Abstract
Purpose
In recent decades operative fracture treatment using elastic stable intramedullary nails (ESINs) has mainly taken precedence over conservative alternatives in children. The development of biodegradable materials that could be used for ESINs would be a further step towards treatment improvement. Due to its mechanical and elastic properties, magnesium seems to be an ideal material for biodegradable implant application. The aim of this study was therefore to investigate the cellular reaction to biodegradable magnesium implants in vitro.
Methods
Primary human growth plate chondrocytes and MG63 osteoblasts were used for this study. Viability and metabolic activity in response to the eluate of a rapidly and a slower degrading magnesium alloy were investigated. Furthermore, changes in gene expression were assessed and live cell imaging was performed.
Results
A superior performance of the slower degrading WZ21 alloy’s eluate was detected regarding cell viability and metabolic activity, cell proliferation and morphology. However, the ZX50 alloy’s eluate induced a favourable up-regulation of osteogenic markers in MG63 osteoblasts.
Conclusions
This study showed that magnesium alloys for use in biodegradable implant application are well tolerated in both osteoblasts and growth plate chondrocytes respectively.
Electronic supplementary material
The online version of this article (doi:10.1007/s00264-013-2163-3) contains supplementary material, which is available to authorized users.
Keywords: Biodegradable magnesium, Orthopaedics, Immature skeleton, Growth, Biocompatibility
Introduction
Fracture treatment in children has changed over the last few decades. Before the early 1980s, most paediatric fractures were treated conservatively with casting and immobilisation. Elastic stable intramedullary nails (ESIN), developed by French surgeons [1], changed these treatment strategies. They are easily applicable and fulfil their purpose in a simple way. Advantages of ESIN are early load bearing, avoidance of a cast and thereby more adapted to the children’s requirements [1]. However, further surgery is needed for implant removal, risking harm to the surrounding soft tissue and further morbidity. Major and minor complications are reported for implant removals [2].
Implants manufactured from biodegradable materials render implant removal unnecessary and thereby could be a more reasonable alternative to traditional implants by eliminating the pain, disability, risks, inconvenience and costs of a second operation and possible hospitalisation. Moreover, children might be offered the possibility to return to play sooner.
A number of investigators have recently focussed on magnesium and its alloys for use in fracture fixation [3–5]. Magnesium is a natural element in the human body. It shows good biocompatibility without systemic inflammatory reaction or affecting the cellular blood composition [6, 7]. The elastic properties of magnesium resemble those of human bone thus avoiding a stress shield reaction as its Young’s modulus is close to that of human bone [8]. During its degradation in bone, several investigators [9, 10] have observed high mineral apposition rates and increased bone mass. Furthermore, bone-implant interface strength and osseointegration are reportedly greater for magnesium than for conventional titanium materials [11].
One of its major drawbacks is its rapid degradation in aqueous solutions producing high amounts of hydrogen gas [12]. Although these levels of gas apparently do not harm the bone [13], they render rapidly degrading magnesium alloys inappropriate for fracture fixation as the amounts of gas might disturb the process of bone healing. Several strategies have been used to address this problem: First, adding rare earth elements leads to a decrease in degradation by simultaneously stabilising the implant [7]. However, rare earth components are toxic substances that must be avoided—particularly in a child’s body. Second, surface modifications have been reported to reduce degradation [14, 15].
Although children’s bones have an amazing capacity to heal and a high potential for remodelling, their bone may be damaged irreversibly if the growth plate is injured by trauma [16] or by surgery in the case of improperly used implants; therefore, special regard has to be paid to the growth plate, among further considerations in individual cases. Moreover, a material must be selected that will not harm an immature growing body. However, the specific cell reaction to biodegradable magnesium-based implants has not yet been investigated.
The aim of this study was therefore to investigate the specific reaction of human growth plate chondrocytes (hGPC) and osteoblasts (i.e. the cell line MG63 osteoblasts) to biodegradable magnesium-based implants in vitro. Two different magnesium-based alloys were tested: the slower degrading WZ21 alloy and the faster degrading ZX50 alloy. Both were compared to an untreated control group. The study hypothesis was that magnesium alloys for use in biodegradable implant application are well tolerated in both osteoblasts and growth plate chondrocytes respectively.
