Bone marrow-derived mesenchymal stem cell-conditioned medium ameliorates diabetic foot ulcers in rats

Xu, Yi-Feng; Wu, Yan-Xiang; Wang, Hong-Mei; Gao, Cui-Hua; Xu, Yang-Yang; Yan, Yang

doi:10.1016/j.clinsp.2023.100181

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Original articles • Clinics 78 • 2023 • https://doi.org/10.1016/j.clinsp.2023.100181 linkcopy

Bone marrow-derived mesenchymal stem cell-conditioned medium ameliorates diabetic foot ulcers in rats

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Objectives: This study aimed to explore the effects of bone marrow-derived Mesenchymal Stem Cell-Conditioned Medium (MSC-CM) treating diabetic foot ulcers in rats.

Methods: Models of T2DM rats were induced by a high-fat diet and intraperitoneal injection of STZ in SD rats. Models of Diabetic Foot Ulcers (DFUs) were made by operation on hind limbs in diabetic rats. Rats were divided into four groups (n = 6 for each group), i.e., Normal Control group (NC), Diabetes Control group (DM-C), MSC-CM group and Mesenchymal Stem Cells group (MSCs). MSC-CM group was treated with an injection of conditioned medium derived from preconditioned rats’ bone marrow MSCs around ulcers. MSCs group were treated with an injection of rats’ bone marrow MSCs. The other two groups were treated with an injection of PBS. After the treatment, wound closure, re-epithelialization (thickness of the stratum granulosums of the skin, by H&E staining), cell proliferation (Ki67, by IHC), angiogenesis (CD31, by IFC), autophagy (LC3B, by IFC and WB; autoly-sosome, by EM) and pyroptosis (IL-1β, NLRP3, Caspase-1, GSDMD and GSDMD-N, by WB) in ulcers were evaluated.

Results: After the treatment wound area rate, IL-1β by ELISA, and IL-1β, Caspase-1, GSDMD and GSDMD-N by WB of MSC-CM group were less than those of DM group. The thickness of the stratum granulosums of the skin, proliferation index of Ki67, mean optic density of CD31 and LC3B by IFC, and LC3B by WB of MSC-CM group were more than those of DM group. The present analysis demonstrated that the injection of MSC-CM into rats with DFUs enhanced the wound-healing process by accelerating wound closure, promoting cell proliferation and angiogenesis, enhancing cell autophagy, and reducing cell pyroptosis in ulcers.

Conclusions: Studies conducted indicate that MSC-CM administration could be a novel cell-free therapeutic approach to treat DFUs accelerating the wound healing process and avoiding the risk of living cells therapy.

Keywords: Diabetic Foot Ulcers; Conditioned-Medium; Mesenchymal Stem Cells; Therapy

HIGHLIGHTS

BMMSC-CM therapy on rats with DFUs enhanced the wound healing process.

It accelerated wound closure and promoted cell proliferation and angiogenesis.

It enhancd cell autophagy and reduced cell pyroptosis in ulcers.

Group	0 d (g)	7 d (g)	14 d (g)
NC	352±29^a a p < 0.05 compared with the other three groups.	381±36^a a p < 0.05 compared with the other three groups.	391±42^a a p < 0.05 compared with the other three groups.
DM-C	401±25	407±27	416±24
MSC-CM	403±19	411±22	420±23
MSCs	398±26	406±23	414±18

Group	0 d (%)	3 d (%)	7 d (%)	10 d (%)	14 d (%)
NC	100	51±4	31±5	18±4	4±1
DM-C	100	70±6^a a p < 0.05 compared with the other three groups.	58±6	32±5^a a p < 0.05 compared with the other three groups.	15±4^a a p < 0.05 compared with the other three groups.
MSC-CM	100	55±6	38±7	22±4	8±3
MSCs	100	54±5	34±6	21±5	7±2

Group	0 d (mmoL/L)	7 d (mmoL/L)	14 d (mmoL/L)
NC	4.88±0.55^a a p < 0.05 compared with the other three groups.	5.12±0.46^a a p < 0.05 compared with the other three groups.	5.07±0.62^a a p < 0.05 compared with the other three groups.
DM-C	10.43±2.75	11.47±2.33	10.84±2.54
MSC-CM	11.32±2.88	10.57±2.34	9.97±2.18
MSCs	10.68±2.23	10.12±2.59	9.65±2.37

Group	Thickness of stratum granulosums (μm)	PI (%)	CD31	LC3B	IL-1β (pg/mL)
NC	22.5±3.4	5.4±1.2	0.11±0.04	0.08±0.02	17.1±2.5
DM-C	15.4±3.8^a a p < 0.05 compared with the other three groups.	1.5±0.3^a a p < 0.05 compared with the other three groups.	0.02±0.01^a a p < 0.05 compared with the other three groups.	0.01±0.002^a a p < 0.05 compared with the other three groups.	34.3±5.8^a a p < 0.05 compared with the other three groups.
MSC-CM	20.9±3.2	3.1±0.6	0.05±0.02	0.05±0.01	22.7±2.8
MSCs	20.4±3.5	2.8±0.5	0.04±0.01	0.04±0.01	21.3±2.5

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[TFN1] ^a
p < 0.05 compared with the other three groups.