Additive manufacturing of PCL 3D scaffolds

Orientadores: Rubens Maciel Filho, Jorge Vicente Lopes da SilvaDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia QuímicaResumo: Em decorrência da grande demanda de transplantes de órgãos e tecidos no Brasil, ha estímulos para criação de terapias alternativas como o desenvolvimento de substitutos biológicos temporários, isto e, scaffolds, através da Bioengenharia e Engenharia Tecidual, descartando a necessidade de doadores. Neste trabalho, foi utilizado o polímero policaprolactona (PCL) para estruturar scaffolds 3D por meio da plataforma experimental de manufatura aditiva Fab@CTI, a qual apresenta um cabeçote de extrusão intercambiável construído para entrada de material em forma de filamento. Uma nova proposta de orientação de raster foi criada para o design dos scaffolds. Foram estruturados scaffolds com raster regular e randômico com poros de 0.25, 0.5 e 1 mm. Analises de Difração de raios-x (DRX), Calorimetria exploratória diferencial (DSC) e Espectroscopia de absorção no infravermelho com transformada em Fourier (FTIR) foram realizadas para verificar possíveis mudanças nas propriedades térmicas do material durante o processamento. Microscopia eletrônica de varredura (MEV) foi feita para checar a morfologia dos scaffolds e medir o diâmetro dos filamentos extrudados na Fab@CTI alem do espaçamento entre eles. Também foram realizadas analises de citotoxicidade e viabilidade celular com células tronco mesenquimais de tecido adiposo. Ainda, utilizando o software modeFRONTIER, foi feita uma simulação de valores de modulo de compressão do PCL possíveis de serem obtidos. Os resultados de DRX e FTIR não mostraram degradação do PCL e o DSC revelou algumas alterações no ponto de fusão e diminuição da cristalinidade. Os scaffolds não se mostraram tóxicos e os modelos com poros de 0.25 mm e raster regular e randômico foram os que apresentaram viabilidade celularAbstract: Due to the demand for organs and tissues transplants in Brazil, is indeed the creation of new therapies as the development of temporary biological substitutes, ie, scaffolds, through Bioengineering and Tissue Engineering, discarding the require for donors. In this work, we used the polymer polycaprolactone (PCL) to structure 3D scaffolds by the additive manufacturing experimental platform, Fab@CTI, which presents an interchangeable extrusion head build to filaments materials input. A new proposal for random raster was presented to design the scaffolds. Were structured scaffolds with regular and random raster and pores of 0.25, 0.5 and 1 mm. Analysis of X-Ray Diffraction (XRD), Differential Scanning Calorimetry (DSC) and Fourier Transform Spectroscopy (FTIR) were conducted to check for possible changes in thermal properties of the material during the processing. Scanning Electron Microscopy (SEM) was done to check the morphology of scaffolds and measure the diameter of filaments extruded at Fab@CTI beyond the distance between them. Analyses of cytotoxicity and cell viability with mesenchymal stem cells from adipose tissue were also performed. In addition, using the software modeFRONTIER, a simulation of compressive modulus of PCL was done with values possible to obtain. The result of the XRD and FTIR showed no degradation of PCL and DSC revealed some changes in melting point and decrease of crystallinity. The scaffolds were not toxic and models of 0.25 mm pores and both regular and random raster showed cell viabilityMestradoDesenvolvimento de Processos QuímicosMestre em Engenharia Químic

L-Lactic Acid Production by Lactobacillus rhamnosus

Lactic acid has been shown to have the most promising application in biomaterials as poly(lactic acid). L. rhamnosus ATCC 10863 that produces L-lactic acid was used to perform the fermentation and molasses was used as substrate. A solution containing 27.6 g/L of sucrose (main composition of molasses) and 3.0 g/L of yeast extract was prepared, considering the final volume of 3,571 mL (14.0% (v/v) inoculum). Batch and fed batch fermentations were performed with temperature of 43.4°C and pH of 5.0. At the fed batch, three molasses feed were applied at 12, 24, and 36 hours. Samples were taken every two hours and the amounts of lactic acid, sucrose, glucose, and fructose were determined by HPLC. The sucrose was barely consumed at both processes; otherwise the glucose and fructose were almost entirely consumed. 16.5 g/L of lactic acid was produced at batch and 22.0 g/L at fed batch. Considering that lactic acid was produced due to the low concentration of the well consumed sugars, the final amount was considerable. The cell growth was checked and no substrate inhibition was observed. A sucrose molasses hydrolysis is suggested to better avail the molasses fermentation with this strain, surely increasing the L-lactic acid

