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Adiponectin attenuates osteolysis in aseptic loosening of total hip replacements
Accepted Manuscript Adiponectin Attenuates Osteolysis in Aseptic Loosening of Total Hip Replace‐ ments Stefan Landgraeber, S. Putz, M. Schlattjan, Lars P. Bechmann, Martin Totsch, Florian Grabellus, Gero Hilken, M. Jäger, A. Canbay PII: DOI: Reference:
S1742-7061(13)00423-6 http://dx.doi.org/10.1016/j.actbio.2013.08.031 ACTBIO 2876
To appear in:
Acta Biomaterialia
Received Date: Revised Date: Accepted Date:
18 June 2013 16 August 2013 20 August 2013
Please cite this article as: Landgraeber, S., Putz, S., Schlattjan, M., Bechmann, L.P., Totsch, M., Grabellus, F., Hilken, G., Jäger, M., Canbay, A., Adiponectin Attenuates Osteolysis in Aseptic Loosening of Total Hip Replacements, Acta Biomaterialia (2013), doi: http://dx.doi.org/10.1016/j.actbio.2013.08.031
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Adiponectin Attenuates Osteolysis in Aseptic Loosening of Total Hip Replacements Stefan Landgraeber1*, S. Putz1, M. Schlattjan2, Lars P. Bechmann2, Martin Totsch3, 4, Florian Grabellus3, Gero Hilken5, M. Jäger1, A. Canbay2
1
Department of Orthopaedics, University Hospital Essen, University of Duisburg-Essen, Hufelandstr. 55, 45122 Essen, Germany
2
Department of Gastroenterology and Hepatology, University Hospital Essen, University of Duisburg-Essen, Germany
3
Institute of Pathology and Neuropathology, University Hospital Essen, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany
4
Institute of Cytology, University Hospital of Graz, University of Graz, Auenbruggerplatz 20, 8036 Graz, Austria
5
Central Animal Laboratory, University Hospital Essen, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany
*Corresponding Author: Stefan Landgraeber, M.D. Department of Orthopaedics University of Duisburg-Essen Hufelandstr. 55 45122 Essen Germany Phone +49-201-4089-2146 Fax +49-201-4089-2722 Email [email protected] Running Title: Adiponectin and apoptosis in aseptic hip loosening
Abbreviations used: APN: Adiponectin; AdipoR: Adiponectin receptor; HMDM: Human monocyte derived macrophages; KO: Knockout; P: Particles; WT: Wildtype
Abstract Background: Joint replacements have a longer durability in patients with high serum levels of adiponectin (APN) than in patients with low levels. We aimed to characterize the unknown pathophysiological effects of APN on wear particle-induced inflammation, apoptosis and osteolysis. Methods: Immunohistochemistry was performed to detect APN, its receptors and apoptosis in patients with and without aseptic loosening. Additionally, APN knockout mouse (KO) studies and pharmacological intervention of APN were performed in an established calvarial mice model. Osteolysis and inflammation were quantified by histomorphometry and micro-CT, apoptosis by immunohistochemistry and TUNEL assay. In a cell culture model human monocyte derived macrophages (HMDM) were incubated with or without metal wear debris particles and partially treated with APN. Results: Expression of APN, AdipoR1 and calreticulin in specimens from patients with aseptic loosening were significantly higher than in patients without aseptic loosening. Administration of APN in mice significantly reduced wear particle-induced inflammation, osteolysis and the number of caspase-3 positive macrophages. The cell culture model showed that APN leads to significantly lower values of TNF-α. Conclusion: These findings support a prominent role of APN in the development of particle-induced osteolysis and APN may be therapeutically useful in patients with aseptic loosening.
1. Introduction Periprosthetic osteolysis is a serious long-term complication after hip joint replacement which can lead to implant instability and failure. It is initiated by an aseptic inflammatory response to phagocytosis of implant wear particles which results in increased proliferation and differentiation of osteoclast precursors into mature osteoclasts [1]. The complexity and relevance of this topic are reflected by the large number of published studies on particleinduced bioreactivity and by the increasing number of hip replacement procedures. The osteolytic cascade initiated by cytokine release from macrophages has been studied extensively. However, knowledge about the role of apoptosis in this context is limited. Previous studies have demonstrated that apoptosis in macrophages and giant cells during particle engulfment is partially responsible for osteolysis in aseptic loosening of joint implants [2-4]. We demonstrated by means of a murine calvarial model of wear particle-induced osteolysis that inhibition of apoptosis leads to decreasing bone resorption by osteoclasts [5]. This paradoxical effect of apoptosis may be due to apoptotic cell remnants which induce inflammation unless they are broken down within an adequate period of time [6-8]. Adiponectin (APN), an adipocytokine, has been found to influence apoptosis and inhibit inflammation. It also plays a role in glucose utilization, lipid synthesis, insulin sensitivity, and energy homeostasis in several mammalian species [9-11]. In a previous study we demonstrated that aseptic loosening in the first ten years after implantation is associated with decreased serum adiponectin [12]. So far, three receptors for adiponectin have been discovered: Adiponectin receptor 1 (AdipoR1), adiponectin receptor 2 (AdipoR2), and Tcadherin. AdipoR1 is abundant in the skeletal muscle, whereas AdipoR2 is predominantly expressed in the liver [13, 14]. The third receptor is T-cadherin, expressed on endothelial and muscle cells. T-cadherin lacks a cytoplasmatic domain and is presumed to act only as a coreceptor [9]. Interestingly, the binding of adiponectin to calreticulin on the cell surface of macrophages increases the removal of apoptotic cell remnants [15]. It is well known that
calreticulin plays an important role in the uptake of apoptotic debris through its interactions with CD91. This process is independent of the specific adiponectin receptors AdpoR1 and AdipoR2 [15-17]. The aim of the current study was to elucidate whether (i) adiponectin is present in capsules and interface membranes of patients with aseptic hip prosthesis loosening, (ii) expression of AdipoRs and calreticulin is induced by wear particle-phagocytizing cells, (iii) adiponectin influences wear particle-induced apoptosis, (iv) inflammatory response and (v) osteolysis. To answer these questions we performed studies with human samples, cell culture and used an established mice model. The hypothesis of this study is that APN decreases the amount of wear particle-induced osteolysis through its influence on apoptotic and inflammatory pathways.
2. Materials and Methods 2.1. Human Samples Tissue specimens from forty-three patients treated at the Department of Orthopaedics of the University of Duisburg-Essen for aseptic loosening of a total hip replacement were investigated. The specimens were harvested from the newly formed hip joint capsules as well as from the interface membranes of the acetabular and femoral periprosthetic regions after removal of the loosened prostheses. None of the patients had previously undergone implant exchange. The articulations of all the loosened prostheses were a metal femoral head and a polyethylene cup. The reason for revision surgery was aseptic loosening of a hip implant. The presence of infection was excluded by analysis of inflammatory markers in the blood (Creactive protein, leucocytosis), by culture of joint fluid obtained by aspiration from the hip joint for twenty-one days and by histology of hip joint capsules and interface membrane. Bone defects were classified according to Paprosky's method [18].
Specimens from sixteen patients with primary osteoarthritis retrieved during primary total hip arthroplasty for osteoarthritis at the Department of Orthopaedics of the University of Duisburg-Essen served as control groups. Some of these samples were used in a former study [19]. The study was approved by the local Ethics Committee. Details are given Table 1.
2.2. Animal Model of Particle-Induced Osteolysis A well-established calvarial model of ultra-high-molecular-weight polyethylene (UHMWPE) particle-induced osteolysis recently introduced by our group [20] and based on the original model of calvarial osteolysis [21] was utilized in 28 specific-pathogen-free 12week-old male C57BL/6J wildtype mice (WT) (Harlan Laboratories, NM Horst, Netherlands) and 14 adiponectin knockout (KO) mice on a C57BL/6 (The Jackson Laboratory, Bar Harbor, Maine, USA) background. The animals were treated according to official guidelines and the experiments were approved by the university and the local government. A 1.0 x 1.0 cm area of periosteum was exposed by a 10 mm midline sagittal incision over the calvarium anterior to the line connecting both external ears. In the sham controls (-P) the incision was closed without any further intervention. In the other animals the exposed periosteum was covered uniformly with 30 µl dried pure UHMWPE polyethylene particles (+P) at a concentration of 2 x 108 particles/ml after decontamination from endotoxins (Ceridust VP 3,610, Clariant, Gersthofen, Germany). The KO mice were randomized equally into two groups, KO-P and KO+P, consisting of seven mice each. The group size of seven was determined in a power analysis indicating that for a two-sided test at alpha = 0.05 there was 85% power to detect a significant difference between the groups. The adiponectin levels of the WT mice in groups WT+Ad-P and WT+Ad+P were enhanced by daily intraperitoneal injection of APN (Fa. PROSPEC, East Brunswick, USA), at a dose of 2 mg/kg body weight, starting from one day before surgery until sacrifice. During surgery an additional dose of APN was injected into the tissue next to the incision before implantation of the particles. The WT-Ad-P and WT-Ad+P
groups represented the WT group with a normal APN value and received, like the KO mice, a daily i.p. injection of phosphate-buffered saline instead of APN. The WT were also randomized into groups (Table 2). Twelve days postoperatively, the animals were sacrificed and an elliptical plate of the calvarial caps was removed from the region between the foramen magnum, auditory canals, and orbits [22].
