Journal of Materials Science & Technology, 2020, 47(0): 202-215 DOI: 10.1016/j.jmst.2019.10.045

Research Article

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

Rui Liua,b,1, Yulong Tangc,1, Hui Liua,d,1, Lilan Zenge, Zheng Maa, Jun Lia,d, Ying Zhao,e,*, Ling Ren,a,*, Ke Yanga

aInstitute of Metal Research, Chinese Academy of Sciences, Shenyang 110016, China

bInstitute of Molecular Medicine, State Key Laboratory of Oncogenes and Related Genes, Shanghai Institute of Cancer, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200011, China

cDepartment of Stomatology, General Hospital of Shenyang Military Command, Shenyang 110016, China

dSchool of Materials Science and Engineering, University of Science and Technology of China, Hefei 230026, China

eCenter for Human Tissues and Organs Degeneration, Shenzhen Institute of Advanced Technology, Chinese Academy of Science, Shenzhen 518055, China

Corresponding authors: * Corresponding authors. E-mail addresses:ying.zhao@siat.ac.cn(Y. Zhao),lren@imr.ac.cn(L. Ren).

First author contact: 1These authors contributed equally to this work.

Received: 2019-09-5   Accepted: 2019-10-30   Online: 2020-06-15

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.

Keywords: Ti-Cu ; SLA ; Peri-implantitis ; Osseointegration

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Cite this article

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. Journal of Materials Science & Technology[J], 2020, 47(0): 202-215 DOI:10.1016/j.jmst.2019.10.045

1. Introduction

Dental implants made of commercially pure titanium (Ti) and titanium alloys are currently the main treatment choice for replacing defective and absent teeth [1]. Titanium dental implant has been used in approximately 300,000 people each year, with some individuals receiving more than 12 implants [2]. A rapidly established, strong and long-lasting bond at the implant-bone interface is essential for the successful clinical application of implants. However, deficient or poor early bone healing at the interface of bone/implant often occurred in the early post-implantation period [3]. In addition, as regards the complexity of oral cavity there are hundreds of bacteria which can readily colonize on different types of surfaces especially immigrant implants and then evolve to form biofilms leading to the development of peri-implant diseases [4]. Therefore, it is essentially important to improve the bioactivity of dental implant materials, which should possess not only osteoconductivity (for guidance of new bone growth), the ability to stimulate osteogenesis (for promoting new bone formation), angiogenesis (for inducing vascularization), but also antimicrobial activity [[5], [6], [7], [8]].

Copper (Cu), an alloying element in metal materials, which is also an essential element in human body, has been proved to promote osteogenesis, angiogenesis and antimicrobial ability [9,10]. It has been utilized to chemically modify biomaterials for enhanced bioactivity. Wu et al. prepared Cu-containing mesoporous bioactive glass (Cu-MBG) scaffolds and the results indicated that the incorporation of Cu ions into MBG scaffolds significantly enhanced hypoxia-like tissue reaction leading to the coupling of angiogenesis and osteogenesis, and offered the scaffolds antimicrobial ability as well [4]. Recently, a novel class of integral Cu-bearing metallic biomaterials have been developed, including Cu-bearing stainless steels, Cu-bearing titanium alloys (Ti-Cu and Ti6Al4V-Cu) [[11], [12], [13], [14]], among which Ti-Cu alloy was previously proved to inhibit proliferation of oral bacteria including Streptococcus mutans and Porphyromonas gingivalis with good cytocompatibility [14], so it can be used potentially as dental implant material. Meanwhile, it was also proved that dental implants made of Ti-Cu alloy showed antibacterial ability in vivo to inhibit the bone resorption resulted from bacterial infection, which is ascribed to the addition of Cu [15].

Besides the above-mentioned chemical modification, topographical modification is another important approach to improving the biological performance of dental and orthopedic implants materials. The beneficial effects of micro- and nanoscale structure on osteoblast activity and osteoconductivity have been proved by many studies in vitro and in vivo [[16], [17], [18], [19], [20], [21]]. Among these, sandblasting and acid etching (SLA), inducing micro-submicron hybrid structures into the surface of materials, have been used to effectively stimulate cellular response and promote bone formation ability in vivo [1,22]. For example, commercial dental implants with SLA modified surfaces have been widely used in clinic, such as Straumann® Ti SLA® dental implant.

Based upon the above analyses, the Ti-Cu alloy was further modified by using SLA technique, hoping that combination of chemical design (Cu addition) and topographical modification (SLA) would offer significantly improved effects of biological activities, including osteogenesis, angiogenesis and antimicrobial ability as well. Thus, such improved biological activities and the related analysis of SLA treated Ti-Cu alloy (Ti-Cu/SLA) from both in vitro and in vivo were comprehensively explored, which will help this novel dental implant gain application in clinic.

2. Experimental

2.1. Materials and surface treatments

The Ti-Cu alloy was fabricated in a 30 kg consumable electrode vacuum arc-melting furnace. Then the Ti-Cu alloy ingot, with composition of Cu 5 (wt.%) and Ti in balance, was hot-forged to bars and heat treated at 850 °C for 2 h followed by cooling in air. Ti-Cu alloy was cut into disks (with a diameter of 10 mm and a thickness of 2 mm.) that were polished with SiC paper up to 2000 grits. Half of the Ti-Cu samples were sandblasted by Al2O3 (Φ250-500 μm) at a pressure of 3 atm. Blasted disks were treated in a mixture of HCl and H2SO4 (18% HCl:48% H2SO4 = 1:1, v/v) at 60 °C for 5 min and then cleaned adequately (denoted as Ti-Cu/SLA in this work). Pure Ti as the control was treated with the same sandblast and acid etching treatment (denoted as Ti/SLA in this work). All the samples were ultrasonically cleaned in acetone, ethyl alcohol and distilled water for three times in turn and finally dried in air. Prior to the experiment, all the samples were sterilized in 75% (v/v) ethyl alcohol and washed in sterile PBS.

2.2. Characterization of surfaces

The surface morphology of samples was examined by a scanning electron microscope (SEM, SIGMA500, ZEISS, Germany) and a confocal laser scanning microscopy (CLSM, OlympusFV10-ASW, Japan). The surface roughness (Ra) was measured by surface profilometry (ALpha-step IQ, KLA-Tencor, America). Three samples for each type of materials and five different tracks on each sample were measured. The Ra values were expressed as means ± standard deviations (SD) (n = 15).

