Physicochemical and Tissue Response of PLA Nanofiber Scaffolds Sterilized by Different Techniques / Evaluación fisicoquímica y respuesta tisular de andamios nanofibrilares de PLA esterilizados por diferentes técnicas

ABSTRACT In recent years, tissue engineering has evolved considerably, due to the problems in the biomedical area concerning tissue regeneration therapies. Currently, work has been focused on the synthesis and physicochemical characterization of poly lactic acid scaffolds, a synthetic polyester that has been extensively study for its excellent biocompatibility and biodegradability. Moreover, sterilization strategies of scaffold are a crucial step for its application in tissue regeneration, however, the sterilization process have to maintain the structural and biochemical properties of the scaffold. Therefore, it is very important to carry out studies on the sterilization methods of the sample's material, since translational medicine is intended for in vivo applications. The aim of the present study was designed to analyze the effects of different sterilization techniques, i.e. ethylene oxide (ETO), gamma radiation (GR) and hydrogen peroxide- based plasma (H2O2) in biodegradable PLA scaffolds, and to determine the best sterilization technique to render a sterile product with minimal degradation and deformation, and good tissue response. Analysis of surface morphology showed that ETO and GR modified the PLA scaffolds without any change in its chemical composition. Moreover, the histological response showed that the scaffolds are biocompatible and those sterilized by GR showed a more severe inflammatory response, accompanied with the presence of giant foreign body cells. In conclusion, the results show that among sterilization techniques used in the preset study, the best results were observed with H2O2 sterilization, since it did not significantly modify the surface structure of the PLA fibers and their in vivo response did not cause an unfavorable tissue reaction.

RESUMEN En los últimos años, la ingeniería de tejidos ha evolucionado considerablemente, debido a las incógnitas en las terapias de regeneración en el área biomédica. Actualmente, se ha trabajado en la síntesis y caracterización fisicoquímica de andamios de poliácido láctico, el cual es un polímero sintético que se ha estudiado para aplicaciones en ingeniería de tejidos, debido a su biocompatibilidad y biodegradabilidad. El proceso de esterilización es un paso crucial en la aplicación de andamios en terapias de regeneración, sin embargo, la técnica de esterilización debe mantener las propiedades estructurales y bioquímicas del andamio. Por lo tanto, es muy importante realizar estudios sobre los métodos de esterilización de dichos andamios, ya que la medicina traslacional está diseñada para aplicaciones in vivo. El objetivo del presente estudio fue analizar los efectos de diferentes técnicas de esterilización como óxido de etileno (ETO), radiación gamma (GR) y plasma a base de peróxido de hidrógeno (H2O2) en andamios biodegradables de PLA, y determinar la mejor técnica de esterilización con mínima degradación y deformación, así como una respuesta tisular favorable. La estructura de la superficie de los andamios de PLA se modificó principalmente con las técnicas de óxido de etileno y radiación gamma, sin embargo, ninguna técnica modificó su composición química. Con la respuesta histológica se demostró que los andamios de PLA son biocompatibles y que los esterilizados por radiación gamma desencadenan una mayor respuesta inflamatoria y la formación de células gigantes de cuerpo extraño. En conclusión, los resultados muestran que las técnicas de esterilización utilizadas pueden modificar la morfología del andamio, sin embargo; los mejores resultados se observaron con la esterilización por plasma a base de peróxido de hidrógeno, ya que no modificó significativamente la estructura de la superficie de las fibras de PLA y su respuesta in vivo no provocó una reacción desfavorable en el tejido.

Behavior of Mesenchymal Stem Cells Obtained From Dental Tissues: A Review of the Literature / Comportamiento de células mesenquimales obtenidas de tejidos dentales: Revisión de literatura

Abstract 20. The success of tissue engineering in combination with tissue regeneration depends on the behavior and cellular activity in the biological processes developed within a structure that functions as a support, better known as scaffolds, or directly at the site of the injury. The cell-cell and cell-biomaterial interaction are key factors for the induction of a specific cell behavior, together with the bioactive factors that allow the formation of the desired tissue. Mesenchymal Stem Cells (MSC) can be isolated from the umbilical cord and bone marrow; however, the behavior of Dental Pulp Stem Cells (DPSC) has been shown to have a high potential for the formation of bone tissue, and these cells have even been able to induce the process of angiogenesis. Advances in periodontal regeneration, dentin-pulp complex, and craniofacial bone defects through the induction of MSC obtained from tooth structures in in vitro-in vivo studies have permitted the obtaining of clinical evidence of the achievements obtained to date.

