The application of atomic force microscopy in the surface analysis of polymeric biomaterials

When a polymeric biomaterial is employed within a living system an interface is created between the solid surface of the polymer and an aqueous environment. The processes that occur at this interface will determine if the biomaterial is accepted by the patient and often will determine if the specific function of the biomaterial can be achieved. Increasingly, novel biomaterials are expected to perform more sophisticated functions and, therefore, their surfaces must be designed to realize precise interfacial events, such as specific interactions with proteins and cells or controlled biodegradation. To design polymeric biomaterials with specific surface properties it is necessary to develop surface analytical techniques that can accurately characterize these properties. The work described in this thesis has aimed to investigate the potential contribution of the atomic force microscope (AFM) to this characterization. The advantages of utilizing AFM in the study of polymeric biomaterials lie in the ability of the instrument to visualize insulating surfaces at a high resolution within a variety of environments, including gaseous and liquid environments. Therefore, it is possible to image the nanoscopic organization of polymeric biomaterials within environmental conditions that are similar to the conditions encountered within living systems. Initial studies have concentrated on imaging the surface morphology of poly(ethylane oxide) (PEG) samples in air. These studies highlighted the high resolution capability of the AFM on untreated polymer samples. On sphemlitic samples, the AFM has visualized the lamellar organization of crystalline fibres. These lamellae had widths of between 10 and 30 nm and height variations of less than 15 nm. The ability of the AFM to resolve such structures, without the introduction of an etching or staining procedure required by transmission electron microscopy, relies on the sensitivity of the instrument to changes in the height of the topography. This sensitivity has been further utilized to image polymer strands with recorded widths of 8 nm. This width represents an overestimation of the true dimensions of the strand due to the finite size of the AFM probe apex and using the circular probe model it has been calculated that the strands have true widths of less than 0.8 nm, indicating that they are composed of one or two PEG molecules. Further studies on PEG have demonstrated the ability to control polymer surface morphology through changes in the temperature of thin film preparation and changes in the method of polymer solution deposition. The work on PEG surface morphology acts as the foundation for the remaining studies, which employ the AFM to study biodegradable polymers within aqueous environments. This in situ application of the AFM has recorded the changes in surface morphology that occur to poly(sebacic anhydride) (PSA) during surface erosion in alkaline conditions. These studies have visualized the preferential degradation of amorphous regions of sphemlites over the crystalline fibres for solution cast and melt-crystallized samples. It has been found that rapid cooling during the solidification of PSA increases the amount of amorphous material at the surface of samples. However, once this outer layer has been eroded the underlying material is dominated by crystalline fibres. In situ AFM studies have also demonstrated the pH dependence of the rate of PSA surface erosion. The AFM techniques developed to visualize the evolution of surface changes during PSA erosion have then been employed to investigate the degradation of immiscible blends of PSA and the polyester poly(DL-lactic acid) (PLA). PLA degrades at a slower rate than PSA and therefore, as these blends eroded the surface morphology became dominated by PLA, revealing the phase separation of the material. For solution cast samples on mica substrates it was found that at high PSA content the PSA formed a continuous network around islands of PLA. However, as the relative content of PLA increased the morphology reversed and the PLA formed the network around islands of PSA. The interest in studying biodegradable polymers is derived from their application in surface eroding drug delivery systems. Having demonstrated the potential of the AFM to visualize dynamic interfacial changes occurring to these polymeric biomaterials, the in situ studies were extended to investigate the release of a model protein drug from a degrading polymer film. The system under investigation was a poly(ortho ester) film containing particles of bovine serum albumin. The AFM visualized the initiation of dissolution of some protein particles within minutes of the exposure of the sample to a pH 6 environment. Other particles, however, displayed retarded dissolution behaviour and did not appear to dissolve until the sample had been exposed to the pH 6 environment for over 1 hour. To assist the interpretation of these studies computational methods of calculating changes in volume during polymer degradation and protein dissolution have been developed on the Genesis II system. In the final experiments of this thesis, the application of a novel combined atomic force microscopy/surface plasmon resonance instrument is described. This instrument allows the simultaneous acquisition of topographical data by the AFM and kinetic data by the surface plasmon resonance instrument (SPR). The instrument is first applied to a simple poly(ortho ester) system to demonstrate that the changes surface morphology and polymer film thickness can be simultaneously monitored. Then, the PSA/PLA blends were re-analysed. This analysis highlighted the synergistic information obtained by the combined AFM/SPR and revealed new data on the relationship between polymer phase separation and biodegradation kinetics. NB. This ethesis has been created by scanning the typescript original and may contain inaccuracies. In case of difficulty, please refer to the original text

