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The authors determined the effect of the GLP-1 receptor agonist liraglutide on endothelial surface expression of vascular cell adhesion molecule (VCAM)-1 in murine apolipoprotein E knockout atherosclerosis. Contrast-enhanced ultrasound molecular imaging using microbubbles targeted to VCAM-1 and control microbubbles showed a 3-fold increase in endothelial surface VCAM-1 signal in vehicle-treated animals, whereas in the liraglutide-treated animals the signal ratio remained around 1 throughout the study. Liraglutide had no influence on low-density lipoprotein cholesterol or glycated hemoglobin, but reduced TNF-α, IL-1β, MCP-1, and OPN. Aortic plaque lesion area and luminal VCAM-1 expression on immunohistology were reduced under liraglutide treatment., Competing Interests: This work is supported by grants 310030-169905 and 310030-197673 from the Swiss national Science Foundation and from the Swiss Heart Foundation to Dr Kaufmann. This work was funded in part by Novo Nordisk A/S, Bagsværd, Denmark. The authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2023 The Authors.)

Excessive alcohol consumption is a global healthcare problem with enormous social, economic, and clinical consequences. While chronic, heavy alcohol consumption causes structural damage and/or disrupts normal organ function in virtually every tissue of the body, the liver sustains the greatest damage. This is primarily because the liver is the first to see alcohol absorbed from the gastrointestinal tract via the portal circulation and second, because the liver is the principal site of ethanol metabolism. Alcohol-induced damage remains one of the most prevalent disorders of the liver and a leading cause of death or transplantation from liver disease. Despite extensive research on the pathophysiology of this disease, there are still no targeted therapies available. Given the multifactorial mechanisms for alcohol-associated liver disease pathogenesis, it is conceivable that a multitherapeutic regimen is needed to treat different stages in the spectrum of this disease.

Background: Congenital pseudarthrosis of the tibia (CPT) is an uncommon congenital deformity and a special subtype of bone nonunion. The lower ability of osteogenic differentiation in CPT-derived mesenchymal stem cells (MSCs) could result in progression of CPT, and miR-30a could inhibit osteogenic differentiation. However, the role of miR-30a in CPT-derived MSCs remains unclear., Methods: The osteogenic differentiation of CPT-derived MSCs treated with the miR-30a inhibitor was tested by Alizarin Red S staining and alkaline phosphatase (ALP) activity. The expression levels of protein and mRNA were assessed by Western blot or quantitative reverse transcription-polymerase chain reaction (RT-qPCR), respectively. The interplay between miR-30a and HOXD8 was investigated by a dual-luciferase reporter assay. Chromatin immunoprecipitation (ChIP) was conducted to assess the binding relationship between HOXD8 and RUNX2 promoter., Results: CPT-derived MSCs showed a lower ability of osteogenic differentiation than normal MSCs. miR-30a increased in CPT-derived MSCs, and miR-30a downregulation promoted the osteogenic differentiation of CPT-derived MSCs. Meanwhile, HOXD8 is a direct target for miR-30a, and HOXD8 could transcriptionally activate RUNX2. In addition, miR-30a could inhibit the osteogenic differentiation of CPT-derived MSCs by negatively regulating HOXD8., Conclusion: miR-30a inhibits the osteogenic differentiation of CPT-derived MSCs by targeting HOXD8. Thus, this study might supply a novel strategy against CPT., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

Introduction: The role of osteopontin (OPN) following severe injury remains to be elucidated, especially its relationship with type I collagen (encoded by the Col1a1 gene) secretion by newly-differentiated odontoblast-like cells (OBLCs). In this study, we examined the role of OPN in the process of reparative dentin formation with a focus on reinnervation and revascularization after tooth replantation in Opn knockout (KO) and wild-type (WT) mice., Methods: Maxillary first molars of 2- and 3-week-old- Opn KO and WT mice ( Opn KO 2W, Opn KO 3W, WT 2W, and WT 3W groups) were replanted, followed by fixation 3-56 days after operation. Following micro-computed tomography analysis, the decalcified samples were processed for immunohistochemistry for Ki67, Nestin, PGP 9.5, and CD31 and in situ hybridization for Col1a1 ., Results: An intense inflammatory reaction occurred to disrupt pulpal healing in the replanted teeth of the Opn KO 3W group, whereas dental pulp achieved healing in the Opn KO 2W and WT groups. The tertiary dentin in the Opn KO 3W group was significantly decreased in area compared with the Opn KO 2W and WT groups, with a significantly low percentage of Nestin-positive, newly-differentiated OBLCs during postoperative days 7-14. In the Opn KO 3W group, the blood vessels were significantly decreased in area and pulp healing was disturbed with a failure of pulpal revascularization and reinnervation., Conclusions: OPN is necessary for proper reinnervation and revascularization to deposit reparative dentin following severe injury within the dental pulp of erupted teeth with advanced root development., Competing Interests: The authors declare no conflicts of interest related to this study., (© 2022 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

