Engenharia tecidual e biotecnologia aplicadas no desenvolvimento de scaffolds multifuncionais para estimular o reparo do complexo dentina-polpa
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Data
2021-02-02
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Universidade Estadual Paulista (Unesp)
Resumo
Este trabalho teve por objetivo empregar diferentes técnicas de engenharia tecidual e biotecnologia para o desenvolvimento de scaffolds biomiméticos em associação com moléculas com reconhecido potencial osteogênico, visando seu emprego em estratégias cell-homing para reparo do complexo dentina-polpa. A primeira estratégia foi baseada na formulação de scaffolds de quitosana (CH) contendo hidróxido de cálcio (Ca) e beta-glicerofosfato de sódio (βGP), sendo realizada sua caracterização físico-química (MEV, EDS, FTIR, perda de massa, grau de porosidade) e biológica in vitro (ensaios de contato direto e extrato). O potencial cellhoming foi testado em um modelo experimental pulp-in-a-chip, onde os scaffolds sem células foram cultivados justapostos a uma cultura 3D das células pulpares humanas sob pressão intra-pulpar simulada. Viabilidade celular (Alamar blue e Live/Dead), adesão/espalhamento (F-actina), migração celular (transwell), deposição cálcio/matriz mineralizada (o-cresolftaleína/Alizarin red), atividade de ALP (ensaio ponto final) e expressão de DSP (imunofluorescência) foram avaliados (n=6; ANOVA/Tukey; α=5%). A partir da incorporação de Ca e βGP em uma formulação específica foi possível desenvolver um scaffold poroso de quitosana contendo nanoglóbulos de cálcio e fosfato depositados sobre sua superfície (CH-Ca-βGP), com grau de degradabilidade controlada. Este scaffold permitiu adequada interação com as células pulpares semeadas sobre os mesmos, apresentando efeito bioestimulador sobre a viabilidade e expressão de marcadores de diferenciação odontogênica (ALP e mineralização), bem como foi capaz de modular a quimiotaxia e diferenciação celular à distância, de forma significativamente superior às demais formulações. Finalmente, o ensaio pulp-in-a-chip demonstrou seu potencial em mobilizar células da cultura 3D para sua superfície, e induzir a expressão de DSP e matriz mineralizada na ausência de suplementação osteogênica. A segunda estratégia foi baseada na formulação de um hidrogel fotopolimerizável de gelatina e metacrilato de metila (GelMA), ao qual foram adicionados nanotubos de haloisita (HNT) contendo dexametasona (DEX), criando-se um sistema de liberação controlada. Estes hidrogéis foram caracterizados quanto a morfologia (MEV), composição física e química (MET/FTIR), degradação enzimática e resistência compressiva. A avaliação biológica foi realizada por meio do contato direto com células pulpares humanas de dentes decíduos (SHEDs), e em modelo de co-cultura a distância com transwells sob estímulo inflamatório (LPS). Foram realizadas análises de viabilidade celular (MTS), atividade de ALP (AnaSpec) e deposição de matriz mineralizada (Alizarin red). A biocompatibilidade foi avaliada por teste de implantação em subcutâneo de ratos, e o potencial bioativo em defeitos críticos em calvária (ANOVA Two-way/Tukey; α=5%). Foi possível desenvolver um hidrogel macro-poroso com adequado módulo de compressão e degradabilidade, capaz de promover liberação controlada da DEX. As células semeadas sobre a superfície do GelMA contendo 5% de HNT-DEX 10% e em modelo de co-cultura sob estímulo inflamatório apresentaram os maiores valores de atividade de ALP e deposição de matriz mineralizada. Este biomaterial não promoveu reação inflamatória tecidual, e foi capaz de aumentar intensamente a deposição óssea in vivo. A partir dos resultados obtidos, podemos concluir que ambos os sistemas desenvolvidos são promissores para aplicação na regeneração dentinária, pois são capazes de interagir positivamente com as células pulpares e estimulam a expressão do fenótipo odontoblástico in vitro e in vivo.
