Met. Mater. Int., Vol. 18, No. 2 (2012), pp. 243~247 doi: 10.1007/s12540-012-2007-5 Published 27 April 2012
Bone Formation Within the Vicinity of Biodegradable Magnesium Alloy Implant in a Rat Femur Model 1
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Hyung-Seop Han , Young-Yul Kim , Yu-Chan Kim , Sung-Youn Cho , Pil-Ryung Cha , Hyun-Kwang Seok3, *, and Seok-JoYang1,* 1
Department of Mechatronics Engineering, Chungnam National University, Daejeon 305-764, Korea 2 Department of Orthopedic Surgery, The Catholic University, Daejeon 520-2, Korea 3 Biomedical Research Institute, Korea Institute of Science & Technology, Seoul 136-650, Korea 4 Department of Mechanical Engineering, Korea University, Seoul 136-713, Korea 5 School of Advanced Materials Engineering, Kookmin University, Seoul 136-702, Korea (received date: 3 May 2011 / accepted date: 17 July 2011) The purposes of this preliminary study were to investigate the effect of increased Ca contents (5-10 wt% Ca) in Mg-Ca alloy on the mechanical properties and osseous healing rate in a standard rat defect model. Mechanical tests were performed using a compression system followed by qualitative histological analysis using the hemotoxylin and eosin (H&E) staining method and quantitative reverse transcriptase polymerase chain reaction (reverse transcriptase PCR). Mg-Ca alloy degraded fast in vivo while displaying a high level of the bone formation markersOC and ALP. Favorablemechanical strength properties were displayed as Ca content increased from 5 wt% to 10 wt% to show its potential to be considered as a load bearing implant material. The resultfrom this study suggests that the developed Mg-Ca alloy has the potential to serve as a biocompatible load bearing implant material that is degradable and possibly osteoconductive. Key words: biomaterials, casting, mechanical properties, bone, compression test
1. INTRODUCTION Magnesium is one of the most common compositionalelements in the human body. It is a lightweight, biocompatible metalwith mechanical properties such as its elastic modulus and compressive yield strength that are much closer to those ofnatural bone than other commonly used metallic implant materials [1]. Magnesium has a unique characteristic of dissolving readily in an aqueous solution that contains chloride ions [2]. It has been reported that magnesium accelerates the bone formation rate and forms a soluble and non-toxic oxide in body fluids that is harmlessly excreted with the urine [3,4]. However, the mechanical properties of pure magnesium are not sufficient for it to be considered as an implant material. The degradationprocess in a physiological system causes further loss of mechanical integrity before the hosting tissue can sufficiently heal, and ultimately results in implant failure [1,5-7]. Due to these unique characteristics, there has been a significant increase in research forthe development of magnesium-based alloys into a new biodegradable orthopedic material [8-22]. One method to improve the mechanical *Corresponding author:
[email protected] and
[email protected] ©KIM and Springer
propertiesand degradation rate is alloying the pure magnesium. In this study, calcium was selected as an alloying element due to its abundance in the human body, especially in 3 bones. Its low density (1.55 g/cm ) ensuresanalloy system with similar density to bone [23]. A beneficialbone healing effect could be achieved by the co-release of magnesium and calcium through degradation since magnesium plays an important role in bone metabolism [2]. Nie et al. suggested that the Mg-Ca system has a good potential in the sense that it has a useful combination of strength, creep resistance, castability, corrosion resistance, and cost because the equilibrium intermetallic phase (Mg2Ca) is structurally analogous with the magnesium matrix. The Mg2Ca phase has a melting point of 715 °C and is relatively stable at elevated temperatures. Its hexagonal crystal structure, which is similar to that of the magnesium matrix phase, has the potential for good lattice matching. Consequently, microstructures containing thermally stable, coherent precipitates are uniformly distributed within the matrix phase, providing enhanced mechanical properties for the Mg-Ca alloy [24]. Li et al. reported minor improvements in the mechanical properties ofbinary Mg-Ca alloy with low Ca contents (1-3 wt% Ca) [2]. However, they have failed to produce an alloy with higher Ca contents (5-10 wt% Ca) to show the broad alloying effect
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of Cacontent in binary Mg-Ca alloy. The purposes of this preliminary study were to investigate the effect of increased Ca contents (5-10 wt% Ca) in Mg-Ca alloy and to compare the mechanical propertiesand osseous healing rates of the Ca-enhanced alloy in a standard rat defect model.
