Resumen de Nanofibrous membranes obtained by electrospinning for bone tissue engineering and wound dressing applications
The current Doctoral Thesis work has been performed under a co-supervision agreement between University of Zaragoza (Home University), University of Calabria (Host University) and Faculty of Sciences and Technology of the NOVA University of Lisbon (FCT NOVA) (Host University). This research has been carried out inside the Erasmus Mundus Doctorate in Membrane Engineering program (EUDIME), (FPA 2011-0014), funded by the European Union.
This thesis focused mainly on the use of the electrospinning technique to produce different kind of membranes for biomedical applications. In particular, it described the synthesis and production of inorganic and organic nanoparticles to be used as fillers or as carriers (drug delivery system) as well as the production of electrospun nanofibrous membranes. This work was carried out within the Institute of Nanoscience of Aragon (INA), specifically in the Nanostructured Films and Particles (NFP) group under the supervision of the Professor Silvia Irusta and Dr Gracia Mendoza. Also an important part of the physico-chemical characterization was done at INA.
The study of different biological signals and the use of specific techniques for membrane characterization were acquired at the University of Calabria under the supervision of Dr. Loredana De Bartolo in the Institute on Membrane Technology of the National Research Council of Italy (ITM-CNR). On the other hand, the mobility carried out at the Faculty of Sciences and Technology (FCT NOVA) of Universidade NOVA (FCT NOVA) under the supervision of Professor Ana Isabel Aguiar-Ricardo, allowed a total characterization of two asymmetric membranes following different International Standards to accomplish testing for primary wound dressing.
The development of novel membranes loaded with morphogenetic proteins or antibiotic are of great interest in the field of bone tissue engineering. To promote the cellular viability and extracellular matrix production, electrospun membranes with enhanced porosity and micro-scale pores could be beneficial since increased porosity and pore size can provide a three-dimensional (3D) environment that not only facilitates cell seeding/diffusion but also provides better diffusion of nutrients and waste throughout the membranes. The addition of calcium phosphate ceramics has been extensively investigated to fabricate highly porous membranes to bone tissue engineering due to their close similar composition of bone, including excellent biocompatibility, osteoinductive and osteoconductive properties. A homogeneous distribution of the bone morphogenetic protein-loaded particles along the entire membrane could be ensuring a continuous release of the growth factor to provide the necessary biochemical cues for bone repair and regeneration.
Antibiotic-loaded membranes may provide drug targeted and sustained release, avoiding the long-term oral and intravenous systematic multidrug administration, which implies toxic side effects, low delivery to the target site and low patient adherence to the treatment. Therefore, membranes loaded with antibiotics can overcome the drawbacks of the traditional therapy sustaining enhanced osteogenic properties for the successful regeneration of the bone.
Another interesting biomedical application of electrospun membranes is the fabrication of efficient smart dressings for the treatment of wounds. A rapid wound healing requires developing appropriate membranes with interconnected pores that allow the oxygen diffusion and transport of metabolic waste, as well as an adequate pore size to prevent rapid dehydration and bacteria penetration. A high absorption capacity and adequate water vapor transmission will be necessary to keep a moist environment in the wound bed. Besides, if the electrospun membrane has some bactericidal properties will be better for the healing process.
The main goal of this thesis was the development of fibrous membranes by electrospinning with the appropriate characteristics to be used in bone tissue engineering or as wound dressing materials. To achieve this target, several specific objectives were defined, which are described in Chapters II to V.
The thesis was divided in the following sections: CHAPTER I, is an introduction where the concepts of biomaterials, scaffolds and tissue engineering and the main target of drug delivery systems are described. The chapter includes the classification of biomaterials according to the origin of the materials and tissue engineering is also described as well as all the factors that must be taken into account to develop and properly apply a wound dressing are discussed. Different kind of techniques used in the literature to produce scaffolds or membranes for bone tissue engineering and wound dressings are mentioned, focusing on the use of electrospinning and electrospray to produce them.
