BACKGROUND: Reconstruction of large eyelid defects remains challenging due to the lack of suitable eyelid tarsus tissue substitutes. We aimed to evaluate a novel bioengineered chitosan scaffold for use as an eyelid tarsus substitute. METHODS: Three-dimensional macroporous chitosan hydrogel scaffold were produced via cryogelation with specific biomechanical properties designed to directly match characteristics of native eyelid tarsus tissue. Scaffolds were characterized by confocal microscopy and tensile mechanical testing. To optimise biocompatibility, human eyelid skin fibroblasts were cultured from biopsy-sized samples of fresh eyelid skin. Immunological and gene expression analysis including specific fibroblast-specific markers were used to determine the rate of fibroblast de-differentiation in vitro and characterize cells cultured. Eyelid skin fibroblasts were then cultured over the chitosan scaffolds and the resultant adhesion and growth of cells were characterized using immunocytochemical staining. RESULTS: The chitosan scaffolds were shown to support the attachment and proliferation of NIH 3T3 mouse fibroblasts and human orbital skin fibroblasts in vitro. Our novel bioengineered chitosan scaffold has demonstrated biomechanical compatibility and has the ability to support human eyelid skin fibroblast growth and proliferation. CONCLUSIONS: This bioengineered tissue has the potential to be used as a tarsus substitute during eyelid reconstruction, offering the opportunity to pre-seed the patient's own cells and represents a truly personalised approach to tissue engineering.