Background: Osteoporosis (OP) is a systemic metabolic bone disease characterized by decreased bone mass and destruction of bone microstructure, which tends to result in enhanced bone fragility and related fractures. The postmenopausal osteoporosis (PMOP) has a relatively high proportion, and numerous studies reveal that estrogen-deficiency is related to the imbalance of gut microbiota (GM), impaired intestinal mucosal barrier function and enhanced inflammatory reactivity. However, the underlying mechanisms remain unclear and the existing interventions are also scarce., Methods: In this study, we established a mouse model induced by ovariectomy (OVX) and conducted fecal microbiota transplantation (FMT) by gavage every day for 8 weeks. Subsequently, the bone mass and microarchitecture of mice were evaluated by the micro computed tomography (Micro-CT). The intestinal permeability, pro-osteoclastogenic cytokines expression, osteogenic and osteoclastic activities were detected by the immunohistological analysis, histological examination, enzyme-linked immunosorbent assay (ELISA) and western blot analysis accordingly. Additionally, the composition and abundance of GM were assessed by 16S rRNA sequencing and the fecal short chain fatty acids (SCFAs) level was measured by metabolomics., Results: Our results demonstrated that FMT inhibited the excessive osteoclastogenesis and prevented the OVX-induced bone loss. Specifically, compared with the OVX group, FMT enhanced the expressions of tight junction proteins (zonula occludens protein 1 (ZO-1) and Occludin) and suppressed the release of pro-osteoclastogenic cytokines (tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β)). Furthermore, FMT also optimized the composition and abundance of GM, and increased the fecal SCFAs level (mainly acetic acid and propionic acid)., Conclusions: Collectively, based on GM-bone axis, FMT prevented the OVX-induced bone loss by correcting the imbalance of GM, improving the SCFAs level, optimizing the intestinal permeability and suppressing the release of pro-osteoclastogenic cytokines, which may be an alternative option to serve as a promising candidate for the prevention and treatment of PMOP in the future., The Translational Potential of This Article: This study indicates the ingenious involvement of GM-bone axis in PMOP and the role of FMT in reshaping the status of GM and ameliorating the bone loss in OVX-induced mice. FMT might serve as a promising candidate for the prevention and treatment of PMOP in the future., Competing Interests: The authors have no financial or potential competing interests or affiliations with any institution, organization, or company relating to this current manuscript., (© 2022 The Authors.)

Because of the heterogeneity of lower-grade gliomas (LGGs), patients show various survival outcomes that are not reliably predicted by histological classification. The tumour microenvironment (TME) contributes to the initiation and progression of brain LGGs. Identifying potential prognostic markers based on the immune and stromal components in the TME will provide new insights into the dynamic modulation of these two components of the TME in LGGs. We applied ESTIMATE to calculate the ratio of immune and stromal components from The Cancer Genome Atlas database. After combined differential gene expression analysis, protein-protein interaction network construction and survival analysis, CD44 was screened as an independent prognostic factor and subsequently validated utilizing data from the Chinese Glioma Genome Atlas database. To decipher the association of glioma cell CD44 expression with stromal cells in the TME and tumour progression, RT-qPCR, cell viability and wound healing assays were employed to determine whether astrocytes enhance glioma cell viability and migration by upregulating CD44 expression. Surprisingly, M1 macrophages were identified as positively correlated with CD44 expression by CIBERSORT analysis. CD44 + glioma cells were further suggested to interact with microglia-derived macrophages (M1 phenotype) via osteopontin signalling on the basis of single-cell sequencing data. Overall, we found that astrocytes could elevate the CD44 expression level of glioma cells, enhancing the recruitment of M1 macrophages that may promote glioma stemness via osteopontin-CD44 signalling. Thus, glioma CD44 expression might coordinate with glial activities in the TME and serve as a potential therapeutic target and prognostic marker for LGGs., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Background: Amitriptyline is a tricyclic antidepressant drug accustomed to treat depressive disorders. It recorded many side effects on different tissues., Objective: To investigate reaction of Albino rats' periodontium after oral administration of Amitriptyline histologically and radiographically., Methods: Fourteen adult male albino rats (150-200 g) were divided into two groups, control and experimental. Rats of experimental group received 10 mg⁄kg⁄day of Amitriptyline hydrochloride by oral gavage for four weeks. Mandibles were prepared for hematoxylin and eosin (H&E) and anti-osteopontin (Anti-OPN) immunohistochemistry staining. Bone mineral density was measured in mandibular alveolar bone. Statistical analysis for Anti-OPN and relative Hounsfield unit value (HU value) was performed using independent-samples t -test., Results: Gingiva of experimental group showed epithelial degeneration with pyknotic nuclei and disintegration in lamina propria. Areas of separation in alveolar bone and degeneration of some regions in cementum were seen with apparent increase in periodontal ligament (PDL) thickness and its detachment from bone and cementum at some regions. Immunohistochemical examination of experimental group showed apparently increased immunopositivity in gingiva, cementocytes, osteocytes, cementum, bone matrices, fibroblasts and PDL fibers when compared to control group. Statistical analysis revealed insignificant difference of Anti-OPN area% in gingiva between both studied groups. While there was statistical significant increase of Anti-OPN area% in the other periodontium tissues and high statistical significant decrease of relative HU value in experimental group when compared to control., Conclusions: Amitriptyline has destructive effect on periodontal tissues and statistically increases the expression of Anti-OPN in all periodontal tissues except gingiva and decreases bone mineral density., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Abstract: Zinc is an essential trace element that is involved in diverse metabolic and signaling pathways. Zinc deficiency is associated with retardation of bone growth. Previous in vitro studies have suggested a direct effect of zinc on both the proliferation and differentiation of osteoblast-like cells. However, the mechanisms for uptake of zinc into osteoblasts have not been examined in detail. Several families of zinc transporters have previously been characterized in mammalian cells; such transporters function in the uptake, intracellular sequestration or efflux of zinc. In the current study, we examined zinc transport in osteoprogenitor cells and have attempted to define a functional role for a zinc transport mechanism in osteogenic differentiation. We identified at least two zinc transporters in both human mesenchymal stem cells (MSCs) and in osteoblastic cells—the ubiquitous zinc transporter, ZIP1, and LIV-1, which was previously characterized as a protein that is expressed in breast cancer cells. The subcellular localization of both these zinc transporters suggested distribution in both the plasma membrane and also diffusely in the cytoplasm. During the differentiation process of pluripotent MSCs into osteoblast-like cells, both zinc uptake and expression of the ZIP1 protein were increased. An adenoviral-mediated overexpression of ZIP1 in MSCs resulted in Alizarin-red-positive mineralization and also increased expression of specific osteoblast-associated markers, such as alkaline phosphatase, and of several osteoblast differentiation genes, including osteopontin, Cbfa1/Runx2, promyelocytic leukemia zinc finger and bone sialoprotein. An siRNA-mediated reduction of ZIP1 protein expression in MSCs caused decreased zinc uptake and inhibition of osteoblastic differentiation under osteogenic culture conditions. Finally, following overexpression of ZIP1 in MSCs, cDNA microarray analysis revealed differential regulation of several genes associated with the proliferation of osteoprogenitor cells and osteoblast differentiation. In conclusion, these studies provide important insights into the role of a plasma membrane zinc transporter in the initiation of an osteogenic lineage from MSCs. [Copyright &y& Elsevier]

