Phase III clinical trial of autologous CD34 + cell transplantation to accelerate fracture nonunion repair.

BACKGROUND: We previously demonstrated that CD34 + cell transplantation in animals healed intractable fractures via osteogenesis and vasculogenesis; we also demonstrated the safety and efficacy of this cell therapy in an earlier phase I/II clinical trial conducted on seven patients with fracture nonunion. Herein, we present the results of a phase III clinical trial conducted to confirm the results of the previous phase studies using a larger cohort of patients. METHODS: CD34 + cells were mobilized via administration of granulocyte colony-stimulating factor, harvested using leukapheresis, and isolated using magnetic cell sorting. Autologous CD34 + cells were transplanted in 15 patients with tibia nonunion and 10 patients with femur nonunion, who were followed up for 52 weeks post transplantation. The main outcome was a reduction in time to heal the tibia in nonunion patients compared with that in historical control patients. We calculated the required number of patients as 15 based on the results of the phase I/II study. An independent data monitoring committee performed the radiographic assessments. Adverse events and medical device failures were recorded. RESULTS: All fractures healed during the study period. The time to radiological fracture healing was 2.8 times shorter in patients with CD34 + cell transplantation than in the historical control group (hazard ratio: 2.81 and 95% confidence interval 1.16-6.85); moreover, no safety concerns were observed. CONCLUSIONS: Our findings strongly suggest that autologous CD34 + cell transplantation is a novel treatment option for fracture nonunion. TRIAL REGISTRATION: UMIN-CTR, UMIN000022814. Registered on 22 June 2016.

Human Non-Hypertrophic Nonunion Tissue Contains Osteoblast Lineage Cells and E-BMP-2 Activates Osteogenic and Chondrogenic Differentiation.

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.

Utilization of Mechanical Stress to Treat Osteoporosis: The Effects of Electrical Stimulation, Radial Extracorporeal Shock Wave, and Ultrasound on Experimental Osteoporosis in Ovariectomized Rats.

Current treatment options for osteoporosis primarily involve pharmacotherapies, but they are often accompanied by undesirable side effects. Utilization of mechanical stress which can noninvasively induce bone formation has been suggested as an alternative to conventional treatments. Here, we examined the efficacy of mechanical stress induced by electrical stimulation, radial extracorporeal shock waves, and ultrasound for estrogen-deficient osteoporosis. Female Wistar rats were divided into following five groups: sham-operated group, untreated after ovariectomy, and treated with electrical stimulation, radial extracorporeal shock wave, or ultrasound starting at 8 weeks after ovariectomy for 4 weeks. Trabecular bone architecture of the femur was assessed by micro-CT and its biomechanical properties were obtained by mechanical testing. The femurs were further evaluated by histochemical, immunohistochemical, and real-time PCR analyses. Radial extracorporeal shock wave and ultrasound treatment improved trabecular bone microarchitecture and bone strength in osteoporotic rats, but not electrical stimulation. The shock wave decreased osteoclast activity and RANKL expression. The exposure of ultrasound increased osteoblast activity and ß-catenin-positive cells, and they decreased sclerostin-positive osteocytes. These findings suggest that mechanical stress induced by radial extracorporeal shock wave and ultrasound can improve estrogen-deficient bone loss and bone fragility through promoted bone formation or attenuated bone resorption.

Clinical experience of the use of reamer irrigator aspirator in Japanese patients: A report of the first 42 cases.

