Development of chitosan-xanthan scaffolds for pontential application in periosteum-guided bone tissue regeneration

Orientadores: Ângela Maria Moraes, Diego MantovaniTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia QuímicaResumo: O periósteo é uma importante estrutura do tecido ósseo e se mostra fundamental no processo de cura de lesões ósseas, fornecendo compostos celulares e biológicos essenciais para sua regeneração. Scaffolds, capazes de mimetizar o periósteo são particularmente atraentes, pois podem melhorar significativamente a regeneração óssea em lesões graves. Estes dispositivos servem como suporte para a proliferação de células capazes de se diferenciar em linhagens ósseas e podem ser constituídos de polímeros naturais. A quitosana é um polímero biocompatível e biodegradável, que facilita a proliferação celular, e por isso é muito utilizada na produção de scaffolds. Para melhorar sua osteoindutividade, a introdução de grupos fosfato em sua estrutura, ou fosforilação, pode ser realizada. A xantana é um polissacarídeo capaz de interagir com a quitosana, resultando em complexos com propriedades superiores às dos polímeros isolados. Neste trabalho, scaffolds de quitosana (fosforilada ou não modificada) combinada com xantana foram produzidos, na presença ou ausência de um agente porogênico, o tensoativo Kolliphor® P188, e do gel de silicone Silpuran® 2130 A/B. A caracterização dos materiais produzidos foi realizada para validação de sua potencial aplicação como substitutos do periósteo. Materiais altamente porosos foram obtidos e a presença do silicone reforçou mecanicamente as matrizes, não impactando negativamente em suas propriedades físicas, físico-químicas e biológicas. A fosforilação da quitosana levou à obtenção de scaffolds com propriedades similares às dos materiais produzidos com o polímero não modificado, com potencial para concentrar proteínas morfogenéticas ósseas nativas in vivo, o que foi inferido pela maior adsorção de citocromo C apresentada por essas formulações. Além disso, não foram observados efeitos citotóxicos dos scaffolds, que mostraram, como vantagem adicional, cinética de degradação apropriada para a aplicação proposta. A análise do cultivo in vitro de células tronco derivadas do tecido adiposo nos scaffolds e de sua diferenciação em osteoblastos mostraram que modificações ou tratamentos adicionais na superfície dos materiais podem trazer benefícios quanto ao desempenho celular nos mesmos. Além disso, elevada deposição de minerais e formação de matriz extracelular foram observadas. Ensaios complementares são necessários para investigar de forma mais aprofundada a resposta gerada pelos materiais in vivo. Como conclusão geral, os resultados obtidos fornecem indícios de que as formulações propostas neste trabalho são promissoras para a aplicação na engenharia de tecido periosteal e reparo ósseoAbstract: The periosteum is an important structure of the bone tissue and is fundamental in the healing process of bone lesions, providing cellular and biological compounds essential for tissue repair. Tissue engineered scaffolds able to mimic the periosteum are particularly attractive because they can significantly improve bone regeneration in severely injured tissues. These devices function as support for cells that are able to proliferate and differentiate into bone cells and can be constituted of natural polymers, such as xanthan gum and chitosan. Xanthan gum is a polysaccharide that may interact with chitosan, a biocompatible and biodegradable polymer, resulting in complexes with improved properties in comparison to those of matrices produced with each polysaccharide alone. Chemical modifications, such as the phosphorylation of chitosan, can be performed to enhance the osteoinductivity of the resulting scaffold. In this work, chitosan (phosphorylated or chemically unmodified) scaffolds combined with xanthan were produced, in the presence or not of a porogenic agent, the surfactant Kolliphor® P188, and the silicone rubber Silpuran® 2130 A/B. The scaffolds were characterized to validate their potential application as periosteum substitutes. Highly porous materials were obtained and the presence of silicon mechanically reinforced the matrices, not negatively impacting their physical, physicochemical and biological properties. Formulations produced using phosphorylated chitosan presented similar properties to formulations produced with the unmodified polymer, with potential to concentrate native bone morphogenetic proteins in vivo, which was inferred by the higher adsorption of cytochrome C presented by these formulations. In addition, no cytotoxic effects of the scaffolds were observed, and they presented, as an additional advantage, degradation kinetics appropriate for the proposed application. The analysis of adipose derived stem cells culture n vitro on the scaffolds produced and of cell differentiation into osteoblasts showed that additional scaffold surface treatment or modification procedures may contribute to improve cell performance on the materials. Moreover, high deposition of minerals and formation of extracellular matrix were observed. Complementary studies are required to further investigate the in vivo response generated by these materials. As a general conclusion, the obtained results indicate that the formulations proposed in this work are promising for the application in periosteal tissue engineering and bone repairDoutoradoEngenharia QuímicaDoutora em Engenharia Quimica33003017034P8CAPE

