Zengwu Shao

Cell death in intervertebral disc degeneration

Degeneration of intervertebral disc (IVD) is mainly a chronic process of excessive destruction of the extracellular matrix (ECM), and also is thought to be the primary cause of low back pain. Presently, however, the underlying mechanism of IVD degeneration is still not elucidated. Cellular loss from cell death has been believed to contribute to the degradation of ECM and plays an important role in the process of IVD degeneration, but the mechanisms of cell death in degenerated IVD remain unclear. Apoptosis, a very important type of IVD cell death, has been considered to play a crucial role in the process of degeneration. Autophagy, a non-apoptosis death type of programmed cell death, has been considered extensively involved in many pathological and physiological processes, including the degenerative diseases. Thus, the research on cell death in IVD degeneration has become a new focus recently. In this review, by analyzing the available literature pertaining to cell death in IVD and discussing the inducing factors of IVD degeneration, NP cells and ECM in IVD degeneration, apoptotic signal transduction pathways involved in IVD cell death, the relationship of cell death with IVD degeneration and potential therapeutic strategy for IVD degeneration by regulating cell death, we conclude that different stimuli induce cell death in IVD via various signal transduction pathways, and that cell death may play a key role in the degenerative process of IVD. Regulation of cell death could be a potential and attractive therapeutic strategy for IVD degeneration.

Repair of full-thickness articular cartilage defects by cultured mesenchymal stem cells transfected with the transforming growth factor beta1 gene.

Articular cartilage repair remains a clinical and scientific challenge with increasing interest focused on the combined techniques of gene transfer and tissue engineering. Transforming growth factor beta 1 (TGF-beta(1)) is a multifunctional molecule that plays a central role in promotion of cartilage repair, and inhibition of inflammatory and alloreactive immune response. Cell mediated gene therapy can allow a sustained expression of TGF-beta(1) that may circumvent difficulties associated with growth factor delivery. The objective of this study was to investigate whether TGF-beta(1) gene modified mesenchymal stem cells (MSCs) could enhance the repair of full-thickness articular cartilage defects in allogeneic rabbits. The pcDNA(3)-TGF-beta(1) gene transfected MSCs were seeded onto biodegradable poly-L-lysine coated polylactide (PLA) biomimetic scaffolds in vitro and allografted into full-thickness articular cartilage defects in 18 New Zealand rabbits. The pcDNA(3) gene transfected MSCs/biomimetic scaffold composites and the cell-free scaffolds were taken as control groups I and II, respectively. The follow-up times were 2, 4, 12 and 24 weeks. Macroscopical, histological and ultrastructural studies were performed. In vitro SEM studies found that abundant cartilaginous matrices were generated and completely covered the interconnected pores of the scaffolds two weeks post-seeding in the experimental groups. In vivo, the quality of regenerated tissue improved over time with hyaline cartilage filling the chondral region and a mixture of trabecular and compact bone filling the subchondral region at 24 weeks post-implantation. Joint repair in the experimental groups was better than that of either control group I or II, with respect to: (1) synthesis of hyaline cartilage specific extracellular matrix at the upper portion of the defect; (2) reconstitution of the subchondral bone at the lower portion of the defect and (3) inhibition of inflammatory and alloreactive immune responses. The transfected MSCs overexpressed their TGF-beta(1) gene products for at least 4 weeks in vivo. The control defects were filled with a mixture of fibrous and fibrocartilaginous tissue. The TGF-beta(1) gene transfected MSCs/poly-L-lysine coated PLA composite allografts used in this study are effective for articular cartilage repair. This novel TGF-beta(1) gene enhanced tissue engineering strategy may be of potential benefit to enhancing the repair of damaged articular cartilage, especially such damage caused by degenerative disease.

Bone regeneration with active angiogenesis by basic fibroblast growth factor gene transfected mesenchymal stem cells seeded on porous β-TCP ceramic scaffolds

Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focused on combining gene transfer with tissue engineering techniques. Basic fibroblast growth factor (bFGF) is one of the most prominent osteogenic growth factors that has the potential to accelerate bone healing by promoting the proliferation and differentiation of mesenchymal stem cells (MSCs) and the regeneration of capillary vasculature. However, the short biological half-lives of growth factors may impose severe restraints on their clinical usefulness. Gene-based delivery systems provide a better way of achieving a sustained high concentration of growth factors locally in the defect and delivering a more biologically active product than that achieved by exogenous application of recombinant proteins. The objective of this experimental study was to investigate whether the bFGF gene modified MSCs could enhance the repair of large segmental bone defects. The pcDNA3-bFGF gene transfected MSCs were seeded on biodegradable porous beta tricalcium phosphate (beta-TCP) ceramics and allografted into the 15 mm critical-sized segmental bone defects in the radius of 18 New Zealand White rabbits. The pcDNA3 vector gene transfected MSCs were taken as the control. The follow-up times were 2, 4, 6, 8, 10 and 12 weeks. Scanning electron microscopic, roentgenographic, histologic and immunohistological studies were used to assess angiogenesis and bone regeneration. In vitro, the proliferation and differentiation of bFGF gene transfected MSCs were more active than that of the control groups. In vivo, significantly more new bone formation accompanied by abundant active capillary regeneration was observed in pores of the ceramics loaded with bFGF gene transfected MSCs, compared with control groups. Transfer of gene encoding bFGF to MSCs increases their osteogenic properties by enhancing capillary regeneration, thus providing a rich blood supply for new bone formation. This new bFGF gene enhanced tissue engineering strategy could be of potential benefit to accelerate bone healing, especially in defects caused by atrophic nonunion and avascular necrosis of the femoral head.

miR-145 suppresses osteogenic differentiation by targeting Sp7

Osteogenesis depends on a coordinated network of transcription factors including Sp7. Emerging evidence indicates that microRNAs (miRNAs) act as pivotal regulators in various biological processes including osteoblast proliferation and differentiation. Here, we investigated the effect of miR‐145 on osteogenic differentiation. miR‐145 was decreased during osteogenic differentiation, which could suppress the osteogenic differentiation of C2C12 and MC3T3‐E1 cells confirmed by gain‐ and loss‐of‐function experiments. Moreover, bioinformatic analysis combined with luciferase reporter assay, and Western blot validated that miR‐145 negatively regulated Sp7 expression. Inhibition of Sp7 showed similar effect with miR‐145 on osteogenic differentiation, whereas overexpression of Sp7 attenuated this effect. Collectively, these data indicate that miR‐145 is a novel regulator of Sp7, and it suppresses the osteogenic differentiation of C2C12 and MC3T3‐E1 cells.

A causal role for circulating miR-34b in osteosarcoma.

PURPOSE To investigate the associations between plasma miR-34b/c expression levels and osteosarcoma (OS). SUBJECTS AND METHODS A case-control study was conducted in 133 patients with OS and 133 controls. MiR-34b/c levels were detected by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays. Genotyping of SNP rs4938723 was done using the TaqMan assay. The causal association was examined by mendelian randomization analysis. RESULTS Plasma miR-34b level was significantly lower in OS patients than in controls (P = 0.001). Expression levels of miR-34b in OS tissues decreased (P = 3.22 × 10(-4)) and was significantly related with its expression in plasma (r = 0.21, P = 0.004). Compared with wild-type TT genotype, the variant genotypes of rs4938723 TC/CC were significantly associated with increased OS risk (TC vs. TT: OR, 1.97 [95% CI: 1.40-2.55], P = 0.021; CC vs. TT: OR, 2.76 [95% CI: 2.00-3.53], P = 0.009; TC + CC vs. TT: OR, 2.16 [95% CI: 1.61-2.70], P = 0.006), consistent with its decreased effect on plasma miR-34b (TC vs. TT: -0.32 (-0.43, -0.21), P < 0.001; CC vs. TT: -0.70 (-0.84, -0.56), P < 0.001; TC + CC vs. TT: -0.42 (-0.53, -0.32), P < 0.001). Adjustment for miR-34b completely abolished the association between SNP rs4938723 and OS risk (P > 0.05). In addition, plasma expression levels of miR-34b were significantly decreased in the metastatic patients compared with that in the non-metastatic ones (P = 0.004). CONCLUSION Plasma miR-34b was causally associated with OS risk and related with its metastatic status, suggesting that plasma miR-34b might be a novel biomarker and a potential treatment target for OS.

