Three-dimensional collagen/chitosan scaffolds fabricated by freeze-drying technique in 96-well polystyrene and PDMS plates were optimized during this study. Surface tension is, by and large, one of the most limiting factors in fabricating freeze-dried scaffolds in small format well plates. Traditionally, bowl-shaped top surfaces of collagen/chitosan scaffolds were common in polystyrene 96-well plate; whereas for PDMS 96-well plate, dome-shaped surfaces were formed. These surface tension phenomena are not desirable in cell studies especially during initial cell seeding. A combination of surface treatment and change of freeze-drying regime were developed to mitigate the surface tension problem in PS and PDMS 96-well plates respectively. Collagen/chitosan scaffolds of varying concentration and composition were experimented in both polystyrene and PDMS 96-well plates. Thin water film treatment with UV cross-linking was successfully used to eliminate meniscus in PS well plates; pre-cooling, on the other hand, was utilised to treat scaffold solutions in PDMS well plates. The resultant matrices all had flat top surfaces and average thickness of 1 mm. As expected, scaffolds with lower overall polymer concentration or, from a compositional perspective, scaffolds with high chitosan content generally had larger pores. Microscopic observation by multi-photon microscope was performed and chemical analyses were conducted to characterize the surface-treated scaffolds. In addition, scaffolds were tested in vitro using DLD-1 cells, hMSCs and fibroblasts for their biological performance. The purpose of this study was to address the problem of using small format culture wells for the fabrication of freeze-dried collagen-based scaffolds for studies of cell growth in 3D culture and in microfluidic perfusion bioreactors.

Hydrogels aim to mimic the natural living environment by entrapping large amount of water or biological fluids in their polymeric network. There has been growing interest in the development of peptide and protein hydrogels, due to their improved biocompatibility, biodegradability and biological properties in comparison to purely synthetic polymer hydrogels. Under the appropriate conditions, biomacromolecular protein hydrogels can self-assemble into ordered meso- to macroscopic supramolecules with better resulting networks that promote tissue development. The work presented here mainly focuses on producing protein hydrogels with controlled physical properties useful for tissue regeneration process and drug delivery applications. Hen egg white lysozyme (HEWL) hydrogels were studied in the presence of water and different reducing agents forming three HEWL systems including HEWL/water, HEWL/DTT and HEWL/TCEP gels. Strong, self-supporting HEWL gels were successfully prepared in the range of pH 2 to 7, using a temperature of 85°C. At pH 2, the protein denaturation in water was relatively slow resulting in a high percentage of turn structure (~50%) that promotes HEWL gelation after 3 days of heating. No lysozyme gelation in water was observed at pH 3, 4 and 7 even after 21 days of heating. A small quantity of DTT (~20 mM) was added to encourage lysozyme unfolding and HEWL/DTT samples formed gels at higher pH including at physiological pH. The pH 2 HEWL/water gel was found to be stronger but more brittle than pH 7 HEWL/DTT gel. It was observed there were some irregularities in the distribution of pH 2 fibrils (~7µm in length) that form large pore sizes within the network. The pH 7 sample contained shorter and stiff fibrils with repetitive polygon-shaped mesh network. The use of TCEP, which is a stronger reductant than DTT, led to the formation of self-supporting HEWL gels between pH 3.5 and 5.5. The highest storage modulus was observed at pH 5, which is related to the high β-sheet content of the sample (~45%). In addition, a promising strategy has been devised to form thermoresponsive HEWL hydrogels by synthesising and incorporating a small fraction of lysozyme-PNIPAAm bioconjugates into the major protein matrix. Results show the thermoresponsive nature of PNIPAAm was conferred to HEWL protein that exhibits higher storage stability in response to changing temperature.