BACKGROUND: A reamer irrigator aspirator (RIA) can be used to harvest substantial amounts of autologous bone and debride the intramedullary canal. Clinical experience using reamer irrigator aspirators in Japan is very limited. The applicability of the reamer irrigator aspirator head with a minimum diameter of 12 mm for Japanese people is often questioned as the Japanese are smaller than Americans and Europeans. There are no reports of complications in Japanese patients. This study aimed to retrospectively review clinical cases and describe reamer irrigator aspirator use in Japanese patients. METHODS: All patients for whom a reamer irrigator aspirator was used during surgery at our hospital between January 2014 and September 2018 were included. The patients' clinical and radiographic data were retrospectively reviewed. RESULTS: Data of 40 patients (42 cases) were collected. The reamer irrigator aspirator was used for bone graft harvesting in 32 cases, intramedullary debridement and irrigation in 9 cases, and reaming for exchange nailing in 1 case. The diameter of the reamer irrigator aspirator reamer head was 12 mm in 22 cases (53.7%), 12.5 mm in 4 cases (9.8%), 13 mm in 9 cases (22.0%), 13.5 mm in 1 case (2.4%), 14.0 mm in 1 case (2.4%), 14.5 mm in 1 case (2.4%), and 15 mm in 4 cases (9.8%). Mean intraoperative bleeding volume was 1158.6 mL (range, 100-3800 mL). We experienced no difficulty inserting the reamer irrigator aspirator into the intramedullary canals and no cases of insertion-related intraoperative fracture. Five cortical perforations (11.9%) were observed on postoperative computed tomography scans, although no patient was symptomatic. One case (2.4%) of postoperative femur fracture occurred. CONCLUSIONS: Reamer irrigator aspirators can be used in Japanese patients. Smaller reamer head sizes were mainly used in our experience. We should manage complications as in previous reports from Western countries.

Migration Measurement of Pins in Postoperative Recovery of the Proximal Femur Fractures Based on 3D Point Cloud Matching.

Background and objectives: Internal fixation is one of the most effective methods for the treatment of proximal femur fractures. The migration of implants after the operation can seriously affect the reduction of treatment and even cause complications. Traditional diagnosis methods can not directly measure the extent of displacement. Methods: Based on the analysis of Hansson pins, this paper proposes a measurement method based on three-dimensional matching, which uses computerized tomography (CT) images of different periods of patients after the operation to analyze the implants' migration in three-dimensional space with the characteristics of fast speed and intuitive results. Results and conclusions: The measurement results show that the method proposed in this paper has more minor errors, more flexible coordinate system conversion, and more explicit displacement analysis than the traditional method of manually finding references in CT images and measuring displacement.

Percutaneous CO2 Treatment Accelerates Bone Generation During Distraction Osteogenesis in Rabbits.

BACKGROUND: 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 CO2 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 CO2 therapy on distraction osteogenesis has been reported. QUESTIONS/PURPOSES: We aimed to investigate the effect of transcutaneous CO2 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 CO2 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 CO2 on the operated leg using a CO2 absorption-enhancing hydrogel was performed five times per week in the CO2 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 CO2 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 CO2 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 CO2 group had a larger cartilage area compared with those in the control group (2.09 ± 1.34 mm [95% CI 1.26 to 2.92 mm] versus 5.10 ± 3.91 mm [95% CI 2.68 to 7.52 mm]; p = 0.011). More newly formed bone was observed in the CO2 group than the control group at Week 4 (68.31 ± 16.32 mm [95% CI 58.19 to 78.44 mm] versus 96.26 ± 19.37 mm [95% CI 84.25 to 108.26 mm]; 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 CO2 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 [95% CI 232.58 to 340.49] versus 410.24 ± 55.29 /mm [95% CI 361.78 to 458.71]; p < 0.001; surrounding tissue at Week 0 395.09 ± 68.16/mm [95% CI 335.34 to 454.83] versus 589.75 ± 174.42/mm [95% CI 436.86 to 742.64]; p = 0.003; at Week 2 271.22 ± 169.42 /mm [95% CI 122.71 to 419.73] versus 508.46 ± 49.06/mm [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 CO2 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 CO2 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 CO2 accelerated bone generation in a distraction osteogenesis model of rabbit tibias. As demonstrated in previous studies, CO2 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 CO2 may open new possibilities for shortening healing time in patients with distraction osteogenesis. However, a deeper insight into the mechanism of CO2 in the local tissue is required before it can be used in future clinical practice.

Transcutaneous carbon dioxide application inhibits muscle atrophy after fracture in rats.

