Bioactivity of a sol–gel-derived hydroxyapatite coating on titanium implants in vitro and in vivo
Article Category: Brief communication (original)
Published Online: Dec 31, 2018
Page range: 35 - 44
DOI: https://doi.org/10.1515/abm-2018-0029
Keywords
© 2018 Jintamai Suwanprateeb et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.
Hydroxyapatite has been widely used as bioactive coating on metallic implants to promote the bioactivity of the underlying implants by creating the direct chemical bonding between bone and implant [1]. Examples of the coating applications include hip implants, knee implants, pedicle screws, fixation pins, and dental implants [2, 3]. Such bioactive coating can increase the stability and decrease the prevalence of fixation failure of the implant [4, 5]. The improvement of fixation induced by bioactive coating has been observed under stable-unloaded, stable-loaded, and unstable mechanical conditions [6]. Besides enhanced bone bonding, hydroxyapatite coating was shown to convert a motion-induced fibrous membrane into a bony anchorage [7].
Typically, hydroxyapatite coating is applied using a plasma spraying process that involves the use of extremely high temperatures resulting in the phase heterogeneity and coating delamination, which could lead to instability and even the failure of coated implants [3]. Alternatively, low-temperature sol–gel coating has shown to offer several advantages, including better control of the chemical composition of the coating, homogeneity of coatings, uniform coating microstructure, low processing temperature, low-cost equipment, and non-line-of-sight coating. Therefore, sol–gel techniques are considered to be a flexible and commercially promising for preparation of hydroxyapatite coatings on medical devices [8, 9]. Recently, a new sol–gel system for coating was developed by authors in which the sol solution used for coating was modified to improve the coating uniformity and had a long storage time without premature gel formation compared with typical sol gel systems [10]. The present study aimed to evaluate the performance of the hydroxyapatite coating produced by this new sol–gel system in vitro and in vivo. Titanium alloy was used as the substrate because of its increasing use in medical implants as a result of its several advantages compared with other metals, for example the higher strength-to-weight ratio, greater fatigue resistance, lower rigidity, and greater corrosion resistance. For studies in vitro, biocompatibility in terms of osteoblast proliferation and cell matrix mineralization on the prepared coating was examined and compared with that of uncoated titanium. For studies in vivo, a rabbit model was used whereby self-tapping coated screws were inserted in the trabecular bone of the femur and tibia. New Zealand white rabbits were used because they provide sufficient bony mass to insert the screws and determine extraction torques. We conducted examinations of extraction torques, surfaces, and histology of the samples after 7 weeks, 12 weeks, and 24 weeks of implantation.
Forty titanium alloy (Ti6Al4V) disks (10 mm in diameter and 0.5 mm thick) and 36 screws (3 mm in diameter and 1.5 cm long) were used. Coating sol with calcium to phosphorus molar ratios (Ca/P) of 1.67 using ammonium hydrogen carbonate as a gel retarding agent was prepared as described previously [10]. Briefly, precursors were prepared by separately mixing calcium nitrate tetrahydrate (Sigma-Aldrich) and phosphorus pentoxide (BDH) with ethyl alcohol (BDH) in a laboratory atmosphere for 30 min. The precursors were then mixed together, and 10 mL of 25% ammonium hydrogen carbonate (Sigma-Aldrich) was added and further stirred for 8 h to produce a sol. Samples (20 disks and 18 screws) were coated by dipping them into the prepared sol using a dip coater (PTL-200; MTI Corporation) with a dipping and withdrawing rate of 2 mm
Osteoblasts had been isolated from human donor cancellous bone after Institutional Review Board approval and written informed consent provided by the donors. The isolates had been anonymized such that we could not identify any connection with the donor. In brief, human donor cancellous bone immediately after surgery was stored in Dulbecco’s modified Eagle medium (DMEM; BioWhittaker) and brought to a laboratory where they were maintained at 37°C in a humidified incubator under an atmosphere of 5% CO2 for 48 h and further washed in DMEM to remove erythrocytes and adipose cells. The cleaned bone was then crushed into small pieces and placed with DMEM in a culture dish. The bone fragments (BFs) were further incubated at 37°C in a humidified incubator under an atmosphere of 5% CO2. The primary human osteoblasts started growing out from the fragments of bone after a few days, and the cells almost reached confluence within 2 weeks with changes in the medium 2 times per week. For each isolation, the cells were tested for mycoplasma contamination and bacterial contamination, and examined for typical osteoblast morphology before cryopreservation. We used primary subculture human osteoblasts (passage No. 3) from the in-house cryopreserved stock. Before use, they were again tested for mycoplasma contamination and bacterial contamination, and examined for typical osteoblast morphology.
