毛囊混合细胞在海藻酸钠支架上诱导毛囊重建

Reconstruction of hair follicles in three-dimensional alginate scaffolds with mixed cells of hair folli- cles

目的探讨原代培养的毛囊外根鞘细胞、毛乳头细胞和成纤维细胞，采用不同接种顺序在体外和体内诱导毛囊形成。方法将培养的毛囊外根鞘细胞（ORSC）、毛乳头细胞（DPC）和丝裂霉素干预后的成纤维细胞按一定比例制成细胞悬液，并按不同的接种顺序接种于海藻酸钠3D细胞支架上，构建毛囊的体外三维模型并培养8周，行HE染色光镜观察毛囊形成的情况；同时将该三维模型移植人bal／bcl裸鼠皮下体内培养8周，移植部位取材后分别行HE染色、免疫组化和电镜观察毛囊形成的情况。结果体外构建的毛囊i维模型未见到角化物质及毛囊样结构；裸鼠皮下移植物HE染色可见细胞聚集成团，有呈环状排列的毛囊样结构，CKl4，CK15、B1整合素和波形蛋白染色阳性；电镜下模型中可见贴附在支架上的毛囊细胞和红细胞。先接种用丝裂霉素干预的成纤维细胞于海藻酸钠3D细胞支架上培养1周后再接种DPC：ORSC（1：5）细胞悬液，裸鼠体内移植物HE染色不仅可见明显的毛囊样结构形成，而且形成毛囊样结构的数目较多。结论模型中DPC保持了诱导毛囊形成的能力，ORSC保持了毛囊干细胞的特点。并明确了诱导毛囊形成的最佳细胞组合、接种顺序和比例，为体外构建含有毛囊的人工皮肤奠定了一定基础。

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, immunohistoehemical 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 ceils and erythrocytes adhering to the scaffolds. One-week precuhure of mitomycin-c-treated 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.