Abstract: Using a cDNA microarray, we identified osteopontin (OPN) as one of the genes upregulated in cultured activated hepatic stellate cells (HSCs). Northern and western blot analyses showed that OPN was increasingly expressed during the progressive activation of cultured rat HSCs, and a significant increase in OPN was observed in carbon tetrachloride-induced rat liver fibrosis. In biliary atresia, OPN protein was predominantly expressed in Kupffer cells and HSCs in the necrotic areas. Incubation of HSCs with recombinant OPN-induced significant proliferative and migratory effects, and induced matrix metalloproteinase 2 production and activation. Moreover, OPN increased type I collagen production and type II transforming growth factor-β receptor mRNA and protein. In conclusion, this study shows that OPN is expressed in activated HSCs and suggests that the upregulation of OPN might be a central pathway of HSC activation. [Copyright &y& Elsevier]

The transcription factors C/EBPα and Sp1 functionally interact to induce expression of specific genes during myeloid and epithelial cell differentiation. The C/EBPα-Sp1 transcription factor “module” binds to enhancer elements within the upstream regulatory sequences of target genes. In our previous study we identified mouse TPA inducible sequence 7 (TIS7) as a novel co-repressor in epithelial cells undergoing loss of polarity. Increased levels of TIS7 down-regulate the transcription of a specific set of genes. Using bioinformatic analysis we identified a common binding site for the C/EBPα-Sp1 transcription factor module within the upstream regulatory regions of TIS7-regulated genes. The inhibitory effect of TIS7 on C/EBPα-Sp1-mediated transcription was confirmed by reporter assays. Our data showed that the TIS7 effect was mediated through specific interference with Sp1 transcriptional activity. Furthermore, TIS7 prevented formation of a complex between Sp1 protein and its consensus DNA binding site. Data presented here further specify the mechanism of action of the transcriptional co-repressor TIS7 as well as document the strength of a bioinformatic approach for the prediction and analysis of transcription factor modules. [Copyright &y& Elsevier]

