Background
Bacterial bone infections due to trauma with subsequent delayed or impaired fracture healing are highly feared complications in orthopedics and traumatology and represent a great challenge. Open fractures in particular exhibit an incidence of osteitis as high as 55% [
1]. Bacteria colonizing implants can interfere with physiological bone formation and remodeling mechanisms and lead to a higher risk of impaired fracture healing [
1,
2]. In most cases,
Staphylococcus aureus (
S. aureus) is responsible for infection. The bacterium can infiltrate osteoblasts, builds biofilms and mutates into its small colony variants form, all leading to a high antibiotic resistance [
1,
3,
4]. Therefore currently available therapeutic options must involve not only antibiotic treatment but also repeated surgical debridement, potentially exacerbating extensive bone defects [
4]. Furthermore, prolonged treatment of delayed fracture healing may have a profound effect on both the medical and emotional condition of the patient as well as his financial and professional security [
5].
Bone morphogenetic proteins (BMPs), already established as a useful addition to classic therapy in cases of non-union and delayed fracture union, have not been sufficiently investigated in infected fractures of the long bones [
6]. Only rhBMP-2 was evaluated on surgical infections in a rabbit posterolateral lumbar fusion model showing an insignificant trend toward improved fusion rate and less mortality [
7]. BMP-application is officially not indicated in the situation of an infected non-union of the long bones, because scientific data in the literature is insufficient. Currently, BMPs can only be used off-label in the situation of an infection.
BMPs are naturally responsible for the induction of osteogenic differentiation of mesenchymal stem cells and for angiogenesis and show limited expression during delayed fracture healing [
8‐
10]. Recombinant(rh)BMP-2 and rhBMP-7 have been proven to promote fracture healing both in delayed fracture healing and non-union animal models [
11‐
14]. RhBMP-2 has shown high potential in the treatment of open fractures in clinical trials, leading to faster union establishment, lower infection rate and lower number of surgeries needed [
15‐
17]. Similarly, rhBMP-7 could enhance the efficacy and the treatment success of non-union in the combined therapy with autologous cancellous bone [
18].
The outcome of BMP-enhanced treatment of delayed fracture healing in the situation of an ongoing low-grade bone infection has not yet been sufficiently explored.
Therefore, the goal of this study was to investigate the impact of rhBMP-7 and rhBMP-2 on fracture healing in an animal model of delayed osseous union secondary to chronic bacterial osteitis [
19]. The hypothesis was that rhBMP-7 and rhBMP-2 may improve bone healing in the situation of an infection without significant differences between both factors. Primary objective was the fracture torque in biomechanical evaluations, secondary objectives were radiological and histological outcome.
Discussion
In this study, we have evaluated the effect of recombinant human bone morphogenetic proteins − 7 and − 2 for the treatment of delayed osseous union secondary to bacterial osteitis in our animal model using micro-CT examinations, qualitative histology, histomorphometric evaluations, and biomechanical investigations. To our knowledge this is the first study, which compared recombinant human bone morphogenetic proteins − 7 and − 2 in an in vivo animal model of delayed osseous union in the situation of an ongoing bacterial bone infection.
We have chosen rhBMP-7 and -2 in our study setting, because these growth factors have been established clinically in the treatment of non-union [
15,
27‐
29]. Giannoudis et al. [
29] could show a synergistic effect of autograft and bone morphogenetic protein − 7 in the therapy of atrophic humeral, femoral and tibial non-union with a healing rate of 100% in 45 patients. Govender et al. [
15] investigated rhBMP-2 in the treatment of open tibial fractures and showed better fracture- and wound-healing and reduced infection rate. Similar effects have been described by others [
30,
31]. Significant differences in the effectiveness of both factors or synergistic effects have not been described.
