Wanshun Liu

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.

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.

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.

Chitooligosaccharides and N-acetyl-D-glucosamine stimulate peripheral blood mononuclear cell-mediated antitumor immune responses

The aim of the present study was to evaluate the anticancer effects of chitooligosaccharides (COS) and N-acetyl-D-glucosamine (NAG), as well as to investigate the possible mechanisms involved. MTT assay and flow cytometry were used to evaluate the effect of various concentrations of COS and NAG on the proliferation and differentiation of peripheral blood mononuclear cells (PBMCs). In addition, sarcoma 180 cells were transplanted into mice to establish a tumor model. COS and NAG were administered by gavage of various doses. The tumor inhibition rate, thymus and spleen indexes, natural killer (NK) cell activity, and interleukin-2 (IL-2) and interferon-γ (IFN-γ) serum levels were detected. Vascular endothelial growth factor (VEGF) expression levels, an important marker of angiogenesis, were also detected. As shown by immunohistochemistry, VEGF mRNA expression was decreased following treatment with COS and NAG, indicating that COS and NAG have an inhibitory effect on the expression of VEGF. The results from this study indicate that COS administered at a dose of 100 mg/kg and NAG at a dose of 300 mg/kg or 500 mg/kg can not only promote the differentiation of PBMCs and the secretion of IL-2 and IFN-γ, but can also inhibit the expression of VEGF mRNA in sarcoma 180 tumors. Our results show that the antitumor and immunoregulatory effects of COS and NAG are dose-dependent. Furthermore, the antitumor effect is achieved by the improvement of immunoregulation indirectly.

Mechanical properties of biodegradable small-diameter chitosan artificial vascular prosthesis.

Initial clinical feasibility with the small-diameter chitosan artificial vascular prosthesis has been reported previously. Here, we present the results of mechanical properties of artificial vascular prosthesis with 2, 3, and 4 mm inner diameter (ID) and compare some of the properties with the native blood vessel of dog femoral artery. Thickness wall measurement demonstrated the average wall thickness of the artificial vascular prosthesis with 2, 3, and 4 mm ID and the native blood vessel of the dog femoral artery with 3 mm ID were 0.54 ± 0.022 mm, 0.71 ± 0.032 mm, 0.79 ± 0.026 mm, and 0.67 ± 0.22 mm (n = 20). Water absorption rate of 226.02% ± 8.17%, 216.13% ± 4.86%, and 205.69% ± 4.34% were obtained from 2-, 3-, and 4-mm-diameter artificial vascular prosthesis (n = 12), respectively. Water osmotic pressure of the 2-, 3-, and 4-mm-diameter artificial vascular prosthesis was 39.25 ± 3.35 mmHg, 34.2 ± 4.54 mmHg, and 28.00 ± 2.72 mmHg (n = 20), respectively. Water osmotic amount of the 2-, 3-, and 4-cm-diameter artificial vascular prosthesis (n = 20) was 4.90 ± 0.47 mL/(min cm(2) ), 5.51 ± 0.21 mL/(min cm(2) ), and 6.24 ± 0.71 mL/(min cm(2) ), respectively. The ruptured stretching rate of the artificial vascular prosthesis (n = 20) with 2, 3, and 4 mm ID and the native blood vessel was 1.59% ± 0.14%, 1.99% ± 0.24%, 2.52% ± 0.21%, and 32.16% ± 2.15%, respectively. The longitudinal tensile strength of the artificial vascular prosthesis (n = 20) with 2, 3, and 4 mm ID and the native blood vessel was 8.58 ± 1.98 N, 19.75 ± 4.07 N, 22.92 ± 3.85 N, and 18.76 ± 2.05 N, respectively. The suture retention of the artificial vascular prosthesis (n = 20) with 2, 3, and 4 mm ID and the native blood vessel in dry condition is 5.80 ± 0.51 N, 7.01 ± 0.32 N, 8.49 ± 0.56 N, and 7.92 ± 0.39 N, respectively. The suture retention of the artificial vascular prosthesis (n = 20) with 2, 3, and 4 mm ID in wet condition is 3.87 ± 0.43 N, 4.73 ± 0.37 N, 5.63 ± 0.36 N, and 7.92 ± 0.39 N, respectively. The compliance of the artificial vascular prosthesis with 2, 3, and 4 mm ID and the native blood vessel was 6.5% ± 2.6%/100 mmHg, 5.2% ± 1.5%/100 mmHg, 4.7% ± 1.3%/100 mmHg, and 10.3% ± 2.3%/100 mmHg, respectively. The data reported here fulfill the quality requirement of clinical use of this kind of biodegradable small diameter artificial vascular prosthesis.