2. EXPERIMENTAL PROCEDURES The binary Mg-Ca alloys were prepared throughmelting and casting under an Argon (Ar) atmosphere inside avacuum and inert gas atmosphere furnace. The oxygen and moisture −3 in the furnace were removed using a vacuum to 10 torr before the Ar gas was released into the furnace. The STS430 crucible filled with high purity Mg (99.98%) and Ca (99.95%) was kept at 850 °C for 120 min in order to melt the alloy completely. The molten alloy was then castinto a STS430 steel mold after stirring for 5 minutes. Casting samples were machined to a cylindrical shape of Ø3×6 mm and tested using −3 −1 a compression system with a strain rate of 0.6×10 s . Four samples for each condition were tested. Adult male Sprague Dawleyrats weighing approximately 400 g were used (Orient Bio, Gyunggi, South Korea). This study was approved by the Animal Care and Use Committee of Chungnam National University. Rats were anesthetized with 0.8 ml ketamine with 0.01 ml lumpun. Skin preparation was done and a non-contaminateddrapewas applied to prevent bacterial contamination. A skinincision was made at the lateral aspect of the knee and the patella bone was medially retracted exposing the distal femoral condyle. An osteochondraldefect, 2 mm in diameter and 6 mm to 7 mm in the depth, was made through the articular surface into the subchondral bone of the distal femoral condyle of the rats using an electrical burr as illustrated in Fig. 1. The defectwas filled with cylindrical rodsof Mg10 wt%Ca alloy, pure magnesium, AZ91,orautograft,alternatively, for each rat bone harvested
from the posterior iliac bone of the specimens. The ratswere sacrificed andtissue response was examined at 1, 2, 4, 8, and 12 weeks after the surgery. A qualitative histological analysis was carried out using the hemotoxylin and eosin (H&E) staining method,and samples were acquired from the implanted material (2 mm of the outer surface was measured from the center of the original margin) for detecting the cell osteogenesis potential. Aquantitative real time polymerase chain reaction (reverse transcriptase PCR) was performed. 2.1. H&E Staining Femoral condylesfilled with implanted specimens were harvested and fixed in 80 % alcohol. The hardtissue sectioning technique was used to preserve metallic specimens left inside the condylar bony tissue.Sample tissues were fixed with alcohol to determine the transient zone between alloy and bone tissue. A dehydrationprocedure followed with 80 % alcohol,and theosteo-bed resins were changed twice every 48 hours. Benzoyl peroxide was added and changesof the resin were made 3 times. Embedded samples were trimmed andgroundwitha Technovit 4000. An EXAKT BS-3000N was then used to produce sections. The thicknessof the sectioned samples was carefully inspected before staining with hematoxylin and eosin. 2.2. Reverse Transcriptase PCR The same margin from the center of the sample (2 mm) was checked using optical microscopy to accommodate the fact that the magnesium alloy’s margin was changed to bone-like tissue. For reverse transcriptase PCR analysis, the total RNA was extracted using the Trizol (Invitrogen. Carlsbad, CA.) reagent according to the manufacturer’s instructions. Single-stranded complementary DNA (cDNA) was prepared from 1 µg of total RNA, 8 µl of 5 X RT buffer (250 mMTris-HCl, pH 8.3, 250 mMKCl, 50 mM MgCl2, 2.5 mM spermidine, 50 mM DTT), 4 µl of 2.5 mMdNTP, 1 µl of oligo-dT (100 pmol/µl), 2 µl of Rnase inhibitor (4 nit/µl), and 2 µl of AMV reverse transcriptase (5 unit/µl). The sequences of each primer set used are given in Table 1. Established marker genes for bone formation, alkaline phosphatase (ALP) and osteocalcin (OC) were used. PCR was carried out in a 20 µl mixture containing 5 µl of 10 X buffer (100 mMTris-HCl, pH 8.3, 400 mMKCl, 15 mM MgCl2, 10 mM DTT), 5 µl of 2.5 mMdNTP, 5 µl of each primer (10 pmoles/µl), 2 µl of Taq polymerase (0.5 unit/µl), and 26 µl of DEPC-treated water. Table 1. Primer and probe sequence in RT-PCR reactions Primer Osteocalcin (199bp)
Fig. 1. Illustration of osteochondral defects on femoral condyle of adult Sprague Dawleyratfilled with cylindrical rods.
