ANALYSIS OF THE W4 MAGNESIUM IMPLANT IN VIVO

Alicia Kolling and Dr. Frank Witte Laboratory for Biomechanics and Biomaterials, Department of Biomaterials Hannover Medical School, Hannover, Germany Email: [email protected], Web: http://www.lbb-mhh.de/

INTRODUCTION federal welfare legislation. Guidelines for the care and use of A biomaterial is a material considered to be suitable for laboratory animals (NIH Publication No. 85-23 Rev. 1985) contact with tissues or blood. To be accepted, it is required that have been observed. Five W4 scaffolds were surgically inserted the biomaterial be biocompatible [1]. This means the material into each rabbit. The scaffolds were created to be 60% porosity does not have toxic or injurious effects on biological function. using sintering; then cut to be 3mm in diameter and 5mm in Biomaterials are important in many medical applications length using a laser. Two were placed subcutaneously, two including joint replacements, artificial organs, prostheses and intramuscularly, and one in the femur of each rabbit bone plates/rods. In part of their critical role in fracture healing, (alternating left and right femur per rabbit). One, seven and two improved biomaterials continue to be an area of extensive animals were sacrificed at 2, 6 and 12 weeks, respectively. Two research. blood samples were drawn from each rabbit. The first sample Currently used biomaterials include various metals, was taken just before scaffold implantation and the second was polymers, ceramics and resins. Metallic biomaterials, such as just before sacrifice. Each sample was kept at room temperature stainless steel, titanium and chromium-cobalt alloys, are strong and underwent same-day analysis. enough for load-bearing applications, but are not degradable in The organ samples collected were fixed in paraffin wax vivo. Because these are permanent fixtures, a second surgery is while the femur samples were fixed in Technovit 9100 New. required if implant removal is desired, resulting in unnecessary Once embedded, both the paraffin wax and Technovit 9100 risk to the patient. Resorbable polymers are just the opposite. New samples were cut into 5µm thin sections and fixed on These materials can degrade within the body; however, they poly-L-lysine-coated glass slides for staining. The Technovit lack the mechanical properties suitable for hard tissue 9100 New samples were stained using toluidine blue and von application [2-4]. This gap in biomaterials has increased Kossa techniques, while the paraffin samples were stained research with magnesium, a metal which has a natural presence according to Mayer’s hematoxylin and eosin (H&E) procedure. in the human body. Magnesium has both the capability to The H&E staining procedure provided an overview of the corrode in vivo and the mechanical properties that resemble tissue by staining the basophilic structures (nuclei) blue/purple those of natural bone [3]. Additionally, it has been shown to and the acidophilic structures (cytoplasm) pink/red. The enhance osteoblast activity in the region adjacent to the toluidine blue technique was used to easily detect osteoblasts implanted material [5]. A disadvantage of magnesium being on the bone surface and the von Kossa technique was used to implemented as a biomaterial is that pure magnesium has an mark the mineralized bone mass by staining it brown/black. extremely fast corrosion rate in vivo. This can cause a Photomicrographs of the each staining were taken with a Zeiss subcutaneous buildup of hydrogen gas, a product of magnesium Axioskop 40 microscope and set saved for further analysis. corrosion. The reaction below summarizes the corrosion of A eudiometer is a testing device used to measure the magnesium: change in volume of a gas due to a chemical change. This was done to record the amount of hydrogen gas produced during Mg(s) + 2H20  Mg(OH)2(s) + H2(g) corrosion of a W4 magnesium scaffold. Twelve W4 scaffolds In order to slow this reaction rate, biocompatible magnesium were placed in a simulated body fluid medium to track this surface treatments and magnesium alloys are currently being production in an environment similar to the body. The medium researched [2-3]. used was Dulbecco’s Modified Eagle Medium (DMEM). In this project, a material composed of magnesium alloyed Before testing, this solution was degassed in a 37 C ultrasonic with 4% yttrium (W4) was used to form a scaffold to implant. water bath for 15 minutes. An initial pH was recorded to The purpose of this project was to observe the effects of the W4 compare against a normal body pH and track the change⁰ in pH magnesium alloy in vivo and evaluate its potential as a that occurs with gas production. The scaffold was then attached biomaterial. This was done through the analysis of blood to the assembled eudiometer glassware and the medium was samples pre- and post-implantation, examination of organ pipetted inside. The inner and outer medium levels of the tissues, evaluation of bone growth and remodeling around the eudiometer column were recorded once an hour for 24 hours. area of implantation, and assessment of the hydrogen gas Afterwards, the final pH of the medium was again recorded and production rate during magnesium corrosion in vitro. the scaffold was massed. It was hypothesized that the W4 magnesium alloy would be biocompatible, causing no harmful effects in vivo. Based on RESULTS previous research, the alloy would instead be beneficial to the Blood parameters were tested to see if values stayed within body by enhancing the mineralized bone growth and osteoblast normal body ranges in the presence of a W4 implant. Some of activity adjacent to the implant. these parameters included enzymes, nutrients, erythrocytes, and leukocytes. The blood work from each rabbit appeared normal MATERIALS AND METHODS both before implantation and just before sacrifice. All of the Ten adult New Zealand White Rabbits were used for results fell within each parameter’s acceptable range at both implantation. The animal experiments were conducted under an time points. ethic committee approved protocol in accordance with German 1 The tissues of the heart, intestine, kidney, liver, lung, entire 24 hours, it appears as though the medium enables pancreas and spleen were observed under a microscope. At 2, 6 production of hydrogen at a rate within the body’s absorption and 12 weeks after W4 implantation, all organ tissues appeared limit at 0.0940mL/h. Additionally, this value remains within normal in structure. No differences were found in the tissues the absorption rate when looking at the hour-by-hour results. between the specified time intervals. The greatest volume of hydrogen gas was produced within the Through the von Kossa staining (Figure 1), the amount of first hour of testing, 0.768±0.239mL. Although these results mineralized bone area directly surrounding the W4 scaffold suggest the W4 scaffold corrodes slowly enough for the body to was determined. The percentage of bone area per tissue area harmlessly dispose of the excess hydrogen gas, a biological varied between the 2, 6 and 12 week time points. At 2 weeks, environment cannot be matched exactly with a simulated the bone area per tissue area was found to be 75.52%. The medium; therefore, in vivo testing is necessary. average bone area per tissue area surrounding the implant at 6 As seen in Figure 2, the open porous structure of the weeks was 73.25% and 80.79% at 12 weeks. scaffold enabled the bone to grow through the scaffold rather than in a circular pattern surrounding the implant region. The W4 scaffold also showed the ability to corrode in vivo. The corrosion over time can be seen when comparing the amount of scaffold present after 6 and 12 weeks.

