A new sequential animal model for infection-related non-unions with segmental bone defect.

BACKGROUND: The treatment of fracture-related infections (FRI) is still a challenge for orthopedic surgeons. The prevalence of FRI is particularly high in open fractures with extensive soft-tissue damage. This study aimed to develop a new two-step animal model for non-unions with segmental bone defects, which could be used to evaluate new innovative bone substitutes to improve the therapeutic options in humans with FRI and bone defects. METHODS: After randomization to infected or non-infected groups, 30 Sprague-Dawley rats underwent a transverse osteotomy of the mid-shaft femur with a 5 mm defect. Additionally, the periosteum at the fracture zone was cauterized at both sides. After intramedullary inoculation with 103 CFU Staphylococcus aureus (infected group) or PBS (non-infected group), a fracture stabilization was done by intramedullary K-wires. After 5 weeks, the bone healing process was evaluated, and revision surgery was performed in order to obtain increased bone healing. The initial K-wires were removed, and debridement of the osteotomy-gap was done followed by a more stable re-osteosynthesis with an angle-stable plate. After further 8 weeks all rats were euthanized and the bone consolidation was tested biomechanically and the callus formation quantitatively by micro-CT analysis. RESULTS: We developed and presented a new two-stage non-union animal model through a targeted S. aureus infection. After 5 weeks, all animals showed a non-union irrespective of assignment to the infected and non-infected group. Lane and Sandhu score showed a higher callus formation in the infected group. In all infected animals, the inoculated S. aureus strain was detected in the revision surgery. The second surgery did not improve bone healing, as shown by the Lane Sandhu score and in the µ-CT analysis. Similarly, biomechanical testing showed in both groups a significantly lower maximum torque as compared to the contralateral side (p < 0.0001). CONCLUSIONS: We were able to successfully develop a new two-stage non-union animal model, which reflects a genuine clinical situation of an infection-related non-union model with segmental bone defects. This model could be used to evaluate various therapeutic anti-infectious and osteoinductive strategies in FRIs.

Bone morphogenetic proteins - 7 and - 2 in the treatment of delayed osseous union secondary to bacterial osteitis in a rat model.

BACKGROUND: Bone infections due to trauma and subsequent delayed or impaired fracture healing represent a great challenge in orthopedics and trauma surgery. The prevalence of such bacterial infection-related types of delayed non-union is high in complex fractures, particularly in open fractures with additional extensive soft-tissue damage. The aim of this study was to establish a rat model of delayed osseous union secondary to bacterial osteitis and investigate the impact of rhBMP-7 and rhBMP-2 on fracture healing in the situation of an ongoing infection. METHODS: After randomization to four groups 72 Sprague-Dawley rats underwent a transverse fracture of the midshaft tibia stabilized by intramedullary titanium K-wires. Three groups received an intramedullary inoculation with Staphylococcus aureus (103 colony-forming units) before stabilization and the group without bacteria inoculation served as healing control. After 5 weeks, a second surgery was performed with irrigation of the medullary canal and local rhBMP-7 and rhBMP-2 treatment whereas control group and infected control group received sterile saline. After further 5 weeks rats were sacrificed and underwent biomechanical testing to assess the mechanical stability of the fractured bone. Additional micro-CT analysis, histological, and histomorphometric analysis were done to evaluate bone consolidation or delayed union, respectively, and to quantify callus formation and the mineralized area of the callus. RESULTS: Biomechanical testing showed a significantly higher fracture torque in the non-infected control group and the infected rhBMP-7- and rhBMP-2 group compared with the infected control group (p < 0.001). RhBMP-7 and rhBMP-2 groups did not show statistically significant differences (p = 0.57). Histological findings supported improved bone-healing after rhBMP treatment but quantitative micro-CT and histomorphometric results still showed significantly more hypertrophic callus tissue in all three infected groups compared to the non-infected group. Results from a semiquantitative bone-healing-score revealed best bone-healing in the non-infected control group. The expected chronic infection was confirmed in all infected groups. CONCLUSIONS: In delayed bone healing secondary to infection rhBMP treatment promotes bone healing with no significant differences in the healing efficacy of rhBMP-2 and rhBMP-7 being noted. Further new therapeutic bone substitutes should be analyzed with the present rat model for delayed osseous union secondary to bacterial osteitis.

