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Si-doped porous TiO2 coatings enhanced in vitro angiogenic behavior of human umbilical vein endothelial cells
Accepted Manuscript Title: Si-doped porous TiO2 coatings enhanced in vitro angiogenic behavior of human umbilical vein endothelial cells Authors: Zhihong Ding, Yuqin Qiao, Feng Peng, Chao Xia, Shi Qian, Tao Wang, Junying Sun, Xuanyong Liu PII: DOI: Reference:
S0927-7765(17)30526-X http://dx.doi.org/doi:10.1016/j.colsurfb.2017.08.010 COLSUB 8766
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
14-5-2017 30-7-2017 3-8-2017
Please cite this article as: Zhihong Ding, Yuqin Qiao, Feng Peng, Chao Xia, Shi Qian, Tao Wang, Junying Sun, Xuanyong Liu, Si-doped porous TiO2 coatings enhanced in vitro angiogenic behavior of human umbilical vein endothelial cells, Colloids and Surfaces B: Biointerfaceshttp://dx.doi.org/10.1016/j.colsurfb.2017.08.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Si-doped porous TiO2 coatings enhanced in vitro angiogenic behavior of human umbilical vein endothelial cells Zhihong Ding a,b, Yuqin Qiao c,, Feng Peng c, Chao Xia c, Shi Qian c, Tao Wang a, Junying Sun a,*, Xuanyong Liu c, a
Orthopaedic Department, The First Affiliated Hospital of Soochow University, 188
Shizi Street, Suzhou, Jiangsu 215006, P.R. China b
Orthopedics of Shanghai Pudong New area Gongli Hospital, Shanghai 200135, P.R.
China c
State Key Laboratory of High Performance Ceramics and Superfine Microstructure,
Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, P.R. China
Author to whom correspondence would be addressed
Both Zhihong Ding and Yuqin Qiao are equal contribution to this work. Tel.: +86 21 52412409; fax: +86 21 52412409; E-mai: [email protected] (X. Y. Liu), [email protected] (J. Y. Sun)
Graphical Abstract
Small amount of silicon doping significantly enhanced angiogenic activity of HUVECs.
Highlights Silicon-doped porous TiO2 coatings were fabricated via plasma electrolyte oxidation method (PEO) successfully. Si doping induced dose-dependent stimulation in angiogenic activities of HUVECs. Larger amount of Si results in reduced angiogenic responses.
Abstract Recent evidence demonstrates that vessel involvement is crucial in various bone remodeling situations, indicating that blood vessel formation within or surrounding the implant is essential for establishment of rigid implant fixation. In this work, the ability of the silicon-doped porous TiO2 coatings fabricated via plasma electrolyte
oxidation method (PEO) to enhance the angiogenic potential of human umbilical vein endothelial cells (HUVECs) were investigated. The cellular responses of HUVECs on the silicon-doped porous TiO2 coatings were studied through cell proliferation, vascular endothelial growth factor (VEGF) secretion, and angiogenic-associated gene (VEGF, HIF-1 and HGF) expression analysis. The results show that small amount of silicon significantly enhanced angiogenic activity of HUVECs, while larger amount of silicon appears excessive. Hence, the silicon-doped TiO2 coating offers a potential solution to improve bone vascularization to achieve efficient osseointegration and restoration of function after implantation.
Keywords : Si, porous TiO2 coatings, HUVECs, angiogenic potential
1. Introduction More recently, increasing evidence has demonstrated that osteogenesis is coupled with angiogenesis during bone regeneration and remodeling [1]. Upon implantation, angiogenesis occurs rapidly in response to inflammation of host tissue to develop new blood vessels from pre-existing vessels [2]. The vasculature ensures the sufficient
supply of circulating cells, nutrients, oxygen and metabolic wastes essential for maintaining osteoblastic bone matrix synthesis and mineralization [3, 4]. In contrast, insufficient vascularization in bone defects cannot meet the growing tissue nutrient delivery and clearance needs, leading to inadequate osseointegration of implants. Hence, the importance of angiogenesis as an essential component in the design of orthopedic and dental implants to enhance the process of skeletal tissue engineering should be brought to the forefront. The process of angiogenesis involves a series of steps including basement membrane degradation, endothelial cell migration and proliferation, and capillary tube formation mediated by various angiogenic factors [5]. During bone development and remodeling, endothelial cells invade from the perichondrial tissues to form blood vessels for nutrient supply and serve as a scaffold for new bone formation [6-8]. Capillaries also merge to form a network to maintain long-term bone viability. Notably, recent studies suggest that the reciprocal crosstalk between endothelial and bone cells can further modulate the bone behavior and angiogenesis [9-12]. Endothelial cells have been found to induce the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs) and stimulate the expression of osteogenic markers through direct contact co-culture [9]. Accordingly, BMSCs exhibited
a
supportive
expression
of
angiogenic
function
and
enhanced
vascularization for endothelial cells [9]. Thus, designing biomaterials with combined osteogenic and angiogenic potential remains a rational and challenging strategy for the bone healing and remodeling.
Titanium (Ti) and its alloys have been widely used for dental and orthopedic implants device because of their good biocompatibility and mechanical properties [13]. Over the years, increasing efforts have been focused on promoting osteogenic activities to enhance osseointegration [14]. However, this cannot fulfill all the requirements of a successful bone grafting [15]. To promote angiogenesis and vascularization in Ti and its alloys, a number of strategies have been explored to tailor their physicochemical properties (i.e., surface topography and chemical composition) [16-18]. Among these properties, surface topography is known to have a great influence on protein adsorption and cell behaviors [19-21]. Topographical dimensions ranging from nanoscale to micron have been reported to enhance angiogenesis by stimulating growth factors secretion and up-regulating the expression of angiogenic-associated genes [22-24]. Bone growth and development involves a variety of trace elements, which have been shown to increase osteogenesis and/or neovascularization. Tuning chemical compositions of biomaterials by doping vascularization-favorable elements (e.g., Cu, Mg, Sr, Co, etc.) also seems an efficient approach to improve angiogenesis [24-27]. To achieve the bifunctional purpose, incorporating elements combining angiogenesis potential and osteostimulation to biomaterials seems to be a promising therapy strategy for bone healing and remodeling. Silicon (Si) is one of the elements which play multiple functions in bone formation and maintenance, vascular health, and wound healing [28, 29]. In this sense, Si has the potential to be a candidate of interest added to biomaterials for dental and
orthopedic implant applications. In our previous studies, we have found Si doping can obviously enhance the osteogenic potential of TiO2 coatings [30-32]. Therefore, the objective of this study is to investigate the angiogenic capability of Si-doped porous TiO2 coatings. Si-free and Si-doped TiO2 porous coatings were fabricated using the plasma electrolyte oxidation (PEO) method as reported previously, and the influence of silicon elements on the functionality of human umbilical vein endothelial cells (HUVECs) was evaluated by examining the cell viability and morphology, migration assay, tube formation assay, VEGF secretion and expression of angiogenic-associated genes.
