The amelioration of cartilage degeneration by photo-crosslinked GelHA hydrogel and crizotinib encapsulated chitosan microspheres

Abstract

The present study aimed to investigate the synergistic therapeutic effect of decreaseing cartilage angiogenesis via exposure to crizotinib encapsulated by chitosan microspheres and photo-crosslinked hydrogel, with the goal of evaluating crizotinib as a treatment for osteoarthritis. First, we developed and evaluated the characteristics of hydrogels and chitosan microspheres. Next, we measured the effect of crizotinib on the cartilage degeneration induced by interleukin-1β in chondrocytes. Crizotinib ameliorated the pathological changes induced by interleukin-1β via its anti-angiogenesis function. In addition, we surgically induced osteoarthritis in mice, which were then injected intra-articularly with crizotinib-loaded biomaterials. Cartilage matrix degradation, expression of vascular endothelial growth factor and extracellular signal-regulated kinases 1/2 were evaluated after surgery. Treatment with the combination of crizotinib-loaded biomaterials retarded the progression of surgically induced osteoarthritis. Crizotinib ameliorated cartilage matrix degradation by promoting anti-angiogenesis and impeding extracellular signal-regulated kinases 1/2 signaling pathway. Our results demonstrate that the combination of photo-crosslinked hydrogel and crizotinib-loaded chitosan microspheres might represent a promising strategy for osteoarthritis treatment.

Keywords: angiogenesis; hydrogel; osteoarthritis.

Figure 1. Preparation and characterization of GelHA hydrogels

(A) Molecule structures and synthesis of polymer precursors of gelatin methacrylate (GelMA) and hyaluronic acid methacrylate (HAMA) hydrogels. (B) Microscopic structure of the hydrogels (low and high magnification). (C) Swelling kinetics of the hydrogels. n = 3, *p < 0.05 (one way ANOVA). (D) In vitro degradation profile of the hydrogels. n = 3, *p < 0.05 (one way ANOVA). (E) The compressive moduli for the hydrogels. n = 3, *p < 0.05 (one way ANOVA). GelHA hydrogel, hybrid hydrogel with the two components of GelMA and HAMA.

Figure 2. In vitro crizotinib release in chitosan microspheres

(A) Scanning electron micrographs of crizotinib-loaded chitosan microspheres. (B) Fourier transform infrared (FTIR) spectroscopy for chitosan microspheres. (C) Absorption curve of crizotinib. (D) Standard curve of crizotinib. (E) Controlled release profile of chitosan microspheres.

Figure 3. Expression of VEGF in cartilage samples from human patients with OA and mice with surgically induced OA

(A) Immunofluorescence staining and quantification of VEGF-positive cells of VEGF in normal and OA human cartilage samples. Scale bars = 100 μm. (B) Immunofluorescence staining and quantification of VEGF-positive cells of VEGF in normal and OA mouse cartilage samples. Scale bars = 100 μm. n = 3, *p < 0.05 (Student's t-test). (C) Western blot analysis of VEGF level in human OA and normal cartilage (cropped blots are displayed). n = 3, *p < 0.05 (Student's t-test). (D) Western blot analysis of VEGF level when human OA chondrocytes treated with IL1β (10 ng/mL) and measurement of relative density of VEGF band (cropped blots are displayed). n = 3, *p < 0.05 (Student's t-test).

Figure 4. Effect of crizotinib in IL1-β-stimulated chondrocytes

(A) The effects of crizotinib (10 μM) on mRNA transcript levels of MMP13, COL2A1, ADAMTS-5, and aggrecan after treatment with IL1β (10 ng/mL) for 48 h. n = 3, *p < 0.05 (one way ANOVA). (B) Western blot analysis of MMP13, COL2A1, ADAMTS-5, aggrecan, and VEGF protein expression levels after treatment with crizotinib [with or without IL1β (10 ng/mL) stimulation, cropped blots are displayed]. (C) Immunocytochemistry detection of VEGF in chondrocytes. Scale bars = 30 μm. (D) Effect of crizotinib on chondrocytes calcification visualised by Alizarin Red staining. Scale bars = 30 μm. n = 3, *p < 0.05 (Student's t-test).

Figure 5. Effect of crizotinib on cartilage matrix degeneration in an ex vivo model

(A, B) Crizotinib significantly attenuated loss of proteoglycan in cartilage induced by IL-1β, assayed by Safranin O staining. Cartilage samples were postmortem from human subjects with no history of OA and cultured with 10 ng/mL IL-1β, in presence or absence of 10 μM crizotinib. Scale bars=100 μm. n = 3, *p < 0.05 (one way ANOVA). (C) The effects of crizotinib on mRNA transcript levels of MMP13, COL2A1, ADAMTS-5, and aggrecan. n = 3, *p < 0.05 (one way ANOVA). (D, E) Immunocytochemistry was performed to assess expression of VEGF. Scale bars=100 μm. n = 3, *p < 0.05 (one way ANOVA).

Figure 6. Efficacy of the combination of crizotinib-loaded chitosan microspheres and GelHA hydrogel as a treatment for OA

Eight-week-old male C57BL/6 mice were used. Safranin-O staining of cartilage samples 4 (A) and 8 weeks (B) after OA induction. Scale bars = 100 μm. (C, D) OARSI scores of samples 4 and 8 weeks after OA induction. n = 8, *p < 0.05; **p < 0.01 (Kruskal-Wallis ANOVA). Group I, OA mice; group II, OA mice treated with GelHA hydrogel and chitosan microspheres (without crizotinib); group III, OA mice treated with crizotinib alone; group IV, OA mice treated with crizotinib-loaded chitosan microspheres and GelHA hydrogel.

Figure 7. Effect of crizotinib on cartilage matrix degradation in vivo

(A) Immunohistochemistry of MMP13, COL2A1, ADAMTS-5, and aggrecan (4 weeks). Scale bars = 100 μm. (B, C, D, E) Quantification of MMP13-positive (B), COL2A1-positive (C), ADAMTS-5-positive (D) and aggrecan-positive-cells (E) within cartilage samples at 4 weeks post surgery. n = 8, *p < 0.05, **p < 0.01 (one way ANOVA). (F) Immunohistochemistry of MMP13, COL2A1, ADAMTS-5, and aggrecan (8 weeks). Scale bars = 100 μm. (G, H, I, J) Quantification of MMP13-positive (G), COL2A1-positive (H), ADAMTS-5-positive (I) and aggrecan-positive-cells (J) within cartilage samples at 8 weeks post surgery. n = 8, *p < 0.05, **p < 0.01 (one way ANOVA).

Figure 8. Effect of crizotinib on chondrocyte angiogenesis in vivo

Immunocytochemistry was performed for VEGF after 4 (A, B) or 8 weeks (C, D). Scale bars = 100 μm. n = 8. *p < 0.05, **p < 0.01 (one way ANOVA).

Figure 9. Mechanism of crizotinib in decreasing cartilage matrix degradation

(A) Immunocytochemistry detection of ERK1/2 and p-ERK1/2 in mouse primary chondrocytes after stimulating with IL1β (10 ng/mL) with or without crizotinib (10 μM) for 1 h. Scale bars = 30 μm. (B) Western blot analysis of ERK1/2 signalling in mouse primary chondrocytes after IL1β treatment (cropped blots are displayed). (C) Immunocytochemistry was performed for ERK1/2 and p-ERK1/2 in OA mouse cartilage treated with or without crizotinib for 8 weeks. Scale bars = 100 μm. (D) A proposed model for the role of crizotinib in osteoarthritis treatment.