dissertation presentation - mcl-scl pha blend films for cardiovascular tissue engineering

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MCL-SCL PHA Blend films for Cardiovascular Tissue Engineering

By

ANUBHAV SARKARCardiovascular 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)

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 materials 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

PolyhydroxyalkanoatesPolyhydroxyalkanoates (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 bacterial cell walls

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

Aims of the ProjectProduction 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

Production of P(3HB) using Bacillus subtilis OK2Steps involved in P(3HB) production:

Polymer CharacterisationSeed Culture(Nutrient broth)Production stageK-R mediaBiomass Harvest and LyophilisationPolymer ExtractionPolymer production

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

POLYMER CHARACTERISATION

FTIR ( Fourier Transform Infrared Spectroscopy).

GC-MS (Gas Chromatography Mass spectroscopy)

Thermal properties of the were measured by DSC (Differential Scanning Calorimetry).

G:\final yr project\FTIR results\FTIR.jpgIR 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

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

G:\final yr project\DSC results\PHB students.jpgThermal properties of obtained polymer carried out using DSC

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 .

G:\final yr project\images\IMAG2403.jpgMicro-patterningMicro-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.

http://www.oxfordlasers.com/wp/wp-content/uploads/2012/01/patterning-banner-120x120.jpg

- DSCTensile test

SEM (Scanning Electron Microscopy) image of surface topography

P(3HB)/PHA 10:90 POLYMER BLEND CHARACTERISATION

DSC results of the blend

D:\final yr project\DSC results\DSC polymer blend.jpgMechanical properties of obtained P(3HB)/PHA1 10:90 blend

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

ConclusionsSuccessful 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.

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 WorkAcknowledgementsProf. Ipsita Roy

Dr. Pooja Basnett

Dr. Rinat Nigmatulin

Barbara Lukasiewicz

All members of C7.01, University of Westminster

Thank you

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dissertation presentation - mcl-scl pha blend films for cardiovascular tissue engineering

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