Development of implants composed of bioactive materials for bone repair

The purpose of this Ph.D. research was to address the clinical need for synthetic bioactive materials to heal defects in non-loaded and loaded bone. Hollow hydroxyapatite (HA) microspheres created in a previous study were evaluated as a carrier for controlled release of bone morphogenetic protein-2 (BMP2) in bone regeneration. New bone formation in rat calvarial defects implanted with BMP2-loaded microspheres (43%) was significantly higher than microspheres without BMP2 (17%) at 6 weeks postimplantation. Then hollow HA microspheres with a carbonate-substituted composition were prepared to improve their resorption rate. Hollow HA microspheres with ~12 wt. % of carbonate showed significantly higher new bone formation (73 ± 8%) and lower residual HA (7 ± 2%) than stoichiometric HA microspheres (59 ± 2% new bone formation; 21 ± 3% residual HA). The combination of carbonate-substituted hollow HA microspheres and clinically-safe doses of BMP2 could provide promising implants for healing non-loaded bone defects. Strong porous scaffolds of bioactive silicate (13-93) glass were designed with the aid of finite-element modeling, created by robocasting and evaluated for loaded bone repair. Scaffolds with a porosity gradient to mimic human cortical bone showed a compressive strength of 88 ± 20 MPa, a flexural strength of 34 ± 5 MPa and the ability to support bone infiltration in vivo. The addition of a biodegradable polylactic acid (PLA) layer to the external surface of these scaffolds increased their load-bearing capacity in four-point bending by ~50% and dramatically enhanced their work of fracture, resulting in a ductile mechanical response. These bioactive glass-PLA composites, combining bioactivity, high strength, high work of fracture and an internal architecture conducive to bone infiltration, could provide optimal implants for structural bone repair --Abstract, page iv

A Review

This work is co-financed by FEDER, European Funds, through the COMPETE 2020 POCI and PORL, National Funds through FCT—Portuguese Foundation for Science and Technology, and POR Lisboa2020, under the projects PIDDAC (POCI-01-0145-FEDER-007688, Reference UIDB/50025/2020-2023) and PTDC/CTMCTM/30623/2017 (DREaMM). P.S. also acknowledges the individual contract CEECIND.03189.2020. C.T. acknowledges i3N for the Ph.D. grant with reference UI/BD/151541/2021. Publisher Copyright: © 2022 by the authors.In recent decades, new and improved materials have been developed with a significant interest in three-dimensional (3D) scaffolds that can cope with the diverse needs of the expanding biomedical field and promote the required biological response in multiple applications. Due to their biocompatibility, ability to encapsulate and deliver drugs, and capacity to mimic the extracellular matrix (ECM), typical hydrogels have been extensively investigated in the biomedical and biotechnological fields. The major limitations of hydrogels include poor mechanical integrity and limited cell interaction, restricting their broad applicability. To overcome these limitations, an emerging approach, aimed at the generation of hybrid materials with synergistic effects, is focused on incorporating nanoparticles (NPs) within polymeric gels to achieve nanocomposites with tailored functionality and improved properties. This review focuses on the unique contributions of clay nanoparticles, regarding the recent developments of clay-based nanocomposite hydrogels, with an emphasis on biomedical applications.publishersversionpublishe

3D bioactive composite scaffolds for bone tissue engineering

Bone is the second most commonly transplanted tissue worldwide, with over four million operations using bone grafts or bone substitute materials annually to treat bone defects. However, significant limitations affect current treatment options and clinical demand for bone grafts continues to rise due to conditions such as trauma, cancer, infection and arthritis. Developing bioactive three-dimensional (3D) scaffolds to support bone regeneration has therefore become a key area of focus within bone tissue engineering (BTE). A variety of materials and manufacturing methods including 3D printing have been used to create novel alternatives to traditional bone grafts. However, individual groups of materials including polymers, ceramics and hydrogels have been unable to fully replicate the properties of bone when used alone. Favourable material properties can be combined and bioactivity improved when groups of materials are used together in composite 3D scaffolds. This review will therefore consider the ideal properties of bioactive composite 3D scaffolds and examine recent use of polymers, hydrogels, metals, ceramics and bio-glasses in BTE. Scaffold fabrication methodology, mechanical performance, biocompatibility, bioactivity, and potential clinical translations will be discussed