According to micro-CT-scans and histology, treatment with rhBMPs was unable to achieve a complete union in the situation of infection during the follow-up period, but we could find positive effects on bone healing in biomechanical evaluations as well as in the semi-quantitative bone-healing-score and in callus histology. Biomechanical investigations and scoring of the micro-CT images revealed significantly more stability and healing in the infected rhBMP groups equal to non-infected controls if compared to the infected control group. Thus, the findings indicate that rhBMP treatment is possibly able to improve bone healing in the setting of infection, although it has no direct effect on the infection with
S. aureus [
32]. In this regard, our investigations supported the results in the literature [
33,
34]. All infected groups, no matter if rhBMP therapy was added or not showed significantly increased mineralized callus (BV) and soft-tissue callus (TV), but a significantly decreased bone volume fraction (BV/TV) in quantitative micro-CT evaluation as well as larger periosteal callus areas and smaller mineralized periosteal bone areas of the periosteal total callus area in histology. This may be interpreted as a hypertrophic callus, explaining less stability compared with the non-infected control group. In agreement with other studies, Schmidmaier et al. [
28] and Bode et al. [
35] postulated that an infection or an instable fixation are the cause of a hypertrophic non-union formation. They recommend repeated debridements in case of infected, especially open fractures, with stable osteosynthesis depending on the type of the fracture to prevent non-union. In the present study, histology further showed some qualitative differences of the callus. In rhBMP specimens, we saw increased signs of fracture remodeling with only moderately remaining cartilage and fibrous tissue. These changes seemed less pronounced in infected controls without rhBMP treatment. In contrast to the study of Chen et al. [
33,
34], which used a rat femur model with a segmental chronically infected bone defect, we were not able to achieve significantly more newly mineralized callus compared to infected controls in our animal model. A possible reason for this better mineralization might be the additional antibiotic treatment in the study of Chen et al.
As a limitation of our study, we did not analyze longer follow-up times, so further effects might have been detectable after the analyzed duration of 5 weeks after rhBMP treatment. Furthermore, we were not able to specifically identify or quantify the underlying mechanisms for the increased stability, possibly due to technical limitations as the bone specimen had to be collected, removed from soft tissue, further prepared and fixed before cutting into 6 μm sections for histological slides. Hence, semi-quantitative histological assessment of the callus architecture was not possible. Nevertheless, signs of partial fracture remodeling were mainly found in rhBMP specimen contrary to more fibroblasts and remaining cartilage in infected controls.
Clinical studies such as the BESTT study (BMP-2 Evaluation in Surgery and Tibial Trauma) have presented both an accelerated healing process and a lower infection rate after treatment with rhBMP-2 [
15]. Similarly successful results were obtained by a combined two-step non-union therapy with rhBMP-7 after osteomyelitis [
36]. However, it should be taken into consideration that clinical routine therapy of bone infections also involves debridement of necrotic bone, soft tissue management and antibiotic therapy [
4,
37]. These procedures lead to reduction of bacterial load and open the way for osteoprogenitor cells from the periosteum, blood vessels and soft tissue into the defect [
38,
39]. Similar procedure was used in the rhBMP-2 and rhBMP-7 studies in a rat model of Chen et al. [
33]. In the experimental setting of our study only intramedullary irrigation with sterile saline, rhBMPs application and k-wire replacement were performed, which will have maintained a high bacterial burden in the bone. Most clinical cases of unsuccessful non-union treatment reveal subclinical infection [
36]. This clinical experience, together with the findings of the current animal study suggest that an ongoing bone infection will hamper bone healing with potentially reduced effectiveness of the rhBMPs. Further in-vivo studies might help to get further information on this topic. RhBMP application in an animal model together with differently concentrated bacterial suspensions and without could be analyzed, to determine potential bacterial-dose-dependent effects. Also adverse effects have been described in the literature for rhBMP therapy [
40‐
43] but were not found in the present animal study at least in the short term, as we did not find rhBMP associated side-effects or complications such as heterotopic ossifications or induction of neoplasms [
42,
43].
Future studies should investigate later time-points of bone healing in order to compare the biomechanical, histological and radiological results with the present study. Other growth factor application systems as well as other antibacterial or bone stimulating substances and osteosynthesis techniques [
44] could also be analyzed with the present animal model considering their effectiveness in case of infection. The long-term goal would be to establish a material or a combination of materials and techniques that have osteoinductive and osteoconductive but also antimicrobial effects to provide bone healing within a short operation and healing period.
Acknowledgements
The present study was financially supported by the Center for Orthopedics, Trauma Surgery and Spinal Cord Injury, Heidelberg University Hospital. The authors thank our institutional statistician Simone Gantz for statistical analyses.