Investigation into the effects of leukemia inhibitory factor on the bone repair capacity of BMSCs-loaded BCP scaffolds in the mouse calvarial bone defect model.

Leukemia inhibitory factor (LIF) is known to play a major role in bone physiology. In the present study, we examined the in vitro effects of LIF on osteoblast differentiation of bone marrow stem cells (BMSCs) and explored in vivo effects of LIF on the bone repair capacity of BMSCs-loaded biphasic calcium phosphate (BCP) scaffolds in mouse calvarial bone defect model. The mRNA and protein expression levels in the BMSCs were determined by quantitative real-time PCR and western blot, respectively; the in vitro osteoblast differentiation of the BMSCs was evaluated by using Alizarin Red S staining. The bone volume and bone density in the repaired calvarial bone defect were determined by Micro-CT. Bone regeneration was also histologically evaluated by hematoxylin and eosin staining and Masson's trichrome staining. Hypoxia treatment induced the up-regulation of Lif mRNA and LIF protein in the BMSCs. Lif overexpression up-regulated the mRNA expression levels of osteopontin and Runt-related transcription factor 2, and increased intensity of Alizarin Red S staining in the BMSCs; while Lif silence exerted the opposite effects. The in vivo studies showed that implantation of Lif-overexpressing BMSCs-loaded BCP scaffolds significantly increased the bone volume and bone density at 4 and 8 weeks after transplantation, and promoted the regeneration of bone tissues in the mouse calvarial bone defect at 8 weeks after transplantation when compared to the BMSCs-loaded BCP scaffolds group; while Lif-silencing BMSCs-loaded BCP scaffolds had the opposite effects. The present study for the first time demonstrated that LIF promoted the in vitro osteoblast differentiation of hypoxia-treated BMSCs; and further studies revealed that LIF exerted enhanced effects on the bone repair capacity of BMSCs-load BCP scaffolds in mouse calvarial bone defect model. However, future studies are warranted to determine the detailed mechanisms of LIF in the large-scale bone defect repair.

Correction to: CBFA2T2 is associated with a cancer stem cell state in renal cell carcinoma.

[This corrects the article DOI: 10.1186/s12935-017-0473-z.].

The Role of Forkhead Box 1 (FOXO1) in the Immune System: Dendritic Cells, T Cells, B Cells, and Hematopoietic Stem Cells.

Forkhead box-O (FOXO) transcription factors have a fundamental role in the development and differentiation of immune cells. FOXO1 and FOXO3 are FOXO members that are structurally similar and bind to the same conserved consensus DNA sequences to induce transcription. FOXO1 has been studied in detail in the activation of dendritic cells (DCs), where it plays an important role through the regulation of target genes such as ICAM-1, CCR7, and the integrin αvß3. FOXO1 is activated by bacteria challenge in DCs and promotes DC bacterial phagocytosis, migration, homing to lymph nodes, DC stimulation of CD4+ T cells and resting B cells, and antibody production. Deletion of FOXO1 in DCs enhances susceptibility to bacteria-induced periodontal disease. FOXO1 and FOXO3 maintain naive T cell quiescence and survival. FOXO1 and FOXO3 enhance the formation of regulatory T cells and inhibit the formation of T-helper 1 (Th1) and Th17 cells. FOXO1 promotes differentiation, proliferation, survival, immunoglobulin gene rearrangement, and class switching in B cells, but FOXO3 has little effect. Both FOXO1 and FOXO3 are important in the maintenance of hematopoietic stem cells by protecting them from oxidative stress. This review examines FOXO1/FOXO3 in the adaptive immune response, key target genes, and FOXO inhibition by the phosphoinositide 3-kinase/AKT pathway.

