型胶原在聚-L-乳酸(PLLA)表面的化学接枝和涂层及其对软骨细胞生长的促进作用

采用接枝-涂层技术在聚-L-乳酸(PLLA)膜表面接枝聚甲基丙烯酸(PMAA)得到稳定的PLLA-g-PMAA胶原涂层.利用水溶性碳化二亚胺作为缩合剂,使PLLA-g-PMAA表面的羧基和胶原分子中的氨基发生缩合反应,从而将型胶原接枝在PLLA表面;同时保留材料表面物理涂层的胶原溶液,获得了稳定的胶原涂层.XPS谱图证实了接枝反应的发生.分别用比色法和茚三酮法测定了PLLA-g-PMAA表面的羧基密度和PLLA膜表面胶原的接枝量和涂层量.细胞培养结果表明,改性后的PLLA膜表面软骨细胞的铺展性能和增长速度明显改善.     

Synthesis and Application of Chitosan‐g‐PLLA Copolymers

The purpose of this paper is to study the synthesis and application of a new type of chitosan‐g‐poly(L‐lactide) copolymer with different grafting percentage in the presence of triethylamine. FTIR and ¹H NMR results indicate that grafting percentage of graft copolymers increases with the molar feeding ratio of L‐lactide to chitosan. The measurement of XRD and TG shows that graft copolymer exhibits low crystallinity and thermal degradation temperature. Static water contact angle testing suggests that graft copolymer has superior hydrophilicity compared with PLLA, which can be very useful for biomedical applications. 5‐Fluorouracil loaded copolymer microspheres were prepared by phase separation method. The size and distribution of microspheres were measured by a Laser particle analyzer. The microspheres with LLA:CS feeding molar rotio (15∶1) have a mean diameter of 332 nm with a narrow unimodal distribution. The spherical microspheres were observed by transmission electron microscopy (TEM). The microspheres shows good releasing property from drug release in vitro, and the drug release rate decreases as the increase of microspheres size.     

The influence of chitosan on in vitro properties of Eudragit RS microspheres

Eudragit RS microspheres containing chitosan hydrochloride were prepared by the solvent evaporation method using acetone/liquid paraffin solvent system and their properties were compared with Eudragit RS microspheres without chitosan, prepared in our previous study. Different stirring rates were applied (400-1200 rpm) and drug content, Higuchi dissolution rate constant, surface and structure characteristics of the microspheres were determined for each size fraction. An increase in average particle size with a reduction of stirring rate appeared in limited interval in both series. The average particle size of microspheres without chitosan, prepared at the same stirring rate, was smaller. Pipemidic acid content increased with increasing fraction particle size, but not with increasing stirring rate as it was observed for microspheres without chitosan. We presume that high pipemidic acid content in larger microspheres is a consequence of cumulation of undissolved pipemidic acid particles in larger droplets during microspheres preparation procedure. Pipemidic acid release was faster from microspheres with chitosan and no correlation between Higuchi dissolution rate constant and stirring rate or fraction particle size was found, though it existed in the system without chitosan. Structure and surface characteristics of microspheres observed by scanning electron microscope (SEM) were not changed significantly by incorporation of chitosan. But in contrast with microspheres without chitosan, the surface of chitosan microspheres was more porous after three hours of dissolution. It is supposed that the influence of particle size fraction and stirring rate on release characteristics is expressed to a great extent through porosity and indirectly through total effective surface area, but the incorporation of highly soluble component i.e. chitosan salt hides these effects on drug release. In conclusion, changes in biopharmaceutical properties due to varying stirring rate and fraction particle size exhibited the same direction as those reported for the microspheres without chitosan, although they are less expressed because of increased experimental variability, likely caused by chitosan.     

