Baoqin Han

An in situ formed biodegradable hydrogel for reconstruction of the corneal endothelium.

Biodegradable hydrogels are important biomaterials for tissue engineering and drug delivery. For the purpose of corneal regenerative medicine, we describe an in situ formed hydrogel based on a water-soluble derivative of chitosan, hydroxypropyl chitosan (HPCTS), and sodium alginate dialdehyde (SAD). Periodate oxidized alginate rapidly cross-links HPCTS due to Schiffs base formation between the available aldehyde and amino groups. Hydrogel cytotoxicity, degradability and histocompatibility in vivo were examined. The potential of the composite hydrogel for corneal endothelium reconstruction was demonstrated by encapsulating corneal endothelial cells (CECs) to grow on Descemets membranes. The results demonstrate that the composite hydrogel was both non-toxic and biodegradable and that CECs transplanted by the composite hydrogel could survive and retain normal morphology. These results provide an opportunity for corneal endothelium reconstruction based on tissue engineering by the in situ formed composite hydrogel.

Effects of carboxymethyl chitosan on the blood system of rats.

Carboxymethyl chitosan (CM-chitosan), a derivative of chitosan, was extensively studied in the biomedical materials field for its beneficial biological properties of hemostasis and stimulation of healing. However, studies examining the safety of CM-chitosan in the blood system are lacking. In this study CM-chitosan was implanted into the abdominal cavity of rats to determine blood indexes at different times and to evaluate the effects of CM-chitosan on the blood system of rats. Coagulation function was reflected by thrombin time (TT), prothrombin time (PT), activated partial thromboplatin time (APTT), fibrinogen (FIB) and platelet factor 4 (PF4) indexes; anti-coagulation performance was assessed by the index of antithrombinIII (ATIII); fibrinolytic function was reflected by plasminogen (PLG) and fibrin degradation product (FDP) indexes; and blood viscosity (BV) and plasma viscosity (PV) indexes reflected hemorheology. Results showed that CM-chitosan has no significant effects on the blood system of rats, and provides experimental basis for CM-chitosan to be applied in the field of biomedical materials.

Fabrication and characters of a corneal endothelial cells scaffold based on chitosan

A novel chitosan-based membrane that made of hydroxyethyl chitosan, gelatin and chondroitin sulfate was used as a carrier of corneal endothelial cells. The characteristics of the blend membrane including transparency, equilibrium water content, ion and glucose permeability were determined. The results showed that the optical transparency of the membrane was as good as the natural human cornea. The water content of this scaffold was 81.32% which was remarkably close to the native cornea. The membrane had a good ion permeability and its glucose permeability was even higher than natural human cornea. The cultured rabbit corneal endothelial cells formed a monolayer on the membrane. The results demonstrated that the membrane was suitable for corneal endothelial cells to attach and grow on it. In addition, the membranes in vivo could be degraded steadily with less inflammation and showed a good histocompatibility. These results demonstrated that the hydroxyethyl chitosan-chondroitin sulfate-gelatin blend membrane can potentially be used as a carrier for corneal endothelial cell transplantation.

Toll like receptor 4 (TLR4) mediates the stimulating activities of chitosan oligosaccharide on macrophages.

The in vivo and in vitro immunostimulating properties of chitosan oligosaccharide (COS) prepared by enzymatic hydrolysis of chitosan and the mechanisms mediating the effects were investigated. Our data showed that the highly active chitosanase isolated could hydrolyze chitosan to the polymerization degree of 3-8. The resulting COS was an efficient immunostimulator. COS markedly enhanced the proliferation and neutral red phagocytosis by RAW 264.7 macrophages. The production of nitric oxide (NO) and tumor necrosis factor alpha (TNF-α) by macrophages was significantly increased after incubation with COS. Oral administration of COS in mice could increase spleen index and serum immunoglobin G (IgG) contents. COS was labeled with FITC to study the pinocytosis by macrophages. Results showed that FITC-COS was phagocyted by macrophages and anti-murine TLR4 antibody completely blocked FITC-COS pinocytosis. RT-PCR indicated that COS treatment of macrophages significantly increased TLR4 and inducible nitric oxide synthase (iNOS) mRNA levels. When cells were pretreated with anti-murine TLR4 antibody, the effect of COS on TLR4 and iNOS mRNA induction was decreased. COS-induced NO secretion by macrophages was also markedly decreased by anti-murine TLR4 antibody pretreatment. In conclusion, the present study revealed that COS possesses potent immune-stimulating properties by activating TLR4 on macrophages.

