Effect of tourniquet application on postoperative outcomes in sinus tarsi approach for intra-articular calcaneus fractures.

INTRODUCTION: Tourniquets are commonly used during foot and ankle surgery to provide a bloodless operative field and increase surgical comfort, despite the potential risks associated with it. This study compared postoperative outcomes of tourniquet-assisted and non-tourniquet-assisted operative fixation of calcaneal fractures via the sinus tarsi approach. MATERIALS AND METHODS: A retrospective study from March 2015 to December 2018 revealed 131 patients with closed calcaneal fractures who underwent minimally invasive surgery at our hospital. Visualization, operating time, blood loss, and postoperative pain were collected. Patients in the tourniquet group (n = 62) were compared with patients in the non-tourniquet group (n = 69). RESULTS: The visibility of the surgical field was fair/poor in 2 cases in the tourniquet group and fair/poor in 19 cases in the non-tourniquet group (P < 0.05). The mean operative time was 64.7 ± 3.5 min in the tourniquet group and 76.0 ± 6.1 min in the non-tourniquet group (P < 0.05). The estimated intraoperative and postoperative blood loss was 56.6 ± 33.3 and 100.0 ± 25.3 mL, respectively, in the tourniquet group and 205.0 ± 31.6 and 38.3 ± 19.8 mL, respectively, in the non-tourniquet group (P < 0.05). The VAS pain scores 24 h, 48 h, and 72 h postoperatively were 4.3 ± 1.8, 3.1 ± 1.2, and 2.0 ± 0.5 points, respectively, in the tourniquet group and 2.1 ± 1.1, 1.6 ± 1.0, and 1.0 ± 0.3 points, respectively, in the non-tourniquet group (P < 0.05). CONCLUSION: Tourniquet application during the sinus tarsi approach for calcaneal fractures can significantly improve surgical visualization and reduce intraoperative blood loss. However, adverse events associated with the use of tourniquets include increased postoperative pain and bleeding. Due to increased postoperative bleeding and pain, more attention should be given to the postoperative phase in patients treated with tourniquets.

Pseudomyogenic Hemangioendothelioma of the Talocalcaneal Coalition: A Case Report.

Pseudomyogenic hemangioendothelioma is a rare soft tissue tumor most often found in the lower extremities and predominantly occurring in males. The talocalcaneal coalition is an anatomic anomaly that develops between the talus and calcaneus bones, can cause hindfoot pain and subtalar joint stiffness, and has a prevalence of less than 1%. We present what is to our knowledge the first case report of a 17-year-old male with pseudomyogenic hemangioendothelioma occurring within a talocalcaneal coalition. The patient was treated with local excision of the tumor and the coalition. His American Orthopaedic Foot & Ankle Society ankle/hindfoot score went from 70 preoperatively to 92 at 1 year postoperatively, and he has had no evidence of recurrence at 1 and 3 years postoperatively. These tumors have suggestive but not diagnostic computed tomography, magnetic resonance imaging, and histopathological findings, and they are associated with a uniquely characteristic immunophenotype, including immunochemical reactivity to CD31, FLi-1, INI-1, ERG, and FOSB. Primary treatment of pseudomyogenic hemangioendothelioma most often involves local excision (but can require amputation) and may include adjuvant radiotherapy and/or chemotherapy. It has a relatively favorable prognosis, with a moderate risk of local recurrence and a low risk of metastases. Because metastases have been reported many years after treatment, long-term follow-up is necessary.

Open Curettage With Bone Augmentation for Symptomatic Tumors and Tumor-like Lesions of Calcaneus: A Comparison of Bioactive Glass Versus Allogeneic Bone.