The application of atomic force microscopy in the surface analysis of polymeric biomaterials

When a polymeric biomaterial is employed within a living system an interface is created between the solid surface of the polymer and an aqueous environment. The processes that occur at this interface will determine if the biomaterial is accepted by the patient and often will determine if the specific function of the biomaterial can be achieved. Increasingly, novel biomaterials are expected to perform more sophisticated functions and, therefore, their surfaces must be designed to realize precise interfacial events, such as specific interactions with proteins and cells or controlled biodegradation. To design polymeric biomaterials with specific surface properties it is necessary to develop surface analytical techniques that can accurately characterize these properties. The work described in this thesis has aimed to investigate the potential contribution of the atomic force microscope (AFM) to this characterization. The advantages of utilizing AFM in the study of polymeric biomaterials lie in the ability of the instrument to visualize insulating surfaces at a high resolution within a variety of environments, including gaseous and liquid environments. Therefore, it is possible to image the nanoscopic organization of polymeric biomaterials within environmental conditions that are similar to the conditions encountered within living systems. Initial studies have concentrated on imaging the surface morphology of poly(ethylane oxide) (PEG) samples in air. These studies highlighted the high resolution capability of the AFM on untreated polymer samples. On sphemlitic samples, the AFM has visualized the lamellar organization of crystalline fibres. These lamellae had widths of between 10 and 30 nm and height variations of less than 15 nm. The ability of the AFM to resolve such structures, without the introduction of an etching or staining procedure required by transmission electron microscopy, relies on the sensitivity of the instrument to changes in the height of the topography. This sensitivity has been further utilized to image polymer strands with recorded widths of 8 nm. This width represents an overestimation of the true dimensions of the strand due to the finite size of the AFM probe apex and using the circular probe model it has been calculated that the strands have true widths of less than 0.8 nm, indicating that they are composed of one or two PEG molecules. Further studies on PEG have demonstrated the ability to control polymer surface morphology through changes in the temperature of thin film preparation and changes in the method of polymer solution deposition. The work on PEG surface morphology acts as the foundation for the remaining studies, which employ the AFM to study biodegradable polymers within aqueous environments. This in situ application of the AFM has recorded the changes in surface morphology that occur to poly(sebacic anhydride) (PSA) during surface erosion in alkaline conditions. These studies have visualized the preferential degradation of amorphous regions of sphemlites over the crystalline fibres for solution cast and melt-crystallized samples. It has been found that rapid cooling during the solidification of PSA increases the amount of amorphous material at the surface of samples. However, once this outer layer has been eroded the underlying material is dominated by crystalline fibres. In situ AFM studies have also demonstrated the pH dependence of the rate of PSA surface erosion. The AFM techniques developed to visualize the evolution of surface changes during PSA erosion have then been employed to investigate the degradation of immiscible blends of PSA and the polyester poly(DL-lactic acid) (PLA). PLA degrades at a slower rate than PSA and therefore, as these blends eroded the surface morphology became dominated by PLA, revealing the phase separation of the material. For solution cast samples on mica substrates it was found that at high PSA content the PSA formed a continuous network around islands of PLA. However, as the relative content of PLA increased the morphology reversed and the PLA formed the network around islands of PSA. The interest in studying biodegradable polymers is derived from their application in surface eroding drug delivery systems. Having demonstrated the potential of the AFM to visualize dynamic interfacial changes occurring to these polymeric biomaterials, the in situ studies were extended to investigate the release of a model protein drug from a degrading polymer film. The system under investigation was a poly(ortho ester) film containing particles of bovine serum albumin. The AFM visualized the initiation of dissolution of some protein particles within minutes of the exposure of the sample to a pH 6 environment. Other particles, however, displayed retarded dissolution behaviour and did not appear to dissolve until the sample had been exposed to the pH 6 environment for over 1 hour. To assist the interpretation of these studies computational methods of calculating changes in volume during polymer degradation and protein dissolution have been developed on the Genesis II system. In the final experiments of this thesis, the application of a novel combined atomic force microscopy/surface plasmon resonance instrument is described. This instrument allows the simultaneous acquisition of topographical data by the AFM and kinetic data by the surface plasmon resonance instrument (SPR). The instrument is first applied to a simple poly(ortho ester) system to demonstrate that the changes surface morphology and polymer film thickness can be simultaneously monitored. Then, the PSA/PLA blends were re-analysed. This analysis highlighted the synergistic information obtained by the combined AFM/SPR and revealed new data on the relationship between polymer phase separation and biodegradation kinetics. NB. This ethesis has been created by scanning the typescript original and may contain inaccuracies. In case of difficulty, please refer to the original text

