Abstract: Induced membrane (IM) is the key component of Masquelet reconstruction surgery for the treatment of bone defects. IM is formed around the cement spacer and is known to secrete growth factors and osteoinductive factors. However, there is limited evidence available concerning the presence of osteoinductive factors in IM. This study aimed to investigate the existence of bone morphogenetic proteins (BMPs) in IM harvested from patients during the treatment of bone defects using the Masquelet technique. Methods This study involved six patients whose bone defects had been treated using the Masquelet technique. The affected sites were the femur ( n  = 3) and the tibia ( n  = 3). During the second-stage surgery, 1 cm 2 pieces of IM were harvested. Histological sections of IM were immunostained with anti-BMP-4, 6, 7, and 9 antibodies. Human bone tissue served as the positive control. Results The presence of BMP-4, 6, 7, and 9 was observed in all IM samples. Further, immunolocalization of BMP-4, 6, 7, and 9 was observed in blood vessels and fibroblasts in all IM samples. Immunolocalization of BMP-4, 6, 7, and 9 was also observed in bone tissue within the IM in one sample, in which osteogenesis inside the IM was observed. Conclusions This study showed that osteoinductive factors BMP-4, 6, 7, and 9 were present in the IM harvested from patients, providing evidence indicating that the Masquelet technique effectively contributes to healing large bone defects. Therefore, it may be possible for surgeons to omit the addition of BMPs to bone grafts, given the endogenous secretion of BMPs from the IM.

Abstract: Diabetes mellitus (DM) negatively affects fracture repair by inhibiting endochondral ossification, chondrogenesis, callus formation, and angiogenesis. We previously reported that transcutaneous CO 2 application accelerates fracture repair by promoting endochondral ossification and angiogenesis. The present study aimed to determine whether CO 2 treatment would promote fracture repair in cases with type I DM. Research design and methods A closed femoral shaft fracture was induced in female rats with streptozotocin-induced type I DM. CO 2 treatment was performed five times a week for the CO 2 group. Sham treatment, where CO 2 was replaced with air, was performed for the control group. Radiographic, histologic, genetic, and biomechanical measurements were taken at several time points. Results Radiographic assessment demonstrated that fracture repair was induced in the CO 2 group. Histologically, accelerated endochondral ossification and capillary formation were observed in the CO 2 group. Immunohistochemical assessment indicated that early postfracture proliferation of chondrocytes in callus was enhanced in the CO 2 group. Genetic assessment results suggested that cartilage and bone formation, angiogenesis, and vasodilation were upregulated in the CO 2 group. Biomechanical assessment revealed enhanced mechanical strength in the CO 2 group. Conclusions Our findings suggest that CO 2 treatment accelerates fracture repair in type I DM rats. CO 2 treatment could be an effective strategy for delayed fracture repair due to DM.

Abstract: In our hospital, cases of bone and soft tissue infections have been treated with continuous local antibiotics perfusion that allows for continuous circulation of antibiotics throughout the infected lesion. We termed this treatment “intramedullary antibiotics perfusion (iMAP)” for bone infection such as fracture-related infection (FRI) and “intrasoft tissue antibiotics perfusion” for soft tissue infection. Many cases are treated with both modalities. To introduce iMAP, this study focused on the patients with FRI treated with iMAP and reviewed their treatment outcomes. Methods We included 10 patients with FRI treated with iMAP between 2004 and 2017. The iMAP needles were inserted near the infected lesion, and an aminoglycoside antimicrobial was continuously administered. Patient characteristics, pathogenic bacteria, administered antibiotics, duration of administration, concentrations of antibiotics in blood and leachate fluid, fracture union rate, implant retention rate, and complications were studied. Results The mean age of patients was 59.9 years, and the mean follow-up period was 2.5 years. Affected bones were the tibia ( n = 8), humerus ( n = 1), and fibula ( n = 1). Deep infections developed on average 29.9 days after osteosynthesis. Pathogenic bacteria were methicillin-susceptible Staphylococcus aureus ( n = 6), methicillin-resistant S. aureus ( n = 2), and unknown ( n = 2). Average iMAP duration was 17.1 days. In all patients, infection was eradicated while preserving the implants, and fracture union was achieved without complications. Conclusion iMAP is a novel local drug delivery system allowing high concentrations of antibiotics to be administered without complications and is useful in the treatment of FRI.

