Tese de doutoramento em Engenharia de MateriaisIn the orthopedics field, one of the major hurdles... more Tese de doutoramento em Engenharia de MateriaisIn the orthopedics field, one of the major hurdles that surgeons face on daily basis is the need for bone replacing materials to restore defects that lose capability for self repair. To date, autograft materials remain as the “gold standard” for bone repair; however donor-site morbidity and limitations on the amount of tissue that can be collected have led scientists to search for new materials capable to induce bone repair. The main objective of this thesis was the conception of membranes that comprise pivotal characteristics and properties to impart bone repair. The first part of this thesis addressed the production of membranes that have the capacity of osteointegration, i.e., that possess an adequate bone-bond ability. We hypothesized that, in some situations, the two sides of the membranes are in contact with distinct biological environments in which one side faces a region in which osteointegration should be ideally promoted. For the proof of concept, the first work describes the development and characterization of poly(D,L-lactic acid)/Bioglass® (PDLLA/BG) composite membranes with asymmetric bioactivity. Such asymmetry was obtained by an adjusted solvent casting method that promoted a nonuniform distribution of the inorganic component along the membrane thickness. The membranes presented a good integration between the polymeric and inorganic fractions. Moreover, only the inorganic-rich face promoted the deposition of bone-like apatite. Additionally, the composite membranes were found to be stiffer compared with the pure polymer. The results indicate that the proposed asymmetric PDLLA/BG membranes could have potential to be used in guided bone regeneration therapies. This work was validated by the use of synthetic PDLLA polymer, a material widely used in bone repair but biologically inert. The subsequent studies of this thesis make use of natural polymers once they are biologically active possessing similarities with the bone extracellular matrix. In this context, the bone-bonding ability was also demonstrated by the preparation of chitosan/Bioglass® (CHI/BG) composite membranes where their potential to induce a bone-like layer was demonstrated upon immersion in simulated body fluids (SBF). Although in literature the bioactivity (capacity to form a bonelike apatite layer under physiological-like conditions) of such chitosan-based systems has been reported, such studies never demonstrate what happens with the mechanical properties of the material during this process. For that, dynamic mechanical analysis (DMA) experiments were performed in real time while the samples were immersed in simulated body fluid at 37ºC, being subjected to different tension loads. It was possible to follow the formation of a bone-like apatite using microscopic and spectroscopic techniques, and, simultaneously, the variations of the mechanical/viscoelastic properties were also evaluated. Moreover, as in in-vivo conditions bone tissues are subjected to mechanical stimuli with distinct intensities, we demonstrated that the membranes can present different calcification kinetics by varying the dynamic strain amplitude. With the same reasoning, we also developed CHI/BG composite membranes with nano-sized BG particles (CHI/nBG). The results demonstrated that CHI/nBG membranes possess enhanced mechanical properties and higher bioactivity in comparison with the CHI/BG membranes that contained micron-sized BG particles. Although bone-bonding ability is an important characteristic, it is not the only requirement for inducing bone healing upon implanting a device for such purposes. The second part of this thesis focused on the preparation of membranes that hold osteoconductive and osteoinductive properties. To achieve this goal, we transpose the Layer-by-Layer (LbL) technology for the production of nanostructured bioactive free-standing (FS) polymeric membranes that could be directly used to cover bone defects and actively assist bone healing. To this end, an adequate protocol was used to detach the multilayer films from lowsurface energy substrates in mild conditions. The FS membranes, prepared using chitosan and alginate, were crosslinked at various extents and their biocompatibility was validated. Additionally, in order to enhance osteoinductive properties of the membranes, bone morphogenetic protein 2 (BMP-2) – an osteogenic growth factor already approved to be used in the clinic - was loaded in the membranes. The BMP-2 incorporation was proven successful, once improved osteogenesis was detected in cells when cultured in presence of membranes leaded with the growth factor. Taken altogether, distinct methods and strategies to tailor membranes with topographical, chemical and biological signals were obtained in this thesis. The developed systems present valuable properties for the enhancement of bone healing and have characteristics that make them suitable to be potentially used in the…
Vascularization is crucial process for the success of tissue engineered constructs. Without a pro... more Vascularization is crucial process for the success of tissue engineered constructs. Without a proper vascular network, implants, both in vivo and in vitro, suffer from hypoxia, lack of nutrients and accumulation of waste products that ultimately result in cell death. This problem can be circumvented by promoting enhanced blood vessel ingrowth into the implant. To this end, we created an in vitro system using Endothelial Progenitor Cells (EPCs) and a PLA/ calcium phosphate glass composite (PLA/G5) developed in our group for bone regeneration (Charles-Harris et al, 2005). These scaffolds show a high porosity (95%) and a pore size larger than 500 um with very open interconnected porosity. We expected G5 particles to introduce bioactivity in the composite, improving bone and vascular tissue regeneration. EPCs are stem cells that can be isolated from bone marrow. They show expression of several endothelial and stem cell markers (CD31, CD34, VEGFR-2, vWF) and have strong vasculogenic prop...