Effects of divinylbenzene‐maleic anhydride copolymer hollow microspheres on crystallization behaviors,mechanical properties and heat resistance of poly(l‐lactide acid)

Divinylbenzene‐maleic anhydride copolymer hollow microspheres (DMs) were used as novel organic nucleating agents to promote crystallization of poly(l‐lactide acid) (PLLA). The effects of these DMs on crystal behaviors of the PLLA were investigated by differential scanning calorimeter (DSC), polarizing optical microscopy (POM), and wide angle X‐ray diffraction (WAXD). Both isothermal and non‐isothermal processes in DSC demonstrated that the DMs significantly altered the crystal behaviors of PLLA as both crystallization velocity and degree of crystallinity increased with increasing DM loadings from 0 to 3%. Our POM results also indicated that as nucleating agents, the DMs promoted nucleating densities and decreased spherulitic sizes. In addition, WAXD suggeted that the addition of DMs did not induce new types of crystals. Finally, our results showed that the ductility of the PLLA was enhanced by a small amount of DMs during the PLLA crystallization process since 0.5% DMs added to the PLLA resulted in 1.4‐fold increase in the elongation at break in comparision with the neat PLLA.     

Microscale control over collagen gradient on poly(L-lactide) membrane surface for manipulating chondrocyte distribution

A collagen gradient was constructed to interrogate cell adhesion on a poly(L-lactide) (PLLA) membrane surface. Utilizing a microinfusion pump, gradients of amino groups were generated on the PLLA surface by an aminolysis method. Immobilization of collagen onto the gradient surfaces was performed by glutaraldehyde (GA) coupling to form the collagen gradients. The -NH(2) and immobilized collagen density profiles on the PLLA membranes were quantitatively determined by ninhydrin and hydroproline (Hyp) analysis, respectively. By using fluorescein isothiocyanate (FITC) labeled collagen (FITC-Col), the profile of the as-prepared collagen gradient was directly monitored by a fluorescence microscope. The scanning force microscopy and water contact angle studies revealed that the morphology and wettability of the modified membranes changed progressively as a function of position along the gradient surface. Rabbit auricular chondrocytes were cultured on the collagen gradient membranes to test their cellular response. The attachment and spreading behaviors of the chondrocytes were dependent on the surface collagen density. These results indicate a surface on which the variation of collagen gradient strongly modifies the biological response of chondrocytes.     

Microencapsulation of DNA Within alginate microspheres and crosslinked chitosan membranes for in vivo application

Calf thymus DNA was microencapsulated within crosslinked chitosan membranes, or immobilized within chitosan-coated alginate microspheres. Microcapsules were prepared by interfacial polymerization of chitosan, and alginate microspheres formed by emulsification/ internal gelation. Diameters ranged from 20 to 500 Μm, depending on the formulation conditions. Encapsulated DNA was quantifiedin situ by direct spectrophotometry (260 nm) and ethidium bromide fluorimetry, and compared to DNA measurements on the fractions following disruption and dissolution of the microspheres. Approximately 84% of the DNA was released upon core dissolution and membrane disruption, with 12% membrane bound. The yield of encapsulation was 96%. Leakage of DNA from intact microspheres/capsules was not observed. DNA microcapsules and microspheres were recovered intact from rat feces following gavage and gastrointestinal transit. Higher recoveries (60%) and reduced shrinkage during transit were obtained with the alginate microspheres. DNA was recovered and purified from the microcapsules and microspheres by chromatography and differential precipitation with ethanol. This is the first report of microcapsules or microspheres containing biologically active material (DNA) being passed through the gastrointestinal tract, with the potential for substantial recovery.     

Characterization and Caco-2 Cell Transport Assay of Chito-Oligosaccharides Nano-Liposomes Based on Layer-by-Layer Coated

Chito-oligosaccharides (COSs) were encapsulated by the film-ultrasonic method into three nano-liposomes, which were uncoated liposomes (COSs-Lip), chitosan-coated liposomes (CH-COSs-Lip), and sodium alginate (SA)/chitosan (CH)-coated liposomes (SA/CH-COSs-Lip). The physicochemical and structural properties, as well as the stability and digestive characteristics, of all three nano-liposomes were assessed in the current study. Thereafter, the characteristics of intestinal absorption and transport of nano-liposomes were investigated by the Caco-2 cell monolayer. All nano-liposomes showed a smaller-sized distribution with a higher encapsulation efficiency. The ζ-potential, Z-average diameter (Dz), and polydispersity index (PDI) demonstrated that the stability of the SA/CH-COSs-Lip had much better stability than COSs-Lip and CH-COSs-Lip. In addition, the transport of the nano-liposomes via the Caco-2 cell monolayer indicated a higher transmembrane transport capacity. In summary, the chitosan and sodium alginate could serve as potential delivery systems for COSs to fortify functional foods and medicines.     