2.3. Tissue Processing and Staining The retrieved human samples and mice calvaria were processed with undecalcified hard-tissue techniques and neutral EDTA solution for 12 hours in an ultrasonic decalcifying bath (Medite USE 33, MEDITE GmbH, Burgdorf, Germany). Tissue samples were cut from the mice calvaria in the coronal plane from the centre of the area where the particles were deposited. Sections were cut to a thickness of 2 µm and mounted on protein-coated glass slides. After dewaxing in xylene and rehydration in a series of alcohols, Hematoxylin Eosin (HE), TRAP and immunohistochemical staining as well as TUNEL reaction were performed according to the manufacturer’s instructions (Suppl.). Since apoptotic markers have already been analyzed in human capsules and interface membranes of patients with and without aseptic loosening in prior studies [2, 3], BAK, Bcl-2, caspase-3 cleaved, BAD and FAS/CD95 were only stained in the mice calvaria samples. Adiponectin, AdipoR1, AdipoR2 and Calreticulin stainings were performed in both the human and mice samples. For double immunohistochemistry (IHC) we used the same antibodies against caspase3 cleaved and APN as for single staining. Firstly, caspase-3 labeling with an immunohistochemical staining technique based on a horseradish peroxidase (HRP) labeled polymer that is conjugated to secondary antibodies (ZytoChemPlus HRP Polymer Kit, Zytomed Systems) was performed. Staining was completed by incubation with 3,3’diaminobenzidine (DAB)+ substrate-chromogen (Zytomed Systems). Secondly, adiponectin
labeling was performed with an alkaline phosphatase (AP) labeled polymer (ZytoChemPlus AP Polymer Kit, Zytomed Systems) and developed with a Permanent Red chromogenic substrate system (Zytomed Systems). At the end of the procedure nuclei were counterstained with hematoxylin (Hämalaun Mayer, Merck, Darmstadt Germany) for one minute.
2.4. Histological assessment to detect the presence of adiponectin and its receptors, apoptotic and inflammatory reactions in human and animal samples The sections were coded and blinded prior to analysis. Four independent observers, a consultant pathologist (F.G.), a consultant orthopaedic surgeon (S.L.), a biologist (M.S.) and a postgraduate student (doctoral candidate S.P.) specially trained in histology for this project, evaluated the stainings under a light microscope at a magnification of 400x. The results were equalized by a consensus read-out. Macrophages and giant cells were identified according to histomorphological criteria. In case of doubt, immunohistochemical staining with CD68 was applied to identify macrophages and giant cells (data not shown). The sections were scored semi-quantitatively for the respective immunohistochemical reactions using a modified intensity percentage score (IP-score) as used by Waltregny et al. and van den Brule et al. [23, 24]. In the caspase-3 cleaved / globular adiponectin double-stained sections the number of macrophages and giant cells without a positive result, but with positivity for caspase-3 cleaved as well as for adiponectin, respectively positive for just one of the two markers, were counted separately in five fields of vision. We then determined the percentage ratio of these four staining groups. The human sections were scored semi-quantitatively for the presence of microscopicsized polyethylene particles and metal particles (for example titanium) on a 0-5 score: 0 = no particles, 1 = small number of particles in only a few regions, 2 = several particles in some
regions, 3 = quite a large number of particles in a large number of regions, 4 = many particles in most regions, 5 = many particles in the whole tissue.
2.5. Assessment of osteolysis in the animal model and the influence of adiponectin Using the standard high-quality light microscope Nikon Eclipse 80i (Nikon, Düsseldorf, Germany) the HE stained specimens were photographed with the digital camera Nikon CCD1300 (Nikon, Düsseldorf, Germany). Histomorphometric measurements were performed as described previously with a system consisting of a PC and image analysis software NIS Elements AR 3.0 (Nikon, Düsseldorf, Germany) [5, 25]. We measured osteolysis and calculated the relative ratio of osteolysis to the unaffected bone and determined the suture size, the number of osteoclasts per high power field and the area of inflammation around the particles. The mean values per animal of each available section (min. 2 to max. 4 sections per animal) were calculated. In addition, a high-resolution micro CT (Skyscan 1072; Skyscan, Aartselaar, Belgium) was used for qualitative and quantitative analyses of murine calvarial bone. Previous studies by our group reported that this method is suitable for obtaining evidence of the degree of osteolysis in mouse skulls [5] [26]. We used the protocol described in these studies. To accurately quantify the micro-architecture of mouse skulls, we employed CT-Analyser (SkyScan, Aartselaar, Belgium) which enabled a three-dimensional analysis and calculation of specific morphometric parameters including Bone Volume (BV). To produce a visual representation of the results, images of the mouse skulls were developed using CT-Volume and Analyze software (BIR, Mayo Clinic, USA).
2.6. Cell-culture model to assess the influence of adiponectin on wear-particle phagocytizing macrophages, expression of adiponectin associated receptors, apoptosis and inflammation
To initiate cell differentiation of human monocyte cells (THP-1) (American Type Culture Collection, USA) into macrophages, cells were placed at a concentration of 2 x 104 in six-well plates filled with a growth medium containing RPMI-1640 classic cell culture medium (PAA) supplemented with 10% heat-inactivated FCS, 100U/ml penicillin, 0.1mg/ml streptomycin and 2mM L-glutamine and addition of Phorbol-12-myristat-13-acetat (PMA; 8 nM in DMSO) for 24 to 48h at 37°C. Further incubation was performed in 12-well culture plates (Greiner, Germany) of the following: (a) Differentiated cells without particles; (b) differentiated cells with particles and without APN; (c) differentiated cells with particles and with APN. (density of 200,000 cells per well in 2 mL of the growth medium (10% FCS)) + wear particles without APN (R&D) respectively with 0.25ng/ml or 0.50ng/ml APN. The wear particles were tested using titanium particles by Continuum Blue Technology (Caerfilly, UK) incubated in ethanol for one hour. Ninety percent of the particles were less than 1µm in diameter. Non-cytotoxicity and an endotoxin content of less than 0.25 EU/mg were guaranteed by the manufacturer. Five milligrams of the particles were re-suspended in 500µl RPMI and transferred into 10ml RPMI. 200µl of the suspension was placed in 6-well plates before the cells were added. The different apoptotic and inflammatory markers were analyzed by quantitative real time PCR using Hypoxanthin-Phosphoribosyl-Transferase 1 (HPRT1) as housekeeping gene after 24h incubation time because the highest levels of tumor necrosis factor alpha (TNFα) and other inflammatory reactions were previously observed in cell culture models of macrophages cultured with particles between 18 and 24 h [27]. 2.7. Statistical Analysis Summary statistics of data were expressed as mean
SD. The Kolmogorov-Smirnov-
Test was used to test for normal distribution of the data (p≤ 0.05 => not normal distribution). The normally distributed data was tested with the Student`s t-test and the non-normally
distributed data with the u-test. A p-value of 0.05 was considered as statistically significant. The software SPSS 19 (IBM, Ehningen, Germany) was used to carry out the statistical computations.
3. Results 3.1. Expression of adiponectin and its related receptors in patients with and without aseptic loosening of a hip replacement Samples from patients with aseptic loosening of a hip implant showed varying quantities of wear debris, including metal and polyethylene particles (Table 1), and a cellular infiltrate of particle-engulfing macrophages and giant cells (Figure 1). In the capsule samples from patients with osteoarthritis macrophages were seen in smaller numbers and giant cells were rarely observed. Comparison of the groups regarding the expression of APN, AdipoR1 and calreticulin in these cells revealed significantly higher IP-scores in the aseptic loosening group, and caspase-3 cleaved was detectable only in this group (Figure 1, Table 3). The interface membranes of patients with aseptic hip loosening showed staining similar to that in the capsules (Table 3). AdipoR2 was not detectable in any of the tissue samples. Determination of the caspase-3 cleaved / APN ratio by double staining revealed that beside the predominant proportion of macrophages and giant cells that was negative for both, there was a greater percentage of APN-negative / caspase-positive than caspase-negative / APNpositive and only a fractional part was positive for both (Figure 2). In view of the absence of caspase-3 cleaved and the rare and weak staining of APN, double staining was not meaningful in the group of patients without aseptic loosening.
