Elsevier

Life Sciences

Volume 97, Issue 2, 3 March 2014, Pages 96-106
Life Sciences

Titanium and zirconia particle-induced pro-inflammatory gene expression in cultured macrophages and osteolysis, inflammatory hyperalgesia and edema in vivo

https://doi.org/10.1016/j.lfs.2013.11.008Get rights and content

Abstract

Aims

The biological reaction to wear debris is critical to the osteolysis underlying aseptic loosening of joint prosthetic implants. In an attempt to reduce aseptic loosening, ceramics have been introduced. This study was designed to evaluate, compare and correlate the expression of Toll-like receptors (TLRs), their intracellular adaptors and proinflammatory cytokines in cultured macrophages challenged with titanium or zirconia particles, as well as particle-induced osteolysis in calvaria and hyperalgesia and edema in hind paw.

Main methods

TLRs and their adaptors were evaluated at the mRNA level by RT-PCR, and cytokine expression was evaluated at the mRNA and protein levels. Osteolysis and hyperalgesia and edema were evaluated in vivo, in calvaria and hind paw, respectively.

Key findings

Cultured macrophages challenged with zirconia or titanium particles expressed increased mRNA for TLRs 2, 3, 4 and 9, and their adaptors MyD88, TRIF and NF-κB and cytokines TNF-α, IL-1β and IL-6, which were also increased at protein level. Quantitative differences are evident and, in general, zirconia particle-induced pro-inflammatory gene expression was lower than that induced by titanium particles. In in vivo experiments, exposition to titanium or zirconia particles induced osteolysis in calvaria and hyperalgesia and edema in hind paw; however those induced by zirconia particles were significantly lower. There is a strong and positive correlation between the expressions of mRNA for TLR4, NF-κB, TNF-α, IL-1β and IL-6.

Significance

Collectively, our data suggest that zirconia ceramic particles are less bioactive than titanium particles.

Introduction

Current estimates indicate that approximately 1.5 million joint replacement surgeries are performed annually worldwide (Schwarz et al., 2000). Although the survival rate of these prostheses is increasing, long-term outcomes are often limited by aseptic loosening induced by local bone resorption surrounding the implants, a process known as osteolysis (Looney et al., 2006). Although some promising strategies to prevent or inhibit periprosthetic osteolysis are under investigation (Chen et al., 2012, Geng et al., 2010, Mao et al., 2012), to date, aseptic loosening of prostheses can only be overcome by revision surgery. The key stimulus for osteolysis appears to be the phagocytosis of wear particles by macrophages and the resultant production of osteolytic inflammatory cytokines (Ingham and Fisher, 2005). In fact, macrophages represent at least 70% of all cells in the interface membranes around aseptic loose periprosthetic tissues (Tamaki et al., 2009). The precise nature of cellular interaction with wear debris and the subsequent production of inflammatory mediators remain unknown; however, Toll-like receptors (TLRs) appear to be promising candidates to facilitate this interaction (Pajarinen et al., 2009, Pajarinen et al., 2010, Takagi et al., 2007). TLRs are transmembrane proteins of inflammatory cells, which recognize well-conserved pathogen and endogenous danger-associated molecular patterns (Kawai and Akira, 2007). After ligand binding, TLRs stimulate the recruitment of a set of intracellular adaptors, such as myeloid differentiation primary response protein 88 (MyD88) and TIR domain-containing adapter inducing interferon-ß (TRIF, also known as TICAM1). MyD88 is a universal adapter that activates inflammatory pathways; it is shared by all TLRs with the exception of TLR3. TRIF is recruited by TLR3 and TLR4, and activates an alternative pathway (MyD88-independent pathway), leading to interferon (IFN)-β production. Both pathways culminate in the activation of NF-κB, which is the master regulator of TLR induced responses. Activation of this strong proinflammatory transcription factor leads to production of several proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, responsible for periprosthetic bone resorption (Masui et al., 2005, Xu et al., 2009). Therefore, although not directly demonstrated, TLRs may mediate cytokine production associated with osteolysis.

Titanium is one of the most used joint surface materials due to its biocompatibility, lower modulus and corrosion resistance. However, these attractive properties are counterbalanced by a number of reports about aseptic prosthetic failure (Lombardi et al., 1989, Harris, 2001, Maurer et al., 2000). Although studies about the biological reaction to titanium particles have been accumulated, contradictions still exist. For example, it was suggested that titanium particles stimulateTLR expression in aseptic loose periprosthetic tissues (Tamaki et al., 2009, Lahdeoja et al., 2010), while decrease it in cultured macrophages (Pajarinen et al., 2009, Takagi et al., 2007). In an attempt to reduce aseptic prosthetic failure, ceramics have been introduced with relative successful results (Kennedy et al., 2008, Innocenti et al., 2010). Among ceramic materials, zirconia has been the object of extensive investigations and is recognized for its good mechanical properties. However, information about the biological reaction to zirconia particles is still limited and the response of macrophages, which are known as the major mediators of osteolysis, to ceramic particles, is poorly understood. It is also not known whether the simple exposition to titanium or zirconia particles is able to initiate an immune and inflammatory response leading to pain and edema, two common features in patients with aseptic prosthetic loosening. Therefore, in this study we used cultured macrophages, largely used as an in vitro representation of the response to wear debris, to evaluate, compare and correlate titanium and zirconia particle-induced expression of TLRs, their intracellular adaptors and proinflammatory cytokines. Particle-induced osteolysis was evaluated in mice calvaria and particle-induced hind paw edema and painful response were evaluated and compared to those induced by carrageenan, a widely used inflammatory agent.