L-lactic acid production process development obtained from sugars fermentation : kinetic data obtaining and separation processes evaluation

Orientadores: Maria Regina Wolf Maciel, Rubens Maciel FilhoTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia QuímicaResumo: O uso de substratos alternativos em fermentações biotecnológicas é uma alternativa viável em substituição àqueles provenientes de fontes não-renováveis. O melaço é um exemplo e pode ser usado para produzir L-ácido láctico o qual tem se mostrado promissor na síntese de poli-L-ácido láctico (PLLA) para aplicação na área médica. Para este trabalho, três bactérias produtoras de L-ácido láctico e consumidoras de melaço foram selecionadas na literatura para ensaios em shaker: Lactobacillus (L.) rhamnosus ATCC 7469, L. rhamnosus ATCC 10863 e L. delbrueckii ATCC 9649. Por meio de um planejamento fatorial 23 do tipo estrela, foi selecionada a bactéria que mais produziu L-ácido láctico, a L. rhamnosus ATCC 10863 com produção de 16,5 g/L. As condições de operação foram temperatura de 43? C e concentração de sacarose (açúcar com maior porcentagem no melaço) e de extrato de levedura de 27,6 g/L e 3,0 g/L, respectivamente. O comportamento dos açúcares mostrou que a glicose e frutose foram praticamente totalmente consumidos, enquanto a sacarose foi pouco consumida. Em biorreator, aquela mesma cepa com as condições de operação mencionadas, além de pH 5,0, foi usada em dois processos fermentativos de batelada alimentada (processos 1 e 2) e dois de batelada (processos 3 e 4) com o melaço sem pré-tratamento (virgem). Dentre estes processos, a sacarose ainda foi pouco convertida, atingindo no máximo 22,5 % no processo de batelada alimentada 1. Neste mesmo processo, o máximo de frutose foi convertido, 98,4%, e o máximo de conversão da glicose foi no processo de batelada 4, 91,7 %. O pico máximo de L-ácido láctico produzido foi no processo de batelada alimentada 2, 22,0 g/L. Logo, posteriormente, foi realizado outro processo fermentativo de batelada (processo 5) com pré-tratamento do melaço, a hidrólise da sacarose em glicose e frutose utilizando a enzima invertase, resultando em praticamente 100 %. No processo fermentativo 5, a glicose foi convertida em 98,2 % e a frutose em 99,3 % e foram gerados 35,5 g/L de ácido láctico (pico máximo). A produtividade máxima foi de 2,0 g/Lh, o crescimento da biomassa foi de 5,0 g/L, o rendimento foi de 97,5 % e foram detectados 98,5 % do isômero L(+) do ácido láctico no caldo da fermentação ao final do processo. Portanto, este processo foi identificado como o "ótimo" deste trabalho. Em seguida, o processo de separação do L-ácido láctico foi feito no equipamento de cromatografia líquida de alta eficiência (HPLC) com coletor de frações. Foi usada a coluna semi-preparativa ZORBAX Eclipse XDB-C18 para separar o L-ácido láctico de todos os outros componentes restantes no caldo da fermentação, porém o ácido acético presente na solução enzimática anteriormente usada foi também coletado no mesmo tempo. Após, foi usada a coluna analítica Aminex HPX-87H que isolou e coletou o L-ácido láctico, obtendo 11 ml de solução de fase móvel e ácido diluído, com taxa de recuperação de 53,0 %. A título de demonstração de viabilidade de uso desta técnica para separar o L-ácido láctico proveniente de fermentação, esta metodologia foi válidaAbstract: The use of alternative substrates in biotechnological fermentations is a viable alternative to substitute those derived from non-renewable sources. The molasses is an example and can be used to produce L-lactic acid that has shown to be promising to produce poly-L-lactic acid (PLLA) for application in medical field. For this work, three L-lactic acid producing bacteria and also molasses consumers were selected from literature for shaker trials: Lactobacillus (L.) rhamnosus ATCC 7469, L. rhamnosus