Bioprinting: From Technique to Application in Tissue Engineering and Regenerative Medicine.

Among the different approaches present in regenerative medicine and tissue engineering, the one that has attracted the most interest in recent years is the possibility of printing functional biological tissues. Bioprinting is a technique that has been applied to create cellularized three-dimensional structures that mimic biological tissues and thus allow their replacement. Hydrogels are interesting materials for this type of technique. Hydrogels based on natural polymers are known due to their biocompatible properties, in addition to being attractive biomaterials for cell encapsulation. They provide a threedimensional aqueous environment with biologically relevant chemical and physical signals, mimicking the natural environment of the extracellular matrix (ECM). Bioinks are ink formulations that allow the printing of living cells. The controlled deposition of biomaterials by bioinks needs to maintain cell viability and offer specific biochemical and physical stimuli capable of guiding cell migration, proliferation, and differentiation. In this work, we analyze the theoretical and practical issues of bioprinting, citing currently used methods, their advantages, and limitations. We present some important molecules that have been used to compose bioinks, as well as the cellular responses that have been observed in different tissues. Finally, we indicate future perspectives of the method.

Development of a chitosan and hyaluronic acid hydrogel with potential for bioprinting utilization: A preliminary study.

Bioprinting is a technique that has been applied in the areas of tissue engineering and regenerative medicine (TERM). Natural polymer-based hydrogels are known for their favorable biocompatible properties, as well as attractive biomaterials for cell encapsulation. These hydrogels provide an aqueous three-dimensional environment with biologically relevant chemical and physical signals, mimicking the natural environment of the extracellular matrix (ECM). Chitosan (CHI) and hyaluronic acid (HA) have been widely researched for biomedical applications. Bioinks are "ink" formulations, usually hydrogels, that allow the printing of living cells. This work proposes the development of a low cost and simple chitosan CHI-AH hydrogel with potential to become a bioink. At physiological temperature, the biomaterials form a hydrogel. The material developed was characterized by the analysis of morphology, cytotoxicity, and cell viability. FTIR showed the characteristic vibrational bands of chitosan and HA. No difference in swelling was observed between the different formulations studied, although SEM showed architectural differences between the hydrogels obtained. Extract cytotoxicity testing showed that the hydrogel is not cytotoxic. The direct toxicity test also revealed the absence of toxicity, but the cells had difficulty migrating into the gel, probably because of its density. These data were confirmed by SEM. Further testing are ongoing to better understand the gel's characteristics to improve the limitations found so far.

Production of fibrous polymer scaffolds for tissue engineering using an automated solution blow spinning system

Abstract Introduction Solution blow spinning (SBS) and airbrushing are two techniques that can be used as alternatives to electrospinning in the production of fibrous scaffolds for tissue engineering (TE). SBS seems particularly interesting due to its versatility, however, it has not been much explored and no automated SBS systems were found in the literature. Therefore, the present work aimed to develop such equipment and compare the results to those found for airbrushing, considering the same set of parameters. Methods A new SBS set up, composed of a specially designed nozzle with automated movement, a syringe pump and a compressor, was used to produce fibrous poly (ε-caprolactone) (PCL) mats. The airbrushed fibers were produced under the same conditions, and samples of both types of mats were imaged using scanning electron microscopy (SEM) to compare them in terms of microstructure and fiber diameter. Results The SBS system was robust and performed well, in terms of movement and fiber deposition. In comparison to airbrushing's, SBS mats presented different microstructural characteristics (considering the parameters used). Conclusion The biggest advantage over airbrushing may be its versatility and simple automation, which may improve sample reproducibility, especially considering scaled up processes. To further improve this apparatus, a better understanding of how process variables interfere in the microstructure is needed, as well as more sophisticated interface and operation.

Evaluation of cell interaction with polymeric biomaterials based on hyaluronic acid and chitosan.

