Regulation Of Stem Cell Fate By Inorganic Nanomaterials Constructed Physical/Chemical Microenvironments

Because of the excellent self-renewal ability,the potential to differentiate into multiple cell types,and easy to be extracted from adults,stem cells have been widely used as seed cells in stem cell therapy,tissue engineering,and regenerative medicine.However,how to realize the regulation of the fate of stem cells,especially the regulation of the stem cell differentiation into a specific type of cells(for example,osteoblast)for repair and regeneration of defective tissues,is a difficulty in related research and has been attracting growing interests in these areas.In the human body,each type of cells are located in a three-dimensional dynamic microenvironment,which is composed of the extracellular matrix(ECM),cell-to-cell communication,soluble growth factors,and mechanical stimuli.The specific physical,chemical,and biological signals in the microenvironment directly influence and even determine a variety of cell behaviors and functions such as cell attachment,migration,self-renewal,and differentiation.Based on this fact,design of artificial microenvironment to mimic the physical,chemical,and biological factors in natural extracellular microenvironment for regulation of stem cell fate has been a common strategy in the field of implant research,tissue engineering,and regenerative medicine.Most recently,nanomaterials have been widely used to construct the specific physical/chemical microenvironments in vitro to mimic the natural extracellular microenvironment.Using nanomaterials to construct bio-mimic physical/chemical microenvironments for regulation of stem cell fate has several advantages.Firstly,cells in the human body are naturally located in a microenvironment having a diversity of nanoscale features.The physical/chemical signals provided by these nanoscale features have an essential influence on the cell behaviors and functions.For example,collagen nanofibers,a mainly composition in the ECM,not only provide multiple adhesion sites to cells but also influence their migration and even differentiation.Secondly,the subunits and compositions of cells,such as filopodia and transmembrane proteins,have the ability to sense the physical/chemical signals from nanostructures and transfer them to the cells through specific signal pathways.Thirdly,the physical/chemical microenvironments constructed by nanomaterials,especially by inorganic nanomaterials,have better stability than biological growth factors.Thus,it's possible to realize the long-term regulation of stem cell fate by nanomaterials constructed physical/chemical microenvironments.Fourthly,nanomaterials generally perform their effect on regulation of stem cell fate through a contact-interaction mode,while have negligible influence on the non-contact cells.The contact-interaction mode provides a beneficial to achieve the location-committed regulation of stem cell fate.Fifthly,various morphologies and structures constructed by nanomaterials,especially by inorganic nanomaterials,dramatically enrich the diversity of the bio-mimic extracellular microenvironments,which would provide more strategies for regulation of stem cells fate.Based on the above facts,nanomaterials constructed microenvironments featured with nano-topography,mechanical strength and chemical composition,have shown significant advantages and success in regulation of the fate of stem cells in the past decade.However,most of the relevant researches are still focused on construction of nano-featured microenvironments on two-dimensional planar models.Although the performances are good and sometimes are better than conventional growth factors,difficult to translate these two-dimensional models into complex three-dimensional models limits their use in clinical applications.In addition,in the field of implants research and tissue engineering,it is often necessary to construct spatially variable physical/chemical microenvironments to achieve location-committed differentiation of stem cells.For example,when implant material contacts different types of tissues in vivo,the implant should have the ability to induce stem cells to specifically differentiate into corresponding cells at the contact areas to achieve better biological binding to surrounding tissues.Although the contact-interaction mode between nanomaterials and stem cells provides an excellent platform for achieving the spatially controlled differentiation of stem cells,there are few reports about it.On the other hand,as more and more