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PubMed1992

Cloning of poly(3-hydroxybutyric acid) synthase genes of Rhodobacter sphaeroides and Rhodospirillum rubrum and heterologous expression in Alcaligenes eutrophus.

From genomic libraries of the purple non-sulfur bacteria Rhodospirillum rubrum Ha and Rhodobacter sphaeroides ATCC 17023 in the broad-host range cosmid pVK100, we cloned a 15- and a 14-kbp HindIII restriction fragment, respectively. Each of these fragments restored the ability to accumulate poly(3-hydroxybutyrate) (PHB), in the PHB-negative mutant Alcaligenes eutrophus PHB-4. These hybrid cosmids also complemented PHB-negative mutants derived from wild-type R. rubrum or R. sphaeroides. Both fragments hybridized with the PHB synthase structural gene of A. eutrophus H16 and conferred the ability to express PHB synthase activity. Only the 15-kbp HindIII fragment from R. rubrum conferred on the mutant PHB-4 the ability to form large PHB granules (length up to 3.5 microns).

Acyltransferases↗
PubMed1992

Ultrastructural and chemical assessment of poly-beta-hydroxybutyric acid in the marine cyanobacterium Trichodesmium thiebautii.

We report here on the occurrence and quantities of poly-beta-hydroxybutyric acid (PHB) in natural populations of the marine cyanobacterium Trichodesmium thiebautii. A diurnal variation in the shape and size of PHB granules and in PHB content was observed. The highest PHB levels (2.3 +/- 0.8 mg g-1 dry wt) were recorded in the early morning and the values decreased thereafter with a minimum at night (1.6 +/- 0.9 mg g-1 dry wt). Our data suggest that PHB is a prominent cell constituent in T. thiebautii and that its synthesis takes place in the early morning whereas it is utilized during the rest of the day.

Cyanobacteria↗
PubMed1992

Differentiation of polyphosphate and poly-beta-hydroxybutyrate granules in an Acinetobacter sp. isolated from activated sludge.

Cells containing polyphosphate 71 micrograms P (mg protein)-1 and no poly-beta-hydroxybutyrate showed metachromatic granules but no lipid granules; cells containing poly-beta-hydroxybutyrate (15% of dry weight) showed fluorescence lipid granules but no metachromatic granules; whereas cells containing both polyphosphate and poly-beta-hydroxybutyrate showed both types of granules. These observations, together with a critical review of the literature, show a clear distinction between metachromatic (or volutin) granules and lipid granules.

Acinetobacter↗
PubMedDate not supplied

Growth rate and some physiological features of Deleya halophila CCM 3662 at different salt concentrations.

The growth rate of the type strain of the new, moderately halophilic species Deleya halophila (Deleya halophila CCM 3662) at different salt concentrations was determined and its optimal marine salt concentration for growth was established. Oxygen uptake and some cellular chemical composition characteristics of cells grown at their optimal salt concentration (7.5% w/v) and at a higher concentration (15% w/v) were compared, showing that both protein and carbohydrate intracellular contents decreased when the external medium salt concentration was increased, with the carbohydrate content reaching 50% of the value presented by cells cultured at optimal salinity conditions. Oxygen uptake and poly-beta-hydroxybutyric acid content were not significantly affected by changes in external salinity.

Bacterial Proteins↗
PubMedDate not supplied

Studies on bone marrow stromal cells affinity of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate).

The objective of this study is to investigate the biocompatibility of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) with bone marrow stromal cells in vitro. The adsorption of fibronectin on the material was studied by enzyme-linked immunosorbent assay. After bone marrow stromal cells were seeded and cultured on PHBHHx, their proliferation was investigated by MTT. Differentiation of the cells was assessed by measuring alkaline phosphatase activity and by histochemical assay. The wettability and thermal property of PHBHHx films were also studied by contact angle goniometer, thermogravimetry and differential scanning calorimetry, respectively. The results show that bone marrow stromal cells can attach, proliferate and differentiate into osteoblasts on PHBHHx films. These results suggest that PHBHHx has good affinity with bone marrow stromal cells and may have potential applications in bone tissue engineering.