A new animal model for delayed osseous union secondary to osteitis.

BACKGROUND: The treatment of infection-related delayed bone unions is still very challenging for the orthopedic surgeon. The prevalence of such infection-related types of osteitis is high in complex fractures, particularly in open fractures with extensive soft-tissue damage. The aim of this study was to develop a new animal model for delayed union due to osteitis. METHODS: After randomization to infected or non-infected groups 20 Sprague-Dawley rats underwent a transverse fracture of the midshaft tibia. After intramedullary inoculation with staphylococcus aureus (10(3) CFU) fracture stabilization was done by intramedullary titanium K-wires. After 5 weeks all rats were euthanized and underwent biomechanical testing to evaluate bone consolidation or delayed union, respectively. Micro-CT scans were additionally used to quantitatively evaluate the callus formation by the score of Lane and Sandhu. Blood samples were taken to analyze infectious disease markers (day 1, 14 and 35). RESULTS: Biomechanical testing showed a significant higher maximum torque in the non-infected group 5 weeks postoperatively compared with the infected group (p < 0.001). According to the Lane and Sandhu score a significantly higher callus formation was found in the non-infected group (p < 0.001). Similarly, the leucocyte count in the infected group was significantly higher than in the non-infected group (p < 0.05). CONCLUSIONS: Here we have established a new animal model for delayed osseous union secondary to osteitis. The animal model appears to be appropriate for future experimental studies to test new therapeutic strategies in these difficult to treat bone healing complications.

Establishment of a new methicillin resistant Staphylococcus aureus animal model of osteomyelitis.

PURPOSE: The increase in methicillin-resistant Staphylococcus aureus (MRSA) infections is currently a major health care problem. Vancomycin is still often the first-line anti-microbiological agent for treating such infections; however, a recent decline in efficacy of vancomycin in MRSA infections has raised concerns and accelerated the search for new antibiotics. The aim of this study was to establish a MRSA peri-implant osteomyelitis animal model for future testing of new anti-microbiological agents under typical MRSA infection conditions. METHODS: Eighteen randomised NZW-rabbits underwent a standardised surgical procedure with the insertion of a femoral bone implant. Animals were then divided into group 1 (MRSA inoculation, no antibiotics; M/N), group 2 (MRSA inoculation, Vancomyin; M/V), and group 3 (no MRSA inoculation, no antibiotics; N/N). The primary study outcome parameters were animal leucocyte count, animal weight, and animal body temperature at one, seven, and 42 days after surgery. Additionally, a histo-morphometrical score was established and adjusted to a modified histological Smeltzer score. RESULTS: Macroscopic and histo-morphometrical findings showed a peri-implant osteomyelitis in group 1 with both increased acute and chronic infection parameters in M/N, as compared to M/V and N/N, indicating that vancomycin treatment prevented typical morphological changes of MRSA peri-implant osteomyelitis. Similarly, there was a reduction in animal weight and increase in leucocyte count and body temperature in group 1 (each p < 0.005). Vancomycin treatment again resulted in significantly reduced leucocyte count and body temperature, and increased animal body weight. CONCLUSIONS: Here we have established a peri-implant MRSA osteomyelitis model that successfully combined clinical and laboratory outcome parameters of infection with histo-morphometrical results; this model appears to be valuable for future experimental use and therapeutic monitoring of new anti-microbiological MRSA drugs.

Use of Autologous Bone Graft with Bioactive Glass as a Bone Substitute in the Treatment of Large-Sized Bone Defects of the Femur and Tibia.

