A new scaffold material composed of extracellular matrix （ECM） and thermal sensitive hydrogel （HG）, and evaluated its biocompatibility were investigated. We cultured bladder smooth muscle cells with this compound ...A new scaffold material composed of extracellular matrix （ECM） and thermal sensitive hydrogel （HG）, and evaluated its biocompatibility were investigated. We cultured bladder smooth muscle cells with this compound material, and then observed with phase contrast microscopy and scanning electron microscope （SEM） to assess the cell growth and morphology. The cell adhesion and proliferation were detected with MTT assay and cell count. Results show the ECM/HG compounds appeared as a net-like and red-stained construction with enough meshes and without any cellular fragments. 6 h after implantation, cells were observed adhere on the compounds and extend spurious along the fibers 12 h later. Under SEM even some ECM was observed to be secreted. MTT assay shows there was obvious statistic difference among 3 groups （P〈0.05）. ECM/HG compound materials show a good biocompatibility, which confirms that it would be an ideal tissue engineering scaffolds.展开更多
Background Damage to the central nervous system(CNS)usually leads to the activation of astrocytes,followed by glial scar formation.For years,glial scar has been thought as a major obstacle for successful axon regenera...Background Damage to the central nervous system(CNS)usually leads to the activation of astrocytes,followed by glial scar formation.For years,glial scar has been thought as a major obstacle for successful axon regeneration.However,increasing evidence suggests a beneficial role for this scar tissue as part of the endogenous local immune regulation and repair process.Surprisingly,in contrast to scars in other tissues,glial scars(mainly consist of reactive astrocytes)in both rat cortex and spinal cord were recently found to be significantly softer than healthy CNS tissues.Naive astrocytes have been found to change their phenotype to reactive astrocytes and gradually into scar-forming astrocytes,upregulating the astrocyte marker glial fibrillary acidic protein(GFAP),vimentin,and inflammatory proteins in almost all known brain disorders.Such phenotype transformation process has been widely thought unidirectional or irreversible.However,recent research revealed the environment-dependent plasticity of astrocyte phenotypes,with reactive astrocytes could revert in retrograde to naive astrocytes in proper microenvironment.In consideration of the important roles of mechanical cues in CNS and the unique softening behavior of glial scars,it is of great interesting to study the effects of dynamic changes of matrix stiffness on astrocyte phenotypic switch.Materials&methods Primary astrocyes were isolated from the cortex of SpragueDawley(SD)rats at PI.After cultured for 2 weeks,astrocytes were encapsulated into a set of three-dimensional(3D)hybrid hydrogel system composed of type I collagen and alginate.Immunofluorescence and Western blot expression analysis were applied for characterizting cell responses to different and dynamically changed matrix stiffness.A molecular dynamics model was developed for simulation.Results&discussion In this work,we established an in-vitro model to study the effects of dynamic changes of matrix stiffness on astrocyte phenotypic switchings in 3D.To simulate native cellular environment,we fabricated a set of hybrid hydrogel system composed of type I collagen and alginate.The stiffness of the hybrid hydrogels was demonstrated to be dynamically changed by adding calcium chloride or sodium citrate to crosslink or decrosslink alginate,respectively.Using 3D culture models,we showed that the decrease of matrix stiffness could promote astrocyte activation,with upregulated GFAP and IL-1β.In addition,3D cultured astrocytes spread greater with decreasing matrix stiffness.Moreover,we surprisingly found that astrocyte phenotype could be switched by dynamically changing matrix stiffness.Specifically,matrix stiffening reverted the activation of astrocytes,whereas matrix softening induced astrocyte activation.We further demonstrated that matrix stiffness-induced astrocyte activation was mediated through cytoskeletal tension and YAP protein.To some extent,YAP inhibition enhanced the responses of astrocytes to matrix stiffness.These may guide researchersto re-examine the role of matrix stiffness in reactive astrogliosis in vivo,and inspire the development of novel therapeutic approach for reducing glial scar following injury,enabling axonal regrowth and improving functional recovery by exploiting the benefits of mechanobiology studies.Conclusions Taken together,our results clarify the effects of matrix stiffness and its dynamic changes on phenotypic swtich of astrocytes in three dimensions and reveal environmental factors that regulate astrocytic phenotype transformation process,which may provide potential therapeutic approach for CNS injury.展开更多
基金the Natural Science Foundation of Hubei Province (No.2003ABA187)Hubei Education Bureau (No.2003X123)
文摘A new scaffold material composed of extracellular matrix （ECM） and thermal sensitive hydrogel （HG）, and evaluated its biocompatibility were investigated. We cultured bladder smooth muscle cells with this compound material, and then observed with phase contrast microscopy and scanning electron microscope （SEM） to assess the cell growth and morphology. The cell adhesion and proliferation were detected with MTT assay and cell count. Results show the ECM/HG compounds appeared as a net-like and red-stained construction with enough meshes and without any cellular fragments. 6 h after implantation, cells were observed adhere on the compounds and extend spurious along the fibers 12 h later. Under SEM even some ECM was observed to be secreted. MTT assay shows there was obvious statistic difference among 3 groups （P〈0.05）. ECM/HG compound materials show a good biocompatibility, which confirms that it would be an ideal tissue engineering scaffolds.