BACKGROUND: Muscle atrophy causes difficulty in resuming daily activities after a fracture. Because transcutaneous carbon dioxide (CO2) application has previously upregulated oxygen pressure in the local tissue, thereby demonstrating its potential in preventing muscle atrophy, here we investigated effects of CO2 application on muscle atrophy after femoral shaft fracture. METHODS: Thirty fracture model rats were produced and randomly divided into a no treatment (control group) and treatment (CO2 group) groups. After treatment, the soleus muscle was dissected at post-fracture days 0, 14, and 21. Evaluations were performed by measuring muscle weight and performing histological examination and gene expression analysis. RESULTS: Muscle weight was significantly higher in the CO2 group than in the control group. Histological analysis revealed that the muscle fiber cross-sectional area was reduced in both groups. Nevertheless, the extent of atrophy was lesser in the CO2 group. Muscle fibers in the control group tended to change into fast muscle fibers. Vascular staining revealed that more capillary vessels surrounded the muscle fibers in the CO2 group than in the control group. Messenger RNA (mRNA) analysis revealed that the CO2 group had a significantly enhanced expression of genes that were related to muscle synthesis. CONCLUSION: Transcutaneous CO2 application may be a novel therapeutic strategy for preventing skeletal muscle atrophy after fracture.

Effects of the duration of transcutaneous CO2 application on the facilitatory effect in rat fracture repair.

BACKGROUND: Carbon dioxide therapy has been reported to be effective in treating certain cardiac diseases and skin problems. Although a previous study suggested that transcutaneous carbon dioxide application accelerated fracture repair in association with promotion of angiogenesis, blood flow, and endochondral ossification, the influence of the duration of carbon dioxide application on fracture repair is unknown. The aim of this study was to investigate the effect of the duration of transcutaneous carbon dioxide application on rat fracture repair. METHODS: A closed femoral shaft fracture was created in each rat. Animals were randomly divided into four groups: the control group; 1w-CO2 group, postoperative carbon dioxide treatment for 1 week; 2w-CO2 group, postoperative carbon dioxide treatment for 2 weeks; 3w-CO2 group, postoperative carbon dioxide treatment for 3 weeks. Transcutaneous carbon dioxide application was performed five times a week in the carbon dioxide groups. Sham treatment, where the carbon dioxide was replaced with air, was performed for the control group. Radiographic, histological, and biomechanical assessments were performed at 3 weeks after fracture. RESULTS: The fracture union rate was significantly higher in the 3w-CO2 group than in the control group (p < 0.05). Histological assessment revealed promotion of endochondral ossification in the 3w-CO2 group than in the control group. In the biomechanical assessment, all evaluation items related to bone strength were significantly higher in the 3w-CO2 group than in the control group (p < 0.05). CONCLUSIONS: The present study, conducted using an animal model, demonstrated that continuous carbon dioxide application throughout the process of fracture repair was effective in enhancing fracture healing.

Ras associated with diabetes may play a role in fracture nonunion development in rats.

BACKGROUND: Rad is the prototypic member of a subfamily of Ras-related small G-proteins and is highly expressed in the skeletal muscle of patients with type II diabetes. Our previous microarray analysis suggested that Rad may mediate fracture nonunion development. Thus, the present study used rat experimental models to investigate and compare the gene and protein expression patterns of both Rad and Rem1, another RGK subfamily member, in nonunions and standard healing fractures. METHODS: Standard healing fractures and nonunions (produced via periosteal cauterization at the fracture site) were created in the femurs of 3-month-old male Sprague-Dawley rats. At post-fracture days 7, 14, 21, and 28, the fracture callus and fibrous tissue from the standard healing fractures and nonunions, respectively, were harvested and screened (via real-time PCR) for Rad and Rem1 expression. The immunolocalization of both encoded proteins was analyzed at post-fracture days 14 and 21. At the same time points, hematoxylin and eosin staining was performed to identify the detailed tissue structures. RESULTS: Results of real-time PCR analysis showed that Rad expression increased significantly in the nonunions, compared to that in the standard healing fractures, at post-fracture days 14, 21, and 28. Conversely, immunohistochemical analysis revealed the immunolocalization of Rad to be similar to that of Rem1 in both fracture types at post-fracture days 14 and 21. CONCLUSIONS: Rad may mediate nonunion development, and thus, may be a promising therapeutic target to treat these injuries.