nanoparticles and nanotechnologies are used in the biomedical area,researchers start to realize that the dispersed nanoparticles in cell microenvironment could be endocytosed by stem cells,which will finally lead to the changes of cell behaviors and functions.However,how the endocytosis interaction between nanoparticles and stem cell commits its influence on the stem cell fate remains unclear.Thus,understanding the mechanism and trying to make it usable in the biomedical applications still face a lot of challenges.Thus,in this work we aim to construct physical/chemical microenvironments by inorganic nanomaterials with the ability to be used in three-dimensional implants for regulation of stem fate.Meanwhile,we try to construct a spatially variable physical/chemical microenvironments to achieve the spatially regulation of stem cell fate by means of contact interaction between nanomaterials and stem cells.On the other hand,we also attempt to study the regulation of stem cell fate by endocytosis interaction between nanoparticles and stem cells and try to understand the related underlying regulation mechanism.The main contents of this study are as follows:(1)TiO2 nanorods array constructed nanotopographic microenvironment for the realization of spatially controlled osteogenic differentiation of stem cellsA layer of rutile TiO2 nanorods array was vertically grown on the surface of a variety of substrates such as FTO conductive glass,silicon wafer,titanium plate,titanium dioxide ceramic,and even three-dimensional porous titanium foam by a hydrothermal method,where a typical titanium dioxide nanorod had a length of about 1.5 ?m and a diameter of about 100 nm with a density of 36 per ?m2.Cell proliferation experiment show that the titanium dioxide nanorod array had good biocompatibility.Cytoskeleton staining and scanning electron microscopy results demonstrate that the nano-topographic microenvironment constructed by TiO2 nanorods array promoted rat bone marrow-derived mesenchymal stem cells(rBMSCs)to grow more filopodium and enhanced their adhesion ability.The real-time quantitative polymerase chain reaction(q-PCR)results show that the expression level of osteogenic related genes,Runx-2,osteopontin(OPN),and osteocalcin(OCN),of rBMSCs on TiO2 nanorods array were up-regulated by 4.7,2.2,and 3.7 folds,respectively,compared to cells on TiO2 ceramic with a smooth surface.Alkaline phosphatase(ALP)kit and immunofluorescence staining results show that rBMSCs on TiO2 nanorods array had a higher expression level of alkaline phosphatase(ALP),OPN,and OCN protein,compared to cells on TiO2 ceramic.Alizarin red staining results confirm that TiO2 nanorods array promoted the calcium deposition ability of rBMSCs compared to TiO2 ceramic.The above results demonstrate that the nano-topographic microenvironment constructed by TiO2 nanorods array promoted the osteogenic differentiation of rBMSCs.In addition,a patterned TiO2 nanorods array was prepared on a TiO2 ceramics substrate by a micro/nanofabrication technology combined with hydrothermal reaction,which realized the spatially controlled differentiation of rBMSCs based on the spatially varied surface nano-topographic microenvironment.These work provides a new strategy for the surface modification of implants to improve the biological interaction between implants with mutil-tissues in clinical applciations.(2)Gradient hydroxyapatite nanoparticles constructed physical/chemical microenvironment for spatially controlled osteogenic differentiation of stem cellUnder heating at 80 ?,the suspension of hydroxyapatite(HAp)nanoparticles was added in the cubic close packed(ccp)array composed of 200 ?m-gelatin beads,and the HAp nanoparticles would diffuse into the void of the ccp array driven by the gravitational force.Meanwhile,the gelatin beads in the ccp array became soft and sticky,and the adjacent gelatin beads would gradually be fused with each other under heating,which gradually limited the diffusion of the HAp nanoparticles,finally resulting in a top-to-down gradient distribution of HAp in the ccp array.Taking this ccp array as a template,an inverse opal scaffold,poly(lactic-glycolic acid)copolymer/HAp(PLGA/HAp),together with a gradient distribution of HAp nanoparticles could be obtained.Optical microscopy and scanning electron microscopy results indicate that the PLGA/HAp inverse opal scaffold had a uniform interconnected porous structure,where the pore size was 179.4 ± 1.7 ?m,and the window size was 53.9 ± 2.3 ?m.The uniform interconnected porous structure endows the PLGA/HAp