Abstract: Fracture-related infections are difficult to treat because of the formation of biofilms around implants. Systemic antibiotics are notoriously ineffective against biofilms due to their insufficient penetration of tissues with poor vascularity. The goal of treating fracture-related infections is to achieve bone union while retaining the implant. Our proposal of continuous local antibiotic perfusion is a sustained local delivery system of sufficient antibiotics to bone and soft tissue infection sites, including to bone marrow via needles as intra-medullary antibiotics perfusion and to soft-tissue via double-lumen subcutaneous tubes as intra-soft tissue perfusion. Methods In this study, we examined the outcomes of 40 patients treated for fracture-related infections using continuous local antibiotic perfusion between 2015 and 2021 at Steel Memorial Hirohata Hospital, Himeji, Japan. Result The antibiotic used for continuous local antibiotic perfusion was gentamicin in all cases. Implant removal was required in five patients. Two patients required toe amputation and knee arthrodesis, while the remaining 38 patients achieved fracture union. Only one case of transient acute renal injury as a systemic side effect was observed, but it soon resolved. The blood concentration of gentamicin could be adjusted to less than the trough level. Conclusions Continuous local antibiotic perfusion is a novel local drug delivery system that has the potential of delivering sufficient concentrations of antibiotics with few systemic side effects; it is a useful option for the treatment of fracture-related infections.

Abstract: In this study, we examined the proliferation capability and osteogenic and chondrogenic differentiation potential of non-hypertrophic nonunion cells (NHNCs), and the effect of Escherichia coli-derived BMP-2 (E-BMP-2) on them. We enrolled five patients with non-hypertrophic nonunion. NHNCs isolated from nonunion tissue sampled during surgery were cultured, passaged, counted every 14 days, and analyzed. NHNCs were homogenous fibroblastic adherent cells and long-lived through at least 10 passages, with a slight decline. The cells were consistently positive for mesenchymal stem cell-related markers CD73 and CD105, and negative for the hematopoietic markers CD14 and CD45. NHNCs could differentiate into osteoblast lineage cells; however, they did not have strong calcification or sufficient chondrogenic differentiation capability. E-BMP-2 did not affect the proliferative capability of the cells but improved their osteogenic differentiation capability by increasing alkaline phosphatase activity and upregulating the gene expression of osterix, bone sialoprotein, and osteocalcin. E-BMP-2 enhanced their chondrogenic differentiation capability by upregulating the gene expression of aggrecan and collagen type II. We showed, for the first time, that NHNCs have the capacity to differentiate into osteoblast-lineage cells, although the chondrogenic differentiation potential was poor. Local application of E-BMP-2 with preservation of nonunion tissue is a potential treatment option for non-hypertrophic nonunion.

Abstract: For the prevention of surgical site infection (SSI), continuous disinfection could be helpful. Short wavelength ultraviolet radiation C (UVC) is highly bactericidal but shows cytotoxicity. Radiation of UVC with a wavelength of 222 nm to the skin is considered to be safe because it only reaches the stratum corneum. However, the safety of 222 nm irradiation to the surgical field not covered with skin is unknown. The purpose of this study was to examine the safety of 222 nm UVC irradiation on a surgical field in a rabbit model. Five types of tissue were surgically exposed and irradiated with 222 or 254 nm UVC. Immunohistological assessment against cyclobutane pyrimidine dimer (CPD), an index of DNA damage by UVC, was performed. The CPD‐positive cell rate was significantly higher in the 254 nm group than in the other groups in all tissues. A 222 nm group showed significantly more CPD than control in fat tissue, but no significant difference in all other tissues. In fat tissue collected 24 h after irradiation, the 254 nm group showed higher CPD than the other groups, while the 222 nm group had reduced to the control level. These data suggest that 222 nm UVC irradiation could be a new method to safely prevent SSI.

