Cytotoxicity assessment of bone marrow mesenchymal stem cells-cuttlebone composite scaffold / 中国组织工程研究

BACKGROUND: Repair materials for bone tissue engineering should hold good biocompatibility and degradability. There are various related studies, but the Chinese medicine composite cellular bioscaffolds are little reported. OBJECTIVE: To detect the in vitro cytotoxicity of rabbit bone marrow mesenchymal stem cells-cuttlebone bioscaffold based on the Biological Evaluation of Medical Device, and assess its cytotoxicity level in order to provide the theoretical support for its clinical application. METHODS: Bone marrow mesenchymal stem cells-cuttlebone bioscaffold extract was prepared according to an ISO standard — material area: extraction medium volume = 3-6 cm2:1 mL. L-929 cell suspension was prepared, and the cells were then cultured with a density of 1×107/L. There were three groups: positive group (DMEM medium containing phenol), experimental group (material extract), and negative group (DMEM culture medium). The absorbance value of L-929 cells was detected by MTT assay after 24, 48 and 72 hours of culture. The relative proliferation rate of cells was then calculated and the toxicity level was valued in each group. RESULTS AND CONCLUSION: The absorbance values in the experimental, negative and positive groups were not exactly same at different time points (P=0.000 < 0.01). The absorbance values in the experimental and negative groups were significantly higher than those in the positive group (P < 0.01).The cytotoxicity of bone marrow mesenchymal stem cells-cuttlebone bioscaffold was grade 1. To conclude, the bone marrow mesenchymal stem cells-cuttlebone bioscaffold has no obvious toxic effects, and meets the requirements of biomaterial application.

Proteomics and peptidomics analysis of Sepiae Endoconcha / 中国中药杂志

Shotgun based proteomics and peptidomics analysis were used to investigate the proteins and peptides in marine traditional Chinese medicine(TCM) Sepiae Endoconcha(cuttlebone). Peptides were extracted from cuttlebone by acidified methanol, and then strong cation exchange(SCX) resin was used to enrich those peptides. Also, proteins from cuttlebone were extracted and digested by trypsin. nano-LC Q Exactive Orbitrap mass spectrometry was used to analyze proteins and peptides from cuttlebone. As a result, a total of 16 proteins and 168 peptides were identified by protein database search, and 328 peptides were identified by De novo sequencing. The identified proteins were hemocyanin, enolase, myosin, actin, calmodulin, etc., and the identified peptides were derived from actin, histone, and tubulin. All these proteins and peptides were important components in cuttlebone, which would provide important theoretical and research basis for marine TCM cuttlebone investigations.

Preparation technology of Haimeng Zhisuan sustained release tablets / 中草药

Objective: To optimize the formulation of Haimeng Zhisuan gastric floating sustained-release tablets and to investigate its in vitro release properties. Methods: Plackett-Burman design was used to screen three most influential factors, i.e. matrices, floating material, and fillers which had significant impact on the preparation of floating sustained-release tablet by evaluating the cumulative release rate of CaCO3 at 2, 4, and 12 h; The levels of non-important factors were also determined. A 3-factor, 3-level Box-Behnken design was led to optimize the formulation, using cumulative release rate as index. The experiment result was optimized and the drug release kinetics equation was fitted by response optimizer. Results: The optimized tablet formulation was as follows: cuttlebone 225.0 mg, Montmorillonite 25.0 mg, HPMC K100M 100.0 mg, MCC 48.3 mg, EC 25.0 mg, searyl alcohol 74.7 mg, and 5% PVP-K30 ethanol solution as adhesive. The experimental data of optimized tablet were similar to the predicted values. The floating duration was over 12 h in simulated gastric fluid. Data of the in vitro release were fitted to the first-order equation. Conclusion: Formulation of Haimeng Zhisuan floating sustained-release tablet is found to be reasonable. The preparation technology is feasible. The tablet shows good in vitro release properties within 12 h.

