Nupr1 deficiency downregulates HtrA1, enhances SMAD1 signaling, and suppresses age-related bone loss in male mice.

Nuclear protein 1 (NUPR1) is a stress-induced protein activated by various stresses, such as inflammation and oxidative stress. We previously reported that Nupr1 deficiency increased bone volume by enhancing bone formation in 11-week-old mice. Analysis of differentially expressed genes between wild-type (WT) and Nupr1-knockout (Nupr1-KO) osteocytes revealed that high temperature requirement A 1 (HTRA1), a serine protease implicated in osteogenesis and transforming growth factor-ß signaling was markedly downregulated in Nupr1-KO osteocytes. Nupr1 deficiency also markedly reduced HtrA1 expression, but enhanced SMAD1 signaling in in vitro-cultured primary osteoblasts. In contrast, Nupr1 overexpression enhanced HtrA1 expression in osteoblasts, suggesting that Nupr1 regulates HtrA1 expression, thereby suppressing osteoblastogenesis. Since HtrA1 is also involved in cellular senescence and age-related diseases, we analyzed aging-related bone loss in Nupr1-KO mice. Significant spine trabecular bone loss was noted in WT male and female mice during 6-19 months of age, whereas aging-related trabecular bone loss was attenuated, especially in Nupr1-KO male mice. Moreover, cellular senescence-related markers were upregulated in the osteocytes of 6-19-month-old WT male mice but markedly downregulated in the osteocytes of 19-month-old Nupr1-KO male mice. Oxidative stress-induced cellular senescence stimulated Nupr1 and HtrA1 expression in in vitro-cultured primary osteoblasts, and Nupr1 overexpression enhanced p16ink4a expression in osteoblasts. Finally, NUPR1 expression in osteocytes isolated from the bones of patients with osteoarthritis was correlated with age. Collectively, these results indicate that Nupr1 regulates HtrA1-mediated osteoblast differentiation and senescence. Our findings unveil a novel Nupr1/HtrA1 axis, which may play pivotal roles in bone formation and age-related bone loss.

A novel role of helix-loop-helix transcriptional factor Bhlhe40 in osteoclast activation.

The basic helix-loop-helix transcriptional factor, Bhlhe40 has been shown as a crucial regulator of immune response, tumorigenesis, and circadian rhythms. We identified Bhlhe40 as a possible regulator of osteoclast differentiation and function by shRNA library screening and found that Bhlhe40 was required for osteoclast activation. Bhlhe40 expression was induced in bone marrow macrophages (BMMs) by RANKL, whereas the expression of its homolog Bhlhe41 was decreased in osteoclastogenesis. µCT analysis of tibias revealed that Bhlhe40 knockout (KO) mice exhibited increased bone volume phenotype. Bone morphometric analysis showed that osteoclast number and bone resorption were decreased in Bhlhe40 KO mice, whereas significant differences in the osteoblast parameters were not seen between wild-type (WT) and Bhlhe40 KO mice. In vitro culture of BMMs showed that Bhlhe40 deficiency did not cause difference in osteoclast formation. In contrast, bone resorption activity of Bhlhe40 KO osteoclasts was markedly reduced in comparison with that of WT osteoclasts. Analysis of potential target genes of Bhlhe40 using data-mining platform ChIP-Atlas (http://chip-atlas.org) revealed that predicted target genes of Bhlhe40 were related to proton transport and intracellular vesicle acidification. We then analyzed the expression of proton pump, the vacuolar (V)-ATPases which are responsible for bone resorption. The expression of V-ATPases V1c1 and V0a3 was suppressed in Bhlhe40 KO osteoclasts. In addition, Lysosensor yellow/blue DND 160 staining demonstrated that vesicular acidification was attenuated in vesicles of Bhlhe40 KO osteoclasts. Furthermore, analysis with pH-sensitive fluorescent probe showed that proton secretion was markedly suppressed in Bhlhe40 KO osteoclasts compared to that in WT osteoclasts. Our findings suggest that Bhlhe40 plays a novel important role in the regulation of acid production in osteoclastic bone resorption.

