Investigation on the mechanisms for biocompatibility of chitosan using agarose/chitosan blended hydrogels as a model / 国际生物医学工程杂志

ObjectiveTo investigate the possible mechanisms for biocompatibility of chitosan material using agarose/chitosan blended hydrogels as a model.Methods A series of agarose/chitosan blended hydrogels with different chitosan content were prepared by the blending method.The chemical groups of the blended hydrogels were analyzed by the Fourier transform infrared (FTIR) spectroscopy.The blending compatibility between the agarose and chitosan was evaluated with the fluorescein-4-isothiocyanate (FITC) staining method.The charge of the blended hydrogels was determined by the zeta potential measurement.The adsorption of total fetal bovine serum (FBS) proteins and bovine serum albumin (BSA) on the blended hydrogels was measured by the bicinchoninic acid (BCA) method.The adsorption of fibronectin (FN) on the blended hydrogels was measured with ELISA.Cell culture experiment adopted human microvascular endothelial cell line (HMEC-1) as the model.The cytocompatibility was studied by evaluating adhesion,proliferation,and morphology of the cells on the blended hydrogels.Results Characteristic chemical groups of chitosan could be detected in the agarose/chitosan blended hydrogels.The chitosan had a good blending compatibility with the agarose.The amino groups of chitosan were uniformly distributed in the blended hydrogels.The blended hydrogels were strongly positively charged at acidic pH (pH 3.0),however,the zeta potentials of all the hydrogels were reduced to nearly 0 mV at neutral pH (pH 7.4).There were no significant differences in the adsorption of total FBS proteins and BSA between the blended hydrogel groups.However,the adsorption of FN on the hydrogels significantly increased with the increase of chitosan content.Cell culture experiment indicated that the cytocompatibihty of the blended hydrogels was significantly improved with the increase of chitosan content.The HMECs exhibited higher levels of adhesion,spreading,and proliferation on the hydrogels with higher chitosan content.ConclusionResults in this study indicated that the chitosan component preferentially adsorbed FN compared to the other serum proteins,leading to adhesion and spreading of the cells on the blended hydrogels.In contrast to prevailing views,it was found in the present study that the biocompatibility of chitosan did not relate to its positive charge.

Human Schwann cells from normal nervous tissue cultured in vitro / 中国组织工程研究

BACKGROUND:Peripheral nerve tissue engineering needs a large number of Schwann cells.In previous studies,lack of normal human nervous tissue,so animal(rat,rabbit,et al)nervous tissues are commonly used to isolate Schwann cells,but as xeno-cells it is limited in clinical application.OBJECTIVE:To investigate an effective technique for isolation,cultivation and purification of human Schwann cells of normal peripheral nerves cultured in vitro.METHODS:Normal peripheral nerves were obtained from the surgery of cerebral palsy patients.Schwann cells were cultured with enzymatic digestion culture method and differential attachment method.Tissues were cut into pieces and incubated in medium supplemented with fetal bovine serum,collagenase and Oispase enzyme,centrifuged.Tissue blocks were placed in the medium,triturated into monoplast suspension,and then moved into a DMEM Petri dish containing polylysine,supplemented with basic fibroblast growth factor.When adherent cells were confluent about 85% 90%,cells could subculture.Schwann cells were counted by Trypan Blue coloring method at 2,3,4,5,6,7,8,9,10.The purity of Schwann cells was identified through S-100 protein immunohistochemistry staining.RESULTS AND CONCLUSION:More than 0.5×10~8/L Schwann cells were detected after four days under a microscope.Following the third passage,the number of Schwann cells was over 9×10~8/L.The purity of Schwann cell population was up to 85%.Results suggested that plenty and purified human Schwann cells could be obtained by enzymatic digestion culture and differential attachment methods in a short time,which can be used for the source of peripheral nerve tissue engineering.

Adipose derived mesenchymal stem cell transplantation for Alzheimer's disease / 中国组织工程研究

Alzheimer's disease is an irreversible neurodegenerative disease characterized by progressive neuronal loss. To date, there has been no effective medicine or therapy for neurodegenerative disease. With development of stem cell technique and theory, neural stem cell transplantation has been found to be prospective in Alzheimer's disease treatment. However, it was challenged by the deficiency of autologous neural stem cell, which can bypass immunological barrier. Compared with neural stem cells, mesenchymal stem cells exhibit extensive resources, such as liver, bone marrow and adipose, and multiple differentiations into bone, muscle or adipose. Considering the easy access, the minor trauma to the patients, and the neuron differentiation potential of adipose derived mesenchymal stem cells (A-MSC), we hypothesize that A-MSC graft is a potential and innovative strategy for the treatment of Alzheimer's disease.

Effects of extract of Ginkgo biloba on apoptosis of neurocytes and expression of inducible nitricoxide synthuse after spinal cord injury in rats / 中华创伤杂志

Objective To investigate the effect of extract of Ginkgo biloba (Egb) on apoptosis of nerve cells and its mechanism after spinal cord injury (SCI) in rats. Methods Forty eight adult SD rats weighing 200-230 g were divided equally and randomly into Egb group and normal saline (NS) group. After hemisectian of spinal cord at T9 vertebrae level, rats in Egb group were lavaged with 2 ml EGB (20 mg) daily and those in NS group with 2 ml NS daily. Tissue sections were collected and stained with Nissl's staining, myelin sheath staining, and inducible nitric oxide synthase (iNOS) immunohisto-chemistry as well as terminal deoxynueleotidyl transferase-mediated dUTP nick end lebeling (TUNEL) at days 1,7, 14 and 21 respectively to evaluate the injured spinal cord tissues after six rats from each group were sacrificed Results Nissl's staining manifested less swelling of the nerve cells near the injury epi-center ( rostral and caudal ), smaller cavity and demyelinated area and higher ratio of bilateral anterior horn neurons of transection side to normal side in Egb group, compared with NS group ( P <0.05). Ap-optotie index (AI) and expression of iNOS in NS group were higher than those in Egb group ( P <0.01 or P <0. 05). Furthermore, the rate of iNOS-positive cells was positively correlated with the AI (r = 0.729, P<0.01) after SCI. Conclusion Egb can prevent nerve cells from apoptosis after SCI in rats, as may be related with inhibition of expression of iNOS.