3.2. Expression of adiponectin and its related receptors in the calvarial mice model The animals which received UHMWPE particles on the calvarium (+P) exhibited a large number of macrophages in the vicinity of the particles, whereas the histological slides of controls (sham controls -P) showed regular calvarium tissue with a small number of macrophages. Immunostaining of Group 4 WT mice samples showed, that the presence of wear particles induces a significantly increased occurrence of APN, AdipoR1 and calreticulin in macrophages in comparison to the sham controls. This finding corresponded to the results
from the human samples. The levels of APN and calreticulin were again significantly higher in Group WT+Ad+P, whereas AdipoR1 was expressed in a non-significant lower value (Figure 3A). Group KO+P mice lacked expression of APN and AdipoR1 and showed significantly lower levels of calreticulin compared to Group WT+Ad+P but not in comparison to WT-Ad+P. AdipoR2 was not detectable in any group, which again corresponded to the results from the human samples.
3.3. Attenuation of wear particle-induced apoptosis by adiponectin in the calvarial mice model of osteolysis As in the human samples, caspase-3 was significantly increased in the mice macrophages which had contact with wear particles. Furthermore, the additional TUNEL assay and apoptotic molecules BAK, BAD, FAS/CD95 and Bcl-2 were significantly upregulated (Figure 3B and 4). Comparison of the particle-treated groups with each other revealed that the samples from animals treated with APN (WT+Ad+P) had a significantly smaller number of cells with a positive TUNEL reaction and significantly lower IP scores for Bcl-2 and caspase-3 cleaved staining than samples of untreated WT and KO mice. In comparison to Group WT-Ad+P mice the differences were also significant for FAS/CD95 (Figure 3B). Comparison of the apoptotic markers between Group KO+P and WT-Ad+P showed significant differences only for Bcl-2, BAK and TUNEL. A valid number of apoptotic macrophages were found only in the mice which received UHMWPE particles, therefore determination of the caspase-3 cleaved / APN ratio was only meaningful in these groups. Most cells were negative for both APN and caspase-3 cleaved. As the cells of the KO mice lacked expression of APN, the number of caspase-negative / APNpositive and both positive cells was significantly smaller than in WT mice. Comparison of the APN-treated and -untreated WT mice regarding caspase-negative / adiponectin-positive cells and both positive cells revealed no significant differences. However, the number of caspase-3
positive / adiponectin negative macrophages and giant cells in the APN-treated group of WT+Ad+P mice was significantly decreased compared to untreated KO and WT mice (Figure 3C).
3.4. Attenuation of wear particle-induced osteolysis and inflammation by adiponectin in the calvarial mice model of osteolysis We observed significant differences for all parameters that indicate bone resorption (TRAP, histomorphometry and µ-CT) in all animals that received UHMWPE particles on the calvaria (+P) in comparison to the respective control groups (-P) (Figure 5A-D). Comparison of the particle groups revealed the best results and therefore the least bone destruction for the APN-treated WT mice (WT+Ad+P). In comparison to Group WT-Ad+P mice without application of APN all differences were significant. In comparison to the KO mice (Group KO+P) differences were also significant for histomorphometrically determined osteolysis and the osteolysis ratio, as well as for TRAP staining. The suture size and BVTV and bone volume measured by µCT revealed no significant differences. Expression of RANKL was observed in macrophages and osteoclasts and was significantly lower in Group WT+Ad+P than in Groups KO+P or WT-Ad+P (Figure 5E). The area of inflammation around the particles was significantly smaller in APNtreated mice compared to untreated WT and KO mice, which in turn did not significantly differ (Figure 5B). Therefore, administration of APN significantly attenuates UHMWPE particle-induced osteolytic and inflammatory reactions.
3.5. The role of adiponectin on wear particle-engulfing macrophages regarding expression of adiponectin associated receptors, apoptosis and inflammation in the cellculture model The interaction of macrophages with the particles induced a significantly increased expression of caspase-3, calreticulin, and TNF-α, whereas AdipoR1 did not significantly differ. Implementation of the experiment in the presence of 0.25ng/ml APN resulted in a significantly increased expression of AdipoR1 respectively significantly lower values of calreticulin, whereas the other markers remained unchanged. Interestingly, a higher dose of 0.5ng/ml APN resulted in a significant decrease in TNF-α and an increase in AdipoR1, calreticulin and caspase-3 (Figure 6).
4. Discussion The aim of the current study was to determine the role of adiponectin (APN) in particle-induced osteolysis and other accompanying effects that result in aseptic loosening of total hip replacements. The presence of polyethylene and metal debris in capsules and interface membranes from aseptically loosened total hip implants is associated with significantly increased expression of APN, AdipoR1 and calreticulin in wear particle-engulfing cells. To investigate the possible relevance of these findings for the induction of osteolysis, we additionally used a well-established murine calvarial model of wear particle-induced osteolysis [28]. We observed that the application of wear particles induces a significantly increased expression of APN, AdipoR1 and calreticulin in WT mice and even higher levels after application of APN. This implies that wear debris induces a higher requirement for APN, and if it is available in higher quantities it is also consumed by the macrophages. It is even more interesting that administration of APN counteracted wear particleinduced osteolysis in WT+Ad+P mice. This was demonstrated by all methods used to
evaluate bone resorption. However, the absence of APN in KO+P mice does not definitely result in higher bone resorption compared to WT-Ad+P mice. Consequently, only higher levels of APN were beneficial. One indication for the protective effect of APN might be the decrease in particle-induced inflammation. In our animal model we found a significantly smaller inflammatory area and in the cell culture model a lower TNF- level after administration of APN. This is a highly interesting observation as periprosthetic osteolysis is initiated by an aseptic inflammatory response to phagocytosis of implant wear particles which results in increased proliferation and differentiation of osteoclast precursors into mature osteoclasts [1]. The anti-inflammatory effect of APN has also been observed in other diseases, e.g. diabetes, coronary and liver diseases [29]. Beside the inflammatory reactions we also focused on the influence of APN on apoptosis, which is another factor responsible for wear particle-induced osteolysis [5]. The animal model supports our previous observations in patients with aseptic hip loosening, as wear particles activate the apoptotic pathway including the common key regulatory checkpoint caspase-3 [2]. Double staining of samples from patients with aseptic loosening as well as from the mice calvaria that received wear particles showed that caspase-3 cleaved positive macrophages were mostly negative for APN. APN administration leads to a decrease in these cells, whereas the numbers of cells that are positive for APN and caspase-3 remain at a low level. We assume that APN-positive apoptotic cells are removed quickly. Accordingly, double staining with caspase-3 cleaved and APN of the human specimens revealed only a small portion of caspase-3 cleaved negative and APN-positive cells and a considerably higher number of APN-negative / caspase-3 cleaved positive cells. The removal of apoptotic cells by adiponectin depends on calreticulin [15], which is also detectable in large quantities in the tissues of patients with aseptic loosening and is increased by APN application in animal and cell culture. Therefore, the removal of apoptotic bodies by contributing calreticulin may be a
major task of APN. Consequently, the lower levels of caspase-3 and TUNEL-positive cells in the calvaria model after administration of APN may be an expression of faster resorption of apoptotic cell debris. Promoting the clearance of early apoptotic cells by phagocytizing cells in the presence of calreticulin is important in that if the phagocytic clearance system is impaired or overwhelmed by massive waves of apoptosis, uncleared apoptotic cell remnants could undergo secondary necrosis which results in release of their inflammatory and cytotoxic contents [30-32]. The increased excretion of cytokines may in turn be responsible for the induction of osteolysis [33]. We observed that decreased levels of FAS/CD95 after administration of APN and direct down-regulation of the death receptor FAS/CD95 by binding of APN result in inhibition of the key regulator caspase-3, a phenomenon which has also been described in studies on other diseases [34]. The results in the mice model also suggest that APN has antiapoptotic characteristics. However, we also observed increased expression of AdipoR1 in the human, animal and cell culture models which could have anti-apoptotic as well as proapoptotic consequences [34]. The strength of AdipoR1 expression depends on the presence of wear particles as well as on the APN value. In cell culture pro-apoptotic effects were associated with higher doses of APN. As calreticulin increases simultaneously and TNFdecreases, we suppose that calreticulin compensates for a possibly enhanced formation of apoptotic bodies. This would explain why pro-apoptotic influences were not detectable in the mice model. Pro-apoptotic effects could be also induced by enhancement of the anti-apoptotic bcl-2, the level of which decreases as the level of APN increases. The influence of APN on bcl-2 and the resulting apoptosis were also observed in other studies [35]. The different regulatory effects of APN on wear particle-induced apoptosis may also explain why wear particles do not induce higher osteolysis, inflammation and apoptosis in KO mice than in WT mice without administration of APN.
Finally, the protective effect of adiponectin against wear particle-induced osteolysis, beside regulating inflammatory and apoptotic pathways, may be due to the inhibition of osteoclastogenesis and down-regulation of RANKL-enhanced expressions of osteoclastogenic regulators including NFAT2, TRAF6, cathepsin K, and tartrate-resistant acid phosphatase [36]. In fact, decreased RANKL expression was also observed in this study. In conclusion, this is the first study which demonstrates that APN inhibits the development of particle-induced osteolysis by enhancing the consequences of particleinduced apoptosis. Its anti-inflammatory and possibly anti-RANKL effect also contribute to this process. It is not possible to clarify all aspects of the role of APN in aseptic loosening in one study, therefore further research will be necessary to verify the dose-dependent effects of APN in connection with aseptic hip loosening and determine the optimal level of APN. In conclusion, a high serum level of APN seems to be beneficial.
