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.

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.

Multicenter study on atypical femoral fractures in patients with bone metastases taking bone- modifying agents.

Bone-modifying agents (BMAs), with bone-resorptive inhibitory effects, such as zoledronic acid and denosumab, are widely used at higher doses for bone-related events caused by bone metastasis of malignant tumors. These drugs have been suggested to be associated with atypical femoral fractures (AFFs), and the relationship between BMAs and AFFs has attracted attention. To investigate the clinical features including bone union time of AFFs in patients administered BMA for bone metastasis, we conducted a retrospective multicenter study. Thirty AFFs from 19 patients were enrolled in this study. Thirteen patients had bilateral AFFs, and nineteen AFFs had prodromal symptoms. Eighteen AFFs underwent surgery after complete fracture, three failed to achieve bone union and required nonunion surgery, and 11 AFFs that achieved bone union had an average period until bone union of 16.2 months, which was much longer than that previously reported for ordinary AFFs. Seven patients discontinued the BMAs, but not due to AFFs. Stopping BMAs in patients with bone metastasis would make it difficult to secure their performance of activities of daily living, and AFF with BMA administration might require a longer time for union. Therefore, it would be important to prevent incomplete AFF from becoming complete AFF via prophylactic internal fixation.

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.

Nonunion of Hoffa Fracture Successfully Treated by Total Knee Arthroplasty: A Case Report.

A Hoffa fracture is a rare type of femoral fracture that is difficult to treat. Nonoperative treatments usually result in failure; hence, in most cases, surgical treatments are essential. Nonunion following Hoffa fracture appears to be relatively uncommon, and there are limited reports in the literature about this type of nonunion. These reports suggest that open reduction and rigid internal fixation is the standard treatment for this type of nonunion. This study reports the case of a 61-year-old male patient who suffered from left lateral Hoffa fracture after falling from a truck bed. At the former hospital, open reduction and internal fixation were performed with plates and screws at 8 days post-injury. Postoperatively, displacement of the lateral proximal fragment was observed, and the patient reported left knee pain. Therefore, a revision open reduction and internal fixation was performed 4 months post-surgery. However, 6 months after the revision surgery, the patient reported instability and pain in the left knee, and subsequent radiography revealed nonunion of the fracture in the lateral condyle. The patient was referred to our hospital for further treatment. Treatment by re-revision open reduction and internal fixation was deemed challenging, and so rotating hinge knee (RHK) arthroplasty was performed as a salvage treatment. At 3 years post-surgery, no significant problems were observed, and the patient could walk without any assistance. The range of motion of the left knee was 0 to 100° without extension lag, and there was no lateral instability. Standard treatment for Hoffa fracture nonunion is commonly anatomical reduction and rigid internal fixation. However, total knee arthroplasty may be a better option for the treatment of Hoffa fracture nonunion in older patients.

Effect of low-intensity pulsed ultrasound on osteogenic differentiation of human induced membrane-derived cells in Masquelet technique.

INTRODUCTION: The Masquelet technique is a relatively new method for large bone defect treatment. In this technique, grafted bone tissue is used, and after the cement is removed, the induced membrane (IM; that form around the cement spacers placed in the bone defect region) is thought to play an important role in promoting bone formation. On the other hand, low-intensity pulsed ultrasound (LIPUS) is known to promote fracture healing and angiogenesis through mechanical stimulation. This study aimed to investigate the in vitro effects of LIPUS on the osteogenic differentiation of human induced membrane-derived cells (IMCs). METHODS: Seven patients who had been treated using the Masquelet technique were enrolled. The IM was harvested during the second stage of the technique. IMCs were isolated, cultured in growth medium, and then divided into two groups: (1) control group, IMCs cultured in osteogenic medium without LIPUS, and (2) LIPUS group, IMCs cultured in osteogenic medium with LIPUS treatment. Adherent cells from the IM samples were harvested after the first passage and evaluated for cell surface protein expression using immunostaining. A cell proliferation assay was used to count the number of IMCs using a hemocytometer. Osteogenic differentiation capability was assessed using an alkaline phosphatase (ALP) activity assay, Alizarin Red S staining, and real-time reverse transcription-polymerase chain reaction. RESULTS: Cell surface antigen profiling revealed that the IMCs contained cells positive for the mesenchymal stem cell-related markers CD73, CD90, and CD105. No significant difference in cell numbers was found between the control and LIPUS groups. The ALP activity of IMCs in the LIPUS group was significantly higher than that in the control group on days 7 and 14. Alizarin red S staining intensity was significantly higher in the LIPUS group than in the control group on day 21. Runx2 and VEGF expression was significantly upregulated on days 7 and 14, respectively, compared with levels in the control group. CONCLUSION: We demonstrated the significant effect of LIPUS on the osteogenic differentiation of human IMCs. This study indicates that LIPUS can be used as an additional tool for the enhancement of the healing process of the Masquelet technique.

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.

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.

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.