2. Materials and methods 2.1 Preparation of Si-doped TiO2 coatings Commercially pure titanium plates (Cp Ti, TA1, purity > 99.85%) with dimensions of 10 mm 10 mm 1 mm were used in cellular experiments, and 20 mm 20 mm 1 mm in size were used in the molecular experiments to extract sufficient nucleic acid samples. They were ground with 400# abrasive papers and then ultrasonically washed with acetone, ethyl alcohol and distilled water in an ultrasonic bath prior to plasma electrolyte oxidation (PEO) treatment. PEO was conducted in electrolytes composed of 0.1 mol/L calcium acetate monohydrate (CA, C4H6O4CaH2O), 0.05 mol/L glycerophosphate disodium salt pentahydrate (GP, C3H7Na2O6P5H2O) and various amounts of disodium silicate (Na2SiO3) to prepare Si-free (S0) and Si-doped coatings (S1 and S2). By adjusting the concentrations of disodium silicate (Na2SiO3) 0.01 and 0.02 mol/L) in the electrolytes, coatings designated as S1 and S2 were produced.
During the PEO treatment, a Ti plate was used as an anode, and a spiral steel pipe was used as a cathode and a dwelling water pipe to sustain the temperature of the electrolyte < 30C in the electrolytic cell. A magnetic stirrer was employed to keep the components and temperature of the electrolytes uniform. After PEO treatment, the samples were boiled for 4 min, washed with deionized water and air dried. 2.2 Characterization of Si-doped TiO2 coatings Scanning electron microscopy (S-3400N, Hitachi, Japan) was used to examine the surface morphology of the coatings and energy-dispersive X-ray spectrometry (EDS) attached to an electron probe X-ray microanalysis system (EPMA, JAX-8100, Japan) was performed to measure the elemental compositions. X-ray diffraction (XRD, D8 ADVANCE, Bruker) was employed to analyze their phase compositions. 2.3 Si ion release The Si-doped samples were immersed in 10 ml PBS at 37C for 7 days, and Si concentration of the buffer solution at different time points (1, 4, 7 and 14 days) was measured by an inductively coupled plasma/optical emission spectroscopy (ICP-OES; Vista AX, Varian, USA). 2.4 Cultivation of HUVECs Human umbilical vein endothelial cells (HUVECs, ScienCell) were cultured in complete Endothelial Cell Growth Medium (EGM) supplemented with 10% fetal bovine serum (FBS), endothelial cell growth supplement and penicillin/streptomycin (ScienCell, CA) at 37C in an incubator of 5% CO2. The culture medium was changed every 2 days.
2.5 Cell proliferation and morphologies AlamarBlue assay was performed to evaluate the cell proliferation. Briefly, specimens with size of 10 10 1 m3 were placed in 24-well plates, HUVECs were then seeded at a density of 5 104/well. After culturing for 1, 4, and 7 days, the cells were incubated in culture medium supplemented with 10% AlamarBlue (Invitrogen, USA) for 2 h at 37 C. Post reaction medium aliquots (100 l) were transferred to 96-well solid black plates and the fluorescence intensity was recorded using Cytation 5 Multi-Mode Reader (Biotek, USA) at 560 nm (Ex)/590 nm (Em). The morphologies of HUVECs on different samples at day 7 then was stained using live/dead viability/cytotoxicity kit (Invitrogen, USA). 2.6 Cell Migration HUVECs were seeded at a density of 5 104/well on samples with size of 10 10 1 m3. After 1 d adhesion, lines crossing the cells were drawn by 20-200 l pipet tips. After cultivation for 8 h, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 1% BSA in PBS, and then stained with Rhodamine-phalloidin and DAPI at room temperature. After each step, the samples were washed 3 times for 5 min each with PBS. The images were captured on a fluorescence microscope (Olympus IX 71, Olympus, Japan). 2.7 Matrigel tube formation assay In vitro angiogenesis was assessed by the endothelial tube formation assay according to the manufacturer’s instructions. Prior to the assay, samples were autoclaved and later immersed in 1 ml complete cell growth medium and in the
incubator for 24 h. The extracted solutions were then collected. The Growth factor reduced matrigelTM (BD Biosciences) was thawed out at 4C, followed by adding 140 l of thawed gel solution to each well of a prechilled 48-well plate. After the Matrigel was allowed to gel in the incubator for 1 h, HUVECs were seeded at a density of 3 104 cells/well in complete cell growth medium. While cell adhesion was completed after 3 h, cell growth medium was removed and changed to extracted solutions. The assay plate was then incubated for 12 h, and stained with Calcein AM (Sigma-Aldrich, US) for 30 min in the incubator. The tube formation of HUVECs was observed and analyzed using an Olympus fluorescence microscope and imaged at 10 magnification. 2.8 Quantification of VEGF protein secretion The production of vascular endothelial growth factor (VEGF) proteins was quantified using the Enzyme-linked Immunosorbent Assay (ELISA) Kit (Abcam, USA) in accordance to the manufacturer’s instruction. Briefly, HUVECs were cultured on specimens for the prescribed time period, and proteins from cell culture supernatant were collected and quantified using the ELISA kit. 2.9 RNA extraction and real-time quantitative RT-PCR analysis The total RNA was extracted using the Trizol reagent (Invitrogen Life Technologies) at days 4, 7 and 14 according to the manufacture’s protocols. The RNA concentration and purity were determined spectrophotometrically by Take3TM Micro-Volume Plate of Cytation 5 Multi-Mode Reader (Biotek, USA). In the cDNA synthesis, 1 g of total RNA was reversely transcribed using Transcriptor First Strand cDNA Synthesis
Kit (Roche) and oligo dT18 primers (Roche). The real-time polymerase chain reaction (RT-PCR) was used to quantify the gene expression of the angiogenic related genes. In brief, the 10-fold diluted cDNA was used as the template in the 10-l qRT-PCR reactions using the SYBR Green Matermix and Roche LightCycler 480 System (Roche). The level of each target gene was calculated by the CT method and GAPDH gene expression was used as an endogenous control for normalization. Each sample was analyzed in triplicates and the reaction mixture without the cDNA was used as a negative control in each run. The PCR primer sequences used in this project are listed in Supplementary Information (Supplementary Table S1). 2.10 Statistical analysis Data were expressed as means standard deviations (SD) from experiments performed in quintuplicate. Statistical analysis was performed using the one-way analysis of variance (ANOVA) with Tukey-Kramer Multiple comparison post-test using GraphPad Instant Software (GraphPad Prism Software, Inc., USA) with p < 0.05 being considered to be statistically significant.