scaffold good nutrient transport and metabolic waste transfer capabilities.Micro-CT and scanning electron microscopy confirm that HAp nanoparticles in the PLGA/HAp inverse opal scaffold had a gradient distribution.Atomic force microscopy results demonstrate that the mechanical properties of the scaffold exhibited a gradient distribution consistent with the distribution of HAp nanoparticles.Live/dead staining results show that rat adipose-derived mesenchymal stem cells(rAMSCs)were uniformly distributed inside the PLGA/HAp inverse opal scaffold with a survival rate of over 95%.Immunofluorescence staining and alizarin red staining results demonstrate that the osteogenic differentiation markers alkaline phosphatase(ALP),osteocalcin(OCN),and calcium deposition content of rAMSCs in PLGA/HAp scaffolds showed a gradient expression consistent with the distribution of HAp nanoparticles,indicating that the spatially varied physical(mechanical strength)and chemical(mineral)microenvironment constructed by gradient HAp resulted in the gradient osteogenic differentiation of rAMSCs in the PLGA/HAp scaffold.This PLGA/HAp scaffold would have great potential application in the area of interfacial tissue engineering.(3)Influence of the endocytosis interaction between graphene quantum dot and stem cells on their proliferation and differentiation and the related mechanismThe endocytosis interaction between graphene quantum dots(GQDs)and rat bone marrow-derived mesenchymal stem cells(rBMSCs)was directly observed under fluorescence microscopy taking advantages of the intrinsic fluorescent properties of GQDs.The GQDs dispersed in the microenvironment were mainly concentrated in the cell cytoplasm after endocytosis by rBMSCs.Cell activity experiments show that under a low concentrations(less than 50 ?g/mL),the endocytosis interaction between GQDs and rBMSCs had no significant negative effect on the cell activity.Q-PCR results show that the osteogenic differentiation genes,Runx-2 and OCN,of rBMSCs were up-regulated by 13.9 and 9.2-folds,respectively,when they were exposed to GQDs at a concentration of 50 ?g/mL.Alkaline phosphatase(ALP)kit and immunofluorescence staining results show that rBMSCs had a higher expression level of ALP,OPN,and OCN protein when they were incubated with GQDs at a concentration of 50 ?g/mL.Alizarin red staining results confirm that rBMSCs had a better calcium deposition ability when they were incubated with GQDs at a concentration of 50 ?g/mL.The above results demonstrate the endocytosis interaction between graphene quantum dots and rBMSCs promoted the osteogenic differentiation of rBMSCs.To make clear the underlying mechanism about the promoting effect of the endocytosis interaction of GQDs on the osteogenic differentiation of rBMSCs,the involved genes,and signaling pathways were analyzed with the help of gene microarray technology.Based on the gene microarray results,a possible underlying mechanism was proposed,namely,during the endocytosis process of GQDs by rBMSCs;the mechanical stress would be generated because of the contraction and movement of the cell skeleton actin,which would promote the osteogenic differentiation of rBMSCs through related signal pathways.In addition,oil red staining results indicate that the endocytosis interaction between GQDs and rBMSCs had no negative effect on their adipogenic differentiation potential.This work will facilicite the understanding of the endocytosis interaction between nanoparticles and stem cells,and would broad the applications of GQDs in the area of stem cell labelling,imaging,drug delivery,stem therapy,and tissue engineering.In summary,we realized the spatially controlled osteogenic differentiation of MSCs by the inorganic nanomaterials,such as TiO2 nanorods array and HAp nanoparticles,constructed spatially varying nanotopographic,mechanical,and chemical microenvironments through a contact interaction mode.This work provides a new strategy for improvement of the biological interaction between the tissues and implant,when they contact with multiple-tissues,as well as a new route for repair of the transition tissues.It would have an important application in the field of implant research and interfacial tissue engineering.Meanwhile,we also investivated endotytosis interaction between inorganic nanomaterials GQDs and MSCs,and its influence on the fate of MSCs.This work opens a new insight to understand the mechanism of the endotytosis interaction between nanoparticles and MSCs,which would facilicate the better use of nanomaterials in the biomedical area.