1. Preparation and characterisation of polyhydroxyalkanoate-based macrodiols as
building blocks for biomedical polyesterurethanes
ABSTRACT:
Nowadays, research in the biomedical field has reached a high quality level of results in materials for
application in tissue engineering and regenerative medicine. Polymers actually have a very interesting
and innovative role in the employment of new materials in medicine. It is possible to define two main
different classes of polymers in the market: synthetic or natural. The first group present relevant
qualities, such as the possibility to easily process them into many different shapes to achieve
particular physical or thermal properties or the feasibility of functionalization with biomolecules. On
the other hand, they also show some shortcomings, concerning poor biocompatibility and
biodegradability. Natural polymers are produced by living organisms, and this fact can potentially
overcome these drawbacks. In this work a specific family of natural polymers, called
polyhydroxyalkanoates (PHAs), has been investigated. They are polyesters based on
hydroxycarboxylate monomers, primarily produced by bacteria, as Gram-negative as Gram-positive,
for energy reserves under limiting survival conditions, such as excess of carbon and shortage of
oxygen or nitrogen. In nature, it is possible to find two different types of polyhydroxyalkanoates,
depending on the length of the carbon atoms chain: short chain length (scl) (from 3 to 5 atoms) and
medium chain length (mcl) (from 5 to 14 or more). PHAs can be promising materials in the near future
for tissue engineering applications, in view of some interesting properties, such as their good
biocompatibility and biodegradability. Moreover, because of their versatility, PHAs can be also added
to other materials actually present in the market to modify their behaviour and improve their
characteristics. In this sense, polyurethanes (PUs) are very attractive materials for tissue engineering
and regenerative medicine purposes. They are segmented block copolymers, made by three building
blocks, a diol, a diisocyanate and a chain extender. PU chemical versatility gives the possibility of
tuning their properties accordingly to the application required. Therefore, it is possible to find
examples of PUs for hard tissue repair, such as bone tissue, but also PUs for soft tissue regeneration,
like skin or muscles. In such a challenging context, the goal of this thesis was the exploitation of
PHAs as building blocks for PU synthesis, with the final aim of designing innovative polymers with
improved physico-chemical properties for application in tissue engineering/regenerative medicine.
Hence, two different PHAs diols, one scl, poly(3-hydroxybutyrate) and one mcl, poly(3-
hydroxyoctanoate-co-3-hydroxydecanoate-co-3-hydroxydodecanoate) , have been produced and
characterised at the University of Westminster in London, under the supervision of Prof. Ipsita Roy.

2. While for scl PHA diol production, the biopolymer was purchased and properly purified prior to use,
for mcl PHA diol, the starting raw material was synthesised by Pseudomonas family in prof. Roy’s
lab. For both of these polymers a reaction of transesterification was carried out in order to make them
diols. The synthesised diols were characterised trough Differential Scanning Calorimetry (DSC),
Proton Nuclear Magnetic Resonance (1H-NMR), Carbon Nuclear Magnetic Resonance (13C-NMR)
and Size Exclusion Chromatography (SEC). With the aim to determine the quantity of hydroxyl
groups present at the end of the reaction a novel method based on Fourier Transform Infrared
spectroscopy (FTIR) was introduced to assess the hydroxyl number of the synthesised PHAs diols.
Characterisation highlighted the repeatability of the transesterification reaction. In fact, PHAs diols
showed low polydispersity, no signs of degradation or impurities and good consistency in hydroxyl
groups content. The diols produced in London were finally exploited in the synthesis of PUs at the
Biomedical Laboratory of Politecnico di Torino. A library of polyurethanes was synthesised, differing
in the composition of their soft segment (macrodiols), and thoroughly characterised by Attenuated
Total Reflectance Fourier Transform Infra Red (ATR FT-IR), SEC, contact angle test and tensile
strength test. PUs produced showed hydrophobic behaviour and improvements in molecular weight
and mechanical properties. In addition, feasibility of making scaffold by Thermally Induced Phase
Separation (TIPS) was investigated and relative morphology was studied through Scanning Electron
Microscopy (SEM). The introduction of PHAs based macrodiols in PU backbone allowed the
fabrication of more structurally ordered scaffold, with a medium pore size of 65µm. Therefore, it is
reasonable studying in deep these new type of polyurethanes for subsequent biomedical applications.