(C-112) Assessing Role of Keratin Additive in Fibroblast Adhesion and Infiltration

Introduction:: Dermal adhesion and ingrowth are essential processes in tissue engineering, such that scaffold attachment to the host tissue/wound bed ensures stability, nutrient exchange, and reduced infection potential. Additionally, ingrowth of dermal tissue within a scaffold supports native tissue infiltration, leading to vascularization, extracellular matrix (ECM) deposition, host tissue integration, and tissue maturation. Both processes are crucial for creating successful tissue-engineered constructs that improve and promote tissue functionality and repair/regeneration, respectively. There are various clinical scenarios in which a metal implant is meant to interface directly with dermal tissue (e.g., osseointegration and dental implants). Here, effective dermal adhesion ensures a secure connection between a metal implant and the surrounding tissue; this minimizes the risk of implant failure by establishing a functional interface for improved load distribution and reduced migration or dislocation. Despite this need, dermal adhesion remains complex, and additional strategies to improve tissue/implant integration should be explored.

Keratin, a fibrous protein found in the epidermis, has been shown to promote tissue attachment to scaffolding materials, providing stability, increasing nutrient exchange, and reducing infection potential. Therefore, the incorporation of keratin as an additive holds significant promise for enhancing dermal adhesion and ingrowth. By leveraging the observed benefits of keratin, we hypothesize that tissue-engineered scaffolds with varying concentrations of keratin will result in improved dermal adhesion and functionality, promoting more efficient overall tissue repair and regeneration.

Materials and Methods:: This research aims to demonstrate the efficacy of the additive keratin for inducing two critical tissue behaviors: infiltration (macroporous cryogels) and adhesion (microporous electrospun meshes). Chitosan-gelatin (1:4) cryogel and 5% polycaprolactone (PCL; wt/v%) electrospun scaffolds were both doped with varying concentrations of keratin (0, 1, 2, 3 wt%). Initial scaffold formation verification included scanning electron microscopy (SEM) and pore/fiber analysis, as applicable. All scaffolds (+/- keratin) were then seeded with human dermal fibroblasts (HDF; 50,000 cells/scaffold). Cryogel HDF infiltration was assessed via fixed scaffold sections imaged on days 1, 3, 7, and 14 via confocal imaging, with MTS assay providing proliferation quantification. Similarly, HDF adhesion to the electrospun scaffolds was assessed via an adhesion strength assay on days 1, 3, 7, and 14.

Results, Conclusions, and Discussions:: Initial analysis assessed the changes in scaffold formation based on the inclusion of increasing concentrations of keratin (0, 1, 2, 3 wt%). In PCL electrospun scaffolds, the average pore area decreased, while overall structure homogeneity increased, with the addition of keratin additives at all concentrations (Figure 1). Changes in the porous matrix as a result of adding keratin, however, were not found to be significantly affected compared to the 0% scaffold, indicating usability of keratin meshes for cell studies (Figure 1A-D; P < 0.05). Further, initial data demonstrates fibroblast cellular compatibility with both PCL electrospun meshes and chitosan/gelatin cryogel scaffolds, with ongoing work quantifying changes in adhesion with the addition of varying concentration so keratin. Cryogels were also successfully formed with increasing amounts of keratin additive, with ongoing work quantifying changes in pore sizing, and cellular compatibility.

Ultimately, the addition of keratin additive to varying forms of tissue-engineered polymer scaffolds (i.e., cryogel and electrospun scaffold) has the potential to advance the biomaterials field and dermal regeneration. Overall, the addition of keratin to PCL electrospun scaffolds resulted in a more uniform porous mesh, making the keratin-enhanced dermal scaffolds optimal for fibroblast cellular adhesion. Similar results were noted with cryogel scaffolds, where both types of scaffolds provide regenerative advantages in dermal environments. This initial data also demonstrates cellular compatibility, regardless of keratin concentration, leading to potentially improved outcomes in clinical translation of these scaffolds at implant interface. The outcomes of this research will contribute valuable insights into the effectiveness of keratin additives in dermal tissue engineering applications, especially as it relates to improved skin adhesion and infiltration.

Acknowledgements (Optional): : NH BioMade is funded by NSF Award #IIA 1757371 administered by NH EPSCoR at UNH
We would like to thank Sreejith Panicker for electrospinning training and Daniel Cullen for SEM use and training

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