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Polyethylene particles in joint fluid and osteolysis in revision total knee arthroplasty
The Knee 21 (2014) 402–405
Contents lists available at ScienceDirect
The Knee
Polyethylene particles in joint fluid and osteolysis in revision total knee arthroplasty Ignasi Piñol ⁎, Alberto Torres, Gabriel Gil, Eva Prats, Lluis Puig-Verdier, Pedro Hinarejos Hospital del Mar. Servei de COT., Passeig Maritim 25-29, Barcelona CP: 08003, Spain
a r t i c l e
i n f o
Article history: Received 11 April 2013 Received in revised form 6 September 2013 Accepted 22 October 2013 Keywords: Polyethylene joint fluid osteolysis total knee arthroplasty
a b s t r a c t Background: One of the most frequent reasons for total knee arthroplasty late failure is osteolysis. It has been related to foreign body reaction to polyethylene particles. The aim of this study is to analyse the number, size and morphology of polyethylene particles in synovial fluid in total knee arthroplasty revision and correlate them to the pathology and the degree of osteolysis. Methods: Synovial fluid was obtained in 12 patients before the revision total knee arthroplasty. Polyethylene particles were isolated and analysed through scanning electron microscopy. Samples of synovial tissue were analysed with optical microscopy while considering the parameters of particles and histiocytic infiltration. Osteolysis was analysed with plain radiography and the macroscopic aspect during surgery. Results: The statistical analysis showed a significant correlation between a high concentration of polyethylene particles in synovial fluid and a high degree of osteolysis. The concentration of particles in synovial fluid also showed a significant correlation with a high degree of particles and histiocytes in the histological analysis. There was a relationship between the size of particles and the degree of osteolysis. No relationship was found between the shape of the particles and the histological findings or the degree of osteolysis. Conclusions: In an “in vivo” TKA scenario, the presence of a high concentration of polyethylene particles in the synovial fluid seems to be the cause of a highly active foreign body histological reaction, with an increased number of histiocytes, which seems to be the cause of a significant degree of osteolysis around the implant. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Aseptic loosening secondary to osteolysis is the main cause for total knee arthroplasty (TKA) failure in the long-term period [1]. Periprosthetic osteolysis has been related to foreign body reaction to polyethylene particles, which are in the knee joint fluid due to material fatigue mechanism from the insert [2]. When polyethylene particles are phagocytised by macrophages and giant cells, many cytokines are secreted. Some interleukins (IL), as IL-1β, IL-6 and alpha-necrosis tumoral factor cause inflammation and activate osteoclasts, the main cells responsible for osteolysis [3–5]. Moreover, cytokines inhibit bone formation by osteoblasts [6]. The size of the polyethylene particles is an important issue as those between 1 and 10 μm bring about greater biological activity and more osteolysis than particles greater than 10 μm, which cannot be phagocytised and cause less of an inflammatory reaction [7]. Over the last decade, some studies have described the technique, which permits studying polyethylene wear and its effects “in vivo”, for joint fluid analysis of polyethylene particles [8]. The main aim of this study was to analyse the number, size and shape of polyethylene particles in knee synovial fluid in TKA that are ⁎ Corresponding author. Tel.: +34 932483196; fax: +34 932483332. E-mail address: [email protected] (I. Piñol). 0968-0160/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.knee.2013.10.013
being revised and to analyse the relationship between these particles in joint fluid, radiological osteolysis around TKA. The findings during revision surgery and the histological findings in the tissues around the implant were also analysed. 2. Material and methods A prospective study was designed. The inclusion criteria were patients with an indication for a first revision TKA after a cruciate-retaining (CR) or a posterior-stabilised (PS) primary TKA that had been implanted at least 1 year before the revision. The exclusion criteria were the presence of an infection and the impossibility of obtaining at least 3 ml of synovial fluid previous to revision surgery. In all cases, the revision surgery was done by the same surgeon (PH). The preoperative variables analysed were: age, gender, weight, height, body mass index (BMI), femoro-tibial angle (FTA) measured in a long weight-bearing X-ray, time between primary and revision surgeries, cause of revision, severity of radiographic osteolysis (quantified as none, mild, moderate or severe), and the type of primary TKA. Twelve patients were included in the study. The mean age was 70.6 (61–90) years. There were two males and 10 females. The mean weight was 72.83 (62–92) kg, and the mean BMI was 29.99 (24.21– 41.44) kg/m2. Nine patients had a normal FTA, two patients had more than 3 degrees of valgus and one patient showed more than 3
I. Piñol et al. / The Knee 21 (2014) 402–405
