Introduction

Brachial plexus blocks are used widely to provide intra-operative and post-operative analgesia. Their efficacy is well established, but little is known about discharging patients with a numb or weak arm. We need to quantify the risk of complications for improved informed consent.

Unlike metal-on-Polyethylene, metal-on-metal (MoM) implants seem to affect the adaptive immune response as evident from the associated perivascular infiltrate containing lymphocytes and plasma cells. This is more pronounced in implant failure secondary to aseptic loosening, and may represent the failure mode. A reduction in CD8+ T lymphocyte counts has also been described with Hip Resurfacing. MoM articulations produce a much smaller order of size of wear particles (nanoparticles) than metal-on- Polyethylene, which may be responsible for the observed adaptive immune system effects. We therefore analyzed the effects of CoCr nanoparticles (CoCrNP) on Dendritic Cells, T cells & B cells.

We produced CoCrNP using repetitive short spark discharges between electrodes of prosthetic CoCr alloy. Electron micrography and Brunauer-Emmet-Teller method both confirmed nanoparticle size. The following experiments were then undertaken.

Dendritic Cells were cultured from mouse bone marrow and incubated with CoCrNP of varying concentrations for 24hrs, or lipopolysaccharide as a positive control. Activation status was then characterized by CD40 expression on fluorescence activated cell sorting (FACS) analysis.

We found CoCrNP did not significantly increase CD40 expression on DCs, neither did it alter CD40 or CD86 expression on B cells. Using a sublethal concentration of CoCrNP as determined from the viability tests, CoCrNP inhibited CD3 & CD3/CD28 dependent T-cell proliferation. This would indicate CoCrNP reduces T cell proliferation and/or survival, which may explain the observed reduction in CD8+ count with hip resurfacing. Understanding the development of the Peri-vascular infiltrate associated with MoM implants will however, probably require more complex (most likely in vivo) models.

Background: Increased use of metal on metal (MOM) hip replacements has stimulated interest in immunological effects of chronic CoCr elevation. Unlike metal-on-polyethelylene, MOM implants are associated with a perivascular infiltrate of lymphocytes & plasma cells. This may be the mode of failure of MOM implants. A reduction in CD8+ T lymphocyte counts associated with MOM implants has previously been described. CoCr therefore seems to affect the adaptive immune response even though it is not a proteinous antigen.

We therefore analyzed the effects of CoCr particles on T cells & B cells. We also analyzed it effects on dendritic cells, which are the key antigen presenting cells to T helper cells.

Methods: CoCr nano particles were produced by repetitive short spark discharges between electrodes of prosthetic CoCr alloy. Electron micrography & BET both confirmed nanoparticle size.

Dendritic cells (DCs) were harvested from mouse bone marrow & cultured in medium supplemented with GM-CSF for 6 days, generating DCs typically 80–90% CD11c+. These were incubated with CoCr in concentrations of 25, 10 & 2.5 μg/ml, for 24 hours, or lipopolysaccharide 1 μg/ml as a positive control. Following incubation, activation status of CD11c+ DCs was characterized by MHC Class II, CD40, CD80 & CD86 expression by FACS analysis.

T-Lymphocytes were harvested from mouse lymph nodes & cultured in medium without phenol red. These were incubated at 5 ×105 cells/well with either CoCr, conA (positive control) or CoCr + conA & repeated using 2.5 ×105 cells/well. Other positive controls (CD3 & CD 28) were studied in repeating the experiment. At 48 hours Almar Blue was added & further incubation for 24 hrs. Light absorbance at 570nm & 600nm was then used to determine T cell proliferation

B-Lymphocytes were harvested from the lymph nodes of mice which were only able to mount a B-cell reaction to Hen egg Lysozyme (HEL). These were incubated with medium with CoCr, HEL (positive control) or CoCr+ HEL. The concentration of the CoCr was varied between 25, 10 & 2.5 μg/ml. FACS analysis for markers of B cell regulation was performed after 48 hours incubation..

Results: CoCr did not significantly increase CD 40 expression on DCs, although such expression was increased significantly by lipopolysaccaride CoCr did not significantly up or down regulate B cells as compared to the effects of HEL. CoCr did inhibit proliferation of T-cells & this was more pronounced where the ratio of CoCr/cell density was higher.

Conclusion: Both dendritic & B cells are unaffected by CoCr in vitro. However, CoCr inhibited T cell proliferation. This demonstrates the observed reduction in CD + T cells are probably due to a direct effect of CoCr, & not mediated through another cell type. The perivascular response to MOM implants on the other hand probably requires cell interaction in an in vivo environment.

Introduction: Metal on Metal articulations produce Cobalt Chromium nanoparticles (CoCrNP) which seems to affect the adaptive immune system, as evident from the perivascular infiltrate of lymphocytes & plasma cells found around some implants, and the reduced CD8+ count described with hip resurfacing. We therefore analyzed effects of CoCrNP on Dendritic Cells, T cells & B cells.

Methods: CoCrNP were produced by repetitive short spark discharges between electrodes of prosthetic CoCr alloy. Electron micrography & BET both confirmed nanoparticle size.

Dendritic Cells were cultured from mouse bone marrow and incubated with CoCrNP of varying concentrations, for 24hrs, or lipopolysaccharide as a positive control. Activation status was then characterized by CD40 expression on FACS analysis.

Results: CoCrNP did not significantly increase CD 40 expression on DCs or Cd 40/ Cd 86 expression on B cells. At subletal concentrations, CoCrNP inhibited ∝CD3 & ∝CD28 dependent T-cell proliferation.

Discussion: CoCrNP reduces both signal 1 & signal 2 dependent T cell proliferation, which may explain the observed reduction in CD 8+ count with hip resurfacing.