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Quan R.,Xiaoshan Traditional Chinese Medical Hospital | Tang Y.,Xiaoshan Traditional Chinese Medical Hospital | Huang Z.,Xiaoshan Traditional Chinese Medical Hospital | Xu J.,Xiaoshan Traditional Chinese Medical Hospital | And 2 more authors.
Ceramics International | Year: 2012

To evaluate the effects of hydroxyapatite/Zirconiumoxide (HA/ZrO 2) composite powder on proliferation and osteogenic differentiation of rabbit mesenchymal stem cells (MSCs) by using molecular biology methods in vitro. HA/ZrO 2 composite powder prepared by using powder of HA and ZrO 2 with different proportions sintered at 1600 °C were compared with pure HA powder and pure ZrO 2 powder. The effects of the composite powder suspensions on the proliferation of the rabbit MSCs were measured by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay), alkaline phosphatase (ALP) activities were measured by the ALP colorimetric assay, and the cellular expression levels of Collagen I, osteocalcin and osteopontin mRNAs were determined by Reverse Transcription Polymerase Chain Reaction (RT-PCR). The HA phase was transformed into the β-Ca 3(PO 4) 2(β-TCP), α-Ca 3(PO 4) 2 and CaZrO 3 phases in the composite powder after sintering at 1600 °C, with a positive correlation between the contents of the HA phase and the new phases. The MTT assay showed that both pure HA powder and HA containing composite powder were able to promote cellular proliferation, but pure ZrO 2 powder had no effect in stimulating cell proliferation (P<0.05). Vonkossa staining revealed that the composites and pure HA powder were capable of reducing the percentages of positively stained cells. The ALP colorimetric assay demonstrated that the ALP activities of cells maintained in culture media with HA and HA containing composite powder were significantly higher than that of cells cultured in regular media or media containing pure ZrO 2 (P<0.05). RT-PCR results showed that the composite powder were able to stimulate the expression of Collagen I and osteocalcin. The HA/ZrO 2 composite powder can also facilitate osteogenic differentiation. © 2012 Elsevier Ltd and Techna Group S.r.l.


Quan R.,Xiaoshan Traditional Chinese Medical Hospital | Tang Y.,Xiaoshan Traditional Chinese Medical Hospital | Huang Z.,Xiaoshan Traditional Chinese Medical Hospital | Xu J.,Xiaoshan Traditional Chinese Medical Hospital | And 2 more authors.
Materials Science and Engineering C | Year: 2013

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). © 2012 Published by Elsevier B.V.


PubMed | Xiaoshan Traditional Chinese Medical Hospital, Zhejiang University, Yunnan University of Traditional Chinese Medicine, Shanghai University and Zhejiang Chinese Medical University
Type: Journal Article | Journal: Journal of biomaterials applications | Year: 2016

To evaluate the effects of porous gradient composites with hydroxyapatite/zirconia and autologous iliac in repair of lumbar vertebra body defects in dogs.(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.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 m. 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.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.


PubMed | Xiaoshan Traditional Chinese Medical Hospital, Zhejiang University, Xiamen University and Zhejiang Chinese Medical University
Type: Journal Article | Journal: Acupuncture in medicine : journal of the British Medical Acupuncture Society | Year: 2014

Spinal cord injury (SCI) can be caused by a variety of pathogenic factors. In China, acupuncture is widely used to treat SCI. We previously found that acupuncture can reduce apoptosis and promote repair after SCI. However, the antiapoptotic mechanisms by which acupuncture exerts its effects on SCI remain unclear. Our aim was to investigate the role of the PI3K/Akt and extracellular signal-regulated kinases (ERK)1/2 signalling pathways in acupuncture treatment of acute SCI. Eighty pure-bred New Zealand rabbits were randomly divided into the following five groups (n=16 per group): control; model; elongated needle electroacupuncture (EA); EA+LY294002; and EA+PD98059. We established a spinal cord contusion model of SCI in all experimental groups except controls, in which only a laminectomy was performed. After SCI, three of the groups received EA once daily for 3days. One hour before SCI, the two drug groups received LY294002 (Akt inhibitor; 10g, 20L) or PD98059 (ERK inhibitor; 3g, 20L) via intrathecal injection. At 48h after SCI, animals were killed and spinal cord tissue samples were collected for transferase dUTP nick end labelling (TUNEL) assays, immunohistochemistry and western blot assays. EA significantly increased p-Akt and p-ERK1/2 expression, reduced cytochrome c and caspase-3 expression and inhibited neuronal apoptosis in the injured spinal cord segment. The opposite effects were seen after using Akt and ERK inhibitors. Acupuncture promotes the repair of SCI, possibly by activation of the PI3K/Akt and ERK1/2 signalling pathways and by inhibition of the mitochondrial apoptotic pathway.