Few studies have characterized the clinical outcomes of 45S5 Bioglass® applied as a bone graft to that of allogeneic bone applied in calcaneal open curettage. Therefore, the purpose of the present investigation was to compare the outcomes of patients with calcaneal tumors and tumor-like lesions treated by open curettage with 45S5 Bioglass® or allogeneic bone. Of the 31 patients who underwent open curettage (18 cases of unicameral bone cysts, 7 cases of aneurysmal bone cysts, and 6 cases of intraosseous lipoma), 16 (52%) received grafts with 45S5 Bioglass® and 15 (48%) with allogeneic bone. All the feet achieved bone fusion according to the modified Neer radiographic classification system at the last follow-up examination. The mean bone ingrowth time for the grafts with 45S5 Bioglass® versus allogeneic bone was 3.71 ± 0.86 versus 4.46 ± 1.04 months (p = .038), the mean bone healing time was 4.86 ± 0.93 versus 5.73 ± 1.07 months (p = .021), and the mean incision drying time was 7.2 ± 1.8 versus 8.2 ± 1.5 days (p = .047), respectively. No differences were found in the postoperative American Orthopaedic Foot and Ankle Society ankle-hindfoot scale scores between the 2 groups (p = .213). These results show that 45S5 Bioglass® can better facilitate the formation of new bone with a faster drying time of the incision than allogeneic bone. Although both materials can benefit the clinical outcomes of calcaneal tumors and tumor-like lesions, further studies are needed to observe the long-term complications and lesion recurrence rates.

NIPA2 regulates osteoblast function via its effect on apoptosis pathways in type 2 diabetes osteoporosis.

Type 2 diabetes osteoporosis has recently become a hot topic in the study of diabetic complications, but the specific mechanism of its development remains unclear. Non-imprinted in Prader-Willi/Angelman syndrome region protein 2 (NIPA2), a highly-selective magnesium ion transporter, has been found to be associated with type 2 diabetes. In this study we aimed to investigate the specific role and mechanism of NIPA2 in the pathogenesis of type 2 diabetes osteoporosis. We first used western blotting, PCR, immunofluorescence, and magnesium ion probes to detect changes of NIPA2 and intracellular magnesium levels in osteoblasts at different concentrations of advanced glycation end products (AGEs). We then up- or down-regulated NIPA2 using a lentivirus and analyzed apoptotic biomarkers as well as the osteogenic ability of osteoblasts. We found that AGEs dose-dependently down-regulated the expression of NIPA2 in osteoblasts. NIPA2 also regulated osteoblast apoptosis by affecting the intracellular magnesium level and further affecting the osteogenic capacity of osteoblasts. Our study revealed the changes of NIPA2 in response to AGEs in the environment, as well as its function and mechanism in osteoblasts, demonstrating its important role in the pathogenesis of type 2 diabetes osteoporosis. The study suggests that NIPA2 is a potential target for the treatment of type 2 diabetes osteoporosis.

High glucose downregulates the effects of autophagy on osteoclastogenesis via the AMPK/mTOR/ULK1 pathway.

Diabetes is a chronic disease that disrupts the balance between bone formation and bone desorption, which can lead to osteoporosis, increasing the risk of fracture. However, compared with osteoblasts, the biological effects of hyperglycemia on osteoclastogenesis remain to be elucidated. Therefore, we investigated the impact of glucose at different concentrations (5.5, 10.5, 15.5, 20.5, 25.5, and 30.5 mM) on osteoclastogenesis using RAW264.7 cells. Cell proliferation was measured with the cell counting kit-8 assay, and osteoclastogenesis was detected with tartrate-resistant acid phosphatase staining and bone resorption assays, as well as protein cathepsin K expression. Compound C, the AMP-activated protein kinase (AMPK) pathway inhibitor, was used to examine the relationship between the AMPK/mTOR/ULK1 signaling pathway and autophagy in osteoclasts. Autophagy was evaluated with transmission electron microscopy and immunofluorescence microscopy and associated proteins were detected with western blotting. The pharmacological autophagic reagents bafilomycin A1, 3-methyladenine, and rapamycin were used to determine the effect of autophagy on osteoclastogenesis. Our results showed that glucose negatively affected osteoclast formation and function but did not affect the proliferation of RAW264.7 cells. Suppression of the AMPK/mTOR/ULK1 signaling axis decreased autophagy in glucose-mediated osteoclast. Furthermore, High levels of glucose decreased autophagy level in osteoclasts. Additionally, interfering with autophagy affected osteoclast formation and function. These findings clarify the mechanisms underlying the effects of glucose-mediated osteoclastogenesis and will help identify novel therapeutic strategies for the protection of skeletal health in diabetic osteoporosis.

