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

Immunomodulatory effects exerted by extracellular vesicles from <i>Staphylococcus epidermidis</i> and <i>Staphylococcus aureus</i> isolated from bone-anchored prostheses

Staphylococcus aureus and Staphylococcus epidermidis are the bacteria that most frequently cause osteomyelitis. This study aimed to determine whether staphylococci isolated from osteomyelitis associated with septic loosening of orthopedic prostheses release extracellular vesicles (EVs) and, if so, to determine tentative immunomodulatory effects on the human monocytic cell line THP-1. EVs were isolated from bacterial cultures using filtration and ultracentrifugation and characterized by scanning electron microscopy, nanoparticle tracking analysis and Western Blot. The cytotoxic effect of EVs was analyzed by NucleoCounter and lactate dehydrogenase (LDH) analyses. Confocal laser scanning microscopy was employed to visualize the uptake of EVs by THP-1 cells. Activation of the transcription factor nuclear factor-κB (NF-κB) was determined in THP1-Blue™ NF-κB cells, and the gene expression and secretion of cytokines were determined by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. All investigated strains, irrespective of their biofilm formation ability, were able to secrete EVs in vitro. The S. aureus strains produced significantly more EVs than the S. epidermidis strains. Both S. aureus-derived EVs and S. epidermidis-derived EVs were internalized by THP-1 cells, upregulated Toll-like receptor 3 (TLR3) gene expression, activated NF-κB, and promoted the gene expression and secretion of interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, matrix metallopeptidase (MMP)-9 and IL-10. Whereas EVs from both staphylococcal species upregulated the proapoptotic DNA damage-inducible transcript 4 (DDIT4) gene and downregulated the antiapoptotic B-cell lymphoma 2 (Bcl-2) gene, cytolysis was preferentially induced in S. aureus EV-stimulated cells, possibly related to the expression of cytolytic proteins predominantly in S. aureus EVs. In conclusion, staphylococcal EVs possess potent cytolytic and immunomodulatory properties

Monocyte exosomes stimulate the osteogenic gene expression of mesenchymal stem cells.

Inflammation and regeneration at the implant-bone interface are intimately coupled via cell-cell communication. In contrast to the prevailing view that monocytes/macrophages orchestrate mesenchymal stem cells (MSCs) and progenitor cells via the secretion of soluble factors, we examined whether communication between these different cell types also occurs via exosomes. LPS-stimulated human monocytes released exosomes, positive for CD9, CD63, CD81, Tsg101 and Hsp70, as determined by flow cytometry and Western blot. These exosomes also contained wide size distribution of RNA, including RNA in the size of microRNAs. The exosomes were shown to interact with human mesenchymal stem cells. After 24 h of culture, a considerable portion of the MSCs had internalised PKH67-labelled exosomes. Furthermore, after 72 h, the gene expression of the osteogenic markers runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein-2 (BMP-2) had increased in comparison with control medium, whereas no significant difference in osteocalcin (OC) expression was demonstrated. The present results show that, under given experimental conditions, monocytes communicate with MSCs via exosomes, resulting in the uptake of exosomes in MSCs and the stimulation of osteogenic differentiation. The present observations suggest that exosomes constitute an additional mode of cell-cell signalling with an effect on MSC differentiation during the transition from injury and inflammation to bone regeneration

Mesenchymal stem cell-derived exosomes have altered microRNA profiles and induce osteogenic differentiation depending on the stage of differentiation

<div><p>Human mesenchymal stem cell (hMSC)-derived exosomes have shown regenerative effects, but their role in osteogenesis and the underlying mechanism are yet to be determined. In this study, we examined the time-course secretion of exosomes by hMSCs during the entire process of osteogenic differentiation. Exosomes derived from hMSCs in various stages of osteogenic differentiation committed homotypic cells to differentiate towards osteogenic lineage, but only exosomes from late stages of osteogenic differentiation induced extracellular matrix mineralisation. Exosomes from expansion and early and late stages of osteogenic differentiation were internalised by a subpopulation of hMSCs. MicroRNA profiling revealed a set of differentially expressed exosomal microRNAs from the late stage of osteogenic differentiation, which were osteogenesis related. Target prediction demonstrated that these microRNAs enriched pathways involved in regulation of osteogenic differentiation and general mechanisms how exosomes exert their functions, such as “Wnt signalling pathway” and “endocytosis”. Taken together, the results show that MSCs secrete exosomes with different biological properties depending on differentiation stage of their parent cells. The exosomal cargo transferred from MSCs in the late stage of differentiation induces osteogenic differentiation and mineralisation. Moreover, it is suggested that the regulatory effect on osteogenesis by exosomes is at least partly exerted by exosomal microRNA.</p></div

Differentially expressed microRNAs in exosomes derived from hMSCs during expansion and osteogenic differentiation.

<p>Differentially expressed microRNAs in exosomes derived from hMSCs during expansion and osteogenic differentiation.</p

Characterisation of exosomes by transmission electron microscopy (A) and nanoparticle tracking analysis (B).

<p>(A) A1, A4 and A7: Survey transmission electron micrographs of exosomes from the Exo_P6, Exo_D3 and Exo_D21 groups. Exosomes are approximately 30–150 nm and mainly display a rounded shape. A4-A9: Details of selected exosomes without (A2, A5 and A8) or with (A3, A5 and A9) labelling of 10 nm gold-labelled anti-CD63 antibodies. Scale bar: top panel (A1, A4 and A7), 100 nm; bottom panel (A2-A3, A5-A6 and A8-A9), 50 nm. (B) B1-B3: Distribution of particles from all nine captures of three biological replicates in each group of exosomes. B4-B6: Average concentration/size distribution of all nine captures in each group of exosomes. B7-B8: Mean and mode of particle size of each group of exosomes. B9: Mean concentration of particles in each group of exosomes. Error bars in B7-B9 denote standard error mean, SEM; n = 3 biological replicates.</p

Internalisation of exosomes in hMSCs.

<p>Confocal micrographs of hMSCs incubated for 24h with A) PBS (negative control); B) Exo_P6; C) Exo_D3 and D) Exo_D21. PKH67-stained exosomes are detected mainly in the cytoplasm of some of the cells. The intensity varied between individual cells. No PKH67-stained material was found in the negative control. A1-D1, blue channel; A2-D2, green channel; A3-D3, transmission (TD) channel; A4-D4, merged channels. Blue, DAPI-stained nucleus; Green, PKH67-stained exosomes. Scale bar: 20 μm.</p

Summary of biological pathways enriched by differentially expressed microRNAs.

<p>Summary of biological pathways enriched by differentially expressed microRNAs.</p

Evaluation of osteogenic differentiation of hMSCs after exosome treatment.

<p>(A) ALP activity 14 d after treatment with exosomes or the appropriate controls; (B) Quantification of calcium in ECM after 21 d; (C) Quantification of phosphate in ECM after 21 d. In (A-C), bars indicate mean values whereas error bars denote standard errors of the mean (SEM). Small letters a, b and c represent statistical significance when compared with NCtrl_D0, NCtrl_D14/D21 and Sexo_Ctrl respectively, based on Bonferroni-corrected p value < 0.05. n = 3 independent experiments; (D) Alizarin red staining after 21 d.</p