Picosecond Laser Ablation of Polyhydroxyalkanoates (PHAs): Comparative Study of Neat and Blended Material Response

Polyhydroxyalkanoates (PHAs) have emerged as a promising biodegradable and biocompatible material for scaffold manufacturing in the tissue engineering field and food packaging. Surface modification is usually required to improve cell biocompatibility and/or reduce bacteria proliferation. Picosecond laser ablation was applied for surface micro structuring of short- and medium-chain length-PHAs and its blend. The response of each material as a function of laser energy and wavelength was analyzed. Picosecond pulsed laser modified the surface topography without affecting the material properties. UV wavelength irradiation showed halved ablation thresholds compared to visible (VIS) wavelength, revealing a greater photochemical nature of the ablation process at ultraviolet (UV) wavelength. Nevertheless, the ablation rate and, therefore, ablation efficiency did not show a clear dependence on beam wavelength. The different mechanical behavior of the considered PHAs did not lead to different ablation thresholds on each polymer at a constant wavelength, suggesting the interplay of the material mechanical parameters to equalize ablation thresholds. Blended-PHA showed a significant reduction in the ablation threshold under VIS irradiation respect to the neat PHAs. Picosecond ablation was proved to be a convenient technique for micro structuring of PHAs to generate surface microfeatures appropriate to influence cell behavior and improve the biocompatibility of scaffolds in tissue engineerin

Biosynthesis of polyhydroxyalkanoates, their novel blends and composites for biomedical applications

Polyhydroxyalkanoates (PHAs) are a family of polyhydroxyesters of 3-, 4-, 5- and 6- hydroxyalkanoic acids produced by bacterial fermentation in a nutrient limiting conditions with excess carbon. They can be produced easily using renewable carbon sources. They are biodegradable and biocompatible in nature. Their physical properties are highly tailorable and a range of desired properties can be achieved based on the type of application. Owing to these properties, there has been a considerable interest in the commercial exploitation of PHAs, particularly for biomedical applications. The main aim of this research project was to produce MCL-PHAs from Pseudomonas mendocina and use them for biomedical applications. In this study, an economical production of MCL-PHAs using renewable and cheap carbon sources such as sugarcane molasses, biodiesel waste and pure glycerol was carried out. Maximum PHA yield of 43.2% dcw was obtained in the media containing biodiesel waste. The results demonstrated the successful utilisation of these cheap carbon sources by P. mendocina for the economical production of MCL-PHAs. One of the main objectives of this project was to utilize the PHAs produced for biomedical applications. Multifunctional novel 2D P(3HO)/bacterial cellulose composite films were developed for their potential use in tissue engineering applications. Chemically modified bacterial cellulose microcrystals were used as the reinforcing agent to improve the properties of P(3HO). Mechanical properties such as the Young’s modulus and tensile strength values of the P(3HO)/bacterial cellulose composite films were significantly higher in comparison to the neat P(3HO) film. Also, the composite film had a rougher and more hydrophilic surface compared to the neat P(3HO) film. It is known from literature that surface roughness and hydrophilicity affects protein adsorption on the surface of the biomaterial. Protein adsorption, in turn, plays an important role in determining the biocompatibility of a material being used for medical applications (Das et al., 2007). In this study, protein adsorption was higher in the P(3HO)/25% bacterial cellulose composite film compared to the neat P(3HO) film. In vitro biocompatibility studies using Human microvascular endothelial cells (HMEC-1) was carried out. Both neat and composite films were able to support the proliferation of HMEC-1 cells. However, the biocompatibility of the P(3HO)/25% bacterial cellulose composite films had increased. The cell proliferation significantly higher on the P(3HO)/25% bacterial cellulose composite film as compared to the neat P(3HO) film on day 7. In addition, multifunctional 2D P(3HO)/P(3HB) blend films with varying percentages of P(3HO) and P(3HB) were developed and assessed for their suitability in the development of biodegradable stents. Mechanical, thermal and microstructural properties of the P(3HO)/P(3HB) blends were characterised. The results highlighted the role of P(3HB) in enhancing the mechanical properties and thermal stability of the blend films compared to the neat P(3HO) films. However, the results suggested that the mechanical properties of the P(3HO)/P(3HB) had to be further improved to meet the desired values required for the development of a biodegradable stent. The overall protein adsorption and % cell viability was significantly higher in the blend films compared to the neat P(3HO) film. Hydrolytic degradation was faster in the blend films and the degradation rate could potentially be tailored to achieve the optimum rate required for a particular medical application. From the literature, it is known that the surface topography determines the compatibility of a biomaterial by governing important processes such as wettability, protein adsorption, cell adhesion and proliferation (Duncan et al., 2007). In this part of the study, P(3HO)/P(3HB) 50:50 blend films were micropatterned using the laser micropatterning technique to improve their biocompatibility. The results demonstrated an increase in hydrophilicity and protein adsorption on the micropatterned blend films compared to the plain P(3HO)/P(3HB) 50:50 blend films. Cell attachment, proliferation and alignment was significantly higher on the micropatterned blend films compared to the P(3HO)/P(3HB) 50:50 blend films which was a desirable outcome. Furthermore, an investigation of the P(3HO)/P(3HB) 50:50 2D films as the base material for the development of a drug eluting biodegradable stent was carried out by incorporating aspirin within the film. The percentage viability of the HMEC-1 cells was higher in the blend films with aspirin compared to the blend films without aspirin indicating an increased biocompatibility of the P(3HO)/P(3HB) 50:50 blend film containing aspirin. Controlled release of aspirin was observed without any burst release and 96.6% release was achieved within 25 days, ideal for the development of biodegradable drug eluting stents. Finally, a drug delivery system for the controlled delivery of aspirin was successfully developed. In this part of the study, 2D solvent cast films and microspheres (average size=30 μm) were developed using P(3HB). Drug release pattern from P(3HB) films as well as P(3HB) microspheres were monitored. The results demonstrated that the P(3HB) films with aspirin were suitable for sustained long term drug release whereas P(3HB) microspheres with aspirin were more suitable for fast release. In conclusion, this project has led to the successful production of PHAs, and their utilisation in the development of a range of composites, blends and drug elution structures with promising potential medical applications

