Probing cytocompatibility, hemocompatibility and quantitative

Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Probing Cytocompatibility, Hemocompatibility, and Quantitative Inflammatory Response in Mus musculus toward Oxide Bioceramic Wear Particulates and a Comparison with CoCr Nitu Bhaskar,† Debasish Sarkar,‡ and Bikramjit Basu*,†,§ †

Laboratory for Biomaterials, Materials Research Centre, Indian Institute of Science, Bangalore-560012, India Department of Ceramic Engineering, National Institute of Technology, Rourkela, Odisha 769004, India § Center for Biosystems Science and Engineering, Indian Institute of Science, Bangalore-560012, India ‡

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ABSTRACT: Peri-prosthetic bone resorption and loosening of artificial joints have been widely recognized to limit the performance of the load-bearing implants. Therefore, the present study probes into the cyto-, hemo-, and histocompatibility of the small sized wear particulates of ZrO2-toughened Al2O3 (ZTA). In order to develop a comprehensive, yet clinically relevant understanding, a comparison is made with two baseline ceramics (Al2O3 and ZrO2) and metallic material (CoCr), all in particulate forms. While in vitro cytotoxicity assessment was carried out with mouse osteoblast cells, preclinical testing of intraarticularly injected particulates up to the concentration 25 wt % in PBS over the period of 12 weeks was performed in mouse model. Interestingly, mouse osteoblast cells, cultured in media with ZTA, Al2O3, and ZrO2 particulates of three different concentrations (0.25, 2.5, and 25 mg/mL) exhibited uncompromised cell viability and considerable cell spreading, up to the time frame of 72 h. In contrast, similar experiments with CoCr particulates demonstrated significant decrease in cellular growth with drastic change in osteoblast proliferation behavior. Furthermore, the decrease in RBC damage after contact with ZTA, Al2O3, and ZrO2 particulates at 25 mg/mL of dose level illustrated 4.4, 5.8, and 1.25% hemolysis, respectively, confirming clinically acceptable hemocompatibility. However, hematic activity of CoCr particles was reflected with 15.6% of hemolysis. In vivo, the absence of any significant effect of intra-articularly injected ceramic (Al2O3, ZrO2, ZTA) as well as metallic (CoCr) particulates on complete hemogram and serum biochemistry of Balb/C mice was recorded at all the time points up to 12 weeks. The extensive histological analysis confirmed the absence of any signature of the tissue-level toxicity at all time points. The proinflammatory cytokine analysis using TNF-α and IL-1β markers provided complementary evidence toward nongranulomatous and nonimmunogenic response of synovial membrane of knee joint and other vital organs of mice that were exposed to Al2O3, ZrO2, ZTA, and CoCr particulates. Taken together, our results establish the nontoxic nature of oxide ceramic particulates to bone cells, in vitro, as well as to periprosthetic tissue, in vivo. KEYWORDS: Ceramics, ZrO2-toughened-Al2O3, wear debris, cytotoxicity, in vivo, intra-articular, cytokines, total joint replacement, arthroplasty increase in human life expectancy.5,6 However, the longevity and performance of these orthopedic implants can not be guaranteed beyond 12 to 15 years due to the complex problem

1. INTRODUCTION In recent years, a steady growth has been noticed in the number of total hip arthroplasties due to increased global burden of disease associated with osteoarthritis and inflammatory rheumatoid arthritis in younger patients.1−5 Both total hip and knee arthroplasties, involving the patient’s joint being replaced by an implant, have reduced the aging process with an © XXXX American Chemical Society

Received: May 19, 2018 Accepted: July 26, 2018 Published: July 26, 2018 A

DOI: 10.1021/acsbiomaterials.8b00583 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

CoCr metal alloy powder (150 μm, Goodfellow Cambridge Ltd., England, Catalog No. CK266010) were procured for the competitive in vivo analysis. To obtain phagocytable size range of CoCr, a lloy particles were ball milled (agate balls of size 10 mm) using isopropyl alcohol as milling medium in a planetary ball mill (FRITSCH, Puloerisette 1583, Germany) at 300 rpm. The powder to ball ratio (by weight) was kept to be 1:10. The ball milled powders were dried overnight, crushed, and sieved for further experimental usage. Alpha modified Eagle’s Medium (MEM-alpha), fetal bovine serum (FBS), antibiotic antimycotic solution (10 UmL−1 penicillin and 0.1 mg mL−1 streptomycin), and trypsin−EDTA (0.25% trypsin, 0.53 mM EDTA) were purchased from Gibco, Invitrogen Bioservices, India. All other chemicals and reagents were of analytical grade, purchased from commercial sources, and used as received. 2.2. Characterization of ZTA Particulates. Zirconia-toughened alumina (ZTA) wear debris were generated after continuous interactive motion of two articulating surfaces of acetabular socket and femoral head, both made of ZTA [alumina (95 wt %)−zirconia (5 wt %)−800 ppm MgO]. The final stage of prototype development involved the multistage sintering with final holding at 1600 °C for 6 h in air.13,17 The wear debris particles were generated through a tailormade assembly, wherein the unpolished ZTA femoral head was mounted upward on the vertical spindle, rotated anticlockwise at 100 rpm. The femoral head was firmly pressed against ZTA acetabular socket, attached with a spring-loaded clockwise rotating and swinging device under a load of 100 kgf. The finer debris particles were collected carefully, and the particle sizes were reduced to clinically relevant size, for in vitro and in vivo experimentation. The phase composition of ZTA wear particles was determined using X-ray diffraction (XRD; X’Pert Pro, PANalytical, Netherland). The Cu Kα (λ = 0.15456 nm at 40 kV and 30 mA) target was used as a X-ray source radiation and diffraction patterns were recorded with 2θ ranging from 20° to 90° with step size of 0.02°. Transmission electron microscopy (TEM, Model-2100F, JEOL) was performed at an accelerating voltage of 200 kV to assess the finer scale morphology and aggregation state. For TEM analysis, the particles were suspended in ethanol and sonicated for 30 min in order to reduce agglomeration. The particles were mounted by drop-coating (droplets of 3 μL) on TEM copper grids and vacuum-dried overnight. The size of the particles and agglomerates in supplemented ethanol was also measured by dynamic light scattering (DLS, Zetasizer nano ZS model, Malvern Instruments Ltd., Malvern, U.K.). 2.3. Eluate Preparation. The particulate eluates of three different concentrations (0.25, 2.5, and 25 mg/mL) for Al2O3, ZrO2, CoCr, and ZTA were prepared by suspending the respective nanoparticles in sterile phosphate buffer saline (PBS; pH 7.4). These dispersed mixtures were ultrasonicated for 30 min for complete dispersion of particles. Further, the eluates were steam autoclaved at 121 °C for 20 min, followed by ultraviolet (UV) sterilization for 30 min. The eluates were ultrasonicated again, once before conducting in vitro and in vivo experiments, to ensure complete homogeneous dispersion of particles. 2.4. In Vitro Cytotoxicity. 2.4.1. MC3T3-E1 Cell Culture. The mouse osteoblast precursor cell line (MC3T3-E1), derived from Mus musculus calvaria were used for the in vitro cytotoxicity experiments. Importantly, MC3T3-E1 is a widely used osteoblast cell line, with a list of advantages such as (a) ease of maintenance (b) unlimited number of cells because of active replication, and (c) relative osteoblast phenotypic stability during initial phase of culture.18,19 Prior to seeding, the cells were procured from cryo-preserved stock and expanded in tissue culture graded 25 cm2 flask (Eppendorf, Germany) in the presence of complete growth media, supplemented with alpha modified Eagle’s medium (α-MEM; Gibco, India), 15% (v/v) fetal bovine serum (FBS; Gibco, India), and 1% (v/v) antibiotic antimycotic solution (10 UmL−1 penicillin and 0.1 mg mL−1 streptomycin; Sigma-Aldrich, India). The osteoblast cells were maintained at 37 °C in 95% humidified atmosphere of 5% CO2 in an incubator (Sanyo, MCO-18AC, USA). The culture flask was fed every 3 days and split at 80−90% monolayer cultured confluency using 0.05 (w/v) % trypsin-EDTA (Gibco, India) solution for conducting the cell culture experiments.