Quan R.,Xiaoshan Traditional Chinese Medical Hospital | Zheng X.,Xiaoshan Traditional Chinese Medical Hospital | Zheng X.,Zhejiang Chinese Medical University | Xu S.,Xiaoshan Traditional Chinese Medical Hospital | And 2 more authors.
Stem Cell Research and Therapy | Year: 2014

Introduction: In the field of skin tissue engineering, gelatin-chondroitin-6-sulfate-hyaluronic acid (Gel-C6S-HA) stents are a suitable bio skin substitute. The purpose was to investigate the effect of genetically-modified hair follicle stem cells (HFSCs), combined with Gel-C6S-HA scaffolds, on the vascularization of tissue-engineered skin. Methods: Three-dimensional (3D) Gel-C6S-HA scaffolds were prepared by freeze-drying. Vascular endothelial growth factor (VEGF) 165 gene-modified rat HFSCs (rHFSCs) were inoculated into the scaffolds and cultured for 7 days. Two bilateral full-thickness skin defects were created on the back of 18 Sprague-Dawley rats. Rats were randomly divided into four groups: Group A, HFSCs transduced with VEGF165 seeded onto Gel-C6S-HA scaffolds; Group B, HFSCs transduced with empty vector seeded onto Gel-C6S-HA scaffolds; Group C, Gel-C6S-HA scaffold only; Group D, Vaseline gauze dressing. These compositions were implanted onto the defects and harvested at 7, 14 and 21 days. Wound healing was assessed and compared among groups according to hematoxylin-eosin staining, CD31 expression, alpha smooth muscle actin (α-SMA) and major histocompatibility complex class I (MHC-I) immunohistochemistry, and microvessel density (MVD) count, to evaluate the new blood vessels. Results: SEM revealed the Gel-C6S-HA scaffold was spongy and 3D, with an average pore diameter of 133.23±43.36 μm. Cells seeded on scaffolds showed good adherent growth after 7 days culture. No significant difference in rHFSC morphology, adherence and proliferative capacity was found before and after transfection (P >0.05). After 14 and 21 days, the highest rate of wound healing was observed in Group A (P <0.05). Histological and immunological examination showed that after 21 days, MVD also reached a maximum in Group A (P <0.05). Therefore, the number of new blood vessels formed within the skin substitutes was greatest in Group A, followed by Group B. In Group C, only trace amounts of mature subcutaneous blood vessels were observed, and few subcutaneous tissue cells migrated into the scaffolds. Conclusions: Tissue-engineered skin constructs, using 3D Gel-C6S-HA scaffolds seeded with VEGF165-modified rHFSCs, resulted in promotion of angiogenesis during wound healing and facilitation of vascularization in skin substitutes. This may be a novel approach for tissue-engineered skin substitutes. © 2014 Quan et al.; licensee BioMed Central Ltd.


PubMed | Xiaoshan Traditional Chinese Medical Hospital and Zhejiang Chinese Medical University
Type: Journal Article | Journal: Cytotechnology | Year: 2016

The purpose of this study was to establish methods for isolation, culture, expansion, and characterization of rat hair follicle stem cells (rHFSCs). Hair follicles were harvested from 1-week-old Sprague-Dawley rats and digested with dispase and collagenase IV. The bulge of the hair follicle was dissected under a microscope and cultured in Dulbeccos modified Eagles medium/F12 supplemented with KnockOut Serum Replacement serum substitute, penicillin-streptomycin, L-glutamine, non-essential amino acids, epidermal growth factor, basic fibroblast growth factor, polyhydric alcohol, and hydrocortisone. The rHFSCs were purified using adhesion to collagen IV. Cells were characterized by detecting marker genes with immunofluorescent staining and real-time polymerase chain reaction (PCR). The proliferation and vitality of rHFSCs at different passages were evaluated. The cultured rHFSCs showed typical cobblestone morphology with good adhesion and colony-forming ability. Expression of keratin 15, integrin 6, and integrin 1 were shown by immunocytochemistry staining. On day 1-2, the cells were in the latent phase. On day 5-6, the cells were in the logarithmic phase. Cell vitality gradually decreased from the 7th passage. Real-time PCR showed that the purified rHFSCs had good vitality and proliferative capacity and contained no keratinocytes. Highly purified rHFSCs can be obtained using tissue culture and adhesion to collagen IV. The cultured cells had good proliferative capacity and could therefore be a useful cell source for tissue-engineered hair follicles, vessels, and skin.


PubMed | Xiaoshan Traditional Chinese Medical Hospital, Zhejiang University and Zhejiang Chinese Medical University
Type: | Journal: Journal of the mechanical behavior of biomedical materials | Year: 2014

