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

Satisfactory healing of soft tissue wounds and bone fracture requires activation and response of the immune system. Our previously reported laboratory observations revealed that proper healing of the long bone fractures corresponds with an increased mass of lymph nodes (LNs) draining the site of injury when compared with contralateral limb, whereas delayed fracture healing is characterized by decreased mass of the regional LNs. Aim of the study was to investigate whether an image of LNs may be an indicator of the kinetics of fracture healing.

Patients & Methods. Observations were carried out in two groups of adult patients with closed not infected fractures of lower extremities healing uneventfully (n=15) and fractures with delay healing (n=17, diagnosed clinically and with X-ray and CT scans). Patients with venous thromboses were excluded. Morphologic structures of the lymphatic system of lower extremities were visualized on lymphoscintigraphies with 99mTc-Nanocol. Lymphoscintigrams were scanned and evaluated quantitatively. The surface of iliac lymph nodes of the fractured and contralateral limb served to calculate of LNs size index. Differences between groups were evaluated using Student-T-test.

Results. The surfaces of inguinal LNs of the fractured limb in patients with uneventful healing fractures were larger than in contralateral limbs (index=1.67±0.57), whereas in patients with non-union the inguinal LNs surface was smaller (index=0.49±0.45, p=0,00000031).

Conclusions. Lymphoscintigraphic pictures of the lymph nodes draining the site of injury reflect to a certain extent regenerative processes. Speculatively, observed changes depend upon molecular mediators drained with the lymph from fracture gap. The method can be used for non-invasive monitoring of the fracture repair process.