10 research outputs found
Fabricating delivery platforms for wound management and tissue regeneration
Skin provides the protective surface for animals and humans and is therefore prone to physical, chemical, and biological injuries. In all but superficial wounds, the capacity to repair by regeneration is lost and the mechanisms involved in wound closure are unable to restore the skin’s original functions. In this context, skin repair is achieved using surgical techniques including skin grafts, and a range of synthetic or biological scaffolds. Wounds impact millions of patients every year and represent a serious cause of morbidity and mortality worldwide. The increase in need for better skin repair, in part due to issues such as the aging population coupled with chronic conditions has driven the development of products to enhance therapeutic outcomes, yet current treatment outcomes are far from ideal and complete replication of the cellular structure and tissue functional requirements of skin remains a challenge.
General aims: Address the major drawbacks of available skin substitutes and delivery system platforms. Herein two approaches are proposed, the first one (Chapters 2-3) involves the development and initial in vitro characterization of a 3D multifunctional bioprinted platform based on platelet lysate, which could be used to deliver cells and growth factors to the wound site while providing a supportive network that mimics the native ECM for skin cells to infiltrate and thrive. This system was designed with the aim of providing an advanced alternative to current skin grafts and skin substitutes available for clinical use. The second system proposed (Chapter 4) is based on an electrofluidic approach for control of bioactive molecule delivery into soft tissue model using threads and surgical sutures which was designed with the aim of being used in sutures for surgical wound closure.
Methods: (Chapter 2-3) 3D printed HDF-PLGMA bioink were fabricated using a pneumatic extrusion-based 3D Bioplotter. The epidermal-dermal model was fabricated by seeding HaCaT on top of 3D printed HDF-PLGMA constructs. The innervated dermal model was fabricated by seed hNSC H9 neurospheres to the bottom of 3D printed HDF-PLGMA constructs. (Chapter 4) Commonly employed surgical sutures were used to create an adequate fluid connection between the electrodes and a tissue-like 3D hydrogel support. The platform consisted of two reservoirs into which the ends of the thread/suture were immersed. The anode and cathode were placed separately into each reservoir. The thread/suture was taken from one reservoir to the other through the gel. When the current was applied, biomolecules loaded onto the thread/suture were directed into the gel, and the rate of movement of the biomolecules was dependent on the magnitude of the current.
Results: (Chapter 2) Briefly, the work described in this chapter relates to the development of a multifunctional bioink consisting of PL and GelMA (PLGMA) and the biofabrication of a 3D printed dermal-like structure. The data presented shows that the proposed PLGMA bioink meets essential requirements of printability in terms of rheological properties and shape fidelity. Moreover, its mechanical properties can be readily tuned to achieve stiffness that is equivalent to native skin tissue. Biologically relevant factors were successfully released in a sustainable manner and the bioavailability of those factors was demonstrated by high cell viability, good cell attachment, and improved proliferation of printed dermal fibroblasts, as well as by upregulation of ECM synthesis by dermal fibroblasts. (Chapter 3) Continuing the work described in chapter 2, chapter 3 relates the fabrication of a more complex skin equivalent based on the PLGMA platform previously established. Bilayer skin model: The expression of general keratinocyte differentiation markers was used to confirm the capacity of the platform to promote normal epithelial morphogenesis and differentiation of keratinocytes. Innervated skin model: The expression of development neuronal markers, as well as a general neuronal marker, was used to demonstrate that the proposed platform supported hNSC-H9 neurosphere neurite outgrowth and neuronal differentiation. Challenges faced in the co-incorporation of HaCaT and hNSC-H9 neurospheres to HDF-PLGMA construct that need to be considered to progress with this research will also be presented. (Chapter 4) A novel electrofludic system for the controlled release of bioactive molecules such as small molecules, drugs, polysaccharides, or proteins via an electric field is described for the first time. In a proof-of-concept study, the controlled delivery of dexamethasone 21-phosphate disodium salt (DSP), a clinically relevant anti-inflammatory prodrug, as well as other molecules were used to demonstrate the feasibility of the proposed system. Despite being peripherally related to the overall PhD theme; this system could be potentially integrated in the PLGMA matrix by printing the 3D skin cells-PLGMA on top of the suture for precise delivery of growth factors and cells to sutured wounds.
