Aim. The time to onset of symptoms after fracture fixation is still commonly used to classify fracture-related infections (FRI). Early infections (<2 weeks) can often be treated with debridement, systemic antibiotics, irrigation, and implant preservation (DAIR). Late infections (>10 weeks) typically require implant removal as mature, antibiotic-tolerant biofilms have formed. However, the recommendations for delayed infections (2–10 weeks) are not clearly defined. Here, infection healing and bone healing in early and delayed FRI is investigated in a rabbit model with a standardized DAIR procedure. Method. Staphylococcus aureus was inoculated into 17 rabbits after plate osteosynthesis in a humerus osteotomy. The infection developed either one week (early group, n=6) or four weeks (delayed group, n=6) before a standardized DAIR procedure and microbiological analysis were performed. Systemic antibiotics were administered for six weeks (two weeks: Nafcillin+Rifampin, four weeks: Levofloxacin+Rifampin). A control group (n=5) also underwent a revision operation (debridement and irrigation) after four weeks, but received no antibiotic treatment. Rabbits were euthanized seven weeks after the revision operation. Bone healing was assessed using a modified radiographic union score for tibial fractures (mRUST). After euthanasia, a quantitative microbiological examination of the entire humerus, adjacent soft tissues, and implants was performed. Results. All animals were infected at the time of revision surgery, with the bacterial load in the early group (especially in soft tissues) being greater than in the delayed group and control group. This indicates infiltration of bacteria into areas that are more difficult to reach after four weeks of debridement. The infection was eradicated in all animals in both the early and delayed groups at euthanasia, but not in the control group (CFU median (IQR): 2.1×10. 7. (1.3×10. 7. -2.6×10. 7. ). The osteotomy healed in the early group, while bone healing was significantly impaired in both the delayed group and control group (mRUST median (IQR): early group: 16 (14–16), delayed group: 7.5 (6–10), control: 7 (5.5–9); early vs. delayed: p=0.0411, early vs. control p=0.0065). Conclusion. The maturation of the infection between the first and fourth week does not affect the success of infection eradication in this rabbit FRI model. However, bone healing appears to be impaired with increasing duration of infection

Aim. To classify Fracture-related Infection (FRI) allowing comparison of clinical studies and to guide decision-making around the main surgical treatment concepts. Method. An international group of FRI experts met in Lisbon, June 2022 and proposed a new FRI classification. A core group met during the EBJIS Meeting in Graz, 2022 and on-line, to determine the preconditions, purpose, primary factors for inclusion, format and the detailed description of the elements of an FRI Classification. Results. Historically, FRI was classified by time from injury alone (early, delayed or late). Time produces pathophysiological changes which affect the bone, the soft-tissues and the patient general health, over a continuum. No definitive cut-off is therefore possible. Also, in several studies, time was not identified as an independent predictor of outcome. The most important primary factors were characteristics of the fracture (F), relevant systemic co-morbidities of the patient (R) and impairment of the soft-tissue envelope (I). These factors determine FRI severity, choice of treatment method and are predictors of outcome. For the fracture (F), the state of healing, the potential for bone healing and the presence or absence of a bone defect are critical factors. Co-morbidities are listed and the degree of end-organ damage is important (R). The ability to close the wound directly or the need for soft tissue reconstruction determines the impairment of the soft tissue component (I). Hence the FRI Classification was designed. The final proposal of the FRI Classification is presented here. The new classification has five stages; from simple cases of infected healed fractures, in healthy individuals with good soft tissues (Stage 1), through unhealed fractures with variable potential for bone healing (Stages 2, 3 or 4) to Stage 5, with no limb-sparing or reconstructive options. For instance, the need for a free flap (I4), over a well-healed fracture (F1), in a patient with 2 co-morbidities (R2) gives a classification of F1R2I4 for that patient. Conclusions. This novel approach to FRI classification builds on previous work in osteomyelitis, PJI and chronic medical conditions. It focusses attention on the elements of the disease which need treatment. It now requires validation in large patient cohorts. On behalf of the FRI Classification Consensus Group

Introduction. Frame configuration for the management of complex tibial fractures is highly variable and is dependent not only on fracture pattern and soft tissue condition but also surgeon preference. The optimal number of rings to use when designing a frame remains uncertain. Traditionally, larger, stiffer constructs with multiple rings per segment were thought to offer optimal conditions for bone healing, however, the concept of reverse dynamisation questions this approach. Materials & Methods. We compared clinical outcomes in 302 consecutive patients with tibial fractures treated in our unit with either a two-ring circular frame or a three-or-more-ring (3+) frame. The primary outcome measure was time spent in frame. Secondary outcomes were the incidence of malunion and the need for further surgical procedures to achieve bone union. The groups were evenly matched for age, co-morbidities, energy of injury mechanism, fracture classification, post-treatment alignment and presence of an open fracture. Results. The mean time in frame was 168 days for the 2-ring group and 200 days for the 3+ rings group (p=0.003). No significant difference was found in the rate of malunion (p=0.428) or the requirement for secondary surgical intervention to achieve union (p=0.363). No significant difference in time in frame was found between individual surgeons. Conclusions. This study finds that 2-ring frame constructs are a reliable option associated with significantly shorter duration of treatment and no increase in rates of adverse outcomes compared with larger, more complex frame configurations. Although this study cannot identify the underlying cause of the difference in treatment time between frame designs, it is possible that differences in mechanical stability lead to a more favourable strain environment for fracture healing in the 2-ring group