Three-dimensional structures obtained by complexation of chitosan with other polysaccharides for application in vascular tissue engineering

Orientadores: Ângela Maria Moraes, Diego MantovaniTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia QuímicaResumo: Doenças cardiovasculares decorrentes da aterosclerose, tais como isquemia cardíaca e doenças vasculares periféricas, são a principal causa de mortes no mundo. Intervenções cirúrgicas para o reparo dos vasos sanguíneos danificados são frequentemente necessárias, incluindo a substituição parcial do vaso ou a utilização de remendos vasculares (patches). Existe uma demanda de materiais capazes de atuar de maneira eficaz como substitutos vasculares, especialmente no caso de vasos sanguíneos de pequeno calibre (diâmetro menor que 6 mm), tendo em vista que os normalmente utilizados levam muitas vezes à ocorrência de complicações pós-cirúrgicas que acarretam na reoclusão do vaso. Recentes avanços no desenvolvimento de substitutos vasculares são consequência de abordagens no âmbito da engenharia de tecidos, com a cultura de células vasculares em scaffolds biodegradáveis. A quitosana (Ch), um polímero natural biocompatível, é bastante usada na produção de scaffolds e sua combinação com outros polissacarídeos, como o alginato (A) e a pectina (P) contribui para a melhoria das propriedades dos biomateriais. Neste trabalho, matrizes de geometria plana foram produzidas com quitosanas de três diferentes massas molares em combinação com A ou P, na presença ou ausência do agente porogênico Kolliphor® P188 (K) e do gel de silicone Silpuran® 2130 A/B (S), e caracterizadas para verificação da influência da composição das matrizes em suas propriedades. Caracterizações complementares foram feitas para validação de sua potencial aplicação como patches na engenharia de tecidos vasculares. O uso de P e Ch de alta massa molar em combinação com S resultou em materiais com comportamento poroviscoelástico e maior resistência mecânica dentre as formulações testadas. Matrizes de Ch-P se destacaram por apresentar maior estabilidade e melhor resposta ao contato com sangue, devido à presença de plaquetas em estágios menos avançados de ativação e menores níveis de trombogenicidade, além de melhor adesão, proliferação e maior viabilidade de células musculares lisas nelas inoculadas. Scaffolds de geometria tubular constituídos de Ch-P foram fabricados com sucesso utilizando uma nova metodologia desenvolvida, dando origem a dispositivos homogêneos, estáveis e altamente porosos. A utilização de gel de colágeno para encapsulação de células musculares lisas facilitou a introdução e distribuição homogênea das células na estrutura tridimensional tubular dos scaffolds. O presente estudo evidenciou que o uso de pectina complexada com quitosana, uma combinação ainda pouco usada na engenharia de tecidos, tem potencial para ser melhor explorada para este fim. Além disso, o estudo mostrou que as estruturas tridimensionais obtidas em diferentes geometrias são promissoras para a aplicação na engenharia de tecidos vascularesAbstract: Cardiovascular diseases such as coronary artery and peripheral vascular diseases are the leading cause of death worldwide. Surgical interventions for the repair of damaged blood vessels are often necessary, including partial replacement of the vessel or use of vascular patches. There is a need for materials capable of acting effectively as vascular substitutes, especially in the case of small-caliber blood vessels (diameter smaller than 6 mm), since the materials regularly used often lead to postoperative complications and vessel reocclusion. Recent advances in the development of vascular substitutes are a consequence of tissue engineering approaches, with the culture of vascular cells on biodegradable scaffolds. Chitosan (Ch), a natural biocompatible polymer, is widely used in the production of scaffolds and its combination with other polysaccharides such as alginate (A) and pectin (P) contributes to the improvement of biomaterial properties. In this work, matrices with flat geometry were produced using chitosan of three different molar weights in combination with A or P in the presence or absence of the surfactant Kolliphor® P188 (K) and silicone gel Silpuran® 2130 A/B (S), and characterized to assess the influence of composition on matrix¿s properties. Additional characterizations were performed to validate their potential application as tissue-engineered vascular patches. The use of chitosan of high molar weight and pectin, in combination with S, resulted in materials with poroviscoelastic behavior and higher mechanical resistance when comparing all formulations tested. Ch-P matrices presented higher stability and better response to blood contact, due to the presence of platelets in the less advanced stages of activation and lower levels of thrombogenicity, as well as better adhesion, proliferation and greater viability of smooth muscle cells. Devices with tubular geometry prepared with Ch-P were successfully fabricated using a new methodology, resulting in homogeneous, stable and highly porous scaffolds. The use of collagen gel for entrapment of smooth muscle cells facilitated the introduction and homogeneous distribution of the cells in the three-dimensional tubular structure. The present study evidenced that the use of chitosan complexed with pectin, a combination still underexplored in tissue engineering, has the potential to be further investigated for this purpose. In addition, this study demonstrated that the three-dimensional structures obtained in different geometries are promising for the use in the reconstruction and regeneration of vascular tissuesDoutoradoEngenharia QuímicaDoutora em Engenharia Quimica2013/26534-1, 2017/01858-0FAPES