Topical cutaneous application of carbon dioxide via a hydrogel for improved fracture repair: results of phase I clinical safety trial.

BACKGROUND: Clinicians have very limited options to improve fracture repair. Therefore, it is critical to develop a new clinically available therapeutic option to assist fracture repair biologically. We previously reported that the topical cutaneous application of carbon dioxide (CO2) via a CO2 absorption-enhancing hydrogel accelerates fracture repair in rats by increasing blood flow and angiogenesis and promoting endochondral ossification. The aim of this study was to assess the safety and efficacy of CO2 therapy in patients with fractures. METHODS: Patients with fractures of the femur and tibia were prospectively enrolled into this study with ethical approval and informed consent. The CO2 absorption-enhancing hydrogel was applied to the fractured lower limbs of patients, and then 100% CO2 was administered daily into a sealed space for 20 min over 4 weeks postoperatively. Safety was assessed based on vital signs, blood parameters, adverse events, and arterial and expired gas analyses. As the efficacy outcome, blood flow at the level of the fracture site and at a site 5 cm from the fracture in the affected limb was measured using a laser Doppler blood flow meter. RESULTS: Nineteen patients were subjected to complete analysis. No adverse events were observed. Arterial and expired gas analyses revealed no adverse systemic effects including hypercapnia. The mean ratio of blood flow 20 min after CO2 therapy compared with the pre-treatment level increased by approximately 2-fold in a time-dependent manner. CONCLUSIONS: The findings of the present study revealed that CO2 therapy is safe to apply to human patients and that it can enhance blood flow in the fractured limbs. TRIAL REGISTRATION: This study has been registered in the UMIN Clinical Trials Registry (Registration number: UMIN000013641, Date of registration: July 1, 2014).

Topical cutaneous application of CO2 accelerates bone healing in a rat femoral defect model.

BACKGROUND: Bone defects may occur because of severe trauma, nonunion, infection, or tumor resection. However, treatments for bone defects are often difficult and have not been fully established yet. We previously designed an efficient system of topical cutaneous application of carbon dioxide (CO2) using a novel hydrogel, which facilitates CO2 absorption through the skin into the deep area within a limb. In this study, the effect of topical cutaneous application of CO2 on bone healing was investigated using a rat femoral defect model. METHODS: In this basic research study, an in vivo bone defect model, fixed with an external fixator, was created using a rat femur. The affected limb was shaved, and CO2 was applied for 20 min/day, 5 days/week. In the control animals, CO2 gas was replaced with air. Radiographic, histological, biomechanical, and genetic assessments were performed to evaluate bone healing. RESULTS: Radiographically, bone healing rate was significantly higher in the CO2 group than in the control group at 4 weeks (18.2% vs. 72.7%). The degree of bone healing scored using the histopathological Allen grading system was significantly higher in the CO2 group than in the control group at 2 weeks (1.389 ± 0.334 vs. 1.944 ± 0.375). The ultimate stress, extrinsic stiffness, and failure energy were significantly greater in the CO2 group than in the control group at 4 weeks (3.2 ± 0.8% vs. 38.1 ± 4.8%, 0.6 ± 0.3% vs. 41.5 ± 12.2%, 2.6 ± 0.8% vs. 24.7 ± 5.9%, respectively.). The volumetric bone mineral density of the callus in micro-computed tomography analysis was significantly higher in the CO2 group than in the control group at 4 weeks (180.9 ± 43.0 mg/cm3 vs. 247.9 ± 49.9 mg/cm3). Gene expression of vascular endothelial growth factor in the CO2 group was significantly greater than that in the control group at 3 weeks (0.617 ± 0.240 vs. 2.213 ± 0.387). CONCLUSIONS: Topical cutaneous application of CO2 accelerated bone healing in a rat femoral defect model. CO2 application can be a novel and useful therapy for accelerating bone healing in bone defects; further research on its efficacy in humans is warranted.