of aseptic loosening, initiated by adverse cellular inflammatory response to wear particles generated primarily at articulating surfaces.7,8 These debris particulates stimulate the periprosthetic bone resorption (osteolysis), that results in mechanical instability between the implant and the surrounding tissue, ultimately leading to implant failure.5 Therefore, it is important to develop new biomaterials and to comprehensively investigate the biocompatibility properties, which are expected to mitigate the risks of osteolysis.5 At present, the focus of intensive research is to fabricate various hard-on-hard articulating bearings (i.e., metal-on metal or ceramic-on-ceramic), which generally produce less amount of debris than hard-on-soft (metal-on-polymer) prostheses.2,9 It is worthwhile to mention that ceramic-on-polymer is also clinically used as an articulating joint in THR surgery. Although the metal implants have good mechanical properties, due to low wear and corrosion resistance, their biocompatibility often gets compromised resulting from the release of metallic ions.10 Therefore, the best choice for clinical applications of implants appears to be the ceramics, which possess high chemical stability and excellent osteoconductive properties.10 However, the biocompatibility properties of the finer (nano- to submicrometer-sized) ceramic particles are of particular concern, clinically.11 For example, Affatato et al. noted the absence of any significant difference between the wear behavior and primary osteoblast proliferation onto ZTA nanocomposites in comparison to commercial alumina. In another study, Roualdes et al. reported the in vitro and in vivo biocompatibility of an alumina−zirconia ceramic composite with particular focus on cell proliferation and extracellular matrix formation by osteoblasts and fibroblasts.12 A moderate and nonspecific granulomatous response of the synovial membrane without any major inflammation was recorded in Sprage Dawley rats, injected with these powders around the knee articulation. In our recent research, we have reported a unique manufacturing strategy to develop dense ZTA-based femoral head and acetabular socket prototypes.13−16 A number of performance limiting properties, including burst strength and sliding wear resistance were also determined. In the above backdrop, we present an in vitro and in vivo approach for evaluating the biocompatibility of finer particles of ZTA. The immunological as well as systemic synovial microcirculation effects caused by small sized ZTA wear particulates were then compared with baseline material (Al2O3 and ZrO2). More importantly, a comparison is also made with metallic alloy (cobalt−chromium; CoCr), which is clinically used as orthopedic implant. In preclinical testing, all the particles were injected intra-articularly into mice knee joint to simulate the release of wear debris into the joint cavity. In order to evaluate the potential injury in vital organs, we observed changes in the hematologic and serum biochemical parameters along with effects on the ultrastructure of the knee joint, liver, kidney, lung, heart, and spleen collected at different time intervals, postinjection. The induction of proinflammatory cytokines, Interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) markers in serum as well as in the synovium were also determined to assess inflammatory response in knee joint, quantitatively.

2. MATERIALS AND METHODS 2.1. Materials. Commercial grade of virgin particles of Al2O3 (120 nm, Sumitomo, Japan), ZrO2 (50 nm, Tosoh, Japan), and atomized B


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Figure 1. Schematic depicting the biological significance of the present study and experimental design to investigate the inflammatory response of lab-scale generated wear particles of ZTA after intra-articular treatment in Balb/C mice. 2.4.2. Cell Proliferation. At 24 and 72 h of culture, the osteoblast viability was analyzed by MTT assay (3(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide, Sigma-Aldrich, India).20 The cells were cultured in 96 well plates at approximate density of 3 × 103 cells/well with further incubation for 24 h. After the desired period of incubation, the culture media was aspirated and replaced with fresh medium containing different test particles at the concentration of 0, 0.25, 2.5, and 25 mg/mL. The medium was harvested at 24 and 72 h later and washed twice with PBS to remove the particle layer. Furthermore, the cells were incubated with 0.5 mg MTT/mL reagent prepared in DMEM without serum. After 4 h of incubation at 37 °C, unreacted MTT dye was removed, and formazan crystals were solubilized by addition of 200 μL dimethyl sulfoxide (DMSO). The optical density (OD) was quantified spectrophotometrically in an ELISA microplate reader (iMark, Bioradlaboratories, India) at 595 nm. The results were expressed as percentage viability, calculated as follows, % cell viability =

OD of the sample × 100 OD of the control

coagulation. First, 3 mL of blood was centrifuged at 1500 rpm for 10 min. The plasma and surface layer were discarded. The remaining RBC pellet was washed five times with 6 mL of PBS solution and RBCs were diluted in 25 mL of PBS solution. Then, 0.8 mL of different particle eluates suspended in PBS at concentration of 25 mg/ mL were added to 0.2 mL of RBC suspension. Also, positive and negative control samples were prepared by adding distilled water and PBS, respectively to 0.2 mL of RBC solution, respectively. Then, the samples were incubated at room temperature for 2 h. After stipulated time period of incubation, each sample were then inverted to mix the contents homogeneously and centrifuged at 3000 rpm for 5 min (Eppendorf 5810R, USA). Supernatant was carefully collected and transferred to 96 well plates for measuring absorbance by an ELISA microplate reader (iMark, Biorad laboratories, India) at 570 nm. The percentage hemolysis was calculated as follows:

hemolysis (%) =

OD(test) − OD(negative control) OD(positive control) − OD(negative control) × 100

(1)

2.6. In Vivo Study. All animal experiments were performed in compliance with animal care guidelines provided by the “Committee for the Purpose of Control and Supervision of Experiments on Animals” (CPCSEA, India) and preapproved by Institutional ethical committee of laboratory animals, Indian Institute of Science, Bangalore (Approval No. CAF/Ethics/478/2016). Female Balb/C mice (M. musculus), provided by Central Animal Facility, Indian Institute of Science, Bangalore, India, aged 6−8 weeks and weighing 20−22 g were used in the experiments. Every five mice were housed in stainless steel cages containing sterile paddy husk as bedding in ventilated animal rooms. They were acclimated in the standard controlled environment (22 ± 1 °C room temperature, 60 ± 10% relative humidity, and 12 h light/dark cycle) with free access to water and a commercial laboratory complete food. 2.6.1. Intra-Articular Exposure Assay. For the present work, sixtythree mice were randomly assigned into seven treatment groups comprising one control group and six experimental groups for administration of different test particles. These were as follows: Group I, 0 mg/mL of particles (control/vehicle); Group II, 25 mg/ mL of CoCr particles; Group III, 25 mg/mL of Al2O3 particles; Group IV, 25 mg/mL of ZrO2 particles; Group V, 0.25 mg/mL of ZTA wear particles; Group VI, 2.5 mg/mL of ZTA; Group VII, 25 mg/mL of ZTA particles. On the basis of our previous in vivo study on hydroxyapatite (HA)−40 wt % barium titanate (BaTiO3), the right-knee of all the animals were shaved prior to intra-articular treatment.11 The anesthesia used for all experiments were 90 mg/kg ketamine and 4.5 mg/kg xylazine, administered by intraperitoneal injections. All the mice received sterile intra-articular eluate injections of approximately

2.4.3. Cell Morphology. In order to analyze the cell morphology, mouse osteoblast cell (MC3T3-E1) were plated in 24 well plates (5 × 103 cells/well) and incubated for 24 h. The finer particles of Al2O3, ZrO2, CoCr, and ZTA were introduced separately into cells with 25 mg/mL concentration in complete growth medium. Cell culture in the medium without adding any particles were taken as control. At 24 and 72 h of culture, the cells were washed thrice quickly with PBS to remove particles and then fixed with 4% paraformaldehyde (PFA; SD Fine-Chem Limited, India) in PBS for 30 min at room temperature. The cells were again washed with PBS and permeabilized with 0.1% Triton X solution for 5−10 min. To prevent nonspecific binding of dye, the cells were blocked with 1% bovine serum albumin (BSA) for 1 h after a two time wash with PBS. Cells were immunostained for 30 min with Hoechst stain 33342 (Life technologies, India) and Alexa Fluor 488 (Life technologies, India) to observe nuclei and actin filaments, respectively. The cells were observed under fluorescence microscope (Nikon LV 100D, Japan) to study their morphological/ phenotypical behavior. 2.5. Hemocompatibility Assessment. Hemolysis experiments with CoCr, Al2O3, ZrO2, and ZTA particles were conducted according to the protocol mentioned in ASTM E2524-08(2013), Standard Test Method for Analysis of Hemolytic Properties of Nanoparticles, ASTM International, West Conshohocken, PA, 2013. Also, the guidelines and policies of Institutional Animal Ethics Committee of laboratory animals, Indian Institute of Science, Bangalore (Approval No. CAF/ Ethics/414/2014) were followed. Approximately, 5 mL of healthy whole blood was drawn from New Zealand white rabbits and mixed with a 9:1 volumetric ratio of (3.8% w/v) sodium citrate to prevent C