Coated and uncoated titanium disks (20 each) were sterilized by autoclaving and placed in the tissue culture plate. We placed 0.1 mL of suspended human osteoblasts in DMEM (BioWhittaker) supplemented with 15% fetal calf serum and 1% penicillin/streptomycin solution (1 × 105 cells/mL) onto each sample. The samples were then incubated at 37°C under an atmosphere of 5% CO2 and 95% relative humidity for 1, 3, 7, 14, and 21 days. The cell survival and proliferation were determined using a methyl thiazolyl tetrazolium (MTT; (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2
Cultured samples were washed with phosphate-buffered saline and fixed with cold methanol for 30 min. They were then washed with sterile deionized water, stained with 1% Alizarin red S (Color Index 58005) for 5 min, and washed again to remove the residual Alizarin red S until the rinses were clear. Subsequently, 10% cetylpyridinium chloride (Sigma-Aldrich) was added, and the supernatant was transferred to a 96-well plate. The absorbance of the mixture was measured at 570 nm using a UVM 340 microplate reading spectrophotometer (Easys) to quantify the cell staining, which corresponded to the amount of calcification.
The morphology of cells was determined by fixing the osteo-blasts using glutaraldehyde solution for 2 h at room temperature, dehydrated by series of ethanol and observed by a scanning electron microscope (JEOL JSM-5410) at the accelerating voltage of 20 kV.
The animal experimental protocol was reviewed and approved by the Chulalongkorn University Animal Care and Use Committee (approval No. 0831080), and all procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Washington, DC: National Academy Press; 1996) and the Animals for Scientific Purposes Act, BE 2558 (AD 2015) (Thailand). Three adult male New Zealand white rabbits weighing approximately 3 kg each (mean standard deviation: 3.1 ± 0.14 kg) were obtained from the Faculty of Veterinary Science, Chulalongkorn University, and used in this study. Each rabbit was housed in a room with controlled temperature (20 ± 2°C) and humidity (60 ± 10%) under a 12–12 h light–dark cycle and allowed access to a rabbit-specific diet and water ad libitum. The rabbits were anesthetized with isoflurane by inhalation. Once under anesthesia, the level of anesthesia was monitored and maintained by a veterinarian. Surgery was performed using standard aseptic techniques. Bones were exposed, and 18 uncoated and 18 coated screws were inserted into 3 locations (proximal femur, distal femur, and proximal tibia). The wound was then closed with monofilament sutures (Supramid 3-0; Braun).
Postoperatively, carprofen (Rimadyl (Pfizer), 2.2 mg.kg–1) and enrofloxacin (Baytril (Bayer), 12 mg.kg–1) were daily given for 7 days. Visual observations of animal conditions and soft tissue healing were made for 24 weeks. After reaching the specified periods (7 weeks, 12 weeks, and 24 weeks), the rabbits were euthanized with an intravenous overdose of phenobarbital followed by cervical dislocation. The tibiae and femurs were dissected en bloc and further resected using a hand saw near screw locations. The samples were divided into two groups. One group was used to measure extraction torque, while another was placed in 40% w/v buffered formalin for histology.
The resected bone with screws in place was clamped and tested using a torsion tester (MT2; Instron) to measure the extraction torque at 23°C and 50% relative humidity. Elemental analysis was conducted using energy dispersive spectroscopy (Oxford Instruments). The surfaces of the tested screws after extraction were sputtered with gold before observation with a JSM 5410 scanning electron microscope (JEOL).
Bone samples were dehydrated through series of increasing ethanol concentrations and dried using a critical point dryer (CPD 020; Balzers). They were then embedded in epoxy resin under vacuum and cured for 12 h. Sections were cut, ground, and polished into thin slices (400CS; Exakt). They were then decalcified using 10% formic acid for 48 h and stained with hematoxylin and eosin (Varistain Gemini NS; Thermo Scientific). Photomicrographs of sections were obtained using a light microscope (Olympus DX41).