Preparation and characterization of biodegradable poly(l-lactide)/chitosan blends

In order to improve the properties of chitosan and obtain new fully biodegradable materials, blends of poly(l-lactide) (PLLA) and chitosan with different compositions were prepared by precipitating out PLLA/chitosan from acetic acid-DMSO mixtures with acetone. The blends were characterized by Fourier transform infrared analysis (FTIR), X-ray photoelectron spectroscopy (XPS), differential scanning calorimetry (DSC), ¹³C solid-state NMR and Wide-angle X-ray diffraction (WAXD). FTIR and XPS results showed that intermolecular hydrogen bonds existed between two components in the blends, and the hydrogen bonds were mainly between carbonyls of PLLA and amino groups of chitosan. The melting temperatures, cold crystallization temperatures and crystallinity of the PLLA component decreased with the increase in chitosan content. Blending chitosan with PLLA suppressed the crystallization of the PLLA component. Although the crystal structure of PLLA component was not changed, the crystallization of the blends was affected because of the existence of hydrogen bonds between two components, which was proved by WAXD results.     

Core‐shell microspheres with surface grafted poly(vinyl alcohol) as drug carriers for the treatment of hepatocellular carcinoma

Core(polyvinyl neodecanoate‐ethylene glycol dimethacrylate)‐shell(polyvinyl alcohol) (core (P(VND‐EGDMA))‐shell(PVA)) microspheres were developed by seeded polymerization with the use of conventional free radical and RAFT/MADIX mediated polymerization. Poly(vinyl pivalate) PVPi was grafted onto microspheres prepared via suspension polymerization of vinylneodecanoate and ethylene glycol dimethacrylate. The amount of grafted polymer was found to be independent from the technique used with conventional free radical polymerization and MADIX polymerization resulting into similar shell thicknesses. Both systems—grafting via free radical polymerization or the MADIX process—were found to follow slightly different kinetics. While the free radical polymerization resulted in a weight gain linear with the monomer consumption in solution the growth in the MADIX controlled system experienced a delay. The core‐shell microspheres were obtained by hydrolysis of the poly(vinyl pivalate) surface grafted brushes to form poly(vinyl alcohol). During hydrolysis the microspheres lost a significant amount of weight, consistent with the hydrolysis of 40–70% of all VPi units. Drug loading was found to be independent of the shell layer thickness, suggesting that the drug loading is governed by the amount of bulk material. The shell layer does not appear to represent an obstacle to the drug ingress. Cell testing using colorectal cancer cell lines HT 29 confirm the biocompatibility of the empty microspheres whereas the clofazimine loaded particles lead to 50% cell death, confirming the release of the drug. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 3256–3272, 2007     

单模微波辐照下壳聚糖-左旋聚乳酸接枝共聚物的酶催化合成

以猪胰脂肪酶(PPL)代替传统的有机金属作为催化剂,在单模微波辐照下利用左旋丙交酯(L-LA)的开环聚合制备壳聚糖-左旋聚乳酸(CS-g-PLLA)接枝共聚物.考察了反应温度及酶用量对接枝率的影响.以此为基础,利用DTG、XRD和3T3成纤维细胞培养对产物的物理性能及细胞相容性进行分析.结果显示,猪胰酶可有效催化接枝反应的进行.反应温度和酶用量对产物的接枝率有较大影响.在单模微波作用下,较低的反应温度(50℃)可获得具有较高接枝率(178.8%)的接枝产物.与纯壳聚糖相比,接枝产物的结晶度和热稳定性降低,说明PLLA的引入破坏了壳聚糖的高结晶性.产物具有良好的细胞相容性,可作为优良的组织工程支架材料.     

