6 research outputs found
Development of new chitosan based biodegradable blends for bone and cartilage tissue engineering
Tese de Doutoramento em Ciência e Tecnologia de Materiais - Engenharia de Tecidos - Materiais HíbridosA number of natural origin polymers are being employed in tissue engineering strategies. Natural origin
polymers offer the advantage of being similar to macromolecular substances, which the biological
environment is prepared to recognize and to deal with metabolically. Another attractive characteristic of
natural polymers is their ability to be degraded by naturally occurring enzymes, implying that the implant
will be degraded and eventually metabolized by physiological mechanisms. Chitosan, a
mucopolysaccharide, is the alkaline deactylated product of chitin and has structural similarities to
glycosaminoglycans, thus, mimicking their functional behaviour. Moreover, it is reported to be typically
non-toxic, biodegradable, and biocompatible. Chitosan exhibit a range of properties that make it suitable
for use as alternatives to currently used biomaterials, but its mechanical properties and processability
are less than ideal which precludes its use as material in load bearing applications. The development of
hybrid materials that combine naturally occurring polymers with biocompatible synthetic polymers is
expected to be able to minimize the mismatch of mechanical properties and to preserve the functional
biocompatibility. However, little research has been conducted in the melt blending of synthetic polyester
and chitosan. The main objectives of this thesis were to: i) compound and evaluate the properties of
chitosan and biodegradable aliphatic polyesters blends and composites processed using melt based
technologies; ii) study the water absorption and degradation characteristics of the produced blends and
composites; iii) produce chitosan-based scaffolds with different architectures by melt based
technologies.; iv) evaluate their potential cytotoxic behaviour and to conduct direct contact assays
aiming to validate their potential use in bone or cartilage tissue engineering applications.
The first step of this thesis experimental work was to melt blend chitosan with poly-ε-caprolactone
(PCL), poly(butylene succinate) (PBS), poly(lactic acid) (PLA), poly(butylene terephthalate adipate)
(PBTA), and poly(butylene succinate adipate) (PBSA). For the chitosan/PBS blend, the amount of
chitosan was varied from 25% to 70% by weight. The remaining polyesters had 50% of chitosan by
weight. The morphology, tensile and thermal properties of the produced blends were evaluated.
Composites consisting of hydroxyapatite, chitosan, and aliphatic polyester were also compounded using
a twin-screw extruder. The polyesters used were the same applied in blends preparation. The mass
fraction of chitosan ranged from 17.5 to 45%, while that of HA ranged from 10 to 30%. The tensile properties (tensile strength and tensile modulus), thermal properties, and morphological properties of
the composites were evaluated. Ternary blends often lead to complex morphology and poor properties.
The water absorption and degradation of the developed chitosan-based blends and composites were
also evaluated, since these properties are critical for their possible applications as biomaterials. These
studies revealed that chitosan content significantly affects the water uptake, weight loss and mechanical
properties of the developed blends. The weight loss also directly correlates with the HA content being
higher for higher HA amount present in the composite.
Novel scaffolds made from chitosan-based blends and composites were produced by the melt-based
routine involving compression molding and salt leaching. The scaffolds were made with different pore
sizes and porosity, aiming at applications in bone and cartilage tissue engineering. Cytotoxicity
evaluation were carried out using standard tests, revealing that L929 cells had comparable metabolic
activities to that obtained for the negative control.
Chitosan based fibers and chitosan fiber mesh scaffolds could also be prepared by melt processing
(solvent-free) to be used as tissue engineering templates. Biological assessment was performed in
direct contact assays, using a human osteosarcoma cell line (SaOs-2). Cells showed remarkable cell
colonization, not only at the surface but also in the inner porous structure of the different scaffolds.
Furthermore, the cells showed high indexes of viability in all the scaffold types.
The motivation for this thesis was to develop and to produce a new range of chitosan based scaffolds
using novel polymeric blends and composites in an established and reproducible melt processing
technology. Most of the approaches reported in the literature to produce scaffolds from chitosan-based
blends or composites involve the use of solvents, being this work the first that reports the successful use
of a melt-based route. Moreover, the methodologies used are very versatile and avoid the drawbacks
associated with solvents and allows for producing a large variety of scaffolds with a wide range of
porosities and pore morphologies using different polymers.Um número cada vez maior de polímeros de origem natural tem sido utilizado em estratégias de
engenharia de tecidos. Estes oferecem a vantagem de serem similares a substâncias
macromoleculares, cujo sistema biológico está preparado para reconhecer e lidar metabolicamente.
