Abstract： Objective To induce the formation of hair follicles in vivo and in vitro with primary human hair follicle outer root sheath cells (ORSCs),human hair dermal papilla cells (DPCs) and fibroblasts seeded on three-dimensional alginate scaffolds at different sequences.Methods Second-passage ORSCs,DPCs and mitomycin-c-treated human fibroblasts were seeded into three-dimensional alginate scaffolds at different sequences and a certain ratio to reconstruct hair follicles in vitro.After 8 weeks of culture,some of the substitutes were subjected to hematoxylin and eosin (HE) staining,and some were transplanted into the subcutaneous tissue of bal/bcl nude mice followed by additional culture for 8 weeks.Then,the mice were sarcrificed and transplants were harvested and subjected to HE staining,immunohistochemical staining and transmission electron microscopy for the observation of hair follicle formation.Results After 8 weeks of culture in vitro,hair follicle cells were evenly scattered in three-dimensional scaffolds,with no keratinized material or hair follicle formation.When the substitutes were cultured in vivo for 8 weeks,DPCs and ORSCs gathered together to form cell aggregates,with the visualization of hair follicle-like structure in circular arrangement,which was positive for cytokeratin (CK) 14,CK15,β1-integrin and vimentin.Electron microscopy showed hair follicle cells and erythrocytes adhering to the scaffolds.One-week preculture of mitomycin-c-trcatcd human fibroblasts in the scaffolds followed by the seeding of the suspension of ORSCs and DPCs at a ratio of 1∶5 resulted in increased follicular like structures with more typical appearance in mice compared with the other strategies for cell inoculation and culture.Conclusions In the reconstructed hair follicle models,DPCs keep the capability to induce the formation of hair follicles,and ORSCs keep the characteristics of hair follicle stem cells.The optimal cell density,ratio and process were determined in the experiment,which may lay a methodological and theoretical basis for hair follicle reconstruction.