Ti-Cu/SLA and Ti-Cu surfaces were used to investigate the content and existing form of the Cu element by X-ray photoelectron spectroscopy (XPS, ESCALAB250, Thermo VG, USA). The binding energy of copper was analyzed according to the NIST XPS database. The binding energy of C 1s (284.6 eV) was used as the reference to calibrate the results. According to the settings of the experimental parameters, the sputtering depth is 0.1 nm per second of sputtering time.

Static contact angles on three kinds of surfaces were measured at room temperature by the sessile drop method using 1 μL of DI water droplets in a contact angle-measuring device (Theta Lite, Biolin Scientific, Sweden). Three samples in each stage were used to provide an average and standard deviation.

2.3. Ionic concentrations analysis

After sterilized and dried, according to the ISO 10,993-12 standard, Ti-Cu/SLA and Ti-Cu samples were respectively immersed in the 0.9% (m/v) NaCl solution for various periods at 37 °C without stirring, with a ratio of extraction solution/sample surface area of 1.25 cm2/1 mL. The amounts of released Cu ions at 1, 3, 7, 14, 21 and 28 days after immersion were detected by an inductively-coupled plasma mass spectrometry (ICP-MS, Thermo, America).

2.4. In vitro antibacterial tests

2.4.1. Bacteria culture

Streptococcus mutans (S. mutans ATCC 25,175 and Porphyromonas gingivalis (P. gingivalis, ATCC33277) were provided by the Laboratory Center in China Medical University (Shenyang, China) and were cultured on fresh Brain Heart Infusion and blood agar plates (BHI, Oxoid, supplemented with 5 mg/mL yeast extract, 5 mg/mL hemin and 0.2 mg/mL menadione) under standard anaerobic conditions (80% N2, 10% H2 and 10% CO2, at 37 °C). The samples were respectively cultured with 1 × 107 cfu/mL S. mutans or 1 × 108 cfu/mL P. gingivalis for 24 h under standard anaerobic conditions. The antibacterial activities of Ti-Cu/SLA and Ti-Cu were determined by RT-PCR and Live/Dead staining.

2.4.2. Quantitative real-time PCR (RT-PCR)

After cultured in bacterial suspension for 24 h, the samples with adhered bacteria were taken out, gently washed in PBS and then vortexed within 2 mL fresh PBS in order to elute the adhered bacteria. Then the washed PBS was centrifuged and then the supernatant was discarded. Afterwards the DNA was extracted using a Bacterial DNA extraction kit (Takara, Japan). The concentration and purity of DNA were measured by a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA), and real-time PCR analysis was performed using a real time PCR system (ABI 7500, US). The default cycling program was used with the following cycling conditions: 95 °C for 30 s; 40 cycles at 95 °C for 5 s, 60 °C for 34 s; 95 °C for 15 s; 60 °C for 1 min and finally 95 °C for 15 s. Triplicate reactions were prepared with 20 μL of PCR mixture containing 10 μL of SYBR Premix Ex Taq Ⅱ, 2 μL of cDNA, 0.8 μL of PCR forward primer and 0.8 μL of PCR reverse primer, 6 μL of sterile distilled water and 0.4 μL of ROX Reference Dye Ⅱ for calibration. The primer sequences of 16 s rRNA, kgp for P. gingivalis and 16 s rRNA, brp and gtf for S. mutans were given in Table 1. Kgp is one of protease genes related to inactivation of host defense mechanisms, tissue destruction, and nutrient acquisition [23]. S. mutans produces glucosyltransferases (gtf), which synthesise glucan polymers from sucrose [24]. Adhesive glucans mediate the attachment of bacteria to the tooth surface. Brp is one of regulatory genes responsible for biofilm formation [25]. The real-time PCR produced a linear quantitative detection range over concentrations spanning seven exponential values, with a detection limit of a few copies of genomic DNA per reaction tube. Three independent experiments were performed for each sample, and data analyses were conducted by using LightCyclerR Software 3.5. The antibacterial rate (R) was calculated by Eq. (1), R ≥ 99% meaning strong antibacterial activity and R ≥ 90% antibacterial activity.

R = (Ccontrol - Cmaterial) / Ccontrol ×100%

where Ccontrol and Cmaterial are the average concentrations of adhered bacterial DNA for the control (Ti) and Ti-Cu samples, respectively.

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

GenesPrimer (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
OPNF: CTCCATTGACTCGAACGACTC; R: CAGGTCTGCGAAACTTCTTAGAT
ALPF: CCTTGTAGCCAGGCCCATTG; R: GGACCATTCCCACGTCTTCAC
Collagen IF: CTGACCTTCCTGCGCCTGATGTCC; R: GTCTGGGGCACCAACGTCCAAGGG
Runx 2F: TTACCTACACCCCGCCAGTC; R: TGCTGGTCTGGAAGGGTCC
VEGFF: ACTCGCCCTAATCCTCTTCC; R: TCAACACACTCACACACACAAC
MMP 2F: CCCACTGCGGTTTTCTCGAAT; R: CAAAGGGGTATCCATCGCCAT
KDRF: AGCCAGCTCTGGATTTGTGGA; R: CATGCCCTTAGCCACTTGGAA
FLT 1F: GCGCTTCACCTGGACTGACA; R: GAAACTGGGCCTGCTGACATC
FAKF: CCCCACCAGAGGAGTATG; R: CCAGGTCAGAGTTCAATAGCT
β-actinF: CATGTACGTTGCTATCCAGGC; R: CTCCTTAATGTCACGCACGAT

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2.4.3. Analysis of biofilm

After cultured for 24 h, the samples with biofilms were immersed in 800 μL PBS and stained with 50 μL of a mixed staining solution (SYTO-9:PI:PBS = 3:3:1000) using SYTO-9 (green) for staining alive and PI (red) for staining dead bacteria cells, according to the instruction of LIVE/DEAD Bacterial Viability Kit (Invitrogen Inc, USA). Afterwards, the samples were washed in PBS for three times and immediately observed under a confocal laser scanning microscopy (CLSM, OlympusFV10-ASW, Japan). The acquired images were quantitatively analyzed using the ImageJ software and three-dimension imaging was reconstructed by using the NIS Viewer software.

