Disheng Yang

Size effect of hydroxyapatite nanoparticles on proliferation and apoptosis of osteoblast-like cells

Nano-hydroxyapatite (nano-HAP) may be a better candidate for an apatite substitute of bone in biomedical applications than micro-sized hydroxyapatite (m-HAP). However, size control is always difficult when synthesizing well-defined nano-HAP particles. In this study, nano-HAP particles with diameters of approximately 20nm (np20) and approximately 80nm (np80) were synthesized and characterized. The size effects of these nano-HAPs and m-HAP were studied on human osteoblast-like MG-63 cells in vitro. Our results demonstrate that both cell proliferation and cell apoptosis are related to the size of the HAP particles. Np20 has the best effect on promotion of cell growth and inhibition of cell apoptosis. This work provides an interesting view of the role of nano-HAPs as ideal biomedical materials in future clinical applications.

Response of osteogenic sarcoma to neoadjuvant therapy: evaluated by 18F-FDG-PET

ObjectiveThe aim of this study was to evaluate the potential role of F-18-fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET) in assessing the chemotherapy response of osteosarcoma when compared with histologically assessed tumor necrosis.MethodsFifteen patients were examined with whole-body FDG-PET prior to and following neoadjuvant therapy. The maximum standard uptake value (SUVmax) of tumor and tumor to background ratio (TBR) prior to and following chemotherapy was used for semiquantitative PET imaging analysis. The SUVmax of prechemotherapy and post-chemotherapy was recorded as SUV1 and SUV2. TBR1 and TBR2 represented prechemotherapy and post-chemotherapy TBR. TBR was calculated by drawing an identical region of interest over the tumor and the contralateral normal limb or pelvis. Tumor necrosis was classified according to Salzer-Kuntschik’s criteria.ResultsEight patients with more than 90% tumor necrosis were classified as showing good responses and seven patients with less than 90% tumor necrosis as showing poor responses. SUV2/SUV1, TBR2/TBR1, and TBR2 were significantly correlated with the tumor necrosis degree (P < 0.01, P < 0.001, P < 0.001). TBR2/TBR1 were below 0.46 in all the patients with favorable responses, and higher than 0.49 in all the patients with unfavorable responses. However, it was difficult to distinguish good responses from poor responses by SUV2/SUV1.ConclusionsFDG-PET is a promising tool to assess the chemotherapy response of osteosarcoma noninvasively. The TBR was better than SUVmax in evaluating the chemotherapy response in this study.

Caspase‐8 dependent osteosarcoma cell apoptosis induced by proteasome inhibitor MG132

Many researchers have reported that proteasome inhibitors could induce apoptosis in a variety of cancer cells, such as breast cancer cell, lung cancer cell, and lymphoma cell. However, the effect of proteasome inhibitors on osteocsarcoma cells and the mechanisms are seldom studied. In this study, we found proteasome inhibitor MG132 was an effective inducer of apoptosis in human osteosarcoma MG‐63 cells. On normal human diploid fibroblast cells, MG132 did not show any apoptosis‐inducing effects. Apoptotic changes such as DNA fragment and apoptotic body were observed in MG132‐treated cells and MG132 mostly caused MG‐63 cell arrest at G2–M‐phase by cell cycle analysis. Increased activation of caspase‐8, accumulation of p27Kip1, and an increased ratio of Bax: Bcl‐2 were detected by RT—PCR and Western blot analysis. Activation of caspase‐3 and caspase‐9 were not observed. This suggests that the apoptosis induced by MG132 in MG63 cells is caspase‐8 dependent, p27 and bcl‐2 family related.

