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Basic fibroblast growth factor (bFGF) was immobilized onto quartz slides and collagen films by assembly with chondroitin sulfate (CS) in a layer-by-layer (LBL) manner. First, the LBL-deposition process on the amino-silanized quartz slides was monitored by UV–vis spectroscopy and water contact angle measurement. By substituting the normal bFGF with rhodamine-labeled one (Rd-bFGF), a linear increase of the absorbance versus bilayer number was recorded. The water contact angle oscillated between the odd CS and the even bFGF layers, demonstrating the alternating change of the surface chemistry. Scanning force microscopy (SFM) revealed that the surface topography was altered slightly after multilayer assembly. In vitro incubation of the CS/bFGF multilayers in PBS showed that ∼30% of the incorporated bFGF was released within 8 days. In vitro cell culture found that the fibroblasts showed star-like morphology with plenty of pseudopods on the bFGF-incorporated collagen film after cultured for 1 day, and the collagen films assembled with bFGF possess improved bioactivity than that of the virgin one and the bFGF control. Since the immobilized growth factors can maximally retain their bioactivity, the LBL assembly would be a potential approach to construct a bioactive substrate for biomedical applications. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2007

A thermoresponsive copolymer, trimethyl chitosan-g-poly(N-isopropylacrylamide) (TMC-g-PNIPAAm), was synthesized by coupling PNIPAAm-COOH to TMC. Their molecular structures were characterized by 1HNMR. The lower critical solution temperature (LCST) of TMC-g-PNIPAAm in PBS was measured as 32 degrees C by dynamic light scattering (DLS) and UV-vis spectroscopy, regardless of the grafting ratios. Upon mixing with DNA, TMC/DNA particles were formed, whose size and morphology were investigated by DLS and transmission electron microscopy, respectively. The particle size ranged from 200 to 900 nm depending on the N/P ratio and was less influenced by the temperature variation. The majority of the particles have spherical morphology. The zeta potentials of these particles were increased along with the N/P ratio. At a given N/P ratio, the zeta potentials were almost constant at 25 degrees C regardless of the existence of serum proteins. However, the values were significantly decreased at 37 degrees C in a solution containing serum protein. The affinity between DNA and TMC was examined by ethidium bromide competitive binding assay. TMC-g-PNIPAAm has stronger ability to combine with DNA at 40 degrees C when the PNIPAAm chain is collapsed. Gel electrophoresis results reveal that the vectors/DNA complexes can be formed regardless of the incubation temperature. HEK293 cell line was chosen as a model to study cellular uptake of the TMC-g-PNIPAAm/DNA particles, gene transfection and cytotoxicity. The grafting of PNIPAAm will not affect cellular uptake of the particles at 37 degrees C. The level of gene transfection could be thermally controlled. By using a temperature variation protocol, i.e. incubation of the cultured cells at 25 degrees C for a while, the gene transfection efficiency was significantly improved. Finally, the optimized gene transfection efficiency achieved by TMC-g-PNIPAAm is comparable to Lipofectamine 2000. No obvious cytotoxicity was detected for the TMC-g-PNIPAAm/DNA particles. These results suggest that TMC-g-PNIPAAm is an effective thermoresponsive gene carrier with minimal cytotoxicity, which has great promise for practical applications.

The interaction between mesoscopic colloids and cells is largely dependent on the particle size and surface properties. Under a mild reaction condition, gold particles with an average diameter of approximately 100 nm were prepared by incubating poly(dimethylsiloxane) film in HAuCl4/acetic acid solution. The particles were then transferred into a polycaprolactone (PCL) film by thermal pressing. Bare and PCL-coated particles were obtained by control over the extent of rinsing. The bare and PCL-coated gold particles were co-cultured with ECV-304 cells to examine the particle internalization and their influence on the cell morphology and cytotoxicity. Transmission electron microcopy observed the subcellular distribution of the gold particles, which were found in the cell compartments (endosomes or lysosomes), cytoplasm, nucleic envelope, and even nucleus regardless of the existence of PCL coating. However, scanning electron microscopy and beta-tubulin staining revealed a significant change in terms of the cell morphology and cytoskeleton caused by the bare gold particles. Higher cytotoxicity was also determined for the bare gold particles. By contrast, no significant difference of the cell morphology and cytoskeleton change was caused by the PCL-coated gold particles, which have also shown lower cytotoxicity.

Poly(ethylene terephthalate) (PET) film and PET vascular graft were modified by layer-by-layer (LBL) assembly of collagen and chondroitin sulfate (CS) to mimic the natural composition and structure of extracellular matrix. PET materials were first hydrolyzed to endow the surfaces with negative charge. Alternative assembly of positively charged collagen and negatively charged CS was then conducted in an LBL manner to produce biological multilayers. X-ray photoelectron spectroscopy confirmed the occurrence of hydrolysis and existence of collagen and CS. The stepwise layer growth on the PET film was monitored by UV-vis spectroscopy. The multilayers had sufficient stability to pass through the cell culture period (8 days). In vitro endothelial cell culture revealed that the incorporated biomacromolecules could significantly improve the cell attachment, proliferation, and viability. Cells showed more spreading morphology with preserved phenotype, as illustrated by scanning electron microscopy and factor VIII-related antigen immunocytochemistry, respectively.

Multilayer microcapsules were fabricated by layer-by-layer (LbL) assembly of natural polysaccharides onto CaCO3 particles, following with core removal. The micron-sized CaCO3 particles were synthesized by reaction between Ca(NO3)2 and Na2CO3 solutions in the existence of carboxylmethyl cellulose (CMC). The incorporated amount of CMC in the CaCO3 particles was found to be 5.3 wt% by thermogravimetric analysis. Two biocompatible polysaccharides, chitosan and sodium alginate were alternately deposited onto the CaCO3(CMC) templates to obtain hollow microcapsules. Regular oscillation of surface charge as detected by zeta potential demonstrated that the assembly proceeded surely in a LbL manner. The stability of the microcapsules was effectively improved by cross-linking of chitosan with glutaraldehyde. The chemical reaction was verified by infrared spectroscopy. The microcapsules thus fabricated could be spontaneously filled with positively charged low molecular weight substances such as rhodamine 6G and showed good biocompatibility, as detected by in vitro cell culture.

