Biocompatibility analysis of titanium bone wedges coated by antibacterial ceramic-polymer layer | Scientific Reports

Oct 20, 2024

Scientific Reports volume 14, Article number: 23085 (2024) Cite this article

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This paper presents the surface treatment results of titanium, veterinary bone wedges. The functional coating is composed of a porous oxide layer (formed by a plasma electrolytic oxidation process) and a polymer poly(sebacic anhydride) (PSBA) layer loaded with amoxicillin (formed by dip coatings). The coatings were porous and composed of Ca (4.16%-6.54%) and P (7.64%-9.89% determined by scanning electron microscopy with EDX) in the upper part of the implant. The titanium bone wedges were hydrophilic (54° water contact angle) and rough (surface area (Sa):1.16 μm) The surface tension determined using diiodomethane was 68.6 ± 2.0° for the anodized implant and was similar for hybrid coatings: 60.7 ± 2.2°. 12.87 ± 0.91 µg/mL of amoxicillin was released from the implants during the first 30 min after immersion in the phosphate-buffered saline (PBS) solution. This concentration was enough to inhibit the Staphylococcus aureus ATCC 25923, and Staphylococcus epidermidis ATCC12228 growth. The obtained inhibition zones were between 27.3 ± 2.1 mm–30.7 ± 0.6 mm when implant extract after 1 h or 4 h immersion in PBS was collected. Various implant biocompatibility analyses were performed under in vivo conditions, including pyrogen test (3 rabbits), intracutaneous reactivity (3 rabbits, 5 places by side), acute systemic toxicity (20 house mice), and local lymph node assay (LLNA) (20 house mice). The extracts from implants were collected in polar and non-polar solutions, and the tests were conducted according to ISO 10993 standards. The results from the in vivo tests showed, that the implant’s extracts are not toxic (mass body change below 5%), not sensitizing (SI < 1.6), and do not show the pyrogen effect (changes in the temperature 0.15ºC). The biocompatibility tests were performed in a certificated laboratory with a good laboratory practice certificate after all the necessary permissions.

Implants play a very important role in different areas of medicine, and antibacterial properties more often play a key role in designing them because of the bacterial tendency to attack the implant surface, where they usually form a biofilm1. The biofilm leads to septic inflammation, which is getting more difficult to treat due to increasing bacterial resistance to antibiotics2.

Bone implants are usually recognized as long-term solutions. Thus, their surface is widely modified to enhance the osseointegration process. Titanium alloys are widely used as long- or short-term implants. In the last 10 years (from 2014 to February 2024), the Scopus database identified more than 74,900 research studies covering manufacturing, characterization, and coatings formation with titanium alloys as keywords. Titanium alloy (Ti-6Al-4 V) is recognized as a biomaterial with good biocompatibility3. There are doubts regarding the corrosion products of titanium alloyed with vanadium, but this material still meets the standards for implantation materials. PSBA is a poly(ester-anhydride) that easily degrades in water. The hydrolysis of PSBA is much faster compared with the FDA-approved biodegradable polymer poly(lactide-co-glycolide), which degraded around 4–5 weeks in water4.

The surface morphology of the titanium implant might be covered using a porous oxide layer formed during the plasma electrolytic oxidation process (PEO). This is an anodization process carried out between ~ 150–500 V or higher. When spark discharges occur and form a plasma due to oxide layer breakdown, then the anodization process is classified as PEO. This phenomenon was discussed in detail in papers5,6. A porous oxide layer is formed on the complicated shapes of implants such as dental implants, bone wedges, or bone plates. During the anodization process, antibacterial agents like ceramic particles could be incorporated, such as silver oxide, silver phosphates, copper, or zinc-based species7,8,9,10. The insoluble compounds incorporated in the ceramic coatings have extended contact with tissue and may show cytotoxicity. The main goal of the antibacterial properties of the surface is to protect it from bacteria adhesion and biofilm formation within the first 24 h. In our previous study11, we presented the method to form a ceramic-polymer layer on the dental implant’s surface. The implant surface was anodized and covered by poly(sebacic anhydride) (PSBA) polymer with amoxicillin. We found the processing parameters to obtain a bacteriostatic layer with very good cytocompatibility. The coating was formed on the dental implant and the in vitro characterization was carried out. In this work, we have found how the coatings make on the bigger implant with complicated shapes like in the bone wedges. Especially, when septic inflammation is still a crucial problem after surgery, and bacteria infection could cause painful reoperation and damage to the bone tissue. It was reported that more than 40% of orthopedic infections are caused by Gram-positive bacteria Staphylococcus aureus12. Pseudomonas aeruginosa and Escherichia coli are the most Gram-negative bacteria that cause the infections.