Escherichia coli-derived BMP-2-absorbed ß-TCP granules induce bone regeneration in rabbit critical-sized femoral segmental defects.

PURPOSE: This study investigated whether Escherichia coli-derived bone morphogenetic protein (BMP)-2 (E-BMP-2) adsorbed onto ß-tricalcium phosphate (ß-TCP) granules can induce bone regeneration in critical-size femoral segmental defects in rabbits. METHODS: Bone defects 20 mm in size and stabilized with an external fixator were created in the femur of New Zealand white rabbits, which were divided into BMP-2 and control groups. E-BMP-2-loaded ß-TCP granules were implanted into defects of the BMP-2 group, whereas defects in the controls were implanted with ß-TCP granules alone. At 12 and 24 weeks after surgery, radiographs were obtained of the femurs and histological and biomechanical assessments of the defect area were performed. Bone regeneration was quantified using micro-computed tomography at 24 weeks. RESULTS: Radiographic and histologic analyses revealed bone regeneration in the BMP-2 group but not the control group; no fracturing of newly formed bone occurred when the external fixator was removed at 12 weeks. At 24 weeks, tissue mineral density, the ratio of bone volume to total volume, and volumetric bone mineral density of the callus were higher in the BMP-2 group than in control animals. In the former, ultimate stress, extrinsic stiffness, and failure energy measurements for the femurs were higher at 24 weeks than at 12 weeks. CONCLUSION: E-BMP-2-loaded ß-TCP granules can effectively promote bone regeneration in long bone defects.

Profiling microRNA expression during fracture healing.

BACKGROUND: The discovery of microRNA (miRNA) has revealed a novel type of regulatory control for gene expression. Increasing evidence suggests that miRNA regulates chondrocyte, osteoblast, and osteoclast differentiation and function, indicating miRNA as key regulators of bone formation, resorption, remodeling, and repair. We hypothesized that the functions of certain miRNAs and changes to their expression pattern may play crucial roles during the process of fracture healing. METHODS: Standard healing fractures and unhealing fractures produced by periosteal cauterization at the fracture site were created in femurs of seventy rats, with half assigned to the standard healing fracture group and half assigned to the nonunion group. At post-fracture days 3, 7, 10, 14, 21, and 28, total RNA including miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was performed with miRNA samples from each group on post-fracture day 14. For further analysis, we selected highly up-regulated five miRNAs in the standard healing fracture group from the microarray data. Real-time PCR was performed with miRNA samples at each time point above mentioned to compare the expression levels of the selected miRNAs between standard healing fractures and unhealing fractures and investigate their time-course changes. RESULTS: Microarray and real-time polymerase chain reaction (PCR) analyses on day 14 revealed that five miRNAs, miR-140-3p, miR-140-5p, miR-181a-5p, miR-181d-5p, and miR-451a, were significantly highly expressed in standard healing fractures compared with unhealing fractures. Real-time PCR analysis further revealed that in standard healing fractures, the expression of all five of these miRNAs peaked on day 14 and declined thereafter. CONCLUSION: Our results suggest that the five miRNAs identified using microarray and real-time PCR analyses may play important roles during fracture healing. These findings provide valuable information to further understand the molecular mechanism of fracture healing and may lead to the development of miRNA-based tissue engineering strategies to promote fracture healing.

Clinical and radiological results of locking plate fixation for periprosthetic femoral fractures around hip arthroplasties: a retrospective multi-center study.

