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. 2021 Jul 19:9:718255.
doi: 10.3389/fbioe.2021.718255. eCollection 2021.

Endowing Orthopedic Implants' Antibacterial, Antioxidation, and Osteogenesis Properties Through a Composite Coating of Nano-Hydroxyapatite, Tannic Acid, and Lysozyme

Guofeng Wang  1 , Yaxin Zhu  2 , Xingjie Zan  2 , Meng Li  1
Affiliations

Endowing Orthopedic Implants' Antibacterial, Antioxidation, and Osteogenesis Properties Through a Composite Coating of Nano-Hydroxyapatite, Tannic Acid, and Lysozyme

Guofeng Wang et al. Front Bioeng Biotechnol. .

Abstract

There is a substantial global market for orthopedic implants, but these implants still face the problem of a high failure rate in the short and long term after implantation due to the complex physiological conditions in the body. The use of multifunctional coatings on orthopedic implants has been proposed as an effective way to overcome a range of difficulties. Here, a multifunctional (TA@HA/Lys)n coating composed of tannic acid (TA), hydroxyapatite (HA), and lysozyme (Lys) was fabricated in a layer-by-layer (LBL) manner, where TA deposited onto HA firmly stuck Lys and HA together. The deposition of TA onto HA, the growth of (TA@HA/Lys)n, and multiple related biofunctionalities were thoroughly investigated. Our data demonstrated that such a hybrid coating displayed antibacterial and antioxidant effects, and also facilitated the rapid attachment of cells [both mouse embryo osteoblast precursor cells (MC3T3-E1) and dental pulp stem cells (DPSCs)] in the early stage and their proliferation over a long period. This accelerated osteogenesis in vitro and promoted bone formation in vivo. We believe that our findings and the developed strategy here could pave the way for multifunctional coatings not only on orthopedic implants, but also for additional applications in catalysts, sensors, tissue engineering, etc.

Keywords: hydroxyapatite; multi-functionality; orthopedic coatings; osteogenesis; polyphenol.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer JS declared a shared affiliation, with no collaboration, with the authors YZ and XZ to the handling editor at the time of the review.

