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Poly(3-hydroxyalkanoates) (PHAs) are structurally simple macromolecules. PHAs accumulate as discrete granules to levels as high as 90% of the cell dry weight and are generally believed to play a role as a sink for carbon and reducing equivalents [35, 37]. Most of the well identified PHAs are linear, head-to-tail polyesters composed of 3-hydroxy fatty acid monomers (Figure 2.3). In these polymers, the carboxyl group of one monomer forms an ester bond with the hydroxyl group of another monomer. In 1976, Imperial Chemical Industries (ICI) in England started to produce P(3HB) by fermentation. In 1993, Zeneca Bioproducts started their production business, later in 1996 Monsanto bought the production business from Zeneca. Using R. eutropha about 800 tons per year of P(3HB-co-3HV) produced under the trademark Biopol^TM. Monsanto terminated its activities in this area by the end of 1998. Today many other companies remain still active in research and development of PHAs like Procter and Gamble as well as Metabolix (USA) and others all over the world. Biodegradable materials are often used within the biomedical field as implants or as drug carrier systems. Early inteest in environmentally friendly bioplastics was by the European environmental legislation in 2005

Figure 2.3 Chemical structures of PHAs. (1) General structural of PHAs. (2) The copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) as example of short chain length polymer (SCL). (3) P(HHX-co-HO-co-HD-co-HDD-co-….) as an example for PHA_MCL.

[37, 214]. PHA was proved to be biocompatible and can be used in tissue engineering, implantations, and so on. Retinal pigment epithelium cells grow well on P(3HB-co-3HV) as a monolayer for their subretinal transplantation. PHA can be melted or solution processed into a variety of forms. Salt leaching, dip coating and thermally induced phase separation were used to produce scaffolds for cardiovascular tissue engineering. When seeded with cells and cultured in vitro, these scaffolds were used to create living tissue implants [215].