Disclosures All authors state that they have no conflicts of interest related to the work reported in the manuscript.
Acknowledgements This study was supported by a Grant from the German Research Foundation (DFG): LA 2619/3-1.
We thank Mrs. Nicole Cramer, Mrs. Claudia Lodewick and Mrs. Sara Lask for technical assistance.
Figure Legends Fig. 1: A small number of macrophages and giant cells was detected in the capsules of patients without aseptic loosening. They were only sporadically positive for APN, AdipoR1 and calreticulin, whereas in the samples from patients with aseptic loosening the staining for these markers was frequent and intensive. The images show AdipoR1 as an example.
Fig. 2: (A) Double staining of the joint capsules with adiponectin (red) and caspase-3 cleaved (brown) revealed that beside a predominant proportion that was negative for both, a greater percentage was positive for caspase-3 cleaved but not for adiponectin. The vessel-cells serve as internal control. Magnifications are at original x 1000. (B) However, a representative proportion of APN-positive cells was also visible, whereby only a fractional part was positive for both caspase-3 cleaved and APN. (C) The bar diagram shows the relative distribution of the afore-mentioned double-stained cells in capsules and interface membranes (C: caspase-3 cleaved / A: APN).
Fig. 3: (A) In comparison to the control groups, the administration of UHMWPE particles led to a stronger reaction of APN, AdipoR1 and calreticulin in Group WT-Ad+P. After administration of APN, the levels of APN and calreticulin were again significantly higher in Group WT+Ad+P, whereas AdipoR1 was expressed in a non-significant lower value. Group KO+P mice lacked expression if APN and AdipoR1 and showed significantly lower levels of calreticulin compared to Group WT+Ad+P, but not in comparison to Group WT-Ad+P. (B) Immunohistochemical analysis revealed activation of the apoptotic parameters BAK, BAD, Bcl-2, FAS/CD95 and Caspase-3 only in samples from the mice which received particles on the calvaria. Only Bcl-2, Caspase-3 and FAS/CD95 were significantly decreased by APN. (*=p<0.05, **=<0.01, ***=<0.001).
(C) Double staining revealed a significantly smaller number of caspase-3 positive / adiponectin negative macrophages in APN-treated WT mice in comparison to the untreated KO and WT mice. The KO mice lacked expression of adiponectin positive cells. The other combinations, did not significantly differ between the groups which received UMHWPE particles. (*=p<0.05, **=<0.01, ***=<0.001).
Fig. 4: TUNEL reaction showed a highly significant particle-induced increase in apoptosis in Group KO+P, Group WT-Ad+P and Group WT+Ad+P in comparison to the sham controls. In comparison to the non-specifically treated WT and KO mice there was a significant decrease after administration of APN in Group WT+AD+P. (*=p<0.05, **=<0.01, ***=<0.001).
Fig. 5: (A) Histomorphometry of the Hematoxylin Eosin (HE) stained coronal sections of calvaria at the sagittal suture showed that addition of UHMWPE particles without any further specific intervention induced pronounced bone resorption in the WT and KO mice compared to the sham controls. Treatment with APN diminished these adverse UHMWPE particlemediated effects and led to a smaller medial suture area (*) and decreased osteolysis ( ) in the WT+Ad+P mice in comparison to the untreated KO and WT-mice. (B) The effect of APN was quantitatively assessed by determining osteolysis and the ratio of osteolysis to the unaffected bone. It was significantly lower in comparison to the untreated KO and WT mice. The evaluation of the midline suture size revealed better results for the APN-treated mice only in comparison to the WT mice. The size of the inflammatory area around the particles was also significantly smaller in comparison to both groups. (*=p<0.05, **=<0.01, ***=<0.001). (C) TRAP staining was only positive in the particle-treated groups and showed a significantly smaller number of osteoclasts in the APN-treated animals than in the untreated WT or KO
mice. The images show representative samples of WT-Ad+P and WT+Ad+P. (*=p<0.05, **=<0.01, ***=<0.001). (D) Micro-CT showed pronounced bone resorption in the UHMWPE particle-treated groups compared to the sham control groups. Comparison of the particle groups with each other revealed a significantly larger bone volume and BVTV ratio in the APN-treated mice than in the non-treated WT or KO mice. The examples of the 3D pictures show that the skull surface of the animals that underwent particle implantation and were treated with APN appears less lacerated than the skulls of the untreated mice. (*=p<0.05, **=<0.01, ***=<0.001). (E) RANKL staining showed significantly lower staining of macrophages and osteoclasts in the APN-treated animals in comparison to the untreated KO or WT mice. (*=p<0.05, **=<0.01, ***=<0.001).
Fig. 6: The cell culture model showed that addition of particles to the macrophages induced a significantly increased expression of caspase-3, calreticulin and TNF-α, whereas AdipoR1 did not differ significantly. Implementation of the experiment in the presence of 0.25ng/ml APN leads to a significantly increased expression of AdipoR1, respectively significantly lower values of calreticulin, whereas the other markers remained unchanged. Administration of 0.5ng/ml APN leads to a significant decrease in TNF-α and an increase in AdipoR1, calreticulin and caspase-3. (*=p<0.05, **=<0.01, ***=<0.001).
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[20] von Knoch M, Jewison DE, Sibonga JD, Sprecher C, Morrey BF, Loer F, et al. The effectiveness of polyethylene versus titanium particles in inducing osteolysis in vivo. J Orthop Res 2004;22:237-43. [21] Merkel KD, Erdmann JM, McHugh KP, Abu-Amer Y, Ross FP, Teitelbaum SL. Tumor necrosis factor-alpha mediates orthopedic implant osteolysis. Am J Pathol 1999;154:203-10. [22] Schwarz EM, Lu AP, Goater JJ, Benz EB, Kollias G, Rosier RN, et al. Tumor necrosis factor-alpha/nuclear transcription factor-kappaB signaling in periprosthetic osteolysis. J Orthop Res 2000;18:472-80. [23] van den Brule FA, Waltregny D, Liu FT, Castronovo V. Alteration of the cytoplasmic/nuclear expression pattern of galectin-3 correlates with prostate carcinoma progression. Int J Cancer 2000;89:361-7. [24] Waltregny D, Bellahcene A, Van Riet I, Fisher LW, Young M, Fernandez P, et al. Prognostic value of bone sialoprotein expression in clinically localized human prostate cancer. J Natl Cancer Inst 1998;90:1000-8. [25] Parfitt AM. Bone histomorphometry: proposed system for standardization of nomenclature, symbols, and units. Calcif Tissue Int 1988;42:284-6. [26] Wedemeyer C, Xu J, Neuerburg C, Landgraeber S, Malyar NM, von Knoch F, et al. Particle-induced osteolysis in three-dimensional micro-computed tomography. Calcif Tissue Int 2007;81:394-402. [27] Smith RA, Hallab NJ. In vitro macrophage response to polyethylene and polycarbonateurethane particles. J Biomed Mater Res A 2010;93:347-55. [28] von Knoch M, Jewison DE, Sibonga JD, Sprecher C, Morrey BF, Loer F, et al. The effectiveness of polyethylene versus titanium particles in inducing osteolysis in vivo. J Orthop Res 2004;22:237-43. [29] Wree A, Kahraman A, Gerken G, Canbay A. Obesity affects the liver - the link between adipocytes and hepatocytes. Digestion 2011;83:124-33. [30] Savill J, Haslett C. Granulocyte clearance by apoptosis in the resolution of inflammation. Semin Cell Biol 1995;6:385-93. [31] Wang L, Antonini JM, Rojanasakul Y, Castranova V, Scabilloni JF, Mercer RR. Potential role of apoptotic macrophages in pulmonary inflammation and fibrosis. J Cell Physiol 2003;194:215-24. [32] Canbay A, Guicciardi ME, Higuchi H, Feldstein A, Bronk SF, Rydzewski R, et al. Cathepsin B inactivation attenuates hepatic injury and fibrosis during cholestasis. J Clin Invest 2003;112:152-9. [33] Wang JT. The role of particulate orthopaedic implant materials in peri-implant osteolysis. In: Morrey BF, editor. Biological, material, and mechanical considerations of joint replacement. New York: Raven Press, Ltd.; 1993. p. 122. [34] Sun Y, Xun K, Wang C, Zhao H, Bi H, Chen X, et al. Adiponectin, an unlocking adipocytokine. Cardiovasc Ther 2009;27:59-75. [35] Akifusa S, Kamio N, Shimazaki Y, Yamaguchi N, Nishihara T, Yamashita Y. Globular adiponectin-induced RAW 264 apoptosis is regulated by a reactive oxygen species-dependent pathway involving Bcl-2. Free Radic Biol Med 2009;46:1308-16. [36] Tu Q, Zhang J, Dong LQ, Saunders E, Luo E, Tang J, et al. Adiponectin inhibits osteoclastogenesis and bone resorption via APPL1-mediated suppression of Akt1. J Biol Chem 2011;286:12542-53.