Leukemia/lymphoma-related factor (LRF) or osteoclast zinc finger protein (OCZF) overexpression promotes osteoclast survival by increasing Bcl-xl mRNA: A novel regulatory mechanism mediated by the RNA binding protein SAM68.

RANKL induces NFATc1, a key transcriptional factor to induce osteoclast-specific genes such as cathepsin K, whereas transcriptional control of osteoclast survival is not fully understood. Leukemia/lymphoma-related factor (LRF) in mouse and osteoclast zinc finger protein (OCZF) in rat are zinc finger and BTB domain-containing protein (zBTB) family of transcriptional regulators, and are critical regulators of hematopoiesis. We have previously shown that differentiation and survival were enhanced in osteoclasts from OCZF-Transgenic (Tg) mice. In the present study, we show a possible mechanism of osteoclast survival regulated by LRF/OCZF and the role of OCZF overexpression in pathological bone loss. In the in vitro cultures, LRF was highly colocalized with NFATc1 in cells of early stage in osteoclastogenesis, but only LRF expression persisted after differentiation into mature osteoclasts. LRF expression was further enhanced in resorbing osteoclasts formed on dentin slices. Osteoclast survival inhibitor such as alendronate, a bisphosphonate reduced LRF expression. Micro CT evaluation revealed that femurs of OCZF-Tg mice showed significantly lower bone volume compared to that of WT mice. Furthermore, OCZF overexpression markedly promoted bone loss in ovariectomy-induced osteolytic mouse model. The expression of anti-apoptotic Bcl-xl mRNA, which is formed by alternative splicing, was enhanced in the cultures in which osteoclasts are formed from OCZF-Tg mice. In contrast, the expression of pro-apoptotic Bcl-xs mRNA was lost in the culture derived from OCZF-Tg mice. We found that the expression levels of RNA binding splicing regulator, Src substrate associated in mitosis of 68 kDa (Sam68) protein were markedly decreased in OCZF-Tg mice-derived osteoclasts. In addition, shRNA-mediated knockdown of Sam68 expression increased the expression of Bcl-xl mRNA, suggesting that SAM68 regulates the expression of Bcl-xl. These results indicate that OCZF overexpression reduces protein levels of Sam68, thereby promotes osteoclast survival, and suggest that LRF/OCZF is a promising target for regulating pathological bone loss.

PMEPA1 and NEDD4 control the proton production of osteoclasts by regulating vesicular trafficking.

Osteoclast bone resorption activity is critically regulated to maintain bone homeostasis. Osteoclasts resorb bone by producing protons and acid hydrolase via lysosomal secretion, however, a detailed mechanism remains elusive. PMEPA1 is a vesicular membrane protein, which binds to the NEDD4 family member of ubiquitin ligases. We have previously reported that Pmepa1 is highly expressed in bone resorbing osteoclasts, and regulates bone resorption. Here, we investigated the mechanism of bone resorption regulated by PMEPA1. Mutant mice lacking NEDD4-binding domains of PMEPA1 displayed enhanced bone volume, and reduced bone resorption activity in comparison with those of WT mice. Analysis with pH-sensitive fluorescence probe revealed that proton secretion from osteoclasts significantly decreased in Pmepa1 mutant osteoclasts. Immunofluorescence analysis revealed that PMEPA1 was colocalized with NEDD4, V0A3, and V0D2 subunits of vacuolar ATPase, which regulate the proton production of osteoclasts. In addition, Nedd4 knockdown reduced bone resorption and proton secretion of osteoclasts. Furthermore, Pmepa1 mutation and Nedd4 knockdown altered the cytoplasmic distribution of components of V-ATPase and expression of autophagy-related proteins, suggesting that lysosomal secretion is affected. Collectively, these findings indicate that PMEPA1 controls proton secretion from osteoclasts via NEDD4 by regulating vesicular trafficking, and NEDD4 is an important regulator of bone resorption.

