[Objective]To explore a method for isolating and culturing bone marrow mesenchymal stem cell(BMSCs) in vitro,and improve the cellular conglutination ability of biomaterial poly-lactide-co-glycolic acid(PLGA),and observer the adhesion of BMSCs cultured on PLGA modified with Ⅱ collagen.[Method]BMSCs were isolated and cultured in vitro.The cell exterior antigen,cell livingness and cell cycle were analyzed by flow cytometry method,and cellular configuration was observed continually under invertd phase-contrast microscope.PLGA was made by phase separation,and was modified with Ⅱ collagen.The third era BMSCs were cultured on PLGA,and its adhesion with biomaterial was observed scan electron microscope.[Result]BMSCs could be isolated and cultured in vitro,and express CD29,CD44,CD106,but not express CD34,CD45.The cell livingness was 88.96%,cells in G0-Glperiod are 90.32%.The cell morphology was spindle.The average diameter of PLGA modified with Ⅱ collagen was 100 ?m,and PLGA has good adhesion with BMSCs.[Conclusion]BMSCs could be cultured stability in vitro for long time,and is good seminal cell of tissue engineering.Ⅱ collagen can improve the cellular adhesion of PLGA,PLGA modified with Ⅱ collagen is the good biomaterial of tissue engineering.

Transcription factor NF-κB has attracted attention due to its important role in the regulation of the expression of a number of cellular genes involved in host defense, inflammation/immune responses, cytokine and growth factor regulation, cell survival, proliferation, and also in embryonic development and programmed cell death. In this review, we provide an overview of established TNF-α, TLR/IL-1R, TCR and BCR signaling pathways to NF-κB, the alternative NF-κB pathway, and the regulation of NF-κB transcriptional activity. Furthermore, we discuss on emphasis the cross-talks between NF-κB and other signaling pathways (TGF-β, p53, nuclear receptor family, Ras/MEK/MAPK...), which are very complex and remain to be understood. The activation or inhibition of NF-κB has effect on other signaling pathways. They compose a complex signaling network, in which many regulatory signals integrate systematically and coordinate transcriptional responses to the stimulations. So when NF-κB is used as an important target of drugs in human diseases, the pathological and physiological functions of related signal pathways also should be understood sufficiently.

Transcription factor NF-?B has attracted attention due to its important role in the regulation of the expression of a number of cellular genes involved in host defense,inflammation/immune responses,cytokine and growth factor regulation,cell survival,proliferation,and also in embryonic development and programmed cell death. In this review,we provide an overview of established TNF-?,TLR/IL-1R,TCR and BCR signaling pathways to NF-?B,the alternative NF-?B pathway,and the regulation of NF-?B transcriptional activity. Furthermore,we discuss on emphasis the cross-talks between NF-?B and other signaling pathways (TGF-?,p53,nuclear receptor family,Ras/MEK/MAPK...),which are very complex and remain to be understood. The activation or inhibition of NF-?B has effect on other signaling pathways. They compose a complex signaling network,in which many regulatory signals integrate systematically and coordinate transcriptional responses to the stimulations. So when NF-?B is used as an important target of drugs in human diseases,the pathological and physiological functions of related signal pathways also should be understood sufficiently.

