Liquid Crystal Modified Polylactic Acid Improves Cytocompatibility and M2 Polarization of Macrophages to Promote Osteogenesis

Abstract

Liquid crystalline phases (LC phases) are widely present in an organism. The well-aligned domain and liquidity of the LC phases are necessary for various biological functions. How to stabilize the floating LC phases and maintain their superior biology is still under study. In addition, it is unclear whether the exogenous LC state can regulate the immune process and improve osteogenesis. In this work, a series of composite films (PLLA/LC) were prepared using cholesteryl oleyl carbonate (COC), cholesteryl pelargonate (CP), and polylactic acid (PLLA) via a controlled facile one-pot approach. The results showed that the thermo-responsive PLLA/LC films exhibited stable LC phases at human body temperature and the cytocompatibility of the composites was improved significantly after modification by the LC. In addition, the M2 polarization of macrophages (RAW264.7) was enhanced in PLLA/LC films, and the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) was improved as co-cultured with macrophages. The in vivo bone regeneration of the materials was verified by calvarial repair, in which the amount of new bone in the PLLA-30% LC group was greater than that in the PLLA group. This work revealed that the liquid crystal-modified PLLA could promote osteogenesis through immunomodulation.

Keywords: bone regeneration; cytocompatibility; liquid crystal; macrophages; polylactic acid.

FIGURE 1

Thermal properties of the composite LC. (A) DSC curve of COC/CP LC; (B) DSC curve of PLLA/LC composite films; (C) blended LC LC-30% CP LC texture observed under POM; and (D) LC texture of the PLLA-30% LC composite films observed under POM.

FIGURE 2

(A)Cell proliferation in COC/CP LC and (B) PLLA-LC composites for 7 days * refer to statistically significant proliferation p < 0.05. (C) AO-EB staining of MC3T3-E1 cells seeded on pure PLLA, PLLA-10% LC, PLLA-30% LC, and PLLA-50% LC films.

FIGURE 3

(A)SEM images of morphologies of MC3T3-E1 cells culturing on pure PLLA, PLLA-10% LC, PLLA-30% LC, and PLLA-50% LC films for 3 days; (B) CLSM images of morphologies of MC3T3-E1 cell culturing on pure PLLA, PLLA-10% LC, PLLA-30% LC, and PLLA-50% LC films for 1 day.

FIGURE 4

(A) Cell count of osteoblasts migrating in the transwell chamber for 24 h; (B) quantitative analysis of osteoblasts migrating in the transwell chamber for 72 h; (C) crystal violet staining of osteoblasts migrating in the transwell chamber for 24 and 72 h; and (D) distribution of cells on LC-30% CP.

FIGURE 5

Polarization of macrophages on the material.

FIGURE 6

(A) ALP quantitative analysis; (B) Alizarin Red staining quantitative analysis; (C) and (D) BMSC-related osteogenic gene expression on different materials; (E) ALP staining; and (F) Alizarin Red dyeing.

FIGURE 7

(A) Bone volume analysis; (B) bone volume density analysis; (C) micro-CT image of an SD rat skull defect 4 weeks after operation; and (D) HE staining photograph of an SD rat skull defect.