Metal-Assisted and Microwave-Accelerated Decrystallization of Pseudo-Tophus in Synthetic Human Joint Models

Abstract

In this paper, we tested a hypothesis that the metal-assisted and microwave-accelerated decrystallization (MAMAD) technique, based on the combined use of low-power medical microwave heating (MWH) and gold nanoparticles (Au NPs), can be used to decrystallize laboratory-prepared monosodium urate monohydrate crystal aggregate (pseudo-tophus) placed in three-dimensional (3D) synthetic human joint models. To simulate a potential treatment of chronic tophaceous gout using the MAMAD technique, we used three different 3D synthetic human joint models and assessed the percent mass reduction (PMR, i.e., decrystallization) of pseudo-tophus and microwave-induced synthetic skin patch damage after MAMAD sessions (a MAMAD session = 120 s of MWH in the presence of Au NPs). Our three synthetic joint models are: Model 1: Application of seven MAMAD sessions in a closed synthetic joint with a pseudo-bursa containing a pseudo-tophus submerged in a solution of 20 nm Au NPs followed by dehydration of pseudo-tophus after each MAMAD session to assess PMR. Model 2: Application of seven MAMAD sessions in a closed or open synthetic joint with a pseudo-bursa containing a pseudo-tophus submerged in a solution of Au NPs followed by intermittent dehydration of pseudo-tophus after seven MAMAD sessions to assess PMR. Model 3: Application of 18 MAMAD sessions in a rotated closed synthetic joint (three sides are heated separately) with a pseudo-bursa containing a pseudo-tophus submerged in a solution of Au NPs followed by dehydration after every three MAMAD sessions to assess PMR. After a single MAMAD session, pseudo-tophus exposed to MWH and Au NPs had an average PMR of 8.30% (up to an overall PMR of 15%), and microwave-induced damage to the synthetic skin can be controlled by the use of a sacrificial skin sample and by adjusting the duration and the number of the MAMAD sessions. Computational electromagnetic simulations predict a 10% absorption of electric field by the pseudo-tophus placed in the synthetic joint models, which led us to conclude that a medical microwave source with higher power than 20 W can potentially be used with the MAMAD technique.

Introduction

Gout is a crystal deposition disease commonly associated with metabolic syndrome and without proper management and therapy can cause permanent damage to an afflicted human joint.¹⁻³ Hyperuricemia, a precursor to the development of gout, occurs when supersaturated systemic uric acid crystals precipitate and nucleate in human joints.⁴⁻⁶ Hyperuricemia (2%) occurs when supersaturated systemic uric acid produced from the catabolism of purines is excreted from the body in urine, and 98% of the ionic form remains in circulation.⁷⁻¹⁰ Monosodium urate monohydrate (MSUM), the mono-protonated ionic form of hyperuricemia occurs when supersaturated systemic uric acid, circulates in the blood plasma and synovial fluid of joints. The presence of MSUM crystals in soft tissues is indicative of acute gout, and the accumulation of intradermal, intra-articular deposits of nodular MSUM crystals (i.e., tophus) in the synovial fluid of joints is an indicator of chronic tophaceous gout.

Corticosteroids (anti-inflammatory agents), colchicine (an antipyretic agent), and nonsteroidal anti-inflammatory drugs (analgesic agents) are currently used for the management of acute gout.¹¹⁻¹⁵ Management of chronic gout can involve surgical removal of tophus, a procedure that is invasive and can lead to permanent joint damage. Numerous adverse effects associated with the use of current pharmaceuticals for the treatment of acute and chronic gout necessitate the development of minimally invasive new treatments for gout and, potentially, other crystal deposition diseases.

In response to the need for treatments for gout, our research group has developed a technique, called metal-assisted and microwave-accelerated decrystallization (MAMAD),^16,17 which combines the use of Au nanoparticles (NPs) in solution and microwave heating (MWH), as a potential therapeutic aid. In the MAMAD technique, MWH of target crystals in the presence of Au NPs is used to create a temperature gradient between the Au NPs, water, and the crystals in solution, increasing the kinetic energy of the Au NPs. When the kinetic energy of the Au NPs increases with MWH, Au NPs act as “nanobullets,”¹⁸ resulting in increased collisions between the Au NPs and the target crystals, which causes break up and eventually decrystallization of the target crystals.¹⁹ To date, we have demonstrated the application of the MAMAD technique on planar surfaces using uric acid crystals as a model for small crystals and l-alanine crystals as a model for large crystals. Recently, we have established a synthetic human finger joint model using three-dimensional (3D)-printed plastic bones and synthetic skin.²⁰ We have previously shown that large-sized (>400 μm) l-alanine crystals in a solution of Au NPs could be decrystallized entirely by low-power MWH through a synthetic skin patch.¹⁸ However, l-alanine was used as a model crystal in our two-dimensional (2D) model, which is not representative of the tophus seen in individuals with tophaceous gout, which motivated us to employ our MAMAD technique for the decrystallization of pseudo-tophus in 3D models that mimic gout tophus in human joints.

In this proof-of-principle study, we tested the hypothesis that our MAMAD technique can be used to decrystallize MSUM pseudo-tophus using an 8 GHz low-power (maximum power: 20 W) medical microwave source in the presence of Au NPs using three synthetic human joint models. To mimic tophaceous gout in a human joint, a pseudo-tophus was submerged in a solution of Au NPs, enclosed in a pseudo-bursa made of a thin plastic enclosure, and embedded in the articular space of our closed synthetic joint covered with a synthetic skin patch or in an open synthetic joint covered with a glass coverslip. In each synthetic human joint model used, we determined the percent mass reduction (PMR) of each pseudo-tophus after each MAMAD session. Throughout these experimental sessions, the temperatures of the synthetic joints, exposed to our MAMAD technique, were monitored via an interjoint fiber optic temperature probe to determine whether MWH caused temperature increases in the synthetic joints that exceeded the physiological range. We also qualitatively assessed the integrity of the synthetic skin patches used to cover the closed synthetic joints for microwave-induced damage via optical microscopy. We carried out finite-difference time-domain (FDTD) simulations of the electric field distribution throughout the synthetic joint models. These results offer a closer step to the applicability of the MAMAD technique as a potential alternative treatment for gout or other crystal deposition diseases in humans after animal trials.