Water-soluble chitosan having double bonds (CS-MA-LA) was synthesized by sequential grafting of methacrylic acid (MA) and lactic acid (LA) via the reaction between amino groups and carboxyl groups under the catalysis of carbodiimide. Its molecular structure was verified by FTIR and (1)H NMR characterizations. Elemental analysis measured grafting ratios of 19% and 10.33% for MA and LA, respectively. CS-MA-LA was readily soluble in pure water and did not precipitate till pH 9. Gelation of the CS-MA-LA was realized by thermal treatment at body temperature under the initiation of a redox system, ammonium persulfate (APS)/N, N,N',N'-tetramethylethylenediamine (TEMED). The gelation time could be mediated in a wide range, e.g. from 6 to 20 min, by reaction temperature and/or initiator's concentration. 3T3 fibroblast culture showed that the cytotoxicity of the hydrogel extractant was dependent on the cell seeding number and the initiator's concentration. With enough number of cells (>2.5 x 10(4)) and low initiator's concentration (5 mM), the cytotoxicity introduced by the initiator is very minimal and negligible. Although the hydrogel could cause acute inflammation and foreign body reaction, no tissue necrosis and malignant infection were evidenced in vivo, demonstrating that the material has better histocompatibility. These features have endowed the chitosan with great opportunity as injectable biomaterials, which may find wide applications in the rapidly developed fields such as tissue engineering and orthopedics.

Multilayer microcapsules showing unique charge-controlled permeability have been successfully fabricated by employing poly(styrene sulfonate) (PSS)-doped CaCO3 particles as templates. Encapsulation of the PSS molecules is thus achieved after core removal. Scanning force microscopy (SFM), UV-vis, Raman spectroscopy, and zeta-potential confirm the existence of the PSS molecules in the CaCO3 particles and the resultant microcapsules, which are initially incorporated during the core fabrication process. A part of these additionally introduced PSS molecules interacts with PAH molecules residing on the inner surface of the multilayer wall to form a stable complex, while the other part is intertwined in the capsule wall or in a free state. Capsules with this structure possess many special features, such as highly sensitive permeability tuned by probe charge and environmentally controlled gating. They can completely reject negatively charged probes, but attract positively charged species to form a higher concentration in the capsule interior, as evidenced by confocal microscopy. For example, the capsules completely exclude dextran labeled with fluorescein isothiocyanate (FITC-dextran), but are permeable for dextran labeled with tetramethylrhodamine isothiocyanate (TRITC-dextran) having similar molecular mass (from 4 to 70 kDa), although there are only few charged dyes in a dextran chain. By reversing the charge of the probes through pH change, or by suppressing charge repulsion through salt addition, the permeation can be readily switched for proteins such as albumin or small dyes such as fluorescein sodium salt.

Porous poly(l-lactic acid) (PLLA) scaffolds fabricated by a gelatin particle-leaching technique have good mechanical property and cytocompatibility, as demonstrated by a previous in vitro study. Here we investigate further the in vitro degradation of the scaffolds in terms of weight loss, water uptake, weight-average molecular weight, thermal behavior and morphology during a 39 week period in phosphate-buffered saline. The water uptake decreased dramatically during the initial stage due to release of the remaining gelatin, and then increased slightly with degradation time. The weight-average molecular weight decreased linearly as a function of time, while the crystallinity steadily increased with slightly decreased melting temperature. After degradation, many defects and big holes were seen in the scaffolds by scanning electron microscopy. Cartilage regeneration and scaffold disappearance in vivo were compared by implanting the construct into nude mice for 30-120 days. While the scaffolds maintained their intact pore structure after 23 weeks of degradation in vitro, they almost disappeared in vivo at the same time, implying a faster degradation rate in vivo. By 120 days after implantation, the scaffolds were hardly seen in the newly formed cartilage-like tissue. The regenerated cartilages could not maintain their predesigned shape after a long period of in vivo culture due to the weakening of the mechanical strength of the constructs as a result of PLLA degradation. The regions occupied initially by PLLA scaffold were filled later by collagen type II secreted by the chondrocytes, but with no evident basophilic proteoglycan.

Skin loss or damage affects severely the life quality of human being and can even cause death in many cases. We report here a bilayer dermal equivalent (BDE) composed of collagen-chitosan porous scaffold and silicone membrane, which can effectively induce the regeneration of dermis in an animal model of full thickness skin loss. The in vitro biosecurity test showed that the BDE had no cytotoxicity, and no remarkable sensitization and irritability. In vitro cell culture proved that the BDE had good biocompatibility to support the proliferation of fibroblasts. Animal test was performed on Bama miniature pig skin. Gross view and histological sections found plenty of fibroblasts and extracellular matrix in the regenerative scaffold after transplantation of the BDE for 4 weeks. Immunohistochemistry results proved that the BDE has the ability to support the angiogenesis of the regenerated dermis. All these results indicate that the BDE might be a promising equivalent in treating dermal loss.

To construct three kinds of collagen-chitosan porous scaffolds with enhanced bio-stability and to investigate the histocompatibility of the scaffolds in vivo.Collagen-chitosan porous scaffolds were fabricated by freeze-drying method, cross-linked using dehydrothermal treatment and glutaraldehyde, respectively. The morphology of the uncross-linked scaffold (scaffold 1), dehydrothermal cross-linked scaffold (scaffold 2) and glutaraldehyde cross-linked scaffold (scaffold 3) was studied by scanning electron microscopy. Three kinds of scaffolds were embedded subcutaneously on dorsal surface of 12 rabbit ears. The general and local responses were recorded daily. The bio-stability and histocompatibility of the scaffolds were observed by using HE staining after 3, 7, 14 and 28 days of operation.The scaffolds had three-dimensional porous structures with a porosity of more than 90%, and possessed pore sizes of 120 +/- 10 microm, 80 +/- 15 microm and 170 +/- 20 microm, respectively. All experimental rabbits survived with good general condition during the study. All skin incisions healed well without obvious reactive red or swelling. Histological study showed that scaffold 1 was degraded rapidly with obvious inflammation. The degradation of scaffold 2 was slower than that of scaffold 1 and the inflammation of scaffold 2 was also milder than that of scaffold 1. Scaffold 3 possessed slow degradation property with slight inflammatory reaction, and rapid tissue regeneration.The collagen-chitosan porous scaffolds have three-dimensional porous structures that are suitable for tissue regeneration. The bio-stability and histocompatibility of the scaffolds are enhanced after cross-linked. Glutaraldehyde cross-linked is better than dehydrothermal cross-linked, which can facilitate dermal tissue reconstruction.