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Figure 2. Toxicity and inflammatory response largely depends on wear particle chemistry and size distribution. (a) X-ray diffraction pattern showing peaks of both alumina and zirconia; (b) bright field transmission electron microscopic images showing particle round morphology; (c) particle size distribution determined using DLS. Most of the ZTA wear particles diameter were in the range 170−220 nm. ZTA: zirconia-toughened alumina 50 μL of different test particles suspended in PBS (at the dose concentrations mentioned above), in right knee of mice, once a week for a period of 8 weeks (Figure 1). The injections were made with insulin syringes with ultrafine 6 mm × 31G needle. Following the exposure, the animals were held for postexposure period of 4 weeks to notice the recovery of animals, toward any lethal effects caused by administration of different test particulates. All the animals were euthanatized at either the fourth or eighth week (during the injection period) or at the 12th week (without any eluate dose injections in the preceding weeks after 8 weeks) to collect the blood samples for hemotological and serum estimation, and various vital organs along with the right knee joint for histological observations. All the animals were examined daily for survival and evidence of behavioral aspects. Mice were also weighed every week to observe any change in their body weight. 2.6.2. Haematological and Serum Biochemical Analysis. Blood was drawn for hematology analysis (EDTA collection tube) using a standard orbital sinus blood collection technique. A number of hematology markers were analyzed using a hematological autoanalyzer (Sysmex KX-21N, USA). These include red blood cell count (RBC), hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, neutrophils, lymphocytes, monocytes, and white blood cells (WBC) count. Whole blood collected via orbital sinus or by cardiac puncture (0.5−0.8 mL) was centrifuged twice at 3000 rpm for 10 min in order to separate serum. Using a biochemical autoanalyzer (Erba Mannheim, Erba Diagnostics, Inc. USA), serum biochemical analysis was carried out to determine the serum levels of total protein and

total cholesterol, which are the important parameters for studying the influence of oxidative stress caused by a toxic material. Total bilirubin (TBIL), alanine aminotransferase (ALT/SGPT), aspartate aminotransferase (AST/SGOT), and alkaline phosphatase (ALP) were measured to evaluate liver function. The levels of blood urea nitrogen (BUN) and creatinine (CR) were determined to assess nephrotoxicity. Also, Albumin (ALB) was assayed as one parameter of damage of tissue or inflammation. The effect on cardiac activity was assayed by determining the level of the enzyme, lactate dehydrogenase (LDH). 2.6.3. Histological Analysis. A small piece of a vital organ such as heart, liver, kidney, spleen, lung, and knee joint from the sacrificed animal was recovered, fixed in 10% neutral buffer formalin (NBF) and dehydrated using ascending grade of ethanol series (70, 80, 90, and 100%). The following sequential stages were then followed, and those include embedding of the sample into paraffin blocks, sectioning them into 5−7 μm thickness, and mounting the resulting sample on the glass microscope slides using standard histological techniques.21 The histology sections were stained with a standard Hematoxylin and Eosin (H & E) stain. The bright-field images were taken using observed using optical microscope (Nikon LV100D, Japan), equipped with a Nikon DS-Fi1 color CCD camera. Histological changes, such as synovial hyperplasia, inflammatory cells (macrophages/leukocytes) infiltration, and articular cartilage erosion, were also scored from stained sections of knee joint of mice to evaluate the severity of the inflammation and immune cell infiltrations. The scoring of histological changes was done as follows: (−) normal; (+) mild; (++) moderate; (+++) severe. In addition, the thickness of synovial membrane was measured at five random locations in each histological section using Image-J software. D


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Figure 3. Shape and size of particles plays critical role in toxicity assessment in vitro/in vivo. Bright field transmission electron microscopic images of (a) CoCr, (b) Al2O3, and (c) ZrO2. Particles distribution curve determined from DLS, showing the distribution of the (d) CoCr, where the majority of particles were in the size range of 188−254 nm; (e) Al2O3 in the size range of 165−298 nm; (f) ZrO2 with size range of 220−600 nm. CoCr: Cobalt−chrome. 2.6.4. ELISA Quantification of Pro-Inflammatory Cytokines. The release of IL-1β and TNF-α markers was measured in synovium homogenate, as well as in serum, by use of a quantitative enzyme linked immunosorbent assay kit (Platinum ELISA kit, eBioscience, Austria). Synovium homogenate was prepared by lavaging the knee joint first with PBS (pH 7.4) with further mincing in ice cold buffer, followed by centrifugation at 3000 rpm for 15 min at 4 °C. The supernatant was kept at −80 °C (153 K) until use. The ELISA assays were performed according to the manufacturer’s protocols. Photometric measurements in pg/mL were conducted at 450 nm by using an ELISA microplate reader (iMark, Biorad Laboratories, India). 2.7. Statistical Analysis. The data were expressed as mean ± standard error (S.E.). For statistical analysis, the experimental values were compared to corresponding control ones. A one-way analysis of variance (ANOVA) with posthoc Tukey HSD calculated in SPSS software was used to illustrate the significant difference between the experimental group and the control. The significant difference was considered to be p ≤ 0.05 and p ≤ 0.01.

(ICDD) with JCPDS card numbers 01-074-1081 and 01-0751863 for Al2O3 and 00-050-1089 for ZrO2. The morphology and hydrodynamic particle diameter (size) distributions of Al2O3, ZrO2, CoCr, and ZTA particulates were characterized by TEM and DLS, respectively. From the TEM bright field images, we observed that the commercially available virgin Al2O3 and ZrO2 particles were nearly round to polygonal in shape (see Figure 3a,b). Dynamic light scattering measurement showed the average size of 220 and 340 nm for Al2O3 and ZrO2 particles, respectively (Figure 3e,f). Furthermore, the hydrodynamic particle size distribution of CoCr particles showed mean size of 221 nm, where most of the particles ranged from 188−220 nm (Figure 3d). TEM images also revealed that CoCr particles in aggregates were close to granular in shape with more polygonal morphology (Figure 3a) . The ZTA wear particles have a mean size of 191 nm with narrow size distribution from 165- 220 nm. Representative bright field TEM images of ZTA particles confirmed their regular spherical to round polygonal shapes (Figure 2b,c). The larger irregular shaped particles of both CoCr and ZTA suggested that the particles aggregated in the aqueous medium. Summarizing, the above results imply that both ZTA and CoCr have similar particle size distribution, and therefore, any difference in the toxicity response should be attributed to compositional differences only. Also, the size distribution of ZTA and alumina is also comparable with that of zirconia particles in slightly larger sizes with broad distribution. 3.2. In Vitro Cytocompatibility of ZTA Wear Particles. 3.2.1. Effect on Cell Viability. In order to assess the cytocompatibility of ZTA wear particles, in vitro study with MC3T3-E1 murine osteoblast cells was performed, and cellular response was compared with commercially available particles of Al2O3 and ZrO2 and metallic CoCr. The results of cell

3. RESULTS In this section, we shall first describe the particle morphology and size distribution of the investigated wear debris particles. This will be followed by cyto-, hemo-, and histocompatibility assessment of such particles, when tested as per the protocols, described in the preceding section. A critical analysis of the results will follow in the next section. 3.1. Characterization of Clinically Relevant ZTA Wear Particulates. The phase and morphology of the wear debris particles were initially analyzed using XRD, TEM, and DLS techniques. Figure 2(a) shows the XRD pattern of the ZTA wear particles, confirming the presence of both alumina (Al2O3) and zirconia (ZrO2) phases. The peak positions of the constituent phases were observed to match with the standard XRD patterns from the International Crystallographic database E


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spreading, and density appeared to be normal, when incubated with ceramic eluates of Al2O3, ZrO2, and ZTA, up to 24 h of incubation (Figure 5c,d). Most of the cells adopted flat to

viability, i.e., the proliferation of murine osteoblast cells with different doses (0.25, 2.5, and 25 mg/mL) of biomaterial particulates at 24 and 72 h of time points, are presented in Figure4. At both the time points, the mitochondrial viable cells