P222 (EI0323) SUBCRITICAL SINTERING OF AN ALIPHATIC POLYESTER ENRICHED WITH ULVAN CAPSULES LOADED... more P222 (EI0323) SUBCRITICAL SINTERING OF AN ALIPHATIC POLYESTER ENRICHED WITH ULVAN CAPSULES LOADED WITH A BIOACTIVE AGENT A. Alves1, A.R.C. Duarte1, J.F. Mano1, R.A. Sousa1, R.L. Reis1 13B’s Research Group Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Caldas das Taipas, Guimarães, Portugal
a 3B ́s Research Group Biomaterials, Biodegradables and Biomimetic, Department of Polymer Enginee... more a 3B ́s Research Group Biomaterials, Biodegradables and Biomimetic, Department of Polymer Engineering, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine. b IBB Institute for Biotechnology and Bioengineering, PT Government Associated Laboratory AvePark, Zona Industrial da Gandra, S. Cláudio do Barco, 4806-909 Caldas das Taipas, Guimarães, Portugal E-mail Address: [email protected]
In the orthopedics field, one of the major hurdles that surgeons face on daily basis is the need ... more In the orthopedics field, one of the major hurdles that surgeons face on daily basis is the need for bone replacing materials to restore defects that lose capability for self repair. To date, autograft materials remain as the “gold standard” for bone repair; however donor-site morbidity and limitations on the amount of tissue that can be collected have led scientists to search for new materials capable to induce bone repair. In this context, we make use of composite membranes composed of chitosan (CHI) in combination with Bioglass® (CHI/BG) where their potential to induce a bone-like layer was demonstrated upon immersion in simulated body fluids (SBF). Moreover, we also intended to recreate several environments to predict both the evolution of the membranes properties upon implantation and to have more insight about the mineralization process. Therefore the mineralization process is monitored, in-real time, where the viscoelastic/mechanical properties are followed simultaneously as an apatite layer is formed using dynamic mechanical analysis (DMA)
Revealing the potential of squid chitosan-based structures for biomedical applications This artic... more Revealing the potential of squid chitosan-based structures for biomedical applications This article has been downloaded from IOPscience. Please scroll down to see the full text article.
L u c i a n a S o u s a , R i t a M a g a l h ã e s , S o f i a C a r i d a d e , I l í d i o B r... more L u c i a n a S o u s a , R i t a M a g a l h ã e s , S o f i a C a r i d a d e , I l í d i o B r a n d ã o INTRODUÇÃO: A vitamina B12 é um elemento essencial na eritropoiese, devendo o seu défice ser interrogado quando na presença de eritrócitos macrocíticos com ou sem anemia, existência de pancitopenia ou aparecimento de sintomas ou sinais neurológicos inexplicados. O diagnóstico é obtido através da confirmação directa da sua diminuição ou indirectamente pela presença de precursores aumentados. Os factores etiológicos são os mais diversos, podendo variar desde factores dietéticos ou iatrogénicos, até doenças hereditárias ou então infecção ou colonização por bactérias. O tratamento passa pela correcção da causa e reposição oral ou intramuscular (IM) da vitamina.