Materials and methods
Alloys
In this study Mg-Zn alloy discs of two different types were used. They were 20 mm in diameter and had a thickness of 8.8 mm. Their nominal compositions are illustrated in detail in Table 1. The alloys were produced using direct chill casting and direct extrusion. Billets of 300 mm length and 109 mm in diameter were preheated to 310 °C and pressed into cylindrical profiles with a diameter of 20 mm, corresponding to an extrusion ratio of 30:1. From the profiles, discs with 20 mm diameter and 8.8 mm thickness were machined. The faster degrading ZX50 alloy typically exhibits a yield stress of 210 MPa, an ultimate tensile strength of 295 MPa, a uniform elongation of 18 % and an elongation at fracture of 26 %. The slower degrading WZ21 alloy features a yield stress of 150 MPa, an ultimate tensile strength of 250 MPa, a uniform elongation of 20 % and an elongation at fracture of 28 %.
Table 1.
Nominal chemical composition in wt % of the two magnesium alloys used
| Alloy | Mg | Zn | Ca | Mn | Y |
|---|---|---|---|---|---|
| ZX50 | Balance | 5 | 0.25 | 0.15 | – |
| WZ21 | Balance | 1 | 0.25 | 0.15 | 2 |
Alloy preparation
Each disc was grounded with 2400 grit SiC abrasive paper, cleaned in acetone and then sterilised in 70 % ethanol for five minutes. Then each specimen was washed three times in simulated body fluid (SBF) (for composition see supplementary Table 1) and subsequently immersed in ten millilitres SBF at 37 °C for 40 hours with agitation at 180 rpm. The ion concentration of SBF after 48 hours of incubation is given as supplementary material (supplementary Table 2). SBF was chosen as the immersion solution because it most appropriately reflects the conditions in the human body [7]. The resulting eluate was then used to conduct the in vitro cytocompatibility studies. All solutions used for cell testing were sterile filtered in advance using filters with a pore size of 0.2 μm.
Cells and cell culture
Ethical approval for this study was obtained from the Ethics Committee of the Medical University of Graz (Austria). All experiments were performed with both MG63 osteoblasts and primary hGPC. We used these two cell types to conduct our study as they are the ones that are in contact with ESINs following a bony fracture in paediatric patients. MG63 osteoblasts (doubling time 23 hours [17]) were obtained from the European Collection of Cell Cultures (ECACC) and cultured in Eagle’s minimum essential medium with Earle’s balanced salt solution (Sigma-Aldrich, Vienna, Austria) supplemented with 2 mM glutamine, 1 % non-essential amino acids and 10 % foetal bovine serum (FBS) (all reagents Invitrogen, Carlsbad, CA, USA).
Primary hGPC (doubling time 40 hours) were isolated from the growth plate of the supernumerary digit of children with polydactylism at the time of surgical excision [18]. Growth plate chondrocytes from six different donors were selected for this study. Ages ranged from 0.25 to 2.3 years (mean age ± SD 1.14 ± 0.68 years) with no significant difference between female and male subjects. To isolate the chondrocytes growth plate tissue was digested in 2 mg/ml collagenase B (150 units/mg collagenase B, Worthington Biochemical Corp., Lakewood, NJ, USA) in a shaking water bath at 37.5 °C overnight. The cell suspension was passed through two layers of nylon grid (40 μm mesh size) and then expanded in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 supplemented with 5 % FBS and 2 mM glutamine (all reagents Invitrogen). Environmental parameters were kept at 37 °C, 5 % CO2 in a humidified atmosphere at all times. The medium was changed three times weekly.
Assessment of cell viability and metabolic activity: MTT and neutral red test
Cytocompatibility of the alloys was studied by analysing the cell viability based on the physical uptake of neutral red and the metabolic activity based on 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT) conversion as described previously [7].
