Factors that are responsible for fecal leakage include a disrupted external and/or internal anal sphincter (IAS) and changes in bowel motility, rectal capacity, and sensation.1,2 Current cell-based animal research has focused on repairing a defect of the anal sphincters.3–10 Although none of the studies have evaluated muscle morphology, most studies have reported filling of small-to-moderate defects with muscle.5,7,10–12 Some studies evaluated muscle tensile strength in the regenerated muscle.6,9 All of these studies, however, have reported findings in the setting of an acute injury. However, fecal incontinence most often manifests many years after an injury, which may have occurred after childbirth, surgical trauma, radiation, or neurologic etiologies, among others, and hence there is a need to research regeneration of deficient muscle long after the injury.1,13
Current therapies to treat fecal leakage address a sphincter defect by a sphincter repair, which has poor long-term results.14–16 Alternate therapies include sacral neuromodulation, which is minimally invasive and has success rates of ≈60%,17 injection of bulking agents (Solesta) with similar success rates,18 and invasive options like the magnetic anal sphincter19 (Fenix), which has limited application. New noninvasive anal plugs, such as Renew and Procon20 and the Eclipse21 vaginal insert can provide treatment options for milder cases of fecal incontinence. Most devices are expensive, have suboptimal results, and their efficacy in the long term remains to be proven.22,23 Therefore, there is a need to find a simple and effective solution.
In treatment of fecal incontinence, an ideal regenerative therapy would regenerate muscle in the setting of a chronic injury and would have a sustained regenerative effect that is also functionally effective. To achieve this goal, we have evaluated the tissue environment that occurs after an acute injury and have reported on the cytokines, stromal cell-derived factor 1 (SDF-1) and monocytic chemotactic protien 3, which are upregulated after injury and decline 3 weeks later.24 We have also evaluated the regenerative potential of bone marrow–derived mesenchymal stem cells (MSCs) in the setting of an acute injury and have postulated that regeneration may be the result of paracrine effects of the MSCs, as we have not shown evidence of survival of exogenously implanted MSCs.7,8 In the chronic setting we have shown early regeneration of muscle 4 weeks after treatment with a plasmid encoding for SDF-1 with and without MSCs given 3 weeks after a large anal sphincter defect.25
In this study we aimed to evaluate whether this effect is sustained and to study tissue morphology 8 weeks after treatment. We hypothesized that the plasmid encoding for SDF-1 regenerates both smooth and skeletal muscles with sustained functional improvement in a rat model of a chronic anal sphincter injury 8 weeks after treatment.
MATERIALS AND METHODS
This research protocol was approved by the Cleveland Clinic Institutional Animal Care and Use Committee. We used age- and weight-matched virgin female Sprague–Dawley rats (250 to 300 g) for this study.
SDF-1–Encoded Nonviral Plasmid
The SDF-1–encoded plasmid was obtained from Juventas Therapeutics, Inc (Cleveland, OH), as was used in the previous study.12 This study used the SDF-1 plasmid in dextrose solution with a concentration of 2 mg/mL.
MSC Harvesting, Culture, and Identification
The harvesting and sorting protocol for rat bone marrow–derived MSCs was followed as per our previous study.12 In brief, the tibia and femoral bone marrow was harvested and the cells were cultured in MSC culture medium made from Dulbecco’s Modified Eagle Medium (Invitrogen, Carlsbad, CA), 12.5% fetal bovine serum (GIBCO, Invitrogen), and 1% antibiotic and antimycotic solution (GIBCO, Invitrogen). The cells were passaged when they reached 70% to 80% confluence using trypsin-EDTA (GIBCO, Invitrogen). Intracellular adhesion molecule 1 antibody (Abcam, Cambridge, United Kingdom) was used for cell sorting with the concentration of 5 μl/106 cells for 30 minutes at room temperature. The sorting of intracellular adhesion molecule 1–positive MSCs was performed with an LSRII flow cytometer (BD, Franklin Lakes, NJ). MSCs at passage 8 to 12 were used for this study.
MSC lineage analysis was performed using a rat MSC functional identification kit (R&D Systems, Inc, Minneapolis, MN) following the manufacturer’s instruction. Immunocytochemistry was used to define mature phenotypes of adipocytes, chondrocytes, and osteocytes with antibodies to fatty acid binding protein 4, aggrecan, and osteocalcin, separately.