Angiogenesis, which is essential for tumor growth, is regulated by various angiogenic factors. Osteopontin (OPN) is expressed in various human tumors and is postulated to be involved in tumor progression. We have recently reported that culture medium with murine neuroblastoma C1300 cells transfected with OPN gene significantly stimulates human umbilical vein endothelial cell migration and induces neovascularization in mice by dorsal air sac assay. However, the effect of OPN on tumorigenesis as an angiogenic factor remains to be clarified. In this study, we injected the OPN-transfected C1300 cells and control cells into the nude mice subcutaneously. OPN-overexpressing C1300 cells significantly formed rapidly growing tumor as compared to the control cells in mice, although in vitro and in vivo cell growth rates were similar. In vivo tumorigenecity of these cells correlated with the amount of secreted OPN protein. In addition, neovascularization of OPN-transfected tumor was significantly increased in comparison with those of control cells by immunohistochemistry for CD31. In vitro chemoinvasiveness and gene expression of proteases including uPA, MMP2, 9, MT1-MMP, and cathepsin B, D, L, were not different between OPN-transfected and control cells determined with matrigel invasion assay and cDNA expression macroarray, respectively. Conclusively, these results strongly imply that OPN plays an important role in tumor growth through the enhancement of angiogenesis in vivo. [Copyright &y& Elsevier]

A characteristic peculiarity of the trigeminal sensory system is the presence of two distinct populations of primary afferent neurons. Most of their cell bodies are located in the trigeminal ganglion (TG) but part of them lie in the mesencephalic trigeminal nucleus (MTN). This review compares the neurochemical content of central versus peripheral trigeminal primary afferent neurons. In the TG, two subpopulations of primary sensory neurons, containing immunoreactive (IR) material, are identified: a number of glutamate (Glu)-, substance P (SP)-, neurokinin A (NKA)-, calcitonin gene-related peptide (CGRP)-, cholecystokinin (CCK)-, somatostatin (SOM)-, vasoactive intestinal polypeptide (VIP)- and galanin (GAL)-IR ganglion cells with small and medium-sized somata, and relatively less numerous larger-sized neuropeptide Y (NPY)- and peptide 19 (PEP 19)-IR trigeminal neurons. In addition, many nitric oxide synthase (NOS)- and parvalbumin (PV)-IR cells of all sizes as well as fewer, mostly large, calbindin D-28k (CB)-containing neurons are seen. The majority of the large ganglion cells are surrounded by SP-, CGRP-, SOM-, CCK-, VIP-, NOS- and serotonin (SER)-IR perisomatic networks. In the MTN, the main subpopulation of large-sized neurons display Glu-immunoreactivity. Additionally, numerous large MTN neurons exhibit PV- and CB-immunostaining. On the other hand, certain small MTN neurons, most likely interneurons, are found to be GABAergic. Furthermore, NOS-containing neurons can be detected in the caudal and the mesencephalic-pontine junction portions of the nucleus. Conversely, no immunoreactivity to any of the examined neuropeptides is observed in the cell bodies of MTN neurons but these are encircled by peptidergic, catecholaminergic, serotonergic and nitrergic perineuronal arborizations in a basket-like manner. Such a discrepancy in the neurochemical features suggests that the differently fated embryonic migration, synaptogenesis, and peripheral and central target field innervation can possibly affect the individual neurochemical phenotypes of trigeminal primary afferent neurons. [Copyright &y& Elsevier]

Objectives: Angiotensin II (Ang II) induces fibroblast proliferation and collagen synthesis in the myocardium, but its precise mechanisms of action in human hearts are still unknown. Therefore, we investigated whether Ang II directly affects the collagen mRNA content in the human myocardium and in isolated human cardiac fibroblasts or whether the growth factors TGFβ-1 and osteopontin are involved in this process. Methods and results I: In a first set of experiments, the direct effect of Ang II on collagen I, TGFβ-1 and osteopontin mRNA content in fresh samples of human atrial myocardium was determined by the use of a short stimulation period. After 4 h, Ang II-stimulated atrial samples gave a significantly higher expression of both TGFβ-1 (183±21% of control, p<0.05) and osteopontin mRNA (275±58%, p<0.02) than the controls. In contrast, the expression of collagen I mRNA was unchanged (95±8%). Stimulation with TGFβ-1 led to an increase in collagen I and III mRNA (127±10%, p<0.05; 140±15%, p<0.02). Methods and results II: In a second protocol, to assess the effects of longer stimulation periods, we determined the effects of Ang II and its potential mediator TGFβ-1 on collagen I, III and fibronectin mRNA expression and on proliferation of cultured human cardiac fibroblasts. Ang II caused a dose-dependent stimulation of proliferation but did not affect collagen I, III or fibronectin mRNA content after 24 h. In contrast, TGFβ-1 stimulation significantly increased collagen I and III mRNA expression (124±5% and 128±5%, p<0.002). Conclusions: In the human heart, Ang II does not directly increase collagen or fibronectin mRNA, but it does increase TGFβ-1 and osteopontin mRNA expression. Since TGFβ-1 induces collagen I and III mRNA in atrial samples and in isolated cardiac fibroblasts, it may represent a necessary mediator of the Ang II effects in the human heart. [ABSTRACT FROM PUBLISHER]