BACKGROUND: Managing bone defects in non-union surgery remains challenging, especially in cases of large defects exceeding 5 cm in size. Historically, amputation and compound osteosynthesis with a remaining PMMA spacer have been viable and commonly used options. The risk of non-union after fractures varies between 2% and 30% and is dependent on various factors. Autologous bone grafts from the iliac crest are still considered the gold standard but are limited in availability, prompting consideration of artificial grafts. OBJECTIVES: The aims and objectives of the study are as follows: 1. To evaluate the radiological outcome of e.g., the consolidation and thus the stability of the bone (three out of four consolidated cortices/Lane-Sandhu-score of at least 3) by using S53P4-type bioactive glass (BaG) as a substitute material for large-sized bone defects in combination with autologous bone using the RIA technique. 2. To determine noticeable data-points as a base for future studies. METHODS: In our clinic, 13 patients received bioactive glass (BaG) as a substitute in non-union therapy to promote osteoconductive aspects. BaG is a synthetic material composed of sodium, silicate, calcium, and phosphate. The primary endpoint of our study was to evaluate the radiological consolidation of bone after one and two years. To assess bone stabilization, we used a modified Lane-Sandhu score, considering only radiological criteria. A bone was considered stabilized if it achieved a minimum score of 3. For full consolidation (all four cortices consolidated), a minimum score of 4 was required. Each bone defect exceeded 5 cm in length, with an average size of 6.69 ± 1.92 cm. RESULTS: The mean follow-up period for patients without final bone consolidation was 34.25 months, with a standard deviation of 14.57 months, a median of 32.00 months and a range of 33 months. In contrast, patients with a fully consolidated non-union had an average follow-up of 20.11 ± 15.69 months and a range of 45 months. Overall, the mean time from non-union surgery to consolidation for patients who achieved final union was 14.91 ± 6.70 months. After one year, six patients (46.2%) achieved complete bone consolidation according to the Lane-Sandhu score. Three patients (23.1%) displayed evident callus formation with expected stability, while three patients (23.1%) did not develop any callus, and one patient only formed a minimal callus with no expected stability. After two years, 9 out of 13 patients (69.2%) had a score of 4. The remaining four patients (30.8%) without expected stability either did not heal within two years or required a revision during that time. CONCLUSIONS: Bioactive glass (BaG) in combination with autologous bone (RIA) appears to be a suitable filler material for treating extensive non-unions of the femur and tibia. This approach seems to show non-inferiority to treatment with Tricalcium Phosphate (TCP). To ensure the success of this treatment, it is crucial to validate the procedure through a randomized controlled trial (RCT) with a control group using TCP, which would provide higher statistical power and more reliable results.

Matched-Pair Analysis: Large-Sized Defects in Surgery of Lower Limb Nonunions.

BACKGROUND: The treatment of large-sized bone defects remains a major challenge in trauma and orthopaedic surgery. Although there are many treatment options, there is still no clear guidance on surgical management, and the influence of defect size on radiological and clinical outcome remains unclear due to the small number of affected patients. The aim of the present study was to determine the influence of defect size on the outcome of atrophic and infected nonunions of the tibia or the femur based on the diamond concept in order to provide recommendations for treatment guidance. PATIENTS AND METHODS: All medical records, surgical reports, laboratory data and radiological images of patients treated surgically for atrophic or infected nonunions of the lower limbs (femur or tibia) between 1 January 2010 and 31 December 2020 were examined. Patients with proximal, diaphyseal or distal nonunions of the femur or tibia who were surgically treated at our institution according to the "diamond concept" and attended our standardised follow-up program were included in a database. Surgical treatment was performed as a one- or two-step procedure, depending on the type of nonunion. Patients with a segmental bone defect ≥5 cm were matched with patients suffering a bone defect <5 cm based on five established criteria. According to our inclusion and exclusion criteria, 70 patients with a bone defect ≥5 cm were suitable for analysis. Two groups were formed by matching: the study group (bone defect ≥5 cm; n = 39) and control group (bone defect <5 cm; n = 39). The study was approved by the local ethics committee (S-262/2017). RESULTS: The mean defect size was 7.13 cm in the study and 2.09 cm in the control group. The chi-square test showed equal consolidation rates between the groups (SG: 53.8%; CG: 66.7%). However, the Kaplan-Meier curve and log-rank test showed a significant difference regarding the mean duration until consolidation was achieved, with an average of 15.95 months in the study and 9.24 months in the control group (α = 0.05, p = 0.001). Linear regression showed a significant increase in consolidation duration with increasing defect size (R2 = 0.121, p = 0.021). Logistic regression modelling showed a significant negative correlation between consolidation rate and revision performance, as well as an increasing number of revisions, prior surgeries and total number of surgeries performed on the limb. Clinical outcomes showed equal full weight bearing of the lower extremity after 5.54 months in the study vs. 4.86 months in the control group (p = 0.267). CONCLUSION: Surprisingly, defect size does not seem to have a significant effect on the consolidation rate and should not be seen as a risk factor. However, for the treatment of large-sized nonunions, the follow-up period should be prolonged up to 24 months, due to the extended time until consolidation will be achieved. This period should also pass before a premature revision with new bone augmentation is performed. In addition, it should be kept in mind that as the number of previous surgeries and revisions increases, the prospects for consolidation decrease and a change in therapeutic approach may be required.