A new class of bioactive thin films using growth factors as building blocks has been fabricated via layer-by-layer assembly (LBL) technique. Acid fibroblast growth factor (aFGF) in the presence of heparin was used as negatively charged polyelectrolytes, while poly(ethyleneimine) (PEI) was chosen as a positively charged counterpart. The self-deposition process and surface morphology of the resultant multilayers were monitored and detected by UV-vis absorbance spectra, advanced contact angle measurements, and scanning force microscopy (SFM) observations. Cell culture was performed to assess the efficiency of the growth factors. The fibroblasts proliferated faster on the surface assembled with five bilayers of (aFGF/heparin)/PEI with apparent higher cytoviability than on those surfaces modified by one bilayer of (aFGF/heparin)/PEI, five bilayers of aFGF/PEI, or five bilayers of heparin/PEI, and tissue culture polystyrene. Enhanced secretion of collagen type I and interleukin 6 (IL-6) by the fibroblasts seeded on the five bilayers of (aFGF/heparin)/PEI was also verified by immunohistochemical examination. The bioactivity of the (aFGF/heparin)/PEI multilayers could be largely preserved when stored at -20 degrees C.

Crosslinked polysaccharide and composite polysaccharide capsules with diameters ranging from 200 nm to several microns and wall thicknesses of several tens of nanometers have been fabricated by interfacial polymerization of methacrylated N , N -diethylaminoethyl dextran (DdexMA) and DdexMA-vinyl terminated polylactide macromonomers (PLAM). In this method, chloroform droplets or PLAM-containing chloroform droplets were dispersed in water, on which water soluble DdexMA was polymerized to form closed shell structure. Their hollow nature was confirmed by confocal laser scanning microscopy and transmission electron microscopy. Dynamic light scattering revealed that these capsules possess good stability against coagulation during storage. Fourier transform infrared and elemental analysis found that the DdexMA capsules were actually composed of crosslinked DdexMA, while the DdexMA–PLAM capsules were composed of the crosslinked DdexMA–PLAM copolymers and PLAM. By dissolution of ibuprofen in the chloroform droplets, drug-loaded capsules were also fabricated. It was found that the loaded drug could be released again in a sustained manner for up to 100 h. The capsule walls had a prominent effect in slowing down the drug release rate, particularly for the DdexMA–PLAM capsules.

Poly(ethylene glycol) (PEG), a hydrophilic and repulsive polymer to non-specific adsorption, was stamped onto carboxylic acid-enriched polymer surfaces using the micro-contact printing technique. The patterns are stabilized via hydrogen bonds. Areas printed with PEG were then shown to be non-adsorbed with fluorescein isothiocyanate (FITC)-labeled dextran, while the poly(methacrylic acid) (PMAA) regions could via hydrogen bonding. Due to this contrast, well defined dextran patterns were obtained. Tuned with pH and temperature, the PEG molecules could be detached from the surfaces, erasing the template. Moreover, ionization of PMAA at higher pH induced an abrupt transition to an extended conformation, weakening the interactions between PMAA and dextran. Not only the dextran patterns lose their spatial selectivity, but also the overall adsorption amount is much lower. The pH sensitivity was in a quite narrow range, i.e. around pH 5. As the hydrogen bonds are also temperature sensitive, the attach points of PEG molecules on the surfaces disappeared at higher temperature. For poly(acrylic acid) (PAA) photografted surfaces, the pH sensitivity was more complicated due to the formation of the compact complexes of PEG and PAA molecules.

A novel crosslinking method was adopted to modify the porous collagen scaffolds by using a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) and N-hydroxysuccinimide (NHS) in the presence of lysine, which functions as a crosslinking bridge. In vitro biodegradation tests proved that in the presence of lysine the biostability of the EDAC crosslinked scaffolds was greatly enhanced. The biostability of the resultant scaffolds was also elucidated as a function of the concentrations of lysine and EDAC/NHS. Compared to the Col-DHT, the ability of the Col-EDAC and the Col/Lys to resist cell-mediated contraction (CMC) was greatly enhanced. Yet no obvious difference between the Col-EDAC and the Col/Lys was found with respect to CMC. SEM observations showed that the microstructure of the crosslinked scaffolds could be largely preserved after fibroblast seeding. As a result, MTT assays proved that the fibroblasts in the Col/Lys scaffolds proliferated faster compared to the DHT-treated one on the assumption that the cell viability was preserved to a similar level. Histological section results indicated that the Col/Lys scaffolds had the ability to accelerate the cell infiltration and proliferation. All these results demonstrated that this novel crosslinking method is an effective way to achieve a collagen scaffold with improved biostability and a more stable structure, which can resist cell-mediated contraction. (c) 2004 Wiley Periodicals, Inc. J Biomed Mater Res 71A: 334-342, 2004.

A novel pathway for ciprofloxacin hydrochloride delivery system based on spontaneous deposition mechanism was introduced with respect to encapsulation, quantitative drug loading and sustained release. Layer-by-layer assembly of oppositely charged polyelectrolytes onto melamine formaldehyde (MF) colloidal particles, followed by removal of the cores at low pH has yielded hollow microcapsules having a unique property to induce spontaneous deposition of various water-soluble substances. Observations under scanning electron microscopy, atomic force microscopy and transmission electron microscopy provided direct proofs of the spontaneous deposition. The quantitative drug loading and sustained release properties were elucidated. Results show that the loaded drug is proportional to drug feeding concentrations, temperature and salt concentrations, demonstrating tailorable deposition behavior that is crucial for the drug carrier. The deposited ciprofloxacin hydrochloride could be again released in a sustained manner and exhibited a significant antiseptic activity with high biocompatibility.