Regulation of DMT1 on autophagy and apoptosis in osteoblast.

Iron overload has recently been associated with the changes in the bone microstructure that occur in osteoporosis. However, the effect of iron overload on osteoblasts is unclear. The purpose of this study was to explore the function of divalent metal transporter 1 (DMT1) in the pathological processes of osteoporosis. Osteoblast hFOB1.19 cells were cultured in medium supplemented with different concentrations (0, 50, 100, 200, 300, 400, 500 µmol/L) of ferric ammonium citrate (FAC) as a donor of ferric ions. We used western blotting and immunofluorescence to determine the levels of DMT1 after treatment with FAC. Apoptosis was evaluated by detecting the levels of cleaved caspase 3, BCL2, and BAX with western blotting. Autophagy was evaluated by detecting the levels of LC3 with western blotting and immunofluorescence. Beclin-1 expression was also assessed with western blotting. The autophagy inhibitor 3-methyladenine was used to determine whether autophagy affects the apoptosis induced by FAC. Our results show that FAC increased the levels of DMT1, upregulated the expression of BCL2, and downregulated the apoptosis-related proteins cleaved caspase 3 and BAX. Both LC3I/LC3II levels and beclin-1 were also increased, indicating that FAC increases the accumulation of autophagosomes in hFOB1.19 cells. FAC-induced autophagy was increased by the apoptosis inhibitor 3-MA but was reduced in DMT1 shRNA hFOB1.19 cells. These results suggest that the increased expression of DMT1 induces iron overload and iron overload induces osteoblast autophagy and apoptosis, thus affecting the pathological processes of osteoporosis. Clarifying the mechanisms underlying the effects of DMT1 will allow the identification of novel targets for the prevention and treatment of osteoporosis.

Advanced Glycation End Products Affect Osteoblast Proliferation and Function by Modulating Autophagy Via the Receptor of Advanced Glycation End Products/Raf Protein/Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase/Extracellular Signal-regulated Kinase (RAGE/Raf/MEK/ERK) Pathway.

The interaction between advanced glycation end products (AGEs) and receptor of AGEs (RAGE) is associated with the development and progression of diabetes-associated osteoporosis, but the mechanisms involved are still poorly understood. In this study, we found that AGE-modified bovine serum albumin (AGE-BSA) induced a biphasic effect on the viability of hFOB1.19 cells; cell proliferation was stimulated after exposure to low dose AGE-BSA, but cell apoptosis was stimulated after exposure to high dose AGE-BSA. The low dose AGE-BSA facilitates proliferation of hFOB1.19 cells by concomitantly promoting autophagy, RAGE production, and the Raf/MEK/ERK signaling pathway activation. Furthermore, we investigated the effects of AGE-BSA on the function of hFOB1.19 cells. Interestingly, the results suggest that the short term effects of low dose AGE-BSA increase osteogenic function and decrease osteoclastogenic function, which are likely mediated by autophagy and the RAGE/Raf/MEK/ERK signal pathway. In contrast, with increased treatment time, the opposite effects were observed. Collectively, AGE-BSA had a biphasic effect on the viability of hFOB1.19 cells in vitro, which was determined by the concentration of AGE-BSA and treatment time. A low concentration of AGE-BSA activated the Raf/MEK/ERK signal pathway through the interaction with RAGE, induced autophagy, and regulated the proliferation and function of hFOB1.19 cells.

Regulation of DMT1 on Bone Microstructure in Type 2 Diabetes.

Diabetic osteoporosis is gradually attracted people's attention. However, the process of bone microstructure changes in diabetic patients, and the exact mechanism of osteoblast iron overload are unclear. Therefore, the present study aimed to explore the function of DMT1 in the pathological process of diabetic osteoporosis. We build the type two diabetes osteoporosis models with SD rats and Belgrade rats, respectively. Difference expression of DMT1 was detected by using the method of immunohistochemistry and western blotting. Detection of bone microstructure and biomechanics and iron content for each group of samples. We found that DMT1 expression in type 2 diabetic rats was higher than that in normal rats. The bone biomechanical indices and bone microstructure in the rat model deficient in DMT1 was significantly better than that in the normal diabetic model. The loss of DMT1 can reduce the content of iron in bone. These findings indicate that DMT1 expression was enhanced in the bone tissue of type 2 diabetic rats, and plays an important role in the pathological process of diabetic osteoporosis. Moreover, DMT1 may be a potential therapeutic target for diabetic osteoporosis.