Live cell imaging: Cell-IQ®
To investigate the influence of the alloys’ eluates on cell proliferation and cellular morphology, continuous live cell imaging was performed using the Cell-IQ V2 MLF Cell Imaging and Analysis System (Chip-Man Technologies, Tampere, Finland). This instrument is a label-free system, which uses Machine Vision Technology for the automatic identification, analysis and quantification of morphological features. Cells were inoculated in 24-well plates at a density of 1.2 × 105 cells/well. After an incubation period of 24 hours cells were treated with the alloys’ eluates and pure SBF. The eluate volume corresponded to 4 % of the total amount of liquid. This eluate concentration was chosen because it was the highest eluate concentration tolerated by both cell types without any significant decrease in cell viability and metabolic activity. The cells were then observed for 48 hours. This short observation period was chosen for two reasons: first to make the results comparable to those of our previous work [7] and second to minimise the risk of dedifferentiation in primary hGPC [19–21].
Gene expression analysis: quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR)
To analyse possible changes in the expression of osteogenic marker genes (alkaline phosphatase and osteocalcin) of MG63 osteoblasts and osteogenic and chondrogenic marker genes (alkaline phosphatase, osteocalcin, sox-9 and type II collagen) of primary hGPC in response to the alloys’ eluates total RNA was extracted from the cells after 48 hours of incubation with the respective eluates or pure SBF (4 % eluate volume of the total amount of liquid) as described previously [18]. The selected markers are well-known markers for either chondrogenic or osteogenic lineage differentiation [22, 23]. Ribonucleic acid was further extracted from untreated controls that had been incubated for the same period. Real-time RT-PCR reactions were performed and monitored using the Bio-Rad CFX96 instrument (Bio-Rad, Vienna, Austria). Sequences of primers for the reference gene (GAPDH) and genes of interest (alkaline phosphatase, osteocalcin, sox-9, type II collagen) are listed in Table 2. All primers were provided by MWG (Ebersberg, Germany). Results were calculated using the comparative CT method. All graphs demonstrate the fold change and its range of each experimental group when the control group was normalised as 100 % expression.
Table 2.
Human primer pairs used for real-time RT-PCR
| Gene | Primer |
|---|---|
| Alkaline phosphatase | Forward: 5-CACCAACGTGGCTAAGAATG-3 |
| Reverse: 5-ATCTCCAGCCTGGTCTCCTC-3 | |
| Osteocalcin | Forward: 5-GGCGCTACCTGTATCAATGG-3 |
| Reverse: 5-TCAGCCAACTCGTCACAGTC-3 | |
| sox-9 | Forward: 5-AGACCTTTGGGCTGCCTTAT-3 |
| Reverse: 5-TAGCCTCCCTCACTCCAAGA-3 | |
| Type II collagen | Forward: 5-CTATCTGGACGAAGCAGCTGGCA-3 |
| Reverse: 5-ATGGGTGCAATGTCAATGATGG-3 | |
| GAPDH | Forward: 5-AAGGTCGGAGTCAACGG-3 |
| Reverse: 5-ACCAGAGTTAAAAGCAGCCCT-3 |
Statistical analysis
For statistical analysis SPSS® 19.0 software (SPSS Inc., Chicago, IL, USA) was used. To determine statistically significant differences between the experimental groups compared to the untreated controls the Wilcoxon signed rank test was performed; t tests were used for parametric data sets and Mann-Whitney U tests for non-parametric data. A p value <0.05 was considered as statistically significant.
Results
Cell viability and metabolic activity: MTT and neutral red test
hGPC viability significantly decreased when the added ZX50 eluate accounted for 10 % or more of the total amount of liquid (p < 0.001, Fig. 1a). Simultaneously, hGPC metabolic activity based on MTT conversion also decreased significantly (p < 0.001, Fig. 1b). The WZ21 eluate as well as pure SBF neither influenced hGPC viability nor cell metabolic activity (p value >0.005).
Fig. 1.
a, b Cell viability (a) determined by lysosomal uptake of neutral red and metabolic activity based on MTT conversion (b) of hGPC significantly decreased when the ZX50 eluate volume accounted for 10 % of the total amount of liquid. hGPC treated with the WZ21 eluate or with pure SBF neither showed any significant changes in cell viability nor metabolic activity compared to the untreated control (ctrl.)