3-Hydroxybutyric Acid↗
PubMed2005

Effect of composition of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) on growth of fibroblast and osteoblast.

Films made of poly (3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate- co-3-hydroxyhexanoate) (PHBHHx) consisting of 5%, 12% and 20% hydroxyhexanoate (HHx), respectively, were evaluated for biomedical application in comparison with poly (L-Lactide) (PLA). With the increase of HHx content in PHBHHx, the polymer surface properties changed accordingly. P(HB-co-20%-HHx) had the smoothest surface while PHB surface was most hydrophilic among the evaluated PHB and all the PHBHHx. All PHBHHx also showed strong protein affinity and biocompatibility. It was found that fibroblast and osteoblast had different responses to these polymers: fibroblast cells favored P(HB-co-20%-HHx), yet osteoblast cells preferred P(HB-co-12%-HHx). PHB and all PHBHHx appeared to have better biocompatibility for fibroblast and osteoblast compared with PLA. Polymers possessing different surface properties may help meet different cellular requirements. Combined with their good mechanical properties for elongation and adjustable biocompatibility, PHBHHx may meet the needs of growth requirements of different tissues and cells.

3-Hydroxybutyric Acid↗
PubMed2005

Evaluation of three-dimensional scaffolds made of blends of hydroxyapatite and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) for bone reconstruction.

Hydroxyapatite (HAP) was blended into poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) to make films and scaffolds. After HAP blending, mechanical properties of PHB including compressive elastic modulus and maximum stress showed improvement and osteoblast responses including cell growth and alkaline phosphatase activity were also strengthened. On the other hand, scaffolds made of PHBHHx blended with HAP had an adverse effect. No remarkable change on degradation of PHB or PHBHHx blended with HAP, respectively, was observed in simulated body fluid. Scanning electron microscopy examination revealed that osteoblast responses to HAP incorporation may be related to surface morphology and to the exposed HAP particles on polymer surface. All these results indicated that the blending of HAP particles into PHBHHx scaffolds fabricated by salt leaching was unable to either strengthen its mechanical properties or enhance osteoblast responses. Although HAP is bioactive and osteoconductive, its blending with PHBHHx did not generate a better performance on bone reconstruction.

3-Hydroxybutyric Acid↗
PubMed2005

Effects of crystallization of polyhydroxyalkanoate blend on surface physicochemical properties and interactions with rabbit articular cartilage chondrocytes.

As a new member of polyhydroxyalkanoate (PHA) family, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) has better mechanical properties compared with poly(3-hydroxybutyrate) (PHB). Still, many properties of PHBHHx and its blend with PHB may be exploited for a wide range of applications, such as tissue engineering and drug delivery. In this study, PHBHHx was found to be completely miscible with PHB in their blends. Crystallization behavior of these polyester blends played an important role in determination of the surface physicochemical properties including surface free energy, chemical states and polarity, and could subsequently govern the cellular responses. Interaction between PHB and PHBHHx influenced the non-dispersion surface free energy component, which governed the total surface free energy. It was shown that the blend with a 1:1 ratio of PHB/PHBHHx possessed the highest surface free energy, which was the most optimal material for chondrocytes adhesion. After 24 h, the amount of chondrocytes adhered on films of PHB/PHBHHx (1:1) was 2.1 x 10(4)cells/cm(2), 5-times more compared with that on the PHB films (0.4 x 10(4)cells/cm(2)). The polarity of the blends increased with decreasing crystallinity. After 8 days of cultivation, the chondrocytes attached on PHB films were surrounded by both collagen II and collagen X, the amount of extracellular collagen X decreased with increasing polarity contributed by increasing PHBHHx content in the blend, while chondrocytes changed shapes from spherical to flat with increasing polarity. It indicated that endochondral ossification of chondrocytes was remarkably influenced by the crystallinity of the polyesters.

3-Hydroxybutyric Acid↗
PubMed2005

Tissue engineered cartilage on collagen and PHBV matrices.