T-Gelatin Hydrogel Scaffold Preparation
The type-1 collagen-based scaffold was obtained courtesy of Dr. Anthony Calabro (Cleveland Clinic Lerner Research Institute). It contains tyramine-substituted gelatin (T-Gelatin) using a novel enzymatic cross-linking method in the presence of hydrogen peroxide, as used in our previous study.26
Rat Model of a Chronic Large Anal Sphincter Defect
The animals were anesthetized with a ketamine (100 mg/kg intraperitoneal) and xylazine (10 mg/kg intraperitoneal) mixture before undergoing an excision of 50% of the circumference of the ventral portion of the anal sphincter. All of the procedures were carried out by a single operator. The animals were allowed to recover for 3 weeks. At this 3-week time point they were randomly allocated to 4 groups, as follows: group IA included injury without any treatment; group P included 50 µL of SDF-1 plasmid solution (100 μg) injected at the ends of the defect; group P+MSC included MSCs injected at the ends of the defect 3 days after injection of SDF-1 plasmid; and group P+S&MSC included gelatin scaffold and MSC mixture injected into the site of the defect 3 days after injection of SDF-1 plasmid in the same area. All of the MSC-treated animals received 800,000 cells in 50 µL of phosphate buffered saline solution (16 × 106 cells per mL). Function (resting anal sphincter pressures) and histology were evaluated 8 weeks after treatment (n = 8 per group). A separate group of rats (n = 6 per group outlined below) was used to investigate cytokine expression 7 days after treatment.
Sample Size Estimation
Based on that our previous work with a 0.8 power and adjusted significance level of 0.5 with a conservative Tukey–Kramer method for multiple comparisons, a sample size of 8 animals per group (3 treatments and 3 time points) was calculated for functional outcomes.
Resting anal pressure (RP) was assessed before excision, as well as before and 8 weeks after treatment, as per our previous protocol.12 Under anesthesia, a 7-F T-Doc air-charged catheter (Laborie Medical Technologies, Mississauga, Ontario, Canada) was inserted into the anal canal and connected to a Goby Anorectal Manometry System (Laborie Medical Technologies) for RP recording. Eight typical RP waves (a stable baseline pressure) were collected for analysis at each of the 3 investigational time points. The average of the 8 measured RPs in each animal was used for additional group analysis.
Anal sphincter histology was evaluated 8 weeks after treatment. Masson trichrome staining was performed as described previously.12 After euthanasia, the anal tissues were harvested and fixed with 10% formalin before being paraffin embedded for histology. For each specimen, Masson trichrome staining was performed on serial transverse sections (5 μm thick, 50 μm apart) along the 1.5 mm-long anal canal tissue starting at the anal verge. The sections were viewed and scanned by an observer blinded to group assignments under a bright-field microscope at ×20 magnification using the Leica SCN400 Slide Scanner (Leica Microsystems Inc, Buffalo Grove, IL). The site of injury was recognized by the disruption of the anal sphincter complex in cross-section.
Quantification of muscle and connective tissue was performed using Image-Pro Plus 7.0 software (Media Cybernetics, Rockville, MD). Volumetric analysis of each section was done as described previously.25 Briefly, in the same histological section, we assessed the percentage of muscle (circular red fiber-like bundles, usually disorganized, in the area of the created defect on Masson staining) and connective tissue (collagen-rich fibrosis). The proportion of muscle in the region of the defect was calculated as muscle area divided by the area of muscle plus the area of connective tissue in the region of the defect. The proportion of connective tissue in the region of the defect was calculated as connective tissue area divided by the area of muscle plus the area of connective tissue in the region of the defect. The proportion of muscle in the intact region was calculated as muscle area divided by the area of muscle plus the area of connective tissue in the intact region. The proportion of connective tissue in the intact region was calculated as connective tissue area divided by the area of muscle plus the area of connective tissue in the intact region. Results are reported as the ratio of muscle and connective tissue at the defect to that in the intact area.
