Engineered Caf1 Constructs Encoding Laminin-332 Motifs: A Novel Approach for Enhanced Cutaneous Wound Healing

Laminin-332 is a key extracellular matrix (ECM) protein that supports keratinocyte adhesion, migration, and re-epithelialisation during wound healing. Its loss, as in Junctional Epidermolysis Bullosa (JEB) disrupts re-epithelialization, which highlights its therapeutic relevance. However, direct application of full-length laminin or its fragments is limited by poor stability and high production costs. As an alternative, biomaterial scaffolds are being developed. The Capsular Antigen F1 (Caf1) is one such scaffold, offering a stable, customisable platform for presenting bioactive motifs in a more practical format.

To develop stable Caf1 scaffolds displaying laminin-332 motifs and evaluate their therapeutic potential in promoting epithelial repair under both normal and laminin-deficient conditions, with the goal of enabling clinically translatable wound-healing strategies.

Laminin-332-derived peptide motifs were identified through a systematic literature review and ranked using a scoring system. Leading candidates were engineered into Caf1 and expressed in Escherichia coli. Keratinocyte migration, motility, adhesion and pSMAD2 signalling were assessed in vitro using normal human epidermal keratinocytes and LAMA3-knockdown cells cultured on Caf1-coated plates. An ex vivo human skin wound model was used to assess translational relevance: wounded skin explants were treated with Caf1 constructs and cultured at the air-liquid interface for 7 days. Re-epithelialization and epithelial thickness were evaluated by haematoxylin and eosin staining.

Caf1 proteins displaying laminin-332-derived motifs were expressed in E. coli and purified. Among the engineered constructs, Caf1-J3 and Caf1-J4 significantly enhanced keratinocyte migration and single-cell motility in vitro. Combined application further accelerated wound closure, suggesting complementary effects. In LAMA3-deficient keratinocytes, these coatings restored adhesion, promoted actin cytoskeletal organisation, and reduced nuclear pSMAD2 levels. In ex vivo wounded human skin, they also promoted re-epithelialisation and supported homogeneous neo-epidermis formation.

These findings show that short laminin-332-derived motifs, when presented on the Caf1 scaffold, can mimic and partially recover key basement membrane functions in epithelial repair. Caf1-J3 and Caf1-J4 function as minimal bioactive units that restore keratinocyte cell adhesion and modulate key signalling pathways involved in wound healing. This modular protein platform offers a clinically relevant strategy for chronic wounds and ECM-deficient skin, and provides a foundation for next-generation regenerative therapies.