Table 1: Patient Characteristics
Characteristics
Age (years) Median Range
No Arthroplasty
64.8 44 to 75
Aseptic loosening
72.9 45 to 93
Sex Male Female
6 10
19 24
Loosening of Acetabulum only Stem only Both
22 12 9
Cemented Fixation No Stem only Acetabulum and Stem
23 3 17
Survivorship of endoprosthesis (years) Median Range
11.98 1 to 24
Extent of osteolysis, acetabular (Paprosky score) 1 2a 2b 2c 3a 3b
3 7 8 5 2 6
Extent of osteolysis, femoral (Paprosky score) 1 2a 2b 2c 3
4 2 7 4 4
Polyethylene debris (semi-quantitative score) Median Range
2.0 1 to 3
Metal debris (semi-quantitative score) Median Range
2.1 1 to 5
Table 2: Animal Model (Groups) Group KO-P KO+P WT-Ad-P WT-Ad+P WT+Ad-P WT+Ad+P
Mice Knockout Knockout Wildtype Wildtype Wildtype Wildtype
Particles No Yes No Yes No Yes
Adiponectin No No No No Yes Yes
Table 3: Humans No Arthroplasty Capsules APN AdipoR1 Calreticulin Caspase-3 cleaved Interface membranes APN AdipoR1 Calreticulin Caspase-3 cleaved
0.25 (0 to 1; SD 0.43) 0.25 (0 to 1; SD 0.43) 0.25 (0 to 1; SD 0.43) 0
Aseptic Loosening 2.0 (1 to 4; SD 1.04) 4.37 (1 to 12; SD 2.49) 5.59 (1 to 9; SD 2.75) 2.80 (1 to 12; SD 2.49)
2.1 (1 to 4; SD 0.78) 4.96 (1 to 9; SD 2.32) 4.93 (1 to 9; SD 2.95) 3.18 (1 to 9; SD 2.32).
Results are given as mean with range and standard deviation (SD)
p-value <0.001 <0.001 <0.001 <0.001
S1742-7061(13)00423-6 http://dx.doi.org/10.1016/j.actbio.2013.08.031 ACTBIO 2876
To appear in:
Acta Biomaterialia
Received Date: Revised Date: Accepted Date:
18 June 2013 16 August 2013 20 August 2013
Please cite this article as: Landgraeber, S., Putz, S., Schlattjan, M., Bechmann, L.P., Totsch, M., Grabellus, F., Hilken, G., Jäger, M., Canbay, A., Adiponectin Attenuates Osteolysis in Aseptic Loosening of Total Hip Replacements, Acta Biomaterialia (2013), doi: http://dx.doi.org/10.1016/j.actbio.2013.08.031
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Adiponectin Attenuates Osteolysis in Aseptic Loosening of Total Hip Replacements Stefan Landgraeber1*, S. Putz1, M. Schlattjan2, Lars P. Bechmann2, Martin Totsch3, 4, Florian Grabellus3, Gero Hilken5, M. Jäger1, A. Canbay2
1
Department of Orthopaedics, University Hospital Essen, University of Duisburg-Essen, Hufelandstr. 55, 45122 Essen, Germany
2
Department of Gastroenterology and Hepatology, University Hospital Essen, University of Duisburg-Essen, Germany
3
Institute of Pathology and Neuropathology, University Hospital Essen, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany
4
Institute of Cytology, University Hospital of Graz, University of Graz, Auenbruggerplatz 20, 8036 Graz, Austria
5
Central Animal Laboratory, University Hospital Essen, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany
*Corresponding Author: Stefan Landgraeber, M.D. Department of Orthopaedics University of Duisburg-Essen Hufelandstr. 55 45122 Essen Germany Phone +49-201-4089-2146 Fax +49-201-4089-2722 Email [email protected] Running Title: Adiponectin and apoptosis in aseptic hip loosening
Abbreviations used: APN: Adiponectin; AdipoR: Adiponectin receptor; HMDM: Human monocyte derived macrophages; KO: Knockout; P: Particles; WT: Wildtype
Abstract Background: Joint replacements have a longer durability in patients with high serum levels of adiponectin (APN) than in patients with low levels. We aimed to characterize the unknown pathophysiological effects of APN on wear particle-induced inflammation, apoptosis and osteolysis. Methods: Immunohistochemistry was performed to detect APN, its receptors and apoptosis in patients with and without aseptic loosening. Additionally, APN knockout mouse (KO) studies and pharmacological intervention of APN were performed in an established calvarial mice model. Osteolysis and inflammation were quantified by histomorphometry and micro-CT, apoptosis by immunohistochemistry and TUNEL assay. In a cell culture model human monocyte derived macrophages (HMDM) were incubated with or without metal wear debris particles and partially treated with APN. Results: Expression of APN, AdipoR1 and calreticulin in specimens from patients with aseptic loosening were significantly higher than in patients without aseptic loosening. Administration of APN in mice significantly reduced wear particle-induced inflammation, osteolysis and the number of caspase-3 positive macrophages. The cell culture model showed that APN leads to significantly lower values of TNF-α. Conclusion: These findings support a prominent role of APN in the development of particle-induced osteolysis and APN may be therapeutically useful in patients with aseptic loosening.
1. Introduction Periprosthetic osteolysis is a serious long-term complication after hip joint replacement which can lead to implant instability and failure. It is initiated by an aseptic inflammatory response to phagocytosis of implant wear particles which results in increased proliferation and differentiation of osteoclast precursors into mature osteoclasts [1]. The complexity and relevance of this topic are reflected by the large number of published studies on particleinduced bioreactivity and by the increasing number of hip replacement procedures. The osteolytic cascade initiated by cytokine release from macrophages has been studied extensively. However, knowledge about the role of apoptosis in this context is limited. Previous studies have demonstrated that apoptosis in macrophages and giant cells during particle engulfment is partially responsible for osteolysis in aseptic loosening of joint implants [2-4]. We demonstrated by means of a murine calvarial model of wear particle-induced osteolysis that inhibition of apoptosis leads to decreasing bone resorption by osteoclasts [5]. This paradoxical effect of apoptosis may be due to apoptotic cell remnants which induce inflammation unless they are broken down within an adequate period of time [6-8]. Adiponectin (APN), an adipocytokine, has been found to influence apoptosis and inhibit inflammation. It also plays a role in glucose utilization, lipid synthesis, insulin sensitivity, and energy homeostasis in several mammalian species [9-11]. In a previous study we demonstrated that aseptic loosening in the first ten years after implantation is associated with decreased serum adiponectin [12]. So far, three receptors for adiponectin have been discovered: Adiponectin receptor 1 (AdipoR1), adiponectin receptor 2 (AdipoR2), and Tcadherin. AdipoR1 is abundant in the skeletal muscle, whereas AdipoR2 is predominantly expressed in the liver [13, 14]. The third receptor is T-cadherin, expressed on endothelial and muscle cells. T-cadherin lacks a cytoplasmatic domain and is presumed to act only as a coreceptor [9]. Interestingly, the binding of adiponectin to calreticulin on the cell surface of macrophages increases the removal of apoptotic cell remnants [15]. It is well known that
calreticulin plays an important role in the uptake of apoptotic debris through its interactions with CD91. This process is independent of the specific adiponectin receptors AdpoR1 and AdipoR2 [15-17]. The aim of the current study was to elucidate whether (i) adiponectin is present in capsules and interface membranes of patients with aseptic hip prosthesis loosening, (ii) expression of AdipoRs and calreticulin is induced by wear particle-phagocytizing cells, (iii) adiponectin influences wear particle-induced apoptosis, (iv) inflammatory response and (v) osteolysis. To answer these questions we performed studies with human samples, cell culture and used an established mice model. The hypothesis of this study is that APN decreases the amount of wear particle-induced osteolysis through its influence on apoptotic and inflammatory pathways.
2. Materials and Methods 2.1. Human Samples Tissue specimens from forty-three patients treated at the Department of Orthopaedics of the University of Duisburg-Essen for aseptic loosening of a total hip replacement were investigated. The specimens were harvested from the newly formed hip joint capsules as well as from the interface membranes of the acetabular and femoral periprosthetic regions after removal of the loosened prostheses. None of the patients had previously undergone implant exchange. The articulations of all the loosened prostheses were a metal femoral head and a polyethylene cup. The reason for revision surgery was aseptic loosening of a hip implant. The presence of infection was excluded by analysis of inflammatory markers in the blood (Creactive protein, leucocytosis), by culture of joint fluid obtained by aspiration from the hip joint for twenty-one days and by histology of hip joint capsules and interface membrane. Bone defects were classified according to Paprosky's method [18].
Specimens from sixteen patients with primary osteoarthritis retrieved during primary total hip arthroplasty for osteoarthritis at the Department of Orthopaedics of the University of Duisburg-Essen served as control groups. Some of these samples were used in a former study [19]. The study was approved by the local Ethics Committee. Details are given Table 1.