BACKGROUND: Osteosynthesis of periprosthetic femoral fractures around hip arthroplasties is challenging, and locking plate fixation has been found to be a reasonable treatment. However, there is a paucity of evidence of the extent to which patients recover their activities of daily living (ADL). The purpose of this study was to study the clinical results, particularly recovery of ADL, radiological results, and potential complications arising from the use of locking plate fixation for periprosthetic femoral fractures around hip arthroplasties. METHODS: We conducted a retrospective, multi-center study. Patients with periprosthetic femoral fractures around hip arthroplasties who were treated by osteosynthesis with use of locking plates and who underwent follow-up for at least 6 months postoperatively were enrolled in the study. For each patient, recovery of ADL, in terms of social and ambulatory recovery and Parker mobility score, were compared before fracture and at last follow-up. Postoperative complications were investigated. Bony union, loss of reduction, and malunion were assessed radiologically. RESULTS: Thirty-two patients were enrolled in this study. Mean follow-up was 25.1 months. For 84.4, 68.8, and 53.1% of patients, respectively, social status, ambulation, and Parker mobility score at last follow-up were determined to be equal to that before the fracture. Bony union was observed for 30 patients within the follow-up period. Four patients had not achieved bony union 6 months postoperatively. There was no loss of reduction, malunion, or implant breakage, and no infection. For one patient each, partial pullout of the locking screws and a supracondylar fracture at the plate end were observed, and additional surgery was required. CONCLUSIONS: Our results reveal that locking plate fixation provided sufficient stability for satisfactory recovery of ADL for most elderly patients with periprosthetic femoral fractures around hip arthroplasties.

Effects of high antibiotic concentrations applied to continuous local antibiotic perfusion on human bone tissue-derived cells.

Aims: Continuous local antibiotic perfusion (CLAP) has recently attracted attention as a new drug delivery system for orthopaedic infections. CLAP is a direct continuous infusion of high-concentration gentamicin (1,200 µg/ml) into the bone marrow. As it is a new system, its influence on the bone marrow is unknown. This study aimed to examine the effects of high-concentration antibiotics on human bone tissue-derived cells. Methods: Cells were isolated from the bone tissue grafts collected from six patients using the Reamer-Irrigator-Aspirator system, and exposed to different gentamicin concentrations. Live cells rate, apoptosis rate, alkaline phosphatase (ALP) activity, expression of osteoblast-related genes, mineralization potential, and restoration of cell viability and ALP activity were examined by in vitro studies. Results: The live cells rate (the ratio of total number of cells in the well plate to the absorbance-measured number of live cells) was significantly decreased at ≥ 500 µg/ml of gentamicin on day 14; apoptosis rate was significantly increased at ≥ 750 µg/ml, and ALP activity was significantly decreased at ≥ 750 µg/ml. Real-time reverse transcription-polymerase chain reaction results showed no significant decrease in the ALP and activating transcription factor 4 transcript levels at ≥ 1,000 µg/ml on day 7. Mineralization potential was significantly decreased at all concentrations. Restoration of cell viability was significantly decreased at 750 and 1,000 µg/ml on day 21 and at 500 µg/ml on day 28, and ALP activity was significantly decreased at 500 µg/ml on day 28. Conclusion: Our findings suggest that the exposure concentration and duration of antibiotic administration during CLAP could affect cell functions. However, further in vivo studies are needed to determine the optimal dose in a clinical setting.

Comparative Study of Different Entry Spots on Postoperative Gluteus Medius Muscle Cross-Sectional Area in Patients With Intertrochanteric Fractures Nailing.

