As matrizes acelulares são utilizadas na engenharia de tecidos com o objetivo da reconstrução ou substituição de tecidos danificados. Nesse trabalho matrizes acelulares foram preparadas a partir de pericárdio porcino e pericárdio bovino, por possuírem uma grande quantidade colágeno tipo I, usando-se ao tratamento de hidrólise alcalina nos tempos de 12 e 24 horas. Essas matrizes foram desenvolvidas para serem utilizadas como barreiras de proteção no processo de cicatrização de feridas como também na indução de formação de tecidos ósseos. Foram submetidas ao teste de biocompatibilidade in vivo, com a implantação das matrizes no subcutâneo de 65 ratos Wistar, machos, adultos que sofreram eutanásia aos 7, 14, 21, 28 e 35 dias de pós-operatório. A avaliação microscópica no 7º dia mostrou existência da matriz e uma grande quantidade de infiltrado inflamatório em volta do material, sendo que ao 14º dias além do infiltrado inflamatório, foi observado uma neovascularização. Aos 21º e 28º dias foi observado uma diminuição gradual do infiltrado inflamatório, crescimento celular no local em que as fendas se formaram nas matrizes e uma grande revascularização. Ao 35º dias a matriz de pericárdio bovino e porcino foram reabsorvidas totalmente. Comparando o pericárdio bovino com o porcino tratado por 12 e 24 horas em relação ao tempo de degradação, foi observado que as matrizes tanto bovina como a porcina tratadas por 24 horas degradaram mais rápido que as tratadas por 12 horas. Portanto as matrizes de pericárdio bovino e porcino são biocompativeis, pois não causaram reações citotóxicas, reações inflamatórias e nem imunológica adversa. / Acellular matrices are used in tissue engineering for reconstruction or replacement of damaged tissues .In this study acellular matrices were developed using porcine pericardium and bovine pericardium, which contain a large quantity of type I collagen, using an alkaline hydrolysis treatment at the times of 12 hours and 24 hours. The matrices were developed to be used as protective barriers in the process of wound healing, and also in bone tissue inducing formation. In the test of in vivo biocompatibility, matrices were implanted subcutaneously in 65 adult male Wistar rats, sacrificed at 7, 14, 21, 28 and 35 days postoperatively. The microscopic evaluation on day 7 showed a large amount of inflammatory infiltrate around the material; at the 14th day, beyond the inflammatory infiltrate was observed a neovascularization. At 21st and 28th days were observed a gradual decrease in the inflammatory infiltrate, cellular growth and a large neovascularization. By day 35, porcine and bovine pericardium matrices were completely resorbed. Comparing the degradation time between the matrices treated with 12 and 24 hours, it was observed that both matrices treated for 24 h, degraded faster than those treated for 12 hours. Both matrices are biocompatible, do not cause cytotoxic reactions, inflammatory or immune adverse reactions.

As matrizes acelulares são utilizadas na engenharia de tecidos com o objetivo da reconstrução ou substituição de tecidos danificados. Nesse trabalho matrizes acelulares foram preparadas a partir de pericárdio porcino e pericárdio bovino, por possuírem uma grande quantidade colágeno tipo I, usando-se ao tratamento de hidrólise alcalina nos tempos de 12 e 24 horas. Essas matrizes foram desenvolvidas para serem utilizadas como barreiras de proteção no processo de cicatrização de feridas como também na indução de formação de tecidos ósseos. Foram submetidas ao teste de biocompatibilidade in vivo, com a implantação das matrizes no subcutâneo de 65 ratos Wistar, machos, adultos que sofreram eutanásia aos 7, 14, 21, 28 e 35 dias de pós-operatório. A avaliação microscópica no 7º dia mostrou existência da matriz e uma grande quantidade de infiltrado inflamatório em volta do material, sendo que ao 14º dias além do infiltrado inflamatório, foi observado uma neovascularização. Aos 21º e 28º dias foi observado uma diminuição gradual do infiltrado inflamatório, crescimento celular no local em que as fendas se formaram nas matrizes e uma grande revascularização. Ao 35º dias a matriz de pericárdio bovino e porcino foram reabsorvidas totalmente. Comparando o pericárdio bovino com o porcino tratado por 12 e 24 horas em relação ao tempo de degradação, foi observado que as matrizes tanto bovina como a porcina tratadas por 24 horas degradaram mais rápido que as tratadas por 12 horas. Portanto as matrizes de pericárdio bovino e porcino são biocompativeis, pois não causaram reações citotóxicas, reações inflamatórias e nem imunológica adversa. / Acellular matrices are used in tissue engineering for reconstruction or replacement of damaged tissues .In this study acellular matrices were developed using porcine pericardium and bovine pericardium, which contain a large quantity of type I collagen, using an alkaline hydrolysis treatment at the times of 12 hours and 24 hours. The matrices were developed to be used as protective barriers in the process of wound healing, and also in bone tissue inducing formation. In the test of in vivo biocompatibility, matrices were implanted subcutaneously in 65 adult male Wistar rats, sacrificed at 7, 14, 21, 28 and 35 days postoperatively. The microscopic evaluation on day 7 showed a large amount of inflammatory infiltrate around the material; at the 14th day, beyond the inflammatory infiltrate was observed a neovascularization. At 21st and 28th days were observed a gradual decrease in the inflammatory infiltrate, cellular growth and a large neovascularization. By day 35, porcine and bovine pericardium matrices were completely resorbed. Comparing the degradation time between the matrices treated with 12 and 24 hours, it was observed that both matrices treated for 24 h, degraded faster than those treated for 12 hours. Both matrices are biocompatible, do not cause cytotoxic reactions, inflammatory or immune adverse reactions.