Cartilage engineering is a very novel approach to tissue repair through use of implants. Matrices of collagen containing calcium phosphate (CaP-Gelfix), and matrices of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) were produced to create a cartilage via tissue engineering. The matrices were characterized by scanning electron microscopy (SEM) and electron diffraction spectroscopy (EDS). Porosity and void volume analysis were carried out to characterize the matrices. Chondrocytes were isolated from the proximal humerus of 22 week-old male, adult, local albino rabbits. For cell type characterization, Type II collagen was measured by Western Blot analysis. The foams were seeded with 1x10(6) chondrocytes and histological examinations were carried out to assess cell-matrix interaction. Macroscopic examination showed that PHBV (with or without chondrocytes) maintained its integrity for 21 days, while CaP-Gelfix was deformed and degraded within 15 days. Cell-containing and cell-free matrices were implanted into full thickness cartilage defects (4.5 mm in diameter and 4 mm in depth) at the patellar groove on the right and left knees of eight rabbits, respectively. In vivo results at 8 and 20 weeks with chondrocyte seeded PHBV matrices presented early cartilage formation resembling normal articular cartilage and revealed minimal foreign body reaction. In CaP-Gelfix matrices, fibrocartilage formation and bone invasion was noted in 20 weeks. Cells maintained their phenotype in both matrices. PHBV had better healing response than CaP-Gelfix. Both matrices were effective in cartilage regeneration. These matrices have great potential for use in the repair of joint cartilage defects.

Absorbable Implants↗
PubMed2005

The application of polyhydroxyalkanoates as tissue engineering materials.

Polyhydoxyalkanoates (PHA) are polyesters produced by microorganisms under unbalanced growth conditions. They are generally biodegradable and thermoprocessable, making them attractive as biomaterials for applications in both conventional medical devices and tissue engineering. Over the past years, PHA, particularly poly 3-hydroxybutyrate (PHB), copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), poly 4-hydroxybutyrate (P4HB), copolymers of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) and poly 3-hydroxyoctanoate (PHO) and its composites have been used to develop devices including sutures, repair devices, repair patches, slings, cardiovascular patches, orthopedic pins, adhesion barriers, stents, guided tissue repair/regeneration devices, articular cartilage repair devices, nerve guides, tendon repair devices, bone marrow scaffolds, and wound dressings. The changing PHA compositions also allow favorable mechanical properties, biocompatibility, and degradation times within desirable time frames under specific physiological conditions. This paper reviews what have been achieved in the PHA tissue engineering area and concluded that the PHA prospective will look very bright in the near future.

3-Hydroxybutyric Acid↗
PubMed2005

Enhanced vascular-related cellular affinity on surface modified copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx).

Random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate, short as PHBHHx, was surface modified by ammonia plasma treatment and/or fibronectin coating, respectively. The improved results were demonstrated by better growth of human umbilical vein endothelial cells (HUVECs) and rabbit aorta smooth muscle cells (SMCs) on the surface of ammonia plasma-treated PHBHHx coated with fibronectin (PFn-PHBHHx), compared with the fibronectin-coated (Fn-PHBHHx) or uncoated PHBHHx, respectively, although XPS analysis and ELISA demonstrated higher fibronectin adsorption on Fn-PHBHHx than on PFn-PHBHHx. Confocal microscopy observation showed that the specific co-localization of fibronectin with F-actin was impaired on PFn-PHBHHx, while it was almost lost on Fn-PHBHHx compared with pristine PHBHHx or plasma-treated PHBHHx (P-PHBHHx). These were attributed to the generation of new nitrogen- and oxygen-containing groups on the PHBHHx surface by the ammonia plasma treatment, which led to increased polar components that enhanced polymer surface energy and hydrophilic properties on P-PHBHHx. The most prominent effect of PFn-PHBHHx was its stimulation of HUVECs proliferation. HUVECs on PFn-PHBHHx formed a confluent monolayer after 3 days of incubation, while SMCs were unable to form a sub-confluent layer. The above evidences revealed that PFn-PHBHHx would benefit endotheliazation rather than SMCs proliferation. We therefore believed that PFn-PHBHHx would be a promising material as a luminal surface of vascular grafts.

3-Hydroxybutyric Acid↗
PubMed2005

The in vitro hydrolysis of poly(ester urethane)s consisting of poly[(R)-3-hydroxybutyrate] and poly(ethylene glycol).

