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Evaluation of 3D-Printing Scaffold Fabrication on Biosynthetic Medium-Chain-Length Polyhydroxyalkanoate Terpolyester as Biomaterial-Ink.

ABSTRACT: Currently, the selection of materials for tissue engineering scaffolds is still limited because some tissues require flexible and compatible materials with human cells. Medium-chain-length polyhydroxyalkanoate (MCL-PHA) synthesized in microorganisms is an interesting polymer for use in this area and has elastomeric properties compatible with the human body. MCL-PHAs are elastomers with biodegradability and cellular compatibility, making them an attractive material for fabricating soft tissue that requires high elasticity. In this research, MCL-PHA was produced by fed-batch fermentation that Pseudomonas Putida ATCC 47054 was cultured to accumulate MCL-PHA by using glycerol and sodium octanoate as carbon sources. The amounts of dry cell density, MCL-PHA product per dry cells, and MCL-PHA productivity were at 15 g/L, 27%, and 0.067 g/L/h, respectively, and the components of MCL-PHA consisting of 3-hydroxydecanoate (3HD) 64.5%, 3-hydroxyoctanoate (3HO) 32.2%, and 3-hydroxyhexanoate (3HHx) 3.3%. The biosynthesized MCL-PHA terpolyester has a relatively low melting temperature, low crystallinity, and high ductility at 52 °C, 15.7%, and 218%, respectively, and considering as elastomeric polyester. The high-resolution scaffold of MCL-PHA terpolyester biomaterial-ink (approximately 0.36 mm porous size) could be printed in a selected condition with a 3D printer, similar to the optimum pore size for cell attachment and proliferation. The rheological characteristic of this MCL-PHA biomaterial-ink exhibits shear-thinning behavior, leading to good shape fidelity. The study results yielded a condition capable of fabricating an elastomer scaffold of the MCL-PHA terpolyester, giving rise to the ideal soft tissue engineering application.

SUBMITTER: Panaksri A

PROVIDER: S-EPMC8309464 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Transcriptomics analysis of biopolymer (medium chain length polyhydroxyalkanoate) producing strain P.putida LS46 cultured with biodiesel derived waste carbon sources: studies of cellular adaptation to the industrial waste streams and metabolic profiling under the polymer producing conditions. We are reporting RNAseq analysis data here as part of our multi-level Omics study of medium chain length polyhydroxyalkanoate (mcl-PHA) producing strain P.putida LS46 culture with biodiesel derived waste glycerol and waste fatty acids. The data presented here will be used in two separate manuscripts. The objectives of this study are a): to evaluate cellular responses of P.putida LS46 under industrial waste stream. b): to study gene expression profile under two selected mcl-PHA producing conditions of P.putida LS46. Comparative multi-level Omics study: for objective a): Exponential P.putida LS46 cell from waste glycerol culture compared against reagent grade pure glycerol culture. For objective b): Two mcl-PHA producing conditions, namely stationary phase waste glycerol culture and exponential phase waste fatty acid culture of P.putida LS46, were compared against exponential phase waste glycerol culture of P.putida LS46. Major results from objective a): The waste glycerol substrate induced expression of a large number of genes putatively involved in heavy metal tolerance, including three gene clusters: a putative cusABC transcript unit and two copies of copAB, which are usually involved in copper resistance and tolerance to other monovalent heavy metals. A local gene relocation was observed in cluster 1 consisting cusABC and copAB relative to the KT2440 type strain according to the phylogenetic and gene neighbourhood analyses on various P. putida strains. P. putida LS46 also contains 11 putative MerR family regulators, which sense various environmental stimuli including heavy metals. MerR-1 is an ortholog of the copper response regulator of other gram-negative bacteria, and was highly up-regulated in waste glycerol cultures. Finally, a number of genes involved in cell responses to high extra-cellular Na+ concentrations, and genes of the fatty acid beta-oxidation pathway were up-regulated in waste glycerol cultures Major results from objective b): Regardless to the type of substrates, up-regulation of two mcl-PHA synthase (PhaC1 and PhaC2), and two phasin proteins (PhaF and PhaI) are the most common genotype under mcl-PHA production conditions. PhaG and possible PhaJ4 connect fatty acid de novo synthesis to mcl-PHA in waste glycerol culture. Interestingly, expression of gene, fabZ, in production of unsaturated fatty acid from fatty acid de novo synthesis was only observed in waste glycerol culture. On the other hand, PhaJ1 and PhaJ4 derived mcl-PHA production via fatty acid beta-oxidation was observed under waste fatty acid culture. These results would help to explain observed different production kinetics and monomer distribution of the polymer. Although under active mcl-PHA production condition, depression on the expression of glpF genes in glycerol transportation system prevent further channelling extra-cellular glycerol into the cell. Waste glycerol culture also triggers trahalose synthesis pathway, a potential competing pathway during mcl-PHA synthesizing. In waste fatty acid culture, the intermediates (acyl-CoA and 3-hydroxyacyl-CoA) of fatty acid beta-oxidation were used for mcl-PHA production and were also likely hydrolysed to their free acid forms via an up-regulated thioesteras coding gene, tesA. Acetyl-CoA cleaved from the pathway was clearly channeled into glyoxylate shut for C2 carbon assimilation over spillage as CO2 through TCA cycle or used in fatty acid biosynthesis pathway.

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Evaluation of 3D-Printing Scaffold Fabrication on Biosynthetic Medium-Chain-Length Polyhydroxyalkanoate Terpolyester as Biomaterial-Ink.

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