Novel automatically stratified fibrotic 3D cutaneous spheroids for studying fibrotic mechanisms in hypertrophic scars

Summary: Abstract Body: Background: Hypertrophic scarring (HTS) is marked by excessive fibrosis and structural disorganization. Traditional 2D scar models fail to mimic the structural and biological complexity of HTS. Recent 3D bioprinted models are innovative but labor-intensive, limiting their practicality for fibrosis studies. Methods: Fibroblasts, keratinocytes, and vascular endothelial cells were co-cultured in a multicellular model using Molecular Assembly-based Macromolecular Crowding (MAMC) technology. This approach enabled stratified spheroid formation with a dermal core and epidermal layer, inducing myofibroblast differentiation. Fibrotic features and multicellular interactions were analyzed using TEM, qPCR, and Western blot. Results: MAMC-derived spheroids displayed key HTS features, including a 100 µm-thick epidermal crust and increased collagen/SMA accumulation in the dermal core. Surface keratinocytes exhibited tight connections, replicating the HTS's mechanical microenvironment. Gene analysis revealed elevated TWIST and SNAIL levels during fibroblast activation, highlighting their role in multicellular interactions driving fibrosis. The dermal core showed more complex fibril structures and increased fibril diameter, validating the model's accuracy in simulating pathological conditions. Conclusion: This novel 3D cutaneous spheroid model, featuring automatic stratification, offers a scalable and biologically relevant platform for studying HTS pathology with a focus on multicellular interactions. By incorporating three cell types, it more accurately replicates the scar's mechanical microenvironment than single-cell-type models. qPCR results highlight its utility in exploring fibrosis mechanisms and its potential value for research. Liang Hua^{1, 2}, Anastasiya Gorkun¹, Kyung W. Yoo¹, Pin Lyu¹, Meimei Wan¹, Weixin Zhao¹, Henry White³, Amy McMichael⁴, Steven R. Feldman⁴, Anthony Atala¹, Xu Han^{1, 3} 1. Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, United States. 2. Dermatology, Shanghai University of Traditional Chinese Medicine Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai, Shanghai, China. 3. CryoCrate LLC, Winston Salem, NC, United States. 4. Wake Forest University School of Medicine, Winston-Salem, NC, United States. Stem Cell Biology, Tissue Regeneration and Wound Healing