Infections regularly complicate orthopaedic procedures loosing implant stability and impairing bone union. Nevertheless, the question whether infection is caused by pathogens transposed intraoperatively, infiltrating the implant with blood stream or lymph, or dwelling in clinically healthy tissues, remains unanswered. The AIM of our study was to validate the hypothesis that pathogens may residue deep tissue.

Material and Methods: Skin, subcutaneous fat, muscle and fracture gap callus were obtained from 155 adult patients operated on due to closed comminuted fractures of tibia or femur, 75 because of non-alignment of bone axis and 80 due to delayed fracture healing.

Results: Aerobic bacteria were isolated from gap callus of 12% healing and 31% non-healing fractures, but also from deep soft tissues. No anaerobic bacteria were detected. PCR amplifications of 16s rRNA were found positive in 40% of callus specimens proving presence of bacterial DNA even when no isolates were found. The 95% similarity of the genetic pattern of some strains from foot skin and callus, estimated with RAPD technique, suggested their foot skin origin.

Conclusions: The colonizing bacteria and their DNA were detected in fracture callus and deep soft tissues. Contamination was precluded by lack of isolates in disinfected skin and materials used for sampling cultured after surgery. Our results point out that bacterial cells residing clinically non-infected deep tissues may be a source of infection, when activated by mechanical trauma and/or orthopaedic implant insertion.

We previously reported the presence of the bacterial genetic material (16S rRNA) and viable pathogens in fracture gaps specimens, which suggests an impaired pathogen recognition and/or elimination. The aim of study was to validate the hypothesis that patients with delayed bone fracture healing express the higher frequency of TLR4 mutations. Observations were performed in 295 patients treated due to closed fractures of the long bones of the lower extremity; in 151 with delayed bone union (Group A), and in 144 with uneventful healing (Group B). Control group consisted of 125 healthy blood donors from ethnically the same as investigations groups polish population. Fracture gaps and deep tissue biopsies served for microbiological studies, and DNA isolated from venous blood leukocytes was used for analysis of mutations of TLR4 gene at Asp299Gly (1/W) and Thr399Ile (2/W).

Results: Microbiological studies revealed positive isolates in 31.5% fracture gaps in Group A and 16.4% in Group B (p< 0.05). The most frequent isolates were S. epidermidis, S. aureus and S. warneri, capitis, sciuri and lentus, in lower percentage micrococci and enterococci. Amplification of 16S rRNA was positive in 56.8 and 65.2% of fracture gaps in both groups respectively. The frequency of occurrence of 1/W was significantly higher (p< 0.05) in subgroups of patients with non-healing infected vs. sterile fractures. In all subgroups with viable pathogens isolated from fracture gaps the frequency of 1/W allele was higher when compared with subgroups, where fracture gaps occurred sterile.

Discussion: Performed investigations supported our previously reported observations that gaps of closed bone fractures are not sterile and are positive for 16S rRNA. Genetic predisposal to infection and inflammatory response evoked by a single TLR4 mutation may be one of the factors affecting bone union. Observed coexistence of bacterial colonization with decreased inflammatory reaction observed in individuals bearing TLR4 mutations have to be mentioned as a possible, etiologic factor responsible for delayed healing

Edema and infection represent serious complications of blunt extremity trauma. It is important to differentiate between pathophysiological changes within tissues proximal and within distal to the site of trauma. The aim was to investigate the effects of soft tissue trauma on the microcirculation of the mouse lower limb. Endothelial leakage and leukocyte accumulation proximal and distal to the site of trauma were studied using intravital fluorescence microscopy.

Low-energy trauma to the lower limb was defined in previous experiments as a trauma transferring 50% of the energy required to produce tibial fracture. The trauma was inflicted under general anesthesia by an accelerator, hitting the mid-section of the calf in a perpendicular direction. 5, 90, and 180 minutes after trauma, the following microcirculatory parameters were measured: diameter of arterioles, venules, functional capillary density (FCD), extravasation of FITC-dextrane, and leukocyte-endothelial cell-interactions. Two groups (control and trauma) were studied proximal to, distal to and at the site of trauma. Skin, subcutaneous tissue and muscle were investigated individually in the trauma and the control groups (each group n=7).

At the site of trauma, distinct extravasation and edema formation in all tissues was observed. In subcutaneous and muscle tissue, microvascular thrombosis as well as edema were detected proximal and distal to the trauma. FCD was reduced in muscle and fat tissue. The numbers of rolling and adherent leukocytes were enhanced 5 minutes after trauma and throughout the observational period.

Our results demonstrate endothelial leakage and extravasation early after low-energy soft tissue trauma in all soft tissues proximal and distal to the site of trauma. In addition, we found high accumulation of leukocytes in all locations, especially in soft tissues. The model presented is ideally suited for the in vivo investigation of new therapeutic strategies for edema and thrombosis prevention in animals with soft tissue trauma.