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

Date Published: March 14, 2019

Publisher: Public Library of Science

Author(s): Lisa Oezel, Carina Büren, Armin O. Scholz, Joachim Windolf, Ceylan D. Windolf, Hiroyuki Tsuchiya.

http://doi.org/10.1371/journal.pone.0213590

Abstract

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.

Partial Text

Osteitis is described as an infection of the bone with a concomitant inflammation involving the bone marrow and the surrounding tissues [1]. These infections can originate from many different mechanisms, whereby a common cause of osteitis is a bacterial incorporation during or after surgical intervention as well as in case of open fractures caused by trauma [2]. Besides the marked progress in operating standards and amelioration of perioperative measures, especially implant-associated and perioperative infections in trauma and orthopedic surgery still represent significant complications [3]. In literature, the incidence of infection shows a wide range from 1% in primary fracture stabilization up to 55% in the treatment of open fractures [4]. Apart from that the development of an osteitis depends on individual risk factors provided by the patient such as obesity, anemia and diabetes [5]. So overall, the interaction of implants with incorporated bacteria and individual defense capacities lead to a localized infection. Regarding medical device-associated osteitis the most frequent germs are counted to be Staphylococcus aureus (SA) and Staphylococcus epidermidis [1,6]. SA has developed multiple strategies to escape from the host’s immune defense. Among those, generating a protective biofilm with resistance against systemic antibiotic substances, as well as the invasion of immune host cells present fundamental mechanisms [7]. Moreover, the segregation of cytotoxic molecules, like proteases, together with onward infectious process detains fracture healing [8]. Further, these mechanisms lead to an obstructed activation of the innate immune system which consequently hamper fracture healing, e. g. the activation of polymorphonuclear neutrophils (PMN) represents an important mechanism of bacterial defense [9]. Appropriate treatment with the attempt of eradication of osteitis is necessary to prevent life threatening complications such as sepsis [10]. Osteitis is chronological distinguished into an acute and a chronic type. Time borders are set distinctly, from 4 to 8 weeks describing an acute event and all exceeding that characterizing a chronic process [11]. Both osteitis types are addressed by surgical intervention in terms of radical debridement, lavage and removal of implants as the gold standard of therapy, combined with systemic or oral antibiotic treatment [12]. Detriments of intravenous or oral antibiotic treatment are systemic side effects of antibiotics as well as potentially low effects and concentrations at the local infection site. Furthermore, impairment of local vascularity can decrease the effects of oral or parenteral antibiotic administration [13]. Application of local antibiotics can result in an increase of concentrations at the infection site. Moreover, trauma associated fractures or osteitis itself can often leave large bone defects that do not have self-healing potential and lead to even pronounced infect reactions often requiring stabilization by a bone void filler [14]. The use of autologous bone grafts is often subordinate, first in order to avoid donor site morbidity in general, secondary because most of these accrued defects are big in size leaving this option ineligible [15]. Respectively, the use of bone graft substitutes presents a reasonable option. Properties like biocompatibility, biodegradability as well a positive effect on osteoconduction and osteoinduction have high importance as they allow “one-stage” operative procedures and impel bone healing. Cerament (BoneSupport AG, Lund, Sweden) is a synthetic, bioresorbable bone substitute, composed of 60% calcium sulfate and 40% hydroxyapatite. Hereby, the calcium sulfate is intended to be quickly replaced by newly formed bone and the hydroxyapatite is supposed to act as a template to allow further bone ingrowth [16]. CAS/HA biocomposites are often used as bone graft in delayed- and non-unions after fractures [15]. Besides, several clinical studies have demonstrated the efficacy of antibiotic infused CAS/HA with gentamicin sulfate or vancomycin in the treatment of infected bone defects as well as the successful use as a coating on implants [16–19]. McNally et al. for example, present the effective treatment of chronic osteomyelitis with gentamicin loaded CAS/ HA spacer in patients [19]. Limitations of these studies seem to be that they either describe a primary infect prevention or the treatment of a chronic osteomyelitis and results from these studies in general show a specific risk of bias [20]. Moreover, molecular analysis of bone healing, infection progress and immune response during treatment with CAS/HA has not been fully dissected in these studies. A recent animal study suggested an increased bone formation as well as a decreased rate of detectable infection using CAS/HA impregnated with gentamicin in a rat model of osteomyelitis [21]. This study analyzes the effect of CAS/HA in a plain bone defect model with no critical defect as well as no implanted device. Both criteria, namely the existence of a critical size defect (CSD) as well as the association with an implanted orthopedic device present the most challenging aspects of current septic surgery. Especially the presence of foreign surfaces like implants or prosthesis significantly increases the risk for the development of an infection [22]. Related to that, the implant type as well as the size of the bone defect might additionally influence bone healing or inflammatory processes during osteitis.

Implant-associated infections remain one of the most feared complications in orthopedic and trauma surgery [25]. The current gold standard is radical surgical debridement along with removal of the infected osteosynthesic or prosthetic devices. Besides, initiation of systemic antibiotic therapy as well as application of local antibiotic substances complement the common therapy algorithm. In most cases, osteitis can leave a large, critical defect that requires stabilization and prevent the dispersal or recurrence of infection. Respectively, the use and development of calcium-based bone grafts acting as void fillers that feature bone-building qualities such as autografts and also serve as vehicles for antibiotic substances, is a main target in septic surgery. A systematic review by Hake et al. presents a wide range of various synthetic calcium-based bone substitutes either acting as bone grafts or as local antibacterial carriers [26]. Antibiotic-loaded substances serve plenty advantages, as high local concentrations of antibiotics are delivered to the infection site, devoid of all systemic detriments of a vascular therapy [27]. Moreover, bone graft substitutes can provide profitable attributes as biodegradability, osteoconduction as well as osteoinduction [20]. CAS/HA biocomposite is an injectable and moldable bone substitute, which is intended to be transformed to host bone within a few months and likewise serves as a scaffold for further bone remodeling. The use of CAS/HA was previously evaluated in studies, either in terms of a bone graft related to mere bone defects but also with respect to its potential as an antibiotic vehicle in bone infections. The aim of this present experimental study was to evaluate the use of antibiotic impregnated CAS/HA in an implant associated osteitis model to assess its effects on fracture healing, infection progress and inflammatory response on molecular level.

We acknowledge limitations to our present study. First, our study was supposed to consist of a 12- week mice cohort next to the 6- week cohort. Mice of the 12-week cohort were supposed to be euthanized on day 84 to simulate a long-term process of bone consolidation and thus to evaluate and compare results regarding our experimental project.

The present osteitis model is sufficient to study fracture healing, infection progress and immune response following an acute implant-associated SA-mediated osteitis in mice. Moreover, this study supports a use of CAS/HA as void filler after infect-convalescence or in non-infectious environments. Application of CAS/HA in acute implant- associated infections, as shown in our model obviously does not lead to limitation of infection and can therefore not be recommended. Nonetheless, the use of CAS/HA in chronic septic environments combined with debridements and if necessary systemic antibiotic therapy, as described in stated studies, could serve as a suggestive tool in trauma and orthopedic surgery.

 

Source:

http://doi.org/10.1371/journal.pone.0213590

 

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