Osteogenic Differentiation Facilitation Effect Of Iron Oxide Nanoparticles In Mesenchymal Stem Cells

Regeneration and functional reconstruction of injured tissues and organs is one of the major issues in biomedical research,meanwhile regenerative medicine and tissue engineering attracts much concerns with the progress in stem cells sciences and biomaterials technologies.Tissue engineering can promote the regeneration and repair of the defect site by introducing the seed cells into the damaged parts under the guiding and supporting of scaffold biomaterials.MSCs is a kind of pluripotent stem cell with multiple differentiation potential,which can be differentiated into many stromal cells under different induce conditions,such as osteoblasts,adiocytes,myocytes,et al.MSCs exist in multiple tissues and play an important role during the tissue damage repair process.Among them,bone marrow derived MSCs is an important source of osteoblasts during bone reconstruction process.Recently,many studies have reported that external magnetic field can facilitate the repair of bone injury,and the research progress about magnetic bone scaffold materials developed rapidly.However,whether magnetic materials can affect or how they influence MSCs differentiation ability are poorly understood.In this dissertation,we employed the clinical approved IONPs developed by our research group to study the assoicated nanobiological effects to human bone marrow derived MSCs and the related mechanisms,which is expect to provide guidance for the further study of IONPs in the treatment of bone tissue regeneration.In detail,main work in the dissertation including these points:(1)Cytotoxicity study of IONPs in MSCsIONPs was gradually uptaken by cells within the co-incubation of 72 hours,and mainly distributed in the endosomes or lysosomes.The uptake of IONPs was both time-and dosedependent after quantitative analysis.For acute cytotoxicity,CCK-8 assay was performed to determine the cell viability after 24 hours co-incubation,cell viability decreased by 5.69%,7.45% and 8.65% respectively under the concentration of 100,200 and 400 ?g/m L.For long-term cytotoxicity,the LDH leakage increased only by 2.87%,and the apoptotic rates were below 5% after co-incubation of 21 days.(2)Genomics study of IONPs interaction with MSCsThe variation of gene expression in IONPs-treated cells was analyzed by employing gene microarray tests.After blasting to the known database,2092 up-regulated coding genes and 1631 down-regulated coding genes were found,which was the bases to study the complex interaction mechnisms between IONPs and MSCs.After bioinformatics analysis,the influence rules of gene functional annotations and KEGG signaling transduction pathways were obtained,which showed IONPs might promote osteogenic differentiation of MSCs.(3)IONPs facilitate osteogenic differentiation of MSCsIONPs with different concentration were co-incubate with MSCs.The results showed 100 ?g/m L IONPs can improve the cellular ALP activity after 7 to 14 days,and the extracellular matrix calcification nodules can stained red after 21 days.SEM detection was further performed to observe the morphological variation,and western blot to detect the differentiation marker,which confirmed the osteogenic differentiation facilitation effect of IONPs.Moreover,by contrast with the ferric ion and coating material PSC,this facilitation effect was owing to the whole particle.MAPK signaling pathway is enriched in KEGG pathways analysis,which suggest this pathway might be a potential key factor in regulating IONPs-facilitated osteogenic differentiation.After detection of phosphorylated kinases,results suggested that this pathway was activated during IONPs treatment,and then subsequently promote osteogenic genes transcription.(4)Participation of lnc RNAs in IONPs-facilitated osteogenic differentiationLnc RNAs microarray was performed to analyze the variation of lnc RNAs expression in IONPs-treated cells,and 411 up-regulated lnc RNAs and 605 down-regulated lnc RNAs were identified.To obtain more comprehensive information about lnc RNAs regulation in IONPspromoted osteogenic differentiation for MSCs,the coding-noncoding co-expression network was constructed.After functional annotation of co-expressed coding genes,it could be concluded that lnc RNAs would influence RTKs/MAPK pathway,BMPs/Smads pathway,magnetic response and cytoskeleton rearrangement to regulate osteogenic differentiation via their co-expressed coding genes.(5)Mechanism of INZEB2 in regulating IONPs-facilitated osteogenic differentiationAmong the differentially expressed lnc RNAs,LOC105373660 was up-regulated during IONP exposure.Using the sequence and genomic location for BLAST searches,the putative transcript is transcribed from the partial opposite strand of ZEB2's intron 2,therefore,it was named for INZEB2(intronic ZEB2).Meanwhile,knockdown of INZEB2 inhibited the osteogenesis phenotype of IONPs treated MSCs,and caused a significant increase of ZEB2 protein level which was reduced by IONPs treatment.Furthermore,we demonstrated that INZEB2 interacted with the intron 2 of ZEB2 pre-m RNA which leading to the splicing of its complementary sequence within this intron.As a result,a premature translation termination occurred and viable ZEB2 protein level was reduced.Therefore,TGF-?/BMPs/Smadsmediated osteogenic differentiation was reactivated.Taken together,these results illustrated INZEB2 responds to IONPs and modulates ZEB2 expression by influencing its pre-m RNA splicing,which results in the facilitation of osteognenic differentiation in MSCs.(6)The role of magnetism in IONPs-facilitated osteogenic differentiationHelmholtz coils were used to produce uniform adjustable electromagnetic field for cell magnetic stimulus,it was found that weak static magnetic fields(SMFs)could enhance the osteogenic differentiation facilitation effect of IONPs.Particles with different magnetization were incubated with cells,which suggested that the facilitation effect of IONPs on osteogenic differentiation was dependent on its own magnetism.Interestingly,this facilitation effect of both IONPs and SMFs were all dependent on the expression of magnetoreceptor(Mag R)protein complex.Furthermore,proteomics analysis suggested proteins associated with focal adhesion and extracellular matrix(ECM)-receptor interaction were changed during SMFs/IONPs-mediated osteogenesis.As a result,cellular elasticity was increased with actin stress fiber altered from relax to tension status,downstream signal events were simultaneously activated to promote osteogenic differentiation.