Formation the coatings with bacteriostatic properties is a promising way to avoid the infections. For example, the titanium surface could be enriched with doxycycline13, novobiocin14 to reduce possibility to adhere methicillin-resistant S. aureus (MRSA). The next step to prove the safety and antibacterial properties of the ceramic-polymer implant is in vivo biocompatibility tests, necessary for the final proof of antibacterial ceramic-polymer properties.

In vivo tests may prove the properties and quality of the biomaterials in living organisms. There are several techniques and methods to consider for biocompatibility analysis, including animal model selection. For example, biocompatibility of Ti and Ti-6Al-4 V after alkali or heat treatment was evaluated after implantation in the abdominal connective tissue of mice15. They exhibited good biocompatibility properties and were proposed as a potential candidate for the new medical implants. For polymeric biomaterials, polyurethane materials treated with cold plasma and proteins were evaluated in vivo using a rat model16. An advanced biocompatibility test refers to an in vivo model of bacterial contamination to analyze the effectiveness of the proposed system in treating bacterial infection17. Biocompatibility tests designed for long-term implants include local lymph node assay (LLNA), intracutaneous reactivity, acute systemic toxicity, pyrogen test, and implantation to the bone for 13 weeks. These tests are carried out only when the preliminary results show that the developed biomaterials have a high probability of being used and bringing technology to the market. The main challenge of comparing biocompatibility testing results is accounting for differences in animal model, test, and time of animal breeding.

In this study, we present the biocompatibility test results of a ceramic-polymer layer containing an antibiotic formed on the titanium veterinary implant (bone wedge). The surface was modified using PEO and then covered by PSBA with amoxicillin. The three steps of surface treatment and proposed analysis are presented in Fig. 1. The coatings were characterized in detail (i.e., surface morphology, oxide layer thickness, chemical composition, surface roughness and wettability, and drug release). The biological experiments involved microbial analysis and cytocompatibility. According to ISO10993 standard, the biocompatibility tests were performed in a certified laboratory and included: LLNA, intracutaneous reactivity test, acute systemic toxicity, and pyrogen test.

Scheme of the bone wedge surface treatment to obtain the ceramic- polymer layer and methods of the implant surface and biocompatibility analysis. Created in BioRender (2024). BioRender.com/y62v618

The titanium bone wedges were purchased from IWET (Poland), which produces implants for animals. The steps of coatings formation and parameters of the anodization process and polymer layer formation are presented in Fig. 2. The bone wedges were cleaned in isopropyl alcohol for 5 min, etched in 100 g/L oxalic acid for 1 h, then anodized in 0.1 M Ca(H2PO2)2 solution at 300 V for 5 min, using the controllable power supply (Kikusui, Japan). The anodized bone wedges were immersed in polymer solution composed of 1 wt.% poly(sebacic anhydride) with 2.5 wt.% amoxicillin in dichloromethane. The speed of immersion and withdrawal was 100 mm/min. The chemical reagents were purchased from Avantor (Poland). Only the PSBA was synthesized according to a published procedure18. Figure 3 presents the images of the as-received investigated bone wedges (i.e., without a coating) and bone wedges after the anodization process and formation of a polymer layer composed of PSBA and amoxicillin.

The steps and parameters of the coating formation on titanium bone wedges.

Anodized bone wedge (IM-A) and its morphology (A) surface roughness (C,E), and bone wedge and morphology of anodized bone wedge covered by polymer with amoxicillin (IM-A-P) (B) and analyzed surface roughness (D,F).

Surface morphology and chemical composition of the anodized implant (IM-A) were analyzed using a Phenom ProX scanning electron microscope (SEM) equipped with energy dispersive X-ray (EDX) spectroscopy at an accelerating voltage of 15 kV. The anodized implant with a polymer coating (IM-A-P) was analyzed using an SEM with an Everhart–Thornley detector (EDT) (Nanores, Poland).