Polysaccharide‐based membranes loaded with erythromycin for application as wound dressings

In this study, the antibiotic erythromycin (Ery) was incorporated into chitosan (Ch)–alginate (A) and Ch–xanthan (X) membranes with the aim of using them as bioactive wound dressings. Drug incorporation was performed by direct addition (DA) to the polysaccharide mixture and by membrane impregnation in solution (IS). A higher incorporation efficiency was obtained for DA, but higher amounts of drug were loaded into membranes by the IS method (maxima ≈ 2.1 and 0.7 g/g for Ch–X and Ch–A, respectively) because the initial concentration of drug could be higher than that in the DA method. Ery release in phosphate‐buffered saline was slow, reaching about 12 and 32 mg of drug/g of membrane in 60 h for Ch–X and 4 and 16 mg/g for Ch–A by the DA and IS methods, respectively. With formulations prepared with IS, the required therapeutic dosage was reached within 60 h, whereas for those incorporating the drug by DA, prolonged use would be required. Both membrane types behaved as drug reservoirs, providing continuous antibiotic release to the wound site. Formulations with higher drug contents showed effective antibacterial activity against two species of bacteria commonly found in skin lesions, Staphylococcus aureus and Pseudomonas aeruginosa , and were thus potentially capable of protecting the wound site from bacterial attack13322CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESsem informaçã

Analysis of the performance of polysaccharide membranes in aqueous media as a tool to assist wound‐dressing selection

The behavior of hydrophilic matrixes in the presence of aqueous media plays a pivotal role in the selection of materials that come into contact with body fluids. Because polysaccharides have proven benefits in the treatment of skin lesions, the performance of membranes produced with chitosan combined with alginate [chitosan–alginate (Ch–A)], xanthan [chitosan–xanthan (Ch–X)], or guar gum [chitosan–guar gum (Ch–G)] after exposure to different aqueous solutions and humidity levels was analyzed with the aim of directing their applications as dressings in wounds with different exudate productions. The Ch–X membranes presented a high fluid‐uptake capacity and water‐vapor transmission rate (WVTR); this was attributed to ramifications in the xanthan structure, and the membranes were then recommended for moderately to highly exuding wounds. The Ch–G membranes showed a dense structure and presented low fluid‐uptake capacity; they were more appropriate for low‐exuding wounds or wounds in the advanced stage of cicatrization. Both the Ch–A and Ch–G membranes presented adequate mechanical properties in a wide range of relative humidity conditions and could be considered suitable for use in all body parts. However, as the Ch–A formulation showed limited WVTR, its use should be restrictedly to, at most, moderately exuding wounds. In all cases, the assessment of the wound type by a professional would be required to define the final dressing formulation13440CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESsem informaçã

Incorporation and release kinetics of alpha-bisabolol from pcl and chitosan/guar gum membranes

Alpha-bisabolol, an anti-inflammatory and antioxidant compound extracted from candeia trees (Eremanthus erythropappus), was incorporated into hydrophobic polycaprolactone (PCL) and hydrophilic chitosan/guar gum (Ch-G) membranes aiming at the production of bioactive wound dressings. The incorporation efficiency achieved a maximum of ca. 18% (1 gram of alpha-bisabolol per gram of membrane) for Ch-G membranes. For PCL membranes, all of the active compound added was retained (0.2 gram of alpha-bisabolol per gram of membrane). Alpha-bisabolol release in phosphate-buffered saline was relatively slow in both cases, reaching around 6% and 24% after 120 hours respectively for PCL and Ch-G membranes presenting equivalent initial alpha-bisabolol/membrane mass ratios. Both formulations were capable of releasing alpha-bisabolol in the typically recommended topical dose range (from 1 to 10 grams of alpha-bisabolol per gram of vehicle). The extended release periods observed are advantageous, allowing less frequent dressing changes and contributing to turn the treatment more comfortable for the patient333453467CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESsem informação20th Brazilian Congress of Chemical Engineering (COBEQ