CBFA2T2 is associated with a cancer stem cell state in renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) is the most common kidney cancer, accounting for approximately 80-90% of all primary kidney cancer. Treatment for patients with advanced RCC remains unsatisfactory. Rare cancer stem cells (CSCs) are proposed to be responsible for failure of current treatment. METHODS: OncoLnc was used as a tool for interactively exploring survival correlations. Gene manipulation and expression analysis were carried out using siRNA, RT-PCR and Western blotting. Wound healing and invasion assays were used for phenotypical characterization. Aldefluor assay and FACS sorting Sphere culture were used to determine the "stemness" of CSCs. Co-Immunoprecipitation (Co-IP) was used to examine the interaction between OCT4 and CBFA2T2. Student's t-test and Chi square test was used to analyze statistical significance. RESULTS: CBFA2T2 expression can significantly predict the survival of RCC patients. Knocking-down of CBFA2T2 can inhibit cell migration and invasion in RCC cells in vitro, and reduce ALDHhigh CSCs populations. CBFA2T2 expression is necessary for sphere-forming ability and cancer stem cells marker expression in RCC cell lines. CONCLUSIONS: Our data suggest that CBFA2T2 expression correlates with aggressive characteristics of RCC and CBFA2T2 is required for maintenance of "stemness" through regulation of stem cells factors, thereby highlighting CBFA2T2 as a potential therapeutic target for RCC treatment.

LIF Aggravates Pulpitis by Promoting Inflammatory Response in Macrophages.

Leukemia inhibitory factor (LIF) has been recognized as a novel inflammatory modulator in inflammation-associated diseases. This study aimed to investigate the modulation of LIF in dental pulp inflammation. Experimental pulpitis was established in wild-type (WT) and Lif-deficient (Lif-/-) mice. Histological and immunostaining analyses were conducted to assess the role of LIF in the progression of pulpitis. Mouse macrophage cell line (RAW264.7) was treated with LPS to simulate an inflammatory environment. Exogenous LIF was added to this system to examine its modulation in macrophage inflammatory response in vitro. Primary bone marrow-derived macrophages (BMDMs) from WT and Lif-/- mice were isolated and stimulated with LPS to confirm the effect of Lif deletion on macrophage inflammatory response. Supernatants from LIF and LPS-treated human dental pulp cells (hDPCs) were collected and added to macrophages. Macrophage chemotaxis was assessed using transwell assays. The results showed an increased expression of LIF and LIFR with the progression of pulpitis, and LIFR was highly expressed in macrophages. Lif deficiency alleviated experimental pulpitis with the reduction of pro-inflammatory cytokines and macrophage infiltration. Exogenous LIF promoted inflammatory response of LPS-induced macrophages through a STAT3/p65-dependent pathway. Consistently, Lif deletion inhibited macrophage inflammatory response in vitro. Supernatants of LIF-treated hDPCs enhanced macrophage migration in LPS-induced inflammatory environment. Our findings demonstrated that LIF aggravates pulpitis by promoting macrophage inflammatory response through a STAT3/p65-dependent pathway. Furthermore, LIF plays a crucial role in driving the recruitment of macrophages to inflamed pulp tissue by promoting chemokine secretion in DPCs.

Leukemia inhibitory factor protects against experimental periodontitis through immuno-modulations of both macrophages and periodontal ligament fibroblasts.

BACKGROUND: To explore the role of leukemia inhibitory factor (LIF) in periodontitis via in vivo and in vitro experiments. METHODS: The second upper molar of LIF knockout mice and their wild-type littermates were ligated for 8 days. Micro-computed tomography (micro-CT), histological analysis, and quantitative real-time polymerase chain reaction (qRT-PCR) were performed. The expression levels of proinflammatory cytokines were examined in mouse bone marrow derived macrophages and human periodontal ligament fibroblasts (HPDLFs) after lipopolysaccharide (LPS) treatment. RESULTS: LIF deficiency promoted alveolar bone loss, inflammatory cells infiltration, osteoclasts formation and collagen fiber degradation in ligature-induced mouse, along with higher expressions of proinflammatory cytokines, including interleukin-6 (IL6), IL-1ß (IL1B), tumor necrosis factor-α (TNFA), matrix metalloproteinase 13 (MMP13), and RANKL/OPG ratio. Additionally, LIF deletion led to higher expression levels of these proinflammatory cytokines in mouse bone marrow-derived macrophages from both femur and alveolar bone and HPDLFs when treated with LPS. Administration of recombined LIF attenuated TNFA, IL1B, and RANKL/OPG ratio in HPDLFs. CONCLUSIONS: These findings indicate that LIF deficiency promotes the progress of periodontitis via modulating immuno-inflammatory responses of macrophages and periodontal ligament fibroblasts, and the application of LIF may be an adjunctive treatment for periodontitis to resolute inflammation.