The oxide layer thickness was determined based on cross-sectional analysis. The IM-A sample was fixed with conductive epoxy resin (VersoCit-2, Struers) for 24 h. Then, the sample was ground using an automatic polishing machine (Forcipol 2 V, Metkon) and water abrasive paper with granulation of up to 1000 and diamond paper with granulation of 1000. The cross-section was determined using a SEM (Hitachi TM-3000, accelerating voltage = 15 kV).

All the samples were dried and coated with a thin gold layer using a gold sputtering machine (Cressington Sputter Coater 108 Auto, Cressington Scientific Instruments, UK) before SEM analyses.

Surface roughness testing was performed using a 3D Surface Metrology Microscope Leica DCM8 optical profilometer. The surface scans were processed in Leica Map software, resulting in average surface area (Sa, the extension of Ra – arithmetical mean height of a line to a surface) values and 3D surface visualizations at a magnification of 20x. Sa were measured on an area of 0.63 mm2, and Ra on a line of 0.88 mm.

The water contact angle measurements were conducted using a goniometer (DataPhysics, OCA 15EC, Germany). The 0.2 µL of deionized water was dropped onto the surfaces, and the contact angle was measured in different places in triplicate and the results are presented as an average with standard deviations. To determine the surface tension, diiodomethane was used at the same volume of water that was dropped.

Implants with the oxide polymer coatings (I-AM-P) were immersed in 15 mL of the phosphate buffer saline (PBS) solution and shaken at 80 rpm for 1 h, 4 h, 6 h, 8 h, 24 h, and 48 h. Three independent series of samples, in triplicate, were prepared, and two times all the solution were injected to the HPLC. The results are presented as an average concentration with standard deviations. After the appropriate immersion time, 100 µL of the solution was collected and filtered by a nylon filter with a pore diameter of 0.22 µm. Then, 100 µL of fresh PBS saline was added to the falcon tube with implants.

The concentration of amoxicillin released from the implant was determined using high-performance liquid chromatography (HPLC, Shimadzu chromatograph with Diode Array Detector). The analysis was conducted under isocratic conditions using a mixture of 0.05% CF3COOH in a water and acetonitrile solution (90:10 v/v) with 0.8 mL/min flow and a C18 column, 150 mm × 4.6 mm with a particle size of 5 µm (Arion, Poland). The determined equation of the calibration curve was as follows:

where: y is the calculated concentration, and x is the determined area identified.

The analytical wavelength for amoxicillin concentration analysis was set to 230 nm. The temperature of the incubator, chromatographic column, cells, and autosampler was the same: 37 °C11.

Modified surfaces of the bone wedges (IM-P-A) were immersed in PBS solution for 1 and 4 h to collect the extracts and assess their ability to inhibit bacteria growth. Analyses were performed using the Gram-positive bacteria (Staphylococcus aureus (S. aureus) ATCC 25923 and Staphylococcus epidermidis (S. epidermidis) ATCC 12228). The following tests were conducted:

analysis of bacteria inhibition in a liquid solution, where the extracts were mixed with the tryptic soy broth (bacteria culture media) in a 1:1 v/v proportion. The initial concentration of bacteria added to the solution was ~ 5·106 CFU/mL. The optical density of the solution was measured before solution incubation and after 18 h of culture in an incubator at 37 °C.

bactericidal effect of the extracts collected from the IM-A-P implants. In this case, 100 µL of solution was spread on the agar plates after 18 h of bacteria culture with the extracts, as described in Sect. 2.4a.

minimal inhibition zones were determined using the collected extracts from the implants, and 100 µL of the solution was added into the hole in agar plates and cultured in an incubator for 18 h. The bacteria were spread on the agar plates at an initial concentration of ~ 5·108 CFU/mL.

The detailed procedures for the above experiments have been published in our previous paper11. The experiment was carried out using the six independent samples in two series, and the results are presented as an average result with standard deviations.

All the procedures performed during the animal study were approved by the 1st Local Ethical Committee for Animal Experiments in Warsaw (Faculty of Biology, University of Warsaw, Ilji Miecznikowa Street 1, 02–096 Warsaw, Poland) no. 1406/2022 and the National Ethical Commission for Animal Experiments (Ministry of Science and Higher Education, Wspólna 1/3 Street, 00–529 Warsaw, Poland) no. 79/2020, 80/2020, 81/2020, 53/2021. The animal procedures were performed at the European Biomedical Institute (Warsaw, Poland), an accredited testing laboratory (PCA No. AB 1763). All experiments were performed in accordance with relevant guidelines and regulations. The animals were maintained in temperature- and humidity-controlled animal quarters, under standard light/dark conditions (12L/12D), with food and water ad libitum. The temperature and humidity were checked daily (18–21 °C and 45–57%). Animal care and handling were performed following the principles of the 3Rs (Replacement, Reduction, and Refinement) and we confirm that the experiments were carried out in agreement with ARRIVE recommendations, guidelines and regulations. All the implants were sterilized before implantation by an irradiation technique (28 kGy) at the Radiation Sterilization Plant of Medical Devices and Allografts, Institute of Nuclear Chemistry and Technology, Warsaw, Poland. The test implants were stored at 2–8 °C.

