Microscopy‐guided laser ablation for the creation of complex skin models with folliculoid appendages

Abstract

Engineering complex tissues requires the use of advanced biofabrication techniques that allow the replication of the tissue's 3D microenvironment, architecture and cellular interactions. In the case of skin, the most successful strategies to introduce the complexity of hair follicle (HF) appendages have highlighted the importance of facilitating direct interaction between dermal papilla (DP) cells and keratinocytes (KCs) in organotypic skin models. In this work, we took advantage of microscopy‐guided laser ablation (MGLA) to microfabricate a fibroblast‐populated collagen hydrogel and create a subcompartment that guides the migration of KCs and lead their interaction with DP cells to recreate follicular structures. Upon definition of the processing parameters (laser incidence area and power), MGLA was used to create 3D microchannels from the surface of a standard organotypic human skin model up to the aggregates containing DP cells and KCs, previously incorporated into the dermal‐like fibroblast‐collagen layer. Analysis of the constructs showed that the fabricated microfeatures successfully guided the fusion between epidermal and aggregates keratinocytes, which differentiated into follicular‐like structures within the organotypic human skin model, increasing its functionality. In summary, we demonstrate the fabrication of a highly structured 3D hydrogel‐based construct using MGLA to attain a complex skin model bearing folliculoid structures, highlighting its potential use as an in vitro platform to study the mechanisms controlling HF development or for the screening of bioactive substances.

Layer-by-Layer Analysis of In Vitro Skin Models

“…IHC markers K10, K14, K15, fibronectin, vimentin, involucrin, and β-catenin confirmed that the keratinocytes infiltrated the biofabricated channels, and a structure resembling an immature hair bulb formed. However, there was no hair shaft formation …”

Innovative Approaches and Advances for Hair Follicle Regeneration

“…The system also directed the interaction between keratinocyte and DPCs to reconstruct the HF structure. 138 Human keratinocytes derived from outer root sheath cells and DPCs cultured in Matrigel (3D network of ECM proteins and collagen) form tubule-like structures in the skin in vitro. They organize into epidermal-like cyst-like spheres rather than form the intact HFs.…”

“…Abreu et al used microscopy-guided laser ablation to microprocess fibroblast-filled collagen hydrogels and create a sub compartment to guide the migration of keratinocyte. The system also directed the interaction between keratinocyte and DPCs to reconstruct the HF structure . Human keratinocytes derived from outer root sheath cells and DPCs cultured in Matrigel (3D network of ECM proteins and collagen) form tubule-like structures in the skin in vitro.…”

Recreation of a hair follicle regenerative microenvironment: Successes and pitfalls

“…While epithelial invagination and formation of an epithelial column are required for HF morphogenesis, DP spheroids rapidly dissociate when placed within collagen matrices 83 , which might be compromising DP cell inductivity and model progression. Bearing this in mind, we were able to create a more complex skin model with folliculoid appendages by incorporating, within the dermal equivalent layers, aggregates with compartmentalized DP cells and keratinocytes 161 . Microscopy-guided laser ablation was used to fabricate a channel connecting the constructs' surface and the aggregates, to support epithelial cell migration.…”

“…Bearing this in mind, we were able to create a more complex skin model with folliculoid appendages by incorporating, within the dermal equivalent layers, aggregates with compartmentalized DP cells and keratinocytes. 161 Microscopy‐guided laser ablation was used to fabricate a channel connecting the constructs' surface and the aggregates, to support epithelial cell migration. Follicle‐like structures with differenced concentric epithelial layers and mimicking the hair bulb architecture were obtained, though a hair shaft was not formed.…”