|Title||Biosynthesis of polyhydroxyalkanoates for cardiac tissue engineering applications|
As a result of the enormous clinical need, cardiac tissue engineering has become a prime focus of research within the field of tissue engineering. In this project, Poly(3- hydroxyoctanoate), P(3HO), a medium chain length (mcl-PHAs) biodegradable,
biocompatible and elastomeric polyhydroxyalkanoate, was studied as a potential material for cardiac tissue engineering. Mcl-PHAs are an alternative source of polymers produced by Pseudomonas sp. As Gram-negative bacteria, Pseudomonas sp. contains lipopolysaccharides in the membrane, which are co-purified with PHAs and may cause immunogenic reactions. This limits the biomedical applications of the mcl-PHAs in several cases. In this work, the Pseudomonas mendocina PHA synthase gene (phaC1) was expressed in the LPS free, GRAS, Gram-positive microorganism, Bacillus subtilis so as to produce LPS-free mcl-PHAs. Our results showed that the recombinant Bacillus subtilis containing the phaC1 gene produced poly(3-hydroxybutirate), P(3HB), with a maximum yield of 32.3 % DCW, an unexpected result. This result thus revealed the unusually broad substrate specificity of the PHA synthase from P. mendocina, which is able to catalyse both medium and short chain length PHAs biosynthesis depending on the metabolic pool available in the host organism. Sequence comparison of this PHA synthase with stringent mcl-PHA synthases revealed possible residues influencing the substrate specificity of PHA synthases.
As studies on mcl-PHAs remain limited mainly because of the lack of availability of mcl- PHAs in large quantities, the capacity to scale-up P(3HO) production from 2 L to 20 L and 72 L pilot plant bioreactors, based on constant oxygen transference, was studied. The interaction of freshly isolated rat cardiomyocytes with the P(3HO) polymer, during contraction, was studied when cells were stimulated at a range of frequencies of electrical pulses or calcium concentrations. These results showed that P(3HO) did not have any deleterious effects on the contraction of adult cardiomyocytes. P(3HO) cardiac patches nonporous, porous or with P(3HO) electrospun fibres deposited on their surface were developed. Our results showed that the mechanical properties of the final constructs were close to that of the cardiac structures, with a Young’s modulus value of 0.41±0.03 MPa. Myoblast (C2Cl2) cell proliferation was studied on the different constructs showing an enhanced cell adhesion and proliferation when both porous and fibrous structures were incorporated together. Finally, for further enhancement of the cardiac patch function, VEGF and RGD peptide were incorporated. Results obtained in this project showed that the P(3HO) multifunctional cardiac patches were potentially promising constructs for efficient cardiac tissue engineering.