Polycytosine RNA-binding protein 1 regulates osteoblast function via a ferroptosis pathway in type 2 diabetic osteoporosis.

BACKGROUND: Recently, type 2 diabetic osteoporosis (T2DOP) has become a research hotspot for the complications of diabetes, but the specific mechanism of its occurrence and development remains unknown. Ferroptosis caused by iron overload is con-sidered an important cause of T2DOP. Polycytosine RNA-binding protein 1 (PCBP1), an iron ion chaperone, is considered a protector of ferroptosis. AIM: To investigate the existence of ferroptosis and specific role of PCBP1 in the development of type 2 diabetes. METHODS: A cell counting kit-8 assay was used to detect changes in osteoblast viability under high glucose (HG) and/or ferroptosis inhibitors at different concentrations and times. Transmission electron microscopy was used to examine the morphological changes in the mitochondria of osteoblasts under HG, and western blotting was used to detect the expression levels of PCBP1, ferritin, and the ferroptosis-related protein glutathione peroxidase 4 (GPX4). A lentivirus silenced and overexpressed PCBP1. Western blotting was used to detect the expression levels of the osteoblast functional proteins osteoprotegerin (OPG) and osteocalcin (OCN), whereas flow cytometry was used to detect changes in reactive oxygen species (ROS) levels in each group. RESULTS: Under HG, the viability of osteoblasts was considerably decreased, the number of mitochondria undergoing atrophy was considerably increased, PCBP1 and ferritin expression levels were increased, and GPX4 expression was decreased. Western blotting results demonstrated that infection with lentivirus overexpressing PCBP1, increased the expression levels of ferritin, GPX4, OPG, and OCN, compared with the HG group. Flow cytometry results showed a reduction in ROS, and an opposite result was obtained after silencing PCBP1. CONCLUSION: PCBP1 may protect osteoblasts and reduce the harm caused by ferroptosis by promoting ferritin expression under a HG environment. Moreover, PCBP1 may be a potential therapeutic target for T2DOP.

Prolonged Cadmium Exposure and Osteoclastogenesis: A Mechanistic Mouse and in Vitro Study.

BACKGROUND: Cadmium (Cd) is a highly toxic and widespread environmental oxidative stressor that causes a myriad of health problems, including osteoporosis and bone damage. Although nuclear factor erythroid 2-related factor 2 (NRF2) and its Cap 'n' Collar and basic region Leucine Zipper (CNC-bZIP) family member nuclear factor erythroid 2-related factor 1 (NRF1) coordinate various stress responses by regulating the transcription of a variety of antioxidant and cytoprotective genes, they play distinct roles in bone metabolism and remodeling. However, the precise roles of both transcription factors in bone loss induced by prolonged Cd exposure remain unclear. OBJECTIVES: We aimed to understand the molecular mechanisms underlying Cd-induced bone loss, focusing mainly on the roles of NRF2 and NRF1 in osteoclastogenesis provoked by Cd. METHODS: Male wild-type (WT), global Nrf2-knockout (Nrf2-/-) and myeloid-specific Nrf2 knockout [Nrf2(M)-KO] mice were administered Cd (50 or 100 ppm) via drinking water for 8 or 16 wk, followed by micro-computed tomography, histological analyses, and plasma biochemical testing. Osteoclastogenesis was evaluated using bone marrow-derived osteoclast progenitor cells (BM-OPCs) and RAW 264.7 cells in the presence of Cd (10 or 20 nM) with a combination of genetic and chemical modulations targeting NRF2 and NRF1. RESULTS: Compared with relevant control mice, global Nrf2-/- or Nrf2(M)-KO mice showed exacerbated bone loss and augmented osteoclast activity following exposure to 100 ppm Cd in drinking water for up to 16 wk. In vitro osteoclastogenic analyses suggested that Nrf2-deficient BM-OPCs and RAW 264.7 cells responded more robustly to low levels of Cd (up to 20 nM) with regard to osteoclast differentiation compared with WT cells. Further mechanistic studies supported a compensatory up-regulation of long isoform of NRF1 (L-NRF1) and subsequent induction of nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1 (NFATc1) as the key molecular events in the Nrf2 deficiency-worsened and Cd-provoked osteoclastogenesis. L-Nrf1 silenced (via lentiviral means) Nrf2-knockdown (KD) RAW cells exposed to Cd showed dramatically different NFATc1 and subsequent osteoclastogenesis outcomes compared with the cells of Nrf2-KD alone exposed to Cd, suggesting a mitigating effect of the Nrf1 silencing. In addition, suppression of reactive oxygen species by exogenous antioxidants N-acetyl-l-cysteine (2 mM) and mitoquinone mesylate (MitoQ; 0.2µM) mitigated the L-NRF1-associated effects on NFATc1-driven osteoclastogenesis outcomes in Cd-exposed Nrf2-KD cells. CONCLUSIONS: This in vivo and in vitro study supported the authors' hypothesis that Cd exposure caused bone loss, in which NRF2 and L-NRF1 responded to Cd and osteoclastogenic stimuli in a cooperative, but contradictive, manner to coordinate Nfatc1 expression, osteoclastogenesis and thus bone homeostasis. Our study suggests a novel strategy targeting NRF2 and L-NRF1 to prevent and treat the bone toxicity of Cd. https://doi.org/10.1289/EHP13849.

