Substance P combined with epidermal stem cells promotes wound healing and nerve regeneration in diabetes mellitus.

Exogenous substance P accelerates wound healing in diabetes, but the mechanism remains poorly understood. Here, we established a rat model by intraperitoneally injecting streptozotocin. Four wounds (1.8 cm diameter) were drilled using a self-made punch onto the back, bilateral to the vertebral column, and then treated using amniotic membrane with epidermal stem cells and/or substance P around and in the middle of the wounds. With the combined treatment the wound-healing rate was 100% at 14 days. With prolonged time, type I collagen content gradually increased, yet type III collagen content gradually diminished. Abundant protein gene product 9.5- and substance P-immunoreactive nerve fibers regenerated. Partial nerve fiber endings extended to the epidermis. The therapeutic effects of combined substance P and epidermal stem cells were better than with amniotic membrane and either factor alone. Our results suggest that the combination of substance P and epidermal stem cells effectively contributes to nerve regeneration and wound healing in diabetic rats.

[Effect of substance P combined with epidermal stem cells on wound healing and nerve regeneration in rats with diabetes mellitus].

OBJECTIVE: To observe the effect of sensory neuropeptide substance P combined with epidermal stem cells (ESC) on wound healing and nerve regeneration in diabetic rats. METHODS: ESC that had been isolated from SD rats were identified and cultured in vitro, and they were inoculated onto nourishing layer of amniotic membrane to construct amniotic membrane-ESC. Four full-thickness skin wounds were produced on the back of each of 48 diabetic rats. The resulted 192 wounds were randomly divided into ESC + substance P group, ESC group, substance P group, and control group according to the lottery method, with 48 wounds in each group. Wounds in ESC + substance P group and ESC group were transplanted with amniotic membrane-ESC, and those in substance P group and control group were transplanted with amniotic membrane. After transplantation, 250 µL substance P in the concentration of 1 × 10(-7) mol/L was injected around and into the middle of the wounds in ESC + substance P group and substance P group, 2 times a day, and continued for 4 days, while 250 µL PBS solution was injected in the above-mentioned position in ESC group and control group as control, 2 times a day, and continued for 4 days. On post injury day (PID) 4, 7, 10, 14, 17, and 23, the wound healing rate (with 8 wounds at each time point) was observed and determined, and changes in wound tissue structure were observed with HE staining. On PID 4, 7, and 10, collagen distribution in wound tissue was observed with Masson staining, and type I and type III collagen deposition in wound tissue was respectively observed after immunohistochemical staining. The distribution of protein gene product 9.5 (PGP 9.5) and regeneration of substance P positive nerve fibers in wound tissue were observed with immunohistochemical staining on PID 14 and 23. Data were processed with one-way analysis of variance and t test. RESULTS: (1) The wound healing rate in ESC + substance P group reached 100.0% on PID 14, which was obviously earlier than that in ESC group, substance P group, and control group, healing was respectively observed on PID 17, 17, and 23. The wound healing quality in ESC + substance P group was better than that in the other three groups as shown by HE staining. (2) On PID 10, collagen that was darkly stained and widely distributed was observed in wound tissue of ESC + substance P group and substance P group, while collagen in the other two groups was lightly stained and narrowly distributed. Deposition quantity of type I collagen gradually increased, and that of type III collagen gradually decreased in the wounds of each group over time. On PID 4, 7, and 10, distribution amount of type I collagen in wound tissue of ESC + substance P group was significantly higher than that in ESC group (with t value respectively 32.72, 118.21, 26.71, P values all below 0.01) and control group (with t value respectively 44.37, 22.76, 30.32, P values all below 0.01), while there was no significance between ESC + substance P group and substance P group. On PID 4, 7, and 10, distribution amount of type III collagen in wound tissue of ESC + substance P group was significantly higher than that in ESC group (with t value respectively 32.27, 28.68, 14.51, P values all below 0.01) and control group (with t value respectively 35.68, 22.52, 22.24, P values all below 0.01). (3) A large amount of PGP 9.5 and regeneration of substance P positive nerve fibers, and some peripheral nerve fibers in deep wound extending to epidermis were observed in wound tissue of ESC + substance P group and substance P group. A small amount of PGP 9.5 and regeneration of substance P positive nerve fibers without peripheral nerve fibers extending to epidermis were observed in deep wound tissue of ESC group and control group. On PID 14, 23, ratios of area of PGP 9.5 positive nerve fiber in the wounds of ESC + substance P group were (3.86 ± 0.25)% and (7.03 ± 0.28)%, and they were significantly higher than those of ESC group [(1.48 ± 0.30)%, (3.01 ± 0.43)%, with t value respectively 23.95, 30.27, P values all below 0.01] and control group [(1.46 ± 0.23)%, (2.84 ± 0.29)%, with t value respectively 27.35, 40.32, P values all below 0.01]. On PID 14, 23, ratios of substance P positive nerve fiber area in the wounds of ESC + substance P group were (2.01 ± 0.14)% and (1.19 ± 0.11)%, which were obviously higher than those of ESC group [(0.85 ± 0.17)%, (1.34 ± 0.21)%, with t value respectively 20.50, 2.60, P < 0.05 or P < 0.01] and control group [(0.74 ± 0.15)%, (1.30 ± 0.17)%, with t value respectively 23.98, 2.41, P < 0.05 or P < 0.01]. CONCLUSIONS: Joint application of substance P and ESC can effectively promote healing of wound and nerve regeneration in diabetic rats.

[Experimental study on the differentiation of human induced pluripotent stem cells into epidermal-like stem cells].

OBJECTIVE: To investigate the feasibility of differentiation of human induced pluripotent stem cells (iPSCs) into epidermal-like stem cells. METHODS: (1) Human strain of iPSCs were plated on-to trophoblast of inactivated Fb strain of mouse embryos and cultured in complete medium of embryonic stem cells, iPSCs were subcultured by collagenase IV digestion method. The morphology and growth of iPSCs were observed under inverted phase contrast microscope, and the cells were stained with alkaline phosphatase (AKP). iPSCs were cultured in incomplete medium of embryonic stem cells to observe the ability of embryoid body formation. (2) Human iPSCs were inoculated onto 6-well plate covered with human amniotic membrane to culture as induction group. Other iPSCs were cultured on 6-well plate without human amniotic membrane as control group. Morphological changes in iPSCs in two groups were observed. Expressions of integrin beta1 and CK19 of iPSCs in two groups were determined by immunocytochemical staining. RESULTS: Human iPSCs showed a typical stem cell clone-like growth with a clear boundary, and they proliferated vigorously in complete medium of embryonic stem cells. These cells were AKP-positive. iPSCs formed embryoid body in trophoblast-free and suspension culture conditions. After 4 days of co-culture, stem cell clones were formed on the surface of amniotic membrane in induction group, and part of the cells were integrin beta1 and CK19 positive. Most of the cells died, and no integrin beta1 and CK19 positive cells were found in control group. CONCLUSIONS: Human iPSCs can be differentiated into epidermal-like stem cells by amniotic membrane induction, and it lays an experimental basis for providing new source of seed cells of skin tissue engineering.