PHBV Research Articles

Effect of Additives on Characteristics of Poly(3-Hydroxybutyrate) Microspheres

Poly(3-hydroxybutyrate) (PHB) based microspheres were prepared via double emulsion solvent evaporation using polyethylene glycol (PEG), poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), polylactide (PLA), poly(dl-lactic-co-glycolic acid) (PLGA) or chitosan (CTS) as the additive of wall polymers. It was found that additives had distinct effect on the properties of microspheres, such as the yield, drug loading, average diameter, crystallization states microstructure and surface morphological characters. PHB based microspheres using PEG as the additive had the lowest yield, the smallest average diameter, and the highest drug loading which reached 12.2% thereinto. At the same time it had the lowest crystallinity of PHB, and the diameter of the crystal particles was only 11.44 nm. It was feasible to prepare PHB based microspheres using PEG and PHBV as additives, which had relatively high protein loading but different microstructures and surface morphologies, and they were anticipated to have a good effect of controlled release.

Production of Polyhydroxybutyrate (Bioplastic) and its Biodegradation by Pseudomonas Lemoignei and Aspergillus Niger

The biodegradation of polyhydroxybutyrate (PHB) and its copolymer polyhydroxy butyrate‐co‐hydroxyvalerate (PHB‐co‐HV) was studied. Bacterial as well as fungal isolates were isolated from the local industrial ecosystem. All these isolates were tested for the degradation of the above polymers in assay agar medium as well in liquid cultures. The culture biomass and the clear zone around the colonies were measured to evaluate the activity of these isolates. In all, the fungal isolates were found to degrade these polymers more rapidly when compared to bacteria, due to their versatile depolymerase activities.

Effects of Wollastonite on Proliferation and Differentiation of Human Bone Marrow-derived Stromal Cells in PHBV/Wollastonite Composite Scaffolds

In this study, the effects of wollastonite on proliferation and differentiation of human bone marrow-derived stromal cells (hBMSCs) have been investigated based on a polyhydroxybutyrate-co-hydroxyvalerate (PHBV)/ wollastonite (W) composite scaffolds system. Cell morphology, proliferation, and differentiation were measured. The results showed that the incorporation of wollastonite benefited hBMSCs adhesion, proliferation, and differentiation rate. In addition, an increase of proliferation and differentiation rate was observed when the wollastonite content in the PHBV/W composite scaffolds increased from 10 to 20 wt%. Based on our previous studies on PHBV/W composite discs, the differentiation measurements in this paper further proved that the wollastonite itself can stimulate the hBMSCs to differentiate toward osteoblasts without any osteogenic medium, and the ionic products (Ca and Si) released from wollastonite might contribute to this advantage. It is also suggested that the osteogenic differentiation of the hBMSCs can be affected by adjusting the wollastonite content in the composite scaffolds.

Biodegradation Of Polyhydroxyalkanoates

The biodegradation of Polyhydroxybutyrate (PHB) and its copolymer Polyhydroxybutyrate-co-hydroxyvalerate (PHB-co-HV) was studied. Bacterial as well as fungal isolates were isolated from 9 different ecosystems. All these isolates were tested for the degradation of the above polymers in assay agar medium as well as in liquid cultures. The culture biomass and the clear zone around the colonies were measured to evaluate the activity of these isolates. In all, the fungal isolates were found to degrade these polymers more rapidly when compared to bacteria, due to their versatile depolymerase activities.

In vitro biocompatibility assessment of PHBV/Wollastonite composites

Biodegradable and biocompatible materials are the basis for tissue engineering. As an initial step for developing bone tissue engineering scaffolds, the in vitro biocompatibility of degradable and bioactive composites consisting of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and wollastonite (W) was studied by culturing osteoblasts on the PHBV/W substrates, and the cell adhesion, morphology, proliferation, and alkaline phosphatase (ALP) activity were evaluated. The results showed that the incorporation of wollastonite benefited osteoblasts adhesion and the osteoblasts cultured on the PHBV/W composite substrates spread better as compared to those on the pure PHBV after culturing for 3 h. In the prolonged incubation time, the osteoblasts cultured on the PHBV/W composite substrates revealed a higher proliferation and differentiation rate than those on the pure PHBV substrates. In addition, an increase of proliferation and differentiation rate was observed when the wollastonite content in the PHBV/W composites increased from 10 to 20 wt%. All of the results showed that the addition of wollastonite into PHBV could stimulate osteoblasts to proliferate and differentiate and the PHBV/W composites with wollastonite up to 20 wt% were more compatible than the pure PHBV materials for bone repair and bone tissue engineering.