Morphology and thermal properties of poly(L‐lactide)/poly(butylene succinate‐co‐butylene adipate) compounded with twice functionalized clay

Poly(L ‐lactide) (PLLA)/poly(butylene succinate‐co‐butylene adipate) (PBSA) blends were compounded with Cloisite 25A® (C25A) and C25A functionalized with epoxy groups, respectively. Epoxy groups on the surface of C25A were introduced by treating C25A with (glycidoxypropyl)trimethoxy silane (GPS) to produce so called Twice Functionalized Organoclay (TFC). Variation of morphology and properties of PLLA/PBSA/C25A composites was investigated before and after the treatment with GPS. The morphological structure of the composites was analyzed by using X‐ray diffractometry (XRD) and transmission electron microscopy (TEM). The silicate layers of PLLA/PBSA/TFC were exfoliated to a larger extent than PLLA/PBSA/C25A. Incorporation of the epoxy groups on C25A improved significantly elongation at break as well as tensile modulus and tensile strength of PLLA/PBSA/C25A. The larger amount of exfoliation of the silicate layers in PLLA/PBSA/TFC as compared with that in PLLA/PBSA/C25A was attributed to the increased interfacial interaction between the polyesters and the clay due to chemical reaction. Thermo gravimetric analysis revealed that both T_5%, which was the temperature corresponding to 5% weight loss, and activation energy of thermal decomposition of PLLA/PBSA/TFC were far superior to those of PLLA/PBSA/C25A as well as to those of PLLA/PBSA, indicating that the composites with exfoliated silicate layers were more thermally stable than those with intercalated silicate layers. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 478–487, 2005     

Preparation and Drug Release of PVA Composite Nanofibers Loaded Chitosan Microsphere

In this paper, we reported the fabrication of poly(vinyl alcohol)-chitosan (PVA-CS) microspheres composite nanofibers by electrospinning technique. The chitosan microspheres were firstly prepared by electrospray with the solution of chitosan and combretastatin A4. The morphology and size distribution of chitosan microspheres were analyzed by scanning electron microscopy. The influencing factors including the concentrations of both PVA and CS microspheres were studied. The physical properties of the composite nanofibers were characterized by X-ray diffraction (XRD). The drug release rate, MTT toxicity test, and cell culture were also investigated in detail. Results indicate that the chitosan microsphere-loaded composite nanofibers can be prepared when the PVA concentration is 120 mg/mL. The continuity of the nanofibers was influenced by the concentration of CS microspheres. The characteristic peaks of CS or PVA were not observed in the diffractograms after the CS and PVA were processed using the high-voltage electrostatic technique. In addition, the drug release rate showed that nanofibers induce an obvious slow-release effect. Composite nanofibers were non-toxic to fibroblasts cells, and the fibroblasts cells could proliferate on the nanofiber mat.     

A hybrid scaffold of poly(lactide-<Emphasis Type="Italic">co</Emphasis>-glycolide) sponge filled with fibrin gel for cartilage tissue engineering

The poly(lactide-co-glycolide)(PLGA) sponge fabricated by a gelatin porogen leaching method was filled with fibrin gel to obtain a hybrid scaffold for chondrocytes culture in vitro.The fibrin gel evenly distributed in the hybrid scaffold with visible fibrinogen fibers after drying.In vitro culture it was found that in the hybrid scaffold the chondrocytes distributed more evenly and kept a round morphology as that in the normal cartilage.Although the chondrocytes seeded in the control PLGA sponges showed similar proliferation behavior with that in the hybrid scaffolds,they were remarkably elongated,forming a fibroblast-like morphology.Moreover,a larger amount of glycosaminoglycans was secreted in the hybrid scaffolds than that in the PLGA sponges after in vitro culture of chondrocytes for 4 weeks.The results suggest that the fibrin/PLGA hybrid scaffold may be favorably applied for cartilage tissue engineering.     