T cells and macrophages jointly modulate osteogenesis of mesenchymal stromal cells.

Approximately 5%-10% of fractures go on to delayed healing and nonunion, posing significant clinical, economic, and social challenges. Current treatment methods involving open bone harvesting and grafting are associated with considerable pain and potential morbidity at the donor site. Hence, there is growing interest in minimally invasive approaches such as bone marrow aspirate concentrate (BMAC), which contains mesenchymal stromal cells (MSCs), macrophages (Mφ), and T cells. However, the use of cultured or activated cells for treatment is not yet FDA-approved in the United States, necessitating further exploration of optimal cell types and proportions for effective bone formation. As our understanding of osteoimmunology advances, it has become apparent that factors from anti-inflammatory Mφ (M2) promote bone formation by MSCs. Additionally, M2 Mφ promote T helper 2 (Th2) cells and Treg cells, both of which enhance bone formation. In this study, we investigated the interactions among MSCs, Mφ, and T cells in bone formation and explored the potential of subsets of BMAC. Coculture experiments were conducted using primary MSCs, Mφ, and CD4+ T cells at specific ratios. Our results indicate that nonactivated T cells had no direct influence on osteogenesis by MSCs, while coculturing MSCs with Mφ and T cells at a ratio of 1:5:10 positively impacted bone formation. Furthermore, higher numbers of T cells led to increased M2 polarization and a higher proportion of Th2 cells in the early stages of coculture. These findings suggest the potential for enhancing bone formation by adjusting immune and mesenchymal cell ratios in BMAC. By understanding the interactions and effects of immune cells on bone formation, we can develop more effective strategies and protocols for treating bone defects and nonunions. Further studies are needed to investigate these interactions in vivo and explore additional factors influencing MSC-based therapies.

Mechanisms of tissue degeneration mediated by periostin in spinal degenerative diseases and their implications for pathology and diagnosis: a review.

Periostin (POSTN) serves a dual role as both a matricellular protein and an extracellular matrix (ECM) protein and is widely expressed in various tissues and cells. As an ECM protein, POSTN binds to integrin receptors, transduces signals to cells, enabling cell activation. POSTN has been linked with various diseases, including atopic dermatitis, asthma, and the progression of multiple cancers. Recently, its association with orthopedic diseases, such as osteoporosis, osteoarthritis resulting from cartilage destruction, degenerative diseases of the intervertebral disks, and ligament degenerative diseases, has also become apparent. Furthermore, POSTN has been shown to be a valuable biomarker for understanding the pathophysiology of orthopedic diseases. In addition to serum POSTN, synovial fluid POSTN in joints has been reported to be useful as a biomarker. Risk factors for spinal degenerative diseases include aging, mechanical stress, trauma, genetic predisposition, obesity, and metabolic syndrome, but the cause of spinal degenerative diseases (SDDs) remains unclear. Studies on the pathophysiological effects of POSTN may significantly contribute toward the diagnosis and treatment of spinal degenerative diseases. Therefore, in this review, we aim to examine the mechanisms of tissue degeneration caused by mechanical and inflammatory stresses in the bones, cartilage, intervertebral disks, and ligaments, which are crucial components of the spine, with a focus on POSTN.

C-C Motif Chemokine Ligand 2 Enhances Macrophage Chemotaxis, Osteogenesis, and Angiogenesis during the Inflammatory Phase of Bone Regeneration.