The factors controlling the microstructure and properties of collagen-based bioactive artificial dermis are reviewed. The second component, the pore diameter and porosity, the thickness of scaffold, the bioactive factors as well as the cross-linking density that are important parameters of artificial dermis should be carefully researched and designed. Experiment methods controlling these parameters are suggested.

The layer-by-layer (LBL) self-assembly technique has been diversely applied to fabricate multilayer ultrathin organic or hybrid films with various properties since it was introduced by Decher et al. Oppositely charged polyelectrolytes from dilute aqueous solution are deposited consecutively onto solid substrates by utilizing electrostatic attraction and complex formation between polyanions and polycations. Recently, LBL assembly of oppositely charged polyelectrolytes onto removable colloidal particles has been utilized to create novel hollow nanoand microcapsules with customized physicochemical properties. These nanoand micron-sized capsules are of both scientific and technological interest because of their potential applications as new colloidal structures in areas such as medicine, drug delivery, artificial cells (viruses), and catalysis. Many efforts have been devoted to the nanoscale encapsulation of drugs, minerals, dyes, proteins, enzymes and genes. Materials such as enzymes or drugs can be transformed into a core template or can be attached to core template particles prior to core decomposition so as to trap them inside hollow capsules. Hollow polyelectrolyte capsules have been filled with low molecular weight dyes such as 6-carboxyfluorescein (6CF) and rhodamine 6G (Rd6G) by modifying the solubility of a compound by variation of the pH value. Macromolecules such [14] C. P. Collier, J. O. Jeppesen, Y. Luo, J. Perkins, E. W. Wong, J. R. Heath, J. F. Stoddart, J. Am. Chem. Soc. 2001, 123, 12632 ± 12641. [15] T. J. Huang, H.-R. Tseng, L. Sha, W. Lu, A. H. Flood, B. Brough, B. Yu, P. C. Celestre, P. J. Chang, J. F. Stoddart, C.-M. Ho, unpublished results. [16] I. C. Lee, C. W. Frank, T. Yamamoto, H. R. Tseng, A. H. Flood, J. F. Stoddart, unpublished results. [17] For a discussion of first-principles computational methods which indicate that, when the CBPQT ring surrounds the DNP ring system, the switch is on, whereas, when it surrounds the TTF unit, the switch is off, see: W. Deng, R. P. Muller, W. A. Goddard III, J. Am. Chem. Soc. , in press. See also a report on TMComputational Nanotechnology∫ by E. K. Wilson, Chem. Eng. News 2003, April 28, pp. 27 ± 29. [18] In a prescient communication published back in 1993, Gokel and Kaifer showed that TMit is possible to utilize noncovalent intermolecular interactions, such as stacking forces between aromatic -donor and acceptor subunits, to build a catenane-like structure supported on gold surfaces∫. See: T. Lu, L. Zhang, G. W. Gokel, A. E. Kaifer, J. Am. Chem. Soc. 1993, 115, 2542 ± 2543. [19] a) For a recent monograph and reviews on supramolecular electrochemistry, see: A. E. Kaifer, M. Go¬mez-Kaifer, Supramolecular Chemistry, Wiley-VCH, Weinhem, 1999 ; b) P. L. Boulas, M. Go¬mez-Kaifer, L. Echegoyen, Angew. Chem. 1998, 110, 226 ± 258; Angew. Chem. Int. Ed. 1998, 37, 216 ± 247; c) A. Niemz, V. M. Rotello, Acc. Chem. Res. 1999, 32, 44 ± 52; d) A. E. Kaifer, Acc. Chem. Res. 1999, 32, 62 ± 76; e) J. H. R. Tucker, S. Collinson, Chem. Soc. Rev. 2002, 31, 147 ± 156. [20] a) In solution, the metastability of a bistable [2]catenane has been reported. See: M. Asakawa, P. R. Ashton, V. Balzani, A. Credi, C. Hamers, G. Mattersteig, M. Montalti, A. N. Shipway, N. Spencer, J. F. Stoddart, M. S. Tolley, M. Venturi, A. J. P. White, D. J. Williams, Angew. Chem. 1998, 110, 357 ± 361; Angew. Chem. Int. Ed. 1998, 37, 333 ± 337; b) V. Balzani, A. Credi, G. Mattersteig, O. A. Matthews, F. M. Raymo, J. F. Stoddart, M. Venturi, A. J. P. White, D. J. Williams, J. Org. Chem. 2000, 65, 1924 ± 1936. [21] Detecting the minor translational isomer by H NMR spectroscopy in either CD3CN or CD3COCD3 for bistable [2]rotaxanes of this type has been an elusive exercise; see ref.s [8d,13b,c] . In essence, translational isomerism is fast on the H NMR timescale at room temperature and so only averaged signal information is available. At low temperature, where the isomerism becomes TMfrozen out∫, it would appear that the major translational isomer is overwhelmingly preferred at equilibrium and so no evidence for the minor isomer can be found. [22] The irreversible oxidation process associated with the DNP ring system in SSD is located at 1100 mV. The same process for SSR is moved to even higher potential ( 1500 mV) because of the encirclement of the DNP ring system by the CBPQT ring following the oxidation of the TTF unit. [23] A. J. Bard, Electrochemical Methods: Fundamentals and Applications, Wiley, New York, 2001. [24] In studies on the dumbbell control SSD, the surface cover was found to be 0.35 molecule nm , that is, a footprint on gold of the order 2.8 nm. [25] a) C. P. Collier, G. Mattersteig, E. W. Wong, Y. Luo, K. Beverly, J. Sampaio, F. M. Raymo, J. F. Stoddart, J. R. Heath, Science 2000, 289, 1172 ± 1175; b) A. R. Pease, J. O. Jeppesen, J. F. Stoddart, Y. Luo, C. P. Collier, J. R. Heath, Acc. Chem. Res. 2001, 34, 433 ± 444; c) M. R. Diehl, D. W. Steuerman, H.-R. Tseng, S. A. Vignon, A. Star, P. C. Celestre, J. F. Stoddart J. R. Heath, ChemPhysChem 2003, 5, 1335 ± 1339. [26] The first-order equation for the decay of the metastable state is Equation (1)