Resumen 24. El éxito de la ingeniería de tejidos en combinación con la regeneración de tejidos depende del comportamiento y la actividad celular en los procesos biológicos desarrollados dentro de una estructura que funciona como soporte mejor conocida como andamio o directamente en el sitio de la lesión. La interación célula-célula y célula-biomaterial son factores claves para la inducción a un comportamiento célular específico junto con factores bioactivos que permitan la formación del tejido deseado. Las células troncales mesenquimales (MSCs) pueden ser aisladas del cordón umbilical y de la medula ósea, sin embargo, el comportamiento de las células troncales de pulpa dental (DPSCs) han demostrado tener un alto potencial para la formación de tejido óseo e incluso han logrado inducir el proceso de angiogénesis. Avances en la regeneración periodontal, complejo dentino-pulpar y defectos óseos craneofaciales a travez de la inducción de MSCs obtenidas de estructuras de dientes en estudios in vitro-in vivo han permitido obtener evidencia clínica de los logros obtenidos hasta el momento.

Dental Pulp Regeneration: Insights from Biological Processes / Regeneración pulpar: Perspectivas desde los procesos biológicos

Abstract One of the major approaches on dental research in this century is the development of biological strategies (tissue engineering) to regenerate/biomineralize lost dental tissues. During dentin- pulp regeneration, the interaction between stem cells, signaling molecules, biomaterials and the microenvironment in the periapical area drives the process for pulp tissue engineering. Understanding the signaling mechanisms and interactions involved with the biological process for the formation of a new tissue is essential. The knowledge of the micro-environment is the key for the application of tissue engineering. The present article is a short review of the current state of this topic, with the purpose of showing insights of pulp regeneration.

Resumen Actualmente la investigación en odontología se orienta al desarrollo de estrategias basadas en principios biológicos (ingeniería de tejidos) para la regeneración/biomineralización de estructuras dentales perdidas. El proceso de regeneración del complejo dentino-pulpar está guiado por la compleja interacción entre las células indiferenciadas de origen dental (DTSC), moléculas de señalización y biomateriales con el microambiente donde se va a restablecer. Es esencial comprender detalladamente, los mecanismos de señalización e interacciones involucradas en los procesos biológicos para la formación de un nuevo tejido, además de la identificación de los componentes presentes en los tejidos dentales implicados en este proceso (características del microambiente), ya que representan la base sobre la cual se debe emplear la ingeniería de tejidos. El presente articulo es una breve revisión del estado actual del tema, con el fin de entender el proceso de regeneración pulpar, basado en la comprensión de los fundamentos biológicos.

Síntese e caracterização de scaffolds de Pcl/Biovidro: estudo in vitro da biocompatibilidade e da diferenciação celular / Characterization and synthesis of Pcl/Bioglass scaffolds: An in vitro study about biocompatibility and celular diferenciation

As membranas reabsorvíveis tem sido utilizadas para promover o crescimento ósseo em defeitos causados por doenças periodontais, periapicais ou por exodontia. Essas membranas possuem diversas composições, podendo ter em sua estrutura polímeros como Policaprolactona (PCL) que combinado com o biovidro que facilita a neoformação óssea. O objetivo desta pesquisa foi desenvolver um scaffold de PCL com 10% de biovidro obtido por meio de duas rotas, Fusão e SolGel. Foram preparadas soluções de 15% PCL dissolvidos em acetona pura e incorporados 10% de biovidro de acordo com meio de obtenção das partículas, resultando em 3 soluções: Controle: somente PCL, e dois grupos experimentais: PCL/Biovidro, divididas de acordo com o tipo de produção das partículas de biovidro: PCL/biov. fusão - solução de PCL com 10% de Biovidro por fusão e PCL/biov. Sol-Gel - solução de PCL com 10% de Biovidro Sol-Gel. A solução foi eletrofiada com os seguintes parâmetros: 12 kv, 12 cm de distância ponta/coletor e fluxo de 1ml/h. As mantas obtidas tiveram suas propriedades físico/químicas e biológicas analisadas por: Morfologia (MEV e FEG), propriedades físico-químicas; (Análise do diâmetro médio das fibras, Ângulo de contato, EDS, FTIR) e Celular: MTT, Fosfatase alcalina, Adesão celular, Proteína total e Formação de nódulos de mineralização. Os dados obtidos foram analisados estatisticamente pelos testes ANOVA e Tukey (5%) e os resultados apresentados em gráficos e tabelas. Foi evidenciado a incorporação das partículas de biovidro nas fibras de PCL, tal incorporação proporcionou características como maior porcentagem de fosfatase alcalina (ALP) e formação de matriz óssea quando comparado a fibra de PCL sem a presença de biovidro. O sccaffold de PCL proporcionou um meio favorável para adesão e proliferação celular, e a adição das partículas de biovidro intensificaram o processo de formação de matriz óssea, principalmente o biovidro obtido pelo método sol-gel, atuando de forma ativa no processo de regeneração e favorecendo uma reparação adequada no defeito ósseo(AU)