Local cell therapy has recently gained attention for the treatment of joint diseases and fractures. Mesenchymal stem cells (MSCs) are not only involved in osteogenesis and angiogenesis, but they also have immunomodulatory functions, such as inducing macrophage migration during bone regeneration via macrophage crosstalk. C-C motif chemokine ligand 2 (CCL2), a known inflammatory mediator, is associated with the migration of macrophages during inflammation. This study examined the utility of CCL2 as a therapeutic target for local cell therapy. Using lentiviral vectors for rabbit MSCs, genetically modified CCL2 overexpressing MSCs were generated. Osteogenic differentiation assays were performed using MSCs with or without macrophages in co-culture, and cell migration assays were also performed. Additionally, co-cultures were performed with endothelial cells (ECs), and angiogenesis was evaluated using a tube formation assay. Overexpression of CCL2 did not affect bone formation under monoculture conditions but promoted chemotaxis and osteogenesis when co-cultured with macrophages. Furthermore, CCL2-overexpression promoted tube formation in co-culture with ECs. These results suggest that CCL2 induces macrophage chemotaxis and osteogenesis by promoting crosstalk between MSCs and macrophages; CCL2 also stimulates ECs to induce angiogenesis. These findings indicate that CCL2 may be a useful therapeutic target for local cell therapy in areas of bone loss.

Vertebral Osteomyelitis and Infective Endocarditis Co-Infection.

Many cases of vertebral osteomyelitis (VO) and infective endocarditis (IE) co-infection have been reported, and it has been recognized that attention should be paid to the possibility of both diseases co-existing during diagnosis and treatment. However, the incidence, clinical status, and outcomes of IE in patients with VO remain unclear. For this study, the eligibility criteria for patient recruitment included all cases of VO at the five medical university hospitals. Patients with a history of spinal surgery were excluded from this study. Echocardiography was routinely performed for all patients with VO. IE was diagnosed according to the modified Duke criteria for definite endocarditis. We analyzed demographic data, underlying conditions, clinical features, laboratory data, echocardiography, radiologic images, treatments, and outcomes. VO was diagnosed in 59 patients and IE was diagnosed in seven patients (12%). There were no significant differences in the clinical features, microorganisms, or radiographic status between the VO-IE co-infection and VO-only groups. In this study, using routine echocardiography for VO, the IE prevalence was 12%. The lack of specific clinical features and laboratory findings may hamper the diagnosis of IE. Therefore, clinicians are always required to suspect IE in patients with VO.

Comparing the usefulness of a fluoroscopic navigation system in femoral trochanteric fracture for orthopaedic residents with the conventional method.

INTRODUCTION: Lag screw insertion into the ideal position is essential to obtain good results in open reduction and internal fixation for femoral trochanteric fracture. Tip-apex distance (TAD) is a widely adopted method for evaluating the risk of lag screw cut-out. Adaptive positioning technology (ADAPT) is a fluoroscopic computer-assisted surgery system that enables orthopaedic surgeons to guide the screw into a proper position intraoperatively. A randomized control study concluded that ADAPT resulted in excellent TAD. However, it was not significantly better than conventional methods when performed by fellowship-trained traumatologists. Therefore, we hypothesised that ADAPT would be useful to orthopaedic residents and evaluated this usefulness. METHODS: We reviewed 102 patients who underwent open reduction and internal fixation for femoral trochanteric fracture from May 2017 to March 2019 using Gamma-3 intertrochanteric nails. Two residents performed all procedures; 51 patients underwent surgery using ADAPT and the others underwent surgery without navigation. The number of attempts to drill guide-wire, operation time, lag screw insertion time, radiation exposure time, TAD, and lag screw position were evaluated for each surgeon. RESULTS: In one resident, when using the ADAPT system, the number of attempts to drill guide-wire (p=0.001), lag screw insertion time (p=0.000), radiational exposure time (p=0.009) and TAD (p=0.007) were lower, and the percentage of ideal lag screw position (p=0.035) were better than that in the conventional method. However, there was no significant difference in the performance of another resident with respect to the aforementioned factors, whether using ADAPT or not. CONCLUSION: One resident showed better results with the ADAPT system than with conventional osteosynthesis. However, another resident received no benefit from ADAPT. The efficiency may not apply to everyone as individual competence can influence efficiency when using ADAPT system. Therefore, as a new device, it must be used cautiously because skill or experience may influence its use, especially by orthopaedic residents.

Analysis of a Cementless Femoral Stem Neck Fracture Using Scanning Electron Microscopy and the Finite Element Method.