Results and Discussion

Since this study contains three different synthetic joint models, it is important to first introduce these models and summarize the differences between them in the first few paragraphs below and then present our findings. Detailed information on the synthetic joint models is provided in the Materials and Methods. Figure 1 shows model 1, where seven MAMAD sessions (5 W; 1 MAMAD session = 30 s × 4 intermittent MWH = 120 s; 7 MAMAD sessions = 840 s of MWH) were applied to a closed synthetic joint (covered by a sacrificial synthetic skin patch) embedded with a pseudo-bursa, containing a pseudo-tophus submerged in a 500 μL solution of Au NPs (20 nm in diameter) and dehydrated for 1 h after each MAMAD session. In these experiments, we used the closed synthetic joint as the more stringent model compared to the open synthetic joint (microwave heating is applied to tophus in the absence of a sacrificial synthetic skin patch). That is, if pseudo-tophus decrystallization occurred under a synthetic skin patch, then it would occur under a glass coverslip. From these experiments, we learned that the pseudo-tophus should be submerged in a larger volume of Au NP solution, and that dehydration after every MAMAD sessions is time consuming without additional benefits. It is important to note that the choice of 20 nm Au NPs is based on the ease of human body to clear Au NPs at these sizes, and the fact that Au NPs are Food and Drug Administration approved for use in humans.

Figure 1.

Model 1: application of seven MAMAD sessions in a closed synthetic joint with a pseudo-bursa containing a pseudo-tophus submerged in Au NPs with dehydration after each MAMAD session. Schematic representation of a pseudo-bursa (plastic pouch) containing a MSUM pseudo-tophus, submerged in a 500 μL solution of Au NPs embedded in a closed synthetic joint covered by a synthetic skin patch, and exposed to MWH (5 W) for seven MAMAD sessions, applied at 30 s intervals (1 MAMAD session = 4 × 30 s = 120 s × 7 sessions = 840 s of MWH) followed by dehydration for 1 h after each session. After each MAMAD session, the pseudo-tophus was removed from the pseudo-bursa, dehydrated, and weighed to determine the PMR. The internal temperature of the closed synthetic joint was monitored with an interjoint fiber optic temperature probe. Microwave-induced damage to the synthetic skin patch was assessed after each MAMAD session.

Figure 2 shows model 2, where seven MAMAD sessions (5 W; 120 s × 7 MAMAD sessions = 840 s of MWH) or an equivalent duration of ambient temperature (AT, no microwave heating, 17–18 °C) were applied to a closed or an open synthetic joint (covered by a 18 mm × 18 mm, 0.13 mm thick glass coverslip) embedded with a pseudo-bursa, containing a pseudo-tophus submerged in an 800 μL solution of Au NPs (20 nm in diameter, note the increase in volume of Au NP solution based on our observations in model 1 as described in the text) or deionized (DI) water and dehydrated for 1 h after seven MAMAD sessions. In these experiments, we compared the extent of pseudo-tophus decrystallization in closed and open synthetic joints. Although the Au NPs were suspended in a mild citrate buffer, containing a proprietary surfactant as a stabilizer, we chose to use deionized water as the Au NP control. From these experiments, we learned that dehydration step after seven MAMAD sessions is preferable to minimize experimental error in assessing the PMR.

Figure 2.

Model 2: application of seven MAMAD sessions in a closed or open synthetic joint with a pseudo-bursa containing a pseudo-tophus submerged in Au NPs or deionized water and dehydrated after seven MAMAD session. Schematic representation of a pseudo-bursa containing a pseudo-tophus, submerged in 800 μL solutions of Au NPs or deionized water embedded in a closed synthetic joint or in an open synthetic joint covered by a glass coverslip, exposed to MWH (5 W) for 21 MAMAD sessions applied at 30 s intervals or an equivalent duration of ambient temperature (17–18 °C) followed by dehydration for 1 h after 7 MAMAD session. The pseudo-tophus was removed from the pseudo-bursa, dehydrated, and weighed to determine the PMR. Internal temperatures of the closed and open synthetic joints were monitored with an interjoint fiber optic temperature probe. Microwave-induced damage to the synthetic skin patch was assessed after each MAMAD session.

Figure 3 shows model 3, where 18 MAMAD sessions (5 W; 18 MAMAD sessions = 3 × 120 s MAMAD sessions (on the right, top, and left sides of the closed synthetic joint) = 360 s × 6 trials = 2160 s of MWH) were applied to a rotated closed synthetic joint (MWH on three sides of joint) embedded with a pseudo-bursa, containing a pseudo-tophus submerged in an 800 μL solution of Au NPs (20 nm) and dehydrated for 1 h after every three MAMAD sessions (after each side). In the experiments for model 3, we focused on the closed synthetic joint because we wanted to determine if MWH is applied to the closed synthetic joint on three sides, will fewer MAMAD sessions may be required. Our observations and detailed discussions on the application of the MAMAD technique to each synthetic human joint model are described below.

Figure 3.

Model 3: application of 18 MAMAD sessions in a rotated closed synthetic joint (MWH on right, top, and left sides) with a pseudo-bursa containing a pseudo-tophus submerged in Au NPs with dehydration after complete rotation, three MAMAD sessions (360 s). (a–c) Real-color images of a rotated closed synthetic joint, microwave applicator tip, and an interjoint fiber optic temperature probe. First, second, and third MAMAD sessions, MWH on the right, top, and left sides of the closed synthetic joint for 18 MAMAD sessions (5 W; 18 MAMAD sessions = 3 MAMAD sessions on the right, top, and left sides of the closed synthetic joint × 6 trials). A MSUM pseudo-bursa containing pseudo-tophus, submerged in 800 μL solutions of Au NPs or deionized water, embedded in a closed synthetic joint, exposed to MWH for total of 18 MAMAD sessions followed by dehydration for 1 h after every three MAMAD sessions. The pseudo-tophus was removed from the pseudo-bursa, dehydrated, and weighed to determine the PMR. The internal temperature of the closed synthetic joint was monitored with an interjoint fiber optic temperature probe.

