Elsevier

Biomaterials

Volume 23, Issue 2, January 2002, Pages 517-526
Biomaterials

Inflammatory responses to orthopaedic biomaterials in the murine air pouch

https://doi.org/10.1016/S0142-9612(01)00134-XGet rights and content

Abstract

An in vivo model of the inflammatory response to orthopaedic biomaterials was used to examine cellular and cytokine responses to polymer particles of ultra high molecular weight polyethylene (UHMWPE) and polymethylmethacrylate (PMMA), and metal particles of cobalt–chrome (Co–Cr) and titanium alloy (Ti–6Al–4V). Responses were determined separately and in combinations, to examine interactions between different forms of biomaterials. Murine air pouches were injected with particle suspensions, and reactions evaluated using histological, immunological, and molecular techniques. All particulate biomaterials caused significant increases in membrane thickness compared with control (saline) air pouches, with the highest reaction seen in response to Ti–6Al–4V particles. A synergistic increase in membrane thickness was observed when PMMA was combined with UHMWPE, suggesting that multiple biomaterial stimuli markedly increase the inflammatory reaction. Cellular analysis indicated that all particles increased the absolute number and the percentage of macrophages in the membrane over the control level, with the most pronounced increase due to individual biomaterial occurring with UHMWPE particles. Cytokine analysis revealed that biomaterials provoked a strong IL-1 response. Ti–6Al–4V stimulated the highest IL-6 gene transcription and the lowest IL-1 gene transcription. The data suggest that synergism in the inflammatory response to biomaterials may be important in adverse responses to orthopaedic wear debris.

Introduction

A critical aspect of adverse responses to the biomaterials used in the construction of orthopaedic prosthetic components is the stimulation of cells in the peri-prosthetic tissue by particles resulting from wear of the prosthesis. The peri-prosthetic tissue serves as an interface between the prosthesis and the bone, and contains cells of the macrophage/monocyte lineage. Thus small particles are readily phagocytosed by the resident cells of the peri-prosthetic membrane. Plastics and metal are impervious to enzymatic destruction, and wear particles disturb the biological degradative function of phagocytes that ingest the debris [1]. Repeated phagocytosis of the particles results in activated cells that secrete both proteolytic enzymes and high levels of the inflammatory cytokines IL-1 and TNFα, which may in turn contribute to the osteolytic process by providing activation signals to osteoclasts [2]. Biomaterial debris also appears to provoke other biological effects, including granuloma formation and inflammatory cell influx, which may also contribute to bone resorption, osteolysis and the eventual loss of the prosthesis support. The tissue from the areas of osteolysis shows a synovial-like layer at the cement surface, and the presence of macrophages and foreign-body giant cells invading the femoral cortices. This appearance shares some histological characteristics of both rheumatoid arthritis (RA) and a foreign body reaction. Microscopic examination of specimens obtained at revision surgery of failed hip replacements has revealed a varied cellular composition of the pseudosynovium, with histiocytes, giant cells, lymphocytes, plasma cells and neutrophils all present, with the areas around the loosened prostheses characterized as aggressive granulomatous lesions consisting of well organized tissue containing histiocytic–monocytic and fibroblastic reactive zones [3]. Furthermore, immunohistological evaluation has revealed the presence of multinucleated giant cells and C3bi-receptor bearing monocyte–macrophages [4]. Particles of acrylic cement and shards of polyethylene appear incorporated into the histiocyte/macrophage or giant cell population, resulting in “foci” of cellular activity within the synovial-like membrane [5].

It is recognized that any type of particulate debris, including that generated by poor surgical technique, loss of mechanical fixation of the polymethylmethacrylate (PMMA) bone cement, or wear at the ultra-high molecular weight polyethylene (UHMWPE)–metal interface, may lead to the aseptic loosening process. However, the critical aspects of biomaterial debris that provoke the most severe cellular responses remain unclear, and quantitative analysis of cell types present in this periprosthetic tissue revealed considerable heterogeneity between tissues from different individuals. Hence a predictable model of the inflammatory response to biomaterials in particulate form would be useful to examine the molecular pathogenesis of the steps leading to the osteolytic process. We have adapted a murine model of inflammation that closely resembles the peri-prosthetic tissue encountered in aseptically loosened prosthetic components to evaluate the cellular response to biomaterials in vivo. The rodent air pouch has been identified as a useful model for the evaluation of the response to orthopaedic biomaterials [6], [7], providing cellular infiltration and mediators of inflammation that appear to closely resemble the pseudosynovium associated with aseptic loosening. In this study we have examined the cellular and cytokine reactions to both metal and polymeric biomaterials, and demonstrate that the model is sensitive differences in the material composition of the particles under investigation.

Section snippets

Murine air pouches

Air pouches were generated according to the methods of Sedgewick et al. [8] in groups of 10–15 female BALB/c mice. An area of the dorsal skin (2 cm2) was cleaned with alcohol and shaved to provide the pouch site. A subcutaneous injection of 3 ml of air was injected at a single site with 25-gauge needle and 3 ml syringe. The air pouches were injected with 1 ml of air on alternate days for 5 days to establish a definitive fluid filled pouch. On day 6, the pouches were injected with 500 μl of

Air pouch membrane

The histological appearance of the control (saline) air pouch membrane is illustrated by Fig. 2. The control membrane was characterized by an outer fibrous layer populated mainly by fibroblastic cells and an inner inflammatory layer predominantly comprised of macrophages. The mean thickness of control pouch membranes was 62.0 μm (S.D. 11.6 μm) while the number of nucleated cells was 960/mm2 with a standard deviation of 54 cells. The differential count revealed the control membrane was comprised

Discussion

In order to evaluate the mechanisms of the biological response to orthopaedic biomaterials, it is important to establish accurate models of the disease process. We have utilized the murine air pouch model, initially developed to investigate the response to orthopaedic materials by Schumacher et al. [6], [7], for the investigation of the inflammatory response to particulate debris. This model exhibits cellular infiltration and mediators of inflammation that appear to closely resemble the

Acknowledgments

The authors gratefully acknowledge the contributions of W. Dwayne Lawrence, M.D. and the Pathology Image Analysis laboratory at Hutzel Hospital in establishing the image techniques used in this study, and the grant support from the Veterans Administration (Rehabilitation Study Section) and the Arthritis Foundation.

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