Bilateral cadaveric Achilles tendon graft in reconstruction after Achilles tendon tumor resection.

The standard approach to reconstruction after resection of a diffuse-type tenosynovial giant cell tumor is a local patch with free flaps. However, in cases in which the Achilles tendon involvement is extensive, and the entire tendon must be removed, an autologous flap graft might not be adequate to allow a return to function. We report a case of a 52-year-old female patient who developed bilateral tumors of the Achilles tendon, with a 10-year duration. By the time, she sought medical help, both Achilles tendons required removal. We chose to use Achilles tendon allografts to replace the Achilles tendons. Postoperatively, the patient did well. The allograft shortened the recovery time, and the patient regained full ankle range of motion.

Exosomes derived from platelet-rich plasma promote diabetic wound healing via the JAK2/STAT3 pathway.

Diabetic non-healing wounds are bringing a heavy burden on patients and society. Platelet-rich plasma (PRP) has been widely applied in tissue regenerating for containing various growth factors. Recently, PRP-derived exosomes (PRP-Exos) have been proved to be more effective than PRP in tissue regeneration. However, few studies have investigated the therapeutic potential of PRP-Exos in diabetic wound healing to date. Therefore, we extracted and identified exosomes derived from PRP and tested its promoting effect on diabetic wound healing in vivo and in vitro. We found that high glucose (HG) inhibited cell proliferation and migration and induced apoptosis through ROS-dependent activation of the JNK and p38 MAPK signaling pathways. PRP-Exos can stimulate fibroblast functions and accelerate diabetic wound healing. The benefits of PRP-Exos may be attributed to its capability to prevent HG-induced ROS-dependent apoptosis via the PDGF-BB/JAK2/STAT3/Bcl-2 signaling pathway. This illustrates the therapeutic potential of PRP-Exos in diabetic wounds.

Cell apoptosis induced by zinc deficiency in osteoblastic MC3T3-E1 cells via a mitochondrial-mediated pathway.

Deficiency of zinc plays an important role in the pathogenesis of osteoporosis; however, the underlying mechanism is not well understood. Apoptosis of osteoblast causing the loss of bone mass is an important event in the osteoporosis. In this article, we investigated whether zinc deficiency would induce cell apoptosis in MC3T3-E1 cells and ask if it is involved in mitochondrial-mediated pathway. Significant increased apoptosis were observed in zinc deficiency group (ZnD: 5 µM TPEN and 1 µM zinc) compared with untreated control or zinc adequacy group (ZnA: 5 µM TPEN and 15 µM zinc). The mitochondrial membrane potential was strikingly reduced in ZnD group. Furthermore, we observed that the levels of Bax in mitochondria fraction and cyto c, AIF, and cleaved caspase-3/-9 in cytosol fraction were increased in ZnD group. We proposed that zinc deficiency would induce the translocation of Bax into mitochondria, which could lead to the reduction in mitochondrial membrane potential as well as the increase in mitochondrial membrane permeability. In addition, cyto c and AIF were released from mitochondria into the cytosol, which finally activated caspase-dependent and caspase-independent apoptosis processes in MC3T3-E1 cells. Our findings suggested that zinc deficiency is capable of inducing apoptosis through a mitochondria-mediated pathway in osteoblastic cells.

