Acta Biomaterialia 66 (2018) 109–117

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Acta Biomaterialia

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The influence of biodegradable magnesium implants on the growth plate

https://doi.org/10.1016/j.actbio.2017.11.0311742-7061/� 2017 Published by Elsevier Ltd on behalf of Acta Materialia Inc.

⇑ Corresponding author at: Medical University Graz, Auenbruggerplatz 34, 8036Graz, Austria.

E-mail address: tanja.kraus@medunigraz.at (T. Kraus).

Tanja Kraus a,⇑, Stefan Fischerauer b, Stefan Treichler c, Elisabeth Martinelli c, Johannes Eichler c,Anastasia Myrissa c, Silvia Zötsch d, Peter J. Uggowitzer e, Jörg F. Löffler e, Annelie M. Weinberg c

aDepartment of Paediatric Orthopaedics, Medical University Graz, 8036 Graz, AustriabDepartment of Trauma Surgery, Medical University Graz, 8036 Graz, AustriacDepartment of Orthopaedics, Medical University Graz, 8036 Graz, AustriadDepartment of Paediatric and Adolescent Surgery, Medical University Graz, 8036 Graz, Austriae Laboratory of Metal Physics and Technology, Department of Materials, ETH Zurich, 8093 Zurich, Switzerland

a r t i c l e i n f o

Article history:Received 30 July 2017Received in revised form 18 November 2017Accepted 20 November 2017Available online 23 November 2017

Keywords:MagnesiumBone fixationGrowth platemCTIn-vivo degradation

a b s t r a c t

Mg-based biodegradable materials are considered promising candidates in the paediatric field due totheir favourable mechanical and biological properties and their biodegrading potential that makes a sec-ond surgery for implant removal unnecessary. In many cases the surgical fixation technique requires acrossing of the growth plate by the implant in order to achieve an adequate fragment replacement orfracture stabilisation. This study investigates the kinetics of slowly and rapidly degrading Mg alloys ina transphyseal rat model, and also reports on their dynamics in the context of the physis and consecutivebone growth. Twenty-six male Sprague–Dawley rats received either a rapidly degrading (ZX50; n = 13) ora slowly degrading (WZ21; n = 13) Mg alloy, implanted transphyseal into the distal femur. The contralat-eral leg was drilled in the same manner and served as a direct sham specimen. Degradation behaviour,gas formation, and leg length were measured by continuous in vivo micro CT for up to 52 weeks, andadditional high-resolution mCT (HRS) scans and histomorphological analyses of the growth plate wereperformed. The growth plate was locally destroyed and bone growth was significantly diminished bythe fast degradation of ZX50 implants and the accompanying release of large amounts of hydrogengas. In contrast, WZ21 implants showed homogenous and moderate degradation performance, and theeffect on bone growth did not differ significantly from a single drill-hole defect.

Statement of Significance

This study is the first that reports on the effects of degrading magnesium implants on the growth plate ina living animal model. The results show that high evolution of hydrogen gas due to rapid Mg degradationcan damage the growth plate substantially. Slow degradation, however, such as seen for WZ21 alloys,does not affect the growth plate more than drilling alone, thus meeting one important prerequisite fordeployment in paediatric osteosynthesis.

� 2017 Published by Elsevier Ltd on behalf of Acta Materialia Inc.

1. Introduction

Growth plates – located close to the ends of long bones – areresponsible for longitudinal bone growth in immature bodies. Aslong as the growth plates are open, length growth occurs as resultof an enchondral ossification process that gradually converts carti-lage into bony tissue [1]. Any injury involving the growth plate canresult in growth disturbances such as length discrepancies or axial

deviations. Physicians must therefore monitor the immaturepatient carefully to ensure that growth continues in an appropriatefashion. In the context of paediatric trauma, 15 to 30% of paediatricinjuries to long bones involve the growth plate [2,3].

Metal wires or screws are frequently used for bone fixation inthe immature skeleton. They provide temporary mechanical sup-port during the healing of the injured tissue. Currently they aremade of stainless steel or titanium alloys, which require implantremoval when the bone has healed. This means repeated surgeryfor the patient, and is associated with increased morbidityand higher healthcare costs [4,5]. Therefore, research over thelast few decades has focused on the development of various

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biodegradable implants [6–12]. Such self-degrading devices rendersubsequent surgical intervention for implant removal unnecessary[13–15]. They are of crucial interest, particularly in paediatricorthopaedics, because even the procedure for implant removalcan cause an arrest in growth.

