Recombinant fibroin containing the rgd motif enhances limbal mesenchymal stromal cell adhesion and proliferation

Elham Nili ahmadabadi,1,* Neil richardson,2 Damien harkin,3 Shuko suzuki,4 Damien harkin,5

2. School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland
3. School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland, Australia
4. Queensland Eye Institute
5. School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland, Australia

Abstract

Introduction

At the periphery of the cornea, in a region called the limbus, exists a population of stromal progenitor cells termed limbal mesenchymal stromal cells (l-msc). l-msc likely contribute to maintenance of the corneal stroma. moreover, l-mscs have anti-inflammatory and immunomodulatory effects. as such, there is widespread interest in exploiting the properties of l-msc for the treatment of corneal disease. for example, it has been proposed that l-mscs improve wound healing in the cornea by enhancing epithelial cell growth and repair of the stromal tissue. as a consequence, studies have been initiated in our laboratory to optimise techniques for the isolation and cultivation of l-msc. in parallel studies we have demonstrated that silk fibroin (sf) has potential as a scaffold for supporting the growth and implantation of l-msc. in the current study we report an attempt to optimise l-msc culture on recombinant silk fibroin containing the rgd motif (rgd-sf), a recognized ligand for cell attachment. our hypothesis is that l-msc display enhanced attachment and growth on rgf-sf compared to sf thus providing a superior scaffold for clinical use.

Methods

Human l-msc cultures derived from four donors (n=4) were seeded onto either tissue culture plastic, rgd-recombinant fibroin or conventional fibroin for 90 minutes, 6 and 10 days, at a density of 1.5*10⁴ cells/cm². differences in l-msc adhesion and growth were determined photographically and quantified using the picogreen assay (measures dsdna).

Results

At all time points examined, l-mscs displayed evidence of superior attachment and proliferation on rgd-sf coated surfaces compared with control fibroin as indicated by both morphological examination and picogreen assay. furthermore, morphological examination revealed that while l-mscs cultured on conventional fibroin generally formed sparsely scattered clusters, those cultured on rgd-sf were more uniform in adherence to sf.

Conclusion

The results of the current study suggest that rgd-sf offers superior performance as a scaffold material for l-msc cultures compared to conventional fibroin. based on such data we suggest that rgd-sf has potential as a biomaterial for improving strategies for the treatment of corneal disease.

Keywords

Cornea, limbus, msc, stem cell, biomaterial