BACKGROUND: Chitosan and sodium alginate are the good natural materials for microcapsule, and also used widely in tissue engineering. Our research teams have made thorough work at anticoagulant materials, but these materials are inert or simulate the liquid crYstal form of blood vessel wall. While in this experiment, on the base of our previous study, we microencapsulated heparin with biotic anticoagulation activity and other specific performances in order to enable microcapsule to have a long time releasing effect of medicine.OBJECTIVE: To microencapsulate the low molecular heparin so as to ensure the stability of heparin in vivo and analyze the effect of content of chitosan on the performance of heparin microcapsules basing on the natural chitosan and sodium alginate as the enwrapped materials of microcapsules.DESIGN: Open experiment.SETTING: Department of Material Science and Engineering, Jinan University.MATERIALS: The experiment was performed at the laboratory of Department of Material Science and Engineering, Jinan University from October 2004 to June 2005. Heparin, with relative molecular mass＜ 5 000, was provided by Shandong Freda Biochem Co., Ltd.,; Chitosan was provided by Shanghai Bio Life Science & Technology Co., Ltd, DD≥90%, η＜ 100 cps;Sodium alginate was provided by Qingdao Bright Moon Seaweed Industrial Co., Ltd. Emulsions were Span80, and CaCl2, which were both made in China.METHODS: ①Preparation of heparin/chitosan microcapsules (HCM):Some heparin aqueous solution was emulsified in liquid paraffin. The reaction system was stirred fully and presented emulsion. Then the whole reaction system was warmed to be at 50 ℃ and maintained for 20 minutes. Afterwards, 20 g/L chitosan solution was added slowly, subsequently with raising the temperature to be at 60 ℃ and then glutaraldehyde was dropwised keeping the reaction system at 80 ℃ for 1hour. Centrifugation, filtration and washing followed by washing with kerosene fully, remain organic was extracted by dehydrated alcohol with extractor were performed.Drying and xeransis in vacuum were done at last. ② Preparation of heparin-sodium alginate-chitosan microcapsules (HSCM) :Heparin aqueous solution and sodium alginate were emulsified in paraffin, and the reaction system was stirred into emulsion at room temperature for 20 minutes, then 3% CaCl2 solution containing different concentrations of chitosan was added slowly. 30 minutes later, Microcapsules were separated, washed and dried as the treatments as before. ③ Drug content and envelope efficiency were measured, heparin standard curve was determined and in vitro releasing effect of heparin microcapsules was also measured.MAIN OUTCOME MEASURES: ①Effect of chitosan solution concentration on preparation of heparin-chitosan microcapsules; ② Effect of glutaraldehyde dosage on preparation of heparin-chitosan microcapsules; ③Effect of sodium alginate concentration on hepatin-sodium alginate; ④Effect of chitosan concentration on hepatin-sodium alginate-chitosan microcapsules. ⑤ In vitro release of heparin microcapsules enwrapped by different materials. ⑥Measurement of heparin content and envelope efficiency. ⑦ Observation of heparin microcapsule under scanning electron microscope RESULTS: ①With the increasing concentration of chitosan, the color of production changed from yellow to dark, and microcapsules were increscent, but the microcapsules uniformity and property of balling were increased. ②The increasing content of glutaraldehyde led darker production.Increase of glutaraldehyde content made production bond each other severely. The glutaraldehyde, which did not react with chitosan, can solidify itself and presented anomalous microcapsules forming. ③There was not obvious balling property of the production with the change of concentration of sodium alginate. ④The balling property of microsphere was good with increasing concentration of chitosan. However, microcapsules conglutinated with each other. 2% chitosan would be better. ⑤With the increase of chitosan content, the releasing speed ofheparin became slow. ⑥The envelope efficiency was about 58% when microcapsule contained 20%(wt) of chitosan, and used chitosan only the envelope efficiency could approach to 79.9%. ⑦ The surface of microcapsules with chitosan was very compact,and with increasing of content of glutaraldehyde, microcapsules would bond each other.CONCLUSION: Chitosan at certain concentration will affect the uniformity and balling property of microcapsules. Chitosan dosage can alter the envelope efficiency of heparin. Envelope efficiency of heparin is increased and releasing speed of heparin is decreased with the increase of content of chitosan.

BACKGROUND:Biocompatibility is a key parameter in drug delivery systems. In general, to obtain a proper microparticle carrier, the materials themselves should have excellent biocompatibility. Besides, spherical degree and surface smoothness both have significant influence on biocompatibility.OBJECTIVE: To obtain spherical and smooth poly (lactide-co-glycolide)microparticles, so as to improve the property of biocompatibility.DESIGN: Open experiment.SETTING: Research Laboratory of Biomaterials, Jinan University.MATERIALS: The experiment was conducted in the Research Laboratory of Biomaterials, Jinan University, between June 2004 and January 2005.The materials included poly(lactide-co-glycolide), lysozyme and poly (vinyl alcohol). The other reagents were analytical pure. The instruments included a homogenizer, a mechanical stirrer, an ultrasonic cleaning instrument, a scanning electron microscope and an atomic force microscope.METHODS: ① Preparation of microparticles: Lysozyme was selected as a model protein for encapsulation into poly(lactide-co-glycolide) using a dou-ble emulsion solvent extraction/evaporation method. Three separation methods, namely direct vacuum freeze-drying, filtration and centrifugation,were investigated and compared. ② Observation under the scanning electron microscope: We observed the effect of three separation methods on the shape of microparticles. All the samples were attached to copper mounts and coated with gold, and then were observed with an electron microscope.③ Observation under the atomic force microscope: The morphological structure of the surface was analyzed with atomic force microscope.RESULTS: ① Observation results of scanning electron microscope: Compared with direct vacuum freeze-drying and filtration, centrifugation method was more efficacious in obtaining spherical and smooth microparticles based on the scanning electron microscope pictures. But results also warned us to be more careful when we prepared scanning electron microscope samples using ultrasonic to separate the aggregates. ② Atomic force microscope results indicated that the surface was smooth with the average roughness of 48.55 nm.CONCLUSION: By investigating the influence of different downstream processes, we can obtain spherical and smooth products. Besides, a new one-step method is put forward in constructing some microparticle-combined polymer based scaffolds because the combined scaffolds and microparticles are formed synchronously.

