Osteogenic and anti-inflammatory effect of the multifunctional bionic hydrogel scaffold loaded with aspirin and nano-hydroxyapatite

Abstract

Although tissue engineering offered new approaches to repair bone defects, it remains a great challenge to create a bone-friendly microenvironment and rebuild bone tissue rapidly by a scaffold with a bionic structure. In this study, a multifunctional structurally optimized hydrogel scaffold was designed by integrating polyvinyl alcohol (PVA), gelatin (Gel), and sodium alginate (SA) with aspirin (ASA) and nano-hydroxyapatite (nHAP). The fabrication procedure is through a dual-crosslinking process. The chemical constitution, crystal structure, microstructure, porosity, mechanical strength, swelling and degradation property, and drug-release behavior of the hydrogel scaffold were analyzed. Multi-hydrogen bonds, electrostatic interactions, and strong "egg-shell" structure contributed to the multi-network microstructure, bone tissue-matched properties, and desirable drug-release function of the hydrogel scaffold. The excellent performance in improving cell viability, promoting cell osteogenic differentiation, and regulating the inflammatory microenvironment of the prepared hydrogel scaffold was verified using mouse pre-osteoblasts (MC3T3-E1) cells. And the synergistic osteogenic and anti-inflammatory functions of aspirin and nano-hydroxyapatite were also verified. This study provided valuable insights into the design, fabrication, and biological potential of multifunctional bone tissue engineering materials with the premise of constructing a bone-friendly microenvironment.

Keywords: aspirin; multifunctional hydrogel scaffold; nano-hydroxyapatite; sustained release; tissue engineering.

SCHEME 1

Schematic illustration of the procedures of the dual-crosslinking hydrogel scaffolds. Effects on MC3T3-E1 cells bioactivity, osteogenic differentiation, and anti-inflammatory capacity.

FIGURE 1

Chemical constitution and crystal structure analysis of the raw powders and the composited hydrogel scaffolds. (A) The FTIR spectra of nanohydroxyapatite, aspirin, sodium alginate (SA), gelatin (Gel), and polyvinyl alcohol (PVA). (B) The FTIR spectra of ASA/PVA/Gel/SA (ASA group), nHAP/PVA/Gel/SA (nHAP group), and ASA-nHAP/PVA/Gel/SA (ASA-nHAP group) hydrogel scaffold. (C) The XRD pattern of nanohydroxyapatite, aspirin, sodium alginate (SA), gelatin (Gel), and polyvinyl alcohol (PVA). (D) The XRD pattern of ASA/PVA/Gel/SA (ASA group), nHAP/PVA/Gel/SA (nHAP group), and ASA-nHAP/PVA/Gel/SA (ASA-nHAP group) hydrogel scaffold.

FIGURE 2

Microstructure of hydrogel scaffolds. (A) SEM images of the ASA group, nHAP group, and ASA-nHAP group. The scale bar for low-magnification images is 100 μm; the scale bar for high-magnification images is 10 μm. (B–D) Pore size distribution pattern of the ASA group, nHAP group, and ASA-nHAP group.

FIGURE 3

Characterization of hydrogel scaffolds. (A) The porosity of hydrogel scaffolds. (B) The stress-strain curve of hydrogel scaffold. (C) The compressive strength of hydrogel scaffold. (D) The compressive modulus of hydrogel scaffold. (E) The swelling performance of hydrogel scaffolds. (F) The degradation performance of hydrogel scaffolds. (G) The drug release performance of hydrogel scaffolds. (**p < 0.01).

FIGURE 4

Growth ability of MC3T3-E1 cells cultured in vitro. (A) Live/dead staining images of MC3T3-E1 cells cultured with the hydrogel extractions for 1, 3, and 5 d. (B) The OD value of the control group, ASA group, nHAP group, and ASA-nHAP group from the CCK-8 assay (*p < 0.05, **p < 0.01). (C, D) Semi-quantitative analysis and laser confocal images of MC3T3-E1 cells on the ASA group, nHAP group, and ASA-nHAP group. Blue represents the nucleus stained by DAPI and red represents the cytoskeleton stained by FITC phalloidin at a scale bar of 40 μm.

FIGURE 5

Osteogenic differentiation ability of MC3T3-E1 cells cultured in vitro. (A) Digital and microscopic images of MC3T3-E1 cells in the control group, ASA group, nHAP group, and ASA-nHAP group after 2, 7, and 14 days to assess ALP activity, the scale bar is 200 μm. (B) Digital and microscopic images of MC3T3-E1 cells in the control group, ASA group, nHAP group, and ASA-nHAP group after 7, 14, and 21 days to assess the expression of calcium nodules, scale bar is 200 μm. (C) Semi-quantitative analysis of the expression of calcium nodules in the control group, ASA group, nHAP group, and ASA-nHAP group after 7, 14, and 21 days (**p < 0.01). (D–F) Expression of osteogenic genes of RUNX2, OCN, and OPN after 14 days (**p < 0.01).

FIGURE 6

Expression of the inflammatory mediators of MC3T3-E1 cells in the inflammatory environment after 1, 3, and 7 d (A) TNF-α. (B) IL-6. (C) IL-8. (**p < 0.01).