基金This work was supported by National Natural Science Foundation of China(31171654)National High-tech R&D Program(863 Program)of China(2011AA100805-2).
基金financially supported by the National Natural Science Foundation of China ( 11872298, 11602191,1161101223,11532009)the China Postdoctoral Science Foundation ( 2018M631141)the Shaanxi Postdoctoral Science Foundation,and the Fundamental Research Funds for the Central Universities ( Z201811336)
文摘Background Damage to the central nervous system(CNS)usually leads to the activation of astrocytes,followed by glial scar formation.For years,glial scar has been thought as a major obstacle for successful axon regeneration.However,increasing evidence suggests a beneficial role for this scar tissue as part of the endogenous local immune regulation and repair process.Surprisingly,in contrast to scars in other tissues,glial scars(mainly consist of reactive astrocytes)in both rat cortex and spinal cord were recently found to be significantly softer than healthy CNS tissues.Naive astrocytes have been found to change their phenotype to reactive astrocytes and gradually into scar-forming astrocytes,upregulating the astrocyte marker glial fibrillary acidic protein(GFAP),vimentin,and inflammatory proteins in almost all known brain disorders.Such phenotype transformation process has been widely thought unidirectional or irreversible.However,recent research revealed the environment-dependent plasticity of astrocyte phenotypes,with reactive astrocytes could revert in retrograde to naive astrocytes in proper microenvironment.In consideration of the important roles of mechanical cues in CNS and the unique softening behavior of glial scars,it is of great interesting to study the effects of dynamic changes of matrix stiffness on astrocyte phenotypic switch.Materials&methods Primary astrocyes were isolated from the cortex of SpragueDawley(SD)rats at PI.After cultured for 2 weeks,astrocytes were encapsulated into a set of three-dimensional(3D)hybrid hydrogel system composed of type I collagen and alginate.Immunofluorescence and Western blot expression analysis were applied for characterizting cell responses to different and dynamically changed matrix stiffness.A molecular dynamics model was developed for simulation.Results&discussion In this work,we established an in-vitro model to study the effects of dynamic changes of matrix stiffness on astrocyte phenotypic switchings in 3D.To simulate native cellular environment,we fabricated a set of hybrid hydrogel system composed of type I collagen and alginate.The stiffness of the hybrid hydrogels was demonstrated to be dynamically changed by adding calcium chloride or sodium citrate to crosslink or decrosslink alginate,respectively.Using 3D culture models,we showed that the decrease of matrix stiffness could promote astrocyte activation,with upregulated GFAP and IL-1β.In addition,3D cultured astrocytes spread greater with decreasing matrix stiffness.Moreover,we surprisingly found that astrocyte phenotype could be switched by dynamically changing matrix stiffness.Specifically,matrix stiffening reverted the activation of astrocytes,whereas matrix softening induced astrocyte activation.We further demonstrated that matrix stiffness-induced astrocyte activation was mediated through cytoskeletal tension and YAP protein.To some extent,YAP inhibition enhanced the responses of astrocytes to matrix stiffness.These may guide researchersto re-examine the role of matrix stiffness in reactive astrogliosis in vivo,and inspire the development of novel therapeutic approach for reducing glial scar following injury,enabling axonal regrowth and improving functional recovery by exploiting the benefits of mechanobiology studies.Conclusions Taken together,our results clarify the effects of matrix stiffness and its dynamic changes on phenotypic swtich of astrocytes in three dimensions and reveal environmental factors that regulate astrocytic phenotype transformation process,which may provide potential therapeutic approach for CNS injury.