Section snippets

Animals

Male C57BL/6 mice (8–10 weeks old) were obtained from the Multidisciplinary Center for Biological Investigation at the University of Campinas (CEMIB-UNICAMP). All animal experimental procedures were approved by the Committee on Animal Research of the University of Campinas (protocol number 1312-1) and their guidelines for animal care were followed. The animals were maintained on a temperature-controlled room (± 23 °C) and were housed in plastic cages with soft bedding on a 12:12 h light cycle with

TLR mRNA expression in macrophages challenged with titanium and zirconia particles

TLR2 mRNA expression (Fig. 1A): zirconia and titanium challenge significantly increased TLR2 mRNA expression at 2 h and 24 h (as indicated by the symbol +). The expression increased up to 2 h and did not significantly change between 2 h and 24 h after zirconia or titanium challenge. Compared with titanium, zirconia particles induced a significantly higher increase in TLR2 mRNA expression at 2 and 24 h (as indicated by the symbol *). There is a statistically significant interaction between material

Discussion

This study demonstrated that cultured macrophages challenged with zirconia or titanium particles expressed increased mRNA levels for TLRs, their intracellular adaptors and pro-inflammatory cytokines, which were also increased at protein level. The in vivo exposition to zirconia or titanium particles induced significant osteolysis in calvaria and hyperalgesia and edema in the hind paw. These data show that the simple exposition to zirconia, as well as to titanium particles, in the absence of any

Conclusion

In summary, this study demonstrated that cultured macrophages challenged with zirconia or titanium particles express increased levels of TLRs, their intracellular adaptors and inflammatory cytokines. In vivo, both particles induced osteolysis in calvaria and hyperalgesia and edema in hind paw. Compared with titanium particles, zirconia particles induced, in general, lower expression of TLRs, its intracellular adaptors and osteolytic cytokines in cultured macrophages and induced lower osteolysis

Conflict of interest

The authors have no conflict of interest.

Acknowledgment

This study was supported by Grants 07/58796-4 and 07/56731-2 from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). L.F. is a recipient of a fellowship from the Fundação Araucária, Governo do Estado do Paraná, Brazil.

References (44)

  • M.C. Trindade et al.

    In vitro reaction to orthopaedic biomaterials by macrophages and lymphocytes isolated from patients undergoing revision surgery

    Biomaterials

    (2001)
  • A. Cerami

    Inflammatory cytokines

    Clin Immunol Immunopathol

    (1992)
  • E.M. Schwarz et al.

    Anti-TNF-alpha therapy as a clinical intervention for periprosthetic osteolysis

    Arthritis Res

    (2000)
  • R.J. Looney et al.

    Periprosthetic osteolysis: an immunologist's update

    Curr Opin Rheumatol

    (2006)
  • D. Chen et al.

    Inhibition of p38 mitogen-activated protein kinase down-regulates the inflammatory osteolysis response to titanium particles in a murine osteolysis model

    Inflammation

    (2012)
  • X. Mao et al.

    Inhibition of titanium particle-induced inflammation by the proteasome inhibitor bortezomib in murine macrophage-like RAW 264.7 cells

    Inflammation

    (2012)
  • Y. Tamaki et al.

    Increased expression of Toll-like receptors in aseptic loose periprosthetic tissues and septic synovial membranes around total hip implants

    J Rheumatol

    (2009)
  • J. Pajarinen et al.

    Profile of Toll-like receptor-positive cells in septic and aseptic loosening of total hip arthroplasty implants

    J Biomed Mater Res A

    (2010)
  • J. Pajarinen et al.

    Titanium particles modulate expression of Toll-like receptor proteins

    J Biomed Mater Res A

    (2010 Mar 15)
  • M. Takagi et al.

    Toll-like receptors in the interface membrane around loosening total hip replacement implants

    J Biomed Mater Res A

    (2007)
  • A.V. Lombardi et al.

    Aseptic loosening in total hip arthroplasty secondary to osteolysis induced by wear debris from titanium-alloy modular femoral heads

    J Bone Joint Surg Am

    (1989)
  • W.H. Harris

    Wear and periprosthetic osteolysis: the problem

    Clin Orthop Relat Res

    (2001)
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