Manganese Enhances the Osteogenic Effect of Silicon-Hydroxyapatite Nanowires by Targeting T Lymphocyte Polarization.

Biomaterials encounter considerable challenges in extensive bone defect regeneration. The amelioration of outcomes may be attainable through the orchestrated modulation of both innate and adaptive immunity. Silicon-hydroxyapatite, for instance, which solely focuses on regulating innate immunity, is inadequate for long-term bone regeneration. Herein, extra manganese (Mn)-doping is utilized for enhancing the osteogenic ability by mediating adaptive immunity. Intriguingly, Mn-doping engenders heightened recruitment of CD4+ T cells to the bone defect site, concurrently manifesting escalated T helper (Th) 2 polarization and an abatement in Th1 cell polarization. This consequential immune milieu yields a collaborative elevation of interleukin 4, secreted by Th2 cells, coupled with attenuated interferon gamma, secreted by Th1 cells. This orchestrated interplay distinctly fosters the osteogenesis of bone marrow stromal cells and effectuates consequential regeneration of the mandibular bone defect. The modulatory mechanism of Th1/Th2 balance lies primarily in the indispensable role of manganese superoxide dismutase (MnSOD) and the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK). In conclusion, this study highlights the transformative potential of Mn-doping in amplifying the osteogenic efficacy of silicon-hydroxyapatite nanowires by regulating T cell-mediated adaptive immunity via the MnSOD/AMPK pathway, thereby creating an anti-inflammatory milieu favorable for bone regeneration.

Matrix mechanics regulate the polarization state of bone marrow-derived neutrophils through the JAK1/STAT3 signaling pathway.

Matrix mechanics regulate essential cell behaviors through mechanotransduction, and as one of its most important elements, substrate stiffness was reported to regulate cell functions such as viability, communication, migration, and differentiation. Neutrophils (Neus) predominate the early inflammatory response and initiate regeneration. The activation of Neus can be regulated by physical cues; however, the functional alterations of Neus by substrate stiffness remain unknown, which is critical in determining the outcomes of engineered tissue mimics. Herein, a three-dimensional (3D) culture system made of hydrogels was developed to explore the effects of varying stiffnesses (1.5, 2.6, and 5.7 kPa) on the states of Neus. Neus showed better cell integrity and viability in the 3D system. Moreover, it was shown that the stiffer matrix tended to induce Neus toward an anti-inflammatory phenotype (N2) with less adhesion molecule expression, less reactive oxygen species (ROS) production, and more anti-inflammatory cytokine secretion. Additionally, the aortic ring assay indicated that Neus cultured in a stiffer matrix significantly increased vascular sprouting. RNA sequencing showed that a stiffer matrix could significantly activate JAK1/STAT3 signaling in Neus and the inhibition of JAK1 ablated the stiffness-dependent increase in the expression of CD182 (an N2 marker). Taken together, these results demonstrate that a stiffer matrix promotes Neus to shift to the N2 phenotype, which was regulated by JAK1/STAT3 pathway. This study lays the groundwork for further research on fabricating engineered tissue mimics, which may provide more treatment options for ischemic diseases and bone defects. STATEMENT OF SIGNIFICANCE.

An update on the role of tumor necrosis factor alpha stimulating gene-6 in inflammatory diseases.

At present, the occurrence and development of inflammatory diseases are closely related to the abnormal changes of the content and function of many cytokines. At the same time, targeting related cytokines to prevent and treat diseases has also achieved good results. Therefore, it is very important to explore the role of various cytokines in inflammatory diseases. As an inflammation related protein, Tumor necrosis factor alpha stimulating gene-6 (TSG-6) has attracted more and more attention. In the process of disease, it's like a double-edged sword, showing different responses. It is constitutively expressed in some tissues with high metabolic activity and barrier protection. The diversity of its functions depends on the binding of TSG-6 with a variety of ligands, including matrix molecules, autoimmune regulatory factors and growth factors, participating in extracellular matrix remodeling and regulating protease network. This paper reviews the structure, biological function and research progress of TSG-6 in inflammatory diseases, in order to provide reference for drug development in the future.