3. Results and discussion Fig. 1 shows the SEM surface micrographs of the cp Ti, Si-free and Si-doped coatings. It can be seen that silicon doping does not alter the main structural characteristics of plasma electrolyte oxidized TiO2 coatings including the porous morphology, pore size ( 10 m), and homogeneous distribution of pores. The strong Si signals in the inserted EDS spectra of S1 and S2 indicate successful Si doping by PEO. The surface EDS maps in
supplementary information (Fig. S1) clearly illustrate that the coatings have uniform distributions of the major nutritional elements (Ca, P and Si). XRD patterns of the S0, S1 and S2 coatings are shown in Fig. 2. All these coatings display similar patterns, which mainly consist of TiO2 with pure anatase phase. It is obviously that the doping of Si does not change the phase compositions of the TiO2 coatings and no feature peaks of Si-containing compounds were detected in these samples. Table 1 summarizes the elemental compositions of the S0, S1 and S2 coatings detected on their interior coatings by EDS. The Si contents in the TiO2 coatings increase with increasing concentrations of disodium silicate in the electrolytes (S1 < S2). With the increase of Si content in the coatings, the P content varies < 10% (from 10.9 0.2 wt.% to 9.6 0.3 wt.%), and the Ca content varies < 20% (from 8.2 0.2 wt.% to 8.9 0.3 wt.%) compared to S0. It should be noted that the differences in Ca and P contents can be neglected compared to the large amounts of additives (> 100 ppm of Ca and P) in endothelial cell growth medium. Moreover, the Si-free TiO2 coating (S0) which contains similar amounts of Ca (7.5 0.9 wt.%) and P (10.6 0.5 wt.%) was used as a negative control in all in vitro experiments. Therefore, difference among porous TiO2 coatings is mainly attributed to the varying doping amount of Si. To further investigate the relationship between the various contents of Si in the coatings and their biological activities, the amount of Si released from the S1 and S2 coatings were measured by ICP-OES, and the results are shown in Fig. 3a. When seen over a time scale of 14 days, cumulative profiles show similar extended release characteristics. To a certain extent, the Si release profiles are direct reflections of the
Si contents throughout the entire coating with the following order: S1 < S2. This trend is consistent with the aforementioned EDS analysis about the zinc contents in the different coatings. The roles of surface properties such as surface wettability, topography, phase composition, and chemistry in modulating angiogenesis activity have been recognized [33, 34]. Therefore, characterization of the surface properties of different coatings is essential to elucidate how to control angiogenesis by surface characteristics. As shown in the Fig. S2, the contact angles ( 40) before and after Si doping remain approximately constant, indicating that surface wettability makes negligible contribution to angiogenic responses of different TiO2 coatings. Moreover, all porous TiO2 coatings (S0, S1 and S2) show similar surface microstructures, CaP additives and phase compositions, implying that different biological response can only be attributed to the variance of doping amount of silicon. However, the characteristic differences between Ti and porous TiO2 coatings not only exist in surface microstructures, but also due to CaP additives that also have the potential to affect angiogenesis [17]. Proliferation of endothelial cell is one step in the multi-step process of angiogenesis necessary for the extension of the vasculature [35]. In this study, AlamarBlue assay was performed after 1, 4, and 7 days to investigate the effects of Si-doped porous TiO2 coatings on proliferation of HUVECs. As shown in Fig. 3b, HUVECs showed a progressive growth with time for all surfaces and proliferate faster on porous TiO2 coating than on the cp Ti at days 4 and 7. With regard to the
Si-doped coatings, larger proliferation rate was observed on S1 with smaller Si content compared to S2 (S1 vs S2, p < 0.05), which suggests that proliferation of HUVECs does not positively correlate with the Si contents, and larger Si amount in S2 appears to be excessive. The morphological responses of HUVECs to all surfaces were investigated through live/dead viability kit after 7 days of culture (Fig. 4). The results showed that HUVECs were flattened and partly presented a rounded morphology on cp Ti surface. However, HUVECs adhered well and exhibited a spindle-shape morphology on all the porous TiO2 coatings (S0, S1 and S2), showing extended filopodia connected to each other nearly achieve a continuous cell layer. In general, S1 exhibited enhanced cell proliferation and morphology compared to the other coatings. Endothelial cell migration has been shown to play an important role in the formation of blood vessels and wound healing after biomaterials implanted [36]. In this study, motility of endothelial was evaluated using an in vitro scratch assay. Fig. 5 shows the images of the created wound gaps in HUVECs monolayer and HUVECs migration rate on different samples. After 8 h, HUVECs migrated on all samples and all the gaps were significantly narrower. It is clear that the porous TiO2 nanostructures (S0, S1 and S2) were more beneficial for cell migration than titanium. However, the fastest HUVECs migration took place on S1 coating, while HUVECs migrated slower on S0 and S2 because of the existed small gaps. Tube formation of endothelial cells is one of the key steps of angiogenesis[37]. An in vitro angiogenesis assay was conducted to evaluate the angiogenic abilities of
