Daming Dong

The promotion of neural progenitor cells proliferation by aligned and randomly oriented collagen nanofibers through β1 integrin/MAPK signaling pathway.

In regenerative medicine, accumulating evidence demonstrates that the property of substrates monitors neural stem cells behavior. However, how stem cells sense and interpret biochemical and topographical cues remains elusive. This study aimed to explore the mechanism how nanofibrous scaffold modulated stem cells behavior. Spinal cord derived neural progenitor cells (NPCs) were cultured on electrospun aligned and randomly oriented collagen nanofibrous scaffolds. A 30% increase in proliferation and an elevation of BrdU incorporation were observed in NPCs on collagen nanofibers, compared to that on collagen-coated surface. In particular, NPCs expanded faster on aligned nanofibers in comparison with that on randomly oriented nanofibers. Moreover, an alteration in cell cycle progression with a reduced percentage of cells in G0/G1 phase and increased cell proliferation index (S phase plus G2/M phase) was also detected in NPCs cultured on collagen nanofibers. Incubating NPCs with anti-β1 integrin antibody or U1026 (an inhibitor of mitogen-activated protein kinase kinase, MEK) eliminated the altered cell cycle dynamics and BrdU incorporation induced by collagen nanofibers. In addition, cyclin D1 and cyclin dependent kinase 2 (CDK2), downstream genes of β1 integrin/mitogen-activated protein kinase (MAPK) pathway that control G1/S phase transition, were correspondingly regulated by nanofibers. Collectively, these data suggested that the property of substrate modulated NPCs proliferation by promoting cell cycle through β1 integrin/MAPK pathway. Our findings provide a better understanding of the interaction between NPCs and the substrate and therefore will pave way for regenerative medicine.

Association between the -1306C/T polymorphism of matrix metalloproteinase-2 gene and lumbar disc disease in Chinese young adults

Matrix metalloproteinase-2 (MMP-2) has been shown to play a pivotal role in the pathophysiology of lumbar disc disease (LDD). Increased expression and activity of MMP-2 has been documented in degenerative discs. The polymorphism -1306C/T in the promoter region of MMP-2 gene was reported to influence gene transcription and expression. The objective of this study was therefore to investigate the possible association of MMP-2 -1306C/T polymorphism with the occurrence and the clinical characteristics of LDD. MMP-2 genotypes were determined by polymerase chain reaction (PCR) and direct DNA sequencing in a case-control study involving 162 younger patients with LDD and 318 age- and sex-matched healthy adults. The results showed that the frequency of MMP-2 -1306CC genotype was significantly higher in LDD patients when compared with controls. Subjects with the CC genotype had nearly threefold increased risk for LDD (odds ratio 3.08; 95% confidence interval 1.84–5.16) compared with subjects carrying at least one variant T allele. Furthermore, this genotype was found to correlate with more severe grades of disc degeneration observed on magnetic resonance imaging scan. These findings suggest that MMP-2 -1306C/T polymorphism may be a genetic risk factor related to LDD susceptibility in the young adult population.

Promotion of peripheral nerve regeneration of a peptide compound hydrogel scaffold

Background Peripheral nerve injury is a common trauma, but presents a significant challenge to the clinic. Silk-based materials have recently become an important biomaterial for tissue engineering applications due to silk’s biocompatibility and impressive mechanical and degradative properties. In the present study, a silk fibroin peptide (SF16) was designed and used as a component of the hydrogel scaffold for the repair of peripheral nerve injury. Methods The SF16 peptide’s structure was characterized using spectrophotometry and atomic force microscopy, and the SF16 hydrogel was analyzed using scanning electron microscopy. The effects of the SF16 hydrogel on the viability and growth of live cells was first assessed in vitro, on PC12 cells. The in vivo test model involved the repair of a nerve gap with tubular nerve guides, through which it was possible to identify if the SF16 hydrogel would have the potential to enhance nerve regeneration. In this model physiological saline was set as the negative control, and collagen as the positive control. Walking track analysis and electrophysiological methods were used to evaluate the functional recovery of the nerve at 4 and 8 weeks after surgery. Results Analysis of the SF16 peptide’s characteristics indicated that it consisted of a well-defined secondary structure and exhibited self-assembly. Results of scanning electron microscopy showed that the peptide based hydrogel may represent a porous scaffold that is viable for repair of peripheral nerve injury. Analysis of cell culture also supported that the hydrogel was an effective matrix to maintain the viability, morphology and proliferation of PC12 cells. Electrophysiology demonstrated that the use of the hydrogel scaffold (SF16 or collagen) resulted in a significant improvement in amplitude recovery in the in vivo model compared to physiological saline. Moreover, nerve cells in the SF16 hydrogel group displayed greater axon density, larger average axon diameter and thicker myelin compared to those of the group that received physiological saline. Conclusion The SF16 hydrogel scaffold may promote excellent axonal regeneration and functional recovery after peripheral nerve injury, and the SF16 peptide may be a candidate for nerve tissue engineering applications.