Model 1: Application of Seven MAMAD Sessions in a Closed Synthetic Joint with a Pseudo-Bursa (Plastic Pouch) Containing a Pseudo-Tophus Submerged in Au NPs and Dehydrated after Each MAMAD Session

Figure 4a shows real-color images of one of the model 1 three different pseudo-tophus that was submerged in a 500 μL solution of Au NPs (20 nm) in a pseudo-bursa, which was embedded in a closed synthetic joint covered by a synthetic skin patch, after the application of seven MAMAD sessions or at AT (i.e., no MWH). Figure 4b shows the average PMR calculated for three different pseudo-tophus, after each session, pseudo-tophus samples were exposed to the MAMAD technique, microwaves in the absence of Au NPs and at AT in the presence of Au NPs or deionized water. The pseudo-tophus shown in Figure 4 (initial mass: 486 mg) had a PMR of 8.30% (31 mg) after seven MAMAD sessions. The pseudo-tophus was dehydrated for 1 h after seven MAMAD sessions and weighed. The PMR of 5.40% (44 mg) was observed in the control sample (initial mass: 582 mg) at AT in the presence of Au NPs. The PMR of 5.51% (9 mg) was observed in the control sample (initial mass: 167 mg) with MWH in the absence of Au NPs. The PMR of 4.45% (9 mg) was observed in the control sample (initial mass: 205 mg) at AT in the absence of Au NPs.

Figure 4.

Model 1: real-color images of one of three different pseudo-tophus in Au NP solution after seven MAMAD sessions. (a) Real-color images of one of the three MSUM pseudo-tophus that was submerged in a solution of Au NPs in a pseudo-bursa, which was embedded in a closed synthetic joint covered by a 2 cm synthetic skin patch, after the application of seven MAMAD sessions or at ambient temperature. After each MAMAD session, pseudo-tophus was removed from the pseudo-bursa, dehydrated, and weighed. (b) The table shows the average PMR calculated for four different pseudo-tophus sample groups, after each 120 s session (MAMAD, controls: 5 W in the absence of Au NPs, at ambient temperature in the presence of Au NPs or deionized (DI) water).

Figure 5a shows real-color images of four different model 1 pseudo-tophus samples, in pseudo-bursas, dehydrated Au NPs solutions and pseudo-tophus samples, and microwave-induced synthetic skin patch damage. Figure 5b shows the change in the temperature of the four closed synthetic joints after 120 s of MWH. After each MAMAD session, each pseudo-tophus was removed from the pseudo-bursa, dehydrated, and weighed to determine the PMR. The internal temperature of each closed synthetic joint was monitored with a temperature probe. Qualitative microwave-induced damage to the synthetic skin patch was assessed using a digital camera after each MAMAD session. The increases in temperature for the four closed synthetic joints, during MWH were, 6.8, 5.6, 1.7, and 4.9 °C.

Figure 5.

Model 1: real-color images of four different pseudo-tophi aggregates exposed to seven MAMAD sessions (5 W) with dehydration after every MAMAD session. (a) Real-color images of four different MSUM pseudo-tophus in pseudo-bursas, dehydrated Au NPs solutions, and pseudo-tophus samples, and microwave-induced synthetic skin patch damage. (b) The graph shows change in the temperature of the four closed synthetic joints after 120 s of MWH.

Figure S1a shows measured mass of pseudo-tophus, pseudo-tophus + Au NPs, wet and dry pseudo-tophus, the pseudo-tophus mass difference, and the average PMR after seven MAMAD sessions. The pseudo-tophus (initial mass: 305 mg) before the initiation of the MAMAD sessions and the addition of 500 μL solution of Au NPs gradually decreased the mass of the pseudo-tophus via the dissolution process. After the pseudo-tophus was removed from the pseudo-bursa and dehydrated, the mass decreased to 293 mg (3.93%). Figure S1b shows a graph of microwave-induced temperature changes recorded at 30 s intervals during seven MAMAD sessions. The largest change in the temperature of the closed synthetic joint was during MAMAD session 5 between 30 and 60 s. Figure S1c shows the duration for microwave-induced synthetic skin patch damage during each MAMAD session.

Model 2: Application of Seven MAMAD Sessions in a Closed or Open Synthetic Joint with a Pseudo-Bursa Containing a Pseudo-Tophus Submerged in Au NPs and Dehydrated after Seven MAMAD Sessions

Table 1 summarizes the calculated PMR from three different pseudo-tophus submerged in 800 μL solutions of Au NPs or deionized water in pseudo-bursas, which were embedded in closed synthetic joints covered by synthetic skin patches (Table 1a) or in open synthetic joints covered by glass coverslips (Table 1b). Pseudo-tophus samples were exposed to ambient temperature (AT; 17–18 °C) or MWH for seven sessions in the presence of Au NPs or deionized water (7 MAMAD sessions = 840 s of MWH in the presence of Au NPs) with dehydration for 1 h after seven MAMAD sessions. For each of the three pseudo-tophus, the initial pseudo-tophus mass (mg), the PMR (%) after seven MAMAD sessions, the final pseudo-tophus mass (mg), the mass reduction (mg), and the PMR (%) of pseudo-tophus and the average PMR (%) of all three pseudo-tophus were recorded under four conditions: control: AT + Au NPs; MAMAD: MWH + Au NPs; control: AT + deionized water; and control: MWH + deionized water. Model 2 pseudo-tophus were dehydrated for 1 h after seven MAMAD sessions. Control pseudo-tophus samples were kept at AT in the presence of Au NPs or deionized water and exposed to MWH in the presence of deionized water for the duration equal to that of the seven MAMAD sessions.

Table 1. Model 2: Seven MAMAD Sessions in a Closed or Open Synthetic Joint with Three Different Pseudo-Bursae Each Containing a Pseudo-Tophus Submerged in Au NPs and Dehydrated after Seven MAMAD Sessions^a.