The aim of this study was to apply tissue engineering and biotechnology techniques to develop biomimetic scaffolds in association with osteogenic molecules, as a cellhoming therapy for dentin regeneration. The first strategy was based on chitosan (CH) scaffolds containing calcium hydroxide (Ca) and beta-glycerophosphate (βGP). The scaffolds were subjected to physical-chemical (MEV, EDS, FTIR, degradation, porosity degree) and in vitro biological characterizations (direct contact and extract assay). Cell-homing potential was evaluated in a pulp-in-a-chip experimental model, in which cell-free scaffolds were cultivated in intimate contact with 3D cultures of dental pulp cells under simulated intra-pulpal pressure. Cell viability (Alamar blue and Live/Dead), adhesion/spread (F-actin), cell migration (transwell), calcium/mineralized matrix deposition (o-cresolftaleína/Alizarin red), ALP activity (end point assay) and DSP expression (immunofluorescence) were evaluated (n=6; ANOVA/Tukey; α=5%). The incorporation of Ca and βGP in a specific formulation created a stable porous chitosan scaffold containing calcium phosphate nano-globules on its surface (CH-Ca- βGP). This scaffold allowed adequate interaction with pulp cells and presented biostimulator effect on cell viability and odontogenic markers expression (ALP and mineralization) on cells seeded onto its surface and at distance, along with chemotactic potential. These cell effects were significantly higher than the other formulation. Finally, pulp-in-a-chip assay showed that CH-Ca-βGP mobilized cells from 3D culture to its surface, and induced DSP expression and mineralized matrix deposition in absence of osteogenic supplementation. In the second strategy, a photopolymerized hydrogel composed by gelatin and methyl metacrylate (GelMA) was associated with halloysite nanotubes (HNT) functionalized with dexamethasone (DEX), creating a drug delivery system. Hydrogel morphology (MEV), physical and chemical composition (MET, FTIR), enzymatic degradation and compressive strength were assessed. Biological analysis was performed by seeding stem cells from human exfoliated deciduous teeth (SHEDs) onto hydrogens, and by means of a co-culture model at distance using transwells under inflammatory stimulus (LPS). Cell viability (MTS), ALP activity (AnaSpec) and mineralized matrix deposition (Alizarin red) were determined. Hydrogel biocompatibility was evaluated after rat’s subcutaneous implantation and the bioactive potential was investigated on critical calvarial defects (ANOVA Two-way/Tukey; α=5%). It was possible to create a porous hydrogel with adequate compressive modulus and degradability, capable to promote a controlled release of DEX. Cells seeded on GelMA surface containing 5% of HNT-DEX 10% and in co-culture model under inflammatory stimulus presented the highest values of ALP activity and mineralized matrix deposition. This biomaterial did not elicit inflammatory reaction and was capable to promote an intense increase on bone deposition in vivo. Based these results, it was possible conclude that both systems are promising for application in dentin regeneration, since they are capable to interact positively with pulp cells and also stimulate the odontoblastic phenotype expression in vitro and in vivo.
The aim of this study was to apply tissue engineering and biotechnology techniques to develop biomimetic scaffolds in association with osteogenic molecules, as a cellhoming therapy for dentin regeneration. The first strategy was based on chitosan (CH) scaffolds containing calcium hydroxide (Ca) and beta-glycerophosphate (βGP). The scaffolds were subjected to physical-chemical (MEV, EDS, FTIR, degradation, porosity degree) and in vitro biological characterizations (direct contact and extract assay). Cell-homing potential was evaluated in a pulp-in-a-chip experimental model, in which cell-free scaffolds were cultivated in intimate contact with 3D cultures of dental pulp cells under simulated intra-pulpal pressure. Cell viability (Alamar blue and Live/Dead), adhesion/spread (F-actin), cell migration (transwell), calcium/mineralized matrix deposition (o-cresolftaleína/Alizarin red), ALP activity (end point assay) and DSP expression (immunofluorescence) were evaluated (n=6; ANOVA/Tukey; α=5%). The incorporation of Ca and βGP in a specific formulation created a stable porous chitosan scaffold containing calcium phosphate nano-globules on its surface (CH-Ca- βGP). This scaffold allowed adequate interaction with pulp cells and presented biostimulator effect on cell viability and odontogenic markers expression (ALP and mineralization) on cells seeded onto its surface and at distance, along with chemotactic potential. These cell effects were significantly higher than the other formulation. Finally, pulp-in-a-chip assay showed that CH-Ca-βGP mobilized cells from 3D culture to its surface, and induced DSP expression and mineralized matrix deposition in absence of osteogenic supplementation. In the second strategy, a photopolymerized hydrogel composed by gelatin and methyl metacrylate (GelMA) was associated with halloysite nanotubes (HNT) functionalized with dexamethasone (DEX), creating a drug delivery system. Hydrogel morphology (MEV), physical and chemical composition (MET, FTIR), enzymatic degradation and compressive strength were assessed. Biological analysis was performed by seeding stem cells from human exfoliated deciduous teeth (SHEDs) onto hydrogens, and by means of a co-culture model at distance using transwells under inflammatory stimulus (LPS). Cell viability (MTS), ALP activity (AnaSpec) and mineralized matrix deposition (Alizarin red) were determined. Hydrogel biocompatibility was evaluated after rat’s subcutaneous implantation and the bioactive potential was investigated on critical calvarial defects (ANOVA Two-way/Tukey; α=5%). It was possible to create a porous hydrogel with adequate compressive modulus and degradability, capable to promote a controlled release of DEX. Cells seeded on GelMA surface containing 5% of HNT-DEX 10% and in co-culture model under inflammatory stimulus presented the highest values of ALP activity and mineralized matrix deposition. This biomaterial did not elicit inflammatory reaction and was capable to promote an intense increase on bone deposition in vivo. Based these results, it was possible conclude that both systems are promising for application in dentin regeneration, since they are capable to interact positively with pulp cells and also stimulate the odontoblastic phenotype expression in vitro and in vivo.
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Medicina Regenerativa, Engenharia Tecidual, Dentina, Regenerative medicine, Tissue engineering, Dentin