Wound pH-Responsive Sustained Release of Therapeutics from a Poly(NIPAAm-co-AAc) Hydrogel

Wound pH strongly influences residence time and activity of various growth factors during wound healing. Hence, a pH-responsive sustained release growth factor delivery system could be beneficial for effective treatment of wound. In this context, an effort was made to evaluate the potential of a poly(N-isopropylacrylamide-co-acrylic acid) hydrogel as pH-sensitive sustained release system for wound-pH-dependent therapeutics delivery. The polymer was synthesized via radical copolymerization and influence of pH on lower critical solution temperature (LCST), microarchitechture and swelling of the hydrogel was evaluated. Results showed a pH-dependent variation in the physical properties of the hydrogel within the wound pH range. Fluorescence recovery after photobleaching (FRAP) analysis endorsed a pH dependent restricted diffusion of the BSA in the hydrogel. Later, release of bovine serum albumin (BSA), vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) (each 5%, w/v) from the hydrogel within the range of wound pH (pH 6.7–7.9) were examined. Analysis showed non-Fickian release of therapeutics from the hydrogel with a significant variation in release rate and cumulative release with the increase in pH. Retention of the bioactivity of the released EGF was confirmed by studying murine dermal fibroblast cell proliferation in vitro. Finally, a growth factor (EGF or VEGF)-loaded hydrogel was applied on a murine excisional wound model and showed augmentation of wound healing in comparison to conventional sustained release growth factor therapy.     

Albumin and urea production by hepatocytes cultured on extracellular matrix proteins-conjugated poly (vinyl alcohol) membranes

Production of albumin and urea by mouse hepatocytes on poly(vinylalcohol-co-ethylamine) (PVA-EA) membranes containing immobilized extracellular matrix (ECM) proteins was investigated for 7 days. The amount of ECM proteins (collagen, vitronectin and laminin) immobilized on PVA-EA and PVA-ECM membranes was determined to range from 1.09 μg/cm² to 1.60 μg/cm². Hepatocytes cultured on PVA-ECM membranes in serum-free media showed higher albumin production than those cultured on PVA-EA membranes after a 7-day incubation under the conditions in this study. Urea production by hepatocytes on PVA-ECM membranes was also determined to be higher than that on PVA-EA membranes up until day 5 of incubation in serum-free media, whereas no difference of urea production by hepatocytes on different PVA-ECM membranes and PVA-EA membranes was observed at 7 days of incubation. The effect of ECM proteins in PVA-ECM membranes on hepatocyte function (such as albumin and urea production) was observed in hepatocytes cultured in serum-free media up to day 5 of incubation. The ECM proteins immobilized on the PVA-ECM membranes contributed not only to the long-term stable production of albumin and urea by hepatocytes, but also the improved survival (viability) of hepatocytes on PVA-ECM membranes.     

Mechanical properties and biocompatibility of soluble eggshell membrane protein/poly(vinyl alcohol) blend films

Poly(vinyl alcohol) (PVA) has been blended with soluble eggshell membrane protein (SEP) to improve the mechanical properties of SEP film that is brittle. Tensile strength and elongation-at-break increase with increasing amount of PVA. When the SEP/PVA proportion is 1:1, a strong and flexible film is obtained, having a tensile strength of 22.7 MPa and elongation-at-break of 106%. Although scanning electron microscopy observation of the freeze-fractured cross-section shows microphase separation, interaction between SEP and PVA exists, as revealed by FT-IR. NIH3T3 cell culture demonstrates that SEP/PVA blend films with up to 50% of PVA show biocompatibility comparable to pure SEP film.     