A simple method to detect human intraosseous vascular structures: using the calcaneus as an example.

BACKGROUND: Intraosseous vessels play an important role in regeneration of bone. However, the anatomy of the intraosseous vessels in humans has not been clearly delineated due to inadequate method of stereoscopically investigating vessels surrounded by bone tissues. PURPOSE: This study was to investigate the feasibility of simple CT scanning with barium sulphate perfusion to detect intraosseous vessels in humans. METHODS: Two freshly obtained feet from a patient who required a double amputation were used in this study. One foot was perfused with barium sulfate and then scanned by CT (CT method). The other foot was processed using vascular corrosion casting (traditional method). Intraosseous vessels in both specimens were compared. RESULTS: The anatomical distributions of the calcaneal intraosseous vessels were similar as assessed by the CT and traditional methods. However, in comparison to traditional method, the CT method allows the preservation of the surrounding bone tissue, which is important for analyzing the relationship between intraosseous vessels and the surrounding bone structures, and the visualization of a special vascular structure called the sinusoid cluster. CONCLUSION: Simple CT scanning with barium sulfate perfusion may be a practical and adequate method for stereoscopically detecting the morphology and distribution of the intraosseous vessels.

Development and validation of a gene signature predicting the risk of postmenopausal osteoporosis.

AIMS: We aimed to develop a gene signature that predicts the occurrence of postmenopausal osteoporosis (PMOP) by studying its genetic mechanism. METHODS: Five datasets were obtained from the Gene Expression Omnibus database. Unsupervised consensus cluster analysis was used to determine new PMOP subtypes. To determine the central genes and the core modules related to PMOP, the weighted gene co-expression network analysis (WCGNA) was applied. Gene Ontology enrichment analysis was used to explore the biological processes underlying key genes. Logistic regression univariate analysis was used to screen for statistically significant variables. Two algorithms were used to select important PMOP-related genes. A logistic regression model was used to construct the PMOP-related gene profile. The receiver operating characteristic area under the curve, Harrell's concordance index, a calibration chart, and decision curve analysis were used to characterize PMOP-related genes. Then, quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the expression of the PMOP-related genes in the gene signature. RESULTS: We identified three PMOP-related subtypes and four core modules. The muscle system process, muscle contraction, and actin filament-based movement were more active in the hub genes. We obtained five feature genes related to PMOP. Our analysis verified that the gene signature had good predictive power and applicability. The outcomes of the GSE56815 cohort were found to be consistent with the results of the earlier studies. qRT-PCR results showed that RAB2A and FYCO1 were amplified in clinical samples. CONCLUSION: The PMOP-related gene signature we developed and verified can accurately predict the risk of PMOP in patients. These results can elucidate the molecular mechanism of RAB2A and FYCO1 underlying PMOP, and yield new and improved treatment strategies, ultimately helping PMOP monitoring.Cite this article: Bone Joint Res 2022;11(8):548-560.

Mitochondrial Ferritin Deficiency Promotes Osteoblastic Ferroptosis Via Mitophagy in Type 2 Diabetic Osteoporosis.

