Osteogenic and antibacterial dual functions of a novel levofloxacin loaded mesoporous silica microspheres/nano-hydroxyapatite/polyurethane composite scaffold

Abstract

Lev/MSNs/n-HA/PU has been proved to be a novel scaffold material to treat bone defect caused by chronic osteomyelitis. We have previously identified that this material can effectively treat chronic osteomyelitis caused by Staphylococcus aureus in vivo. However, the potential mechanisms of antibacterial and osteogenic induction properties remain unclear. Thus, for osteogenesis property, immunohistochemistry, PCR, and Western blot were performed to detect the expression of osteogenic markers. Furthermore, flow cytometry and TUNEL were applied to analyze MC3T3-E1 proliferation and apoptosis. For antibacterial property, the material was co-cultivated with bacteria, bacterial colony forming units was counted and the release time of the effective levofloxacin was assayed by agar disc-diffusion test. Moreover, scanning electron microscope was applied to observe adhesion of bacteria. In terms of osteogenic induction, we found BMSCs adherently grew more prominently on Lev/MSNs/n-HA/PU. Lev/MSNs/n-HA/PU also enhanced the expression of osteogenic markers including OCN and COL1α1, as well as effectively promoted the transition from G1 phase to G2 phase. Furthermore, Lev/MSNs/n-HA/PU could reduce apoptosis of MC3T3-E1. Besides, both Lev/MSNs/n-HA/PU and n-HA/PU materials could inhibit bacterial colonies, while Lev/MSNs/n-HA/PU possessed a stronger antibacterial activities, and lower bacterial adhesion than n-HA/PU. These results illustrated that Lev/MSNs/n-HA/PU composite scaffold possess favorable compatibility in vitro, which induce osteogenic differentiation of MSCs, promote proliferation and differentiation of MC3T3-E1, and inhibit apoptosis. Moreover, clear in vitro antibacterial effect of Lev/MSNs/n-HA/PU was also observed. In summary, this study replenishes the potential of Lev/MSNs/n-HA/PU composite scaffold possess dual functions of anti-infection and enhanced osteogenesis for future clinical application.

Keywords: Antibacterial; Composite scaffold; Levofloxacin; Nanobiomaterial; Osteogenesis.

Figure 1

Effect of novel Lev/MSNs/n-HA/PU composite scaffold materials on biocompatibility and proliferation of BMSCs. (A) SEM micrographs of the BMSCs adhesion in different materials for 7 days. (B) MTT assay for proliferation of BMSCs cultured with different materials at 2, 4 and 7 days. Each experiment was repeated three times. “*” means P < 0.05.

Figure 2

Immunohistochemistry assay of osteogenic markers for composite scaffold materials. BMSCs were cultured on different scaffold materials for 7 days and next on slides for 3 days. They were subjected to immunohistochemistry assay of the expression of ALP (A), OCN (B) and OPN (C). The negative control was added with PBS instead of primary antibody (D). Black bar represents 100 μm.

Figure 3

Q-PCR and Western-Blot assay of osteogenic markers for composite scaffold materials. BMSCs were cultured on different scaffold materials for 14 days. RNA was isolated and subjected to qPCR analysis of the expression of OCN and COL1α1 (A) in osteoblast cells. The expression of COL1α1 was detected by Western-Blot (B, C). “*” means P < 0.05.

Figure 4

Effect of novel Lev/MSNs/n-HA/PU composite scaffold materials on proliferation and apoptosis of MC3T3-E1. (A) MC3T3-E1 was cultured on different scaffold materials for 14 days and digested for assay of flow cytometry. (B) The percentage of cell proliferation cycles treated with different materials. (C) TUNEL assay for evaluation of apoptosis of MC3T3-E1 co-cultured with different materials for 14 days. (D) The percentage of TUNEL positive cell treated with different materials, and black arrows represent positive cells. Each experiment was repeated three times. “*” means P < 0.05.

Figure 5

The antibacterial effect of novel Lev/MSNs/n-HA/PU composite scaffold materials. (A) Bacterial colony formation after treatment with material extracts at 1 st and 12 th day, and (B) Number of bacterial colony units. (C) The zone of inhibition (ZOI) around n-HA/PU and Lev/MSNs/n-HA/PU scaffold materials. (D) The diameter of ZOI around the materials. Each experiment was repeated three times. Representative results are shown. “*” means P < 0.05 compared with n-HA/PU group.

Figure 6

The adhesive effect of novel Lev/MSNs/n-HA/PU composite scaffold materials on E. coli and S. aureus. Scanning electron microscope (SEM) was used to detect the adhesion of Escherichia coli (upper) and Staphylococcus aureus (lower) to the composite scaffold materials n-HA/PU (A-B), and the adhesion to novel composite scaffold materials Lev/MSNs/n-HA/PU (C-D). Representative results are shown.