A previously reported "grafting and coating" method (J. Biomed. Mater. Res. (Appl. Biomater.) 63 (2002) 838) was modified and used to introduce stable collagen layer and incorporate basic fibroblast growth factor (bFGF) on PLLA scaffold surface to prepare tissue engineering scaffold with improved biocompatibility. To make the modification of the 3-D porous PLLA scaffold possible, grafting of polymethacrylic acid (PMAA) onto the PLLA surface was initiated by the -OOH/Fe2+ system instead of the UV light used in the former method. Water soluble carbodimmide chemistry was applied to graft collagen onto the PLLA scaffold surface, followed by physical coating of the collagen solution with or without basic fibroblast growth factor (bFGF). Surface modification of 2-D PLLA membrane was also done for fundamental understanding of the modification. The -COOH density on/in the PMAA grafted PLLA membrane/scaffold was measured by colorimetric method and the collagen content on/in the collagen immobilized PLLA membrane/scaffold was measured by ninhydrin method. Chondrocyte culturing on the collagen immobilized PLLA surfaces showed significantly improved cell spreading and growth. Incorporation of fibroblast growth factors in the collagen layer further enhanced the cell growth. This convenient and effective method can be used to prepare bioactive scaffolds with extra cellular matrix (ECM)-mimic composition for tissue engineering.

Using the recently developed surface modification technique, free amino groups have been introduced onto polyester-type polyurethane (PU) scaffolds. The introduction of these free amino groups increases the surface energy and provides a convenient way to further immobilize bioactive species such as gelatin, collagen or chitosan, etc. on the scaffold surface by employing glutaraldehyde as a coupling agent. These modifications are advantageous to enhance cell–material interaction. The culture of human umbilical vein endothelial cells (HUVECs) in vitro proved that the cell proliferation ratio of both the aminolyzed and the biomacromolecules-immobilized PU membranes was improved greatly comparing with the control PU. Scanning electron microscopy and confocal laser scanning microscopy observations displayed that the gelatin-immobilized PU vascular scaffold had formed a monolayer of endothelial intima on its luminal surface after HUVECs were cultured for 6 d. Therefore, the aminolysis and the following biomacromolecule immobilization is a promising way to enhance the cell–PU interaction that can accelerate the endothelium regeneration, which is crucial for blood vessel tissue engineering.

A biodegradable scaffold for skin-tissue engineering was designed using collagen and chitosan, which are common materials for biomedical application. The scaffolds containing different amounts of chitosan were prepared by mixing the collagen and chitosan solutions followed by removal of the solvent using a freeze-drying method. The cross-linking treatment of these scaffolds was performed using the dehydrothermal treatment (DHT) method or glutaraldehyde (GA) to increase their biostability. The effect of the chitosan concentration and the cross-linking methods on the morphology of these scaffolds was studied by SEM. The water retention and the biodegradability in vitro of various collagen-chitosan scaffolds were investigated. Finally the biocompatibility of the collagen-chitosan (10 wt% chitosan) scaffold treated with different cross-linking methods was evaluated using a in vivo animal test. A mild inflammatory reaction could be detected in the early stages, and GA treatment can decrease the inflammatory reaction in a long-term implantation. After implantation for four weeks, all kinds of scaffolds, especially the GA-treated scaffolds (Col-GA) were filled with a large number of fibroblasts and were vascularized to a certain extent. These results suggest that the GA-treated scaffold has an increased biostability and excellent biocompatibility. It can be a potential candidate for skin-tissue engineering.

Three-dimensional poly(L-lactic acid) (PLLA) scaffolds with high porosity and pore size ranging from 150 to 700 microm were conveniently prepared with paraffin spheres used as porogen. PLLA/1,4-dioxane solution containing a given amount of paraffin spheres was frozen at -25 degrees C to obtain a solidified mixture, followed with freeze drying and subsequent leaching with hexane to remove the 1,4-dioxane and paraffin spheres, respectively. The fabricated PLLA scaffolds were highly porous with evenly distributed and interconnected pores. The microstructures of the PLLA scaffolds as a function of paraffin-sphere size, paraffin-sphere dosage, and PLLA concentration were characterized by confocal laser scanning microscopy (CLSM) and scanning-electronic microscopy (SEM). To improve the cytocompatibility of the bioinert material, a hybrid PLLA scaffold containing Type I collagen was prepared by pressing the collagen solution into the scaffold under reduced pressure. The amounts of the collagen introduced in the scaffolds were detected by ninhydrin method. The distribution of the collagen in the scaffolds was studied with CLSM. Finally, in vitro cell culture was performed by injecting a chondrocyte suspension into the scaffolds. The results showed that the chondrocytes were more evenly distributed and more spread out in the collagen-modified PLLA scaffolds than in the unmodified ones.

As regeneration scaffolds or templates in tissue engineering, polymeric materials play key roles in inducing tissue regeneration, and mediating cell growth and differentiation, i.e. functioning as artificial extracellular matrix. The polymeric materials including biological derived biopolymers and synthetic polymers, which are usually employed in tissue engineering, are reviewed in this paper.

Collagen porous scaffolds have been widely employed as a dermal equivalent to induce fibroblasts infiltration and dermal regeneration. To eliminate the disadvantageous drawback of the fast degradation speed, a cross-linking method was adopted by using a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) and N-hydroxysuccinimide (NHS) in the presence of amino acids (glycin, glutamic acid or lysine), which function as cross-linking bridge between collagen molecular chains. In vitro assessment of the biological stability of the cross-linked collagen scaffolds found that the collagenase biodegradation degree was greatly decreased when lysine was added, resulting in a more biological stable scaffold. On the other hand, the biodegradation degree was accelerated compared with the purely cross-linked when glutamic acid was added, while less influenced by glycin addition. By comparing the biodegradation degree of the scaffolds added with amino acids and their model compounds, i.e. adipic acid and hexane diamine, the key factor influencing the biological stability was further investigated. The results indicated that the crucial factor is dependent on the ratio of amino groups to carboxyl groups in the cross-linking system. At optimal ratio the lowest biodegradation degree is achieved. Scanning electron microscopy measurements prove that the three-dimensional structure of the scaffolds was largely preserved. Preliminary in vitro culture of fibroblasts in the collagen scaffold cross-linked with EDAC/NHS in the presence of lysine has shown that the original good cytocompatibility of collagen was retained.