The Cavendish Living lab - a multidisciplinary, vertically integrated project focused on sustainability

Colleagues from the School of Life Sciences will present findings from The Cavendish Living Lab’: a 2 year vertically integrated project (VIP) that focuses on co-creating sustainable solutions with the student participants from various disciplines and levels. Through applied research and learning within an authentic setting, the ‘Living Lab’ approach uses our university campus as the laboratory, and a platform for the students to partner with various stakeholders to address real world issues and develop innovative, sustainable solutions to problems such as food waste, plastic waste, and waste water

Comparison of the Influence of 45S5 and Cu-Containing 45S5 Bioactive Glass (BG) on the Biological Properties of Novel Polyhydroxyalkanoate (PHA)/BG Composites

Polyhydroxyalkanoates (PHAs), due to their biodegradable and biocompatible nature and their ability to be formed in complex structures, are excellent candidates for fabricating scaffolds used in tissue engineering. By introducing inorganic compounds, such as bioactive glasses (BGs), the bioactive properties of PHAs can be further improved. In addition to their outstanding bioactivity, BGs can be additionally doped with biological ions, which in turn extend the functionality of the BG-PHA composite. Here, different PHAs were combined with 45S5 BG, which was additionally doped with copper in order to introduce antibacterial and angiogenic properties. The resulting composite was used to produce scaffolds by the salt leaching technique. By performing indirect cell biology tests using stromal cells, a dose-depending effect of the dissolution products released from the BG-PHA scaffolds could be found. In low concentrations, no toxic effect was found. Moreover, in higher concentrations, a minor reduction of cell viability combined with a major increase in VEGF release was measured. This result indicates that the fabricated composite scaffolds are suitable candidates for applications in soft and hard tissue engineering. However, more in-depth studies are necessary to fully understand the release kinetics and the resulting long-term effects of the BG-PHA composites

Chemical modification of bacterial cellulose for the development of an antibacterial wound dressing

Bacterial cellulose is a bacterially derived polymer with great potential for application in wound healing due to its innate properties such as high biocompatibility and biodegradability. In addition to this, it is naturally biosynthesized by bacteria as a hydrogel, which makes it an optimal substrate for the treatment of dry wounds, where additional moisture is required to facilitate the healing process. However, this polymer lacks antibacterial properties. As bacterial infections are becoming increasingly common and difficult to treat due to antimicrobial resistance, it is of crucial importance to develop strategies for the modification of cellulose to ensure protection against bacterial contamination. In this study, a green-chemistry approach was proposed for the functionalization of cellulose to introduce antibacterial functional groups. Two different active agents, namely glycidyl trimethylammonium chloride and glycidyl hexadecyl ether, were used for the covalent derivatization of the hydroxyl groups of glucose through a heterogeneous reaction in basic aqueous conditions. The modified material was chemically and mechanically characterized by solid-state techniques and rheological measurements. A biological assessment was then carried out both using bacterial cells and human keratinocytes. It was observed that the functionalization performed induced a reduction of approximately half of the bacterial population within 24 h of direct contact with Staphylococcus aureus subsp. aureus Rosenbach 6538PTM and Escherichia coli (Migula) Castellani and Chalmers ATCC® 8739TM (respectively, a reduction of 53% and 43% in the cell number was registered for the two strains). In parallel, cytotoxicity studies performed on keratinocytes (HaCaT cell line) showed cell viability in the range of 90 to 100% for up to 6 days of direct contact with both unmodified and modified samples. The morphology of the cells was also visually evaluated, and no significant difference was noted as compared to the control. Finally, the in vitro scratch assay evidenced good wound closure rates in the presence of the samples, with complete coverage of the scratched area after 5 days for both the modified cellulose and the positive control (i.e., keratinocytes growth medium). Overall, the modified hydrogel showed promising features, confirming its potential as an alternative substrate to develop a sustainable, antibacterial and biocompatible wound dressing