Multicentric Epithelioid Angiosarcoma of Bone

Bone epithelioid angiosarcoma is rare and generally shows positive immunostaining for epithelial markers. Multicentric bone epithelioid angiosarcoma is easily misdiagnosed as carcinoma, including metastatic carcinoma, multiple myeloma, and multiple lymphoma of bone. This article describes a case of multicentric bone epithelioid angiosarcoma. The patient was first misdiagnosed as having metastatic carcinoma. Examination showed osteolytic lesions in the bilateral heels and the lower left humerus. The diagnosis was confirmed postoperatively and corrected after immunohistochemical analysis of the biopsy. The immunohistochemical analysis revealed that the tumor mass was strongly positive for CD31, factor VIII, vimentin, and neuron-specific enolase. The patient refused chemotherapy and died of lung metastasis 4 months postoperatively.Most bone epithelioid angiosarcomas are immunopositive for epithelial markers (ie, keratin, cytokeratin, high-molecular-weight keratin, and epithelial membrane antigen), vascular endothelial markers (ie, CD31, CD34, and von Willebrand factor), and factor VIII-associated antigen. Bone epithelioid angiosarcoma shows a relatively high degree of malignancy. Patients often die of distant metastasis, including those found in the lung and lymph node tissue. A wide excision of epithelioid angiosarcoma should be performed during the operation of the primary tumor. A better understanding of the clinicopathologic features of this disease may help to clarify the confusion, provide better treatment, and improve the clinical prognosis.

The construction of the eukaryotic expression plasmid pcDNA3.1/azurin and the increased apoptosis of U2OS cells transfected with it

In our previous study, we demonstrated that azurin could selectively trigger apoptosis in human osteosarcoma cell line U2OS cells. However, the rate of apoptosis (35.8 ± 3.2%) is not very high, and azurin is too expensive to obtain readily. To solve these problems, we constructed a eukaryotic expression plasmid containing the azurin gene with an influenza virus haemagglutinin 9 peptide HA epitope tag, and transfected the recombinant plasmid pcDNA3.1(+)/azurin into U2OS cells. RT-PCR and Western blot analysis validated the successful transfection and the expression of the azurin-HA protein. Conspicuous apoptosis of the transfected cells was detected by flow cytometry (FCM) and the DNA ladder test. The apoptosis rate reached 64.3 ± 13.1%. The transcriptional levels of the Bax and p53 genes increased significantly in U2OS cells transfected with pcDNA3.1(+)/azurin, but the Bcl-2 mRNA level decreased. There was no difference in the levels of Bcl-xl mRNA and Survivin mRNA. We propose that the transfection of the recombinant plasmid pcDNA3.1(+)/azurin can significantly induce apoptosis in U2OS cells. This is closely associated with the up-regulation of the transcriptional level of the Bax and p53 genes, and the down-regulation of that of the Bcl-2 gene.

Study on the genotoxicity of HA/ZrO2 composite particles in vitro

To evaluate the genotoxicity of the HA/ZrO2 composite particles by using the micronucleus test (MNT) in vitro. HA/ZrO2 composite particles prepared by sintering at high temperature and pressure, that used powder of HA and ZrO2 of different proportions, were compared with pure HA particles and pure ZrO2 particles. The effect of the composite particles on cell proliferation of rabbit mesenchymal stem cells, and its the genotoxicity to rabbit mesenchymal stem cells were detected by MNT method. The MTT test showed that both pure HA particles and composite particles which contained HA promoted cell proliferation of rabbit mesenchymal stem cells, while pure ZrO2 particles did not, and there was a significant difference (P<0.05). The MNT test showed no significant difference between the HA group and the negative control group (P>0.05), but a significant difference between the HA group and the positive control group (P<0.05). The difference between the ZrO2 group and the negative control group was significant (P<0.01), while the difference between the ZrO2 group and the positive control group was insignificant (P>0.05). The genotoxicity of the HA/ZrO2 composite particle increased with a higher proportion of ZrO2 and an increase in the concentration of the composite, and the 30 wt.% HA/70% ZrO2 composite with 200 μg/mL concentration showed significant genotoxicity (P<0.01).