HUVECs. It can be seen from Fig. 6 that, after 12 h, HUVECs were cultured on matrigelTM in all extracted solutions developed well-formed tube networks with branched nodes and mesh-like circles. And the HUVECs in S1 and S2 extracts exhibited more notable angiogenic patterns than those cultured in other extracts (Ti and S0). Moreover, enhanced networks of tubes with higher number of branching nodes and longer tube lengths were observed in S1 extracts, implying that S1 coating possesses greater angiogenic ability in inducing differentiated endothelial cell migration. Many research results underline that VEGF is a key regulator of angiogenesis which triggers endothelial cell proliferation, migration and tube formation [38, 39]. The secretion levels of the VEGF protein over 7 days of cell culture were determined quantitatively using ELISA assay. As demonstrated in Fig. 7a, there was no difference in VEGF expression levels among these surfaces at day 1, while HUVECs on Si-doped TiO2 coatings secreted higher levels than those on cp Ti and S0 at day 3. At days 5 and 7, it was observed that the level of VEGF protein was found to be significantly higher on S1 surface than that on the cp Ti, S0 and S2 surfaces. Moreover, VEGF exhibits a higher expression on S0 compared to cp Ti and a decreased expression on S2 compared to S1. In comparison, HUVECs cultured on S1 surfaces produced highest secreted level of VEGF protein than those cells cultured on the cp Ti, S0 and S2, implying that S1 enhances the ability of HUVECs in producing angiogenic factors in vitro. To further examine the influence of the surface properties on the differentiation
of HUVECs at the molecular level, the mRNA expressions of angiogenicassociated genes, including VEGF, hepatocyte growth factor (HGF) and hypoxia-inducible factor 1-alpha (HIF-1) were characterized quantitatively by real-time PCR after cultured for 4, 7 and 14 days, and the results are depicted in Fig. 7b-d. As illustrated, the transcription level of HIF-1 is significantly up-regulated on S1 compared to the other samples at each time intervals (P < 0.001). However, no statistically significant mRNA variations of HGF and VEGF were observed among different samples at earlier stage (day 4). The significant difference in mRNA variations of VEGF was detected at day 14, and that of HGF was detected at days 7 and 14, in which S1 induced the highest mRNA levels. VEGF and HGF are the major pro-angiogenic growth factors which are known to participate in blood vessel formation [40]. HIF-1 is a transcription factor that regulates a spectrum of angiogenic factors, thus elevated HIF-1 can stimulate new blood vessel formation and invasion by transactivating these target genes such as VEGF [41]. As demonstrated, all the expressions of HGF, VEGF and HIF-1 were significantly up-regulated on S1 than on the other surfaces. Substantial evidence from literature shows that some elements can cause toxicity beyond a certain dose despite their stimulatory effects on biological responses [42, 43]. In present study, Si doping clearly induced dose-dependent stimulation in angiogenic gene expression, that is, TiO2 coating with 2% Si doping leading to enhanced angiogenic activities, while 3% Si doping giving rise to reduced angiogenic responses. Many elements (e.g., Ca, Mg, Zn, Cu, Fe, Si, etc.) have been found to play vital
roles in the bone growth, development and remodeling [25]. Therefore, incorporation of trace elements into biomaterials provides an efficient strategy for offering multi-functionalization in tissue engineering, which is also considered to be with low cost and risk compared to growth factor and cell delivery therapies. However, this also raised concerns about the long-term safety of ions brought by excessive ions concentration. Hence, exploring the dose-dependent effect is essential for efficient incorporation. In this work, we have demonstrated that S2 doped with larger amount of Si may be excessive for HUVECs resulting in decreased angiogenic responses compared to S1, but elevated or similar levels of responses compared to Ti or S0. Such observation indicates that the amount of Si is not restricted in a narrow range for HUVECs, which offering more safety in clinical applications.
4. Conclusion In the present study, we examined the angiogenic potential of Si-doped porous TiO2 coatings by evaluating the in vitro cellular response of HUVECs. Significant enhancement of endothelial cell proliferation, migration and tube formation of HUVECs on Si-doped porous TiO2 coatings was observed. Moreover, Si doping significantly stimulates VEGF protein secretion and expression levels of angiogenic-associated genes (VEGF, HGF, HIF-1). All together, our results verified that, the Si-doped TiO2 coating S1, which possesses the smaller content of Si, shows the best angiogenic characteristics and prominent vascularization capacity.
Acknowledgments This work was jointly supported by the National Natural Science Foundation of China
(31570973, 81472060 and 51401234) and Shanghai Science and Technology R&D Fund under grant (15441904900).
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induction of vascular endothelial growth factor: the case for paracrine amplification of angiogenesis, Circulation 97(4) (1998) 381-90. [41] C.W. Pugh, P.J. Ratcliffe, Regulation of angiogenesis by hypoxia: role of the HIF system, Nat Med 9(6) (2003) 677-84. [42] Y.Q. Qiao, W.J. Zhang, P. Tian, F.H. Meng, H.Q. Zhu, X.Q. Jiang, X.Y. Liu, P.K. Chu, Stimulation of bone growth following zinc incorporation into biomaterials, Biomaterials 35(25) (2014) 6882-6897. [43] C. Liu, X. Fu, H. Pan, P. Wan, L. Wang, L. Tan, K. Wang, Y. Zhao, K. Yang, P.K. Chu, Biodegradable Mg-Cu alloys with enhanced osteogenesis, angiogenesis, and long-lasting antibacterial effects, Sci Rep 6 (2016) 27374.
Figure captions Fig. 1. SEM morphologies of the as-prepared Ti, S0, S1 and S2 samples.
Fig. 2. XRD patterns of S0, S1 and S2 coatings.
Fig. 3. (a) Cumulative profiles of Si ion release from the S1 and S2 coatings in PBS. (b) Cell proliferation of HUVECs on cp Ti, Si-free and Si-doped TiO2 coatings. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significant statistical difference (p > 0.05).
Fig. 4. Morphologies of HUVECs cultured on cp Ti, Si-free and Si-doped TiO2
coatings at day 7 stained using live/dead assay.
Fig.
5.
HUVECs
migration
on
Ti,
S0,
S1
and
S2
after
8
h.
Fig. 6. In vitro angiogenesis study of HUVECs cultured with different media on ECMatrix gel.
Fig. 7. (a) VEGF proteins produced level of HUVECs cultured on cp Ti, Si-free and
Si-doped TiO2 coatings at days 1, 3, 5 and 7. (b) Expression of angiogenic-related genes in HUVECs cultured on cp Ti, Si-free and Si-doped TiO2 coatings was measured by quantitative real-time RT-PCR. The results were normalized to GAPDH and expressed as fold increase relative to cp Ti values. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significant statistical difference (p > 0.05).