Delivery of definable number of drug or growth factor loaded poly(dl-lactic acid-co-glycolic acid) microparticles within human embryonic stem cell derived aggregates

Embryoid bodies (EBs) generated from embryonic stem cells are used to study processes of differentiation within a three dimensional (3D) cell environment. In many instances however, EBs are dispersed to single cell suspensions with a subsequent monolayer culture. Moreover, where the 3D integrity of an EB is maintained, cytokines or drugs of interest to stimulate differentiation are often added directly to the culture medium at fixed concentrations and effects are usually limited to the outer layers of the EB. The aim of this study was to create an EB model with localised drug and or growth factor delivery directly within the EB. Using poly(DL-lactic acid-co-glycolic acid) microparticles (MPs) with an average diameter of 13 μm, we have demonstrated controllable incorporation of defined numbers of MPs within human ES cell derived EBs, down to 1 MP per EB. This was achieved by coating MPs with human ES cell lysate and centrifugation of specific ratios of ES cells and MPs to form 3D aggregates. Using MPs loaded with simvastatin (pro or active drug) or BMP-2, we have demonstrated osteogenic differentiation within the 3D aggregates, maintained in culture for up to 21 days, and quantified by real time QPCR for osteocalcin. Immunostaining for RUNX2 and osteocalcin, and also histochemical staining with picrosirius red to demonstrate collage type 1 and Alizarin red to demonstrate calcium/mineralisation further demonstrated osteogenic differentiation and revealed regional staining associated with the locations of MPs within the aggregates. We also demonstrated endothelial differentiation within human ES cell-derived aggregates using VEGF loaded MPs. In conclusion, we demonstrate an effective and reliable approach for engineering stem aggregates with definable number of MPs within the 3D cellular structure. We also achieved localised osteogenic and endothelial differentiation associated with MPs releasing encapsulated drug molecules or cytokines directly within the cell aggregate. This provides a powerful tool for controlling and investigating differentiation within 3D cell cultures and has applications to drug delivery, drug discovery, stem cell biology, tissue engineering and regenerative medicine

Supercritical carbon dioxide: putting the fizz into biomaterials

This paper describes recent progress made in the use of high pressure or supercritical fluids to process polymers into three-dimensional tissue engineering scaffolds. Three current examples are highlighted: foaming of acrylates for use in cartilage tissue engineering; plasticization and encapsulation of bioactive species into biodegradable polyesters for bone tissue engineering; and a novel laser sintering process used to fabricate three-dimensional biodegradable polyester structures from particles prepared via a supercritical route

Thermoresponsive magnetic colloidal gels via surface-initiated polymerisation from functional microparticles