Effect of Tricalcium Phosphate on Healing of Non-Unions: An Observational Study of over 400 Non-Unions.

Purpose: A central aspect of the treatment of non-unions is the filling of bone defects. The quantity of available autologous bone for this purpose is limited. Alternatively, or additionally, bone substitutes may be used. The aim of this retrospective, single-center study including 404 non-unions in 393 patients is to investigate the effect of tricalcium phosphate (TCP) on the healing of non-unions. Furthermore, the influence of gender, age, smoking status, comorbidities, type of surgical procedure, presence of infection, and length of treatment was investigated. Methods: We evaluated three groups of patients. Group 1 received TCP + BG, group 2 received BG alone and group 3 received no augmentation. Bone stability was assessed 1 and 2 years after non-union revision surgery through analysis of radiographs using the Lane Sandhu Score. Scores ≥3 were rated as stable Other influencing factors were collected from the electronic medical record. Results: In 224 non-unions, bone defects were filled with autologous bone and TCP (TCP+BG). In 137 non-unions, bone defects were filled with autologous bone (BG), and in 43 non-unions presenting non-relevant defects, neither autologous bone nor TCP were used (NBG). After 2 years, 72.7% of the TCP+BG patients, 90.1% of the BG patients and 84.4% of the NBG patients achieved a consolidation score ≥3. Advanced age, presence of comorbidities and longer treatment period had a significantly negative effect on consolidation 1 year after surgery. Longer treatment periods also showed a negative significant effect after 2 years. It is notable that larger defects, mainly treated with the combination of autologous bone and TCP, showed similar healing rates to that of smaller defects after 2 years. Conclusion: The combination of TCP and autologous bone-grafts shows good results in the reconstruction of complicated bone-defects, but patience is required since the healing period exceeds 1 year in most patients.

Late activation of stress-activated protein kinases/c-Jun N-terminal kinases triggered by cisplatin-induced DNA damage in repair-defective cells.

Although stress-activated protein kinases/c-Jun N-terminal kinases (SAPK/JNK) are rapidly activated by genotoxins, the role of DNA damage in this response is not well defined. Here we show that the SEK1/MKK4-mediated dual phosphorylation of SAPK/JNK (Thr-183/Tyr-185) correlates with the level of cisplatin-DNA adducts at late times (16-24 h) after drug treatment in both human and mouse cells. Transfection of platinated plasmid DNA also caused SAPK/JNK activation. A defect in transcription-coupled nucleotide excision repair resting on a mutation in Cockayne syndrome group B protein promoted the late SAPK/JNK activation following cisplatin exposure. Signaling to SAPK/JNK was accompanied by activation of Ataxia telangiectasia mutated- and Rad3-related kinase, replication protein A, and checkpoint kinases as well as by the formation of DNA double strand breaks (DSBs). Ionizing radiation-induced DSBs did not provoke SAPK/JNK activation, and inhibition of transcription also failed to provoke this response. Late activation of SAPK/JNK stimulated by cisplatin-induced DNA lesions was reduced in the absence of specific DNA repair proteins, such as xeroderma pigmentosum protein C, pointing to an essential function of individual repair factors in DNA damage signaling to SAPK/JNK. Collectively, the data indicate that late SAPK/JNK activation is triggered by non-repaired cisplatin adducts in transcribed genes and involves replication-associated events, DSBs, tyrosine kinases, Rho GTPases, and specific repair factors.

99mTc-polyphosphonate labelling - Enhancement of a novel method for the quantification of osteogenic differentiation of MSCs in vitro.