Introduction. The presence of pluripotent mesenchymal cells in the periosteum along with the growth factors produced or released following injury provides this tissue with an important role in bone healing. Utilising this property, vascularised periosteal flaps may increase the union rates in recalcitrant atrophic long bone non-union. The novel chimeric fibula-periosteal flap utilises the periosteum raised on an independent periosteal vessel, thus allowing the periosteum to be inset freely around the osteotomy site, improving bone biology. Materials & Methods. Ten patients, with established non-union, underwent fibula-periosteal chimeric flaps (2016–2022) at the Canniesburn Plastic Surgery Unit, UK. Preoperative CT angiography was performed to identify the periosteal branches. A case-control approach was used. Patients acted as their own controls, which obviated patient specific risks for non-union. One osteotomy site was covered by the chimeric periosteal flap and one without. In two patients both the osteotomies were covered using a long periosteal flap. Results. Union rate of 100% (11/11) was noted with periosteal flap osteotomies, versus those without flaps at 28.6% (2/7) (p = 0.0025). Time to union was also reduced in the periosteal flaps at 8.5 months versus 16.75 months in the control group (p = 0.023). Survival curves with a hazard ratio of 4.1, equating to a 4 times higher chance of union with periosteal flaps (log-rank p = 0.0016) was observed. Conclusions. The chimeric fibula-periosteal flap provides an option for atrophic recalcitrant non-unions where use of vascularised fibula graft alone may not provide an adequate biological environment for consolidation

Fractures of the ankle are common, and they mostly affect young adults. Wound complications are not uncommon following the fixation of these fractures. This study evaluated the impact of HIV on wound healing after plate osteosynthesis in patients with closed ankle fractures. This is an observational retrospective study of patients operated on at a tertiary level hospital. We reviewed hospital records for patients above 18 years of age who presented with wound breakdown following ankle open reduction and internal fixation. The patients’ hospital records were retrieved to identify all the patients treated for closed ankle fractures and those who developed wound breakdown. Patients with Pilon fractures were excluded. The National Health Laboratory System (NHLS) database was accessed to retrieve the CD4 count, viral load, haematology study results, and biochemistry results of these patients at the time of surgery and subsequent follow-up. The x-rays were retrieved from the electronic picture archiving system (PACS) and were assessed for fracture union at a minimum of 3 months follow-up. We reviewed the medical records of 172 patients with closed ankle fractures treated from 2018 to 2022. Thirty-one (18.0%) developed wound breakdown after surgery, and they were all tested for HIV. Most of the patients were male (58.0%), and the average age of the cohort was 43.7 years (range: 21 years to 84 years). Ten of these patients (32.2%) were confirmed HIV positive, with CD4 counts ranging from 155 to 781. Viral load levels were lower than detectable in 40% of these patients. All patients progressed to fracture union at a minimum of 3 months follow-up. We observed no difference between HIV-positive and HIV-negative patients in terms of wound breakdown and bone healing post-plate osteosynthesis for closed ankle fractures

Aims. As an alternative to external fixators, intramedullary lengthening nails (ILNs) can be employed for distraction osteogenesis. While previous studies have demonstrated that typical complications of external devices, such as soft-tissue tethering, and pin site infection can be avoided with ILNs, there is a lack of studies that exclusively investigated tibial distraction osteogenesis with motorized ILNs inserted via an antegrade approach. Methods. A total of 58 patients (median age 17 years (interquartile range (IQR) 15 to 21)) treated by unilateral tibial distraction osteogenesis for a median leg length discrepancy of 41 mm (IQR 34 to 53), and nine patients with disproportionate short stature treated by bilateral simultaneous tibial distraction osteogenesis, with magnetically controlled motorized ILNs inserted via an antegrade approach, were retrospectively analyzed. The median follow-up was 37 months (IQR 30 to 51). Outcome measurements were accuracy, precision, reliability, bone healing, complications, and patient-reported outcome assessed by the Limb Deformity-Scoliosis Research Society Score (LD-SRS-30). Results. A median tibial distraction of 44 mm (IQR 31 to 49) was achieved with a mean distraction index of 0.5 mm/day (standard deviation 0.13) and median consolidation index of 41.2 days/cm (IQR 34 to 51). Accuracy, precision, and reliability were 91%, 92%, and 97%, respectively. New temporary range of motion limitations occurred in 51% of segments (34/67). Distraction-related equinus deformity treated by Achilles tendon lengthening was the most common major complication recorded in 16% of segments (11/67). In 95% of patients (55/58) the distraction goal was achieved with 42% unplanned additional interventions per segment (28/67). The median postoperative LD-SRS-30 score was 4.0 (IQR 3.6 to 4.3). Conclusion. Tibial distraction osteogenesis using motorized ILNs inserted via an antegrade approach appears to be a reliable and precise procedure. Temporary joint stiffness of the knee or ankle should be expected in up to every second patient. A high rate and wide range of complications of variable severity should be anticipated. Cite this article: Bone Joint J 2024;106-B(3):293–302