The Effect of Hydrolysis Degree on the Properties of Antibacterial Polymeric Films Based on Poly(vinyl alcohol) and Zinc Sulphate for Biomedical Applications

Fully and partially hydrolyzed poly(vinyl alcohol) (PVA) was modified with zinc sulphate in the concentration range from 0 to 9 wt% of recalculated zinc content using the solvent cast technique. The resulting polymeric films were characterized by optical microscopy, stress–strain analysis, differential scanning calorimetry and Fourier transform infrared spectroscopy (FT-IR–ATR). In addition, agar diffusion test and dilution and spread plate technique were used for determination of antibacterial properties of the films against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) bacterial strains. A mathematical model was applied on the measured data and parameters characterizing the antibacterial efficiency of the material were calculated and discussed. The results revealed that the PVA hydrolysis degree can play an important role in all studied properties, including antibacterial activity of the all PVA-based materials.     

In vivo degradation and biocompatibility of a new class of alternate poly(ester-anhydrides) based on aliphatic and aromatic diacids

The degradation, tissue compatibility, and toxicology of a novel class of alternate poly(ester-anhydrides) were assessed in rats. It was observed that the degradation rate of the polymers in vivo was slower than that in vitro. In addition, erosion and intact zone were observed for all the polymers. IR and SEM analysis of the outer erosion and inner intact zone revealed that the outer zone degraded more rapidly than the inner zone. Such results were similar to that in vitro. All the studied poly(ester-anhydrides) produced mild inflammatory reactions and tissue encapsulation by layers of fibroblastic cells in vivo. Observation of liver and kidney tissue by light microscopy suggested the hydrolytic products of the studied poly(ester-anhydrides) had no harmful effects on the normal tissue/organs. In addition, the polymer and the breakdown products were found to be non-mutagenic by examination of micronucleus in bone marrow.     

Amoxicillin-Loaded Sponges Made of Collagen and Poly[(methyl vinyl ether)-co-(maleic anhydride)] for Root Canal Treatment: Preparation,Characterization and In Vitro Cell Compatibility

The difficulty of eliminating Enterococcus faecalis and other bacteria infecting dental root canals makes it desirable to develop formulations capable of sustained release of antibiotics within the canal. With this function in view, in this work we compared the mechanical, drug release and biocompatibility properties of amoxicillin-loaded collagen (CL) and CL complexed with poly[(methyl vinyl ether)-co-(maleic anhydride)] (PVMMA), with or without glutaraldehyde (GTA) or the natural product genipin (GN) as cross-linker. Collagen was not denatured by complexation with PVMMA. Only CL–PVMMA–GN sponges did not disintegrate during 7 days exposure to cell culture medium (un-cross-linked CL disintegrated within 24 h and un-cross-linked CL–PVMMA within 4 days), and CL–PVMMA–GN sponges also exhibited the most appropriate combination of mechanical properties (hardness, modulus of deformability and plasticity). CL–PVMMA–GN sponges absorbed aqueous medium faster than other cross-linked formulations, but their maximum uptake was less; and drug release from CL–PVMMA–GN sponges tended to be faster than from any other, except un-cross-linked CL–PVMMA, maximum release taking about 4 days. No formulation significantly altered the viability of L929 fibroblast-like mouse connective tissue cells, but cells growing on sponges showed signs of non-adherence. It is concluded that genipin-cross-linked CL–PVMMA sponges merit further investigation as antibiotics vehicles and aids to tissue regeneration in the dental root canal.     

聚乙烯醇水凝胶及其复合物的研究与在人工软骨替代材料上的应用

聚乙烯醇水凝胶是一种具有良好生物相容性和力学性能的高弹性材料。本文介绍了聚乙烯醇水凝胶的成型方法，复合改性技术的研究进展，着重对聚乙烯醇水凝胶作为替代材料在关节软骨损伤修复中的研究现状和存在问题进行了综述，并概述了其应用前景和发展方向。     

In Vivo Validation of Biological Responses of bFGF Released from Microspheres Formulated by Blending Poly-Lactide-co-Glycolide and Poly(ethylene glycol)-Grafted-Chitosan in Hamster Cheek Pouch Microcirculatory Models