Novel magnetothermally responsive core-shell microparticles have been synthesized. The aqueous suspensions of these particles exhibit fast thermoreversible fluid-to-gel transitions and retain good magnetic properties. Rheological measurements demonstrated that the viscoelasticity of the prepared particle gels can be tuned, enabling these gels to have the mechanical properties that should facilitate their applications as 3D cell scaffolds for in vitro expansion of cells. Also, it was found that the responsive particles could be used in repeated heating-cooling cycles without marked changes in gel elasticity. Presto Blue viability assays of 3T3 fibroblasts and human mesenchymal sem cells cultured within the colloidal gel showed that the cells remained viable and proliferated, with significant increases in cell numbers over extended culture times. Confocal microscopy images of 3T3 cells cultured within the colloidal gel demonstrated that cells adhered, spread and retained their normal morphologies during proliferation.. Furthermore, magnetic separation allowed efficient recovery of cells after their expansion in vitro without need for enzyme-mediated release steps. Trypsin-free cell passages were performed allowing multiple growth, separation and reloading of cells within the colloidal gels. Overall, the results suggest this colloidal gel has potential as a 3D scaffold for in vitro expansion of a variety of cell types and for enzyme free cell harvesting

Odontogenic differentiation of human dental pulp stem cells on hydrogel scaffolds derived from decellularized bone extracellular matrix and collagen type I

ObjectivesThe aim of this study was to evaluate the level of odontogenic differentiation of dental pulp stem cells (DPSCs) on hydrogel scaffolds derived from bone extracellular matrix (bECM) in comparison to those seeded on collagen I (Col-I), one of the main components of dental pulp ECM.MethodsDPSCs isolated from human third molars were characterized for surface marker expression and odontogenic potential prior to seeding into bECM or Col-I hydrogel scaffolds. The cells were then seeded onto bECM and Col-I hydrogel scaffolds and cultured under basal conditions or with odontogenic and growth factor (GF) supplements. DPSCs cultivated on tissue culture polystyrene (TCPS) with and without supplements were used as controls. Gene expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP-1) and matrix extracellular phosphoglycoprotein (MEPE) was evaluated by quantitative reverse transcription-polymerase chain reaction (qRT PCR) and mineral deposition was observed by Von Kossa staining.ResultsWhen DPSCs were cultured on bECM hydrogels, the mRNA expression levels of DSPP, DMP-1 and MEPE genes were significantly upregulated with respect to those cultured on Col-I scaffolds or TCPS in the absence of extra odontogenic inducers. In addition, more mineral deposition was observed on bECM hydrogel scaffolds as demonstrated by Von Kossa staining. Moreover, DSPP, DMP-1 and MEPE mRNA expressions of DPSCs cultured on bECM hydrogels were further upregulated by the addition of GFs or osteo/odontogenicmedium compared to Col-I treated cells in the same culture conditions.SignificanceThese results demonstrate the potential of the bECM hydrogel scaffolds to stimulate odontogenic differentiation of DPSCs

Targeted protein delivery: carbodiimide crosslinking influences protein release from microparticles incorporated within collagen scaffolds.

Tissue engineering response may be tailored via controlled, sustained release of active agents from protein-loaded degradable microparticles incorporated directly within three-dimensional (3D) ice-templated collagen scaffolds. However, the effects of covalent crosslinking during scaffold preparation on the availability and release of protein from the incorporated microparticles have not been explored. Here, we load 3D ice-templated collagen scaffolds with controlled additions of poly-(DL-lactide-co-glycolide) microparticles. We probe the effects of subsequent N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride crosslinking on protein release, using microparticles with different internal protein distributions. Fluorescein isothiocyanate labelled bovine serum albumin is used as a model protein drug. The scaffolds display a homogeneous microparticle distribution, and a reduction in pore size and percolation diameter with increased microparticle addition, although these values did not fall below those reported as necessary for cell invasion. The protein distribution within the microparticles, near the surface or more deeply located within the microparticles, was important in determining the release profile and effect of crosslinking, as the surface was affected by the carbodiimide crosslinking reaction applied to the scaffold. Crosslinking of microparticles with a high proportion of protein at the surface caused both a reduction and delay in protein release. Protein located within the bulk of the microparticles, was protected from the crosslinking reaction and no delay in the overall release profile was seen.This work was supported by the European Research Council [ERC Advanced Grant 320598 3D-E] and was also funded by a grant from the Medical Research Council, Arthritis Research UK, Reumafonds and the UKRM