degrees of varus. The reason for the revision was aseptic loosening in five patients (41.7%), painful arthroplasty with a scintigraphy suggesting loosening in three patients (25%), instability in two patients (16.7%), knee stiffness in one case (8.3%) and polyethylene wear in another patient (8.3%). Time elapsed between primary and revision surgeries averaged 5.08 years (2–12). Fifty percent of the primary TKAs were PS and 50% were CR. The FTA was measured by the same person (IP) in a preoperative digitalised full-length X-ray with the iPACS, software version 5.0. The radiographic osteolysis was measured prior to revision surgery by the same person in all cases (PH) [9]. In all cases, an arthrocentesis in sterile conditions was done immediately before revision TKA surgery and the maximum possible quantity of synovial fluid was aspirated. The average volume of synovial fluid was 14.08 (3–50) ml. During revision surgery, tissue samples of synovial tissue and periprosthetic membranes were taken and sent to the pathology laboratory. These samples were analysed by the same person (NJ) who measured the quantity of polyethylene particles in the samples and the degree of histiocytic infiltration with a modification of the Mirra et al. grading scale [3,10,11] (Fig. 1). Macroscopic osteolysis was graded as none, mild, moderate or severe by the surgeon during surgery. Other variables analysed were the addition of radiographic and intraoperative osteolysis and the addition of histiocytic infiltration and the quantity of particles in the tissue sample. The samples of synovial fluid, after identification, were preserved in a sterile tube in a freezer at −20 °C until the day of processing these samples in the following weeks. The samples were processed by the same person (IP) following the technique described by Minoda et al. [8]. It consists in the digestion of the sample in sodium hydroxide at 65 °C for 12 h and the application to a sucrose-density gradient in a 14 ml tube which is ultra-centrifuged at 28,000 rpm for 3 h at 4 °C. The highest level of the tube is applied to an isopropanolol–water density gradient in a 40 ml tube, which is also ultracentrifuged at 28,000 rpm for 1 h at 20 °C to group the polyethylene particles. The interface line is filtered through a 0.1 μm polycarbonate filter to retain the polyethylene particles. This filter is attached to a Petri plate. In the scanning electron microscope (SEM) department, the filter is set on a brass layer and coated with a gold layer of 40 nm for SEM analysis (Quanta 200 FEI, Co.). In the SEM analysis, 10 random 10,000 augments fields were studied by the same person (EP). If there was a doubt about the composition of a particle, a study with energy-dispersive spectrometry (EDS) was performed so as to confirm that it was a polyethylene and
Fig. 1. Microscopic analysis of synovial tissue: moderate concentration of histiocytes with focal foreign body reaction of giant cells and polyethylene particles (hematoxylin and eosin, original magnification ×20).
403
Fig. 2. Polyethylene particles of different sizes and shapes (scanning electron microscope × 10,000).
not a contaminant particle. In each random field, the number, size and shape of the particles were analysed (Fig. 2). With that data, we found out the polyethylene particles concentration per ml of synovial fluid and the total quantity of particles in each patient.
2.1. Statistical analysis Data were analysed using SPSS for Windows version 17.0 (SPSS Inc., Chicago, IL, USA). For the statistical correlations of the studied variables, the Spearman's rank correlation coefficient rho was used. Statistical significance was set at a p value of b 0.05. 3. Results In the preoperative X-rays, osteolysis was graded as none in six patients, mild in zero cases, moderate in two and severe in four patients. In the intraoperative assessment, osteolysis was graded as none in five patients, mild in two cases, moderate in one case and severe in four patients. The pathological study of the samples of the joint membranes showed six cases with no polyethylene particles, one patient with a low number of particles, three cases with moderate and two cases with a high number of particles. In these samples, there were no cases without histiocyte infiltration. It was mild in four cases with isolated histiocytes. There were five moderate cases with few groups of histiocytes and three severe cases with many groups of histiocytes. The electron microscope analysis of the processed synovial fluid showed polyethylene particles in different concentrations in all samples, an average concentration of particles that was 1.55 ± 1.34 × 106 particles/ml and the total average of polyethylene particles in the samples was 16.9 ± 10.58 × 106. The percentage of particles greater than 1 μm was 19.4%, and the analysis of the shape showed that 27.4% of the particles were not round. The statistical analysis showed a significant correlation between a higher concentration of polyethylene particles in synovial fluid and a higher degree of osteolysis assessed intraoperatively or the addition of both radiological and intraoperative osteolysis. The concentration of particles in synovial fluid also showed a significant correlation with a higher degree of particles in the histological analysis or the addition of particles and histiocytes in the tissue samples (Table 1). Both, a greater histiocyte infiltration in the tissue and a higher number of polyethylene particles in the tissue samples were significantly correlated with a higher osteolysis assessed both in the preoperative X-rays and in the intraoperative macroscopic aspect of the bone (Table 2). When analysing the size of the particles, there was a positive correlation between a higher percentage of particles greater than 1 μm and greater osteolysis both in the radiological and in the intraoperative assessment. No relationship was found between the shape of the particles (percentage of round particles) and the histological findings or the degree of osteolysis.