Here, magnesium (Mg)-based alloys seem to be promising can-didates for use in orthopaedic load-bearing applications [11,15,16].Because Mg is a natural element in the human body, it shows goodbiocompatibility with no systemic inflammatory reaction or effectson cellular blood composition. Its degradation products are alsoexpected to be nontoxic to the surrounding tissue [12,17,18]. Addi-tionally, the mechanical properties of Mg-based implants are ofadvantage, because of their high specific strength and a Young‘smodulus that is comparable to that of cancellous bone. Thus therisk of stress shielding in orthopaedic applications is greatlyreduced with Mg alloys [7,19].

However, specific properties must be present if biodegradableimplants are to be deployed as material for osteosynthesis in agrowing skeleton. Key issues include high levels of biocompatibil-ity, adequate stability during fracture healing, full regeneration ofthe bone structure, and moderate, homogeneous degradation per-formance in balance with the bone‘s healing process [15]. For usearound the growth plate it is also essential that the materialsthemselves do not affect the ongoing growth negatively or evenstop it. Nevertheless, information on magnesium‘s degradationbehaviour within the growth plate and its functional influenceare lacking. We therefore investigated the behaviour of the rapidlydegrading alloy ZX50 and the slowly degrading alloy WZ21, andtheir morphological and functional dynamics and effects on thegrowth plates in a living animal model.

2. Materials and methods

2.1. Implants

Two different types of turned cylindrical Mg pins (ZX50 andWZ21), each with a diameter of 1.6 mm and a length of 8 mm,were deployed in this study. ZX50 is alloyed with 5% Zn, 0.25%Ca, and 0.15% Mn (in wt%). The alloy exhibits excellent mechanicalproperties (see Table 1), with a yield strength of 210 MPa, an ulti-mate tensile strength of 295 MPa, a uniform elongation of 18%, andan elongation at fracture of 26%. Its microstructure is fine-grained,with a mean grain size of �4 mm. WZ21 is alloyed with 2% Y, 1% Zn,0.25% Ca, and 0.15% Mn (in wt%), and is known to degrade moder-ately. It also shows good mechanical properties, with a yieldstrength of 150 MPa, an ultimate tensile strength of 250 MPa, a

Table 1Measured chemical composition, main impurity content, mean grain size, tensileyield strength (TYS), ultimate tensile strength (UTS), elongation to fracture (Af), andmean in vitro degradation rate (estimated from hydrogen evolution data [11]) of thealloys ZX50 and WZ21.

ZX50 WZ21

Composition (wt%) Mg: BalanceZn: 5.21Ca: 0.29Mn: 0.16

Mg:BalanceY: 1.65Zn: 0.85Ca: 0.25Mn: 0.17

Typical main impurities (wt ppm) Si: 430Fe: 30Cu + Ni + Co: 20

Si: 45Fe: 25Cu + Ni + Co: <10

Mean grain size (lm) 4 7TYS (MPa) 210 150UTS (MPa) 295 250Af (%) 26 28Mean in vitro degradation rate in SBF

(mm/year)1.3 0.16

uniform elongation of 20%, and an elongation at fracture of 28%.Its mean grain size is approximately 7 mm. Table 1 summarizesthe mechanical properties and lists the actual chemical composi-tions, typical main impurities, and estimated mean in vitro degra-dation rates (in mm/year) for ZX50 and WZ21. More details onalloy processing and microstructure can be found in Refs. [11,15].All pins were dry-machined with clean tools. After machining theywere cleaned in a cascade of pure ethanol in an ultrasonic bath anddried in warm air.

2.2. Experimental design

Twenty-six immature male Sprague–Dawley rats (5 weeks ofage, 140–160 g body weight) were randomly assigned to twogroups (n = 13/group). Each rat received either a ZX50 or a WZ21implant, placed orthograde to the growth plate in its right femoralbone (Fig. 1). The contralateral femoral bones were also drilled intoand served as shams.

All rats underwent micro-focus computer tomography (mCT)evaluation at different time points over a total observation periodof 12 months. For the histological investigations two rats fromeach group were sacrificed at three different time points (4, 24,and 52 weeks). At 24 weeks, two additional rats (n = 1 withWZ21 and n = 1 with ZX50 implant) were sacrificed for HRS(high-resolution mCT). An overview of the experimental design isgiven in Table 2.