The implant coating extracts were prepared, by immersing them in a solution of 0.9% sodium chloride for injection (polar extract, PE) for all tests (only for local lymph node assay the solution of 0.5% hydroxyethylocelulose in sodium chloride was used), and acetone in olive oil (4:1 v/v) for local lymph node assay (LLNA) test, or cottonseed oil for intracutaneous reactivity and acute toxicity test (non-polar extract, NPE), at a ratio of 1 mL per 3 cm2 of the implant surface, for 72 ± 2 h at 50 ± 2 °C. Collected extracts were stored in the 5 ± 3ºC for less than 24 h before use. The sodium chloride (polar control, PC) and olive oil or cottonseed oil (non-polar control, NPC), accordingly, were used as vehicle controls.

The test was conducted in compliance with the following standards: ISO 10,993–12:2021(E), Biological evaluation of medical devices – Part 12: Sample preparation and reference materials19, ISO 10,993–10:2021, and Biological evaluation of medical devices – Part 10: Tests for skin sensitization20.

Local lymph node assay (LLNA) was performed in adult, albino, healthy house mice (Mus musculus), BALB/C strain. 20 female nulliparous and non-pregnant mice, 8–12 weeks old, weighing 17.3–22.1 g, were chosen for the LLNA test. To perform the LLNA test, mice were randomly assigned to two experimental groups: PE (n = 5) and NPE (n = 5), and two vehicle control groups: PC (n = 5) and NPC (n = 5). The 25 μL/day of tested solution was applied on the dorsal side of both mouse ears for three consecutive days. On the 5th day from the beginning of the LLNA test, each mouse was injected intraperitoneally with 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) in PBS solution. 24 ± 1 h after BrdU injection, animals were humanely euthanized, and auricular lymph nodes from each mouse’s ear were excised and processed separately in PBS. A single-cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation of tissue through a 70 μm nylon mesh cell strainer. In each case, the final volume of the LNC suspension was adjusted to 15 mL with PBS.

Abcam’s BrdU Cell Proliferation ELISA Kit (colorimetric) was used according to the manufacturer’s protocol. The colored reaction product was quantified using a μQuant spectrophotometer with a dual wavelength of 450/550 nm. The results were expressed as the mean spectral intensity (SI). For this test, the acceptance criteria were as follows: SI of Positive control (PC) > 1.6, SI of test item ≤ 1.6 non-sensitizing, SI of test item > 1.6 sensitizing.

The intracutaneous reactivity test was conducted according to the ISO 10993-23:2021(E) standard. 3 adult, healthy, female, non-pregnant New Zealand white rabbits (Oryctolagus cuniculus), weighing 3.5–4.9 kg were used for the test. 17 h before animal treatment, the test area of fur on the animal’s back on both sides of the spinal column was closely clipped, avoiding mechanical irritation and trauma. The left side of the rabbit’s body was injected 20 times intracutaneously with 200 μL of PE or NPE extracts or PE, NPE control solutions. The one side of the same rabbit’s body was injected 5 times intracutaneously with 200 μL of PC or NPC, on the other side rabbit’s body was injected 5 times with 200 μL of the control PC or NPC solutions. All animals were observed immediately after injection, 24 ± 2, 48 ± 2, and 72 ± 2 h after the treatment. Injection sites were examined for evidence of any tissue reaction, such as erythema, edema, and eschar. For erythema, eschar and edema formation the tested and control sites were scored according to the grading system presented in Table 1:

Acute systemic toxicity was conducted according to ISO 10,993–11:2017(E): Biological evaluation of medical devices – Part 11: Test for systemic toxicity21. The toxicity test was performed in adult, healthy house mice (Mus musculus). The 20 female nulliparous and non-pregnant mice, between 9–10 weeks old, weighing 19.0–22.6 g, were chosen for the toxicity test. To perform the toxicity test, mice were randomly assigned to four experimental groups of 5 animals (n = 4 × 5 mice). The animals were injected with the same dose of extracts or controls (50 mL/kg of body weight), but in a different manner: NPE and NPC were injected intraperitoneally, whereas PE and PC were injected intravenously. Then, animals underwent a clinical examination and were weighed 24 ± 2 h, 48 ± 2 h, and 72 ± 2 h after injection. After the toxicity test, the mice were euthanized.