The protective effect of leukemia inhibitory factor on apoptosis of BMSCs induced by hypoxia and serum-deprivation.

OBJECTIVES: Bone marrow-derived mesenchymal stem cells (BMSCs) - based tissue engineering is an important strategy for treatment of bone defects. However, the ischemia environment limits the survival and biological functions of BMSCs. The present study aimed to investigate the effect of leukemia inhibitory factor (LIF) on the apoptosis of BMSCs induced by hypoxia and serum-deprivation (H&SD) as well as the underlying pathway mechanism. METHODS: Mitochondrial membrane potential (MMP) was determined by flow cytometry. The apoptotic phenomenon of nuclear morphology was detected by fluorescence microscope. The ratio of apoptotic BMSCs was investigated by Annexin V/propidium iodide (PI) double staining and flow cytometric analysis. The expression of apoptosis-related molecules was detected by quantitative polymerase chain reaction (qPCR) and western blotting. RESULTS: H&SD treatment induced a series of apoptotic phenotypes, including the downregulation of MMP, the apoptotic phenomenon of nuclear morphology, the increased rate of BMSCs at early and late apoptotic stage, and the reduced B-cell lymphoma-2 (Bcl-2)/Bcl-2-associated X (Bax) ratio. Administration of recombinant LIF alleviated the apoptosis of BMSCs induced by H&SD, which was reflected in recovery of MMP, morphology of nuclei, rate of apoptotic cells and inhibition of cleaved Caspase-3. The results of western blot demonstrated that phosphorylation of janus kinase (JAK) 1 and signal transducer and activator of transcription (STAT) 3 was inhibited by H&SD treatment, which was upregulated by LIF administration. JAK1-specific inhibitor GLPG0634 or STAT3-specific inhibitor S3I-201 eliminated the protective effects of LIF on the apoptosis of BMSCs. CONCLUSION: These data indicated that LIF played a protective role in apoptosis of BMSCs induced by ischemia via activating JAK1/STAT3 signaling pathway.

Key role of exosomes derived from M2 macrophages in maintaining cancer cell stemness (Review).

Cancer stem cells (CSCs) constitute a specific subset of cells found within tumors that are responsible for initiating, advancing and resisting traditional cancer treatments. M2 macrophages, also known as alternatively activated macrophages, contribute to the development and progression of cancer through their involvement in promoting angiogenesis, suppressing the immune system, supporting tumor growth and facilitating metastasis. Exosomes, tiny vesicles released by cells, play a crucial role in intercellular communications and have been shown to be associated with cancer development and progression by influencing the immune response; thus, they may serve as markers for diagnosis and prognosis. Currently, investigating the impact of exosomes derived from M2 macrophages on the maintenance of CSCs is a crucial area of research with the aim of developing novel therapeutic strategies to target this process and improve outcomes for individuals with cancer. Understanding the biological functions of exosomes derived from M2 macrophages and their involvement in cancer may lead to the formulation of novel diagnostic tools and treatments for this disease. By targeting M2 macrophages and the exosomes they secrete, promising prospects emerge for cancer treatment, given their substantial contribution to cancer development and progression. Further research is required to fully grasp the intricate interactions between CSCs, M2 macrophages and exosomes in cancer, and to identify fresh targets for cancer therapy. The present review explores the pivotal roles played by exosomes derived from M2 cells in maintaining the stem­like properties of cancer cells.

Leukemia Inhibitory Factor Facilitates Self-Renewal and Differentiation and Attenuates Oxidative Stress of BMSCs by Activating PI3K/AKT Signaling.

