Abstract
Background and purpose Magnesium (Mg) has a biomechanical character resembling bone, with mechanical strength exceeding that of ceramics, but has a high corrosion rate. One method to reduce the corrosion level of Mg is to mix it with other materials or coatings. Carbonate apatite (CA) was chosen as a Mg composite mixture because of its good osteoconductivity, and this study aimed to evaluate the biodegradability of MgxCA composite implants made by the extrusion technique in Sprague Dawley (SD) rats. Methods This study was a post-test only in vivo experiment in SD rats from July to December 2021. Thirty SD rats were divided into five treatment groups: Mg0CA, Mg5CA, Mg10CA, Mg15CA, and titanium plates. The examination included implant weight changes, postoperative gas formation, and local and systemic histopathological analyses on days 15 and 30. Results There was a significant difference in gas formation on day 15 and implant degeneration between groups (p < 0.05). However, in post-hoc analysis, we found no significant differences in implant weight difference or implant gas production between pre- and post-sacrifice in the MgxCA composites (p > 0.05). Histopathological examination revealed no significant local or systemic inflammatory response differences between groups (P > 0.05). Conclusion The combination of magnesium with apatite carbonate from extruded fabrication techniques is a biodegradable implant with biocompatibility and nontoxic properties, either locally or systemically.
Keywords: Biodegradable, Magnesium Carbonate, Carbonate apatite, Extrusion techniques
Subject terms: Biotechnology, Medical research
Introduction
Biodegradable orthopaedic implants have rapidly developed, reducing removal morbidity. Magnesium, a biomechanical material with human bone properties, is a viable option but is highly corrosive .(1) Mixing magnesium alloy with other materials reduces the corrosion rate.(2) Carbonate apatite is commonly used in orthopaedic implants because of its non-organic, osteoconductive nature. (3) Despite the advantages of the magnesium carbonate apatite (MgCA) alloy, the alloy is degraded rapidly, thus leading to the weakening of the implant.(3) The corrosion process of MgCA alloy is related to the low density of MgCA alloy.(4) Modification in fabrication techniques through milling, sintering, and extrusion can improve MgCA alloy density. Extrusion offers dense compaction and a lower corrosion rate, while milling technique shows good biocompatibility.(5).
Results from an in vitro study of MgCA alloy wire produced using the extrusion technique have low toxicity to normal human cells.(6) However, results regarding biodegradation from in vitro and in vivo studies may differ. A study by Antoniac et al. comparing the biodegradability of Mg-1Ca magnesium alloys in vitro and in vivo found contradicting finding regarding the corrosion and cytotoxicity results.(7) There has been no prior in vivo biodegradability study of MgCA. This study aimed to evaluate the biodegradability and bioavailability of MgCA alloy fabricated using the extrusion technique as an implant in SD rats.
Results
In this study, we conducted an experimental test in 30 SD rats divided into five research groups. The sacrifice period for histopathological examination was conducted in two stages, namely at 15 days post-operation and 30 days post-operation. Characteristics of the samples included a median body weight of 295.5 g (lowest weight of 228 g and highest weight of 360 g). The average implant weight was 30 mg, with a minimum weight of 20 mg and a maximum weight of 125 mg. Complications were observed in 17 (56.6%) research samples, with the majority experiencing mild non-suppurative nephritis (60%), followed by mild focal portal hepatitis (6.7%), and mild white pulp hyperplasia (3.3%) on histopathological examination.(Table 1).
Table 1.
Sample characteristics.
| Group | n(%) / Median (min-max) |
|---|---|
| Titanium | 6 (20) |
| Mg0%CA | 6 (20) |
| Mg5%CA | 6 (20) |
| Mg10%CA | 6 (20) |
| Mg15%CA | 6 (20) |
| Body weight (gram) | 295.5 (228–360) |
| Implant weight (mg) | 30 (20–125) |
| Sacrifice Time | |
| Day 15 | 15 (50) |
| Day 30 | 15 (50) |
| Complication | 17 (56.6) |
| Mild non-suppurative interstitial nephritis | 18 (60) |
| Mild focal portal hepatitis | 2 (6.7) |
| Mild white pulp hyperplasia | 1 (3.3) |
Post-operative hematological tests for systemic inflammation results showed no significant difference between groups. (Table 2) The local histopathological examination of inflammatory cell infiltration, connective tissue scoring, neovascularization scoring, number of osteoclast cells, and osteoblast activity showed no significant differences between the groups (Table 3).