2.5. In vitro cytocompatibility evaluation

2.5.1. Cell culture

Human bone marrow derived mesenchymal stem cells (HBMSCs, Cyagen, China) were cultured in MEM Alpha Modification (αMEM, Hyclone, USA) with 10% fetal bovine serum (FBS, Corning, USA) and 1% penicillin/streptomycin and were used for the following experiments after 4th to 8th passages. For the osteogenic differentiation, after cultured for 3 days, cells were sequentially cultured in the osteogenic differentiation medium (α-MEM including 10% FBS, 1% penicillin/streptomycin, 100 nM dexamethasone, 50 μg/mL ascorbate and 10 mM β-glycerophosphate).

Human umbilical vein endothelial cells (HUVECs, Sciencell, USA) were cultured in the endothelial cell medium (ECM, Sciencell, USA) including 10% FBS, 1% penicillin/streptomycin and 1% VEGF growth factor. Only cells of 4-10 passages were used in the experiments.

All the cells were cultured at 37 °C in a 5% CO2 incubator, and the medium was replaced two or three times a week. The pretreated samples were put into the 48-well plates and immersed by 200 μL fresh medium, and then a 500 μL cell suspension with a density of 2 × 104 cells/mL was seeded onto each sample surface.

2.5.2. CCK-8 test

The cytotoxicity of Ti-Cu/SLA and Ti-Cu were detected using a CCK-8 kit (Beyotime, China) according to the ISO 10993-5 standard. After cultured for 1, 3 and 7 days, respectively, the samples were moved into new 48-well plates and were washed with PBS slightly, and then 10% CCK-8 solutions diluted with αMEM without FBS (diluted with ECM for HUVECs) were added to every well and incubated for 4 h at 37 °C in the incubator. After that, 100 μL of the solution from each well was transferred into a 96-well plate and the optical density (OD) was detected by ELIASA (Infinite 200 PRO, China) at 450 nm.

2.5.3. Cell morphology

After cultured for 4 and 24 h, respectively, the samples with adhered cells were washed with PBS twice, fixed with 4% (m/v) paraformaldehyde (PFA, Sigma, USA) for 10 min at room temperature, permeabilized with 0.1% (v/v) Triton X-100 (Amresco, USA) for 7-8 min, washed twice again, and then stained with rhodamine phalloidin (Invitrogen, USA) for 40 min without light and further stained with DAPI (Sigma, USA) for 5 min. The cell nuclei and F-actin were observed by a fluorescence microscope (FM-600, China).

2.5.4. Alkaline phosphatase (ALP) activity assay of HBMSCs

After cultured for 3 days, cells on the samples were sequentially cultured in the osteogenic differentiation medium for other 3 and 7 days respectively. After each time point, the cells on the surfaces were washed for three times with PBS and lysed in 0.2% (v/v) Triton X-100. Then the lysate was further incubated with Para-nitrophenyl phosphate (p-NPP, Beyotime, China) for 30 min at 37 °C. The absorbance of p-nitrophenol formed was measured at 405 nm. Meanwhile, the total protein content was determined using a BCA protein assay kit (Thermo, USA). Therefore, the ALP levels were normalized to the total protein content.

2.5.5. ECM mineralized nodule staining of HBMSCs

After cultured for 3 days, cells were sequentially cultured in the osteogenic differentiation medium for 14 and 21 days. When closing to the time point, the differentiation medium needed to refresh every two days. After each time point, the cells on the samples were fixed with 4% PFA for 15 min and further incubated with 20 mg/mL Alizarin Red staining (pH 4.1-4.3) for 30 min at 37 °C. After rinsing off the redundant stain with PBS, the mineralization was dissolved with 10% cetyl pyridinium chloride in 10 mM sodium phosphate. The optical density was measured at 620 nm.

2.5.6. Wound healing test of HUVECs

The migration of HUVECs in the extracts of Ti/SLA, Ti-Cu/SLA and Ti-Cu was evaluated using the wound-healing assay. The samples with only one surface were immersed in serum-free ECM (with no VEGF) for 10 days with a ratio of medium/sample surface area as 1.25 cm2/1 mL to obtain the extract solutions. Cells at a density of 3 × 104 cells/well were cultured on the 48-well cell culture plates for 24 h with the extract solutions (with 10% serum), and at this time the cells could reach confluence. After that, the scratches (cell-free gaps) were wounded using a micro-pipette tip (10 μL white tip), gently rinsed with PBS buffer, and then cultured in the same assay media (serum-free) for another 10, 20 and 24 h. The live cells and gap area changes in situ were photographed at every time point, and then measured using the ImageJ software. The results were discussed by the percentage of relative migration area.

2.5.7. Tube formation test of HUVECs

The tube formation test was used to evaluate the effect of released Cu ions on angiogenesis in vitro. In this assay, Matrigel provides for endothelial cells to create tubule structures, which is a multiple process in angiogenesis, including cell adhesion, migration and cell proliferation [26]. Each well in the 96-well plate was covered with 100 μL liquid Matrigel (Corning, USA), which was placed to solidify at 37 °C for 1 h. Cells with a density of 2 × 104 well/100 μL in extract solutions were cultured on the solid Matrigel at 37 °C. After 4 h, the formed tube-like structure was observed and photographed by an inverted microscope.

2.5.8. Quantitative RT-PCR analysis

The expressions of osteogenesis and angiogenesis related genes were evaluated by RT-PCR. HBMSCs and HUVECs at a certain density were respectively cultured on various sample surfaces for different time points. Cellular total RNA was gathered by a Trizol reagent (Invitrogen, USA) and reversely transcripted to cDNA using a PrimeScript cDNA synthesis kit (TaKaRa, Japan). The genes listed in Table 1 including OPN, ALP, collagen I and Runx 2 for hBMSCs and MMP 2, KDR, FLT 1 and FAK for HUVECs were quantified by Real-time PCR (Biorad CFX96, USA). The relative mRNA expression levels of genes were respectively normalized to β-actin.

2.6. In vivo experiments

2.6.1. Samples preparation

The Ti-Cu and Ti dental implants (3.5 mm in diameter and 10 mm in length) were manufactured according to the design drawings [27] and manufactured with surface configuration, sand blasting and acid etching (SLA), as shown in Fig. 1. Then all the implants were orderly cleaned with acetone, deionized water and ethyl alcohol in ultrasonic. Each implant was individually packaged, sterilized by ethylene oxide and stored at room temperature.

Fig. 1.

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.