^aEach of three different pseudo-tophus was submerged in 800 μL solutions of Au NPs or deionized water in pseudo-bursa, which were embedded (a) in closed synthetic joints with synthetic skin patches or (b) in open synthetic joints with glass coverslips and exposed to ambient temperature (17–18 °C) to seven MAMAD sessions (5 W; 840 s of MWH) with dehydration for 1 h after seven MAMAD sessions. For each of the three pseudo-tophus, the initial pseudo-tophus mass (mg), the PMR (%) after seven MAMAD session, the final pseudo-tophus mass (mg), the mass reduction (mg), and the PMR (%) of pseudo-tophus and the average PMR (%) of all three pseudo-tophus were recorded under four conditions: control: ambient temperature + Au NPs; MAMAD: MWH + Au NPs; control: AT + deionized water and MWH + deionized water.

For the closed synthetic joint, the largest PMR occurred using the MAMAD technique: MWH + Au NPs treatment group with an initial average mass of 187 ± 53.8 mg. The PMR, after the seven MAMAD sessions for all three closed synthetic joints, was 11.5 ± 1.82%; Moreover, the pseudo-tophus in the open joint had an initial mass of 311 ± 124 mg, before the commencement of seven MAMAD sessions and a final average mass of 294 ± 120 mg after the MAMAD sessions concluded. The PMR after the initial seven MAMAD sessions for all three open synthetic joints was 5.89 ± 1.59%. The treatment groups exposed to MWH + Au NPs for seven MAMAD sessions had the overall mass of pseudo-tophus in the closed synthetic joint and the open synthetic joint reduced by 11.5 ± 1.82 and 5.89 ± 1.59%, respectively.

The pseudo-tophus in the control group in the presence of Au NPs and at AT (no MWH) in the closed joint had an initial mass of 416 ± 61.5 mg and a mass of 391 ± 57.7 mg after the duration of seven MAMAD sessions. The PMR after the duration of seven MAMAD sessions for all three closed synthetic joints was 6.08 ± 0.01%. The pseudo-tophus in the presence of Au NPs and at AT (no MWH) in the open joint had an initial mass of 472 ± 117 mg before and a final mass of 460 ± 114 mg after the duration of seven MAMAD sessions. The PMR after seven MAMAD sessions for all three open synthetic joints was 2.57 ± 1.13%. The control group exposed to AT + Au NPs for seven MAMAD sessions had the overall mass of pseudo-tophus in the closed synthetic joint and the open synthetic joint reduced by 6.08 ± 0.01 and 2.57 ± 1.12%, respectively.

The control pseudo-tophus submerged in deionized water (without Au NPs) in the closed joint at AT had an initial mass of 205 ± 13.5 mg before and a final mass of 197 ± 13.0 mg after the duration of seven MAMAD sessions. The PMR after the duration of seven MAMAD sessions for all three closed synthetic joints was 3.91 ± 0.42%. The pseudo-tophus at AT (no MWH) + deionized water in the open joint had an initial mass of 173 ± 20 mg before and a final mass of 164 ± 15.6 mg after the duration of seven MAMAD sessions. The PMR after the seven MAMAD sessions for all three open synthetic joints was 5.42 ± 2.43%. The treatment groups exposed to AT + deionized water for seven MAMAD sessions had the overall mass of pseudo-tophus in the closed synthetic joint and the open synthetic joint reduced by 3.91 ± 0.42 and 5.42 ± 2.43%, respectively.

The control pseudo-tophus submerged in deionized water (without Au NPs) in the closed joint exposed to MWH had an initial mass of 154 ± 11.7 mg before and a final mass of 146 ± 10.4 mg after the duration of seven MAMAD sessions. The PMR after the duration of seven MAMAD sessions for all three closed synthetic joints was 5.17 ± 0.91%. The pseudo-tophus at MWH + deionized water in the open joint had an initial mass of 132 ± 0.58 mg before and a final mass of 124 ± 0.28 mg after the duration of seven MAMAD sessions. The PMR after the seven MAMAD sessions for all three open synthetic joints was 6.07 ± 0.06%. The treatment groups exposed to MWH + deionized water for seven MAMAD sessions had the overall mass of pseudo-tophus in the closed synthetic joint and the open synthetic joint reduced by 3.91 ± 0.42 and 5.42 ± 2.43%, respectively.

Figure 6a shows real-color images of an open synthetic joint (model 2), a temperature probe, and an embedded pseudo-tophus before and after seven MAMAD sessions. Figure 6b shows real-color images of the weight (in milligram) of the pseudo-tophus after removal from the Au NP solution and after dehydration for 1 h following upon completion of seven MAMAD sessions. The initial mass of pseudo-tophus was 428 mg, and after seven MAMAD sessions and dehydration, the pseudo-tophus weighed 401 mg, which is a 21.5 mg decrease from the initial mass. Without dehydration after each MAMAD session (as was done in model 1), the weight of the pseudo-tophus increased with each MAMAD session.

Figure 6.

Model 2: real-color images of a pseudo-tophus in an open synthetic joint exposed to seven MAMAD sessions followed by dehydration of pseudo-tophus after seven MAMAD sessions. (a) Real-color images of a pseudo-tophus that was submerged in an 800 μL solution of Au NPs, which was in a pseudo-bursa embedded in an open synthetic joint with an interjoint temperature probe, before and after seven MAMAD sessions. (b) Real-color images of the weight of a pseudo-tophus after each MAMAD session and after dehydration (note: the weight of the pseudo-tophus increases with each MAMAD session).

Figure S2a shows the time (in seconds) to microwave-induced damage to synthetic skin patches from three closed synthetic joints during exposure to seven MAMAD sessions. Figure S2b shows microwave-induced temperature changes in a closed synthetic joint during seven MAMAD sessions at 30 s intervals. It is important to note that the synthetic skin patch was changed after each MAMAD session to simulate the use of sacrificial synthetic skin samples to alleviate potential microwave-induced pain in humans. The change in temperature was also monitored per MAMAD session in the closed and open synthetic joints. Figure S3 shows the internal temperature changes for three different model 2 closed and open synthetic joints, as monitored via an interjoint temperature probe during exposure to seven MAMAD sessions. It is interesting to note that the final temperatures of both the closed and open synthetic joints remained in the initial temperature ranges, which demonstrates that MWH does not cause significant increases in temperature. This is an important observation for the future potential use of the MAMAD technique in humans with gout.

