Publicação
Development of an injectable dextrin hydrogel for bone regeneration
| Resumo: | Bone is a dynamic, highly vascularized tissue that remodels itseif continuousiy over an individual lifetime. However, this regenerative capacity is limited and, as in the case of large bone defects, where the template for an orchestrated regeneration is absent, surgical procedures are needed. In this context, bone tissue engineering is a very chailenging and promising field given the limitations of traditional methods and the need to create substitutes that mimics bone mechanical, structural, physicai-chemical and biological features, achieving better clinical outcomes. The general concept consists in the deveiopment of 3D scaffolds, from biocompatibie materiais (natural or synthetic), which confer temporary support for the regeneration of bone tissue, while the scaffold itself will be resorbed and replaced by newiy formed tissue. Hydrogels are 3D hydrated, biocompatible, polymeric networks, which mimics native extraceilular matrix and could promote a suitable environment for celis or unstable drugs. These features make them attractive materiais in the biomedicai fieid for celi encapsulation, drug or gene delivery or to act as an interface between tissue and materiais. Dextrin is a iow molecular weight carbohydrate, generaily regarded as safe (GRAS), obtained from partiai hydroiysis of starch or glycogen. It is a glucose poiymer linked by a-1,4 glycosidic linkages with some degree of branching due to the presence of ct-1,6 bonds. It is usually biocompatible and non-immunogenic, degradable by a-amyiases and can undergo renal clearance avoiding tissue accumulation. This work aims to develop an injectable multifunctionai dextrin-based hydrogel through dextrin oxidation (oxDex) fotlowed by crossiinking with a dihydrazide molecuie, intended to be ciinically used as carrier for Bonelike® a synthetic bone graft (Biosckin, SA). The oxDex hydrogei performance as a muitifunctional matrix incorporating dextrin nanogeis, native ECM and ceils was also evaluated. The structural characterization of several commercial avaiiabie dextrins aiiowed the determination of the polymerization and branching degrees, which ranged from 6 to 17 glucose residues and 2 to 13%, respectively. The combination of hydrogel with a dextrin nanogel and urinary bladder matrix was achieved without compromising the mechanical properties or morphoiogy. These features support the proposai of this material as a piatform for drug delivery with improved bioactivity. The cytotoxicity of the free dihydrazide was evaivated and only a miid inhibitory effect on ceil proliferation was observed for the concentration used in the hydrogei crossiinking. The encapsulation of cells, preserving its viabiiity, confirmed the biocompatibiiity of the injectable hydrogeis, which have therefore great potential for biomedicai applications. One key aspect when developing a biomateriai for clinical use is its susceptibility to sterilisation. Optimization of this process is essential to avoid degradation or modification, especialiy for sensitive natural materiais. The effects of autoclaving and ionizing irradiation (í3 and y) on oxDex soiutions were investigated through rheoiogical analysis. Heat treatment has proven to be an inadequate method for the steriiization of this material, since its effects on dextrin chain prevented hydrogei formation. According to the rheologicai behaviour observed, ali materiais remained gelabie after 0 or y-irradiation. Both kinds of radiation induced slight differences in hydrogei storage modulus, suggesting the occurrence of chain scission/crossiinking effects on the dextrin chains. Nevertheiess, these effects did not seem to be dose or temperature dependent for the tested conditions; furthermore, those differences could not be attributed to free radicais generation since its presence was not detected. ionizing radiation seems to be a safe method for the steriiization of oxidised dextrin without compromising of hydrogei formation or its rheologicai properties. In vivo performance of oxDex hydrogel was evaivated, according to 150 10993-6, for inflammatory response assessment in subcutaneous implants. The effects of the combination with human mesenchymal stem ceiis (hMSCs), urinary biadder matrix (UBM) and bone graft granules (Bonelike®) were also evaluated. Histological anaiysis after 3 and 15 days showed typical acute and chronic infiammatory