2.6.2. Implantation surgery

The animal surgical procedures involved in this work were approved by the Animal Care and Experiment Committee of General Hospital of Shenyang Military Area. Model in beagle dogs was used, because of their likeness to human mandible bone in size and ease of handling. Three male beagle dogs of approximately 1 year old were used as an osseointegration model to evaluate the osteogenic effect and biocompatibility of Ti-Cu/SLA implant in vivo. After sedation with acepromazine (0.17 mg/kg body weight), the dogs were anesthetized with 21.5 mg/kg thiopental-sodium. For all the surgical procedures, inhalation anesthesia was administered using oxygen, nitrous oxide and isoflurane. Each underwent bilateral extraction of mandibular premolars and first molars, and three months later, implantation surgeries were performed and the implant sites were marked as shown in Fig. 2 (L1 indicating Ti/SLA implant, R1 indicating Ti-Cu/SLA implant and R2 indicating Ti-Cu implant). The cavity for implantation was prepared at mandibular by a twist drill, with diameter of 3.3 mm. Subsequently different implants (Ti/SLA, Ti-Cu/SLA and Ti-Cu) were implanted to a similar depth with coronal margin at the level of the alveolar bone crest. Every implant received a 3 mm custom-made healing abutment, which was sutured and covered by soft tissue. Normal soft diet was taken after surgery. Two weeks after implantation, the sutures were removed. Three months after implantation, the dogs were euthanized by an intravenous lethal overdose of sodium pentobarbital injection. The mandibles were retrieved and immersed in 10% formalin.

Fig. 2.

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).


In order to evaluate the anti-infection ability of Ti-Cu/SLA implant in vivo, a modified ligature-induced peri-implantitis model was built. Four male beagle dogs (1 year old, 13-16 kg in weight) were prepared. Tooth extraction and implantation surgeries were same as the above scheme. Every implant received a healing abutment. Cotton floss ligatures were placed around the abutments. Flaps were sutured through the abutments. Peri-implantitis was initiated by ligature placement and followed high-sugar soft diet (500 g/per dog/day). Healing was evaluated monthly and ligatures were made sure to be still in the whole process. After three months, the dogs were treated with lethal injection, and the mandibles were retrieved and stored in the formalin.

2.6.3. Radiographic inspection

At 0 month and 3 months after the treatment, sets of radiographs were obtained respectively. The radiographs were analyzed using an Olympus SZH10 stereo macroscope (Olympus, Tokyo, Japan) and digital images obtained with a Leica DFC280 camera (Leica, Wetzlar, Germany). The vertical bone resorption level, referring to the vertical distance between the implant shoulder and the marginal bone level, was assessed at the mesial and distal aspects of each implant using the QWin software (Leica Qwin Standard V3.2.0, Leica Imaging Systems Ltd., Cambridge, UK).

2.6.4. Micro-CT

After the mandibles were removed and stored in the fixative, the implants were scanned by local micro-computed tomography (Inveon, Siemens, Germany) at a scanning resolution of 14.97 μm to evaluate the changes in peri-implant bone tissue including bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp). The regions of interest including the trabecular compartment around implant were selected. The trabecular compartment region was defined as a ring with a radius of 0.5 mm from the implant surface.

2.6.5. Histomorphometric and histological analysis

After micro-CT imaging, the specimens were immersed in 10% buffered formalin solution at room temperature (25 °C). Then the tissue blocks containing the implant and the surrounding soft and hard tissues were dissected using a diamond saw (Exakts, Kulzer, Germany) and processed for ground sectioning according to the methods described by Donath & Breuner (1982). The blocks were embedded in Technovit 4000 VLC-resin (Kulzer, Friedrichsdorf, Germany). Then the blocks were cut in a buccal-lingual direction using a cutting grinding unit (Exakt, Apparatebau, Norderstedt, Germany). From each block, two buccal-lingual sections including the implant were prepared. All the sections were reduced to a thickness of 20-50 μm by micro-grinding and polishing using a micro-grinding unit (Exacts Cutting, System, Apparatebau Gmbh). These sections were stained by a Masson's trichrome staining kit. The histological examinations were performed using a LeicaS (Typ 007) camera (Leica, Wetzlar, Germany) and a Leica DM-RBE microscope (Leica, Heidelberg, Germany) with 100× magnification. The bone-implant contact (BIC) ratio and bone resorption values were identified and used for the linear measurement.

2.7. Statistical analysis

All the in vitro experiments were carried out in triplicate and three parallel samples were performed in every experiment. For each set, the relevant data was summarized as the mean standard deviation. Statistical significance was determined using SPSS 13.0 (SPSS Inc., Chicago, IL). The results were considered statistically significant at *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.

3. Results

3.1. Surface characterization and Cu ions release

The sandblasting and acid etching (SLA) process successfully created multiscale structures on both Ti/SLA and Ti-Cu/SLA surfaces, featured in micro-submicron hybrid as shown in Fig. 3. Different colors represent different depths of the pits in range of 5-10 μm in Fig. 3(A). In SEM photographs of Fig. 3(B), there were lots of large pits with size of 5-20 μm and in which there evenly distributed small holes on the surfaces of Ti/SLA and Ti-Cu/SLA. Under a high magnification, submicron-sized pits with size less than 1 μm can be observed in Fig. 3(C). The surface roughness (Ra) of Ti/SLA and Ti-Cu/SLA were (1.78 ± 0.82) and (2.04 ± 0.57) μm, respectively. As for Ti-Cu without SLA, there were only scratches of mechanical grinding on its surface, and the corresponding Ra was only (0.23 ± 0.03) μm.

Fig. 3.

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) depicts the XPS results of Ti-Cu/SLA and Ti-Cu surfaces, from which we can see that with increase of the sputtering depth, the Cu Concentrations of Ti-Cu surface steadily climbed upto the Cu content in base, while Cu distribution of Ti-Cu/SLA surface was stable. Meanwhile, the XPS spectra of Cu2p for Ti-Cu/SLA shows that the oxides of copper existed in the passive film on the surface. Fig. 4(C) displays the water contact angle changes of Ti/SLA, Ti-Cu/SLA and Ti-Cu surfaces. In Fig. 4(C), with increase of the exposure time in air, the contact angles of all the three surfaces increased. At the same time, we can see that contact angles were in range of 100°-120° for Ti/SLA and Ti-Cu/SLA, while about 90° for Ti-Cu, which indicates a much better wettability of Ti-Cu (non-SLA-treated). The amount of Cu ions released from the surfaces of Ti-Cu/SLA and Ti-Cu at different time points are presented in Fig. 4(D). With time going on, the amount of Cu ions released from either Ti-Cu/SLA or Ti-Cu surface were growing up. However, the release amount of Cu ions from Ti-Cu/SLA at every point was higher than that from Ti-Cu, meaning that the SLA modified surface promoted the release of Cu ions from Ti-Cu alloy. Meanwhile, the concentration of released Cu ions revealed a linear trend, and the slope was defined to be the average daily release rate of Cu ions [12]. The slope for Ti-Cu/SLA was 0.0256 μg/mL/day, higher than that for Ti-Cu (0.0134 μg/mL/day), further indicating that SLA treatment could increase the Cu ions release from Ti-Cu alloy.

