to the site of injury. Although local engraftment and cell differentiation was observed in some experimental protocols, in a number of reports functional improvement was observed with minimal MSC engraftment at the site of injury.1215 Therefore, it has been postulated that MSC have significant paracrine effects and thereby secrete factors that enhance regeneration without direct incorporation into the site of injury.13

Here, we utilized a rat model to extend our previous work on direct injection of myogenic stems into the anal sphincter. Specifically, we compared the effect of intravenous and direct administration of mesenchymal stems cells on functional recovery of the repaired anal sphincter after injury. Further, we compared gene expression profiles of several proinflammatory and anti-inflammatory mediators in the anal sphincter at various time points in the healing process, both with and without different modes of MSC delivery.

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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), 204 young, nulliparous, female Lewis rats were randomly assigned using a random number table to one of three treatment groups (Fig. 1). Twenty additional animals served as unoperated controls and 27 animals were used to identify stem cells in the sphincter complex after injury. Anal sphincter transections and repairs were performed as previously described1618 by the same investigator (S.D.P.). Briefly, a 7-mm incision was made with dissecting scissors through the anal sphincter complex. Rectal mucosa was reapproximated with single interrupted stitches (1 mm apart) of 5-0 braided polyglactin suture (Vicryl, Ethicon). A second layer of single interrupted stitches of the same suture was used as a reinforcing layer. The external anal sphincter was reapproximated with two single interrupted stitches of 5-0 braided polyglactin suture.

Fig. 1
Fig. 1
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All animals received both intravenous and direct injections. Specifically, the control group received intravenous and direct injections of phosphate-buffered saline (PBS); the intravenous MSC group received intravenous MSCs, and the direct PBS and the direct MSC groups received intravenous PBS and direct MSCs (Fig. 1). Immediately before direct injection, 4×106 cells were suspended in PBS (50 microliters). The direct route consisted of two 20-microliter injections into either side of the transected anal sphincter using a 25-microliter Hamilton syringe. Immediately before intravenous injection, 4×106 cells were suspended in PBS (500 microliters). Intravenous injections (400 microliters) were performed through the tail vein using a 30-gauge syringe. Rats were killed with pentobarbital (50 mg/kg) intraperitoneally and sphincters were harvested 24 hours, 48 hours, 7 days, and 21 days after injury.

Rat bone–derived mesenchymal stromal cells were obtained from the Center for the Preparation and Distribution of Adult Stem Cells located at Texas A & M University. Cells were cultured in alpha-minimal essential medium with 20% fetal bovine serum, 200 nM glutamine, and 1% antibiotic-antimycotic. To maintain the early progenitor phenotype, cells were maintained at preconfluence at all times. Forty-eight hours before injection, cells were treated with an adenoviral construct of green fluorescent protein (100 multiplicity of infection/cell). Infection of stem cells with this construct has been shown to maintain green fluorescent protein fluorescence for 60 days in vitro (Victor Lin, PhD, University of Texas Southwestern, personal communication).

Tissues were immediately snap-frozen in liquid nitrogen. Specimens were pulverized while frozen and homogenized in 4 mol/L guanidinium isothiocyanate buffer, layered over 5.7 mol/L cesium chloride and centrifuged overnight at 237,000 g to extract RNA. Reverse-transcription reactions were conducted with 2 micrograms of total RNA in a reaction volume of 20 microliters. Each reaction contained 10 mM DTT, 0.5 mM dNTPs, 0.015 micrograms/microliters random primers, 40 units of RNase inhibitor (Invitrogen 10777–019; Invitrogen), and 200 units of reverse-transcriptase (Invitrogen 18064–014, Invitrogen). Primer sequences for amplifications were chosen using published cDNA sequences and the Primer Express program (Applied Biosystems). Primers were chosen such that, when possible, the resulting amplicons would cross an exon junction, thereby eliminating any possible false-positive signals from genomic DNA contamination (Table 1). SYBR green was used for amplicon detection. Gene expression was normalized to expression of the housekeeping gene cyclophilin. Positive controls (heart, lung, spleen) were performed on each plate as appropriate and all assays included no template controls. All primer sets were tested to ensure that efficiency of amplification over a wide range of template concentrations was equivalent to that of cyclophilin. The reverse-transcription product from 50 ng RNA was used as template and reaction volumes (30 microliters) contained 1× Master Mix (Applied Biosystems 4309155, Applied Biosystems). Primer concentrations were 900 nM. Cycling conditions were 2 minutes at 50°C, followed by 10 minutes at 95°C, then 40 cycles of 15 sec at 95°C, and 1 minute at 60°C. A pr-programmed dissociation protocol was used after amplification to ensure that all samples exhibited a single amplicon. Levels of mRNA were determined using the ddCt method (Applied Biosystems) and were expressed relative to an external calibrator on each plate.

