Hyaluronan and Derivatives: An In Vitro Multilevel Assessment of Their Potential in Viscosupplementation

Abstract

In this research work, viscosupplements based on linear, derivatized, crosslinked and complexed HA forms were extensively examined, providing data on the hydrodynamic parameters for the water-soluble-HA-fraction, rheology, sensitivity to enzymatic hydrolysis and capacity to modulate specific biomarkers' expression in human pathological chondrocytes and synoviocytes. Soluble HA ranged from 0 to 32 mg/mL and from 150 to 1330 kDa MW. The rheological behavior spanned from purely elastic to viscoelastic, suggesting the diversity of the categories that are suitable for restoring specific/different features of the healthy synovial fluid. The rheological parameters were reduced in a diverse manner upon dilution and hyaluronidases action, indicating different durations of the viscosupplementation effect. Bioactivity was found for all the samples, increasing the expression of different matrix markers (e.g., hyaluronan-synthase); however, the hybrid cooperative complexes performed better in most of the experiments. Hybrid cooperative complexes improved COLII mRNA expression (~12-fold increase vs. CTR), proved the most effective at preserving cell phenotype. In addition, in these models, the HA samples reduced inflammation. IL-6 was down-regulated vs. CTR by linear and chemically modified HA, and especially by hybrid complexes. The results represent the first comprehensive panel of data directly comparing the diverse HA forms for intra-articular injections and provide valuable information for tailoring products' clinical use as well as for designing new, highly performing HA-formulations that can address specific needs.

Keywords: OA biomarkers; human chondrocytes; human sinoviocytes; hyaluronan; hyaluronidase; intra-articular injection; rheology.

Figure 1

Mechanical spectra for the HA-based formulas as commercialized (a–c) and after 1:2 dilution in physiological medium (a’–c’). Measurements were performed at 37 °C.

Figure 2

Tan delta and complex viscosity as function of frequency for the HA-based formulas as commercialized (a–c) and after 1:2 dilution in physiological medium (a’–c’). Measurements were performed at 37 °C.

Figure 3

Degradation during incubation with BTH 10 U/mL for the gels (a). Reduction in complex viscosity during 25 min incubation with BTH 10 U/mL for the 1st group gel (a), the 2nd group gels (b,c) and the 3rd group formula (d). For each gel, the complex viscosity values recorded during incubation with PBS, measured under the same conditions (control), are also reported. Residual complex viscosity (% with respect to the control) at 5, 10, 15, 20, and 25 min of incubation with the enzyme (e). The value at time zero (control) is also reported. All measurements were performed at 37 °C, 2% strain and 2.5 Hz frequency.

Figure 4

Cell image panels in the presence of different HA hydrogels were compared to untreated chondrocytes (a) and synoviocytes (c). Quantification of cell viability was performed using CCK8 staining for both chondrocytes (b) and synoviocytes (d).

Figure 5

Gene expression analyses normalized with respect to pathological untreated cells (CTR) for COLII, HAS-1, MMP-13 (a), AGN and SOX-9 for chondrocytes (c), and HAS-1, MMP-13 (b), AGN and COL-I for synoviocytes (d). In addition, specific inflammation biomarkers (COMP-2, TNF-α, and IL-6) were accomplished for both chondrocytes (e) and synoviocytes (f). Comparative analyses were performed between a linear HA (Hyalubrix^®), two chemically modified HAs (HyMovis^® and Jonexa Hyalastan SGL-80™), and an HA hybrid cooperative complex (Sinovial HL^®). The data show the averages to be ± S.D. The statistical significance was analyzed through one-way ANOVA and the Tukey post hoc test for comparison of a family of 5 estimates: * p < 0.01 vs. untreated-cells (CTR); # p < 0.01 vs. HyMovis^®, § p <0.01 vs. Jonexa Hyalastan SGL-80™; ° p < 0.01 vs. Sinovial HL^®; + p < 0.01 vs. Hyalubrix^®.

Figure 6

Evaluation of COMP-2 and NF-kB protein levels in pathological chondrocytes treated for 48 h with HA-based gels. Densitometric analysis was performed, normalizing COMP-2 and NF-kB protein expression with respect to TUBULIN: * p < 0.05. A t-test compared the significance of each treatment with respect to CTR.

Figure 7

Immunofluorescence staining of COMP-2 (a) in treated and untreated primary human chondrocytes; HAS-1 (b) in treated and untreated primary human synoviocytes. In blue nuclei, actin fibers of cytoskeleton were stained with tritc phalloidin, and fitc-green antibodies were used for COMP-2 and HAS-1, respectively. Arrows indicate COMP-2 and HAS-1 expression in treated and untreated human primary cells. Pictures were from one representative experiment. Magnification = 40×.