Intra-articular delivery of sinomenium encapsulated by chitosan microspheres and photo-crosslinked GelMA hydrogel ameliorates osteoarthritis by effectively regulating autophagy
Introduction
Osteoarthritis (OA) is a destructive joint disease that leads to degeneration of cartilage and changes in the subchondral bone and synovium, followed by bone damage and morphological changes [1]. Age, genetic predisposition, hereditary factors, obesity, mechanical injuries, and joint trauma are risk factors of OA [2], [3]. Structure-modifying medications and nutraceuticals may be effective therapeutic agents for osteoarthritis and merit further investigation [4].
Autophagy is a lysosomal degradation pathway and a homeostatic cellular mechanism that is essential for survival, differentiation, development, and homeostasis [5]. Meanwhile, autophagy is a protective mechanism in normal cartilage [6]. Reduced expression of autophagy regulators was observed in pathological cartilage in humans and mice [7]. Activation of autophagy by rapamycin treatment reduced the severity of OA in experimental models [8]. Therefore, autophagy activation may be a novel therapeutic target for OA treatments. Microtubule-associated protein 1A/1B-light chain 3 (LC3) is a soluble protein that is distributed ubiquitously in mammalian tissues and cultured cells and is used as a specific marker to monitor autophagy [9]. During autophagy, LC3 is recruited to the autophagosomal membrane. When autophagy is induced, LC3-I is conjugated to phosphatidylethanolamine to form LC3-II, which reflects autophagic activity [9], [10].
Sinomenium (9α,13α,14α)-7,8-didehydro-4-hydroxy-3,7-dimethoxy-17-methyl-morphinane-6-one, SIN) is a natural alkaloid extracted from the Chinese medicinal plant Sinomenium acutum. SIN has several pharmacological effects, including anti-rheumatism and immunomodulation [11]. The hydrochloride salt of SIN is widely used in the clinic as a treatment for patients with rheumatoid arthritis (RA) [12]. SIN has the protective capacity to antagonize cartilage degradation [13], and it reduces protein levels of the cartilage degradation marker matrix metallopeptidase 13 (MMP-13, also known as collagenase 3) in rats and blocks the pathogenesis of collagen-induced arthritis via NF-κB signaling and down-regulates MMP13 expression [14], [15]. In addition, by regulating autophagy in animal models of sepsis, SIN improved survival, reduced organ damage, and attenuated the release of inflammatory cytokines [16]. These clues suggest that SIN protects chondrocytes by facilitating autophagy and preventing cartilage degradation. However, few investigations have been carried out to evaluate the therapeutic efficacy of SIN as a treatment for patients with OA.
Chitosan microspheres (CMs) are an efficient biopolymeric drug delivery system capable of delivering therapeutic agents in a controlled and/or sustained manner. CMs are characterized by biocompatibility, nontoxicity, a lack of allergenicity, and biodegradability, all of which have contributed to their successful utilization for site-specific drug delivery [17]. In this study, gelatin methacrylate (GelMA) hydrogel was used as a vehicle to facilitate intra-articular injection of chitosan. Compared with other hydrogels, GelMA crosslinks, when exposed to light irradiation to form hydrogel with tunable physical properties, have essential properties resembling native extracellular matrix, and show promise for cartilage tissue engineering applications [18], [19]. However, thus far, no investigation has been carried out to evaluate its therapeutic efficacy in animal models of OA, making its suitability for this purpose uncertain. In this study, we report the results of experiments in which GelMA hydrogels containing chitosan microspheres encapsulating SIN were injected into mouse joints, with the goal of producing sustained SIN release and ameliorated OA changes.
To assess the therapeutic potential of microsphere-encapsulated SIN as a treatment for patients with OA, we developed and evaluated the characteristics of a photo-crosslinked GelMA hydrogel, after which chitosan microspheres were utilized to produce controlled SIN release. Finally, we investigated the effects of SIN on OA cartilage degeneration. We hypothesized that the combination of chitosan microsphere-encapsulated SIN (CM-SIN) with our newly developed photo-crosslinked GelMA hydrogel would synergistically prevent the progression of degenerative changes in surgically induced OA.
Section snippets
Materials
Gelatin (Type A, 300 bloom from porcine skin) and methacrylic anhydride (MA) were purchased from Sigma–Aldrich (St. Louis, MO, USA). All other chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA) unless specifically mentioned.
Methacrylated gelatin synthesis
Type A porcine skin gelatin was mixed into phosphate-buffered saline (PBS) at 60 °C and stirred until fully dissolved at a concentration of 10% (w/v). MA was added to the gelatin solution at a rate of 0.5 mL/min until the target volume was reached, stirred at
Preparation and characterization of GelMA hydrogels and chitosan microspheres
The GelMA hydrogels were formed by exposing the prepolymer to UV light for 2 min. Scanning electron microscopy of the gel cross-sections showed the porosity of the gels. Disordered pores of 50–150 μm were uniformly distributed throughout the gel. The GelMA hydrogels showed a loose, honeycomb-like structure (Fig. 1c). The swelling ratios of the hydrogels in PBS are shown in Fig. 1d. The hydrogels exhibited fast swelling and reached equilibrium within 36 h. In tests of the degradation profile of
Discussion
In this study, we developed a new strategy for treating OA, in which CM-SIN combined with GelMA hydrogel were applied. The three major findings of this study were as follows: first, autophagy was down-regulated in cartilage samples from mice with surgically induced OA and human patients with OA; second, SIN ameliorated cartilage degeneration through autophagy in normal chondrocytes; third, the combination of CM-SIN and GelMA hydrogel slowed the progression of degenerative changes in OA.
The
Conclusion
In this study, we demonstrated that the combination of SIN encapsulated by CMs and photo-crosslinked GelMA hydrogel ameliorated the progression of surgically-induced osteoarthritis. These results demonstrate the importance of autophagy as a therapeutic target for OA prevention and treatment.
Financial interests
The authors hereby declare that no conflict of interest exists.
Acknowledgments
The study is sponsored by National Nature Science Fund of China (81171739, 81101378, 81271971, 81271972, 31270997), Natural Science Fund of Zhejiang Province (Y2110372), funds of science and technology department of Zhejiang Province (2009C03014-1), and Zhejiang provincial program for the cultivation of high-level innovative health talents.
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Authors contributed equally to this work.