A novel technique to introduce free amino groups onto polyester scaffolds via aminolyzing the ester groups with diamine has been developed recently. Positively charged chitosan was then deposited onto the aminolyzed poly(L-lactic acid) (PLLA) membrane surface in a layer-by-layer assembly manner using poly(styrene sulfonate, sodium salt) (PSS) as a negatively charged polyelectrolyte. The layer-by-layer deposition process of PSS and chitosan was monitored by UV-vis absorbance spectroscopy, energy transfer by fluorescence spectroscopy, and advancing contact angle measurements. The existed chitosan obviously improved the cytocompatibility of PLLA to human endothelial cells. The cell attachment, activity, and proliferation on the PLLA membranes assembled with three or five bilayers of PSS/chitosan with chitosan as the outermost layer were better than those with one bilayer of PSS/chitosan or the control PLLA. The cells also showed morphology of an elongated shape with abundant cytoplasm, and a confluent cell layer was reached after being cultured for 4 days. Measurement of von Willebrand factor secreted by these endothelial cells (ECs) verified the endothelial function. Hence, better ECs compatible PLLA were produced.

Amino groups were covalently introduced onto a polycaprolactone (PCL) surface by the reaction between 1,6-hexanediamine and the ester groups of PCL. The occurrence of the aminolysis and the introduction of free NH(2) groups were verified qualitatively by fluorescence spectroscopy, where rhodamine B isothiocyanate was employed to label NH(2) groups, and quantitatively by absorbance spectroscopy, where ninhydrin was used to react with NH(2) to generate a blue product. Due to the presence of deep pores on the PCL membrane, the aminolysis reaction could penetrate as deep as 50 microm to yield NH(2) density as high as 2 x 10(-7) mol/cm(2). By use of the NH(2) groups as active sites, biocompatible macromolecules such as gelatin, chitosan, or collagen were further immobilized on the aminolyzed PCL membrane via a cross-linking agent, glutaraldehyde. X-ray photoelectron spectroscopy (XPS) and surface wettability measurements confirmed the coupling of the biomacromolecules. The endothelial cell culture proved that the cytocompatibility of the aminolyzed PCL was improved slightly regardless of the NH(2) amount on the surface. After immobilization of the biomacromolecules, however, the cell attachment and proliferation ratios were obviously improved and the cells showed a similar morphology to those on tissue culture polystyrene. Measurement of the von Willebrand factor (vWF) secreted by these endothelial cells (ECs) verified the endothelial function. Hence, a better EC-compatible PCL was produced.

With the use of a grafting-coating method, three kinds of natural macromolecules, that is, gelatin, collagen, or chitosan, were immobilized on poly-L-lactic acid (PLLA) membrane surfaces with the goal of improving of cellular interactions. Attenuated total reflectance infrared spectroscopy (ATR-IR), x-ray photoelectron spectroscopy (XPS) and surface morphology analysis using scanning electronic microscopy (SEM) confirmed that the natural macromolecule layers adhered tightly to the hydrophobic PLLA membrane surfaces. Chondrocyte culture showed that the modified PLLA membranes had higher cell attachment, higher cell proliferation rate, and higher cell activity than the control PLLA membrane. Moreover, the chondrocytes were more spread out on the modified PLLA membranes than on the control PLLA membranes.

Horseradish peroxidase (HRP) was encapsulated in pre-formed polyelectrolyte multilayer (PEM) microcapsules by spontaneous deposition with remarkably improved stability and catalytic activity.

Chitosan and chitosan-amino acid derivatives were explored to engineer poly(D,L-lactic acid) (PDL-LA) as an extracellular matrix-like surface to promote cell adhesion and growth. Four kinds of chitosan-amino acid derivatives were prepared to mimic the carbohydrate moieties of cell matrix glycoprotein. The chitosan-amino acid derivatives were characterized by using Fourier transform infrared and ultraviolet spectra. The amino acid content on chitosan-amino acid derivatives was determined by using a ninhydrin-ultraviolet method. A new strategy, entrapment, was therefore used to modify the PDL-LA membrane with chitosan and chitosan-amino acid derivatives. The results of X-ray photoelectron spectroscopy, attenuated total reflectance-Fourier transform infrared, and contact angle confirmed that a stable thin film of chitosan and its derivatives can be entrapped on the surface of the PDL-LA membrane. From the results of chondrocyte cytocompatibility, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assays, and cell morphology, the chitosan-amino acid derivative modified PDL-LA membranes were shown to promote chondrogenesis. The novel surface treatment method combines the good mechanical property of PDL-LA with the good cytocompatibility of chitosan derivatives, which may have potential for tissue engineering.

The schematic structure model, materials selection and microstructure modulation are introduced for the design of artificial dermis equivalent. The artificial skin should also possess a bilayer structure that imitates then natural skin, i.e. the top layer functions as a temporary epidermis which is composed of polymer elastomer that is permeable for moisture but not for water, the bottom layer is the skin regeneration template employing collagen based sponge. In addition to collagen, polysaccharides like glycosaminoglycan is also used in the artificial dermis equivalent in order to simulate the natural extracellular matrix of skin and to modulate the degradation rate. The pore size and morphology of collagen porous membranes can be controlled by variation of the pH value, concentration and freezing temperature. Hence, the microstructure of the dermis equivalent can be optimized. The collagen based artificial dermis equivalent thus fabricated may be an option to skin graft in the clinical treatment of full skin injuries and ulcers.

Three types of stearyl poly(ethylene oxide) (SPEO) with Mn of 2,300, 6,000 and 12,000 were synthesized; accordingly, three types of amphiphilic coupling-polymer SPEO-MDI-SPEO (MSPEO) were prepared by the reactions with 4,4'-methylene diphenyl diisocyanate (MDI). As the surface-modifying additives (SMA), MSPEOs were coated onto the outer wall of the medical guiding catheters. Due to the lack of stability, when coated, MSPEO blended with the film building agent (FBA), poly(ether urethane) (PEL). The process of coating was performed with a lifter. With invariable speed, the PU guiding catheter was vertically dipped into the coating mixture of SMA-MSPEO and FBA-PEL. The surface analysis was carried out by ATR-FTIR and contact angle measurements. It was proved that the surface enrichment of PEO on water interface was much higher than that on air interface. Three kinds of static clotting time tests, PRT, PT and TT, as well as the static platelet adhesion experiment were performed. The results indicated that the coated surface could resist the blood coagulation effectively. In order to test the blood compatibility of the coated catheters under a shear of blood flow, the dynamic experiment was performed with a closed-loop tubular system under a shear rate of 1,500 s(-1). The blood regular testing was carried out on the samples taken out at six different times (0, 5,10, 20, 30 and 60 min). The results were ideal. Finally, the SMA-MSPEO was proved to be non-acute-toxic by LD50 test.