Implant fracture is one of the rarest complications of total hip arthroplasty (THA). A 57-year-old woman experienced a fracture of the femoral stem (AHFIX Q, KYOCERA, Japan) about five years after THA. We examined the broken stem by digital microscopy, scanning electron microscopy, and finite element method. The anterolateral corner of the stem's neck was found to be the origin point of the fracture. Finite element method analysis revealed that the stress concentration was highest in the corner of the hollow for apparatus attachment. The stem's design has been considered one of the risk factors for stem fracture. In this patient, multiple risk factors, including thin stem (the smallest size, NAR #1), use of the long neck (+3 mm), obesity (body mass index: 27.3), and adjacent osteoarthritis (contralateral THA loosening and knee osteoarthritis), were present. To our knowledge, this is the first reported case of an AHFIX Q stem fracture. Surgeons must keep in mind that fracture of the femoral stem in patients with several risk factors is possible even several years after THA.

Ipsilateral stress fracture of the proximal fibula after total knee arthroplasty in a patient with severe valgus knee deformity on a background of Rheumatoid arthritis.

INTRODUCTION: Previous studies have reported a lower extremity stress fracture after total knee arthroplasty (TKA). However, a fibular fracture after TKA is quite rare. We report a case of proximal fibula fracture after TKA in a patient with rheumatoid arthritis (RA). PRESENTATION OF CASE: A 45 year old woman with RA had severe knee and foot pain with an antalgic gait disturbance. There was a significant joint deformity in many of lower limb joints. Interval bilateral TKAs were performed two weeks apart. Right TKA was performed using a constraint-type prosthesis, through lateral parapatellar approach. Left TKA was performed using a posterior-stabilized (PS) prosthesis through the more commonly employed, medial parapatellar approach. Seven weeks after the right TKA, the patient was found to have an atraumatic proximal fibular fracture. The fracture went on to heal conservatively. DISCUSSION: The fracture was considered to have occurred after the TKA. The callus appeared eleven weeks after the TKA. The factors that contributed to the fracture were thought to be overload of the fragile bone secondarily to disuse osteopaenia, RA or potentially the significant valgus malalignment correction. The surgical approach, the implant or implantation or the persisting joint deformity, were thought to be contributing factors to the aetiology of the stress fracture. The resultant change in clinical outcome/course is outlined in this case report. CONCLUSION: A stress fracture of the proximal fibula has the potential in the aetiology of may cause other stress fractures, joint other instability, and/or malalignment of the total lower extremity.

Accuracy analysis of computer-assisted surgery for femoral trochanteric fracture using a fluoroscopic navigation system: Stryker ADAPT® system.

PURPOSE: ADAPT is a fluoroscopic computer-assisted surgery system which intraoperatively shows the distance from the tip of the screw to the surface of the femoral head, tip-to-head-surface distance (TSD), and the tip-apex distance (TAD) advocated by Baumgaertner et al. The study evaluated the accuracy of ADAPT. PATIENTS AND METHODS: A total of 55 patients operated with ADAPT between August 2016 and March 2017 were included as subjects. TSD and TAD were measured postoperatively using computed tomography (CT) and X-rays. The intraclass correlation coefficient (ICC) was checked in advance. The error was defined as the difference between postoperative and intraoperative measurement values of ADAPT. Summary statistics, root mean square errors (RMSEs), and correlations were evaluated. RESULTS: ICC was 0.94 [95% CI: 0.90-0.96] in TSD and 0.99 [95% CI: 0.98-0.99] in TAD. The error was -0.35 mm (-1.83 mm to 1.12 mm) in TSD and +0.63 mm (-5.65 mm to 4.59 mm) in TAD. RMSE was 0.63 mm in TSD and 1.53 mm in TAD. Pearson's correlation coefficient was 0.79 [95% CI: 0.66-0.87] in TSD and 0.83 [95% CI: 0.72-0.89] in TAD. There were no adverse events with ADAPT use. CONCLUSION: ADAPT is highly accurate and useful in guiding surgeons in properly positioning the screws.