responses, respectiveiy, with the Bonelike® groups exhibiting a more severe reaction and the hydrogel was abie to better stabiiize Boneiike granules in the impiant site. The presence of viable hMSCs couid be confirmed by HuNu ceiis and by severai infiammatory and immunomoduiatory cytokines and growth factors in the peripheral biood. Dextrin hydrogei was scored as siight irritant after 3 days of impiantation, simiiarly to its combination with UBM and as non-irritant after 15 days. Successfui encapsuiation of hMSCs into hydrogei matrix was achieved and its presence seemed to modulate infiammatory response by acceierating its progression when compared to the aceliuiar groups. Pre-ciinicai triais in New Zeaiand rabbits demonstrated that oxDex hydrogei did not induce any adverse effects in regeneration of bone defects. Complete regeneration was observed after 8 weeks in non-criticai tibiae defects, both for sham control and in the presence of tested formuiations (hydrogei; hydrogei/Boneiike®; hydrogei/UBM). Bone regeneration of caivaria modeis (criticai and non-critcai) was improved by oxDex hydrogel, comparing with sham control, since areas of trabecuiar extensions were observed, proionging from the defect edges and composed by dense and organized coiiagen fibers. Better outcomes were obtained with Boneiike® aione and its combination with oxDex hydrogei in criticai size defects. in both cases, despite complete regeneration being stiil absent, areas of interconnected dense bone tissue were observed in the center of the defect. Furthermore, UM seemed to improve the bone matrix apposition when compared with controi and oxDex hydrogei aione. This injectabie dextrin-based hydrogei provides a system that can carry and stabiiize ceils, nanogeis, Boneiike® granules and other biomoiecuies. Although further investigation is needed for the assessment of hydrogei in vivo performance during bone regeneration, it seems a promising biomateriai due to its biocompatibiiity, easiness of processing, steriiization and handling, and potentiai to promote an adequate environment to moduiate infiammatory response, by hMSCs encapsulation. |
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| Autores principais: | Silva, Dina Maria Morais da |
| Assunto: | Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
| Ano: | 2016 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Bone is a dynamic, highly vascularized tissue that remodels itseif continuousiy over an individual lifetime. However, this regenerative capacity is limited and, as in the case of large bone defects, where the template for an orchestrated regeneration is absent, surgical procedures are needed. In this context, bone tissue engineering is a very chailenging and promising field given the limitations of traditional methods and the need to create substitutes that mimics bone mechanical, structural, physicai-chemical and biological features, achieving better clinical outcomes. The general concept consists in the deveiopment of 3D scaffolds, from biocompatibie materiais (natural or synthetic), which confer temporary support for the regeneration of bone tissue, while the scaffold itself will be resorbed and replaced by newiy formed tissue. Hydrogels are 3D hydrated, biocompatible, polymeric networks, which mimics native extraceilular matrix and could promote a suitable environment for celis or unstable drugs. These features make them attractive materiais in the biomedicai fieid for celi encapsulation, drug or gene delivery or to act as an interface between tissue and materiais. Dextrin is a iow molecular weight carbohydrate, generaily regarded as safe (GRAS), obtained from partiai hydroiysis of starch or glycogen. It is a glucose poiymer linked by a-1,4 glycosidic linkages with some degree of branching due to the presence of ct-1,6 bonds. It is usually biocompatible and non-immunogenic, degradable by a-amyiases and can undergo renal clearance avoiding tissue accumulation. This work aims to develop an injectable multifunctionai dextrin-based hydrogel through dextrin oxidation (oxDex) fotlowed by crossiinking with a dihydrazide molecuie, intended to be ciinically used as carrier for Bonelike® a synthetic bone graft (Biosckin, SA). The oxDex hydrogei performance as a muitifunctional matrix incorporating dextrin nanogeis, native ECM and ceils was also evaluated. The structural characterization of several commercial avaiiabie dextrins aiiowed the determination of the polymerization