Microspheres formulated from blending poly(lactide-co-glycolide) (PLGA) and poly(ethylene glycol)-grafted-chitosan (PEG-g-CHN), using a modified in-emulsion-solvent-evaporation method, were investigated for the delivery of protein. A model protein, bovine serum albumin (BSA), was incorporated into the PLGA/PEG-g-CHN microspheres and both initial burst and release kinetics could be modulated by varying the PEG-g-CHN content. Basic fibroblast growth factor (bFGF) was formulated into the microspheres containing 5% PEG-g-CHN and the bFGF contents in the releasates were determined by a receptor-based ELISA with their in vitro bioactivities validated by fibroblast cell culture. The in vivo effect of the bFGF microspheres formulation was evaluated in a hamster cheek pouch model using a 7 day exposure (e.g., before significant vascular remodeling was expected). Using intravital microscopy, the tissue showed no evidence of inflammation with any formulation; deliberate activation of a preconditioning response linked to inflammation was attenuated by BSA microspheres alone. Vasoactive responses (receptor-dependant and independent constriction and dilation) linked to nitric oxide were attenuated, and constriction to endothelin was enhanced in bFGF and not BSA containing microspheres. PLGA/PEG-g-CHN blended microspheres were also demonstrated to be non-inflammatory and non-thrombogenic in vivo by observing the vascular changes in the cheek pouch. In conclusion, the addition of PEG-g-CHN to PLGA microspheres can serve as a sustained delivery vehicle for bFGF and the released protein provides vasoactive changes consistent with chronic bFGF exposure.     

Swelling Behaviors of Poly(N‐isopropylacrylamide) Nanosized Hydrogel Particles/Poly(vinyl alcohol)/Water Systems: Effect of the Degree of Hydrolysis of PVA

Using poly(vinyl alcohol) as a drug model, the swelling behaviors of thermosensitive poly(N‐isopropylacrylamide) gel with various concentrations of PVA are investigated. The swelling ratio in PVA9000 is larger than that of PVA89000 at the same composition because the fully hydrolyzed PVA acts as a steric hindrance. A lattice‐based molecular thermodynamic model is used to obtain interaction parameters and then directly applied to describe the swelling behaviors of the gel. Using only one adjustable parameter, the PVA selectivity can be theoretically predicted and the ternary phase diagrams of PNIPA/water/PVA system can be generated. Lastly, the calculated results are compared with the swelling data for PNIPA/PVA copolymer gel, and are found to be in good agreement with the proposed model.

     

Application of MS-Based Proteomics to Study Serum Protein Adsorption/Absorption and Complement C3 Activation on Poly(ethylene glycol) Hydrogels

Although the interaction between cells and poly(ethylene glycol) (PEG) hydrogels is well documented, there lacks a thorough investigation into the adsorption of blood proteins on these surfaces which dictates the observed cellular and in vivo host response. Thus, a clear understanding of how surface-bound proteins mediate the unique biological property of PEG hydrogels is fundamentally important. The information obtained will also provide insights into future biomaterial design. In this study, several mass-spectrometrybased proteomic tools coupled with complementary immunoassays were employed to survey the complex surface-bound serum proteome. The adsorption of vitronectin, thrombin, fibrinogen and complement component C3 was significantly lower on PEG hydrogels than on tissue culture polystyrene (TCPS). Although PEG hydrogels mediated lower C3 adsorption than TCPS, the extent of C3 activation between the two surfaces was comparable. Adherent monocyte density was also significantly lower on PEG hydrogels as compared to TCPS. Taken together, these results support the critical role of the complement C3 in mediating monocyte adhesion on biomaterials. Thus we conclude that the biocompatibility of PEG hydrogels both in vitro and in vivo can be partly contributed to their limited C3 interaction and monocyte activity.     