ATCC 10863 and L. delbrueckii ATCC 9649. Through a star configuration 23 experimental planning, the bacterium that produced more L-lactic acid was selected, which was the L. rhamnosus ATCC 10863, producing 16.5 g/L. The operation conditions were 43? C as temperature and concentrations of 27.6 g/L of sucrose (sugar with higher percentage at molasses) and 3.0 g/L of yeast extract, respectively. The sugars behavior showed that glucose and fructose were basically completely consumed while sucrose was barely consumed. At bioreactor, that bacterium with the operation conditions just mentioned, besides the pH 5.0, was used at two fed batch fermentation processes (processes 1 and 2) and two batch fermentation processes (processes 3 and 4) with the molasses without pre-treatment (virgin). Among these processes, the sucrose was still scarcely consumed, reaching the maximum of 22.5 % at fed batch process 1. At this same process, the maximum of fructose was converted, 98.4 %, and the maximum of glucose converted was at batch process 4, 91.7 %, The maximum peak of L-lactic acid produced was at fed batch process 2, 22.0 g/L. Thus, after, it was performed another batch fermentation process (process 5) with molasses pre-treatment, the sucrose hydrolysis into glucose and fructose using the enzyme invertase, resulting in 100 %. At fermentative process 5, the glucose was converted in 98.2 % and fructose in 99.3 %, generating 35.5 g/L of L-lactic acid (maximum peak). The maximum productivity was 2.0 g/Lh, the biomass growth was 5.0 g/L, the yield was 97.5 % and it was detected 98.5 % of the isomer L(+) of lactic acid at fermentation broth at the end of process. Therefore, this process was identified as the "optimum" of this work. Afterwards, the L-lactic acid separation process was done at the high performance liquid chromatography (HPLC) equipment with fraction collector. It was used the semi-preparative column ZORBAX Eclipse XDB-C18 to separate the L-lactic acid from all components remained at fermentation broth, but the acetic acid at the enzymatic solution previously used was collected at the same time. Subsequently, it was used the analytical column Aminex HPX-87H that isolated and collected the L-lactic acid, obtaining 11 mL of solution of mobile phase and diluted acid, with recovery rate of 53.0 %. Intending to demonstrate the viability of use of this technique to separate L-lactic acid from fermentation, this methodology was validDoutoradoDesenvolvimento de Processos QuímicosDoutora em Engenharia Quimica2011/11742-2FAPES

L-Lactic Acid Production by Lactobacillus rhamnosus ATCC 10863

Lactic acid has been shown to have the most promising application in biomaterials as poly(lactic acid). L. rhamnosus ATCC 10863 that produces L-lactic acid was used to perform the fermentation and molasses was used as substrate. A solution containing 27.6 g/L of sucrose (main composition of molasses) and 3.0 g/L of yeast extract was prepared, considering the final volume of 3,571 mL (14.0% (v/v) inoculum). Batch and fed batch fermentations were performed with temperature of 43.4 ∘ C and pH of 5.0. At the fed batch, three molasses feed were applied at 12, 24, and 36 hours. Samples were taken every two hours and the amounts of lactic acid, sucrose, glucose, and fructose were determined by HPLC. The sucrose was barely consumed at both processes; otherwise the glucose and fructose were almost entirely consumed. 16.5 g/L of lactic acid was produced at batch and 22.0 g/L at fed batch. Considering that lactic acid was produced due to the low concentration of the well consumed sugars, the final amount was considerable. The cell growth was checked and no substrate inhibition was observed. A sucrose molasses hydrolysis is suggested to better avail the molasses fermentation with this strain, surely increasing the L-lactic acid