Lysostaphin-coated titan-implants preventing localized osteitis by Staphylococcus aureus in a mouse model.

The increasing incidence of implant-associated infections induced by Staphylococcus aureus (SA) in combination with growing resistance to conventional antibiotics requires novel therapeutic strategies. In the current study we present the first application of the biofilm-penetrating antimicrobial peptide lysostaphin in the context of bone infections. In a standardized implant-associated bone infection model in mice beta-irradiated lysostaphin-coated titanium plates were compared with uncoated plates. Coating of the implant was established with a poly(D,L)-lactide matrix (PDLLA) comprising lysostaphin formulated in a stabilizing and protecting solution (SPS). All mice were osteotomized and infected with a defined count of SA. Fractures were fixed with lysostaphin-coated locking plates. Plates uncoated or PDLLA-coated served as controls. All mice underwent debridement and lavage on Days 7, 14, 28 to determine the bacterial load and local immune reaction. Fracture healing was quantified by conventional radiography. On Day 7 bacterial growth in the lavages of mice with lysostaphin-coated plates showed a significantly lower count to the control groups. Moreover, in the lysostaphin-coated plate groups complete fracture healing were observed on Day 28. The fracture consolidation was accompanied by a diminished local immune reaction. However, control groups developed an osteitis with lysis or destruction of the bone and an evident local immune response. The presented approach of terminally sterilized lysostaphin-coated implants appears to be a promising therapeutic approach for low grade infection or as prophylactic strategy in high risk fracture care e.g. after severe open fractures

Effect of antibiotic infused calcium sulfate/hydroxyapatite (CAS/HA) insets on implant-associated osteitis in a femur fracture model in mice.

Cerament (Bonesupport Holding, Lund, Sweden) is a bioresorbable synthetic bone substitute consisting of calcium sulfate and hydroxyapatite which is successfully used as a bone graft in bone defects or in delayed and non-unions after fractures. Besides, calcium sulfate/ hydroxyapatite (CAS/HA) could have, attributed to its composition and osteoinductive properties, have great importance in the treatment of bone infections with critical size defects (CSD). Aim of the study was to evaluate the effects of antibiotic infused CAS/HA on inflammation and bone healing in an implant-associated osteitis mice model. In a standardized murine model, the left femur of 72 BALB/c mice were osteotomized, generating a CSD (2,5 mm) with stabilization through a 6-hole titanium locking plate. Osteitis has been induced through inoculation of Staphylococcus aureus (SA) into the fracture gap. To analyze the effect of CAS/HA, following groups were generated with either CAS/HA, CAS/HA with gentamycin (CAS/ HA-G) or CAS/HA with vancomycin (CAS/HA-V) insets placed into the osteotomy. Debridément and lavages were progressed on day 7 and 42 to determine the local bacterial growth and the immune reaction. Fracture healing was quantified on day 7 and 42 by x-ray and bone healing markers from blood samples. Progression of infection was assessed by estimation of colony-forming units (CFU) and immune response was analyzed by determination of Interleukin (IL)- 6 and polymorphonuclear neutrophils (PMN) in lavage samples. Osteitis induced higher IL-6 and PMN-levels in the lavage samples on day 7. Both parameters showed a reduction in all groups on day 42. CAS/HA-V revealed a significant reduction of CFU and PMNs in lavage samples on day 42. A positive effect on bone healing could only be shown in non-infected mice. Whereas, application of mere CAS/HA in infected mice did show tendencies of bone destruction and lysis, independent of impregnation with antibiotics or not. Thus, application of CAS/HA in acute implant-associated infections is not recommended. In non-infectious environments or after infect-convalescence CAS/HA could albeit serve as a suggestive tool in trauma and orthopedic surgery

Detection of leukocytes in lavage fluid.

<p>Significantly lower counts of leukocytes in lavages of both mice groups with lysostaphin-coated plates (<i>n</i> = 52) versus control groups (mice with uncoated plates, and mice with only PDLLA coated plates; <i>n</i> = 49). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001. Statistical analysis was performed using two-tailed Student's t-test and Mann-Whitney-test.</p

Percentage distributions of neutrophils in lavage fluid.

<p>In mice groups with lysostaphin-coated plates (<i>n</i> = 52) are less neutrophils than in the control groups (<i>n</i> = 49). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001. Statistical analysis was performed using two-tailed Student's t-test and Mann-Whitney-test.</p

Lysostaphin-coating of titanium discs prevents bacterial growth.

<p>Titanium discs were incubated with in SA strain ATCC 29213 for 48 h. 36 of 42 cultures with lysostaphin-coated discs were sterile and thus only the six positive cultures are shown. The median CFU of cultures with uncoated discs (n = 50) was 5.62E+07 (<i>p</i><0.0001). CFU are presented as box plots with median and whiskers min to max. Statistical analysis was performed using two-tailed Mann-Whitney-test.</p

Score of fracture healing.

<p>Standardized radiographic quantification of fracture healing from mice groups with lysostaphin-coated plates (<i>n</i> = 51) vs. control groups (<i>n</i> = 50) on Days 7, 14, and 28 after osteotomy, fracture fixation and inoculation of SA. Significant differences on Day 7: not coated vs. lysostaphin +40 kGy-β (<i>p</i> = 0.0340), PDLLA vs. lysostaphin (<i>p</i> = 0.0211), PDLLA vs. lysostaphin +40 kGy-β (<i>p</i> = 0.0017). On Day 14: PDLLA vs. lysostaphin (<i>p</i> = 0.0009), PDLLA vs. lysostaphin +40 kGy-β (<i>p</i> = 0.0014). On Day 28: not coated vs. lysostaphin (<i>p</i> = 0.0186), not coated vs. lysostaphin +40 kGy-β (<i>p</i> = 0.0186), PDLLA vs. lysostaphin (<i>p</i> = 0.0014), PDLLA vs. lysostaphin +40 kGy-β (<i>p</i> = 0.0022). Radiographic quantifications are presented as mean and SEM. Statistical analysis was performed using one-tailed Mann-Whitney-test.</p

The coating method of the titan discs was transferable to the MouseFix plate.

<p>In 4 of 5 cultures of each group (MouseFix plates coated with lysostaphin and MouseFix plates coated with lysostaphin and sterilized with 40 kGy beta irradiation) no bacterial growth was measured after 48 h cultivation in a basic culture of SA with 1.00E+04 CFU/ml and thus only one positive culture of each group is shown.</p

Detection of IL-6 in lavage fluids.

<p>IL-6 in lavage of both mice groups with lysostaphin-coated plates (<i>n</i> = 52) were only detectable to a minor degree in contrast to the control groups (mice with uncoated plates, and mice with only PDLLA coated plates; <i>n</i> = 49). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001. Statistical analysis was performed using two-tailed Student's t-test and Mann-Whitney-test.</p

Representative x-rays of the fracture zone on Day 28.

<p>A consolidation of the fracture gap on Day 28 in both mice groups with lysostaphin-coated plates in contrast to the control groups (mice with uncoated plates, and mice with only PDLLA coated plates) after osteotomy, plate fixation and inoculation of SA with an average CFU of 1.94E+03/µl is demonstrated.</p