The biodegradability and when functionalised biocompatibility of aliphatic polycarbonates (APCs) makes them an attractive class of materials for biomedical applications such as tissue engineering scaffolds and drug-delivery carriers. One route to accessing a wide-range of well-defined and functional APCs is the controlled ring-opening polymerisation (ROP) of cyclic carbonates. In turn, these would ideally be prepared by the direct coupling of CO2 with diols to give water as the only by-product. In this way, the combination of CO2 and sugar-derived diols draws upon two natural renewable building blocks for the construction of polycarbonates that are anticipated to show good biocompatibility properties. Chapter 2 develops a simple and mild alternative to the traditional use of phosgene derivatives for the synthesis of six-membered cyclic carbonates from 1,3-diols and CO2. DFT calculations highlighted the need to lower both the CO2-insertion and ring-closing kinetic barriers to cyclic carbonate formation. Organic superbase, 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) enabled the formation of carbonate species at 1 atm CO2 pressure whereas, the introduction of a leaving group strategy lowered the cyclisation barrier. Mechanistic considerations suggested a kinetic preference for ring- closing via a nucleophilic addition-elimination pathway rather than a SN2-like intramolecular cyclisation. Chapter 3 applies the procedure with CO2 to the preparation of a novel monomer from natural sugar, ᴅ-mannose. ROP was carried out via an organocatalytic approach and a preference for head-tail linkages in the polycarbonate backbone indicated by NMR spectroscopy and supported by DFT calculations. Chapter 4 utilises CO2 to invert the natural stereochemistry of sugars and create a thymidine-based monomer. The thermodynamic parameters of the ROP with 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) catalyst are determined and the properties of the polycarbonates investigated to include preliminary cell attachment studies. Finally, chapter 5 details the synthesis of cyclic carbonates from 2- deoxy-ᴅ-ribose and the investigation into the different ROP behaviour of the α- and β- anomers. The ability to tune the polymer properties through copolymerisation with trimethylene carbonate (TMC) is also discussed.

Tissue engineering is a multidisciplinary field that is rapidly emerging as a promising new approach in the restoration and reconstruction of tissues. In this approach, three dimensional (3D) scaffolds are of great importance. Scaffolds function both as supports for cell growth and depot for sustained release of required active agents (e.g. enzymes, genes, antibiotics, growth factors). Scaffolds should possess certain properties in accordance with usage conditions. Wet-spinning is a simple technique that has been widely used for the fabrication of porous scaffolds for tissue engineering applications. Natural polymers can effectively be used in scaffold fabrication due to their biocharacteristics. Among natural polymers, chitosan and alginate are two of the most studied ones in tissue engineering and drug delivery fields because of being biologically renewable, biodegradable, biocompatible, non-antigenic, non-toxic and biofunctional. In this study, two kinds of porous scaffolds were produced as chitosan and alginate coated chitosan fibrous scaffolds by wet-spinning technique In order to investigate the delivery characteristics of the scaffolds, loading of gentamicin as a model antibiotic and bovine serum albumin (BSA) as a model protein was carried out in different loading models. Resultant scaffolds were characterized in terms of their structural formation, biodegradation, biomineralization, water uptake and retention ability and mechanical properties. Additionally, release kinetics of gentamicin and BSA were examined. Efficiency of gentamicin on Escherichia coli (E.coli) was examined. Characterization of scaffolds revealed their adequacy to be used in bone tissue engineering applications and capability to be employed as bioactive agent delivery systems.

This study investigates the effect on: 1) the bulk surface; and 2) the three-dimensional non-woven microfabric scaffolds of poly(N-isopropylacylamide)-CNT-polyaniline on growth and viability of  mice fibroblast cells L929. The poly(N-isopropylacylamide)-CNT-polyaniline was prepared using coupling chemistry and electrospinning was then used for the fabrication of responsive, nonwoven microfabric scaffolds. The electrospun microfabrics were assembled in regular three-dimensional scaffolds with OD: 400-500 mm; L: 6-20 cm. Mice fibroblast cells L929 were seeded on the both poly(N-isopropylacylamide)-CNT-polyaniline bulk surface as well as non-woven microfabric scaffolds. Excellent cell proliferation and viability was observed on poly(N-isopropylacylamide)-CNT-polyaniline non-woven microfabric matrices in compare to poly(N-isopropylacylamide)-CNT-polyaniline bulk and commercially available Matrigel™ even with a range of cell lines up to 168 h. Temperature dependent cells detachment behaviour was observed on the poly(N-isopropylacylamide)-CNT-polyaniline scaffolds by varying incubation at below lower critical solution temperature (LCST) of poly(N-isopropylacylamide). The results suggest that poly(N-isopropylacylamide)-CNT-polyaniline non-woven microfabrics could be used as a smart matrices for applications in tissue engineering. / European Commission FP7 (PIIF-GA-2009-254955), JSPS, JST-CREST and MEXT

