Effects of combined chemical design (Cu addition) and topographical modification (SLA) of Ti-Cu/SLA for promoting osteogenic, angiogenic and antibacterial activities

doi:10.1016/j.jmst.2019.10.045

Abstract

Abstract:

Cu has been proved to possess various beneficial biological activities, while sandblasting and acid etching (SLA) is widely used to modify the commercial dental implant in order to improve osseointegration. Based on the above, a novel antimicrobial dental implant material, Ti-Cu alloy, was treated with SLA, to combine chemical design (Cu addition) and topographical modification (SLA). In this work, the effects of SLA treated Ti-Cu alloys (Ti-Cu/SLA) on osteogenesis, angiogenesis and antibacterial properties were evaluated from both in vitro and in vivo tests, and Ti/SLA and Ti-Cu (without SLA) were served as control groups. Benefiting by the combined effects of chemical design (Cu addition) and micro-submicron hybrid structures (SLA), Ti-Cu/SLA had significantly improved inhibitory effects on oral anaerobic bacteria (P. gingivalis and S. mutans) and could induce upregulation of osteogenic-related and angiogenic-related genes expression in vitro. More importantly, in vivo studies also demonstrated that Ti-Cu/SLA implants had wonderful biological performance. In the osseointegration model, Ti-Cu/SLA implant promoted osseointegration via increasing peri-implant bone formation and presenting good bone-binding, compared to Ti/SLA and Ti-Cu implants. Additionally, in the peri-implantitis model, Ti-Cu/SLA effectively resisted the bone resorption resulted from bacterial infection and meanwhile promoted osseointegration. All these results suggest that the novel multiple functional Ti-Cu/SLA implant with rapid osseointegration and bone resorption inhibition abilities has the potential application in the future dental implantation.

Key words: Ti-Cu, SLA, Peri-implantitis, Osseointegration

Rui Liu, Yulong Tang, Hui Liu, Lilan Zeng, Zheng Ma, Jun Li, Ying Zhao, Ling Ren, Ke Yang. Effects of combined chemical design (Cu addition) and topographical modification (SLA) of Ti-Cu/SLA for promoting osteogenic, angiogenic and antibacterial activities[J]. J. Mater. Sci. Technol., 2020, 47: 202-215.

Figures/Tables 14

Table 1 Primer sequences used for quantitative RT-PCR analysis.

Genes	Primer (5′-3′)
16 s rRNA (P. gingivalis)	F: TGTAGATGACTGATGGTGAAA; R: ACTGTTAGCAACTACCGATGT
kgp (P. gingivalis)	F: AGCTGACAAAGGTGGAGACCAAAGG; R: TGTGGCATGAGTTTTTCGGAACCGT
16 s rRNA (S. mutans)	F: CCTACGGGAGGCAGCAGTAG; R: CAACAGAGCTTTACGATCCGAAA
brp (S. mutans)	F: GGAGGAGCTGCATCAGGATTC; R: AACTCCAGCACATCCAGCAAG
Gtf (S. mutans)	F: AGCCATGCGCAATCAACAGGTT; R: CGCAACGCGAACATCTTGATTAG
OPN	F: CTCCATTGACTCGAACGACTC; R: CAGGTCTGCGAAACTTCTTAGAT
ALP	F: CCTTGTAGCCAGGCCCATTG; R: GGACCATTCCCACGTCTTCAC
Collagen I	F: CTGACCTTCCTGCGCCTGATGTCC; R: GTCTGGGGCACCAACGTCCAAGGG
Runx 2	F: TTACCTACACCCCGCCAGTC; R: TGCTGGTCTGGAAGGGTCC
VEGF	F: ACTCGCCCTAATCCTCTTCC; R: TCAACACACTCACACACACAAC
MMP 2	F: CCCACTGCGGTTTTCTCGAAT; R: CAAAGGGGTATCCATCGCCAT
KDR	F: AGCCAGCTCTGGATTTGTGGA; R: CATGCCCTTAGCCACTTGGAA
FLT 1	F: GCGCTTCACCTGGACTGACA; R: GAAACTGGGCCTGCTGACATC
FAK	F: CCCCACCAGAGGAGTATG; R: CCAGGTCAGAGTTCAATAGCT
β-actin	F: CATGTACGTTGCTATCCAGGC; R: CTCCTTAATGTCACGCACGAT

Fig. 1. Photographs and SEM images of different dental implants: (a-c) photographs of Ti/SLA, Ti-Cu/SLA and Ti-Cu implants; (d-f) SEM images of three implant surfaces.

Fig. 2. (A) Study outline: three months before the implantation, each dog underwent bilateral extraction of mandibular premolars and first molars. Ligatures were placed with implant and removed at 3 months. Animals were euthanized at 3 months; (B) tooth implant: screw-shaped implant, with diameter of 3.5 mm and length of 10 mm; (C) dog mandible diagram: L1-L2 and R1-R2 are the left- and right-mandible implantation sites; (D) implantation depth; (E) bone resorption (peri-implantitis).

