Effects Of Matrix Elasticity And Cell Density On Human Mesenchymal Stem Cells Osteo/Chondro Differentiation And Mechanism Study

Bone marrow-derived mesenchymal stem cells (BMSCs) are an importantmultipotent adult stem cells with the potential to differentiate into a variety of cell types,including neural cells, vascular smooth muscle cells (SMCs), osteoblasts, chondrocytesand adipocytes. Therefore, MSCs are an important cell source for tissue repair andregeneration. Currently, rehabilitation study shows that MSCs are valuable inmyocardial damage repair and regeneration; they also can directionally differentiate intonerve cells in spinal injury regeneration and new bone production after bone injury.However, it is not yet well understood how the directional differentiation of MSCsrespond to the physical characteristics of biomaterial and microenvironment aroundcells, especially matrix elasticity.The differentiation of MSCs is especially important in tissue repair andregeneration. There is evidence that the tissue microenvironment includes not only thewidely studied biochemical cues, but also the biophysical factors that deliver signals forMSC differentiation. It is not clear whether and how matrix elasticity and cell seedingdensity collectively regulate MSCs. In this study, we investigated the collective effectsof substrate elasticity and cell seeding densities on ostogenesis and chondrogenesis. Inaddition, we studied the underlying mechanism during osteogenesis induced by matrixelasticity. The main experiments and results are as follows:1. Morphology and proliferation of MSCs on different matrix elasticity with differentcell densitiesTo investigate the role of matrix elasticity in the proliferation and differentiation ofMSCs, we utilize tunable elasticity polyacrylamide hydrogels which can be manipulatedby adjusting the relative concentrations of acrylamide and bis-acrylamide. The physicalelasticities can be controlled between0.5kPa and100kPa. We cultured MSCs on softmatrix(1.6±0.3kPa for5%/0.05%acryl/bis) and hard matrix(40±3.6kPa for10%/0.5%acryl/bis), following by the observation of cell morphology and spreading area. Theresults showed that cells cultured on soft matrix have restricted spreading area, which isonly approximately40%of that on hard matrix24hours after culture. To determine theeffects of cell density and matrix elasticity on hMSC proliferation, the cell cycledistribution was determined. We also detected the cell cycle positive and negativeregulatory factor Phospho-Rb, P-P70S6kinase and P-27. The result showed that soft matrix with low cell density inhibited MSCs’ proliferation, and obviously decreased theexpression of P-Rb, P-P70and up-regulated p-27. However, both soft and hard matricescaused significant decreases cell percentage in the synthetic phase in high-density.These results suggest that the effect of matrix elasticity on hMSCs proliferation dependson cell density and that high cell density was able to override theproliferation-promotion effect of hard matrix.2. Effects of matrix elasticity and cell density on osteogenesis and chondrogenesisTo determine the role of matrix elasticity on MSCs differentiation, we culturedMSCs on soft and hard hydrogel in growth medium. The result showed that hard matrixincreased the expression of osteogenic marker genes including Alkaline Phosphatase(ALP)，Type I collagen, alpha1(Col1α1) and runt-related transcription factor2(Runx2).This demonstrated that hard matrix promote osteogenesis of MSCs, in both inductionand growth medium. In addition, we found that chondrogenesis was promoted on softmatrix(1.6±0.3kPa). Western blotting confirmed that SOX-9was significantly higher onsoft matrix. Alcian blue staining and collagen II immunostaining also confirmed theresult, the mRNA expressions of SOX-9, ACAN, Collagen II and Collagen X weresignificantly higher on soft matrix. There are studies indicating that cell densityinfluence MSC differentiation, so we detected the MSCs differentiation with diversecell density on soft and hard hydrogels. At high cell density (20,000/cm2), the cellsshowed no significant difference in osteogenic marker expressions between hard andsoft matrices; matrix elasticity affects chondrogenic process at both high and low cellseeding density (1,000/cm2). This finding illustrated that low cell density and hardmatrix are benefit for osteogenesis, however, matrix rigidity affects chondrogenicprocess at both high and low cell seeding density.3. The role of RhoA/ROCK and Paclitaxel in MSCs proliferation and differentiationon matrixTo observe the MSCs morphology, proliferation and marker genes expressionvariation, we utilized RhoAV14(constitutively active form of RhoA with GST tag), Rhokinase inhibitor Y-27632and microtubule inhibitor Paclitaxel. RhoAV14transfectionup regulated ERK phosphorylation, MSCs proliferation and osteogenic marker genesexpression on soft matrix. Y-27632inhibited osteogenic marker genes expression andup regulated neurocyte marker gene β3tubulin expression, F-actin fluorescenceintensity from averaged at each fractional distance across the cell to a preferential distribution in the peripheral cortical layer. The MSCs’ proliferation difference wasdisappeared and the cell viability test showed that cell apoptosis on soft matrix wasmore than those on hard matrix with Paclitaxel treatment. Runx2mRNA expression wassignificantly down regulated on soft matrix, not on hard matrix. There were nosignificantly difference of TUBβ3mRNA expression between control and Paclitaxeltreatment. Based on above, the cell differentiation process which regulated by substrateelasticity exist cytoskeleton like microfilament, microtubule, RhoA and ROCKparticipation.4. Hard matrix induced Smad1/5/8, ERK, and AKT activation in osteogenesisTo further investigate the matrix elasticity influence on MSCs differentiation, wefound that the phosphorylation of Smad1/5/8, ERK and AKT were up regulated, whileP-38was down regulated on hard matrix, in contrast, our results showed that ERK andAKT activations were not affected by matrix elasticity during chondrogenesis,suggesting that the phosphorylation of Smad1/5/8、ERK and AKT may play importantrole in osteogenesis induced by matrix elasticity, other signal networks may be involvedin chondrogenesis. To further test the roles of ERK phosphorylation in matrix-regulatedosteogenesis, recombinant adenoviruses with constitutive active Ras (RasV12) anddominant negative Ras (RasN17) were used to modulate the signaling cascades.RasN17markedly inhibited the phosphorylation of Smad1/5/8, ERK, and AKT, RasV12up-regulated ERK activation, but had little effect on Smad1/5/8and AKTphosphorylations. The expressions of osteogenic markers, such as Runx2, Col1α1andALP mRNA, were significantly down-regulated after RasN17infection. However,RasV12did not cause any up-regulation of osteogenic markers. These results suggestthat there should be other signals besides Ras-Smad1/5/8/ERK/AKT involved inosteogenesis which induced by matrix elasticity.In summary, this study used hydrogel model which physical elasticity can becontrolled to culture the MSCs, to investigate the MSCs osteogenesis andchondrogenesis on soft and hard hydrogel, as well as the underlying signal transductionin osteogenesis. We found that cell cytoskeleton, RhoA-ROCK paticipate in MSCsdifferentiation and the phosphorylation of Smad1/5/8、ERK and AKT are involved inosteogenesis which determined by matrix elasticity. Our results demonstrated that thecollective effects of matrix elasticity and cell seeding density on MSCs differentiationare lineage dependent. Elucidation of the roles of lineage-specific extracellular cues will improve the control of stem cell fate to generate the desired cell types for potentialtherapeutic usage.