Table 1
Table 1
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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.19 Twitch tension, tetanic force generation, and fatigue were conducted at 30°C. Twitch tension was determined after stimulation with one square 0.4-msec pulse of 50 volts. For tetanic force generation, the muscle was allowed to recover and then force-frequency curves were determined by stimulation at 10–120 Hz, 50 volts, and 0.4-msec pulse duration for 300 msec. After each frequency, muscles were stimulated with maximal stimulation of 50 volts and 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. Fatigue was determined by maximally stimulating the muscle for 30 sec at 50 volts and 150 Hz. Force generation at 30 sec was expressed relative to initial maximal force. Maximal responses to field stimulation were then determined at 37°C.

Tissues were fixed in neutral buffer formalin (10%) for 24 hours. The 5-micrometer cross-sections of formalin-fixed, paraffin-embedded tissues were obtained at 100-micrometer intervals throughout the entire sphincter complex (average of 10–12 sections per sphincter). Tissues were stained with hematoxylin and eosin and analyzed with a Nikon Eclipse E1000N microscope. Sections identified as having complete circumferential external anal sphincter were destained and restained with picrosyrius red, which binds specifically to collagen fibrils of varying diameter. Collagen content then was quantified using ImageJ software (http://rsbweb.nih.gov/ij/). Images of sphincters were converted to grayscale, the green channel was used to segment red-stained collagen from the tissue, and thresholding was automatic. Area of interest was designated as a radial length of 5 mm from the center of injury. Area of localized injury (ie, granuloma, fibrous capsule, walled-off suture) was then subtracted. Fractional area of collagen was then determined and divided by total tissue area.

Anesthetized animals were perfused with ice-cold PBS until clear and then with paraformaldehyde. Thereafter, the sphincter complex was frozen in optimum cutting temperature and stored at −20°C. Cryosections of the anal sphincter complex were immunostained with antigreen fluorescent protein antibody (1:200 dilution, Living Colors JL-8, Clontech) using a mouse-on-mouse HRP detection kit (Biocare Medical).

Statistical comparisons between groups were conducted by one-way analysis of variance using Dunnet comparison of means with the PBS group used as the control. A two-factor analysis of variance was conducted on studies involving both time and treatment as variables, and a two-way repeated measures analysis of variance was used to analyze force-frequency curves. Differences in collagen quantification were determined using Kruskal Wallis testing and Dunn multiple comparison test. P≤.05 was considered significant. Based on the variability determined from previous studies6 after anal sphincter repair, an estimated sample size of seven animals per time period in each study group was required to detect a 30% change in stem cell-induced recovery of sphincter physiology with a power of 0.80 and an alpha of 0.05. Pilot studies of mRNA quantification of the anal sphincter complex indicated that eight animals in each group would be required to detect a twofold change at each time point. Because it was anticipated that up to 30% of RNA samples would be degraded or lost in sample preparation, 10 animals at each time point were used for molecular studies.

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RESULTS

To estimate the effect of intravenous or direct injection of MSCs on function of the external anal sphincter after injury and repair, neurophysiologic studies were conducted on freshly isolated tissues 21 days after injury (Fig. 2). Results were compared with unoperated littermate controls. Twitch tension and maximal field-stimulated force generation were decreased significantly in PBS-injected sphincters 21 days after injury and repair. The external anal sphincter was also more susceptible to fatigue in this group, suggesting that the external anal sphincter had not recovered 21 days after injury. In contrast, twitch tension, maximal force generation, and fatigue of sphincters in which MSCs were injected directly were virtually identical to sphincters from unoperated controls (Fig. 2). Data from sphincters isolated 21 days after intravenous MSC infusion appeared intermediate between unoperated controls and direct injections.

