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

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

A methodological platform to study molecular biocompatibility of biomaterials. Experimental and clinical studies

The aim of this project was to develop a methodological platform in order to advance our scientific understanding of the mechanisms of osseointegration. Screw-shaped, titanium implants, with different surface properties, were inserted in the rat tibia, or incubated in mono- or co-culture of human monocytes and MSCs. After different time points, the implant-adherent cells or the peri-implant bone were harvested and processed for different analyses. For in-vivo studies, qPCR, immunohistochemistry, histomorphometry, electron microscopy and removal torque analyses were used. In the in-vitro study, FACS, qPCR, ELISA and protein profiling were applied. Finally, qPCR was employed in a clinical study to analyse the abutment-adherent cells of osseointegrated fixtures. At the early time points in vivo, a higher gene expression of MSC recruitment and adhesion factors (CXCR4 and integrin-β1) was found in cells adhering to the oxidised compared to machined implant. This was corroborated by predominance of MSCs at the oxidised surface, as judged by immunohistochemistry and SEM. At the later time points, cells adhering to oxidised implants retained a higher expression of bone formation (ALP and OC) and bone remodelling (TRAP and CatK) genes. The qPCR findings correlated with histomorphometric, electron microscopy and removal torque measurements, revealing progressively increasing bone-implant contact and bone bonding and, as a result, an increase in the biomechanical stability of the oxidised implant. The enhanced RANKL/OPG expression ratio corresponded to the remodelling phase at the bone-implant interface. The qPCR analysis of FACS-sorted cells showed that the co-existence of monocytes and MSCs on the implant surface, in vitro, upregulates the gene expression of some cytokines in a cell-specific manner. The clinical study showed that bacterial colonisation was frequently detected on the skin, the abutment and in the bone canal. A higher expression of TNF-α was associated with positive cultures of S. aureus, whereas fixture loss was associated with lower expression of OC and IL-10. In conclusion, the present methodological platform enables detailed analyses of the events at the bone-implant interface. Employing this platform demonstrated that implant surface properties elicit a cellular and molecular cascade for rapid cell recruitment and enhanced bone formation and remodelling, which accelerates bone maturation and implant stability. Finally, the results of the thesis provide a first line of information on factors that could affect the performance of percutaneous implants