Introduction: The hair follicle (HF) is a complex feature of the skin, and its origins and mechanisms are not fully understood. Additionally, the HF is an intricate niche for epidermal stem cells, in which the epidermal cells interact with specialized mesenchymal cells known as dermal papillae (DP) cells. Studies have shown that DP cells are required to induce the HF fate'1', but the mechanisms are as yet unknown. In this study, a hydrogel with surface features similar in structure to the HF is used as the scaffold for a co-culture of neonatal mouse dermal fibroblasts and keratinocytes. Materials and Methods: Norbornene-succinimidyl ester was synthesized by EDC coupling of norbornene carboxylic acid and NHS, and used to functionalize multi-arm amine-terminated poly (ethylene glycol) (PEG) and gelatin (100 bloom from porcine skin). Acrylamide functionated PEG and gelatin was similarly synthesized using n-acryloxysuccinimide. Extent of functionalization was determined using proton NMR for PEG-norbomene and a TNBSA assay for gelatin to determine the number of free amines before and after functionalization. Multi-functional PEG-thiol was used to crosslink norbornene functionalized polymers. Thiolated RGD peptide and tetrazine functkxialized laminin were added to impart adhesivity to PEG hydrogels. Gels were reacted using 2 mM lithium acyl-phosphinate (LAP) as a photoinitiator and 10 mW/cm2 365 nm light. Gelation was monitored using a TA Instruments Ares Rheometer, and shear storage modulus at 100s was reported as the final modulus. To create surface patterns in the gel, soft lithography was used. An SU-8 photoresist template was polymerized using a custom chrome photomask, after which a PDMS mold was created with evenly spaced arrays of cylinders (D=50,100, or 200 μm, H=50 or 100 μm). Fibroblast aggregates were formed in the surface wells using a previously described technique , and keratinocytes were seeded evenly across the surface of the resulting gel. Results and Discussion: Gelatin and PEG macromers were functionalized with norbomene and acrylamide functional groups corresponding to 80% (gelatin) and 95% (PEG) conversion of primary amines. Photopolymerization occurred in less than 10 seconds for thiol-ene hydrogels and -100 seconds for acrylamide hydrogels. Importantly, pattern fidelity was retained on the hydrogel surface (Fig 1 .c), allowing for fibroblast aggregate formation within surface microwells (Fig 1 .d). In each case, it was possible to vary the bulk shear elastic modulus from -0.5 to ＞40 kPa by increasing the relative amount of higher functionality PEG precursor (data not shown). In contrast to PEG-based gels, primary keratinocyte adhesion was greatly enhanced on gelatin-based hydrogels, with cells reaching confluency within 5 days (Fig 1.e). Conclusion: Surface microwells were used to capture and aggregate primary dermal fibroblasts, mimicking the dermal papillae of the HF. Gelatin hydrogels promoted creation of a uniform monolayer of keratinocytes. Co-culture experiments are ongoing with a Sox9GFP reporter mouse line to determine whether keratinocytes can adopt the hair follicle fate in vitro. This system is a promising platform to study stem cells in the hair follicle niche.
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