Chitin from Cuttlebone Activates Inflammatory Cells to Enhance the Cell Migration

Our previous report showed that the extract from cuttlebone (CB) had wound healing effect in burned lesion of rat and the extract was identified as chitin by HPLS analysis. We herein investigated the morphology in CB extract using scanning electron microscope (SEM). Chitin was used as a control. There is no difference in morphology between CB extract and chitin. We also assessed the role of CB extract on the production of inflammatory mediators using murine macrophages and the migration of inflammatory cells. The extract induced the production of nitric oxide (NO) in macrophages. While the extract of CB itself stimulated macrophages to increase the expression of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6, CB extract suppressed the production of those cytokines by LPS. CB extract also induced the production of mouse IL-8 which is related to the cell migration, and treatment with CB enhanced fibroblast migration and invasion. Therefore, our results suggest that CB activates inflammatory cells to enhance the cell migration.

Chitin from the Extract of Cuttlebone Induces Acute Inflammation and Enhances MMP1 Expression

We previously reported that the extract from cuttlebone (CB) has wound healing effect in burned lesion of rat. In present study, the main component of CB extract was analyzed and its wound healing activity was evaluated by using in vitro acute inflammation model. The extract of CB stimulated macrophages to increase the production of TNF-alpha. The extract also enhanced the production of TGF-beta and VEGF, which were involved in angiogenesis and fibroblast activation. The treatment with CB extract enhanced proliferation of murine fibroblast. CB extract also induced the activation of fibroblast to increase the secretion of matrix metalloproteases 1 (MMP1). The constituent of CB extract which has wound healing activity was identified as chitin by HPLC analysis. The mechanism that the CB extract helps to promote healing of burned lesion is associated with that chitin in CB extracts stimulated wound skins to induce acute inflammation and to promoted cell proliferation and MMP expression in fibroblast. Our results suggest that CB or chitin can be a new candidate material for the treatment of skin wound such as ulcer and burn.

Screening of antimicrobial potential of polysaccharide from cuttlebone and methanolic extract from body tissue of Sepia prashadi Winkworth, 1936

Objective: To evaluate the antimicrobial activity of polysaccharide from cuttlebone and methanolic extract from body tissue of Sepia prashadi, against ten human pathogenic bacteria and five fungi. Methods:The activity of polysaccharide and methanolic extract was investigated against Vibrio cholerae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Vibrio alginolyticus, Staphylococcus aureus, Vibrio parahaemolyticus, Streptococcus sp., Streptococcus pneumoniae, Salmonella sp. and Escherichia coli, and five fungal strains such as Alternaria alternata, Candida tropicalis, Penicillium italicum, Fusarium equiseti and Candida albican using disc diffusion method and minimum inhibitory concentration (MIC) were also calculated. Results:Both polysaccharide and methanolic extract was active against gram positive than that of gram negative pathogenic bacteria but inactive against fungi. The MIC of both the extract ranging from 60 to 100 mg/mL. Conclusions: These results suggest that cephalopod polysaccharide and methanolic extract possess relatively good antibacterial activity.

Extraction of Cuttlebone polysaccharides and purification of their active component CPS-1 / 第二军医大学学报

Objective:To extract the active polysaccharides from traditional Chinese herb-cuttlebone and to purify cuttlebone polysacchride salts 1(CPS-1),an active component of crude polysaccharides,so as to obtain refined natural active polysaccharides.Methods: Hot-water extraction method was optimized by orthogonal designing and was used to extract crude polysaccharides from cuttlebone.The total sugar contents of crude polysaccharides were determined.DEAE Sepharose F.F column and Sepharose CL-6B column were applied to separate CPS-1 from the crude polysaccharides.The active components were determined by animal experiments and Sephacryl S-300 column was used for further purification of CPS-1.HPLC was used to determine the purity of refined CPS-1 and the molecular weight of CPS-1 was determined by polysaccharides standard curve.Results: Crude polysaccharides were successfully extracted from cuttlebone.After purification with DEAE Sepharose F.F column,Sepharose CL-6B column and Sephacryl S-300 column and activity study,refined CPS-1 with average molecular weight(1?10~6) was obtained,and the sugar content reached 93.6%.Conclusion: Different extraction conditions have different extraction results for crude polysaccharides and different separation materials can separate the mixed polysaccharides by their molecular weights or charge characteristics.CPS-1 is a natural active polysaccharide extracted from cuttlebone.