Figure 4. Cell viability against a material in particulate form is different from its bulk composition. MTT assay results to probe into eluate dosage dependent cell proliferation of mouse osteoblasts (MC3T3-E1) at 24 and 72 h of culture. The data are expressed as percentages values (mean ± S.E., n = 9) where 100% corresponds to control untreated cells. ** represents significant difference at p ≤ 0.01 from control using one way Anova followed by a post hoc Tukey test. ZTA: zirconia-toughened alumina. CoCr: cobalt−chrome.

were measured to any reduction in cell growth by MTT assay (3(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide). The oxidization of the MTT dye by mitochondrial succinate dehydrogenase enzyme, which is specifically present in mitochondria, results in the formation of blue-violet colored formazan crystals.22 After 24 h of exposure, the cell proliferation was not affected by the presence of CoCr, ZrO2, and ZTA particles at any of the eluate dosage. Indeed, no significant difference (p ≥ 0.05) was observed in the cell proliferation between cultures with or without particles, as indicated by ANOVA followed by a comparison with Tukey’s posthoc test. However, a highly significant (p ≤ 0.01) increase in osteoblasts viability by 48 and 40% was recorded when cultured in the presence of 2.5 and 25 mg/mL of Al2O3 particles, respectively. At 72 h of cell culture, the osteoblasts, when incubated with CoCr particulates, exhibited a marked decrease (p ≤ 0.01) with 53%, 53%, and 44% at the respective doses of 0.25, 2.5, and 25 mg/mL, compared to control. In contrast, such growth rate was not affected significantly by Al2O3, ZrO2, and ZTA particles at any of the treated concentrations, when compared to control. Summarizing, ceramic particulates of Al2O3, ZrO2, and ZTA were well tolerated by proliferating osteoblasts with no evidence of significant toxicity in contrast to metallic CoCr particles, which caused significant decrease in cell viability. 3.2.2. Changes in Cell Morphology. The morphology of proliferating cells on any material substrate is one of the important indicator for the status of cells. The phenotypical changes after exposure to 25 mg/mL of particulate concentration were recorded to demonstrate the effect of CoCr, Al2O3, ZrO2, and ZTA particles on morphology and spreading behavior of MC3T3-E1 murine osteoblasts. As compared to control (without particles), cell morphology,

Figure 5. Cell morphological changes depend on eluate treatment. Fluorescence microscopy images of Hoechst 33342 (actin filament, green)/ DAPI (nucleus, blue) stained mouse osteoblast cells (MC3T3-E1) exposed to 25 mg/mL of particle eluates of (a) control, (b) CoCr, (c) Al2O3, (d) ZrO2, and (e) ZTA at 24 h of post exposure; (f) control, (g) CoCr, (h) Al2O3, (i) ZrO2, and (j) ZTA at 72 h post exposure. Cells with a distinct morphological changes can be easily observed at 24 (b) and 72 h (g) after incubating with CoCr particles. ZTA: zirconia-toughened alumina. CoCr: cobalt−chrome.

polygonal shape with elongated thread-like projections (filopodial extension of cytoskeletal actin). Such observations indicate firm attachment of healthy cells to the culture plates, in the presence of particles. Furthermore, at 72 h of cell culture, the cells began to aggregate and the neighboring cells appeared to maintain physical contact with each other by multiple extensions. A marked elevation in cell density was F


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articular injection of CoCr, Al2O3, ZrO2, and ZTA particulates for 8 weeks did not cause any adverse effect on animal health status, because there was no significant change observed in the experimental animal’s body weight (Figure S1). The size, color, and morphology in tissue architecture also remain unchanged with no evidence of congestion, atrophy, or inflammation after intra-articular treatment with Al2 O 3 , ZrO 2 , and ZTA particulates. But, in the case of CoCr, visual inspection of mice showed granular deposit around the patella during the course of treatment (until the eighth week), corresponding to the accumulation of CoCr particles in the knee joint tissue. However, such influence was reduced 4 weeks postexposure (at 12 weeks). 3.4.2. Changes in Hematological and Serum Biochemical Parameters. The hematological studies were carried out to assess changes on the levels of RBC, WBC, Hb, Hct, MCV, MCH, MCHC, neutrophils, lymphocytes with other immune cells, and platelets. Representative complete blood count results indicated that measured factors were within normal ranges. Also, none of these parameters showed any statistical difference (p ≥ 0.05) between the control and the particulate treated groups (Figure 7). From Figure 7, it can be clearly seen that at week 8 and week 12, the WBC and differential neutrophils count in particulate treated animals showed the elevation (p ≥ 0.05) in their mean values, when compared with that of fourth week of necropsy. However, the level of neutrophils again decreased at the 12th week and was found to be similar to that of the fourth week. Additionally, to determine if particles of ceramic (Al2O3, ZrO2, and ZTA) and metal (CoCr) produce any physiological changes in the animals, different types of serum biochemical parameters were determined. The level of total protein and cholesterol in the serum are assessed to know the effects of stressors on animal’s metabolic pathway. At eighth week of time point, the total protein level in mice treated with CoCr and Al2O3 particles showed a significant decrease (p ≤ 0.05) by 30% and 31% respectively, when compared with control. However, total cholesterol and ALB in serum remain unaffected in the animals, when treated with either of these material particles (Figure 8). For renal toxicity, the levels of BUN and CR (metabolites associated with the functionality of the kidney) were measured. Also, the levels of TBIL, ALT, AST, and ALP in blood were tested to measure of hepatic and biliar functionality. A detailed analysis of all these metabolites of kidney and liver in serum of animals treated with CoCr, Al2O3, ZrO2, and ZTA could not reveal any statistically significant differences (p ≥ 0.05) as compared to controls (Figure 8). However, at eighth week, the levels of ALT and ALP showed inclination in the mice exposed with CoCr particles, but difference was not significant (p ≥ 0.05) compared to control. In all the experimental groups, the release of LDH in serum was also similar to that of control with no statistically significant difference. 3.4.3. Qualitative Observations of Histological Changes in Tissues. The potential translocation and toxicity of metallic (CoCr) as well as different ceramic (Al2O3, ZrO2, and ZTA) particulates after 8 weeks of intra-articular exposure to the major organs such as liver, kidney, spleen, heart, and knee joint were evaluated at week 4, 8, and 12 of animal sacrifice. 3.4.3.1. Liver. Because of its relatively large size, high metabolic activity, and anatomical location, the liver serves as the prime site for xenobiotic metabolism and detoxification.23 Liver sections are good indicators of chemical toxicity, which

exhibited, when compared to the images of 24 h (Figure 5h− j). This increase in cell number was similar to that of control. After incubating with CoCr particles, osteoblasts appear to be internalized/endocytosed the particles. This resulted in a marked shuffle of cell morphological features with a change in cytoskeletal organization and an extensive decrease in cell numbers. The osteoblast cells after CoCr exposure, showed spindle shaped morphology, rather than the normal shape (Figures 5b and 5g). The length of one axis of the cells appeared to be larger than that of control. Also, the cultured cells exhibited a marked shrinkage with increased irregularity, which might have resulted probably due to an increase in number of microvilli on the cell surface. All these changes can be seen at 24 h of particulate contact and become clearly prominent after 72 h. In general, the results of the cell morphological analysis was consistent with the results of the mitochondrial viability (MTT) assays. 3.3. Effect on Hemolytic Activity. All the particles that enter the blood get in contact with erythrocytes, and therefore, it is important to explore the safety of particles in the bloodstream. In order to assess such impact on red blood cells (RBC), hemolysis test was performed, in vitro. The particles of CoCr, Al2O3, ZrO2, and ZTA were incubated with rabbit whole blood to evaluate their capacity to induce hemolysis. Such an effect was quantified using spectrophotometric measurement of hemoglobin release. The measurement with CoCr particles showed 15.6% of hemolysis, whereas at the same concentration of 25 mg/mL, the ceramic particles of Al2O3, ZrO2, and ZTA caused 5.8, 1.3, and 4.4% hemolysis, respectively (Figure 6). It can be seen that the hemolysis by