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I. Piñol et al. / The Knee 21 (2014) 402–405
Table 1 Correlations between and statistical significance of polyethylene particle concentration in synovial fluid and the studied parameters of osteolysis, volume of synovial fluid and histological analysis. Particles/ml (concentration) n = 12
Spearman's rho
p
Radiological osteolysis Macroscopic osteolysis Total osteolysis Synovial fluid volume Particles in tissue sample Histiocytes in tissue sample Particles + histiocytes in tissue sample
0.412 0.620 0.599 0.743 0.724 0.417 0.677
0.183 0.032 0.040 0.052 0.008 0.177 0.016
4. Discussion The main finding of our study is the clear correlation between a higher concentration of particles in the synovial fluid in the patient joint and the greater histiocytic reaction to a foreign body in the tissue samples and the higher degree of osteolysis in the bone surrounding the failed knee arthroplasty. As far as we know, this is the first study that tries to correlate the concentration of polyethylene particles in synovial fluid and pathological findings in a failed TKA. Osteolysis around the knee components is in many cases related to aseptic loosening of the TKA in the long term due to foreign body reaction to polyethylene particles and, in this process, the tissue macrophages activate bone resorption. These polyethylene particles are in the synovial fluid before they are in the surrounding tissue, so the particles can be isolated either from the synovial fluid or in the tissues. To study the concentration of particles, a joint puncture is enough, with very few risks and without a more aggressive procedure as a synovial biopsy that is required to study the particles in the tissue. The adverse biological reactions caused by osteolysis are dependant not only on the amount of the wear, but also on the concentration and size of the polyethylene particles [12]. Particles less than 10 μm have been related to a greater capacity to cause osteolysis because particles greater than that size are not phagocytised by the macrophages [13,14]. In the present study, a higher concentration of particles greater than 1 μm has been found in knees with more severe osteolysis. However, there were very few particles greater than 10 μm. Thus, a relationship between osteolysis and the biggest particles could not be found. There is some evidence about the fact that long particles cause more inflammation than the round ones [7]. However, no relationship between the shape of the particles and the pathological reaction to foreign body or a greater osteolysis could be found in this study. The results of the present study confirm that a high concentration of polyethylene particles in the synovial fluid is related to a high concentration of particles and histiocytes in the tissue. The inflammatory response to a foreign body would be the cause of a severe osteolysis and the aseptic loosening of the TKA [3,4,15]. A simple X-ray is the most frequently used image study for osteolysis, but this method can only obtain images in two dimensions [16]. Using a CT-scan or MRI images would allow for a better study of osteolysis around TKA as the TKA structure would be studied in 3-dimensions [17–19]. In our study, a better correlation between the particles concentration and Table 2 Correlation with Spearman's rho (and statistical significance) between the osteolysis grade and the histological findings (particles, histiocytes and both) in the tissue samples. n = 12 Radiological osteolysis Macroscopic osteolysis Total osteolysis
Particles
Histiocytes
Particles + histiocytes
0.79 (p = 0.002)
0.732 (p = 0.007)
0.766 (p = 0.004)
0.808 (p = 0.001)
0.662 (p = 0.019)
0.743 (p = 0.006)
0.804 (p = 0.002)
0.650 (p = 0.022)
0.729 (p = 0.007)