Table 2.
Comparison of hematological results of Mg0CA, Mg5CA, Mg10CA, Mg15CA, and titanium groups.
| Implant | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Titanium | p | Mg0CA | Nilai p | Mg5CA | Nilai p | Mg10CA | Nilai p | Mg15CA | Nilai p | ||
| Hb | Pre | 14.9 (± 1.82) | 0,898 | 14.9 (± 0.99) | 0,033 | 14.5 (± 0.25) | 0,466 | 15.1 (± 0.65) | 0,295 | 14.6 (± 1.36) | 0.981 |
| Post | 13.8 (± 1.51) | 14.6 (± 0.83) | 14.1 (± 0.56) | 13.4 (± 1.52) | 14.5 (± 0.99) | ||||||
| RBC | Pre | 8.42 (± 1.22) | 0,797 | 8.28 (± 0.62) | 0,292 | 8.56 (± 0.52) | 0,639 | 8.67 (± 0.28) | 0,963 | 8.09 (± 0.60) | 0.637 |
| Post | 7.53 (± 0.69) | 8.00 (± 0.30) | 8.04 (± 0.68) | 7.52 (± 0.71) | 7.95 (± 0.54) | ||||||
| Ht | Pre | 46.7 (± 7.51) | 0,735 | 46.4 (± 2.36) | 0,979 | 47.1 (± 3.14) | 0,081 | 47.5 (± 2.14) | 0,247 | 45.5 (± 3.49) | 0.763 |
| Post | 40.8 (± 3.65) | 45.1 (± 2.02) | 43.6 (± 1.74) | 40.5 (± 4.41) | 44.5 (± 2.92) | ||||||
| MCV | Pre | 55.3 (± 1.27) | 0,373 | 57.3 (± 1.81) | 0,275 | 55.1 (± 2.61) | 0,415 | 54.8 (± 2.07) | 0,371 | 56.3 (± 1.88) | 0.597 |
| Post | 54.3 (± 0.45) | 56.5 (± 1.49) | 54.5 (± 2.93) | 53.9 (± 1.04) | 56.1 (± 0.87) | ||||||
| MCH | Pre | 17.7 (± 0.75) | 0,308 | 18.0 (± 0.98) | 0,457 | 16.9 (± 1.07) | 0,156 | 17.3 (± 0.97) | 0,310 | 18.1 (± 1.37) | 0.931 |
| Post | 18.2 (± 0.86) | 18.3 (± 0.88) | 17.5 (± 1.30) | 17.4 (± 0.58) | 16.6 (± 4.48) | ||||||
| MCHC | Pre | 32.1 (± 1.43) | 0,081 | 31.6 (± 1.56) | 1,000 | 30.9 (± 1.93) | 0,049 | 31.8 (± 2.25) | 0,114 | 32.2 (± 2.91) | 0.939 |
| Post | 33.7 (± 1.39) | 32.5 (± 1.00) | 32.3 (± 0.94) | 32.9 (± 0.95) | 32.6 (± 1.18) | ||||||
| RDW | Pre | 13.2 (± 1.29) | 0,182 | 13.3 (± 2.99) | 0,364 | 12.6 (± 1.09) | 0,006 | 12.9 (± 1.02) | 0,300 | 13.1 (± 1.42) | 0.284 |
| Post | 12.8 (± 1.09) | 13.5 (± 2.01) | 13.1 (± 0.59) | 14.0 (± 0.92) | 13.1 (± 0.97) | ||||||
| Thrombocyte | Pre | 898.33 (± 585.64) | 0,053 | 1129.5 (± 510.82) | 0,327 | 1316.6 (± 316.23) | 0,131 | 1244.6 (± 206.14) | 0,127 | 1116 (± 200.51) | 0.527 |
| Post | 668.83 (± 351.89) | 974.8 (± 184.29) | 1019.3 (± 348.47) | 567.16 (± 446.56) | 981 (± 238.84) | ||||||
| PCT | Pre | 0.36 (± 0.16) | 0,915 | 0.53 (± 0.21) | 0,659 | 0.60 (± 0.06) | 0,154 | 0.56 (± 0.08) | 0,947 | 0.59 (± 0.07) | 0.639 |
| Post | 0.34 (± 0.18) | 0.50 (± 0.11) | 0.52 (± 0.16) | 0.55 (± 0.59) | 1.31 (± 2.09) | ||||||
| MPV | Pre | 5.38 (± 0.21) | 0,183 | 5.38 (± 0.25) | 0,118 | 5.36 (± 0.58) | 0,096 | 5.15 (± 0.33) | 0,096 | 5.6 (± 0.57) | 0.278 |