CHAPTER II, focuses on the development of enhanced 3D membranes able to promote efficient bone regeneration together with targeted antibiotic release to prevent bacteria colonization. The aim of this work was to synthesize and characterize a drug delivery system consisting of polycaprolactone (PCL) electrospun nanofibers decorated with rifampicin (RFP) loaded into poly(lactic-coglicolic acid) (PLGA) particles. This material would promote bone repair avoiding the impairment of the membrane mediated by infection. The bactericidal ability of the synthesized electrospun material was assessed In vitro against gram positive (Staphylococcus aureus) and gram negative (Escherichia coli) bacteria, as well as its cytocompatibility in human osteoblasts 3D cultures. These results are included in the accepted article entitled “Composite scaffold obtained by electro-hydrodynamic technique for infection prevention and treatment in bone repair”. Javier Aragon, Sergio Feoli, Gracia Mendoza, Silvia Irusta. International Journal of Pharmaceutics.
CHAPTER III, describes the synthesis and characterization of core-shell membranes of PCL and polyvinyl acetate (PVAc) obtained by electrospinning. The fibers were loaded with synthetic hydroxyapatite nanoparticles (HAn) to increase the bioactivity of the materials. The prepared membranes were then treated by laser ablation to create desired microscale topographical features in order to favor cell adhesion and growth.
All prepared membranes exhibited a three-dimensional network structure with interconnected pores; the laser treatment has modified the structural characteristics of the membrane causing an increase the cell viability and cell density. The materials biocompatibility is affected by the structural properties of the membranes, indeed smaller micropore sizes favor cell adhesion and proliferation. These results are published in the article entitled “Laser-treated electrospun fibers loaded with nano-hydroxyapatite for bone tissue engineering”. Javier Aragon, Nuria Navascues, Gracia Mendoza, Silvia Irusta. International Journal of Pharmaceutics 525,112–122, 2017. DOI:10.1016/j.ijpharm.2017.04.022.
CHAPTER IV, refers to the development of a composite electrospun membrane of PCL or PCL/PVAc core–shell fibers loaded with synthetic HAn. These fibers were decorated with bone morphogenetic protein 2 (BMP2) loaded in/into PLGA particles via simultaneous electrospraying and coaxial electrospinning. The aim of this study was to evaluate the structural and physico-chemical properties and biodegradation processes of the newly developed membranes assessing their ability to address the architectural, biochemical, and functional features of bone tissue. For this purpose, the membrane bioactivity was tested by culturing human osteoblasts on the membranes and by monitoring cell viability up to 4 weeks. The In vitro osteogenic activity of cells seeded onto the membranes was evaluated by assessing alkaline phosphatase (ALP) activity and the expression of osteogenic proteins osteocalcin (OCN) and osteopontin (OPN). These results are published in the article “Polymeric electrospun scaffolds for bone morphogenetic protein 2 delivery in bone tissue engineering”. Javier Aragón, Simona Salerno, Loredana De Bartolo, Silvia Irusta and Gracia Mendoza. Journal of Colloid and Interface Science, 531 (2018) 126–137. DOI:10.1016/j.jcis.2018.07.029.
CHAPTER V, describes the synthesis of an antimicrobial wound dressing material, with appropriate mechanical resistance avoiding rapid dehydration and absorbing exudates. PCL/PVAc asymmetric membranes loaded with carvacrol (CRV) were prepared by electrospinning and electrospraying simultaneously. The membranes consist of two layers: the first is an electrospun PCL sheet, the second a PVAc sheet that would be in contact with the skin releasing the antimicrobial compound. The use of different solvents results in different morphologies for the PVAc-CRV layer. The membranes exhibit mechanical properties with strain to failure values that are in the range of human skin, being adequate to be deposited over a wound surface. The samples present Water Vapor Transmission (WVTR) values in the required range to keep good moisture balance with water loss from the wound at the optimal rate. In the first week, more than 60 % of the loaded CRV was released while after three weeks membranes released between 85 to 100 % of the loaded CRV through a Fickian diffusion and diffusion due to polymer relaxation. The synthesized membranes are potential candidates to be used for wound dressing applications. The manuscript summing up these results has been submitted to a scientific journal and is currently under review.
GENERAL CONCLUSIONS, summarizes the conclusions of the thesis work.
APPENDIX 1, describes the main characterization techniques and the methods to evaluate different properties according to the possible applications.
APPENDIX 2, summarizes the articles published and the participation in scientific forums during the thesis period.
