Details of thermal behavior of spin-coated film of biodegradable poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer studied by principal component analysis-based two-dimensional (PCA2D) correlation spectroscopy

Principal component analysis-based two-dimensional (PCA2D) correlation spectroscopy was applied to the temperature-dependent infrared-reflection absorption (IRRAS) spectra of a spin-coated film of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(HB-co-HHx)) (HHx=7.2 mol%) copolymer. In asynchronous PCA2D correlation spectra, we clearly captured the existence of two components in the crystalline band of the CO stretching mode, well-ordered primary crystals observed at lower wavenumber and less ordered secondary crystals observed at higher wavenumber, which is not readily detectable in the original 1D spectra. Furthermore, the intensity changes of bands at 1298 and 1280 cm(-1) are significantly different in the temperature ranges below and above the transition temperature around 140 degrees C identified by the 2D first derivatives plot. The result further confirms that the sequence of intensity changes with increasing temperature is such that bands for less ordered crystalline components of P(HB-co-HHx) (HHx=7.2 mol%) are changing first at an earlier (i.e., lower temperature) stage.     

Improvement of mechanical properties of poly(dl-lactide) films by blending of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and poly(dl-lactide) (PDLLA) were blended at different ratios in an attempt to form a biomaterial with suitable properties for nerve regeneration. FT-IR and X-ray analysis showed that the blending of the PDLLA component did not alter the helical structure of PHBHHx, but did lead to a reduction of crystallinity. Differential scanning calorimetry (DSC) analysis indicated that the two polymers were immiscible in the melted state. The mechanical properties of certain composite films were more desirable than those of unblended PDLLA films. Blends consisting of PDLLA and PHBHHx at ratios of 2:1 and 1:2 exhibited a lower elastic modulus and a higher elongation at break compared to unblended PDLLA. ELISA results indicated that the amount of fibronectin adsorbed on composite films was much higher than the amount adsorbed on PDLLA film. The results of this study demonstrate the feasibility of using PDLLA/PHBHHx blended materials for biomedical applications.     

Crystallization behaviors and morphology of biodegradable poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

Crystallization behaviors and spherulitic morphology of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] with different 4-hydroxybutyrate (4HB) molar fraction were investigated by differential scanning calorimetry and polarized optical microscopy. Crystallization behaviors of P(3HB-co-4HB) are significantly affected by 4HB molar fraction. The melting temperature (T _m), glass transition temperature (T _g), and crystallinity (X _c) decrease with the increase of 4HB molar fraction. Banded spherulites are observed in poly (3-hydroxybutyrate) (PHB) and P(3HB-co-4HB) copolymers. The band spacing decreases with the increase of 4HB molar fraction. The morphology and growth rate of the spherulites strongly depend on 4HB molar fraction and the crystallization temperatures. The introduction of 4HB unit can inhibit the emergence of cracks in PHB spherulites.     

Improved mechanical property and biocompatibility of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) for blood vessel tissue engineering by blending with poly(propylene carbonate)

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and poly(propylene carbonate) (PPC) were blended by solvent casting method into films at various weight ratios in order to obtain materials with properties more suitable for blood vessel tissue engineering than pure PHBHHx alone. FTIR and XRD analysis indicated the crystal structure of PHBHHx was not altered but the crystallinity was reduced by the interfusion of PPC. Mechanical properties of the films were improved significantly by blending with PPC. A lower elastic modulus and a higher elongation at break were obtained with the increase of PPC content. Wettability, fibronectin adsorption and adhesion of rabbit aorta smooth muscle cells (RaSMCs) on blend films were similar to or better than that on PHBHHx film. All these results showed promises of PHBHHx/PPC blended materials as scaffold material for blood vessel tissue engineering.     

Thermal and mechanical properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) plasticized by biodegradable soybean oils

The effects of soybean oil (SO) and epoxidized soybean oil(ESO) as biodegradable plasticizers for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were studied using thermal and mechanical analyses. PHBV/SO and PHBV/ESO blends were prepared by evaporating solvent from blend solutions. The levels of additive in the blend varied from 5% to 30%. As a plasticizer for PHBV, ESO was more effective than SO in depression of the glass transition temperature as well as in increasing the elongation at break and the impact strength of the films with increasing levels of additive. Biodegradation of the plasticized PHBV films was carried out by accelerated compost method. The degradation rates of the blend films with SO or ESO were found to be faster than that of PHBV film. From the thermogravimetric analysis, it was found that the thermal reaction between the epoxide groups of ESO and PHBV fragments with carboxylic chain ends, occurred during the degradation of PHBV/ESO blends.     