Vertebroplasty using poly(methyl methacrylate) (PMMA) bone cement is the most common method to treat osteoporotic vertebral fractures. However, several questions of interest remain to be clarified, including how does PMMA affect the cement-bone interface area and surrounding bone tissue, can damaged bone tissues be repaired, how will PMMA change the bone interface over the long-term, and what happens to PMMA itself? The purpose of this study is to investigate these concerns and provide a basis for clinical evaluation. We made bone defects in the lumbar vertebrae of New Zealand rabbits as a model of osteoporosis and injected them with bone cement. A mechanical testing machine was used to perform axial compression, three-point bending, and twisting resistance tests to observe and investigate the short- and long-term biomechanical properties of PMMA after implantation. Optical, fluorescence, scanning electron microscopy, and nanoindentation were used to observe the changes in the interface microstructure. PMMA can rapidly establish the strong support with stable function in the near future. Biomechanical experiments showed that biomechanical property of bone cement group was significantly higher than those in the other two groups (P<0.05) biomechanical property of bone cement group may decline with the time, but its still better than that of OP in the control group (P<0.05). Histomorphological observation result shows that under osteoporosis state the bone grows slower, also bones rebuilding time extended. And in the later period, main bones continuous osteoporosis has some impact on the interface. Nano-indentation testing shows that the young modulus and stiffness of the interface among bone, material and interface were significantly differences (P<0.05). Bone cement had gave the best nano indentation hardness, then was interface and bone tissue. PMMA bone cement was able to quickly support and stabilize the defect in the short term, and bone growth restarted at the bone interface and was tightly integrated. However, over the long-term, fluorescent signal was weakened, osteoclasts appeared, the mechanical indicators for both the interface and the whole vertebra decreased, and bone resorption was eventually greater than bone formation, resulting in bone loss. Therefore, vertebroplasty is not the end of treatment, and we need to further study ways to improve the bone cement material, which is crucial for long-term vertebroplasty efficacy, to better treat osteoporosis.


PubMed | Xiaoshan Traditional Chinese Medical Hospital and Zhejiang University
Type: Journal Article | Journal: Molecular medicine reports | Year: 2016

Monotropein, the primary iridoid glycoside isolated from Morindacitrifolia, has been previously reported to possess potent antioxidant and antiosteoporotic properties. However, there is no direct evidence correlating the antiosteoporotic effect of monotropein with its observed antioxidant capacity, and the molecular mechanisms involved in mediating these processes remain unclear. Therefore, the aim of the present study was to investigate the protective effects of monotropein against oxidative stress in osteoblasts and the mechanisms involved in mediating this process. Osteoblast viability was evaluated using the MTT assay. The mitochondrial membrane potential and reactive oxygen species were detected by flow cytometry analyses. Western blotting and enzymelinked immunosorbent assays were performed to detect protein expression levels. A significant reduction in osteoblast viability was observed at 24h following exposure to various concentrations (1001,000M) of H2O2 compared with untreated osteoblasts. The cytotoxic effect of H2O2 was notably reversed when osteoblasts were pretreated with 110g/ml monotropein. Pretreatment with 1-10g/ml monotropein increased the mitochondrial membrane potential and reduced the generation of reactive oxygen species in osteoblasts following exposure to H2O2. In addition, the H2O2induced increase in apoptotic markers (caspase-3 and caspase-9) and H2O2-induced reduction in sirtuin 1 levels were significantly reversed following pretreatment of cells with monotropein. Furthermore, monotropein significantly reduced H2O2induced stimulation of NFB expression, in addition to the expression of a number of proinflammatory mediators. These results indicate that monotropein suppresses apoptosis and the inflammatory response in H2O2induced osteoblasts through the activation of the mitochondrial apoptotic signaling pathway and inhibition of the NFB signaling pathway.


Quan R.,Xiaoshan Traditional Chinese Medical Hospital
Scandinavian journal of rheumatology | Year: 2013

To evaluate the effects of the proteasome inhibitor MG-132 on the expression of nuclear factor (NF)-κB p65, inhibitor (I)-κB, tumour necrosis factor (TNF)-α, and interleukin (IL)-1β in the cartilage and synovial tissues of rats with osteoarthritis (OA), and to investigate the role that the ubiquitin/proteasome system (UPS) plays in the OA process. A total of 144 adult male Sprague Dawley rats were randomly assigned to four groups: anterior cruciate ligament transaction (ACLT) + MG-132 (ACLT/M), ACLT + dimethylsulfoxide (ACLT/D), sham surgery (Sham), and naïve + MG-132 (naïve/M). Pathological morphology was undertaken. mRNA expression levels of NF-κB p65, I-κB, TNF-α, and IL-1β were determined using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The activities of the 20S proteasome chymotrypsin-like and peptidylglutamyl-peptide hydrolase-like enzymes were measured using fluorospectrophotometry. The Mankin scores at all time points in ACLT/M rats were significantly lower than those in ACLT/D rats (p < 0.05). Despite the NF-κB p65 in the synovial tissue at 2 weeks after surgery and IL-1β in the cartilage tissue at 12 weeks after surgery, mRNA expression levels of NF-κB p65, IL-1β, and TNF-α at other time points in ACLT/M were significantly lower than those in ACLT/D (p < 0.05). mRNA levels of I-κB in the cartilage tissue in ACLT/M were significantly higher than those in ACLT/D at 2 weeks after surgery (p < 0.05). mRNA levels of I-κB in the synovial tissue in ACLT/M were higher than those in ACLT/D at all time points, and the difference was significant at 4 weeks after surgery (p < 0.05). MG-132 decreased the activities of the 20S proteasome chymotrypsin-like and peptidylglutamyl-peptide hydrolase-like enzymes in the cartilage and synovial tissues of rats. The proteasome inhibitor MG-132 delays the progress of OA by alleviating synovial inflammation and protecting the articular cartilage tissue.

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