In this study, we synthesized and characterized a series of macromers based on poly( N-isopropylacrylamide) that undergo thermally induced physical gelation and, following chemical modification, can be chemically cross-linked. Macromers with number average molecular weights typically ranging from 2000-3500 Da were synthesized via free radical polymerization from, in addition to N-isopropylacrylamide, pentaerythritol diacrylate monostearate, a bifunctional monomer containing a long hydrophobic chain, acrylamide, a hydrophilic monomer, and hydroxyethyl acrylate, a hydrophilic monomer used to provide hydroxyl groups for further chemical modification. Results indicated that the hydrophobic-hydrophilic balance achieved by varying the relative concentrations of comonomers used during synthesis was an important parameter in controlling the transition temperature of the macromers in solution and stability of the resultant gels. Storage moduli of the macromers increased over 4 orders of magnitude once gelation occurred above the transition temperature. Furthermore, chemical cross-linking of these macromers resulted in gels with increased stability compared to uncross-linked controls. These results demonstrate the feasibility of synthesizing poly( N-isopropylacrylamide)-based macromers that undergo tandem gelation and establish key criteria relating to the transition temperature and stability of these materials. The data suggest that these materials may be attractive substrates for tissue engineering and cellular delivery applications as the combination of mechanistically independent gelation techniques used in tandem may offer superior materials with regard to gelation kinetics and stability.
Hydrogels that solidify in response to a dual, physical and chemical, mechanism upon temperature increase were fabricated and characterized. The hydrogels were based on N-isopropylacrylamide, which renders them thermoresponsive, and contained covalently crosslinkable moieties in the macromers. The effects of the macromer end group, namely acrylate or methacrylate, and the fabrication conditions were investigated on the degradative and swelling properties of the hydrogels. The hydrogels exhibited higher swelling below their lower critical solution temperature (LCST). When immersed in cell culture media at physiological temperature, which was above their LCST, hydrogels showed constant swelling and no degradation over eight weeks, with methacrylated hydrogels having higher swelling than their acrylated analogs. In addition, hydrogels immersed in cell culture media under the same conditions showed lower swelling as compared to phosphate buffered saline. The interplay between chemical crosslinking and thermally induced phase separation affected the swelling characteristics of hydrogels in different media. Mesenchymal stem cells encapsulated in the hydrogels in vitro were viable over three weeks and markers of osteogenic differentiation were detected when the cells were cultured with osteogenic supplements. Hydrogel mineralization in the absence of cells was observed in cell culture medium with the addition of fetal bovine serum and β-glycerol phosphate. The results suggest that these hydrogels may be suitable as carriers for cell delivery in tissue engineering.
Mesenchymal stem cells (MSCs) are capable of differentiating into a variety of lineages, including bone, cartilage, or fat, depending on the inducing stimuli and specific growth and differentiation factors. It is widely acknowledged that basic fibroblast growth factor (bFGF) modulates chondrogenic and osteogenic differentiation of MSCs, but thorough investigations of its effects on adipogenic differentiation are lacking. In this study, we demonstrate on the cellular and molecular level that supplementation of bFGF in different phases of cell culture leads to a strong enhancement of adipogenesis of MSCs, as induced by an adipogenic hormonal cocktail. In cultures receiving bFGF, mRNA expression of peroxisome proliferator-activated receptor c2 (PPARc2), a key transcription factor in adipogenesis, was upregulated even prior to adipogenic induction. In order to investigate the effects of bFGF on PPARc ligand-induced adipogenic differentiation, the thiazolidinedione troglitazone was administered as a single adipogenic inducer. Basic FGF was demonstrated to also strongly increase adipogenesis induced by troglitazone, that is, bFGF clearly increased the responsiveness of MSCs to a PPARc ligand.
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