The imbalance of bone homeostasis is the root cause of osteoporosis. However current therapeutic approaches mainly focus on either anabolic or catabolic pathways, which often fail to turn the imbalanced bone metabolism around. Herein we reported that a SIRT-1 agonist mediated molecular therapeutic strategy to reverse the imbalance in bone homeostasis by simultaneously regulating osteogenesis and osteoclastogenesis via locally sustained release of SRT2104 from mineral coated acellular matrix microparticles. Immobilization of SRT2104 on mineral coating (MAM/SRT) harnessing their electrostatic interactions resulted in sustained release of SIRT-1 agonist for over 30 days. MAM/SRT not only enhanced osteogenic differentiation and mineralization, but also attenuated the formation and function of excessive osteoclasts via integrating multiple vital upstream signals (β-catenin, FoxOs, Runx2, NFATc1, etc.) in vitro . Osteoporosis animal model also validated that it accelerated osteoporotic bone healing and improved osseointegration of the surrounding bone. Overall, our work proposes a promising strategy to treat osteoporotic bone defects by reversing the imbalance in bone homeostasis using designated small molecule drug delivery systems., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2022 The Authors.)

Hepatocellular carcinoma (HCC) is the predominant primary cancer arising from the liver and is one of the major causes of cancer-related mortality worldwide. The cellular origin of HCC has been a topic of great interest due to conflicting findings regarding whether it originates in hepatocytes, biliary cells, or facultative stem cells. These cell types all undergo changes during liver injury, and there is controversy about their contribution to regenerative responses in the liver. Most HCCs emerge in the setting of chronic liver injury from viral hepatitis, fatty liver disease, alcohol, and environmental exposures. The injuries are marked by liver parenchymal changes such as hepatocyte regenerative nodules, biliary duct cellular changes, expansion of myofibroblasts that cause fibrosis and cirrhosis, and inflammatory cell infiltration, all of which may contribute to carcinogenesis. Addressing the cellular origin of HCC is the key to identifying the earliest events that trigger it. Herein, we review data on the cells of origin in regenerating liver and HCC and the implications of these findings for prevention and treatment. We also review the origins of childhood liver cancer and other rare cancers of the liver., Competing Interests: The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2021 The Authors.)

Purpose: The development of novel and efficient biomarkers for primary bone cancers is of grave importance., Methods: The expression pattern of osteopontin (OPN) was investigated in the 153 patients with benign (n = 72) and malignant (n = 81) primary bone cancers. Both local and circulating OPN mRNA expression levels and their protein concentration in serum and tumor site were assessed using real-time qRT-PCR, ELISA, and immunohistochemistry techniques, respectively. As a control, 29 healthy individuals were considered. The number of 153 tumor tissue specimens and the 153 paired margins were taken on surgical resection from the patients. 153 blood samples were also drained from all participants, then peripheral blood mononuclear cells (PBMC) and sera were separated., Results: The mean mRNA expression was significantly higher in all of the cancerous tissues than the paired margins and the PBMC of the patients than the controls. Consistently, the protein concentrations of OPN in serum and tumor tissues were significantly higher in the patients. Furthermore, the malignant cases had significantly elevated the mRNA levels and the protein compared to the benign cases. OPN could potentially differentiate the patients from the controls with 100% sensitivity and specificity in serum. Moreover, OPN could predict some of the malignant cases' clinicopathological features, including metastasis, recurrence, grade, and response to chemotherapy., Conclusions: In conclusion, OPN might be involved in the pathogenesis of primary bone tumors and can be considered as a potential biomarker to bone cancer diagnosis., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier GmbH.)

