Supercritical technology applied to the development of drug delivery systems for bone regeneration

Bone is the second most transplanted tissue, only behind blood transfusions, with over 2 million of procedures worldwide each year. This number is expected to increase, given that ageing is a risk factor for fractures and fracture delayed union and non-union, that currently reach up to 10 % of cases. Different approaches have been proposed with the purpose of obtaining synthetic scaffolds that can be used as bone grafts. In this Thesis, supercritical fluids were employed to optimize the preparation of synthetic scaffolds made of two biocompatible and biodegradable polymers, poly(lactic-co-glycolic acid) (PLGA) and poly(e-caprolactone) (PCL), with the incorporation of aerogel microparticles and anti-inflammatories as bioactive compounds in order to improve their properties for their application as bone grafts. The scaffolds were tested regarding their textural, phisicochemical and mechanical properties, degradation rates, drug release profiles, stability in storage and performance in an animal model

Subcritical carbon dioxide foaming of polycaprolactone for bone tissue regeneration

Accepted manuscriptThe preparation of three-dimensional polycaprolactone scaffolds using dense CO2 as foaming agent, without supercritical conditions, was evaluated in this study towards future applications in bone repair. Herein, 3D foams were obtained at 5.0 MPa and 45 °C. To induce bioactivity, β-tricalcium phosphate (β-TCP, 10 wt%) and dexamethasone (5 and 10 wt%) were dispersed in the scaffolds. Foams revealed a pore size range of 164–882 μm, 73–99% porosity and 79–99% interconnectivity, assessed by micro-computed tomography, and a Young modulus of 1.76–2.92 MPa. Dexamethasone did not impair morphology of the matrices in comparison with PCL+β-TCP, which presented a water uptake of nearly 100% after 14 days. A sustained release of dexamethasone was achieved over 35 days in physiologic solution. This study reports the feasibility of using dense CO2 to produce in one-step a porous matrix loaded with active agents opening new possibilities towards injectable systems for in situ foamingEuropean Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number REGPOT-CT2012-316331-POLARIS. It was also funded by the project “Novel smart and biomimetic materials for innovative regenerative medicine approaches” (RL1-ABMR-NORTE-01-0124-FEDER-000016) co-financed by North Portugal Regional Operational Programme (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF) and the project NORTE‐01‐0145‐FEDER‐000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement. The authors would like to acknowledge the funding of the project Associate Laboratory ICVS/3B’s, under grant agreement number POCI-01-0145-FEDER-007038 supported by FEDER, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT)info:eu-repo/semantics/publishedVersio

Osteogenic and Chondrogenic Differentiation of rBMSCs on Microsphere-Based Scaffolds Sintered Using Subcritical CO2

Large bone defects remain a major clinical orthopedic challenge. It has been predicted that osteoarthritis will affect over 100 million adults in the United States by the year 2030. Current treatments for repairing bone defects include the use of bone grafts (autologous and allogenic) or implants (polymeric or metallic). These approaches have significant limitations due to insufficient supply, potential disease transmission, rejection, cost and the inability to integrate with the surrounding host tissue. The engineering of bone and cartilage tissue offers new therapeutic strategies to treat bone defects. Several scaffold-based approaches have been used in the past. However, this thesis presents a novel microsphere-based scaffold approach, sintered using subcritical carbon dioxide for osteogenic and chondrogenic tissue regeneration. As a next step in the fabrication of three-dimensional tissue engineered scaffolds, this thesis primarily focused on subcritical carbon dioxide sintering for forming scaffolds, performance of these scaffolds in culture for 6 weeks, and evaluation of two different polymers in osteogenic and chondrogenic differentiation. In this investigation, both temperature and pressure (along with time) were necessary to control during the CO2 sintering of PCL (higher temperature and pressure conditions with longer exposure time), as opposed to PLGA, which was sintered at ambient temperature and pressure conditions (for 1 hour exposure). The results obtained showed the feasibility of using these constructs for bone and cartilage tissue regeneration. Biochemical analysis, gene expression and histological staining were used to analyze the data. The mechanical integrity of the constructs was evaluated at the beginning and end of the culture period. The onset of PLGA degradation for the CO2 sintered microspheres in this study appeared at 1.5 weeks which affected chondrogenesis. With osteogenesis, the Osteogenic PLGA group showed greater calcium content value over the Osteogenic PCL group while PCL retained its shape, size and mechanical integrity and had twice as many cells per construct at 6 weeks. In conclusion, this thesis lays a foundation to explore numerous applications using subcritical carbon dioxide sintering for tissue engineering applications

