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. 2019 Apr 30:14:3043-3054.
doi: 10.2147/IJN.S198583. eCollection 2019.

Antibacterial and osteogenesis performances of LL37-loaded titania nanopores in vitro and in vivo

Xinkun Shen  1 , Mohammed A Al-Baadani  1 , Hongli He  1 , Lina Cai  1 , Zuosu Wu  1 , Litao Yao  1 , Xinghai Wu  1 , Shuyi Wu  1 , Mengyu Chen  1 , Hualin Zhang  2   3 , Jinsong Liu  1
Affiliations

Antibacterial and osteogenesis performances of LL37-loaded titania nanopores in vitro and in vivo

Xinkun Shen et al. Int J Nanomedicine. .

Abstract

Background: Many studies have shown that the size of nanotube (NT) can significantly affect the behavior of osteoblasts on titanium-based materials. But the weak bonding strength between NT and substrate greatly limits their application. Purpose: The objective of this study was to compare the stability of NT and nanopore (NP) coatings, and further prepare antibacterial titanium-based materials by loading LL37 peptide in NP structures. Methods: The adhesion strength of NT and NP layers was investigated using a scratch tester. The proliferation and differentiation of MC3T3-E1 cells on different substrates were evaluated in vitro by CCK8, alkaline phosphatase activity, mineralization and polymerase chain reaction assays. The antibacterial rates of NP and NP/LL37 were also measured by spread plate method. Moreover, the osteogenesis around NP and NP/LL373 in vivo was further evaluated using uninfected and infected models. Results: Scratch test proved that the NP layers had stronger bonding strength with the substrates due to their continuous pore structures and thicker pipe walls than the independent NT structures. In vitro, cell results showed that MC3T3-E1 cells on NP substrates had better early adhesion, spreading and osteogenic differentiation than those of NT group. In addition, based on the drug reservoir characteristics of porous materials, the NP substrates were also used to load antibacterial LL37 peptide. After loading LL37, the antibacterial and osteogenic induction abilities of NP were further improved, thus significantly promoting osteogenesis in both uninfected and infected models. Conclusion: We determined that the NP layers had stronger bonding strength than NT structures, and the corresponding NP materials might be more suitable than NT for preparing drug-device combined titanium implants for bone injury treatment.

Keywords: LL37 peptide; antibacterial; nanopores; nanotubes; osteogenesis; titanium.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
(A) Scanning electron microscopy (SEM) and atomic force microscopy (AFM) images of NT and NP substrates; statistics of pore size (B), wall thickness (C) and length (D) of surface nanotubes and nanopores; (E) statistics of surface roughness according to AFM images; (F) water contact angle and (G) X-ray diffraction (XRD) patterns of NT and NP substrates.
Figure 2
Figure 2
(A) Representative scratch track image of NT and NP; (B) statistics of critical loads of cohesion (Lc1), adhesion (Lc2) and breakthrough (Lc3). Error bars represent mean±SD for n=4, *p<0.05.
Figure 3
Figure 3
(A) Morphology of MC3T3-E1 cells on NT and NP substrates; statistics of cell aspect ratio (B) and area (C) according to morphology images; (D) early adhesion of MC3T3-E1 cells at 0.5 and 2 hrs; (E) cell viability, (F) ALP activity, (G) mineralization level and (H) osteogenic genes expression of MC3T3-E1 cells at 7 and/or 14 d. Error bars represent mean±SD for n=6, *p<0.05.
Figure 4
Figure 4
(A) Scanning electron microscopy (SEM) images and (B) water contact angle of NP and NP/LL37 substrates; (C) release profile of LL37 from NP/LL37 sample at different time. Error bars represent mean ± SD for n = 6.
Figure 5
Figure 5
(A) Bacterial concentrations of Staphylococcus aureus (S. aureus, A and C) and Methicillin-resistant Staphylococcus aureus (MRSA, B and D) on different substrates (A and B) or in medium (C and D) at 24 h. Error bars represent mean ± SD for n = 6, *p < 0.05.
Figure 6
Figure 6
(A) Cell viability of MC3T3-E1 cells on Ti, NP and NP/LL37 substrates at 1, 3 and 7 d; (B) ALP activity and (C) mineralization level of MC3T3-E1 cells at 7 and/or 14 d; gene expression of ALP (D), COL Ⅰ(E), OCN (F) and OPG (G) at 7 d. Error bars represent mean±SD for n=6, *p<0.05.
Figure 7
Figure 7
(A) 3D images of new bone in normal and infected NP and NP/LL37 groups after implantation for 8 weeks; statistics of bone volume fraction (BV/TV) (B), trabecular number (Tb.N) (C), trabecular thickness (Tb.Th) (D), trabecular separation (Tb.Sp) (E) and connectivity density (Conn-Dens) (F). Error bars represent mean ± SD for n = 8, *p < 0.05.
Scheme 1
Scheme 1
Schematic illustration of the preparation processes and/or biological functions of different substrates.
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