Background: Wounds with embedded metal fragments are an unfortunate consequence of armed conflicts. In many cases the exact identity of the metal(s) and their long-term health effects, especially on the kidney, are not known., Aim of Study: The aim of this study was to quantitate the urinary levels of metals solubilized from surgically implanted metal pellets and to assess the effect of these metals on the kidney using a battery of biomarker assays., Materials and Methods: Using a rodent model system developed in our Institute to simulate embedded fragment injuries, eight metals considered likely components of an embedded fragment wound were individually implanted into the gastrocnemius muscle of male Sprague-Dawley rats. The rats were followed for 12 months post-implantation with urine collected prior to surgery then at 1-, 3-, 6-, 9-, and 12-months post-implantation to provide a within-subjects cohort for examination. Urinary metal levels were determined using inductively coupled plasma-mass spectrometry and urinary biomarkers assessed using commercially available kits to determine metal-induced kidney effects., Results: With few exceptions, most of the implanted metals rapidly solubilized and were found in the urine at significantly higher levels than in control animals as early as 1-month post-implantation. Surprisingly, many of the biomarkers measured were decreased compared to control at 1-month post-implantation before returning to normal at the later time points. However, two metals, iron and depleted uranium, showed increased levels of several markers at later time points, yet these levels also returned to normal as time progressed., Conclusion: This study showed that metal pellets surgically implanted into the leg muscle of Sprague-Dawley rats rapidly solubilized with significant levels of the implanted metal found in the urine. Although kidney biomarker results were inconsistent, the changes observed along with the relatively low amounts of metal implanted, suggest that metal-induced renal effects need to be considered when caring for individuals with embedded metal fragment wounds., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Effects of high magnetic fields [MFs, ≥ 1 T (T)] on osteoblastic differentiation and the orientation of cells or matrix proteins have been reported. However, the effect of low MFs (< 1 T) on the orientation of bone formation is not well known. This study was performed to verify the effects of low MFs on osteoblastic differentiation, bone formation, and orientation of both cells and newly formed bone. An apparatus was prepared with two magnets (190 mT) aligned in parallel to generate a parallel MF. In vitro , bone marrow-derived stromal cells of rats were used to assess the effects of low MFs on cell orientation, osteoblastic differentiation, and mineralization. A bone morphogenetic protein (BMP)-2-induced ectopic bone model was used to elucidate the effect of low MFs on microstructural indices, trabecula orientation, and the apatite c -axis orientation of newly formed bone. Low MFs resulted in an increased ratio of cells oriented perpendicular to the direction of the MF and promoted osteoblastic differentiation in vitro . Moreover, in vivo analysis demonstrated that low MFs promoted bone formation and changed the orientation of trabeculae and apatite crystal in a direction perpendicular to the MF. These changes led to an increase in the mechanical strength of rhBMP-2-induced bone. These results suggest that the application of low MFs has potential to facilitate the regeneration of bone with sufficient mechanical strength by controlling the orientation of newly formed bone., Competing Interests: This study was funded by the Pip Corporation, Japan., (© 2021 The Author(s).)

Bone biomineralization is a complex process in which type I collagen and associated non-collagenous proteins (NCPs), including glycoproteins and proteoglycans, interact closely with inorganic calcium and phosphate ions to control the precipitation of nanosized, non-stoichiometric hydroxyapatite (HAP, idealized stoichiometry Ca 10 (PO 4 ) 6 (OH) 2 ) within the organic matrix of a tissue. The ability of certain vertebrate tissues to mineralize is critically related to several aspects of their function. The goal of this study was to identify specific NCPs in mineralizing and non-mineralizing tissues of two animal models, rat and turkey, and to determine whether some NCPs are unique to each type of tissue. The tissues investigated were rat femur (mineralizing) and tail tendon (non-mineralizing) and turkey leg tendon (having both mineralizing and non-mineralizing regions in the same individual specimen). An experimental approach ex vivo was designed for this investigation by combining sequential protein extraction with comprehensive protein mapping using proteomics and Western blotting. The extraction method enabled separation of various NCPs based on their association with either the extracellular organic collagenous matrix phases or the inorganic mineral phases of the tissues. The proteomics work generated a complete picture of NCPs in different tissues and animal species. Subsequently, Western blotting provided validation for some of the proteomics findings. The survey then yielded generalized results relevant to various protein families, rather than only individual NCPs. This study focused primarily on the NCPs belonging to the small leucine-rich proteoglycan (SLRP) family and the small integrin-binding ligand N-linked glycoproteins (SIBLINGs). SLRPs were found to be associated only with the collagenous matrix, a result suggesting that they are mainly involved in structural matrix organization and not in mineralization. SIBLINGs as well as matrix Gla (γ-carboxyglutamate) protein were strictly localized within the inorganic mineral phase of mineralizing tissues, a finding suggesting that their roles are limited to mineralization. The results from this study indicated that osteocalcin was closely involved in mineralization but did not preclude possible additional roles as a hormone. This report provides for the first time a spatial survey and comparison of NCPs from mineralizing and non-mineralizing tissues ex vivo and defines the proteome of turkey leg tendons as a model for vertebrate mineralization., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Published by Elsevier Inc.)

Objective: The antibacterial activity of copper (Cu)-alloy biomaterials has shown a great potential in clinical application. Here, we evaluated the osteogenesis and antibacterial effects of Ti6Al4V-6.5wt%Cu alloy in an in vivo model of infected bone defects and determine their responsible proteins and pathways using proteomics., Methods: After bone defects were filled with Ti6Al4V and Ti6Al4V-6.5wt%Cu implants for 6 week, the tissue and bone samples around the implants were harvested for radiographic, micro-CT, histological, and bone-related gene expression analyses. An iTRAQ-based protein identification/quantification approach was used to analyze the osteogenic and antibacterial effects of Ti6Al4V-6.5wt%Cu alloy., Results: Imaging and histological results showed Ti6Al4V alloy induced a stronger inflammatory response than Ti6Al4V-6.5wt%Cu alloy; imaging results and osteogenic protein levels showed Ti6Al4V-6.5wt%Cu alloy exerted a stronger osteogenic effect. In vitro experiment, we found the Ti6Al4V-6.5wt%Cu had significant antibacterial effects and inhibited the activity of Staphylococcus aureus in the early stage. In addition, the bacterial biofilm formed in Ti6Al4V-6.5wt%Cu group was significantly lower than that in Ti6Al4V group. Proteomic screening of 4279 proteins resulted in 35 differentially expressed proteins for further examination which were mainly associated with the cellular process, metabolic process, stimulus response, and cellular component organization. In further exploration of the mechanism of osteogenic mineralization of Ti6Al4V-6.5wt%Cu alloy, we found out SDC4 and AGRN were the top two target proteins associated with osteogenic differentiation and bone mineralization., Conclusion: Ti6Al4V-6.5wt%Cu alloy shows a great potential as a bone implant material due to its positive effects against bacterial infection and on bone formation., The Translational Potential of This Article: At present, titanium alloys and other non-antibacterial metal materials are used in orthopedic internal fixation operations. Our study demonstrates that Ti6Al4V-6.5wt%Cu alloy has good antibacterial and osteogenic effects in vivo and in vitro. This means that Ti6Al4V-6.5wt%Cu alloy may become a new kind of antimicrobial metallic material as internal fixation material to continuously exert its antimicrobial effects and reduce the infection rate after clinical internal fixation., Competing Interests: The authors have no conflicts of interest to disclose in relation to this article., (© 2020 The Author(s).)