Introduction. Frame HI is the #Days for device removal/cm. IM Nail HI is less relevant (31–45 D/cm). Albizzia HI was 33 D/cm (1991–2003). Patients felt fine approximately 1M after end of lengthening (EoL), resuming normal life and sports. This sometimes resulted in implants fractures (e.g. skying before bone fusion). Ideally, the full fusion should occur at the EoL. We decided to shorten the HI to reach this target, optimising all parameters. Materials & Methods. The evolution of care has been monitored over a 32-year clinical experience with a fully weight-bearing nails (Albizzia then G-nail). Monitoring was with X-rays, DEXA, blood bone activity, and in London with special 5G CBCT Scans. We implemented several changes in the Care of patients and measured them according to the ‘Five Principles’ (stability, function, ‘Roads-vascular supply’, ‘Materials-calories’ and ‘Workers-BFC’, with actions on food intake, activity levels and on muscle and bone vascular growths. Results. Preop: training (vascularity, muscle force). Op & Postop: spine morphine, IM sawing preserving BFC, controlled hypo-pressure, low hydration, 50 cm leg elevation, walking, resistance bike, full motion (drainage, muscle reactivation), discharge 3–4h postop (including bilateral). Postop daily intense gym training. POD07-21: Distraction increased to fight non-linear hyper-ossification (44–50 mm gain at POD30) +/- aided by NSAIDs. HI decreased to 12–20D/cm, sometimes 8D/cm with some ‘soft fusion’ during lengthening, hardening within 1W after EoL. Conclusions. The surgeon is not a passive X-rays observer, but has an active role in changing the healing speed and decreasing HI for patient safety. Electro/Magnetic nails (torque 1 Nm) may be clocked by bone fusion, which does not occur with the G-Nail (19 Nm). An holistic vision for patients and treatments at several levels is essential to accelerate bone healing, and to return fast to full normal life, after a short ‘lengthening parenthesis’

Abstract. Introduction. Vitamin D deficiency in the UK is well documented − 30–40% of the population. It is an essential component of calcium metabolism and adequate levels are important for bone healing. Studies have demonstrated an overall prevalence of vitamin D deficiency/insufficiency at 77% in trauma patients aged >18, deficiency alone was 39%. Adequate vitamin D levels have a positive effect on bone mineral density and callus formation at fracture sites. Methods. We conducted a retrospective consecutive case series of all patients aged 0–50 undergoing surgical management for any fracture in October 2021 to March 2022. We assessed if vitamin D levels were checked and if patients were prescribed replacement as per local guidelines. Results. A total of 131 patients were identified, (mean 29 years; 83 male and 48 female). Most cases were upper limb fractures (n=78, 60%), as opposed to lower limb (n=53, 40%). Only 20 (15%) had their levels checked, of which 13 (65%) were insufficient/deficient (10 insufficiency, 2 deficiency, 1 severe deficiency). Of these 13 patients, only 3 (23%) were prescribed replacement therapy. Conclusions. Only a small proportion of patients had their levels checked, however the majority were insufficient/deficient. The prevalence in our study is consistent with larger epidemiology studies, which reflect a higher rate of deficiency in fracture patients compared to the general population. Thus, we propose that all patients in this age group should undergo a vitamin d level check upon time of clerking and this should be accurately treated as per trust guidance

Aim. Staphylococcus aureus is the leading pathogen in fracture-related infection (FRI). Virulence factors vary between different strains, which may have a decisive influence on the course of infection. Previous in vitro experiments, in vivo testing in wax moth larvae, and genomic analysis of S. aureus isolates from FRI identified a low- and high-virulent strain. These findings correlated with the acute course of FRI induced by the high-virulent pathogen, whereas the low-virulent strain caused a chronic FRI in its human host. However, the role of bacterial virulence in FRI is not completely understood. Therefore, the present study aimed to compare the identified high- and low-virulent S. aureus isolates in a murine FRI model. Method. Skeletally mature C57Bl/6N mice received a femoral osteotomy stabilized by titanium locking plates. FRI was established by inoculation of either high-virulent S. aureus EDCC 5458 or low-virulent S. aureus EDCC 5464 in the fracture gap. Mice were euthanized 4 and 14 days after surgery, respectively. Severity and progression of infection were assessed in terms of clinical presentation, quantitative bacteriology, semiquantitative histopathologic evaluation, and serum cytokine profile. Results. Quantitative bacteriological results 4 days after surgery revealed a higher bacterial load in soft tissue samples in high-virulent infected animals (p =0.026). Mice infected with the high-virulent strain also displayed higher rates of organ dissemination (24/36 organs in high-virulent, versus 5/36 organs in low-virulent infected animals; p <0.0001). In the histopathological assessment, bacterial agglomerations at the fracture ends were present to a greater extent in the high-virulent cohort and barely detectable in low-virulent infected mice. In both cohorts, no bone healing was observed after 4 days. On day 14, bone healing at the fracture site was visible in low-virulent infected animals, whereas callus formation was observed in only one animal from the high-virulent infected cohort. Furthermore, osteonecrosis and osteolysis were increased in high-virulent infected animals. Regarding serum cytokines, innate immune markers were elevated in both groups at day 4. By day 14, a more pronounced proinflammatory response indicated by increased serum cytokine levels of IFN-γ, IL-1β, and IL-6 was observed in high-virulent infected animals. Conclusions. The present study demonstrated distinct bacteriological and histopathological differences between two different virulent S. aureus strains previously shown to have different courses in human patients. While host physiology is often considered to have a major impact on the course of FRI, this study highlights the critical influence of the invading pathogen and its virulence characteristics