The paraffin-embedded, formalin-fixed rat anal canal samples were sectioned at 5 µm. Immunohistochemistry staining was performed using a Discovery ULTRA automated stainer (Ventana Medical System Inc, Tuscon, AZ). In brief, antigen retrieval was performed using a tris/borate/EDTA buffer (Discovery CC1, Ventana Medical Systems, Inc, Oro Valley, AZ)(pH 8.0 to 8.5) for 64 minutes at 95°C. Slides were incubated with Desmin antibody-1 (D33) at a 1:40 dilution (MS-376-S; Thermo Scientific, Fremont, CA) for 1 hour at room temperature. The primary antibody was visualized using the OmniMap antimouse horseradish peroxidase secondary antibody (Ventana Medical Systems) and the ChromoMap DAB detection kit (Ventana Medical Systems). Lastly, the slides were counterstained with hematoxylin and eosin.
The smooth muscle IAS and striated muscle external anal sphincter (EAS) were identified based on analysis of Desmin-stained sections (Fig. 1). The EAS was identifiable as dark-brown stained tissue with striations, whereas the IAS was identifiable as light-brown staining without the striated structure. Volumetric analysis was performed as described previously.25 Sections were viewed and scanned under a bright-field microscope at ×20 magnification using the Leica SCN400 Slide Scanner. Quantification of each muscle was performed using Image-Pro Plus 7.0 software. We assessed the volume of both IAS and EAS muscles at the area of defect and in the intact area separately and evaluated the volume as a percentage of the total muscle at the site of the defect and the intact area. We then compared the individual muscles as a ratio of that percentage at the defect with that in the intact area.
Seven days posttreatment, anal canal tissue of 5 mm length was harvested before it was preserved in liquid nitrogen. Tissue was prepared using a lysate solution in a 1:10 ratio of tissue weight:lysate volume (which includes 10% cell lysis buffer No. 9803, Cell Signaling Technology, Danvers, MA; and 1% (milligrams per milliliter) protease inhibitor tablet No. 8820, Sigma–Aldrich, St. Louis, MO). Western blots were performed using primary antibodies to CXCR4 (No. ab124824, Abcam, Cambridge, MA) and Myf5 (No. ab125078, Abcam), with anti-β-actin (No. sc47778, Santa Cruz Biotechnology, Santa Cruz, CA) as the endogenous control. Fluorescence dye–labeled secondary antibodies (LI-COR, Lincoln, NE) were mixed with IRDye 800CW Donkey antirabbit (No. 926–32213, green) for both CXCR4 and Myf5 and IRDye 680RD Donkey antimouse for β-actin (No. 926–68072, red). An Odyssey CLx infrared imaging system (LI-COR) was used for band imaging and quantification of cytokine expression. The ratio of CXCR4 and Myf5 to endogenous β-actin of each sample was calculated, and the final result is shown normalized to the mean of the injury alone group (n = 6).
Parametric group comparisons for anal manometry over time with respect to resting pressure and measures of change are performed using ANOVA with pairwise group comparisons using a t test with a Bonferroni correction such that p < 0.0083 was regarded as significant. Quantification for histology, immunohistochemistry, and cytokine expression was performed using a 1-way ANOVA followed by a Tukey test in SigmaPlot 11.0 (Systat Software Inc, San Jose, CA), with p < 0.05 indicating a statistically significant difference in all comparisons. Results are presented as mean ± SD of data from 6 (Western blot) or 8 (manometry and histology) animals per group.
There was no mortality during the course of the study.
The multipotent features of the MSC was confirmed by their differentiation capability into adipogenic, chondrogenic, and osteogenic cells ex vivo.
Before the injury, there was no significant difference in resting pressure among the 4 groups (IA, 10.4 ± 5.08 cm H2O; P, 10.0 ± 2.85 cm H2O; P+MSC, 11.4 ± 3.27 cm H2O; P+S&MSC, 11.5 ± 4.97 cm H2O; p > 0.0083). Three weeks after injury and before treatment, no significant difference was noted between the groups (IA, 5.0 ± 1.98 cm H2O; P, 7.2 ± 2.20 cm H2O; P+MSC, 6.9 ± 2.24 cm H2O; P+S&MSC, 5.0 ± 1.06 cm H2O). Eight weeks after treatment, all of the SDF-1 plasmid-treated groups had significantly higher pressures than the IA group (IA, 3.4 ± 0.96 cm H2O; P, 10.6 ± 3.70 cm H2O, p = 0.001; P+MSC, 13.1 ± 7.07 cm H2O, p < 0.001; P+S&MSC, 10.9 ± 2.11 cm H2O, p < 0.001; Fig. 2). No significant differences in anal pressure were noted between animals receiving the SDF-1 plasmid and those receiving the plasmid and cells or scaffold.