BACKGROUND:Polylactic acid(PLA)surface is hydrophobic and there are no nataral recognition sites,so its application is limited.Many different strategies have been studied such as composition and chemical Drafting,to produce hydrophilic groups.However,these traditional methods are always involved in complex process and many organic reagents,resulting in decrease of biocompatibility.OBJECTIVE:To conduct the surfacr modification of PL.A and improve its hydrophilicity with low temperature plasma treatment.DESIGN:Controlled observation.SETTING:Department of Materials Science and Engineering in Jinan University.MATERIAIS:The experiment was processed from October 2004 to October 2005 at Guangzhou Research Center of Artificial Organs and Materials Engineering(Ministry of Education).The mainly used materials were:Poly-D,L-lactic acid(Mr 2.9×104,Shandong Medical Instrument Institute),N-vinyl-pyrrolidone(NVP,Acros Company,purified before use).METHODS:The materials were treated in the plasma reactor under different conditions(power=150 W,t=3,2,1 minutes;power=30 W,t=10,8,5,3,1 minutes)to choose the optimal condition,and then PLA were grafted with NVP by gas phase polymerization and liquid polymerization,respectively.MAIN OUTCOME MEASURES:THe surface morphology of the films was observed with scanning electron microscopy and atomic force microscopy.The hydrophilicity of native and treated material surfaces was measured using a water contact angle meter.Surface composition was detected with Fourier transform infrared spectrometer.Mass changesmeasurement was applied to characterize the grafting efficiency.RESULTS:Scanning electron microscopy and atomic force microscopy showed that the plasma medified materials possessed coarse surface with pores and notches.Water contact angles decreased obviously from 78°to 50°after grafting,Fourier transform infrared spectrometer showed a new absorption peak at 1 637.19 cm-1,which corresponded to the carbonyl stretch vibration absorption of amide group in poly-N-vinyl-pyrrolidone.After the plasma treatmerlt at low temperature.the mass change ratio of PLA film material was-0.6.CONCLUSION:The plasma treatment and polymerization can improve the hydrophilicity and cause coarsr surface of PLA material,which will be helpful for the cell attachment.

BACKGROUND: Microspheric injectable scaffold has a perspective in cartilage tissue engineering; however, it is still limited by in vivo hard forming and microsphere transmigration.OBJECTIVE: To investigate the feasibility of self-assembly scaffolds by attaching negatively charged calcium alginate microspheres and positively charged chitosan microspheres by electrostatic force. METHODS: The calcium alginate and chitosan microspheres were prepared by emulsion-internal gelation and spray drying technique, respectively. The characterizations of the microspheres were determined by means of scanning electron microscopy, optical microscopy and zeta potential analysis methods. Self-assembly scaffolds were fabricated by mixing the aqueous suspensions of the microspheres with oppositely charged surfaces. The phenomenon of electrostatic attachment was characterized by optical microscopy and scanning electron microscopy, and the elastic compress modulus of the scaffolds was also investigated.RESULTS AND CONCLUSION: The average diameter of the calcium alginate microspheres was 52.5 μm, and the chitosan one was 4.1 μm, respectively. The zeta-potential of the calcium alginate microspheres was 23.5 mv, and the chitosan one was +9.8 mv, respectively. The microspheres were spherical and smooth. The small size chitosan microspheres could attach to the surface of the calcium alginate microspheres and anchor the calcium alginate microspheres together. The elastic compress modulus increased with the increase of solid content of the microspheres, but decreased with the increase of the ionic strength. The elastic compress modulus increased firstly and then decreased with the increase of the mass ratio of m (CHI):m (ALG), and it showed the highest elastic compress modulus when m (CHI):m (ALG) was 2:1. The positively charged chitosan microspheres could attach to the negatively calcium alginate microspheres to form a self-assembly scaffold.

