Poly(3-hydroxybutyrate) (P(3HB)) is a stiff, brittle bacterial polyester whose progressive embrittlement during ambient storage — driven by secondary crystallization and physical ageing of the amorphous phase — limits its use in soft tissue engineering. Lukasiewicz et al. (Acta Biomaterialia 71, 2018, 225–234) showed that an oligomeric medium-chain-length polyhydroxyalkanoate (an acid-hydrolyzed mcl-PHA of Mw ≈ 10 kDa, termed oligo-HA) strongly plasticizes P(3HB), but uncovered a puzzling non-monotonicity: after 7 weeks of ambient ageing, the 90/10 blend is simultaneously stiffer, stronger and more ductile than the 95/5 blend despite containing less P(3HB). The authors hypothesized that the rigid amorphous fraction (RAF) may change non-monotonically with oligo-HA content, offered crystallite-size differences as the alternative explanation, and stated the effect requires further investigation; the mobile/rigid amorphous partitioning of these blends was never quantified. Plasticizer partitioning between the mobile and rigid amorphous phases has so far been quantified (Barbosa et al., ACS Omega 2021) only for unaged PHBV/oligomeric-polyester blends, not for P(3HB)/oligo-HA blends. The problem is therefore open: establish the crystalline/RAF/mobile-amorphous composition of these blends as a function of oligo-HA content and ageing time, and identify the microstructural factor that causes the non-monotonic aged mechanical response.

Background

Polyhydroxyalkanoates (PHAs) are bacterial storage polyesters, and poly(3-hydroxybutyrate) (P(3HB)) is their most-studied member: a biodegradable, biocompatible thermoplastic melting at about 170–180 °C. Its biomedical use is limited by stiffness and brittleness: P(3HB) is highly crystalline, and because its glass transition lies near room temperature, stored articles embrittle progressively. This ageing is attributed to two coupled processes — secondary crystallization (growth and perfection of lamellar crystals) and physical ageing of the amorphous phase (densification of the glass toward equilibrium). The amorphous phase of semicrystalline polymers is conventionally described by the three-phase model: besides the crystalline fraction, the amorphous phase splits into a mobile amorphous fraction (MAF), which has bulk-like mobility and a normal glass transition, and a rigid amorphous fraction (RAF), the interlamellar material whose chains are constrained by crystal surfaces and which vitrifies and devitrifies differently from the bulk. For P(3HB) specifically, calorimetry shows that roughly two thirds of the amorphous phase is rigid (Schick, Wurm and Mohamed, 2001); crystal and amorphous fractions evolve measurably over weeks of storage (Di Lorenzo and Righetti, 2013; Righetti, Tombari and Di Lorenzo, 2013); and stiffness scales with the combined crystalline-plus-RAF content while ductility scales with the MAF (Righetti et al., 2019). Plasticization is the standard remedy for P(3HB) brittleness. Prior work on P(3HB) with a low-molecular-mass atactic PHB-diol showed that an oligomeric plasticizer does not distribute homogeneously: the MAF is richer in plasticizer than the RAF, and both amorphous populations could be resolved through their glass transitions (El-Taweel et al., 2004). Studies of P(3HB) with low-molar-mass plasticizers further showed that plasticizers slow physical ageing but can themselves phase-segregate during ageing or annealing (Kurusu et al., 2015; Crétois et al., 2016). Against this backdrop, Lukasiewicz, Basnett, Nigmatullin, Matharu, Knowles and Roy (2018) prepared the first fully-PHA plasticized P(3HB) materials. A medium-chain-length PHA copolymer, P(3HHx-3HO-3HD-3HDD) — a random copolyester of 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate and 3-hydroxydodecanoate units — biosynthesized by Pseudomonas mendocina CH50 from waste frying oil, was depolymerized by acid hydrolysis to a waxy, nearly amorphous oligomer of Mw ≈ 10 kDa (abbreviated oligo-HA, for oligomeric hydroxyalkanoate — the name has no connection to hyaluronic acid) and solvent-cast (i.e., films cast from polymer solution) into blends with P(3HB) at 95/5, 90/10 and 80/20 wt%. The oligomer sharply reduced crystallinity and stiffness and improved ductility, and C2C12 myoblasts (an immortalized mouse muscle-precursor cell line) proliferated well on all blends. Two observations, however, were left mechanistically unexplained. First, over 7 weeks of ambient ageing, the crystallinity measured by differential scanning calorimetry (DSC, the standard technique for quantifying crystalline fraction and glass transitions from heat-flow signals) stabilized after about two weeks while tensile properties kept evolving — implicating physical ageing of an amorphous phase that constitutes more than half of the material, whose internal structure the paper did not resolve beyond total crystallinity. Second, and most strikingly, the aged 90/10 blend showed higher Young's modulus and tensile strength than the aged 95/5 blend, together with a larger elongation at break (6.2% versus 3.8%), despite containing less P(3HB) — a non-monotonic composition dependence of every aged mechanical property. Because RAF was never measured in these blends, the authors could only hypothesize that the rigid amorphous fraction might change non-monotonically with oligo-HA content, with differences in crystallite sizes as the alternative explanation, explicitly stating that such an effect of relatively small oligo-HA amounts on the mechanical properties of aged samples requires further investigation.