| Post | 5.35 (± 0.64) | 5.12 (± 0.42) | 5.23 (± 0.47) | 5.13 (± 0.40) | 6.88 (± 4.48) | ||||||
| PDW | Pre | 16.2 (± 0.20) | 0,938 | 16.1 (± 0.25) | 0,903 | 16.1 (± 0.24) | 0,916 | 15.9 (± 0.19) | 0,825 | 15.5 (± 1.75) | 0.876 |
| Post | 16.4 (± 1.15) | 15.9 (± 0.21) | 16.0 (± 0.27) | 16.3 (± 0.64) | 15.6 (± 1.28) | ||||||
| WBC | Pre | 12.0 (± 4.40) | 0,603 | 13.1 (± 6.20) | 0,691 | 15.9 (± 5.72) | 0,243 | 13.4 (± 5.15) | 0,092 | 14.1 (± 6.59) | 0.744 |
| Post | 11 (± 3.36) | 12.3 (± 3.61) | 13.2 (± 3.34) | 9.61 (± 3.47) | 11.3 (± 3.24) | ||||||
| Lymphocyte | Pre | 7.63 (± 3.42) | 0,350 | 8.38 (± 4.72) | 0,225 | 8.68 (± 3.10) | 0,036 | 6.95 (± 2.72) | 0,520 | 8.7 (± 5.17) | 0.322 |
| Post | 6.58 (± 1.69) | 7.24 (± 3.00) | 7.33 (± 2.15) | 5.16 (± 0.94) | 5.96 (± 3.42) | ||||||
| Monocyte | Pre | 0.38 (± 0.13) | 0,589 | 0.43 (± 0.22) | 0,870 | 0.46 (± 0.23) | 0,923 | 0.41 (± 0.21) | 0,034 | 0.46 (± 0.22) | 0.153 |
| Post | 0.36 (± 0.15) | 0.4 (± 0.14) | 1.36 (± 2.37) | 0.31 (± 0.13) | 1.13 (± 1.75) | ||||||
| Granulocyte | Pre | 4.06 (± 1.20) | 0,507 | 4.36 (± 2.18) | 0,367 | 6.76 (± 3.54) | 0,172 | 6.03 (± 2.68) | 0,969 | 4.95 (± 2.40) | 0.448 |
| Post | 4.05 (± 1.94) | 4.66 (± 1.29) | 4.55 (± 2.94) | 4.13 (± 2.55) | 6.56 (± 5.25) | ||||||
| ALP | Pre | 29.6 (± 16.1) | 0,314 | 26.6 (± 9.15) | 0,075 | 26.5 (± 10.0) | 0,002 | 37.5 (± 18.9) | 0,781 | 27.5 (± 8.61) | 0.986 |
| Post | 38 (± 4.47) | 36.5 (± 12.1) | 37.2 (± 5.03) | 38.1 (± 3.54) | 35.5 (± 2.73) | ||||||
| SGPT | Pre | 23.6 (± 5.64) | 0,033 | 32.8 (± 10.4) | 0,868 | 39.6 (± 14.7) | 0,024 | 37.1 (± 16.6) | 0,185 | 40.8 (± 14.5) | 0.048 |
| Post | 26.5 (± 5.64) | 37.8 (± 15.5) | 41.8 (± 9.88) | 34.1 (± 14.4) | 38.1 (± 12.9) | ||||||
| SGOT | Pre | 55.1 (± 23.5) | 0,026 | 69.6 (± 30.9) | 0,692 | 78.1 (± 28.8) | 0,829 | 79.3 (± 38.9) | 0,390 | 61.8 (± 11.5) | 0.868 |
| Post | 40.8 (± 17.2) | 63.8 (± 14.0) | 59 (± 7.94) | 50.3 (± 30.6) | 45.5 (± 8.24) | ||||||
| Total Protein | Pre | 5.88 (± 0.33) | 0,375 | 6.36 (± 0.73) | 0,709 | 6.26 (± 0.67) | 0,312 | 6.45 (± 0.71) | 0,233 | 6.18 (± 0.54) | 0.539 |
| Post | 5.71 (± 0.80) | 6.23 (± 0.45) | 6.2 (± 0.50) | 6.36 (± 0.58) | 6.38 (± 0.51) | ||||||
| Glucose | Pre | 94.1 (± 35.5) | 0,581 | 83.1 (± 30.2) | 0,919 | 71.8 (± 21.2) | 0,179 | 141. (± 128.) | 0,586 | 84.3 (± 8.80) | 0.605 |
| Post | 90.3 (± 29.6) | 95.1 (± 31.5) | 87 (± 31.0) | 90.6 (± 26.9) | 97.5 (± 33.8) | ||||||
| Ureum | Pre | 18.5 (± 3.83) | 0,684 | 20.1 (± 4.99) | 0,085 | 19.1 (± 4.62) | 0,766 | 18 (± 6.03) | 0,815 | 18.3 (± 3.44) | 0.384 |
