Regulate The Cell's Fate By The Microenvironment Of Nanomaterials

In the field of modern medicine,the repair of tissues or organs still poses great difficulties.In order to solve this problem,tissue engineering,a discipline based on engineering science and life sciences,which can be used for clinical diagnosis and treatment has attracted more researchers'attention.Tissue engineering contains three elements,i.e.,stem cells,scaffold,and grow factors.Stem cells are widely used in the fields of tissue engineering and regenerative medicine to repair tissues or organs,because they are easy to extract from the human body and have the characteristics of self-renewal and multilineage differentiation.However,how to induce directional differentiation and regulate the fate of stem cells autonomously has always been an urgent problem for researchers.The environment in which cells live in an organism is a three-dimensional dynamic microenvironment.Multiple factors,such as chemical,physical,and biological signals generated by mechanical stimulation,extracellular matrix and intercellular interactions,affect and control cells'migration,proliferation and differentiation.Among them,stimulation from chemical,physical,and biological signals is currently the most simple and effective method to regulate the fate of stem cells.In addition,the extracellular matrix contains a large number of collagen fibers and other micro/nanostructures,which together with the surrounding physical and chemical microenvironment affect the cell's function and behaviours.Therefore,in the fields of regenerative medicine and tissue engineering,simulating the chemical,physical,and biological signals in the extracellular microenvironment by using nano/micromaterials is a feasible method to regulate the fate of stem cells,which is favored by researchers.Up to now,most researchers have successfully utilized the physical signals generated by nanomaterials'micro/nanotopographies to conduct directed differentiation.Compared with chemical and biological signals,physical signals have better stability,which can regulate the fate of stem cells for a long time.In addition,the cells'pseudopodium and transmembrane proteins on the cell membrane can better receive signals from the outside of the cell through contact with the material,which is beneficial to promote stem cells'differentiation.So far,most researchers have focused on synthetic two-dimensional planar materials,to regulate the fate of stem cells.However,the synthesis process of materials is often limited by factors such as economic conditions and controllability,which makes the materials unable to be massively produced.In nature,there are some plant-derived materials which have unique and ordered micro/nanostructures,such as leaves,petals,and wood.These materials are rich in sources and easy to obtain,have certain biocompatibility and biodegradability,which provides a new material idea and template for tissue engineering.Compared with the traditional two-dimensional planar materials,the three-dimensional materials'structures are more similar to the local network structures in the human body.Moreover,in the same space,the three-dimensional materials avail more stem cells'proliferation and differentiation.For example,the ferroelectric polyvinylidene fluoride?PVDF?fiber membrane fabricated by electrospinning technology has a certain three-dimensional structure,which can achieve three-dimensional cell culture.In addition,the electrical stimulation generated by PVDF has also been proved to be a practical and effective way to regulate the fate of stem cells.Therefore,in the field of tissue engineering and regenerative medicine,using the physical and chemical properties of three-dimensional materials to induce stem cell differentiation is closer to practical clinical applications.In this study,the contact interaction between stem cells and nano/micromaterials was utilized to regulate the fate of stem cells in the extracellular physical and chemical microenvironment constructed by three-dimensional nano/micromaterials.The specific content of this paper includes the following two parts:1.Topographical regulation of human adipose-derived stem cells?hADSCs?osteogenic differentiation by plant-derived micro/nanostructures.In this study,we used plant-derived raffia which has different micro/nanostructures on its different surfaces as tissue engineering scaffold,hADSCs as seed cells to regulate the fate of stem cells.The results of this research are as follows:?1?It was found that the surfaces of natural plant-derived raffia had different nano/microtopographies.The front surface of raffia is a nanorod array structure with a diameter ranging from 50-100 nm,and the back is a honeycomb micro/nanostructure with a diameter of about 10-15?m.?2?Raffia had good cell compatibility.When cultured for 5 days,the number of cells cultured on the front and back of raffia were 80%and 60%of the number of cells on the TCPs,respectively,showing a good proliferation tendency.After 2 days of culture,the results of live/dead cell staining confirmed that the cells cultured on different surfaces of raffia maintained good cell activity.Moreover,F-actin immunofluorescence staining and SEM images of dehydrated cells revealed that the cells cultured on the different surfaces of raffia had good adhesion at 2 days and there was a tendency