Carboxymethyl chitosan represses tumor angiogenesis in vitro and in vivo.

Carboxymethyl chitosan (CMCS), with potent water solubility, biocompatibility, and non-toxicity, has emerged as a promising candidate for biomedical applications. In this study, the anti-tumor angiogenesis effects of CMCS were evaluated in vitro and in vivo. Our results showed that CMCS could inhibit the 2-dimensional and 3-dimensional migration of human umbilical vein endothelial cells (HUVECs) in vitro. CMCS significantly inhibited the growth of mouse hepatocarcinoma 22 tissues and could promote tumor cell necrosis as suggested by pathological observations. The CD34 expression in H22 tumor tissue, the levels of vascular endothelial growth factor and tissue inhibitor of metalloproteinase 1 in serum was regulated by CMCS treatment. CMCS could significantly improve thymus index, spleen index, tumor necrosis factor α and interferon γ level. In a conclusion, CMCS possessed potent anti-tumor effects by inhibiting tumor angiogenesis, stimulating immune functions. Our date provide more foundation for application of CMCS in biomedicine or biomaterials for targeted anticancer drugs delivery.

Investigation of the skin repair and healing mechanism of N-carboxymethyl chitosan in second-degree burn wounds

N‐carboxymethyl chitosan (NCMC) was synthesized with the modification of chitosan; the substitution degree was measured by titration. The biocompatibility and degradability of the NCMC were studied in vivo and the results showed that the NCMC was nontoxic and biocompatible. The in vivo degradation rate of NCMC in musculature was faster than that in subcutaneous tissue due to the relatively high lysozyme concentration. The NCMC was used as biomaterial to heal deep second‐degree burn wounds. The wound size reduction, histological examination, and the quantification of transforming growth factor‐β1, tumor necrosis factor‐α and interleukin‐8 protein levels, and Smad3 gene expression were measured to evaluate the healing effects. The results demonstrated that the NCMC was efficient in accelerating wound healing via activating transforming growth factor‐β1/Smad3 signaling pathway.

Antitumor Activities of D-glucosamine and Its Derivatives

The growth inhibitory effects of D-glucosamine hydrochloride (GlcNH2·HCl), D-glucosamine (GlcNH2) and N-acetyl glucosamine (NAG) on human hepatoma SMMC-7721 cells in vitro were investigated. The results showed that GlcNH2·HCl and GlcNH2 resulted in a concentration-dependent reduction in hepatoma cell growth as measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. This effect was accompanied by a marked increase in the proportion of S cells as analyzed by flow cytometry. In addition, human hepatoma SMMC-7721 cells treated with GlcNH2·HCl resulted in the induction of apoptosis as assayed qualitatively by agarose gel electrophoresis. NAG could not inhibit the proliferation of SMMC-7721 cells. GlcNH2·HCl exhibited antitumor activity against Sarcoma 180 in Kunming mice at dosage of 125∼500 mg/kg, dose of 250 mg/kg being the best. GlcNH2·HCl at dose of 250 mg/kg could enhance significantly the thymus index, and spleen index and could promote T lymphocyte proliferation induced by ConA. The antitumor effect of GlcNH2·HCl is probably host-mediated and cytocidal.

Structural insights into the substrate-binding mechanism for a novel chitosanase.