Dual-layer dual-energy CT for improving differential diagnosis of squamous cell carcinoma from adenocarcinoma at gastroesophageal junction.

Objective: To examine the clinical values of dual-energy CT parameters derived from dual-layer spectral detector CT (SDCT) in the differential diagnosis of squamous cell carcinoma (SCC) and adenocarcinoma (AC) of the gastroesophageal junction (GEJ). Methods: Totally 66 patients with SCC and AC of the GEJ confirmed by pathological analysis were retrospectively enrolled, and underwent dual-phase contrast-enhancement chest CT with SDCT. Plain CT value, CT attenuation enhancement (△CT), iodine concentration (IC), spectral slope (λHU), effective atomic number (Zeff) and 40keV CT value (CT40keV) of the lesion in the arterial phase (AP) and venous phase (VP) were assessed. Multivariate logistic regression analysis was performed to evaluate the diagnostic efficacies of different combinations of dual-energy CT parameters. Receiver operating characteristic (ROC) curves were used to analyze the accuracy of dual-energy CT parameters and Delong test was used to compare AUCs. Results: IC, λHU, Zeff and CT40keV in AP and VP and △CT in VP were significantly higher in the AC group than those in the SCC group (all P<0.05). ROC curve analysis showed that IC, λHU, Zeff and CT40keV in VP had high diagnostic performances, with AUCs of 0.74, 0.74, 0.79 and 0.78, respectively. Logistic regression showed the combination of ICVP, λHU VP, CT40keV VP and Zeff VP had the highest AUC (0.84), with a threshold of 0.40, sensitivity and specificity in distinguishing SCC and AC were 93.1% and 73.0%, respectively. Delong test showed that the AUC of △CTVP was lower than other AUCs of dual-energy CT parameters. Conclusion: Dual-energy CT parameters derived from SDCT provide added value in the differential diagnosis of SCC and AC of the GEJ, especially the combination of IC, λHU, CT40keV and Zeff in VP. Advances in knowledge: Dual-energy CT parameters derived from dual-layer spectral detector CT provide added value to differentiate AC from SCC at the GEJ, especially the combination of effective atomic number, spectral slope, iodine concentration and 40keV CT value in VP.

Histone demethylase JMJD3 downregulation protects against aberrant force-induced osteoarthritis through epigenetic control of NR4A1.

Osteoarthritis (OA) is a prevalent joint disease with no effective treatment strategies. Aberrant mechanical stimuli was demonstrated to be an essential factor for OA pathogenesis. Although multiple studies have detected potential regulatory mechanisms underlying OA and have concentrated on developing novel treatment strategies, the epigenetic control of OA remains unclear. Histone demethylase JMJD3 has been reported to mediate multiple physiological and pathological processes, including cell differentiation, proliferation, autophagy, and apoptosis. However, the regulation of JMJD3 in aberrant force-related OA and its mediatory effect on disease progression are still unknown. In this work, we confirmed the upregulation of JMJD3 in aberrant force-induced cartilage injury in vitro and in vivo. Functionally, inhibition of JMJD3 by its inhibitor, GSK-J4, or downregulation of JMJD3 by adenovirus infection of sh-JMJD3 could alleviate the aberrant force-induced chondrocyte injury. Mechanistic investigation illustrated that aberrant force induces JMJD3 expression and then demethylates H3K27me3 at the NR4A1 promoter to promote its expression. Further experiments indicated that NR4A1 can regulate chondrocyte apoptosis, cartilage degeneration, extracellular matrix degradation, and inflammatory responses. In vivo, anterior cruciate ligament transection (ACLT) was performed to construct an OA model, and the therapeutic effect of GSK-J4 was validated. More importantly, we adopted a peptide-siRNA nanoplatform to deliver si-JMJD3 into articular cartilage, and the severity of joint degeneration was remarkably mitigated. Taken together, our findings demonstrated that JMJD3 is flow-responsive and epigenetically regulates OA progression. Our work provides evidences for JMJD3 inhibition as an innovative epigenetic therapy approach for joint diseases by utilizing p5RHH-siRNA nanocomplexes.

