| The hydrogel is a kind of polymer network structure with water as dispersing medium,and they are widely used in various tissue repair,such as wound healing,cartilage repair,bone defect repair materials.The hydrogels are made from natural polymer materials,such as gelatin(Gel),chitosan(CS),hyaluronic acid(HA),and sodium alginate(SA),which possess better biocompatibility compare with synthetic polymers.Through the modification of natural polymer chains,a three-dimensional(3D)cell culture platform similar to the natural extracellular matrix(ECM)can be performed in vitro.At present,most cell culture methods are based on a two-dimensional(2D)plane.Although it can easily and massively expand the required cells,it will cause some cells to show abnormal biological behavior(compared with in vivo).Traditional cell culture in vitro is mostly based on polystyrene and glass plates.The elastic modulus of these materials is not in the physiological range,so it will cause cells to show abnormal behavior,such as flat shape,abnormal polarization,abnormal response to small molecule drugs,and phenotypic changes.The reason for these abnormal cellular responses is not only the substrate stiffness but also the distribution of integrins.Under the natural microenvironment,cells can receive chemical/physical signals from the extracellular matrix,and the receiving range is 360 degrees,which is different from the signal transmission in the 2D plane which is only limited to the dorsal and ventral surface of cells.Therefore,in vitro cell culture system based on hydrogel(polymer network structure with high water content)is one of the best choices.Natural polymer hydrogel can not only be used as a platform for cell culture in vitro but also a powerful tool to reveal the mechanism of extracellular matrix physical properties regulating cell behavior.In recent years,many studies have revealed that the physical factors of the extracellular matrix play an important role in the regulation of cells,such as stiffness,topological structure,viscoelasticity,porosity,and degradation rate.It is undeniable that the physical properties of the extracellular matrix play an important role in the same way as soluble chemical factors.Natural polymer hydrogel not only has high biocompatibility but also has many unique physical properties of natural tissues.However,a hydrogel-based 3D culture system will introduce more physical variables for basic cell research.These physical properties are often interrelated and dependent on each other.How to control the change of a single physical property is the difficulty of current research.Alginate hydrogel,as one of the natural polymer hydrogels,has good biocompatibility and the possibility of artificial modification.With its characteristics of chelating and crosslinking with calcium ions,which was named ?egg-box? crosslinking,we can adjust the stiffness or viscoelasticity alone without affecting the internal pore structure.Exploring the underlying mechanics of matrix physical characteristics and cell response would deeply affect the utilization of natural polymers.Due to superior multipotential differentiation,high availability,low immunogenicity,and no ethical issues,bone marrow mesenchymal stem cells(BMSCs)have become one of the most important seed cells in the process of tissue engineering repair.The niche of BMSCs is very complex,involving the synergistic effect of a variety of chemical and physical factors.Under normal physiological conditions,the bone marrow microenvironment will constantly be reconstituted to stabilize stem cells and keep them at the lowest energy consumption.When the injury occurred in vivo,stem cells will migrate to the injured site through short/long distance after mobilization,so as to participate in tissue repair,and this program is named ?stem cell homing?.This process is very complex and amount of chemical and physical factors are involved.In the past few decades,the biological functions(migration,proliferation,paracrine,and differentiation)of stem cells which were influenced by chemical factors have been paid much attention and studied.In recent years,the physical properties of ECM have been paid more attention and studied deeply.However,the current research on the physical properties of ECM and the biological response of stem cells is mostly based on two-dimensional(2D)culture conditions.However,the cell would present a different or even opposite response under 3D conditions,and this limitation hind the design of biomaterials.In view of this,our research aims to explore the response of stem cells to the ECM physical properties depending on alginate hydrogel.Using this hydrogel cultural system,we tried to elucidate the relationship between matrix properties and cell functions,and provide a theoretical basis for the design and optimization of the hydrogel.At the same time,it also provides a more physiological culture model for the study of stem cell biological behavior in vitro.