A stable repaired fracture is the key factor responsible for the recovery of a damaged bone. The iron-based implant is one of the biodegradable metals that have been proven safe as a fracture fixation device. The objective of our experimental approach was to examine the potential of the iron-based implant as a biodegradable metal in tibia shaft fracture in sheep chronically. The samples used for this experiment were iron-based and stainless steel implants. Each had a diameter of 5 mm. These samples were analyzed through 3 phases which are material characterization, in vitro and in vivo examination. The samples were examined using a scanning electron microscope with energy dispersive spectrometer and X-ray diffraction. Based on the analysis carried out, the samples contained 90,02% and 60,81% Fe for iron-based implant and stainless implant, respectively. Both implants maintained high viability when being in contact with calf pulmonary artery endothelial cells, indicating that both implants had a minimum response to the cell in a hemocytometer and methyl tetrazolium (MTT) assay. The systemic effect of the implants was observed using hematology and blood chemistry examination. Data collection also shows that both implants also had a minimum response to the erythrocytes, leucocytes, blood chemistry, and blood mineral during the period of observation. Therefore, it could be concluded that the iron-based implant is tolerable for a period of 9 months. It also has the potential to be used as a biodegradable orthopedic implant.
The main factor that influenced the bone healing is a stable fracture repaired. While the structure of the bone healing is unstable, the intramedullary nail has long been used as a fracture fixation device for tibia shaft fracture. The intramedullary nail is still being used in the veterinary field as needed [
Nondegradable metals have been used for decades as orthopedic implants for their high strength, ductility, tenacity, hardness, fracture toughness, corrosion resistance, formability, and biocompatibility [
Iron is a normal component of heme and nonheme enzymes and proteins [
Numerous orthopedic research has been conducted using sheep as an animal model due to the physical stature and weight [
Iron-based implant (BjPT 6, Tunggal Jaya Steel®, Indonesia) and stainless steel implant (Steinmann Pin, Sklar®, USA) with 5 mm diameter were cut to 1 mm length for material characterization and in vitro examination and were adapted to the tibia bone length size of sheep for in vivo examination. All the implants were sterilized using dry autoclave 121°C for 6 hours and UV ray for 1 hour before being used in this study.
Surface morphology and metal composition of the implants were observed using a scanning electron microscope (SEM) with an energy dispersive spectrometer (JSM-6510LA, JEOL®) to observe the microstructure of the implants. Iron-based and stainless steel implants were polished using sandpaper until grid # 2000 then etched by using nital (Nital Etch 2%, USA). SEM analysis was taken at an accelerating energy rate of 20 keV. X-ray diffraction with CuK
The viability of the cell was examined using hemocytometer and MTT assay. Haemocytometer was used to observe the interference of the implant directly to the cell, and the MTT assay was used to observe the interference of the degradation solution of the implant to the cell. For direct method, the calf pulmonary artery endothelial cell was cultured 10.000 cells per plate in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) and incubated (Binder, Germany) for 24 hours in 5% CO2 and 37°C. Thereafter, an implant with 1 mm of thickness and 5 mm of diameter was put on the surface of the cells and incubated for 76 hours in 5% CO2 and 37°C. Half of the media culture was removed, then the rest of it was poured into a 15 ml centrifuge tube (Corning, USA) and centrifuged using flexpin bench-top centrifuge (Tomy, Japan) 500 g for 5 min. The supernatant was removed and the cells were counted using hemocytometer. For the indirect method, all of the implants with 3 mm of thickness and 5 mm of diameter were immersed in low glucose DMEM for 7 and 14 days. One hundred
All of the procedures were approved by animal care and use committee of Veterinary Teaching Hospital of Bogor Agricultural University (ACUC RSHP FKH IPB) number 12-2015 RSHP FKH-IPB. Eight male sheep were divided into 2 groups of iron-based and stainless steel implant. Implant with 5 mm of diameter and a length that adapted to the length of tibia bone of the sheep was inserted into the left tibia bone intramedullary.