Bone tissue engineering is a fast-growing field in regenerative medicine. Consequently, there is a high demand for new, fast and reliable methods to track and quantify the osteogenic differentiation of cells. Recently, a novel method was published to non-destructively quantify the hydroxyapatite content of monolayer and 3-dimensional mesenchymal stem cell cultures using the ability of 99mTechnetium-methylene diphosphonate (MDP), a well-established tracer in clinical nuclear medicine, to bind to newly synthesized hydroxyapatite. In the present study, two other commonly used 99mTechnetium tracers, 2,3-dicarboxypropane-1,1-diphosphonate (DPD) and hydroxydiphosphonate (HDP), were evaluated to see if they could also be used for the same purpose. Furthermore, we investigated if labelling at various timepoints influenced the effectiveness of the labelling. The results were analysed using one-factor ANOVA followed by Bonferroni post-hoc testing. This revealed a highly significant difference between the three osteogenic groups at each timepoint compared to their corresponding negative controls. However, there was no statistically significant difference between the three different tracers (MDP, DPD, HDP) in the osteogenic groups. Therefore all three tracers are of similar value when quantifying the extracellular hydroxylapatite content in osteogenic stem cells cultures.

Treatment of Infection-Related Non-Unions with Bioactive Glass-A Promising Approach or Just Another Method of Dead Space Management?

The treatment of infected and non-infected non-unions remains a major challenge in trauma surgery. Due to the limited availability of autologous bone grafts and the need for local anti-infective treatment, bone substitutes have been the focus of tissue engineering for years. In this context, bioactive glasses are promising, especially regarding their anti-infective potential, which could reduce the need for local and systemic treatment with conventional antibiotics. The aim of this study was to investigate the osteoinductive and osteoconductive effects, as well as the anti-infectious potential, of S53P4 using a standardized non-union model, which had not been investigated previously. Using an already established sequential animal model in infected and non-infected rat femora, we were able to investigate bioactive glass S53P4 under realistic non-union conditions regarding its osteoinductive, osteoconductive and anti-infective potential with the use of µCT scans, biomechanical testing and histological, as well as microbiological, analysis. Although S53P4 did not lead to a stable union in the non-infected or the infected setting, µCT analysis revealed an osteoinductive effect of S53P4 under non-infected conditions, which was diminished under infected conditions. The osteoconductive effect of S53P4 remained almost negligible in histological analysis, even 8 weeks after treatment. Additionally, the expected anti-infective effect could not be demonstrated. Our data suggested that S53P4 should not be used in infected non-unions, especially in those with large bone defects.

Efficacy of an Antibiotic Loaded Ceramic-Based Bone Graft Substitute for the Treatment of Infected Non-Unions.

The treatment of non-unions is often complicated by segmental bone defects and bacterial colonization. Because of the limited availability of autologous bone grafts, tissue engineering focuses on antibiotic-loaded bone graft substitutes. HACaS+G is a resorbable calcium sulphate-hydroxyapatite loaded with gentamicin. The osteoinductive, osteoconductive, and anti-infective effect of HACaS+G has already been demonstrated in clinical studies on patients with chronic osteomyelitis. However, especially for the treatment of infected non-unions with segmental bone defects by HACaS+G, reliable clinical testing is difficult and sufficient experimental data are lacking. We used an already established sequential animal model in infected and non-infected rat femora to investigate the osteoinductive, osteoconductive, and anti-infective efficacy of HACaS+G for the treatment of infected non-unions. In biomechanical testing, bone consolidation could not be observed under infected and non-infected conditions. Only a prophylactic effect against infections, but no eradication, could be verified in the microbiological analysis. Using µ-CT scans and histology, osteoinduction was detected in both the infected and non-infected bone, whereas osteoconduction occurred only in the non-infected setting. Our data showed that HACaS+G is osteoinductive, but does not have added benefits in infected non-unions in terms of osteoconduction and mechanical bone stability, especially in those with segmental bone defects.

The Influence of an Occult Infection on the Outcome of Autologous Bone Grafting During Surgical Bone Reconstruction: A Large Single-Center Case-Control Study.