Fig. 2
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These results were confirmed with force-frequency curves (Fig. 3). After direct injection of stem cells, force production in response to various frequencies (10–100 Hz) at 30 volts was similar to that of unoperated controls. In contrast, force production in control sphincters after injury (PBS) was compromised significantly from 30 to 100 Hz (Fig. 3). Intravenous infusion of MSC tended to result in improved force production in response to higher frequencies compared with the PBS group. Taken together, contractility studies revealed that external anal sphincter function was significantly decreased 21 days after injury compared with unoperated controls, direct injection of MSCs at the time of repair resulted in full functional recovery within 21 days, and intravenous injection of MSCs at the time of repair did not fully rescue the compromised sphincter.

Fig. 3
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To study wound healing at the molecular level in the external anal sphincter with and without MSCs, we chose to quantify mRNA levels of two proinflammatory genes highly expressed in wounds after acute injury (cyclooxygenase 2 and interleukin [IL]-6), two anti-inflammatory genes (IL-10 and tumor necrosis factor [TNF]-α–stimulated gene-6), and two genes involved in matrix synthesis and deposition (lysyl oxidase, an enzyme important for synthesis of collagen and elastin, and transforming growth factor (TGF)-β1, an important mediator of matrix deposition by mesenchymal cells) as a function of time after injury and repair (Figs. 4 and 5).

Fig. 4
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Fig. 5
Fig. 5
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As expected, both cyclooxygenase 2 and IL-6 expressions were low in uninjured controls (Fig. 4). Expression of both genes increased dramatically (40-fold to 60-fold) 24 hours after injury, remained elevated for 48 hours, and decreased to lower levels by 7–21 days. The magnitude and time course of change in these two proinflammatory genes in response to anal sphincter injury were not altered by MSCs, either intravenously or directly (Fig. 4). Levels of IL-10 increased 10-fold to 14-fold within 24 hours after sphincter injury and remained elevated for 21 days (Fig. 4C). This response was not altered by MSCs. Likewise, gene expression of the anti-inflammatory glycoprotein, TNF-stimulated gene-6, increased significantly in the external anal sphincter after injury but was not modified by MSCs (Fig. 4D).

Next, we determined mRNA levels of two genes important in collagen, elastin, and matrix synthesis (lysyl oxidase and TFG-β1) in the injured external anal sphincter with or without MSCs (Fig. 5). In PBS-injected sphincters, TFG-β1 increased twofold within 24 hours after injury but increased further (threefold to fourfold) 7–21 days after injury. A similar profile was observed with intravenous MSCs. In contrast, direct MSC injection resulted in rapid increases in TFG-β1 mRNA (threefold to fourfold within 24 hours) and then decreased to almost basal levels by 21 days. Lysyl oxidase gene expression profiles in the external anal sphincter after injury were also altered by MSCs (Fig. 5B). Specifically, lysyl oxidase gene expression did not increase in PBS-treated controls until 7–21 days after injury. Direct injection of MSCs, however, resulted in dramatic and rapid increases in lysyl oxidase gene expression, falling to baseline within 21 days. The intravenous MSC-treated group demonstrated a profile intermediate between that of PBS and direct injection (Fig. 5).

Hematoxylin and eosin staining of serial sections throughout the sphincter were analyzed. In uninjured controls, striated muscle fibers of the external anal sphincter were intact, inflammatory cells were low in number, and fibrous connective tissue was not seen between the muscle fibers (Fig. 6 A, B). The histomorphology of the sphincter was grossly altered 21 days after injury. With PBS injection, an area of acute and chronic inflammation was noted at the injury site (Fig. 6C, D) characterized as an inner core of densely aggregated neutrophils, an outer layer of neutrophils, monocytes, and macrophages, and a thin fibrous rim. Striated muscle fibers of the external anal sphincter lost orientation on either side of the injury and were not seen for more than 0.5 mm from the injury site (Fig. 6C, D). Nuclei of fibers remained eccentric, suggesting that the injury did not involve denervation. This morphologic appearance of the PBS-injected injured sphincter was similar in intravenous MSC-treated animals (Fig. 6E, F). In sphincters from the direct-injected MSC group (Fig. 6G, H), the striated myocytes maintained orientation and traversed a dense fibrous component surrounding the injury.