Outra característica interessante dos polímeros naturais é a possibilidade de serem degradados por
enzimas existentes no organismo, levando a que o implante seja degradado e eventualmente
metabolizado por mecanismos fisiológicos. O quitosano é um produto obtido pela desacetilação alcalina
da quitina e tem similaridades estruturais com os glicosaminoglicanos, ou seja, mimetiza o seu
comportamento funcional. Além disso, é descrito como sendo tipicamente não-tóxico, biodegradável e
biocompatível. O quitosano exibe um espectro de propriedades que o tornam adequado para ser
utilizado como alternativa aos biomateriais usados correntemente. Mas as suas propriedades
mecânicas e processabilidade não são as ideais limitando a sua utilização em aplicações sujeitas a
esforços elevados. É expectável que o desenvolvimento de materiais híbridos, combinando polímeros
de origem natural com polímeros sintéticos biocompatíveis, minimize a incompatibilidade das
propriedades mecânicas e preserve a sua biocompatibilidade funcional. Pouca ou nenhuma
investigação tem sido desenvolvida focando o desenvolvimento de misturas de quitosano com
poliésteres utilizado tecnologias baseadas na fusão. Uma vez identificada esta lacuna, os principais
objectivos desta tese foram: i) produzir e determinar as propriedades de misturas e compósitos à base
de quitosano com poliésteres alifáticos processados por tecnologias baseadas na fusão; ii) estudar as
características de absorção de água e degradação das misturas e compósitos produzidos; iii) preparar
scaffolds à base de quitosano com diferentes arquitecturas utilizando tecnologias baseadas na fusão;
iv) avaliar o seu potencial comportamento citotóxico e conduzir ensaios de contacto directo com o
objectivo de validar o potencial de serem utilizados em aplicações para engenharia de tecidos do osso
ou cartilagem.
O primeiro passo desta tese em termos de trabalho experimental consistiu em misturar, por técnicas de
fusão, quitosano com policaprolactona (PCL), polibutileno succinato (PBS), polibutileno succinato coadipato
(PBSA), polibutileno teraftalato co-adipato (PBTA) e poli (ácido láctico) (PLA). As quantidades
de quitosano utilizadas na preparação das misturas de quitosano/PBS variaram entre 25% e 70% em
peso. Aos restantes poliésteres foi adicionado 50% em peso de quitosano. As propriedades térmicas e
mecânicas e respectiva morfologia das misturas produzidas foram avaliadas. Compósitos contendo hidroxiapatite (HA), quitosano e poliéster alifático foram preparados utilizando
uma extrusora duplo fuso. Os poliésteres utilizados foram os mesmos das misturas anteriormente
preparadas. A fracção mássica do quitosano variou entre 17.5% e 45%, enquanto a da HA variou entre
10% e 30%. As propriedades mecânicas à tracção, propriedades térmicas, e propriedades morfologias
dos compósitos foram determinadas. As misturas ternárias apresentaram uma morfologia complexa e
propriedades mecânicas abaixo do previsto.
O comportamento em termos de absorção de água e degradação das misturas e compósitos à base de
quitosano foi determinado uma vez que estas propriedades são criticas na utilização de biomateriais.
Estes estudos revelaram que a quantidade de quitosano utilizado afectou significativamente a absorção
de água, perda de peso e propriedades mecânicas das misturas produzidas. O perda de peso
apresentou também uma relação directa com a quantidade de HA sendo maior nos compósitos
preparados com maior quantidade de HA.
Foram produzidos scaffolds com as misturas e compósitos à base de quitosano utilizando métodos
baseados na fusão, nomeadamente moldação por compressão com lixiviação de sal. Os scaffolds
foram produzidos com diferentes porosidades e diferentes tamanhos de poros com o objectivo de
serem utilizados em estratégias de engenharia de tecidos do osso ou cartilagem. Foram realizados
ensaios de citotoxicidade usando testes padrão, tendo as células L929 apresentado uma actividade
celular semelhante à do controlo negativo.
