Article pubs.acs.org/journal/abseba
Influence of Hydroxyl Groups on the Cell Viability of Polyhydroxyalkanoate (PHA) Scaffolds for Tissue Engineering Chayatip Insomphun,† Jo-Ann Chuah,† Shingo Kobayashi,‡ Tetsuya Fujiki,‡ and Keiji Numata*,† †
Enzyme Research Team, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Kaneka Corporation, 1-8 Miyamae-cho, Takasago-cho, Takasago, Hyogo 676-8688, Japan
‡
S Supporting Information *
ABSTRACT: Polyhydroxyalkanoates (PHAs) are biopolyesters that have been studied as tissue engineering materials because of their biodegradability, biocompatibility, and low cytotoxicity. In this study, poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-2,3dihydroxybutyrate) [PHBVDB] containing hydroxyl groups was produced by recombinant Ralstonia eutropha. R. eutropha were constructed to express the propionate-coenzymeA transferase (pct) gene from Megasphaera elsdenii, and glycolate was used as the carbon source. Disruption of phaA encoding β-ketothiolase in the phaCAB operon increased 2,3-dihydroxybutyrate (2,3-DHBA) compositions to 3 mol %. The PHBVDB film showed a lower water contact angle compared with other PHA films, indicating increased hydrophilicity due to the hydroxyl groups. The mechanical properties of the PHBVDB scaffold met the requirements for a soft tissue matrix. The effect of hydroxyl groups on cytotoxicity was evaluated with human mesenchymal stem cells. Results of cell proliferation and live/dead assays showed that the PHBVDB scaffold did not exhibit significant cytotoxicity toward the cells. These results indicate that PBVDB containing hydroxyl groups could be applied as a hydrophilicity-controlled scaffold for soft tissue engineering. KEYWORDS: polyhydroxyalkanoate, hydroxyl group, Ralstonia eutropha, scaffold, tissue engineering
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INTRODUCTION Polyhydroxyalkanoates (PHAs) are a family of biodegradable polyesters that are produced intracellularly by many microorganisms as carbon and energy storage compounds under unbalanced growth conditions.1−3 PHAs are also biomass-based thermoplastics, which is attractive for a sustainability.4 To date, a variety of PHAs containing 3-hydroxybutyrate (3HB) and a second monomer unit, such as 4-hydroxybutyrate (4HB), 5-hydroxyvalerate (5HV), 3-hydroxyvalerate (3HV), 3-hydroxyhexanoate (3HHx), or 3-hydroxy-10-undecanoate (3HU), have been produced by using different carbon sources, culture conditions, and bacterial strains.5−9 A combination of various types of monomers in different compositions can also be achieved by metabolic engineering. PHAs have been developed as bioimplantable materials for many medical applications because of their biodegradability, biocompatibility, and low cytotoxicity.10,11 One well-known medical application for PHAs is as a scaffold in tissue engineering. PHA scaffolds have been used to replace or regenerate living tissue by providing support for cell growth and attachment.8,12,13 However, some restrictions, such as hydrophobicity, have limited their use in medical applications. In tissue engineering, © XXXX American Chemical Society
the hydrophilicity of the scaffold surface influences the cell response14,15 because cells adhere and grow easily on moderately hydrophilic materials than on hydrophobic surfaces.16−18 Therefore, it is necessary to adjust the hydrophilicity of PHA to improve and fine-tune a scaffold for tissue engineering. One solution is to combine functional PHA with hydrophilic groups such as carboxylic, amine, or hydroxyl groups. In a previous study, we successfully biosynthesized a new PHA copolymer with pendant hydroxyl groups, poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-2,3-dihydroxybutyrate) (PHBVDB), in Escherichia coli using glycolate as a carbon source.19 The propionate-CoA transferase (pct) gene from Megasphaera elsdenii and the β-ketothiolase (bktB) gene and phaCAB operon from Ralstonia eutropha H16 were introduced into E. coli JM109. A novel monomer containing a hydroxyl group, dihydroxybutyrate (DHBA), was the expected product of the condensation of glycolyl-CoA and acetyl-CoA by BktB. However, PHA production Special Issue: PHA Biomaterials Received: May 22, 2016 Accepted: August 17, 2016
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DOI: 10.1021/acsbiomaterials.6b00279 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
purchased from TAKARA (Shiga, Japan). Ligation mixture was purchased from TOYOBO (Osaka, Japan). A synthetic gene encoding pct from M. elsdenii (DDBJ accession number: LC126829, 1554 bp) with codon optimization was synthesized by Operon Biotechnologies (Tokyo, Japan). The ribosome binding site and linker (AAAGGAGGAACAACC) were added. The synthesized fragment was digested by EcoRI and BamHI and then inserted into pBBR1-MCS2 at the corresponding sites to obtain pBBR-pct. A coding region of bktB (H16_A1446, 1540 bp) was amplified by PCR with genomic DNA of R. eutropha H16 and bktB-FwXhoI/bktB-RvEcoRI as a template and a primer pair, respectively. The amplified fragment was digested by XhoI and EcoRI and then inserted into pBBR-pct at the corresponding sites to obtain pBBR-bktB-pct. pBBR-phaC1Ac and pBBR-pct-phaC1Ac were constructed as follows. A coding region of phaC1Ac was amplified by PCR with pJRDEE32d3 and phaC1Ac-FBamHI/phaC1Ac-RXbaI as a template and a primer pair. The amplified fragment was digested by BamHI and XbaI and then inserted into pBBR1-MCS2 and pBBR-pct at the corresponding sites to obtain pBBR-phaC1Ac and pBBR-pct, respectively. pBBR-phaC1Ps and pBBR-pct-phaC1Ps were constructed as follows. A coding region of phaC1Ps was amplified by PCR with pGEM″CAB and phaC1Ps-FBamHI/phaC1Ps-RXbaI as a template and a primer pair. The amplified fragment was digested by BamHI and XbaI and then inserted into pBBR1-MCS2 and pBBR-pct at the corresponding sites to obtain pBBR-phaC1Ac and pBBR-pct, respectively. For the deletion of phaA from the chromosome of R. eutropha strains, plasmid pK18ΔphaA was constructed as follows. A coding region of phaA (H16_A1438, 1182 bp) along with the upstream and downstream flanking regions (approximately 1 kbp each) was amplified by PCR with genomic DNA of R. eutropha H16 and phaCFw/phaB-Rv as a template and a primer pair, respectively. The amplified fragment was 5′-phosphorylated and inserted into pK18mobsacB at the SmaI site. Inverse PCR using phaA-UpR and phaA-DownF primers was performed to remove the phaA coding region, and the
and DHBA composition were relatively low (PHA content