Erythropoietin promotes spinal cord-derived neural progenitor cell proliferation by regulating cell cycle.

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells by binding to its specific transmembrane receptor (EPOR). The presence of EPO and its receptor in the CNS suggests a different function for EPO other than erythropoiesis. The purpose of the present study was to examine EPOR expression and the role of EPO in the proliferation of neonatal spinal cord-derived neural progenitor cells. The effect of EPO on cell cycle progression was also examined, as well as the signaling cascades involved in this process. Our results showed that EPOR was present in the neural progenitor cells and EPO significantly enhanced their proliferation. Cell cycle analysis of EPO-treated neural progenitor cells indicated a reduced percentage of cells in G0/G1 phase, whereas the cell proliferation index (S phase plus G2/M phase) was increased. EPO also increased the proportion of 5-bromo-2-deoxyuridine (BrdU)-positive cells. With respect to the cell cycle signaling, we examined the cyclin-dependent kinases D1, D2 and E, and cyclin-dependent kinase inhibitors, p21cip1, p27kip1 and p57kip2. No significant differences were observed in the expression of these transcripts after EPO administration. Interestingly, the anti-apoptotic factors, mcl-1 and bcl-2 were significantly increased twofold. Moreover, these specific effects of EPO were eliminated by incubation of the progenitor cells with anti-EPO neutralizing antibody. Those observations suggested that EPO may play a role in normal spinal cord development by regulating cell proliferation and apoptosis.

Serum fatty acid profiles and potential biomarkers of ankylosing spondylitis determined by gas chromatography–mass spectrometry and multivariate statistical analysis

Ankylosing spondylitis (AS) is a common chronic inflammatory rheumatic disease. Early and accurate detection is essential for effective disease treatment. Recently, research has focused on genomics and proteomics. However, the associated metabolic variations, especially fatty acid profiles, have been poorly discussed. In this study, the gas chromatography-mass spectrometry (GC-MS) approach and multivariate statistical analysis were used to investigate the metabolic profiles of serum free fatty acids (FFAs) and esterified fatty acids (EFAs) in AS patients. The results showed that significant differences in most of the FFA (C12:0, C16:0, C16:1, C18:3, C20:4, C20:5, C22:5 and C22:6) and EFA (C12:0, C16:1, C18:0, C18:1, C18:2, C18:3, C20:4 and C22:6) concentrations were found between the AS patients and healthy controls (p < 0.05). Principal component analysis and partial least squares discriminant analysis were performed to classify the AS patients and controls. Additionally, FFAs C20:4, C12:0, C18:3 and EFAs C22:6, C12:0 were confirmed as potential biomarkers to identify AS patients and healthy controls. The present study highlights that differences in the serum FFA and EFA profiles of AS patients reflect the metabolic disorder. Moreover, FFA and EFA biomarkers appear to have clinical applications for the screening and diagnosis of AS.

Suppression of MicroRNA-383 Enhances Therapeutic Potential of Human Bone-Marrow-Derived Mesenchymal Stem Cells in Treating Spinal Cord Injury via GDNF

Background/Aims: Transplantation of bone-marrow-derived mesenchymal stem cells (MSCs) has been used to treat spinal cord injury (SCI) to enhance tissue repair and neural cell regeneration. Glial cell line derived neurotrophic factor (GDNF) is an identified neural growth and survival factor. Here, we examined whether modification of GDNF levels in MSCs may further increase the potential of MSCs in promoting neural cell regeneration and subsequently the therapeutic outcome. Methods: We examined the mRNA and protein levels of GDNF in human MSCs by RT-qPCR and Western blot, respectively. Bioinformatics analyses were done to predict microRNAs (miRNAs) that target GDNF in MSCs. The functional binding of miRNAs to GDNF mRNA was examined by a dual luciferase reporter assay. MSCs were transduced with adeno-associated virus (AAV) carrying null or antisense for miR-383 (as-miR-383), which were transplanted into nude rats that underwent SCI. The intact tissue, cavity volume, and recovery of locomotor activity were assessed. Results: MSCs expressed very low GDNF protein, but surprisingly high levels of GDNF mRNA. Bioinformatics analyses showed that miR-383 inhibited protein translation of GDNF, through binding to the 3’-UTR of the GDNF mRNA. MSCs transduced with AAV-as-miR-383 further increased the intact tissue percentage, decreased cavity volume, and enhanced the recovery of locomotor activity in nude rats that underwent SCI, compared to MSCs. Conclusions: Suppression of miR-383 may increase the therapeutic potential of human bone-marrow-derived MSCs in treating SCI via augmentation of GDNF protein levels.

