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Mariana Garcia, Amit K. Jha, Kevin E. Healy, Christine F. Wildsoet; Optimization Of A Synthetic Biomimetic Hydrogel Intended For Myopia Control Through Scleral Regeneration. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3459.
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© ARVO (1962-2015); The Authors (2016-present)
To optimize the formulation of a synthetic, injectable biomimetic matrix for growing mammalian scleral fibroblasts, with the ultimate goal of developing a material for rehabilitating the myopic sclera. This matrix is a thermoresponsive enzymatically-degradable semi-interpenetrating polymer network (edsIPN) cleavable by native matrix metalloproteinases. The semi-interpenetrating chain is grafted with RGD-containing 15-amino acid sequences derived from bone sialoprotein (bsp-RGD(15)), which serve as integrin binding sites.
A series of edsIPNs was synthesized via redox radical addition polymerization, varying in crosslinker concentration from 1-4 mg/ml, and in bsp-RGD(15) concentration, from 0-210 μM. The complex modulus of each formulation was measured with parallel plate rheology, and their biocompatibility examined using scleral fibroblasts isolated from a 4 day-old pigmented guinea pig, which were seeded on the edsIPNs and cultured for 7 days. Cell viability was qualitatively assessed with CalceinAM and Ethidium homodimer (Invitrogen), and cell morphology was further examined by staining the actin fibers with rhodamine-conjugated phalloidin. Cell proliferation rates were measured with Alamar Blue (AbDserotec).
Mechanical characterization: The edsIPNs underwent phase transitions at ~33 oC. Moduli increased with increasing crosslinker concentration (1 to 4 mg/ml), ranging from ~70 Pa to ~1 kPa at 37 oC. Cell viability, morphology & proliferation: Cells seeded on 0 μM bsp-RGD(15) gels formed clumps and retained a rounded shape, while cells on gels with 105 and 210 μM bsp-RGD(15) spread well and displayed stable cytoskeletons. The same gels lead to better proliferation and morphology with increasing gel stiffness. Gels with an intermediate concentration (105 μM) of bsp-RGD(15) supported the highest number of viable cells.
Stiffer gels with an intermediate concentration of bsp-RGD(15) yielded optimal cell proliferation and spreading. An intermediate crosslinker concentration (2 mg/ml), satisfies our requirement that the gel be injectable while supporting good cell adhesion and proliferation.
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