MG63 osteoblasts tolerated a higher amount of the ZX50 eluate. Their cell viability (Fig. 2a) and metabolic activity (Fig. 2b) did not decrease until the added eluate volume reached a concentration of 20 % of the total amount of liquid (p = 0.006 and p < 0.001 respectively). Moreover, in these cells, the WZ21 eluate and pure SBF did not significantly influence cell viability and metabolic activity of these cells.
Fig. 2.
a, b Both cell viability (a) and metabolic activity (b) of MG63 osteoblasts, assessed by lysosomal neutral red uptake and MTT conversion respectively, decreased significantly when the amount of ZX50 eluate reached 20 % of the total amount of liquid (p = 0.006 and p = 0.000 respectively). This indicates that MG63 osteoblasts tolerate a higher amount of this eluate when compared to hGPC. The WZ21 eluate and pure SBF neither influenced MG63 osteoblast cell viability nor metabolic activity
Live cell imaging: Cell-IQ®
Live cell imaging analysis showed that SBF treatment neither influenced cell proliferation nor cell shape in both cell types (Figs. 3e–f and 4e–f).
Fig. 3.
a–h hGPC treated with the ZX50 eluate (a), the WZ21 eluate (c), pure SBF (e) and untreated hGPC (g) before and after (b, d, f, h) 48 h of incubation. Both the ZX50 (b) and the WZ21 eluate (d) inhibited cell proliferation when compared to the pure SBF (f) and the untreated control cells (h). hGPC incubated with the ZX50 eluate even showed a reduced cell density after 48 h (b). See also supplementary material, videos 1–8
Fig. 4.
a–h MG63 osteoblasts treated with the ZX50 eluate (a), the WZ21 eluate (c), pure SBF (e) and untreated MG63 osteoblasts (g) before and after (b, d, f, h) 48 h of incubation. The ZX50 eluate inhibited cell proliferation and caused cell death in MG63 osteoblasts (b). However, the WZ21 eluate was very well tolerated with no influence on cell density after 48 h of incubation (d) compared to the SBF-treated (f) and the untreated MG63 osteoblasts (h). See also supplementary material, videos 5–8
The ZX50 eluate inhibited cell proliferation and caused cell death of both hGPC and MG63 osteoblasts (Figs. 3a, b and 4a, b). After 48 hours of incubation with this eluate, cell density of both cell types was reduced compared to the starting point (Figs. 3a, b and 4a, b). The WZ21 eluate inhibited cell proliferation only in hGPC (Fig. 3c, d). MG63 osteoblasts proliferated like the untreated control (Fig. 4c, d).
Gene expression analysis: qRT-PCR
After 48 hours of incubation in hGPC, both the ZX50 and the WZ21 eluates increased the expression of the chondrogenic transcription factor sox-9 (Fig. 5a, both p < 0.001), whereas expression of the chondrogenic marker type II collagen was significantly reduced when the cells were treated with these eluates (Fig. 5b, p = 0.000/0.001). These eluates also induced a significantly higher expression of the osteogenic markers alkaline phosphatase and osteocalcin in the experimental samples compared to control (Fig. 5a, b, p = 0.017/0.005, p = 0.000/0.017).
Fig. 5.
a–d Changes in hGPC mRNA expression of chondrogenic (a, b) and osteogenic (c, d) marker genes after 48 h of incubation with the corresponding eluates or pure SBF. Data are expressed as ratios of expression levels of target genes over the internal control gene GAPDH. Data are presented as average ± SEM. Significance was assumed when p < 0.05 (indicated by an asterisk)
Expression of alkaline phosphatase (Fig. 6a, p = 0.027) and osteocalcin (Fig. 6b, p = 0.039) was significantly increased in MG63 osteoblasts only when they were treated with the ZX50 eluate. The WZ21 eluate did not alter the mRNA expression in these cells.
Fig. 6.
a, b Changes in MG63 osteoblast mRNA expression of osteogenic marker genes after 48 h of incubation with the corresponding eluates or pure SBF. Ratios of expression levels of target genes over the internal control gene GAPDH are reported. Data are presented as average ± SEM. Significance was assumed when p < 0.05 (indicated by an asterisk)
Discussion
The aim of this study was to investigate the specific reaction of hGPC and osteoblasts to biodegradable magnesium-based implants. The study hypothesis was that magnesium alloys for use in biodegradable implant application are well tolerated in both osteoblasts and growth plate chondrocytes respectively.