Fig. 6
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The histologic appearance of increased collagen deposition in direct MSC-injected animals led us to quantify the fractional area of collagen at the site of injury in sphincters from PBS-injected and MSC-injected animals using picrosyrius red staining (Fig. 7). Specifically, collagen staining was ascertained within 5 mm circumferentially from the injury site by examiners blinded to treatment group. Collagen fibers surrounding the striated muscle fibers were widely spaced, frothy in appearance, and disorganized in PBS-injected and intravenous MSC-injected animals (Fig. 7A, B). In striking contrast, collagen fibers were densely packed, oriented in bundles, and tightly surrounded the striated muscle fibers of direct MSC-injected animals (Fig. 7C). Fractional collagen area was increased significantly at the site of injury in direct MSC-injected animals (Fig. 7D). Thus, early increases in TFG-β1 and lysyl oxidase mRNA were associated with dramatic increases in collagen deposition and orientation of muscle fibers in direct MSC-injected animals.

Fig. 7
Fig. 7
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Although strong autofluorescence of the external anal sphincter prevented tracking of the stem cells by fluorescence alone, immunostaining with antigreen fluorescent protein antibodies was used to determine the fate of these cells in the anal sphincter complex 48 hours, 7 days, and 21 days after injection. Results for the 48-hour time point are shown in Figure 8. As expected, immunostaining was absent in sphincters from PBS-injected animals (Fig. 8A). Furthermore, we found no green fluorescent protein-positive cells in the sphincter complex from intravenous MSC-injected animals (Fig. 8B). In direct MSC-injected animals, however, green fluorescent protein-positive cells were found throughout the sphincter complex within 1 mm of the injection site. Specifically, immunostained MSCs were found in the rectal mucosa, internal anal sphincter, lining the injury site, and, importantly, incorporated in the external anal sphincter (Fig. 8C, D). Although we found one to two immunostained cells per high-power field in direct-injected sphincters at 48 hours, green fluorescent protein-positive cells were completely absent in all sphincters at 7 and 21 days after injection. Further, we found no green fluorescent protein-positive cells in the lungs of intravenously injected animals, suggesting that the clearance of these cells was rapid using the bolus method of the current study. It should be noted that mortality was also increased in intravenously injected animals in that 9 of 68 intravenously injected animals died within 24 hours after injection compared with 3 of 68 PBS controls and 1 of 68 with direct injections (P=.057). Gross evidence of pulmonary emboli was noted in the intravenously injected group.

Fig. 8
Fig. 8
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DISCUSSION

Wound healing is characterized by cell proliferation, migration, extracellular matrix deposition, resolution, and remodeling. A host of cytokines, growth factors, and proangiogenic molecules orchestrate this response to injury. Although there is no doubt that the type, size, and depth of injury (eg, scalpel incisions compared with burns or excisional wounds) affect the rate and recovery from injury, nevertheless, the host response to injury is surprisingly consistent, not only for the different types of trauma but also for different organs.20 Recently, investigations have focused on potential mechanisms whereby stem cells may alter the wound healing process to improve functional outcomes.915 Here, we determined the effect of direct or intravenously injected mesenchymal-derived stem cells on functional outcomes and healing of the external anal sphincter.