Treatment of segmental bone defects remains a major clinical problem, and innovative strategies are often necessary to successfully reconstruct large volumes of bone. When fractures occur, the resulting hematoma serves as a reservoir for growth factors and a space for cell infiltration, both crucial to the initiation of bone healing. Our previous studies have demonstrated very clear ultrastructural differences between fracture hematomas formed in normally healing fractures and those formed in segmental bone defects. However, there is little information available regarding potential differences in the underlying gene expression between hematomas formed in normal fractures, which usually heal by themselves, and segmental bone defects, which do not. Therefore, the aim of this study was to identify differences in gene expression within hematomas collected from 0.5 mm (normal fracture) and 5 mm (segmental bone defect) fracture sites during the earliest stages of bone healing. Osteotomies of 0.5 and 5 mm in the femur of Fisher 344 rats were stabilized with external fixators (RISystem AG). After 3 days the rats were sacrificed, and the fracture hematomas were collected for RNA-sequencing. Ingenuity pathway analysis (IPA) was used to identify upstream regulators and biological functions that were significantly enriched with differentially expressed genes from the RNA-sequencing analysis. Animal procedures were conducted following the IACUC protocol of the UT Health Science Center San Antonio. Key upstream regulators of bone formation were less active (e.g. TGFB1, FGF2, SMAD3) or even inhibited (e.g. WNT3A, RUNX2, BMP2) in non-healing defects when compared to normally healing fractures. Many upstream regulators that were uniquely enriched in healing defects were molecules recently discovered to have osteogenic effects during fracture healing (e.g. GLI1, EZH2). Upstream regulators uniquely enriched in non-healing defects were mainly involved in an abnormal modulation of hematopoiesis, revealing evidence of impaired maturation of functional macrophages and cytokines (e.g. IL3, CEBPE), both essential for successful bone healing. In addition, the enrichment pattern suggested a dysregulation of megakaryopoiesis (e.g. MRTFA, MRTFB, GATA2), which directly affects platelet production, and therefore fracture hematoma formation. Remarkably, the organization of the ECM was the most significantly enriched biological function in the normally healing fractures, and implies that the defect size directly affected the structural properties within the fracture hematoma. Conversely, genes encoding important ECM components (e.g. BGN, various collagens, IBSP, TNC), cell adhesion molecules, MMPs (MMP2), and TIMPs were all significantly downregulated in non-healing defects. Our most recent findings reveal new important key molecules that regulate defect size-dependent fracture healing. Combined with our previous results, which identified structural differences in fracture hematomas from both types of defects, current findings indicate that differential expression of genes is dictated by the structural properties of the hematomas formed during early fracture healing. Consequently, creating a bioscaffold that mimics the structure of normal fracture hematomas could be the first step towards developing new orthoregenerative treatment strategies that potentiate healing of large bone defects and non-healing fractures

Fracture non-union can be as high as 20% in certain clinical scenarios and has a high associated socioeconomic burden. Boron has been shown to regulate the Wnt/β-catenin pathway in other bodily processes. However, this pathway is also critical for bone healing. Here we aim to demonstrate that the local delivery of boric acid can accelerate bone healing, as well as to elucidate how boric acid, via the regulationtheWnt/β-catenin pathway, impacts theosteogenic response of bone-derived osteoclasts and osteoblasts during each phase of bone repair. Bilateral femoral cortical defects were created in 32 skeletally mature C57 mice. On the experimental side, boric acid (8mg/kg concentration) was injected locally at the defect site whereas on the control side, saline was used. Mice were euthanized at 7, 14, and 28 days. MicroCT was used to quantify bone regeneration at the defect. Histological staining for ALP and TRAP was used to quantify osteoblast and osteoclast activity respectively. Immunohistochemical antibodies, β-catenin and CD34 were used to quantify active β-catenin levels and angiogenesis respectively. Sclerostin and GSK3β were also quantified and are both inhibitors of the wnt signaling pathway via degradation and inactivation of β-catenin. The boron group exhibited higher bone volume and trabecular thickness at the defect site by 28 days on microCT. ALP activity was significantly higher in boron group at 7 days whereas boron had no effect on TRAP activity. Additionally, CD34 staining revealed increased angiogenesis at 14 days in boron treated groups. β-catenin activity on immunohistochemistry was significantly higher in the boron group at 7 days, GSK3β was significantly higher in the boron group at 14 days and Sclerostin was significantly higher in the boron group at 28 days. Boron appears to increase osteoblast activity at the earlier phases of healing. The corresponding early increase in β-catenin along with ALP likely supports that boron increases osteoblast activity via the wnt/β-catenin pathway. Increased angiogenesis at 14 days could be a separate mechanism increasing bone formation independent of wnt/β-catenin activation. Neither GSK3β or Sclerostin levels correlated with β-catenin activity therefore boron likely increases β-catenin through a mechanism independent of both GSK3β and Sclerostin. The addition of this inexpensive and widely available ion could potentially become a non-invasive, cost-effective treatment modality to augment fracture healing and decrease non-union rates in high risk patients