Resorbable membranes have been used to promote bone growth in defects caused by periodontal, periapical or exodontic diseases. These membranes have several compositions, and can have in their structure polymers such as polycaprolactone (PCL) that combined with the bioglass that facilitates the bone formation. The objective of this research was to develop a PCL scaffold with 10% bioglass obtained through two routes, Fusion and Sol-Gel. A solution of 15% PCL dissolved in pure acetone and incorporated in 10% of bioglass according to the medium of the particles was prepared, resulting in 3 solutions: Control: PCL only, and two experimental groups: PCL/Biovidro, divided according to type of production of the bioglass particles: PCL/biov. fusão - PCL solution with 10% of Bioglass by melting and PCL / biov.Sol-Gel - PCL solution with 10% of bio.solgel. The solution was electrophied with the following parameters: 12 kv, 12 cm tip / collector distance and 1ml / h flow. The obtained blankets had their physical / chemical and biological properties analyzed by: Morphology (SEM and FEG), physicochemical properties; (Analysis of mean fiber diameter, contact angle, EDS, FTIR) and Cellular: MTT, alkaline phosphatase, cell adhesion, total protein and formation of mineralization nodules. The data were analyzed statistically by ANOVA and Tukey tests (5%) and the results presented in graphs and tables. It was evidenced the incorporation of the bioglass particles in the PCL fibers, such incorporation provided characteristics as a higher percentage of alkaline phosphatase (ALP) and formation of bone matrix when compared to PCL fiber without the presence of bioglass. The PCL sccaffold provided a favorable medium for adhesion and cell proliferation, and the addition of the bioglass particles intensified the process of bone matrix formation, mainly the bioglass obtained by the sol-gel method, acting actively in the regeneration process and favoring adequate repair of the bone defect. (AU)

Repair of bone defects using adipose-derived stem cells combined with alpha-tricalcium phosphate and gelatin sponge scaffolds in a rat model

Abstract Objectives This study aimed to evaluate the potential of adipose-derived stem cells (ASCs) combined with a modified α-tricalcium phosphate (α-TCP) or gelatin sponge (GS) scaffolds for bone healing in a rat model. Material and Methods Bone defects were surgically created in the femur of adult SHR rats and filled with the scaffolds, empty or combined with ASCs. The results were analyzed by histology and histomorphometry on days seven, 14, 30, and 60. Results Significantly increased bone repair was observed on days seven and 60 in animals treated with α-TCP/ASCs, and on day 14 in the group treated with GS/ASCs, when compared with the groups treated with the biomaterials alone. Intense fibroplasia was observed in the group treated with GS alone, on days 14 and 30. Conclusions Our results showed that the use of ASCs combined with α-TCP or GS scaffolds resulted in increased bone repair. The higher efficacy of the α-TCP scaffold suggests osteoconductive property that results in a biological support to the cells, whereas the GS scaffold functions just as a carrier. These results confirm the potential of ASCs in accelerating bone repair in in vivo experimental rat models. These results suggest a new alternative for treating bone defects.

Effects of epicatechin, a crosslinking agent, on human dental pulp cells cultured in collagen scaffolds

ABSTRACT Objective The purpose of this study was to investigate the biological effects of epicatechin (ECN), a crosslinking agent, on human dental pulp cells (hDPCs) cultured in collagen scaffolds. Material and Method To evaluate the effects of ECN on the proliferation of hDPCs, cell counting was performed using optical and fluorescent microscopy. Measurements of alkaline phosphatase (ALP) activity, alizarin red staining, and real-time polymerase chain reactions were performed to assess odontogenic differentiation. The compressive strength and setting time of collagen scaffolds containing ECN were measured. Differential scanning calorimetry was performed to analyze the thermal behavior of collagen in the presence of ECN. Results Epicatechin increased ALP activity, mineralized nodule formation, and the mRNA expression of dentin sialophosphoprotein (DSPP), a specific odontogenic-related marker. Furthermore, ECN upregulated the expression of DSPP in hDPCs cultured in collagen scaffolds. Epicatechin activated the extracellular signal-regulated kinase (ERK) and the treatment with an ERK inhibitor (U0126) blocked the expression of DSPP. The compressive strength was increased and the setting time was shortened in a dose-dependent manner. The number of cells cultured in the ECN-treated collagen scaffolds was significantly increased compared to the cells in the untreated control group. Conclusions Our results revealed that ECN promoted the proliferation and differentiation of hDPCs. Furthermore, the differentiation was regulated by the ERK signaling pathway. Changes in mechanical properties are related to cell fate, including proliferation and differentiation. Therefore, our study suggests the ECN treatment might be desirable for dentin-pulp complex regeneration.