Fabrication, characterization and long-term in vitro release of hydrophilic drug using PHBV/HA composite microspheres

In this paper, we fabricated polyhydroxybutyrate-co-hydroxyvalerate (PHBV)/Hydroxyapatite (HA) composite microspheres as a long-term drug delivery system. The characterization and in vitro release properties of the system were investigated. It was observed that the PHBV/HA composite microspheres showed very low initial burst that could be neglected, owing to the high affinity and absorbability of nano-HA particles, and the sustained release lasted more than 10 weeks. The in vitro release rate was controlled by diffusion rate of drugs from polymer matrices, and the release profile can be expressed by the Higuchi equation. From our work, it indicated that PHBV/HA composite microsphere could be a promising long-term drug release system.

Superhydrophobic surface directly created by electrospinning based on hydrophilic material

We describe a method to form hydrophobic surfaces using PHBV (Poly (hydroxybutyrate-co-hydroxyvalerate))—a kind of intrinsically hydrophilic material. The concentration of polymer solutions was varied to control the surface morphology and resultant wetting property. The as-prepared films were characterized by micro-scale valley-and-hill structure, which was formed by aggregating of electrospun beads. The bead morphology changed from smooth to porous and popcorn-like with decreased concentrations. The shape of water droplet on these surfaces had contact angles ranging from 110.7 to 158.1°, with a maximum standard deviation of 2.5°. It was found that both the micro and nanostructure were important to create a superhydrophobic surface.

Evaluation of Biological Responses of UMR-106 Cells to Porous PHBV Matrix

ABSTRACTThis paper compared the results of osteoblast-like UMR-106 cells response to 3-D biodegradable porous with nonporous PHBV (poly (3-hydroxybutyrate-co-3-hydroxyvalerate) films. Nonporous PHBV films were prepared by solvent casting and evaporation. Porous PHBV films were prepared by solute leaching of salt/PHBV cast film. Sieved sodium chloride (∼150 μm) was used to create a matrix with high porosity and then leached out by the solvent. Thermo gravimetric analysis showed that over 99% of the salts were leached out by the solvent. Osteoblast-like UMR-106 cells were seeded onto the nonporous and porous PHBV films, respectively. After 6 and 10 days of incubation, growth rate of UMR-106 cells on porous film was higher than on nonporous film. The viable cell proliferation on the porous and nonporous PHBV film was quantified by (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) (MTS) assay. The results of this study suggest that the UMR-106 cells proliferate more on the porous PHBV matrix and porous PHBV films are a promising material for bone tissue engineering applications.

골 생체조직공학을 위한 복합 세라믹 골 지지체의 제조와 생체역학적 특성

Novel porous composite ceramic bone scaffolds composed of biodegradable PHBV(polyhydroxybutyrate-co-hydroxyvalerate) and TA(toothapatite) have been fabricated for bone tissue engineering by a modified solvent casting and particulate leach-ing method with salt-contained heat compression technique. The results of this study suggest that the PHBV-TA composite scaffold, especially the scaffold containing 30 weight of TA may be a good candidate far bone tissue engineering of non-load bearing area in oral and maxillofacial region.

Reduction of Surgery-Induced Peritoneal Adhesions by Continuous Release of Streptokinase from a Drug Delivery System

Postoperative abdominal adhesions may lead to intestinal obstruction and infertility. The effect of continuous release of streptokinase to the peritoneal cavity on postoperative adhesions was examined under experimental conditions. Peritoneal adhesions were induced in rats and the animals were further treated by intraperitoneal administration of streptokinase solution, polyhydroxybutyrate-co-hydroxyvalerate (PHBV) membrane alone and streptokinase loaded PHBV membrane and compared to sham operated and untreated groups. Formation of adhesions was evaluated by quantitative macroscopic grading, histopathologically with light microscopy, on the following week. Streptokinase loaded PHBV prevented postoperative adhesion formation in 90% of the cases. PHBV membrane alone also reduced the severity of adhesions due to its anti-adhesive properties. Histopathological examination revealed limited foreign body reaction due to PHBV. Continuous streptokinase activity in the peritoneal cavity during early post-surgical period prevents postoperative adhesion.

