Abstract
The magnesium alloy was made into orthopedic steel plates to repair tibial fractures of New Zealand white rabbits and to explore the biocompatibility, degradation behavior, and mechanical properties of the magnesium alloy plates in repairing fractures in vivo. Fifty-four rabbits were randomly divided into experimental, control, and sham-operated groups. Tibial fractures in the experimental and the control groups were fixed with magnesium alloy and titanium alloy plates, respectively, and only bone tunnels were established without any implants in the sham-operated group. The concentrations of serum alanine transaminase, creatinine (CREA), creatine kinase (CK), and magnesium ion were measured before and 1 day, 1, 2, 4, 8, and 16 weeks after operation, respectively, to evaluate the biocompatibility of magnesium alloy plates. The corrosion products and components were observed using a scanning electron microscope with an energy-dispersive spectroscopy system, and the corrosion rate was observed by weight loss testing. Then the degradation behavior of magnesium alloy plate was analyzed. Analysis of mechanical properties of magnesium alloy plates was done by four-point bending tests. There were no statistically significant differences in serum alanine transaminase, CREA, or CK at each time point among the three groups (). The degradation behavior of the magnesium alloy plates increased with the longer implantation time. The four-point bending test results indicated that the mechanical properties of magnesium alloy plates decreased gradually during the degradation. The results showed that magnesium alloy plates implanted into rabbit tibias degrade gradually with the implantation time, and the mechanical properties of the magnesium alloy weaken gradually during the degradation. Meanwhile, the magnesium alloy plate had excellent biocompatibility and biosafety in the process of degradation in vivo.
1. Introduction
The history of the use of magnesium and its alloys as medical biomaterials can be traced back to 1878 when Edward used magnesium wire to ligate blood vessels and stop bleeding [1]. Payr put forward the use of pure magnesium as an orthopedic material in 1900 [2], while Lambotte [3] used pure magnesium plates for lower limb fractures in 1907. Early clinical application studies found that magnesium is biologically safe and promotes the healing of bone tissue. However, because of the rapid degradation of magnesium in vivo, fractures do not heal, and internal fixation becomes ineffective. Therefore, magnesium and its alloys were substituted by stainless steel materials which had better mechanical properties [4]. In recent years, magnesium and its alloys have attracted attention once again and have been adopted to form new medical, biological materials [5–11].
Magnesium alloy is an advantageous orthopedic material due to its degradability, good biocompatibility, and appropriate mechanical properties [12–14]. Witte et al. [15, 16] proved in animal experiments that the corrosion layer formed during the degradation of magnesium alloy is always closely connected to the peripheral bone, and there is more bone formation around magnesium alloy bars than around polymer bars. Pietak et al. [17] found that magnesium alloys promoted osteoblast adhesion, differentiation, and proliferation in vitro and bone formation in vivo. In our previous study, the magnesium alloy was made into orthopedic plates and used to repair tibial fractures of New Zealand white rabbits. The results showed that the implantation of magnesium alloy bone splints into rabbit tibia promoted bone scab formation and in vivo osteogenesis [18]. At present, there is no report on the treatment of fractures with magnesium alloy plates in the body, and the degradation behavior and mechanical properties of magnesium alloy plate are uncharted. Within this study, magnesium alloys were made into plates for repairing tibial fractures in New Zealand white rabbits and explored the biocompatible, degradation behavior, and mechanical properties of magnesium alloy plates for repairing fractures in vivo. The results of this research may provide an experimental basis for the in the body application of magnesium alloy plates, leading to the development of a promising degradable biomaterial for orthopedic applications.
2. Materials and Methods
2.1. Materials
Magnesium and titanium alloys were made into plates and screws for internal fixation by Shanghai Puwei Medical Instrument Co., Ltd. (Shanghai, China) (Figure 1).
The length, width, and thickness of the plates were 32, 5, and 1.5 mm, respectively. Each plate contained four holes, with a 2 mm diameter and a 5 mm distance between each hole. The screw’s length and diameter were 7 and 2 mm, respectively. Plates and screws were sterilized and sealed after sterilization with ethylene oxide.
Fifty-four clean, healthy adult New Zealand white rabbits, weighing an average of 2.5 ± 0.3 kg, were supplied by the Shanghai Jiesijie Lab. Animal Co., Ltd., (Shanghai, China; license No.: SCXK (hu) 2010-0026). All procedures were performed in accordance with Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of the People’s Republic of China (2006-09-30) [19].
2.2. Animal Experiments
Fifty-four rabbits were randomly divided into three groups: an experimental group treated with magnesium alloy plates, a control group treated with titanium alloy plates, and a sham-operated group. All animals were housed in cages and supplied with tap water and standard feed, and the experiment was performed after 1 week of adaptation.