New strategies for the production of ready-to-implant bone scaffolds using supercritical fluid technology

This PhD Thesis highlights the advantages of the supercritical CO2 technology for the preparation of sterile scaffolds for regenerative medicine applications. On the one hand, this technology allows the production of polymeric scaffolds in the absence of organic solvents and downstream processes. In addition, the extraction capacity of supercritical CO2 also allows the preparation of bio-based aerogels. On the other hand, the antimicrobial effect of supercritical CO2 can be exploited for the sterilization of thermosensitive materials, including biodegradable scaffolds

Hybrid 3D structure of poly(d,l-lactic acid) loaded with chitosan/chondroitin sulfate nanoparticles to be used as carriers for biomacromolecules in tissue engineering

In the tissue engineering (TE) field, the concept of producing multifunctional scaffolds, capable not only of acting as templates for cell transplantation but also of delivering bioactive agents in a controlled manner, is an emerging strategy aimed to enhance tissue regeneration. In this work, a complex hybrid release system consisting in a three-dimensional (3D) structure based on poly(d,l-lactic acid) (PDLLA) impregnated with chitosan/chondroitin sulfate nanoparticles (NPs) was developed. The scaffolds were prepared by supercritical fluid foaming at 200 bar and 35 "C, and were then characterized by scanning electron microscopy (SEM) and micro-CT. SEM also allowed to assess the distribution of the NPs within the structure, showing that the particles could be found in different areas of the scaffold, indicating a homogeneous distribution within the 3D structure. Water uptake and weight loss measurements were also carried out and the results obtained demonstrated that weight loss was not significantly enhanced although the entrapment of the NPs in the 3D structure clearly enhances the swelling of the structure. Moreover, the hybrid porous biomaterial displayed adequate mechanical properties for cell adhesion and support. The possibility of using this scaffold as a multifunctional material was further evaluated by the incorporation of a model protein, bovine serum albumin (BSA), either directly into the PDLLA foam or in the NPs that were eventually included in the scaffold. The obtained results show that it is possible to achieve different release kinetics, suggesting that this system is a promising candidate for dual protein delivery system for TE applications

Solvent-Free Processing of Drug-Loaded Poly(ε-Caprolactone) Scaffolds with Tunable Macroporosity by Combination of Supercritical Foaming and Thermal Porogen Leaching

Demand of scaffolds for hard tissue repair increases due to a higher incidence of fractures related to accidents and bone-diseases that are linked to the ageing of the population. Namely, scaffolds loaded with bioactive agents can facilitate the bone repair by favoring the bone integration and avoiding post-grafting complications. Supercritical (sc-)foaming technology emerges as a unique solvent-free approach for the processing of drug-loadenu7d scaffolds at high incorporation yields. In this work, medicated poly(ε-caprolactone) (PCL) scaffolds were prepared by sc-foaming coupled with a leaching process to overcome problems of pore size tuning of the sc-foaming technique. The removal of the solid porogen (BA, ammonium bicarbonate) was carried out by a thermal leaching taking place at 37 °C and in the absence of solvents for the first time. Macroporous scaffolds with dual porosity (50–100 µm and 200–400 µm ranges) were obtained and with a porous structure directly dependent on the porogen content used. The processing of ketoprofen-loaded scaffolds using BA porogen resulted in drug loading yields close to 100% and influenced its release profile from the PCL matrix to a relevant clinical scenario. A novel solvent-free strategy has been set to integrate the incorporation of solid porogens in the sc-foaming of medicated scaffoldsThis research was funded by Xunta de Galicia [ED431F 2016/010; ED431C 2020/17], MCIUN [RTI2018-094131-A-I00], Agencia Estatal de Investigación [AEI] and FEDER funds. C.A. García-González acknowledges to MINECO for a Ramón y Cajal Fellowship [RYC2014-15239]. V. Santos-Rosales acknowledges to Xunta de Galicia (Consellería de Cultura, Educación e Ordenación Universitaria) for a predoctoral research fellowship [ED481A-2018/014]S