Molecular architecture of poly[(R,S)-3-hydroxybutyrate-co-6-hydroxyhexanoate] and poly[(R,S)-3-hydroxybutyrate-co-(R,S)-2-hydroxyhexanoate] oligomers investigated by electrospray ionization ion-trap multistage mass spectrometry

A series of aliphatic copolyesters was obtained from (R,S)-beta-butyrolactone and two isomeric hydroxy acids, 6-hydroxyhexanoic and (R,S)-2-hydroxyhexanoic acids. The reactions were conducted in bulk without catalyst. Electrospray ionization tandem mass spectrometry (ESI-MSn) was used for molecular characterization of these copolyester oligomers. The mass spectra of the copolyesters studied have enabled identification of their molecular structures including chemical nature of the end groups (hydroxyl and carboxylate). The compositions of the oligocopolyesters and their sequence distributions were determined based on measurement of the relative intensities of the individual oligocopolyester peaks in the ESI mass spectra. The mass spectra (MS1) provided information on composition and sequence distribution, and small deviations from Bernoulli statistics were detected. The arrangement of co-monomer structural units along the oligopolyester chains was verified by MS/MS experiments and investigation of the fragmentation pathways.     

Isothermal crystallization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Isothermal crystallization behavior of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was investigated by means of differential scanning calorimetry and polarized optical microscopy (POM). The Avrami analysis can be used successfully to describe the isothermal crystallization kinetics of PHBV, which indicates that the Avrami exponent n=3 is good for all the temperatures investigated. The spherulitic growth rate, G, was determined by POM. The result shows that the G has a maximum value at about 353 K. Using the equilibrium melting temperature (448 K) determined by the Flory equation for melting point depression together with U∗=1500 cal mol⁻¹, T_∞=30 K and T_g=278 K, the nucleation parameter K_g was determined, which was found to be 3.14 ± 0.07 × 10⁵ (K²), lower than that for pure PHB. The surface-free energy σ=2.55×10⁻² J m⁻² and σ_e=2.70±0.06×10⁻² J m⁻² were estimated and the work of chain-folding (q=12.5±0.2 kJ mol⁻¹) was derived from σ_e, and found to be lower than that for PHB. This implies that the chains of PHBV are more flexible than that of PHB.     

Electrospray ion-trap multistage mass spectrometry for characterisation of co-monomer compositional distribution of bacterial poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) at the molecular level

We report an electrospray ionisation multistage mass spectrometry (ESI-MSn) method that utilises molecular mass information for determination of sequence distribution and chemical structure of mass-selected macromolecules of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biopolyester, PHBH. On the basis of ESI-MSn studies of PHBH oligomers obtained by partial alkaline depolymerisation of natural PHBH containing 13-14 mol% of hydroxyhexanoate (HH) units, the microstructure of this bacterial copolyester was assessed up to the level of 28 repeat units. The subtle structural details of the PHBH were evaluated based on sequencing of individual macromolecular ions thus showing the utility of this technique for the analysis of biological copolyester macromolecules. It was confirmed that both HH and hydroxybutyrate (HB) units of the PHBH copolymer are randomly distributed.     

Effects of metal ion-carbonyl interaction on miscibility and crystallization kinetic of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)/lightly ionized PBS

Poly(butylene succinate) (PBS) and PBS-based ionomers (PBSi) with 1.0 and 3.0 mol% sodium sulfonate ionic group were synthesized and blended with poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHB-HHx) via direct melt compounding. FT-IR analysis demonstrated that the sodium metal ion–carbonyl interaction between PHB-HHx and PBS ionomer increased in strength with the ionic group concentration. Both non-isothermal and isothermal analyses showed the crystallization rates of PHB-HHx to decrease as the strength of the sodium metal ion–carbonyl interaction increased. However, the constant value obtained for the Avrami exponent indicated that the presence of PBS ionomer did not interfere in any way with the nucleation mechanism or the geometry of the crystal growth of PHB-HHx. DMTA analysis confirmed that PBS ionomer reduced the crystallinity of PHB-HHx, and this phenomenon increased in proportion to the ionic group content. As the ionic group concentration increased, the sodium metal–carbonyl interaction between PHB-HHx and PBS ionomer became much stronger, resulting in the improvement of the miscibility for the blend. The interaction parameter obtained by analyzing the equilibrium melting temperature was negative for all bend systems, with the ionomer having ionic group content, displaying a more negative value. Based on the Lauritzen–Hoffman secondary nucleation theory, the regime of the PHB-HHx/PBS ionomer blend remained unchanged throughout the crystallization process. In addition, both the nucleation constant and surface free energy were found to decrease as both ionomer content and ionic group concentration increased.     