The synthesis of short- and medium-chain-length poly(hydroxyalkanoate) mixtures from glucose- or alkanoic acid-grown Pseudomonas oleovorans.

Pseudomonas oleovorans NRRL B-778 accumulated mixtures of poly-3-hydroxybutyrate (PHB) and medium-chain-length poly(hydroxyalkanoates) (mcl-PHAs) when grown on glucose, octanoic acid or oleic acid, whereas growth on nonanoic acid or undecanoic acid resulted in copolymers of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-co-HV). Acetone fractionation verified the presence of PHB/mcl-PHA mixtures. The acetone-insoluble (AIS) fractions of the polymers derived from glucose (PHA-glucose), octanoic acid (PHA-octanoic) and oleic acid (PHA-oleic) were exclusively PHB while the acetone-soluble (AS) fractions contained mcl-PHA composed of differing ratios of 3-hydroxy-acid monomer units, which ranged in chain length from 6 to 14 carbon atoms. In contrast, both the AIS and AS fractions from the polymers derived from nonanoic acid (PHA-nonanoic) and undecanoic acid (PHA-undecanoic) were composed of comparable ratios of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). The unfractionated PHA-glucose, PHA-octanoic and PHA-oleic polymers had melting temperatures (Tm) between 177 and 179 degrees C, enthalpies of fusion (AHf) of 20 cal/g and glasstransition temperatures (Tg) of 3-4 degrees C. This was due to the large PHB content in the polymer mixtures. On the other hand, the PHA-nonanoic and PHA-undecanoic polymers had thermal properties that supported their copolymer nature. In both cases, the Tm values were 161 degrees C, deltaHf values were 7 cal/g and Tg values were - 3 degrees C.

The synthesis of short- and medium-chain-length poly(hydroxyalkanoate) mixtures from glucose- or alkanoic acid-grown Pseudomonas oleovorans

NRRL B-778 accumulated mixtures of poly-3-hydroxybutyrate (PHB) and medium-chain-length poly(hydroxyalkanoates) (mcl-PHAs) when grown on glucose, octanoic acid or oleic acid, whereas growth on nonanoic acid or undecanoic acid resulted in copolymers of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-co-HV). Acetone fractionation verified the presence of PHB/mcl-PHA mixtures. The acetone-insoluble (AIS) fractions of the polymers derived from glucose (PHA-glucose), octanoic acid (PHA-octanoic) and oleic acid (PHA-oleic) were exclusively PHB while the acetone-soluble (AS) fractions contained mcl-PHA composed of differing ratios of 3-hydroxy-acid monomer units, which ranged in chain length from 6 to 14 carbon atoms. In contrast, both the AIS and AS fractions from the polymers derived from nonanoic acid (PHA-nonanoic) and undecanoic acid (PHA-undecanoic) were composed of comparable ratios of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). The unfractionated PHA-glucose, PHA-octanoic and PHA-oleic polymers had melting temperatures (Tm) between 177 and 179°C, enthalpies of fusion (ΔHf) of 20 cal/g and glass transition temperatures (Tg) of 3–4°C. This was due to the large PHB content in the polymer mixtures. On the other hand, the PHA-nonanoic and PHA-undecanoic polymers had thermal properties that supported their copolymer nature. In both cases, the Tm values were 161°C, ΔHf values were 7cal/g and Tg values were −3°C. Journal of Industrial Microbiology & Biotechnology (2002) 28, 147–153 DOI: 10.1038/sj/jim/7000231

Effects of cell fermentation time and biomass drying strategies on the recovery of poly-3-hydroxyalkanoates from Alcaligenes eutrophus using a surfactant–chelate aqueous system