Anal pressure increase from pretreatment to postinjury was significantly increased in all the SDF-1 plasmid-treated groups compared with the IA group, which showed a significant decrease in pressure (IA, –1.6 ± 1.49 cm H2O; P, 3.5 ± 3.39 cm H2O, p = 0.004; P+MSC, 6.2 ± 5.94 cm H2O, p = 0.007; P+S&MSC, 5.9 ± 2.97 cm H2O, p < 0.001). No significant difference was noted among the 3 SDF-1 plasmid-treated groups in the change in pressure after treatment (p > 0.0083).
Histology and Immunohistochemistry
All 3 of the SDF-1 plasmid-treated groups showed filling of the defect with muscle fibers, whereas the area of the defect in the IA group showed disorganized architecture with patchy filling of the defect (Fig. 3). Quantification of the total muscle at the site of injury revealed that, compared with the IA group, the plasmid alone group had significantly more muscle (IA, 0.86 ± 0.06; P, 0.97 ± 0.09; p = 0.03). No significant difference in total muscle quantification was noted on the other intergroup comparisons (P+MSC, 0.95 ± 0.07; P+S&MSC, 0.90 ± 0.05; Fig. 4A).
Significantly less fibrosis was seen in the SDF-1 plasmid alone group compared with the IA and P+S&MSC groups (P, 0.97 ± 0.09; IA, 1.44 ± 0.24, p = 0.018; P+S&MSC, 1.40 ± 0.21, p = 0.03). No significant difference was noted between the other SDF-1 plasmid-treated groups on intergroup comparisons (P+MSC, 1.19 ± 0.22; Fig. 4B). No significant differences were found between the groups in the proportion of muscle in the defect region and intact areas of the IAS muscle (IA, 0.95 ± 0.27; P, 0.72 ± 0.3; P+MSC, 0.99 ± 0.45; P+S&MSC, 1.03 ± 0.64; p = 0.52; Fig. 5A) or the EAS muscle (IA, 1.20 ± 0.47; P, 1.58 ± 0.56; P+MSC, 1.24 ± 0.49; P+S&MSC, 1.57 ± 1.98; p = 0.85; Fig. 5B).
Seven days after treatment, there were no significant differences between the groups in CXCR4 (IA, 1.20 ± 0.47; P, 1.58 ± 0.56; P+MSC, 1.24 ± 0.49; P+S&MSC, 1.57 ± 1.99; p = 0.37) or Myf5 (IA, 1.00 ± 0.43; P, 1.32 ± 0.27; P+MSC, 1.42 ± 0.20; P+S&MSC, 0.98 ± 0.3; Fig. 6).
The quest to regenerate muscles in the region of the anal sphincter is ongoing. Early studies evaluated the concept of anal sphincter regeneration after an acute injury,4–7,9,11 and most recent studies are still centered on this concept.27 Clinical trials using stem cells also use the cells after a repair of the anal sphincter and inject into the cut ends.28 However, the challenge is in regenerating functional muscle at a time when the tissue environment for regeneration is quiescent long after injury.
To change the tissue environment, few options have been studied. We have researched electrical stimulation as a low-grade injury and have reported on retention of exogenous MSCs and regeneration of the muscles.12 One clinical trial has used electrical stimulation for 21 days, followed by injection of cells and reported significant increases in anal pressures and quality-of-life scores with a decrease in incontinence episodes, incontinence scores, and frequency of bowel movements 5 years after treatment.29,30 Other therapies that have been evaluated are shock wave therapy31 and platelet-rich plasma.32
Advances in cellular therapy have not focused on muscle regeneration in the absence of inflammation. Apart from the studies on the anal sphincter, studies involving regeneration of volumetric muscle loss or tendon injuries have shown slow progress because of regulatory issues, poor donor cell viability, and engraftment issues.33 To regenerate an anal sphincter that is deficient in its continuity, the treatment needs not only to fill a defect with muscle but that which is functional.