2.2. Animal Model of Particle-Induced Osteolysis A well-established calvarial model of ultra-high-molecular-weight polyethylene (UHMWPE) particle-induced osteolysis recently introduced by our group [20] and based on the original model of calvarial osteolysis [21] was utilized in 28 specific-pathogen-free 12week-old male C57BL/6J wildtype mice (WT) (Harlan Laboratories, NM Horst, Netherlands) and 14 adiponectin knockout (KO) mice on a C57BL/6 (The Jackson Laboratory, Bar Harbor, Maine, USA) background. The animals were treated according to official guidelines and the experiments were approved by the university and the local government. A 1.0 x 1.0 cm area of periosteum was exposed by a 10 mm midline sagittal incision over the calvarium anterior to the line connecting both external ears. In the sham controls (-P) the incision was closed without any further intervention. In the other animals the exposed periosteum was covered uniformly with 30 µl dried pure UHMWPE polyethylene particles (+P) at a concentration of 2 x 108 particles/ml after decontamination from endotoxins (Ceridust VP 3,610, Clariant, Gersthofen, Germany). The KO mice were randomized equally into two groups, KO-P and KO+P, consisting of seven mice each. The group size of seven was determined in a power analysis indicating that for a two-sided test at alpha = 0.05 there was 85% power to detect a significant difference between the groups. The adiponectin levels of the WT mice in groups WT+Ad-P and WT+Ad+P were enhanced by daily intraperitoneal injection of APN (Fa. PROSPEC, East Brunswick, USA), at a dose of 2 mg/kg body weight, starting from one day before surgery until sacrifice. During surgery an additional dose of APN was injected into the tissue next to the incision before implantation of the particles. The WT-Ad-P and WT-Ad+P
groups represented the WT group with a normal APN value and received, like the KO mice, a daily i.p. injection of phosphate-buffered saline instead of APN. The WT were also randomized into groups (Table 2). Twelve days postoperatively, the animals were sacrificed and an elliptical plate of the calvarial caps was removed from the region between the foramen magnum, auditory canals, and orbits [22].
2.3. Tissue Processing and Staining The retrieved human samples and mice calvaria were processed with undecalcified hard-tissue techniques and neutral EDTA solution for 12 hours in an ultrasonic decalcifying bath (Medite USE 33, MEDITE GmbH, Burgdorf, Germany). Tissue samples were cut from the mice calvaria in the coronal plane from the centre of the area where the particles were deposited. Sections were cut to a thickness of 2 µm and mounted on protein-coated glass slides. After dewaxing in xylene and rehydration in a series of alcohols, Hematoxylin Eosin (HE), TRAP and immunohistochemical staining as well as TUNEL reaction were performed according to the manufacturer’s instructions (Suppl.). Since apoptotic markers have already been analyzed in human capsules and interface membranes of patients with and without aseptic loosening in prior studies [2, 3], BAK, Bcl-2, caspase-3 cleaved, BAD and FAS/CD95 were only stained in the mice calvaria samples. Adiponectin, AdipoR1, AdipoR2 and Calreticulin stainings were performed in both the human and mice samples. For double immunohistochemistry (IHC) we used the same antibodies against caspase3 cleaved and APN as for single staining. Firstly, caspase-3 labeling with an immunohistochemical staining technique based on a horseradish peroxidase (HRP) labeled polymer that is conjugated to secondary antibodies (ZytoChemPlus HRP Polymer Kit, Zytomed Systems) was performed. Staining was completed by incubation with 3,3’diaminobenzidine (DAB)+ substrate-chromogen (Zytomed Systems). Secondly, adiponectin
labeling was performed with an alkaline phosphatase (AP) labeled polymer (ZytoChemPlus AP Polymer Kit, Zytomed Systems) and developed with a Permanent Red chromogenic substrate system (Zytomed Systems). At the end of the procedure nuclei were counterstained with hematoxylin (Hämalaun Mayer, Merck, Darmstadt Germany) for one minute.
2.4. Histological assessment to detect the presence of adiponectin and its receptors, apoptotic and inflammatory reactions in human and animal samples The sections were coded and blinded prior to analysis. Four independent observers, a consultant pathologist (F.G.), a consultant orthopaedic surgeon (S.L.), a biologist (M.S.) and a postgraduate student (doctoral candidate S.P.) specially trained in histology for this project, evaluated the stainings under a light microscope at a magnification of 400x. The results were equalized by a consensus read-out. Macrophages and giant cells were identified according to histomorphological criteria. In case of doubt, immunohistochemical staining with CD68 was applied to identify macrophages and giant cells (data not shown). The sections were scored semi-quantitatively for the respective immunohistochemical reactions using a modified intensity percentage score (IP-score) as used by Waltregny et al. and van den Brule et al. [23, 24]. In the caspase-3 cleaved / globular adiponectin double-stained sections the number of macrophages and giant cells without a positive result, but with positivity for caspase-3 cleaved as well as for adiponectin, respectively positive for just one of the two markers, were counted separately in five fields of vision. We then determined the percentage ratio of these four staining groups. The human sections were scored semi-quantitatively for the presence of microscopicsized polyethylene particles and metal particles (for example titanium) on a 0-5 score: 0 = no particles, 1 = small number of particles in only a few regions, 2 = several particles in some
regions, 3 = quite a large number of particles in a large number of regions, 4 = many particles in most regions, 5 = many particles in the whole tissue.
2.5. Assessment of osteolysis in the animal model and the influence of adiponectin Using the standard high-quality light microscope Nikon Eclipse 80i (Nikon, Düsseldorf, Germany) the HE stained specimens were photographed with the digital camera Nikon CCD1300 (Nikon, Düsseldorf, Germany). Histomorphometric measurements were performed as described previously with a system consisting of a PC and image analysis software NIS Elements AR 3.0 (Nikon, Düsseldorf, Germany) [5, 25]. We measured osteolysis and calculated the relative ratio of osteolysis to the unaffected bone and determined the suture size, the number of osteoclasts per high power field and the area of inflammation around the particles. The mean values per animal of each available section (min. 2 to max. 4 sections per animal) were calculated. In addition, a high-resolution micro CT (Skyscan 1072; Skyscan, Aartselaar, Belgium) was used for qualitative and quantitative analyses of murine calvarial bone. Previous studies by our group reported that this method is suitable for obtaining evidence of the degree of osteolysis in mouse skulls [5] [26]. We used the protocol described in these studies. To accurately quantify the micro-architecture of mouse skulls, we employed CT-Analyser (SkyScan, Aartselaar, Belgium) which enabled a three-dimensional analysis and calculation of specific morphometric parameters including Bone Volume (BV). To produce a visual representation of the results, images of the mouse skulls were developed using CT-Volume and Analyze software (BIR, Mayo Clinic, USA).
2.6. Cell-culture model to assess the influence of adiponectin on wear-particle phagocytizing macrophages, expression of adiponectin associated receptors, apoptosis and inflammation
To initiate cell differentiation of human monocyte cells (THP-1) (American Type Culture Collection, USA) into macrophages, cells were placed at a concentration of 2 x 104 in six-well plates filled with a growth medium containing RPMI-1640 classic cell culture medium (PAA) supplemented with 10% heat-inactivated FCS, 100U/ml penicillin, 0.1mg/ml streptomycin and 2mM L-glutamine and addition of Phorbol-12-myristat-13-acetat (PMA; 8 nM in DMSO) for 24 to 48h at 37°C. Further incubation was performed in 12-well culture plates (Greiner, Germany) of the following: (a) Differentiated cells without particles; (b) differentiated cells with particles and without APN; (c) differentiated cells with particles and with APN. (density of 200,000 cells per well in 2 mL of the growth medium (10% FCS)) + wear particles without APN (R&D) respectively with 0.25ng/ml or 0.50ng/ml APN. The wear particles were tested using titanium particles by Continuum Blue Technology (Caerfilly, UK) incubated in ethanol for one hour. Ninety percent of the particles were less than 1µm in diameter. Non-cytotoxicity and an endotoxin content of less than 0.25 EU/mg were guaranteed by the manufacturer. Five milligrams of the particles were re-suspended in 500µl RPMI and transferred into 10ml RPMI. 200µl of the suspension was placed in 6-well plates before the cells were added. The different apoptotic and inflammatory markers were analyzed by quantitative real time PCR using Hypoxanthin-Phosphoribosyl-Transferase 1 (HPRT1) as housekeeping gene after 24h incubation time because the highest levels of tumor necrosis factor alpha (TNFα) and other inflammatory reactions were previously observed in cell culture models of macrophages cultured with particles between 18 and 24 h [27]. 2.7. Statistical Analysis Summary statistics of data were expressed as mean
SD. The Kolmogorov-Smirnov-
Test was used to test for normal distribution of the data (p≤ 0.05 => not normal distribution). The normally distributed data was tested with the Student`s t-test and the non-normally
distributed data with the u-test. A p-value of 0.05 was considered as statistically significant. The software SPSS 19 (IBM, Ehningen, Germany) was used to carry out the statistical computations.