During the mCT investigations both lower extremities werescanned to examine the implant degradation process and the leglength, in order to determine possible leg length discrepancies.The animal study was conducted according to established guideli-nes on animal care and was authorized by the Austrian Ministry ofScience and Research (accreditation number BMWF-BMWF-66.010/070-II/3b/2011).

2.3. Surgical procedure

All surgical procedures were performed by the same surgeonand according to the previously described conditions (see Ref.[15]). The regio intercondylaris of all the femoral bones was exposedby an incision medial to the patellar ligament followed by lateral

Fig. 1. Schematic representation for the implantation of the Mg-based alloy anddrill-hole defect, respectively. (P = patella; Ip = Implant/drill hole; GP = growthplate; F = femur; T = tibia.)

Table 2Overview of experiments performed within the timeframe of 52 weeks.

Time 1w 4w 8w 12w 24w 36w 52w

mCTLength x x x x x x xDegradation x x x x x xDegradation (HRS) xHistology x x x

Fig. 2. Determination of the femoral length using mCT slices and Mimics� software.P1 was determined at the most proximal point at the trochanter major in all threedimensions. P2 was determined at the most distal point at the lateral femoralcondyle. Both points were connected via Mimics� and the distance was measured.(a) Femur with ZX50 implant; (b) femur with drill-hole defect at contralateral side.

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dislocation of the patella. A drill hole was created by a 1.6-mm drillwith ascending diameter (Synthes�, Paoli, PA) in the longitudinalaxis of the femur. In order to minimize frictional heat and thermalnecrosis, drilling was performed at a low rotational speed of 200rpm with continuous profuse physiological saline irrigation. Thecylindrical implant was inserted into the drill hole by gentle tap-ping, resulting in a uniform press fit. To prevent blocking of theknee during flexion by an excessively long implant, special carewas taken to position the implant appropriately with regard tothe intercondylar area. After implantation, the operating fieldwas rinsed with physiological saline solution and the wound wasclosed in layers. The contralateral side was prepared and drilledin the same way, but no pin implantation followed.

Postoperatively, all animals immediately received 200 mg kg�1

Caprofen (Rimadyl�, Pfizer Corporation, Vienna, Austria) subcuta-neously to ensure analgesia. All animals were allowed to movefreely in their cages with unrestricted weight bearing. During thefirst postoperative week analgesia was maintained by administer-ing 60 mg Piritramid (Dipidolor�, Janssen-Cilag GmbH, Neuss, Ger-many) in 40 ml 5% glucose added to 500 ml drinking water. Dailyclinical observation proceeded throughout the entire study period.

2.4. Preparation of the bone-pin model

The mCT investigation at week 24 was performed at high resolu-tion. Immediately after euthanasia a longitudinal skin incision wasmade medially in each rat’s femur. After transection of the mus-cles, the femur bone was exposed carefully and ex-articulated atthe adjacent joints. The area beneath the implant was leftuntouched and was covered by thin laminas of remaining tissuein order to retain the existing pin-bone conditions. After prepara-tion the bone was wrapped in physiological saline-solution-dipped swab material and transferred to the mCT.

2.5. Microfocus-computed tomography and image reconstruction

Micro-CT scans were performed under general anesthesia (see[15]) with a Siemens Inveon Acquisition Workplace 1.2.2.2 at 70kV voltage, 500 mA current, and 1000 ms exposure time at weeks1, 4, 8, 12, 24, 36 and 52 after implantation. The effective voxel sizewas 35.55 mm. Images were reconstructed using the image pro-cessing software Mimics� (Version 14.12, Materialise NV, Leuven,Belgium). Pins and bubbles of emitted gas were reconstructed as3D models with upper and lower thresholds for the pin at 226and 3071 Hounsfield units (HU), and for the gas at �1000 and�1024 HU.

To determine bone growth discrepancies we used each mCT scanto measure the length of both hind limbs (pin-implanted side andcontrol side). The trochanter major was marked in all three dimen-sions, serving as the proximal reference point (P1). Afterwards thelateral condyle of the distal femur was marked in all three dimen-sions and used as a distal reference point (P2) (Fig. 2). P1 and P2were connected by a line and the length between the points wasdetermined using Mimics�. To take the individual bone growthinto account we related all measurements to the animal’s con-tralateral bone length (sham bone).