A functionally effective anal sphincter depends on both the IAS and EAS complexes being intact and innervated. The large muscle defect that we created disrupted the anal sphincter complex, requiring regeneration and reinnervation of the smooth and striated muscles of the anal sphincters. Previous work has also demonstrated recovery of the neuromuscular system of the anal sphincter in acute,7,8,34 as well as a chronic, injury models.25 Hence, there is a need to focus on regeneration of viable muscle with innervation to achieve continence control.
Bitar et al35 have investigated the possibility of regenerating the IAS from GI cells. Their emphasis is to replace the IAS with an engineered gut sphincter complex, composed of human smooth muscle and neural progenitor cells and engineered on a scaffold, which has been successfully implanted in rodents. They have demonstrated both innervation and ex vivo muscle tensile strength of the regenerated composite.35,36 Kajbafzadeh et al37 have also implanted myogenic cells on a decellularized EAS in a rabbit model. Their results show that a decellularized EAS implanted with myogenic cells from a thigh muscle can improve anal sphincter contractility in the short term, whereas in the long term, 2 years later, both seeded and nonseeded decellularized anal sphincter matrices had equivalent results. Although the authors state that this may be an option for treating fecal incontinence, their model is that of a complete external sphincter excision, and it is unclear whether they are suggesting that the EAS can be augmented without excising it. Clinically, translation of both of these lines of research would fit a clinical indication similar to that of an artificial anal sphincter.
In our previous study we evaluated the same groups at an earlier time point of 4 weeks.25 We demonstrated an increase in anal sphincter pressures in the group treated with the SDF-1 plasmid alone and the group that received the plasmid along with the exogenous MSCs. Histology was significant for a greater reorganization of muscle, and muscle when quantified was increased in the plasmid plus MSC group compared with the injury alone group.
The current study has shown that, at a time when the process of innate regeneration from an injury has waned, a plasmid encoding SDF-1 results in regenerated muscle to bridge an entire hemicircumference and not just a small defect. These effects have been improved since our last study, which evaluated results 4 weeks after treatment25 and have increased the muscle volume in the same proportion as that seen in the uninjured area. In addition, for the first time, a therapy has regenerated both smooth and skeletal muscles after an injury. Not only have both muscles regenerated, but it is in the same ratio as that present in the uninjured area, suggesting that, although the plasmid may be expressed for <30 days,25 there seem to be factors that modulate this effect and may prevent excessive muscle regeneration. However, a longer-term study in rodents should also be performed to evaluate any additional growth of muscle beyond 8 weeks.
Anal sphincter pressure was also increased to near normal values and sustained over 8 weeks. This is one of the criterion required to translate this research, because muscle without tone would be redundant. However, we did not examine the tensile strength ex vivo of the regenerated muscle fibers. Another limitation is that, because we did not block the action of SDF-1, we could not demonstrate that the reported effects were from SDF-1 alone. Future research should also examine muscle innervation and angiogenesis of the regenerated muscle.
CXCR4 is a receptor for the ligand CXCL12 (SDF-1) and has been involved in chemotaxis of leukocytes in specific inflammatory conditions.38 The CXCL12/CXCR4 chemokine axis is involved in the recruitment of stem cells from bone marrow and other tissues and signaling involved in chemotaxis, cell survival, proliferation, increase in intracellular calcium, and gene transcription.39 We have shown previously that there was an increase in satellite cells after plasmid therapy and have inferred that this may be the cause of muscle regeneration.25 MyF5 is one of the factors responsible for myogenic specification and differentiation.40 However, both of these cytokines were not differentially expressed in this study. Hence, we have not conclusively ascertained the molecular mechanisms that could cause this effect. Large animal studies will focus on this aspect.
In a rat model of a large anal sphincter injury, the plasmid encoding for SDF-1 regenerated both smooth and skeletal muscles. Increased muscle regeneration and increased anal sphincter pressures were sustained over 8 weeks. The addition of MSCs with or without a scaffold did not enhance this effect. Future research should be directed toward detecting muscle tensile strength of the regenerated muscle and the molecular mechanisms that could cause and inhibit it.
The authors thank Mathew Kiedrowski of the Penn laboratory, Anna Reitsch of the Damaser laboratory, Kelly Simmerman of Image Core at the Cleveland Clinic, and Dr Anthony Calabro for their expertise and assistance.
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Plasmid; Regeneration; Skeletal muscle; Smooth muscle; Stromal cell derived factor-1
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