Interactions Of Composition And Surface Morphology Of Chitosan With MSCs

Objective:Biomaterials endowed with the function of inducing tissue regeneration have been a hot topic.In this study,chitosan(CS) was first modified by phosphorylation and by addition of calcium phosphate cement to modulate nucleation and crystallization.The products were used to study effects of biomaterials on the proliferation and differentiation of mesenchymal stem cells(MSCs).Secondly CS membranes with different physical morphology were fabricated to study effects of biomaterials on attachment and proliferation of MSCs.They provide experimental basis for developing novel biodegradable materials which have good biocompatibility and osteo-inductivity in guiding bone tissue regeneration.Methods:(1) MSCs were isolated and purified by differential adherence and their biological characteristics were observed.Cells were identified by surface molecules and the ability to differentiate into osteoblasts or adipoctyes.(2) Laser scanning confocal microscope(LSCM) and atomic force microscopy(AFM) were employed to observe changes of MSCs due to osteogenic differentiation.(3) CS was mixed with home-made N-methylene phosphonic chitosan(NMPC) to form CS/NMPC membrane and its performance was then characterized.MSCs were cultured on the CS/NMPC membrane to evaluate its biocompatibility.The osteogenic induction of materials were detected by fluorescence quantitative real-time PCR (FQ-PCR).(4) Tetracalcium phosphate(TTCP) made by co-precipitation and dicalicium phosphate anhydrous(DCPA) made by dehydration were mixed with CS to form CS/hydroxyapatite(CS/HA) membrane and its performance was then detected.To evaluate the biocompatibility and osteogenic induction of CS/HA,MSCs was cultured on the materials for detection of the activity of alkaline phosphatase(ALPase) and osteogenic mRNA.(5) Polystyrene(PS) membranes with pits of different sizes were presented by liquid phase separation and then convex CS membranes were prepared with the PS membranes as templates.The attachment and proliferation of the MSCs on the surface of CS with different microstructures were evaluated.Results:(1) MSCs cultured in vitro demonstrated uniform appearance under the inverted microscope.The morphous of cells was fibroblast shape and swirled when they assembled together.By flow cytometer,surface antigens was positive for CD29, CD44 and negative for CD45.MSCs isolated could be successfully induced into osteoblasts and adipocytes.(2) After osteo-induction,the distribution of Ca²⁺ was changed from uniform to punctiform and massive.The microtubules showed rarefaction and derangement.The morphology of MSCs changed from fusiform to irregular form.The filipodium of cells decreased and nucleus increased after induction.Membrane of cells became rough just like deposited mineralizer.(3) When NMPC was mixed with chitsan,CS/NMPC membrane was formed.The phenotype of membrane showed that the water contact angle decreased from CS's 77±2°to CS/NMPC's 20±10°and the water absorption increased from CS's 128±14%to CS/NMPC's 248±24.6%.The tensile strength and breaking elongation decreased but elastic modulus increased slightly.When immersed into culture medium, the materials could absorb Ca²⁺.Cell culture assay showed that proliferation of MSCs on CS/NMPC was higher than that on pure CS 5,7 and 9 days after inoculation.There were significant differences between the two groups(P＜0.05).After 14 days' growth in basic culture medium on materials,the expression of osteocalcin(OC) and osteopontin(OPN) of MSCs on CS/NMPC was determined as 275.1%and 736.2% respectively of that on CS by FQ-PCR.This indicated that NMPC played an important role in MSCs osteogenic differentiation.(4) Home-made TTCP and DCPA were added into CS solution to form CS/HA membrane.Water contact angle decreased from CS's 77±2°to CS/HA's 57±5°and water absorption increased from CS's 128±14%to CS/HA's 185±15%.The tensile strength,breaking elongation and elastic modulus decreased compared with CS.CS/HA absorbed Ca²⁺ quickly and low crystalline hydroxyapatite deposited on the surface of the membranes.MTT showed that proliferation of stem cells on CS/HA was higher than that on CS 3,5 and 9 days after inoculation.There were significant differences between the two groups after 3 and 5 days of culture(P＜0.05) and extremely significant differences after 9days(P＜0.01).ALPase activity value of MSCs on CS/HA was 6.3 times of that on CS detected by ALPase kit.The expression mRNA levels ofâ… -collagen(Colâ… ),ALPase and OPN of MSCs on CS/HA were determined as 479.0%,377.1%and 597.9%respectively of that on CS by FQ-PCR. These results indicated that hydroxyapatite on CS could induce MSCs to differentiate into osteoblasts,(5) The PS membranes with different morphologies and microstructures were obtained and the correspondingly scaled convex CS films were then prepared based on the template technology.MSCs cultured on the surface of CS with different microstructures showed no significant difference in cell's attachment on the first day. But the proliferation of MSCs was highest on CS 10-50 followed by CS10 and lowest on the surface with smooth CS and CS0.8-1 by MTT.Conclusion:Chemical composition and micro-structure of biomaterials can affect the attachment,growth and differentiation of MSCs.When materials with phosphate radical just as organic NMPC and inorganic HA are added to CS,it not only increases the biocompatibility but also accelerates the differentiation of MSCs into osteoblast.It is a good tissue engineering materials that have favorable osteo-inductivity.The microstructure of CS membrane surface affects the distribution of MSCs and results in increased proliferation of MSCs.