BACKGROUND: Occult infections (OI) lack typical inflammatory signs, making them challenging to diagnose. Uncertainty remains regarding OI's influence on the outcome of autologous bone grafting (ABG), and evidence-based recommendations regarding an appropriate course of action are missing. Thus, we sought to determine the incidence of an OI in patients receiving ABG, evaluate whether it influences the outcome of ABG and whether associated risk factors have a further negative influence. METHODS: This study was designed as a large size single-center case-control study investigating patients treated between 01/01/2010 and 31/12/2016 with a minimum follow-up of 12 months. Patients ≥18 years presenting with a recalcitrant non-union of the lower limb receiving surgical bone reconstruction, including bone grafting, were included. A total of 625 patients were recruited, and 509 patients included in the current study. All patients received surgical non-union therapy based on the "diamond concept" including bone reconstruction using ABG. Additionally, multiple tissue samples were harvested and microbiologically analyzed. Tissue samples were microbiologically evaluated regarding an OI. Bone healing was analyzed using clinical and radiological parameters, patient characteristics and comorbidities investigated and ultimately results correlated. RESULTS: Forty-six out of 509 cases with OI resulted in an incidence of 9.04%. Overall consolidation time was increased by 15.08 weeks and radiological outcome slightly impaired (79.38% vs 71.42%), differences were at a non-significant extent. Diabetes mellitus had a significant negative influence on consolidation time (p=0.0313), while age (p=0.0339), smoking status (p=0.0337), diabetes mellitus (p=0.0400) and increased BMI (p=0.0315) showed a significant negative influence on the outcome of bone grafting. CONCLUSION: Surgeons treating recalcitrant non-unions should be aware that an OI is common. If an OI is diagnosed subsequent to ABG the majority of patients does not need immediate revision surgery. However, special attention needs to be paid to high-risk patients.

Systemic Administration of PTH Supports Vascularization in Segmental Bone Defects Filled with Ceramic-Based Bone Graft Substitute.

Non-unions continue to present a challenge to trauma surgeons, as current treatment options are limited, duration of treatment is long, and the outcome often unsatisfactory. Additionally, standard treatment with autologous bone grafts is associated with comorbidity at the donor site. Therefore, alternatives to autologous bone grafts and further therapeutic strategies to improve on the outcome and reduce cost for care providers are desirable. In this study in Sprague-Dawley rats we employed a recently established sequential defect model, which provides a platform to test new potential therapeutic strategies on non-unions while gaining mechanistic insight into their actions. The effects of a combinatorial treatment of a bone graft substitute (HACaS+G) implantation and systemic PTH administration was assessed by µ-CT, histological analysis, and bio-mechanical testing and compared to monotreatment and controls. Although neither PTH alone nor the combination of a bone graft substitute and PTH led to the formation of a stable union, our data demonstrate a clear osteoinductive and osteoconductive effect of the bone graft substitute. Additionally, PTH administration was shown to induce vascularization, both as a single adjuvant treatment and in combination with the bone graft substitute. Thus, systemic PTH administration is a potential synergistic co-treatment to bone graft substitutes.

Individualized Techniques of Implant Coating with an Antibiotic-Loaded, Hydroxyapatite/Calcium Sulphate Bone Graft Substitute.

BACKGROUND: The treatment of fracture- or non-union-related infections has persistently been a major challenge for both patients and treating surgeons. With rising aging of patients and increasing comorbidities, combined with the heterogeneity of germs and any number of multi-resistance against standard antibiotics, a successful treatment is increasingly difficult. One potential solution could be a custom-made individualized antibacterial coating of standard implants with a biphasic degradable biocarrier (Cerament G/V, supplied by Bonesupport AB, Lund, Sweden) that releases high doses of antibiotics around the bone-implant-interface. Here, we describe our technique of coating intramedullary nails, plates and press-fit shoulder endoprostheses which may prevent bacterial adhesion and biofilm formation. So far, there is very limited experience in individual coating of implants in hip or knee endoprostheses to prevent reoccurrence of surgical-site infection. Currently, no reports are available for coating of stems of shoulder prosthesis and nails or plates for fracture fixation. METHODS: Here, we show our first experiences with a new individualized surgical technique of coating these implants with a resorbable antibiotic-loaded hydroxyapatite/calcium sulphate biocomposite to prevent biofilm formation and thereby recurrence of bone or joint infection. We describe three cases for coating of plates and nails for fracture fixation and coating of stems of a shoulder prosthesis. RESULTS: No adverse events of the resorbable bone graft substitute were observed. In all of the cases, no recurrence of the infection was observed and osseointegration was achieved. After implant coating of the shoulder prosthesis, no radiological signs of loosening were detected. CONCLUSION: We present a new surgical approach of a surface coating of plates, intramedullary nails or prostheses. The osteoconductive- and anti-inflammatory effect of the gentamicin- or vancomycin-loaded hydroxyapatite/calcium sulphate bone graft substitutes shows promising results.