Chitosanase is able to specifically cleave β-1,4-glycosidic bond linkages in chitosan to produce a chito-oligomer product, which has found a variety of applications in many areas, including functional food and cancer therapy. Although several structures for chitosanase have been determined, the substrate-binding mechanism for this enzyme has not been fully elucidated because of the lack of a high-resolution structure of the chitosanase-substrate complex. In the present study we show the crystal structure of a novel chitosanase OU01 from Microbacterium sp. in complex with its substrate hexa-glucosamine (GlcN)6, which belongs to the GH46 (glycoside hydrolyase 46) family in the Carbohydrate Active Enzymes database (http://www.cazy.org/). This structure allows precise determination of the substrate-binding mechanism for the first time. The chitosanase-(GlcN)6 complex structure demonstrates that, from the -2 to +1 position of the (GlcN)6 substrate, the pyranose rings form extensive interactions with the chitosanase-binding cleft. Several residues (Ser27, Tyr37, Arg45, Thr58, Asp60, His203 and Asp235) in the binding cleft are found to form important interactions required to bind the substrate. Site-directed mutagenesis of these residues showed that mutations of Y37F and H203A abolish catalytic activity. In contrast, the mutations T58A and D235A only lead to a moderate loss of catalytic activity, whereas the S27A mutation retains ~80% of the enzymatic activity. In combination with previous mutagenesis studies, these results suggest that the -2, -1 and +1 subsites play a dominant role in substrate binding and catalysis. DSF (differential scanning fluorimetry) assays confirmed that these mutations had no significant effect on protein stability. Taken together, we present the first mechanistic interpretation for the substrate (GlcN)6 binding to chitosanase, which is critical for the design of novel chitosanase used for biomass conversion.

Preparation, characterization and feasibility study of dialdehyde carboxymethyl cellulose as a novel crosslinking reagent

The natural biopolymers usually need to be chemically modified by crosslinking reagents to improve their mechanical properties. In the present research, the feasibility of using the dialdehyde carboxymethyl cellulose (DCMC) as a crosslinking reagent was systematically studied. DCMC was prepared by oxidizing carboxymethyl cellulose using sodium periodate. The formation of dialdehyde groups was confirmed by FTIR and the degree of oxidation was determined. The biocompatibility of DCMC was investigated by evaluating its cytotoxicity to L929 fibroblasts and histocompatibility in rat model via intramuscular and subcutaneous injection. DCMC-crosslinked carboxymethyl chitosan (DCMC-CMCTS) was prepared and characterized using the glutaraldehyde-crosslinked CMCTS (GA-CMCTS) as control. The result demonstrated that DCMC was non-cytotoxic, biodegradable and biocompatible. The DCMC-CMCTS displayed significantly better thermostability, swelling capacity and cyto-compatibility compared with GA-CMCTS. Our data provided experimental basis for the future application of DCMC as a novel crosslinking reagent.

Structural and biochemical insights into the degradation mechanism of chitosan by chitosanase OU01.

BACKGROUND A detailed knowledge about the degradation mechanism of chitosanase hydrolysis is critical for the design of novel enzymes to produce well-defined chito-oligosaccharide products. METHODS Through the combination of structural and biochemical analysis, we present new findings that provide novel insights into the degradation mechanism of chitosanase OU01. RESULTS We have determined the crystal structure of Asp(43)/Ala mutant of OU01, and have trapped the hydrolyzed product of the reaction. This structure reveals the role of the general acid (Glu(25)) in catalysis. Two structural features about the mechanisms of the non-processive chitosanases are described for the first time. 1). Structural comparison reveals that the enzyme goes through an open-closed-open conformational transition upon substrate binding and product release; 2). polar residues constitute the substrate binding cleft. Additional site important for polymeric substrate recognition is identified and a three-step polymeric substrate recognition mechanism is proposed. CONCLUSIONS Detailed substrate recognition mechanism is described for non-processive chitosanase for the first time. GENERAL SIGNIFICANCE These findings provide new structural insights into the understanding of overall hydrolysis mechanism for non-processive chitosanase, and also will facilitate the design of new enzymes used for industrial purpose.