During healing, a provisional fibrin–fibronectin matrix acts as a scaffold for cell adhesion and migration. In the anal sphincter, we found massive accumulation of neutrophils and macrophages at the site of injury after 21 days. Further, evidence of cell migration was found at the injured ends of the external anal sphincter and a fibrous capsule was noted surrounding the area of injury, particularly around sutures. Direct injection of MSCs appeared to accentuate the migration of striated myocytes into the wound. Collagen bundles were more numerous and densely packed in the striated muscle near the site of injury in animals directly injected with MSCs. Transforming growth factor-β1 plays crucial roles in wound healing through induction of matrix deposition, fibroblast transformation to myofibroblasts, and proangiogenesis.21 Because lysyl oxidase is essential for collagen and elastin matrix stability, and because TFG-β1 regulates lysyl oxidase gene expression, these proteins may be involved in the accelerated recovery of direct MSC-injected sphincters. Direct injection, but not intravenous administration, of MSCs resulted in acute and dramatic increases in both TFG-β1 and lysyl oxidase mRNA. These results suggest that MSC-mediated upregulation of these proteins in the injured sphincter may play an important role in increased collagen deposition. We propose that directly injected MSCs, but not intravenously injected MSCs, not only may provide progenitor cells for new myoblasts but also may stimulate matrix production to orient and guide migration of the cells into the sphincter complex.

Limitation of certain aspects of inflammation may theoretically reduce muscle damage as well as signals for muscle scarring.22 Although cyclooxygenase 2 and IL-6 gene expressions were induced dramatically in the sphincter complex within 24 hours after injury, the transient upregulation of these genes was not altered by intravenous or direct MSC injection. Initially, IL-10 was described as a cytokine synthesis inhibitory factor23 and is now known to play an important role in limiting pathologic immune responses to injury. Here, we found that both IL-10 and TNF-stimulated gene-6 were also transiently upregulated in the anal sphincter after injury. The temporal profile, however, was not altered by either intravenous or direct MSCs. This finding is in contrast with those of Lee et al,13 in which intravenously injected MSCs were found to be trapped in the lung, where they secreted a variety of therapeutic chemokines, including TNF-stimulated gene-6, a potent anti-inflammatory glycoprotein. The MSC-derived TNF-stimulated gene-6 was required for stem cell-induced improvement of cardiac function13 in a rat model of myocardial infarction.

Previously, we found that the injured external anal sphincter recovered to baseline function 21 days after injury.16,24 In the current investigation, control PBS-injected injured anal sphincters did not recover to those of unoperated controls at 21 days. There are several possible reasons for this discrepancy. First, animals were of a different strain (Lewis compared with Sprague-Dawley). Second, the animals in this study were younger at the time of surgery. In this study, contractile function of PBS-injected sphincters was compromised at 21 days, whereas direct injection of MSCs resulted in full recovery of the sphincter to baseline levels. These results are consistent with our previous results in which myogenic stem cells improved contractile function 90 days after injury.16

Several reports have documented functional improvement of the anal sphincter after muscle-derived stem cells6 or mesenchymal-derived stem cells.5,7 Moreover, injection of muscle-derived stem cells into the periurethral sphincter was shown to be effective in the rat urinary incontinence model.2529 It is not clear, however, whether the stems cells acted through engraftment or through paracrine mechanisms independent of stem cell engraftment. Here, we found green fluorescent protein-labeled MSCs embedded in the anal sphincter complex 48 hours after direct injection of 3.2×106 MSCs (80% labeled). Interestingly, green fluorescent protein-labeled cells were no longer visible after 7 days. This finding could be attributable to death or autophagy of the stem cells, division of the cells leading to loss of green fluorescent protein incorporation, phagocytosis of stem cells by activated macrophages, or (intravenous) decreased half-life of the green fluorescent protein within the cells. Injection of MSCs led to early incorporation of the cells into the sphincter and increased gene expression of lysyl oxidase and TFG-β1 at early time points. Although it is unclear whether stem cell viability was maintained for more than 48 hours, direct injection and early engraftment may be necessary to augment a collagen scaffold and striated muscle migration into the healing external anal sphincter.

This study provides a preclinical model in which to investigate the potential benefit of stem cell therapy for anal sphincter injuries. Direct injection of MSCs improved contractile function of the sphincter after injury, increased matrix deposition in the external anal sphincter, and increased expression of TFG-β1 and lysyl oxidase in the acute phase after injury.

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