Mesoporous silica thin film has been widely used in various fields, particularly the medical implant coating for drug delivery. However, some drawbacks remain with the films produced by traditional method (evaporation-induced self-assembly, EISA), such as the poor permeability caused by their horizontal aligned mesochannels. In this study, the vertical aligned mesoporous silica thin film (VMSTF) is uniformly grown alongside the walls of titania nanotubes array via a biphase stratification growth method, resulting in a hierarchical two-layered nanotubular structure. Due to the exposure of opened mesopores, VMSTF exhibits more appealing performances, including rapid degradation, efficient small-molecular drug (dexamethasone) loading and release, enhanced early adhesion and osteogenic differentiation of MC3T3-E1 cells. This is the first time successfully depositing VMSTF on nanoporous substrate and our findings suggest that the VMSTF may be a promising candidate for bone implant surface coating to obtain bioactive performances., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.)

Introduction: Obtaining a certain bone volume is an important goal in implantology or orthopedics. Thus, after tooth extraction, quite a lot of horizontal and vertical alveolar bone is lost in time and can be detrimental to the implant treatment outcome, while the treatment of critical bone defects is a considerable challenge for surgery., Objectives: In this study we designed a new in vivo model as an useful experimental tool to assess guided bone regeneration (GBR) using a computer-aided design/manufacturing (CAD-CAM) space-maintaining barrier., Methods: The barrier was 3D printed with three progressive heights, surgically placed on rat femur, and GBR results were analyzed at 2, 4, and 8 weeks by X-ray and bone mineral density analysis, histology/morphometry and by immunofluorescence and immunohistochemistry for osteogenesis and angiogenesis evaluation., Results: The obtained results show that the proposed experimental model provides a real-time useful information on progressive bone tissue formation, which depends on the volume of isolated space created for GBR and on molecular events that lead to satisfactory vertical and horizontal bone augmentation and osteointegration., Conclusion: In conclusion, the proposed customized three-dome space-maintaining barrier is suitable as an experimental tool to assess the potential of using the designed barriers in dentistry and orthopedics to promote the formation of new bone and determine their space- and time-dependent limitations. Meanwhile, guided bone augmentation for dentistry requires subsequent evaluation on an alveolar bone preclinical model followed by clinical implementation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2020 The Authors. Published by Elsevier B.V. on behalf of Cairo University.)

This study sought to investigate whether reactive oxygen species (ROS)-generating reduced nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) contributes to calcific aortic valve disease (CAVD) or whether celastrol, a natural Nox2 inhibitor, may provide potential therapeutic target for CAVD. CAVD is an active and cellular-driven fibrocalcific process characterized by differentiation of aortic valvular interstitial cells (AVICs) toward an osteogenic-like phenotype. ROS levels increase in calcified aortic valves, while the sources of ROS and their roles in the pathogenesis of CAVD are elusive. The roles of Nox2 and the effects of celastrol were studied using cultured porcine AVICs in vitro and a rabbit CAVD model in vivo. Nox2 proteins were significantly upregulated in human aortic valves with CAVD. In vitro, Nox2 was markedly induced upon stimulation of AVICs with osteogenic medium, along with the increases in ROS production and calcium nodule formation. Celastrol significantly decreased calcium deposition of AVICs by 35%, with a reduction of ROS generation. Knockdown of endogenous Nox2 substantially suppressed AVIC calcification by 39%, the inhibitory effect being similar to celastrol treatment. Mechanistically, either celastrol treatment or knockdown of Nox2 significantly inhibited glycogen synthase kinase 3 beta/β-catenin signaling, leading to attenuation of fibrogenic and osteogenic responses of AVICs. In a rabbit CAVD model, administration of celastrol significantly reduced aortic valve ROS production, fibrosis, calcification, and severity of aortic stenosis, with less left ventricular dilatation and better preserved contractile function. Upregulation of Nox2 is critically involved in CAVD. Celastrol is effective to alleviate CAVD, likely through the inhibition of Nox2-mediated glycogen synthase kinase 3 beta/β-catenin pathway in AVICs., (Crown Copyright © 2020 Published by Elsevier on behalf of American College of Cardiology Foundation.)