The rabbits were intramuscularly anesthetized with xylazine (4 mg/kg, Jilin Huamu Animal Health Products Co., Ltd., Jilin, China) and ketamine (80 mg/kg, Fujian Gutian Medicine Co., Ltd., China). The skin was disinfected three times with iodophor before surgery. Hole sheets were paved. A 4 cm longitudinal incision was made in the medial side of the proximal right tibia. Under sterile conditions, the subcutaneous tissue was incised, and the muscles were separated to expose the proximal tibia; a chainsaw was then used to create the fracture. In each group, the appropriate implant, either a magnesium alloy plate or a titanium alloy plate, was fixed onto the proximal tibia to stabilize the fracture, while the sham-operated group did not receive any implants (Figure 2). After saline solution washing, the incision was sewed up tier upon tier. The rabbits can move freely inside the cage and have access to food and water. After surgery, the rabbits were given continuous intramuscular penicillin (400,000 U/day) for 3 days. The dietary, movement, inflammatory response, and trauma status of all animals were registered, and clinical examination of the tibia was performed daily after surgery.
2.3. Blood Tests
Collecting a 5 mL blood sample from all the rabbits in each group before surgery and at 1, 7, 14, 28, and 56 days after surgery. These were used for the measurement of hematological and biochemical parameters by blood analyzer (Hitachi 7600-020, Tokyo, Japan).
2.4. Degradation Behavior of Magnesium Alloy Plate
At 4, 8, or 16 weeks postoperatively, the magnesium alloy plates were removed, and the corrosion products on its surface were monitored by a scanning electron microscope (SEM). The components of the corrosion products were analyzed by energy-dispersive spectroscopy (EDS). Finally, the corrosion products deposited on the surface of the magnesium alloy plate were removed with 200 g/L chromic acid, and the corrosion morphology of the magnesium alloy plate was analyzed by SEM. In addition, the corrosion rate of the magnesium alloy plate was analyzed by weight loss test.
2.5. Mechanical Properties of the Magnesium Alloy Plate
The magnesium alloy plates were removed at 4, 8, or 16 weeks postoperatively, and the corrosion products deposited on the surface of the magnesium plate were cleaned off with 200 g/L chromic acid. A four-point bending test was performed to evaluate the mechanical properties of the degraded implanted plates and the plate before implantation using a mechanics tester (Zwick/Roller Z100; Zwick Roell, Ulm, Germany).
2.6. Statistical Analysis
The data were analyzed by SPSS 13.0 (SPSS, Inc., Chicago, IL, USA) statistical software. Differences between the groups were compared using one-way analysis of variance. A P-value < 0.05 was considered to indicate a statistically significant difference.
3. Results
3.1. General Condition of Experimental Animals
From the 54 rabbits, 51 were finally included for analysis. One rabbit in the experimental and one in the sham-operated group each had a fracture in the surgical area, while one in the control group died of postoperative diarrhea; these three were not included. The remaining rabbits in each group moved well, grew, developed well, and ate normally. None of the surgical sites were infected, swollen, necrotic, or inflamed, and none of the rabbits were febrile.
3.2. Blood Test Results
Figures 3–5 show the alanine aminotransferase (ALT), creatinine (CREA), and creatine kinase (CK) levels in the experimental group, control group, and sham-operated group at different time-points. The differences in ALT, CREA, and CK levels were not statistically significant when comparing the groups before surgery (). There was no statistically significant difference in ALT and CREA levels between the groups at each postoperative time (). There were no significant differences in ALT and CREA levels within any of the groups at different time-points after surgery (). On the first day postoperative, CK levels increased rapidly in each group () but returned to preoperative levels at 4 weeks postoperative (), and the differences in CK levels between the three groups at the same postoperative time points were not statistically significant ().
As shown in Figure 6, there were no significant differences in magnesium ion concentration (MIC) among the three groups before surgery (). On the first postoperative day, there were no significant differences in MIC in the experimental group (). At 1 week after surgery, the MIC was significantly increased () but returned to preoperative levels at 8 weeks postoperatively (). In the control and sham-operated groups, there were no significant changes in MIC at any of the time-points after surgery compared with MIC before operation ().
3.3. Degradation Behavior
Weight loss tests were used to evaluate the degradation behavior of the magnesium alloy plates in the body. The magnesium alloy plates were removed 4, 8, and 16 weeks after implantation into the rabbit tibias. The surface of the magnesium alloy plates had lost their original metallic luster, and the edges were less distinct. As shown in Figure 7(a), under the SEM, a layer of sparse gray–white degradation products was visible on the surface of the experimental group after 8 weeks of implantation of magnesium alloy. EDS analysis showed that the corrosion products formed on the surface of magnesium alloy plates were mainly consisted of oxygen, carbon, nitrogen, magnesium, and phosphorus after 8 weeks of implantation (Figure 7(b)). When the degradation products of the plate were removed with chromic acid, it was found that the surface was rough with irregular corrosion pits with small size and uniform distribution (Figure 8).