Microcapsules in the micrometer size range with walls of nanometer thickness are of both scientific and technological interest, since they can be employed as micro- and nano-containers. Liposomes represent one example, yet their general use is hampered due to limited stability and a low permeability for polar molecules. Microcapsules formed from polyelectrolytes offer some improvement, since they are permeable to small polar molecules and resistant to chemical and physical influences. Both types of closed films are, however, limited by their spherical shape which precludes producing capsules with anisotropic properties. Biological cells possess a wide variety of shapes and sizes, and, thus, using them as templates would allow the production of capsules with a wide range of morphologies. In the present study, human red blood cells (RBC) as well as Escherichia coli bacteria were used; these cells were fixed by glutardialdehyde prior to layer-by-layer (LbL) adsorption of polyelectrolytes. The growth of the layers was verified by electrophoresis and flow cytometry, with morphology investigated by atomic force and electron microscopy; the dissolution process of the biological template was followed by confocal laser scanning microscopy. The resulting microcapsules are exact copies of the biological template, exhibit elastic properties, and have permeabilities which can be controlled by experimental parameters; this method for microcapsule fabrication, thus, offers an important new approach for this area of biotechnology.

Cytocompatible polyurethane (PU) surface was prepared by photo-grafting 2-hydroxyethyl acrylate (HEA) onto the membrane surface. Graft polymerization was conducted by combining the use of the photo-oxidation and irradiation grafting. PU membrane was photo-oxidized to introduce the hydroperoxide groups onto the surface, then the membrane, immersed previously in monomer solution, was irradiated under UV light. The ATR-FTIR spectra, element spectroscopy for chemical analysis (ESCA), scanning electron microscopy (SEM) and water contact angle characterized the grafted copolymers and verified the occurrence of graft polymerization. The results showed that UV irradiation could realize the graft polymerization effectively and the grafting was confined within the surface layer. The grafted membrane showed minimal surface morphology. Human umbilical vein endothelial (HUVE) cells were seeded on the grafted surface. The performance of the surface in cell attachment and growth correlated with the oxygen content and mainly the carbonyl content on the surface. Cells were spread more extensively and grew faster on the surface with a higher oxygen content.

The molecular structure and physicochemical properties of nano- and colloidal materials have a big impact on their intracellular uptake and cell functions. Elucidation of the cellular uptake and cytotoxicity of the colloidal materials has been recognized recently as one of the paramount prerequisites for their biomedical applications. In this review, the influence of important parameters of colloidal biomaterials such as size, shape, chemical and mechanical properties on cellular uptake and its pathways is summarized. The intracellular distribution and transportation of the colloidal particles are then correlated with their physiochemical properties. The impacts on cytotoxicity and cell functions are also discussed. Finally, more attention is suggested to be paid on the basic understanding of cellular uptake and subsequent influence on cells, which is of great significance for the function implementations and safer applications of nano- and colloidal materials.

Specific implants to speedily regenerate critical-sized osteoporotic bone defects (COBDs) are a major clinical need. However, little progress in methods focusing on biological repair has been reported. We developed a biomimetic mineralization method to prepare trace element-codoped calcium phosphate (CaP) particles via hydrothermal treatment of modified simulated body fluid (SBF) with the addition of binary to quaternary trace elements. The morphology, structure, and composition of the particles were characterized by a combination of SEM, TEM, XRD, and FTIR measurements. The quantitative analysis shows that the dopant contents in the solid phase can be regulated by the trace ion concentrations in the aqueous medium. The conditioned cell culture medium from the quaternary Mg/Zn/Sr/Si-co-doped CaP (qCaP) could significantly enhance cell activity and osteogenic differentiation of ovariectomized rat-derived bone marrow mesenchymal stem cells. After injecting the qCaP-loaded chitosan/hyaluronic acid hydrogel into the COBDs, histology and computed tomography scanning revealed that the new bone regeneration was significantly enhanced, and the quantity of mature bone was substantially increased in the rats implanted with qCaP 12 weeks post-operatively in comparison with the defects filled with the CaP obtained from SBF. These results suggest that the biomimetic mineralization of the trace ion-added SBF allows the preparation of highly bioactive trace element-codoped CaP biomaterials and these materials are potential candidates for the biological repair of COBDs.

The novel micro–nano composite poly(sodium styrene sulfonate)/poly(allylamine hydrochloride) multilayer microcapsules containing in situ-formed 1-pyrenecarboxaldehyde nanorods were fabricated and characterized. Fluorescence properties and functions of the 1-pyrenecarboxaldehyde nanorods could be effectively manipulated by electron donor/acceptor interaction within the permeable polyelectrolyte shells. The microcapsule integrated with the nanorods may shed creative light on the design of novel micro–nano structures and materials for nanoscience, and biological and other advanced technologies.

The ability to prepare micro/nano materials with a stable structure is highly demanded in advanced sciences and technologies. In this work, poly(allylamine hydrochloride)-graft-pyrene microcapsules with a stable structure were obtained by glutaraldehyde crosslinking, which could then be employed as microreactors to protrude 1-pyrenecarboxaldehyde nanorods at low pH. The protrusion length of nanorods (NR) was tuned by the pH value, e.g. shorter NRs and a larger covering density or longer NRs and a lower covering density on the microcapsule surface at higher and lower pHs, respectively. The protruded pyrene NRs showed strong fluorescent emissions on the surface of microcapsules. The NRs could be repeatedly protruded by manipulating the reversible Schiff base formation and hydrolysis.