the intraoperative assessment of the osteolysis was found. It suggested that radiological assessment of the osteolysis might not be so precise and this may be a limitation of our study. This study has several limitations like the reduced number of cases and the fact that different TKA models were used in the primary TKA. Another limitation is the type of filters used, with a size of the pores of 0.1 μm, following the technique described by Minoda [8]. Therefore, particles smaller than this size were not retained in the filter. Nevertheless, some studies have shown that most of the particles caused by TKA wear are in the 0.1–1 μm range [15,20] and the particles that provoke a high degree of macrophage activation are those between 0.3 and 10 μm [14,21]. Thus, particles smaller than 0.1 μm, not retained in the filter used, might cause little osteolysis. In this study, as in others previously reported [15,20], most of the isolated particles were in the 0.1–1 μm range. To conclude, we can say that, in an in vivo TKA scenario, the presence of a high concentration of polyethylene particles in the synovial fluid seems to be the cause of a highly active foreign body histological reaction with an increased number of histiocytes, which seems to be the cause of high grade of osteolysis around the implant. 5. Conflict of interest statement 1. Royalties from a company or supplier (the following conflicts were disclosed): none 2. Speakers bureau/paid presentations for a company or supplier (the following conflicts were disclosed): none 3A. Paid employee for a company or supplier (the following conflicts were disclosed): none 3B. Paid consultant for a company or supplier (the following conflicts were disclosed): none 3C. Unpaid consultants for a company or supplier (the following conflicts were disclosed): none 4. Stock or stock options in a company or supplier (the following conflicts were disclosed): none 5. Research support from a company or supplier as a principal investigator (the following conflicts were disclosed): none 6. Other financial or material support from a company or supplier (the following conflicts were disclosed): none 7. Royalties, financial or material support from publishers (the following conflicts were disclosed): none 8. Medical/orthopaedic publications editorial/governing board (the following conflicts were disclosed): none 9. Board member/committee appointments for a society (the following conflicts were disclosed): none Acknowledgement The authors gratefully acknowledge the assistance in the statistical analysis by Sergi Mojal. References [1] Sharkey PF, Hozack WJ, Rothman RH, Shastri S, Jacoby SM. Insall Award paper. Why are total knee arthroplasties failing today? Clin Orthop Relat Res Nov 2002;404:7–13. [2] Baxter RM, Freeman TA, Kurtz SM, Steinbeck MJ. Do tissues from THA revision of highly crosslinked UHMWPE liners contain wear debris and associated inflammation? Clin Orthop Relat Res Aug 2011;469(8):2308–17. [3] Chiba J, Rubash HE. A biochemical, histologic, and immunohistologic analysis of membranes obtained from failed cemented and cementless total knee arthroplasty. Clin Orthop Relat Res Dec 1994;309:278. [4] Ingham E, Fisher J. The role of macrophages in osteolysis of total joint replacement. Biomaterials Apr 2005;26(11):1271–86. [5] Jacobs JJ, Roebuck KA, Archibeck M, Hallab NJ, Glant TT. Osteolysis: basic science. Clin Orthop Relat Res Dec 2001;393:71–7. [6] Gupta SK, Chu A, Ranawat AS, Slamin J, Ranawat CS. Osteolysis after total knee arthroplasty. J Arthroplasty Sep 2007;22(6):787–99. [7] Hallab NJ, Jacobs JJ. Biologic effects of implant debris. Bull NYU Hosp Jt Dis 2009;67(2):182–8.
I. Piñol et al. / The Knee 21 (2014) 402–405 [8] Minoda Y, Kobayashi A, Iwaki H, Miyaguchi M, Kadoya Y, Ohashi H, et al. Polyethylene wear particles in synovial fluid after total knee arthroplasty. Clin Orthop Relat Res May 2003;410:165–72. [9] Mulhall KJ, Ghomrawi HM, Engh GA, Clark CR, Lotke P, Saleh KJ. Radiographic prediction of intraoperative bone loss in knee arthroplasty revision. Clin Orthop Relat Res May 2006;446:51–8. [10] Mochida Y, Bauer TW, Koshino T, Hirakawa K, Saito T. Histologic and quantitative wear particle analyses of tissue around cementless ceramic total knee prostheses. J Arthroplasty Jan 2002;17(1):121–8. [11] Mirra JM, Amstutz HC, Matos M, Gold R. The pathology of the joint tissues and its clinical relevance in prosthesis failure. Clin Orthop Relat Res Jun 1976;117:221–40. [12] Fisher J, Bell J, Barbour PS, Tipper JL, Matthews JB, Besong AA, et al. A novel method for the prediction of functional biological activity of polyethylene wear debris. Proc Inst Mech Eng H 2001;215(2):127–32. [13] Illgen II RL, Forsythe TM, Pike JW, Laurent MP, Blanchard CR. Highly crosslinked vs conventional polyethylene particles — an in vitro comparison of biologic activities. J Arthroplasty Aug 2008;23(5):721–31. [14] Green TR, Fisher J, Stone M, Wroblewski BM, Ingham E. Polyethylene particles of a ‘critical size’ are necessary for the induction of cytokines by macrophages in vitro. Biomaterials Dec 1998;19(24):2297–302.
