**Cytokine Profile in Patients with Aseptic Loosening of Total Hip Replacements and Its Relation to Metal Release and Metal Allergy**

**Rune J. Christiansen 1,2,\*, Henrik J. Münch 3, Charlotte M. Bonefeld 2, Jacob P. Thyssen 4, Jens J. Sloth 5, Carsten Geisler 2, Kjeld Søballe 3, Morten S. Jellesen <sup>1</sup> and Stig S. Jakobsen 3,\***


Received: 19 July 2019; Accepted: 9 August 2019; Published: 20 August 2019

**Abstract:** Metal release from total hip replacements (THRs) is associated with aseptic loosening (AL). It has been proposed that the underlying immunological response is caused by a delayed type IV hypersensitivity-like reaction to metals, i.e., metal allergy. The purpose of this study was to investigate the immunological response in patients with AL in relation to metal release and the prevalence of metal allergy. THR patients undergoing revision surgery due to AL or mechanical implant failures were included in the study along with a control group consisting of primary THR patients. Comprehensive cytokine analyses were performed on serum and periimplant tissue samples along with metal analysis using inductive coupled plasma mass spectrometry (ICP-MS). Patient patch testing was done with a series of metals related to orthopedic implant. A distinct cytokine profile was found in the periimplant tissue of patients with AL. Significantly increased levels of the proinflammatory cytokines IL-1β, IL-2, IL-8, IFN-γ and TNF-α, but also the anti-inflammatory IL-10 were detected. A general increase of metal concentrations in the periimplant tissue was observed in both revision groups, while Cr was significantly increased in patient serum with AL. No difference in the prevalence of metal sensitivity was established by patch testing. Increased levels of IL-1β, IL-8, and TNF-α point to an innate immune response. However, the presence of IL-2 and IFN-γ indicates additional involvement of T cell-mediated response in patients with AL, although this could not be detected by patch testing.

**Keywords:** arthroplasty; replacement; hip; hypersensitivity; contact; allergy and immunology; cytokines; Interleukin-8

#### **1. Introduction**

#### *1.1. Background*

Aseptic loosening (AL) of implants is the most common reason for revision surgeries in patients with total hip replacements (THRs), representing close to 75% of all cases, with serious consequences for patients and healthcare systems [1,2]. Although the etiology of AL is multifactorial and yet to be fully understood, evidence suggest that the predominant cause of AL is due to a macrophage-driven chronic inflammatory response initiated by implant wear [1,3–5]. This adverse tissue reaction is associated

with the innate immune system and can lead to the bone degrading state of osteolysis, subsequently resulting in implant failure by AL.

Cytokines are small messenger molecules that coordinate the immune response by regulating inflammation and modulating cellular activities such as growth, differentiation and survival [6]. Key mediators of osteolysis have been identified as pro-inflammatory cytokines like interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF)-α secreted by activated macrophages. In turn, these cytokines are capable of inducing the differentiation of osteoclast precursor cells into mature, bone resorbing, osteoclasts [1,7–11]. Interferon (IFN)-γ is another important immune regulatory cytokine implicated in bone resorption but also in inflammation progression and cell mediated immunity [12,13].

Macrophages have been established as important mediators of ostolysis, but several other cell types have also been identified in the periimplant tissue of failed implants, including lymphocytic T cells [9,14–16]. T helper (Th) cells, a subtype of T cells, are important regulators of macrophage function and the adaptive immune response, which given rise to the concept of implant-related metal sensitivity. This concept is evolved around a delayed type IV T cell mediated hypersensitivity (DTH), exemplified by allergic contact dermatitis to metal ions (metal allergy). Due to their small size, metal ions are considered to be incomplete antigens, referred to as haptens, and must interact with peptides or proteins to form an antigen able to mount DTH.

In support of the concept above, findings of elevated levels of metal particles and ions have been shown to correlate with an increased prevalence of metal allergy in patients with failing implants [17–21]. Furthermore, some of the most commonly applied alloys for THR like stainless steel (FeCrNiMo), cobalt chromium (CoCrMo) and titanium alloys (Ti6Al4V) contain known sensitizing metals [22].

Immunological studies of AL in THRs, have suggested the involvement of a Th1 cell response, crucial for DTH, due to increased levels of Th1 cell specific cytokines like IFN-γ and IL-2 [4,23]. Previous studies also suggest the involvement of a Th2 and Th17 cell response in AL, which are respectively characterized by the production of IL-4, IL-17 and granulocyte-macrophage colony-stimulating factor (GM-CSF) [24–26]. However, the causal relationship between immune reactions, metal release from implants, and AL is still uncertain.

### *1.2. Aim*

The aim of the present study was to determine and compare levels of THR relevant metals and cytokine profiles from periimplant tissue and blood serum, and to investigate the prevalence of metal allergy in patients undergoing revision surgery due AL, mechanical failure or undergoing primary THR surgery. Periimplant tissue obtained from patients with AL showed a significantly different cytokine profile suggesting the involvement of both innate and adaptive immunity in AL. No prevalence of metal allergy was established in patients with failed implants despite elevated levels of metal ions in the periimplant tissue.

#### **2. Experimental Section**

#### *2.1. Patients and Samples*

We conducted a prospective case study including three patient groups. This study was approved by the Central Denmark Region Committee on Biomedical Research Ethics (Journal number: 1-10-72-90-13). All patients gave their written informed consent before entering the study.

Criteria for inclusion in the AL (+) group were: revision (entirely or partial) due to aseptic loosening, osteolysis, or unexplainable pain that could not be treated conservatively. The AL (−) group; revision (entirely or partial) due to fracture, dislocation, or component failure. The Control group; patients received a primary THR. Implant components are listed in Table 1.


**Table 1.** Implant overview. Implant types and materials used for femoral, head, liner and acetabular components are given for patients in the revision groups. In addition to the implant bulk material, model names and surface finish is also listed. cpTi relates to commercially pure titanium and PS to plasma sprayed coatings. FeCrNiMn is also referred to as Orthinox stainless steel.

Criteria for exclusion: infection (positive Kamme-Lindberg biopsies [27]), use of immunomodulating medication, occupational metal exposure, known metal allergies towards implanted metals or secondary osteoarthritis (fracture, inflammation). The mean age for the AL (+) group was 60.8 years with a gender distribution of 4/2 (M/W). For the AL (−) group, the mean age was 73 years, and the distribution was 4/2 (M/W). The control group had a mean age of 62 years and a distribution of 5/3 (M/W). Tissue samples for cytokine and ICP-MS analysis were snap-frozen in liquid nitrogen and stored at −80 ◦C for later use. Serum obtained from patients blood samples were taken before the operation and stored at −80 ◦C for later cytokine and ICP-MS analysis.