Development of chitosan-xanthan scaffolds for pontential application in periosteum-guided bone tissue regeneration

Orientadores: Ângela Maria Moraes, Diego MantovaniTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia QuímicaResumo: O periósteo é uma importante estrutura do tecido ósseo e se mostra fundamental no processo de cura de lesões ósseas, fornecendo compostos celulares e biológicos essenciais para sua regeneração. Scaffolds, capazes de mimetizar o periósteo são particularmente atraentes, pois podem melhorar significativamente a regeneração óssea em lesões graves. Estes dispositivos servem como suporte para a proliferação de células capazes de se diferenciar em linhagens ósseas e podem ser constituídos de polímeros naturais. A quitosana é um polímero biocompatível e biodegradável, que facilita a proliferação celular, e por isso é muito utilizada na produção de scaffolds. Para melhorar sua osteoindutividade, a introdução de grupos fosfato em sua estrutura, ou fosforilação, pode ser realizada. A xantana é um polissacarídeo capaz de interagir com a quitosana, resultando em complexos com propriedades superiores às dos polímeros isolados. Neste trabalho, scaffolds de quitosana (fosforilada ou não modificada) combinada com xantana foram produzidos, na presença ou ausência de um agente porogênico, o tensoativo Kolliphor® P188, e do gel de silicone Silpuran® 2130 A/B. A caracterização dos materiais produzidos foi realizada para validação de sua potencial aplicação como substitutos do periósteo. Materiais altamente porosos foram obtidos e a presença do silicone reforçou mecanicamente as matrizes, não impactando negativamente em suas propriedades físicas, físico-químicas e biológicas. A fosforilação da quitosana levou à obtenção de scaffolds com propriedades similares às dos materiais produzidos com o polímero não modificado, com potencial para concentrar proteínas morfogenéticas ósseas nativas in vivo, o que foi inferido pela maior adsorção de citocromo C apresentada por essas formulações. Além disso, não foram observados efeitos citotóxicos dos scaffolds, que mostraram, como vantagem adicional, cinética de degradação apropriada para a aplicação proposta. A análise do cultivo in vitro de células tronco derivadas do tecido adiposo nos scaffolds e de sua diferenciação em osteoblastos mostraram que modificações ou tratamentos adicionais na superfície dos materiais podem trazer benefícios quanto ao desempenho celular nos mesmos. Além disso, elevada deposição de minerais e formação de matriz extracelular foram observadas. Ensaios complementares são necessários para investigar de forma mais aprofundada a resposta gerada pelos materiais in vivo. Como conclusão geral, os resultados obtidos fornecem indícios de que as formulações propostas neste trabalho são promissoras para a aplicação na engenharia de tecido periosteal e reparo ósseoAbstract: The periosteum is an important structure of the bone tissue and is fundamental in the healing process of bone lesions, providing cellular and biological compounds essential for tissue repair. Tissue engineered scaffolds able to mimic the periosteum are particularly attractive because they can significantly improve bone regeneration in severely injured tissues. These devices function as support for cells that are able to proliferate and differentiate into bone cells and can be constituted of natural polymers, such as xanthan gum and chitosan. Xanthan gum is a polysaccharide that may interact with chitosan, a biocompatible and biodegradable polymer, resulting in complexes with improved properties in comparison to those of matrices produced with each polysaccharide alone. Chemical modifications, such as the phosphorylation of chitosan, can be performed to enhance the osteoinductivity of the resulting scaffold. In this work, chitosan (phosphorylated or chemically unmodified) scaffolds combined with xanthan were produced, in the presence or not of a porogenic agent, the surfactant Kolliphor® P188, and the silicone rubber Silpuran® 2130 A/B. The scaffolds were characterized to validate their potential application as periosteum substitutes. Highly porous materials were obtained and the presence of silicon mechanically reinforced the matrices, not negatively impacting their physical, physicochemical and biological properties. Formulations produced using phosphorylated chitosan presented similar properties to formulations produced with the unmodified polymer, with potential to concentrate native bone morphogenetic proteins in vivo, which was inferred by the higher adsorption of cytochrome C presented by these formulations. In addition, no cytotoxic effects of the scaffolds were observed, and they presented, as an additional advantage, degradation kinetics appropriate for the proposed application. The analysis of adipose derived stem cells culture n vitro on the scaffolds produced and of cell differentiation into osteoblasts showed that additional scaffold surface treatment or modification procedures may contribute to improve cell performance on the materials. Moreover, high deposition of minerals and formation of extracellular matrix were observed. Complementary studies are required to further investigate the in vivo response generated by these materials. As a general conclusion, the obtained results indicate that the formulations proposed in this work are promising for the application in periosteal tissue engineering and bone repairDoutoradoEngenharia QuímicaDoutora em Engenharia Quimica33003017034P8CAPE