2.6. High-resolution images at week 24 (HRS)

For HRS we used the Siemens Inveon Acquisition Workplace1.2.2.2 and set the scanning parameters to 70 kV voltage, 450 mAcurrent and 1000 ms exposure time. The scans were performedat high magnification with a binning setting of 2, which producedan effective voxel size of 19.17 mm. Using HRS we evaluated thepin-bone model, which included the distal femur epiphysis, thegrowth plate, the metaphyseal area and the distal part of the femurdiaphysis. Particular attention was paid to the influences ofimplant on growth plate and drill hole on growth plate.

2.7. Histology

The bones were cut to the appropriate size and region, placed inembedding cassettes, and fixed in Formalin for 48 h at �4 �C. Afterthis, they were dehydrated using sequential isopropanol gradesand xylene. Then the probes were processed using the Technovit9100 New (Heraeus Kulzer, Wehrheim, Germany) embeddingmethod as described by Willbold et al. [20]. After polymerisationthe bone samples were cut in 5 mm slices using a rotation micro-tome (Histocom, HM 355 S, Vienna, Austria) with a specific D knife(Histocom, 152120, Vienna, Austria) and dried at 37 �C for 3 days.

For Toluidine Blue staining, the sections were deplastified inxylene and 2-Methoxyethyl acetate and rehydrated in graded ser-ies of isopropanol. Then they were stained with 0.1% Toluidine BlueO for 40 s, dehydrated using a series of isopropanol, and mountedin Eukitt� (Sigma-Aldrich Handels-GmbH, Vienna, Austria).

For safranin-O staining, the sections were stained with 0.05%safranin-O (Sigma; Vienna, Austria) for 4 min after the rehydration

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steps, then they were washed in distilled water, dehydrated usinga series of isopropanol and xylol, and finally mounted in Eukitt�

(Sigma-Aldrich Handels-GmbH, Vienna, Austria).For the immunohistochemistry with collagen II the rehydrated

sections were digested for antigen retrieval with proteinase K(Dako, Vienna, Austria) in Tris-buffered saline (TBS) for 20 min at37 �C. After washing in TBS, the sections were pre-incubated witha solution of 0.3% H2O2 in TBS for 30 min to block endogenous per-oxidase activity. Subsequently, they were incubated for 30 min in asolution of 10% normal goat serum (Vector/ZABO-SCANDIC Han-dels GmbH, Vienna, Austria) in TBS to block unspecific antibodybinding. Afterwards they were incubated with the primary anti-body (mouse anti-collagen II, Acris, Vienna, Austria) for 60 min atroom temperature. They were then washed twice in TBS (5 mineach), followed by a peroxidase-labelled secondary antibody(goat-anti-mouse-EnVision, Dako, Vienna, Austria) for 30 min atroom temperature. Peroxidase activity was visualized using a liq-uid diaminobenzidine (DAB) substrate chromogen system (Dako,Vienna, Austria). The sections were afterwards washed twice withTBS and distilled water, stained with Hematoxylin and finallymounted in Aquatex� (Merck, Vienna, Austria). As negative con-trols, the primary antibody was omitted and no staining was seenin these sections [21].

2.8. Data analysis

Statistical analysis was performed using IBM� SPSS� Statistics20.0.0 (IBM Corporation, Armonk, NY, USA). Because the data didnot show a normal distribution according to the Kolmogorov-Smirnov test, statistically significant differences between thegroups at each selected time point were analyzed using theKruskal-Wallis test. The Wilcoxon test was applied to determinesignificant differences within each experimental group. Valueswere expressed as medians, with minimums and maximums. Ap-value < .05 was considered to be statistically significant. To com-pare the volumes and surfaces of the different Mg-based alloys,values were normalized.

Fig. 3. Degradation performance of the two Mg-alloys ZX50 and WZ21. (a) ZX50degrades significantly faster than WZ21, and less than 5% of the (normalized) basevolume is found for ZX50 at week 12. (b) At 4 weeks of degradation a significantincrease in (normalized) surface area is visible for WZ21, with a further hugeincrease by week 12. In contrast, ZX 50 shows an immediate decrease of surface dueto its rapid degradation. (c) Because of its rapid degradation, large amounts of gasrelease are visible at weeks 1 and 4 for ZX50, while WZ21 reveals only moderateand homogeneous gas release over time.