The migration and invasion of cancer cells through 3D confined extracellular matrices is coupled to cell mechanics and the mechanics of the extracellular matrix. Cell mechanics is mainly determined by both the mechanics of the largest organelle in the cell, the nucleus, and the cytoskeletal architecture of the cell. Hence, cytoskeletal and nuclear mechanics are the major contributors to cell mechanics. Among other factors, steric hindrances of the extracellular matrix confinement are supposed to affect nuclear mechanics and thus also influence cell mechanics. Therefore, we propose that the percentage of invasive cells and their invasion depths into loose and dense 3D extracellular matrices is regulated by both nuclear and cytoskeletal mechanics. In order to investigate the effect of both nuclear and cytoskeletal mechanics on the overall cell mechanics, we firstly altered nuclear mechanics by the chromatin de-condensing reagent Trichostatin A (TSA) and secondly altered cytoskeletal mechanics by addition of actin polymerization inhibitor Latrunculin A and the myosin inhibitor Blebbistatin. In fact, we found that TSA-treated MDA-MB-231 human breast cancer cells increased their invasion depth in dense 3D extracellular matrices, whereas the invasion depths in loose matrices were decreased. Similarly, the invasion depths of TSA-treated MCF- 7 human breast cancer cells in dense matrices were significantly increased compared to loose matrices, where the invasion depths were decreased. These results are also valid in the presence of a matrix-metalloproteinase inhibitor GM6001. Using atomic force microscopy (AFM), we found that the nuclear stiffnesses of both MDA-MB- 231 and MCF-7 breast cancer cells were pronouncedly higher than their cytoskeletal stiffness, whereas the stiffness of the nucleus of human mammary epithelial cells was decreased compared to their cytoskeleton. TSA treatment reduced cytoskeletal and nuclear stiffness of MCF-7 cells, as expected. However, a softening of the nucleus by TSA treatment may induce a stiffening of the cytoskeleton of MDA-MB-231 cells and subsequently an apparent stiffening of the nucleus. Inhibiting actin polymerization using Latrunculin A revealed a softer nucleus of MDA-MB-231 cells under TSA treatment. This indicates that the actin-dependent cytoskeletal stiffness seems to be influenced by the TSA-induced nuclear stiffness changes. Finally, the combined treatment with TSA and Latrunculin A further justifies the hypothesis of apparent nuclear stiffening, indicating that cytoskeletal mechanics seem to be regulated by nuclear mechanics.

Besides biochemical and molecular regulation, the migration and invasion of cells is controlled by the environmental mechanics and cellular mechanics. Hence, the mechanical phenotype of cells, such as fibroblasts, seems to be crucial for the migratory capacity in confined 3D extracellular matrices. Recently, we have shown that the migratory and invasive capacity of mouse embryonic fibroblasts depends on the expression of the Rho-GTPase Rac1, similarly it has been demonstrated that the Rho-GTPase Cdc42 affects cell motility. The p21-activated kinase (PAK) is an effector down-stream target of both Rho-GTPases Rac1 and Cdc42, and it can activate via the LIM kinase-1 its down-stream target cofilin and subsequently support the cell migration and invasion through the polymerization of actin filaments. Since Rac1 deficient cells become mechanically softer than controls, we investigated the effect of group I PAKs and PAK1 inhibition on cell mechanics in the presence and absence of Rac1. Therefore, we determined whether mouse embryonic fibroblasts, in which Rac1 was knockedout, and control cells, displayed cell mechanical alterations after treatment with group I PAKs or PAK1 inhibitors using a magnetic tweezer (adhesive cell state) and an optical cell stretcher (non-adhesive cell state). In fact, we found that group I PAKs and Pak1 inhibition decreased the stiffness and the Young’s modulus of fibroblasts in the presence of Rac1 independent of their adhesive state. However, in the absence of Rac1 the effect was abolished in the adhesive cell state for both inhibitors and in their nonadhesive state, the effect was abolished for the FRAX597 inhibitor, but not for the IPA3 inhibitor. The migration and invasion were additionally reduced by both PAK inhibitors in the presence of Rac1. In the absence of Rac1, only FRAX597 inhibitor reduced their invasiveness, whereas IPA3 had no effect. These findings indicate that group I PAKs and PAK1 inhibition is solely possible in the presence of Rac1 highlighting Rac1/PAK I (PAK1, 2, and 3) as major players in cell mechanics.