The glucagon-like peptide-1 receptor agonists (GLP-1RAs) liraglutide and semaglutide reduce cardiovascular risk in type 2 diabetes patients. The mode of action is suggested to occur through modified atherosclerotic progression. In this study, both of the compounds significantly attenuated plaque lesion development in apolipoprotein E-deficient (ApoE -/- ) mice and low-density lipoprotein receptor-deficient (LDLr -/- ) mice. This attenuation was partly independent of weight and cholesterol lowering. In aortic tissue, exposure to a Western diet alters expression of genes in pathways relevant to the pathogenesis of atherosclerosis, including leukocyte recruitment, leukocyte rolling, adhesion/extravasation, cholesterol metabolism, lipid-mediated signaling, extracellular matrix protein turnover, and plaque hemorrhage. Treatment with semaglutide significantly reversed these changes. These data suggest GLP-1RAs affect atherosclerosis through an anti-inflammatory mechanism.

Background & Aims: Uncontrolled liver proliferation is a key characteristic of liver cancer; however, the mechanisms by which this occurs are not well understood. Elucidation of these mechanisms is necessary for the development of better therapy. The oncogene Gankyrin (Gank) is overexpressed in both hepatocellular carcinoma and hepatoblastoma. The aim of this work was to determine the role of Gank in liver proliferation and elucidate the mechanism by which Gank promotes liver proliferation., Methods: We generated Gank liver-specific knock-out (GLKO) mice and examined liver biology and proliferation after surgical resection and liver injury., Results: Global profiling of gene expression in GLKO mice showed significant changes in pathways involved in liver cancer and proliferation. Investigations of liver proliferation after partial hepatectomy and CCl 4 treatment showed that GLKO mice have dramatically inhibited proliferation of hepatocytes at early stages after surgery and injury. In control LoxP mice, liver proliferation was characterized by Gank-mediated reduction of tumor-suppressor proteins (TSPs). The failure of GLKO hepatocytes to proliferate is associated with a lack of down-regulation of these proteins. Surprisingly, we found that hepatic progenitor cells of GLKO mice start proliferation at later stages and restore the original size of the liver at 14 days after partial hepatectomy. To examine the proliferative activities of Gank in cancer cells, we used a small molecule, cjoc42, to inhibit interactions of Gank with the 26S proteasome. These studies showed that Gank triggers degradation of TSPs and that cjoc42-mediated inhibition of Gank increases levels of TSPs and inhibits proliferation of cancer cells., Conclusions: These studies show that Gank promotes hepatocyte proliferation by elimination of TSPs. This work provides background for the development of Gank-mediated therapy for the treatment of liver cancer. RNA sequencing data can be accessed in the NCBI Gene Expression Omnibus: GSE104395.

Histone acetylation is an important epigenetic mechanism that controls expression of certain genes. It includes non-sequence-based changes of chromosomal regional structure that can alter the expression of genes. Acetylation of histones is controlled by the activity of two groups of enzymes: the histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs remove acetyl groups from the histone tail, which alters its charge and thus promotes compaction of DNA in the nucleosome. HDACs render the chromatin structure into a more compact form of heterochromatin, which makes the genes inaccessible for transcription. By altering the transcriptional activity of bone-associated genes, HDACs control both osteogenesis and osteoclastogenesis. This review presents an overview of the function of HDACs in the modulation of bone formation. Special attention is paid to the use of HDAC inhibitors in mineralized tissue regeneration from cells of dental origin.

The isoflavone calycosin-7-O-β-d-glucopyranoside (CG) is a principal constituent of Astragalus membranaceus (AR) and has been reported to inhibit osteoclast development in vitro and bone loss in vivo. The aim of this study was to investigate the osteogenic effects of CG and its underlying mechanism in ST2 cells. The results show that exposure of cells to CG in osteogenic differentiation medium increases ALP activity, osteocalcin (Ocal) mRNA expression and the osteoblastic mineralization process. Mechanistically, CG treatment increased the expression of bone morphogenetic protein 2 (BMP-2), p-Smad 1/5/8, β-catenin and Runx2, all of which are regulators of the BMP- or wingless-type MMTV integration site family (WNT)/β-catenin-signaling pathways. Moreover, the osteogenic effects of CG were inhibited by Noggin and DKK-1 which are classical inhibitors of the BMP and WNT/β-catenin-signaling pathways, respectively. Taken together, the results indicate that CG promotes the osteoblastic differentiation of ST2 cells through regulating the BMP/WNT signaling pathways. On this basis, CG may be a useful lead compound for improving the treatment of bone-decreasing diseases and enhancing bone regeneration.