Introduction In a preliminary study of cephalo-medullary (CM) nailing in patients with femoral intertrochanteric fractures, the authors of this study found a 25% to 30% decrease in muscle strength, especially abduction force, during the postoperative follow-up period. This decline was partially attributed to the entry point for the nail insertion causing damage to the gluteus medius tendon at the junction of the greater trochanter after reaming. Therefore, we assumed that changing the position of nail insertion to a "bald spot (BS)" could mitigate postoperative functional impairment. Automated computed tomography (CT) imaging of skeletal muscle cross-sectional area (CSA) and adipose tissue ratio (ATR) can show pathological changes on the operated side compared with the non-operated side. In this study, the authors quantified the difference in postoperative CSA and ATR of the gluteus medius muscle after bald spot nailing versus nail insertion through the conventional tip of the greater trochanter. It was hypothesized that bald spot nailing could avoid significant injury to the gluteus medius muscle. Materials and methods Patients with femoral intertrochanteric fractures were grouped according to the site of cephalo-medullary nailing: greater trochanteric tip (TIP) in 27 patients (8 men and 19 women, mean age 84.9±5.1 years) and BS in 16 patients (3 men and 13 women, mean age 86.9±6.2 years). The CSA and ATR of the gluteus medius muscles were assessed in three slices (A, B, and C from proximal to distal). Each slice was manually traced and automatically calculated based on its contour. Adipose tissue (-100 to -50 in Hounsfield units) in the designated area was distinguished by a bimodal image histogram resulting from the distribution of CT numbers of adipose tissue and muscle. The body mass index (BMI) was used to correct the CSA in each patient. Results In the TIP group, the mean CSA values (mm2) from the non-operated/operated sides were as follows: slice A, 2180.2 ± 616.5/1976.3 ± 421.2; slice B, 2112.3 ± 535.7/1857.7 ± 386.7; and slice C: 1671.8 ± 460.0/1404.1 ± 404.3 (p<0.01 in slices A, B, and C). In the BS group, slice A was 2044.1 ± 473.0/2016.9 ± 388.4; slice B was 2073.2 ± 540.7/1848.3 ± 411.1; and slice C was 1659.1 ± 477.2/1468.5 ± 341.7 (p=0.34 in slice A, and p<0.05 in slices B and C, respectively). The mean CSA values (mm2) of the non-operated minus operated side between the TIP/BS groups were as follows: slice A, 241.3 ± 424.3/-11.8 ± 285.6; slice B, 290.3 ± 313.0/211.8 ± 333.2; and slice C, 276.4 ± 270.4/162.8 ± 319.3 (p < 0.05 in slice A, 0.45, 0.24 in slices B, C, respectively). The mean adjusted CSA per BMI values (mm2) of the non-operated minus the operated side between the TIP/BS groups were slice A, 10.6 ± 19.7/-0.4 ± 14.8; slice B, 13.3 ± 15.0/10.1 ± 16.3; and slice C, 13.1 ± 13.4/ 8.7 ± 15.3 (p < 0.05 in slice A and 0.54 and 0.36 in slices B and C, respectively). Conclusion Nail insertion at the bald spot resulted in a significantly smaller decrease in the CSA of the gluteus medius muscle compared with the conventional tip entry. In addition, an examination of BMI-adjusted CSA showed that CSA was maintained in some image slices. These results suggest that nailing from the BS of the greater trochanter can reduce damage to the gluteus medius muscle and highlight the importance of imaging beyond the usual assessment of skeletal changes.

Posterior Protrusion Measures (PPM) for Three-Dimensional (3D) CT Classification of Pertrochanteric Fractures.

Introduction We introduced a novel numerical index known as posterior protrusion measures (PPM), derived from lateral plain radiograph images, which effectively serves to distinguish stable from unstable pertrochanteric fractures. The present study aims to scrutinize PPM values among two classified fracture patterns, stable and unstable, within the three-dimensional (3D) CT classification system, establishing a numeric threshold for PPM to differentiate between these groups; explore the potential relationship between the PPM index and unclassified categories; investigate how groups divided by the PPM threshold value can predict fracture stability based on 3D CT. Materials and methods In this study, three observers were tasked with measuring PPM on a single occasion. The chi-square test assessed the association between each demographic parameter on a categorical scale and stable/unstable groups. Continuous variables were also subject to examination. Receiver operating characteristic (ROC) analysis was employed to determine optimal cut-off points of PPM for predicting the presence of stable versus unstable groups. Additionally, the chi-square test examined the linear relation between separated groups based on the defined threshold PPM value and the stable/unstable groups. Results A total of 106 pertrochanteric fractures were identified using CT scan images and plain radiographs in the 3D CT classification system, revealing the stable group of 35 patients and the unstable group of 71 patients. The PPM values for stable/unstable fractures were, on average (± standard deviation), 0.34±0.25/0.50±0.29 for observer 1, 0.31±0.23/0.57±0.31 for observer 2, and 0.41±0.29/0.57±0.26 for observer 3, respectively (p<0.01). We established 0.3 as the cut-off value for PPM. The average PPM value among three observers represented each patient to assess fracture stability. The group with PPM <0.3 included 27 patients (16 stable and 11 unstable), and the group with PPM ≥0.3 group comprised 79 patients (19 stable and 60 unstable; p<0.005). Conclusion The present study revealed a significant difference in PPM values among stable and unstable 3D CT classification groups. Additionally, a threshold PPM value of 0.3 suggests a pivotal point for differentiating fracture stability. This innovative methodology makes a substantial contribution to clinical endeavors, potentially circumventing the necessity for 3D CT scanning.