The differences in MTT assay, Alizarin red S assay, and screw extraction torque results between uncoated and coated samples were analyzed using independent-sample Student
The proliferation of osteoblasts on the surface of the samples was studied by the MTT assays. There is a linear correlation between cell numbers and absorbance by the MTT formazan. Osteoblasts could attach and proliferate on all samples as could be seen from the increase in absorbance with incubation time (
Figure 1
Comparison of cell proliferation on the surfaces between 20 uncoated and 20 coated (gray bars) titanium samples by methyl thiazolyl tetrazolium (MTT) assay (error bars, standard deviation, n = 4 per type per time point). Absorbance by the MTT formazan chromophore at 570 nm, and D indicates the time of incubation in days. *
The ability of cells to produce a mineralized matrix and nodules is important for the development of materials for bone regeneration. Many materials are biocompatible and can support cell proliferation, but cannot induce mineralization, and require addition of growth factors and other agents to improve the cell responses. Mineralization, or calcification generally occurs in the differentiation stage, which follows cell proliferation. Alizarin red S is a dye that binds selectively to calcium salts and is a common histochemical technique used to detect and quantify calcium deposits in mineralized tissues and cultures [12].
Figure 2
Comparison of cell mineralization on the surfaces of 20 uncoated and 20 coated (gray bars) titanium samples by Alizarin red S assay (error bars, standard deviation, n = 4 per type per time point). Absorbance by the Alizarin red S at 570 nm, and D indicates the time of incubation in days. *
increased significantly at days 14 and 21, while that produced by uncoated titanium remained unchanged (
Figure 3
Scanning electron microscopy (SEM) images of osteoblast morphology on uncoated and coated titanium samples. a) uncoated 1 day; b) coated 1 day; c) uncoated 3 days; d) coated 3 days; e) uncoated 7 days; f ) coated 7 days; g) uncoated 14 days; h) coated 14 days; i) uncoated 21 days; and j) coated 21 days (magnification ×1000). Scale bars represent 60 μm
All rabbits tolerated the procedure well and survived throughout the duration of the study. They were healthy with good appetites and had no altered behavior or sign of inflammation, infection, or complication.
Figure 4
Extraction torque from bone of 12 uncoated and 12 coated titanium screws at different implantation periods (wks, weeks) (error bars, standard deviation, n = 4 per type per time point). *
those for the uncoated screws at all implantation periods and tended to increase with increasing period of implantation. The torques of coated screws at 24 weeks postimplantation were significantly greater than those at 7 and 12 weeks (
Figure 5
Scanning electron microscopy (SEM) image of uncoated and coated screws extracted from the bone. No detachment of the coating from the underlying screw is noted. Arrows indicate bone fragments (BFs). The amount and size of the BFs increased with increasing implantation periods. a) uncoated 7 weeks; b) coated 7 weeks; c) uncoated 12 weeks; d) coated 12 weeks; e) uncoated 24 weeks; and f ) coated 24 weeks (magnification ×100). Scale bars represent 1 mm
Figure 6
Histology of bone healing around the hydroxyapatite-coated and -uncoated screws in New Zealand white rabbits at 7 weeks, 12 weeks, and 24 weeks after surgery. Hematoxylin and eosin staining demonstrates bone remodeling comprising bone trabeculae with mixed woven bone and lamellar bone. Mature lamellar bone is only observed in coated sample at 24 weeks of implantation. a) uncoated 7 weeks; b) coated 7 weeks; c) uncoated 12 weeks; d) coated 12 weeks; e) uncoated 24 weeks; and f ) coated 24 weeks (magnification ×100; Ti, titanium; B, bone). Scale bars represent 200 μm
Examination of extracted screws by scanning electron microscopy (
Qualitative histological studies of the hematoxylin and eosin-stained bone slices at all periods postoperatively (
A limitation of the histology performed in this study is that the amount of bone contact was not determined quantitatively. However, screw extraction torques as determined in this study can be used to indicate such bone contact and bonding ability of the screws, at least in part. The findings are consistent with the greater cell mineralization ability of the coated samples compared with that of the uncoated samples seen in vitro. Comparison data for the plasma processing coating are not available in the present study, which limits time-dependent comparisons of the osteoblast cultures and histological observations.
The newly developed hydroxyapatite coating on titanium samples could enhance osteoblast proliferation and mineralization in vitro compared with uncoated samples. Mechanical and histological analyses of the screws inserted into the rabbit bone showed that fixation of the sol–gel-derived hydroxyapatite-coated titanium screws to the bone was stronger and the bone healing around the coated screws was faster than for uncoated titanium screws. Therefore, bioactivity was shown both in vitro and in vivo for the sol–gel-derived hydroxyapatite coating, which could be potentially exploited as alternative to the typical plasma spray coating technique to improve the bone bonding ability of implants.