Fig. 4.

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.


3.2. In vitro antibacterial abilities

The antibacterial activities of different samples were evaluated by RT-PCR based on the expression of biofilm formation involved genes, kgp for P. gingivalis and brp and gtf for S. mutans in Fig. 5. The expressions of the above genes were used to calculate the antibacterial rates (R) of Ti-Cu and Ti-Cu SLA by Eq. (1). In Fig. 5(A), Ti-Cu/SLA was found to have lower gene expressions of the two bacterial species compared to Ti/SLA. Additionally, the antibacterial rates of Ti-Cu/SLA (90%-99%) were higher than that of Ti-Cu group (40%-50%), revealing significantly enhanced antibacterial activity.

Fig. 5.

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.


The antibiofilm ability of samples was evaluated by CLSM as shown in Fig. 5(B). There are obviously less viable bacteria and more dead bacteria on Ti-Cu/SLA and Ti-Cu surfaces compared with Ti/SLA. Meanwhile, Ti-Cu/SLA showed better deteriorating impact on bacterial viability with more significant dispersion of bacteria than Ti-Cu. The numbers of sessile live and dead bacteria shown in Fig. 5(C) and (D) exhibited a consistent trend with the above antibacterial test results. The total numbers of live bacteria regardless of P. gingivalis and S. mutans on the Ti-Cu/SLA surface decreased by at least 90% compared to those on Ti/SLA. Rather, the total numbers of dead bacteria on Ti-Cu/SLA were at least 10 times more than Ti/SLA. Among the three materials, Ti-Cu/SLA presented the best antibacterial properties, followed by Ti-Cu, and Ti/SLA revealed the worst based on the significant different numbers of live/dead bacteria. The three-dimensional reconstructions shown in Fig. 5(B) and (E) further supported that the thickness of biofilms ranks as followed: Ti/SLA>Ti-Cu>Ti-Cu/SLA, which is in agreement with the above results of biofilm viability. Therefore, Ti-Cu/SLA showed the best antibacterial ability among the three materials.

3.3. Cell proliferation and adherence of HBMSCs and HUVECs

Fig. 6(A) shows that after cultured for 4 h, HBMSCs attached on Ti-Cu/SLA and Ti-Cu surfaces with more spreading filopodia extensions compared to Ti/SLA. While after 24 h, the cell morphology showed no significant difference between the three groups, and the spreading of cells on Ti-Cu surface showed specific directivity, which may be in connection with the machined scratches on surface. The HBMSCs on all the surfaces exhibit polygonal shapes with many filopodia and lamellipodia meaning good adherence. The viabilities of HBMSCs cultured on the surfaces for 1, 3 and 7 days were further evaluated using a Cell Counting kit-8 (CCK-8) (Beyotime, China) as shown in Fig. 6(B). There was no difference on optional density (OD) values among the three groups and they all kept increasing within 7 days, indicating that the cells grew well on all the sample surfaces. Thus, two kinds of Ti-Cu samples showed same satisfied cytocompatibility with Ti/SLA for HBMSCs.

Fig. 6.

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. 6(C) and (D) shows the cell adherence and proliferation of HUVECs on sample surfaces. In Fig. 6(C), after culture for 4 h, cell morphologies of HUVECs attached on Ti-Cu/SLA and Ti-Cu surfaces were similar to those on Ti/SLA. While after 24 h, more filopodia extensions could be seen on Ti-Cu/SLA and Ti-Cu surfaces. While for CCK 8 test shown in Fig. 6(D), there was also no difference on cell viability among the three groups. Thus, two kinds of Ti-Cu samples also showed same satisfied cytocompatibility with Ti/SLA for HUVECs.

3.4. Osteogenic differentiation

Besides cytocompatibility, the effect of Ti-Cu/SLA on the osteogenic differentiation of HBMSCs was assessed in terms of ALP activity, ECM mineralization and osteogenesis related gene expression. As shown in Fig. 7(A), the ALP activities of HBMSCs on Ti-Cu/SLA were all higher than those on Ti/SLA at every time point. On day 7, Ti-Cu/SLA presented a lower level of ALP activity than Ti-Cu, but still exhibited significantly enhanced ALP expression compared to Ti/SLA, suggesting good early osteogenic differentiation. As a marker of late osteogenic differentiation, ECM mineralization was examined after 14 and 21 days of HBMSCs incubation, as shown in Fig. 7(B), and the ECM mineralization levels on the Ti-Cu/SLA are all higher than those on Ti/SLA and Ti-Cu after incubation for 14 and 21 days.

Fig. 7.

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. 7(D) shows the gene expression of main osteoblast markers (ALP, Runx2, OPN and Collagen I). ALP was significantly promoted by Ti-Cu/SLA at 3 and 7 days compared with Ti/SLA while Ti-Cu showed a significant increase only at 3 days. Additionally, Ti-Cu/SLA also displayed higher Runx2 expression than Ti/SLA and Ti-Cu at 3 and 7 days. The OPN expressions of Ti-Cu/SLA and Ti-Cu had dramatically higher levels at 7 and 14 days. Therefore, the levels of osteogenesis-related gene expression indicated that Ti-Cu/SLA and Ti-Cu enhanced osteogenic differentiation of HBMSCs and especially Ti-Cu/SLA presented the best osteogenic property among the three materials.

3.5. Pro-angiogenic effects

In order to evaluate the effects of Ti-Cu/SLA on vascularization of HUVECs, angiogenesis-related genes expression was examined after 3 and 5 days of culture and the results were shown in Fig. 8. Ti-Cu/SLA and Ti-Cu significantly enhanced the expressions of FAK and MMP2 genes related to cell migration expression after 3 days of culture. Simultaneously, both expressions of KDR and FLT-1 of Ti-Cu/SLA were significantly higher than those of Ti/SLA at 3 days. High levels of angiogenesis-related genes expression suggest that Ti-Cu/SLA is favorable to promote the vascularization of HUVECs.