Influence of Metal Implants on Quantitative Evaluation of Bone Single-Photon Emission Computed Tomography/Computed Tomography.

When visualizing biological activity at nonunion sites by the radioisotopes, gamma rays are more attenuated if metal implants are placed in the bone. However, the effects of various implant types and their placement on gamma ray attenuation in quantitative evaluation remain unknown. To elucidate these effects, we created a phantom that simulated the nonunion of the femur in this study. The count of gamma rays was measured by single-photon emission computed tomography/computed tomography (SPECT/CT) while considering CT-based attenuation correction (CTAC), metal implant placement, type (intramedullary nail or plate), and position. The count differed significantly with and without CTAC and with and without implants (both types) under CTAC. Significantly different counts were observed between the intramedullary nail and plate placed contralaterally to the lesion (i.e., non-lesion side). No significant difference was observed between the intramedullary nail and plate on the lesion side or between plates on the non-lesion and lesion sides. The measured standardized uptake value (SUV) was closer to the true SUV with CTAC than without. Moreover, the count was higher with implants than without. However, even with implants, it was lower than the actual count, indicating the absence of overcorrection. Implant type and position do not seem to influence the count.

Continuous Local Antibiotic Perfusion (CLAP) for Post-operative Infection of Open Tibial Fractures: A Case Report.

Introduction: We encountered a case of post-operative infection of an open tibial fracture that was controlled by continuous local antibiotic perfusion (CLAP), a novel method of fracture-related infection (FRI) that we have developed. CLAP is a procedure in which a bone marrow needle and a double lumen tube are placed in the infected area, and an appropriate concentration of antimicrobial agent is continuously administered and perfused. Case Report: The patient was a 78-year-old woman. She was hit by a motor vehicle and fell to the farmyard floor. She suffered multiple traumas, including a lower leg open fracture, multiple rib fractures, clavicle fracture, pelvic fracture, mandibular fracture, and liver injury. Her tibial fracture was a Gustilo-Anderson type IIIA open fracture. After debridement and external fixation of the tibial open fracture on the same day, open reduction and internal fixation with an intramedullary nail was performed 3 days after the injury. Twelve days after the injury, local heat and redness were observed at the nail insertion wound and the posteromedial calf, and a purulent clot was discharged from the open wound. We performed curettage of the lesion and retained the implant. CLAP was then constructed to perfuse local antibiotics along the nail and large hematoma area. Locally, the inflammation improved and the inflammatory response became negative 3 weeks after the initiation of CLAP. Six months after surgery, bony union was achieved. At present, 3.5 years after the internal operation, there is no sign of infection, and the patient has returned to her pre-injury life with no abnormalities in motor function. Conclusion: CLAP may be a novel treatment method that can be expected to achieve bone healing while preserving the implant in FRI cases after open tibial fracture.

Continuous local antibiotic perfusion: A treatment strategy that allows implant retention in fracture-related infections.

PURPOSE: 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.

A Case of Nonunion with Ballooning Deformity after Periprosthetic Humeral Fracture.

Introduction: We report a very rare case of nonunion with ballooning deformity of the humeral bone after a periprosthetic humeral fracture nonunion. Case Report: A 79-year-old woman underwent hemiarthroplasty 19 years ago for her proximal humerus fracture. She injured her right humeral diaphysis (stem distal end fracture) 6 years ago. She underwent revision hemiarthroplasty with long stem, but bony union was not obtained, and her right upper limb function was subsequently abolished due to extreme instability and pain in her right upper arm. She was then referred to our hospital for further treatment. X-ray showed nonunion in the humeral diaphysis and a severe ballooning deformity in the distal humeral bone fragment. Due to the advanced age and low activity of the patient, we chose total humerus replacement surgery instead of osteosynthesis. After the surgery, her upper arm pain and instability immediately improved. Three years after the last surgery, there have been no implant failures, and the upper arm is stable and painless. Conclusion: Although there are some reports of total humerus replacement as surgical treatment for humeral tumor and severe periprosthetic fracture, we found no reports of such ballooning deformity around the nonunion site. Total humerus replacement can be one of the treatment options in cases where nonunion surgery is extremely difficult, depending on the patient's age and activities of daily living.

Combined treatment using cross-leg free flap and the Masquelet technique: a report of two cases.

We introduce a treatment that combines the cross-leg free flap with the Masquelet technique and describe two cases using this method for bone and soft tissue reconstruction. Both patients were successfully treated and ambulatory. This novel method can be safely performed using the delay technique, indocyanine-green angiography and near-infrared spectroscopy.

Safety of 222 nm UVC Irradiation to the Surgical Site in a Rabbit Model.

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.

A Piezo1/KLF15/IL-6 axis mediates immobilization-induced muscle atrophy.