| Post | 19 (± 2.52) | 21.8 (± 4.44) | 19.8 (± 3.97) | 18 (± 5.36) | 20 (± 4.19) | ||||||
| Creatinine | Pre | 0.15 (0.1–0.2) | 0.083 | 0.15 (0.1–0.3) | 0.317 | 0.2 (0.1–0.3) | 1.000 | 0.2 (0.1–0.4) | 0.564 | 0.2 (0.1–0.3) | 0.317 |
| Post | 0.2 (0.1–0.3) | 0.2 | 0.2 (0.1–0.2) | 0.2 (0.2–0.3) | 0.2 (0.2–0.3) | ||||||
Mean ± SD; Median (Min-Max).
Table 3.
Local histopathological examination.
| Parameters | None or minimal; n(%) | Mild; n(%) | Moderate; n(%) | Severe; n(%) | Very severe; n(%) | P value |
|---|---|---|---|---|---|---|
| Inflammatory cell infiltration | ||||||
| Titanium | 1 (16.7) | 3 (50.0) | 1 (16.7) | 0 (0) | 1 (16.7) | 0.715 |
| Mg0CA | 0 (0) | 2 (33.3) | 1 (16.7) | 1 (16.7) | 2 (33.3) | |
| Mg5CA | 1 (16.7) | 2 (33.3) | 0 (0) | 0 (0) | 3(50.0) | |
| Mg10CA | 0 (0) | 2 (33.3) | 1 (16.7) | 2 (33.3) | 1 (16.7) | |
| Mg15CA | 0 (0) | 3(50.0) | 2 (33.3) | 0 (0) | 1 (16.7) | |
| Soft tissue scoring | ||||||
| Titanium | 1 (16.7) | 3 (50.0) | 1 (16.7) | 0 (0) | 1 (16.7) | 0.373 |
| Mg0CA | 0 (0) | 0 (0) | 3 (50.0) | 2 (33.3) | 1 (16.7) | |
| Mg5CA | 0 (0) | 3 (50.0) | 0 (0) | 0 (0) | 3 (50.0) | |
| Mg10CA | 0 (0) | 2 (33.3) | 1 (16.7) | 2 (33.3) | 1 (16.7) | |
| Mg15CA | 0 (0) | 1 (16.7 | 5 (83.3) | 0 (0) | 0 (0) | |
| Neovascularization | ||||||
| Titanium | 1 (16.7) | 3 (50.0) | 1 (16.7) | 0 (0) | 1 (16.7) | 0.505 |
| Mg0CA | 0 (0) | 1 (16.7) | 3 (50.0) | 1 (16.7) | 1 (16.7) | |
| Mg5CA | 0 (0) | 3 (50.0) | 0 (0) | 0 (0) | 3 (50.0) | |
| Mg10CA | 0 (0) | 2 (33.3) | 1 (16.7) | 2 (33.3) | 1 (16.7) | |
| Mg15CA | 0 (0) | 3 (50.0 | 2 (33.3) | 0 (0) | 1 (16.7) | |
| Local osteoclast count | ||||||
| Titanium | 5 (83.3) | 1 (16.7) | 0 (0) | 0 (0) | 0 (0) | 0.220 |
| Mg0CA | 2 (33.3) | 2 (33.3) | 1 (16.7) | 1 (16.7) | 0 (0) | |
| Mg5CA | 0 (0) | 3 (50.0) | 1 (16.7) | 2 (33.3) | 0 (0) | |
| Mg10CA | 3 (50.0) | 1 (16.7) | 1 (16.7) | 1 (16.7) | 0 (0) | |
| Mg15CA | 5 (83.3) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
| Osteoblast activity | ||||||
| Titanium | 1 (16.7) | 3 (50.0) | 1 (16.7) | 1 (16.7) | 0 (0) | 0.468 |
| Mg0CA | 0 (0) | 2 (33.3) | 2 (33.3) | 2 (33.3) | 0 (0) | |
| Mg5CA | 0 (0) | 2 (33.3) | 1 (16.7) | 1 (16.7) | 2 (33.3) | |
| Mg10CA | 0 (0) | 2 (33.3) | 2 (33.3) | 1 (16.7) | 1 (16.7) | |
| Mg15CA | 0 (0) | 3 (50.0) | 2 (33.3) | 1 (16.7) | 0 (0) |