Before implantation, all of the sheep were injected with an antitetanus serum (Biostat 1.5®, Biopharma, Indonesia). The implants were implanted under general anesthesia. Anesthesia was induced using ketamine and xylazine combination intravenously (3 mg/kg BB Ketamin®, Kepro BV, Netherland and 0.1 mg/kg BB, Xyla®, Interchemie, Holland) and maintained with ketamine intravenously (3 mg/kg BB). Prior to surgery, the surgical site was shaved and iodine solution was applied. A longitudinal skin incision was made to access the lateral diaphysis of the tibia. A middiaphysis tibia osteotomy was performed, stabilizing with the intramedullary bone implant. The wound was closed in three layers, i.e., the fascial, subcutaneous, and skin layers separately. General antibiotics such as gentamycin (3 mg/kg BB, Bio-genta®, Biopharchemie, Vietnam) and amoxicillin long-acting (20 mg/kg BB, Intramox-150 LA®, Interchemie, Holland) were administered intramuscularly to prevent postsurgery secondary infection for 1 week. Tramadol (2 mg/kg BB, Tramadol®, PT Indofarma, Indonesia) was also administered intravenously as an analgesic for 2 weeks. Blood sampling was performed every month until month 9 after implantation on the jugular vein to analyze the systemic effect of the implants.
Data were analyzed using analysis of variance (ANOVA) and post hoc DUNCAN with the P value of P ≤ 0.05 as statistically significant.
Figure
Surface scanning electron microscopy micrographs of sample (a) overview of iron-based implant, (b) overview of stainless steel implant (1000x mag), (c) EDS spectra of iron-based, and (d) EDS spectra of stainless steel, (e) X-ray diffraction of iron-based, and (f) X-ray diffraction of stainless steel implant.
Calf pulmonary artery endothelial cell viability of iron-based and stainless steel implant is seen in Figure
Calf pulmonary artery endothelial cell viability of iron-based and stainless steel implant using (a) haemocytometer and (b) MTT Assay.
Figure
Mineral concentration of iron-based implant in tibial shaft fracture during 9 months’ observation time after implantation in sheep (a) calcium, (b) phosphorus, and (c) iron concentration. The same superscript letters (A, B) in same group showed no significant differences during observation time,
Normal iron serum in sheep is varied according to the different authors. The iron serum in this study was still in the normal range [
Erythrocyte profile and thrombocyte of iron-based implant compared to stainless steel implant could be seen in Table
Erythrocyte profile and thrombocyte of iron-based implant in tibia shaft fracture in month 0 until month 9 after implantation in sheep.
Mo(s) | Groups | Haemoglobin (g/dL) | Erythrocyte | Haematocrit | Thrombocyte |
---|---|---|---|---|---|
0 | SS | 9.75±0.5 | 3.43±0.10 | 30±1.4 | 201.5±91.27 |
Fe | 10.42 ±1.02 | 3.48±0.21 | 30.75±2.63 | 124.25±30.42 | |
1 | SS | 10.95±2.72 | 3.73±0.88 | 33.25±7.93 | 208.75±57.89 |
Fe | 9.53±1.3 | 3.33±0.46 | 29±4.55 | 132.5±35.27 | |
2 | SS | 11±2.27 | 3.63±0.77 | 33.25±6.9 | 151.75±80.85 |
Fe | 11.77±3.04 | 3.98±0.99 | 36±8.91 | 144.25±56.51 | |
3 | SS | 10.47±1.05 | 3.5±0.37 | 31.75±2.99 | 134.75±43.81 |
Fe | 11±0.67 | 3.57±0.29 | 33±2 | 113.5±9,11 | |
4 | SS | 9.53±0.43 | 3.13±0.13 | 28.5±1.73 | 130.5±61.2 |
Fe | 10.33±0.39 | 3.43±0.13 | 31±1.41 | 107.75±6.9 | |
5 | SS | 10.55±1.03 | 3.53±0.32 | 32.25±2.63 | 112.75±14.75 |
Fe | 10.76±1.4 | 3.58±0.46 | 32.25±4.11 | 148.25±42.28 | |
6 | SS | 10.98±1.19 | 3.65±0.37 | 33±3.37 | 119.5±15.44 |
Fe | 9.98±0.19 | 3.35±0.1 | 29.75±0.5 | 159.5±42.66 | |
7 | SS | 10.25±0.87 | 3.4±0.29 | 31.25±3.3 | 193±122.52 |
Fe | 11.2±1.22 | 3.7±0.44 | 33.33±3.79 | 134.33±32.56 | |
8 | SS | 10.65±0.83 | 3.53±0.25 | 33±2.45 | 168.5±118.49 |
Fe | 11±0.4 | 3.67±0.15 | 33±1 | 145.67±32.35 | |
9 | SS | 10.58±0.71 | 3.5±0.26 | 31.75±2. 22 | 115.75±6.99 |
Fe | 9.67±1.07 | 3.33±0.31 | 29.66±3.06 | 141.67±44.96 |
Description: data is presented in the average ± standard deviation. The same superscript letters (a,b,c) in different rows but in the same group showed no significant differences (P>0.05). The same superscript letters (x) in different rows and the different group showed no significant differences (P>0.05). Mo: month, SS: stainless steel group, Fe: iron-based group.