Enhanced performance of supercritical fluid foaming of natural-based polymers by deep eutetic solvents

Natural deep eutetic solvents (NADES) are defined as a mixture of two or more solid or liquid components, which at a particular composition present a high melting point depression becoming liquids at room temperature. NADES are constituted by natural molecules and fully represent the green chemistry principles. For these reasons, the authors believe that the submit manuscript is a highly valuable contribution to the field of green chemistry and chemical engineering. For the first time, the possibility to use NADES as enhancers of supercritical fluid tecnology is revealed.The research leading to these results has received funding from Fundacao da Ciencia e Tecnologia (FCT) through the project ENIGMA-PTDC/EQU-EPR/121491/2010 and the project PEst-C/EQB/LA0006/2013. The funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no REGPOT-CT2012-316331-POLARIS and from Project "Novel smart and biomimetic materials for innovative regenerative medicine approaches (Ref.: RL1 - ABMR - NORTE-01-0124-FEDER-000016)" cofinanced by North Portugal Regional Operational Programme (ON.2 - O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF) and FEDER are also acknowledged. Marta Martins, Rita Craveiro and Alexandre Paiva are grateful for financial support from Fundacao da Ciencia e Tecnologia (FCT) through the grants BIM/PTDC/EQU-EPR/121491/2010/ENIGMA and SFRH/BPD/44946/2008

Supercritical CO2 technology for one-pot foaming and sterilization of polymeric scaffolds for bone regeneration

Sterilization is a quite challenging step in the development of novel polymeric scaffolds for regenerative medicine since conventional sterilization techniques may significantly alter their morphological and physicochemical properties. Supercritical (sc) sterilization, i.e. the use of scCO2 as a sterilizing agent, emerges as a promising sterilization method due to the mild operational conditions and excellent penetration capability. In this work, a scCO2 protocol was implemented for the one-pot preparation and sterilization of poly(-caprolactone) (PCL)/poly(lactic-co-glycolic acid) (PLGA) scaffolds. The sterilization conditions were established after screening against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) vegetative bacteria and spores of Bacillus stearothermophilus, Bacillus pumilus and Bacillus atrophaeus. The transition from the sterilization conditions (140 bar, 39 °C) to the compressed foaming (60 bar, 26 °C) was performed through controlled depressurization (3.2 bar/min) and CO2 liquid flow. Controlled depressurization/pressurization cycles were subsequently applied. Using this scCO2 technology toolbox, sterile scaffolds of well-controlled pore architecture were obtained. This sterilization procedure successfully achieved not only SAL-6 against well-known resistant bacteria endospores but also improved the scaffold morphologies compared to standard gamma radiation sterilization proceduresThis work was supported by Xunta de Galicia [ED431F 2016/01, ED431C 2020/17], MCIUN [RTI2018-094131-A-I00], MINECO [SAF2017-83118-R], Consellería de Sanidade, Servizo Galego de Saúde, Axencia de Coñecemento e Saúde (ACIS, CT850A-G), Agencia Estatal de Investigación [AEI] and FEDER funds. V. Santos-Rosales acknowledges to Xunta de Galicia (Consellería de Cultura, Educación e Ordenación Universitaria) for a predoctoral research fellowship [ED481A-2018/014]. C.A. García-González acknowledges to MINECO for a Ramón y Cajal Fellowship [RYC2014-15239]S