In this study, the difference in implant weight before and after sacrifice was evaluated. The comparative analysis results showed significant differences in implant weight between the groups (Table 4; Fig. 1). Post-hoc analysis showed a significant difference between MgCA implants and titanium but no significant differences between MgCA compositions.(Table 5). The degradation rate described in this study is as follows: On the 15 days observation, the Mg0CA rate was 5.48 mg/day, Mg5CA was 5.68 mg/day, Mg10CA was 4.55 mg/day, Mg15CA was 3.46 mg/day. While on the 30 days observation, the rate of degradation were slower with Mg0CA of 2.68 mg/day, Mg5CA of 3.01 mg/day, Mg10CA of 2.42 mg/day, and Mg15CA of 1.82 mg/day. From the results, we can see that the higher the CA content, the slower the rate of implant degradation.
Table 4.
Comparison analysis of the weight difference of implants in the Mg0CA, Mg5CA, Mg10CA, Mg15CA, and titanium groups.
| Group | 15 days | 30 days | ||
|---|---|---|---|---|
| Weight difference (gram) |
P value | Weight difference (gram) |
P value | |
| Titanium | 0 | 0.000 | 0 | 0.000 |
| Mg0CA | 82.3 ± 12.5 | 86 ± 13.1 | ||
| Mg5CA | 85.3 ± 10.0 | 90.3 ± 11.0 | ||
| Mg10CA | 68.3 ± 12.0 | 72.6 ± 10.6 | ||
| Mg15CA | 52 ± 26.4 | 54.6 ± 19.1 | ||
Mean ± SD.
Fig. 1.
Implant weight comparison.
Table 5.
Post-hoc analysis of the weight difference of implants in the Mg0CA, Mg5CA, Mg10CA, Mg15CA, and titanium groups post-operation.
| Group | Titanium | Mg0CA | Mg5CA | Mg10CA | Mg15CA |
|---|---|---|---|---|---|
| Titanium | - | 0.000 | 0.000 | 0.000 | 0.003 |
| Mg0CA | 0.000 | - | 1.000 | 1.000 | 0.116 |
| Mg5CA | 0.000 | 1.000 | - | 1.000 | 0.056 |
| Mg10CA | 0.000 | 1.000 | 1.000 | - | 1.000 |
| Mg15CA | 0.003 | 0.116 | 0.056 | 1.000 | - |
Implant gas production was also measured by obtaining the volume of peri-implant gas using radiography (Table 6; Fig. 2). The results showed a significant difference in gas production between groups on day 15. However, post-hoc analysis showed significant differences between MgCA implants and titanium implants, but no significant differences were observed between MgCA compositions.
Table 6.
Implant gas volume.
| Group | Day 1 | Day 15 | Day 30 | |||
|---|---|---|---|---|---|---|
| Gas volume (mm 3 ) | P value | Gas volume (mm 3 ) |
P value | Gas volume (mm 3 ) |
P value | |
| Titanium | 0 | 0,090 | 0,000 | 0,002 | 0,000 | 0,054 |
| Mg0CA | 0 (0–20,98) | 12,9 (0-240,8) | 0 (0–80,2) | |||
| Mg5CA | 2,2 (0–14,7) | 1084,1(20,2-5350,8) | 1241,2 (862,6-1393,9) | |||
| Mg10CA | 26,7 (0-219,6) | 53,3 (0-372,9) | 0 (0-708,2) | |||
| Mg15CA | 2,6 (0–16,0) | 40,7 (21,1-1256,5) | 485,6 (56,01-535,6) | |||
Median (min – max).