Hydrogels are popular materials for biological applications since they exhibit properties like that of natural soft tissue and have tunable properties. Biodegradable hydrogels provide an added advantage in that they degrade in an aqueous environment thereby avoiding the need for removal after the useful lifetime. In this work, we investigated poly(β-amino ester) (PBAE) biodegradable hydrogel systems. To begin, the factors affecting the macromer synthesis procedure were studied to optimize the reproducibility of the resulting hydrogels made and create new methods of tuning the properties. Hydrogel behavior was then tuned by altering the hydrophilic/hydrophobic balance of the chemicals used in the synthesis to develop systems with linear and two-phase degradation profiles. The goal of the research was to better understand methods of controlling hydrogel properties to develop systems for several biomedical applications. Several systems with a range of properties were synthesized, and their in vitro behavior was characterized (degradation, mechanical properties, cellular response, etc.). From these studies, materials were chosen to serve as porogen materials and an outer matrix material to create a composite scaffold for tissue engineering. In most cases, a porous three dimensional scaffold is ideal for cellular growth and infiltration. In this work, a composite with a slow degrading outer matrix PBAE with fast degrading PBAE microparticles was created. First, a procedure for developing porogen particles of controlled size from a fast-degrading hydrogel material was developed. Porogen particles were then entrapped in the outer hydrogel matrix during polymerization. The resulting composite systems were degraded and the viability of these systems as tissue engineering scaffolds was studied. In a second area of work, two polymer systems, one PBAE hydrogel and one sol-gel material were altered through the addition of iron oxide nanoparticles to create materials with remote controlled properties. Iron oxide nanoparticles have the ability to heat in an alternating magnetic field due to the relaxation processes. The incorporation of these nanoscale heating sources into thermosensitive polymer systems allowed remote actuation of the physical properties. These materials would be ideal for use in applications where the system can be changed externally such as in remote controlled drug delivery.

Currently growth plate fractures account for nearly 18.5% of fractures in children and can lead to stunted bone growth or angular deformation. If the body is unable to heal itself a bony bar forms, preventing normal bone growth. Clinical treatment involves removing the bony bar and replacing it with a filler substance, which causes poor results 60% of the time. Using primarily poly(lactic-co-glycolic acid) (PLGA) as the scaffold material, the goal was to develop an implant that would support to the implant site, allow for cell ingrowth, and degrade away over time. Porous scaffolds were fabricated from PLGA microspheres using the salt leaching method. The first part of this work investigated the effect of sintering the microspheres by studying the mechanical properties, degradation and morphology and their potential applications for hard and soft tissue implants. Growth factor or drugs can be encapsulated into PLGA microspheres, which was the second part of this work. Encapsulated insulin-like growth factor I (IGF-I) was able to withstand the scaffold fabrication process without compromising it’s bioactivity and promoted cell proliferation. The next part of this work experimented with the addition of a hydrogel porogen. Porogen particles were made using a quick degrading poly(beta-amino ester) (PBAE) hydrogel and loaded with ketoprofen. The addition of the porogen creates a dual drug-releasing scaffold with a localized delivery system. The final step of this work involved animal studies to determine the effectiveness of the scaffolds in growth plate regeneration and how they compare to the current clinical treatment option. Gross observation, microCT analysis, angular measurement of bone growth and histological methods were employed to evaluate the scaffolds. The goal was to develop a versatile scaffold that could be used for a wide range of tissue engineering applications. The mechanical properties, degradation profiles and drug delivery capabilities can be all tailored to meet the specific needs of an implant site. One specific application was regenerating the native growth plate that can also encourage the endogenous mesenchymal stem cells to follow the desire linage. By regenerating the native growth plate, angular deformation and stunted limb growth were greatly reduced.