Figure 6. Hemocompatibility of ceramic particulate eluates is equally significant as wear particles are translocated from articulating joints to different tissues through bloodstream in physiological systems. Hemolytic effect of different test particulates on blood of New Zealand white rabbits after 2 h incubation with RBCs at 37 °C. The different particles were suspended at the concentration of 25 mg/mL in PBS. Data was generated from three independent experiments and represented as mean ± SE. Particle eluates of Al2O3, ZrO2, and ZTA demonstrated lower hemolytic activity compared to CoCr. ZTA: zirconia-toughened alumina. CoCr: cobalt−chrome.

ceramic particles were less than 10%, which is considered as the safe value for biomaterials according to ISO 10993-4 standard. All the above observations establish hemocompatibility of the investigated ceramic particulates. 3.4. In Vivo Toxicity. 3.4.1. Animal Behavior, Symptoms, and Mortality. No mortality was recorded during the entire study period in experimental and control groups. The intraG


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Figure 7. Animal metabolism can be altered by the toxicity caused by finer particulates, and this can be reflected in changes in the complete hemogram. Complete blood counts of female Balb/C mice, M. musculus, exposed to intra-articular injection of the different particles eluates, once a week for a period of 8 weeks. Data are expressed as mean ± SE (n = 3). No statistically significant (p > 0.05) changes were observed between the groups when compared with control (vehicle) group using ANOVA. ZTA: zirconia-toughened alumina. CoCr: cobalt−chrome.

(Figure 9 and Figures S2 and S3). The ultrastructural observations of liver in all the groups showed a clearly defined liver parenchyma, composed of hepatic cords and sinusoids arranged radially around the central vein. The hepatocytes were polygonal in shape, with round vesicular nuclei located centrally. The blood sinusoids were characterized by narrow

occur frequently toward the intake of foreign body particles. In the present study, liver sections were obtained, showing the hepatocytes, nuclei, hepatic cords, hexagon lobules, sinusoids, central veins, and portal veins of control. Also, mice treated intra-articularly with CoCr, Al2O3, ZrO2, and ZTA particulates were found to be normal at all time points of observations H


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Figure 8. Toxicity by material particulates can influence various organ functionality, which can be indirectly assessed by blood biochemical parameters. Clinical chemistry indexes of female Balb/C mice, M. musculus, following intra-articular injection of particulate of CoCr, Al2O3, ZrO2, and ZTA, once a week for a period of 8 weeks. Data are expressed as mean ± SE (n = 3). The serum protein level of CoCr and Al2O3 groups decreased significantly at week 8 of exposure. * denotes statistical significance difference a p < 0.05 when compared with control (vehicle) group using ANOVA. ZTA: zirconia-toughened alumina. CoCr: cobalt−chrome.

base balance. A toxic material to the kidney therefore could disrupt its functional integrity, leading to adversely affecting total body metabolism.24 In the present study, ultrastructural observation of kidney at week 4, 8 and 12, did not show any renal necrosis (Figure9 and Figures S2 and S3). No marked difference between the histology of the control and mice treated with CoCr, Al2O3, ZrO2, and ZTA particulates was found. In all the histology images, the normal tissue architecture of renal corpuscle, renal tubules, and convoluted tubules were observed. Nearly all of the straight portion of the

branching, separating the cords of the liver cells and anastomising with each other. A closer look at these sections could not reveal any pathological change or particle agglomeration, which can be correlated with an exposure of liver toward particle exposure. 3.4.3.2. Kidney. The kidneys are vital organs involved in total body homeostasis. This is in view of their regenerative capacities to serve against detoxification mechanisms.24 They play a principal role to serve against detoxification mechanisms in the excretion of metabolic wastes and in the regulation of extracellular fluid volume, electrolyte composition, and acid− I


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Figure 9. Changes in tissue architecture can be potentially caused by systemic translocation of wear particles and its toxicity. Histological analysis of vital organ of mouse stained with hematoxylin/eosin (H&E) to assess the potential effects on the organ morphological architecture and cellular damage, after different particulates treatment. Representative images exhibit no signs of toxicity in heart, lung, spleen, kidney, and liver collected from the animals sacrificed at the eighth week (n = 3). The size of the scale bar corresponds to 100 μm. CoCr: cobalt−chrome. ZTA: zirconiatoughened alumina.

Figure 10. Qualitative analysis of local inflammatory response can be assessed by careful analysis of tissues in the vicinity of the delivery site of wear particulates. Histological appearance of the synovial membrane (stained with H&E) of mice knee joints observed at week 8 and 12. The control section showed a normal synovial membrane with no inflammation and intact smooth articular cartilages. Key: AC, articular cartilage; S, synovial membrane; black arrow, synovium with activated dense immune cells infiltration; red arrow, eroded surface of articular cartilages. CoCr: cobalt− chrome. ZTA: zirconia-toughened alumina.

particles. The sections did not show any alteration in the organization of the cardiac tissue. The uninterrupted striated cardiac muscle cells, known as myocardium and cardiomyocytes with one centrally placed oval nucleus, can be noticed in almost all the treated mice, similar to the control group (Figure 9 and Figures S2 and S3). 3.4.3.5. Lung. The histology sections of lung, recovered at 4, 8, and 12 weeks, could not show any remarkable difference among the control and CoCr, Al2O3, ZrO2, and ZTA particletreated groups. They showed normal tissue architecture of lung alveolar, peribranchial, and perivascular regions with uniform alveoli and clear bronchioles lumen. There was no hyperplasia or particulate accumulation in lung parenchyma of mice treated with any of these particles, when compared to that of controls (Figure 9, Figures S2 and S3). 3.4.3.6. Knee Joint. The histological appearance of knee joints of mice showed normal morphological structure of the articular cartilages with intact and smooth articular surfaces after sacrificing at fourth week of exposure, in all the groups

proximal tubules of kidney appeared to have normal tissue architecture (Figure 9 and Figures S2 and S3). 3.4.3.3. Spleen. Histological examination of spleen sections of mice treated with CoCr, Al2O3, ZrO2, and ZTA particulates showed normal architecture without any abnormality when compared with their respective controls (Figure 9 and Figures S2 and S3). All the sections of spleen, collected at the fourth, eighth, and 12th weeks, were characterized by natural parenchymal tissue comprised of red and white pulp. The red pulp is distinguished by a three-dimensional meshworks of vessels and cords. The erythrocytes, leukocytes, hemocytoblasts, and megakaryocytes can easily be seen in the red pulp area. The white pulp region indicates lymphoid tissue, consisting of lymphocytes, macrophages, plasma cells, dendritic cells, arterioles, and capillaries in a reticular framework. 3.4.3.4. Heart. A general observation of cardiac tissue architecture in the histology sections from all the mice treated with CoCr, Al2O3, ZrO2, and ZTA particulates reflected normal appearance without any noticeable presence of eluate J


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eighth and 12th week. The severity of articular cartilage erosion was reduced in ZrO2-treated animals at the 12th week. The surfaces of articular cartilages were found to be very smooth and intact in case of Al2O3- and ZTA-treated animals at all the time intervals of observations, which was very similar to the control. In addition, the measurement of the synovial membrane thickness, as shown in Figure 11, revealed that the synovium

treated with CoCr, Al2O3, ZrO2, and ZTA particles. Synovial membrane was also normal in all these groups with no alteration in its thickness, which reflects hyperplasia. All these observations revealed that mice were able to tolerate all these particles until the fourth week of exposure. In the sections from the eighth and 12th weeks of autopsy (Figure 10), mild foreign-body response with an inflammatory reaction was observed in the synovial membrane of animals exposed to Al2O3 and ZTA particles. The knee joint exposed to CoCr and ZrO2 showed a marked inflammatory reaction with severe erosion of articulating surfaces and synovial hyperplasia percolated with macrophages and foreign body giant cells. The inflammatory reaction seemed to be more vigorous in CoCr group at the 12th week, when compared with animals of other treatment groups (Figure 10). The histological findings did not differ much between ZTA and control groups. Furthermore, no particulate deposition was evident in any of particulates exposed groups at any of the time points. 3.4.4. Semi-quantitative Histological Analysis. In addition to the above-described histological analysis, the semiquantitative approach was also adopted. Histological evaluation as semi-quantitative score, presented in Table 1, indicates Table 1. Semi-Quantitative Scoring of Histological Findings in the Knee Joints of Mice retrieved at Different Time Intervals after Intraarticular Treatments with Different Particles Eluates for a Period of 8 weeksa