and branching degrees, which ranged from 6 to 17 glucose residues and 2 to 13%, respectively. The combination of hydrogel with a dextrin nanogel and urinary bladder matrix was achieved without compromising the mechanical properties or morphoiogy. These features support the proposai of this material as a piatform for drug delivery with improved bioactivity. The cytotoxicity of the free dihydrazide was evaivated and only a miid inhibitory effect on ceil proliferation was observed for the concentration used in the hydrogei crossiinking. The encapsulation of cells, preserving its viabiiity, confirmed the biocompatibiiity of the injectable hydrogeis, which have therefore great potential for biomedicai applications. One key aspect when developing a biomateriai for clinical use is its susceptibility to sterilisation. Optimization of this process is essential to avoid degradation or modification, especialiy for sensitive natural materiais. The effects of autoclaving and ionizing irradiation (í3 and y) on oxDex soiutions were investigated through rheoiogical analysis. Heat treatment has proven to be an inadequate method for the steriiization of this material, since its effects on dextrin chain prevented hydrogei formation. According to the rheologicai behaviour observed, ali materiais remained gelabie after 0 or y-irradiation. Both kinds of radiation induced slight differences in hydrogei storage modulus, suggesting the occurrence of chain scission/crossiinking effects on the dextrin chains. Nevertheiess, these effects did not seem to be dose or temperature dependent for the tested conditions; furthermore, those differences could not be attributed to free radicais generation since its presence was not detected. ionizing radiation seems to be a safe method for the steriiization of oxidised dextrin without compromising of hydrogei formation or its rheologicai properties. In vivo performance of oxDex hydrogel was evaivated, according to 150 10993-6, for inflammatory response assessment in subcutaneous implants. The effects of the combination with human mesenchymal stem ceiis (hMSCs), urinary biadder matrix (UBM) and bone graft granules (Bonelike®) were also evaluated. Histological anaiysis after 3 and 15 days showed typical acute and chronic infiammatory responses, respectiveiy, with the Bonelike® groups exhibiting a more severe reaction and the hydrogel was abie to better stabiiize Boneiike granules in the impiant site. The presence of viable hMSCs couid be confirmed by HuNu ceiis and by severai infiammatory and immunomoduiatory cytokines and growth factors in the peripheral biood. Dextrin hydrogei was scored as siight irritant after 3 days of impiantation, simiiarly to its combination with UBM and as non-irritant after 15 days. Successfui encapsuiation of hMSCs into hydrogei matrix was achieved and its presence seemed to modulate infiammatory response by acceierating its progression when compared to the aceliuiar groups. Pre-ciinicai triais in New Zeaiand rabbits demonstrated that oxDex hydrogei did not induce any adverse effects in regeneration of bone defects. Complete regeneration was observed after 8 weeks in non-criticai tibiae defects, both for sham control and in the presence of tested formuiations (hydrogei; hydrogei/Boneiike®; hydrogei/UBM). Bone regeneration of caivaria modeis (criticai and non-critcai) was improved by oxDex hydrogel, comparing with sham control, since areas of trabecuiar extensions were observed, proionging from the defect edges and composed by dense and organized coiiagen fibers. Better outcomes were obtained with Boneiike® aione and its combination with oxDex hydrogei in criticai size defects. in both cases, despite complete regeneration being stiil absent, areas of interconnected dense bone tissue were observed in the center of the defect. Furthermore, UM seemed to improve the bone matrix apposition when compared with controi and oxDex hydrogei aione. This injectabie dextrin-based hydrogei provides a system that can carry and stabiiize ceils, nanogeis, Boneiike® granules and other biomoiecuies. Although further investigation is needed for the assessment of hydrogei in vivo performance during bone regeneration, it seems a promising biomateriai due to its biocompatibiiity, easiness of processing, steriiization and handling, and potentiai to promote an adequate environment to moduiate infiammatory response, by hMSCs encapsulation. |
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