Fig. 2.
Xray examination (A) immediately postoperative xray; (B) 15 days follow up Xray; (C) 30 days follow up Xray.
Discussion
Progress in the development of implants and orthopaedic devices has led to the need for biodegradable implants with less implant removal morbidity and comfort for patients. Magnesium implants have been recognized as a potential material for biodegradable implants. However, the rapid degradation rate of the material in physiological fluid leads to the weakening of the implant. MgCA is a magnesium alloy that can decrease the degradation rate of magnesium implants. In this study, we aimed to demonstrate the biocompatibility and biodegradability of MgCA implants in vivo using an animal model.
Magnesium is an important chemical macronutrient (comprising 0.2% of the human body weight), which has good biocompatibility and biodegradability, as well as high tensile strength compared to polymers and is less brittle than ceramics. The elastic modulus of magnesium (45 GPa) is closer to the elastic modulus of cortical bone (15–25 GPa) than the elastic moduli of titanium alloys and stainless steel (115–200 GPa).(8) Due to high degenerative rate inside the body, magnesium is combined with carbonate apatite to enhance its strength and decrease its degeneration rate.(2),(9),(10) Magnesium carbonate apatite (MgCA) is chemically related to hydroxyapatite, the major mineral component of bone.(11) Thus, orthopaedic implants composed of materials such as MgCA can aid in bone repair. These compounds have been shown to encourage the development of new bone tissues, implying that they can aid in bone repair and regeneration. Due to its unique characteristics such as biocompatibility, osteoconductivity, and biodegradability, this MgCA is an attractive material to be used in orthopaedic implant.(12) In this study, we conducted an extrusion method of mixing magnesium with carbonate apatite, which has several advantages, including higher implant density, enhanced biocompatibility, and increased resistance to wear and tear.
Biocompatibility profile of MgCA has been described in previous studies which reported high biocompatibility in living tissue.(5) In this study, we observed the local reaction of MgCA with various proportions of carbonate apatite compared to a titanium implant, with no significant difference. Local inflammation is a normal reaction to foreign objects owing to the innate immune response of the body. However, a biocompatible implant should not elicit severe inflammation, which can lead to alterations in bone healing. A previous study by Rahyussalim et al. has reported that the powder form of carbonate apatite cannot be used directly as a bone substitute because it triggers the formation of crystals from the inflammatory response.(5) However, fabrication into a plate using sintering or extrusion method could increase the density of the implant, thus reducing the risks of local inflammation.(11) Unfortunately, the sintering process requires high temperatures, which induce the decomposition of carbonate apatite. In this study, we reported similar results, with no significant difference between local inflammatory reactions in MgCA implants and titanium implants.
Owing to its molecular similarity to hydroxyapatite (HA), MgCA has been reported to have an osteoconductive ability that promotes bone healing. MgCA and HA are both calcium phosphates with comparable chemical and structural structures.(13) HA is the main mineral component of natural bone, and is responsible for its mechanical properties. MgCA has a distinct crystal structure that enables it to disintegrate and regenerate bone tissue.(14) Unlike other biomaterials, MgCA implant does not need to be coated to induce bone development.(15) In this study, we observed osteoblastic and osteoclastic activities both in titanium implant and MgCA implant.(16) Despite no significant difference between the implants, we observed higher osteoblastic activity with moderate to very severe infiltration of osteoblasts in the peri-implant area. However, despite the high activity of osteoblasts and osteoclasts surrounding the implant, the clinical significance of MgCA implants remains to be determined.
Another aspect of biocompatibility of MgCA implant is the systemic effect of the implant to other related organs.(17) In this study, we observed no significant difference in systemic organ complications between MgCA implants with various concentrations of titanium implants. Severe systemic side effects of MgCA has not been reported in prior studies other than hypersensitivity reaction to the implant.(18) In this study, histopathological examination of the kidney revealed mild non-suppurative interstitial nephritis. Kidney injury associated with the use of MgCA implants has not been described in previous studies. The presence of hyperphosphatemia has been related to the development of kidney stones that could lead to nephritis.(19) However, we did not find any significant difference in non-suppurative interstitial nephritis between titanium and MgCA; thus, the reaction could be caused by other factors.