Promotion of cell affinity of porous PLLA scaffolds by immobilization of RGD peptides via plasma treatment

In the present work, RGDS (Arg-Gly-Asp-Ser) was immobilized on PLLA scaffolds with plasma treatment. The amount of immobilization, determined by HPLC, was confirmed to be in the effective order. Results from the culture of rat osteosarcoma (ROS), osteoblastic-like cells, demonstrate that the immobilization of RGDS could effectively enhance the attachment of ROS cells on PLLA and increase the cell density in PLLA scaffolds. In addition, experiments of in vitro mineralization indicate that there were more cells and mineralization focci in the RGDS-immobilized scaffolds, suggesting a tendency to form bone-like tissues, compared with the unmodified PLLA scaffold. On the other hand, the PLLA scaffolds immobilized with RGES (Arg-Gly-Glu-Ser) were much less effective in promotion of ROS attachment, suggesting that the enhancement on cell attachment was mainly due to the recognition of RGDS by the adhesion receptors on the cell membrane. The results presented in this work demonstrate that RGDS could be successfully immobilized on PLLA scaffolds with plasma treatment and such modification can make PLLA scaffolds more suitable for culture of osteoblast-like cells and for generation of bone-like tissues.     

SURFACE OF GELATIN MODIFIED POLY(L-LACTIC ACID)FILM

In this paper, the surface structure of poly(L-lactic acid) (PLLA) film modified with gelatin was investigated. ThePLLA film specimens were treated directly with aqueous alkali solution to provide their surfaces with carboxyl groups, sothat these functional groups could become the reactive sites for gelatin immobilization. The functional groups of the PLLAfilms were identified by ATR-FTIR spectra and XPS spectra, the changes in surface morphology were observed by usingenvironmental scanning electron microscopy (ESEM), and the hydrophilicity of modified PLLA films was examined bywater contact angle measurement. Experimental results showed that the gelatin was immobilized with water-solublecarbodiimide (EDC) onto the PLLA film's surfaces, and the gelatin content on the polymer surface was related to carboxylicgroup formed in the controlled hydrolysis process. Rough surfaces caused by hydrolysis will predominantly favor the adhesion and growth of cell; and the hydrophilicity of these surfaces after the modification procedure is enhanced.     

高压静电法制备多孔磁性壳聚糖微球

以壳聚糖(Chitosan, CS)为基质, 通过共混法引入四氧化三铁磁性颗粒, 以硅胶(Silicagel, S)为致孔剂, 在热的NaOH溶液中溶出硅胶致孔, 采用高压静电法制备磁性壳聚糖微球. 通过SEM观察了微球的结构和形貌, 并对微球结构和形貌的影响因素及其制备工艺进行了系统的研究, 结果表明, 高压静电法制备的磁性硅胶/壳聚糖微球粒径可通过微量进样器的针头大小来控制, 并且粒径分布均匀, 实验重复性及可控性好; 当以质量体积分数为5%的壳聚糖醋酸溶液(体积分数2％, mS∶mCS=4∶1), 用8号针头进样时, 制得直径约为600 μm, 孔洞分布均匀, 孔径约为50 μm的多孔磁性壳聚糖微球. 由于磁性多孔壳聚糖微球中含有大量的活性羟基和氨基, 因此显弱碱性, 对酸性物质和金属离子的吸附作用很好, 且可通过外加磁场进行有效分离. 磁性多孔壳聚糖微球在生物分离及污水中的酸性染料处理方面具有潜在的应用价值.     

香草醛交联壳聚糖载药微球的性能及其成球机理分析

以壳聚糖溶液为水相、液体石蜡为油相形成油包水型乳液, 以香草醛为交联剂, 采用乳化交联法制得壳聚糖微球. 结合IR光谱和XRD测试, 分析了壳聚糖交联固化成球的机理: 壳聚糖和香草醛之间所发生的Schiff碱反应和氢键的形成以及缩醛化反应, 以此为基础共同形成交联结构从而使壳聚糖交联固化成球. 探讨了交联后壳聚糖微球结晶度降低的原因: 壳聚糖固化时分子链未充分进行有序的结晶排列, 交联后的壳聚糖结构较复杂, 从而破坏了原壳聚糖分子的规整性. 选用盐酸小檗碱为模型药物, 制备了香草醛交联的壳聚糖载药微球, SEM结果显示, 载药微球表面致密且球形度好, 微球粒径在5-15 μm之间. 此外, 采用分光光度计对载药微球的载药率、药物包封率和药物体外释放性质进行了测试和分析, 结果表明载药微球缓释效果明显.     