The pyrogen test was conducted according to ISO 10993–11:2017(E): Biological evaluation of medical devices – Part 11: Test for systemic toxicity and European Pharmacopeia (Ph. Eur.). 10th edition21. 3 adult, healthy, female, non-pregnant New Zealand white rabbits (Oryctolagus cuniculus), weighing 3.6–4.4 kg were used. The warm (38.2 °C) PE was injected intravenously into the rabbit’s marginal ear vein at a dose of 10 mL/kg of body weight. The rabbit’s temperature was monitored every 30 min for 3 h after injection.

Figure 2 presents the surface morphology of the anodized titanium implant (Fig. 3A) and anodized implant with a polymer layer with amoxicillin (Fig. 3B). The plasma electrolytic process causes the formation of a porous layer. When the oxide layer is broken, spark discharges occur, causing the formation of open and closed pores. The anodization process easily causes the formation of a ceramic layer covering all of the implant, if there is contact with the anodizing bath. The average surface roughness of the anodized bone wedge analyzed, based on the Sa, was 1.15 µm. Covering the anodized implant using the polymer with amoxicillin did not show significant changes in the Sa parameter (Sa was 1.16 µm). The differences in surface roughness were found when the profiles were analyzed. For the anodized bone wedge, the Ra was 0.99 ± 0.07 µm and increased to 1.12 ± 0.07 µm after the formation of a polymer layer with amoxicillin.

The chemical composition of the anodized titanium bone edge was analyzed using EDX. Depending on the analyzed area (i.e., upper part or internal part of the implant), the calcium (Ca) and phosphorus (P) content was different. In the upper part, Ca content ranged from 4.16 to 6.54%, whereas P content ranged from 7.64 to 9.89%. In the interior parts of the implant, the Ca content was from 0.51–0.94%, whereas the P content was 0.89–4.32%. The oxide layer coatings, determined using a cross-section, were 5.05–8.17 µm thick (Supplementary). The surfaces of both coatings were hydrophilic, and the determined water contact angle for the anodized bone wedge and bone wedge with polymer coatings were 88.79 ± 10.5° and 54.6 ± 1.6°, respectively. The surface tension determined using diiodomethane was 68.6 ± 2.0° for the anodized implant, whereas for the implant with hybrid coatings (i.e., IM-A-P) the contact angle was 60.7 ± 2.2°.

The concentration of the drug (amoxicillin) released from the polymer coatings (i.e., from IM-A-P) surface into a PBS solution was determined using HPLC (Fig. 4). An advantage of this method is the separation and quantification of analytes, in this case, amoxicillin. The bone wedges were immersed in PBS solution for up to 48 h. During the first hour, 12.87 ± 0.91 µg/mL of amoxicillin was released. After 4 h, the amoxicillin concentration slightly decreased to 9.01 ± 0.74 µg/mL. During the first hour, a higher concentration of amoxicillin was released due to drug adsorption and agglomeration on the top part of the coating. Then, the concentration slightly decreased which is related to the decreasing amoxicillin stability in water. These phenomena were recognized and discussed previously22,23. Thus, it is very important to keep the amoxicillin concentration at an appropriate level to effectively inhibit bacteria adhesion and growth, especially during the first 4 h. After 48 h the cumulative concentration of amoxicillin was around 42 μg, it is more than 25% of total drug loaded in the polymer layer.

Concentration of amoxicillin released from the implant surface into the PBS solution. Analysis was performed using HPLC. The total concentration of amoxycillin in the coating was 157.17 ± 3.90 μg (n = 16).

The collected extracts from the IM-A-P samples immersed in PBS solution were used to conduct microbial analysis using Gram-positive bacteria strains (S. aureus ATCC 25923 and S. epidermidis ATCC 12228). Table 2 presents the collected results, where the extract was used to check the inhibition possibility of the bacteria, bactericidal effect, and the inhibition zones using the agar plates.