In the course of poly-3-hydroxyalkanoate (PHAs) recovery from Alcaligenes. eutrophus by a surfactant and chelate method, a number of operating parameters, in addition to dosage of chemical agents, temperature and pH, might influence recovery results. The effects of cell culture time and drying strategy were investigated on poly-3-hydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) recovery. Increasing fermentation time was confirmed to benefit PHB/PHBV accumulation and recovery, but at the late fermentation, both the PHB/PHBV content and the recovered product purity were not clearly enhanced with the increased culture time. Experiments also indicated that the wet or 60° dried biomass could be utilized to recover PHB and PHBV, but the purity and molecular weight of the product were lower than using the lyophilized. By employing one of the following tactics (increasing the surfactant dose, raising the pH, increasing the temperature or lengthening the treatment time), the purity of recovered PHB/PHBV was significantly improved, which suggested another energy-saving strategy for drying biomass instead of freeze drying.

Sulbactam‐cefoperazone polyhydroxybutyrate‐co‐ hydroxyvalerate (PHBV) local antibiotic delivery system: In vivo effectiveness and biocompatibility in the treatment of implant‐related experimental osteomyelitis

In this study, a novel antibiotic carrier system for use in the treatment of implant-related and chronic osteomyelitis was developed. Sulbactam-cefoperazone was introduced to rods of polyhydroxybutyrate-co-hydroxyvalerate (22 mol % HV, w/w), a member of a family of microbial-origin polymer that is biodegradable, biocompatible, and osteoconductive due to its piezoelectric property. The antibiotic-loaded carrier was implanted into the infection site that was induced by Staphylococcus aureus inoculation into the rabbit tibia. The effectiveness of this was assessed macroscopically, radiographically, bacteriologically, and histopathologically. Findings of infection subsided on day 15 and almost complete remission was observed on day 30. The control side that contained antibiotic-free rods, however, worsened. These findings prompted us to conclude that the novel biodegradable antibiotic carrier developed in the present study seems to be a promising candidate for use in the treatment of severe bone infection. © 1999 John Wiley & Sons, Inc. J Biomed Mater Res, 46, 494–503, 1999.

Sulbactam-cefoperazone polyhydroxybutyrate-co-hydroxyvalerate (PHBV) local antibiotic delivery system: in vivo effectiveness and biocompatibility in the treatment of implant-related experimental osteomyelitis.

In this study, a novel antibiotic carrier system for use in the treatment of implant-related and chronic osteomyelitis was developed. Sulbactam-cefoperazone was introduced to rods of polyhydroxybutyrate-co-hydroxyvalerate (22 mol % HV, w/w), a member of a family of microbial-origin polymer that is biodegradable, biocompatible, and osteoconductive due to its piezoelectric property. The antibiotic-loaded carrier was implanted into the infection site that was induced by Staphylococcus aureus inoculation into the rabbit tibia. The effectiveness of this was assessed macroscopically, radiographically, bacteriologically, and histopathologically. Findings of infection subsided on day 15 and almost complete remission was observed on day 30. The control side that contained antibiotic-free rods, however, worsened. These findings prompted us to conclude that the novel biodegradable antibiotic carrier developed in the present study seems to be a promising candidate for use in the treatment of severe bone infection.

Steady shear and dynamic properties of biodegradable polyesters

AbstractThe rheological properties of three biodegradable partially crystalline alipathic polyesters—poly ε carolactone (PCL), polylactic acid (PLA), and polyhydroxybutyrate‐co‐hydroxyvalerate (PHBV)—were investigated. Low shear viscosity (η), first normal stress difference (N1), and dynamic properties (G′ and G″), were measured for each polymer using rotational instruments. High shear viscosity data was obtained using a capillary rheometer. Gel permeation chromatography indicates that both PLA and PHBV undergo degradation during processing. Thermal degradation rate constants for PLA and PHBV were determined from the time‐sweep data. Data indicates that material functions are proportional to the molecular weight for PCL and PLA samples, and directly related to the HV content in the PHBV samples. The relaxation spectra were calculated from the G′ and G″ data using the regularization method and the applicability of the nonlinear Wagner equation was checked on these materials. Results are discussed in terms of variations in the molecular characteristics on rheological properties and their possible influence on the processability.

