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Biocompatibilities and biodegradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)s produced by a model metabolic reaction-based system.

Napathorn SC - BMC Microbiol. (2014)

Bottom Line: The diad and triad sequence distributions indicated that the PHBVs produced via the fed-batch cultivation using two different feed systems resulted in two types of copolymers: random PHBVs and putative block PHBVs.The production of IL-8, which is induced by PHB and PHBVs, may be used to improve and enhance the wound-healing process because of deficiencies of IL-8 in the wound area, particularly in problematic wounds.Taken together, the results support the use of PHB and the random and putative block PHBVs produced in this study as potential biomaterials in tissue engineering applications for connective tissue, bone and dermal fibroblast reconstruction.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok, 10330, Thailand. suchada.cha@chula.ac.th.

ABSTRACT

Background: This study evaluated the biocompatibilities of random and putative block poly(3-hydroxybutyrate-co-3-hydroxyvalerate)s (PHBVs) produced by a metabolic reaction-based system. The produced PHBVs were fractionated, and the copolymer sequence distributions were analyzed using (1)H and (13)C NMR spectroscopy. The thermal properties were analyzed using differential scanning calorimetry (DSC). Mechanical tests were conducted using a universal testing machine. The in vitro cytotoxicities of films composed of random PHBVs and putative block PHBVs were investigated against three types of mammalian cells. The surfaces of the copolymer films and the morphologies of the cells were qualitatively monitored using scanning electron microscopy (SEM) and atomic force microscopy (AFM).

Results: Films composed of poly(3-hydroxybutyrate) (PHB), random PHBVs, putative block PHBVs, polystyrene and polyvinylchloride were prepared and characterized. The diad and triad sequence distributions indicated that the PHBVs produced via the fed-batch cultivation using two different feed systems resulted in two types of copolymers: random PHBVs and putative block PHBVs. The monomer compositions and sequence distributions strongly affected the thermal and mechanical properties. The mechanical integrity and characteristics of the film surfaces changed with the HV content. Notably, the random PHBVs possessed different mechanical properties than the putative block PHBVs. The biocompatibilities of these films were evaluated in vitro against three types of mammalian cells: L292 mouse connective tissue, human dermal fibroblast and Saos-2 human osteosarcoma cells. None of the PHBV films exhibited cytotoxic responses to the three types of mammalian cells. Erosion of the PHA film surfaces was observed by scanning electron microscopy and atomic force microscopy. The production of transforming growth factor-β-1 and interleukin-8 was also examined with regards to the usefulness of PHB and PHBV as biomaterials for regenerative tissue. The production of IL-8, which is induced by PHB and PHBVs, may be used to improve and enhance the wound-healing process because of deficiencies of IL-8 in the wound area, particularly in problematic wounds.

Conclusion: Taken together, the results support the use of PHB and the random and putative block PHBVs produced in this study as potential biomaterials in tissue engineering applications for connective tissue, bone and dermal fibroblast reconstruction.

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The 600 MHz13C NMR spectra of the B1, V1, B2, B3, V2, and V4 carbon resonances of (A) random PHBV with 53 mol% of 3HV (R53) and (B) putative block PHBV with 50 mol% of 3HV (B50).
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Fig1: The 600 MHz13C NMR spectra of the B1, V1, B2, B3, V2, and V4 carbon resonances of (A) random PHBV with 53 mol% of 3HV (R53) and (B) putative block PHBV with 50 mol% of 3HV (B50).

Mentions: This experiment focused on comparing the diad and triad sequence distributions between the random and putative block PHBV samples. The diad and triad sequence distributions were determined from the 600 MHz 13C NMR spectra, which are shown in Figure 1. The obtained relative peak intensities were interpreted in terms of the copolymer sequence distributions. The expanded CO signals of the 3HB unit (B) and of the 3HV unit (V) at 169 to 170 ppm were split into four peaks corresponding to diad sequences of B✳B, B✳V + V✳B, and V✳V. It is clear that the V-centered triad sequences of the random PHBV sample (Figure 1A) had a predominantly random sequence character. The diad and triad sequence distributions of the putative block PHBV samples that were produced using the optimized method and that were well fractionated (Figure 1B) did not show the multiple signals of B✳V + V✳B, and only the V✳V and B✳B signals were detected as a separated signal. Thus, the copolymer sequence distributions of the putative block PHBV samples are different from those of the random PHBVs. It has been reported that the 13C NMR spectra of random and putative block PHBV samples could exhibit a difference around 169–170 ppm and that they are sensitive to the influence of each carbonyl group of every component [28]. Next, the conditional probability for the copolymerization reaction of random PHBV and putative PHBV samples was determined by the nonlinear least squares method using the first-order Markovian model. It has been reported that this model can be applied to analyze random, block and alternative copolymerizations [27,29,30]. The probability derived from the first-order Markovian statistics and the D values of all samples are listed in Table 1. For the completely random PHBV samples, the calculated D values are close to one. For the putative block PHBV samples, the calculated D values are considerably greater than one, and their observed 13C NMR spectra are distinctly different from those of typical random PHBVs.Figure 1


Biocompatibilities and biodegradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)s produced by a model metabolic reaction-based system.