The Masquelet or induced membrane technique (IMT) is a two-stage surgical procedure used for the treatment of segmental bone defects. In this technique, the defect is first filled with a polymethyl methacrylate (PMMA) spacer, which triggers the formation of a membrane that will encapsulate the defect. During the second surgery, the spacer is carefully removed and replaced by autologous bone graft while preserving the membrane. This membrane is vascularized, contains growth factors, and provides mechanical stability to the graft, all of which are assumed to prevent graft resorption and promote bone healing. The technique is gaining in popularity and several variations have been introduced in the clinical practice. For instance, orthopaedic surgeons now often include antibiotics in the spacer to treat or prevent infection. However, the consequences of this approach on the properties of the induce membrane are not fully understood. Accordingly, in a small animal model, this study aimed to determine the impact on the induced membrane of impregnating spacers with antibiotics frequently used in the IMT. We surgically created a five-mm segmental defect in the right femur of 25 adult male Sprague Dawley rats. The bone was stabilized with a plate and screws before filling the defect with a PMMA spacer. Animals were divided into five equal groups according to the type and dose of antibiotics impregnated in the spacer: A) no antibiotic (control), B) low-dose tobramycin (1.2 g/40 g of PMMA), C) low-dose vancomycin (1 g/40 g of PMMA), D) high-dose tobramycin (3.6 g/40 g of PMMA), E) high-dose vancomycin (3 g/40 g of PMMA). The animals were euthanized three weeks after surgery and the induced membranes were collected and divided for analysis. We assessed the expression of selected genes (Alpl, Ctgf, Runx2, Tgfb1, Vegfa) within the membrane by quantitative real-time PCR. Moreover, frozen sections of the specimens were used to quantify vascularity by immunohistochemistry (CD31 antigen), proliferative cells by immunofluorescence (Ki-67 antigen), and membrane thickness. Microscopic images of the entire tissue sections were taken and analyzed using FIJI software. Finally, we measured the concentration of vascular endothelial growth factor (VEGF) in the membranes by ELISA. No significant difference was found among the groups regarding the expression of genes related to osteogenesis (Alpl, Runx2), angiogenesis (Vegfa), or synthesis of extracellular matrix (Ctgf, Tgfb1) (n = four or five). Similarly, the density of proliferative cells and blood vessels within the membrane, as well as the membrane thickness, did not vary substantially between the control, low-dose, or high-dose antibiotic groups (n = four or five). The concentration of VEGF was also not significantly influenced by the treatment received (n = four or five). The addition of tobramycin or vancomycin to the spacer, at the defined low and high doses, does not significantly alter the bioactive characteristics of the membrane. These results suggest that orthopaedic surgeons could use antibiotic-impregnated spacers for the IMT without compromising the induced membrane and potentially bone healing

Impaired bone healing biology secondary to soft tissue deficits and chemotherapy contribute to non-union, fracture and infection following limb salvage surgery in Osteosarcoma patients. Approved bone healing augments such as recombinant human bone morphogenetic protein-2 (rhBMP-2) have great potential to mitigate these complications. rhBMP-2 use in sarcoma surgery is limited, however, due to concerns of pro-oncogenic signalling within the tumour resection bed. To the contrary, recent pre-clinical studies demonstrate that BMP-2 may induce Osteosarcoma differentiation and limit tumour growth. Further pre-clinical studies evaluating the oncologic influences of BMP-2 in Osteosarcoma are needed. The purpose of this study is to evaluate how BMP-2 signalling affects Osteosarcoma cell proliferation and metastasis in an active tumour bed. Two Osteosarcoma cell lines (143b and SaOS-2) were assessed for proliferative capacity and invasion. 143b and SaOS-2 cells were engineered to upregulate BMP-2. In vitro proliferation was assessed using a cell viability assay, motility was assessed with a scratch wound healing assay, and degree of osteoblastic differentiation was assessed using qRT-PCR of Osteoblastic markers (CTGF, ALP, Runx-2 and Osx). For in vivo evaluation, Osteosarcoma cells were injected into the intramedullary proximal tibia of immunocompromised (NOD-SCID) mice and local tumour growth and metastases were assessed using weekly bioluminescence imaging (BLI) and tumour volume measurements for 4–6 weeks. At the experimental end point we assessed radiographic tumour burden using ex-vivo micro-CT, as well as tibial and pulmonary gross and histologic pathology. SaOS-2 was more differentiated than 143b, with increased expression of Runx-2 (p = 0.009), Osx (p = 0.004) and ALP (p = 0.035). BMP-2 upregulation did not stimulate an osteoblast differentiation response in 143b, but stimulated an increase in Osx expression in SaOS-2 (p = 0.002). BMP-2 upregulation in 143b cells resulted in increased proliferation in vitro (p = 0.014), faster in vitro wound healing (p = 0.03), significantly increased tumour volume (p = 0.001) with enhanced osteolysis detected on micro-CT, but did not affect rates of lung metastasis (67% vs. 71%, BMP-2 vs. Control). BMP-2 over-expression in SaOS-2 cells reduced in vitro proliferation when grown in partial osteogenic-differentiation media (p < 0.001), had no effect on in vitro wound healing (p = 0.28), reduced in vivo SaOS-2 tumour burden at 6 weeks (photon counts, p < 0.0001), decreased tumour-associated matrix deposition as assessed by trabecular thickness (p = 0.02), and did not affect rates of lung metastasis (0% vs. 0%). Our results indicate BMP-2 signalling incites a proliferative effect on a poorly differentiated Osteosarcoma cell line (143b), but conditionally reduces proliferative capacity and induces a partial differentiation response in a moderately-differentiated Osteosarcoma cell line (SaOS-2). This dichotomous effect may be due to the inherent ability for Osteosarcoma cells to undergo BMP-2 mediated terminal differentiation. Importantly, these results do not support the clinical application of BMP-2 in Osteosarcoma limb salvage surgery due to the potential for stimulating growth of poorly differentiated Osteosarcoma cells within the tumour bed. Additional studies assessing the effects of BMP-2 in an immune-competent mouse model are ongoing