Development of scaffolds by thermally-induced phase separation from biodegradable poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(butylene succinate)

Aplicat embargament des de la data de defensa fins el 31 de juliol de 2022TIPS process followed by freeze-dtying was used to prepare blodegradable and biocompatible matrices from poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) containing 5 and 12 wt% of 3-hydroxyvalerate (HV) and poly(butylene succinate) (PBS). 1,4 dioxane (DXN) and tetrahydofuran (THF) were used as solvents. The cloud points of the polymer solutions were determíned by turbidimetry method, to predict the locus of binodal curve in the binary phase diagrams. A multidirectional cooling from ~70ºC to 25ºC, and then to -5 ºC or -25 ºC was applied to PHBV solutions in the TIPS process. The effect of the applied thermal gradients and HV molar ratio of PHBV copolymer on phase separation mechanism, morphology and mechanical properties of the scaffolds was studied. Upon high HV contents and fast cooling, the solid-liquid phase separation through crystallization of DXN was the controlling mechanism and generated large pores with well-distinguished walls and great structural continuity. The morphologies ascribed to polymer crystallizatíon, mostly with low structural consistency were further discernible upon slow cooling. An improvement in scaffolds rigidity were observed in low HV and fast cooling conditions, due to the increased polymer crystallinity and the greater structural consistency, respectively. PHBV scaffolds showed a complete biocompatibility towards MDCK and NRK cell adhesion and proliferation. A multidirectional cooling from ~70 ºC to -20 or -74 ºC were applied to PBS-DXN and PBS-THF solutions and a uniaxial cooling from ~70ºC to -74 or -196 ºC to PBS-DXN. 5 and 100 wt% ofcurcumin (CUR) and piperine (PIP) natural drugs were loaded into PBS matrices via a one-step TIPS fabrication/drug loadlng protocot. Utílizing DXN and THF solvents, solid-liquid and liquid-liquid phase separation were respectívely detected as the main mechanisms responsible for creating the porous structures, while the subregions composed of crysta llized PBS were also obse rved . The applied uniaxial thermal gradient enabled DXN solvent to crystallize along the heat transfer direction and form an oriented pare structure. Although the low drug values did not significantly influence the morphology, the high-level drug loading gave rise to the decreased porosity and superficial roughness ofthe scaffolds . A uniform distribution ofprismatic PIP crystals and matrix-integrated CUR aggregation was observed all overthe structure. The integration of CUR which was confirmed by the physicochemical analyses attributed to a possible interaction with the PBS matrix, as it also showed a slower release profile compared to PIP. Oriented matrices showed greater biocompatibility and also retarded drug release from their·dense spherulitic pore walls. Biobased highly rigid polycarbonate and polyesters with terpene oxide units were blended with PBS at different ratios to increase the biocontents and modify the properties. Ali the terpene-derived polymers exhibi ted high Tg, thermal stability biocompatibility and mechanical strength. Their rigid nature and stiff chains led to insignificant hydrolytic and enzymatic biodegradation, while an accelerated degradation in oxidative media was observed. Their blends with PBS were also biocom patible and to sorne extent biodegradable . 