(a)
(b)
Corrosion rates, the weight loss, of magnesium alloy plates were 18.34%, 24.53%, and 27.79% after 4, 8, and 16 weeks of implantation, respectively. The study showed that the degradation behavior of magnesium alloy plates gradually enhanced with the prolongation of in vivo time.
3.4. Mechanical Properties
The bending stiffness (Figure 9) and bending strength (Figure 10) of the magnesium alloy plate before and at 4, 8, and 16 weeks after implantation showed that the bending stiffness and bending strength of the magnesium alloy plate gradually decreased as the degradation process continued.
4. Discussion
The biocompatibility of magnesium alloy plate for fracture repair is very important, and animal experiments in vivo are still the main means of evaluation. Serological results in this study showed no significant differences in ALT and CREA levels among the three groups at the same time-points after surgery and no significant differences in ALT and CREA levels within the groups at different time-points after surgery. These results indicated that the magnesium alloy plates did not affect hematological function in the rabbit. On the first day after surgery, CK levels in the three groups of animals increased rapidly but returned to preoperative levels within 4 weeks after surgery. However, there was no significant difference in CK levels among the three groups at the same time-points after surgery, proving that the increase in CK levels after surgery was caused by surgical factors, not by the implantation of magnesium alloy plates. The MIC levels were measured in each group to evaluate the safety of the implantation of magnesium alloy plates. The results of this study showed that there was no significant change in MIC in the experimental group on the first day after surgery, but on the seventh day after surgery, the MIC had increased and was significantly different both from that before surgery and from the control and sham-operated groups. However, by 8 weeks after surgery, the MIC had returned to preoperative levels. In the control and sham-operated groups, there was no significant change in MIC at 16 weeks postoperatively compared with that before surgery. Comparing the changes in MIC in the three groups, it can be seen that magnesium ions are released during the degradation of the magnesium alloy plates in vivo and are absorbed, which gradually increases the concentration of magnesium ions in the blood. However, as a consequence of metabolic processes and the body’s regulating effect, the MIC returned to the preoperative level within 28 days after surgery. Consequently, the maximum MIC did not exceed the ability of the body to self-regulate and will not cause metabolic disorders.
In addition to exploring the biocompatibility of magnesium alloy sheet, this study also investigated the degradation behavior of magnesium alloy sheet in vivo. SEM was used to observe the surface morphology of magnesium alloy plate before and after implantation in rabbit model. Postoperative weeks 4 and 8, killed the rabbits and removed the plate. Under SEM, the surface of the magnesium alloy plate was covered with a layer of gray corrosion products. After removing the corrosion products from the surface of magnesium alloy sheet, clear, small, and evenly distributed corrosion pits can be seen. Results of the EDS analysis showed that oxygen, carbon, calcium, phosphorus, magnesium, sodium, and chlorine constituted the corrosion products formed on the surface of magnesium alloy plates. In addition, the weight loss rate of magnesium alloy plates gradually increased at 4, 8, and 16 weeks after implantation, indicating that the degradation behavior of magnesium alloy plates in vivo gradually enhanced with the extension of time.
The mechanical properties of the magnesium alloy plates were also studied. After implantation in vivo for 4 weeks, the bending stiffness and bending strength of the magnesium alloy plates decreased to 72.11% and 72.26%, respectively. After 8 weeks of implantation, the bending stiffness and bending strength decreased to 62.68% and 62.16%, respectively, and after 16 weeks of implantation, had decreased to 52.62% and 52.31%, respectively. These results showed that the degradation of the magnesium alloy plates increased with implantation time, and the mechanical properties of the magnesium alloy decreased as the degradation increased.
5. Conclusions
In this study, the biocompatibility, degradation behavior, and mechanical properties of magnesium alloy sheet were studied. The results showed that the magnesium alloy plate had no significant effect on ALT, CREA, CK, and MIC after implantation in rabbit bone, which indicated that the magnesium alloy plate had good biocompatibility and biosafety in vivo. After the magnesium alloy plate was implanted into the rabbit tibia for fracture repair, the degradation of the magnesium alloy plate gradually increased at 4, 8, and 16 weeks, indicating that the magnesium alloy plate would eventually be absorbed by the rabbit bone tissue. In the process of degradation, the mechanical properties decreased gradually with the deepening of degradation. These results indicate that magnesium alloy plate is promising to be a new material for the repair of internal fractures.
Data Availability
The data used to support the findings of this study are included within the article.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Acknowledgments
This study was supported by the Natural Science Foundation of Gansu Province (20JR5RA369), the National Natural Science Foundation of China (81960398), Scientific and Technology Research Projects of Lanzhou (2021-1-79), Research Projects of Gansu Province Health Industry (GSWSKY 2021-009), and Medical Research Improvement Project (lzuyxcx-2022-187).