| Background MSCs possess multiple differentiation potentiality. As an important stem cell category, MSCs are capable of differentiate to bone, cartilage, muscle, nerve, and fat cells. The multiple differentiation potentiality makes MSCs ideal seed cell for tissue engineering. Latest studies show MSCs plays important role in the tissue recovery and regeneration engineering of a lot tissues, for example bone trauma, spine injury. However, the relationship between MSCs and scaffold material is still not fully understand, especially how the stiffness of the contact interface regulate cell behaviors like oriented differentiation. In addition to biochemical factors, physical factors like mechanic transduction also are indispensable for regulating MSCs differentiation. Mimicking the cells’ nature physical microenvironment in order to accelerate cell recovery is a hot point of tissue engineering.Objective The mechanical properties of cells’ microenvironment can regulate cells behaviors including spreading, migration, differentiation etc. The dissertation focused on the effect of interface stiffness on the MSCs bone oriented differentiation. We chose the surface chemical modified PA hydrogel to mimic tissue interface with different stiffness, observing the MSCs growing and bone oriented differentiation by testing the expressing of bone differentiation indicator and key protein in the differentiation related signal pass way. We aimed to verify that the tissue interface stiffness plays an important role on the growing and differentiation of MSCs, in terms of biomolecule biology, hoping to provide a basis for further investigating of mechanism by which stiffness regulate stem cells.Methods:1. BMSCs culturing:under aseptic condition: Take the 4 weeks SD rats hind limbs long bone, and rinse the bone marrow cavity with 10%FBS、DF-12 culture medium, purify the cell using the whole bone marrow adherent cells developing joint differential digestion method.Select P1-P4 generation BMSCs for morphologically observating under dyeing.Tablet cloning experiments was performed to observe the P1, P2, P4 BMSCs self-renewal ability. Respectively by identifying the BMSCs by molecular markers, including FITC-anti-Mouse/Rat CD 44、PE--anti-Mouse/Rat CD45、APC--anti-Mouse/Rat CD90 using immunofluorescence technique, flow cytometry.2. Surface modifying of PA hydrogel: fabricate hydrogel with different stiffness by adjusting ratio of Acrylamide、Bis-Acrylamide. Gel was attached to the chemical modified glass surface. Then Collagen Ⅰ、Fibronectin were modified to the gel surface. Finally, the gel was sterilized by UV light.3. Observation of cell behavior on the surface PA gel of different stiffness: P2, P3 generation cells were chosen to be planted on different PA gel surface at planting density of 1000 a/1000μL, and cultured by complete medium with 10% FBS, DF-12. PA gel were divided into 6 groups by stiffness:group A (4 kpa) and group B (10 kpa), 40 (kpa) group C and group D (80 kpa), group E (1 kpa) and petri dish (stiffness) for control group. Cell morphology, cell spreading area were observed under inverted microscope in different time points, BMSCs growth curve was documented. Immunofluorescence observations P3 generation of BMSCs. We also observed the expression of cytoskeleton, osteopontin and osteocalcin by immunofluorescence, cell proliferation by CCK8.4. Observation of on cells osteogenesis behavior on the PA gels with different stiffness: group A (4 kpa), group B (10 kpa), group C (40 kpa), and medium were chosen for culturing P2 generation BMSCs. on the interface BMSCs differentiation ability was observed by Alizarin red staining method, whichcan detect cells osteogenesis calcium deposits; The kits of alkaline phosphatase (ALP was used to test ALP levels. ELISA was performed to detect BGP (osteocalcin), PICP. Fluorescence quantitative PCR (FQ - PCR) was performed to detect BGP, Runx2, Coll alpha 1 mRNA level in different stiffness cultivation system. Western-blot was performed to detect F-actinand E-cadherin.Results:1. The cell colony of BMSCs increased training after 48 h culturing of PI-P4 generation cells, the overall cell density increased exponentially. After 72 h cells present typical spindle appearance, cell fusion occurred, and radially or spirally growing were oberved. Growth curve presents the s-shaped, consistent with Logistic growth curve. All the in vitro cultured BMSCs have stagnation stage, rapid growth stage, a plateau stage. P1, P2, P4 BMSCs clone formation were 10.4%,9.7% and 8.2%. P2 generation of BMSCs fluorescence immunoassay detection showed that CD90, CD44 positive expression, CD45 were negative. Flow cytometry display:CD45-PE, CD44 - FITC, CD90 - APC expression rate of 1.65%,94.1% and 95.8% respectively. Conform to the typical biological characteristics of mesenchymal stem cells, cultivating system of cells proliferation showed good growth momentum.2. BMSCs growth curve of P2 generation showed:at cell density of 1000/100μL conditions, the low stiffness of PA (1 kpa), inhibit the proliferation of BMSCs compare topetri dish control group, while high stiffness of PA (40,80 kpa) enhanced proliferation of BMSCs; at density of 5 x 106/ml condition, BMSCs growing on B (10 kpa) and C (40 kpa) is significantly faster than (4 kpa) group.Poison test shows that within certain stiffness range, with the increase of interface stiffness, the proliferation of BMSCs is prompted, cell morphology observation results show that: with the stiffness of ascension, cell spreading area increase, typically, cells spreading area the low stiffness interface (1 kpa) is only about 38.8% of high stiffness on the interface (80 kpa). in different cultivation system, cell cytoskeleton of BMSCs always arranged along with the typical longitudinal direction. Expression of osteopontin and osteocalcin shows BMSCs has good osteogenetic differentiation ability in the gel culturing system.3.Pro vide stiffness PA adhesive interface, at cell density of 1000 A/100 mu L conditions, BMSCs have osteogenesis differentiation trend in all groups, includinggroup A (4 kpa) and group B (10 kpa), group C (40kpa). WB experiments shows with stiffness increase, calcium mucin E - cadherin expression increased, whileexpression of actin (actin) is not varied very much. This result indicate reduced expression of calcium protein, which is related tocell spreading and migration,is related to high rigidity can promote cell adhesion. At low density cultivation, Marker gene ALP, Col1 alpha 1 and Runx2 mRNA level increased with stiffness, which is similar to the ELISA test result,indicating larger stiffness value can promote the bone oriented differentiation of BMSCs.Conclusion:1. Bone marrow cell adherent culture combined differential digestion methodcan harvesthigh purity, healthy, rapid growing BMSCs from SD rat.2. Hydrogel with different stiffness, ranging from 0.1 kPa～80 kPa can be fabricated by adjusting the ratio of Acrylamide and Bis-acrylamide. After surface modifying with Collagen Ⅰ、Fibronectin PA hydrogel can provide a good extracellular matrix for MSCs to adhere and spread.3. The spreading area of BMSCs increase with interface stiffness. the spreading area of bmscs on low stiffness interface is about 38.8% of high stiffness interface. BMSCs’ growing in inhibited in the soft gel (1 kpa) compare to petri dish. High stiffness gel on the other hand enhanced BMSCs’ growing in certain degree. Cells behavior varies according to the interface stiffness greatly.4. Physical facors can induce BMSCs differentiate to osteoblast independently in some degree. In the range from 4-80kpa, increasing of stiffness has positive effect on the bone oriented differentiation of BMSCs. Stiffness may regulate BMSCs by take part in the WNT/β-Cateni、MAPKs signal pass way. |
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