Beta-tricalcium phosphate promotes osteogenic differentiation of bone marrow-derived mesenchymal stem cells through macrophages.

Macrophages are vital regulators of skeletal remodeling and osseous repair. Beta-tricalcium phosphate (ß-TCP) is a synthetic ceramic biomaterial that has shown promise as bone substitute. However, whether and how ß-TCP affects osteogenesis-related responses of macrophages has rarely been studied. The aims of this study were to explore (a) the effects of ß-TCP on osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) co-cultured with macrophages and (b) on macrophage polarization as well as macrophage gene and protein expression profiles. BMSC osteogenic differentiation capacity in vitro was enhanced in ß-TCP-induced co-cultured BMSCs compared to that in BMSC monocultures. We also found that macrophages induced with 25 mg ml-1 ß-TCP extract had more significant immune responses and switched to the M2 phenotype. Expression levels of the Wnt signaling pathway modulators wingless-type MMTV integration site family, member 6 (WNT6) and Wnt inhibitory factor 1 (WIF1) were upregulated and downregulated, respectively, in macrophages treated with ß-TCP extract. Our findings suggest that ß-TCP enhances osteogenic differentiation of BMSCs by inducing macrophage polarization and by regulating the Wnt signaling pathway, thereby highlighting its therapeutic potential for bone healing through osteoimmunomodulatory properties.

Transcriptome and proteome profiling reveal complementary scavenger and immune features of rat liver sinusoidal endothelial cells and liver macrophages.

BACKGROUND: Liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs; liver resident macrophages) form the body's most effective scavenger cell system for the removal of harmful blood-borne substances, ranging from modified self-proteins to pathogens and xenobiotics. Controversies in the literature regarding the LSEC phenotype pose a challenge when determining distinct functionalities of KCs and LSECs. This may be due to overlapping functions of the two cells, insufficient purification and/or identification of the cells, rapid dedifferentiation of LSECs in vitro, or species differences. We therefore characterized and quantitatively compared expressed gene products of freshly isolated, highly pure LSECs (fenestrated SE-1/FcγRIIb2+) and KCs (CD11b/c+) from Sprague Dawley, Crl:CD (SD), male rats using high throughput mRNA-sequencing and label-free proteomics. RESULTS: We observed a robust correlation between the proteomes and transcriptomes of the two cell types. Integrative analysis of the global molecular profile demonstrated the immunological aspects of LSECs. The constitutive expression of several immune genes and corresponding proteins of LSECs bore some resemblance with the expression in macrophages. LSECs and KCs both expressed high levels of scavenger receptors (SR) and C-type lectins. Equivalent expression of SR-A1 (Msr1), mannose receptor (Mrc1), SR-B1 (Scarb1), and SR-B3 (Scarb2) suggested functional similarity between the two cell types, while functional distinction between the cells was evidenced by LSEC-specific expression of the SRs stabilin-1 (Stab1) and stabilin-2 (Stab2), and the C-type lectins LSECtin (Clec4g) and DC-SIGNR (Clec4m). Many immune regulatory factors were differentially expressed in LSECs and KCs, with one cell predominantly expressing a specific cytokine/chemokine and the other cell the cognate receptor, illustrating the complex cytokine milieu of the sinusoids. Both cells expressed genes and proteins involved in antigen processing and presentation, and lymphocyte co-stimulation. CONCLUSIONS: Our findings support complementary and partly overlapping scavenging and immune functions of LSECs and KCs. This highlights the importance of including LSECs in studies of liver immunity, and liver clearance and toxicity of large molecule drugs and nano-formulations.