聚羟基丁酸酯共聚物超薄膜中向日葵晶形成的原子力显微镜观察

低维数下的高分子(如在二维薄膜或一维管道及孔洞中)的各种行为由于更加触及高分子的动力学、热力学本质而逐渐成为高分子科学研究的热点之一．实验表明,薄膜中聚合物的玻璃化转变温度、结晶动力学及形貌等与本体有很大的偏离．我们先前的研究结果表明,在基板的作用下,共混物薄膜的相形态会发生逆转;     

Rheo-optical FT-IR spectroscopy of poly(3-hydroxybutyrate)/poly(lactic acid) blend films

The orientation of poly(3-hydroxybutyrate) (PHB) and poly(lactic acid) (PLA) segments in PHB/PLA blend films cast from chloroform solutions with compositions PHB < PLA was studied during uniaxial elongation up to 250% strain at 50 °C by in-situ rheo-optical FT-IR spectroscopy. From the orientation functions of the ν(CO) bands of the blend components, it was derived that the PLA chains orient in the direction of elongation while the PHB chains orient perpendicular to the drawing direction. PHB homopolymer and PHB/PLA blend films with PHB > PLA compositions could only be oriented by cold drawing in ice water after quenching from the melt. The IR-dichroic effects of films drawn under these conditions indicate for both blend components a chain alignment parallel to the drawing direction.     

Macroporous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) matrices for cartilage tissue engineering

Polymer scaffold systems consisting of poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) were investigated for possible application as a matrix for the three-dimensional growth of chondrocyte culture. The PHBV scaffolds were fabricated by a compression moulding, thermal processing and salt particulate leaching method without using organic solvent. The porous structure of the scaffolds was investigated with an optical microscope (OM) and scanning electron microscope (SEM) and the porosity was calculated. Then, the chondrocytes were cultured on the PHBV scaffolds for lone time to investigate whether it can be applied to construct the cartilage tissue in vitro. The results showed that the chondrocytes maintained their activity, fully expressed their phenotype and produced the extracellular matrix after incubation in vitro on the scaffolds for 7 days. In addition, in the prolonged incubation time, the percent of chondrocytes in their nature round morphology increased with an increase in the incubation period and they could synthesize the type II collagen and cartilage-specific proteoglycans. All of these results showed that the PHBV scaffolds had the potential to be used as chondrocytes carrier for cartilage engineering.     

NMR analysis and triad sequence distributions of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Polyhydroxyalkanoates (PHAs) are considered promising “green” alternatives to synthetic polymers because they are bio-derived, biodegradable and biocompatible. The properties of bacterial PHA copolymers depend on their microstructures, which can be modified with the use of different fermentation processes and feed materials. Thus, it is desirable to have an improved testing method for the determination of PHA microstructures. In this work, a detailed NMR analysis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) microstructure was made. Previously only two of the hydroxyvalerate ¹³C NMR peaks have been assigned at the triad level. In this work, three of the ¹³C hydroxyvalerate peaks and two of the hydroxybutyrate peaks were found to be split into four peaks each due to comonomer sequence effects. Using eight copolymer samples with a wide compositional range, we were able to assign all these peaks to B-centered and V-centered triad sequences. Through curve deconvolution, the triad intensities were determined. These triad sequence intensities can then be analyzed via both the first-order Markovian and two-component Bernoullian models to obtain more in-depth information on copolymer composition and comonomer reactivities.     

Assessing the thermoformability of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/poly(acid lactic) blends compatibilized with diisocyanates

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a renewable alternative to conventional barrier packaging polymers due to its thermoplastic properties, biodegradability and gas barrier performance but its potential industrial applications are limited by its high price and difficult processability. A thorough study concerning the thermoforming ability of PHBV, and blends with poly(lactic acid) (PLA) incorporating three different diisocyanates as compatibilizers (hexamethylene diisocyanate, poly(hexamethylene) diisocyanate and 1,4-phenylene diisocyanate) is herein presented after component melt blending. A straightforward universal qualitative method is proposed to assess the thermoformability, based on a visual inspection of a thermoformed specimen and the ability to reproduce the mold shape, and the thermoforming window of the material. The results reveal a significant improvement in the thermoforming capacity and a widening of the thermoforming windows as the correct amounts of diisocyanates are incorporated. The barrier properties and the biodisintegrability of the blends was also studied, confirming a predictable slight decrease of the barrier performance when PLA is added, but without negatively affecting the disintegrability under composting conditions with respect to pristine PHBV.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by metabolically engineered Escherichia coli strains