Cisplatin sensitivity is related to late DNA damage processing and checkpoint control rather than to the early DNA damage response.

The present study aimed at elucidating mechanisms dictating cell death triggered by cisplatin-induced DNA damage. We show that CL-V5B hamster mutant cells, a derivative of V79B, are hypersensitive to cisplatin-induced apoptotic death. CL-V5B cells are characterized by attenuated cisplatin-induced early (2-6 h) stress response, such as phosphorylation of stress-activated protein kinases (SAPK/JNK), ATM and Rad3-related (ATR) protein kinase, histone H2AX and checkpoint kinase-1 (Chk-1). Human FANCC cells also showed a reduced phosphorylation of H2AX and SAPK/JNK at early time point after cisplatin treatment. This was not the case for BRCA2-defective VC-8 hamster cells, indicating that the FA core complex, rather than its downstream elements, is involved in early damage response. The alleviated early response of CL-V5B cells is not due to a general dysfunction in ATM/ATR-regulated signaling. It is rather due to a reduced formation of primary cisplatin-DNA adducts in the hypersensitive mutant as shown by analysis of DNA platination, DNA intra- and interstrand crosslink formation and DNA replication blockage. Despite of lower initial DNA damage and attenuated early DNA damage response (DDR), CL-V5B cells are characterized by an excessive G2/M arrest as well as an elevated frequency of DNA double-strand breaks (DSB) and chromosomal aberrations (CA) at late times (16-24h) after cisplatin exposure. This indicates that error-prone processing of cisplatin-induced lesions, notably interstrand crosslinks (ICL), and the formation of secondary DNA lesions (i.e. DSB), results in a powerful delayed DNA damage response and massive pro-apoptotic signaling in CL-V5B cells. The data provide an example that the initial level of cisplatin-DNA adducts and the corresponding early DNA damage response do not necessarily predict the outcome of cisplatin treatment. Rather, the accuracy of DNA damage processing and late checkpoint control mechanisms determine the extent of cell death triggered by cisplatin-induced DNA lesions.

Initial peri- and postoperative antibiotic treatment of infected nonunions: results from 212 consecutive patients after mean follow-up of 34 months.

PURPOSE: Infected nonunions of the long bones belong to the most feared complications in the field of orthopedic and trauma surgery. Optimal antibiotic therapy should start early with the first revision surgery. Therefore, the aim of this study was to evaluate our peri- and postoperative antibiotic regime in context with the microbial spectrum and antibiotic resistances of patients with infected nonunions and to assess the possible impact on healing rates. METHODS: We included all patients with first revision surgery during 2010-2015 due to nonunion of long bones with a clinical history of infection treated with radical debridement, local application of a gentamicin-impregnated bone cement, and systemic cefuroxime. Mean follow-up was 34.2 months. Data collection was performed retrospectively using a computerized databank with information about microbial species from intraoperatively acquired tissue samples and respective antibiograms. Bone fusion rates were evaluated based on findings of the latest X-rays and computed tomography scans. RESULTS: Two hundred and twelve patients with nonunion and history of infection were selected; 171 patients had positive intraoperative microbial evidence of infection. Bacterial testing was mostly positive in fractures of the tibia (47.4%) and the femur (27.5%). Coagulase-negative Staphylococcus spp. were the most frequently detected (44.4%) followed by mixed infections (18.7%) and Staphylococcus aureus (10.5%). Antibiograms revealed that 62.6% of our cases were cefuroxime sensitive; 87.7% were gentamicin sensitive. Only 10.5% showed resistance to both cefuroxime and gentamicin. There was no statistically significant difference of fusion rates between patients with different microbial species or different antibiograms. CONCLUSION: Our data suggest that besides the high variety of different detected species, initial antibiotic treatment with a combination of systemic cefuroxime and local gentamicin-loaded bone cement is effective and in almost 90% the later determined microbial infection was sensitive to this treatment. Therefore, we recommend initial treatment according to this algorithm until specific antibiograms are available from intraoperatively acquired tissue samples.

The influence of surface coatings of dicalcium phosphate (DCPD) and growth and differentiation factor-5 (GDF-5) on the stability of titanium implants in vivo.