To visually demonstrate the effect of the combined used of Au NPs and MWH on the pseudo-tophus in synthetic joint models, we collected scanning electron microscopy (SEM) images of pseudo-tophus before and after MAMAD sessions and control experiments. Figure 7 shows the SEM images of dry pseudo-tophus, pseudo-tophus in deionized water and pseudo-tophus in Au NPs, control pseudo-tophus or pseudo-tophus without microwave exposure after seven MAMAD sessions. We used dry pseudo-tophus as a baseline for pseudo-tophus in deionized water and pseudo-tophus in Au NPs. The surface of the dry pseudo-tophus appeared smooth without any pores at the largest observation scale (200 μm). However, with higher magnification (i.e., scales marked as 50, 10, and 2 μm), we observed that dry pseudo-tophus were needle-like in structure, similar to those observed in humans (see also Figures S5 and S6 for larger images).

Figure 7.

Model 2: application of seven MAMAD sessions in a closed joint with a pseudo-bursa containing a pseudo-tophus submerged in Au NPs and dehydrated after seven MAMAD sessions. SEM images of pseudo-tophus after application of seven MAMAD sessions in a closed joint with a pseudo-bursa containing a pseudo-tophus submerged in Au NPs and dehydrated after seven MAMAD sessions. Dry tophus is a control sample and shows the surface properties of the tophus before the application of seven MAMAD sessions. See Supporting Information for larger SEM images.

Contrarily, pseudo-tophus in deionized water and pseudo-tophus in Au NPs without application of microwave heating (i.e., AT) displayed different surface properties: pseudo-tophus in deionized water were more porous than dry pseudo-tophus, which indicates a partial dissolving of pseudo-tophus components by water and serves as a baseline for comparison with the MAMAD technique. Pseudo-tophus in Au NPs were also more porous than dry pseudo-tophus, but pseudo-tophus in Au NPs displayed larger pores than pseudo-tophus in deionized water. Both pseudo-tophus in deionized water and pseudo-tophus in Au NPs without application of microwaves displayed needle-like crystals with higher magnification (i.e., 50, 10, and 2 μm). These results imply that the use of Au NPs alone does not remove the sharp corners of MSUM crystals.

After application of MWH, pseudo-tophus in deionized water displayed a porous surface (see image with a scale bar of 200 μm). However, these pores were larger compared to pseudo-tophus in deionized water without exposure to MWH. Sharp corners of MSUM crystals in the pseudo-tophus exposed to MAMAD technique were not as distinct compared to pseudo-tophus in Au NPs without MWH. We observed that the combination of Au NPs and MWH caused the surface of pseudo-tophus to be more porous. Notably, at higher magnification, we did not observe individual MSUM crystals in pseudo-tophus exposed to the MAMAD technique, indicating that the MAMAD technique did indeed cause more characteristic structural changes to pseudo-tophus after the seven MAMAD sessions. These observations visually demonstrate the effectiveness of the MAMAD technique in decrystallization of pseudo-tophus as compared to control experiments in a 3D synthetic human joint model.

Model 3: Application of 18 MAMAD Sessions in a Rotated Closed Synthetic Joint (MWH on Right, Top, and Left Sides) with a Pseudo-Bursa Containing a Pseudo-Tophus Submerged in Au NPs and Dehydrated after Every Three MAMAD Sessions

Table 2 shows the PMR calculated after application of 18 MAMAD sessions (3 × MAMAD sessions on the right, top, and left sides of the closed synthetic joint × 6 trials = 2160 s of MWH), and the controls samples exposed MWH microwaves or at AT. Each of the six trials shown is for three different model 3 pseudo-tophus that were submerged in 800 μL solutions of Au NPs or deionized water in pseudo-bursa, which were embedded in closed synthetic joints covered with glass coverslips. Pseudo-tophus were removed from the pseudo-bursas, dehydrated, and weighed to determine the PMR. Control samples were exposed to MWH or at AT in the presence of 800 μL of Au NPs or 800 μL of deionized water for the duration of time equal to that of the MAMAD sessions. The average PMR after all six trials (18 MAMAD sessions) for all three pseudo-tophus in the rotated closed synthetic joint exposed to MWH and Au NPs was 8.00 ± 2.53%. The pseudo-tophus in the control group in the presence of Au NPs and at AT (no MWH) in the rotated closed synthetic joint had an average PMR of 8.63 ± 0.91% after all six trials. We attribute the similar average PMR observed in control (Au NPs and at AT) and MAMAD sessions to experimental error, especially considering that the PMR variation in the MAMAD session is >30% as compared to 11% in the control experiment. That is, due to multiple evaporation steps involved in evaluation of PMR, measurements are more prone to errors, and average PMR appears to be similar. In addition, there are clear structural differences in pseudo-tophus observed after these experiments as described in Figure 7, demonstrating the superiority of MAMAD over using Au NPs alone at AT. The control pseudo-tophus submerged in deionized water (without Au NPs) in the rotated closed synthetic joint had an average PMR of 2.84 ± 0.82% after all six trials. The control pseudo-tophus exposed to MWH and submerged in deionized water (without Au NPs) in the rotated closed synthetic joint had an average PMR of 6.58 ± 0.67% after all six trials (18 MAMAD sessions). Figure S4 shows the internal temperature changes of the rotated closed joint during exposure to 18 MAMAD sessions and 1 h of dehydration after every three MAMAD sessions. The initial temperature range of the rotated closed synthetic joints was 17–26 °C, and final temperatures remained relatively constant.

