Comparison the effects of chitosan and hyaluronic acid-based thermally sensitive hydrogels containing rosuvastatin on human osteoblast-like MG-63 cells

Abstract

Background and purpose: Bone regeneration can be accelerated by localized delivery of statins. Here, we aimed to evaluate the effect of two thermosensitive hydrogels containing rosuvastatin (RSV) on proliferation and differentiation of human osteoblast-like MG-63 cells.

Experimental approach: Firstly, chitosan (CTS)/glycerophosphate (GP)/gelatin (G) thermosensitive hydrogel was prepared and characterized based on rheological properties, in vitro erosion, and release pattern of RSV and then the optimized mixture was loaded with nanoparticles containing RSV(NRSV). Secondly, the effect of NRSV-embedded in CTS/GP/G on cell viability, alkaline phosphate activity, and cell calcification was evaluated using MG-63 cells and compared with RSV-embedded into hyaluronic acid (HA)/Pluronic^® F127 (PF127) hydrogel.

Findings / results: CTS/GP mixtures with 1 and 1.5 % gelatin existing in solution with low viscosity at 4 °C were solidified at 32-34 °C while the mixture containing 2% gelatin was jellified at room temperature. The gelation times of CTS/GP/G with 1 and 1.5% gelatin were 72 and 44 s, respectively. The hydrogel containing 3% w/v NRSV was also converted to a semisolid upon increasing the temperature to 33-36 °C. Due to the higher gel strength of CTS/GP/G compared to HA/PF127 hydrogel, the release rate of RSV from the NRSV-embedded CTS/GP/G hydrogel was significantly slower than that of HA/PF127 system. As revealed by alkaline phosphatase and mineralization assays, NRSV-embedded in CTS/GP/G hydrogel had the most promotive effect on differentiation of osteoblasts among other mixtures.

Conclusion and implication: NRSV-embedded in CTS/GP/G hydrogel could be efficiently used in the future for bone defects such as osteoporosis and bone fractures.

Keywords: Rosuvastatin; Thermosensitive hydrogel; Tissue engineering.

Fig. 1

(A) Visual observation of CTS/GP/G hydrogel at room temperature (left) and at 37 °C (right), (B) viscosity vs temperature of CTS/GP mixture with 1.5 % G, and (C) viscosity vs time of CTS/GP solutions with 1 and 1.5% G. CTS, chitosan; GP, glycerophosphate; G, gelatin.

Fig. 2

The mass erosion behavior of chitosan/glycerophosphate/gelatin hydrogel system. Data represent the mean ± SD, n = 3.

Fig. 3

In vitro release profiles of RSV from NRSV-embedded in HA/PF127 and NRSV-embedded in CTS/GP/G hydrogels. Data represent the mean ± SD, n = 3. NRSV, Nanoparticles containing rosuvastatin; HA, hyaluronic acid; PF127, Pluronic^® F127; CTS, chitosan; GP, glycerophosphate; G, gelatin.

Fig. 4

(A) In vitro cytotoxicity of the different formulations against human osteoblast-like MG-63 cells and (B) ALK activity of MG-63 cells after 48 and 72 h of culture with NRSV, CTS/CS, NRSV-embedded in CTS/GP/G or CTS/GP/G. Data represent the mean ± SD, n = 3. *P ≤ 0.05 and **P ≤ 0.01 indicate significant differences compared with blank CTS/CS; #P ≤ 0.05 and ^###P ≤ 0.001 show significant differences between defined groups. NRSV, Nanoparticles containing rosuvastatin; HA, hyaluronic acid; PF127, Pluronic® F127; CTS, chitosan; GP, glycerophosphate; G, gelatin; ALK, alkaline phosphatase.

Fig. 5

Photographs of mineral deposition visualized by Alizarin Red staining. Cells were cultured for 48 h with (A) untreated cells, (B) CTS/CS, (C) NRSV, (D) NRSV-embedded CTS/GP/G, (E) NRSV-embedded HA/PF127, (F) CTS/GP/G, and (G) HA/PF127. NRSV, Nanoparticles containing rosuvastatin; HA, hyaluronic acid; PF127, Pluronic^® F127; CTS, chitosan; GP, glycerophosphate; G, gelatin.