Date of Award
Doctor of Philosophy (PhD)
The vitreous humor of the eye is a biological hydrogel principally composed of fibrillary collagen interspersed with semi-flexible polyelectrolyte, hyaluronic acid (HA). Certain pathological conditions necessitate its removal and replacement. Current vitreous substitutes, such as silicone oils and perfluorocarbons, are hydrophobic, do not resemble the properties of the vitreous, and have known complications. An ideal vitreous substitute should have properties of the natural vitreous, perform its functions, and be biocompatible in the eye. Inspired by the structure and composition of the natural vitreous, we used bio-mimicry to develop an injectable two-component hydrogel. The hydrogel is composed of a fibrillary gellan, an analogue of collagen, and a semi-flexible polyelectrolyte poly[methacrylamide-co-(methacrylic acid)], an analogue of hyaluronic acid, both endowed with thiol cross-linkers for reversible covalent linkage. The gellan, in the polymeric mixture, undergoes coil-helix transition near physiological temperature, enabling instantaneous in situ physical gelation of the solution. The thiol cross-linkers that later oxidize to disulfides under physiological conditions, make the hydrogel non-absorbable, non-degradable, and reversible, for facile removal if needed.
We used response surface methodology to investigate the structure-property relationships of eleven two-component hydrogels, and identified two hydrogel formulations that match the primary properties of the vitreous. We determined how each component of the hydrogel affects their optical, mechanical, sol-gel transition temperature, and osmotic swelling properties. All the hydrogels were transparent to visible light, with density and refractive indexes nearly equivalent to those of the natural vitreous. The shear storage moduli of the hydrogels, at 1Hz, ranged from 3 to 358 Pa, and the sol-gel transition temperatures, from 35.5 to 43 °C. In addition, as expected, all the hydrogels swelled in physiological solutions. Interestingly, we discovered that the relatively large swelling capacity of the semi-flexible ionic copolymer was significantly restricted by the minimally swellable fibrillary gellan network. The tightly swollen gel of two dissimilar networks produced Donnan osmotic swelling pressure in physiological solutions, which is also the driving force for re-attachment of the retina. Insights from the biomimetic nature of the gel, led us to propose that the natural vitreous also exhibits controlled swelling, where ionic HA’s swelling capacity is restricted by fibrillary collagen. The Donnan swelling pressure produced by the tightly swollen vitreous gel maintains the delicate internal structure of the eye, and perhaps plays a critical role during the ocular development.
We evaluated the biocompatibility of the two optimized formulations of the hydrogels on different cell lines, and in rabbits. Both hydrogels were found to be biocompatible on primary porcine retinal pigment epithelial cells, human retinal pigment epithelial cells, and fibroblast (3T3/NIH) cells, by electric cell-substrate impedance sensing system. Furthermore, the hydrogels did not impair tight junction formation or affect proliferation of the cells. The hydrogels were also non-degradable in enzymatic solutions and in contact with ocular cell line for four weeks. Judged against silicone oil, a clinically-accepted vitreous replacement, both hydrogel formulations were biocompatible in rabbits for 30 days. Both hydrogels maintained optical clarity, physiological intra-ocular pressure, and intact retinal layers that displayed normal electroretinographs. In two cases of the iatrogenic retinal tear, the hydrogels reattached the retina by producing osmotic swelling pressure. The hydrogels also maintained the low oxygen environment, compared to silicone oil, in the rabbit’s vitreous cavity for 30 days post-surgery.
In conclusion, the two hydrogels reattach the retina via a unique mechanism of osmotic swelling pressure. They overcome the limitations of silicone oil with comparable in-vivo biocompatibility, and merit further evaluations as an artificial vitreous. In addition, the ability to control the mechanical and swelling properties of the two-component hydrogels over a wide range suggests their utility as biomimetic replacements of other soft tissues, such as cornea, nucleus pulposus, and cartilage.
Palghat Ramachandran, Daniel Giammar, Marcus Foston, Srikanth Singamaneni,