Fig. 8.

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.


Since endothelial cell migration is one of the essential processes during angiogenesis, a wound healing assay to identify the effect of Ti-Cu/SLA extract on collective endothelial cell migration was performed and the corresponding results are shown in Fig. 9(A). The black lines are the initial edges of cells, and the red lines are the edges of cells after migration. With increase of the incubating time, cells gradually migrated toward the central part for the three groups and thus the distance between the two red lines was shortened. As compared to Ti/SLA, the distance for Ti-Cu samples was much shortened, meaning better promoting effect on cells migration. Fig. 9(A) presents quantitatively calculated the migration areas for the three groups, and the results show that the migration area of cells for Ti-Cu/SLA was significantly higher than the other two materials, indicating its wonderful effect on cells migration.

Fig. 9.

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.


Tube formation assay using Matrigel was performed to examine the angiogenic effect of Ti-Cu/SLA and the corresponding results are shown in Fig. 9(B-E). After 4 h of culture, the HUVECs in the three extracts obviously entered the angiogenic process, as demonstrated by the formation of nodes and complex morphological characteristics of angiogenesis, including mesh-like circles and tube structures. As shown in Fig. 9(B), the tube formation with capillary-like structures was significantly induced by Ti-Cu/SLA extract compared to Ti/SLA and Ti-Cu. Quantitative measurements further confirmed excellent angiogenesis performance of Ti-Cu/SLA (Fig. 9(C-E)), suggesting that Ti-Cu/SLA also had positive effect on the tube formation of HUVECs.

3.6. In vivo test

An osseointegration dog model with no inductive infection was used to evaluate the osteogenesis effect and biocompatibility of implants, as shown in the part A of Fig. 10, Fig. 11, Fig. 12, Fig. 13. In addition, a modified ligature-induced peri-implantitis model was used to explore the anti-infection ability of samples, as shown in the part B of Fig. 10, Fig. 11, Fig. 12, Fig. 13.

Fig. 10.

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.

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.

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.

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.


3.6.1. Radiographic analysis

At the beginning of implantation (0 month) as shown in Fig. 10(a-f), radiographic photos showed that each implant was implanted, making sure that their coronal margins were at the level of the alveolar bone crest. In Fig. 10(A)(g-i), the Ti/SLA, Ti-Cu/SLA and Ti-Cu implants showed similar excellent osseointegration levels. After 3 months of implantation, there were a few shadows of low density on the bone-implant interface of Ti/SLA and Ti-Cu implants “shoulder” as shown by the red arrows, meaning the occurrence of bone resorption. However, there was scarcely low-density bone shadow around the Ti-Cu/SLA implant. Simultaneously, bone resorption depth (B.R) of Ti/SLA and Ti-Cu implants was in a range of 1.5-1.7 mm as shown in Fig. 10(C), whereas Ti-Cu/SLA implant was only 0.42 mm with satisfactory osseointegration and controllable bone resorption, indicating that Ti-Cu/SLA has better promoting ability on osseointegration during the healing period in comparison with Ti/SLA and Ti-Cu implants in osseointegration dog model.

For the modified ligature-induced peri-implantitis model, 3 months after implant, experimental peri-implantitis was successfully initiated as shown in radiographic photos of Fig. 10(B) (j-l). There was a low-density shadow on the bone-implant interface from the dotted red line to the solid red line of Ti/SLA implant, as shown in Fig. 10(B)(j), meaning serious bone resorption and poor bone bonding around the Ti/SLA implant due to the bacterial infection. Conversely, bone resorption only occurred around “shoulders” area for Ti-Cu/SLA and Ti-Cu implants as shown from red dotted to solid line in Fig. 10(B)(k-l). The amounts of bone resorption (length of red arrows between the dotted line and solid line) were 5.69 ± 1.63 mm for Ti/SLA implant, 2.28 ± 0.13 mm for Ti-Cu/SLA implant and 2.60 ± 0.25 mm for Ti-Cu implant, respectively. This statistically significant difference means when bacteria invasion occurred, Ti-Cu/SLA and Ti-Cu implant could inhibit the bone resorption related to bacterial infection, and meanwhile the two kinds of implants keep better osseointegration than Ti/SLA implant.

3.6.2. Micro-CT analysis

Micro-CT profiles after 3 months of implantation were further taken to evaluate the abilities of osseointegration and anti-infection of different implants. In Fig. 11(A), compact bones grew on the surfaces of the three implants, presented as high green fluorescence intensity. Only mild degree of angular resorptions could be observed around the implant “shoulder” areas, pointed as red arrows and red lines of Ti/SLA and Ti-Cu implants. However, even no resorption could be observed for Ti-Cu/SLA implant indicating its benefit on osseointegration, which is in agreement with the result of X-ray as shown in Fig. 10(A). The micro-CT profiles in Fig. 11(B) obviously demonstrate that after the ligature-induced infection for 3 months, all the implants appeared obvious angular resorption around implant “shoulders” caused by bacterial infections. But the resorption depths (the length of red arrow) of either Ti-Cu/SLA or Ti-Cu implant were far below than that of Ti/SLA. Meanwhile, it was obviously observed that there was close contact between implant and bone tissue for Ti-Cu/SLA and Ti-Cu groups meaning satisfied osseointegration. Conversely, there was a large gap between the surface of Ti/SLA implant and the bone tissue demonstrating a poor osseointegration due to the bacterial infection.

Furthermore, the quantitative micro-CT results of the osseointegration model, as shown in Fig. 11(C-F), including BV/TV, Tb.Th, Tb.N and Tb.Sp, displayed no significant differences among the three kinds of implants. But the quantitative results of the peri-implantitis model showed that BV/TV (%) of the Ti-Cu/SLA and Ti-Cu implants were respectively 57.97 and 51.74, which were much higher than that of Ti/SLA implant (34.01). In addition, the thickness of the newly formed bone surrounding the implant (Tb.Th) was obviously larger for both Ti-Cu/SLA and Ti-Cu implants (0.23 and 0.18) than that for Ti/SLA (0.12). The micro-CT results proved that the bone resorption inhibition and osseointegration for implants were in a sequence of Ti-Cu/SLA > Ti-Cu > Ti/SLA, consistent with the X-ray results.

