Gelation Characteristics and Osteogenic Differentiation of Stromal Cells in Inert Hydrolytically Degradable Micellar Polyethylene Glycol Hydrogels

Abstract

The use of poly(ethylene glycol) (PEG) hydrogels in tissue engineering is limited by their persistence in the site of regeneration. In an attempt to produce inert hydrolytically degradable PEG-based hydrogels, star (SPELA) poly(ethylene glycol-co-lactide) acrylate macromonomers with short lactide segments (<15 lactides per macromonomer) were synthesized. The SPELA hydrogel was characterized with respect to gelation time, modulus, water content, sol fraction, degradation, and osteogenic differentiation of encapsulated marrow stromal cells (MSCs). The properties of SPELA hydrogel were compared with those of the linear poly(ethylene glycol-co-lactide) acrylate (LPELA). The SPELA hydrogel had higher modulus, lower water content, and lower sol fraction than the LPELA. The shear modulus of SPELA hydrogel was 2.2 times higher than LPELA, whereas the sol fraction of SPELA hydrogel was 5 times lower than LPELA. The degradation of SPELA hydrogel depended strongly on the number of lactide monomers per macromonomer (nL) and showed a biphasic behavior. For example, as nL increased from 0 to 3.4, 6.4, 11.6, and 14.8, mass loss increased from 7 to 37, 80, 100% and then deceased to 87%, respectively, after 6 weeks of incubation. The addition of 3.4 lactides per macromonomer (<10 wt % dry macromonomer or <2 wt % swollen hydrogel) increased mass loss to 50% after 6 weeks. Molecular dynamic simulations demonstrated that the biphasic degradation behavior was related to aggregation and micelle formation of lactide monomers in the macromonomer in aqueous solution. MSCs encapsulated in SPELA hydrogel expressed osteogenic markers Dlx5, Runx2, osteopontin, and osteocalcin and formed a mineralized matrix. The expression of osteogenic markers and extent of mineralization was significantly higher when MSCs were encapsulated in SPELA hydrogel with the addition of bone morphogenetic protein-2 (BMP2). Results demonstrate that hydrolytically degradable PEG-based hydrogels are potentially useful as a delivery matrix for stem cells in regenerative medicine.

Experimental and Computational Investigation of the Effect of Hydrophobicity on Aggregation and Osteoinductive Potential of BMP-2-Derived Peptide in a Hydrogel Matrix

“…23,25 Briefly, the residual moisture was removed from the PEG macromer by azeotropic distillation from toluene. The PEG macromer and lactide monomers were heated to 120°C under dry nitrogen atmosphere in a reaction flask equipped with an overhead stirrer.…”

“…The synthesized macromers were characterized by hydrogen nuclear magnetic resonance ( 1 H-NMR) (Varian, Palo Alto, CA) as we previously described. 25 The synthesized macromer is hereinafter referred to as Ln where ''L'' stands for lactide-capped PEG macromer and ''n'' is the number of lactide monomers per chain end.…”

“…The following procedure was used for encapsulation of hMSCs in BMP-2-peptide-incorporated PEGDA hydrogels by UV polymerization, as shown in Figure 1c. 22,25 First, BMP-2 peptide, with or without conjugation to the lactide-capped macromer, was dissolved in a solution of Irgacure-2959 photoinitiator (1 wt% of PEGDA) in PBS. Next, 1 wt% Ac-GRGD adhesive peptide was dissolved in a solution of PEGDA macromer (15 wt%) in PBS to improve cell viability and cell-matrix interaction after encapsulation.…”

“…Double-stranded DNA (dsDNA) content of the homogenized samples was analyzed using the PicoGreen assay as described. 25 Analysis of dsDNA was performed using a Synergy HT plate reader (Bio-Tek, Winooski, VT) with emission and excitation wavelengths of 485 and 528 nm, respectively. ALP activity of the samples was measured using the QuantiChrom ALP assay kit on a Synergy HT plate reader at the wavelength 405 nm as described.…”

“…ALP activity of the samples was measured using the QuantiChrom ALP assay kit on a Synergy HT plate reader at the wavelength 405 nm as described. 25 The measured intensity was correlated to ALP activity in IU/L and normalized to cell numbers. Calcium content of the samples, as a measure of the total mineralized deposit, was measured using the QuantiChrom calcium assay kit as described.…”