The effects of intro-oral parathyroid hormone on the healing of tooth extraction socket: an experimental study on hyperglycemic rats

Abstract Objective To investigate the effects of intro-oral injection of parathyroid hormone (PTH) on tooth extraction wound healing in hyperglycemic rats. Methodology 60 male Sprague-Dawley rats were randomly divided into the normal group (n=30) and DM group (n=30). Type 1 diabetes mellitus (DM) was induced by streptozotocin. After extracting the left first molar of all rats, each group was further divided into 3 subgroups (n=10 per subgroup), receiving the administration of intermittent PTH, continuous PTH and saline (control), respectively. The intermittent-PTH group received intra-oral injection of PTH three times per week for two weeks. A thermosensitive controlled-release hydrogel was synthesized for continuous-PTH administration. The serum chemistry was determined to evaluate the systemic condition. All animals were sacrificed after 14 days. Micro-computed tomography (Micro-CT) and histological analyses were used to evaluate the healing of extraction sockets. Results The level of serum glucose in the DM groups was significantly higher than that in the non-DM groups (p<0.05); the level of serum calcium was similar in all groups (p>0.05). Micro-CT analysis showed that the DM group had a significantly lower alveolar bone trabecular number (Tb.N) and higher trabecular separation (Tb.Sp) than the normal group (p<0.05). The histological analyses showed that no significant difference in the amount of new bone (hard tissue) formation was found between the PTH and non-PTH groups (p>0.05). Conclusions Bone formation in the extraction socket of the type 1 diabetic rats was reduced. PTH did not improve the healing of hard and soft tissues. The different PTH administration regimes (continuous vs. intermittent) had similar effect on tissue healing. These results demonstrated that the metabolic characteristics of the hyperglycemic rats produced a condition that was unable to respond to PTH treatment.

Efficient uptake of blood-borne BK and JC polyomavirus-like particles in endothelial cells of liver sinusoids and renal vasa recta.

Liver sinusoidal endothelial cells (LSECs) are specialized scavenger cells that mediate high-capacity clearance of soluble waste macromolecules and colloid material, including blood-borne adenovirus. To explore if LSECs function as a sink for other viruses in blood, we studied the fate of virus-like particles (VLPs) of two ubiquitous human DNA viruses, BK and JC polyomavirus, in mice. Like complete virions, VLPs specifically bind to receptors and enter cells, but unlike complete virions, they cannot replicate. 125I-labeled VLPs were used to assess blood decay, organ-, and hepatocellular distribution of ligand, and non-labeled VLPs to examine cellular uptake by immunohisto- and -cytochemistry. BK- and JC-VLPs rapidly distributed to liver, with lesser uptake in kidney and spleen. Liver uptake was predominantly in LSECs. Blood half-life (∼1 min), and tissue distribution of JC-VLPs and two JC-VLP-mutants (L55F and S269F) that lack sialic acid binding affinity, were similar, indicating involvement of non-sialic acid receptors in cellular uptake. Liver uptake was not mediated by scavenger receptors. In spleen, the VLPs localized to the red pulp marginal zone reticuloendothelium, and in kidney to the endothelial lining of vasa recta segments, and the transitional epithelium of renal pelvis. Most VLP-positive vessels in renal medulla did not express PV-1/Meca 32, suggesting location to the non-fenestrated part of vasa recta. The endothelial cells of these vessels also efficiently endocytosed a scavenger receptor ligand, formaldehyde-denatured albumin, suggesting high endocytic activity compared to other renal endothelia. We conclude that LSECs very effectively cleared a large fraction of blood-borne BK- and JC-VLPs, indicating a central role of these cells in early removal of polyomavirus from the circulation. In addition, we report the novel finding that a subpopulation of endothelial cells in kidney, the main organ of polyomavirus persistence, showed selective and rapid uptake of VLPs, suggesting a role in viremic organ tropism.

Characteristics of polyomavirus BK (BKPyV) infection in primary human urothelial cells.