Table Table 1. Elemental compositions of the S0, S1 and S2 coatings detected by EDS. Elemental composition (wt.%) Sample Ti
O
Ca
P
Si
S0
25.6 0.7
56.3 0.8
7.5 0.9
10.6 0.5
S1
30.0 0.3
49.1 0.2
8.2 0.2
10.9 0.2
1.8 0.08
S2
29.2 0.7
49.1 0.6
8.9 0.3
9.6 0.3
3.2 0.07
S0927-7765(17)30526-X http://dx.doi.org/doi:10.1016/j.colsurfb.2017.08.010 COLSUB 8766
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
14-5-2017 30-7-2017 3-8-2017
Please cite this article as: Zhihong Ding, Yuqin Qiao, Feng Peng, Chao Xia, Shi Qian, Tao Wang, Junying Sun, Xuanyong Liu, Si-doped porous TiO2 coatings enhanced in vitro angiogenic behavior of human umbilical vein endothelial cells, Colloids and Surfaces B: Biointerfaceshttp://dx.doi.org/10.1016/j.colsurfb.2017.08.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Si-doped porous TiO2 coatings enhanced in vitro angiogenic behavior of human umbilical vein endothelial cells Zhihong Ding a,b, Yuqin Qiao c,, Feng Peng c, Chao Xia c, Shi Qian c, Tao Wang a, Junying Sun a,*, Xuanyong Liu c, a
Orthopaedic Department, The First Affiliated Hospital of Soochow University, 188
Shizi Street, Suzhou, Jiangsu 215006, P.R. China b
Orthopedics of Shanghai Pudong New area Gongli Hospital, Shanghai 200135, P.R.
China c
State Key Laboratory of High Performance Ceramics and Superfine Microstructure,
Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, P.R. China
Author to whom correspondence would be addressed
Both Zhihong Ding and Yuqin Qiao are equal contribution to this work. Tel.: +86 21 52412409; fax: +86 21 52412409; E-mai: [email protected] (X. Y. Liu), [email protected] (J. Y. Sun)
Graphical Abstract
Small amount of silicon doping significantly enhanced angiogenic activity of HUVECs.
Highlights Silicon-doped porous TiO2 coatings were fabricated via plasma electrolyte oxidation method (PEO) successfully. Si doping induced dose-dependent stimulation in angiogenic activities of HUVECs. Larger amount of Si results in reduced angiogenic responses.
Abstract Recent evidence demonstrates that vessel involvement is crucial in various bone remodeling situations, indicating that blood vessel formation within or surrounding the implant is essential for establishment of rigid implant fixation. In this work, the ability of the silicon-doped porous TiO2 coatings fabricated via plasma electrolyte
oxidation method (PEO) to enhance the angiogenic potential of human umbilical vein endothelial cells (HUVECs) were investigated. The cellular responses of HUVECs on the silicon-doped porous TiO2 coatings were studied through cell proliferation, vascular endothelial growth factor (VEGF) secretion, and angiogenic-associated gene (VEGF, HIF-1 and HGF) expression analysis. The results show that small amount of silicon significantly enhanced angiogenic activity of HUVECs, while larger amount of silicon appears excessive. Hence, the silicon-doped TiO2 coating offers a potential solution to improve bone vascularization to achieve efficient osseointegration and restoration of function after implantation.
Keywords : Si, porous TiO2 coatings, HUVECs, angiogenic potential
1. Introduction More recently, increasing evidence has demonstrated that osteogenesis is coupled with angiogenesis during bone regeneration and remodeling [1]. Upon implantation, angiogenesis occurs rapidly in response to inflammation of host tissue to develop new blood vessels from pre-existing vessels [2]. The vasculature ensures the sufficient
supply of circulating cells, nutrients, oxygen and metabolic wastes essential for maintaining osteoblastic bone matrix synthesis and mineralization [3, 4]. In contrast, insufficient vascularization in bone defects cannot meet the growing tissue nutrient delivery and clearance needs, leading to inadequate osseointegration of implants. Hence, the importance of angiogenesis as an essential component in the design of orthopedic and dental implants to enhance the process of skeletal tissue engineering should be brought to the forefront. The process of angiogenesis involves a series of steps including basement membrane degradation, endothelial cell migration and proliferation, and capillary tube formation mediated by various angiogenic factors [5]. During bone development and remodeling, endothelial cells invade from the perichondrial tissues to form blood vessels for nutrient supply and serve as a scaffold for new bone formation [6-8]. Capillaries also merge to form a network to maintain long-term bone viability. Notably, recent studies suggest that the reciprocal crosstalk between endothelial and bone cells can further modulate the bone behavior and angiogenesis [9-12]. Endothelial cells have been found to induce the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs) and stimulate the expression of osteogenic markers through direct contact co-culture [9]. Accordingly, BMSCs exhibited
a
supportive
expression
of
angiogenic
function
and
enhanced
vascularization for endothelial cells [9]. Thus, designing biomaterials with combined osteogenic and angiogenic potential remains a rational and challenging strategy for the bone healing and remodeling.
Titanium (Ti) and its alloys have been widely used for dental and orthopedic implants device because of their good biocompatibility and mechanical properties [13]. Over the years, increasing efforts have been focused on promoting osteogenic activities to enhance osseointegration [14]. However, this cannot fulfill all the requirements of a successful bone grafting [15]. To promote angiogenesis and vascularization in Ti and its alloys, a number of strategies have been explored to tailor their physicochemical properties (i.e., surface topography and chemical composition) [16-18]. Among these properties, surface topography is known to have a great influence on protein adsorption and cell behaviors [19-21]. Topographical dimensions ranging from nanoscale to micron have been reported to enhance angiogenesis by stimulating growth factors secretion and up-regulating the expression of angiogenic-associated genes [22-24]. Bone growth and development involves a variety of trace elements, which have been shown to increase osteogenesis and/or neovascularization. Tuning chemical compositions of biomaterials by doping vascularization-favorable elements (e.g., Cu, Mg, Sr, Co, etc.) also seems an efficient approach to improve angiogenesis [24-27]. To achieve the bifunctional purpose, incorporating elements combining angiogenesis potential and osteostimulation to biomaterials seems to be a promising therapy strategy for bone healing and remodeling. Silicon (Si) is one of the elements which play multiple functions in bone formation and maintenance, vascular health, and wound healing [28, 29]. In this sense, Si has the potential to be a candidate of interest added to biomaterials for dental and
orthopedic implant applications. In our previous studies, we have found Si doping can obviously enhance the osteogenic potential of TiO2 coatings [30-32]. Therefore, the objective of this study is to investigate the angiogenic capability of Si-doped porous TiO2 coatings. Si-free and Si-doped TiO2 porous coatings were fabricated using the plasma electrolyte oxidation (PEO) method as reported previously, and the influence of silicon elements on the functionality of human umbilical vein endothelial cells (HUVECs) was evaluated by examining the cell viability and morphology, migration assay, tube formation assay, VEGF secretion and expression of angiogenic-associated genes.