The beneficial effect of Icariin on bone is diminished in osteoprotegerin-deficient mice

BACKGROUND Osteoprogeterin (OPG) plays an important role in regulating bone homeostasis by inhibiting osteoclastogenesis and bone resorption. Icariin is the major ingredient of Herba Epimedii, which exerts anabolic and anti-resorptive effects on bone, but the mechanism remains unknown. In this study, we evaluated the role of OPG in Icariin-mediated beneficial effects on bone. MATERIALS AND METHODS Twelve-week-old Opg knockout (KO) male mice and their wild type (WT) littermates were orally administered with Icariin (0.3 mg/g) everyday for 8 weeks. Bone mass and microstructure in the right proximal tibiae were analyzed with micro-computed tomography (μCT). Bone remodeling was evaluated with serum biochemical analyses and bone histomorphometry. The colonies of fibroblast and osteoblast from bone marrow derived cells were quantified. The mRNA expressions of osteoblast and osteoclast related genes in trabecular bone from the femora were analyzed by real-time PCR. RESULTS Icariin treatment led to greater trabecular bone volume and trabecular number compared with vehicle treatment in WT mice. Icariin treatment increased bone formation parameters while it decreased bone resorption parameters in WT mice; however, the anabolic response of trabecular bone to Icariin treatment was diminished in KO mice. At cellular and molecular levels, Icariin significantly increased the formation of osteoblast colonies from bone marrow derived cells and the Opg gene expression in trabecular bone of WT mice. CONCLUSIONS These data suggest that Icariin treatment exerted anabolic and anti-resorptive effects on trabecular bone of WT mice, in which the effects were diminished in KO mice. The effects of Icariin treatment on bone are dependent on up-regulation of Opg, therefore, OPG plays an essential role in Icariin-mediated beneficial effects on trabecular bone.

Functionalized self-assembling peptide nanofiber hydrogel as a scaffold for rabbit nucleus pulposus cells.

In this study, a new functionalized peptide RLN was designed containing the bioactive motif link N, the amino terminal peptide of link protein. A link N nanofiber scaffold (LN-NS) was self-assembled by mixing peptide solution of RLN and RADA16. The characterization of LN-NS was tested using atomic force microscopy (AFM). The biocompatibility and bioactivity of this nanofiber scaffold for rabbit nucleus pulposus cells (NPCs) were also evaluated. This designer functionalized nanofiber scaffold exhibited little cytotoxicity and promoted NPCs adhesion obviously. In three-dimensional cell culture experiments, confocal reconstructed images testified that the functionalized LN-NS-guided NPCs migration from the surface into the hydrogel considerably, in which the RADA16 scaffold did not. Moreover, the functionalized LN-NS significantly stimulated the biosynthesis of extracelluar matrices (ECM) by NPCs. Our findings demonstrate that the functionalized nanofiber scaffold containing link N had excellent biocompatibility and bioactivity with rabbit NPCs and could be useful in the nucleus pulposus regeneration.

TNF-a mediated inflammatory macrophage polarization contributes to the pathogenesis of steroid-induced osteonecrosis in mice

The phenotypic polarization of macrophages are involved in steroid-induced osteonecrosis (ON). This study tried to investigate the detrimental and beneficial roles of M1/M2 macrophages associated with TNF-a in ON. Mice ON model was induced by the injection of methylprednisolone. After that, flow cytometry technique, immunohistochemistry, immunofluorescence, ELISA, and RT-PCR methods were used to investigate the expression pattern of macrophages and the expression of inflammatory cytokines. During the progression of ON, massive chronic inflammatory cells infiltrated into the necrotic zone, represented by the infiltration of macrophages. In the early stage of ON, there was high TNF-a activity; and a large population of M1 macrophages infiltrated into the necrotic zone. On the contrary, the expression of TNF-a gradually decreased; simultaneously, a larger M2 cell population presented in the necrotic zone in the late stage of ON. The increased M2 macrophages could be beneficial for resolving inflammation and promoting tissue repair, confirmed by the histologic findings of appositional new bone formation around the necrotic bone. Thus, it showed that TNF-a-mediated alteration of M1/M2 macrophage polarization contributed to the pathogenesis of steroid-induced osteonecrosis. M1-polarized macrophages appeared to be disruptive in the early stage of ON, while M2-polarized macrophages played an important role in the late stage during the pathogenesis of ON.

Effect of microRNA-101 on proliferation and apoptosis of human osteosarcoma cells by targeting mTOR.