In situ forming implants (ISIs) are popular due to their ease of use and local drug delivery potential, but they suffer from high initial drug burst, and release behavior is tied closely to solvent exchange and polymer properties. Additionally, such systems are traditionally viewed purely as drug delivery devices rather than potential scaffold materials due to their poor mechanical properties and minimal porosity. The aim of this research was to develop an injectable ISI with drug release, mechanical, and microstructural properties controlled by micro- and nanoparticle additives. First, an injectable ISI was developed with appropriate drug release kinetics for orthopedic applications. Poly(β-amino ester) (PBAE) microparticles were loaded with simvastatin or clodronate, and their loading efficiency and drug retention after washing was quantified. Drug-loaded PBAE microparticles and hydroxyapatite (HA) microparticles were added to a poly(lactic-co-glycolic acid) (PLGA)–based ISI. By loading simvastatin into PBAE microparticles, release was extended from 10 days to 30 days, and burst was reduced from 81% to 39%. Clodronate burst was reduced after addition of HA, but was unaffected by PBAE loading. Scaffold mass and porosity fluctuated as the scaffolds swelled and then degraded over 40 days. Next, the mechanical properties of these composite ISIs were quantified. Both micro- and nanoparticulate HA as well as PBAE microparticle content were varied. Increasing HA content generally improved compressive strength and modulus, with a plateau occurring at 30% nano-HA. Injectability remained clinically acceptable for up to 10% w/w PBAE microparticles. Ex vivo injections into trabecular bone improved both strength and modulus. Lastly, HA-free ISIs were investigated for drug delivery into the gingiva to treat periodontitis. Doxycycline and simvastatin were co-delivered, with delivery of doxycycline over 1 week accompanied by simvastatin release over 30 days. PBAE-containing ISIs exhibited higher initial and progressive porosity and accessible volume than PBAE-free ISIs over the course of degradation. Additionally, PBAE-containing ISIs provided superior tissue retention within a simulated periodontal pocket. The ISIs investigated here have a wide range of potential applications due to their flexible material and drug release properties, which can be controlled by both the chemistry and concentration of various particulate additives.

No description available.

Additive manufacturing technologies are enabling newfound degrees of geometrical complexity to be realised, particularly with regards to internal structures. All of these manufacturing technologies are dependant on their prior design in an appropriate electronic form, either by reverse engineering, or, primarily, by computer-aided design. Within these emerging applications is the design of scaffolds with an intricate and controlled internal structure for bone tissue engineering. There is a consensus that ideal bone scaffold geometry is evident in biological trabecular structures. In their most basic topological form,these structures consist of the non-linear distribution of irregular interconnecting rods and plates of different size and shape. Complex and irregular architectures can be realised by several scaffold manufacturing techniques, but with little or no control over the main features of the internal geometry, such as size, shape and interconnectivity of each individual element. The combined use of computer aided design systems and additive manufacturing techniques allows a high degree of control over these parameters with few limitations in terms of achievable complexity. However, the design of irregular and intricate trabecular networks in computer aided design systems is extremely time-consuming since manually modelling an extraordinary number of different rods and plates, all with different parameters, may require several days to design an individual scaffold structure. In an attempt to address these difficulties, several other research efforts in this domain have largely focussed on techniques which result in designs which comprise of relatively regular and primitive shapes and do not represent the level of complexity seen biologically. Detailed descriptions of these methods are covered in chapter 1. An automated design methodology for trabecular structures is proposed by this research to overcome these limitations. This approach involves the investigation of novel software algorithms, which are able to interact with a conventional computer aided design program and permit the automated design of geometrical elements in the form of rods, each with a different size and shape. The methodology is described in chapter 2 and is tested in chapter 3. Applications of this methodology in anatomical designs are covered in chapter 4. Nevertheless, complex designed rod networks may still present very different properties compared to trabecular bone geometries due to a lack detailed information available which explicitly detail their geometry. The lack of detailed quantitative descriptions of trabecular bone geometries may compromise the validity of any design methodology, irrespective of automation and efficiency. Although flexibility of a design methodology is beneficial, this may be rendered inadequate when insufficient quantitative data is known of the target structure. In this work a novel analysis methodology is proposed in chapter 5, which may provide a significant contribution toward the characterisation and quantification of target geometries, with particular focus on trabecular bone structures. This analysis methodology can be used either to evaluate existing design techniques or to drive the development of new bio-mimetic design techniques. This work then progresses to a newly derived bio-mimetic automated design technique, driven by the newly produced quantitative data on trabecular bone geometries. This advanced design methodology has been developed and tested in chapter 6. This has demonstrated the validity of the technique and realised a significant stage of development in the context and scope of this work.