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. 2014 May 20;9(5):e97741.
doi: 10.1371/journal.pone.0097741. eCollection 2014.

Bi-functionalization of a calcium phosphate-coated titanium surface with slow-release simvastatin and metronidazole to provide antibacterial activities and pro-osteodifferentiation capabilities

Yunsong Liu  1 , Xiao Zhang  2 , Yang Liu  3 , Xiaoxiao Jin  2 , Cong Fan  2 , Hongqiang Ye  2 , Meng'en Ou  2 , Longwei Lv  2 , Gang Wu  4 , Yongsheng Zhou  5
Affiliations

Bi-functionalization of a calcium phosphate-coated titanium surface with slow-release simvastatin and metronidazole to provide antibacterial activities and pro-osteodifferentiation capabilities

Yunsong Liu et al. PLoS One. .

Abstract

Coating the surface of titanium implants or other bone graft substitute materials with calcium phosphate (Ca-P) crystals is an effective way to enhance the osteoconduction of the implants. Ca-P coating alone cannot confer pro-osteodifferentiation and antibacterial capabilities on implants; however, it can serve as a carrier for biological agents which could improve the performance of implants and bone substitutes. Here, we constructed a novel, bi-functional Ca-P coating with combined pro-osteodifferentiation and antibacterial capabilities. Different concentrations of metronidazole (MNZ) and simvastatin (SIM) were integrated into biomimetic Ca-P coatings on the surface of titanium disks. The biological effects of this bi-functional biomimetic coating on human bone marrow mesenchymal stem cells (hBMMSCs), human adipose derived stromal cells (hASCs), and Porphyromonas gingivalis were assessed in vitro. We observed that Ca-P coatings loaded with both SIM and MNZ display favorable release kinetics without affecting cell proliferation or attachment. In the inhibition zone test, we found that the bi-functional coating showed lasting antibacterial effects when incubated with Porphyromonas gingivalis for 2 and 4 days. Moreover, the osteodifferentiation of hBMMSCs and hASCs were increased when cultured on this bi-functional coating for 7 and 14 days. Both drugs were loaded onto the Ca-P coating at specific concentrations (10(-5) M SIM; 10(-2) M MNZ) to achieve optimal release kinetics. Considering the safety, stability and low cost of SIM and MNZ, this novel bi-functional Ca-P coating technique represents a promising method to improve the performance of metal implants or other bone substitute materials, and can theoretically be easily translated to clinical applications.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Scanning electron microscopy (SEM) observations of the Ca-P coating and drug-loaded Ca-P coating.
(A, B) Ca-P coating. (C, D) Ca-P coating loaded with 10−5 M SIM. (E, F) Ca-P coating loaded with 10−4 M and 10−3 M SIM. (G, H) Ca-P coating loaded with 10−2 M MNZ. (I, J) Ca-P coating loaded with 10−3 M MNZ and 10−4 M MNZ. (K, L) Ca-P coating loaded with 10−2 M MNZ and 10−5 SIM together.
Figure 2
Figure 2. In vitro SIM and MNZ release kinetics in PBS from drug-loaded Ca-P coatings.
(A) SIM release kinetics. (B) MNZ release kinetics.
Figure 3
Figure 3. Energy dispersive X-ray spectroscopy (EDS) analysis of the elementary components of the coating.
(A) The Ca-P coating was mainly composed of calcium, phosphate and oxygen. (B) When loaded with 10−5 M SIM, carbon could also be detected. (C) When loaded with 10−2 M MNZ, carbon and nitrogen could also be detected. (D) When loaded with 10−2 M MNZ and 10−5 M SIM together, the proportion of carbon increased compared with the Ca-P coating loaded with MNZ alone.
Figure 4
Figure 4. Bacterial inhibition capability of the drug-loaded Ca-P coatings.
(A) Inhibition zones formed by different groups of Ti disks. (B) Inhibition zones formed by the Ca-P+MNZ and Ca-P+MNZ+SIM groups after 2 and 4 days exposure to PBS.
Figure 5
Figure 5. Effects of the drug loaded Ca-P coating on cell attachment and proliferation.
(A, B) SEM observation of the cells attached to the bi-functional Ca-P coating. (C, D) Growth curves of the cells cultured on different groups of Ti disks.
Figure 6
Figure 6. Effects of the drug-loaded Ca-P coating on the osteogenic differentiation of human MSCs.
(A) The expression of osteogenic genes in hBMMSCs and hASCs cultured on different groups of Ti disks for 7 days. (B) ALP activity of hBMMSCs and hASCs cultured on different groups of Ti disks for 7 days. (C) The ELISA determination of BMP-2 protein secretion cultured on different groups of Ti disks for 7 days. (D) The expression of osteogenic genes in hBMMSCs and hASCs cultured on different groups of Ti disks for 14 days. (E) ALP activity of hBMMSCs and hASCs cultured on different groups of Ti disks for 14 days. (F) The ELISA determination of BMP-2 protein secretion cultured on different groups of Ti disks for 14 days. *P<0.05 compared with the SLA control group; △ P<0.05 compared with the Ca-P control group.
Figure 7
Figure 7. Immunofluorescent staining for OCN in MSCs cultured on different groups of Ti disks for 14 days.
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