Promotion of Hair Growth by Conditioned Medium from Extracellular Matrix/Stromal Vascular Fraction Gel in C57BL/6 Mice

Shune Xiao, Yurong Deng, Xiaojin Mo, Zhiyuan Liu, Dali Wang, Chengliang Deng, Zairong Wei, Shune Xiao, Yurong Deng, Xiaojin Mo, Zhiyuan Liu, Dali Wang, Chengliang Deng, Zairong Wei

Abstract

Adipose-derived stem cell- (ADSC-) based regenerative medicine has expanded to include the treatment of hair loss. However, stem cell therapy remains a relatively recent technique, and reports of its use for treating alopecia are rare. ADSCs exert biological functions via the paracrine actions of various growth factors and cytokines. Conditioned medium from ADSCs (ADSCs-CM) is a cell-free suspension rich in growth factors and cytokines that has demonstrated a significant role in stimulating hair growth, with encouraging outcomes in terms of hair regeneration and hair growth. Extracellular matrix/stromal vascular fraction gel (ECM/SVF-gel) is an ADSC- and adipose native extracellular matrix-enriched product for cytotherapy. In this study, we compared the effects of CM from ECM/SVF-gel (ECM/SVF-CM) and from stem cells (SVF-CM) on hair growth in mice. ECM/SVF-CM stimulated hair growth more than SVF-CM, through promoting the proliferation of dermal papilla cells and cells in the bulge, neovascularization, and anagen induction. ECM/SVF-CM might, thus, provide an effective and improved strategy for promoting hair growth. These data provide a theoretical foundation for the clinical administration of ECM/SVF-CM for the treatment of hair loss.

Conflict of interest statement

The authors declare no competing financial interests.

Copyright © 2020 Shune Xiao et al.

Figures

Figure 1
Figure 1
Schematic diagram of the preparation of ECM/SVF-CM (a) and SVF-CM (b). ECM: extracellular matrix; SVF: stromal vascular fraction; CM: conditioned medium.
Figure 2
Figure 2
Quantification of growth factor expression. Quantification of VEGF, bFGF, PDGF, and KGF expression in SVF-CM and ECM/SVF-CM. ∗∗∗P < 0.01.
Figure 3
Figure 3
Hair-growth effect of ECM/SVF-CM in hair-loss-induced C57BL/6 mice. The hair was removed from the backs of C57BL/6 mice and the hair-growth rate was monitored for 3 weeks. Mice were injected with ECM/SVF-CM, SVF-CM, and PBS (control), respectively, once per week for 3 weeks. Gross view was observed by photographs (a), and hair-growth scoring was measured (b). Mice in the ECM/SVF-CM injection group (n = 10) showed significantly increased hair regeneration compared with the SVF-CM injection group (∗∗P < 0.05, n = 10) and negative control group (#P < 0.01, n = 10). The SVF-CM injection group also showed significantly increased hair regeneration compared with the control group (∗P < 0.05). The data shown represent one of each group, and experiments were performed three times independently.
Figure 4
Figure 4
Morphology of DPs and DPCs. Immunofluorescence identification of DPCs and proliferation-promoting effect of the two CMs on DPCs. Phase-contrast micrograph showing oval appearance 2 days after inoculation, with most DPs attached with a few DPCs around them (a). DPCs showing spindle-shaped morphology (b). ALP (c) and β-catenin (d) were highly expressed in DPCs as shown by immunofluorescence staining. The viabilities of DPCs in the CM-treated groups were significantly higher than in the control group at days 3 and 5, and the viability in the ECM/SVF-CM-treated group was significantly higher than in the SVF-CM-treated group (e). Scale bar = 100 μm. (∗P < 0.05, ∗∗P < 0.01).
Figure 5
Figure 5
Immunohistochemical staining of Ki-67 in bulge cells. The number of Ki-67-positivity cell was significantly more in both the ECM/SVF-CM- and SVF-CM-treated groups compared with the control group, with the highest cell viability in the ECM/SVF-CM-treated group (∗∗P < 0.05, ∗∗∗P < 0.01). Scale bar = 20 μm.
Figure 6
Figure 6
Stimulatory effect of ECM/SVF-CM on angiogenesis. Vascularization of the hair-regeneration sites was analyzed 2 weeks after injection. The inner skin of the hair-regrowth site showed mature blood vessel branching (a). Among the three groups, the ECM/SVF-CM-injection group showed markedly enhanced formation of mature vessels (a). Immunohistochemical staining of CD31 showed more new blood vessels in the ECM/SVF-CM-injection compared with the SVF-CM-injection and control groups. Arrow indicates blood vessels (b). (∗∗P < 0.05, ∗∗∗P < 0.01). Scale bar = 50 μm.
Figure 7
Figure 7
Involvement of Wnt pathway in the stimulatory effect of hair growth by ECM/SVF-CM. Both ECM/SVF-CM and SVF-CM markedly increased Wnt5a and Wnt10b expression levels compared with the control group, with the highest expression in the ECM/SVF-CM-treated group (∗∗∗P < 0.05).

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