High-level polyomavirus BK (BKPyV) replication in urothelial cells is a hallmark of polyomavirus-associated hemorrhagic cystitis (PyVHC), a painful condition affecting bone marrow transplant recipients. In kidney transplant recipients, replication in tubular epithelial cells is associated with overt disease whereas high-level urothelial replication is clinically silent. We characterized BKPyV replication in primary human urothelial cells (HUCs) and compared it to replication in renal tubular epithelial cells (RPTECs). HUCs were easily infected, as shown by expression of T-antigens, VP1-3, and agnoprotein, and intranuclear virion production. Compared to RPTECs, progeny release was delayed by ≥24h and reduced. BKPyV-infected HUCs rounded up like "decoy cells" and detached without necrosis as shown by delayed cytokeratin-18 release, real-time viability monitoring and imaging. The data show that BKV infection of HUCs and RPTECs is significantly different and support the notion that PyVHC pathogenesis is not solely due to BKPyV replication, but likely requires urotoxic and immunological cofactors.

Fluoroquinolones inhibit human polyomavirus BK (BKV) replication in primary human kidney cells.

Reactivation of human polyomavirus BK (BKV) may cause polyomavirus-associated nephropathy or polyomavirus-associated hemorrhagic cystitis in renal- or bone marrow-transplant patients, respectively. Lack of treatment options has led to exploration of fluoroquinolones that inhibit topoisomerase II and IV in prokaryotes and possibly large T-antigen (LT-ag) helicase activity in polyomavirus. We characterized the effects of ofloxacin and levofloxacin on BKV replication in the natural host cells - primary human renal proximal tubular epithelial cells (RPTECs). Ofloxacin and levofloxacin inhibited BKV load in a dose-dependent manner yielding a ∼90% inhibition at 150 µg/ml. Ofloxacin at 150 µg/ml inhibited LT-ag mRNA and protein expression from 24h post infection (hpi). BKV genome replication was 77% reduced at 48 hpi and a similar reduction was found in VP1 and agnoprotein expression. At 72 hpi, the reduction in genome replication and protein expression was less pronounced. A dose-dependent cytostatic effect was noted. In infected cells, 150 µg/ml ofloxacin led to a 26% and 6% inhibition of cellular DNA replication and total metabolic activity, respectively while 150 µg/ml levofloxacin affected this slightly more, particularly in uninfected cells. Cell counting and xCELLigence results revealed that cell numbers were not reduced. In conclusion, ofloxacin and levofloxacin inhibit but do not eradicate BKV replication in RPTECs. At a concentration of ofloxacin giving ∼90% inhibition in BKV load, no significant cytotoxicity was observed. This concentration can be achieved in urine and possibly in the kidneys. Our results support a mechanism involving inhibition of LT-ag expression or functions but also suggest inhibition of cellular enzymes.

[Modulation of cell proliferation and collagen synthesis of porcine Tenon's fibroblasts by extracts of lens and vitreous with treatment of dexamethasone].

OBJECTIVE: To demonstrate the effects of lens extract (LE), vitreous extract (VE) and dexamethasone on the modulating of the proliferation and collagen synthesis of Tenon's capsule fibroblast. METHODS: Cell proliferation and collagen synthesis of Tenon's fibroblast were measured using MTT and reverse transcription polymerase chain reaction techniques in the presence or absence of LE, VE, or dexamethasone, respectively. RESULTS: The growth of Tenon's fibroblasts was stimulated by LE and VE in dose and time-dependent manners. Dexamethasone significantly inhibited the proliferation of the cells either in the presence or absence of LE and VE. Similarly, LE and VE significantly stimulated the collagen synthesis of the cells. However, dexamethasone showed no significant effect on the LE or VE-enhanced collagen synthesis of the cells. CONCLUSION: These results suggested that there might be some ingredients in LE or VE that could stimulate the cell proliferation and collagen synthesis of Tenon's capsule fibroblasts. These ingredients, if released, may affect the success rate of filtration surgery for glaucoma. The application of glucocorticoid itself might not be sufficient to retard the scar formation following the filtering surgery in refractory glaucoma after cataract surgery or vitrectomy.