Figure 11. Temporal variation of synovium membrane thickness from histological sections of knee joint. Increase in membrane thickness represent the cell layer hyperplasia and immune cell reaction in synovium. The data are represented as mean ± SE of five fields evaluated using using ImageJ software. * Significantly different at p ≤ 0.05 from control using one way Anova followed by a post hoc Tukey test. CoCr: cobalt−chrome. ZTA: zirconia-toughened alumina.

histological findings for eluate treatment time synovium hyperplasia control CoCr Al2O3 ZrO2 ZTA

inflammatory cell infiltration

articular cartilage erosion

8 weeks

12 weeks

8 weeks

12 weeks

8 weeks

12 weeks

− ++ + ++ −

− +++ + +++ +

− ++ ++ + +

− ++ + + ++

− ++ − +++ −

− +++ ++ + −

thickness was more in CoCr particles treated animals at both fourth and eighth weeks, and also in ZrO2 treated animals at eighth week of autopsy. Following statistical analysis, all these changes were found to be nonsignificant, when compared to control. In contrast, the statistical analysis of results showed that eluate suspensions of CoCr and ZrO2 particles caused significantly greater membrane thickening than controls in histology sections of knee joint tissues at 12th week of autopsy. However, membrane thickness was found to be normal in the animals treated with Al2O3 and ZTA particles at all the time points, when compared to the control group. 3.4.5. Quantitative Analysis of Changes in Pro-Inflammatory Cytokines Release. 3.4.5.1. Tumor Necrosis Factor-α. Figure 12 shows that metal (CoCr) as well as ceramic (Al2O3, ZrO2, and ZTA) particles induced TNF-α to the level in knee joint synovium of mice in statistically insignificant level with respect to control, at week fourth of exposure. At 8 weeks, the mice exposed with 25 mg/mL of CoCr (3.6 ± 0.38 pg/mL) showed slightly higher level of TNF-α in their synovium than the control group (2.5 ± 0.66 pg/mL), although this difference was not significant (p ≥ 0.05). Importantly, the biodistribution of ceramic particles of Al2O3, ZrO2, and ZTA did not result in any change in the level of TNF-α, when analyzed at the same time point. In contrast, the mean value of TNF-α was found to be significantly (p ≤ 0.05) low in all the mice exposed intraarticularly with 0.25 mg/mL of ZTA (1.73 ± 0.47 pg/mL), than that of the control group (2.17 ± 0.60 pg/mL) at 12th week of sampling (Figure 12a). However, the delivery of all

a

Key: (−) normal, (+) mild, (++) moderate, and (+++) severe. Values are expressed after evaluating knee joint sections of three animals from each group (n = 3). CoCr: cobalt−chrome. ZTA: zirconia-toughened alumina.

that both CoCr and ZrO2 particles were more inflammatory than Al2O3 and ZTA. This closely corroborates with synovium hyperplasia, sub synovial tissue reaction and erosion of articular cartilage. At eighth week, synovial hyperplasia was found to be moderate in case of the animals treated with CoCr and ZrO2 particulates. In these groups, hyperplasia response in synovial is upregulated to severe at week 12 of necropsy, when compared to other groups (Al2O3, ZTA, and control) exhibiting normal to mild response. Furthermore, at the eighth week, the subsynovial tissue reaction, observed as inflammatory exudates rich in activated macrophages and foreign body giant cells, were recorded within the synovial fluid of knees in all the experimental groups (CoCr, Al2O3, ZrO2, and ZTA). However, the level of leukocyte infiltrations in synovium could be seen only in CoCr and ZTA group at 12th week. In Al2O3and ZrO2-treated animals, these inflammatory exudates were gradually replaced by an inflamed synovium with more pronounced fibroblast-like synoviocyte proliferation. Further evaluation of histological semiquantitative score revealed severely eroded cartilage surfaces found in knee joint of animals treated with particulates of CoCr and ZrO2 at the K


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Figure 12. Quantitative inflammatory response analysis complements the qualitative evidence from histological assessment. The effects of dosage and type of particle eluates (CoCr, Al2O3, ZrO2, and ZTA) on pro-inflammatory cytokine markers (a) TNF-α, (b) IL-1β secretion in local vicinity (synovium) of knee joint, and (c) TNF-α (d) IL-1β in blood serum, measured at different time intervals. The values are represented as mean ± SE (n = 3). * Significantly different at p ≤ 0.05 from control using one way Anova followed by post hoc tukey test. TNF-α: tumor necrosis factor alpha. IL-1β: interleukin 1 beta. ZTA: zirconia-toughened-alumina. CoCr: cobalt−chrome.

Similarly, significant changes (p ≥ 0.05) in IL-1β expressions in the blood serum were not detected at any of the time points, as compared to control, after exposing the animals with particulates of either CoCr or Al2O3, ZrO2, and ZTA (Figure 12d).

other particulates did not result in any significant (p ≥ 0.05) change in TNF-α secretion level in knee joint synovium. At fourth week of sampling, intra-articular treatment of 25 mg/mL Al2O3 (20.7 ± 1.53 pg/mL) and ZrO2 (20 ± 1 pg/ mL), and 0.25 mg/mL of ZTA (20.33 ± 3.21 pg/mL) caused a significant (p ≤ 0.05) decrease in the secretion of blood serum TNF-α of mice, when compared with that of control (23.7 ± 1.53 pg/mL) (Figure 12c). Further, the levels of serum TNF-α of CoCr, Al2O3, ZrO2, and ZTA particulate exposed animal groups were almost same at eighth and 12th weeks, when compared to control with no significant difference (p ≥ 0.05). 3.4.5.2. Interleukin-1β. In Figure 12b, the mice exposed intra-articularly to 25 mg/mL of CoCr, Al2O3, ZrO2, and ZTA particulates for the period of 4 weeks showed elevated expression of synovial IL-1β cytokine level of 11.53 ± 1.56, 11 ± 2.65, 9.9 ± 0.16, and 9.8 ± 1.15 pg/mL, but the differences were found be statistically insignificant (p ≥ 0.05) when compared to control (8.17 ± 0.85 pg/mL). Further, no significant (p ≥ 0.05) variation could be found in the secretions of synovial IL-1β in treated mice when exposed with any of CoCr, Al2O3, ZrO2, and ZTA particulates at the eighth and 12th weeks of study.

4. DISCUSSION Implant-derived particulate wear debris of total joint replacement components have important clinical implications as they can inflame surrounding tissues, leading to periprosthetic bone resorption and aseptic loosing.1,25 The submicron to nanoscale wear particles can be biodistributed to the various vital organs, resulting in toxic effects systemically.26 Therefore, the biological activity of wear particles is considered as a major factor limiting the survival rate of implants and their overall success in patients.27 The understanding of the biological cascade of events resulting from osteolysis requires a number of experimental approaches that closely simulate the potential toxicity of debris generated into the joint cavity by prosthetic implants.25 On this aspect, we planned to comprehensively study in vitro and in vivo effects of clinically relevant wear particulates. The experimental design for the present study was based on earlier reported studies where a L


