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. 2023 Oct 5;12(10):689-706.
doi: 10.1093/stcltm/szad050.

External Application of Human Umbilical Cord-Derived Mesenchymal Stem Cells in Hyaluronic Acid Gel Repairs Foot Wounds of Types I and II Diabetic Rats Through Paracrine Action Mode

Jingan Chen  1 , Yi Liu  1 , Jingwen Zhang  2 , Yuping Yang  1 , Haowei Liang  1 , Ting Li  3 , Li Yan  3   2 , Li Zhou  3 , Letian Shan  3   2 , Hui Wang  1   4
Affiliations

External Application of Human Umbilical Cord-Derived Mesenchymal Stem Cells in Hyaluronic Acid Gel Repairs Foot Wounds of Types I and II Diabetic Rats Through Paracrine Action Mode

Jingan Chen et al. Stem Cells Transl Med. .

Abstract

Diabetic foot ulcer (DFU) is a main diabetic complication with unmet treatment needs. This study applied human umbilical cord-derived mesenchymal stem cells-hyaluronic acid (hucMSCs-HA) gel to treat DFU in a noninvasive external way and investigated its paracrine action and mechanism. In this study, after analyzing the physical and biological properties of HA gel, hucMSCs-HA gel was applied in 2 in vivo models (types I and II DFU), and a molecular mechanism was investigated. To evaluate the paracrine action of hucMSCs, hucMSCs-conditional medium (MSC-CM) was collected to treat 1 in vivo model (type I DFU) and 2 in vitro models (high glucose (HG)-injured human umbilical vein endothelial cells (HUVECs) and human skin fibroblasts (HSFs)). The results indicated that HA gel with a porous microstructure underwent over 90% degradation and swelled to the maximum value within 48 h. In vivo, hucMSCs-HA gel accelerated wound healing of DFU rats by improving re-epithelialization, collagen deposition, and angiogenesis, in which a paracrine action of hucMSCs was confirmed and the phosphorylation of p38, ERK1/2, JNK, and Akt was increased. In vitro, MSC-CM improved cell viability, wound healing, migration, tube formation, cell senescence, and abnormal expressions (TNF-α, IL-1β, IL-6, ET-1, p16 genes, and PCNA protein) of HUVECs, also improved cell viability, wound healing, antioxidant stress, and abnormal expressions (COL1, COL3, COL4, SOD1, SOD2 genes, and PCNA protein) of HSFs. Summarily, noninvasive external application of hucMSCs-HA gel shows great perspective against DFU and exerts wound healing effects through the MAPK and Akt pathways-mediated paracrine mechanism.

Keywords: diabetic foot ulcer; human umbilical cord-derived mesenchymal stem cells; hyaluronic acid gel; noninvasive external application; paracrine; wound healing.

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Conflict of interest statement

