The clinical, radiological, microbiological, and molecular profile of the skin-penetration site of transfemoral amputees treated with bone-anchored prostheses

Abstract

The breach of the skin barrier is a critical issue associated with the treatment of individuals with transfemoral amputation (TFA) using osseointegrated, percutaneous titanium implants. Thirty TFA patients scheduled for abutment exchange or removal were consecutively enrolled. The aims were to determine the macroscopic skin signs, the presence of bacteria and the gene expression in abutment-adherent cells and to conduct correlative and comparative analyses between the different parameters. Redness and a granulation ring were present in 47% of the patients. Bacteria were detected in 27/30 patients, commonly in the bone canal. Staphylococcus aureus, coagulase-negative staphylococci, streptococci, and Enterococcus faecalis were the most common. A positive correlation was found between TNF-α expression and the detection of S. aureus. Staphylococcus aureus together with other bacterial species revealed a positive relationship with MMP-8 expression. A negative correlation was demonstrated between the length of the residual femur bone and the detection of a granulation ring and E. faecalis. A positive correlation was revealed between fixture loosening and pain and the radiological detection of endosteal bone resorption. Fixture loosening was also correlated with the reduced expression of interleukin-10 and osteocalcin. It is concluded that several relationships exist between clinical, radiological, microbiological, and molecular assessments of the percutaneous area of TFAs. Further long term studies on larger patient cohorts are required to determine the precise cause-effect relationships and unravel the role of host-bacteria interactions in the skin, bone canal and on the abutment for the longevity of percutaneous implants as treatment of TFA. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 578-589, 2017.

Keywords: gene expression; infection; osseointegration; percutaneous; transfemoral amputees.

© 2016 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc.

Figures

Figure 1

An overview of the implant components, the sampling procedures and the clinical assessment of the skin. (A, B) Schematic drawing showing the implant components and surrounding tissue. Abutments with a diameter of 11 mm and a length of 72–77 mm were machined from cp‐Ti grade IV (n = 29) and titanium alloy (Ti6Al4V, grade V) (n = 1), cleaned by ultrasonication followed by sterilisation. (C‐E) Bacterial swabs taken from skin‐penetration site (C), abutment (D) and bone canal (E). (F, G) Retrieval of abutment for macroscopic inspection (F) and placement in a fixed volume of RNAlater (G) for subsequent RNA and protein extraction. (H‐K) Representative skin‐penetration sites for the scoring system: H) Score 0: clean, dry, tight; I) Score 2: purple, serous secretion; J) Score 3: granulation ring, redness; K) Score 4: granulation ring, redness, serous secretion.

Figure 2

Radiological changes (A‐F). (A) The early (after 3 years) postoperative X‐ray from patient 1 (P1). (B) The corresponding X‐ray at the 10‐year post‐operative follow‐up of P1. Proximal trabecular buttressing (an increase of the bone density at the proximal end of the fixture) (thick, black arrow) and cancellisation (an increase in the porosity of the cortex surrounding the fixture) (thin, black arrow) have developed. (C) The early postoperative X‐ray (after the first surgery, S1) from patient 2 (P2). (D) At two‐years, a cortical thinning (a decrease in the width of the cortex around the fixture) (white arrow‐head) is evident. (E) An early postoperative X‐ray from patient 3 (P3) after the second surgery, S2. (F). The X‐ray from patient 3 after seven years, demonstrates endosteal bone resorption (thin, white arrow) and distal bone resorption (thick, white arrow). Endosteal bone resorption is the resorption of endosteal bone around the threads of the fixture, while distal bone resorption is the resorption of the bone at the distal end of the fixture.

Figure 3

Western blot analysis of protein A on the abutment. Examples of positive and negative Staphylococcus aureus protein A detection on abutment. P1: One patient of two who did not demonstrate positive detection of protein A. P2‐P4: Four patients (P2‐P4) with positive bands of Staphylococcus aureus protein A (≈50 kDa) detected in 28/30 patients.

Figure 4

Gene expression of (A) osteocalcin (OC) and (B) interleukin‐10 (IL‐10) in the abutment‐adherent cells. The comparison was performed between patients with osseointegrated fixtures (n = 27) and patients with loosened fixtures (n = 3). Bars indicate statistically significant differences (p < 0.05).

Figure 5

Gene expression of tumour necrosis factor‐alpha (TNF‐α) and matrix metalloproteinase‐8 (MMP‐8) in the abutment‐adherent cells. The comparative analyses were performed as following: (A) TNF‐α expression in patients in whom S. aureus was not detected (n = 16) versus patients in whom S. aureus was detected (n = 14); (B) TNF‐α expression in patients in whom S. aureus was not detected (n = 16) versus patients in whom S. aureus was detected alone (n = 6) or detected with other bacteria (n = 8); (C) MMP‐8 expression in patients in whom S. aureus was not detected (n = 16) versus patients in whom S. aureus was detected alone (n = 6) or detected with other bacteria (n = 8). The bars indicate statistically significant differences (p < 0.05).