In recent years, the applications of atomic force microscopy (AFM) have underpinned the fast progress in the area of tissue engineering. Besides the study of surface morphology in the dimension of micro- and nano-, AFM has played an important role in the fabrication of micro- and nano-structure as well as in the investigation of mechanical properties of material and cell. This overview is aimed to introduce the principle of AFM and to review its recent applications in tissue engineering.
Humans ; Microscopy, Atomic Force ; Nanotechnology ; Tissue Engineering ; trends ; Tissue Scaffolds

Objective To study the effect of a novel injectable scaffold material chitosan- beta-TCP combining bone marrow mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) on repairing bone defect of goat. Methods The model of the studies was 12ram diameter circular hole tibia bone defect of goat. 30 Chinese goats were raudomly divided into 5 groups: blank group: nothing was embeded in bone defect; simple material group: the material embeded in bone defect was chitosan-beta-TCP; PRP group: the material was chitesan-beta-TCP combining PRP; MSCs group: the material was chitosan-beta-TCP combining MSCs; PRP/MSCs group:the material was chitosan-beta-TCP combining MSCs and PRP. At 4,8 weeks after operation, the samples were observed, histological and image analysis were used to evaluate the effect of bone regeneration. Results At 8 weeks, the surface of bone defect zone of PRP/MSCs group were coverd by continuous new bones, like normal bone. Histological slice showed the esteoid at boundary of normal bone of MSCs/PRP group obviously increased compare to other groups at the 4th or 8th week after operation respectively. The new bone tissues of bone defect were punctiform or lamellar new bone tissues, in which the proportion of big lamellar new bone tissue obviously increased. Image analysis showed that the areas of balnk group, simple material group, PRP group, MSCs group, PRP/MSCs group were 8.79±3.63,14.49± 3.72,24.18 ± 5.38,24.42 ± 5.10,31.10 ± 3.49 at 4 weeks and 15.41 ± 4.21,25.36 ± 5.37,30.71 ± 4.39, 33.97 ± 4.45,48.60 ± 5.97 at 8 weeks respectively. The effect of bone regeneration of PRP/MSCs group was better than other groups (P < 0.05). Conclusion The injectable tissue-engineering bone constructed with chitosan-beta-TCP, MSCs and PRP possesses good ability on repairing bone defect.

BACKGROUND: Studies have shown that chitooligosaccharides have antitumor effect. However, the influence of chitooligosaccharides on cyclin D1, bcl-2 and bcl-xl remains unclear.OBJECTIVE: To observe the JnhJbJtJon effect of chitooligosaccharides on the proliferation of Hela cells, and the influence on cyclin D1, bcl-xl and bcl-2 mRNA expression.METHODS: Hela cells were stimulated by different concentrations of chitooligosaccharides (0.1, 0.5, 1, 2, 5 g/L). The effects of chitooligosaccharides on Hela cells were detected by CCK8 kit. Using real-time PCR methods, the gene expression of cyclin D1, bcl-xl and bcl-2 mRNA were determined.RESULTS AND CONCLUSION: Chitooligosaccharides inhibited the proliferation of Hela cells. With the concentrations of chitoolJgosaccharides increased from 0.1 g/L to 2 g/L, the inhibition effects on the gene expressJon of cyclin D1, bcl-xl and bcl-2 were enhanced, peaked at 2 g/L, and decreased at high concentration (5 g/L). Antitumor activity of chitooligosaccharides may exert through two aspects: it inhibits cyclin D1 mRNA expression to suppress the proliferation of tumor cells; on the other hand, chitooligosaccharides inhibits the expression of bcl-xl and bci-2 to promote the apoptosis of tumor cells. Moreover, the effects of the former are stronger than the latter.