Abstract: Distraction osteogenesis has been broadly used to treat various structural bone deformities and defects. However, prolonged healing time remains a major problem. Various approaches including the use of low-intensity pulsed ultrasound, parathyroid hormone, and bone morphogenetic proteins (BMPs) have been studied to shorten the treatment period with limited success. Our previous studies of rats have reported that the transcutaneous application of CO 2 accelerates fracture repair and bone-defect healing in rats by promoting angiogenesis, blood flow, and endochondral ossification. This therapy may also accelerate bone generation during distraction osteogenesis, but, to our knowledge, no study investigating CO 2 therapy on distraction osteogenesis has been reported. Questions/purposes We aimed to investigate the effect of transcutaneous CO 2 during distraction osteogenesis in rabbits, which are the most suitable animal as a distraction osteogenesis model for a lengthener in terms of limb size. We asked: Does transcutaneous CO 2 during distraction osteogenesis alter (1) radiographic bone density in the distraction gap during healing; (2) callus parameters, including callus bone mineral content, volumetric bone mineral density, and bone volume fraction; (3) the newly formed bone area, cartilage area, and angiogenesis, as well as the expression of interleukin-6 (IL-6), BMP-2, BMP-7, hypoxia-inducible factor (HIF) -1α, and vascular endothelial growth factor (VEGF); and (4) three-point bend biomechanical strength, stiffness, and energy? Methods Forty 24-week-old female New Zealand white rabbits were used according to a research protocol approved by our institutional ethical committee. A distraction osteogenesis rabbit tibia model was created as previously described. Briefly, an external lengthener was applied to the right tibia, and a transverse osteotomy was performed at the mid-shaft. The osteotomy stumps were connected by adjusting the fixator to make no gap. After a 7-day latency phase, distraction was continued at 1 mm per day for 10 days. Beginning the day after the osteotomy, a 20-minute transcutaneous application of CO 2 on the operated leg using a CO 2 absorption-enhancing hydrogel was performed five times per week in the CO 2 group (n = 20). Sham treatment with air was administered in the control group (n = 20). Animals were euthanized immediately after the distraction period (n = 10), 2 weeks (n = 10), and 4 weeks (n = 20) after completion of distraction. We performed bone density quantification on the plain radiographs to evaluate consolidation in the distraction gap with image analyzing software. Callus parameters were measured with micro-CT to assess callus microstructure. The newly formed bone area and cartilage area were measured histologically with safranin O/fast green staining to assess the progress of ossification. We also performed immunohistochemical staining of endothelial cells with fluorescein-labeled isolectin B4 and examined capillary density to evaluate angiogenesis. Gene expressions in newly generated callus were analyzed by real-time polymerase chain reaction. Biomechanical strength, stiffness, and energy were determined from a three-point bend test to assess the mechanical strength of the callus. Results Radiographs showed higher pixel values in the distracted area in the CO 2 group than the control group at Week 4 of the consolidation phase (0.98 ± 0.11 [95% confidence interval 0.89 to 1.06] versus 1.19 ± 0.23 [95% CI 1.05 to 1.34] ; p = 0.013). Micro-CT demonstrated