Napathorn SC - BMC Microbiol. (2014)

The 600 MHz13C NMR spectra of the B1, V1, B2, B3, V2, and V4 carbon resonances of (A) random PHBV with 53 mol% of 3HV (R53) and (B) putative block PHBV with 50 mol% of 3HV (B50).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4279891&req=5

Fig1: The 600 MHz13C NMR spectra of the B1, V1, B2, B3, V2, and V4 carbon resonances of (A) random PHBV with 53 mol% of 3HV (R53) and (B) putative block PHBV with 50 mol% of 3HV (B50).
Mentions: This experiment focused on comparing the diad and triad sequence distributions between the random and putative block PHBV samples. The diad and triad sequence distributions were determined from the 600 MHz 13C NMR spectra, which are shown in Figure 1. The obtained relative peak intensities were interpreted in terms of the copolymer sequence distributions. The expanded CO signals of the 3HB unit (B) and of the 3HV unit (V) at 169 to 170 ppm were split into four peaks corresponding to diad sequences of B✳B, B✳V + V✳B, and V✳V. It is clear that the V-centered triad sequences of the random PHBV sample (Figure 1A) had a predominantly random sequence character. The diad and triad sequence distributions of the putative block PHBV samples that were produced using the optimized method and that were well fractionated (Figure 1B) did not show the multiple signals of B✳V + V✳B, and only the V✳V and B✳B signals were detected as a separated signal. Thus, the copolymer sequence distributions of the putative block PHBV samples are different from those of the random PHBVs. It has been reported that the 13C NMR spectra of random and putative block PHBV samples could exhibit a difference around 169–170 ppm and that they are sensitive to the influence of each carbonyl group of every component [28]. Next, the conditional probability for the copolymerization reaction of random PHBV and putative PHBV samples was determined by the nonlinear least squares method using the first-order Markovian model. It has been reported that this model can be applied to analyze random, block and alternative copolymerizations [27,29,30]. The probability derived from the first-order Markovian statistics and the D values of all samples are listed in Table 1. For the completely random PHBV samples, the calculated D values are close to one. For the putative block PHBV samples, the calculated D values are considerably greater than one, and their observed 13C NMR spectra are distinctly different from those of typical random PHBVs.Figure 1

Bottom Line: The diad and triad sequence distributions indicated that the PHBVs produced via the fed-batch cultivation using two different feed systems resulted in two types of copolymers: random PHBVs and putative block PHBVs.The production of IL-8, which is induced by PHB and PHBVs, may be used to improve and enhance the wound-healing process because of deficiencies of IL-8 in the wound area, particularly in problematic wounds.Taken together, the results support the use of PHB and the random and putative block PHBVs produced in this study as potential biomaterials in tissue engineering applications for connective tissue, bone and dermal fibroblast reconstruction.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok, 10330, Thailand. suchada.cha@chula.ac.th.

ABSTRACT

Background: This study evaluated the biocompatibilities of random and putative block poly(3-hydroxybutyrate-co-3-hydroxyvalerate)s (PHBVs) produced by a metabolic reaction-based system. The produced PHBVs were fractionated, and the copolymer sequence distributions were analyzed using (1)H and (13)C NMR spectroscopy. The thermal properties were analyzed using differential scanning calorimetry (DSC). Mechanical tests were conducted using a universal testing machine. The in vitro cytotoxicities of films composed of random PHBVs and putative block PHBVs were investigated against three types of mammalian cells. The surfaces of the copolymer films and the morphologies of the cells were qualitatively monitored using scanning electron microscopy (SEM) and atomic force microscopy (AFM).

Results: Films composed of poly(3-hydroxybutyrate) (PHB), random PHBVs, putative block PHBVs, polystyrene and polyvinylchloride were prepared and characterized. The diad and triad sequence distributions indicated that the PHBVs produced via the fed-batch cultivation using two different feed systems resulted in two types of copolymers: random PHBVs and putative block PHBVs. The monomer compositions and sequence distributions strongly affected the thermal and mechanical properties. The mechanical integrity and characteristics of the film surfaces changed with the HV content. Notably, the random PHBVs possessed different mechanical properties than the putative block PHBVs. The biocompatibilities of these films were evaluated in vitro against three types of mammalian cells: L292 mouse connective tissue, human dermal fibroblast and Saos-2 human osteosarcoma cells. None of the PHBV films exhibited cytotoxic responses to the three types of mammalian cells. Erosion of the PHA film surfaces was observed by scanning electron microscopy and atomic force microscopy. The production of transforming growth factor-β-1 and interleukin-8 was also examined with regards to the usefulness of PHB and PHBV as biomaterials for regenerative tissue. The production of IL-8, which is induced by PHB and PHBVs, may be used to improve and enhance the wound-healing process because of deficiencies of IL-8 in the wound area, particularly in problematic wounds.

Conclusion: Taken together, the results support the use of PHB and the random and putative block PHBVs produced in this study as potential biomaterials in tissue engineering applications for connective tissue, bone and dermal fibroblast reconstruction.

Show MeSH
Related in: MedlinePlus