Bioinspired engineering ADSC nanovesicles thermosensitive hydrogel enhance autophagy of dermal papilla cells for androgenetic alopecia treatment

Abstract

Androgenic alopecia (AGA) is a highly prevalent form of non-scarring alopecia but lacks effective treatments. Stem cell exosomes have similar repair effects to stem cells, suffer from the drawbacks of high cost and low yield yet. Cell-derived nanovesicles acquired through mechanical extrusion exhibit favorable biomimetic properties similar to exosomes, enabling them to efficiently encapsulate substantial quantities of therapeutic proteins. In this study, we observed that JAM-A, an adhesion protein, resulted in a significantly increased the adhesion and resilience of dermal papilla cells to form snap structures against damage caused by dihydrotestosterone and macrophages, thereby facilitating the process of hair regrowth in cases of AGA. Consequently, adipose-derived stem cells were modified to overexpress JAM-A to produce engineered JAM-A overexpressing nanovesicles (JAM-A^OE@NV). The incorporation of JAM-A^OE@NV into a thermosensitive hydrogel matrix (JAM-A^OE@NV Gel) to effectively addresses the limitations associated with the short half-life of JAM-A^OE@NV, and resulted in the achievement of a sustained-release profile for JAM-A^OE@NV. The physicochemical characteristics of the JAM-A^OE@NV Gel were analyzed and assessed for its efficacy in promoting hair regrowth in vivo and vitro. The JAM-A^OE@NV Gel, thus, presents a novel therapeutic approach and theoretical framework for promoting the treatment of low cell adhesion diseases similar to AGA.

Keywords: Androgenic alopecia; Autophagy; Hydrogel; JAM-A; Nanovesicles.

Graphical abstract

Fig. 1

Characterization of CNVs and effect of CNVs on hair regeneration in an AGA mouse model. A) Schematic of the preparation of the CNVs. B) Western blotting of exosome markers Calnexin, TSG101, and CD63. C) NanoFlow analysis of CNVs. D) Morphology of CNVs under transmission electron microscopy (TEM); scale bar: 400 nm. E) Fluorescent images of PKH26-labeled CNVs internalized by DPCs stained with phalloidin; scale bar: 40 μm. F) Animal experimental schedule for inducing the AGA mouse model. G) Schematic diagram of animal groupings and interventions. H) Representative optical images of hair growth on days 7, 11, 15 after treatment; scale bar: 2 cm. I) The hair-covered area of the dorsal skin of mice on day 15 after treatment with different formulations. J) Representative H&E staining images of hair follicle regeneration on the dorsal skin of mice on day 21; scale bar: 400 μm. K) Representative Ki67 immunofluorescence staining images of hair follicle regeneration on day 21; scale bar: 100 μm. L) Statistical data on hair follicle number was counted in the same random area. M) Statistical data on the relative Ki67 expression of the different groups. (n = 3 per group, *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 2

Protective effect and potential mechanism of CNVs on DPCs injured by DHT and macrophages. A) Effects of various concentrations of DHT on the viability of DPCs, as shown by CCK-8 assays (n = 3). B) Viability of DHT-injured DPCs treated with CNVs for 48 h (n = 3). C) Statistical data on the proportion of senescent cells in each group was counted in the same random area (n = 4). D) Effect of CNVs on DHT-injured DPC migration at every indicating time; scale bar: 50 μm. E) Relative migration of DHT-injured DPCs in each group (n = 4). F) Relative expression of macrophage chemotaxis, activation, and cytokine production abundances in the frontal alopecia region of AGA patients in GSE212301. G) Schematic diagram of the co-culture of DPCs with LPS-activated Raw 264.7 macrophages. H) Apoptosis cells of macrophage-injured DPCs treated with CNVs for 48 h were determined by an apoptosis kit. I) Statistical data on the proportion of apoptosis cells in each group (n = 3). J) Cellular ultrastructure in each group was detected by TEM, and red arrows indicated autophagosomes; scale bar: 2 μm and 500 nm. K) Change in LC3 and β-catenin expression. L) Relative expression of β-catenin (n = 3). M) Relative expression of LC-3II (n = 3). (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 3