The extracts collected after 1 h and 4 h inhibited the growth of both bacteria species. However, the concentration was not enough to achieve a bactericidal effect. The larger inhibition zones (29–30 mm) were obtained when the extracts were collected after 1 h of the IM-A-P immersion compared to the inhibition zones formed using the extracts collected after 4 h, which were 27–30 mm. Therefore, a lower concentration of amoxicillin was in the solution after 4 h of implant immersion, and these results agree with the HPLC analysis.

The LLNA did not reveal any signs of toxicity or skin irritation in mice from both tested and control groups. The body weight of the animals throughout the test is presented in Table 3. To evaluate the LLNA, the cellular proliferation was determined by measuring the BrdU content in the DNA of lymphocytes. The BrdU was measured by ELISA, and the results are summarized in Table 4. In the murine LLNA, the PEs and NPEs collected from the bone wedges with an oxide-polymer layer didn’t induce the proliferation of lymphocytes in the lymph nodes at the site of the tested extracts and vehicle controls application. For the polar and non-polar extracts the body changes was below 1.4%, and SI value was lower than 1.6. For the non-polar extract the SI value was more than twice lower than the acceptance criteria. Therefore, the test implants were considered as not sensitizing.

An intracutaneous reactivity test was carried out with white rabbits. Table 5 summarizes the results of the experiment, where grade 0 means no changes to the rabbit’s skin, whereas grade 5 indicates a severe reaction of the rabbit’s skin to the tested solution. After 24 h, 48 h and 72 h no signs on the rabbit’s skin were found. Our results showed no intracutaneous reactivity of the tested extracts.

During the observation period, none of the animals treated with the PE or NPEs collected from the implants showed signs of acute systemic toxicity, i.e., no significantly different biological reactivity in comparison to animals treated with the vehicle control was observed. Our results demonstrated that both PEs and NPEs carried no acute systemic toxicity when the tested solutions were administered intraperitoneally or intravenously. Table 6 summarizes the animal’s weight throughout the test. None of the animals lost more than 10% of their body weight. We observed higher weight fluctuations in the NPE group compared to the PE group. However, the NPC, i.e., cottonseed oil, had a similar pattern of body weight fluctuation as the NPE group. Thus, the results may be related solely to the type of NPC vehicle.

A rabbit pyrogen test was conducted using only PE collected from the implants with the coatings. Table 7 summarizes the temperature monitoring throughout the test. The polar extracts did not affect the rabbit’s temperature. The change in the average temperature of the rabbits was between 0 and 0.15 °C. Importantly, the temperature did not exceed 1 °C, which is the top tolerance margin for the pyrogen test. Thus, the implant extract did not show a pyrogen effect.

Formation of the bacteriostatic coating on orthopedic implants is one of the crucial ways to protect the surface against bacteria biofilm formation. Because antibiotics, such as doxycycline or amoxicillin are sensitive to light or decrease their stability over time, the drug delivery system is crucial. Poly(sebacic anhydride) is a crystalline, degradable polymer in water solution. Several methods for PSBA synthesis (22 catalysts have been investigated) result in different molecular weights (Mw) which affects the hydrolysis process duration24. In our previous paper11, we reported the results of a 1 wt.% PSBA blended with 5 wt.% of amoxicillin hydrolysis in PBS. After 1 h of the PSBA immersion, more than 53% of sebacic acid (product of hydrolysis) was found (H1 NMR analysis). Our results indicated that the PSBA caused better water wettability of the bone wedges. The surface tension was also lower for the hybrid (oxide-polymer) layer than for anodized-only implants. Application of the dip coating caused the formation of an additional layer with the loaded drug and better surface wettability. In the literature, we have found that a lower contact angle of polymer is achieved using cold plasma (for polyurethanes or parylene C). Additionally, the surface could have been treated using a sonochemical method to deposit the selected drugs25. These surface functionalization methods are very interesting but need access to specialist apparatuses (plasma), and sonication techniques could make cracks in the oxide layer.

Cytotoxicity analysis confirmed that 0.5 wt.% and 1.0 wt.% PSBA did not decrease the viability of mouse fibroblast L929 cells or osteoblast-like MG-63 cells11. This polymer is biocompatible and used to form nano- or micro-particles or films on metal surfaces. For example, PSBA is loaded with curcumin for pulmonary applications26, chondrocytes to engineer cartilage27, or doxorubicin for cancer treatment28. There are several promising applications of this polymer, such as bacterial infection treatment, where this polymer could be used as a material to deliver a bioactive substance.