Highly efficient intracellular transduction in three-dimensional gradients for programming cell fate

Fundamental behaviour such as cell fate, growth and death are mediated through the control of key genetic transcriptional regulators. These regulators are activated or repressed by the integration of multiple signalling molecules in spatio-temporal gradients. Engineering these gradients is complex but considered key in controlling tissue formation in regenerative medicine approaches. Direct programming of cells using exogenously delivered transcription factors can by-pass growth factor complexity but there is still a requirement to deliver such activity spatio-temporally. We previously developed a technology termed GAG-binding enhanced transduction (GET) to efficiently deliver a variety of cargoes intracellularly using GAG-binding domains to promote cell targeting, and cell penetrating peptides (CPPs) to allow cell entry. Herein we demonstrate that GET can be used in a three dimensional (3D) hydrogel matrix to produce gradients of intracellular transduction of mammalian cells. Using a compartmentalised diffusion model with a source-gel-sink (So-G-Si) assembly, we created gradients of reporter proteins (mRFP1-tagged) and a transcription factor (TF, myogenic master regulator MyoD) and showed that GET can be used to deliver molecules into cells spatio-temporally by monitoring intracellular transduction and gene expression programming as a function of location and time. The ability to spatio-temporally control the intracellular delivery of functional proteins will allow the establishment of gradients of cell programming in hydrogels and approaches to direct cellular behaviour for many regenerative medicine applications

Bioprinting Using Mechanically Robust Core-Shell Cell-Laden Hydrogel Strands

The strand material in extrusion-based bioprinting determines the microenvironments of the embedded cells and the initial mechanical properties of the constructs. One unmet challenge is the combination of optimal biological and mechanical properties in bioprinted constructs. Here, a novel bioprinting method that utilizes core–shell cell-laden strands with a mechanically robust shell and an extracellular matrix-like core has been developed. Cells encapsulated in the strands demonstrate high cell viability and tissue-like functions during cultivation. This process of bioprinting using core–shell strands with optimal biochemical and biomechanical properties represents a new strategy for fabricating functional human tissues and organs

Multi-material 3D bioprinting of porous constructs for cartilage regeneration

© 2020 Elsevier B.V. The current gold standard for nasal reconstruction after rhinectomy or severe trauma includes transposition of autologous cartilage grafts in conjunction with coverage using an autologous skin flap. Harvesting autologous cartilage requires a major additional procedure that may create donor site morbidity. Major nasal reconstruction also requires sculpting autologous cartilages to form a cartilage framework, which is complex, highly skill-demanding and very time consuming. These limitations have prompted facial reconstructive surgeons to explore different techniques such as tissue engineered cartilage. This work explores the use of multi-material 3D bioprinting with chondrocyte-laden gelatin methacrylate (GelMA) and polycaprolactone (PCL) to fabricate constructs that can potentially be used for nasal reconstruction. In this study, we have investigated the effect of 3D manufacturing parameters including temperature, needle gauge, UV exposure time, and cell carrier formulation (GelMA) on the viability and functionality of chondrocytes in bioprinted constructs. Furthermore, we printed chondrocyte-laden GelMA and PCL into composite constructs to combine biological and mechanical properties. It was found that 20% w/v GelMA was the best concentration for the 3D bioprinting of the chondrocytes without comprising the scaffold's porous structure and cell functionality. In addition, the 3D bioprinted constructs showed neocartilage formation and similar mechanical properties to nasal alar cartilage after a 50-day culture period. Neocartilage formation was also observed in the composite constructs evidenced by the presence of glycosaminoglycans and collagen type II. This study shows the feasibility of manufacturing neocartilage using chondrocytes/GelMA/PCL 3D bioprinted porous constructs which could be applied as a method for fabricating implants for nose reconstruction