Impaired bone healing biology secondary to soft tissue deficits and chemotherapy contribute to non-union, fracture and infection following limb salvage surgery in Osteosarcoma patients. Approved bone healing augments such as recombinant human bone morphogenetic protein-2 (rhBMP-2) have great potential to mitigate these complications. rhBMP-2 use in sarcoma surgery is limited, however, due to concerns of pro-oncogenic signalling within the tumour resection bed. To the contrary, recent pre-clinical studies demonstrate that BMP-2 may induce Osteosarcoma differentiation and limit tumour growth. Further pre-clinical studies evaluating the oncologic influences of BMP-2 in Osteosarcoma are needed. The purpose of this study is to evaluate how BMP-2 signalling affects Osteosarcoma cell proliferation and metastasis in an active tumour bed. Two Osteosarcoma cell lines (143b and SaOS-2) were assessed for proliferative capacity and invasion. 143b and SaOS-2 cells were engineered to upregulate BMP-2. In vitro proliferation was assessed using a cell viability assay, motility was assessed with a scratch wound healing assay, and degree of osteoblastic differentiation was assessed using qRT-PCR of Osteoblastic markers (CTGF, ALP, Runx-2 and Osx). For in vivo evaluation, Osteosarcoma cells were injected into the intramedullary proximal tibia of immunocompromised (NOD-SCID) mice and local tumour growth and metastases were assessed using weekly bioluminescence imaging and tumour volume measurements for 4–6 weeks. At the experimental end point we assessed radiographic tumour burden using ex-vivo micro-CT, as well as tibial and pulmonary gross and histologic pathology. SaOS-2 was more differentiated than 143b, with significantly increased expression of the Osteoblast markers Osx (p = 0.004) and ALP (p = 0.035). BMP-2 upregulation did not stimulate an osteoblast differentiation response in 143b, but stimulated an increase in Osx expression in SaOS-2 (p = 0.002). BMP-2 upregulation in 143b cells resulted in increased proliferation in vitro (p = 0.014), faster in vitro wound healing (p = 0.03), significantly increased tumour volume (p = 0.001) with enhanced osteolysis detected on micro-CT, but did not affect rates of lung metastasis (67% vs. 71%, BMP-2 vs. Control). BMP-2 over-expression in SaOS-2 cells reduced in vitro proliferation when grown in osteogenic-differentiation media (p < 0.001), had no effect on in vitro wound healing (p = 0.28), reduced in vivo SaOS-2 tumour burden at 6 weeks (photon counts, p < 0.0001), decreased tumour-associated matrix deposition as assessed by trabecular thickness (p = 0.02), but did not affect rates of lung metastasis (0% vs. 0%). Our results indicate BMP-2 signalling incites a proliferative effect on a poorly differentiated Osteosarcoma cell line (143b), but conditionally reduces proliferative capacity and induces a partial differentiation response in a moderately-differentiated Osteosarcoma cell line (SaOS-2). This dichotomous effect may be due to the inherent ability for Osteosarcoma cells to undergo BMP-2 mediated terminal differentiation. Importantly, these results do not support the clinical application of BMP-2 in Osteosarcoma limb salvage surgery due to the potential for stimulating growth of poorly differentiated Osteosarcoma cells within the tumour bed. Additional studies assessing the effects of BMP-2 in an immune-competent mouse model are ongoing