Fundacao para a Ciencia e Tecnologia (grant SFRH/BPD/95446/2013), ‘‘Fundo Social Europeu’’ and ‘‘... more Fundacao para a Ciencia e Tecnologia (grant SFRH/BPD/95446/2013), ‘‘Fundo Social Europeu’’ and ‘‘Programa Operacional de Potencial Humano’’
In a marine environment, specific proteins are secreted by mussels and used as a bioglue to stick... more In a marine environment, specific proteins are secreted by mussels and used as a bioglue to stick to a surface allowing generate irreversible bonding. Adhesive secreted proteins of mussels present an unusual amino acid 3,4-dihydroxyphenylalanine, DOPA [1]. The outstanding adhesive properties of these materials in the harsh conditions of the sea, impossible to mimic by human efforts until now, have been attributed to the presence of the catechol groups present in DOPA [2]. So, inspired by nature and in particular by the mussel adhesive proteins (MAPs), layer-by-layer films (LbL) with superior adhesive properties in wet environments were developed in this work. These LbL films were based on natural polymers combined with catechol groups, in particular hyaluronic acid and chitosan that have demonstrated adequate properties such as biocompatibility for several biomedical applications, in order to obtain films with enhanced tissue-adhesive properties and improved cell behaviour. Moreover, in order to develop truly multifunctional mussel inspired LbL films, bioactive nanoparticles were incorporated in their composition, besides the presence of the catechol groups. The final goal is to specifically use these films as coatings of a variety of implants for orthopaedic applications in order to improve both cell response and osteoconductivity in a simple and versatile way. Several techniques were used to characterize the properties of the developed films, such as UV spectrophotometry, quartz-crystal microbalance with dissipation monitoring (QCM-D), mechanical tests and SEM
13B's Research Group – Biomaterials, Biodegradables and Biomimetics, University of Minho, Hea... more 13B's Research Group – Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Taipas, Guimarães, Portugal; 2IBB – Institute for Biotechnology and Bioengineering, PT Associated Laboratory, Guimarães, Portugal; 3Department of Health Sciences, Portuguese Catholic University, 3504-505 Viseu, Portugal.
Novel nanostructured free-standing films based on chitosan, alginate and functionalized flake and... more Novel nanostructured free-standing films based on chitosan, alginate and functionalized flake and ribbon-shaped graphene were developed using the layer-by-layer process.
IMAGENS MÉDICAS imaging, leading our thought to a possible foreign body or liposarcoma. 1 During ... more IMAGENS MÉDICAS imaging, leading our thought to a possible foreign body or liposarcoma. 1 During exploratory laparoscopy, a 26 g nodule was extracted, with 4 cm x 3.6 cm x 3.2 cm and a smooth, pale outer surface (Fig. 2A). In the middle we saw a yellowish central area of one cm (Fig. 2B). Histology revealed it had a central adipose tissue with fat necrosis, surrounded by dense fibrous tissue of collagen. During one year of follow-up, the patient remained asymptomatic.
pathogenesis is not clear. Several factors may be associated with this disease. In this case the ... more pathogenesis is not clear. Several factors may be associated with this disease. In this case the suspicion of respiratory and urinary infection associated with acute renal failure may have triggered the ischemic changes. The treatment is not specific with the administration of PPIs, hydration and treatment of the underlying disease and parenteral nutrition. The use of a broad spectrum antibiotic, although there are no data to support its routine use, can be used empirically if the patient's clinical condition warrants. The authors want to demonstrate a clinical case with rare and potentially life threatening situation but with a favorable clinical evolution.
In this work, novel free-standing (FS) films based on chitosan, alginate and graphene oxide (GO) ... more In this work, novel free-standing (FS) films based on chitosan, alginate and graphene oxide (GO) were developed through layer-by-layer assembly.