30 wt% of poly (PA-LO) the copolyester derived from phthalic anhydride and limonene oxide, was blended with PBS and porous matrices were prepared by a one-step TIPS fabrication/blending protocol. Multidirectional cooling to -20 ºC or-74 ºC and uniaxial cooling to -74 ºC or-196 ºC was applied to PBS-Poly (PA-LO)-DX N system. Although the blending did not affect the morphology and pore structure of the random/oriented matrices, could somewhat restrict the crystallization of PBS from the solution during the TIPS process. Accordin gly, thinner polymer leaves upon multídirectional and lower thermal gradient, and smaller, less planar and less integrated spherulites were formed upon high uniaxial gradient.El proceso TIPS seguido de liofilización fue usado para preparar matrices porosas ("scaffolds") biodegradables y biocompatibles a partir del poliéster poli (3-hidroxibutirato-co-3-hidroxivalerato) (PHBV) que contiene 5 y 12 wto/o de 3- hidroxivalerato (HV) y del poliéster poli(butilensuccinato) (PBS). El 1.4 dioxano (DXN) yel tetrahidofurano (THF) fueron los disolventes. En el TIPS para las disoluciones de PHBV se aplicó un enfriamiento multidireccional de 70 a 25ºC y luego a -5 ó -25ºC. Se estudiaron los efectos del gradiente térmico y contenido de HV del copolímero sobre el mecanismo de separación de fases, la morfología y propiedades mecánicas de los scaffolds. La separación de fase sólido-líquido en la cristalización del DXN durante el enfriamiento rápido fue el mecanismo que controla la formación de los scaffolds del copolímero con alto contenido de HV. Los scaffolds mostraron grandes poros con paredes bien formadas y gran integridad estructural. Las morfologías atribuidas a la cristalización del polímero. en su mayoría con poca integridad estructural. fueron obtenidas con el enfriamiento lento. Se observó una mejora en la rigidez y mayor integridad estructural de los scaffolds con bajo HV y enfriamiento rápido. debido al aumento de la cristalinidad del polímero. Los scaffolds de PHBV mostraron gran biocompatibilidad determinada por la adhesión y proliferación de células MDCK y NRK. Las disoluciones de PBS-DXN y PBS-THF fueron enfriadas multidireccionalmente de 70ºC a -20 ó -74ºC. y de manera uniaxial para PBS-DXN de 70ºC a -74 ó -196ºC. Los scaffolds de PBS durante su preparación por TIPS fueron cargados con 5 y 100 wt% de curcumina (CUR) o piperina (PIP). La separación de fases sólido-líquido y líquido-líquido (con los disolventes DXN y THF respectivamente) fueron los principales mecanismos responsables para formar las estructuras porosas y subregiones compuestas por PBS cristalizada. El gradiente térmico uniaxial permitió la cristalización del DXN a lo largo de la dirección de transferencia de calor y la formación de poros orientados. La presencia de los fármacos no influyo significativamente en la morfología de los scaffolds. La gran cantidad del fármaco disminuye la porosidad y la rugosidad superficial en los scaffolds. En los scaffolds se observó una distribución uniforme de cristales de PIP y agregación de CUR. La integración de CUR indico una posible interacción con la matriz de PBS y mostró un perfil de liberación más lento en comparación con PIP. Los scaffolds orientados mostraron una mayor biocompatibilidad y una liberación lenta del fármaco debido a sus densas paredes formadas por esferulitas policarbonatos y poliésteres biobasados altamente rígidos y formados por unidades de óxido de terpeno fueron mezclados con PBS en diferentes proporciones para aumentar su biocontenido y modificar sus propiedades. La Tg. estabilidad térmica, biocompatibilidad y resistencia mecánica son elevadas en los polímeros derivados del terpeno. La biodegradación hidrolítica y enzimática de estos polímeros fue insignificante debido a la rigidez de sus cadenas. mientras una degradación acelerada fue lograda en medios oxidativos. Las mezclas con PBS fueron biocompatibles y algo biodegradables. La mezcla del copoliéster derivado de anhídrido phtalico y óxido de limoneno (poli(PA-LO)) con PBS (30:70 wo/o, respectivamente) fue usada para preparar scaffolds con la metodología TIPS. El enfriamiento multidireccional a -20ºC ó -74ºC y el enfriamiento uniaxial a -74ºC ó -196ºC fue aplicado al sistema PBS-Poly(PA-LO)-DXN. Esta mezcla no influye en la morfología y estructura de los poros de los scaffolds con porosidad orientada o al azar. Durante el proceso TIPS, la cristalización del PBS fue afectada. En consecuencia, el PBS en el menor gradiente térmico multidireccional forma estructuras en hojas más delgadas y con el mayor gradiente uniaxial se formaron esferolitas más pequeñas, menos planas y menos integradas.Postprint (published version