ALP (200bp)
Primer sequences Forward 5’–AGCTCAACCCCAATTGTGAC-3’ Reverse 5’–AGCTGTGCCGTCCATACTTT-3’ Forward 5’–GAGCAGGAACAGAAGTTTGC-3’ Reverse 5’–GTTGCAGGGTCTGGAGAGTA-3’
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Reverse transcriptase PCR was performed in a thermal cycler (Mastercycler gradient, Eppendorf) under the following conditions: denaturation for 3 minutes at 94 °C followed by 30 cycles of denaturation for 30 seconds at 94 °C, primer annealing for 30 seconds at 57 °C, and extension for 1 minute at 72 °C. PCR products were analyzed and confirmed using 2 % agarose gel electrophoresis.
3. RESULTS AND DISCUSSION The previousstudy done by Li et al. showed that the addition of Ca increased the mechanical propertiesof as-cast MgCaalloy when compared to pure Mg [2]. However, their result showed atendency toward decrease in the mechanicalproperties as Cacontent increased from 1 wt% to 3 wt% in Mg-Ca alloys. This was not the case for thecurrent study. Figure 2 shows the yield strength (YS) and ultimate compression strength (UCS) of as-cast Mg-xwt%Ca (x=0, 5, 10) alloys. YS increased significantly from 15 MPa to 165 MPa with the increase of Ca contents. UCS followed a similar pattern by increasing from 145 MPa to 254 MPa with the increase in Ca contents. Results show the Mg-Ca alloy displayed optimal mechanical properties compared to pure magnesium when Ca content increasedfrom 5 wt% Ca to 10 wt% Ca. An animalstudy using SD rats was carried out in order to determine the effect of increased Cacontents in Mg-Ca alloyon osseous healing rate. All the animals survived the surgical procedure without any complications. Furthermore, there were no infections or wound healing problems following the surgery. As shown in Fig. 3, the animal group implanted with pure Mg did not show any immediate degradation of implanted material at the first and second postoperative week. There was a small change in size of the implanted material starting th at the 4 postoperative week. The marginal absorption surface between metal and bone appeared to be more constant and regular than the Mgalloy surface, as shown in Figs. 4 and 5. A noticeablechange in the size and shape of the
Fig. 3. Histological images (40x magnification) of as-cast pure magnesium by using H&E staining method after (a) 2 weeks, (b) 4 weeks, (c) 8 weeks, and (d) 12 weeks.
Fig. 4. Histological images (40x magnification)of as-cast Mg-10 wt%Ca alloy by using H&E staining method after (a) 2 weeks, (b) 4 weeks, (c) 8 weeks, and (d) 12 weeks. th
Fig. 2. Yield strength and ultimate compressive strength of as-cast pure magnesium, as-cast Mg-5wt%Ca alloy, and as-cast Mg-10 wt%Ca alloy.
implanted material was observedat the 8 postoperative week. Eventually, the implanted pure Mg material degraded completely and was replaced with new bone-like tissue at the 12th postoperative week. The introductionof Ca in the Mg-Ca alloy increased the degradation rate in a physiological system. As shown in Fig. 4, histologic analysis of samples from the animal group implanted with Mg alloy indicateddegradation of implanted material starting early in the first postoperative week. There was a significant change in the size of the implanted materialat the 4th postoperative week. A contact between the degrading Mg alloy material and newly formed tissue couldalso be observed at this point. Invasion by the surrounding bony tissues was found and irregular patterns between the contact surface of the Mg alloy and bone were observed. Thissug-
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Fig. 5. Histological images (40x magnification) of as-cast AZ91 by using H&E staining method after (a) 2 weeks, (b) 4 weeks, (c) 8 weeks, and (d) 12 weeks.