Tese de doutoramento em Engenharia de MateriaisIn the orthopedics field, one of the major hurdles... more Tese de doutoramento em Engenharia de MateriaisIn the orthopedics field, one of the major hurdles that surgeons face on daily basis is the need for bone replacing materials to restore defects that lose capability for self repair. To date, autograft materials remain as the “gold standard” for bone repair; however donor-site morbidity and limitations on the amount of tissue that can be collected have led scientists to search for new materials capable to induce bone repair. The main objective of this thesis was the conception of membranes that comprise pivotal characteristics and properties to impart bone repair. The first part of this thesis addressed the production of membranes that have the capacity of osteointegration, i.e., that possess an adequate bone-bond ability. We hypothesized that, in some situations, the two sides of the membranes are in contact with distinct biological environments in which one side faces a region in which osteointegration should be ideally promoted. For the proof of concept, the first work describes the development and characterization of poly(D,L-lactic acid)/Bioglass® (PDLLA/BG) composite membranes with asymmetric bioactivity. Such asymmetry was obtained by an adjusted solvent casting method that promoted a nonuniform distribution of the inorganic component along the membrane thickness. The membranes presented a good integration between the polymeric and inorganic fractions. Moreover, only the inorganic-rich face promoted the deposition of bone-like apatite. Additionally, the composite membranes were found to be stiffer compared with the pure polymer. The results indicate that the proposed asymmetric PDLLA/BG membranes could have potential to be used in guided bone regeneration therapies. This work was validated by the use of synthetic PDLLA polymer, a material widely used in bone repair but biologically inert. The subsequent studies of this thesis make use of natural polymers once they are biologically active possessing similarities with the bone extracellular matrix. In this context, the bone-bonding ability was also demonstrated by the preparation of chitosan/Bioglass® (CHI/BG) composite membranes where their potential to induce a bone-like layer was demonstrated upon immersion in simulated body fluids (SBF). Although in literature the bioactivity (capacity to form a bonelike apatite layer under physiological-like conditions) of such chitosan-based systems has been reported, such studies never demonstrate what happens with the mechanical properties of the material during this process. For that, dynamic mechanical analysis (DMA) experiments were performed in real time while the samples were immersed in simulated body fluid at 37ºC, being subjected to different tension loads. It was possible to follow the formation of a bone-like apatite using microscopic and spectroscopic techniques, and, simultaneously, the variations of the mechanical/viscoelastic properties were also evaluated. Moreover, as in in-vivo conditions bone tissues are subjected to mechanical stimuli with distinct intensities, we demonstrated that the membranes can present different calcification kinetics by varying the dynamic strain amplitude. With the same reasoning, we also developed CHI/BG composite membranes with nano-sized BG particles (CHI/nBG). The results demonstrated that CHI/nBG membranes possess enhanced mechanical properties and higher bioactivity in comparison with the CHI/BG membranes that contained micron-sized BG particles. Although bone-bonding ability is an important characteristic, it is not the only requirement for inducing bone healing upon implanting a device for such purposes. The second part of this thesis focused on the preparation of membranes that hold osteoconductive and osteoinductive properties. To achieve this goal, we transpose the Layer-by-Layer (LbL) technology for the production of nanostructured bioactive free-standing (FS) polymeric membranes that could be directly used to cover bone defects and actively assist bone healing. To this end, an adequate protocol was used to detach the multilayer films from lowsurface energy substrates in mild conditions. The FS membranes, prepared using chitosan and alginate, were crosslinked at various extents and their biocompatibility was validated. Additionally, in order to enhance osteoinductive properties of the membranes, bone morphogenetic protein 2 (BMP-2) – an osteogenic growth factor already approved to be used in the clinic - was loaded in the membranes. The BMP-2 incorporation was proven successful, once improved osteogenesis was detected in cells when cultured in presence of membranes leaded with the growth factor. Taken altogether, distinct methods and strategies to tailor membranes with topographical, chemical and biological signals were obtained in this thesis. The developed systems present valuable properties for the enhancement of bone healing and have characteristics that make them suitable to be potentially used in the…
Vascularization is crucial process for the success of tissue engineered constructs. Without a pro... more Vascularization is crucial process for the success of tissue engineered constructs. Without a proper vascular network, implants, both in vivo and in vitro, suffer from hypoxia, lack of nutrients and accumulation of waste products that ultimately result in cell death. This problem can be circumvented by promoting enhanced blood vessel ingrowth into the implant. To this end, we created an in vitro system using Endothelial Progenitor Cells (EPCs) and a PLA/ calcium phosphate glass composite (PLA/G5) developed in our group for bone regeneration (Charles-Harris et al, 2005). These scaffolds show a high porosity (95%) and a pore size larger than 500 um with very open interconnected porosity. We expected G5 particles to introduce bioactivity in the composite, improving bone and vascular tissue regeneration. EPCs are stem cells that can be isolated from bone marrow. They show expression of several endothelial and stem cell markers (CD31, CD34, VEGFR-2, vWF) and have strong vasculogenic prop...