Toxicological Profile of Nanostructured Bone Substitute Based on Hydroxyapatite and Poly(lactide-co-glycolide) after Subchronic Oral Exposure of Rats

Novel three-dimensional (3D) nanohydroxyapatite-PLGA scaffolds with high porosity was developed to better mimic mineral component and microstructure of natural bone. To perform a final assessment of this nanomaterial as a potential bone substitute, its toxicological profile was particularly investigated. Therefore, we performed a comet assay on human monocytes for in vitro genotoxicity investigation, and the systemic subchronic toxicity investigation on rats being per oral feed with exactly administrated extract quantities of the nano calcium hydroxyapatite covered with tiny layers of PLGA (ALBO-OS) for 120 days. Histological and stereological parameters of the liver, kidney, and spleen tissue were analyzed. Comet assay revealed low genotoxic potential, while histological analysis and stereological investigation revealed no significant changes in exposed animals when compared to controls, although the volume density of blood sinusoids and connective tissue, as well as numerical density and number of mitosis were slightly increased. Additionally, despite the significantly increased average number of the Ki67 and slightly increased number of CD68 positive cells in the presence of ALBO-OS, immunoreactive cells proliferation was almost neglected. Blood analyses showed that all of the blood parameters in rats fed with extract nanomaterial are comparable with corresponding parameters of no feed rats, taken as blind probe. This study contributes to the toxicological profiling of ALBO-OS scaffold for potential future application in bone tissue engineering

Electrospun nanofibers for improved angiogenesis: Promises for tissue engineering applications

Angiogenesis (or the development of new blood vessels) is a key event in tissue engineering and regenerative medicine; thus, a number of biomaterials have been developed and combined with stem cells and/or bioactive molecules to produce three-dimensional (3D) pro-angiogenic constructs. Among the various biomaterials, electrospun nanofibrous scaffolds offer great opportunities for pro-angiogenic approaches in tissue repair and regeneration. Nanofibers made of natural and synthetic polymers are often used to incorporate bioactive components (e.g., bioactive glasses (BGs)) and load biomolecules (e.g., vascular endothelial growth factor (VEGF)) that exert pro-angiogenic activity. Furthermore, seeding of specific types of stem cells (e.g., endothelial progenitor cells) onto nanofibrous scaffolds is considered as a valuable alternative for inducing angiogenesis. The effectiveness of these strategies has been extensively examined both in vitro and in vivo and the outcomes have shown promise in the reconstruction of hard and soft tissues (mainly bone and skin, respectively). However, the translational of electrospun scaffolds with pro-angiogenic molecules or cells is only at its beginning, requiring more research to prove their usefulness in the repair and regeneration of other highly-vascularized vital tissues and organs. This review will cover the latest progress in designing and developing pro-angiogenic electrospun nanofibers and evaluate their usefulness in a tissue engineering and regenerative medicine setting

A perspective on magnetic core–shell carriers for responsive and targeted drug delivery systems