Table 2. Model 3: Percent Mass Reduction (PMR) per Trial for Three Different Pseudo-Tophi in a Rotated Closed Synthetic Joint (Microwave Exposure on the Right, Left, and Top Sides) after 18 MAMAD Sessions (5 W) with Dehydration after Complete Rotation, 3 MAMAD Sessions (360 s)^a.

	trials
rotated open synthetic joint	1	2	3	4	5	6	total PMR (%)
control: + ambient temperature (AT) + Au NPs
pseudo-tophi 1: percent mass reduction (PMR, %) after three MAMAD sessions	4.31	0.82	0.41	1.24	0.00	1.26	8.04
pseudo-tophi 2: PMR (%) after three MAMAD sessions	3.98	0.00	2.59	0.53	0.53	0.54	8.17
pseudo-tophi 3: PMR (%) after three MAMAD sessions	3.70	4.20	0.44	.044	0.44	0.45	9.67
average PMR (%)							8.63 ± 0.91
MAMAD: + MWH (5 W) + Au NPs
pseudo-tophi 1: PMR (%) after three MAMAD sessions	5.00	1.70	0.61	0.00	0.64	0.49	8.44
pseudo-tophi 2: PMR (%) after three MAMAD sessions	3.04	1.02	0.10	0.14	0.84	0.14	5.28
pseudo-tophi 3: PMR (%) after three MAMAD sessions	2.30	4.35	1.70	1.16	0.78	0.00	10.29
average PMR (%)							8.00 ± 2.53
control: + ambient temperature (AT) + deionized water (Dl: no Au NPs)
pseudo-tophi 1: PMR (%) after three MAMAD sessions	1.41	0.36	0.00	0.00	0.00	0.18	1.95
pseudo-tophi 2: PMR (%) after three MAMAD sessions	1.17	0.95	0.48	0.48	0.48	0.00	3.56
pseudo-tophi 3: PMR (%) after three MAMAD sessions	1.49	0.50	0.00	1.01	0.00	0.00	3.00
average PMR (%)							2.84 ± 0.82
control + MWH (5 W) + deionized water (Dl: no Au NPs)
pseudo-tophi 1: PMR (%) after three MAMAD sessions	1.06	2.15	0.00	0.55	0.55	2.78	7.09
pseudo-tophi 2: PMR (%) after three MAMAD sessions	3.59	1.06	0.00	1.08	0.00	1.09	6.82
pseudo-tophi 3: PMR (%) after three MAMAD sessions	4.02	0.00	0.60	0.00	0.00	1.20	5.83
average PMR (%)							6.58 ± 0.67

^aPMR of three different MSUM pseudo-tophus that were submerged in 800 μL solutions of Au NPs or deionized water in pseudo-bursas, which were embedded in closed synthetic joints covered with glass coverslips, was determined after application of 18 MAMAD sessions (three sessions on the right, top, and left sides of the closed synthetic joint × 6 trials = 2160 s of MWH) or at ambient temperature. Pseudo-tophus were removed from the pseudo-bursas, dehydrated, and weighed to determine the PMR.

Finite-Difference Time-Domain Electromagnetic Simulations of Synthetic Joint Models

To determine the extent of electric field distribution throughout the closed synthetic joint models, we have carried out FDTD simulations. Since open joint synthetic joint model did not yield favorable results with the MAMAD technique, we did not carry out FDTD simulations for open joints. FDTD simulations for closed synthetic joint model predicted that 11.6% of the microwave radiation was reflected back into the microwave waveguide by the structure, of which 8.4% is due to the top epidermis/dermis layer (Figure 8). Only 11.4% of the microwave radiation was transmitted to the pseudo-tophus after being absorbed by the top epidermis/dermis, subcutaneous, and muscle layers, where the absorbances of each layer were determined as 61.2, 4.1, and 12.2%, respectively. The transmitted electric field to the bottom muscle layer was predicted to be 0.25% after the pseudo-tophus/synovial fluid structure, which was negligible. These results imply that one can potentially use a microwave source with power higher than 20 W (which is the maximum allowed power of microwave source used in this study). Since no medical microwave device with a microwave applicator used in this study exists yet, we will revisit the microwave power issue as these devices become commercially available. We plan to carry out animal studies to test the applicability of the MAMAD technique, and these results will be reported in due course.

Figure 8.

Model 2: FDTD simulation results of a closed synthetic joint. Dielectric constants for the components of the synthetic joint model were chosen to reflect the actual human skin, synovial fluid, and bones. A monomode microwave point source operating at 8 GHz was used. The left panel depicts the electric field propagation through microwave guide and the closed synthetic joint. The panel on the right shows the dielectric contrast image of the simulated structure.

Conclusions

This proof-of-principle study demonstrates the use of MAMAD technique for the decrystallization of MSUM crystals in three synthetic joint models, which is an important step toward the development an alternative gout treatment based on the MAMAD technique. This paper describes our findings, the limitations of our studies, the lessons learned from these studies, and recommendations made where appropriate as summarized below:

