The document discusses the development of short chain length polyhydroxyalkanoates (scl-PHA) for cardiovascular tissue engineering, highlighting the production of p(3hb) using Bacillus subtilis and the creation of 2D mcl-scl PHA blend films. Key properties of the produced polymers were evaluated through various characterizations including FTIR, GC-MS, and DSC, confirming their suitability for cell attachment and tissue regeneration. Future work includes biocompatibility studies and further optimization of the blend films for enhanced performance in cardiovascular applications.

1. MCL-SCL PHA Blend films for Cardiovascular Tissue
Engineering
By
ANUBHAV SARKAR

2. Cardiovascular tissue engineering
• Cardiovascular diseases (CVD) – leading cause of death; myocardial infarction is the main
cause of CVDs (Bagdadi, 2013)
•
• Cardiac therapies in existence but requires additional improvement to control progression
of disease
•
• WHY CARDIOVASCULAR TISSUE ENGINEERING?
• Lack of organ donors
• Post-operative complications (sepsis, infection, organ rejection)
• Promising alternative method
•
• Synthetic materials used so far
• PLA (polylactic acid)
• PCL (polycaprolactone)
• PGA (polyglycolic acid)
• PLGA (polylactic-glycolic acid)
•
•

3. An ideal biomaterial should have 5 characteristics:
• biocompatible in nature,
• should have similar mechanical properties to the host tissue,
• should have appropriate size and shape to organise cells and repair at
implant site,
• chemistry of the material’s surface should allow cell attachment,
differentiation and proliferation
• the composition of the material should allow biodegradation for tissue
regeneration.
Properties of an “Ideal biomaterial” for tissue engineering scaffold

4. Polyhydroxyalkanoates
• Polyhydroxyalkanoates (PHAs) are bacteria-synthesized intracellularly
accumulated polyesters, produced by both gram positive and gram negative
bacteria in a limiting environment in the presence of excess carbon. (Basnett,
2014)
•
Bacteria containing granules of PHAs inside their

5. • Depending on the number of carbon atoms present in their monomer units,
PHAs can be classified into two main types – short chain length PHA
(SCL-PHA) that have 3-5 carbon atoms and medium chain length PHA
(MCL-PHA) that have 6-14 carbon atoms.
• SCL-PHAs are generally brittle, have very high melting point and crystallinity
and are used in bone tissue engineering, drug delivery, nerve regeneration,
while MCL-PHAs are elastomeric in nature, have low melting temperature
and crystallinity, used mostly for soft tissue engineering. (Rai, et.al, 2011)
•
•
Polyhydroxyalkanoates

6. Aims of the Project
• Production of scl-PHAs (short chain length) named
P(3HB) using Bacillus subtilis OK2
•
• Production of 2D mcl-scl PHA blend films in a ratio of
90:10 by weight and evaluation of the blends for
cardiovascular tissue engineering
•
• Comparison of micro-patterned and non micro-patterned
blend films to study cell-cell communication and
cellular behaviour in multidimensional environments in
vitro

7. Production of P(3HB) using Bacillus subtilis OK2
Steps involved in P(3HB) production:
Polymer
Characterisation
Seed Culture
(Nutrient broth)
Production
stage
K-R media
Biomass Harvest
and
Lyophilisation
Polymer
Extraction

8. Polymer production

9. COMPARISON OF POLYMER YIELD (%DCW) BETWEEN
SHAKEN FLASK AND 5L BIOREACTOR
SHAKEN FLASK
• Weight of Biomass – 8.30 g
• Weight of Polymer – 1.76 g
• Polymer yield (%DCW)
– 21.20%
• Weight of Biomass – 17.64 g
• Weight of Polymer – 6.50 g
• Polymer yield (%DCW)
– 36.84%
BIOREACTOR

10. POLYMER CHARACTERISATION
• FTIR ( Fourier Transform Infrared
Spectroscopy).
• GC-MS (Gas Chromatography – Mass
spectroscopy)
• Thermal properties of the were measured
by DSC (Differential Scanning
Calorimetry).

11. IR spectrum of the polymer showing the presence of two characteristic absorption peaks present in
SCL-PHAs; 1721.50 cm-1 corresponding to the ester carbonyl group and 1278.88 cm-1 corresponding to
the –CH2 group thus confirming that the polymer produced is of a SCL-PHA type.
FTIR spectrum of obtained polymer

12. Gas chromatogram of the P(3HB) . A peak with the retention time (Rt) of 4.096 min corresponds to the
methyl ester of 3-hydroxybutyric acid (3HB). A peak with the retention time (Rt) of 6.425 min
corresponds to methyl benzoate, which was used as an internal standard
GS-MC spectrum of obtained polymer

13. Thermal properties of obtained polymer carried
out using DSC

14. PREPARATION OF NOVEL P(3HB)/PHA1 BLENDS
• PHA1, a MCL-PHA was provided by Dr. Pooja Basnett in order to prepare the solvent cast
films of P(3HB)/PHA1.
•
• P(3HB)/PHA1 blends with an uniform composition of 10:90 and neat PHA1 films were
synthesised using the solvent cast technique. Both the polymers were dissolved in
chloroform in order to obtain a polymer concentration of 5wt% in ratios of 10:90.
•
• The polymer solution was well mixed by using magnetic spinning and then cast in a glass
petri dish. The films were eventually left for air drying .

15. Micro-patterning
• Micro-patterning is the microscopy level of patterning that enables better
attachment of cells.
• There are several different micro-patterning techniques being used to
understand the morphology of the cells such as micro-contact printing,
photo-patterning and laser-patterning (Basnett, 2014).
•
• Laser micro-patterning is one of the well established techniques used for
surface fabrication of scaffolds, stents, and vascular grafts.

16. - DSC
- Tensile test
- SEM (Scanning Electron
Microscopy) image of surface
topography
P(3HB)/PHA 10:90 POLYMER BLEND
CHARACTERISATION

17. DSC results of the blend

18. Mechanical properties of obtained
P(3HB)/PHA1 10:90 blend

19. SEM IMAGES OF SURFACE TOPOGRAPHY OF
P(3HB)/PHA1 10:90 POLYMER BLENDS
Scanning electron microscopy results showing the smooth surface of P(3HB)-PHA1 blends. SEM was
carried out at Eastman Dental College, University College London

20. Conclusions
• Successful production of P(3HB) from Bacillus subtillis OK2 was
carried out. Profiling was also done to measure different
parameters such as optical density, pH, biomass estimation and
glucose concentration.
•
• FTIR, GC-MS and DSC was performed to determine the different
characteristics of the polymer obtained and the results
confirmed that it was a scl-PHA.
•
• Successfully prepared mcl-scl PHA blend films for cardiovascular
tissue engineering. SEM, DSC and tensile testing was done on
the blend films. Based on the different characteristic results
obtained, it looks like mcl-scl PHAs are promising materials for
cardiovascular tissue engineering.

21. • Micropatterning work on P(3HB)/PHA1 10:90 blend films
at Tekniker, Spain
• Biocompatibility study using mouse myoblasts (C2C12)
cell line on blend films with and without patterning –
MTT assay
•
• SEM imaging of the scaffold with seeded cells
•
• Complete characterisation of the blend films – surface
roughness analysis, static wettability studies (water
contact angle)
Future Work

22. Acknowledgements
• Prof. Ipsita Roy
•
• Dr. Pooja Basnett
•
• Dr. Rinat Nigmatulin
•
• Barbara Lukasiewicz
•
• All members of C7.01, University of Westminster
•

23. Thank you