The incidence of type 2 diabetic osteoporosis (T2DOP), which seriously threatens elderly people's health, is rapidly increasing in recent years. However, the specific mechanism of the T2DOP is still unclear. Studies have shown the relationship between iron overload and T2DOP. Mitochondrial ferritin (FtMt) is a protein that stores iron ions and intercepts toxic ferrous ions in cells mitochondria. Ferroptosis, an iron-dependent cell injured way, may be related to the pathogenesis of T2DOP. In this study, we intend to elucidate the effect of FtMt on ferroptosis in osteoblasts and explain the possible mechanism. We first detected the occurrence of ferroptosis in bone tissue and the expression of FtMt after inducing T2DOP rat model. Then we used hFOB1.19 cells to study the influence of high glucose on FtMt, ferroptosis, and osteogenic function of osteoblasts. Then we observed the effect of FtMt on ferroptosis and osteoblast function by lentiviral silencing and overexpression of FtMt. We found ferroptosis in T2DOP rats bone. Overexpression of FtMt reduced osteoblastic ferroptosis under high glucose condition while silent FtMt induced mitophagy through ROS / PINK1/Parkin pathway. Then we found increased ferroptosis in osteoblasts after activating mitophagy by carbonyl cyanide-m-chlorophenyl-hydrazine (CCCP, a mitophagy agonist). Our study demonstrated that FtMt inhibited the occurrence of ferroptosis in osteoblasts by reducing oxidative stress caused by excess ferrous ions, and FtMt deficiency induced mitophagy in the pathogenesis of T2DOP. This study suggested that FtMt might serve as a potential target for T2DOP therapy.

ATF3 Regulates Osteogenic Function by Mediating Osteoblast Ferroptosis in Type 2 Diabetic Osteoporosis.

Purpose: Osteoporosis is a complication of type 2 diabetes, and it is characterized by reduced bone mass, augmented bone fragility, and increased risk of fracture, thus reducing patient quality of life, especially in the elderly. Ferroptosis has been implicated in the pathological process of type 2 diabetic osteoporosis (T2DOP), but the specific underlying mechanisms remain largely unknown. This study clarified the role of activating transcription factor 3 (ATF3) in T2DOP and explored its specific regulatory mechanism, providing a new treatment target for T2DOP. Methods: We cultured hFob1.19 cells in high glucose (HG, 35 mM) and knocked down ATF3 using short hairpin RNA (shRNA). We then measured cell viability, assessed morphology, quantified the expression of ATF3 and glutathione peroxidase 4 (GPX4), detected the levels of reactive oxygen species (ROS) and lipid peroxides, and determined the osteogenic function of osteoblasts. Cystine/glutamate antiporter (system Xc-) activity was evaluated by determining the expression of SLC7A11 and the levels of glutathione (GSH) and extracellular glutamate. We constructed a T2DOP rat model and observed the effect of ATF3 on ferroptosis and T2DOP by knocking down ATF3 using small interfering RNA (siRNA). Then, we evaluated the levels of iron metabolism, lipid peroxidation, and bone turnover in serum, detected the expression of ATF3, SLC7A11, and GPX4 in bone tissues, and assessed bone microstructure using microcomputed tomography. Results: ATF3 expression was increased in osteoblasts under HG condition and in T2DOP rats. Inhibiting the function of ATF3 increased GPX4 levels and reduced the accumulation of ROS and lipid peroxides. These changes inhibited the ferroptosis of osteoblasts and improved osteogenic function. In addition, HG induced ATF3 upregulation, resulting in decreased SLC7A11 expression and lower levels of intracellular GSH and extracellular glutamate. Conclusion: Osteoblast ferroptosis under HG conditions is induced by ATF3-mediated inhibition of system Xc- activity, and these events contribute to T2DOP pathogenesis.

ELAV-like RNA binding protein 1 regulates osteogenesis in diabetic osteoporosis: Involvement of divalent metal transporter 1.

