Jimin Wu

Modification of collagen-chitosan matrix by the natural crosslinker alginate dialdehyde.

In the present study, collagen (Coll) was mixed with the natural crosslinker chitosan (CTS), and then, alginate dialdehyde (ADA) was added to crosslink the mixtures. The properties of these Coll matrix sponges were investigated afterwards. Fourier transform infrared spectroscopy (FTIR) analysis and in vitro fiber formation analysis showed the intact retention of the classical triple-helical structure after crosslinking. Scanning electron microscopy (SEM) showed that microfibril structural interactions between Coll structures became more compact. Significant improvement in the thermostability of the crosslinked mixtures was observed with the pure mixtures of Coll and CTS. Antibacterial activity measurements indicated no affect of ADA on Coll/CTS sponges. In conclusion, the modification of the Coll/CTS mixtures with ADA preserves the classical triple-helical structure, enhances stabilization, maintains good biocompatibility and may pave the way for new medical applications.

Blood clot initiation by mesoporous silica nanoparticles: dependence on pore size or particle size?

Mesoporous silica nanoparticles (MSNs) with controllable pore size and particle size were prepared using a vesicle-organic template method. Transmission electron microscopy (TEM), nitrogen adsorption measurements, X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectra were used to characterize the coagulation-promoting surface chemistry, topologies and porous structure of the MSNs. The clotting blood tests (CBTs) showed that the pore sizes of the MSNs varying from 5 nm to 15 nm greatly affected the blood clot rate of rabbit plasma, while variation of the particle size from 60 nm to 220 nm had little influence on coagulation. Associated with the blood coagulation factor XII (FXII) tests, it could be inferred that the accessibility and diffusion of clotting were mainly dependent on the pore size of the MSNs. Proper pore size could readily promote the blood proteins to contact the huge interior surfaces of the MSNs and then initiate the quick blood clot. Furthermore, the perfect biocompatibility of the MSNs was achieved through a CCK-8 and cellular uptake study, indicating that cell viability could be promoted by MSNs and MSNs with larger pore size showed better biocompatibility. Rapid hemostasis in rabbit femoral artery injury testified the superb hemostatic efficiency of the MSNs. We demonstrated that MSNs with a pore size of 15 nm showed the best hemostatic efficiency and it would be probably an optimal candidate for the first aid of hemorrhage in the field or pre-hospital.

Extracellular Matrix of Mechanically Stretched Cardiac Fibroblasts Improves Viability and Metabolic Activity of Ventricular Cells

Background: In heart, the extracellular matrix (ECM), produced by cardiac fibroblasts, is a potent regulator of heart,s function and growth, and provides a supportive scaffold for heart cells in vitro and in vivo. Cardiac fibroblasts are subjected to mechanical loading all the time in vivo. Therefore, the influences of mechanical loading on formation and bioactivity of cardiac fibroblasts, ECM should be investigated. Methods: Rat cardiac fibroblasts were cultured on silicone elastic membranes and stimulated with mechanical cyclic stretch. After removing the cells, the ECMs coated on the membranes were prepared, some ECMs were treated with heparinase II (GAG-lyase), then the collagen, glycosaminoglycan (GAG) and ECM proteins were assayed. Isolated neonatal rat ventricular cells were seeded on ECM-coated membranes, the viability and lactate dehydrogenase (LDH) activity of the cells after 1-7 days of culture was assayed. In addition, the ATPase activity and related protein level, glucose consumption ratio and lactic acid production ratio of the ventricular cells were analyzed by spectrophotometric methods and Western blot. Results: The cyclic stretch increased collagen and GAG levels of the ECMs, and elevated protein levels of collagen I and fibronectin. Compared with the ECMs produced by unstretched cardiac fibroblasts, the ECMs of mechanically stretched fibroblasts improved viability and LDH activity, elevated the Na+/K+-ATPase activity, sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity and SERCA 2a protein level, glucose consumption ratio and lactic acid production ratio of ventricular cells seeded on them. The treatment with heparinase II reduced GAG levels of these ECMs, and lowered these metabolism-related indices of ventricular cells cultured on the ECMs. Conclusions: Mechanical stretch promotes ECM formation of cardiac fibroblasts in vitro, the ECM of mechanically stretched cardiac fibroblasts improves metabolic activity of ventricular cells cultured in vitro, and the GAG of the ECMs is involved in regulating metabolic activity of ventricular cells.

Comparison of the properties of collagen–chitosan scaffolds after γ-ray irradiation and carbodiimide cross-linking

Abstract The property of collagen–chitosan porous scaffold varies according to cross-linking density and scaffold composition. This study was designed to compare the properties of collagen–chitosan porous scaffolds cross-linked with γ-irradiation and carbodiimide (CAR) for the first time. Eleven sets of collagen–chitosan scaffolds containing different concentrations of chitosan at a 5% increasing gradient were fabricated. Fourier transform infrared spectroscopy was performed to confirm the success of cross-linking in the scaffolds. The scaffold morphology was evaluated under scanning electron microscope (SEM). SEM revealed that chitosan was an indispensable material for the fabrication of γ-ray irradiation scaffold. The microstructure of γ-ray irradiation scaffold was less stable than those of alternative scaffolds. Based upon swelling ratio, porosity factor, and collagenase degradation, γ-ray irradiation scaffold was less stable than CAR and 25% proportion of chitosan scaffolds. Mechanical property determines the orientation in γ-irradiation and CAR scaffold. In vitro degradation test indicated that γ-irradiation and CAR cross-linking can elevate the scaffold biocompatibility. Compared with γ-ray irradiation, CAR cross-linked scaffold containing 25% chitosan can more significantly enhance the bio-stability and biocompatibility of collagen–chitosan scaffolds. CAR cross-linked scaffold may be the best choice for future tissue engineering.

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.

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.