Problem Statement

For solvent-cast blends of P(3HB) with an oligomeric medium-chain-length polyhydroxyalkanoate plasticizer (oligo-HA, Mw ≈ 10 kDa, nearly amorphous), aged at ambient conditions: (1) determine quantitatively how the material partitions into crystalline, rigid amorphous and mobile amorphous fractions — closing the three-phase mass balance — as a function of oligo-HA content across the source's blend series (neat P(3HB) and 95/5, 90/10, 80/20 wt% P(3HB)/oligo-HA) and of ageing time over the source's ambient-ageing window (reported as up to 7 weeks), including the aged 95/5 and 90/10 compositions between which the anomaly appears; in particular, establish whether the rigid amorphous fraction varies non-monotonically with oligo-HA content, as the source hypothesized; and (2) identify which microstructural factor or factors — non-monotonic rigid-amorphous-fraction evolution, differences in crystallite size or lamellar morphology, or related phase-structural features such as oligo-HA distribution or segregation during ageing — cause the non-monotonic composition dependence of the aged mechanical properties, of which the higher stiffness and strength of the aged 90/10 blend relative to the aged 95/5 blend (together with its larger elongation at break) is the defining instance. The overarching objective is the mechanistic link between blend composition, ageing-dependent three-phase microstructure, and the ageing trajectory of stiffness, strength and ductility in these all-PHA materials, including the causal microstructural variable(s) and their quantitative evolution linking blend composition and ageing-dependent three-phase microstructure to the aged mechanical response.