Objective: Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) remains a hopeful therapeutic approach for bone defect reconstruction. Herein, we investigated the effects and mechanisms of leukemia inhibitory factor (LIF) in the function and viability of hypoxic BMSCs as well as bone defect repair. Methods: The effects of LIF on apoptosis (flow cytometry, TUNEL staining), mitochondrial activity (JC-1 staining), proliferation (colony formation, EdU staining), and differentiation (CD105, CD90, and CD29 via flow sorting) were examined in hypoxic BMSCs. LIF, LIFR, gp130, Keap1, Nrf2, antioxidant enzymes (SOD1, catalase, GPx-3), bone-specific matrix proteins (ALP, BSP, OCN), PI3K, and Akt were detected via immunoblotting or immunofluorescent staining. BMSCs combined with biphasic calcium phosphate scaffolds were implanted into calvarial bone defect mice, and the therapeutic effect of LIF on bone defect was investigated. Results: Hypoxic BMSCs had increased apoptosis and oxidative stress and reduced mitochondrial activity. Additionally, LIF, LIFR, and gp130 were upregulated and PI3K/Akt activity was depressed in hypoxic BMSCs. Upregulated LIF alleviated apoptosis and oxidative stress and heightened mitochondrial activity and PI3K/Akt signaling in hypoxic BMSCs. Additionally, LIF overexpression promoted self-renewal and osteogenic differentiation of BMSCs with hypoxic condition. Mechanically, LIF facilitated self-renewal and differentiation as well as attenuated oxidative stress of BMSCs through enhancing PI3K/AKT signaling activity. Implantation of LIF-overexpressed BMSC-loaded BCP scaffolds promoted osteogenesis as well as alleviated oxidative stress and apoptosis through PI3K/Akt signaling. Conclusion: Our findings demonstrate that LIF facilitates self-renewal and differentiation and attenuates oxidative stress of BMSCs by PI3K/AKT signaling.

Chaetocin Promotes Osteogenic Differentiation via Modulating Wnt/Beta-Catenin Signaling in Mesenchymal Stem Cells.

Mesenchymal stemXin cells (MSCs) are a great cell source for bone regeneration. Although combining MSCs with growth factors and scaffolds provides a useful clinical strategy for bone tissue engineering, the efficiency of MSC osteogenic differentiation remains to be improved. Epigenetic modification is related to the differentiation ability of MSCs during osteogenic induction. In this study, we evaluate the effect of Chaetocin, an inhibitor of lysine-specific histone methyltransferases, on the differentiation of MSCs. We found that MSCs treated with Chaetocin demonstrated increased osteogenic ability and reduced adipogenic ability. The expression of osteogenic markers (Runx2 and OPN) was induced in MSCs by Chaetocin during osteogenic induction. Moveover, treatment of Chaetocin in MSCs improves Wnt/ß-catenin signaling pathways and its downstream targets. Finally, we showed increased bone formation of MSC and Wnt/ß-catenin signaling activity by treatment of Chaetocin using in vivo bone formation assays. Our data uncovered a critical role of Chaetocin in MSC osteogenic differentiation and provide new insights into bone tissue regeneration and repair.

Evaluation of the osseointegration of dental implants coated with calcium carbonate: an animal study.

In an attempt to overcome the limitations of titanium in dental and orthopaedic clinical applications, a new method has been developed to prepare calcium carbonate coatings on sandblasted and acid-etched (SA) titanium implants. The purpose of this study was to investigate the effect of calcium carbonate-SA (CC-SA) implants on osseointegration in vivo. The surfaces of SA and CC-SA implants were characterised for surface morphology and surface chemistry. Subsequently, these two kinds of implants were implanted in the femoral condyles of rabbits. The implants were retrieved and prepared for histological and histomorphometric evaluation 1, 2, 4, 8 and 12 weeks after implantation. Significantly higher values of bone-to-implant contact of the entire implant except the gap area (BIC_ALL) and the bone-to-implant contact of the gap area (BIC_GAP) were found in animals with the CC-SA implants than in those with the SA implants at 4 weeks. Higher values of total gap bone were found in those with the CC-SA implants than in those with the SA implants at 1, 2 and 4 weeks. In conclusion, the current findings demonstrate that the calcium carbonate coating can improve and accelerate the early ingrowth of bone and osseointegration at the early healing phase. This may reduce clinical healing times and thus improve implant success rates.

A new implant with solid core and porous surface: the biocompatability with bone.