One of the distinct properties of MgCA implants is their ability to gradually degrade over time without the need for implant removal. While degraded inside the body, the implant reacted to the surrounding tissue, releasing magnesium and carbonate ion into the circulation while releasing hydrogen (H2) gas into the surrounding tissue.(5) In this study, we observed degradation of the MgCA implant with the production of hydrogen gas and decreased implant weight after several weeks of observation compared to titanium implants with no hydrogen gas production. A previous study has reported that the rate of degradation and gas production of magnesium alloy is affected by the composition, microstructure, surface coating, and environmental conditions.(15) Extrusion process in the making of the implant could also affect the density of the alloy, thus affecting the rate of degradation of the implant.(9) From the results of this study, degradation of the MgCA, even though not significant, was fastest in Mg5CA Alloy. This finding is similar to in vitro finding that the addition of CA increases degradation, but higher CA (10% and 15%) content decreases degradation. (16) A similar phenomenon was found with MgHA (magnesium-hydroxyapatite) alloy, which has a similar composition to MgCA, and found that degradation decreased with the addition of HA up to MgHA 10% and then increased at 15%. (20)
In this study, we conducted the first preclinical in vivo study of MgCA alloys fabricated by extrusion as orthopedic implants to test their biocompatibility and biodegradability. We concluded that the MgCA implant fabricated by extrusion is biodegreable with low toxic properties either locally or systemically. This study is a continuation of a study of a preclinical in vitro study of MgCA by Rahyussalim et al.(21) However, we improved the processing method of the alloy mixture using the extrusion method, which allows for increased alloy density, thus overcoming the rapid degradation that occurred in a previous study. The goal of a biodegradable implant is to maintain the balance between implant degradation and implant strength to ensure fixation stability.
The shortcoming and limitation of this study is the lack of parameters to measure the biomechanical properties of the implants. We also did not analyze the surface characteristics of the implant and scanning electron microscope imaging of the implant after removal. Further studies should be conducted to quantify the biomechanical properties and feasibility of biodegradable implants for the treatment of fractures.
Methods
Animals
We conducted an animal study with an in vivo experimental post-test only using a control group. This study is reported in accordance with ARRIVE guidelines. Using the Federer formula, we included 30 pure breeds of male SD rats that were raised and certified by the Animal Hospital, Bogor University, Indonesia. The inclusion criteria were as follows: 12–16 weeks of age, weight 250–350 g, male sex, no abnormalities in the lower extremities, and no congenital abnormalities. The exclusion criteria were as follows: subject’s death and surgical wound infection. The rats were divided into five groups, with six rats randomly allocated to each group and treated with different types of implants, including titanium implants (placebo), pure magnesium implants, 5% carbonate apatite magnesium alloy (Mg5CA), 10% carbonate apatite magnesium alloy (Mg10CA), and 15% carbonate apatite magnesium alloy (Mg15CA). The rats and implants were given random numbers, and the rats were given the corresponding implant. The caregivers, radiologists, and pathologists were blinded to the knowledge of the intervention received from each rat during the experiment, and only the lead researcher was aware of the allocation of the groups. The animals were housed randomly during the experiment. The animals were acclimatized for seven days in the animal laboratory. The animals were raised and maintained in 23 C conditions with 12 h light and dark cycles, and provided with food and water as needed (ad libitum).
After the acclimatization period, the alloy implants were surgically implanted into the rats’ left femur under ketamine-HCl (80 mg/kg) and xylazine (10 mg/kg) anesthesia. The lateral approach was performed by making a longitudinal skin incision along the anterolateral side of the femur. The incision was deepened until the lateral retinaculum was visible. The vastus lateralis and biceps femoris muscles were separated along the direction of their fibers to expose the femoral diaphysis. In each rat, a mini-plate was inserted into the left femoral diaphysis region and fixed using a multifilament vycril suture. Postoperatively, the rats were administered the oral analgesic paracetamol at a dose of 100 mg/kg BW and ampicillin antibiotic at a dose of 100 mg/kg BW for 7 days post-surgery.