2. Materials and methods 2.1 Preparation of Si-doped TiO2 coatings Commercially pure titanium plates (Cp Ti, TA1, purity > 99.85%) with dimensions of 10 mm 10 mm 1 mm were used in cellular experiments, and 20 mm 20 mm 1 mm in size were used in the molecular experiments to extract sufficient nucleic acid samples. They were ground with 400# abrasive papers and then ultrasonically washed with acetone, ethyl alcohol and distilled water in an ultrasonic bath prior to plasma electrolyte oxidation (PEO) treatment. PEO was conducted in electrolytes composed of 0.1 mol/L calcium acetate monohydrate (CA, C4H6O4CaH2O), 0.05 mol/L glycerophosphate disodium salt pentahydrate (GP, C3H7Na2O6P5H2O) and various amounts of disodium silicate (Na2SiO3) to prepare Si-free (S0) and Si-doped coatings (S1 and S2). By adjusting the concentrations of disodium silicate (Na2SiO3) 0.01 and 0.02 mol/L) in the electrolytes, coatings designated as S1 and S2 were produced.
During the PEO treatment, a Ti plate was used as an anode, and a spiral steel pipe was used as a cathode and a dwelling water pipe to sustain the temperature of the electrolyte < 30C in the electrolytic cell. A magnetic stirrer was employed to keep the components and temperature of the electrolytes uniform. After PEO treatment, the samples were boiled for 4 min, washed with deionized water and air dried. 2.2 Characterization of Si-doped TiO2 coatings Scanning electron microscopy (S-3400N, Hitachi, Japan) was used to examine the surface morphology of the coatings and energy-dispersive X-ray spectrometry (EDS) attached to an electron probe X-ray microanalysis system (EPMA, JAX-8100, Japan) was performed to measure the elemental compositions. X-ray diffraction (XRD, D8 ADVANCE, Bruker) was employed to analyze their phase compositions. 2.3 Si ion release The Si-doped samples were immersed in 10 ml PBS at 37C for 7 days, and Si concentration of the buffer solution at different time points (1, 4, 7 and 14 days) was measured by an inductively coupled plasma/optical emission spectroscopy (ICP-OES; Vista AX, Varian, USA). 2.4 Cultivation of HUVECs Human umbilical vein endothelial cells (HUVECs, ScienCell) were cultured in complete Endothelial Cell Growth Medium (EGM) supplemented with 10% fetal bovine serum (FBS), endothelial cell growth supplement and penicillin/streptomycin (ScienCell, CA) at 37C in an incubator of 5% CO2. The culture medium was changed every 2 days.
2.5 Cell proliferation and morphologies AlamarBlue assay was performed to evaluate the cell proliferation. Briefly, specimens with size of 10 10 1 m3 were placed in 24-well plates, HUVECs were then seeded at a density of 5 104/well. After culturing for 1, 4, and 7 days, the cells were incubated in culture medium supplemented with 10% AlamarBlue (Invitrogen, USA) for 2 h at 37 C. Post reaction medium aliquots (100 l) were transferred to 96-well solid black plates and the fluorescence intensity was recorded using Cytation 5 Multi-Mode Reader (Biotek, USA) at 560 nm (Ex)/590 nm (Em). The morphologies of HUVECs on different samples at day 7 then was stained using live/dead viability/cytotoxicity kit (Invitrogen, USA). 2.6 Cell Migration HUVECs were seeded at a density of 5 104/well on samples with size of 10 10 1 m3. After 1 d adhesion, lines crossing the cells were drawn by 20-200 l pipet tips. After cultivation for 8 h, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 1% BSA in PBS, and then stained with Rhodamine-phalloidin and DAPI at room temperature. After each step, the samples were washed 3 times for 5 min each with PBS. The images were captured on a fluorescence microscope (Olympus IX 71, Olympus, Japan). 2.7 Matrigel tube formation assay In vitro angiogenesis was assessed by the endothelial tube formation assay according to the manufacturer’s instructions. Prior to the assay, samples were autoclaved and later immersed in 1 ml complete cell growth medium and in the
incubator for 24 h. The extracted solutions were then collected. The Growth factor reduced matrigelTM (BD Biosciences) was thawed out at 4C, followed by adding 140 l of thawed gel solution to each well of a prechilled 48-well plate. After the Matrigel was allowed to gel in the incubator for 1 h, HUVECs were seeded at a density of 3 104 cells/well in complete cell growth medium. While cell adhesion was completed after 3 h, cell growth medium was removed and changed to extracted solutions. The assay plate was then incubated for 12 h, and stained with Calcein AM (Sigma-Aldrich, US) for 30 min in the incubator. The tube formation of HUVECs was observed and analyzed using an Olympus fluorescence microscope and imaged at 10 magnification. 2.8 Quantification of VEGF protein secretion The production of vascular endothelial growth factor (VEGF) proteins was quantified using the Enzyme-linked Immunosorbent Assay (ELISA) Kit (Abcam, USA) in accordance to the manufacturer’s instruction. Briefly, HUVECs were cultured on specimens for the prescribed time period, and proteins from cell culture supernatant were collected and quantified using the ELISA kit. 2.9 RNA extraction and real-time quantitative RT-PCR analysis The total RNA was extracted using the Trizol reagent (Invitrogen Life Technologies) at days 4, 7 and 14 according to the manufacture’s protocols. The RNA concentration and purity were determined spectrophotometrically by Take3TM Micro-Volume Plate of Cytation 5 Multi-Mode Reader (Biotek, USA). In the cDNA synthesis, 1 g of total RNA was reversely transcribed using Transcriptor First Strand cDNA Synthesis
Kit (Roche) and oligo dT18 primers (Roche). The real-time polymerase chain reaction (RT-PCR) was used to quantify the gene expression of the angiogenic related genes. In brief, the 10-fold diluted cDNA was used as the template in the 10-l qRT-PCR reactions using the SYBR Green Matermix and Roche LightCycler 480 System (Roche). The level of each target gene was calculated by the CT method and GAPDH gene expression was used as an endogenous control for normalization. Each sample was analyzed in triplicates and the reaction mixture without the cDNA was used as a negative control in each run. The PCR primer sequences used in this project are listed in Supplementary Information (Supplementary Table S1). 2.10 Statistical analysis Data were expressed as means standard deviations (SD) from experiments performed in quintuplicate. Statistical analysis was performed using the one-way analysis of variance (ANOVA) with Tukey-Kramer Multiple comparison post-test using GraphPad Instant Software (GraphPad Prism Software, Inc., USA) with p < 0.05 being considered to be statistically significant.