Biosynthesis of polyhydroxyalkanoates (PHAs) consisting of 3-hydroxyalkanoates (3HAs) of 4 to 10 carbon atoms was examined in metabolically engineered Escherichia coli strains. When the fadA and/or fadB mutant E. coli strains harboring the plasmid containing the Pseudomonas sp. 61-3 phaC2 gene and the Ralstonia eutropha phaAB genes were cultured in Luria-Bertani (LB) medium supplemented with 2 g/L of sodium decanoate, all the recombinant E. coli strains synthesized PHAs consisting of C4, C6, C8, and C10 monomer units. The monomer composition of PHA was dependent on the E. coli strain used. When the fadA mutant E. coli was employed, PHA containing up to 63 mol% of 3-hydroyhexanoate was produced. In fadB and fadAB mutant E. coli strains, 3-hydroxybutyrate (3HB) was efficiently incorporated into PHA up to 86 mol%. Cultivation of recombinant fadA and/or fadB mutant E. coli strains in LB medium containing 10 g/L of sodium gluconate and 2 g/L of sodium decanoate resulted in the production of PHA copolymer containing a very high fraction of 3HB up to 95 mol%. Since the material properties of PHA copolymer consisting of a large fraction of 3HB and a small fraction of medium-chain-length 3HA are similar to those of low-density polyethylene, recombinant E. coli strains constructed in this study should be useful for the production of PHAs suitable for various commercial applications.     

Surface vibrational spectroscopy on shear-aligned poly(tetrafluoroethylene) films

Sum-frequency vibrational spectroscopy was used to obtain the first surface vibrational spectra of shear-deposited highly oriented poly(tetrafluoroethylene) (PTFE, Teflon) thin films. The surface PTFE chains appeared to lie along the shearing direction. Vibrational modes observed at 1142 and 1204 cm-1 were found to have the E1 symmetry, in support of some earlier analysis in the long-lasting controversy over the assignment of these modes.     

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Production in Recombinant Aeromonas hydrophila 4AK4 Harboring phbA,phbB and vgb Genes

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), a copolyester consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx), can be synthesized by Aeromonas hydrophila strain 4AK4 using long chain fatty acids as the carbon source. The wild type A. hydrophila 4AK4 accumulated PHBHHx consisting of 12-15 mol% 3HHx regardless of growth conditions. When phbA, phbB and vgb genes encoding β-ketothiolase, acetoacetyl-CoA reductase and vitreoscilla hemoglobin, respectively, were introduced together into A. hydrophila 4AK4, the recombinant strain grew to over 20 g/L cell dry weight (CDW) after 48 h of shake flask cultivation in co-substrates of dodecanoate and gluconate (weight ratio 1:1), and the CDW contained 50% PHBHHx consisting of 9 mol% 3HHx. Under similar conditions, the wild type strain produced only 12 g/L CDW containing 32% PHBHHx with 15 mol% 3HHx. In comparison, recombinant harboring phbA and phbB produced 35% PHBHHx with 9 mol% 3HHx in 15 g/L CDW under the same conditions. The obvious differences in terms of the cell growth and PHBHHx production were attributed to the expression of vgb in A. hydrophila 4AK4, which was clearly observed in carbon monoxide difference spectra. The expression of vgb in the recombinant not only improved cell growth and PHBHHx accumulation, but also increased the plasmid stability during cell growth, especially under low dissolved oxygen tension in fermentors. PHBHHx production could be further increased to over 60% of the CDW by the over expression of phaC and phaJ from Aeromonas caviae encoding PHBHHx synthase and (R)-specific enoyl-CoA hydratase, respectively. Over expression of phaC, phaJ and phaP, alone or in various combinations, also increased the 3HHx content of PHBHHx from 14-34%. The above results showed that A. hydrophila was amenable to genetic manipulation, and that these modifications could be exploited to produce compounds with different properties for commercial and research applications.     

Rheo-optical FT-IR spectroscopy of solid poly(acrylic acid) films

This paper describes experimental work where the mechanical behaviour of relatively dry, solid poly(acrylic acid) films at room temperature was correlated with changes in hydrogen-bonding in the polymer. Hydrogen-bonding between the carbonyl and the hydroxyl groups was followed by FT-IR spectroscopy while the films were stretched uniaxially. Limited mobility of the polymer below its T_g led to small but detectable changes in bonding. A characteristic strain-hardening behaviour was observed if no bound water was detected in the samples.