3. Results 3.1. Expression of adiponectin and its related receptors in patients with and without aseptic loosening of a hip replacement Samples from patients with aseptic loosening of a hip implant showed varying quantities of wear debris, including metal and polyethylene particles (Table 1), and a cellular infiltrate of particle-engulfing macrophages and giant cells (Figure 1). In the capsule samples from patients with osteoarthritis macrophages were seen in smaller numbers and giant cells were rarely observed. Comparison of the groups regarding the expression of APN, AdipoR1 and calreticulin in these cells revealed significantly higher IP-scores in the aseptic loosening group, and caspase-3 cleaved was detectable only in this group (Figure 1, Table 3). The interface membranes of patients with aseptic hip loosening showed staining similar to that in the capsules (Table 3). AdipoR2 was not detectable in any of the tissue samples. Determination of the caspase-3 cleaved / APN ratio by double staining revealed that beside the predominant proportion of macrophages and giant cells that was negative for both, there was a greater percentage of APN-negative / caspase-positive than caspase-negative / APNpositive and only a fractional part was positive for both (Figure 2). In view of the absence of caspase-3 cleaved and the rare and weak staining of APN, double staining was not meaningful in the group of patients without aseptic loosening.
3.2. Expression of adiponectin and its related receptors in the calvarial mice model The animals which received UHMWPE particles on the calvarium (+P) exhibited a large number of macrophages in the vicinity of the particles, whereas the histological slides of controls (sham controls -P) showed regular calvarium tissue with a small number of macrophages. Immunostaining of Group 4 WT mice samples showed, that the presence of wear particles induces a significantly increased occurrence of APN, AdipoR1 and calreticulin in macrophages in comparison to the sham controls. This finding corresponded to the results
from the human samples. The levels of APN and calreticulin were again significantly higher in Group WT+Ad+P, whereas AdipoR1 was expressed in a non-significant lower value (Figure 3A). Group KO+P mice lacked expression of APN and AdipoR1 and showed significantly lower levels of calreticulin compared to Group WT+Ad+P but not in comparison to WT-Ad+P. AdipoR2 was not detectable in any group, which again corresponded to the results from the human samples.
3.3. Attenuation of wear particle-induced apoptosis by adiponectin in the calvarial mice model of osteolysis As in the human samples, caspase-3 was significantly increased in the mice macrophages which had contact with wear particles. Furthermore, the additional TUNEL assay and apoptotic molecules BAK, BAD, FAS/CD95 and Bcl-2 were significantly upregulated (Figure 3B and 4). Comparison of the particle-treated groups with each other revealed that the samples from animals treated with APN (WT+Ad+P) had a significantly smaller number of cells with a positive TUNEL reaction and significantly lower IP scores for Bcl-2 and caspase-3 cleaved staining than samples of untreated WT and KO mice. In comparison to Group WT-Ad+P mice the differences were also significant for FAS/CD95 (Figure 3B). Comparison of the apoptotic markers between Group KO+P and WT-Ad+P showed significant differences only for Bcl-2, BAK and TUNEL. A valid number of apoptotic macrophages were found only in the mice which received UHMWPE particles, therefore determination of the caspase-3 cleaved / APN ratio was only meaningful in these groups. Most cells were negative for both APN and caspase-3 cleaved. As the cells of the KO mice lacked expression of APN, the number of caspase-negative / APNpositive and both positive cells was significantly smaller than in WT mice. Comparison of the APN-treated and -untreated WT mice regarding caspase-negative / adiponectin-positive cells and both positive cells revealed no significant differences. However, the number of caspase-3
positive / adiponectin negative macrophages and giant cells in the APN-treated group of WT+Ad+P mice was significantly decreased compared to untreated KO and WT mice (Figure 3C).
3.4. Attenuation of wear particle-induced osteolysis and inflammation by adiponectin in the calvarial mice model of osteolysis We observed significant differences for all parameters that indicate bone resorption (TRAP, histomorphometry and µ-CT) in all animals that received UHMWPE particles on the calvaria (+P) in comparison to the respective control groups (-P) (Figure 5A-D). Comparison of the particle groups revealed the best results and therefore the least bone destruction for the APN-treated WT mice (WT+Ad+P). In comparison to Group WT-Ad+P mice without application of APN all differences were significant. In comparison to the KO mice (Group KO+P) differences were also significant for histomorphometrically determined osteolysis and the osteolysis ratio, as well as for TRAP staining. The suture size and BVTV and bone volume measured by µCT revealed no significant differences. Expression of RANKL was observed in macrophages and osteoclasts and was significantly lower in Group WT+Ad+P than in Groups KO+P or WT-Ad+P (Figure 5E). The area of inflammation around the particles was significantly smaller in APNtreated mice compared to untreated WT and KO mice, which in turn did not significantly differ (Figure 5B). Therefore, administration of APN significantly attenuates UHMWPE particle-induced osteolytic and inflammatory reactions.
3.5. The role of adiponectin on wear particle-engulfing macrophages regarding expression of adiponectin associated receptors, apoptosis and inflammation in the cellculture model The interaction of macrophages with the particles induced a significantly increased expression of caspase-3, calreticulin, and TNF-α, whereas AdipoR1 did not significantly differ. Implementation of the experiment in the presence of 0.25ng/ml APN resulted in a significantly increased expression of AdipoR1 respectively significantly lower values of calreticulin, whereas the other markers remained unchanged. Interestingly, a higher dose of 0.5ng/ml APN resulted in a significant decrease in TNF-α and an increase in AdipoR1, calreticulin and caspase-3 (Figure 6).
4. Discussion The aim of the current study was to determine the role of adiponectin (APN) in particle-induced osteolysis and other accompanying effects that result in aseptic loosening of total hip replacements. The presence of polyethylene and metal debris in capsules and interface membranes from aseptically loosened total hip implants is associated with significantly increased expression of APN, AdipoR1 and calreticulin in wear particle-engulfing cells. To investigate the possible relevance of these findings for the induction of osteolysis, we additionally used a well-established murine calvarial model of wear particle-induced osteolysis [28]. We observed that the application of wear particles induces a significantly increased expression of APN, AdipoR1 and calreticulin in WT mice and even higher levels after application of APN. This implies that wear debris induces a higher requirement for APN, and if it is available in higher quantities it is also consumed by the macrophages. It is even more interesting that administration of APN counteracted wear particleinduced osteolysis in WT+Ad+P mice. This was demonstrated by all methods used to
evaluate bone resorption. However, the absence of APN in KO+P mice does not definitely result in higher bone resorption compared to WT-Ad+P mice. Consequently, only higher levels of APN were beneficial. One indication for the protective effect of APN might be the decrease in particle-induced inflammation. In our animal model we found a significantly smaller inflammatory area and in the cell culture model a lower TNF- level after administration of APN. This is a highly interesting observation as periprosthetic osteolysis is initiated by an aseptic inflammatory response to phagocytosis of implant wear particles which results in increased proliferation and differentiation of osteoclast precursors into mature osteoclasts [1]. The anti-inflammatory effect of APN has also been observed in other diseases, e.g. diabetes, coronary and liver diseases [29]. Beside the inflammatory reactions we also focused on the influence of APN on apoptosis, which is another factor responsible for wear particle-induced osteolysis [5]. The animal model supports our previous observations in patients with aseptic hip loosening, as wear particles activate the apoptotic pathway including the common key regulatory checkpoint caspase-3 [2]. Double staining of samples from patients with aseptic loosening as well as from the mice calvaria that received wear particles showed that caspase-3 cleaved positive macrophages were mostly negative for APN. APN administration leads to a decrease in these cells, whereas the numbers of cells that are positive for APN and caspase-3 remain at a low level. We assume that APN-positive apoptotic cells are removed quickly. Accordingly, double staining with caspase-3 cleaved and APN of the human specimens revealed only a small portion of caspase-3 cleaved negative and APN-positive cells and a considerably higher number of APN-negative / caspase-3 cleaved positive cells. The removal of apoptotic cells by adiponectin depends on calreticulin [15], which is also detectable in large quantities in the tissues of patients with aseptic loosening and is increased by APN application in animal and cell culture. Therefore, the removal of apoptotic bodies by contributing calreticulin may be a
major task of APN. Consequently, the lower levels of caspase-3 and TUNEL-positive cells in the calvaria model after administration of APN may be an expression of faster resorption of apoptotic cell debris. Promoting the clearance of early apoptotic cells by phagocytizing cells in the presence of calreticulin is important in that if the phagocytic clearance system is impaired or overwhelmed by massive waves of apoptosis, uncleared apoptotic cell remnants could undergo secondary necrosis which results in release of their inflammatory and cytotoxic contents [30-32]. The increased excretion of cytokines may in turn be responsible for the induction of osteolysis [33]. We observed that decreased levels of FAS/CD95 after administration of APN and direct down-regulation of the death receptor FAS/CD95 by binding of APN result in inhibition of the key regulator caspase-3, a phenomenon which has also been described in studies on other diseases [34]. The results in the mice model also suggest that APN has antiapoptotic characteristics. However, we also observed increased expression of AdipoR1 in the human, animal and cell culture models which could have anti-apoptotic as well as proapoptotic consequences [34]. The strength of AdipoR1 expression depends on the presence of wear particles as well as on the APN value. In cell culture pro-apoptotic effects were associated with higher doses of APN. As calreticulin increases simultaneously and TNFdecreases, we suppose that calreticulin compensates for a possibly enhanced formation of apoptotic bodies. This would explain why pro-apoptotic influences were not detectable in the mice model. Pro-apoptotic effects could be also induced by enhancement of the anti-apoptotic bcl-2, the level of which decreases as the level of APN increases. The influence of APN on bcl-2 and the resulting apoptosis were also observed in other studies [35]. The different regulatory effects of APN on wear particle-induced apoptosis may also explain why wear particles do not induce higher osteolysis, inflammation and apoptosis in KO mice than in WT mice without administration of APN.
