Studies of human hair follicle (HF) induction by follicle-derived cells have been limited due to a lack of suitable test systems. In this study, we established a skin organ culture system which supports HF formation by follicle-derived cells. Long-term skin organ cultures were set up from human retroauricular skin specimens and maintained in culture for up to 8 weeks. In vitro expanded human HF-derived cells from the dermal papilla (DP) and the outer root sheath (ORS) were injected together into the skin specimens and evaluated for their ability to induce reorganization of HFs. Macroscopic analysis of the cultured skin specimens demonstrated the growth of velus-like hair after 4 weeks in culture. Histologic evaluation of the cultured skin specimens after 8 weeks of culture revealed multiple miniaturized HFs with sebaceous glands. In addition, cell clusters of various differentiation stages could be demonstrated in serial sections of the cultured skin specimens. Labeling of HF-derived cells with the fluorescence dye CFDA-1 prior to injection suggested a de novo reorganization of HFs out of the injected cells. In conclusion, the study demonstrated HF formation by HF-derived cells in an in vitro skin organ culture model.

Our study demonstrated induction of VEGF mRNA in stored micrografts by AMG. Although the clinical relevance in post-transplant hair growth and wound healing needs further evaluation, the possibility of actively influencing growth factor production in isolated micrografts during the storage period is the basis for the development of hair follicle growth-promoting storage solutions in the future.

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BACKGROUND AND OBJECTIVES. Aminoguanidine (AMG) has been found to inhibit apoptotic cell death in hair follicle micrografts and improves the viability of isolated micrografts during the storage period in hair restoration surgery. In this study, we investigated the effect of AMG on messenger ribonucleic acid (mRNA) synthesis of growth factors in stored micrografts and primary cultures of follicle-derived cell populations.METHOD. Hair follicles were obtained from 10 different patients undergoing routine micrograft transplant and were stored for 5 hours at room temperature in phosphate-buffered saline containing different concentrations of AMG. After a culture period of 72 hours, quantitative changes of mRNA for basic fibroblast growth factor (bFGF), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF) were determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Primary cell cultures of dermal papilla and outer root sheath cells were stimulated for 72 hours with AMG followed by RT-PCR measurement of growth factor mRNA. RESULTS. A dose-dependent induction of VEGF mRNA could be demonstrated in stored micrografts after stimulation with AMG(unstimulated: 1.0 [0.7-2.2]; AMG 10 µg/mL: 5.6 [3.1-9.7], p < .05; AMG 50 µg/mL: 6.9 [5.7-10.0], p < .05; AMG 100 µg/mL: 17.1 [14.1-22.3], p < .001). Expression of bFGF mRNA and IGF-1 mRNA levels was not influenced by AMG stimulation. Stimulation of cultured dermal papilla and outer root sheath cells demonstrated 14-fold induction of VEGF mRNA by AMG in outer root sheath cells (unstimulated: 1.0 [0.8-1.4]; AMG 100 µg/mL: 14.0 [12.5-16.1], p < .01), and no changes in VEGF mRNA levels were detected in dermal papilla cells. CONCLUSIONS. Our study demonstrated induction of VEGF mRNA in stored micrografts by AMG. Although the clinical relevance in post-transplant hair growth and wound healing needs further evaluation, the possibility of actively influencing growth factor production in isolated micrografts during the storage period is the basis for the development of hair follicle growth-promoting storage solutions in the future.