Phosphorylation of chitosan to improve osteoinduction of chitosan/xanthan-based scaffolds for periosteal tissue engineering

The periosteum is a membrane that surrounds bones, providing essential cellular and biological components for fracture healing and bone repair. Tissue engineered scaffolds able to function as periosteum substitutes can significantly improve bone regeneration in severely injured tissues. Efforts to develop more bioactive and tunable periosteal substitutes are required to improve the success of this tissue engineering approach. In this work, a chemical modification was performed in chitosan, a polysaccharide with osteoconductive properties, by introducing phosphate groups to its structure. The phosphorylated polymer (Chp) was used to produce chitosan-xanthan-based scaffolds for periosteal tissue engineering. Porous and mechanically reinforced matrices were obtained with addition of the surfactant Kolliphor® P188 and the silicone rubber Silpuran® 2130A/B. Scaffolds properties, such as large pore sizes (850–1097 μm), micro-roughness and thickness (0.7–3.5 mm in culture medium), as well as low thrombogenicity compared to standard implantable materials, extended degradation time and negligible cytotoxicity, enable their application as periosteum substitutes. Moreover, the higher adsorption of bone morphogenetic protein mimic (cytochrome C) by Chp-based formulations suggests improved osteoinductivity of these materials, indicating that, when used in vivo, the material would be able to concentrate native BMPs and induce osteogenesis. The scaffolds produced were not toxic to adipose tissue-derived stem cells, however, cell adhesion and proliferation on the scaffolds surfaces can be still further improved. The mineralization observed on the surface of all formulations indicates that the materials studied have promising characteristics for the application in bone regeneration143619632CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçãosem informaçãosem informaçã

Comparative study on complexes formed by chitosan and different polyanions : potential of chitosan-pectin biomaterials as scaffolds in tissue engineering

Polyelectrolyte complexes of chitosan (Ch) and pectin (Pc) or alginate (Alg) were produced in the presence or absence of the silicone gel Silpuran® 2130 A/B (Sil) and the surfactant Kolliphor® P188 (Kol). Ch-Pc-Kol-based formulations presented higher porosity (up to 83.3%) and thickness (maximum of 2273.5 μm in PBS). Lower water contact angle was observed for Ch-Alg formulations (minimum of 36.8°) and these formulations presented higher swelling and mass loss in PBS (reaching up to 21.7 g/g and 80.4%, respectively). The addition of Sil to the matrices improved their elastic moduli, reaching a maximum of 4-fold change at 40% strain. The use of pectin instead of alginate augmented the elastic moduli, reaching 66 and 4-fold changes for dense and porous formulations, respectively. Pectin-containing scaffolds presented poroviscoelasticity, a typical mechanical feature of many soft tissues. The suitability of the materials for tissue engineering applications was demonstrated in terms of stability upon degradation in culture medium or lysozyme solution, as well as lack of cytotoxicity. This study evidences the potential of Ch-Pc-based materials to be further explored for this purpose, especially to improve the mechanical properties of chitosan-based scaffolds aiming medical applications132178189CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP307139/2015-80012013/26534-1; 2017/01858-0The authors would like to acknowledge the support to this research by the São Paulo Research Foundation (Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP, Brazil – Grants #2013/26534-1 and #2017/01858-0), National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq, Brazil – Grant # 307139/2015-8), Emerging Leaders in America Program (ELAP, Canada) and Natural Sciences and Engineering Research Council of Canada – Discovery Program (NSERC, Canada). This study was financed in part by the Coordination for the Improvement of Higher Educational Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES, Brazil – Finance Code 001