Properties and morphology of poly(L-lactide). II. Hydrolysis in alkaline solution

Hydrolysis of poly(L -lactide) (PLLA) films in 0.01N NaOH at 37°C was investigated by gel permeation chromatography, differential scanning calorimetry, scanning electron microscopy, and polarizing optical microscopy. The change in molecular weight distribution and surface morphology of PLLA films during hydrolysis revealed that PLLA film hydrolysis in dilute alkaline solution proceeded mainly via the surface erosion mechanism. An insignificant dependence of the rate of weight loss per unit surface area on the PLLA film thickness also supported this conclusion. Etching of the outside of PLLA spherulites resulted in preferred hydrolysis of PLLA chains in the amorphous region. The disorientation of lamella and inhomogeneous erosion in the spherulites implied that hydrolysis of PLLA chains occurred predominantly in the amorphous region between the crystalline regions in the spherulites. The rate of weight loss per unit surface area decreased linearly with the increase in the initial crystallinity of PLLA film, while the radius of spherulites had practically no significant effect on the hydrolysis of PLLA film. The specific low molecular weight of PLLA chains produced by hydrolysis increased with the rise in annealing temperature of the PLLA film, suggesting that the PLLA chains released were the component of one fold in the crystalline region. © 1998 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 36: 59–66, 1998     

Vitamin A and vitamin E interaction behavior on chitosan microspheres

Chitosan is a biodegradable natural polymer with great potential for pharmaceutical applications due to its biocompatibility, high charge density, and non-toxicity. In this study, chitosan microspheres were successfully prepared by an adapted method of coagulation/dispersion. The degree of deacetylation of chitosan powder was obtained by NMR ¹H and FTIR techniques. Chitosan powder and chitosan microspheres were characterized by BET surface area and scanning electron microscopy (SEM). The interactions among the chitosan microspheres and the vitamins A and E were characterized by FTIR. In order to evaluate the ability of interaction of vitamin A and vitamin E with the chitosan microspheres, the thermodynamic parameters were followed by calorimetric titration. Different experimental approaches were applied, such as adsorption isotherms, kinetics and thermodynamics studies. The obtained results showed that the interactions of chitosan microspheres with the vitamins were spontaneous, enthalpically and entropically favorable, indicating that the chitosan microspheres can be used with success in the controlled release of these vitamins.     

Collagen nanofiber-covered porous biodegradable carboxymethyl chitosan microcarriers for tissue engineering cartilage

Nanofibrous collagen-coated porous carboxymethyl chitosan microcarriers (CMC-MCs) were successfully fabricated for use as injectable cell microcarriers. A modified phase separation method combined with temperature controlled freeze-extraction was used for formulating the CMC-MCs. Collagen nanofibers were immobilized onto the surfaces of the CMC-MCs via covalently anchoring some collagen molecules first and more molecules self-assembling into nano-scale fibrous networks afterward. Scanning electron microscopy and hydroxyproline colorimetry analysis revealed that more collagen was immobilized on the CMC-MCs with collagen molecules anchored initially. In vitro cell culture revealed that chondrocytes could adhere, proliferate, and remain differentiated on the nanofiber-coated CMC-MCs. Optical microscopy and confocal laser scanning microscopy showed that chondrocytes grew to confluence on the CMC-MCs within 3 days post-seeding. Subsequently, several confluent CMC-MCs attached to each other, forming tissue-like aggregates after 7 days culture. The mRNA expression of type II collagen was much stronger in chondrocytes cultured on the nanofiber-coated CMC-MCs for 7 days than those cultured in 24-well plates or on CMC-MCs without initial treatment. These porous CMC-MCs could be utilized for cultivating cells and for application in cartilage tissue engineering as injectable scaffolds for cell delivery.