Mechanical stability of implants is usually tested by pull out or push out tests which destroy the interface between the implant and bone. Pull out tests do not ideally reflect the clinical situation. In contrast, applying submaximal load leads to more physiologic micro-displacement between implant and bone. The aim of this study was to evaluate a new non-destructive mechanical testing device on different modifications of titanium implants. In 18 rabbits we investigated the influence of a dicalcium phosphate (DCPD) coating, or of a growth and differentiation factor-5 (GDF-5) coating, or a combination of both on the stability of titanium implants. The stability of implant was assessed by a non-destructive micro-measurement. In the same specimens the interface was investigated by micro-CT and histological evaluation. Surface modifications had a positive effect on the implant stability regarding displacement (p=0.001). Mechanical stability correlated with the quality of peri-implant tissue. Micro-displacement correlated negatively with the bone formation around the implants in histomorphometric evaluation (p=0.02). Amount of peri-prosthetic soft tissue showed a positive correlation with micro-displacement (p=0.01). Our findings indicate the positive effect of DCPD and GDF-5 coatings on stability of titanium implants. Results demonstrate the non-destructive testing to be an effective method to evaluate mechanical stability of implants.

Quantification of TGF-ß1, PDGF and IGF-1 cytokine expression after fracture treatment vs. non-union therapy via masquelet.

INTRODUCTION: Biochemical processes during bone regeneration can be analysed via quantification of peripheral serum cytokine levels. To date, serum levels of cytokines in patients treated with masquelet technique and patients with normal bone healing have not been compared. This comparison is supposed to deliver novel insights into the process of bone regeneration. Our aim was to validate this established method in the monitoring of bone regeneration after non-union treatment in masquelet technique. MATERIALS AND METHODS: Between 04/2008 and 01/2014 three groups were recruited: G1 (10 patients) with long bone non-unions, treated successfully with masquelet therapy, G2 (6 patients) with unsuccessful masquelet therapy and G3 (10 patients) with long bone fractures and normal bone healing. Peripheral blood samples were collected over a period of six months following a standardised time pattern in combination with clinical and radiologic follow up. TGF-ß1, PDGF-AB and IGF-1 were measured using commercially available immunoassays. RESULTS: TGF-ß1 levels in G1 and G2 demonstrated a parallel and lower overall concentration over time compared to G3. G3 showed a significant TGF-ß1 peak 2 weeks after surgery compared to G1 (p=0.0054). PDGF-AB concentrations were always lower in G2 than in G1 and G3. G3 peaked at week 2 with a significant higher value than in G2 (p=0.0177). IGF-1 showed lower overall serum concentrations in G2 than in G1 and G3. G1 had a peak level during the fourth week of follow-up. Compared to G2 this peak was significant (p=0.0015). CONCLUSIONS: This study shows that successful bone regeneration via masquelet technique only partially imitates cytokine expression of physiological bone healing. High expressions of IGF-1 correspond to a successful masquelet therapy while TGF-ß seems to play a minor role. These results assume that objective analysis of an effective non-union therapy with cytokine expression analysis is possible even with a small number of patients.

Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier.

Bone substitute materials can induce bone formation in combination with mesenchymal stem cells (MSC). The aim of the current study was to examine ectopic in vivo bone formation with and without MSC on a new resorbable ceramic, called calcium deficient hydroxyapatite (CDHA). Ceramic blocks characterized by a large surface (48 m2/g) were compared with beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA) ceramics (both ca. 0.5 m2/g surface) and demineralized bone matrix (DBM). Before implantation in the back of SCID mice carriers were freshly loaded with 2x10(5) expanded human MSC or loaded with cells and kept under osteogenic conditions for two weeks in vitro. Culture conditions were kept free of xenogenic supplements. Deposits of osteoid at the margins of ceramic pores occurred independent of osteogenic pre-induction, contained human cells, and appeared in 416 MSC/CDHA composites compared to 216 MSC/beta-TCP composites. ALP activity was significantly higher in samples with MSC versus empty controls (p<0.001). Furthermore, ALP was significantly (p<0.05) higher for all ceramics when compared to the DBM matrix. Compared to previous studies, overall bone formation appeared to be reduced possibly due to the strict human protocol. Ectopic bone formation in the novel biomaterial CDHA varied considerably with the cell pool and was at least equal to beta-TCP blocks.