Tissue engineering involves the development of new materials or devices capable of specific interactions with biological tissues, searching the use of biocompatible materials as scaffolds for in vitro cell growth, and functional tissue development, that is subsequently implanted into patient. The aim of the current study was to evaluate the initial aspects of cell interaction with the polymeric biomaterials blends based on hyaluronic acid with chitosan. The hypothesis approach involves synthesis and analysis of swelling and thermal degradation (thermal gravimetric analysis) of the polymer blend; and Vero cell interaction with the biomaterial, through analysis of cytotoxicity, adhesion and cell morphology. The blend resulted in a biomaterial with a high swelling ratio that can allow nutrient distribution and absorption. The thermal gravimetric analysis results showed that the blend had two stages of degradation at temperatures very close to those observed for pure polymers, confirming that the physical mixing of hydrogels occurred, resulting in the presence of both hyaluronic acid and chitosan in the blend. The evaluation of indirect cytotoxicity showed that the blend was non cytotoxic for Vero cells, and the quantitative analysis performed with the MTT could verify a cell viability of 98%. The cells cultured on the blend showed adhesion, spreading and proliferation on this biomaterial, distinguished from the pattern of the control cells. These results showed that the blends produced from hyaluronic acid and chitosan hydrogels are promising for applications in tissue engineering, aiming at future cartilaginous tissue.

Evaluation of peritoneal adhesions formation and tissue response to polypropylene - poli (2-hydroxyethyl methacrylate)-(polyHEMA) implant on rats' abdominal wall.

PURPOSE: To verify if the composit poli (2-hydroxyethyl methacrylate)-PolyHEMA/polypropylene mesh implanted in the female rat's abdominal wall could be suitable for the prevention of peritoneal adhesions, and for the evaluation of the tecidual response produced by this biomaterial. METHODS: Polypropylene meshes (Group PP, n=20) and polypropylene meshes coated with a layer of poli (2-hydroxyethyl methacrylate)-PolyHEMA (Group PH, n=20) were implanted on the abdominal wall of Wistar female rats. Ten animals from each group were submitted to euthanasia at 15 and 30 days of the postoperative period. RESULTS: The animals from the group PP presented visceral adhesions on the mesh surface, which was not observed in the ones from group PH. At the histopathological examination foreign body response was observed in both groups, whilst there was a greater intensity of inflammatory response in group PH on both moments. CONCLUSION: The poli (2-hydroxyethyl methacrylate) polyHEMA hydrogel associated to polypropylene mesh reduces visceral adhesion formation in rats, although it may be associated to greater inflammatory reaction.

2-hydroxyethyl methacrylate composite - polypropylene mesh for preventing peritoneal adhesions in female dogs.

PURPOSE: To evaluate whether the lining facing the visceral side of polypropylene mesh made with 2-hydroxyethyl methacrylate (p(HEMA)) hydrogel could avoid peritoneal adhesion in female dogs. METHODS: Eight animals (group PP) had a polypropylene mesh implanted to correct a defect in the rectal abdominal muscle, whereas in the other group (group PH) the polypropylene mesh was coated with p(HEMA) composite on the surface facing the peritoneal area. RESULTS: Adhesions were observed on the mesh in 62.5% of the PP group. In the PH group adhesions were present only on the suture lines. CONCLUSION: p(HEMA) hydrogel was well tolerated and effective in avoiding visceral and omental adhesions on the surface of the polypropylene mesh.

Evaluation of peritoneal adhesions formation and tissue response to polypropylene - poli (2-hydroxyethyl methacrylate)-(polyHEMA) implant on rats' abdominal wall / Avaliação da formação de aderências peritoneais e da resposta tecidual ao implante de poli (2-hidroxietil dimetacrilato)-(poliHEMA) na parede abdominal de ratos

PURPOSE: To verify if the composit poli (2-hydroxyethyl methacrylate)-PolyHEMA/polypropylene mesh implanted in the female rat's abdominal wall could be suitable for the prevention of peritoneal adhesions, and for the evaluation of the tecidual response produced by this biomaterial. METHODS: Polypropylene meshes (Group PP, n=20) and polypropylene meshes coated with a layer of poli (2-hydroxyethyl methacrylate)-PolyHEMA (Group PH, n=20) were implanted on the abdominal wall of Wistar female rats. Ten animals from each group were submitted to euthanasia at 15 and 30 days of the postoperative period. RESULTS: The animals from the group PP presented visceral adhesions on the mesh surface, which was not observed in the ones from group PH. At the histopathological examination foreign body response was observed in both groups, whilst there was a greater intensity of inflammatory response in group PH on both moments. CONCLUSION: The poli (2-hydroxyethyl methacrylate) polyHEMA hydrogel associated to polypropylene mesh reduces visceral adhesion formation in rats, although it may be associated to greater inflammatory reaction.