The hydroxyl-substituted carbon atom has R configuration in all characterized PHAs. At the C-3 atom (β position), an alkyl group length can vary from methyl (C1) to tridecyl (C13) [216]. This alkyl side chain is not necessarily saturated. Unsaturated, aromatic, epoxidized, halogenated, and branched monomers were reported as well [217]. Cross-linking of unsaturated bond substations in the side chains of PHAs can be added chemically [218]. Lütke-Eversloh et al. was the first to report on producing biopolymers with thioester linkages in the polymer backbone using C. necator in media containing 3-mercaptopropionate (3MP) or 3-mercaptobutyrate (3MB) in addition to 3-hydroxybutyrate as constituents [219–221]. Many factors affect the PHA’s chemical composition like the microbial strain, the substrate, the cultivation condition, the extraction method, the number of phaC, phaB genes, the regulator phaP (phasin) and the presence of inhibitors. They inhibit different pathways, especially those which supply the synthases with different kinds of monomer or inhibit other pathways, which consume these monomers for their own or degrade it to shorter units like β oxidation pathway. In general, the PHA composition depends on the PHA synthases, the carbon source and the metabolic routes involved. The molecular weights of PHAs were established by light scattering, gel permeation chromatograph and sedimentation analysis. Their monomer composition was determined by gas chromatography (GC), mass spectroscopy (MS) and nuclear magnetic resonance (NMR) analysis [222]. PHAs show material properties that are similar to some common plastics such as polypropylene [223]. The bacterial origins of the PHAs make these polyesters a natural material, and many microorganisms have the ability to degrade these macromolecules [224]. The molecular mass of PHAs varies per PHA producer but is generally in the order of 50 × 10³ to 1 × 10⁶ Da. Inside the cell, P(3HB) exists in a fluid, amorphous state. However, after extraction from the cell with organic solvents, P(3HB) becomes highly crystalline [225] and in this state it is stiff but brittle material. Because of its brittleness, P(3HB) is not very stress resistant. The high melting temperature of P(3HB) (around 170 ^oC) is close to the temperature in which this polymer decomposes thermally and thus limits the ability to process the homopolymer. The incorporation of 3-hydroxyvalerate (3HV) into the P(3HB) resulted in P(3HB-co-3HV) copolymer that is less stiff and brittle than P(3HB), that can be used to prepare films that exert excellent water and gas barrier properties like polypropylene, and that can be processed at lower temperature while retaining most of the other excellent mechanical properties of P(3HB) [226]. (P(3HB-co-3HV)) has also low crystallinity and is more elastic than P(3HB) [227, 228]. The latex-like PHAs (PHA_MCL) display physical properties, which differ significantly from the PHA_SCL, such as P(3HB). Particularly with respect to the melting temperature and the extension to break value the two types of PHAs showed, mainly because of the lower crystallinity of PHA_MCL, striking differences. PHAs have been processed into fibers, which were then used to construct materials such as non-woven fabrics [229]. Moreover P(3HB) and P(3HB-co-3HV) were described as hot-melt adhesives [230]. They are considered for several applications in the packaging industry, medicine, pharmacy, agriculture and food industry or as raw materials for the synthesis of enantiomerically pure chemicals and the production of paints [231]. Possible applications of P(3HB) and copolymers are as packaging materials or agricultural foil [232]. As in the Biopol^TM recovery process, the fermentor contents are heat treated to break down the nucleic acids, and proteases and detergents are added to solubilize the cells. Subsequent washing (i.e., removal of the solubilized cell material) and concentration of the resulting PHA latex is established by cross-flow microfiltration. To produce a coating, the PHA latex is sprayed onto a substrate such as paper. After evaporation of the water, the PHA latex particles readily coalesce into a film [218]. Due to the relatively high cost of PHA production, it is wise to apply PHAs for some cost-effective applications like medicinal instruments. PHAs were proved to be biocompatible in tissue engineering, implantations, etc. Many prokaryotic and eukaryotic organisms are able to produce LMW PHB molecules that are complexed with other biomolecules such as polyphosphates and that are present at low concentrations [233]. Over recent years, PHAs were used to develop many devices and material useful for clinical purposes such as suture fasteners, meniscus repair devices, rivets, tacks, staples, screws, (including interference screws), bone plates and bone plating systems, surgical mesh, repair patches, orthopedic pins (including bone filling augmentation material), adhesion barriers, stents, guided tissue repair regeneration devices, articular cartilage repair devices, nerve guides, tendon repair devices, pericardial patches, bulking and filling agents, vein valves, bone marrow scaffolds, meniscus regeneration devices, ligament, tendon grafts, ocular cell implants, spinal fusion cages, skin substitutes, dural substitutes, bone graft substitutes, bone dowels, wound dressing and hemostats [234–240]. Many biochemical engineering, molecular biology experiments and other tools were used to change the end products of the polyhydroxyalkanoate or to produce copolymers. To assess the biocompatibility of PHB, the structural organization of cellular molecules involved in adhesion was studied using osteoblastic and epithelial cell lines. On PHB, both cell lines revealed a rounded cell shape due to reduced spreading. The filamentous organization of the actin cytoskeleton was impaired. In double immunofluorescence, analyses the co-localization of the fibronectin with the fibril actin was demonstrated [241]. The investigated properties of PHB and PHB-co-PHV films proved to be fundamentally similar [242–246]. PHB-co-PHV film was chosen as a temporary substrate for growing retinal pigment epithelium cells as an organized monolayer before their subretinal transplantation. The surface of the PHB-co-PHV film was rendered hydrophilic by oxygen plasma treatment to increase the reattachment of D407 cells on the film surface. The cells were also grown to confluency as an organized monolayer suggesting PHB-co-PHV film as a potential temporary substrate for subretinal transplantation to replace diseased or damaged retinal pigment epithelium [247]. Tesema et al. and Malm et al. implanted PHB non-woven patches as transannular patches into the right ventricular outflow tract and pulmonary artery in 13 weanling sheep [248–250].

PHB non-woven patches can be used as a scaffold for tissue regeneration in low-pressure systems. The regenerated vessel had structural and biochemical qualities in common with the native pulmonary artery [250]. PHAs were used in tissue engineering, as antibiotic carriers, and many other medicinal applications [238, 251, 252]. Chen and Wu recently reported that PHAs possesses the biodegradability, biocompatibility and thermo-processibility for not only implant applications but also controlled drug release uses. PHAs show a promising future in pharmaceutical application such as drug delivery, which open a new approach. The many possibilities to tailor-make PHAs for medical implant applications have shown that this class of materials has a bright future as tissue engineering materials [253]. Different types of mutagenesis were applied for changing the substrate specificity, study the catalytic residues and to overproduce the PHAs [254–257].