Diabetic osteoporosis (DOP) is a complication of diabetes mellitus (DM) and occurs due to alterations in bone metabolism under hyperglycemic condition. ELAV-like RNA binding protein 1 (ELAVL1) is abnormally up-regulated in diabetes-related diseases. Bioinformatics prediction indicates that divalent metal transporter 1 (DMT1) is a potential target of ELAVL1. To explore the role of ELAVL1 and the involvement of ELAVL1/DMT1 axis in DOP, we established a mouse model of DM by administration of high-fat diet and intraperitoneal injection with streptozotocin (STZ). The expression of ELAVL1 and DMT1 was increased in the bone tissues of DM mice. Knockdown of ELAVL1 reduced iron level and oxidative stress, promoted osteogensis, and prevented bone mass loss, thus mitigating DOP in DM mice. In vitro, mouse pre-osteoblast MC3T3-E1 cells were treated with high glucose (25 mM) and ferric ammonium citrate (FAC, 200 µM). The inhibitory effects of ELAVL1 knockdown on iron accumulation and oxidative stress were evidenced in MC3T3-E1 cells. Knockdown of ELAVL1 enhanced osteoblast viability, differentiation and mineralization. Notably, the expression of DMT1 was positively correlated with that of ELAVL1 in vivo and in vitro. Overexpression of DMT1 abolished the effect of ELAVL1 knockdown on the behaviors of MC3T3-E1 cells, suggesting that ELAVL1 might function through regulating DMT1. In conclusion, knockdown of ELAVL1 likely alleviated DOP by inhibiting iron overload and oxidative stress and promoting osteogenesis, and DMT1 might be involved in this process. These findings provide insights into the pathogenesis of DOP and suggest a potential therapeutic target for DOP treatment.

Glycosaminoglycan-Based Hydrogel Delivery System Regulates the Wound Microenvironment to Rescue Chronic Wound Healing.

Chronic wounds cannot proceed through the normal, orderly, and timely sequence of repair. The adverse cycle between excess reactive oxide species (ROS) and a persistent inflammatory response is an important mechanism of impaired wound healing. Herein, by combining the intrinsic bioactivities of natural polysaccharides and natural drugs, a glycosaminoglycan-based hydrogel delivery system is proposed to regulate the wound microenvironment. Dynamic supramolecular cross-linking enables the hydrogel to easily encapsulate the drug and fully fill the wound area. As the backbone of the hydrogel, heparin captures inflammatory chemokines at the wound site, while hyaluronic acid mimics the function of ECM. The hydrophobic drug curcumin has been ingeniously encapsulated in the hydrogel through micellization, thereby exerting good ROS scavenging ability and anti-inflammatory activity. Evaluations in diabetic mice showed that this antioxidant and anti-inflammatory hydrogel was effective in reducing the influx of immune cells at the wound site and in down-regulating the inflammatory response. Accelerated wound healing was also observed, as evidenced by faster re-epithelialization and better ECM remodeling. The proposed hydrogel can regulate the microenvironment of wounds from multiple aspects and thereby achieve regression of wound repair, which may provide a new therapeutic strategy for chronic wounds.

Acid Sphingomyelinase and Acid ß-Glucosidase 1 Exert Opposite Effects on Interleukin-1ß-Induced Interleukin 6 Production in Rheumatoid Arthritis Fibroblast-Like Synoviocytes.

Acid sphingomyelinase (ASM) and acid ß-glucosidase 1 (GBA1) catalyze ceramide formation through different routes, and both are involved in rheumatoid arthritis (RA) pathogenesis as well as IL-6 production. However, whether ASM and GBA1 regulate IL-6 production in RA remains unknown. Serum ASM, GBA1, and ceramide levels were measured in RA patients and healthy controls by enzyme-linked immunosorbent assay, and their correlations with clinical indicators of patients were evaluated. Pharmacologic inhibitors or small hairpin RNAs of ASM and GBA1 were employed to explore the roles of ASM and GBA1 in IL-6 production, cell behavior, and MAPK signaling in fibroblast-like synoviocytes from RA patients (RAFLS). ASM, GBA1, and ceramide serum levels were significantly elevated in patients with RA. GBA1 and ceramide serum levels were negatively and positively correlated with IL-6 serum level in RA patients, respectively. ASM inhibitor or knockdown of ASM abolished IL-1ß-induced IL-6 expression and secretion. Functionally, ASM inhibitor suppressed IL-1ß-induced cell proliferation, migration, and invasion in RAFLS. Mechanistically, ASM inhibitor or knockdown of ASM effectively countered IL-1ß-induced activation of p38 MAPK signaling. The pharmacologic inhibitor or knockdown of GBA1 exhibited the opposite effects. Importantly, p38 inhibitor blocked IL-1ß-induced IL-6 production in RAFLS. ASM plays a pathogenic role in RA, whereas GBA1 plays a protective role in RA possibly by regulating IL-6 production in RAFLS at least partially via p38 signaling, serving as potential therapeutic targets in RA treatment.