According to our results magnesium alloys for use in biodegradable implant application are well tolerated in both osteoblasts and growth plate chondrocytes respectively. The results of this study indicate a good in vitro cytocompatibility of the tested biodegradable magnesium alloys ZX50 and WZ21 on both MG63 osteoblasts and primary hGPC, confirming the in and ex vivo results from our previous work [7, 13]. In our previous work we demonstrated a good biocompatibility of the same alloys in a growing rat model. Also the mechanical properties of the WZ21 alloy were superior with a ductility of 28 % compared to previously used materials [24].
To our knowledge this is the first time that the biocompatibility of degradable magnesium alloys for orthopaedic implant application has been assessed using primary hGPC and thereby proving their utility for paediatric application. Previous studies—focussing on cell tests—were conducted using animal models, cell lines determined for laboratory use or embryonic stem cells but not on primary human cells [24–27].
The growth plate at the end of long bones is a unique feature of a child’s bone and responsible for longitudinal bone growth. Selection of materials for biodegradable osteosynthesis application without harming this highly orchestrated process of longitudinal growth within the growth plate is therefore indispensable.
A beneficial dose-dependent effect of magnesium has already been reported on articular chondrocytes in terms of proliferation, redifferentiation, gene and protein expression [28]. Magnesium stimulated proliferation and induced matrix production and expression of chondrogenic markers including type II collagen, type IX collagen and aggrecan in these cells in a concentration-dependent manner. However, data about the effect of magnesium on growth plate chondrocytes are limited. Regarding bone cells, favourable biocompatibility has been reported several times for magnesium salts [29, 30] and different magnesium alloys [30–36]. Magnesium can enhance bone cell adhesion on biomaterials via integrin expression [37] and the mitogen-activated protein (MAP) kinase pathway [38].
Our study showed a good biocompatibility of the tested magnesium alloys on both cell types, confirming the in vivo results from our previous work [13]. The ZX50 alloy’s eluate induced a desirable up-regulation of the osteogenic marker genes alkaline phosphatase and osteocalcin in MG63 osteoblasts. A similar effect was also seen for the WZ21 alloys eluate; however, it was not significant (Fig. 6a, b). Elevated alkaline phosphatase levels indicate that active bone formation occurs [39]. Also osteocalcin is a well-known marker for the bone formation process as it is involved in bone mineralisation and calcium ion homeostasis [40].
As did MG63 osteoblasts primary hGPC also expressed a significantly higher amount of the osteogenic markers alkaline phosphatase and osteocalcin when treated with either the ZX50 alloy’s or the WZ21 alloy’s eluate (Fig. 5c, d). In these cells this effect is less desirable as it could be a first step towards premature ossification of the growth plate in vivo. However, besides a significant down-regulation of the chondrogenic marker type II collagen in primary hGPC treated with the alloy’s eluate (Fig. 5b) we also detected a significant up-regulation of the main chondrogenic transcription factor sox-9 (Fig. 5a). This means that the chondrocytes had already started counter-regulation, indicating that they are able to compensate for a transiently elevated magnesium ion concentration. Further studies will be performed to investigate the long-term effect of magnesium ions on growth plate chondrocytes.
In terms of viability and metabolic activity, both primary hGPC and MG63 osteoblasts tolerated a higher amount of the slower degrading WZ21 alloy’s eluate when compared to the faster degrading ZX50 alloy’s eluate (Figs. 1a, b and 2a, b). The live cell imaging study confirmed this finding. After 48 hours of incubation cells treated with the ZX50 alloy’s eluate showed a reduced cell density (Figs. 2a–h and 3a–h). Other researchers working with rapidly degrading magnesium alloys observed similar effects. They attributed these effects to the very high pH caused by fast magnesium corrosion rather then to Mg2+ ion content [35, 41]. However, the effect of pH remains unclear in our study.
From our results we deduce that the WZ21 alloy is the more suitable material as it has a lower impact on cell viability, metabolic activity and gene expression. However, this alloy contains the rare earth element yttrium, which is potentially toxic to the body. Importantly the content of this rare earth element was much lower than in alloys used in other studies for similar applications [25, 27] and we were able to avoid aluminium as an alloying element which is popular but implicated in the onset of different degenerative pathologies, e.g. Alzheimer’s disease, muscle fibre damage and decreased osteoclast viability [5, 25, 26, 42].