Bacterial infections associated with biomaterial implantation have increased over the last few years. In 2021, Ribeiro et al.29 discussed the mechanism of bacteria adhesion to implants and the challenges related to orthopedic implants caused by Staphylococcus sp. Bacterial infection is a leading cause of implant rejection, additional fixation of open-fractured bones, or joint-revision surgeries. Antibacterial surfaces on the implants may restrict bacteria adhesion and colonization. There are several in vitro studies demonstrating the antibacterial effect of ceramic coatings on titanium substrates, where a copper compound composed of hydroxyapatite with Nb2O530 or glass-ZrO29 and glass-ZnO particles31 were applied.

The biocompatibility of the materials influences the chemical composition of the material and physicochemical properties like their surface morphology, roughness, concentration of chemical compounds, and ion release from the implants over time. Usually, the antibacterial coatings are evaluated under in vitro conditions, where the implants with coating are immersed in water-based solutions like PBS or sodium chloride. In this paper, we present a broad spectrum of biocompatibility analysis for the antibacterial properties of implant coating extracts, both in polar and non-polar solutions, using animal models according to ISO standards.

Drug release from the implant surface efficiently inhibits bacteria growth as well as kills bacteria, if their concentration is at a sufficient level. In our previous study32, we reported that the stability of amoxicillin decreased within the first 24 h of immersion in PBS solution. However, the concentration of the drug was enough to inhibit S. aureus (ATCC 25923) growth. In our previous article11, we reported on the antibacterial coating formation process on titanium dental implants. The coating was composed of PSBA with amoxicillin. An advantage of using an anodization process and dip coatings technique for the formation of coatings is the possibility of applying these methods for various implant shapes (the parameters are transferable). The parameters of the dip coatings technique were slightly corrected to produce similar anodization results (chemical composition, pore size, and layer thickness) for the dental implants and bone wedges. For bone wedges, the solution of PSBA contains a lower concentration of amoxicillin (more than 50%). The bone wedges are composed of several plates, and a higher concentration of amoxicillin was deposited on this implant than on the dental implant. Because the main goal of the implant surface treatment is to prevent bacteria adhesion, the concentration of amoxicillin in the solution for polymer layer formation was decreased.

The shape of implants plays a key role in antibacterial coating formation and affects the biocompatibility results. Novel titanium materials recommended as long-term implants are evaluated under in vivo conditions. Animal models used for in vivo studies will differ based on the size and shape of the implants. For example, Sommer et al.33 analyzed the biocompatibility of a cast porous titanium alloy (Ti-6Al-7Nb) and titanium coated with a calcium titanate layer. Two different animal models were chosen. The cylindrical material of the same size was implanted in the femoral condyle of rats and sheep. After 30 days, some abnormalities were observed around the implant in the rats’ bone tissue, whereas, in sheep bone, no significant defects were found in tissue in the 6 months after implantation. The biocompatibility of two materials can also be analyzed differently. For example, a ceramic (zirconium) and titanium implant were tested using the same animal model34. In this case, a pig was chosen to analyze the biocompatibility and osseointegration with bone tissue. Based on the histological analysis, a direct-bone implant contact with both materials was found. Histomorphometry results showed slight differences (~ 5%) in material integration.

In the presented study, we presented the results of a biocompatibility analysis where one type of implant was tested, and the PEs and NPEs were collected. The LLNA analysis did not show any signs of toxicity or skin irritation in the tested animals, both with extracts and vehicle controls. The average value for the BrdU index and SI calculation was below 1.6, which confirmed that the tested extracts were not toxic. When PEs and NPEs were tested, the SI value was higher for the PE. The PSBA polymer easily undergoes hydrolysis. In polar solvents, we measured a higher concentration of amoxicillin compared to non-polar solvents.

When the intracutaneous reactivity of PEs and NPEs was tested, no significant ravages to the rabbits’ skin were observed. Also, in the acute systemic toxicity test, mice did not display any symptoms of the toxicity of the studied extracts. We only observed insignificant fluctuations in the body weight when the NPE was applied. However, the cottonseed oil injections themselves (the NPE vehicle) caused greater fluctuations in body weight when compared to the sodium chloride solution (PE vehicle). The last test we performed is very sensitive and restrictive, where a change in body temperature of more than 1 °C indicates pyrogenicity. In our case, the total rabbit temperature change was 0.30 °C.

