In vitro Scleral Fibroblast Growth on Biomimetic Extracellular Matrices

Abstract

Purpose:: To synthesize and test the biocompatibility of a synthetic extracellular matrix (ECM) that allows independent manipulation of cell adhesion ligand presentation and matrix stiffness. We are exploring this type of biomimetic ECM as an adjunctive treatment for controlling high myopia progression.

Methods:: A synthetic ECM hydrogel system composed of a thermo-responsive semi-interpenetrating polymer network (sIPN) was synthesized by redox radical polymerization. The sIPN consisted of poly(N-isopropylacrylamide-co-acrylic acid) [p(NIPAAm-co-AAc)] crosslinked with an acrylated peptide Gln-Pro-Gln-Gly-Leu-Ala-Lys-NH₂ [(QPQGLAK-NH₂)], with molar ratios of 95:5:0.3 for NIPAAm:AAc:crosslinker. These crosslinks were predominantly cleaved by matrix metalloproteinase-13. The polymer network was interpenetrated by polyacrylic acid-graft-Ac-CGGNGEPRGDTYRAY-NH₂ [p(AAc)-g-RGD] linear chains. The RGD motif provided a binding site for several integrin receptors, thus promoting fibroblast adhesion. sIPNs with 100 uM and 200 uM grafted RGD were made. A parallel plate rheometer was used to determine the complex modulus G* over a frequency range of 0.001-10 Hz. Scleral fibroblasts were isolated for culture from the eyes of 1-wk old White Leghorn chicks and a growth curve assay established to assess scleral fibroblast proliferation. The biocompatibility of sIPNs with 100 uM and 200 uM RGD was assessed in terms of cell counts and cell viability, as determined using Hoescht 33342 and propidium iodide stains. Immunofluorescence staining for collagen type II (mouse anti-chicken collagen II antibody and sheep anti-mouse IgG Rhodamine) was performed.

Results:: The complex modulus G* of the sIPNs was 250±50 Pa, appropriate for an injectable hydrogel that gradually stiffens after implantation. Scleral fibroblast proliferation in culture was relatively rapid, starting at 4x10⁴ cells per 25-cm² on day 1 and stabilizing around 4x10⁶ cells per 25-cm² by day 9. sIPNs with 200uM RGD showed significantly greater cell attachment than sIPNs with 100uM RGD. However, the growth rates did not differ significantly between the 2 sIPNs. Hoechst 33342 and propidium iodide stainings confirmed the viability of cells after seeding. Positive staining for collagen II suggests that some of the scleral fibroblasts were transdifferentiating in vitro.

Conclusions:: We demonstrated that a completely synthetic ECM can support scleral fibroblast attachment and growth, and that the thermo-responsive nature of the matrix allowed for injection and placement, for ophthalmologic applications including control of high myopia.

Keywords: sclera • cell adhesions/cell junctions • myopia