3. Results and discussion Fig. 1 shows the SEM surface micrographs of the cp Ti, Si-free and Si-doped coatings. It can be seen that silicon doping does not alter the main structural characteristics of plasma electrolyte oxidized TiO2 coatings including the porous morphology, pore size ( 10 m), and homogeneous distribution of pores. The strong Si signals in the inserted EDS spectra of S1 and S2 indicate successful Si doping by PEO. The surface EDS maps in
supplementary information (Fig. S1) clearly illustrate that the coatings have uniform distributions of the major nutritional elements (Ca, P and Si). XRD patterns of the S0, S1 and S2 coatings are shown in Fig. 2. All these coatings display similar patterns, which mainly consist of TiO2 with pure anatase phase. It is obviously that the doping of Si does not change the phase compositions of the TiO2 coatings and no feature peaks of Si-containing compounds were detected in these samples. Table 1 summarizes the elemental compositions of the S0, S1 and S2 coatings detected on their interior coatings by EDS. The Si contents in the TiO2 coatings increase with increasing concentrations of disodium silicate in the electrolytes (S1 < S2). With the increase of Si content in the coatings, the P content varies < 10% (from 10.9 0.2 wt.% to 9.6 0.3 wt.%), and the Ca content varies < 20% (from 8.2 0.2 wt.% to 8.9 0.3 wt.%) compared to S0. It should be noted that the differences in Ca and P contents can be neglected compared to the large amounts of additives (> 100 ppm of Ca and P) in endothelial cell growth medium. Moreover, the Si-free TiO2 coating (S0) which contains similar amounts of Ca (7.5 0.9 wt.%) and P (10.6 0.5 wt.%) was used as a negative control in all in vitro experiments. Therefore, difference among porous TiO2 coatings is mainly attributed to the varying doping amount of Si. To further investigate the relationship between the various contents of Si in the coatings and their biological activities, the amount of Si released from the S1 and S2 coatings were measured by ICP-OES, and the results are shown in Fig. 3a. When seen over a time scale of 14 days, cumulative profiles show similar extended release characteristics. To a certain extent, the Si release profiles are direct reflections of the
Si contents throughout the entire coating with the following order: S1 < S2. This trend is consistent with the aforementioned EDS analysis about the zinc contents in the different coatings. The roles of surface properties such as surface wettability, topography, phase composition, and chemistry in modulating angiogenesis activity have been recognized [33, 34]. Therefore, characterization of the surface properties of different coatings is essential to elucidate how to control angiogenesis by surface characteristics. As shown in the Fig. S2, the contact angles ( 40) before and after Si doping remain approximately constant, indicating that surface wettability makes negligible contribution to angiogenic responses of different TiO2 coatings. Moreover, all porous TiO2 coatings (S0, S1 and S2) show similar surface microstructures, CaP additives and phase compositions, implying that different biological response can only be attributed to the variance of doping amount of silicon. However, the characteristic differences between Ti and porous TiO2 coatings not only exist in surface microstructures, but also due to CaP additives that also have the potential to affect angiogenesis [17]. Proliferation of endothelial cell is one step in the multi-step process of angiogenesis necessary for the extension of the vasculature [35]. In this study, AlamarBlue assay was performed after 1, 4, and 7 days to investigate the effects of Si-doped porous TiO2 coatings on proliferation of HUVECs. As shown in Fig. 3b, HUVECs showed a progressive growth with time for all surfaces and proliferate faster on porous TiO2 coating than on the cp Ti at days 4 and 7. With regard to the
Si-doped coatings, larger proliferation rate was observed on S1 with smaller Si content compared to S2 (S1 vs S2, p < 0.05), which suggests that proliferation of HUVECs does not positively correlate with the Si contents, and larger Si amount in S2 appears to be excessive. The morphological responses of HUVECs to all surfaces were investigated through live/dead viability kit after 7 days of culture (Fig. 4). The results showed that HUVECs were flattened and partly presented a rounded morphology on cp Ti surface. However, HUVECs adhered well and exhibited a spindle-shape morphology on all the porous TiO2 coatings (S0, S1 and S2), showing extended filopodia connected to each other nearly achieve a continuous cell layer. In general, S1 exhibited enhanced cell proliferation and morphology compared to the other coatings. Endothelial cell migration has been shown to play an important role in the formation of blood vessels and wound healing after biomaterials implanted [36]. In this study, motility of endothelial was evaluated using an in vitro scratch assay. Fig. 5 shows the images of the created wound gaps in HUVECs monolayer and HUVECs migration rate on different samples. After 8 h, HUVECs migrated on all samples and all the gaps were significantly narrower. It is clear that the porous TiO2 nanostructures (S0, S1 and S2) were more beneficial for cell migration than titanium. However, the fastest HUVECs migration took place on S1 coating, while HUVECs migrated slower on S0 and S2 because of the existed small gaps. Tube formation of endothelial cells is one of the key steps of angiogenesis[37]. An in vitro angiogenesis assay was conducted to evaluate the angiogenic abilities of