This paper reports the study of the complete degradation process for a series of newly synthesized multi-block poly(ester urethane)s consisting of poly[(R)-3-hydroxybutyrate] (PHB) as hard and hydrophobic block and poly(ethylene glycol) (PEG) as soft and hydrophilic segment. The initial stages of degradation of the poly(PHB/PEG urethane)s were monitored by carrying out the degradation experiments at pH 7.4 and 37 degrees C. The weight loss of the copolymer films was traced, and the degraded copolymer films were characterized by GPC, (1)H NMR, TGA, and SEM. The induction phase of the polymer degradation was characterized by a random chain scission of the ester backbone bonds of the PHB segments and an insignificant decline in the weight of the polymer films. An accelerated degradation process was carried out at pH 11.5 and 37 degrees C to investigate the long-term degradation behaviour. The characterization of the degraded polymer films was similar to that for the experiment at pH 7.4. In addition, the water-soluble degradation products were characterized by GPC, (1)H NMR, and FTIR. The main components of the water-soluble degradation products were found to be PEG blocks (monomeric up to quadmeric), 3-hydroxybutyric acid, and crotonic acid. It was found that the copolymer incorporating the highest amount of PEG degraded at the highest rate of all the copolymers studied. The complete degradation of the poly(PHB/PEG urethane)s was monitored using a combination of the physiological and accelerated hydrolytic degradation.

Biocompatible Materials↗
PubMed2005

The mechanical properties and in vitro biodegradation and biocompatibility of UV-treated poly(3-hydroxybutyrate-co-3-hydroxyhexanoate).

Strong mechanical properties and controllable biodegradability, together with biocompatibility, are the important requirement for the development of medical implant materials. In this study, an ultraviolet (UV) radiation method was developed to achieve controlled degradation for bacterial biopolyester poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) which has a low biodegradation rate that limits its application for many implant applications required quick degradation. When UV radiation was applied directly to PHBHHx powder, significant molecular weight (Mw) losses were observed with the powder, Mw reduction depended on the UV radiation time. At the same time, a broad PHBHHx Mw distribution was the result of inhomogeneous radiation. Interestingly, this inhomogeneous radiation helped maintain the mechanical properties of films made of the UV-radiated powder. In comparison, the PHBHHx films subjected to direct UV radiation became very brittle although their degradation was faster than that of the PHBHHx powders subjected to direct UV radiation. After 15 weeks of degradation in simulated body fluid (SBF), films prepared from 8 and 16h UV-treated PHBHHx powders maintained 92% and 87% of their original weights, respectively, while the untreated PHBHHx films lost only 1% of its weight. Significant increases in growth of fibroblast L929 were observed on films prepared from UV-radiated powders. This improved biocompatibility could be attributed to increasing hydrophilic functional groups generated by increasing polar groups C-O and CO. In general, UV-treated PHBHHx powder had a broad Mw distribution, which contributed to fast degradation due to dissolution of low Mw polymer fragments, and strong mechanical property due to high Mw polymer chains. Combined with its improved biocompatibility, PHBHHx is one more step close to become a biomedical implant material.

3-Hydroxybutyric Acid↗
PubMed2005

Fabrication of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) microstructures using soft lithography for scaffold applications.

This paper reports two soft lithographic methods, micromolding and hot embossing, to produce biodegradable poly (3-hydroxybutyrate-co-3-ftydroxyhexanoate) (PHBHHx) arrays of microstructures for hosting and culturing cells in a local microenvironment by controlled shape. Silicon masters with high-aspect-ratio microfeatures were fabricated using KOH and DRIE anisotropic etching. These silicon masters were used as molds to construct PHBHHx microstructures using micromolding and hot embossing. Using silicon rather than conventional PDMS as molds allowed microstructures with feature size of 20 microm and height of 100 microm to be realized. PHBHHx microstructures with different configurations including circles, rectangles, and octagons were fabricated to investigate the effects of topography on cell culture. Mouse fibroblast cell lines L929 were cultured on PHBHHx microstructures in vitro to investigate the biocompatibility. This study demonstrates the feasibility of microfabrication of PHBHHx structures with micro-scale feature size using soft lithography, and the results show that PHBHHx microstructures can be created to mimic cellular microenvironment for cell culture, providing a convenient means to investigate relationships of microstructures and cell functions.