In previous in vivo studies on a polymethylmethacrylate biomaterial, one of three rats in the group developed allergies, and one developed a pyrogen effect, suggesting an existing relationship between allergen and pyrogen tests35. Transient inflammation was observed in rat lung tissue after the application of titanium oxide particles encapsulated in amorphous silica, whereas carbonyl iron particles exhibited a minor inflammatory effect in animals after 24 h of particle inhalation. Therefore, one can conclude that the first hours of an experiment are crucial to observing eventual pathological reactions to powders, solutions, or implanted materials.

This study presents a titanium bone wedge with a successfully modified surface for long-term application in bone tissue. The appropriate parameters of the plasma electrolytic oxidation process and dip coatings technique were found to create a ceramic porous oxide layer and polymer layer with amoxicillin to enhance antibacterial properties. The modified surface released enough amoxicillin into the PBS solution to inhibit S. aureus and S. epidermidis bacteria growth. The large inhibition zones confirmed the antibacterial activity of the extracts collected from the implant.

Several years of experiments led us to invent our implant surface treatment with antibacterial properties. We propose a biocompatible implant with a sufficient antibiotic concentration released within the first hour of material immersion to kill bacteria. PEs and NPEs collected from the implants were used in biocompatibility tests. The advanced analysis according to the ISO 10993 standard confirmed that the extracts are non-allergic, non-toxic, non- sensitizing, and do not show a rabbit pyrogen effect. Moreover, the intracutaneous reactivity test results of the extracts collected from the implants exhibited no effect classified as eurytherms or edema.

All data of measurements and reports are available on request.

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This work was supported by the National Center for Research and Development, Poland, according to the LIDER XI program. Project “Technology of hybrid coatings formation on titanium implants for animals” no. LIDER/12/0048/L-11/19/NCBR/2020. This work was supported by a Rector’s Grant in the field of research and development (Silesian University of Technology, Poland, 04/010/RGJ23/1053). Authors thank MSc Eng. Katarzyna Leśniak-Ziółkowska (Silesian University of Technology, Poland) for SEM analysis (Phenom ProX) and Dr. Monika Śmiga-Matuszowicz (Silesian University of Technology, Poland) for the PSBA synthesis.

Faculty of Chemistry, Silesian University of Technology, B. Krzywoustego Str. 6, 44-100, Gliwice, Poland

Alicja Kazek-Kęsik, Weronika Maciak & Wojciech Simka

Biotechnology Centre, Silesian University of Technology, Krzywoustego Str. 8, 44-100, Gliwice, Poland

Alicja Kazek-Kęsik

Department of Physiology, Faculty of Medical Sciences in Katowice, Medical University of Silesia, Poniatowskiego 15, 40-055, Katowice, Poland

Daria Gendosz de Carrillo

Department of Histology and Cell Pathology, Faculty of Medical Sciences in Zabrze, Medical University of Silesia, Poniatowskiego 15, 40-055, Katowice, Poland

Daria Gendosz de Carrillo

Faculty of Biomedical Engineering, Silesian University of Technology, Roosevelta 40 Street, Zabrze, Poland

Anna Taratuta

European Biomedical Institute, Nałkowskiej Street 5, 05-410, Józefów, Poland

Zuzanna Walas & Damian Matak

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Alicja Kazek-Kęsik, conceptualization hypothesis, writing the paper, analysis and interpretation of data, publication search, graphics, financial source. Daria Gendosz de Carrillo: conceptualization and data analysis, writing the paper. Zuzanna Walas, analysis and interpretation of data. Weronika Maciak: material preparation, analysis and technical support. Anna Taratuta: surface roughness analysis. Damian Matak interpretation of data. managing team for in vivo experiment. Wojciech Simka: anodizing procedure consultation.

Correspondence to Alicja Kazek-Kęsik.

The authors declare no competing interests.

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Kazek-Kęsik, A., de Carrillo, D.G., Maciak, W. et al. Biocompatibility analysis of titanium bone wedges coated by antibacterial ceramic-polymer layer. Sci Rep 14, 23085 (2024). https://doi.org/10.1038/s41598-024-72931-w

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Received: 08 March 2024

Accepted: 11 September 2024

Published: 04 October 2024

DOI: https://doi.org/10.1038/s41598-024-72931-w

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