Edward Michael Greenfield

Fracture-related infection (FRI) can be a devastating complication after fracture surgery and presents many challenges for the treating clinician. A consensus definition including clinical, radiographic, and laboratory components has standardized the diagnosis and investigative efforts into this complex problem. Orthopaedic surgeons managing fractures must be able to appropriately diagnose and treat FRI, while adhering to basic principles to eradicate or suppress infection and promote fracture consolidation. From a multidisciplinary perspective, this review article examines contemporary literature regarding the definition, prevention, patient evaluation, management, and societal effect of FRI.

Background: Biofilms shield bacteria from immune defenses and antibiotics, contributing to implant-associated infections, particularly those caused by Staphylococcus aureus (S. aureus). Halicin, a repurposed antimicrobial with broad-spectrum activity, has shown promise against planktonic and biofilm-residing bacteria. Building on our previous work, we investigated whether halicin enhances the efficacy of conventional antibiotics when used in combination against S. aureus. Methods: The treatment efficacies of combination therapies of Halicin and conventional antibiotics were tested against planktonic S. aureus and S. aureus biofilms grown on titanium substrates of varying maturities. The emergence of antibiotic resistance in treated biofilm bacteria was also characterized. Results: In checkerboard assays against planktonic bacteria, halicin synergized with gentamicin, tobramycin, and cefazolin but not with rifampicin or vancomycin. In biofilms grown on titanium substrates, halicin combined most effectively with rifampicin, reducing viable bacteria by >3 logs in less-mature and >5 logs in more-mature biofilms compared to rifampicin alone. Compared to untreated biofilms, combination therapy reduced viable bacteria by >5 logs in all cases. This combination also prevented rifampicin resistance seen with rifampicin monotherapy. Halicin only modestly enhanced aminoglycoside activity against less-mature biofilms and had no significant effect when combined with vancomycin or cefazolin. Conclusions: These findings highlight that antibiotic-halicin synergy differs between planktonic and biofilm states, underscoring the importance of biofilm-specific studies. Highlighting the translatability of our results, the doses of conventional antibiotics combined with halicin required to affect planktonic and biofilm-residing S. aureus are all achievable systemically and in bone.

The patient-derived cells have numerous genomic amplifications and deletions, consistent with derivation of the cells from the osteosarcoma itself, rather than from bystander cells

Sarcospheres from a subset of patients are sensitive to romidepsin. Metabolic activity of sarcospheres from 18 human (A) and 10 canine (B) patients was measured after exposure for 48 hours to the indicated concentrations of romidepsin. Black curves represent sensitive subset of patients with ED50s 1,000 nmol/L. Each curve represents the mean of at least n = 3 independent experiments with sarcospheres from an individual patient sample. Each experiment included six sarcospheres at each of the indicated doses. Asterisks (*) indicate dose groups (100 and 1,000 nmol/L) that are significantly different (P < 0.05) from the vehicle-treated group for all sensitive samples based on one-way ANOVA with Dunnett multiple comparisons tests. C, Diameters of human patient–derived sarcospheres were measured every 2 hours during exposure to the indicated concentrations of romidepsin or vehicle by the Incucyte S3 Live Cell Analysis System (Sartorius). Each symbol represents mean ± SEM of N = 6 patients (two romidepsin sensitive and four romidepsin resistant). Sarcospheres from each patient were analyzed in 1–3 independent experiments. Each experiment included 3–6 sarcospheres per dose group. D, Representative images of romidepsin-sensitive sarcospheres from experiments depicted in C. Yellow size bars in lower left of images show 600 mm. Time stamp is shown in lower right of each image. E, Representative images from romidepsin-resistant patients from experiments depicted in C. Yellow size bars in lower left of images show 800 mm. Time stamp is shown in lower right of each image.

Clinical information for patients and romidepsin ED50s.

Dose responses and ED50s from secondary screen

Toxicity and therapeutic indices on Normal Human Osteoblasts

Suppliers and Storage Conditions for 13 Drugs in Secondary Screen

The primary effects of romidepsin are a transient block in proliferation for 143B sarcospheres and cytotoxicity for LM7 sarcospheres. 143B (A–D) and LM7 (E–H) sarcospheres were exposed to the indicated concentrations of romidepsin or vehicle for 24 hours (A, B, E, and F) or the indicated times (C, D, G, and H). A and E, Cell cycles were analyzed by flow cytometry after exposure to 10 nmol/L romidepsin or vehicle for 24 hours. Each symbol represents the percentage of the cell population in each phase from an individual experiment with 30 sarcospheres per group. Colored bars show means ± SD of N = 3–4 independent experiments. A one-way ANOVA with a Dunnett multiple comparisons test was used to compare experimental groups with the vehicle control group *, P < 0.05; ***, P < 0.01; ****, P < 0.001. B and F, Recovery from a 24-hour romidepsin exposure was assessed by repeated measurements of metabolic activity on days 2, 4, 8, and 14 after romidepsin addition. All repeated measurements were on the same set of sarcospheres, except that measurements on day 0 were on a separate plate of sarcospheres. Each symbol represents mean ± SD of N = 3 independent experiments. Each experiment included six sarcospheres per dose group. Friedman one-way repeated measures ANOVA by ranks with a Dunn multiple comparisons test was used to compare each experimental group with the vehicle control group over time. ***, P < 0.001. C and G, Sarcosphere diameters were measured every 2 hours during exposure to the indicated concentrations of romidepsin or vehicle by the Incucyte S3 Live Cell Analysis System (Sartorius). Each symbol represents mean ± SEM of N = 3–4 independent experiments. Each experiment included 3–6 sarcospheres per dose group. A Friedman test with a Dunn multiple comparisons test was used to compare each experimental group with the vehicle control group for 143B sarcosphere size experiments: *, P < 0.05 (1 nmol/L group), ***, P < 0.001 (10–10,000 nmol/L groups). D and H, Representative images from experiments depicted in C and G. Yellow size bars on the lower left of images show 800 mm. Timestamp is indicated in the lower right of each image.