3-Hydroxybutyric Acid↗
PubMed2006

Effect of 3-hydroxyhexanoate content in poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) on in vitro growth and differentiation of smooth muscle cells.

In this paper, comprehensive characteristics including cell attachment, cell proliferation status, cell cycle progression and phenotypic changes of smooth muscle cells from rabbit aorta (RaSMCs) were studied on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) containing 0-20% HHx (mol%) in comparison with tissue culture plates (TCPs). Results demonstrated that RaSMCs adhered better on PHBHHx containing 12% HHx (12%HHx) although they proliferated better on 20%HHx-containing PHBHHx films (20%HHx). This was explained by the difference in cell cycle progression observed using flow cytometry, as it was found that only 20%HHx-containing polymer could maintain the normal cell cycle evolution as TCPs did after 3 d incubation. The highest expression level and typical spindle-like distribution of alpha-actin on 20%HHx-containing polymer were characterized as the contractile-like phenotype, suggesting that RaSMCs tended to differentiate rather than proliferate compared to the cells grown on 12%HHx polymer. Results obtained above suggested that 20%HHx was suitable for RaSMCs proliferation, leading to its change to contractile phenotype. This study extends the potential applications of PHBHHx in SMCs-related graft scaffold fabrication for tissue engineering.

3-Hydroxybutyric Acid↗
PubMed2006

In vivo studies of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) based polymers: biodegradation and tissue reactions.

The in vivo tissue reactions and biodegradations of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), poly(lactide) (PLA), poly(3-hydroxybutyrate) (PHB), blends of PHBHHx (X) and poly(ethylene glycol) (PEG) (E) with ratios of 1:1 (E1X1) and 1:5 (E1X5), respectively, were evaluated by subcutaneous implantation in rabbits. Results revealed that the degradation rate increased in the order of PHB < PHBHHx < PLA. During the implantation period, crystallinity of PHBHHx increased from 19% to 22% and then dropped to 14%. Gel permeation chromatography (GPC) displayed increasing polydispersity and typical bimodal distribution from 3 to 6 months. The above results suggested that rapid PHBHHx degradation occurred in amorphous region rather than in crystalline region. While the in vivo hydrolysis of PHB was found to start from a random chain scission both in amorphous and crystalline regions of the polymer matrix, as demonstrated by its hydrolysis process accompanied by a decrease in molecular weight with unimodal distribution and relatively narrow polydispersity. Compared to pure PHBHHx, PHBHHx-PEG blends showed accelerated weight loss of PHBHHx with weak molecular weight reduction. In general, PHBHHx elicited a very mild tissue response during implantation lasting 6 months compared with relative acute immunological reactions observed among PHB and PLA objects, respectively. Pronounced tissue responses were observed in the capsule surrounding E1X1 and E1X5 as characterized by the presence of lymphocytes, eosinophils and vascularization, which might be resulted from the continuous leaching of PEG.

3-Hydroxybutyric Acid↗
PubMed2006

Self-assembled supramolecular hydrogels formed by biodegradable PEO-PHB-PEO triblock copolymers and alpha-cyclodextrin for controlled drug delivery.

A materials design of a new supramolecular hydrogel self-assembled between alpha-cyclodextrin and a biodegradable poly(ethylene oxide)-poly[(r)-3-hydroxybutyrate]-poly(ethylene oxide) (PEO-PHB-PEO) triblock copolymer was demonstrated. The cooperation effect of complexation of PEO segments with alpha-cyclodextrin and the hydrophobic interaction between PHB blocks resulted in the formation of the supramolecular hydrogel with a strong macromolecular network. The in vitro release kinetics studies of fluorescein isothiocyanate labeled dextran (dextran-FITC) model drug from the hydrogel showed that the hydrogel was suitable for relatively long-term sustained controlled release of macromolecular drugs, which many simple triblock copolymer hydrogel systems could not achieve. The hydrogel was found to be thixotropic and reversible, and can be applied as a promising injectable drug delivery system.

Biocompatible Materials↗