Although immobility is a common cause of muscle atrophy, the mechanism underlying this causality is unclear. We here show that Krüppel-like factor 15 (KLF15) and IL-6 are upregulated in skeletal muscle of limb-immobilized mice and that mice with KLF15 deficiency in skeletal muscle or with systemic IL-6 deficiency are protected from immobility-induced muscle atrophy. A newly developed Ca2+ bioimaging revealed that the cytosolic Ca2+ concentration ([Ca2+]i) of skeletal muscle is reduced to below the basal level by immobilization, which is associated with the downregulation of Piezo1. Acute disruption of Piezo1 in skeletal muscle induced Klf15 and Il6 expression as well as muscle atrophy, which was prevented by antibodies against IL-6. A role for the Piezo1/KLF15/IL-6 axis in immobility-induced muscle atrophy was validated in human samples. Our results thus uncover a paradigm for Ca2+ signaling in that a decrease in [Ca2+]i from the basal level triggers a defined biological event.

Immunohistochemical analysis revealed the expression of bone morphogenetic proteins-4, 6, 7, and 9 in human induced membrane samples treated with the Masquelet technique.

BACKGROUND: 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 cm2 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.

Endochondral Bone Tissue Engineering Using Human Induced Pluripotent Stem Cells.

The use of induced pluripotent stem cells (iPSCs) shows potential in bone regenerative strategies. In this study, we investigated whether implantation of chondrogenically differentiated iPSC-derived mesenchymal stem cells (iMSCs) can lead to successful bone regeneration in nude mice with bone defects. Two human iPSC clones (201B7 and 454E2) were used. After generating iMSCs, chondrogenic differentiation was achieved by three-dimensional pellet culture. Thereafter, a 2-mm defect was created in the radius of nude mice, and chondrogenically differentiated iMSC pellets were transplanted in the defect. Microcomputed tomography imaging was performed 8 weeks posttransplantation to assess bone regeneration. All (100%) radii in the 201B7 cell-derived pellet transplantation group and 7 of 10 (70%) radii in the 454E2 cell-derived pellet transplantation group showed bone union. In contrast, 2 of 11 radii (18%) in the control group showed bone union. Thus, the experimental groups showed significantly higher bone union rates than the control group (p < 0.05). Histological analysis 2 weeks postimplantation in the experimental groups revealed hypertrophic chondrocytes within grafted iMSC pellets and the formation of woven bone around them. This hypertrophic chondrocyte transitioning to newly formed bone suggests that the cartilaginous template can trigger endochondral bone ossification (ECO). Four weeks postimplantation, the cartilage template was reduced in size; newly formed woven bone was predominant in the defect site. New vessels were surrounded by a matrix of woven bone, and hypertrophic chondrocytes transitioning to newly formed bone indicated the progression of ECO. Eight weeks postimplantation, the pellets were completely resorbed and replaced by bone; complete bone union was observed. Dense mature bone developed with evidence of lamellar-like bone formation. Collectively, our results suggest that using iMSC-based cartilage grafts recapitulating the morphogenetic process of ECO in the context of embryonic skeletogenesis is a promising strategy for repairing large bone defects. Impact statement We investigated whether implantation of chondrogenically differentiated iPSC-derived mesenchymal stem cells (iMSCs) could lead to the successful regeneration of bone defects in vivo. We implanted two different clones of human induced pluripotent stem cells into a radial bone defect model. Eleven of 11 (100%) and 7 of 10 (70%) radii in the 201B7 and 454E2 cell-derived pellet transplantation groups, respectively, showed bone union, which were significantly higher than those in the control group [only 2 of 11 radii (18%)]. Overall, our results support the use of iMSC-based cartilage grafts recapitulating the morphogenetic process of endochondral bone ossification for repairing large bone defects.

Low-Intensity Pulsed Ultrasound Promotes Osteogenic Differentiation of Reamer-Irrigator-Aspirator Graft-Derived Cells in Vitro.

Recently, reamer-irrigator-aspirator (RIA) systems have been increasingly used to harvest autologous bone grafts. RIA graft materials contain bone marrow, which provides a viable source to derive large numbers of mesenchymal stem cells. Low-intensity pulsed ultrasound (LIPUS) significantly accelerates the differentiation of stem cells derived from bone marrow. This in vitro study investigated the effect of LIPUS on the osteogenic activity and differentiation of RIA graft-derived cells. A small amount of RIA graft was obtained from seven patients. After the cells derived from RIA grafts were cultured, they were divided into two groups: the LIPUS and control groups. LIPUS was applied once daily for 20 min (1.5 MHz, pulse duration: 200 µs, pulse repetition rate: 1 kHz, spatial average-temporal average intensity: 30 mW/cm2). Alkaline phosphatase activity (113.4% and 130.1% on days 7 and 14), expression of osteoblast-related genes (ALP, Runx2) and mineralization (135.2% on day 21) of the RIA graft-derived cells were significantly higher in the LIPUS group than in the control group. However, LIPUS did not affect the cell proliferation of RIA graft-derived cells. This study indicates that LIPUS may enhance the healing of non-union and critical bone defects treated by autologous bone grafting using the RIA system.