Bone regeneration includes a well-orchestrated series of biological events of bone induction and conduction. Among them, the Wnt/β-catenin signaling pathway is critical for bone regeneration. Being involved in several developmental processes, Wnt/β-catenin signaling must be safely targeted. There are currently only few specific therapeutic agents which are FDA-approved and already entered clinical trials. A published work has shown that Tideglusib, a selective and irreversible small molecule non-ATP-competitive glycogen synthase kinase 3-β(GSK-3β) inhibitor currently in trial for Alzheimer's patients, can promote tooth growth and repair cavities. [1]Despite some differences, they are some similarities between bone and tooth formation and we hypothesise that this new drug could represent a new avenue to stimulate bone healing. In this work, we locally delivered Tideglusib (GSK3β inhibitor) in the repair of femoral cortical window defects and investigated bone regeneration. A biodegradable FDA-approved collagen sponge was soaked in GSK-3βinhibitor solution or vehicle only (DMSO) and was implanted in 1 × 2 mm unicortical defects created in femora of 35 adult wild-type male mice. Bone defect repair on control and experimental (GSK-3βinhibitor) groups was assessed after 1 week (n=22), 2 weeks (n=24) and 4 weeks (n=24) with microCT and histological analysis foralkaline phosphatase (ALP, osteoblast activity), tartrate resistant acid phosphatase (TRAP, osteoclasts), and immunohistochemistry to confirm the activation of the Wnt/β-catenin pathway. Our results showed that Tideglusib significantly enhanced cortical bone bridging (20.6 ±2.3) when compared with the control (12.7 ±1.9, p=0.001). Activity of GSK-3β was effectively downregulated at day 7 and 14 resulting in a higher accumulation of active β-catenin at day 14 in experimental group (2.5±0.3) compared to the control (1.1±0.2, p=0.03). Furthermore, the onset of ALP activity appears earlier in the experimental group (day 14, 1.79±0.28), a level of activity never reached at any end-point by the control defects. At 4 weeks treatment, we observed a significant drop in ALP in the experimental group (0.47±0.05) compared to the control (1.01±0.19, p=0.02) and a decrease in osteoclast (experimental=1.32±0.36, control=2.23±0.67, p=0.04). Local downregulation of GSK-3β by tideglusib during bone defect repair resulted in significant increase in amount of new bone formation. The early upregulation of osteoblast activity is one explanation of bone healing augmentation. This is likely the effect of upregulation of β-catenin following pharmaceutical inhibition of GSK-3β since β-catenin activation is known to positively regulate osteoblasts, once committed to the osteoblast lineage. As a GSK-3β inhibitor, Tideglusib demonstrates a different mechanism of action compared with other GSK-3β antagonists as treatment was started immediately upon injury and did not interfere with precursor cells recruitment and commitment. This indicates that tideglusib could be used at the fracture site during the initial intraoperative internal fixation without the need for further surgery. This safe and FDA-approved drug could be used in prevention of non-union in patients presenting with high risk for fracture-healing complications

Introduction. A clinical case of catastrophic ring failure in a 13 year old autistic overweight patient during treatment for tibial lengthening and deformity using a Taylor Spatial Frame is reported. Ring failure was noted during the later stages of bone healing and the frame was removed. The clinical outcome was not affected by the catastrophic ring failure. The photograph of the deformed ring is presented below:. Materials and Methods. The patient's notes and X-rays were reviewed and a macroscopic examination of the deformed ring was performed. Mechanical tests of different Taylor Spatial frame constructs were performed in an attempt to simulate the deformity that was clinically observed. Different constructs of TSF of different ring sizes were fixed to polyurethane cylinders simulating bone, were mechanically tested to failure and load/deflection curves were produced. Results. Macroscopically the ring looked otherwise normal. Gradual mechanical compression tests of Taylor Spatial frame constructs showed that ring deformation increased by increasing the ring diameter and by using jointed rather than full joints without a ring. The ring deformation observed clinically was reproduced at the lab by applying high loads on frame constructs composed of large diameter jointed rings not rigidly fixed to bone. Conclusions. Taylor Spatial frame ring failure during treatment is a serious complication that has not been described in the literature. Possible causes are discussed. Clinicians are advised to use the smaller possible diameter rings. Where large diameter rings are required, these rings should preferably be not jointed. Half rings when used should be carefully and securely joined together by the operating surgeon in order to make a complete ring. For any figures or tables, please contact the authors directly

Aim. Treatment of infected and non-infected non-unions remain a major challenge after orthopedic fracture-related surgery. In clinical practice, several revision surgeries are usually required, including a radical debridement and exchange of implants, to control or even eradicate the infection to finally achieve bone healing. However, a clear treatment algorithm in clinical practice may be difficult to follow due to the heterogeneous patient population. Thus, so controlled settings for research purposes is better achieved in standardized animal studies. So far, there exists no multi-stage animal model that can be realistically transferred to the clinical situation in humans. The importance of such a model is obvious in order to be able to investigate different therapy concepts for infected and non-infected non unions. Methods. In 20 female Sprague-Dawley rats, a critical size defect by a femur osteotomy with 5 mm width was done. The periosteum at the fracture zone was cauterized proximal and distal to the osteotomy to achieve an hypovascularized situation. After randomization, 10 animals were intramedullary infected with a multisensible Staph. aureus strain (10. 3. CFU). After 5 weeks, a second surgery was performed with removing the K-wire, debridement of the osteotomy-gap and re-osteosynthesis with an angle-stable plate. After further 8 weeks all rats were euthanized and underwent biomechanical testing to evaluate bone consolidation or delayed union, respectively. Additional micro-CT analysis, histological, and histomorphometric analysis were done to evaluate bone consolidation or delayed union, respectively, by the score of Lane and Sandhu and to quantify callus formation and the mineralized area of the callus. Results. 5 weeks after the first surgery a non-union had formed in all septic and aseptic animals. According to the Lane and Sandhu score a significantly higher callus formation was found in the infected group. In all infected animals, the inoculated Staph. aureus strain was detected during the revision surgery. 8 weeks after the second surgery no bone healing could be detected in the µ-CT analysis in both groups and biomechanical testing showed a significant lower maximum torque in both groups as compared to the untreated contralateral femura. Conclusion. Here we show first results of a new two-stage pseudarthrosis animal model, which reflects a very realistic clinical situation of an infection-related non-union model. Based on this model, various therapeutic strategies in the treatment of infectious and non-infectious pseudarthrosis, such as the use of bone substitutes, can be evaluated in further studies