Fig. 12 displays 3D micro-CT reconstruction showing the implant (white color) and peri-implant bone (red color) of the osseointegration dog model (Fig. 12(A)) and peri-implantitis model (Fig. 12(B)). In Fig. 12(A), lots of newly formed peri-implant bones can be observed around the middle and root of the three kinds of implants. Certainly, Ti-Cu/SLA and Ti-Cu implants had more newly formed bones than Ti/SLA implant. However, as shown in Fig. 12(B), the peri-implant bone volume around the three implants is significant influenced by bacterial infection. There was more compact formed bone around the Ti-Cu/SLA implant as shown in Fig. 12(e), while the newly formed bone around the Ti/SLA implant was scarce. Furthermore, there was more new bone formed around Ti-Cu implant than that of Ti/SLA.

3.6.3. Histological observations

Fig. 13 shows full pictures of implant-bone histological sections and the bone issues present as blue-violet area. In Fig. 13(A) of osseointegration dog model there was fully new bone bonding around Ti-Cu/SLA implant as well as around Ti/SLA and Ti-Cu implants, meaning that Ti-Cu/SLA implant had same good biocompatibility and osseointegration viability with Ti/SLA and Ti-Cu implants. Bone-implant contact (BIC, %) was calculated as the ratio between the length of the bone in direct contact with the surface of the implant and the length of the implant [26]. The result showed that Ti-Cu/SLA implant had higher BIC rate (63.37%±3.5%) than Ti/SLA implant (48.94%±5.77%) and Ti-Cu implant (59.60%±5.12%), suggesting that Ti-Cu/SLA implant had better osseointegration ability than Ti/SLA implant.

In Fig. 13(B) of peri-implantitis model, the bone resorption around Ti/SLA implant was much stronger than that of Ti-Cu/SLA and Ti-Cu implants, and there was connective tissue (Fig.13(k)) between bone and implant and almost no bone bonding around Ti/SLA implant, meaning that the implantation for Ti/SLA failed after 3 months. However, the Ti-Cu/SLA and Ti-Cu implants showed wonderful bone bonding and only slight bone resorption. Meanwhile, as shown in Fig. 13(C), Ti-Cu/SLA and Ti-Cu implants had significantly higher BIC rates (51.90%±5.33% and 39.57%±2.58%) than Ti implant (2.17%±1.34%). This proved that Ti-Cu/SLA and Ti-Cu implants could reduce the bacterial infection and then had better osseointegration ability than Ti/SLA implant. Meanwhile, Ti-Cu/SLA implant appeared a lower bone resorption ratio (22.7%±10%) than Ti-Cu implant (28%±6.5%) meaning the best anti-infective and osseointegration effect.

4. Discussion

Our early studies [14,15] proved that Ti-Cu alloy exhibited excellent antibacterial ability, thereby inhibiting the bone resorption related to bacterial infection in vivo. However, the previous work was only focused on the Ti-Cu alloy without any surface treatment, just evaluating the antibacterial role of Cu addition. While currently in clinic, variety of commercial dental implants such as Straumann and Dentply implants are modified by SLA treatment in order to have rapid bone healing and osseointegration [27]. Therefore, based on the preliminary work, Ti-Cu alloy with SLA treatment was examined to reveal the effects of both chemical design (Cu addition) and topographical modification (SLA) on biological activities, such as osteogenesis, angiogenesis and antibacterial ability, and meanwhile the relevant mechanism was also analyzed.

4.1. Osseointegration

4.1.1. First comparison: Ti-Cu/SLA with Ti/SLA

Both in vivo and in vitro experimental results reflected that Ti-Cu/SLA had better osseointegration ability than Ti/SLA in the present study. So, what is the key factor leading to this positive effect? Ti-Cu/SLA and Ti/SLA possessed similar surface situation due to the same SLA treatment with hybrid micro-submicron topography. Therefore, the only difference of the Cu addition should be responsible for the positive effect on osseointegration of Ti-Cu/SLA compared with Ti/SLA. The mechanism of Cu promoting osteogenesis has been well studied [12,[28], [29], [30], [31]]. Chengtie Wu et.al proved that Cu ions released from Cu-MBG scaffolds significantly promoted the osteogenic differentiation of HBMSCs by improving their bone-related gene expressions (alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN)) [4]. The result in the present study is in agreement with the above reference results [4] that Ti-Cu/SLA upregulated the expression of bone related genes, and thus presented an excellent osteogenesis role in vivo. Moreover, it is well known that bone vasculature plays a pivotal role in bone growth, remodeling, and homeostasis [32,33]. Previous studies also indicated that Cu could induce angiogenesis by improving vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1a protein secretion as well as angiogenesis-related gene expressions (VEGF, HIF-1a, VEGF receptor 2 (KDR) and endothelial nitric oxide (eNOS)) of human umbilical vein endothelial cells [34]. Our results also showed the pro-angiogenic potential of Cu ions from Ti-Cu/SLA, appearing as outstanding endothelial cell migration and tube formation, as well as enhancing angiogenic-related gene expressions. Consequently, compared with Ti/SLA, Ti-Cu/SLA could promote both angiogenesis and osteogenesis due to continuous release of Cu ions.

4.1.2. Second comparison: Ti-Cu/SLA with Ti-Cu

Compared with Ti-Cu implant, Ti-Cu/SLA implant showed slightly increased bone bonding and significant inhibition on marginal bone loss. So, what is the key factor leading to this positive effect? Ti-Cu/SLA and Ti-Cu had the same composition. However, their surface roughness was significantly different. The roughness of Ti-Cu was 0.23 ± 0.03 μm, but that of Ti-Cu/SLA was greatly increased to 2.04 ± 0.57 μm by SLA treatment. Thus, the different surface roughness should be responsible for the positive role of Ti-Cu/SLA on better osteoconductivity compared with Ti-Cu. Studies [35,36] have indicated that rough surface structure could increase the surface area and thus improve cell adhesion, interaction and proliferation. In addition, the enlarged surface area due to SLA treatment also enhanced the release of Cu ions from Ti-Cu/SLA compared to Ti-Cu as shown in Fig. 4. Therefore, the better osseointegration ability for Ti-Cu/SLA implant can be well explained.

In a word, for the osseointegration dog model, all the Ti/SLA, Ti-Cu/SLA and Ti-Cu implants could have good osseointegration and succeed in implantation. However, Ti-Cu/SLA implant has better osseointegration activity than Ti/SLA and Ti-Cu, resulting from the combined effect by the biological activity of Cu ions and micro-submicron structure.

