To investigate the effects of adipose-derived mesenchymal stem cells (AMSCs) from type 2 diabetes mellitus patients on wound healing of pressure ulcers in mice.
Methods (1) In September 2016, the subcutaneous adipose tissue of a 60-year-old woman with type 2 diabetes mellitus was harvested, and then AMSCs were extracted by collagenase digestion and cultured. The third passage of cells were used for subsequent experiments. The morphology of cells was observed, and their osteogenic, chondrogenic, and adipogenic differentiation abilities were identified. The expressions of cell surface markers CD90, CD105, CD73, and CD34 were detected by flow cytometer (
n=3). (2) Sixteen female C57BL/6 wild-type mice aged 6-8 weeks were selected, and one pressure ulcer wound was created on each side of the spine of each mouse by pressing the skin with two magnets. The two wounds of each mouse were paired and divided into diabetic AMSCs group and negative control group, injected with 100 μL phosphate buffer solution (PBS) containing green fluorescent protein-labeled AMSCs (1×10
6 cells) and 100 μL PBS, respectively. The wound healing status of the two groups within post injection day (PID) 21 was observed, and their wound healing rates on PID 5, 13, and 17 were calculated. Three mice were sacrificed on PID 11 and 21, respectively, and tissue of three wounds was harvested from each group. The skin structure was observed by hematoxylin-eosin staining, the collagen deposition was evaluated by Masson staining, and the positive expression of CD31, i. e., the number of new blood vessels was counted by immunohistochemistry. Wound tissue samples of two groups prepared on PID 21 as above-mentioned were harvested, and the positive cell rate of S100, representing the regeneration of Schwann cells, was detected by immunohistochemistry. Wound tissue samples of diabetic AMSCs group prepared on PID 11 as above-mentioned were harvested, and the colonization of AMSCs was observed by fluorescence tracer method. Data were processed with paired
t test and Bonferroni correction.
Results (1) The third passage of cells isolated and cultured from the subcutaneous adipose tissue of a type 2 diabetes mellitus patient grew adherently to the wall in a long spindle and vortex-like manner. After induction, the cells showed osteogenic, chondrogenic, and lipogenic differentiation abilities. The positive expression rates of CD90, CD105, and CD73 on the cell surface were higher than 90.00%, and the expression rate of CD34 was 0.46%. The cells were identified as AMSCs. (2) The mice wounds of diabetic AMSCs group healed quickly, and all the wounds healed completely on PID 17, while the mice wounds in negative control group were not completely closed at this time, and there was still scab on the surface. On PID 5, 13, and 17, the healing rates of mice wounds of diabetic AMSCs group were (35.6±6.5)%, (87.1±2.5)%, and 100.0%, respectively, significantly higher than (19.8±7.2)%, (66.2±5.2)%, and (86.9±5.3)% of negative control group (
t=6.49, 14.31, 9.73,
P<0.05). Compared with that of negative control group, the inflammatory cell infiltration was reduced in mice wounds tissue of diabetic AMSCs group on PID 11, and thicker epidermis and dermis as well as regenerated skin appendages were observed on PID 21. On PID 11 and 21, the collagen percentages of mice wounds tissue in diabetic AMSCs group was (48.3±1.3)% and (54.1±1.7)%, respectively, significantly higher than (41.4±1.7)% and (50.3±1.2)% of negative control group (
t=6.98, 3.99,
P<0.01). On PID 11 and 21, the numbers of new blood vessels in mice wounds tissue of diabetic AMSCs group were 17.2±1.3 and 18.0±2.1, respectively, significantly more than 8.0±1.4 and 14.0±1.5 of negative control group (
t=10.69, 3.38,
P<0.01). On PID 21, the S100 positive cell percentage in mice wounds tissue of diabetic AMSCs group was (1.76±0.12)%, significantly higher than (0.55±0.03)% of negative control group (
t=21.68,
P<0.001). On PID 11, the colonization of AMSCs in mice wounds tissue of diabetic AMSCs group was observed.
Conclusions Transplantation of AMSCs from type 2 diabetic mellitus patients can accelerate wound healing of pressure ulcers in mice by promoting angiogenesis, collagen deposition, and Schwann cell regeneration.
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