Figures

SCHEME 1
SCHEME 1
Schematic illustration of the molecular structure of (A) lysozyme (Lys), (B) tannic acid (TA), (C) hydroxyapatite (HA), (D) forming process of TApHs@HA, and (E) (TA@HA/Lys)n fabrication: (1) PEI was deposited on the substrate first, followed by the deposition of (2) TA@HA and (3) Lys, (4) repeating (2) and (3) with the desired number.
FIGURE 1
FIGURE 1
(A) X-ray photoelectron spectrometer (XPS) spectra of tannic acid (TA), TA–8.5@HA, and hydroxyapatite (HA). (B) Carbon (C) and oxygen (O) contents and (C) Calcium (Ca) and phosphorus (P) contents in TA, TA–pH@HA, and HA. (D) Peak fitting in O1s of XPS spectra of TA, TA–pH@HA, and HA. (E) C-O and P = O contents in TA, TA–pH@HA, and HA relative to the total O calculated from (D). The line in all figures was used for eye guidance.
FIGURE 2
FIGURE 2
(A) The thickness of (TA@HA/Lys)n–pH deposited at various pH values plotted as the function of a number of bilayers. (B) The thickness of (TA@HA/Lys)6–pH constructed at different pHs. (C) XPS spectra of lysozyme (Lys), (TA@HA/Lys)2–8.5, and (TA@HA/Lys)2.5–8.5. (D) Elemental content ratio of C:N in (TA@HA/Lys)n–8.5 as the function of a number of bilayers. The line in all figures was used for eye guidance.
FIGURE 3
FIGURE 3
SEM images of (A) (TA@HA/Lys)2, (B) (TA@HA/Lys)4, and (C) (TA@HA/Lys)6, and the corresponding magnified images in the insets of the upper right. (D) Surface roughness calculated from the atomic force microscope (AFM) images of (TA@HA/Lys)2 and (TA@HA/Lys)4 coatings, the insets are the corresponding AFM images. The scale bars in (A–C) are 20 μm (200 nm in the insets). The scale bar in (D) is 2 μm.
FIGURE 4
FIGURE 4
(A) Cell viability of mouse embryo osteoblast precursor cells (MC3T3-E1) cultured onto control, (TA@HA/Lys)2, (TA@HA/Lys)4, and (TA@HA/Lys)6 at 1, 3, 5, and 7 days. (B) Fluorescence images and (C) cell numbers of MC3T3-E1 cells cultured onto control, (TA@HA/Lys)2, (TA@HA/Lys)4, and (TA@HA/Lys)6 at day 5. (D) Cell viability of dental pulp stem cells (DPSCs) cultured onto control, (TA@HA/Lys)2, (TA@HA/Lys)4, and (TA@HA/Lys)6 at 1, 3, 5, and 7 days. (E) Fluorescence images and (F) cell numbers of DPSCs cultured onto control, (TA@HA/Lys)2 and (TA@HA/Lys)4 at day 5. All images were using the same scale bar. **p < 0.01, ****p < 0.0001.
FIGURE 5
FIGURE 5
(A) Morphologies of MC3T3-E1 cells and DPSCs cultured on glass coverslips as control (left row), (TA@HA/Lys)2 (middle row), and (TA@HA/Lys)4 (right row) substrates for 5 and 10 h. (B) Statistical cell area per cell and (C) cell number per mm2 of MC3T3-E1 cells. (D) Statistical cell area per cell and (E) cell number per mm2 of DPSCs. All images were using the same scale bar. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
FIGURE 6
FIGURE 6
(A) Antioxidant activities of glass coverslips as control, (TA@HA/Lys)2, and (TA@HA/Lys)4 films evaluated by the fluorescence recovery after photobleaching (FRAP) method. (B) Fluorescence microscopy images of MC3T3-E1 cells cultured on glass coverslips as control, (TA@HA/Lys)2, and (TA@HA/Lys)4 substrates before (top) and after (bottom) 10 μM hydrogen peroxide (H2O2) stimulation. (C) Cell area per cell and (D) cell number per mm2 of MC3T3-E1 cells on the above substrates were determined without/with 10 μM H2O2 stimulation. All images were using the same scale bar. ***p < 0.001, ****p < 0.0001.
FIGURE 7
FIGURE 7
SEM images of (A) Escherichia coli and (B) Staphylococcus aureus after exposure to control (glass coverslips, left row), (TA@HA/Lys)2 (middle row), and (TA@HA/Lys)4 (right row) coatings. Relative live bacteria number of (C) E. coli and (D) S. aureus in above films (the control group as a reference). All images were using the same scale bar.
FIGURE 8
FIGURE 8
OD values at 570 nm after extracting the Alizarin Red S stained for evaluating the osteoblast mineralization of (A) MC3T3-E1 cells and (B) DPSCs cultured on glass coverslips as control, (TA@HA/Lys)2 and (TA@HA/Lys)4 films with/without induction solution for 14 days. Alkaline phosphatase (ALP) activities of (C) MC3T3-E1 cells and (D) DPSCs cultured on glass coverslips as control, (TA@HA/Lys)2 and (TA@HA/Lys)4 films for 7 and 14 days. *p < 0.05, **p < 0.01, ****p < 0.0001.
FIGURE 9
FIGURE 9
Expressions of genes in relation to osteogenic differentiation for DPSCs being inductively cultured on the (TA@HA/Lys)2 and (TA@HA/Lys)4 substrates and glass coverslips as control: (A) runt-related transcription factor 2 (Runx2), (B) osteonectin (ON), and (C) osteocalcin (OCN). **p < 0.01, ****p < 0.0001.
FIGURE 10
FIGURE 10
(A) Bone regeneration images in the rabbit femur were reconstructed by the CT-Volume (CTVol) software in three-dimensional (3D) model, when titanium rods with a blank layer as control, (TA@HA/Lys)2 and (TA@HA/Lys)4 coatings implanted for 4 (top) and 8 weeks (bottom). (B) Bone volume (BV) fraction [BV/total volume (TV)] of control, (TA@HA/Lys)2 and (TA@HA/Lys)4 coatings group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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