Production of purified polyhydroxyalkanoates (PHAs) for applications in contact with blood

Samples of olyhydroxyalkanoates (PHAs), polyhydroxybutyrate (PHB) and copolymers poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) with 4 and 18 mol% hydroxyvalerate, synthesized by the bacteria Ralstonia eutropha B5786, were investigated. PHA films in contact with blood did not activate the hemostasis system at the level of cell response, but they did activate the coagulation system and the complement reaction. To detect biologically-active components in the PHAs, a detailed analysis of the composition of the polymers was conducted. Gas chromatography-mass spectrometry revealed long-chain fatty acids (FAs) in the tested PHAs. Their total concentration in the polymer ranged from tenths of mol% to 2-3 mol%, depending on the purification method. C16:0 constituted the largest proportion, up to 70%. Of the long-chain hydroxy acids, only β-OH-C14:0 was detected and it did not exceed 0.06 mol%. The analysis of the hemocompatibility properties of the PHAs purified by a specialized procedure, including the quantitative and morphological estimation of platelets adherent to the surface of polymer films, the plasma recalcification time and complement activation studies, indicated that PHB and PHBV can be used in contact with blood. It has been found out that the lipopolysaccharides of bacteria producing PHAs, which contain mostly long-chain hydroxy acids, can be the factor activating the hemostasis systems. Thus, the technology of PHA purification must satisfy rather stringent specific requirements.

[1]  H. Mckenzie,et al.  The Kjeldahl determination of nitrogen : a critical study of digestion conditions-temperature, catalyst, and oxidizing agent. , 1954 .

[2]  C. G. Vonk.  Computerization of Ruland's X-ray method for determination of the crystallinity in polymers , 1973 .

[3]  D. J. Finney,et al.  Statistics for biologists , 1974, Nature.

[4]  H. Fujita,et al.  Solution Properties of Poly(D-β-hydroxybutyrate). 1. Biosynthesis and Characterization , 1976 .

[5]  V. Sevastianov,et al.  In vitro assessment of the hemocompatible properties of polymers. , 1983, Artificial organs.

[6]  V. I. Sevast'ianov,et al.  The activation of the complement system by polymer materials and their blood compatibility. , 1984, Journal of biomedical materials research.

[7]  R. Gross,et al.  Ability of the phototrophic bacterium Rhodospirillum rubrum to produce various poly (beta-hydroxyalkanoates): potential sources for biodegradable polyesters. , 1989, International journal of biological macromolecules.

[8]  W. Bonfield,et al.  In vitro and in vivo evaluation of polyhydroxybutyrate and of polyhydroxybutyrate reinforced with hydroxyapatite. , 1991, Biomaterials.

[9]  C. Nathan,et al.  Gram‐negative endotoxin: an extraordinary lipid with profound effects on eukaryotic signal transduction 1 , 1991, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[10]  P. Bentley.  High Performance Biomaterials. A Comprehensive Guide to Medical and Pharmaceutical Applications , 1992 .

[11]  S. Perren,et al.  Tissue response and in vivo degradation of selected polyhydroxyacids: polylactides (PLA), poly(3-hydroxybutyrate) (PHB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/VA). , 1993, Journal of biomedical materials research.

[12]  L. Larsson,et al.  Gas chromatography-mass spectrometry methods for analysis of 2- and 3-hydroxylated fatty acids: Application for endotoxin measurement , 1993 .

[13]  C. Rivard,et al.  [Bio-absorbable synthetic polyesters and tissue regeneration. A study of three-dimensional proliferation of ovine chondrocytes and osteoblasts]. , 1996, Annales de chirurgie.

[14]  Detlef Prof. Dr. Behrend,et al.  Komplementaktivierung und die Freisetzung von Zytokinen aus Monozyten als Indikatoren für die Biokompatibilität von Polyhydroxybuttersäure , 1997 .

[15]  Brian J. Tighe,et al.  A review of biodegradable polymers: uses, current developments in the synthesis and characterization of biodegradable polyesters, blends of biodegradable polymers and recent advances in biodegradation studies , 1998 .

[16]  R. Legras,et al.  Adsorption of albumin, collagen, and fibronectin on the surface of poly(hydroxybutyrate-hydroxyvalerate) (PHB/HV) and of poly (epsilon-caprolactone) (PCL) films modified by an alkaline hydrolysis and of poly(ethylene terephtalate) (PET) track-etched membranes. , 1998, Journal of biomaterials science. Polymer edition.

[17]  David P. Martin,et al.  PHA applications: addressing the price performance issue: I. Tissue engineering. , 1999, International journal of biological macromolecules.

[18]  S. Lee,et al.  Removal of Endotoxin during Purification of Poly(3-Hydroxybutyrate) from Gram-Negative Bacteria , 1999, Applied and Environmental Microbiology.

[19]  Gjalt W. Huisman,et al.  Metabolic Engineering of Poly(3-Hydroxyalkanoates): From DNA to Plastic , 1999, Microbiology and Molecular Biology Reviews.

[20]  I. B. Rozanova,et al.  Carbon Coated Polyethylene: Effect of Surface Energetics and Topography on Human Platelet Adhesion , 2001, ASAIO journal.

[21]  Ekaterina I. Shishatskaya,et al.  Tissue Response to Biodegradable Suture Threads Made of Polyhydroxyalkanoates , 2002 .

[22]  Yong Woo Lee,et al.  Unsaturated fatty acids selectively induce an inflammatory environment in human endothelial cells. , 2002, The American journal of clinical nutrition.