Taken together, although it is still at an early stage of development, the bioprinting platform, as well as the electrofludic systems demonstrated to hold potential as a foundation for the fabrication of complex and physiologically relevant delivery platforms for wound management and tissue regeneration
Quantitative ultrasound imaging of cell-laden hydrogels and printed constructs
In the present work we have revisited the application of quantitative ultrasound imaging (QUI) to cellular hydrogels, by using the reference phantom method (RPM) in combination with a local attenuation compensation algorithm. The investigated biological samples consisted of cell-laden collagen hydrogels with PC12 neural cells. These cell-laden hydrogels were used to calibrate the integrated backscattering coefficient (IBC) as a function of cell density, which was then used to generate parametric images of local cell density. The image resolution used for QUI and its impact on the relative IBC error was also investigated. Another important contribution of our work was the monitoring of PC12 cell proliferation. The cell number estimates obtained via the calibrated IBC compared well with data obtained using a conventional quantitative method, the MTS assay. Evaluation of spectral changes as a function of culture time also provided additional information on the cell cluster size, which was found to be in close agreement with that observed by microscopy. Last but not least, we also applied QUI on a 3D printed cellular construct in order to illustrate its capabilities for the evaluation of bioprinted structures. Statement of Significance: While there is intensive research in the areas of polymer science, biology, and 3D bio-printing, there exists a gap in available characterisation tools for the non-destructive inspection of biological constructs in the three-dimensional domain, on the macroscopic scale, and with fast data acquisition times. Quantitative ultrasound imaging is a suitable characterization technique for providing essential information on the development of tissue engineered constructs. These results provide a detailed and comprehensive guide on the capabilities and limitations of the technique
Surface Enhanced Raman Spectrosocpy (SERS) – an Alternative Tool for the Identification of Pathogenic Bacteria
Label-free NIR-SERS discrimination and detection of foodborne bacteria by in situ synthesis of Ag colloids
Background: Rapid detection and discrimination of bacteria for biomedical and food safety applications remain a considerable challenge. We report a label-free near infrared surface-enhanced Raman scattering (NIR-SERS) method for the discrimination of pathogenic bacteria from drinking water. The approach relies on the in situ synthesis of silver nanoparticles (Ag NPs) within the bacterial cell suspensions. Results: Pre-treatment of cells with Triton X-100 significantly improved the sensitivity of the assay. Using this method, we were able to discriminate several common pathogenic bacteria such as Escherichia coli, Pseudomonas aeruginosa, Methicillin-resistant Staphylococcus aureus (MRSA) and Listeria spp. A comparison of the SERS spectra allowed for the discrimination of two Listeria species, namely L. monocytogenes and L. innocua. We further report the application of the method to discriminate two MRSA strains from clinical isolates. The complete assay was completed in a span of 5 min. Conclusions: The proposed analytical method proves to be a rapid tool for selective and label-free identification of pathogenic bacterium. Pre-treatment of bacterial cells with Triton X-100 resulted in new features on the SERS spectra, allowing for a successful discrimination of common disease related bacteria including E. coli, P. aeruginosa, Listeria and MRSA. We also demonstrate that the spectral features obtained using in situ synthesis of nanoparticles could be could be used to differentiate two species of listeria. By using L.innocua as a model sample, we found the limit of detection of our assay to be 103 CFU/mL. The method can selectively discriminate different bacterial species, and has a potential to be used in the development of point-of-care diagnostics with biomedical and food safety applications
Electrofluidic control of bioactive molecule delivery into soft tissue models based on gelatin methacryloyl hydrogels using threads and surgical sutures
© 2020, The Author(s). The delivery of bioactive molecules (drugs) with control over spatial distribution remains a challenge. Herein, we demonstrate for the first time an electrofluidic approach to controlled delivery into soft tissue models based on gelatin methacryloyl (GelMA) hydrogels. This was achieved using a surgical suture, whereby transport of bioactive molecules, including drugs and proteins, was controlled by imposition of an electric field. Commonly employed surgical sutures or acrylic threads were integrated through the hydrogels to facilitate the directed introduction of bioactive species. The platform consisted of two reservoirs into which the ends of the thread were immersed. The anode and cathode were placed separately into each reservoir. The thread was taken from one reservoir to the other through the gel. When current was applied, biomolecules loaded onto the thread were directed into the gel. Under the same conditions, the rate of movement of the biomolecules along GelMA was dependent on the magnitude of the current. Using 5% GelMA and a current of 100 µA, 2 uL of fluorescein travelled through the hydrogel at a constant velocity of 7.17 ± 0.50 um/s and took less than 8 minutes to exit on the thread. Small molecules such as riboflavin migrated faster (5.99 ± 0.40 μm/s) than larger molecules such as dextran (2.26 ± 0.55 μm/s with 4 kDa) or BSA (0.33 ± 0.07 μm/s with 66.5 kDa). A number of commercial surgical sutures were tested and found to accommodate the controlled movement of biomolecules. Polyester, polyglactin 910, glycolide/lactide copolymer and polyglycolic acid braided sutures created adequate fluid connection between the electrodes and the hydrogel. With a view to application in skin inflammatory diseases and wound treatment, wound healing, slow and controlled delivery of dexamethasone 21-phosphate disodium salt (DSP), an anti-inflammatory prodrug, was achieved using medical surgicryl PGA absorbable suture. After 2 hours of electrical stimulation, still 81.1% of the drug loaded was encapsulated within the hydrogel
Fibrinogen, collagen, and transferrin adsorption to poly(3,4-ethylenedioxythiophene)-xylorhamno-uronic glycan composite conducting polymer biomaterials for wound healing applications
We present the conducting polymer poly (3,4-ethylenedioxythiophene) (PEDOT) doped with an algal-derived glycan extract, Phycotrix™ [xylorhamno-uronic glycan (XRU84)], as an innovative electrically conductive material capable of providing beneficial biological and electrical cues for the promotion of favorable wound healing processes. Increased loading of the algal XRU84 into PEDOT resulted in a reduced surface nanoroughness and interfacial surface area and an increased static water contact angle. PEDOT-XRU84 films demonstrated good electrical stability and charge storage capacity and a reduced impedance relative to the control gold electrode. A quartz crystal microbalance with dissipation monitoring study of protein adsorption (transferrin, fibrinogen, and collagen) showed that collagen adsorption increased significantly with increased XRU84 loading, while transferrin adsorption was significantly reduced. The viscoelastic properties of adsorbed protein, characterized using the ΔD/Δf ratio, showed that for transferrin and fibrinogen, a rigid, dehydrated layer was formed at low XRU84 loadings. Cell studies using human dermal fibroblasts demonstrated excellent cell viability, with fluorescent staining of the cell cytoskeleton illustrating all polymers to present excellent cell adhesion and spreading after 24 h