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[15] Howling GI, Barnett PI, Tipper JL, Stone MH, Fisher J, Ingham E. Quantitative characterization of polyethylene debris isolated from periprosthetic tissue in early failure knee implants and early and late failure Charnley hip implants. J Biomed Mater Res 2001;58(4):415–20. [16] Naudie DD, Ammeen DJ, Engh GA, Rorabeck CH. Wear and osteolysis around total knee arthroplasty. J Am Acad Orthop Surg Jan 2007;15(1):53–64. [17] Berry DJ. Recognizing and identifying osteolysis around total knee arthroplasty. Instr Course Lect 2004;53:261–4. [18] Cadambi A, Engh GA, Dwyer KA, Vinh TN. Osteolysis of the distal femur after total knee arthroplasty. J Arthroplasty Dec 1994;9(6):579–94. [19] Nadaud MC, Fehring TK, Fehring K. Underestimation of osteolysis in posterior stabilized total knee arthroplasty. J Arthroplasty Jan 2004;19(1):110–5. [20] Tipper JL, Galvin AL, Williams S, McEwen HM, Stone MH, Ingham E, et al. Isolation and characterization of UHMWPE wear particles down to ten nanometers in size from in vitro hip and knee joint simulators. J Biomed Mater Res A Sep 1 2006;78(3):473–80. [21] Matthews JB, Besong AA, Green TR, Stone MH, Wroblewski BM, Fisher J, et al. Evaluation of the response of primary human peripheral blood mononuclear phagocytes to challenge with in vitro generated clinically relevant UHMWPE particles of known size and dose. J Biomed Mater Res Nov 2000;52(2):296–307.
Contents lists available at ScienceDirect
The Knee
Polyethylene particles in joint fluid and osteolysis in revision total knee arthroplasty Ignasi Piñol ⁎, Alberto Torres, Gabriel Gil, Eva Prats, Lluis Puig-Verdier, Pedro Hinarejos Hospital del Mar. Servei de COT., Passeig Maritim 25-29, Barcelona CP: 08003, Spain
a r t i c l e
i n f o
Article history: Received 11 April 2013 Received in revised form 6 September 2013 Accepted 22 October 2013 Keywords: Polyethylene joint fluid osteolysis total knee arthroplasty
a b s t r a c t Background: One of the most frequent reasons for total knee arthroplasty late failure is osteolysis. It has been related to foreign body reaction to polyethylene particles. The aim of this study is to analyse the number, size and morphology of polyethylene particles in synovial fluid in total knee arthroplasty revision and correlate them to the pathology and the degree of osteolysis. Methods: Synovial fluid was obtained in 12 patients before the revision total knee arthroplasty. Polyethylene particles were isolated and analysed through scanning electron microscopy. Samples of synovial tissue were analysed with optical microscopy while considering the parameters of particles and histiocytic infiltration. Osteolysis was analysed with plain radiography and the macroscopic aspect during surgery. Results: The statistical analysis showed a significant correlation between a high concentration of polyethylene particles in synovial fluid and a high degree of osteolysis. The concentration of particles in synovial fluid also showed a significant correlation with a high degree of particles and histiocytes in the histological analysis. There was a relationship between the size of particles and the degree of osteolysis. No relationship was found between the shape of the particles and the histological findings or the degree of osteolysis. Conclusions: In an “in vivo” TKA scenario, the presence of a high concentration of polyethylene particles in the synovial fluid seems to be the cause of a highly active foreign body histological reaction, with an increased number of histiocytes, which seems to be the cause of a significant degree of osteolysis around the implant. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Aseptic loosening secondary to osteolysis is the main cause for total knee arthroplasty (TKA) failure in the long-term period [1]. Periprosthetic osteolysis has been related to foreign body reaction to polyethylene particles, which are in the knee joint fluid due to material fatigue mechanism from the insert [2]. When polyethylene particles are phagocytised by macrophages and giant cells, many cytokines are secreted. Some interleukins (IL), as IL-1β, IL-6 and alpha-necrosis tumoral factor cause inflammation and activate osteoclasts, the main cells responsible for osteolysis [3–5]. Moreover, cytokines inhibit bone formation by osteoblasts [6]. The size of the polyethylene particles is an important issue as those between 1 and 10 μm bring about greater biological activity and more osteolysis than particles greater than 10 μm, which cannot be phagocytised and cause less of an inflammatory reaction [7]. Over the last decade, some studies have described the technique, which permits studying polyethylene wear and its effects “in vivo”, for joint fluid analysis of polyethylene particles [8]. The main aim of this study was to analyse the number, size and shape of polyethylene particles in knee synovial fluid in TKA that are ⁎ Corresponding author. Tel.: +34 932483196; fax: +34 932483332. E-mail address: [email protected] (I. Piñol). 0968-0160/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.knee.2013.10.013