The widely used bovine serum albumin (BSA) nanoparticles (NPs) were modified with poly(allylamine hydrochloride) (PAH)/sodium poly(4-styrene sulfonate) (PSS) multilayers and aptamers to improve their suspension stability and targeting ability. For this to occur, a PAH-g-poly(ethylene glycol) (PAH-g-PEG–COOH) layer was further adsorbed onto the (PAH/PSS)2 multilayer-coated BSA NPs and used to covalently bond the aptamer AS1411, which is known to specifically bind the over-expressed nucleolin on cancer cell membranes. The PEGylated multilayer-coated BSA NPs showed good suspension stability in diverse media, in particular in a serum containing medium. By a mechanism of spontaneous deposition, doxorubicin (DOX) was effectively loaded into the pre-formed BSA NPs with both good encapsulation efficiency (98.6%) and loading percentage (9%). The loaded drug showed a pH-dependent release behaviour, i.e. faster at pH 5.5 than at pH 7.4. The multilayer coating did not significantly influence either both drug loading or release. In vitro cell culture demonstrated that the as-prepared BSA NPs could be specifically delivered to liver cancer cells, leading to higher cellular uptake and cytotoxicity.

Metallic and metal oxide nanoparticles (Nps) have a wide range of applications in various settings including household, cosmetics and chemical industries, as well as for coatings. Nevertheless, an in-depth study of the potential toxic effects of these Nps is still needed, in order to fulfill the mandatory requirement of ensuring the safety of workers, patients and the general public. In this study, Quick Cell colorimetric assays were used to evaluate the in vitro toxicity of different metal oxide Nps [Fe(II,III)Ox, TiOx, ZnO and CeO2] in several cell lines. The ZnO Nps were found to be highly toxic, with a lethal dose ≥100 μg/ml for all the cell lines studied. Western blot was also used to test the ability of the different Nps to activate the complement pathway. However, no activation of this cascade was observed when the Nps were added. In addition, the aggregation state and charge of the Nps in culture media was studied by dynamic light scattering (DLS) and measurement of zeta potential. Transmission Electron Microscopy was used to analyze Np uptake and localization at the cellular level.

High efficient loading and controlled release of low-molecular-weight drugs were achieved facilely by heat-shrinkage of polyelectrolyte multilayer capsules with a spontaneous deposition property. The capsules consisting of 5 bilayers of poly(diallyldimethylammonium chloride) and poly(styrene sulfonate) (PSS) were assembled on PSS-doped CaCO3 particles. After core removal, a part of PSS molecules were entrapped, which imparted the capsules with a strong ability to accumulate the positively charged drugs (the so called “spontaneous deposition” property). These capsules also showed temperature-dependent shrinkage, which became prominent above 50 °C, accompanying thickening of the capsule walls. After measuring the encapsulated free PSS and calculation of the net charge ratio in the capsule wall, the shrinkage mechanism was discussed. Using these features, doxorubicin hydrochloride was effectively loaded and its release profile was controlled by the incubation temperature. No detectable cytotoxicity was found for both the original and heat-treated capsules.

Hollow polymeric microcapsules with specific responses to mannose, fructose, glucose and its corresponding polysaccharide, dextran, at the physiological pH range were fabricated from Concanavalin A, a plant lectin, and glycogen, a polysaccharide, based on the lectin–carbohydrate interaction in a layer-by-layer fashion. The growth pattern of the multilayer film was largely influenced by the environmental pH condition. Hollow capsules with variable size and wall thickness were obtained by assembly on CaCO3 microparticles and subsequent template removal. The as-prepared capsules remained stable in the pH range 6–9 for 1 h and could survive 3 M urea treatment for at least 1 h. They could specifically respond to or even be destroyed by mannose, fructose, glucose and dextran (Mw 20 kDa and 40 kDa) at the physiological pH condition, but not by other types of carbohydrates such as lactose and galactose. The responses were dependent on both the molecular weight and the concentration of the carbohydrates regardless of the environmental pH condition (pH 6–9). Finally, the capsules were employed as delivery vehicles for poly(styrene-b-acrylic acid) (PS-b-PAA) micelles, which could be partly released under the triggering of glucose or dextran (Mw 20 kDa). With the combination of good stability and specific responsiveness to various carbohydrates, as well as the possibility of loading and releasing functional nano-objects, these microcapsules could be promising candidates as advanced drug delivery carriers and micro-reactors.

A dermal equivalent having a trilayered structure was designed by combining a silver nanoparticles incorporated chitosan film with a bilayer collagen-chitosan/silicon membrane dermal equivalent (BDE). The silver nanoparticles prepared at different conditions were characterized by UV-Vis and transmission electron microscopy (TEM). The macroscopic sharp and the microstructure of the trilayer dermal equivalent (TDE) were also studied. Then, the in vitro antibacterial property of TDE was evaluated by the antibacterial zone test. The effect of the incorporated silver nanoparticles on the resistance of wound infection was further studied by the in vivo animal test. The results prove that the silver nanoparticles incorporated TDE has a better antibacterial property, thus may be potentially applied to a broader field in skin repair such as full thickness defect and burn.

Numerous colloidal particles have been designed for cellular uptake and intracellular delivery in biological systems. The different kinds of colloidal particle systems and the interactions between the particles and cells are highlighted, with a focus on the parameters governing cellular uptake. A perspective of the research is also proposed.

The morphologies of biomimetic mineralized octacalcium phosphate (OCP) are extremely versatile. Numerous studies have addressed the gel phases, surfactants, polyelectrolytes and thin layers as convenient media to produce OCP nanostructures with various morphologies. However, hardly any studies have to date discussed the concept of controlling the structural evolution of these nanostructures by organic molecule mediation. This paper aims at opening the pathway to such OCP assemblies with levels of morphological expression and evolution from an aqueous solution consisting of highly diluted acidic macromolecule and amphiphilic small molecule as co-mediators. This approach differs in that the usual organic molecules are used to shape the single OCP morphology, and specifically establish an evolutionary relationship between the lamellar clusters and spherical aggregates. We present a possible model that gives qualitative description for the morphology of OCP grown in a variety of molar ratios between acidic macromolecule and small molecule surfactant. We believe that extension of the present approach to other biominerals and inorganic materials will prepare a large variety of highly flexible biomimetic nanostructures with elaborate superstructures across nanoscale to macroscopic dimensions.