A Demographic Survey of Pertrochanteric Fractures Based on the Revised Arbeitsgemeinschaft für Osteosynthesefragen/Orthopedic Trauma Association (AO/OTA) Classification Using 3D CT Scan Images.

Introduction A demographic survey of femoral pertrochanteric fractures provides several important information for the healthcare system of a country since this fracture is commonly seen in the elderly and has a poor postoperative functional prognosis that is a burden on society. The importance of accurately classifying pertrochanteric fractures as stable or unstable cannot be understated. However, the use of plain radiograph images alone is known to underestimate fracture severity with low inter- or intra-observer agreement. Computed tomography (CT) images offer information for a more accurate classification of pertrochanteric fractures. With this three-dimensional (3D) CT-based study using the revised Arbeitsgemeinschaft für Osteosynthesefragen/Orthopedic Trauma Association (AO/OTA) classification, the purpose of this study is to elucidate the epidemiological demography of patients with pertrochanteric fractures. Material and methods We retrospectively collected 808 patients from five hospitals, classified into two groups: stable (A1) or unstable (A2). Age, gender, fracture laterality, and surgery timing were identified as epidemiological variables. Patients with both preoperative plain radiographs and 3D CT scans were included in the study. The exclusion criteria were AO/OTA A3 type fractures, pathological fractures, previous ipsilateral surgery, 60 years old or younger, and conservatively treated patients. The primary outcome involved detailing the total number of fractures based on classification (A1 or A2) and variables. The secondary outcome involved a comparison between the A1 and A2 groups. Results The mean age of patients at the time of surgery was 85 years (range: 61-103 years). There were 637 female and 171 male patients. There were 463 left-sided fractures and 345 right-sided fractures. Of the 808 patients, 371 (45.9%) were classified to have A1 fractures, and 437 (54.1%) had A2 fractures. The age at surgery, gender, fracture laterality, and surgery timing between the A1 and A2 groups were compared. The mean and standard deviation of the age at surgery for patients in the A1 and A2 groups were 84.9±7.7 and 86.9±6.8, respectively. The number of patients for each age distribution of 61-69, 70-74, 75-79, 80-84, 85-89, 90-94, and 95 or older for the A1 and A2 groups was 18 and 7, 18 and 12, 43 and 44, 76 and 82, 107 and 132, 79 and 110, and 30 and 50, respectively, showing that the difference in categorial distribution was statistically significant (p=0.002). Overall, 278 females and 93 males were classified to have A1 fractures compared with 359 females and 78 males with A2 fractures (p=0.01). There were 166 right-sided and 205 left-sided stable A1 fractures and 179 right-sided and 258 left-sided A2 fractures (not significant (NS)). Among the total number of A1 and A2 surgeries by month, the most were in December with 77 surgeries (37 and 40, respectively), and the least was in June with 37 (18 and 19, respectively). The seasonal classification for A1 and A2 surgeries is as follows: spring with 172 (74 and 98, respectively), summer with 150 (70 and 80, respectively), autumn with 193 (90 and 103, respectively), and winter with 208 (97 and 111, respectively) (NS). Conclusion In this demographic study of 808 patients with pertrochanteric fractures classified by 3D CT images, 371 had A1 fractures and 437 had A2 fractures. A2 fractures were significantly more in females with an age peak of 85-89 years.