P222 (EI0323) SUBCRITICAL SINTERING OF AN ALIPHATIC POLYESTER ENRICHED WITH ULVAN CAPSULES LOADED... more P222 (EI0323) SUBCRITICAL SINTERING OF AN ALIPHATIC POLYESTER ENRICHED WITH ULVAN CAPSULES LOADED WITH A BIOACTIVE AGENT A. Alves1, A.R.C. Duarte1, J.F. Mano1, R.A. Sousa1, R.L. Reis1 13B’s Research Group Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Caldas das Taipas, Guimarães, Portugal
a 3B ́s Research Group Biomaterials, Biodegradables and Biomimetic, Department of Polymer Enginee... more a 3B ́s Research Group Biomaterials, Biodegradables and Biomimetic, Department of Polymer Engineering, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine. b IBB Institute for Biotechnology and Bioengineering, PT Government Associated Laboratory AvePark, Zona Industrial da Gandra, S. Cláudio do Barco, 4806-909 Caldas das Taipas, Guimarães, Portugal E-mail Address: [email protected]
In the orthopedics field, one of the major hurdles that surgeons face on daily basis is the need ... more In the orthopedics field, one of the major hurdles that surgeons face on daily basis is the need for bone replacing materials to restore defects that lose capability for self repair. To date, autograft materials remain as the “gold standard” for bone repair; however donor-site morbidity and limitations on the amount of tissue that can be collected have led scientists to search for new materials capable to induce bone repair. In this context, we make use of composite membranes composed of chitosan (CHI) in combination with Bioglass® (CHI/BG) where their potential to induce a bone-like layer was demonstrated upon immersion in simulated body fluids (SBF). Moreover, we also intended to recreate several environments to predict both the evolution of the membranes properties upon implantation and to have more insight about the mineralization process. Therefore the mineralization process is monitored, in-real time, where the viscoelastic/mechanical properties are followed simultaneously as an apatite layer is formed using dynamic mechanical analysis (DMA)
Revealing the potential of squid chitosan-based structures for biomedical applications This artic... more Revealing the potential of squid chitosan-based structures for biomedical applications This article has been downloaded from IOPscience. Please scroll down to see the full text article.
L u c i a n a S o u s a , R i t a M a g a l h ã e s , S o f i a C a r i d a d e , I l í d i o B r... more L u c i a n a S o u s a , R i t a M a g a l h ã e s , S o f i a C a r i d a d e , I l í d i o B r a n d ã o INTRODUÇÃO: A vitamina B12 é um elemento essencial na eritropoiese, devendo o seu défice ser interrogado quando na presença de eritrócitos macrocíticos com ou sem anemia, existência de pancitopenia ou aparecimento de sintomas ou sinais neurológicos inexplicados. O diagnóstico é obtido através da confirmação directa da sua diminuição ou indirectamente pela presença de precursores aumentados. Os factores etiológicos são os mais diversos, podendo variar desde factores dietéticos ou iatrogénicos, até doenças hereditárias ou então infecção ou colonização por bactérias. O tratamento passa pela correcção da causa e reposição oral ou intramuscular (IM) da vitamina.
