Shujie Huang

Proliferative effect and osteoinductive potential of extracellular matrix coated on cell culture plates

Different cell/tissue derived extracellular matrix (ECM) display subtle differences that might provide important cues for proliferation and differentiation of cells in vitro or in vivo. However, the bioactivities of different ECMs in vitro were not fully understood. In this study, osteoblasts-derived and fibroblast-derived ECM-coated cell culture dishes were prepared respectively by culturing osteoblastic MC3T3-E1 cells and rat fibroblast then decellularizing the cultures. We investigated the bioactivities of the two different ECMs coated on cell culture plates using cellular, biochemical and molecular method. The proliferative activity of the bone marrow-derived mesenchymal stem cells (BMSCs) cultured on osteoblast-ECM was lower than for BMSCs grown on fibroblast-ECM. Compared with the BMSCs cultured on fibroblast-derived ECM, the cells grown on osteoblastic ECM showed enhanced alkaline phosphatase (ALP) activity, higher BMP-2 and osteopontin protein levels, increased secreted calcium content, and higher levels of runt-related transcriptional factor 2 (Runx 2) and osteocalcin (OCN) mRNA. Knockdown of BMP-2 or FGF-2 with shRNA transfection hardly effected osteoblastic differentiation or proliferation of MC3T3-E1 seeded on osteoblast-ECM or fibroblast-ECM. Therefore, the osteoblastic ECM had better osteoinductive potential and lower proliferative effect than fibroblastic ECM, and the two ECM presented enough bioactivity, knockdown of growth factors had no significant effect on differentiation and proliferation of re-seeded cells.

Blood coagulation evaluation of N-alkylated chitosan

N-Alkylated chitosan (NACS) may improve the haemostatic efficiency of chitosan (CS). To study its coagulation capability and function, a series of NACS with various carbon chain lengths and substitution degrees (SD) of alkyl groups were synthesized and characterized by FTIR, NMR, and elemental analysis. Haemolysis and toxicity assays revealed that NACS showed good biocompatibility. In vitro blood clotting tests indicated that NACS had better haemostatic activity than CS, of which N-octadecyl CS with 3.85% SD showed the best results. Blood plasma coagulation tests showed that NACS was not favourable for activating coagulation factors. Platelet adhesion, intracellular Ca2+, and CD62p measurements demonstrated that the coagulation properties of NACS were not related to platelet activation. Erythrocyte adhesion examination indicated that blood coagulation of NACS may be attributable to its effects on erythrocytes. This study suggests that NACS is an ideal candidate for clotting.

Effect of Grafted Chain Length on Hemostatic Activity of N-alkylated Chitosan

This study aimed to synthesize N-alkylated chitosan derivates with similar degree of substitution (DS) and alkyl groups of different carbon chain lengths via reductive alkylation, and then to compare their coagulation properties. Structural properties of the N-alkylated chitosan derivates were characterized by Fourier transform infrared spectroscopy, elemental analysis and H nuclear magnetic resonance spectroscopy. Their pro-coagulant properties were evaluated by detecting whole blood clotting time and performing TEG test. Pro-coagulant mechanisms of the Nalkylated chitosan derivates were explored by detecting intracellular Ca concentration in platelets and P-selectin expression on platelets. We successfully prepared N-alkylated chitosan derivates with a DS of 32.88%-39.78%, including NACS6, NACS12 and NACS18. Our experimental findings showed that all N-alkylated chitosan derivates (NACS6, NACS12, NACS18) have outstanding procoagulant effects, and the longer alkyl chain, the better procoagulant ability. However, N-alkylated chitosan cannot activate platelets, and its procoagulant mechanism remains to be further studied in future. Based on the above results, this paper makes a conjecture about the coagulation mechanism of N-alkylation chitosan. Keywords-chitosan; alkylation; blood coagulation

Exploration of Blood Coagulation of N-Alkyl Chitosan Nanofiber Membrane in Vitro

N-Alkylated chitosan (NACS) may improve the blood clotting efficiency of chitosan (CS). To study its blood coagulation capability, a series of NACSs with various carbon chain lengths and degrees of substitution (DS) of alkyl groups were synthesized and characterized by FTIR, NMR, elemental analysis, and X-ray diffraction (XRD). The corresponding NACS nanofiber membranes (NACS-NM) were subsequently fabricated by electronic spinning technique. SEM, XRD, DSC, surface area, porosity, contact angle, blood absorption, and mechanical properties were used to characterize the CS-NM/NACS-NM. Moreover, cytotoxicity, coagulation, activated partial thromboplastin time, plasma prothrombin time, thrombin time, and platelet aggregation tests were performed to evaluate the biocompatibility and blood coagulation properties of NACS-NM. The results showed that NACS-NM was not cytotoxic. NACS-NM with DS of 19.25% for N-hexane CS (CS6b), 17.87% for N-dodecane CS (CS12b), and 8.97% for N-octadecane CS (CS18a) exhibited good blood clotting performance. Moreover, NACS-NMs favored the activation of coagulation factors and platelets. In addition, intracellular Ca2+ was not related to platelet activation. The above results suggested that NACS-NM would be an effective hemostatic agent.