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toxic responses of commercially available CoCr particles, in vitro.26,33−36 During fluorescence microscopic examination, the extensive spreading of osteoblast cells with normal morphological features/cytoskeletal organization were observed, when incubated with either ZTA or Al2O3 and ZrO2 particulate suspensions. At 72 h of incubation period, the substrate was completely covered with the cells, which were connected to each other by multiple extension. In contrary, fluorescence microscopic images revealed disruption in normal morphological features and growth, after incubating the cells with metallic CoCr particle suspension. The most characteristic morphological changes, such as cell shrinkage, formation of long cell processes and microvilli, as observed in the present study, could be attributed to the stress caused by endocytosis of CoCr particles and its metal ions, when released into the culture system.37 All these results indicate the marked difference in cellular compatibility of ceramic and metallic particles. 4.2. Hemocompatibility Response. A systematic investigation of hemolysis was conducted to analyze the hemocompatibility response of ZTA wear particulate to blood, in vitro. Hemolysis is a problem caused by excessive fluid diffusion into the erythrocytes. This causes them to swell and rapture in the presence of any material.22 Also, it reveals the impact of physicochemical characteristics of particles on red blood cells. In general, percentage of hemolysis of less than 10% is considered as a biosafety threshold level. Therefore, all the tested particles are hemocompatible. In particular, the particulates of ZrO2 and ZTA showed significantly lower hemolytic activity (less than 5%) at 25 mg/mL of concentration. At the same dosage, hemolysis rates of Al2O3 particles stayed nearly to 6%, indicating no detectable disturbance of the red blood cell membranes. The hemocompatibility of all the ceramic particles can be attributed to their affinity for blood serum proteins. High affinity to proteins is established through the interplay of hydrophobic interactions between electrostatic force and hydrogen bonding, which effectively prevents both erythrocyte membrane damage and occurrence of blood clotting around particulates.38 In contrast, incubation of CoCr particles with RBCs resulted in high percent destruction of RBC membrane with nearly 15% of hemolysis. Consistent with MTT assay and florescence microscopic observation, CoCr particulates were found to disrupt membrane structure. The reasons for all such in vitro cytotoxic effects can be assigned mainly to interactions of metallic particles with cell membranes, leading to its destruction and subsequent activation of intracellular signal transduction pathways. These aspects however remain controversial in the current literature.39 4.3. Blood Compatibility and Histocompatibility. Since the in vitro results may not reflect the complex interplay of systemic and local metabolic factors in vivo, the present study further validates the in vitro results by carefully evaluating the biological effects of ZTA wear debris in mice, when injected with 0.25, 2.5, and 25 mg/mL concentrations, at the right knee joint. The recovery of the animal was also investigated by keeping the animal for 4 weeks post exposure after continuous injection of particulate suspension with frequency of 1 week for the period of 8 weeks. Also, based on the in vitro cellular toxicity results, only highest dose (25 mg/mL) of Al2O3, ZrO2, and CoCr particulates were selected for comparing the in vivo results in terms of hematology and

similar approach has been used to probe the effects of hydroxyapatite (HA)−40 wt % barium titanate (BaTiO3) biocomposite using in vitro study with mouse myoblast and osteoblast cell lines.11,28 Furthermore, in the same study, the authors reported the histocompatibility of injected particulates of HA-40 wt % BaTiO3 in the right knee joint of Balb/C mice to observe the local as well as systematic cellular responses, in vivo.11 ZTA wear debris used in the present study was isolated from laboratory scale testing of femoral head/acetabular sockets mated surfaces. The particles of CoCr, Al2O3, and ZrO2 were obtained from commercial suppliers, rather than retrieving them from a wear stimulator. As mentioned earlier also, all these particles were used to study baseline toxicity, if any, caused by Al2O3 and ZrO2. Also, attempts were made to compare the results obtained for ZTA with CoCr under similar testing conditions to understand the level of biological toxicity. It is well-known that the size, shape, composition, and concentration/particle load of wear debris play an important role in producing the inflammatory response.11 Also, the particles with a mean size of less than 10 μm are considered to be within the phagocytosable range, eliciting proinflammatory reactions.29 Therefore, care was taken to ensure that all the test particles used for conducting present study have a size range that is reported to be phagocytosable by macrophages, in vivo conditions. It is clear from TEM and DLS investigation that the sizes and morphology of Al2O3, and CoCr particles are comparable Also, zirconia particles are of slightly larger sizes with a broad distribution (see Figure 3c and 3f). Furthermore, our results showed that most of the ZTA wear particles were also varying from 165−220 nm, which is considered to be a clinically relevant size. Furthermore, it is impossible to correlate directly the amount of injected wear particles used for any scientific study with the amount of particles released in the clinical scenario, because the range of debris concentration at the local site has been reported to vary largely depending on the implant type and composition.7,30 In order to mimic such a scenario, three different concentrations of ZTA particulates were chosen to determine the cellular toxicity response ranging from lower to higher exposure level. It is important to reiterate once more that since they are of comparable sizes and morphology, any difference in cytocompatibility, hemocompatibility, and histocompatibility of the investigated partice sizes should be ascribed to compositional differences only. 4.1. Cytocompatibility Response. In order to assess the relative toxicity of the ZTA wear particles, the particles were cultured with MC3T3-E1 osteoblasts. ZTA particles cultured with osteoblasts did not have a significant effect on the cell viability for the period of 72 h at any of the concentration tested. Similarly, Al2O3 and ZrO2 particles, at the same concentrations, were found to be nontoxic without any decrease in cell viability. The present results are also in agreement with the literature reports of very small or weak detrimental biological effects of ceramic debris.12,31,32 In contrary, CoCr particles cultured with osteoblasts, for a period of 72 h, resulted in highly significant reduction in cell viability at all the particle dose concentrations of 0.25, 2.5, and 25 mg/ mL, demonstrating its cytotoxic nature. Although the mechanism of cytotoxicity caused by CoCr is not yet fully understood, cellular toxicity might be attributed either to generation of soluble metallic ion in culture or indeed intracellular dissolution of the particles, when they are internalized by the cells.33 Several studies have reported the M


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membrane thickness showed only small effusion and subsynovial cellular infiltrate of leucocytes in synovial membrane. Also, articular cartilage architecture was found to be intact in the animals, exposed to Al2O3 and ZTA particles. These slight changes in synovium can be correlated with pivotal role of protection elicited toward foreign body particles clearance.12 These events might have also occurred due to complex cellular and molecular interaction due to the injury caused by injection procedure.47 Generally, when particles of a particular size are injected in intra-articular space of joints, they are usually encountered by macrophages. Also, these events trigger the complement system cascade and inflammatory cell response, leading to chemotactic activities.48 Furthermore, the particles of CoCr and ZrO2 were capable of inducing a marked inflammatory reaction with severe erosion of articulating surfaces and thick synovial membrane. This foreign body reaction, which was observed at the eighth week, seemed to be more vigorous in CoCr group at 12 weeks. Similar to the present results, intense macrophage infiltration and focal degeneration of synovial tissues was observed by Howie and Vernon-Roberts, after injection of laboratory-prepared particulate of CoCr alloy particles with a mean size of 3 μm in rat knee joint.30,49 These authors also claimed that CoCr wear particles and the associated macrophage response can persist in the synovium tissues for up to 2 years.30 In the case of ZrO2 particulate exposure, a much lesser number of activated leucocytes with more fibroblast and mast cellslike synoviocyteproliferate, except thickening of the synovial membrane was noticed at 4-week postexposure (12th week of autopsy). These observations confirm the recovery state of synovial tissue as well as damaged articular tissue over a period of time. Interestingly, after repeated injections of different particle suspensions into the knee joint of animals, none of the histological sections showed agglomeration or deposition in the joint cavity, manifesting their endocytosis by macrophages. An absence of particles agglomerates also supports the concept that joints possess the ability to dispose of wear particles via the lymphatic system.49 The degradation of articulating cartilage at knee joint (after treatment with CoCr) are in agreement with those by Wang et al.,50 who suggested that the erosion of cartilage in male Sprague−Dawley rats after intraarticular injection of titanium dioxide nanoparticles could have resulted the inflammatory response in synovium. This might have regulated articular cartilage remodelling and ultimately affected the chondrocytes function. Moreover, as discussed earlier and also contrary to the results with metallic CoCr particles, we neither observed a modification in synovial lining membrane (synovial hyperplasia) nor microscopic evidence of articular damage. These observations clearly demonstrate that ZTA wear particulates could not activate the inflammatory tissue response in the knee joint. 4.3.4. Influence on Pro-Inflammatory Cytokine Secretion. The particulate debris derived from material implants can induce both local and systemic trafficking of immune cells (monocyte−macrophage).5,44 Macrophages located in the interfacial membrane between joint and bone are believed to release proinflammatory cytokines/mediators, such as IL-1β, TNF-α, RANKL, IL-6, and PGE2. All these markers not only activate osteoclasts, but also stimulate the differentiation of osteoclast precursors into mature osteoclasts and increase periprosthetic bone resorption.31,44 Therefore, the evidence of immune response, triggered by wear debris in peri-prosthetic tissue can be documented by proinflammatory marker analysis.