OBJETIVO: Verificar se compósito poli 2-hidroxietil dimetacrilato (PoliHEMA) / tela de polipropileno implantado na parede abdominal de ratas seria adequado para prevenção de aderências peritoneais e avaliar a resposta tecidual desencadeada por este biomaterial. MÉTODOS: Foram implantadas telas de polipropileno - Grupo PP (n=20) e telas de polipropileno revestidas por uma camada de poli 2 (hidroxietil dimetacrilato)-PolyHEMA - Grupo PH (n=20) na parede abdominal de ratas da linhagem Wistar. Dez animais de cada grupo foram submetidos à eutanásia aos 15 e 30 dias de pós-operatório. RESULTADOS: Os animais do grupo PP apresentaram aderências viscerais na superfície da tela, o que não foi observado nos do grupo PH. Observou-se no exame histopatológico resposta tipo corpo estranho nos dois grupos sendo que no grupo PH houve maior intensidade de resposta inflamatória nos dois momentos. CONCLUSÃO: O hidrogel de poliHEMA quando associado à tela de polipropileno reduz a formação de aderências viscerais em ratos, embora possa estar associado à reação inflamatória mais intensa.

2-hydroxyethyl methacrylate composite - polypropylene mesh for preventing peritoneal adhesions in female dogs / Compósito 2-hidroxietil dimetacrilato-tela de polipropileno para prevenção de aderências peritoneais em cadelas

PURPOSE: To evaluate whether the lining facing the visceral side of polypropylene mesh made with 2-hydroxyethyl methacrylate (p(HEMA)) hydrogel could avoid peritoneal adhesion in female dogs. METHODS: Eight animals (group PP) had a polypropylene mesh implanted to correct a defect in the rectal abdominal muscle, whereas in the other group (group PH) the polypropylene mesh was coated with p(HEMA) composite on the surface facing the peritoneal area. RESULTS: Adhesions were observed on the mesh in 62.5 percent of the PP group. In the PH group adhesions were present only on the suture lines. CONCLUSION: p(HEMA) hydrogel was well tolerated and effective in avoiding visceral and omental adhesions on the surface of the polypropylene mesh.

OBJETIVO: Avaliar se o revestimento da face visceral de uma tela de polipropileno com 2-hidroxietil dimetacrilato (p(HEMA)) poderia evitar aderências peritoniais em cadelas. MÉTODOS: Em oito animais (Grupo PP) foram implantadas telas de polipropileno para correção de um defeito do músculo reto abdominal, enquanto em outro grupo (Grupo PH) fez-se a implantação de uma tela de polipropileno cuja face peritonial foi revestida por 2-hidroxietil dimetacrilato (p(HEMA)). RESULTADOS: Foram observados 62,5 por cento de aderências peritoniais sobre a tela nos animais do Grupo PP, enquanto que no Grupo PH estas não ocorreram, exceto sobre a linha se sutura. CONCLUSÃO: O hidrogel de p(HEMA) foi bem tolerado pelos animais e mostrou-se efetivo na prevenção das aderências viscerais e omentais sobre a tela de polipropileno.

Effects of chitosan solution concentration and incorporation of chitin and glycerol on dense chitosan membrane properties.

The aim of this work was to perform a systematic study about the effects induced by chitosan solution concentration and by chitin or glycerol incorporation on dense chitosan membranes with potential use as burn dressings. The membrane properties analyzed were total raw material cost, thickness, morphology, swelling ratio, tensile strength, percentage of strain at break, crystallinity, in vitro enzymatic degradation with lysozyme, and in vitro Vero cells adhesion. While the use of the most concentrated chitosan solution (2.5% w/w) increased membrane cost, it also improved the biomaterial mechanical resistance and ductility, as well as reduced membrane degradation when exposed for 2 months to lysozyme. The remaining evaluated properties were not affected by initial chitosan solution concentration. Chitin incorporation, on the other hand, reduced the membranes cost, swelling ratio, mechanical properties, and crystallinity, resulting in thicker biomaterials with irregular surface more easily degradable when exposed to lysozyme. Glycerol incorporation also reduced the membranes cost and crystallinity and increased membranes degradability after exposure to lysozyme. Strong Vero cells adhesion was not observed in any of the tested membrane formulations. The overall results indicate that the majority of the prepared membranes meet the performance requirements of temporary nonbiodegradable burn dressings (e.g. adequate values of mechanical resistance and ductility, low values of in vitro cellular adhesion on their surfaces, low extent of degradation when exposed to lysozyme solution, and high stability in aqueous solutions).