Ectopic chondrogenesis of nude mouse induced by nano gene delivery enhanced tissue engineering technology

Abstract

Background: Many techniques and methods have been used clinically to relieve pain from cartilage repair, but the long-term effect is still unsatisfactory. Purpose: The objective of this study was to form an artificial chondroid tissue gene enhanced tissue engineering system to repair cartilage defects via nanosized liposomes. Methods: Cationic nanosized liposomes were prepared and characterized using transmission electron microscope (TEM) and dynamic laser light scattering (DLS). The rat mesenchymal stem cells (rMSCs) were isolated, cultivated, and induced by SRY (Sex-Determining Region Y)-Box 9 (Sox9) via cationic nanosized liposomes. The induced rMSCs were mixed with a thermo-sensitive chitosan hydrogel and subcutaneously injected into the nude mice. Finally, the newly-formed chondroid tissue obtained in the injection parts, and the transparent parts were detected by HE, collagen II, and safranin O. Results: It was found that the presently prepared cationic nanosized liposomes had the diameter of 85.76±3.48 nm and the zeta potential of 15.76±2.1 mV. The isolated rMSCs proliferation was fibroblast-like, with a cultivated confluence of 90% confluence in 5-8 days, and stained positive for CD29 and CD44 while negative for CD34 and CD45. After transfection with cationic nanosized liposomes, we observed changes of cellular morphology and a higher expression of SOX9 compared with control groups, which indicated that rMSCs could differentiate into chondrocyte in vitro. By mixing transfected rMSCs with the thermo-sensitive hydrogel of chitosan in nude mice, chondroid tissue was successfully obtained, demonstrating that rMSCs can differentiate into chondrogenic cells in vivo. Conclusion: This study explored new ways to improve the quality of tissue engineered cartilage, thus accelerating clinical transformation and reducing patient pain.

Keywords: Sox9; chondrogenesis; chondroid; gene enhanced tissue engineering; transfection.

Figure 1

Characterization of cationic liposomes. (A) The diameter of cationic liposomes, (B) The zeta potential of cationic liposomes, and (C) TEM of cationic liposomes. * P<0.01.

Figure 2

Isolated rMSCs cultivation and identification. (A) Isolated rMSCs cultivated at different times ( 24 hours, 48 hours, 5–8 day); (B) identification of rMSCs by flow cytometry; (C) growth curve of rMSCs.

Figure 3

In vitro transfection of rMSCs. (A) rMSCs were transfected for 7 days; (B) growth curve of transfected rMSCs; (C) gene transfection of rMSCs with cationic nanosized liposomes at different concentrations of cells (* P<0.05; ** P>0.05). (D) Gene transfection of rMSCs with different formulations.

Figure 4

Sketch of Sox9 gene enhanced tissue engineering in chondrogenesis.

Figure 5

In vivo chondrogenesis of rMSCs. (A) Immunohistochemical stain of different groups. (B) Western blot (WB) analysis of Sox9, collagen II, and collagen IX. (C) Densitometric analysis of Sox9, collagen II, and collagen IX. Group A, untransfected rMSCs with chitosan hydrogel; Group B, transfected rMSCs with chitosan hydrogel. *P<0.05.

Figure S1

Homo sapiens (human) with vector backbone of pcDNA3