At month 4 the number of erythrocytes in the stainless steel group was the lowest from the record gathered and it was significantly different with the iron-based (Table
Differential leucocytes of iron-based implant in tibia shaft fracture in month 0 until 9 after implantation in sheep.
Mo | Group | Eosinophil (%) | Neutrophil band (%) | Neutrophil Segmented (%) | Lymphocyte (%) | Monocyte (%) | Basophil (%) |
---|---|---|---|---|---|---|---|
0 | SS | 0.25±0. | 2±1.4 | 70.25±8.1 | 22.5±6.76 | 4±1.8 | 0±0 |
Fe | 0.75±0.9 | 1.25±1. | 73±10.1 | 22.25±10.05 | 2.75±0.96 | 0±0 | |
1 | SS | 1±0.8 | 2.25±1.7 | 66±13.3 | 28±11.23 | 2.75±1.5 | 0±0 |
Fe | 1±0.8 | 1±1.15 | 62.5±10.2 | 32.25±8.96 | 3.5±0.58 | 0±0 | |
2 | SS | 0.25±0. | 1.25±1.2 | 73.25±3.9 | 21.75±4.35 | 3.5±1.91 | 0±0 |
Fe | 0.5± | 1±1.1 | 75.75±3.3 | 20±2.94 | 2.75±2.22 | 0±0 | |
3 | SS | 0.75±0.9 | 1.75±1.2 | 71± | 24.5±3.70 | 2.5±1.29 | 0±0 |
Fe | 0.5± | 1±1.1 | 62.5±9.9 | 32.75±9.11 | 3.25±2.5 | 0±0 | |
4 | SS | 0± | 0.75±1. | 71.25±5.6 | 25.5±4.20 | 2.5±1 | 0±0 |
Fe | 0.5±0.5 | 1.25±1. | 67.75±5.8 | 28±4.97 | 2.5±0.58 | 0±0 | |
5 | SS | 0.5± | 0.75±0.9 | 73.25±4.6 | 18.5±11.39 | 2±2.83 | 0±0 |
Fe | 0.25±0. | 1.75±1.2 | 69±3.56 | 26±2.94 | 3±1.15 | 0±0 | |
6 | SS | 0.75±0.9 | 1.25±0.9 | 63.75±7.4 | 30±4.97 | 3±0.82 | 0±0 |
Fe | 0.25±0. | 1±1.1 | 64.25±6.1 | 34.25±2.36 | 2.75±1.71 | 0±0 | |
7 | SS | 0.75±0.9 | 1.75±1.2 | 67.25±12.7 | 27.75±10.9 | 2.5±1.29 | 0±0 |
Fe | 1± | 1.33±1.1 | 67.33±12,3 | 27±12.12 | 3.33±1.53 | 0±0 | |
8 | SS | 1.5±0.5 | 2.25±0. | 62±5.4 | 32.5±5.97 | 1.75±0.5 | 0±0 |
Fe | 0.67±0.5 | 2± | 71±1.7 | 23±1 | 3.33±1.15 | 0±0 | |
9 | SS | 0.25±0. | 1±1.1 | 67.75±5.4 | 28.5±3.87 | 2.5±1.73 | 0±0 |
Fe | 0.67±1.1 | 2.33±0.5 | 73±2.65 | 23.67±1.53 | 2.33±0.58 | 0±0 |
Description: data is presented in the average ± standard deviation. The same superscript letters (a,b,c) in different rows but in the same group showed no significant differences (P>0.05). The same superscript letters (x) in different rows and the different group showed no significant differences (P>0.05). Mo: month.
The patterns of erythrocyte, hematocrit, and hemoglobin are alike. An insignificant increase of the patterns indicates light erythropoiesis increases in response to blood loss and inflammation. The increase is caused by the implantation procedures in the early postimplantation, implant-bone tissue interaction, and implant-bone marrow interaction [
Thrombocytes, also known as platelets, are the smallest cells of the blood which play a key role in blood clotting and stimulating bone tissue regeneration [
Leucocytes, also known as white blood cells, defend the body against disease-causing organisms, toxins, and irritants. Figure
The number of leucocytes in tibial shaft fracture during 9 months of observation time after implantation in sheep.