Finally, the protective effect of adiponectin against wear particle-induced osteolysis, beside regulating inflammatory and apoptotic pathways, may be due to the inhibition of osteoclastogenesis and down-regulation of RANKL-enhanced expressions of osteoclastogenic regulators including NFAT2, TRAF6, cathepsin K, and tartrate-resistant acid phosphatase [36]. In fact, decreased RANKL expression was also observed in this study. In conclusion, this is the first study which demonstrates that APN inhibits the development of particle-induced osteolysis by enhancing the consequences of particleinduced apoptosis. Its anti-inflammatory and possibly anti-RANKL effect also contribute to this process. It is not possible to clarify all aspects of the role of APN in aseptic loosening in one study, therefore further research will be necessary to verify the dose-dependent effects of APN in connection with aseptic hip loosening and determine the optimal level of APN. In conclusion, a high serum level of APN seems to be beneficial.
Disclosures All authors state that they have no conflicts of interest related to the work reported in the manuscript.
Acknowledgements This study was supported by a Grant from the German Research Foundation (DFG): LA 2619/3-1.
We thank Mrs. Nicole Cramer, Mrs. Claudia Lodewick and Mrs. Sara Lask for technical assistance.
Figure Legends Fig. 1: A small number of macrophages and giant cells was detected in the capsules of patients without aseptic loosening. They were only sporadically positive for APN, AdipoR1 and calreticulin, whereas in the samples from patients with aseptic loosening the staining for these markers was frequent and intensive. The images show AdipoR1 as an example.
Fig. 2: (A) Double staining of the joint capsules with adiponectin (red) and caspase-3 cleaved (brown) revealed that beside a predominant proportion that was negative for both, a greater percentage was positive for caspase-3 cleaved but not for adiponectin. The vessel-cells serve as internal control. Magnifications are at original x 1000. (B) However, a representative proportion of APN-positive cells was also visible, whereby only a fractional part was positive for both caspase-3 cleaved and APN. (C) The bar diagram shows the relative distribution of the afore-mentioned double-stained cells in capsules and interface membranes (C: caspase-3 cleaved / A: APN).
Fig. 3: (A) In comparison to the control groups, the administration of UHMWPE particles led to a stronger reaction of APN, AdipoR1 and calreticulin in Group WT-Ad+P. After administration of APN, the levels of APN and calreticulin were again significantly higher in Group WT+Ad+P, whereas AdipoR1 was expressed in a non-significant lower value. Group KO+P mice lacked expression if APN and AdipoR1 and showed significantly lower levels of calreticulin compared to Group WT+Ad+P, but not in comparison to Group WT-Ad+P. (B) Immunohistochemical analysis revealed activation of the apoptotic parameters BAK, BAD, Bcl-2, FAS/CD95 and Caspase-3 only in samples from the mice which received particles on the calvaria. Only Bcl-2, Caspase-3 and FAS/CD95 were significantly decreased by APN. (*=p<0.05, **=<0.01, ***=<0.001).
(C) Double staining revealed a significantly smaller number of caspase-3 positive / adiponectin negative macrophages in APN-treated WT mice in comparison to the untreated KO and WT mice. The KO mice lacked expression of adiponectin positive cells. The other combinations, did not significantly differ between the groups which received UMHWPE particles. (*=p<0.05, **=<0.01, ***=<0.001).
Fig. 4: TUNEL reaction showed a highly significant particle-induced increase in apoptosis in Group KO+P, Group WT-Ad+P and Group WT+Ad+P in comparison to the sham controls. In comparison to the non-specifically treated WT and KO mice there was a significant decrease after administration of APN in Group WT+AD+P. (*=p<0.05, **=<0.01, ***=<0.001).
Fig. 5: (A) Histomorphometry of the Hematoxylin Eosin (HE) stained coronal sections of calvaria at the sagittal suture showed that addition of UHMWPE particles without any further specific intervention induced pronounced bone resorption in the WT and KO mice compared to the sham controls. Treatment with APN diminished these adverse UHMWPE particlemediated effects and led to a smaller medial suture area (*) and decreased osteolysis ( ) in the WT+Ad+P mice in comparison to the untreated KO and WT-mice. (B) The effect of APN was quantitatively assessed by determining osteolysis and the ratio of osteolysis to the unaffected bone. It was significantly lower in comparison to the untreated KO and WT mice. The evaluation of the midline suture size revealed better results for the APN-treated mice only in comparison to the WT mice. The size of the inflammatory area around the particles was also significantly smaller in comparison to both groups. (*=p<0.05, **=<0.01, ***=<0.001). (C) TRAP staining was only positive in the particle-treated groups and showed a significantly smaller number of osteoclasts in the APN-treated animals than in the untreated WT or KO
mice. The images show representative samples of WT-Ad+P and WT+Ad+P. (*=p<0.05, **=<0.01, ***=<0.001). (D) Micro-CT showed pronounced bone resorption in the UHMWPE particle-treated groups compared to the sham control groups. Comparison of the particle groups with each other revealed a significantly larger bone volume and BVTV ratio in the APN-treated mice than in the non-treated WT or KO mice. The examples of the 3D pictures show that the skull surface of the animals that underwent particle implantation and were treated with APN appears less lacerated than the skulls of the untreated mice. (*=p<0.05, **=<0.01, ***=<0.001). (E) RANKL staining showed significantly lower staining of macrophages and osteoclasts in the APN-treated animals in comparison to the untreated KO or WT mice. (*=p<0.05, **=<0.01, ***=<0.001).
Fig. 6: The cell culture model showed that addition of particles to the macrophages induced a significantly increased expression of caspase-3, calreticulin and TNF-α, whereas AdipoR1 did not differ significantly. Implementation of the experiment in the presence of 0.25ng/ml APN leads to a significantly increased expression of AdipoR1, respectively significantly lower values of calreticulin, whereas the other markers remained unchanged. Administration of 0.5ng/ml APN leads to a significant decrease in TNF-α and an increase in AdipoR1, calreticulin and caspase-3. (*=p<0.05, **=<0.01, ***=<0.001).
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Table 1: Patient Characteristics
Characteristics
Age (years) Median Range
No Arthroplasty
64.8 44 to 75
Aseptic loosening
72.9 45 to 93
Sex Male Female
6 10
19 24
Loosening of Acetabulum only Stem only Both
22 12 9
Cemented Fixation No Stem only Acetabulum and Stem
23 3 17
Survivorship of endoprosthesis (years) Median Range
11.98 1 to 24
Extent of osteolysis, acetabular (Paprosky score) 1 2a 2b 2c 3a 3b
3 7 8 5 2 6
Extent of osteolysis, femoral (Paprosky score) 1 2a 2b 2c 3
4 2 7 4 4
Polyethylene debris (semi-quantitative score) Median Range
2.0 1 to 3
Metal debris (semi-quantitative score) Median Range
2.1 1 to 5
Table 2: Animal Model (Groups) Group KO-P KO+P WT-Ad-P WT-Ad+P WT+Ad-P WT+Ad+P
Mice Knockout Knockout Wildtype Wildtype Wildtype Wildtype
Particles No Yes No Yes No Yes
Adiponectin No No No No Yes Yes
Table 3: Humans No Arthroplasty Capsules APN AdipoR1 Calreticulin Caspase-3 cleaved Interface membranes APN AdipoR1 Calreticulin Caspase-3 cleaved
0.25 (0 to 1; SD 0.43) 0.25 (0 to 1; SD 0.43) 0.25 (0 to 1; SD 0.43) 0
Aseptic Loosening 2.0 (1 to 4; SD 1.04) 4.37 (1 to 12; SD 2.49) 5.59 (1 to 9; SD 2.75) 2.80 (1 to 12; SD 2.49)
2.1 (1 to 4; SD 0.78) 4.96 (1 to 9; SD 2.32) 4.93 (1 to 9; SD 2.95) 3.18 (1 to 9; SD 2.32).
Results are given as mean with range and standard deviation (SD)
p-value <0.001 <0.001 <0.001 <0.001