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Background Tissue engineering of hair follicles (HFs) has enormous potential for hair loss treatment. However, certain challenges remain, including weakening of the dermal papilla cell (DPC) viability, proliferation, and HF inducibility, as well as the associated inefficient and tedious preparation process required to generate extracellular matrix (ECM)-mimicking substrates for biomolecules or cells. Herein, we utilized gelatin methacryloyl (GelMA) and chitosan hydrogels to prepare scalable, monodispersed, and diameter-controllable interpenetrating network GelMA/chitosan-microcarriers (IGMs) loaded with platelet-rich plasma (PRP) and seeded with DPCs, on a high-throughput microfluidic chip. Results The ECM-mimicking hydrogels used for IGMs exhibited surface nano-topography and high porosity. Mass production of IGMs with distinct and precise diameters was achieved by adjusting the oil and aqueous phase flow rate ratio. Moreover, IGMs exhibited appropriate swelling and sustained growth factor release to facilitate a relatively long hair growth phase. DPCs seeded on PRP-loaded IGMs exhibited good viability (> 90%), adhesion, spreading, and proliferative properties (1.2-fold greater than control group). Importantly, PRP-loaded IGMs presented a higher hair inducibility of DPCs in vitro compared to the control and IGMs group (p < 0.05). Furthermore, DPC/PRP-laden IGMs were effectively mixed with epidermal cell (EPC)-laden GelMA to form a PRP-loaded DPC/EPC co-cultured hydrogel system (DECHS), which was subcutaneously injected into the hypodermis of nude mice. The PRP-loaded DECHS generated significantly more HFs (~ 35 per site) and novel vessels (~ 12 per site) than the other groups (p < 0.05 for each). Conclusion Taken together, these results illustrate that, based on high-throughput microfluidics, we obtained scalable and controllable production of ECM-mimicking IGMs and DECHS, which simulate an effective micro- and macro-environment to promote DPC bioactivity and hair regeneration, thus representing a potential new strategy for HF tissue engineering.

The application of dermal papilla cells to hair follicle (HF) regeneration has attracted a great deal of attention. However, cultured dermal papilla cells (DPCs) tend to lose their capacity to induce hair growth during passage, restricting their usefulness. Accumulating evidence indicates that DPCs regulate HF growth mainly through their unique paracrine properties, raising the possibility of therapies based on extracellular vesicles (EVs). In this study, we explored the effects of EVs from high- and low-passage human scalp follicle dermal papilla cells (DP-EVs) on activation of hair growth, and investigated the underlying mechanism. DP-EVs were isolated by ultracentrifugation and cultured with human scalp follicles, hair matrix cells (MxCs), and outer root sheath cells (ORSCs), and we found low-passage DP-EVs accelerated HF elongation and cell proliferation activation. High-throughput miRNA sequencing and bioinformatics analysis identified 100 miRNAs that were differentially expressed between low- (P3) and high- (P8) passage DP-EVs. GO and KEGG pathway analysis of 1803 overlapping target genes revealed significant enrichment in the BMP/TGF-β signaling pathways. BMP2 was identified as a hub of the overlapping genes. miR-140-5p, which was highly enriched in low-passage DP-EVs, was identified as a potential regulator of BMP2. Direct repression of BMP2 by miR-140-5p was confirmed by dual-luciferase reporter assay. Moreover, overexpression and inhibition of miR-140-5p in DP-EVs suppressed and increased expression of BMP signaling components, respectively, indicating that this miRNA plays a critical role in hair growth and cell proliferation. DP-EVs transport miR-140-5p from DPCs to epithelial cells, where it downregulates BMP2. Therefore, DPC-derived vesicular miR-140-5p represents a therapeutic target for alopecia.

Dermal papillae are a target of androgen action in patients with androgenic alopecia, where androgen acts on the epidermis of hair follicles in a paracrine manner. To mimic the complexity of the dermal papilla microenvironment, a better culture model of human dermal papilla cells (DPCs) is needed. Therefore, we evaluated the inhibitory effect of dihydrotestosterone (DHT)-treated two-dimensional (2D)- and 3D-cultured DPCs on hair follicle growth. 2D- and 3D-cultured DPC proliferation was inhibited after co-culturing with outer root sheath (ORS) cells under DHT treatment. Moreover, gene expression levels of β-catenin and neural cell adhesion molecules were significantly decreased and those of cleaved caspase-3 significantly increased in 2D- and 3D-cultured DPCs with increasing DHT concentrations. ORS cell proliferation also significantly increased after co-culturing in the control-3D model compared with the control-2D model. Ki67 downregulation and cleaved caspase-3 upregulation in DHT-treated 2D and 3D groups significantly inhibited ORS cell proliferation. Sequencing showed an increase in the expression of genes related to extracellular matrix synthesis in the 3D model group. Additionally, the top 10 hub genes were identified, and the expression of nine chemokine-related genes in DHT-treated DPCs was found to be significantly increased. We also identified the interactions between transcription factor (TF) genes and microRNAs (miRNAs) with hub genes and the TF–miRNA coregulatory network. Overall, the findings indicate that 3D-cultured DPCs are more representative of in vivo conditions than 2D-cultured DPCs and contribute to our understanding of the molecular mechanisms underlying androgen-induced alopecia.

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Tissue engineering of hair follicles (HFs) has enormous potential in the treatment of hair loss. HF morphogenesis is triggered by reciprocal interactions between HF germ epithelial and mesenchymal layers. Here, a microfluidic-assisted technology is developed for the preparation of double aqueous microdroplets that entrap double-layer cells and growth factors to ultimately be used for hair regeneration. Mouse mesenchymal cells (MSCs) and epidermal cells (EPCs) are encapsulated in gelatin methacrylate (GelMA) cores and photo-curable catechol-grafted hyaluronic acid (HAD) shells to fabricate GelMA-MSC/HAD-EPC (G/HAD) microspheres. The findings show that the G/HAD microspheres exhibit ultrafast gelation, aqueous phase separation, superior biocompatibility, and favorable wet adhesion properties. G/HAD microspheres can also support cell proliferation and sustain growth factor release. These composite cell microspheres are capable of efficient HF generation upon transplantation into the dorsal dermis of nude mice. This finding facilitates the large-scale preparation of approximately 80 double-layer cell spheres per min. This simple double-layer cell sphere preparation approach is a promising strategy for improving current hairregenerative medicine techniques and can potentially be applied along with other organoid techniques for extended applications.

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