聚乳酸/聚乙二醇-聚乳酸新型亲水支架的制备与研究

利用热致相分离/粒子沥滤结合法,制备了聚乳酸(PDLLA)以及聚乳酸与聚乙二醇-聚乳酸共聚物(PELA)复合的PDLLA/PELA组织工程支架。讨论了聚乙二醇(PEG)分子量、PELA含量及组成比对于支架内部结构、力学性能、降解行为和细胞毒性的影响。结果表明,热致相分离/粒子沥滤结合法制备的支架,具有100~250μm大孔与5~40μm小孔兼备的特殊内部结构,PEG含量愈高、PEG分子量愈小,支架的孔隙率愈大。PDLLA/PELA比率的减小,PEG/PLA比率的增大会引起支架力学性能的下降和降解的加速。材料无细胞毒性。当支架中PDLLA/PELA为3∶1,PEG 5000/PLA为25∶75时,其内部孔形态最为理想。     

Synthesis,Characterization and Properties of a Physically and Chemically Gelling Polymer System Using Poly(NIPAAm-co-HEMA-acrylate) and Poly(NIPAAm-co-cysteamine)

The aim of this work was to develop a simultaneous physically and chemically gelling system using NIPAAm co-polymers. The in situ polymer gel was obtained by synthesizing poly(NIPAAm-co-HEMAacrylate) and poly(NIPAAm-co-cysteamine) through free radical polymerization and further nucleophilic substitution. The purpose of the dual gelation is that physical gelation would take place at higher temperatures as the NIPAAm chains associate, while chemical gelation would occur through a Michael-type addition reaction, resulting in a cross-link forming through a nucleophilic attack of the thiolate on the acrylate. The structure of each co-polymer was then verified using ¹H-NMR and FT-IR spectroscopy. The corresponding lower critical solution temperature and phase transition behavior of each co-polymer was analyzed through cloud point and DSC, while mechanical properties were investigated through rheology. Swelling behavior was also monitored at different temperatures. The resulting polymer system demonstrated properties compatible with physiological conditions, forming a gel at pH 7.4 and at temperatures near body temperature. The hydrogel also showed reduced viscoelastic flow at low frequency stress, and increased strength than purely physical or chemical gels. Swelling behavior was determined to be temperature-dependent; however, no difference was observed in swelling percent beyond 48 h. Having the ability to alter these co-polymers through various synthesis parameters and techniques, this hydrogel can potentially be used as an injectable, waterborne gelling material for biomedical applications such as endovascular embolization.     

生物降解材料聚乳酸－聚乙二醇共聚物体内外降解

本文对ＤＬ－聚乳酸－聚乙二醇共聚物（ＰＥＬＡ）体内外降解进行了研究。采用凝胶渗透色层法（ＧＰＣ）测定分子量，扫描电镜（ＳＥＭ）观察。ＰＥＬＡ在最初第５天出现降解，２月时分子量丧失体内外分别为９７．２８％和９２．０％，而重量仅丧失８％。对其原因进行了讨论，同时将ＰＥＬＡ与ＤＬ－聚乳酸（ＰＬＡ）进行了比较，探讨分析了二者的降解机理。结果表明ＰＥＬＡ是一种新型生物降解材料。     

沉淀法原位复合聚乙烯醇(PVA)/羟基磷灰石(HA)水凝胶的结构与性能研究

研究了制备聚乙烯醇(PVA)，羟基磷灰石(HA)复合水凝胶的沉淀法原位复合技术，对该法制备的复合水凝胶的力学强度、结晶性能和微观形貌进行了分析，并与物理共混法加以比较。结果表明，沉淀法原位复合技术可在PVA水凝胶基体中合成得到粒度细，分散好的晶相HA陶瓷微粒，复合后水凝胶的结晶度和拉伸强度比之基体试样均有大幅度提高。     

Synthesis and Characterization of a Temperature-Responsive Biocompatible Poly(N-vinylcaprolactam) Cryogel: a Step Towards Designing a Novel Cell Scaffold