FIGURE 1 High mineralized bone area adjacent to the implant region is evident by the intense black color visible from the von Kossa staining at 12 weeks.

Using the Toluidine blue staining, the percent of unmineralized bone, or osteoid surface, was calculated from the FIGURE 2 osteoid perimeter (µm) and bone perimeter (µm). As time New Bone Formation within the W4 Implant Region as Scaffold is Undergoing Corrosion – 6 Weeks (left) & 12 Weeks (right) increased, the osteoid surface percentage decreased slightly. Starting at 18.428% at 2 weeks, it decreased to 10.998% at 6 CONCLUSION weeks and 8.530% at 12 weeks. The increased bone area per tissue area and low hydrogen Eudiometric testing was done for 12 W4 implants. The gas volume produced suggest that the yttrium present in the average volume of hydrogen gas produced after 24 hours was magnesium alloy has an enhancing effect on bone growth 2.256mL. This average was calculated from the volume of compared to AZ91D scaffold results published by Witte et al. hydrogen gas produced per scaffold normalized with its surface [6]. This, in combination with the unobserved harmful effects area. It was then important to take a look at the volume of on the blood samples and organ tissues, suggests that the W4 hydrogen gas produced per hour. The largest volume of alloy has the potential to be a biocompatible material for use in hydrogen gas was produced within the first hour of testing, medical applications. However, more research is needed to 0.768±0.239mL. The general trend shows the rate decreased further understand yttrium’s enhancing ability and ensure over the remaining 23 hours staying noticeably lower than the biocompatibility. first hour of testing. ACKNOWLEDGEMENTS DISCUSSION A thank you is extended to Dr. Frank Witte and all those It is essential for a new biomaterial to be biocompatible who work at the Laboratory for Biomechanics and with the body. In addition to immediate symptoms, one must Biomaterials. Additionally, I would like to thank the University look at the body’s response to long term implantation. In of Pittsburgh for funding. general, the blood sample values appeared normal for each rabbit, falling within their respective acceptable ranges. These REFERENCES [1] Zeng R, Dietzel W, Witte F, Hort N, Blawert C. Progress and Challenge results suggest that the W4 scaffold corrosion has no obvious for Magnesium Alloys as Biomaterials. Adv. Eng. Mater. 2008; 10: B3- adverse effects on the body. Observing normal organ tissues at B14. 2, 6 and 12 weeks also support this suggestion. [2] Staiger M, Pietak A, Huadmai J, Dias G. Magnesium and its alloys as A previous study by Witte et al. found that after 3 months orthopedic biomaterials: A review. Biomaterials 2006; 27: 1728-1734. [3] Wang H, Shi Z. In vitro biodegradation behavior of magnesium and of implantation using an AZ91D scaffold, the bone area per magnesium alloy. J Biomed Mater Res Part B 2011; 98: 203-209. tissue area was 45.7% [6]. In comparison, at 12 weeks, or [4] Waizy H, Weizbauer A, Maibaum M, Witte F, Windhagen H, Lucas A, approximately 3 months, the bone area per tissue area was Denkena B, Meyer-Lindenberg A, Thorey F. Biomechanical found to be 80.79% using the W4 scaffold. This higher area of characterisation of a degradable magnesium-based (MgCa0.8) screw. J Mater Sci: Mater Med 2012; 23: 649-655. bone could be a result of the yttrium present in the magnesium [5] Witte F, Ulrich H, Rudert M, Willbold E. Biodegradable magnesium alloy. A high percent osteoid surface percentage suggests that scaffolds: Part I: Appropriate inflammatory response. J Biomed Mater the yttrium may also have an enhanced effect on osteoblast Res Part A 2007; 81: 748-756. proliferation [5]. [6] Witte F, Ulrich H, Palm C, Willbold E. Biodegradable magnesium scaffolds: Part II: Peri-implant bone remodling. J Biomed Mater Res Part Previous research suggests that the body can absorb A 2007; 81: 757-765. hydrogen gas at a rate of 0.954mL/h [7]. When calculating the [7] Piiper J, Canfield R, Rahn H. Absorption of various inert gases from rate of hydrogen production using the total volume over the subcutaneous pockets in rats. J Appl Physiol 1962; 17: 268-274.

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