Properties Of Copolymerized Nanocomposite Hydrogels And The Application In Cell Sheet Detachment

In the present work, nanocomposite hydrogels (NC gel) with excellent mechanicalproperties were synthesized by in situ copolymerization of functional monomers in theaqueous suspension of hectorite clay Laponite. Laponite platelets worked as multifunctionalcross-linkers in the NC gels. The cell proliferation and spontaneous cell sheet detachmentfrom the copolymerized NC gel surface were investigated. First, two kinds of poly(ethyleneglycol) macromonomer with different end groups were individually introduced into thepoly(N-isopropylacrylamide)(PNIPAm) NC gels by copolymerization. The properties of theNC gels were discussed by considering the difference in structure of the macromonomers.Second, the cell culture and cell sheet detachment from the NC gels with the introducing ofhydrophilic or ionic monomers were studied. Third, we prepared NC gels with hydrophobicand hydrophilic polymer chains in the gel network. The main contents and results are asfollows:1. PEGM/NIPAm and PEG/NIPAm copolymerized NC gels were synthesized through insitu copolymerization of PEGM and PEG with NIPAm in the aqueous suspension of hectoriteclay Laponite. Addition of PEG or PEGM macromonomer was found to reduce the viscosityof Laponite suspension, which indicated the adsorption of PEG or PEGM macromonomer onLaponite platelets. The swelling behavior of PEGM/NIPAm and PEG/NIPAm copolymerizedNC gels in deionized water and sodium chloride aqueous solution were investigated. Themain reason for the NC gels performing swelling or deswelling behavior was the change ofosmotic pressure. A small amount of hydrophilic monomers in the NC gels has no adverseeffect on the phase transition behavior. The NC gels can deswell quickly when immersing inwater at50oC. The tensile strength and elongation at break of PEGM/NIPAm copolymerizedNC gels were reduced with the increasing of PEGM content. Increasing PEGM content up to5mol%, the gel became soft and the elongation at break was improved. However, the additionof PEG content induced the decrease of tensile strength and elongation at break for thePEG/NIPAm copolymerized gels. It was hard to clamp the gel with2mol%of PEG, because the gel became fragile. The effective network chain density of PEGM/NIPAm copolymerizedNC gels determined from equilibrium shear modulus Gereduced with the increase of PEGMcontent, while the effective network chain density of PEG/NIPAm copolymerized NC gelsincreased with the addition of PEG content. The end group of PEG monomer can participatein hydrogen bond formation with PNIPAm chains, which enhanced the effective crosslinkingchain. Because the crosslinking network contributed by hydrogen bond was easy to destroy,the tensile strength of PEG/NIPAm copolymerized gels was still lower than PNIPAm NC gels.2. The thermoresponsive NC gels copolymerized by hydrophilic PEG macromonomerand NIPAm were used for promoting cell sheet detachment. Increasing the PEG content led toa small increase in the phase transition temperature of the copolymerized NC gels, due to theenhancement of gel hydrophilicity. However, the phase transition temperature of thecopolymerized NC gels was still below37oC, required for cell culture. The L929celladhesion and proliferation on the surface of these NC gels were not suppressed by theincorporation of hydrophilic PEG side chains. By lowering temperature below the phasetransition temperature, the cell sheet spontaneously detached from the copolymerized NC gelsurface. The time required for a complete cell sheet detachment was~13min for thecopolymerized NC gel with0.5mol%PEG, much shorter than that for PNIPAm NC gel (~23min). The surface morphology and surface wettability of the NC gels were detected by atomforce microscope and contact angle measurement. A rough and hydrophilic surface inducedby a small amount of PEG side chains was found to be favorable to accelerate the cell sheetdetachment, probably due to the enhanced water permeation into the gel-cell sheet interface.3. A series of ionic NC gels were synthesized by in situ copolymerization of twoseparated positively or negatively charged monomers with NIPAm in the suspension ofLaponite aqueous solution. Copolymerized NC gels containing positively charged monomerDMAEMA show almost unchanged phase transition temperature with the increase ofDMAEMA content. The phase transition temperature of the anionic NC gels increased withthe AMPSNa content, but still kept below37oC in the DMEM medium. The ionic NC gelswere favorable for cell culture. As a result of investigations on the culturing of L929cells on the surface of cationic NCgels, it was concluded that the cell proliferation was promoted on the gels containing less than5mol%of DMAEMA, and the cells could not attach on the gel surface with more than7mol%of DMAEMA. By decreasing temperature, accelerated cell sheet detachment wasaccomplished on the gel with1mol%of DMAEMA. However, an intact cell sheet could notbe harvested from the gel surface with more than1mol%of DMAEMA, because theelectrostatic attraction between the cell sheet and gel surface was predominant as compared tothe cytoskeletal tensile forces.As for anionic NC gels, the cell proliferation was slower because of the electrostaticrepulsion between cell and gel surface. The cells could proliferate to form a confluentmonolayer on these gel surfaces. Rapid cell sheet detachment was achieved from the gelsurface with1mol%of AMPANa. The incorporation of a small amount of hydrophilicmonomer plays an important role in promoting cell sheet detachment. The cell sheetdetachment was slowed down from the gels with a large amount of AMPSNa. The hydrophilicsulfonate group in polymer chains led to a fast swelling of NC gels, which hindered theprocess of cell sheet contraction from the edge to central. By adjusting the content of addedionic monomer, we can easily control the cell proliferation and cell sheet detachment, whichindicates the promising application of high mechanical NC gels in cell sheet engineering.4. AAm/MEA/Laponite XLS copolymerized NC gels were synthesized via in situcopolymerization in the aqueous Laponite suspension. Copolymerized NC gels showedexcellent mechanical properties, and the tensile strength was larger than70kPa with theelongation at break larger than1000%. Comparing with PAAm NC gels, the storage modulusG’ and effective network chain density of the copolymerized NC gels were increased. Thecopolymerized NC gels were composed of hydrophobic PMEA and hydrophilic PAAm chains,which broaden the preparation of NC gels.