3-D culture of human umbilical vein endothelial cells with reversible thermosensitive hydroxybutyl chitosan hydrogel.

The aim of this study was to present a non-trypsin 3D cell culture method with a reversible thermosensitive HBCS hydrogel. In this study, hydroxybutyl chitosan (HBCS) was synthesized by grafting hydroxybutyl groups on chitosan molecule chains. The prepared HBCS was water-soluble, and the reversible phase transformation temperature was 26 °C. Scanning electron microscope images illuminated the 3-D network of hydrogel formed irregular porous structure which ranged from 50-250 µm. Cell viability assay indicated that HBCS solution could promote the proliferation of human umbilical vein endothelial cells (HUVECs), and the boost of proliferation was enhanced with the increase of HBCS concentration. HBCS had no harm to the nitric oxide (NO) synthesis functionality of HUVECs. HUVECs could grow and reproduce inside the hydrogel, and showed good vitality after 14-days culture. Meanwhile, cells cultured inside the hydrogel could be passaged successively through the reversible phase transformation process of HBCS. The results revealed that HBCS have the potential to be used for 3-D cell culture without the use of trypsin.

Preparation and evaluation of oleoyl-carboxymethy-chitosan (OCMCS) nanoparticles as oral protein carriers.

Oleoyl-carboxymethy chitosan (OCMCS) nanoparticles based on chitosan with different molecular weights (50, 170 and 820 kDa) were prepared by self-assembled method. The nanoparticles had spherical shape, positive surface charges and the mean diameters were 157.4, 274.1 and 396.7 nm, respectively. FITC-labeled OCMCS nanoparticles were internalized via the intestinal mucosa and observed in liver, spleen, intestine and heart following oral deliverance to carps (Cyprinus carpio). Extracellular products (ECPs) of Aeromonas hydrophila as microbial antigen was efficiently loaded to form OCMCS-ECPs nanoparticles and shown to be sustained release in PBS. Significantly higher (P < 0.05) antigen-specific antibodies were detected in serum after orally immunized with OCMCS-ECPs nanoparticles than that immunized with ECPs alone and non-immunized in control group in carps. These results implied that amphiphilic modified chitosan nanoparticles had great potential to be applied as carriers for the oral administration of protein drugs.

Injectable thermosensitive hydrogel based on chitosan and quaternized chitosan and the biomedical properties.

A novel injectable thermosensitive hydrogel (CS-HTCC/alpha beta-GP) was successfully designed and prepared using chitosan (CS), quaternized chitosan (HTCC) and alpha,beta-glycerophosphate (alpha,beta-GP) without any additional chemical stimulus. The gelation point of CS-HTCC/alpha beta-GP can be set at a temperature close to normal body temperature or other temperature above 25 degrees C. The transition process can be controlled by adjusting the weight ratio of CS to HTCC, or different final concentration of alpha,beta-GP. The optimum formulation is (CS + HTCC) (2% w/v), CS/HTCC (5/1 w/w) and alpha,beta-GP 8.33% or 9.09% (w/v), where the sol-gel transition time was 3 min at 37 degrees C. The drug released over 3 h from the CS-HTCC/alpha,beta-GP thermosensitive hydrogel in artificial saliva pH 6.8. In addition, CS-HTCC/alpha,beta-GP thermosensitive hydrogel exhibited stronger antibacterial activity towards two periodontal pathogens (Porphyromonas gingivalis, P.g and Prevotella intermedia, P.i). CS-HTCC/alpha, beta-GP thermosensitive hydrogel was a considerable candidate as a local drug delivery system for periodontal treatment.

Mussel-inspired antibacterial polydopamine/chitosan/temperature-responsive hydrogels for rapid hemostasis.

The aim of this study was to develop an effective wound dressing using a temperature-responsive hydroxybutyl chitosan (HBC) based hydrogel. The HBC - chitosan (CS) - dopamine (HCS-DOPA) composite hydrogels were prepared by the dopamine self-polymerization at different concentrations (0, 0.5, 1.0 and 2.0 mg/mL), termed as HCS, HCS-DOPA-0.5, HCS-DOPA-1 and HCS-DOPA-2, respectively. The gelling characteristic of HBC hydrogel was not influenced by composite CS and DOPA. The HCS-DOPA composite hydrogels were non-cytotoxic to mouse fibroblast cells (L929), and induced under 5.0% hemolysis rate. In vitro antibacterial studies, composite HCS-DOPA-2 hydrogels exhibited lasting inhibition to S. aureus >8 h. The whole blood test in vitro demonstrated that blood clotting time treated with HCS-DOPA-2 composite hydrogels was shortened to 95.6 s compared with that of HCS in vitro hemostasis. The results suggested that HCS-DOPA-2 composite hydrogels could be applied as a promising wound dressing for hemostasis in vitro.

The temperature-responsive hydroxybutyl chitosan hydrogels with polydopamine coating for cell sheet transplantation.