3. Results

Two different Mg alloys were implanted transepiphyseally intothe distal parts of the 26 femoral rat bones. Thirteen rats receivedZX50 pins, and the other 13 receivedWZ21 pins. The operation wastolerated well by all the rodents, and they all bore full weightimmediately after surgery. No rats died during the operation orbefore the time points designated for euthanasia. Slight rednessand swelling was observed at the operation site in nine of the ratsduring the first postoperative week. Because this condition dimin-ished daily after the operation it was judged physiological. How-ever, despite full weight bearing and inconspicuous scars, slightlimping was observed in the ZX50 group.

Twelve of the rats (n = 6 with WZ21 and n = 6 with ZX50implant) were designated for the continuous mCT investigations.Additional twelve rats (n = 6 with WZ21 and n = 6 with ZX50implant) were assigned to the histological group. These rats weresacrificed at different time points of 4, 24, and 52 weeks (Table 2).After 24 weeks, two other rats (n = 1 with WZ21 and n = 1 withZX50 implant) were sacrificed for HRS.

3.1. Degradation performance, surface area and gas volume

Using the image processing software Mimics� the original vol-ume of the ZX50 implants obtained from lCT was measured tobe 14.4 (±0.76) mm3 for ZX50 and 15.56 (±0.62) mm3 for WZ21.The surface areas determined by lCT were 59.1 (±1.18) mm2 for

ZX50 and 52.44 (±2.76) mm2 for WZ21. These implants exhibitedsignificantly diverging degradation characteristics (Figs. 3 and 5).The ZX50 pins corroded immediately after implantation and werealmost completely degraded after 12 weeks (Fig. 3a). The degrada-tion process was accompanied by strong changes in the surfacearea (Fig. 3b), with correspondingly great increases in thesurface-to-volume ratios from originally 4.13 (±0.22) mm�1 to6.21 (±0.88) mm�1 at 4 weeks and 24.47 (±4.14) mm�1 at 12

Fig. 4. Increase in femoral bone length for ZX50 and WZ21 pin implantation incomparison to that of the sham group. The length increase in % refers to the lengthof 1 week after implantation. Significantly lower length increase is measured for theZX50 group, whereas the growth for theWZ21 group is similar to that of the controlgroup.

T. Kraus et al. / Acta Biomaterialia 66 (2018) 109–117 113

weeks, reflecting the occurrence of severe localized corrosionattacks (Fig. 3b). Correspondingly, massive gas evolution wasobserved during the ZX50 degradation process (Figs. 3c and 5).

WZ21 corroded only slightly within the first 4 weeks and its pinvolume still comprised between 48% and 53% of the initial pin vol-ume after 24 weeks (Fig. 3a). Despite its significant increase in sur-face area (Fig. 3b), the surface-to-volume ratios of WZ21 increasedmuch more moderate, from originally 3.37 (±0.26) mm�1 to 4.99(±2.12) mm�1 at 4 weeks and 11.58 (±4.61) mm�1 at 12 weeks.This less severe increase in surface-to-volume ratio indicates amore homogeneous degradation process for WZ21 compared toZX50 (Fig. 3a and b). Gas release was also considerably less severe

Fig. 5. Bone remodeling of the three groups (ZX50, WZ21 and control) over the observatgas. The physis of the bones implanted with ZX50 was obviously unable to withstand ththe femur length was significant shorter. In contrast, WZ21 degraded moderately, producsignificantly from that of the control (sham) group. The arrows illustrate the more rapid irespectively.

and uniform, with a mean value of 9.2 (±4.58) mm3 in the firstweek and of 5.5 (±0.05) mm3 in the 12th week (Fig. 3c). In bothimplants the degradation was faster in the epiphysis than in themetaphysis. This will be shown further below by presenting themCT images and histological slices (Figs. 5 and 6).

3.2. Length of the femoral bones

The initial mean length of the femoral bones was 34.4 mm (SD:0.92; range: 33.35–35.12 mm). From the 24th week on, the bonesdid not increase significantly in length (p > .05). This indicates thatthe rats were mature at 6 months. As illustrated in Figs. 4 and 5,the rapidly degrading ZX50 interfered significantly with bonegrowth, which generated significantly shorter ipsilateral bonelength. Correspondingly, we observed morphological disturbanceswithin the physeal growth plate in HRS for ZX50 (Fig. 6). The bonegrowth after WZ21 transphyseal implantation did not differ fromthe contralateral sham bone (p = 0.76; Fig. 4), and the defect inthe physis was comparable to that in the sham bone (Fig. 6).

