(M1130-04-21) Rheological Adaptation of Hydrogels as Novel Preclinical In Vitro Model Substitutes for Human Vitreous Body

Purpose: Currently, there is still no ideal preclinical in vitro model for simulating intravitreal injections. Several vitreous substitutes based on hydrogels have already been described in the literature, but their physicochemical properties still need to be adjusted. However, this forms a highly relevant basis for a biorelevant preclinical model. The aim of this study was therefore to adjust these hydrogels to the rheological properties of the vitreous body. Prior to this, the respective relaxation times needed to rebuild the structures after sample preparation and device trimming had to be determined. Subsequently, the viscoelastic properties were assessed in amplitude sweeps, and the viscosities over time through rotational experiments.

Methods: As a surrogate for the human vitreous body, fresh porcine eyes were obtained as an animal by-product from a local slaughterhouse. Phosphate-buffered saline (PBS) pH 7.47 formed the basis of the hydrogels. Gellan gum-hyaluronic acid¹ (Gellan-HA, gellan gum: Kelcogel® F from CP Kelco & hyaluronic acid: Caesar & Loretz) and hyaluronic acid-agar² (HA-Agar, agar: Kobe I, Carl Roth) were tested as gel formers. For the rheological measurements, a 25 mm parallel plate system with a measuring distance of 1 mm (MCR 702e MultiDrive, Anton Paar) was used at an elevated temperature of 35 °C. To determine the necessary relaxation time after trimming, an amplitude sweep was performed at a frequency of 1 Hz with a complex shear strain of 0.01% over 60 minutes. The moving average of the loss factors from the four values before and after each measured point was calculated, and a maximum deviation of 5% from the last moving average value was defined as the limit. Then, an amplitude sweep was conducted at a frequency of 1 Hz with a logarithmically increasing complex shear strain ranging from 0.001% to 100% and also a viscosity test at a low shear rate of 0.5 s^-1.

Results: Figure 1 shows the maximum relaxation times required for the respective gels and porcine vitreous body. Gels with lower concentrations exhibit longer relaxation times, indicating that their structures are more severely disrupted by the removal of the sample and trimming of the rheometer, thus requiring more time to rebuild afterward. The porcine vitreous body requires one of the longest relaxation times, which can be attributed to the additional extraction from the eye. In Figure 2, the viscosities of the gels and the porcine vitreous body are depicted. All exhibit shear-destructive behavior, but the viscosities of the gel concentrations described in the literature (dark colors) consistently differ a lot from those of the porcine vitreous body. Adjusting the gels while maintaining the initial ratios of gel former to hyaluronic acid shows a significant improvement (intermediate colors). A closer examination of the results after 12 seconds, where the porcine vitreous body achieved the lowest standard deviation, reveals that Gellan-HA with 1662 mPa*s and HA-Agar with 1808 mPa*s are very close to the reference value of 1736 mPa*s. Furthermore, comparing these gels in Figure 3 regarding the loss factors, it is evident that this adjustment of gel former concentrations did not improve it. The loss factors in the linear viscoelastic range (LVER) are still lower than those of the porcine vitreous body. Subsequently, attempts were made to further optimize the loss factor of the gels by varying the ratio of gellan gum or agar to hyaluronic acid, with the loss factor increasing as the hyaluronic acid content increased. At the newly optimized gel concentrations of Gellan-HA and HA-Agar, the loss factor for Gellan-HA was adjusted to 0.198 and for HA-Agar to 0.212 to match that of the porcine vitreous body, which is 0.206. The LVER for the optimized HA-Agar gel is also the highest, reaching up to a shear deformation of approximately 10%, indicating the most stable gel structure. When comparing the viscosities of these gels, they still remain very close to those of the porcine vitreous body. However, it is noticeable that for Gellan-HA 0.0340 & 0.2635%, the gellan gum concentration had to be increased compared to the previously viscosity-adjusted concentration, as the increasing amount of hyaluronic acid seems to interfere with the gel formation. In contrast, for HA-Agar 0.2205 & 0.0900%, the agar amount had to be reduced, as here the hyaluronic acid appears to enhance gel formation.

Conclusion: In conclusion, it can be noted that the gels described in the literature, Gellan-HA and HA-Agar, were successfully adjusted to match the rheological properties of the porcine vitreous body. The final optimized concentrations, with 0.0340 & 0.2635 % for Gellan-HA and 0.2205 & 0.0900 % for HA-Agar, exhibit comparable loss factors in the viscoelastic range as well as similar viscosities after a 12 seconds measurement period. To establish the ideal in vitro vitreous body substitute, the diffusion properties should now also be determined. This will form the optimized basis for a biorelevant preclinical vitreous body model.

References: 1. Suri, S. and Banerjee, R. In vitro evaluation of in situ gels as short term vitreous substitutes. Journal of Biomedical Materials Research Part A: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials. 79. 650-664. (2006)
2. Awwad, S.; Lockwood, A.; Brocchini, S. and Khaw, P. T. The PK-Eye: A Novel In Vitro Ocular Flow Model for Use in Preclinical Drug Development. J Pharm Sci. 104. 3330-42. (2015)

Acknowledgements: The authors thank Theo Keinhörster Großschlachterei mit Fleischhandel e.K. for supply of the fresh porcine eyes and CP Kelco for the gellan gum powder.


Figure 1. Maximum relaxation times of porcine vitreous body, gellan gum-hyaluronic acid (Gellan-HA) and hyaluronic acid-agar gels (HA-Agar) at 35 °C, n = 6 (vitreous body) or 3 (hydrogels).


Figure 2. Viscosity tests for porcine vitreous body, gellan gum-hyaluronic acid (Gellan-HA) and hyaluronic acid-agar gels (HA-Agar) at a shear rate of 0.5 s^-1 and 35 °C, n = 6 (vitreous body) or 3 (hydrogels), mean ± sd.


Figure 3.Amplitude sweep for porcine vitreous body, gellan gum-hyaluronic acid (Gellan-HA) and hyaluronic acid-agar gels (HA-Agar) at a frequenzy of 1 Hz and 35 °C, n = 6 (vitreous body) or 3 (hydrogels), mean ± sd.