• (1)In model 1 (closed synthetic joint with a 1 h dehydration period after each of the seven MAMAD sessions) pseudo-tophus exposed to MWH in the presence of Au NPs showed a PMR of 8.3% after a single MAMAD session (120 s), which was a larger decrease than those observed for the control pseudo-tophus at AT in the presence or absence of Au NPs (5.4%). Intermittent dehydration of the solvent to assess the effectiveness of the MAMAD technique for decrystallization was found to be cumbersome and increased the time of MAMAD sessions. Thus, we modified our method of assessment of the effectiveness of decrystallization in models 2 and 3. In these models, we increased the number of MAMAD sessions and decreased the number of dehydration steps. The temperature of the synthetic joints was cooled to a temperature of 17.9 ± 0.4 °C, before the commencement of the MAMAD sessions, and an increase of 4.8 ± 2.2 °C was observed during the MAMAD sessions. These observations imply that the combined use of MWH and Au NPs can decrease the mass of pseudo-tophus in a single MAMAD session without significantly increasing the temperature of the synthetic skin. We also determined that a greater volume than 500 μL of the Au NP solution to avoid evaporation of solvent and keep Au NPs in solution was required. We have previously shown that the size of the Au NPs can increase PMR (increase in size reduces stability of Au NPs in solution), and Au NPs remain in solution in buffer and in synovial fluid during and after microwave heating.²¹
• (2)In model 2 (i.e., closed or open synthetic joint with a 1 h dehydration period after every seven MAMAD sessions experiments), a MSUM pseudo-tophus was submerged in an 800 μL solution of Au NPs or an equal volume of deionized water in a pseudo-bursa and embedded in a closed joint covered by a synthetic skin patch or in an open joint covered by a glass coverslip. The closed joint is the more stringent of the two models, whereby microwave heating must penetrate the depth of the synthetic skin patch for decrystallization to occur. The open joint model was used to determine whether the open joint covered with a glass slide would increase decrystallization.
  • (a)In model 2, pseudo-tophus samples in the closed joint model exposed to seven MAMAD sessions resulted in a more substantial increase in the average PMR of 11.5 ± 1.82% when compared to the pseudo-tophus samples in the control experiments at AT + Au NPs, AT + deionized water, and MWH + deionized water showed an average PMR of 6.08 ± 0.01, 3.91 ± 0.42, and 5.17 ± 0.91% respectively. The open joint model exposed to seven MAMAD sessions had an average PMR of 5.89 ±1.59% and when compared to the closed joint model a difference of 5.61 ± 0.23% was observed. The control experiments in the open joint at AT + Au NPs, AT + deionized water, and MWH + deionized water had an average PMR of 2.57 ± 1.12, 5.42 ± 2.43, and 6.07 ± 0.06% respectively.
  • (b)We also investigated the extent of damage to synthetic skin caused by MWH and the feasibility of using a synthetic skin patch to cover the location of pseudo-tophus during the MAMAD sessions. The integrity of the synthetic skin remained intact for 78 ± 5.1 s of direct microwave exposure before damage occurred. The temperature of the synthetic joint remained in a range that did not exceed AT (0.57 °C was the maximum temperature increase).
• (3)In model 3 (i.e., rotated closed synthetic joint with 18 MAMAD sessions, a 1 h dehydration period after three MAMAD sessions, total MWH time = 2160 s). The total exposure of joint: six MAMAD sessions on the right, left, and top sides of the joint for the duration of experiments. We investigated the feasibility of increasing the efficiency of the MAMAD sessions for large tophus by applying MWH to different sides of the synthetic joint. After a total of 18 MAMAD sessions, a PMR of 8.00 ± 2.53% was observed, as compared to a PMR of the control experiments in the closed joint at AT + Au NPs, AT + deionized water, and MWH + deionized water had an average PMR of 8.63 ± 0.91, 2.84 ± 0.82, and 6.58 ± 0.67%, respectively. We observed that temperature increases in areas exposed to MWH were not significant. The results presented herein are a step closer to the applicability of the MAMAD technique for a future potential treatment of gout and other crystal deposition diseases in humans.

Materials and Methods

Materials and Instrumentation

All chemicals and reagents were used as received unless otherwise described. Sodium hydroxide anhydrous pellets, uric acid powder, chondroitin sulfate, lyophilized powder bovine serum albumin, and phosphatidylcholine, Au NPs (optical density: 1.0; diameter: 20 nm; catalog number: 741965; 7.2 × 10¹¹ particles/mL) were purchased from Sigma-Aldrich (Milwaukee, WI). Acetic acid was purchased from Fisher Scientific (Hampton, NH). Coverslips (microslides, thickness: 0.96–1.06 mm), 20 mL scintillation vials, 150 mL filtration system were obtained from Corning Inc. (Corning, NY). Glass scintillation vials (20 mL), stir bars, and thermometers were purchased from Sigma-Aldrich (St. Louis, MO). Polyurethane plastic pouches were made from 2-mil poly tubing purchased from U-Line (Pleasant Prairie, WI). Synthetic skin was acquired from SynDaver Labs (Tampa, FL). The antialgae solution was manufactured by Clorox (Lawrenceville, GA), and cyanoacrylate was purchased from Gorilla Glue, Inc. (Cincinnati, OH). Aqueous solutions were prepared with deionized water (>18.0 MΩ cm resistivity at 25 °C) obtained from a Millipore Direct Q3 system.

MSUM and Pseudo-Tophus Synthesis

Acicular MSUM crystals used for the synthesis of pseudo-tophus were synthesized according to the method of Burt et al.²² The method involved the dissolution of uric acid in deionized water and adjustment of pH with sodium hydroxide solution and acetic acid. pH and gradual cooling of the uric acid solution at AT facilitated the formation of acicular-shaped MSUM crystals, which were harvested and used to synthesize pseudo-tophus. The pseudo-tophus were synthesized from a mixture of constituents frequently found in the chalky buildup of crystals in joints afflicted with gout with the exclusion of macrophages, mast cells, and other cellular components. Pseudo-tophus were formed from acicular MSUM crystals (4 g), phosphatidylcholine (1 g), chondroitin sulfate (1 g), and lyophilized bovine serum albumin (1 g), reconstituted with 3 mL of deionized water. Premeasured paste-like pseudo-tophus were dehydrated in 5–15 mm batches on watch glasses at 80 °C for 20 s. The mass of each synthesized pseudo-tophus was measured after dehydration (mass range: 100–600 mg) and stored at 25 °C.

Assembly of Synthetic Joint Models

Synthetic skin consisted of layers analogous to human skin layers (i.e., epidermis, dermis, subcutaneous layer, and muscle layers) with a thickness that ranged from 2 to 4 mm. Physicochemical, mechanical, and thermal properties of the synthetic skin (i.e., surface puncture energy, dielectric properties, fiber, heat, and porosity) were validated as analogous to human skin by SynDaver Labs. The synthetic skin was stored in an antialgae solution (60 mL of the antialgae solution in 4500 mL of deionized water and refrigerated at 4 °C) to prevent contamination and maintain the integrity of the synthetic skin. Synthetic skin patches were sized to fit the bones in the metacarpophalangeal joint of the forefinger. Cyanoacrylate was used to secure the 3D-printed second metacarpal and proximal phalange bones to the synthetic skin to form a synthetic joint with a 2 cm intra-articular space for insertion of pseudo-tophus in a pseudo-bursa made from 2-mil poly tubing. The synthetic joint was sutured with a needle and waxed thread and stored at 2 °C.