Fundacao para a Ciencia e Tecnologia (grant SFRH/BPD/95446/2013), ‘‘Fundo Social Europeu’’ and ‘‘... more Fundacao para a Ciencia e Tecnologia (grant SFRH/BPD/95446/2013), ‘‘Fundo Social Europeu’’ and ‘‘Programa Operacional de Potencial Humano’’
In a marine environment, specific proteins are secreted by mussels and used as a bioglue to stick... more In a marine environment, specific proteins are secreted by mussels and used as a bioglue to stick to a surface allowing generate irreversible bonding. Adhesive secreted proteins of mussels present an unusual amino acid 3,4-dihydroxyphenylalanine, DOPA [1]. The outstanding adhesive properties of these materials in the harsh conditions of the sea, impossible to mimic by human efforts until now, have been attributed to the presence of the catechol groups present in DOPA [2]. So, inspired by nature and in particular by the mussel adhesive proteins (MAPs), layer-by-layer films (LbL) with superior adhesive properties in wet environments were developed in this work. These LbL films were based on natural polymers combined with catechol groups, in particular hyaluronic acid and chitosan that have demonstrated adequate properties such as biocompatibility for several biomedical applications, in order to obtain films with enhanced tissue-adhesive properties and improved cell behaviour. Moreover, in order to develop truly multifunctional mussel inspired LbL films, bioactive nanoparticles were incorporated in their composition, besides the presence of the catechol groups. The final goal is to specifically use these films as coatings of a variety of implants for orthopaedic applications in order to improve both cell response and osteoconductivity in a simple and versatile way. Several techniques were used to characterize the properties of the developed films, such as UV spectrophotometry, quartz-crystal microbalance with dissipation monitoring (QCM-D), mechanical tests and SEM
13B's Research Group – Biomaterials, Biodegradables and Biomimetics, University of Minho, Hea... more 13B's Research Group – Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Taipas, Guimarães, Portugal; 2IBB – Institute for Biotechnology and Bioengineering, PT Associated Laboratory, Guimarães, Portugal; 3Department of Health Sciences, Portuguese Catholic University, 3504-505 Viseu, Portugal.
Novel nanostructured free-standing films based on chitosan, alginate and functionalized flake and... more Novel nanostructured free-standing films based on chitosan, alginate and functionalized flake and ribbon-shaped graphene were developed using the layer-by-layer process.
IMAGENS MÉDICAS imaging, leading our thought to a possible foreign body or liposarcoma. 1 During ... more IMAGENS MÉDICAS imaging, leading our thought to a possible foreign body or liposarcoma. 1 During exploratory laparoscopy, a 26 g nodule was extracted, with 4 cm x 3.6 cm x 3.2 cm and a smooth, pale outer surface (Fig. 2A). In the middle we saw a yellowish central area of one cm (Fig. 2B). Histology revealed it had a central adipose tissue with fat necrosis, surrounded by dense fibrous tissue of collagen. During one year of follow-up, the patient remained asymptomatic.
pathogenesis is not clear. Several factors may be associated with this disease. In this case the ... more pathogenesis is not clear. Several factors may be associated with this disease. In this case the suspicion of respiratory and urinary infection associated with acute renal failure may have triggered the ischemic changes. The treatment is not specific with the administration of PPIs, hydration and treatment of the underlying disease and parenteral nutrition. The use of a broad spectrum antibiotic, although there are no data to support its routine use, can be used empirically if the patient's clinical condition warrants. The authors want to demonstrate a clinical case with rare and potentially life threatening situation but with a favorable clinical evolution.
In this work, novel free-standing (FS) films based on chitosan, alginate and graphene oxide (GO) ... more In this work, novel free-standing (FS) films based on chitosan, alginate and graphene oxide (GO) were developed through layer-by-layer assembly.
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