A poly(N-vinylcaprolactam) (PVCl) cryogel and poly(N-vinylcaprolactam)-co-gelatin interpenetrating cryogel network were synthesized and characterized with respect to physical and biological properties. The PVCl cryogel was synthesized in 5% dimethyl sulfoxide (DMSO) containing aqueous medium and PVCl-co-gelatin interpenetrating cryogel network was synthesized in water as solvent. Both these cryogel networks have good physical morphology as confirmed by scanning electron microscopy. The porosity of these cryogels were characterized by various methods like, adsorption of water and cyclohexane and confirmed by analysis on mercury porosimeter and nitrogen adsorption studies. The porosity of PVCl and PVCl-co-gelatin cryogels was 96% and 98%, respectively, and the permeability of the two types of cryogels was 1.01 × 10^?12 m⁴/Ns and 1.66 × 10^?12 m⁴/Ns, respectively. The effective diffusion coefficients (D _eff) of bovine serum albumin (BSA) in PVCl cryogel and PVCl-co-gelatin cryogel were 3.5 × 10^?7 cm²/s and 3.4 × 10^?7 cm²/s, respectively. These materials were further characterized to demonstrate its interaction with biological system. The blood compatibility studies showed minimal hemolysis (4–6%) caused by these materials and a very low adsorption of BSA (0.001–0.002 mg/g dry scaffold). However, the fetal bovine serum (FBS) adsorption studies demonstrate the protein binding at 37°C. Furthermore, cytotoxicity test and the fibroblast cell adhesion studies showed the potential of these PVCl-based cryogels for suitable biomaterial applications.     

Synthesis and In Vitro Biocompatibility Assessment of a Poly(amic acid) Derived from Ethylenediaminetetraacetic Dianhydride

Poly(amic acid) (PAA) derived from ethylenediaminetetracetic dianhydride shows great potential as a biomaterial suitable for biomedical applications. To evaluate this polymer class further, in vitro cell toxicity (WST-1/ECS, ELISA based) and cell compatibility (cell adhesion and cell proliferation) tests were conducted to establish structure–toxicity relationships. PAAs with a number-average molecular weight ranging between 100 to 200 kg/mol were synthesized at 37°C after 24 h. Porcine radial artery cells (RACs) and descending aorta endothelial cells (ECs) were seeded independently in a 96-well cell culture plate at a cell density of 5000 cells/cm² to observe toxic effects. Similarly, RACs and ECs were seeded independently onto PAA coated and uncoated cover slips at a cell density of 7000 cells/cm² to observe growth patterns. Our results showed no toxicity after 96 h of incubation and in addition, both RACs and ECs adhered and proliferated on the PAA films, preserving their phenotype during this time. The tested synthetic material seems promising as a future biomaterial and should elicit a desired cellular response upon implantation.     

Preparation of poly(vinyl alcohol)/DNA hydrogels via hydrogen bonds formed on ultra-high pressurization and controlled release of DNA from the hydrogels for gene delivery

Poly(vinyl alcohol) (PVA) hydrogels interacting with DNA mediated by hydrogen bonds (PVA/DNA hydrogel) were developed using ultra-high pressure (UHP) technology. The goal was to create a new method of gene delivery by controlled release of DNA. Mixed solutions of DNA and PVA at various concentrations were pressurized at 10 000 atmospheres at 37°C for 10 min. PVA/DNA hydrogels with good formability were produced at PVA concentrations of more than 5% w/v. The presence of DNA in the obtained hydrogels was confirmed by spectroscopic analysis and nucleic acid dye staining. DNA release from the hydrogels was investigated using PVA/DNA hydrogel samples of 5% and 10% w/v formed by UHP treatment or by conventional freeze–thaw methods. The DNA release curves from both types of samples showed a rapid phase in the initial 15 h followed by a sustained release phase. However, there was a difference in the amount of DNA released. Less DNA was released by the pressurized hydrogels than by the freeze–thaw hydrogels. Also, the cumulative amount of DNA released decreased as the PVA content in the hydrogels increased. These results indicate that DNA release from the hydrogels can be modulated by changing the preparation method and the PVA content. Furthermore, it was demonstrated that DNA release could be controlled by varying the amount and duration of pressurizing used to form the hydrogels. Intact fractions of plasmid DNA released from the hydrogels were separated by agarose gel electrophoretic analysis. These results suggest that, using controlled release, DNA from PVA/DNA hydrogels formed by UHP treatment can be transfected into cells.