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. 2022 Nov 22:10:1019332.
doi: 10.3389/fchem.2022.1019332. eCollection 2022.

Fabrication of gentamicin loaded Col-I/HA multilayers modified titanium coatings for prevention of implant infection

Le Ma  1 , Jiajia Zong  1 , Xiaowei Xun  1 , Xiaoming Hu  1 , Zejing Chen  1 , Quanchao Zhang  1 , Mengxia Peng  1 , Botao Song  2 , Haiyong Ao  1
Affiliations

Fabrication of gentamicin loaded Col-I/HA multilayers modified titanium coatings for prevention of implant infection

Le Ma et al. Front Chem. .

Abstract

In this study, gentamicin loaded collagen I/hyaluronic acid multilayers modified titanium coating (TC-AA(C/H)6-G) was fabricated via a layer-by-layer (LBL) covalent immobilization method. The drug releasing properties of collagen I/Hyaluronic acid (Col-I/HA) multilayers and the effect of loaded gentamicin on the antibacterial properties and cytocompatibility of modified TC were investigated. The gentamicin release assay indicated that the Col-I/HA multilayers modified TC exhibited agreeable drug-loading amount (537.22 ± 29.66 µg of gentamicin) and controlled-release performance (240 h of sustained release time). TC-AA(C/H)6-G revealed satisfactory antibacterial activity and inhibited the colonization and biofilm formation of S. aureus. Fortunately, the functions of hMSCs on TC-AA(C/H)6-G did not affected by the loaded gentamicin, and TC-AA(C/H)6-G could improve the adhesion, proliferation and osteogenic differentiation of cells, as well as TC-AA(C/H)6. In vivo animal study indicated that TC-AA(C/H)6-G could effectively control intramedullary cavity infection caused by S. aureus and prevent bone destruction.

Keywords: Col-I/HA multilayers; cytocompatibility; drug-release; prevention infection; titanium coatings.

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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.

Figures

FIGURE 1
FIGURE 1
Schematic illustration of the fabrication of gentamicin loaded Col-I/HA multilayers on titanium coating via a LBL covalent immobilization technique.
FIGURE 2
FIGURE 2
SEM of samples (A), Drug loaded amount (B) and cumulative release curves of gentamicin (C) of TC-A-G, TC-AAC-G and TC-AA(C/H)6-G.
FIGURE 3
FIGURE 3
The zone of inhibition (ZOI) of multilayer membrane modified TCs loading gentamycin. (A) The ZOI against S. aureus produced by different specimens at different time points; (B) The ZOI diameters of all specimens.
FIGURE 4
FIGURE 4
Antibacterial properties of TC-AA(C/H)6-G. (A) Bacterial colonization and biofilm formation by the spread plate method; (B) SEM of bacteria on the three different specimens after incubated 6 h and 24 h; (C) The CLSM views of bacterial colonization and biofilm formation on the three kinds of samples, green for living bacteria and red for dead bacteria.
FIGURE 5
FIGURE 5
Cytocompatibility of TC, TC-AA(C/H)6 and TC-AA(C/H)6-G. (A) Attachment of hMSCs on three different samples; (B) Proliferation of hMSCs on three different samples; (C) ALP activity was measured using the pNPP assay and normalized based on the protein content per specimen at days 4, 7 and 14; (D) Colorimetric quantitative analysis of the extracellular matrix mineralization on the samples after 21 days of incubation; (E) ALP staining was performed at day 14; (F) Alizarin red staining. *p < 0.05, there has significant difference between TC-AA(C/H)6-G and TC. #p > 0.05, there has no significant difference between TC-AA(C/H)6-G and TC-AA(C/H)6.
FIGURE 6
FIGURE 6
Postoperative changes in temperature (A) and weight (B) of animals.
FIGURE 7
FIGURE 7
-Gross appearance and scores of longitudinal sections of three kinds of femurs. (A) TC and TC-AA(C/H)6 groups showed signs of purulent infections, while the TC-AA(C/H)6-G group showed no obvious infection signs; (B) The score of the TC-AA(C/H)6-G group was significantly lower than other two groups, *p < 0.05.
FIGURE 8
FIGURE 8
Quantification of bacteria obtained from explanted implants and bone tissue on day of sacrifice. (A) Growth of bacterial colonies of implants on TSA; (B) The number of CFU per implant; (C) Amount of CFU per gram of bone. *p < 0.05 compared with TC-AA(C/H)6-G and other two groups.
FIGURE 9
FIGURE 9
(A) X-ray photograph of left femur in lateral view 1 day, 3 and 6 weeks after operation. Typical signs include osteolytic lesions (black arrowhead) and periosteal new bone formation (white arrowhead); (B) 3D images of overall, longitudinal and transverse sections; (C) Quantitative analysis of the X-ray images; (D) the cortical one mineral density (BMD) of ROI; (E) bone volume/total volume (BV/TV) of ROI. *p < 0.05 compared with TC-AA(C/H)6-G and other two groups.
FIGURE 10
FIGURE 10
Representative photomicrographs of longitudinal sections of femurs. (A) H&E staining; (B) Giemsa staining.
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