Decrystallization of a Pseudo-Tophus Embedded in a Synthetic Joint Using the MAMAD Technique

The synthetic joint was secured to a Universal Multichannel Instrument Stand (FISO Technologies, Inc., Québec, Canada), and a UMI4 fiber optic temperature probe was inserted into the synthetic joint to measure microwave-induced temperature changes that occur with the MAMAD technique. The initial temperature of the synthetic joint was maintained below AT (<20 °C) to mimic the application of a cold pack to an inflamed joint. Pseudo-tophus (100–600 mg) in the pseudo-bursa (2 cm × 2.5 cm polyurethane pouch) containing 20 nm Au NPs was sealed and placed in the synthetic joint. A microscope camera was situated above the synthetic joint to capture images for analysis. The synthetic joint was exposed to a 120 s MAMAD session. The temperature of the synthetic joint with an embedded pseudo-tophus was continuously monitored with the application of the MAMAD technique to ensure that the temperature of the synthetic joint did not exceed the physiological range (36.5–37.5 °C). The pseudo-bursa was covered with a synthetic skin patch in the closed joint, and in the open joint, the pseudo-bursa was covered by a glass coverslip.

Model 1: Application of Seven MAMAD Sessions in a Closed Synthetic Joint with a Pseudo-Bursa (Plastic Pouch) Containing a Pseudo-Tophus Submerged in Au NPs and Dehydrated after Each MAMAD Session

Figure 1 shows the components used to build the synthetic joint model: synthetic skin, pseudo-bursa, and 3D-printed bones and pseudo-tophus. During each MAMAD session, MWH was applied to the synthetic joint with an 8 GHz medical microwave generator via an applicator tip. The pseudo-tophus was submerged in 500 μL of 20 nm Au NPs embedded in pseudo-bursa and placed in a synthetic joint; a 1 cm × 1 cm × 2 cm synthetic skin patch covered the opening of the joint creating a closed synthetic joint model. The synthetic joint was exposed to microwaves for four 30 s intervals of MAMAD sessions (120 s). The microwave-exposed synthetic joints were monitored with an optical microscope camera every 30 s for 120 s with a 10 s delay. The temperature of the synthetic joint was monitored throughout the experiment using an interjoint temperature probe. The pseudo-tophus and Au NPs were removed from the pseudo-bursa after exposure to the MAMAD technique and dehydrated separately at 80 °C for an hour. Thereafter, the mass of the pseudo-tophus was measured and recorded, and each session was repeated after 24 h. The damage that occurred to synthetic skin patch during microwave exposure was assessed after each MAMAD session.

Model 2: Application of 21 MAMAD Sessions in a Closed or Open Synthetic Joint with a Pseudo-Bursa Containing a Pseudo-Tophus Submerged in Au NPs and Dehydrated after Every Seven MAMAD Sessions

The synthetic joint model 2 used in these experiments was similar to the synthetic joint model 1. In Figure 2, the pseudo-tophus was submerged in 800 μL of 20 nm Au NPs and enclosed in a pseudo-bursa. The pseudo-bursa was placed in a synthetic joint (open or closed) and exposed to MWH. The open synthetic joint lacked the 1 cm × 1 cm × 2 cm synthetic skin patch used to cover the opening of the closed joint. The pseudo-tophus, enclosed in the pseudo-bursa within the synthetic joint, was exposed to the MAMAD technique intermittently for seven MAMAD sessions (1 session = 120 s of MWH) without intermittent dehydration for 840 s. The pseudo-tophus and Au NPs were separated from the pseudo-bursa after microwave exposure, dehydrated for 1 h at 80 °C, and weighed for mass loss. The synthetic skin patch, used in the closed joint, was accessed for microwave-induced damage. In the open synthetic joint, the pseudo-bursa containing a pseudo-tophus was covered by a glass coverslip. A Phenom XL desktop SEM was used to view the structural changes on the surface of dehydrated pseudo-tophus samples. SEM was set to 15 kV, and we obtained SEM images magnifications (at scales closer to 200, 50, 10, and 2 μm) to characterize structural damages to the surface of pseudo-tophus.

Model 3: Application of 18 MAMAD Sessions in a Rotated Open Synthetic Joint (MWH on the Right, Top, and Left Sides) with a Pseudo-Bursa Containing a Pseudo-Tophus Submerged in Au NPs and Dehydrated after Every Three MAMAD Sessions

In experiments using synthetic joint models 1 and 2, microwaves were applied to one specific area of each synthetic joint, and changes in the mass (i.e., decrystallization) of the pseudo-tophus were accessed. In experiments using the synthetic joint model 3, microwave heating was applied to three different areas of the synthetic joint (i.e., right, top, and left), containing a pseudo-tophus immersed in 800 μL of 20 nm Au NPs in a pseudo-bursa. The application of microwaves to the top, right, and left sides of the synthetic joint (i.e., rotated closed synthetic joint) increased the likelihood of pseudo-tophus decrystallization by subjecting the synthetic joint to increased microwave exposure. The interjoint temperature of the synthetic joint was monitored via an interjoint temperature probe for the duration of the MAMAD session. Figure 3 shows real-color pictures of the synthetic joint in sequential order.

Finite-Difference Time-Domain Simulations

Finite-difference time-domain (FDTD) electromagnetic simulations were performed to determine the percentage of microwave absorption by each layer of the structure and to visualize the electric field propagation through the structure. MIT’s open source MEEP FDTD software¹ was utilized for the two-dimensional simulations. Dielectric constants of human tissues at 8 GHz microwave frequency were used in all simulations.^2,3 The pseudo-tophus was modeled as a 5 mm diameter round object located between two cortial bones and embedded in synovial fluid. In the electric field visualization simulations, 8 GHz microwave radiation was modeled as a fixed frequency continuous source located on the top part of the simulation cell. As in the experiments, the microwave radiation was transmitted to the structure through a 4.5 mm diameter waveguide, enabling single mode transmission. The field images depicted the propagation of the microwave radiation through the structure (Figure 7).

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b03497.

• Conditions of synthetic skin samples exposed to microwaves, the temperature measurements during microwave heating, the average PMR per MAMAD session for a pseudo-tophus (PDF)

• Short movie showing the FDTD results (MPG)

The authors declare no competing financial interest.