Elevated Levels of Immunoglobulin E May Indicate Steroid Resistance or Relapse in Adult Primary Nephrotic Syndrome, Especially in Minimal Change Nephrotic Syndrome

Immunoglobulin E (IgE) antibodies may play a role in the development of kidney diseases that are related to hypersensitivity reactions. Patients with idiopathic nephrotic syndrome often exhibit increased serum IgE levels and this may be related to sensitivity to steroid treatment. In the present study, the serum IgE levels in 120 patients with different types of primary nephrotic syndrome (PNS) were analysed and found to be significantly elevated in cases of minimal change nephrotic syndrome (MCNS) compared with membranous nephropathy or membranoproliferative glomerulonephritis. No difference in serum IgE level was observed between cases of steroid-sensitive nephrotic syndrome (SSNS) or steroid-resistant nephrotic syndrome, although the serum IgE level was significantly elevated in SSNS patients in relapse compared with SSNS patients in remission. In MCNS patients, 73.6% exhibited SSNS regardless of their serum IgE level at diagnosis. It is concluded that elevated levels of IgE may be a feature of steroid resistance or relapse, indicating prognostic significance in adult PNS, particularly in MCNS.

The study of traditional Chinese medical elongated-needle therapy promoting neurological recovery mechanism after spinal cord injury in rats

ETHNOPHARMACOLOGICAL RELEVANCE Acupuncture is a key part of traditional Chinese medicine, shown to induce favorable neuroplasticity for injuries in the central and peripheral nervous systems. Recent studies report elongated needle therapy (ENT) with BL54 and ST28 may restore acute spinal cord injury (ASCI). However, the precise mechanism for this has not been elucidated. AIM OF THE STUDY In our current study, we investigated the effects of ENT on inflammation and neuronal apoptosis induced by ASCI, and whether PI3K/Akt and MAPK/ERK signaling pathways are involved in the ENT restoration effect. MATERIALS AND METHODS Rat models of moderate SCI were established in accordance with the modified Allens method and were treated with ENT continuously for 7 days. Spontaneous activities were evaluated by the Basso Beattie and Bresnahan locomotor scale. Levels of inflammatory cytokines, such as tumor necrosis factor alpha, interleukin-6, IL-1β, and nuclear factor kappa-β, were determined by enzyme-linked immunosorbent assay. Cell apoptosis was examined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The proportions of cells with positive Bcl-2 and Bax expression were determined by immunohistochemical assays, whilst the expression profiles of p-AKT and p-ERK in spinal cord tissues were evaluated by western blotting. Furthermore, the expression profiles of Cytochrome-C (Cyt-C) and caspase-3 in vivo were analyzed by reverse transcription polymerase chain reaction. The potential inhibitory effects downstream of the Akt and ERK signaling pathways were examined by administration of specific inhibitors LY294002 and PD98059 in vivo. RESULTS As indicated by this study, inflammation as well as PI3K/Akt- and MAPK/ERK signaling pathway-mediated neuronal apoptosis were involved in the course of SCI in rats. The neuro-protective effect of ENT was associated with reduced Bax protein-positive neurons and increased Bcl-2 protein-positive neurons. ENT enhanced recovery of rat activities. Activation of p-Akt and p-ERK in the PI3K/Akt and MAPK/ERK signaling pathways, inhibited expression of the critical component Cyt-C. Cyt-C is required for the mitochondrial apoptosis pathway and cascade of caspase-3, which is involved in activation of neuronal apoptosis through down-regulation of Bax protein and up-regulation of Bcl-2, as determined by TUNEL. The administration of PI3K/Akt and MAPK/ERK signaling pathway specific inhibitors, LY294002 and PD98059, suppressed expression of both p-Akt and p-ERK. CONCLUSION ENT with BL54 and ST28 points can promote the recovery of ASCI. And the neuro-protective effect of ENT during the restoration of SCI may be associated with the suppression of both inflammation and activation of PI3K/Akt and MAPK/ERK signaling pathways, resulting from down-regulation of Bax protein, up-regulation of Bcl-2, and inhibition of the mitochondrial apoptosis pathway.