gests that a rapid absorption area wasbeing formed around the alloy area. The implanted Mg alloy material degradedeth ven furtherat the 8 postoperative week and became comth pletely replaced with new tissue at the 12 postoperative week. Newly formed tissues were not yet in the form of fully matured and mineralized cancellous bonecompared with surrounding mature bone. However, considering the fact that it was not a fibrous material, it could be replaced with bone tissue through the bone remodeling process over time. Histologic analysis of samples from the animal group implanted with AZ91 revealedthat the implanted material remained intact and did not degrade over time, as shown in Fig. 5. The shape of the material remained intact and there was a small change in size of the implanted material at the th 12 postoperative week. The resultfrom the RT-PCR for the Mg alloy showed that there wasa significant change in the level of ALP and OC starting at the 2nd postoperative week. As shown in Fig. 6, the Mg-Caalloy displayed a continuous manifestation of OC and ALP at the implantation site compared with the autograftbone. Although pure Mg displayed a high concentration of both OC and ALP at the early stage of implantation, the level of OC declined at the 4th postoperative week. This suggests that pure Mg did nothave continuous activation potential forosteoblast or bone formation. Normally, if a bone fracture injury occurs, the level of OC will increase when the osteoblasts differentiate into bone matrix formation after 4 weeks with mineralization. ALP is considered as the main marker for bone turn-over and usually shows a peak within 2 to 3 weeks in a normal bone healing process before it starts to continuously decrease over time [25,26]. However, the result from this study showeda continuous increase in the level of OC and ALP over the first 4-week period. This suggested continuous bone turn-over response will occur around the
Fig. 6. Reverse transcriptase PCR analysis of the bone-associated genes OC, ALP and DAPDH for autograft bone, Mg-10wt%Ca alloy, pure Mg, and AZ91. The intensity of each gene band in (A) was quantified and normalized as shown in (B).
Mg alloy and bone. Relatively good results were observed from AZ91.There are several previous studies reporting that the implantation of AZ91 alloy increased the newly formed boneand the accumulation of biological calcium phosphates around the corrosion layer [2]. AZ91 is one of the most commonly studied alloys in the field of Mg research due to its favorable mechanical propertiescompared to pure Mg. There are several animal studies suggesting absorption occurs without major complication in physiological settings. However, the constitutional elements of a magnesium-based alloy should be toxin-free to guarantee the safety of biodegradable orthopedic materials. Aluminum is well known as a neurotoxicant and its accumulation has been suggested to be an associated phenomenon in various neurological disorders such as dementia, senile dementia, and Alzheimer’s disease [2,27,28]. The Mg-Ca alloy degraded fastest in vivo, followed by pure magnesium and AZ91. High levels of the bone formation markers OC and ALP were observed from RT-PCR for Mg alloy throughout the entire 12 weeks of the testing period. The
Bone Formation Within the Vicinity of Biodegradable Magnesium Alloy Implant in a Rat Femur Model
resultfrom this study suggests that the fast degradation of Mg-Ca alloy leads to early and continuousrelease of calcium and magnesium to the physiological setting. This enrichment of Cacan ultimately lead to the formation of new bone material within the implantation site. Hydrogen evolution from the fast degradationremains a key problem to overcome in future works. The Mg-Ca alloy corroded too quickly in a physiological system, producing hydrogen gas in the degradation process at a rate that is too fast to be dealt with by the host tissue. Currently, there are studies being performed to overcome this problem by modifying fabrication methods and adding a third element to the alloy. Real-time RT-PCR will be utilized for more realistic comparison of OC and ALP mRNA in the next experiment.
4. CONCLUSIONS Optimal mechanical properties were displayed as Ca content increased to show its potential to be considered as a load bearing implant material. Qualitative histological analysis and quantitative reverse transcriptase polymerase chain reaction showed that the developed Mg-Caalloy degraded fastest in vivo while displaying a high level of the bone formation markersOC and ALP. The resultfrom this study suggests that the developed Mg-Ca alloy has the potential to serve as a biocompatible load bearing implant material that is degradable and possibly osteoconductive. The limitationof this study was that there were no standard or precise methods to continuously monitor the response. Due to the continuous absorption between the bone and alloy, it was impossible to obtain proteins from the exact margin. Proteins were obtained from hypothetically measured degraded margin based on the shape and size of the implanted cylinder. One could also point out the fact that there were no direct validations of fully matured bone tissue from this study. However, correlation could be drawn fromthe 12-weeks histological response result and bone modeling process, which indirectly suggestspossible osteoconductivity. This study also provides a new measurement method for the analysis of absorbable implants.
ACKNOWLEDGMENTS This study was supported by the KIST Project (2E21950, 2E22710), Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20100028287) and the Seoul R&BD program, Seoul Development Institute, Republic of Korea (SS100008).
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