being revised and to analyse the relationship between these particles in joint fluid, radiological osteolysis around TKA. The findings during revision surgery and the histological findings in the tissues around the implant were also analysed. 2. Material and methods A prospective study was designed. The inclusion criteria were patients with an indication for a first revision TKA after a cruciate-retaining (CR) or a posterior-stabilised (PS) primary TKA that had been implanted at least 1 year before the revision. The exclusion criteria were the presence of an infection and the impossibility of obtaining at least 3 ml of synovial fluid previous to revision surgery. In all cases, the revision surgery was done by the same surgeon (PH). The preoperative variables analysed were: age, gender, weight, height, body mass index (BMI), femoro-tibial angle (FTA) measured in a long weight-bearing X-ray, time between primary and revision surgeries, cause of revision, severity of radiographic osteolysis (quantified as none, mild, moderate or severe), and the type of primary TKA. Twelve patients were included in the study. The mean age was 70.6 (61–90) years. There were two males and 10 females. The mean weight was 72.83 (62–92) kg, and the mean BMI was 29.99 (24.21– 41.44) kg/m2. Nine patients had a normal FTA, two patients had more than 3 degrees of valgus and one patient showed more than 3
I. Piñol et al. / The Knee 21 (2014) 402–405
degrees of varus. The reason for the revision was aseptic loosening in five patients (41.7%), painful arthroplasty with a scintigraphy suggesting loosening in three patients (25%), instability in two patients (16.7%), knee stiffness in one case (8.3%) and polyethylene wear in another patient (8.3%). Time elapsed between primary and revision surgeries averaged 5.08 years (2–12). Fifty percent of the primary TKAs were PS and 50% were CR. The FTA was measured by the same person (IP) in a preoperative digitalised full-length X-ray with the iPACS, software version 5.0. The radiographic osteolysis was measured prior to revision surgery by the same person in all cases (PH) [9]. In all cases, an arthrocentesis in sterile conditions was done immediately before revision TKA surgery and the maximum possible quantity of synovial fluid was aspirated. The average volume of synovial fluid was 14.08 (3–50) ml. During revision surgery, tissue samples of synovial tissue and periprosthetic membranes were taken and sent to the pathology laboratory. These samples were analysed by the same person (NJ) who measured the quantity of polyethylene particles in the samples and the degree of histiocytic infiltration with a modification of the Mirra et al. grading scale [3,10,11] (Fig. 1). Macroscopic osteolysis was graded as none, mild, moderate or severe by the surgeon during surgery. Other variables analysed were the addition of radiographic and intraoperative osteolysis and the addition of histiocytic infiltration and the quantity of particles in the tissue sample. The samples of synovial fluid, after identification, were preserved in a sterile tube in a freezer at −20 °C until the day of processing these samples in the following weeks. The samples were processed by the same person (IP) following the technique described by Minoda et al. [8]. It consists in the digestion of the sample in sodium hydroxide at 65 °C for 12 h and the application to a sucrose-density gradient in a 14 ml tube which is ultra-centrifuged at 28,000 rpm for 3 h at 4 °C. The highest level of the tube is applied to an isopropanolol–water density gradient in a 40 ml tube, which is also ultracentrifuged at 28,000 rpm for 1 h at 20 °C to group the polyethylene particles. The interface line is filtered through a 0.1 μm polycarbonate filter to retain the polyethylene particles. This filter is attached to a Petri plate. In the scanning electron microscope (SEM) department, the filter is set on a brass layer and coated with a gold layer of 40 nm for SEM analysis (Quanta 200 FEI, Co.). In the SEM analysis, 10 random 10,000 augments fields were studied by the same person (EP). If there was a doubt about the composition of a particle, a study with energy-dispersive spectrometry (EDS) was performed so as to confirm that it was a polyethylene and
Fig. 1. Microscopic analysis of synovial tissue: moderate concentration of histiocytes with focal foreign body reaction of giant cells and polyethylene particles (hematoxylin and eosin, original magnification ×20).