JAM-A protein plays a core role in ADSC-NVs-mediated AGA hair regrowth. A) Volcano plot of the GSE184501 microarray data set, and JAM-A significantly downgraded. B) Change in JAM-A expression. C) Relative expression of JAM-A. D) Viability of DPC cell lines treated with DHT for 48 h. E) Statistical data on the proportion of senescent cells in each group was counted in the same random area. F) Effect of DHT on DPC cell line migration at every indicating time; scale bar: 50 μm. G) Relative migration of DHT-injured DPC cell lines in each group. H) Apoptosis cells of DPC cell lines were injured with macrophages for 48 h. I) Statistical data on the proportion of apoptosis cells in each group. J) Change in LC3, AKT, AKT phosphorylation, and p62 expression. K) Relative expression of LC-3II, p62, and AKT phosphorylation. L) Change in Cyclin D1 and β-catenin expression. M) Relative expression of Cyclin D1 and β-catenin. (n = 3 per group, *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 4

Characterization of the JAM-A@NV-enriched thermosensitive hydrogel (JAM-A@NV Gel). A) The viability of DPCs cultured in a common dish or poloxamer 407 hydrogel (Gel) was evaluated by a CCK-8 assay. B) Live/dead staining of DPCs cultured on the gel or common culture dish; scale bar: 50 μm. C) Temperature response behavior of JAM-A@NV Gel using the inversion method. D) Gelation time of JAM-A@NV Gel. E) Injectability of the JAM-A@NV Gel. F, G) Morphology of JAM-A@NV Gel; scale bars: 50 μm and 20 μm. H) Confocal 3D analysis of JAM-A@NV Gel. PKH26 is used to characterize JAM-A@NV (Red). I, J) Rheological properties of JAM-A@NV Gel were analyzed with temperature changes. K) The JAM-A@NV release behavior of JAM-A@NV Gel. (n = 3 per group, ns = not significant).

Fig. 5

Protective effect of JAM-A^OE@NV Gel in the AGA mouse model. A) Photographs were taken on days 7, 11, 15, and 19 after depilation (n = 8). B) The hair coverage area proportion of the dorsal skin on days 7, 11, 15, and 19 (n = 8). C, D) Statistical data of the hair coverage area proportion of the dorsal skin on days 15 and 19, respectively (n = 8). E) Representative H&E staining images of hair follicle regeneration on the dorsal skin of mice on day 21; scale bar: 2 mm. F) Statistical data on hair follicle number was counted in the same random area (n = 8). G) Statistical data on the average diameter of hair follicle base enlargement (n = 15). H) Representative Ki67 immunofluorescence staining images of hair follicle regeneration on day 21. I) Statistical data on the relative Ki67 expression of the different groups (n = 8). (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 6

Protective effect of JAM-A^OE@NV on JAM-A-downregulated DPC cell line injured by DHT and macrophages. A) Change in JAM-A expression. B) Relative expression of JAM-A. C) Viability of the DHT-injured, downregulated DPC cell line treatment with JAM-A@NV for 48 h. D) Statistical data on the proportion of senescent cells in each group was counted in the same random area. E) Effect of JAM-A@NV on the DHT-injured, downregulated DPC cell line migration at 24 h; scale bar: 50 μm. F) Relative migration of the DHT-injured, downregulated DPC cell line in each group. G) Apoptosis cells of the macrophages injured by downregulated DPC cell line treatment with JAM-A@NV for 48 h. H) Statistical data on the proportion of apoptosis cells in each group. I) Change in LC3, AKT, AKT phosphorylation, and p62 expression. J) Relative expression of LC-3II, p62, and AKT phosphorylation. K) Change in Cyclin D1 and β-catenin expression. L) Relative expression of Cyclin D1 and β-catenin. (n = 6 per group, *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 7

Overview diagram of the mechanism of JAM-A^OE@NV Gel promoting AGA hair regeneration.