Dendritic Polyglycerol Sulfate Hydrogels as a Potential Viscosupplement for Treatment of Osteoarthritis

Abstract

The aim of this thesis was to investigate the potential of a fully synthetic, slowly degradable, heparin sulfate mimetic hydrogel as an alternative viscosupplement for OA management and thereby compare it to the current standard viscosupplement HA. A previous study revealed a short half-life of HA, ranging from half a day up to 9 days in vivo.[65] To avoid several injections, which may incur higher costs and infection risks, a fully synthetic dendritic polyglycerol sulfate (dPGS) hydrogel was evaluated for its bioorthogonality. The rheological properties of this slow-degradable hydrogel were then investigated to determine a suitable concentration for intra-articular injections that mimicked HA in terms of its viscoelastic and mechanical properties. Therefore, different concentrations of dPGS ranging from 3.6 to 4.8 wt% were investigated by means of oscillating and flow rheology, thereby yielding storage (G') and loss modulus (G''), as well as yield stress and shear viscosity. Additionally, blends of commercially available HAs, which varied in respect to their molecular weight, were used as references. As a result, a pronounced coupling of the molecular weight and the rheological properties for the HAs was observed. The zero shear viscosity of the studied HAs ranged between 5 and 1600 Pa⋅s, depending strongly on the molecular weight. Besides, all four HA samples exhibited pronounced shear thinning behavior. Furthermore, the dPGS hydrogel formed more compact networks with increasing concentrations. From a broader comparison, the current findings suggest that an overall polymer concentration of 4.0 wt% dPGS has viscoelastic properties that are comparable to HA in the medically relevant frequency range. The third part of the thesis was focused on the evaluation of dPGS effects on normal and OA-like tissue-engineered cartilage. To overcome the low availability of human primary tissue and high costs of animal models an established in vitro OA model has been used. It is based on porcine cartilage sources and offers a high-throughput analysis of potential active substances in a reproducible and very well characterized approach under standardized conditions.[140, 144] In this model, micromass cultures were treated with 2.5 wt% dPGS hydrogel for 7 days under normal and OA conditions (treated with TNF-α). Live/dead staining of micromasses revealed a majority of viable cells embedded in ECM after 7 days of treatment with the hydrogel in normal and OA conditions. This confirmed previous findings and suggested that dPGS was not harmful for different cell types and even in vivo.[145] Safranin-O staining demonstrated a typical depletion of GAGs in OA-like micromasses but not in the presence of the dPGS hydrogel. There was no distinct difference in immunolabeling for type II collagen. The microarray data showed that rheumatoid arthritis and TNF signaling pathways were downregulated in hydrogel-treated OA-like micromasses in comparison to non-treated OA-like micromasses. Furthermore, the dPGS hydrogel alone did not affect genes related to OA such as ANPEP, COMP, CXCL12, COX2, and TNFSF10, but it could prevent their regulation caused by TNF-α. These findings proved the potential of this hydrogel to prevent the development of TNF-α-induced OA with regard to PG loss and TNF-α-induced expression pattern without additional signs of differentiation and inflammation. In the fourth part of this work, the HA-related modifications were investigated on cellular and molecular level in the same in vitro system to serve as a control for comparisons with the dPGS hydrogel. The data showed no inhibiting or activating effect of HA on normal or OA-like tissue-engineered cartilage on cellular level. Microarray data demonstrated a minor impact of HA on gene expression level. The upregulation of VEGFA and ANKRD37 genes confirmed the chondroprotective potential of HA. It could regulate the cartilage anabolism by stabilizing the chondrocyte phenotype in pathological conditions. In conclusion, the evaluation of the dPGS hydrogel showed that it is a potential alternative for HA as an intra-articular injectable lubricant for osteoarthritis. Moreover, in contrast to HA, dPGS can prevent the development of TNF-α-induced OA with regard to proteoglycan loss and TNF-α-induced expression pattern. Although interactions of dPGS-hydrogels with biological systems have been elucidated to a certain extent, still a lot of open questions remain, especially concerning the in vivo effect on synovial joints. To follow up these promising results, further investigation needs to be performed in animal models. In particular, the localization of this hydrogel in the synovial joint should be further investigated by fluorescent dye conjugation and its anti-inflammatory properties by measuring the related cytokine ratios in the synovial fluid. Since it is known that hydrogels can be used as a delivery system, this hydrogel can also be further optimized with biologics to trigger in situ regeneration.