Caracterização e avaliação da citocompatibilidade de nanocompósitos baseados em celulose bacteriana e fibroína para aplicação em regeneração tecidual / Characterization and cytocompatibility evaluation of cellulose and fibroin based nanocomposites for tissue regeneration applications

A engenharia de tecidos tem como perspectiva desenvolver opções terapêuticas para quadros clínicos com prognóstico limitado, objetivando substituir ou regenerar os tecidos lesados por meio do uso de biomateriais. A metodologia baseia-se no cultivo de células sobre uma matriz ou scaffold que deve ser otimizado para minimizar o risco de infecções e permitir a liberação das moléculas bioativas no local pretendido. As propriedades dos diversos biomateriais são essenciais para o sucesso da metodologia e estes podem ser manipulados de forma a mimetizar a arquitetura da matriz nativa. Assim, nos propomos a desenvolver um novo biocompósito baseado em celulose bacteriana (CB)/ fibroína (FB) visando aplicações em regeneração tecidual na odontologia, em 3 concentrações diferentes (CB/FB25%, CB/FB50% e CB/FB75%). Estes foram caracterizados por análise termogravimétrica, Espectroscopia Vibracional na Região do Infravermelho (FT-IR), Difração de Raios-X, e Microscopia Eletrônica de Varredura (MEV), demonstrando boa estabilidade físico-química, inclusive resitência à degradação térmica em altas temperaturas. A investigação da citocompatibilidade foi conduzida por meio de testes in vitro apenas com as amostras de CB/FB50%. Os testes aplicados foram MTT, azul de tripano, adesão e proliferação celular. As análises estatísticas foram realizadas por meio do software Graph Prism 5.0. Os resultados demonstraram que o material desenvolvido é biocompatível e não citotóxico. As imagens de MEV revelaram um número muito maior de células aderidas à superfície dos scaffolds de CB/FB quando comparado ao de CB pura. Com base nos dados obtidos é possível sugerir que o nanocompósito baseados em CB/FB50% configura uma excelente alternativa como scaffold para a reparação de tecidos.

Tissue engineering has the purpose of developping therapeutic options especially designed to be used on limited clinical conditions, aiming to replace or regenerate damaged tissues applying biomaterials for that. The method is based on the cultivation of cells on a matrix or scaffold that should be optimized to offer minimum risk of infections and allow the release of bioactive molecules at the desired location. The success of the methodology depends on Biomaterials' properties that can be manipulated to mimic the three-dimensional architecture of native tissues extracellular matrix. Thus, we purpose to prepare a new nanocomposite based on bacterial cellulose (BC) / fibroin (SF) aiming at applications in the process of tissue regeneration in dentistry. Three different material's compositions were prepared (BC/SF 25 % BC/SF 50 % and BC/SF 75 %) and all of them were characterized by thermogravimetric analysis, Vibrational Spectroscopy in the Infrared Region (FT-IR), X-ray diffraction, and Scanning Electron Microscopy (SEM). The material showed good physical and chemical stability including resistance to thermal degradation at high temperatures. The investigation of the cytocompatibility was realized just using the samples BC/SF 50 % and the in vitro tests conducted were MTT, Trypan Blue, cell adhesion and proliferation assays. The statistical analysis was done applying the software Graph Pad Prism 5.0. The results showed that the developed nanocomposite is biocompatible and non-cytotoxic. SEM images revealed a greater number of cells attached to the scaffold CB / FB surface when compared to pure CB scaffolds. Based on the data obtained, it is possible to suggest that the nanocomposite based on CB/FB50% configures an excellent alternative as a scaffold for tissue repair

In vitro evaluation of Malaysian natural coral porites bone graft substitutes (CORAGRAF) for bone tissue engineering: a preliminary study

Aim: An approach for three-dimensional (3D) bone tissue generation from bone marrow mesenchymal adult stem cells (BMSC-AS) was investigated. Methods: The BMSC-AS cells were induced to differentiate into osteogenic precursors, capable of proliferating, and subsequently differentiating into bone-forming cells. The differentiated cells were seeded on the surface of coral discs with a mean diameter 10 (±2) mm and a mean thickness 1 (±0.5) mm. The seeded scaffolds were characterized using von Kossa and Alizarin Redstaining, electron and confocal microscopy and RT-PCR analysis. Results: The results demonstrated thatBMSC-AS derived bone-forming cells attached to and colonized into coral scaffolds. Furthermore, these cells produced bone nodules when grown for 3-4 weeks in mineralization medium containing ascorbic acid and beta-glycerophosphate both in tissue culture plates and in scaffolds. The differentiated cells also expressed osteospecific markers when grown both in the culture plates and in 3D scaffolds. Osteogenic cells expressed alkaline phosphatase, osteocalcin, and osteopontin, but not a BMSC-AS cell-specific marker, oct-4. Conclusion: These findings suggest that Malaysian Natural coral Porites bone graft substitutes (CORAGRAF) with BMSC-AS cells can be used for in vitro tissue engineering to cultivation of graftable skeletal structures.