Conclusively WZ21 might be well tolerated as osteosynthesis material in adults, but there are severe concerns in using rare earth elements in degradable implant solutions for children. It has been shown that rare earth elements can influence cellular processes in terms of apoptosis and expression of inflammatory markers [30, 43] in vitro. Whether any adverse effects also occur in vivo is less clear.
It is recommended that the influence of rare earth elements on bone and cartilaginous tissue as in the growth plate is studied in detail, particularly since these tissues exhibit very slow release rates of such substances [44, 45] and the tolerance limits in children are small.
Conclusion
Our study has demonstrated that in terms of cellular reactions magnesium is a suitable material for biodegradable implant solutions for use in paediatric orthopaedics. In this context the slower degrading WZ21 alloy showed a superior performance. However, it contains the potentially toxic rare earth element yttrium, whose effects on a child’s body are largely unknown. We therefore suggest either the development of a rare earth element-free magnesium-based alloy with similar degradation characteristics as the WZ21 alloy or detailed studies on the effects of yttrium in humans, especially in children.
Electronic supplementary material
Concentrations of chemical substances used for the SBF (concentrations are indicated in mmol/L). (DOC 33.5 kb)
Element concentration after 48 h of implant incubation given in mg/ml SBF. (DOC 39.5 kb)
Videos show the investigated cells treated with the different alloys’ eluates, SBF and untreated control cells during an incubation period of 48 h.
hGPC treated with the ZX50 eluate. (MPEG 2.66 MB)
hGPC treated with the WZ21 eluate. (MPEG 2.64 MB)
hGPC treated with SBF. (MPEG 2.35 MB)
Untreated hGPC. (MPEG 2.30 MB)
MG63 osteoblasts treated with the ZX50 eluate. (MPEG 2.70 MB)
MG63 osteoblasts treated with the WZ21 eluate. (MPEG 2.71 MB)
MG63 osteoblasts treated with SBF. (MPEG 2.71 MB)
Untreated MG63 osteoblasts. (MPEG 2.64 MB)
Acknowledgments
The authors appreciate support from the Laura Bassi Center of Expertise BRIC (Bioresorbable Implants for Children; FFG – Austria) and from the Staub/Kaiser Foundation, Switzerland. Furthermore, they would like to thank Mr. Rudolf Schmied for his valuable technical assistance in art work preparation and Ms. Aranka Schauer for her help in carrying out some of the experiments.
Footnotes
Karin Pichler and Tanja Kraus contributed equally to this work.
Contributor Information
Karin Pichler, Phone: +43-316-3807606, FAX: +43-316-3809625, Email: karin.pichler@medunigraz.at.
Tanja Kraus, Email: tanja.kraus@medunigraz.at.
Elisabeth Martinelli, Email: elisabeth.martinelli@medunigraz.at.
Patrick Sadoghi, Email: patrick.sadoghi@medunigraz.at.
Giuseppe Musumeci, Email: g.musumeci@unict.it.
Peter J. Uggowitzer, Email: peter.uggowitzer@mat.ethz.ch
Annelie M. Weinberg, Email: annelie.weinberg@t-online.de
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Concentrations of chemical substances used for the SBF (concentrations are indicated in mmol/L). (DOC 33.5 kb)
Element concentration after 48 h of implant incubation given in mg/ml SBF. (DOC 39.5 kb)
hGPC treated with the ZX50 eluate. (MPEG 2.66 MB)
hGPC treated with the WZ21 eluate. (MPEG 2.64 MB)
hGPC treated with SBF. (MPEG 2.35 MB)
Untreated hGPC. (MPEG 2.30 MB)
MG63 osteoblasts treated with the ZX50 eluate. (MPEG 2.70 MB)
MG63 osteoblasts treated with the WZ21 eluate. (MPEG 2.71 MB)
MG63 osteoblasts treated with SBF. (MPEG 2.71 MB)
Untreated MG63 osteoblasts. (MPEG 2.64 MB)