Evaluation of porous gradient hydroxyapatite/zirconia composites for repair of lumbar vertebra defect in dogs

Objective To evaluate the effects of porous gradient composites with hydroxyapatite/zirconia and autologous iliac in repair of lumbar vertebra body defects in dogs. Methods (1) New porous gradient hydroxyapatite/zirconia composites were prepared using foam immersion, gradient compound and high temperature sintering; (2) A total of 18 adult beagle dogs, aged five to eight months and weighted 10–13 kg, were randomly assigned into two subgroups, which were implanted with new porous gradient hydroxyapatite/zirconia composites (subgroup A in 12) or autologous iliac bone (subgroup B in 6); (3) The post-operative data were analyzed and compared between the subgroups to repair the vertebral body defect by roentgenoscopy, morphology and biomechanics. Results The porosity of new porous gradient hydroxyapatite/zirconia composites is at 25 poles per inch, and the size of pores is at between 150 and 300 µm. The post-operative roentgenoscopy displayed that new-bone formation is increased gradually, and the interface between composites and host-bone becomes became blur, and the new-bone around the composites were integrated into host-bone at 24 weeks postoperatively in subgroup A. As to subgroup B, the resorption and restructure were found at six weeks after the surgery, and the graft-bone and host-bone have been integrated completely without obvious boundary at 24 weeks postoperatively. Histomorphologic study showed that the amount of bone within pores of the porous gradient hydroxyapatite/zirconia composites increased continuously with a prolonged implantation time, and that partial composites were degradated and replaced by new-bone trabeculae. There was no significant difference between subgroups (P > 0.05) in the ultimate compressive strengths. Conclusion New porous gradient hydroxyapatite/zirconia composites can promote the repair of bony defect, and induce bone tissue to ingrow into the pores, which may be applied widely to the treatment of bony defect in the future.

Evaluation of a Novel HA/ZrO2-Based Porous Bioceramic Artificial Vertebral Body Combined with a rhBMP-2/Chitosan Slow-Release Hydrogel.

A new HA/ZrO2-based porous bioceramic artificial vertebral body (AVB), carried a recombinant human bone morphogenetic protein-2 (rhBMP-2)/chitosan slow-release hydrogel was prepared to repair vertebral bone defect in beagles. An ionic cross-linking was used to prepare the chitosan hydrogel (CS gel) as the rhBMP-2 slow-release carrier. The vertebral body defects were implanted with the rhBMP-2-loaded AVB in group A, or a non-drug-loaded AVB in group B, or autologous iliac in group C. The encapsulation rate of rhBMP-2 in rhBMP-2-loaded CS gel was 91.88±1.53%, with a drug load of 39.84±2.34 ng/mg. At 6, 12, 24 weeks postoperatively, radiography showed that the bone calluses gradually increased with time in group A, where the artificial vertebral body had completely fused with host-bone at 24 weeks after surgery. In group C, an apparent bone remodeling was occurred in the early stages, and the graft-bone and host-bone had also fused completely at 24 weeks postoperatively. In group B, fusion occurred less than in groups A and C. At 24 weeks after surgery, micro-computed tomography (Micro-CT) revealed that the volume of newly-formed bone in group A was significantly more than in group B (p<0.05). At 24 weeks after surgery, ultra-compressive strengths of the operated segments were 14.03±1.66 MPa in group A, 8.62±1.24 MPa in group B, and 13.78±1.43 MPa in group C. Groups A and C were both significantly higher than group B (p < 0.05). At 24 weeks postoperatively, the hard tissue sections showed that the AVB of group A had tightly fused with host bone, and that pores of the AVB had been filled with abundant nearly mature bone, and that the new bone structured similarly to a trabecular framework, which was similar to that in group C. In contrast, implant fusion of the AVB in group B was not as apparent as group A. In conclusion, the novel HA/ZrO2-based porous bioceramic AVB carried the rhBMP-2-loaded CS gel can promote the repair of bony defect, and induce bone tissue to grow into the pores, which may replace iliac bone grafts as commonly applied in clinical practice.