The Effect of Mesenchymal Stem Cells, Adipose Tissue Derived Stem Cells, and Cellular Stromal Vascular Fraction on the Repair of Acute Anal Sphincter Injury in Rats

Abstract

Background: Anal sphincter incontinence (ASI) can cause a serious decline in the quality of life and can cause a socioeconomic burden. Studies have shown that bone marrow mesenchymal stem cells (MSC) have significant therapeutic effects on ASI, but the cost and risk of MSC harvest limit their further application. In contrast, adipose tissue derived stem cells (ADSC) and cellular stromal vascular fraction (CSVF) as stem cell sources have multipotency and the advantage of easy harvest.

Objective: Here we aim to investigate the effects of ADSC and CSVF on treating ASI and compare them to that of bone marrow MSC.

Methods: Bone marrow MSC, ADSC, and CSVF were obtained and labeled with green fluorescent protein (GFP), and CSVF was labeled with DIL. Sprague Dawley (SD) rats were divided into 5 groups. Four groups were injected with 0.2 mL phosphate buffer saline (PBS), 1 × 10⁷/0.2 mL of MSC, ADSC, or CSVF, respectively, after model establishment. The control group received no treatment. The repair was assessed by anal functional tests and immunostaining on day 5 and day 10 after injection.

Results: MSC, ADSC, and CSVF significantly promoted tissue repair and the recovery of muscle contraction and electromyographic activity in ASI. The generation of myosatellite cells by injected MSC, ADSC, and CSVF was found in the wounded area. On day 5, CSVF showed highest therapeutic effect, while on day 10, MSC and ADSC showed higher therapeutic effects than CSVF. When comparing the effects of MSC and ADSC, ADSC was slightly better than MSC in the indexes of anal pressure, etc. Conclusion: ADSC and CVSF are alternative stem cell sources for ASI repair.

Keywords: ADSC; ASI; CSVF; MSC; anal sphincter incontinence.

Figure 1

Differentiation of BMSC and ADSC into adipocytes, chondrocytes and osteoblasts. Representative images showed the cell morphology in culture after induction (top row) and the staining results of oil red stain for adipogenesis, alcian blue stain for chondrogenesis, and alizarin red stain for osteogenesis.

Figure 2

Cell morphology and fluorescent labeling of MSC, ADSC and CSVF. (A) Morphology of MSC and ADSC in culture and morphology of CSVF after extraction and change of culture medium. (B) The first row shows the phase contrast images of MSC, ADSC in the culture dish ready for sorting and the image of CSVF extracted from rat adipose tissue. The second row shows the fluorescence images of MSC, ADSC, and DIL-stained CSVF after transfection with lentivirus in order. (C) the flow cytometric analysis of MSC and ADSC.

Figure 3

Anal pressure of control, PBS, MSC, ADSC and CSVF groups after modeling and after 5 and 10 days of treatment. (A) The first row shows the anal pressure graphs of 70 s recorded in control, PBS, MSC, ADSC and CSVF groups after modeling, and the anal pressure decreased significantly after modeling; the second row shows the anal pressure graphs of 70 s recorded in each group after 5 days of treatment; the third row also shows the anal pressure graphs of each group after 10 days of treatment. (B) Percentage of anal pressure basal value (a), anal pressure peak (b) in different treatment groups normalized to pre-treatment healthy controls. Data in each bar graph are expressed as mean ± standard deviation. An *, **, *** or **** without a horizontal line indicates a statistical difference in comparison to the control group, and an *, ** or *** with a horizontal line indicates a statistical difference between the groups at the horizontal ends. Significant differences between groups were expressed as **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05.

Figure 4

EMG of control, PBS, MSC, ADSC, CSVF groups after modeling and after 5 and 10 days of treatment. (A) The first row shows the EMG of control, PBS, MSC, ADC and CSVF groups after modeling, the second row shows the EMG of each group after 5 days of treatment, and the third row shows the EMG of each group after 10 days of treatment. (B) Percentage of EMG frequency (a), EMG peak (b) in different treatment groups normalized to pre-treatment healthy controls. Data in each bar graph are expressed as mean ± standard deviation. An **, *** or **** without a horizontal line indicates a statistical difference in comparison to the control group, and an *, *** or **** with a horizontal line indicates a statistical difference between the groups at the horizontal ends. Significant differences between groups were expressed as **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05.

Figure 5

Wound healing in the PBS, MSC, ADSC and CSVF groups on day 0, 5 and 10 after treatment. The first row shows the pictures of the incision in each group of PBS, MSC, ADSC, and CSVF after modeling, the second row shows the healing after 5 days of treatment; the third row shows the healing of the incision after 10 days of treatment. The white box in the figure indicates the injury site.

Figure 6

(A) Masson Trichrome staining of the injury site in the control group, PBS group, MSC group, ADSC group and CSVF group after 5 and 10 days of treatment. Yellow and orange arrows represent the external anal sphincter and internal anal sphincter, respectively, green arrows point to regenerated connective tissue, while black arrows point to regenerated muscle. (B) Percentage of fibers in the control group, PBS group, MSC group, ADSC group, and CSVF group after 5 and 10 days of treatment. An **, *** or **** without a horizontal line indicates a statistical difference in comparison to the control group, and an **, *** or **** with a horizontal line indicates a statistical difference between the groups at the horizontal ends. Significant differences between groups were expressed as **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05.

Figure 7

Fluorescence of DIL labeling after 5 and 10 days of treatment in CSVF group. Cell nucleus was stained in blue with DAPI, and DIL-labeled CSVF was visualized by red fluorescence.

Figure 8

ADSC and MSC co-expressed GFP and PCNA after 5 and 10 days of injection. The 1st to 7th row represents staining of GFP and PCNA in the control group, PBS group on the 5th and 10th day, MSC and ADSC group on the 5th day, and MSC and ADSC on the 10th day, after the treatment.

Figure 9

ADSC and MSC co-expressed GFP and MYOD after 5 and 10 days of injection. The 1st to 7th row represents staining of GFP and MyoD in the control group, PBS group on the 5th and 10th day, MSC and ADSC group on the 5th day, and MSC and ADSC on the 10th day, after the treatment.

Figure 10

ADSC and MSC co-expressed GFP and α-SMA after 5 and 10 days of injection. The 1st to 7th row represents staining of GFP and α-SMA in the control group, PBS group on the 5th and 10th day, MSC and ADSC group on the 5th day, and MSC and ADSC on the 10th day, after the treatment.