HUVECs. It can be seen from Fig. 6 that, after 12 h, HUVECs were cultured on matrigelTM in all extracted solutions developed well-formed tube networks with branched nodes and mesh-like circles. And the HUVECs in S1 and S2 extracts exhibited more notable angiogenic patterns than those cultured in other extracts (Ti and S0). Moreover, enhanced networks of tubes with higher number of branching nodes and longer tube lengths were observed in S1 extracts, implying that S1 coating possesses greater angiogenic ability in inducing differentiated endothelial cell migration. Many research results underline that VEGF is a key regulator of angiogenesis which triggers endothelial cell proliferation, migration and tube formation [38, 39]. The secretion levels of the VEGF protein over 7 days of cell culture were determined quantitatively using ELISA assay. As demonstrated in Fig. 7a, there was no difference in VEGF expression levels among these surfaces at day 1, while HUVECs on Si-doped TiO2 coatings secreted higher levels than those on cp Ti and S0 at day 3. At days 5 and 7, it was observed that the level of VEGF protein was found to be significantly higher on S1 surface than that on the cp Ti, S0 and S2 surfaces. Moreover, VEGF exhibits a higher expression on S0 compared to cp Ti and a decreased expression on S2 compared to S1. In comparison, HUVECs cultured on S1 surfaces produced highest secreted level of VEGF protein than those cells cultured on the cp Ti, S0 and S2, implying that S1 enhances the ability of HUVECs in producing angiogenic factors in vitro. To further examine the influence of the surface properties on the differentiation
of HUVECs at the molecular level, the mRNA expressions of angiogenicassociated genes, including VEGF, hepatocyte growth factor (HGF) and hypoxia-inducible factor 1-alpha (HIF-1) were characterized quantitatively by real-time PCR after cultured for 4, 7 and 14 days, and the results are depicted in Fig. 7b-d. As illustrated, the transcription level of HIF-1 is significantly up-regulated on S1 compared to the other samples at each time intervals (P < 0.001). However, no statistically significant mRNA variations of HGF and VEGF were observed among different samples at earlier stage (day 4). The significant difference in mRNA variations of VEGF was detected at day 14, and that of HGF was detected at days 7 and 14, in which S1 induced the highest mRNA levels. VEGF and HGF are the major pro-angiogenic growth factors which are known to participate in blood vessel formation [40]. HIF-1 is a transcription factor that regulates a spectrum of angiogenic factors, thus elevated HIF-1 can stimulate new blood vessel formation and invasion by transactivating these target genes such as VEGF [41]. As demonstrated, all the expressions of HGF, VEGF and HIF-1 were significantly up-regulated on S1 than on the other surfaces. Substantial evidence from literature shows that some elements can cause toxicity beyond a certain dose despite their stimulatory effects on biological responses [42, 43]. In present study, Si doping clearly induced dose-dependent stimulation in angiogenic gene expression, that is, TiO2 coating with 2% Si doping leading to enhanced angiogenic activities, while 3% Si doping giving rise to reduced angiogenic responses. Many elements (e.g., Ca, Mg, Zn, Cu, Fe, Si, etc.) have been found to play vital
roles in the bone growth, development and remodeling [25]. Therefore, incorporation of trace elements into biomaterials provides an efficient strategy for offering multi-functionalization in tissue engineering, which is also considered to be with low cost and risk compared to growth factor and cell delivery therapies. However, this also raised concerns about the long-term safety of ions brought by excessive ions concentration. Hence, exploring the dose-dependent effect is essential for efficient incorporation. In this work, we have demonstrated that S2 doped with larger amount of Si may be excessive for HUVECs resulting in decreased angiogenic responses compared to S1, but elevated or similar levels of responses compared to Ti or S0. Such observation indicates that the amount of Si is not restricted in a narrow range for HUVECs, which offering more safety in clinical applications.
4. Conclusion In the present study, we examined the angiogenic potential of Si-doped porous TiO2 coatings by evaluating the in vitro cellular response of HUVECs. Significant enhancement of endothelial cell proliferation, migration and tube formation of HUVECs on Si-doped porous TiO2 coatings was observed. Moreover, Si doping significantly stimulates VEGF protein secretion and expression levels of angiogenic-associated genes (VEGF, HGF, HIF-1). All together, our results verified that, the Si-doped TiO2 coating S1, which possesses the smaller content of Si, shows the best angiogenic characteristics and prominent vascularization capacity.
Acknowledgments This work was jointly supported by the National Natural Science Foundation of China
(31570973, 81472060 and 51401234) and Shanghai Science and Technology R&D Fund under grant (15441904900).
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Figure captions Fig. 1. SEM morphologies of the as-prepared Ti, S0, S1 and S2 samples.
Fig. 2. XRD patterns of S0, S1 and S2 coatings.
Fig. 3. (a) Cumulative profiles of Si ion release from the S1 and S2 coatings in PBS. (b) Cell proliferation of HUVECs on cp Ti, Si-free and Si-doped TiO2 coatings. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significant statistical difference (p > 0.05).
Fig. 4. Morphologies of HUVECs cultured on cp Ti, Si-free and Si-doped TiO2
coatings at day 7 stained using live/dead assay.
Fig.
5.
HUVECs
migration
on
Ti,
S0,
S1
and
S2
after
8
h.
Fig. 6. In vitro angiogenesis study of HUVECs cultured with different media on ECMatrix gel.
Fig. 7. (a) VEGF proteins produced level of HUVECs cultured on cp Ti, Si-free and
Si-doped TiO2 coatings at days 1, 3, 5 and 7. (b) Expression of angiogenic-related genes in HUVECs cultured on cp Ti, Si-free and Si-doped TiO2 coatings was measured by quantitative real-time RT-PCR. The results were normalized to GAPDH and expressed as fold increase relative to cp Ti values. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significant statistical difference (p > 0.05).
Table Table 1. Elemental compositions of the S0, S1 and S2 coatings detected by EDS. Elemental composition (wt.%) Sample Ti
O
Ca
P
Si
S0
25.6 0.7
56.3 0.8
7.5 0.9
10.6 0.5
S1
30.0 0.3
49.1 0.2
8.2 0.2
10.9 0.2
1.8 0.08
S2
29.2 0.7
49.1 0.6
8.9 0.3
9.6 0.3
3.2 0.07