The authors have declared that no competing interest exists.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
Therapeutic effects of the hucMSCs-HA gel on foot wound healing of type I and II diabetic (T1DM and T2DM) rats at different time points. (A) Representative digital images of wound healing surfaces in type I and II diabetic rats, scale bar = 2 mm. (B) Line graph of average wound surface healing rate in type I and II diabetic rats. *P < .05 and **P < .01 vs the control group, #P < .05 and ##P < .01 vs the model (T1DM or T2DM) group, $P < .05 and $$P < .01 vs the HA group.
Figure 2.
Figure 2.
Histopathological features of diabetic foot wound closure in type I diabetic (T1DM) rats on day 21. (A) Representative micrographs of HE staining, scale bar = 500 μm (40×) and 50 μm (400×). (B) Representative micrographs of Masson’s trichrome stained sections, scale bar = 500 μm (40×) and 50 μm (400×). (C) Histogram of collagen deposition from Masson’s trichrome. (D) Representative micrographs of VEGFA antibody immunohistochemistry and blood vessels labeled by yellow allows, scale bar = 500 μm (40×) and 50 μm (400×). (E) Quantification of immunohistochemical staining intensity (VEGFA antibody). (F) Quantification of average number of blood vessels. Data were mean ± SD. *P < .05 and **P < .01 vs the control group, #P < .05 and ##P < .01 vs the model group, $P < .05 and $$P < .01 vs the HA group.
Figure 3.
Figure 3.
Histopathological features of diabetic foot wounds closure in type II diabetic (T2DM) rats on day 21. (A) Representative micrographs of H&E staining, scale bar = 500 μm (40×) and 50 μm (400×). (B) Representative micrographs of Masson’s trichrome stained sections, scale bar = 500 μm (40×) and 50 μm (400×). (C) Histogram of collagen deposition from Masson’s trichrome. (D) Representative micrographs of VEGFA antibody immunohistochemistry and blood vessels labeled by yellow allows, scale bar = 500 μm (40×) and 50 μm (400×). (E) Quantification of immunohistochemical staining intensity (VEGFA antibody). (F) Quantification of average number of blood vessels. Data were mean ± SD. *P < .05 and **P < .01 vs the control group, #P < .05 and ##P < .01 vs the model group, $P < .05 and $$P < .01 vs the HA group.
Figure 4.
Figure 4.
Therapeutic effects of the hucMSCs-HA and MSC-CM-HA gels on healing of type I diabetic (T1DM) foot wounds at different time points. (A) Representative digital images of wound healing surfaces in SD rats on days 0, 3, 6, 9, 12, 15, 18, and 21, scale bar = 2 mm. (B) Representative micrographs of H&E staining and Masson’s trichrome stained sections of the wounds on day 21 after wound induction, scale bar = 500 μm (40×) and 50 μm (400×). (C) Line graph of average wound surface healing rate in SD rats. (D) Quantification of collagen deposition from Massonʹs trichrome staining results. Data were mean ± SD. *P < .05 and **P < .01 vs the control group, #P < .05 and ##P < .01 vs the model (T1DM) group.
Figure 5.
Figure 5.
The effects of MSC-CM on the cell viability, wound healing, and cell migration of HUVECs and HSFs. (A) Cell viability of HUVECs was detected by MTT method. (B) Cell viability of HSFs was detected by MTT method. (C) and (D) showed the number of cell migration and the representative pictures of HUVECs in the transwell experiment (scale bar = 50 μm). (E) and (F) showed wound closure rate and representative pictures in the scratch test of HUVECs at 0, 12, 24, 36, and 48 h (scale bar = 50 μm). (G) and (H) showed wound healing rates and representative pictures in the scratch test of HSFs at 0, 12, 24, 36, and 48 h (scale bar = 50 μm). Data were mean ± SD, **P < .01 vs control level, and ##P < .01 vs model level.
Figure 6.
Figure 6.
The role of MSC-CM treatment in mRNA and protein expressions of high glucose-induced HUVECs and HSFs. (A) mRNA expressions of inflammation (TNF-α, IL-1β, and IL-6), tube formation (ET-1), and senescence (p16) related genes in HUVECs. (B) mRNA expressions of collagen synthesis (COL1, COL3, and COL4), oxidative stress (SOD1 and SOD2) related genes in HSFs. (C) Representative protein expression images of PCNA (HUVECs and HSFs). (D) Relative protein levels of PCNA/β-actin (HUVECs and HSFs). Data was presented as mean ± SD, *P < .05 and **P < .01 vs control level, #P < .05 and ##P < .01 vs model level.
Figure 7.
Figure 7.
Involvement of MAPK and Akt signaling pathways in the pro-healing property of hucMSCs-HA. Proteins were extracted from skin tissues in type I diabetic rats (including the control, T1DM, HA, and MSCHA groups). (A) Total and phosphorylated p38, JNK, ERK1/2, and Akt were assessed by Western blotting assay. Among them, representative images from 3 individual rats (marked as 1#, 2#, and 3#) were shown. (B) Relative protein levels were also analyzed. Data were mean ± SD. *P < .05 and **P < .01 vs the control group, #P < .05 and ##P < .01 vs the T1DM group, $P < .05 and $$P < .01 vs the HA group.
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