403
Fig. 2. Polyethylene particles of different sizes and shapes (scanning electron microscope × 10,000).
not a contaminant particle. In each random field, the number, size and shape of the particles were analysed (Fig. 2). With that data, we found out the polyethylene particles concentration per ml of synovial fluid and the total quantity of particles in each patient.
2.1. Statistical analysis Data were analysed using SPSS for Windows version 17.0 (SPSS Inc., Chicago, IL, USA). For the statistical correlations of the studied variables, the Spearman's rank correlation coefficient rho was used. Statistical significance was set at a p value of b 0.05. 3. Results In the preoperative X-rays, osteolysis was graded as none in six patients, mild in zero cases, moderate in two and severe in four patients. In the intraoperative assessment, osteolysis was graded as none in five patients, mild in two cases, moderate in one case and severe in four patients. The pathological study of the samples of the joint membranes showed six cases with no polyethylene particles, one patient with a low number of particles, three cases with moderate and two cases with a high number of particles. In these samples, there were no cases without histiocyte infiltration. It was mild in four cases with isolated histiocytes. There were five moderate cases with few groups of histiocytes and three severe cases with many groups of histiocytes. The electron microscope analysis of the processed synovial fluid showed polyethylene particles in different concentrations in all samples, an average concentration of particles that was 1.55 ± 1.34 × 106 particles/ml and the total average of polyethylene particles in the samples was 16.9 ± 10.58 × 106. The percentage of particles greater than 1 μm was 19.4%, and the analysis of the shape showed that 27.4% of the particles were not round. The statistical analysis showed a significant correlation between a higher concentration of polyethylene particles in synovial fluid and a higher degree of osteolysis assessed intraoperatively or the addition of both radiological and intraoperative osteolysis. The concentration of particles in synovial fluid also showed a significant correlation with a higher degree of particles in the histological analysis or the addition of particles and histiocytes in the tissue samples (Table 1). Both, a greater histiocyte infiltration in the tissue and a higher number of polyethylene particles in the tissue samples were significantly correlated with a higher osteolysis assessed both in the preoperative X-rays and in the intraoperative macroscopic aspect of the bone (Table 2). When analysing the size of the particles, there was a positive correlation between a higher percentage of particles greater than 1 μm and greater osteolysis both in the radiological and in the intraoperative assessment. No relationship was found between the shape of the particles (percentage of round particles) and the histological findings or the degree of osteolysis.
404
I. Piñol et al. / The Knee 21 (2014) 402–405
Table 1 Correlations between and statistical significance of polyethylene particle concentration in synovial fluid and the studied parameters of osteolysis, volume of synovial fluid and histological analysis. Particles/ml (concentration) n = 12
Spearman's rho
p
Radiological osteolysis Macroscopic osteolysis Total osteolysis Synovial fluid volume Particles in tissue sample Histiocytes in tissue sample Particles + histiocytes in tissue sample
0.412 0.620 0.599 0.743 0.724 0.417 0.677
0.183 0.032 0.040 0.052 0.008 0.177 0.016
4. Discussion The main finding of our study is the clear correlation between a higher concentration of particles in the synovial fluid in the patient joint and the greater histiocytic reaction to a foreign body in the tissue samples and the higher degree of osteolysis in the bone surrounding the failed knee arthroplasty. As far as we know, this is the first study that tries to correlate the concentration of polyethylene particles in synovial fluid and pathological findings in a failed TKA. Osteolysis around the knee components is in many cases related to aseptic loosening of the TKA in the long term due to foreign body reaction to polyethylene particles and, in this process, the tissue macrophages activate bone resorption. These polyethylene particles are in the synovial fluid before they are in the surrounding tissue, so the particles can be isolated either from the synovial fluid or in the tissues. To study the concentration of particles, a joint puncture is enough, with very few risks and without a more aggressive procedure as a synovial biopsy that is required to study the particles in the tissue. The adverse biological reactions caused by osteolysis are dependant not only on the amount of the wear, but also on the concentration and size of the polyethylene particles [12]. Particles less than 10 μm have been related to a greater capacity to cause osteolysis because particles greater than that size are not phagocytised by the macrophages [13,14]. In the present study, a higher concentration of particles greater than 1 μm has been found in knees with more severe osteolysis. However, there were very few particles greater