3.3. Histology

As illustrated in Fig. 7, all histological slices show an interrup-tion of the growth plate, indicating a defect due to either drillingor implantation of the alloy. Different stainings with Toluidine BlueO (Fig. 7a) and Safranin-O (Fig. 7b) generated equal results. Tolu-idine Blue O staining (Fig. 7a) shows the general bone morphologyand the growth plate is coloured in dark blue. In Safranin-O stain-ing (Fig. 7b), which gives similar information on bone morphology,the growth-plate is coloured pink.

The histological results were verified with the help of immuno-histochemistry (Fig. 8), in which collagen type II is determined byantibodies. Immunohistochemistry with Collagen II antibodies dif-ferentiates clearly the growth plate (coloured brown) of the distal

ion period of 52 weeks. ZX50 degraded rapidly, thereby producing large amounts ofis irritation and was completely destroyed. Bone growth subsequently stopped anding low amounts of gas. Bone growth continued and the femur length did not differmplant degradation in the physis after 4 weeks in ZX50 and after 24 weeks in WZ21,

Fig. 6. Longitudinal high-resolution mCT slices at week 24 after ZX50 and WZ21 pin implantation in comparison to the control group. In the case of ZX50 the physis issignificantly disturbed, whereas the physis defect after WZ21 implantation is comparable to the one obtained by drilling in the control group.

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femoral epiphysis from the adjacent bone tissue (Fig. 8). Collagen IIis considered a core element of the growth plate [22] and is one ofthe major collagens expressed early in chondrogenesis – the mostimportant process for growing. Alterations in collagen type II gen-erate either qualitative or quantitative results but always occur inconjunction with reduced length growth [22].

As can be seen in Figs. 7 and 8, the ZX50 alloy was completelydegraded by week 24, with a total destruction of the growth plate.The defect was of such large dimensions that the growth plate didnot recover by the end of the study (52 weeks). In contrast, theWZ21 implant was still visible after 24 weeks and remnants ofthe WZ21 implant were still visible by the end of the study (Figs. 7and 8). Because of this moderate implant degradation, the growthplate was able to recover and its defect size was comparable to thatof the sham. All histological results, also verified by immunohisto-chemistry, were found to agree with the mCT investigations and themCT leg length determinations.

4. Discussion

An important aim of bone fixation in children is to protect thegrowth plate and thus to avoid severe growth disturbances suchas length discrepancies or axial deviations. However, in manycases, to achieve adequate bone fixation, the implanted materialmust be positioned close to the physis or even across it. Mg-based implants have received frequent mention as promising can-didates for osteosynthetic application in paediatrics [23]. To thebest of our knowledge, however, a discussion regarding the possi-ble influences of Mg implants on the growth plate is so far lacking.

In our study, implantation was performed transphyseal, andbones implanted with two different Mg alloys (ZX50 and WZ21)were compared with the contralateral sham bone, which receiveda drill-hole of the same dimensions as the implants. The femoralbones implanted with ZX50 grew significantly shorter than thoseof the control group, and the rodents with the shortened legsshowed a slight limp over time. We rate the comparison to a shambone as particularly important, because mere drilling can itselfharm the growth plate and cause growth arrest [24]. Twopathophysiological mechanisms are known here: (1) formation ofepi-diaphyseal bone bridges after invasion of osteoblasts andvascularization originating from the adjacent bone [25]; and (2)ischemic necrosis of the epiphyseal cartilage occurs due to

trauma-related impairment of the blood circulation [26,27]. Inaddition, the drilling in our study was performed from the epiphy-seal side, because it was shown that growth disturbances wereonly observed when fixation was performed from this side [27].