Fig. 3. CLM topography 3D maps and SEM images of different surfaces: (A) CLM topography 3D maps of Ti/SLA, Ti-Cu/SLA and Ti-Cu; (B) SEM images of three surfaces; (C) SEM images of three surfaces under high magnification.

Fig. 4. (A, B) XPS analysis of Ti-Cu/SLA and Ti-Cu; (C) Water contact angle of Ti, Ti-Cu/SLA and Ti-Cu as a function of time; (D) Cumulative Cu ions concentration curve released from Ti-Cu/SLA and Ti-Cu in 0.9% NaCl solution at 37 °C, grey lines standing for the fitting lines.

Fig. 5. PCR results and fluorescent images of P. gingivalis and S. mutans on surfaces of Ti/SLA, Ti-Cu/SLA and Ti-Cu after incubation for 24 h: (A) antibacterial rates of Ti-Cu/SLA and Ti-Cu compared to Ti/SLA by RT-PCR; (B) images of live bacteria (green) and dead bacteria (red) on surfaces; (C) quantity of live bacteria on the surfaces; (D) quantity of dead bacteria on the surfaces; (E) thickness of P. gingivalis and S. mutans biofilms on the surfaces. #p < 0.05, ##p < 0.01 and ###p < 0.001 compared to Ti-Cu, and **p < 0.01 and ***p < 0.001 compared to Ti/SLA.

Fig. 6. Adhesion of cells on samples and results of CCK-8 test: (A) HBMSCs stained with Rhodamine-phalloidin / DAPI after incubation for 4 and 24 h; (B) CCK-8 assay of HBMSCs cultured for 1, 3 and 7 days on different surfaces; (C) HUVECs stained with Rhodamine-phalloidin/DAPI after incubation for 4 and 24 h; (D) CCK-8 assay of HUVECs cultured for 1, 3 and 7 days on different surfaces.

Fig. 7. Osteogenic differentiation of HBMSCs on surfaces of Ti/SLA, Ti-Cu/SLA and Ti-Cu: (A) ALP activity of HBMSCs on different surfaces after 3 and 7days; (B) quantitative assay for ECM mineralization of HBMSCs on different surfaces after 14 and 21 days; (C-F) osteogenic differentiation by measuring mRNA expression level of ALP, OPN, Runx2 and collagen I after 3, 7 and 14 days. The value was normalized to β-actin. #p < 0.05 compared to Ti-Cu, and *p < 0.05, **p < 0.01 and ***p < 0.001 compared to Ti/SLA.

Fig. 8. Expressions of angiogenesis related genes by measuring mRNA expression level including FAK (A), MMP 2 (B), KDR (C) and FLT (D) after 3 and 5 days. The value was normalized to β-actin. ###p < 0.001 compared to Ti-Cu, and *p < 0.05, **p < 0.01 and ***p < 0.001 compared to Ti/SLA.

Fig. 9. Wound healing test and angiogenesis assay of HUVECs cultured in Ti/SLA, Ti-Cu/SLA and Ti-Cu extracts: (A) photographs of the wound and quantitative assay for the relative migration area at 0, 10, 20 and 24 h; (B) processed images of HUVECs cultured on Matrigel in the extracts for 4 h; the statistics of the number of branching points (C), loops (D) and tube length (E) formed in the culture after 4 h. #p < 0.05 and ##p < 0.01 compared to Ti-Cu, and *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the control Ti/SLA.

Fig. 10. Radiographs illustrating the bone levels at implants at 0 month (at the time of ligature placement) and 3 months (at the time of mandible movement) in the osseointegration dog model (A) and the peri-implantitis model (B), red arrows indicating the bone resorption levels; (C) Vertical bone resorption depth of the three kinds of implants in the radiographs.

Fig. 11. Micro-CT 2D reconstructions of Ti/SLA, Ti-Cu/SLA and Ti-Cu implants after 3 months implantation in the osseointegration dog model (A) and the peri-implantitis model (B), red arrows and red lines indicating the level of bone resorption; (C-F) parameters of bone around three kinds of implants after 3 months implantation in the two kinds of modeled dogs, BV/TV: bone volume/total volume, Tb.Th: trabecular thickness, Tb.N: trabecular number, Tb.Sp: trabecular separation.

Fig. 12. Micro-CT 3D reconstructions exhibiting various views of mandible and the implants (white in color) and bone response (red in color) after 3 months implantation in the osseointegration dog model (A) and the peri-implantitis model (B).

Fig. 13. Histological observations of Ti/SLA, Ti-Cu/SLA and Ti-Cu implants after 3 months implantation in the osseointegration dog model (A) and the peri-implantitis model (B), black sections standing for the implants, and blue violet sections standing for bone, OB: the old original bone, NB: the newly formed bone, CT: connective tissue, Im: the implant, yellow arrows indicating newly formed Haversian system; (C) BIC values of different dental implants after 3 months implantation in the two kinds of modeled dogs.

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