Inhibition of IL-18-mediated myeloid derived suppressor cell accumulation enhances anti-PD1 efficacy against osteosarcoma cancer

Myeloid derived suppressor cells (MDSC) are very important in tumor immune evasion and they dramatically increased in peripheral blood of patients with osteosarcoma cancer. The association between MDSC and various cytokines has been studied in the peripheral blood. However, little is known about the mechanism drawing MDSC into tumor parenchyma. This study was to analyze the correlation between MDSC subsets and interleukin 18 (IL-18) level in osteosarcoma tumor model and its effect on the immunotherapy. MDSC were isolated from the blood and parenchyma and analyzed in the osteosarcoma tumor model. IL-18 levels were detected by enzyme-linked immunosorbent assay (ELISA) assay, real-time PCR, western blot and flow cytometry. Moreover, combination treatment with IL-18 inhibition and anti-PD1 was conducted to assess the therapeutic effects of IL-18 blockade. Results showed MDSC levels had a positive correlation with IL-18, suggesting IL-18 may attract MDSC into the parenchyma. IL-18 gene and protein expression significantly increased in blood and tumor lysates of tumor-bearing mice. Anti-IL-18 treatment significantly decreased G-MDSC and M-MDSC in the peripheral blood and tumor. Furthermore, combination therapy decreased the tumor burden and increased CD4+ and CD8+ T cell infiltration, as well as the production of interferon gamma (IFNγ) and granzyme B. Our study revealed a possible correlation between MDSC subsets and IL-18 inducing MDSC migration into the tumor tissue, in addition to provide the potential target to enhance the efficacy of immunotherapy in patients with osteosarcoma.

Promotion of cell growth and adhesion of a peptide hydrogel scaffold via mTOR/cadherin signaling

Understanding neurite outgrowth, orientation, and migration is important for the design of biomaterials that interface with the neural tissue. However, the molecular signaling alternations have not been well elucidated to explain the impact of hydrogels on cell morphology. In our previous studies, a silk fibroin peptide (SF16) hydrogel was found to be an effective matrix for the viability, morphology, and proliferation of PC12 rat pheocrhomocytoma cells. We found that PC12 cells in the peptide hydrogel exhibited adhesive morphology compared to those cultured in agarose or collagen. Moreover, we identified that cell adhesion molecules (E‐ and N‐cadherin) controlled by mTOR signaling were highly induced in PC12 cells cultured in the SF16 peptide hydrogel. Our findings suggest that the SF16 peptide might be suitable to be a cell‐adhesion material in cell culture or tissue engineering, and mTOR/cadherin signaling is required for the cell adhesion in the SF16‐peptide hydrogel.

Association of Estrogen Receptor Alpha Gene Polymorphisms and Risk of Fracture

The association between estrogen receptor alpha (ESR1) gene polymorphisms and risk of fracture is still controversial and ambiguous. The objective of this study was to evaluate the effect of PvuII polymorphisms of the ESR1 gene on fracture risk in Chinese patients. A population-based control study of elderly subjects was conducted in 120 fracture patients and 120 controls. The PvuII pp genotype of the ESR1 gene was determined by using a polymerase chain reaction-restriction fragment length polymorphism assay. There was no relationship between ESR1 gene PvuII polymorphism and fracture risk. When stratifying by fracture type, it was found that vertebral fracture cases had a significantly higher frequency of the PvuII pp genotype (odds ratio=2.00, 95% confidence interval=1.03, 3.88; p=0.04) than controls. This study suggested that there was a modest but statistically significant association between the PvuII pp genotype of the ESR1 gene and vertebral fracture in Chinese patients. The molecular mechanism underlying this association needs further study.

Comprehensive Effects of Suppression of MicroRNA-383 in Human Bone-Marrow-Derived Mesenchymal Stem Cells on Treating Spinal Cord Injury

Background/Aims: Transplantation of bone-marrow-derived mesenchymal stem cells (MSCs) promotes neural cell regeneration after spinal cord injury (SCI). Recently, we showed that suppression of microRNA-383 (miR-383) in MSCs increased the protein levels of glial cell line derived neurotrophic factor (GDNF), resulting in improved therapeutic effects on SCI. However, the overall effects of miR-383 suppression in MSCs on SCI therapy were not determined yet. Here, we addressed this question. Methods: We used bioinformatics tools to predict all miR-383-targeting genes, confirmed the functional bindings in a dual luciferase reporter assay. The effects of alteration of candidate genes in MSCs on cell proliferation were analyzed by MTT assay and by Western blotting for PCNA. The effects on angiogenesis were assessed by HUVEC assay. The effects on SCI in vivo were analyzed by transplantation of the modified MSCs into nude rats that underwent SCI. Results: Suppression of miR-383 in MSCs not only upregulated GDNF protein, but also increased vascular endothelial growth factor A (VEGF-A) and cyclin-dependent kinase 19 (CDK19), two other miR-383 targets. MiR-383-suppression-induced increases in CDK19 resulted in a slight but significant increase in MSC proliferation, while miR-383-suppression-induced increases in VEGF-A resulted in a slight but significant increase in MSC-mediated angiogenesis. Conclusions: Upregulation of CDK19 and VEGF-A by miR-383 suppression in MSCs further improve the therapeutic potential of MSCs in treating SCI in rats.