Magnetic core–shell nanocarriers have been attracting growing interest owing to their physicochemical and structural properties. The main principles of magnetic nanoparticles (MNPs) are localized treatment and stability under the effect of external magnetic fields. Furthermore, these MNPs can be coated or functionalized to gain a responsive property to a specific trigger, such as pH, heat, or even enzymes. Current investigations have been focused on the employment of this concept in cancer therapies. The evaluation of magnetic core–shell materials includes their magnetization properties, toxicity, and efficacy in drug uptake and release. This review discusses some categories of magnetic core–shell drug carriers based on Fe2O3 and Fe3O4 as the core, and different shells such as poly(lactic-co-glycolic acid), poly(vinylpyrrolidone), chitosan, silica, calcium silicate, metal, and lipids. In addition, the review addresses their recent potential applications for cancer treatment.The authors would like to acknowledge Qatar University for funding the project: GCC Co-Fund Program Grant #GCC-2017-001 and student grant QUST-1-CAS-2019-36. The publication of this article was funded by the Qatar National Library.Scopu

Development & Characterisation of Nanocomposites for Bone Tissue Engineering

The aim of this thesis was to develop a bioactive and resorbable nanoscale composite that mimics the properties of bone and will have the potential to regenerate bone. In conventional composites, the polymer phase can mask the bioactive phase and often degrades faster than the ceramic phase due to the weak interfacial bonding between the polymer and ceramic. Here in this thesis an organic/inorganic nanocomposite with stronger interfacial bonding between the two phases has been produced using the sol-gel route. Glasses containing SiO2 and CaO were used as the inorganic while the amino acid poly-γ−glutamic acid (γ−PGA) was used as the organic. This is the first time an inorganic/organic hybrid with enzymatically degradable polymer covalently crosslinked to the inorganic has been produced. Several factors contributed to the homogeneity of the nanocomposites; most important of all was the extent of integration (homogeneity and phase miscibility) of the organic into the inorganic sol. The main focus of this thesis was to synthesise this new material and to develop an understanding of the nanoscale interactions of the two phases. The chemical structure of the nanocomposites were characterised with Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR) and the nanostructure was characterised with scanning and transmission electron microscopy (SEM and TEM). Bioactivity studies of the nanocomposites in simulated body fluid (SBF) showed that the nanocomposites containing calcium were bioactive. Initial in vitro cell response studies also showed that the nanocomposites were not toxic to cells. Nanocomposites were also foamed to create the first porous bioactive inorganic/organic scaffolds with covalent bonding between the organic and inorganic. Micro-computed tomography (μCT) was used to non-destructively image and quantify the internal pore structure of the bioactive nanocomposite scaffolds. The three-dimensional images of the scaffolds show that the nanocomposites have large macropores with multiple connections between them giving a suitable pore structure for tissue engineering

Scaffolding strategies for tissue engineering and regenerative medicine applications

During the past two decades, tissue engineering and the regenerative medicine field have invested in the regeneration and reconstruction of pathologically altered tissues, such as cartilage, bone, skin, heart valves, nerves and tendons, and many others. The 3D structured scaffolds and hydrogels alone or combined with bioactive molecules or genes and cells are able to guide the development of functional engineered tissues, and provide mechanical support during in vivo implantation. Naturally derived and synthetic polymers, bioresorbable inorganic materials, and respective hybrids, and decellularized tissue have been considered as scaffolding biomaterials, owing to their boosted structural, mechanical, and biological properties. A diversity of biomaterials, current treatment strategies, and emergent technologies used for 3D scaffolds and hydrogel processing, and the tissue-specific considerations for scaffolding for Tissue engineering (TE) purposes are herein highlighted and discussed in depth. The newest procedures focusing on the 3D behavior and multi-cellular interactions of native tissues for further use for in vitro model processing are also outlined. Completed and ongoing preclinical research trials for TE applications using scaffolds and hydrogels, challenges, and future prospects of research in the regenerative medicine field are also presented.This research was funded by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) (NORTE-01-0145-FEDER-000023) and by the Portuguese Foundation for Science and Technology ((M-ERA-NET/0022/2016), Transitional Rule DL 57/2016 (CTTI-57/18-I3BS(5)), and (IF/01285/2015))