Production of a novel medium chain length Poly(3-hydroxyalkanoate) using unprocessed biodiesel waste and its evaluation as a tissue engineering scaffold

This study demonstrated the utilisation of unprocessed biodiesel waste as a carbon feedstock for Pseudomonas mendocina CH50, for the production of PHAs. A PHA yield of 39.5% CDM was obtained using 5% (v/v) biodiesel waste substrate. Chemical analysis confirmed that the polymer produced was poly(3-hydroxyhexanoate-co-3-hydroxyoctanoate-co-3- hydroxydecanoate-co-3-hydroxydodecanoate) or P(3HHx-3HO-3HD-3HDD). P(3HHx-3HO- 3HD-3HDD) was further characterised and evaluated for its use as a tissue engineering scaffold (TES). This study demonstrated that P(3HHx-3HO-3HD-3HDD) was biocompatible with the C2C12 (myoblast) cell line. In fact, the % cell proliferation of C2C12 on the P(3HHx-3HO-3HD-3HDD) scaffold was 72% higher than the standard tissue culture plastic confirming that this novel PHA was indeed a promising new material for soft tissue engineering

Binary Polyhydroxyalkanoate Systems for Soft Tissue Engineering

Progress in tissue engineering is dependent on the availability of suitable biomaterials. In an effort to overcome the brittleness of poly(3-hydroxybutyrate), P(3HB), a natural biodegradable polyester, and widen its biomedical applications, plasticising of P(3HB) with oligomeric substances of related structure has been studied. A biosynthesised medium-chain-length polyhydroxyalkanoate (mcl-PHA) copolymer, the plasticizer precursor, was obtained using vegetable waste frying oil as a sole carbon source. The mcl-PHA was transformed into an oligomeric derivative by acid hydrolysis. The plasticising effect of the oligomeric mcl-PHA on P(3HB) was studied via characterisation of thermal and mechanical properties of the blends in the course of ageing at ambient conditions. Addition of oligomeric mcl-PHA to P(3HB) resulted in softer and more flexible materials based entirely on PHAs. It was shown that the oligomeric mcl-PHA transformed highly crystalline P(3HB) into materials with a dominant amorphous phase when the content of oligomeric mcl-PHA exceeded 10wt%. In vitro biocompatibility studies of the new binary PHA materials showed high viability and proliferation of C2C12 myoblast cells. Thus, the proposed approach for P(3HB) plasticisation has the potential for the generation of more pliable biomaterials based on P(3HB) which can find application in unique soft tissue engineering applications where a balance between stiffness, tensile strength and ductility is required

Dual production of polyhydroxyalkanoates and antibacterial/antiviral gold nanoparticles

Gold nanoparticles (AuNPs) have been explored for their use in medicine. Here, we report a sustainable, and cost-effective method to produce AuNPs using a bacterial strain such as Pseudomonas mendocina CH50 which is also known to be a polyhydroxyalkanoate (PHA) producer. A cell-free bacterial supernatant, which is typically discarded after PHA extraction, was used to produce spherical AuNPs of 3.5 ± 1.5 nm in size as determined by Transmission Electron Microscopy (TEM) analysis. The AuNPs/PHA composite coating demonstrated antibacterial activity against Staphylococcus aureus 6538P, and antiviral activity, with a 75% reduction in viral infectivity against SARS-CoV-2 pseudotype virus

Biosynthesis and characterization of a novel, biocompatible medium chain length polyhydroxyalkanoate by Pseudomonas mendocina CH50 using coconut oil as the carbon source

This study validated the utilization of triacylglycerides (TAGs) by Pseudomonas mendocina CH50, a wild type strain, resulting in the production of novel mcl-PHAs with unique physical properties. A PHA yield of 58% dcw was obtained using 20 g/L of coconut oil. Chemical and structural characterisation confirmed that the mcl-PHA produced was a terpolymer comprising of three different repeating monomer units, 3-hydroxyoctanoate, 3-hydroxydecanoate and 3-hydroxydodecanoate or P(3HO-3HD-3HDD). Bearing in mind the potential of P(3HO-3HD-3HDD) in biomedical research, especially in neural tissue engineering, in vitro biocompatibility studies were carried out using NG108-15 (neuronal) cells. Cell viability data confirmed that P(3HO-3HD-3HDD) supported the attachment and proliferation of NG108-15 and was therefore confirmed to be biocompatible in nature and suitable for neural regeneration