This research investigated osteogenic potencies of Farthing-Fray-Chen Titanium (FFcTi) implant with transitional porous-solid structure. The material characteristics, biomechanical property, osteogenic performances were assessed. FFcTi showed similar roughness as sand-blasted and acid etched titanium (SA), but was more hydrophilic than SA and machined commercial pure titanium (MA). Young's modulus of FFcTi implant in compressive tests was 15.8 ± 6.3 GPa, which was close to bone. In vitro observations manifested excellent spreading abilities of MC3T3-E1 cell on FFcTi and SA. Adhesion rates of MC3T3-E1 cells at 4 h gradually decreased on MA, SA, and FFcTi surfaces (MA > SA, p < 0.01; SA > FFcTi, p < 0.05), while cell proliferation ability on FFcTi was weaker than MA during 1-6 days (p < 0.01) and similar to MA and SA in day 11. ALP activity of cells on FFcTi at 14 day was higher than MA and lower than SA (p < 0.01). In a bone defect model of rabbits, BIC and bone volum ratio within 50 µm were significantly higher for FFcTi than MA (BIC, p < 0.01; BT0.05, p < 0.05) while bone volume ratio within 100 and 500 µm were of no differences. Micro CT analysis also showed similar results to the histomorphometric data. Thus, we conclude that FFcTi with melting sphere based multiporous structure has a hydrophilic, rough surface, and close modulus to bone. In vitro, its low proliferation and ALP activity promotion were similar to other micro scale roughed surface. In vivo test showed better osteogenesis ability when compared with MA at least in 2 weeks. Thus, this Farthing-Fray-Chen Titanium implant seems to hold considerable potential for bone implant applications.

Expression of LIF and LIFR in periodontal tissue during orthodontic tooth movement.

OBJECTIVES: To test the hypothesis that leukemia inhibitor factor (LIF) and LIF receptor (LIFR) are expressed in periodontal tissue and that their expression may be upregulated during orthodontic tooth movement. MATERIALS AND METHODS: Forces of 0.3 N were applied to move the upper left first molars mesially in 24 rats. These forces were kept constant for 3, 7, and 14 days and followed by animal sacrifice. The contralateral molars served as control. The rate of tooth movement was measured by Image J software. Paraffin-embedded sections of the upper jaws were prepared for histological and immunohistochemical analysis to test the LIF and LIFR expression. RESULTS: Loaded teeth showed a significantly higher rate of tooth movement. The periodontium of the moved teeth experienced tissue remodeling, while there was no obvious change in the contralateral controls. Furthermore, LIF and LIFR were expressed in the periodontal tissue, and there were statistically significant differences between the loaded and unloaded teeth at 3 and 14 days. LIF presented significantly higher expression on the tension side compared with the pressure side at 3 days. CONCLUSION: Both LIF and LIFR exist in the periodontal tissue, and continuous orthodontic forces induce the upregulation of LIF/LIFR production, suggesting that LIF/LIFR may play important roles in periodontium remodeling.

Effects of leukemia inhibitory factor on proliferation and odontoblastic differentiation of human dental pulp cells.

INTRODUCTION: The purpose of this study was to determine whether the leukemia inhibitory factor (LIF) is expressed in human dental tissue and exerts its effect on proliferation and odontoblastic differentiation of the dental pulp cells (DPCs). METHODS: An immunohistochemical assay was used to detect the expression of LIF and leukemia inhibitory factor receptor (LIFR) in the human dental pulp. The proliferation of DPCs was examined by culturing human primary DPCs in the presence of LIF with different doses or the neutralizing antibody to LIF. Western blot was performed to assay the phosphorylation of Janus kinase 2 (Jak2) and signal transducer and activator of transcription 3 (Stat3) in the presence or absence of LIF and/or AG 490, a specific inhibitor of Jak2. The odontoblastic differentiation of DPCs was determined using the alkaline phosphatase (ALP) activity assay, quantification of bone sialoprotein (BSP) and dentin sialophosphoprotein (DSPP) gene expression, and mineralization nodule formation. RESULTS: LIF and LIFR were present in the odontoblasts and DPCs. LIF induced proliferation of DPCs, which was inhibited by the LIF neutralizing antibody and AG 490. LIF induced phosphorylation of Jak2 and Stat3 but not in the presence of the AG490. ALP activity of DPCs, in the absence or presence of mineralization induction medium, was inhibited by LIF. Furthermore, the mineralization nodule formation and the expression of BSP and DSPP were inhibited by LIF. This inhibition on differentiation was attenuated by the AG490. CONCLUSIONS: LIF and LIFR are expressed in the human dental pulp. LIF promotes the proliferation of DPCs, and the odontoblastic differentiation is inhibited via the Jak2-Stat3 signaling pathway.