Synthesis and mechanical properties of interpenetrating networks of polyhydroxybutyrate- co-hydroxyvalerate and polyhydroxyethyl methacrylate

Naturally occurring, biocompatible, and biodegradable polyhydroxybutyrate- co-hydroxyvalerate (PHBV), and synthetic, nondegrading polyhydroxyethylmethacrylate (PHEMA) membranes were prepared and their mechanical properties were studied. Their performances were compared with the interpenetrating networks (IPN) prepared by photopolymerization of HEMA in the presence of PHBV. The modulus of elasticity, failure stress and failure strain indicated that the IPNs are viscoelastic with properties closer to PHEMA but much stronger than PHEMA homopolymers. Incorporation of PHBV (7, 14 and 22% HV) affected the mechanical properties positively. Increasing the PHBV content increased the modulus of elasticity and failure stress nearly in all samples tested. PHBV (7, 14, and 22% HV, 300 mg) samples showed an approximately 17–30 fold increase in terms of modulus of elasticity and 7–10 fold increase in terms of failure stress. The scanning electron micrographs of the membranes showed that the PHEMA membra-nes are more porous than the PHBV membranes but the IPN structure displayed channels on the membrane surface indicating that HEMA polymerization was achieved by using the PHBV as a scaffold. With the use of the present technique, it is possible to synthesize supramolecular structures from molecules that are not compatible and miscible with each other.

A spectrophotometric method for detection of enzymatic degradation of thin polymer films

An assay method has been developed for monitoring the enzymatic degradation of thin films of translucent polymers. The method was based on the observation that when a solution-cast film of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was exposed to a solution of a depolymerase fromPseudomonas lemoignei, the surface of the film roughened and the film became visibly turbid. This increase in turbidity could be measured spectrophotometrically and was reproducible during the initial stage of degradation. Turbidity correlated very closely with film weight loss early in the degradation but reached a maximum value before extensive degradation had taken place. For a given set of films, this correlation was independent of the concentration of the enzyme used, although it did vary with the mode of enzyme exposure. The turbidity was associated with the exposure of crystalline domains due to the removal of amorphous material from the film surface. The increase in crystallinity at the surface was verified by attenuated total reflectance infrared spectroscopy (ATRIR). In conjunction with SEM, weight loss, and ATRIR, the film turbidity assay provided much semiquantitative insight into the mechanism of the enzymatic degradation reaction. This assay was used to study the enzymatic degradation of films of PHBV solution blended with cellulose acetate esters (CAE). The presence of only 25% of CAE of degree of substitution 2.9 severely hampered the enzymatic degradability of PHBV, a result which is consistent with the environmental degradation of these same samples exposed to activated sludge.

Anaerobic degradation of poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-3-hydroxyvalerate.

The anaerobic degradation of the polyesters poly-3-hydroxybutyrate (PHB) and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) was investigated with special regard to intermediate products, kinetics, and yields. During the degradation of PHBV acetate, propionate, n-butyrate, and n-valerate were detected. Additionally, 3-hydroxybutyrate and 3-hydroxyvalerate and four dimeric esters of these two molecules were identified by GC-MS measurements. Three different test systems for the anaerobic degradation of polyesters were studied. It was not possible to get reproducible results by means of the Anaerobic Sturm-test, a simple system based on carbon dioxide measurement. Secondly, a system based on the GC measurement of accumulated organic acids was investigated. A degradation of 90% in two days was calculated by a carbon balance. Best results were reached with the third test system based on the measurement of methane with a gas meter. A degradation of 99% was observed within 30 days.

Starch-Filled PHB and PHB/HV Copolymer

The structure and mechanical properties of melt-pressed sheets of bacterially produced poly-3-hydroxybutyrate (PHB) and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB/HV) filled with various amounts of particulate maize starch granules have been investigated. The experimental methods included stress–strain measurements, differential thermal analysis to determine heats of fusion and melting temperatures, scanning electron microscopy, and optical microscopy to obtain information on crystallization behaviour and spherulite growth.