than 10 μm. Thus, a relationship between osteolysis and the biggest particles could not be found. There is some evidence about the fact that long particles cause more inflammation than the round ones [7]. However, no relationship between the shape of the particles and the pathological reaction to foreign body or a greater osteolysis could be found in this study. The results of the present study confirm that a high concentration of polyethylene particles in the synovial fluid is related to a high concentration of particles and histiocytes in the tissue. The inflammatory response to a foreign body would be the cause of a severe osteolysis and the aseptic loosening of the TKA [3,4,15]. A simple X-ray is the most frequently used image study for osteolysis, but this method can only obtain images in two dimensions [16]. Using a CT-scan or MRI images would allow for a better study of osteolysis around TKA as the TKA structure would be studied in 3-dimensions [17–19]. In our study, a better correlation between the particles concentration and Table 2 Correlation with Spearman's rho (and statistical significance) between the osteolysis grade and the histological findings (particles, histiocytes and both) in the tissue samples. n = 12 Radiological osteolysis Macroscopic osteolysis Total osteolysis
Particles
Histiocytes
Particles + histiocytes
0.79 (p = 0.002)
0.732 (p = 0.007)
0.766 (p = 0.004)
0.808 (p = 0.001)
0.662 (p = 0.019)
0.743 (p = 0.006)
0.804 (p = 0.002)
0.650 (p = 0.022)
0.729 (p = 0.007)
the intraoperative assessment of the osteolysis was found. It suggested that radiological assessment of the osteolysis might not be so precise and this may be a limitation of our study. This study has several limitations like the reduced number of cases and the fact that different TKA models were used in the primary TKA. Another limitation is the type of filters used, with a size of the pores of 0.1 μm, following the technique described by Minoda [8]. Therefore, particles smaller than this size were not retained in the filter. Nevertheless, some studies have shown that most of the particles caused by TKA wear are in the 0.1–1 μm range [15,20] and the particles that provoke a high degree of macrophage activation are those between 0.3 and 10 μm [14,21]. Thus, particles smaller than 0.1 μm, not retained in the filter used, might cause little osteolysis. In this study, as in others previously reported [15,20], most of the isolated particles were in the 0.1–1 μm range. To conclude, we can say that, in an in vivo TKA scenario, the presence of a high concentration of polyethylene particles in the synovial fluid seems to be the cause of a highly active foreign body histological reaction with an increased number of histiocytes, which seems to be the cause of high grade of osteolysis around the implant. 5. Conflict of interest statement 1. Royalties from a company or supplier (the following conflicts were disclosed): none 2. Speakers bureau/paid presentations for a company or supplier (the following conflicts were disclosed): none 3A. Paid employee for a company or supplier (the following conflicts were disclosed): none 3B. Paid consultant for a company or supplier (the following conflicts were disclosed): none 3C. Unpaid consultants for a company or supplier (the following conflicts were disclosed): none 4. Stock or stock options in a company or supplier (the following conflicts were disclosed): none 5. Research support from a company or supplier as a principal investigator (the following conflicts were disclosed): none 6. Other financial or material support from a company or supplier (the following conflicts were disclosed): none 7. Royalties, financial or material support from publishers (the following conflicts were disclosed): none 8. Medical/orthopaedic publications editorial/governing board (the following conflicts were disclosed): none 9. Board member/committee appointments for a society (the following conflicts were disclosed): none Acknowledgement The authors gratefully acknowledge the assistance in the statistical analysis by Sergi Mojal. References [1] Sharkey PF, Hozack WJ, Rothman RH, Shastri S, Jacoby SM. Insall Award paper. Why are total knee arthroplasties failing today? Clin Orthop Relat Res Nov 2002;404:7–13. [2] Baxter RM, Freeman TA, Kurtz SM, Steinbeck MJ. Do tissues from THA revision of highly crosslinked UHMWPE liners contain wear debris and associated inflammation? Clin Orthop Relat Res Aug 2011;469(8):2308–17. [3] Chiba J, Rubash HE. A biochemical, histologic, and immunohistologic analysis of membranes obtained from failed cemented and cementless total knee arthroplasty. Clin Orthop Relat Res Dec 1994;309:278. [4] Ingham E, Fisher J. The role of macrophages in osteolysis of total joint replacement. Biomaterials Apr 2005;26(11):1271–86. [5] Jacobs JJ, Roebuck KA, Archibeck M, Hallab NJ, Glant TT. Osteolysis: basic science. Clin Orthop Relat Res Dec 2001;393:71–7. [6] Gupta SK, Chu A, Ranawat AS, Slamin J, Ranawat CS. Osteolysis after total knee arthroplasty. J Arthroplasty Sep 2007;22(6):787–99. [7] Hallab NJ, Jacobs JJ. Biologic effects of implant debris. Bull NYU Hosp Jt Dis 2009;67(2):182–8.
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