Studies have proved that microRNA-101 (miR-101) functions as a tumor suppressor and is associated with growth and apoptosis of various human cancers. However, the role of miR-101 in osteosarcoma and the possible mechanism by which miR-101 affects the tumor growth and apoptosis have not been fully elucidated. In this study, we found that the expression of miR-101 was down-regulated in osteosarcoma tissues and Saos-2 cell line as compared with that in adjacent non-neoplastic bone tissues and the osteoblastic cell line. To better characterize the role of miR-101 in osteosarcoma, we used a gain-of-function analysis by transfecting human osteosarcoma cell line Saos-2 with chemically synthesized miR-101 mimics. The results showed that overexpression of miR-101 inhibited the proliferation and promoted the apoptosis of Saos-2 cells. Meanwhile, bioinformatic analysis demonstrated that mTOR gene was a direct target of miR-101. Overexpression of miR-101 significantly decreased the expression of mTOR at both mRNA and protein levels in Saos-2 cells, consequently inhibiting Saos-2 cells proliferation and promoting cells apoptosis in an mTOR-dependent manner. Taken together, these data suggest that miR-101 may act as a tumor suppressor, which is commonly downregulated in both osteosarcoma tissues and cells. mTOR plays an important role in mediating miR-101 dependent biological functions in osteosarcoma. Reintroduction of miR-101 may be a novel therapeutic strategy by down-regulating mTOR expression.SummaryStudies have proved that microRNA-101 (miR-101) functions as a tumor suppressor and is associated with growth and apoptosis of various human cancers. However, the role of miR-101 in osteosarcoma and the possible mechanism by which miR-101 affects the tumor growth and apoptosis have not been fully elucidated. In this study, we found that the expression of miR-101 was down-regulated in osteosarcoma tissues and Saos-2 cell line as compared with that in adjacent non-neoplastic bone tissues and the osteoblastic cell line. To better characterize the role of miR-101 in osteosarcoma, we used a gain-of-function analysis by transfecting human osteosarcoma cell line Saos-2 with chemically synthesized miR-101 mimics. The results showed that overexpression of miR-101 inhibited the proliferation and promoted the apoptosis of Saos-2 cells. Meanwhile, bioinformatic analysis demonstrated that mTOR gene was a direct target of miR-101. Overexpression of miR-101 significantly decreased the expression of mTOR at both mRNA and protein levels in Saos-2 cells, consequently inhibiting Saos-2 cells proliferation and promoting cells apoptosis in an mTOR-dependent manner. Taken together, these data suggest that miR-101 may act as a tumor suppressor, which is commonly downregulated in both osteosarcoma tissues and cells. mTOR plays an important role in mediating miR-101 dependent biological functions in osteosarcoma. Reintroduction of miR-101 may be a novel therapeutic strategy by down-regulating mTOR expression.

Ropivacaine- and bupivacaine-induced death of rabbit annulus fibrosus cells in vitro: involvement of the mitochondrial apoptotic pathway

OBJECTIVE The purposes of this study were to assess whether local anesthetics (LAs), such as ropivacaine and bupivacaine, could induce apoptosis of rabbit annulus fibrosus (AF) cells in vitro and further to explore the possible underlying mechanism. METHODS Rabbit AF cells at second passage were treated with saline solution and various concentrations of LAs. Apoptosis of AF cells were examined by cell counting kit-8 (CCK-8), Annexin V assays, Hoechst 33342 staining, and Caspase-3, -9 activity assays. The expression of apoptosis-related markers was detected by real-time PCR (RT-PCR) and Western Blot. The JC-1 staining was used to evaluate the change of mitochondrial membrane potential (MMP). Moreover, the levels of reactive oxygen species (ROS) were determined with fluorescent probe DCFH-DA. RESULTS The results of flow cytometry indicated that LAs could induce apoptosis of rabbit AF cells in a dose-dependent manner. Apoptosis was confirmed by cell morphology, condensed nuclei and activation of Caspase-3 and -9. In addition, the molecular data showed that LAs could significantly up-regulate the expression of Bax, accompanied by a significant down-regulation of Bcl-2 expression. Furthermore, we also observed that LAs resulted in alteration of MMP and accumulation of intracellular ROS in AF cells. Blockade of ROS production by N-acetyl-l-cysteine (NAC) inhibited LAs-induced apoptosis. CONCLUSIONS These findings suggest that LAs in clinically relevant concentrations could induce apoptosis of rabbit AF cells in vitro, and the mitochondrial pathway was, at least in part, involved in the LAs-mediated apoptosis. Further investigations focusing on the potential cytotoxicity of LAs on IVD cells are needed.