(1)Matrix promotes MSCs migration via improved deformation and switched migration strategyBy adjusting the molecular weight of sodium alginate and concentration of calcium ion of alginate hydrogel,combined with Transwell chamber,we have successfully constructed a 3D cell chemotaxis migration model in vitro.Depending on this model,we have explored the effect of various ECM matrix stiffness on stem cell migration.We found that the stiffer matrix can promote the migration of stem cells under 3D conditions.However,accompanied with the down-regulation of integrins and matrix metalloproteinases(MMP),the expression of SDF-1 and CXCR4 were significantly up-regulated by the stiffer matrix.These results showed that stem cells got a strong migration ability in the stiffer matrix,but this migration strategy was not dependent on the hydrolysis of ECM by protease and integrin.In addition,the addition of ROCK inhibitor could be striking inhibit the cell migration promoted by stiffer matrix.The results indicate that the stiffer extracellular matrix may change the migration strategy of stem cells to obtain stronger motility.In this process,the stiffer matrix can also significantly down-regulate the expression of nucleoskeleton protein lamin A / C,thus softening the nucleus and making the cells obtain greater deformability.Then,a softer nucleus would help the cells to pass through the narrow gap in the 3D matrix.In conclusion,stem cells can smartly switch their migration strategy to cope with the changes of extracellular matrix mechanical properties,so as to obtain the optimal exercise ability.(2)The dynamic matrix can potentiate MSCs paracrine via an effective mechanical doseBy adjusting the different stiffening time points of alginate hydrogel,a dynamic extracellular matrix was constructed on time scales to investigate the effects of different stiffening time points on the paracrine function of stem cells.Our study found that compared with the static extracellular matrix,the dynamic extracellular matrix can improve the paracrine level of stem cells,especially in the third day stiffening extracellular matrix,the paracrine level of stem cells is more vigorous than other groups.Subsequent studies have shown that a soft extracellular matrix can effectively promote the expansion of stem cells,making a more effective combination between cells and ECM.This combination was conducive for stem cells to transmit mechanical signals of the extracellular matrix into intracellular chemical signals,and ultimately regulate gene expression.Finally,effectively improve the paracrine level of stem cells.In this study,we propose that the physical properties of extracellular matrix were means two concepts for stem cells,"total mechanical dose" and "effective mechanical dose".The total mechanical energy exerted by the extracellular matrix on stem cells is transformed into an effective mechanical dose through the nucleocytoplasmic shuttle of YAP protein to regulate gene expression.(3)MSCs can maintain cell division under stiff matrix via activating cortical layerThe physical limitation of the extracellular matrix has a great influence on cell division.We studied the proliferative activity of stem cells in the various stiffness matrix.Through the transcriptome sequencing of stem cells,we found that the stiffer extracellular matrix can effectively promote the gene expression of stem cells,and the different genes between the two are mainly enriched in cell cycle-related signaling pathways.Subsequent studies have shown that a stiff extracellular matrix can effectively promote the proliferation of stem cells.Our study suggests that in the three-dimensional mitosis of stem cells,the increased expression of Ras and Myosin can enhance the cortical toughness of stem cells,which is conducive to stem cells to resist the physical restriction of extracellular matrix during the process of division,thus protecting the normal progress of mitosis.In this process,the down-regulation of integrin expression in stem cells may be beneficial to effectively remove the matrix and round the cells during mitosis,providing the space for the synthesis of DNA and protein.(4)Matrix viscoelasticity can balance self-renewal and quiescence of MSCs via PI3K/AKT-CDK1axisIn this study,we used different ratios of molecular weight and calcium concentration to obtain sodium alginate hydrogel with similar stiffness but different viscoelasticity.By analyzing the transcriptome of stem cells,we found that different stress relaxation can significantly change the gene expression of stem cells,and significantly affect the self-renewal ability of stem cells.Our study suggests that in three-dimensional culture,stem cells maintain self-renewal through PI3 K / AKT-CDK1 axis.However,when the extracellular matrix is insufficient to provide the space for stem cell division,stem cells can enter the quiescence state by downregulating the activity of the PI3 K / AKT-CDK1 axis,and the stem cells reactivated from the quiescence state have higher proliferation/differentiation activity. |
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