References

  1. Barbara Lukasiewicz, Pooja Basnett, Rinat Nigmatullin, Rupy Matharu, Jonathan C. Knowles, Ipsita Roy. 'Binary polyhydroxyalkanoate systems for soft tissue engineering.' Acta Biomaterialia 71 (2018) 225–234. DOI: 10.1016/j.actbio.2018.02.027. https://doi.org/10.1016/j.actbio.2018.02.027
  2. C. Schick, A. Wurm, A. Mohamed. 'Vitrification and devitrification of the rigid amorphous fraction of semicrystalline polymers revealed from frequency-dependent heat capacity.' Colloid & Polymer Science 279 (2001) 800–806. DOI: 10.1007/s003960100507. https://doi.org/10.1007/s003960100507
  3. S.H. El-Taweel, G.W.H. Höhne, A.A. Mansour, B. Stoll, H. Seliger. 'Glass transition and the rigid amorphous phase in semicrystalline blends of bacterial polyhydroxybutyrate PHB with low molecular mass atactic R,S-PHB-diol.' Polymer 45 (2004) 983–992. DOI: 10.1016/j.polymer.2003.12.007. https://doi.org/10.1016/j.polymer.2003.12.007
  4. Maria Laura Di Lorenzo, Maria Cristina Righetti. 'Evolution of crystal and amorphous fractions of poly[(R)-3-hydroxybutyrate] upon storage.' Journal of Thermal Analysis and Calorimetry 112 (2013) 1439–1446. DOI: 10.1007/s10973-012-2734-3. https://doi.org/10.1007/s10973-012-2734-3
  5. Maria Cristina Righetti, Elpidio Tombari, Maria Laura Di Lorenzo. 'The Role of the Crystallization Temperature on the Nanophase Structure Evolution of Poly[(R)-3-hydroxybutyrate].' The Journal of Physical Chemistry B 117 (2013) 12303–12311. DOI: 10.1021/jp4063127. https://doi.org/10.1021/jp4063127
  6. Maria Cristina Righetti, Laura Aliotta, Norma Mallegni, Massimo Gazzano, Elisa Passaglia, Patrizia Cinelli, Andrea Lazzeri. 'Constrained Amorphous Interphase and Mechanical Properties of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate).' Frontiers in Chemistry 7 (2019) 790. DOI: 10.3389/fchem.2019.00790. https://doi.org/10.3389/fchem.2019.00790
  7. R. Crétois, J.-M. Chenal, N. Sheibat-Othman, A. Monnier, C. Martin, O. Astruz, R. Kurusu, N.R. Demarquette. 'Physical explanations about the improvement of PolyHydroxyButyrate ductility: Hidden effect of plasticizer on physical ageing.' Polymer 102 (2016) 176–182. DOI: 10.1016/j.polymer.2016.09.017. https://doi.org/10.1016/j.polymer.2016.09.017
  8. Rafael Salles Kurusu, Chris Alexandra Siliki, Éric David, Nicole Raymonde Demarquette, Catherine Gauthier, Jean-Marc Chenal. 'Incorporation of plasticizers in sugarcane-based poly(3-hydroxybutyrate)(PHB): Changes in microstructure and properties through ageing and annealing.' Industrial Crops and Products 72 (2015) 166–174. DOI: 10.1016/j.indcrop.2014.12.040. https://doi.org/10.1016/j.indcrop.2014.12.040
  9. Rodrigo T. Umemura, Maria Isabel Felisberti. 'Modeling of the properties of plasticized poly(3-hydroxybutyrate) as a function of aging time and plasticizer content.' Materials Today Communications 25 (2020) 101439. DOI: 10.1016/j.mtcomm.2020.101439. https://doi.org/10.1016/j.mtcomm.2020.101439
  10. Jacqueline L. Barbosa, Giovanni B. Perin, Maria Isabel Felisberti. 'Plasticization of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with an Oligomeric Polyester: Miscibility and Effect of the Microstructure and Plasticizer Distribution on Thermal and Mechanical Properties.' ACS Omega 6 (2021) 3278–3290. DOI: 10.1021/acsomega.0c05765. https://doi.org/10.1021/acsomega.0c05765
  11. Denis Mihaela Panaitescu, Adriana Nicoleta Frone, Cristian-Andi Nicolae, Augusta Raluca Gabor, Dana Maria Miu, Mariana-Gratiela Soare, Bogdan Stefan Vasile, Irina Lupescu. 'Poly(3-hydroxybutyrate) nanocomposites modified with even and odd chain length polyhydroxyalkanoates.' International Journal of Biological Macromolecules 244 (2023) 125324. DOI: 10.1016/j.ijbiomac.2023.125324. https://doi.org/10.1016/j.ijbiomac.2023.125324
  12. Rosa Turco, Gabriella Santagata, Iolanda Corrado, Cinzia Pezzella, Martino Di Serio. 'In vivo and Post-synthesis Strategies to Enhance the Properties of PHB-Based Materials: A Review.' Frontiers in Bioengineering and Biotechnology 8 (2021) 619266. DOI: 10.3389/fbioe.2020.619266. https://doi.org/10.3389/fbioe.2020.619266
  13. Caroline S. Taylor, Mehri Behbehani, Adam Glen, Pooja Basnett, David A. Gregory, Barbara B. Lukasiewicz, Rinat Nigmatullin, Frederik Claeyssens, Ipsita Roy, John W. Haycock. 'Aligned Polyhydroxyalkanoate Blend Electrospun Fibers as Intraluminal Guidance Scaffolds for Peripheral Nerve Repair.' ACS Biomaterials Science & Engineering 9 (2023) 1472–1485. DOI: 10.1021/acsbiomaterials.2c00964. https://doi.org/10.1021/acsbiomaterials.2c00964