that bone volume fraction in the CO 2 group was higher than that in the control group at Week 4 (5.56 ± 3.21 % [95% CI 4.32 to 6.12 %] versus 11.90 ± 3.33 % [95% CI 9.63 to 14.25 %] ; p = 0.035). There were no differences in any other parameters (that is, callus bone mineral content at Weeks 2 and 4; volumetric bone mineral density at Weeks 2 and 4; bone volume fraction at Week 2). At Week 2, rabbits in the CO 2 group had a larger cartilage area compared with those in the control group (2.09 ± 1.34 mm 2 [95% CI 1.26 to 2.92 mm 2 ] versus 5.10 ± 3.91 mm 2 [95% CI 2.68 to 7.52 mm 2 ]; p = 0.011). More newly formed bone was observed in the CO 2 group than the control group at Week 4 (68.31 ± 16.32 mm 2 [95% CI 58.19 to 78.44 mm 2 ] versus 96.26 ± 19.37 mm 2 [95% CI 84.25 to 108.26 mm 2 ]; p 〈 0.001). There were no differences in any other parameters (cartilage area at Weeks 0 and 4; newly formed bone area at Weeks 0 and 2). Immunohistochemical isolectin B4 staining showed greater capillary densities in rabbits in the CO 2 group than the control group in the distraction area at Week 0 and surrounding tissue at Weeks 0 and 2 (distraction area at Week 0, 286.54 ± 61.55 /mm 2 [95% CI 232.58 to 340.49] versus 410.24 ± 55.29 /mm 2 [95% CI 361.78 to 458.71]; p 〈 0.001; surrounding tissue at Week 0 395.09 ± 68.16/mm 2 [95% CI 335.34 to 454.83] versus 589.75 ± 174.42/mm 2 [95% CI 436.86 to 742.64]; p = 0.003; at Week 2 271.22 ± 169.42 /mm 2 [95% CI 122.71 to 419.73] versus 508.46 ± 49.06/mm 2 [95% CI 465.45 to 551.47]; p 〈 0.001 respectively). There was no difference in the distraction area at Week 2. The expressions of BMP -2 at Week 2, HIF1-α at Week 2 and VEGF at Week 0 and 2 were greater in the CO 2 group than in the control group (BMP -2 at Week 2 3.84 ± 0.83 fold [95% CI 3.11 to 4.58] versus 7.32 ± 1.63 fold [95% CI 5.88 to 8.75] ; p 〈 0.001; HIF1-α at Week 2, 10.49 ± 2.93 fold [95% CI 7.91 to 13.06] versus 20.74 ± 11.01 fold [95% CI 11.09 to 30.40] ; p 〈 0.001; VEGF at Week 0 4.80 ± 1.56 fold [95% CI 3.43 to 6.18] versus 11.36 ± 4.82 fold [95% CI 7.13 to 15.59] ; p 〈 0.001; at Week 2 31.52 ± 8.26 fold [95% CI 24.27 to 38.76] versus 51.05 ± 15.52 fold [95% CI 37.44 to 64.66] ; p = 0.034, respectively). There were no differences in any other parameters (BMP-2 at Week 0 and 4; BMP -7 at Weeks 0, 2 and 4; HIF-1α at Weeks 0 and 4; IL-6 at Weeks 0, 2 and 4; VEGF at Week 4). In the biomechanical assessment, ultimate stress and failure energy were greater in the CO 2 group than in the control group at Week 4 (ultimate stress 259.96 ± 74.33 N [95% CI 167.66 to 352.25] versus 422.45 ± 99.32 N [95% CI 299.13 to 545.77] ; p 〈 0.001, failure energy 311.32 ± 99.01 Nmm [95% CI 188.37 to 434.25] versus 954.97 ± 484.39 Nmm [95% CI 353.51 to 1556.42] ; p = 0.003, respectively). There was no difference in stiffness (216.77 ± 143.39 N/mm [95% CI 38.73 to 394.81] versus 223.68 ± 122.17 N/mm [95% CI 71.99 to 375.37] ; p = 0.92). Conclusion Transcutaneous application of CO 2 accelerated bone generation in a distraction osteogenesis model of rabbit tibias. As demonstrated in previous studies, CO 2 treatment might affect bone regeneration in distraction osteogenesis by promoting angiogenesis, blood flow, and endochondral ossification. Clinical Relevance The use of the transcutaneous application of CO 2 may open new possibilities for shortening healing time in patients with distraction osteogenesis. However, a deeper insight into the mechanism of CO 2 in the local tissue is required before it can be used in future clinical practice.