An inflammatory response of iron-based implant to the body could be observed using leucocyte differentiation analysis which is expressed in Table
Neutrophils, the first cells recruited to sites of inflammations [
Bone fracture, as well as the interruption of blood supply and platelet aggregation, induces the release of platelet-derived proinflammatory cytokines, interleukin-6 (IL-6), IL-1, IL-2, and tumor necrosis factor-alpha (TNF-
Monocyte can differentiate several subtypes of macrophage depending on environmental circumstances. Fractured bone releases the monocyte chemoattractant-1 (MCP-1) [
Blood chemistry examination including serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, blood urea nitrogen, and creatinine could be seen in Table
Blood chemistry results of iron-based implant in tibia shaft fracture in month 0 until 9 after implantation in sheep.
Mo(s) | Groups | SGPT | SGOT | BUN | Creatinine |
---|---|---|---|---|---|
0 | SS | 30.25±14.93 | 25±13.4 | 39.75±2.36 | 1.1±0.48 |
Fe | 23.5±12.50 | 31±7.87 | 25±10.39 | 0.7±0.22 | |
1 | SS | 27.25±10.78 | 27.25±10.18 | 28.25±5.12 | 0.95±0.26 |
Fe | 35±6.21 | 32.75±3.77 | 2925±6.02 | 0.65±0.19 | |
2 | SS | 39.5±5.20 | 28±4.24 | 36±3.65 | 1.05±0.3 |
Fe | 33±8.49 | 29.75±10.97 | 30±7.26 | 0.45±0.21 | |
3 | SS | 26.25±8.5 | 30±12.94 | 23.25±5.67 | 0.53±0.15 |
Fe | 29±9.27 | 36.75±2.87 | 28.75±8.18 | 0.75±0.44 | |
4 | SS | 31.75±12.26 | 25±8.49 | 33,75±4.35 | 1.18±0.3 |
Fe | 32±6.53 | 27.75±4.86 | 23.5±8.27 | 0.6±0.22 | |
5 | SS | 36±6.06 | 28.75±11.41 | 28.5±2.08 | 0.6±0.18 |
Fe | 26.5±12.29 | 36.25±10.63 | 27.75±6.55 | 0.6±0.18 | |
6 | SS | 30±16.35 | 30.5±7.14 | 21.75±9.46 | 0.6±0.24 |
Fe | 33.75±7.27 | 29.25±9.98 | 24.25±4.79 | 0.68±0.22 | |
7 | SS | 29±3.46 | 32.25±3.20 | 25±6.78 | 0.75±0.33 |
Fe | 33.67±8.74 | 29±11.53 | 22±8.19 | 0.57±0.15 | |
8 | SS | 25.25±5.32 | 31.25±9.11 | 23±8.04 | 0.88±0.40 |
Fe | 31.67±14.57 | 36.67±11.15 | 36±2 | 0.7±0.2 | |
9 | SS | 24.75±6.75 | 30.5±11.79 | 29±3.16 | 0.6±0.16 |
Fe | 34±10.58 | 27.67±11.72 | 32±9.16 | 0.87±0.31 |
Description: the data is presented in the average ± standard deviation. The same superscript letters (a,b,c) in different rows but in the same group showed no significant differences (P>0.05). The same superscript letters (x) in different rows and the different group showed no significant differences (P>0.05). Mo: month, SS: stainless steel, Fe: iron-based implant, SGPT: serum glutamic pyruvic transaminase, SGOT: serum glutamic oxaloacetic transaminase, BUN: blood urea nitrogen.
Majority of metabolic products are excreted via the kidney. Most of the iron in circulation is bound to transferrin, which in the kidney can be taken up via transferrin receptor 1 [
Interestingly, there are contradictory results regarding the creatinine index in this study. Creatinine has no useful function and is eliminated by renal glomerular filtration and to a small extent by renal tubular secretion [
In this study, the iron-based implant had maintained high cell viability and had induced minimum erythrocyte profile, leucocyte number, leucocyte differential count, and also blood chemistry responses. Thus, it can be concluded that iron-based implant has potential as an intramedullary nail in tibia shaft fracture of sheep.
The data used to support the findings of this study are included within the article.
The authors declared no conflicts of interest.
The research was funded by the Indonesia Ministry of Research, Technology and Higher Education in international research collaboration and scientific publication.