biochemical parameters and histological changes in tissues of vital organs. The general approach was to explore the local and systemic response toward ceramic as well as metal particulates at prosthetic joints, initially at the knee joint and later at distant tissues after their translocation.11 4.3.1. Complete Blood Count and Serum Biochemistry. The assessment of complete blood count and serum biochemical parameters of animals can be considered as a diagnostic tool to determine the physiological status of cells or tissues.40 Toxic agents, once distributed into the circulating system, are generally known to interfere with important biochemical and enzymatic processes of metabolism that regulate the normal physiology of animals.41 Any type of alteration in these processes can affect normal functioning of vital organs and disrupt overall homeostasis of organism. In the present study, hematological and biochemical analysis reports at week 4, 8, and 12 of necropsy could not reveal any noticeable change for all groups with respect to control in all the parameters, except serum protein level. The presently observed depletion in serum protein content in mice, observed at eighth week exposure with CoCr and Al2O3 particles, can be attributed to the metabolic processes to compensate for particles generated ions induced stress conditions.42 Further, the insignificant enhancement in ALP and AST activity levels, observed at week 8 of autopsy in the animals exposed to CoCr, may indicate particle mediated injury in cellular membrane of liver tissue at initial period of exposure. The increase of these plasma nonfunctional enzymes, which are normally localized within the cells, is a good bioindicator of liver dysfunction and its cellular membrane death.43 Despite these potential toxicological possibilities, as observed in present study, the association of metal wear particles with any metabolic and immunologic toxicity remains speculative.44 4.3.2. Effect of Particles on Organ Ultrastructure. The histological examination of vital organs such as kidney, liver, spleen, and heart of mice treated with particulate eluates of CoCr, Al2O3, ZrO2, and ZTA could not reveal any indication of gross pathological lesions in any of these tissue obtained at different time points. All the organs showed normal architecture without any noticeable presence of particles. An absence of evidence of particle dissemination or any cellular level immunological response can be attributed either to their local removal through lymphatic systems at the knee joint’s synovium tissue or to their rapid elimination by animal metabolic systems, involving biotransformation and excretion processes, if particles are translocated systemically to various organs.45 In addition, another aspect, which might contribute to an absence of particle mediated pathological changes is the presence of resident macrophages or range of other immunological cells in various tissues. This would result in predisposition for rapid recognition and clearance of particulate matter.46 Macrophages, for example Kupffer cells present in the liver, play a pivotal role in defense against foreign particles (ceramic/metal) by eliminating the particles by phagocytosis.44 In a consistent manner with present results, with present results, Hallab et al.44 also claimed that the effects of systemically elevated amounts of wear particles in various vital organs have not been associated with remote toxicological pathology to date. 4.3.3. Potential Damage of ZTA Particulates on Knee Joint. In the present study, differential histological response to the various types of intra-articular particles were observed. Histological analysis of knee joint and measurement of N


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ACS Biomaterials Science & Engineering IL-1β and TNF-α are important cytokines that are secreted in the early stage of the osteolysis process, which are regarded to regulate bone resorption and are released from particlestimulated macrophages and fibroblastic cells.31,47 In the present study, the exposure to CoCr, Al2O3, ZrO2, and ZTA particulates did not result in any significant change in synovium TNF-α and IL-1β secretion levels, when injected intra-articularly into mice knee joint. The lack of proinflammatory mediator production during particulate exposure might be due to either passive lack of stimulation by wear debris or from an active suppressive mechanism involving the autocrine/paracrine action of TGF-β, PGE2, and PAF (bone forming cytokines) which are involved in inflammatory signaling cascades.51 The present results on ZTA are in accordance with reports demonstrating weak inflammatory reaction induced by ceramic particles after their subcutaneous, intraarticular and peritoneal administration.31,52 In contrary, the increased level of cytokines has been reported in various immune cells incubated with smaller to larger sized CoCr particles, in vitro.53−55 However, no in vivo study is available to conclusively prove that CoCr particles are more proinflammatory in nature. In contrast to TNF-α secretion in synovium, intra-articular treatment of ceramic particulates of Al2O3, ZrO2, and ZTA (at lowest dose), interestingly, caused a significant decrease in the level of serum TNF-α. The clinical relevance of this imbalance of cytokine release is not clear and needs to be further investigated, since this alteration in balance of TNF-α release would have not caused any toxicity in vital organs of animals exposed with any of these material particles.36 Furthermore, no significant measurable difference was found in the levels of IL-1β cytokine in particles treated mice, reflecting that CoCr, Al2O3, ZrO2, and ZTA have not elicited any foreign body or inflammatory response, even when translocated systemically. These results of pro-inflammatory cytokine level analysis in serum can be correlated well with the histological finding of vital organs with normal architecture.

quantitatively detected for all the oxide ceramic particulates and CoCr, in vivo. (d) The delivery of oxide ceramic and CoCr wear particulates at intra-articular joints in the mouse model did not cause inflammation or any other morphologically lethal effects in treated animals, even at the highest dosage of 25 mg/mL. The histological appearances of heart, lung, liver, spleen, and kidney were normal with no evidence of pathological lesion or systemic immune response due to translocation of oxide ceramic/CoCr particulates. (e) The ultramicroscopic observation of knee joint sections showed that ZTA, Al2O3, and ZrO2 particles did not show any potential immune response, which could result in granuloma tissue formation. A mild inflammatory response (synovial hyperplasia) was exhibited initially by all the experimental animals, because of repeated exposure of particles for a period of 8 weeks. In contrast, CoCr particles induced a marked inflammatory reaction with severe erosion of articulating surfaces and thick synovial membrane showing hyperplasia. (f) Importantly, no statistically significant difference in expression of TNF-α and IL-1β was noted either with ZTA, Al2O3, and ZrO2, or CoCr particulates, when analyzed both in synovium and in serum.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.8b00583. Figure S1, change in body weight of female Balb/C mice following intra-articular injection of particulates of CoCr, Al2O3, ZrO2, and ZTA; Figure S2, effect of different particulate treatments on the histology of major organs; and Figure S3, histological analysis of various organs stained with hematoxylin/eosin to assess the potential effects on the organ morphological architecture and cellular damage, after different particulates treatment (PDF)

5. CONCLUSIONS In the light of the anticipated concern over potential toxicological properties of wear debris, this work aimed to evaluate and compare the biological effects of ZTA particles with Al2O3, ZrO2, and CoCr particles, in vitro and in vivo. The morphology and size distribution of the investigated debris particles are comparable. From the present study, the following conclusions can be drawn: (a) The results from the in vitro study demonstrated that phagocytosable ceramic particulates of ZTA (mean size, 191 nm) as well as Al2O3 and ZrO2 did not cause any significant reduction in MC3T3-E1 murine osteoblast cell growth for the period of 24 and 72 h at 0.25, 2.5, and 25 mg/mL of concentrations. (b) Morphological analysis revealed good cellular adhesion, proliferation, and phenotypical features of osteoblast cells, confirming nontoxic potential of the oxide wear particles. CoCr was found to be toxic to osteoblasts, as those markedly reduced the proliferation rate as well as induced stress-mediated phenotypical changes. (c) A better hemocompatibility of ZTA, Al2O3, and ZrO2 with minimized erythrocytes destruction is established as compared with CoCr particles, in vitro. Also, nonsignificant changes in blood serum parameters were

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AUTHOR INFORMATION

Corresponding Author

*(B.B.) E-mail: [email protected]. ORCID

Bikramjit Basu: 0000-0002-9154-5553 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Dr. Abhay Pandit, Network of Excellence for Functional Biomaterials (NFB), National University of Ireland Galway, for his fruitful suggestions. The authors would like to acknowledge the help received from Ravikumar and P. Ranjithkumar in XRD analysis. The authors wish to thank Subbaraj Karunakaran (Organic chemistry department, IISc) for the help provided during TEM and DLS characterization of particulates. N.B. is thankful to the University Grants Commission (UGC, Delhi) for providing financial support for this research work under the Dr. D. S. Kothari Postdoctoral Fellowship scheme (UGC Award No.F.4O


Article


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2/2006 (BSR)/BL/14-15/0340). The authors are grateful to the Department of Biotechnology (DBT; Grant No. BT/PR 13466/COE/34/26/2015), Government of India and “Centre of Excellence and Innovation in Biotechnology” scheme through the Center of excellence project - Translational Center on Biomaterials for Orthopedic and Dental Applications. The authors are grateful to the Central Animal Facility (CAF), Indian Institute of Science, Bangalore, India, for the assistance provided during in vivo experiments.

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Probing cytocompatibility, hemocompatibility and quantitative

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