The rats were observed daily for four weeks to monitor the condition of the wounds and signs of inflammation or infection. Anteroposterior (AP) and lateral radiographs were obtained from all the rats immediately after surgery to confirm the implant position. The animals were sacrificed during the fourth week by an intravenous injection of phenobarbital (10 mg/kg). This study was conducted between July 2021 and December 2021 and was approved by the Ethics Committee of the Faculty of Medicine, University of Indonesia, and Bogor Agricultural University based on the ethical approval letter number: Ethical Approval/IPB/195–2021. All procedures were performed in accordance with the relevant guidelines and regulations, as stipulated by the approved institution.
Implant preparation
MgCA implants were produced by combining commercial magnesium powder and carbonate apatite powder (varied 5, 10, and 15% wt CA) using a planetary ball mill for 5 h at 200 revolutions per minute. The mixed powder was then compacted using a die with a diameter of 1 cm by a hydraulic press with a load of 350 MPa at 350 °C in a muffle furnace. (22) After forming a cylindrical block, the sample is cut using a diamond cutter to create a mini-plate measuring 6 mm × 3 mm × 1 mm with two screw holes. The implant was prepared by the Faculty of Engineering, University of Indonesia.
Implant degradation rate
The weight of the implant was measured before implantation and after the implant was obtained from sacrificed rats. The MgCA alloy was measured in grams and compared before and after implantation to quantify the implant degeneration. The rate of degradation was also measured between the groups.
Radiographic examination
X-ray examination of the left femur was conducted 2 times for each rat, once immediately after surgery to ascertain the positioning of the implant. Additional X-rays were obtained after surgery (on days 15 and 30) to observe the degradation process and detect gas formation in the cavities.
Hematologic examination
Blood samples were collected and analyzed before and after the operation to determine the concentration of serum magnesium and calcium ions and to observe any systemic inflammatory reactions.
Histopathological examination
Histological analysis of the bone samples from each rat was conducted at the Animal Hospital Laboratory, Bogor Agricultural University, Indonesia. The bone sample was decalcified using nitric acid (HNO3 10%), cut into transverse sections with a thickness of 4–5 μm, and stained with Hematoxylin and Eosin (HE). Visceral organ tissue was also stained with HE. The bone samples were examined using light microscopy. A semiqualitative scoring for local reaction was created based on the level of neovascularization, level of fibrosis/fibroblast infiltration, and number of osteoblasts and osteoclasts. The scoring criteria were + 0 (none/minimal), + 1 (mild), + 2 (moderate), + 3 (severe), and + 4 (very severe). (Fig. 3) The scoring was validated and performed by two pathologists in a blinded manner. Validation of the scoring system for each parameter was performed by measuring interobserver agreement using the κ value, calculated as the score agreement between observers (observer agreement) divided by 100. (23) The resulting scoring had κ values ranging from 0.84 to 0.93. Histopathological analysis of the liver, intestines, kidneys, and spleen was also performed to monitor systemic toxicity at various time points after implantation.
Fig. 3.
Local Histopathological Examination
Statistical analysis
Statistical analysis was conducted using SPSS version 20 for Windows. The normality of the data was measured using the Shapiro-Wilk test. Repeated or one-way ANOVA will be conducted according to the normally distributed data, followed by Bonferroni or Tukey post hoc analysis. Nonparametric test will be conducted using Friedman or Kruskall Wallis analysis followed by Mann Whitney or Wilcoxon test as a post hoc analysis.
Acknowledgements
We would like to express our gratitude to all the individuals and organizations that have contributed to the publication of this research paper.We would like to thank our attending veterinarian, Arni Diana Fitri, VMD and her team for invaluable support throughout the research process and keeping the subject healthy. We are also grateful to Prima Rizky Oktari, MD , Ilham Suryo, MD and Randhi Maulana, MD for the help through the data collecting process and their valuable insights.
Author contributions
A.K., E.D., and A.R. conceived the concept and visualization, and also reviewed the manuscript. A.K., E.D., S.S., and A.R. conceived the methodology and E.D., S.S., I.S., and V.J. catered project administration and organized the resources and A.K., E.D., S.S., I.S., V.J., A.R. conducted the experiment(s), analyzed the results and conceived draft preparation and A.K. and A.R. undertook supervision.
Data availability
All data generated or analysed during this study are included in this published article [and its supplementary information files] titled ORTHOREVFINAL.sav and Data compile biodegradable.xlsx.
Declarations
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
All data generated or analysed during this study are included in this published article [and its supplementary information files] titled ORTHOREVFINAL.sav and Data compile biodegradable.xlsx.