Introduction. Circular frame fixation has become a cornerstone of non-union and deformity management since its inception in the 1950s. As a consequence of modularity and heterogenous patient and injury factors, the prediction of the mechanobiological environment within a defect is subject to wide variations in practice. Given these wide range of confounding variables, clinical and cadaveric experimentation is close to impossible and frame constructs are based upon clinician experience. The Finite Element Analysis (FEA) method provides a powerful tool to numerically analyse mechanics. This work aims to develop an FEA model of a tibial defect and predict the mechanical response within the construct. Materials and Methods. The geometry of a tibia was acquired via CT and a series of bone defects were digitally created in the tibial diaphysis. A 4-ring, 10-wire Ilizarov fixator was constructed using 180mm stainless steel rings and 1.8mm stainless steel wires tensioned to 1200N. An axial load (800N) was applied to simulate single leg stance of an 80kg patient. The magnitude of displacement was measured for defects with varying sizes (5–40mm). A numerical analysis was performed in large-strain regime using open-source FEA library (MoFEM). Results. Defect size did not effect displacement, but significantly influenced strain. Measured displacements were 5.72–5.78mm, however strain ranged from 14.5–100%. Moreover, it was found that bone material properties also have no significant impact on the results. Conclusions. Accounting for FEA assumptions, this model predicted a strain environment which was above expected favourable range for bone healing. The addition of graft within the environment is likely to change the mechanobiological environment which warrants further investigation. We plan to develop this model to answer further research questions in the limb reconstruction discipline and validate its accuracy with mechanical data. We believe the presented approach can be a useful tool for investigating the performance circular frames

Introduction. Management of open fractures is challenging and requires a multidisciplinary team approach. Gustilo Anderson Type IIIB fractures reportedly have a higher infection rate (up to 52%) and up to 16% amputation rate. This study aims to evaluate outcomes of using Adjuvant Local Antibiotic Hydroxyapatite Bio-Composite in management of Open Gustilo-Anderson IIIB fractures. Materials and Methods. We reviewed a prospective data of 80 patients who presented with Gustilo Anderson Type IIIB Open Fracture to a single ortho-plastic centre. Only patients who were managed with single-stage “Fix and Flap” along with intra-operative Adjuvant Local Antibiotic Bio-Composite were included. Results. Mean follow-up time was 22 months. The mean time from injury until definitive surgery was 7.73 days (1–30 days). Primary union achieved in 88.3% within 32 weeks on average. The delayed union reported in 7.8% of patients, for the bone healing stimulated by injecting the fracture site with Autologous Bone Marrow Aspirate Concentrate. Subsequent follow-up showed signs of successful fracture healing at 60 weeks post-injury. Three patients (3.9%) had non-union. Limb salvage rate was 96.25%, and only 1.25% deep infection rate. Conclusions. Our results highlight that low infection rates, high limb salvage rates and high union rates can be achieved in these complex injuries with a combined OrthoPlastic approach, MDT input, meticulous technique and the use of adjuvant local antibiotic bio-composite. Delay in definitive surgery, gentamicin resistance and smoking were not associated with any increased deep infection or non-union in our series. At 22 months of follow-up, deep infection rate was 1.25%, limbs salvage rate was 96.25%, fracture union rate was 96.1%, and reoperation rate 18.75%

Introduction. To determine the advantages and risks of plating after lengthening (PAL) of tibia in children and adolescents. Materials and Methods. 35 consecutive tibial lengthenings were done for limb length discrepancy (LLD) in 26 patients. Gradual lengthening by an external fixator from a tibial (usually diaphyseal) osteotomy was followed by internal fixation with a lateral tibial submuscular plate. The mean age at the time of the lengthening was 10.3 years (4.8 – 16.8 years). The aetiology for LLD was congenital in 21, acquired in 3, and developmental in 2 patients. The mean follow-up was 4.3 years (8 months – 9.9 years). Results. The mean lengthening was 5cm (3–8.6cm) or 19.1% (10.8 – 35.2%) of the initial length of tibia. It took 78.8 days to reach the target length at a lengthening rate of 0.75mm/day. The mean time to plate substitution after cessation of lengthening was 24.7days/109 days after osteotomy. This led to an average external fixation index (EFI) of 23.1days/cm. Optimisation of this technique by judicious estimation of timing of plate substitution would reduce the EFI. Consolidation was recorded at 192 days after osteotomy. Bone healing index (BHI) was 39.8days/cm and was age dependent: <12 year olds = 37.5 days/cm; 12 years = 44.7 days/cm. Using the estimated consolidation time if treatment was solely by external fixator, calculated by tripling the time taken to reach target length after osteotomy, the BHI in this series would have been 52.9 days/cm (p < 0.001). Knee flexion recovery to > 90 degrees was noted at 153.5 days after plating. One greenstick fracture occurred 116 days after plate insertion, 1 tibial shaft fracture occurred 315 days post removal of plate - both following injury and were treated conservatively. Six episodes of sepsis, 5 superficial and 1 deep were treated with antibiotic suppression. The plates were removed from 28 tibiae, 437.4 days after insertion. Conclusions. Plating after lengthening not only reduces the fixator time but appears to achieve consolidation faster than if treatment was by external fixation alone. This facilitates early recovery of joint motion and limb function