of osteogenic differentiation.Futhermore,the cytoskeleton of the cells cultured on the honeycomb micro-nano structure showed an osteoblast-like polygonal shape.?3?Micro/nanostructures of raffia can affect the differentiation of stem cells.The results of real-time quantitative polymerase chain reaction?q-PCR?revealed that the micro/nano structures on different surfaces of raffia promoted the expression of Runx2,osteopontin?OPN?and osteocalcin?OCN?genes of hADSCs,especially the OCN gene.The OCN expression levels of the cells cultured on the front and back surfaces increased by approximately 55-fold and 36-fold,respectively.The results of alkaline phosphatase?ALP?kit and immunofluorescence staining confirmed that the micro/nanostructures on different surfaces of raffia can promote the expression of alkaline phosphatase,OPN,and OCN.The results of alizarin redstaining proved that different micro/nanostructures of raffia can promote the up-regulation of calcium deposition ability in hADSCs.Therefore,the abovementioned results confirmed that compared with the nanorod structure on the front of raffia,the honeycomb micro/nano structure on the back of raffia can effectively promote the osteogenic differentiation of hADSCs.?4?In this study,it was the first utilization of natural plant-derived materials to regulate the stem cells'fate.This research not only confirmed the effect of natural structure on stem cell differentiation,but also provided important design ideas for designing tissue engineering scaffold.2.Ultrasonic-wave-powered electrical stimulation for enhancing neural differentiation of mesenchymal stem cells on iron hydroxide oxide/polyvinylidene fluoride fibrous film.?1?Electrospinning technology was used to fabricate a PVDF fibrous film with a single fiber diameter of 600 nanometers,and a layer of nanorod-like?-FeOOH was successfully assembled on this film to fabricate FeOOH/PVDF scaffold through hydrothermal method.The results of SEM,EDS,XRD,and FTIR tests showed that FeOOH growed evenly on the PVDF fibrous film.Moreover,PFM test confirmed that the FeOOH/PVDF fibrous film had good piezoelectricity.?2?FeOOH/PVDF fibrous film had good cell compatibility.The results of CCK-8 confirmed that the rat bone marrow-derived mesenchymal stem cells?rBMSCs? cultured on FeOOH/PVDF fibrous film had good proliferation under different ultrasonic conditions.On the 3rd and 5th days,the number of cells cultured on the FeOOH/PVDF fibrous film accounted for 53%-112%and 78%-107%of the number of cells cultured on TCP.Furthermore,the results of CCK-8 revealed that the cells cultured on the films under 400 W ultrasonic treatment had a similar proliferation trend as the cells cultured on the TCPs.After 2 days of culture,the results of live/dead staining showed that the cells cultured on different sample had good cell activity.Moreover,cytoskeletal staining and dehydration electron microscopy results proved that cells cultured on different samples had good adhesion under ultrasonic conditions,and compared with the cytoskeleton at 2 days,the cytoskeleton at 10 days tended to be more neuronal-like shape.?3?The external ultrasonic wave stimulus was used to make the piezoelectric FeOOH/PVDF fibrous film generate electrical signal stimulations for inducing neural differentiation of stem cells.The results of q-PCR proved that under ultrasonic condition,piezoelectric materials?PVDF and FeOOH/PVDF film?can promote the expression of Nestin,microtubule-associated protein 2?MAP2?,and class?-Tubulin III?Tuj1?genes of rBMSCs.In particular,after 21 days of culture,compared with TCPs,the Nestin,MAP2,and Tuj1 genes of cells cultured on FeOOH/PVDF increased by 89-fold,220-fold,and 127-fold,under 400 W ultrasonic condition,respectively.Furthermore,the results of immunofluorescence staining confirmed that under 400 W ultrasonic condition,FeOOH/PVDF fibrous film can promote the expression of Nestin,MAP2,and Tuj1 protein.Therefore,the abovementioned results confirmed that under 400 W ultrasonic condition,the FeOOH/PVDF fibrous film can effectively promote the neural-like differentiation of rBMSCs.?4?Therefore,these results revealed that the piezoelectric materials can locally generate electrical signal stimulations and under the action of ultrasonic wave,which can be transmitted to the inside of the cell through the direct contact between the material and the stem cells.Thereby,the special signal stimulations promoted the expression of related genes,which in turn induced the neuronal differentiation of rBMSCs.Moreover,the unique nanostructure of FeOOH/PVDF fibrous film and the positive effect of Fe³⁺on brain-derived neurotrophic factor?BDNF?synthesis also accelerated the process of neural differentiation of stem cells.This study confirmed the influence of nanotopography on the regualtion of stem cells fate on the basis of piezoelectricity,thus providing a model structure for neural tissue engineering,which has important significance for nerve repair and regeneration.This paper aims to solve the problems encountered in the repair and reconstruction of tissues and organs.Using the properties of the extracellular nano/microenvironment constructed by the theoretical knowledge of tissue engineering and micro/nanomaterials to regulate the fate of stem cells,which provides novel ideas or theoretical methods for the field of clinical medicine.