4.2. Anti-infection

4.2.1. First comparison: Ti-Cu/SLA with Ti/SLA

Based on the above osseointegration results, bacterial infection was introduced to acquire peri-implantitis to further explore the infection and osseointegration effects of different dental implants. In the peri-implantitis model induced by ligature placement, serious bone resorption happened to Ti/SLA implant after 3 months implantation, with almost no bone bonding or new bone formation around the Ti/SLA implant, which can be recognized as implantation failure. However, the implantation of Ti-Cu/SLA implant was successful with higher BIC standard even under the peri-implantitis. The main reason for the implantation failure is that Ti/SLA implant has no self-protection against the bacterial invasion. What is worse, rough surface is also in favor of bacterial attachment and bacterial biofilm formation, which then would cause more severe infection. Previous studies recognized that implants with high surface roughness were beneficial to bacterial biofilm formation compared with the smooth group at early stage of implantation [37,38]. Consequently, rough surface of Ti/SLA should be of benefit to bacterial adhesion like a warm “harbor” which readily formed biofilm and then easily resulted in occurrence of infection. Although Ti-Cu/SLA also possessed the rough surface, it was more like a “trap” to provide more chances for adhered bacteria to contact with Cu ions, and then Cu ions would kill them to prevent bacteria from forming a biofilm structure on Ti-Cu/SLA surface and hence reduce infection. Thus, Ti-Cu/SLA implant could effectively inhibit the bacteria-related bone resorption.

4.2.2. Second comparison: Ti-Cu/SLA with Ti-Cu

Compared with Ti-Cu, Ti-Cu/SLA has increased surface area for more Cu ions release as shown in Fig. 4. Hence, Ti-Cu/SLA exhibited superior antibacterial property to Ti-Cu. Therefore, in the peri-implantitis model, with bacterial infection for 3 months, lower bone resorption occurred on Ti-Cu/SLA implant than that on Ti-Cu implant, meaning that Ti-Cu/SLA implant could more effectively restrain the bone resorption caused by bacterial infection. Moreover, even if under infection conditions, Ti-Cu/SLA implant still held better surrounding bone bonding and more excellent osteogenesis, which was also beneficial from the osseointegration ability of Ti-Cu/SLA mentioned above.

In summary, when introducing a bacterial infection model, Ti/SLA implant had serious consequences for failure whereas Ti-Cu/SLA and Ti-Cu implants could still resist the infection and keep good osseointegration. What’s more, in terms of infection inhibition and osseointegration ability, Ti-Cu/SLA has better performance than Ti-Cu, which means that the novel Ti-Cu/SLA implant was proved to have excellent anti-infection property and favorable osseointegration, due to the combination of Cu ions release and micro-submicron structure.

More importantly, considering the “race for the surface” between the tissue cell integration and bacterial adhesion, if the surface of implant has wonderful osseointegration ability, by promoting cell integration and rapid bone formation, it could also reduce the bacterial adhesion. The results in this study indicated that without induced infection, Ti-Cu/SLA implant had the best osseointegration activity. While when infection occurred, Ti-Cu/SLA improves resistance of infection, benefiting from combining of the pro-osseointegration and antibacterial effects of the surface. That means the multifunctional surface of Ti-Cu/SLA gives host cells a leg up in winning the race for the surface, via both discouraging bacterial adhesion and biofilm growth and promoting bone integration simultaneously.

5. Conclusion

In this study, the bio-functions of Ti/SLA, Ti-Cu/SLA and Ti-Cu were examined. On account of the combined effect of Cu addition and topographical modification, Ti-Cu/SLA exhibited better performance than Ti/SLA and Ti-Cu, appearing to have better antibacterial, osteogenesis and angiogenesis activities in vitro as well as osseointegration and anti-infection abilities in vivo. All in all, the multifunctional Ti-Cu/SLA implant with excellent osteoinductivity, pro-osseogenic, pro-angiogenic and antibacterial ability, has great potential to be used as a new generation dental implant in clinical application.

Acknowledgements

This work was financially supported by National Natural Science Foundation (Nos. 51631009, 51811530320 and 81572113), National Key Research and Development Program of China (Nos. 2018YFC1106600 and 2016YFC1100600), Innovation Fund Project of Institute of Metal Research, Chinese Academy of Sciences (No. 2017-ZD01), Key Projects for Foreign Cooperation of Bureau of International Cooperation Chinese Academy of Sciences (No.174321KYSB2018000) and Shenzhen Science and Technology Research Funding (No. JCYJ20160608153641020).

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Biocompatible synthetic scaffolds with enhanced osteogenic and angiogenic capacity are of great interest for the repair of large (critical size) bone defects. In this study, we investigated an approach based on the controlled delivery of copper (Cu) ions from borate bioactive glass scaffolds for stimulating angiogenesis and osteogenesis in a rodent calvarial defect model. Borate glass scaffolds (pore size = 200-400 mum) doped with varying amounts of Cu (0-3.0 wt% CuO) were created using a polymer foam replication technique. When immersed in simulated body fluid (SBF) in vitro, the scaffolds released Cu ions into the medium at a rate that was dependent on the amount of Cu in the glass and simultaneously converted to hydroxyapatite (HA). At the concentrations used, the Cu in the glass was not cytotoxic to human bone marrow derived stem cells (hBMSCs) cultured on the scaffolds and the alkaline phosphatase activity of the hBMSCs increased with increasing Cu in the glass. When implanted in rat calvarial defects for 8 weeks, the scaffolds doped with 3 wt% CuO showed a significantly better capacity to stimulate angiogenesis and regenerate bone when compared to the undoped glass scaffolds. Together, these results indicate that the controlled delivery of Cu ions from borate bioactive glass implants is a promising approach in healing bone defects.

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Bone vasculature plays a vital role in bone development, remodeling and homeostasis. New blood vessel formation is crucial during both primary bone development as well as fracture repair in adults. Both bone repair and bone remodeling involve the activation and complex interaction between angiogenic and osteogenic pathways. Interestingly studies have demonstrated that angiogenesis precedes the onset of osteogenesis. Indeed reduced or inadequate blood flow has been linked to impaired fracture healing and old age related low bone mass disorders such as osteoporosis. Similarly the slow penetration of host blood vessels in large engineered bone tissue grafts has been cited as one of the major hurdle still impeding current bone construction engineering strategies. This article reviews the current knowledge elaborating the importance of vascularization during bone healing and remodeling, and the current therapeutic strategies being adapted to promote and improve angiogenesis.

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