Mechanisms for biocompatibility of chitosan: A new viewpoint / 中国组织工程研究

OBJECTIVE:In recent years, chitosan has been widely used as tissue engineering scaffolds. In this paper we reviewed the research progress in chitosan biocompatibility and gave a hypothesison possible mechanism of interactions between cells and chitosan. A model system to test this hypothesis was also discussed. DATA SOURCES: Literatures about chitosan biocompatibility were retrieved with computer in Medline, Pubmed and Elsevier from January 1998 to December 2006 with the key words of."chitosan, biocompatibility, surface charge, cell adhesion" in English.STUDY SELECTION: Literatures about chitosan biocompatibility and interactions between chitosan and cells, especially the influence of chitosan charges on cell attachment, were included, whereas repeated experiments were excluded.DATA EXTRACTION: Totally 374 literatures were collected. Among which, 30 were admitted and reviewed.DATA SYNTHESIS: Many mammalian cells can adhere, spread and proliferate on chitosan materials. It is widely accepted that the biocompatibility of chitosan is due to the electrostatic attractive force between positively charged amino groups on chitosan chains and negatively charged cell membranes. However, the pKa value of chitosan amino groups is 6.2-6.8 and the positive charge of chitosan chains is largely decreased under physiological condition as a result of amino groups unprotonation. Thus whether the chitosan's biocompatibility is due to its positive charge remains doubtful and needs further study.CONCLUSION: Based on prior studies, we hypothesize that the positive charge of amino groups on chitosan chains might not be the major factor in biocompatibility of chitosan material. Agarose/chitosan blending hydrogels is supposed to be an appropriate model system to test this hypothesis.

Proliferation and differentiation of MC 3T3-E1 cells cultured on nanohydroxyapatite/chitosan composite scaffolds / 生物工程学报

with chitosan in situ using a chemical method and a porous structure obtained was then lyophilized. Preosteoblast MC 3T3-E1 the scaffolds was examined after staining it with Wright's stain. Their proliferation was assessed using MTZ assay. After being Abstract Nanohydroxyapatite/chitosan composite scaffolds were fabricated and the proliferation and differentiation of preosteoblast MC 3T3-E1 on them were examined for the assessment of their biocompatibility. Nanohydroxyapatite was combined with chitosan in situ using a chemical method and a porous structure obtained was then lyophilized. Preosteoblast MC 333-E1 cells were inoculated into the porous composite scaffolds and chitosan scaffolds, respectively. The morphology of cells cultured on the scaffolds was examined after staining it with Wright's stain. Their proliferation was assessed using MTT assay. After being cultured in conditioned medium for 30 days, the cells' alkaline phosphatase activities on the scaffolds were studied in situ to compare their differentiation levelabout. Moreover, the alkaline phosphatase activities were assessed with a kit. The expression level of characteristic osteogenic gene was evaluated using Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The results indicated that MC 3T3-E1 cells grown on the composite scaffolds showed a higher proliferation rate and spread better than that on chitosan scaffolds. The alkaline phosphatase stain results showed that the alkaline phosphatase activity of cells on composite scaffolds was significantly higher than that on the chitosan scaffolds. In addition, the quantitative examination of alkaline phosphatase activity indicated that the cells cultured on the composite scaffolds expressed an activity level about 8 times higher than that on chitosan scaffolds. Simultaneously, the osteogenic gene osteopontin (OPN) of cells cultured on composite scaffolds showed a higher expression level than that on chitosan scaffolds. Another osteogenic gene osteocalcin (OC) was expressed in cells cultured on composite scaffolds, whereas it was not detected in cells on chitosan scaffolds. The addition of nanohydroxyapatite in the scaffolds improved not only the proliferation but also the differentiation of preosteoblast cultured on them. The composite scaffolds showed good biocompatibility and bioactivity. These scaffolds would be promising in bone tissue engineering.

Apoptosis and expression of iNOS after spinal cord injury in rats / 中国康复理论与实践

@# ObjectiveTo study the characteristic of apoptosis and the expression of inducible nitric oxide synthase(iNOS),as well as relation between them after spinal cord injury (SCI) in rats.Methods84 adult SD rats were randomly divided into three groups,and made into mild,moderate and severe acute SCI models.The rats were killed at 4h,8h,1d,3d,7d,14d and 21d after surgery.The histopathology changes in spinal cord tissue were observed with HE staining microscopic examination.The expression of iNOS was determined by immunohistochemistry and the apoptosis was labeled by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL).ResultsThe histopathology changes in spinal cord tissue deteriorated with increasing in injury degree.A little iNOS immunoreactivity (iNOS-IR) was detected in nerve cells,gliocytes,ependymocytes and blood vessel endotheliocytes of normal and vertebra shelf operated controls,increasing at 8 h, and in flood tide in 7 d. The expression of Bax and the rates of TUNEL positive cells were similar with that of iNOS after SCI. There was positive correlation between expression of iNOS and degree of primary SCI ,apoptosis index(r=0.414,P<0.01;r=0.854,P<0.01).Conclusions Expression of iNOS and Bax increase and a large number of TUNEL positive cells present after SCI in rats. There is positive correlation between expression of iNOS and degree of primary SCI,apoptosis index.