In accordance with our previous study [15], ZX50 and WZ21exhibited different degradation behaviour. Alloy ZX50 degradedquickly and very inhomogeneously, whereas alloy WZ21 showedmore homogeneous degradation at a moderate rate. At this pointit should be emphasised again that ZX50 was deliberately chosenas a representative of rapidly degrading Mg alloys, in order toinvestigate ”worst-case scenarios” of maximum harm to thegrowth plate. As discussed by Hofstetter et al. [28], the microstruc-ture of ZX50 is characterized by the presence of electrochemicallynoble Zn-rich intermetallic particles (Ca3MgxZn15�x, 4.6 � x � 12).These may be responsible for severe micro-galvanic corrosion,leading to the above-mentioned local corrosion attack, and in turnto rapid degradation with massive gas formation according to thechemical reaction Mg + 2H2O ?Mg(OH)2 + H2. As shown by thehigh-resolution CTs at week 24 (Fig. 6), in this study the largeamounts of gas obviously caused the destruction of the entiregrowth plate, even though the gas has already disappeared at week24 (Fig. 3c), with the result of diminished bone growth (Fig. 4).

In comparison to ZX50, the microstructure of WZ21 is designedto contain intermetallic particles of type Mg12YZn [29], which donot cause pronounced galvanic corrosion [12]. With the lowerdegradation rate and correspondingly reduced gas formation, goodhealing results were achieved and the effect on the growth platedid not differ from that of the contralateral drill-hole lesion. Thedegradation performance of alloy WZ21 thus satisfied an impor-tant prerequisite for application in paediatric osteosyntheticdevices. The exact limit of tolerable gas without harm for thegrowth plate may be the subject of further investigations, but thisstudy shows that it lies between the hydrogen gas generated dur-ing the degradation of WZ21 and that of ZX10.

Both alloys also underwent more rapid degradation within theepiphysis. This may be well explained by an increased perfusionof the epiphysis compared to the meta- and diaphysis [30] andsubsequent faster disappearance of the degraded alloy particles.

As to the transferability of the results of this study to humans,we need to discuss the size defect of the growth plate. In the cur-rent animal study, drilling was rather extensive and exceeded thecritical size defect tolerated by the physis (>20%; [31]). This and

Fig. 7. Histological slices of the two pin groups (ZX50, WZ21) and the control group at different time points of 4, 24, and 52 weeks. (a) All slices (ZX50, WZ21 and the controlgroup) were stained with Toluidine Blue O, which colours the epiphyseal chondrocytes dark blue (GP = growth plate, I = implant, D = drill hole). (b) All slices were stainedwith Safranin-O, which colours the epiphyseal chondrocytes pink (A = Artefact due to the cutting or staining procedure; Ir = implant remnant). The arrows indicate the edgesof the growth plate and its interruption due to the implant insertion. (For interpretation of the references to colour in this figure legend, the reader is referred to the webversion of this article.)

T. Kraus et al. / Acta Biomaterialia 66 (2018) 109–117 115

Fig. 8. Immunohistochemistry of bone slices of the two pin groups ZX50 and WZ21. Collagen II is detected in the growth plate as brown colour (GP = growth plate,I = implant). The edges of the growth plate and its interruption due to implant insertion are indicated by arrows.

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the diameter of the implant itself may have generated growth dis-turbances. Thus, additional experiments on larger bones (e.g.,sheep bones) are needed in order to be closer to clinical applica-tion. Nevertheless it should be emphasized that this first studyonMg-implant degradation in the growth plate provides importantguidelines for the design of Mg-based alloys in orthopaedicpaediatrics.

5. Conclusions

Mg-based alloys are promising candidates for implant materialsin the paediatric orthopaedic field due to their attractive biologicaland mechanical properties, and the fact that Mg-based implantsrequire no removal surgery. Bone fixation in paediatric patientsmust be performed with particular care with regard to the growthplate, in order to avoid associated growth disturbances. The Mg-based biodegradable implant WZ21 showed moderate, homoge-neous degradation characteristics with low amounts of gas release.Its effect on the growth plate did not significantly differ from thatof a single drill-hole lesion. The degradation characteristics ofmoderately corroding Mg-alloys (e.g. WZ21) thus seem to satisfythe prerequisites for use in paediatrics when fixation across orclose to the growth plate is required. For clinical applications, how-ever, we recommend using rare-earth-free Mg-alloys [11] becausetemporary incorporation of Mg and Y into the cortical bone wasobserved in Ref. [32], and potentially toxic long-term effects ofreleased rare-earth elements, such as Y, were reported in Ref. [33].

Acknowledgments

The authors acknowledge support from the Laura BassiCenter of Expertise BRIC (Bioresorbable Implants for Children;FFG—Austria). The authors also thank PD Dr. Elmar Willbold fromthe Medical School of Hannover for his support on the Technovitembedding method and the Core Facility Microscopy group ofthe Medical University of Graz for general help.

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