The aim of this study was to develop an effective cell sheet translocation method using a cell adhesive and temperature-responsive hydroxybutyl chitosan hydrogel (HBC). The polydopamine (PD)-coated HBC hydrogels were prepared by the dopamine self-polymerization on the surface of HBC hydrogel with different coating time, termed as P30, P60 and P120, respectively. Gelling property of HBC was not affected by PD coating. The PD-coated HBC hydrogels promoted the attachment and proliferation of mouse fibroblast cells (L929) and human umbilical vein endothelial cells (HUVECs), and allowed formation of monolayer cell sheet. In vitro translocation of HUVECs sheet could be obtained successively through phase transition of PD coated HBC hydrogel from gel to sol, and the cells sheet transferred from P30 hydrogel to a round cell coverglass maintained relatively complete monolayer and normal cell morphology. The results showed that P30 hydrogel has the potential to be used for cell transplantation therapy.

Biocompatibility, cellular uptake and biodistribution of the polymeric amphiphilic nanoparticles as oral drug carriers.

Oleoyl-carboxymethyl-chitosan (OCMCS) was synthesized and were soluble at neutral pH. The critical micelle concentration (CMC) of OCMCS in deionized water was 0.021 mg/ml. OCMCS nanoparticles were successfully prepared via self-assembly with mean diameter of 215.34 nm, zeta potential of 19.26 mV and an almost spherical shape as determined by electron microscopy. The OCMCS nanoparticles showed low erythrocyte membrane-damaging effect. The MTT survival assay indicated no significant cytotoxicity to Caco-2 cells and MEFs cells. The uptake of FITC labeled OCMCS nanoparticles by Caco-2 cells was confirmed via confocal laser scanning microscope (CLSM). In vivo toxicity assays were performed via histopathological evaluation, and no specific anatomical pathological changes or tissue damage was observed in the tissues of carps. The extent of tissue distribution and retention following oral administration of FITC-OCMCS nanoparticles was analyzed for 3 days. After 3 days, the nanoparticles remained detectable in the muscle, heart, kidney, liver, intestine, and spleen. The results showed that 34.32% of the particles were localized in the liver, 18.79% in the kidney, and 17.36% in the heart. The lowest percentage was observed in the muscle. These results implied that OCMCS nanoparticles had great potential to be applied as safe carriers for the oral administration of protein drugs.

[Effects of surface roughness of bone cements on histological characteristics of induced membranes].

OBJECTIVE: To explore surface roughness of bone cement and surround tissue on histological characteristic of induced membranes. METHODS: Bone cements with smooth and rough surface were implanted in radius bone defect, intramuscular and subcutaneous sites of rabbits, and formed induced membranes. Membranes were obtained and stained (HE) 6 weeks later. Images of membrane tissue were obtained and analyzed with an automated image analysis system. Five histological parameters of membranes were measured with thickness,area,cell density,ECM density and microvessel density. Double factor variance analysis was used to evaluate the effect of the two factors on histological characteristics of induced membranes. RESULTS: Membranes can be induced by each kind of bone cement and at all the three tissue sites. In histological parameters of thickness,area and micro vessel,there were significant differences among the membranes induced at different tissue sites (P = 0.000, P = 0.000, P = 0.000); whereas, there were no significant differences in histological parameters of cell density and ECM density (P = 0.734, P = 0.638). In all five histological parameters of membranes, there were no significant differences between the membranes induced by bone cements with different surface roughness (P = 0.506, P = 0.185, P = 0.883, P = 0.093, P = 0.918). CONCLUSION: Surround tissue rather than surface roughness of bone cements can affect the histological characteristics of induced membranes. The fibrocystic number, vascularity, mechanical tension and micro motion of the surround tissue may be closely correlated with the histological characteristics of induced membranes.

Preparation of biocompatible chitosan grafted poly(lactic acid) nanoparticles.

Chitosan grafted poly(lactic acid) (CS-g-PLA) copolymer was synthesized and characterized by FT-IR and elemental analysis. The degree of poly(lactic acid) substitution on chitosan was 1.90 ± 0.04%. The critical aggregation concentration of CS-g-PLA in distilled water was 0.17 mg/ml. Three methods of preparing CS-g-PLA nanoparticles (diafiltration method, ultrasonication method and diafiltration combined with ultrasonication method) were investigated and their effect was compared. Of the three methods, diafiltration combined with ultrasonication method produced nanoparticles with optimal property in terms of size and morphology, with size ranging from 133 to 352 nm and zeta potential from 36 to 43 mV. Also, the hemolytic activity and cytotoxicity of the CS-g-PLA based nanoparticles was tested, and results showed low hemolysis rate (<5%) and no significant cytotoxicity effect of these nanoparticles.

A facile method for preparing biodegradable chitosan derivatives with low grafting degree of poly(lactic acid).

Chemical modification of chitosan by grafting with PLA (CS-g-PLA) was developed via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) mediated coupling reaction. The introduction of PLA disrupted the crystalline structure of chitosan, improved its solubility and thermal stability. Low degree of PLA substitution showed better degradation efficiency than chitosan and PLA. Weight loss of CS-g-PLA6 and CS-g-PLA4 was 87% and 94%, respectively, in 7 days enzymatic degradation study. CS-g-PLA2 was totally degraded in 1 day. Self-assembly behavior was studied using pyrene fluorescence dye technique and found to be PLA grafting level dependent. CS-g-PLA with low grafting degree showed hydrophilic, self-assembling properties and controllable biodegradability that may widen its applications.