Fibras e scaffolds, à base de fibras, foram também desenvolvidas por tecnologias baseadas na fusão
(sem solventes) para serem utilizados em estratégias de engenharia de tecidos. Foram realizados
ensaios biológicos de contacto directo utilizando uma linha celular derivada de osteosarcoma humano
(SaOs-2). As células apresentaram uma colonização notável, não apenas na superfície mas também
no interior dos scaffolds. Além disso, as células apresentaram altos índices de viabilidade celular em
todo o tipo de scaffolds.
A motivação desta tese foi o desenvolvimento e produção de um novo tipo de scaffolds à base de
quitosano utilizando novas misturas e compósitos com recurso a tecnologias baseadas na fusão. A
maior parte das estratégias descritas na literatura para produção de scaffolds à base de quitosano
envolve o uso de solventes, sendo este o primeiro trabalho que descreve o uso com sucesso de
tecnologias apenas baseadas na fusão. Além disso, as metodologias usadas demonstraram ser muito
versáteis evitando as desvantagens normalmente associadas aos solventes e permitiram a produção
de scaffolds uma variabilidade alargada de porosidades e morfologia de poros.Portuguese Foundation for Science and Technology for the financial support through a PhD grant (SFRH/BD/22455/2005)EU project GENOSTEM (LSHB-CT-2003-503161
Biodentine for Furcation Perforation Repair: An Animal Study with Histological, Radiographic and Micro-Computed Tomographic Assessment
Introduction: Biodentine has been scarcely studied as a furcation perforation (FP) repair material, mostly by in vitro methodologies. This animal study aimed to compare the histological responses, radiographic, and micro-computed tomographic (micro-CT) outcomes after FP repair with Biodentine or ProRoot MTA (MTA) in dogs’ teeth. Methods and Materials: Fifty teeth from five dogs were divided into 4 groups: MTA (n=20, FP repaired with ProRoot MTA), BDT (n=20, FP repaired with Biodentine), PC (n=5, positive control, FP without repair) and NC (n=5, negative control, without perforation). The animals were euthanized after 4 months. Histological assessment included inflammatory cell infiltration, hard tissue resorption, hard tissue repair, and cement repair in the furcation area. Immediate postoperative and 4months follow-up radiographs were compared for radiolucency in the furcation region. The volume of extruded material was quantified using micro-CT images. Results: The tested materials showed equivalent radiographic response, together with similar hard tissue resorption and repair but, BDT group showed significantly less inflammation, lower volume of extruded material and higher cement repair than MTA group. Conclusion: The outcomes of this study, taken together with other favorable results in literature, are highly suggestive that Biodentine is a promising biomaterial to be used for FP repair.Keywords: Biodentine; Biomaterial; Endodontics; Furcation Perforation; Imaging; Micro-Computed Tomograph
Adhesion, proliferation, and osteogenic differentiation of a mouse mesenchymal stem cell line (BMC9) seeded on novel melt-based chitosan/polyester 3D porous scaffolds
The aim of the present work was to study the biological behavior of a mouse mesenchymal stem cell line
when seeded and cultured under osteogenic conditions onto novel processed melt-based chitosan scaffolds.
Scaffolds were produced by compression molding, followed by salt leaching. Scanning electron microscopy
(SEM) observations and lCT analysis showed the pore sizes ranging between 250 and 500 lm and the
interconnectivity of the porous structure. The chitosan–poly(butylenes succinate) scaffolds presented high
mechanical properties, similar to the ones of trabecular bone (E1%*75 MPa). Cytotoxicity assays were
carried out using standard tests (accordingly to ISO/EN 10993 part 5 guidelines), namely, MTS test with a
24 h extraction period, revealing that L929 cells had similar metabolic activities to that obtained for the
negative control. Cell culture studies were conducted using a mouse mesenchymal stem cell line (BMC9).
Cells were seeded onto the scaffold and allowed to proliferate for 3 weeks under osteogenic conditions.
SEM observations demonstrated that cells were able to proliferate and massively colonize the scaffolds
structure. The cell viability assay MTS demonstrated that BMC9 cells were viable after 3 weeks of culture.
The cells clearly evidenced a positive differentiation toward the osteogenic lineage, as confirmed by the
high ALP activity levels. Moreover, energy dispersive spectroscopy (EDS) analysis revealed the presence of
Ca and P in the elaborated extracellular matrix (ECM). These combined results indicate that the novel
melt-based chitosan/polyester scaffolds support the adhesion, proliferation, and osteogenic differentiation
of the mouse MSCs and shows adequate physicochemical and biological properties for being used as
scaffolds in bone tissue engineering–related strategies