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

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-<i>co</i>-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-<i>co</i>-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

Viability of the cells encapsulated in PEGDA gel.

<p>Representative images of live (green) and dead (red) 4T1 cells encapsulated in PEGDA gels with 5 kPa modulus and cultured in stem cell culture medium for 2 (a), 6 (b) and 12 (c) days. Cells were stained with cAM/EthD for live (green) and dead (red) cell imaging. The insets in (a) to (c) are live/dead images of 4T1 cells in PEGDA gels with 70 kPa modulus after 2, 6, and 12 (day), respectively.</p

Effect of CD44BP on tumorsphere formation and CSC marker expression.

<p>Representative fluorescent images of the tumorsphere size and distribution for 4T1 cells encapsulated in PEGDA gels (1.4×10⁵ cells/ml) conjugated with CD44BP (a, conj CD44BP), conjugated with a scrambled sequence of CD44BP (b, conj s-CD44BP), CD44BP dissolved in the gel (c, dis CD44BP), and s-CD44BP dissolved in the gel (d, dis s-CD44BP) and cultured in the stem cell culture medium for 9 days. Effect of CD44BP on cell number density (e) and tumorsphere number density (f) for 4T1 tumor cells encapsulated in PEGDA hydrogel and incubated in the stem cell culture medium for 9 days. Effect of CD44BP conjugation on CD44 (g), CD24 (h), ABCG2 (i) and SCA1 (j) mRNA marker expression for 4T1 tumor cells encapsulated in PEGDA gel and incubated in the stem cell culture medium for 9 days. RNA levels of the cells were normalized to those at time zero. A star indicates a statistically significant difference (p<0.05) between the test group and “Ctrl”. Two stars indicates a significant difference (p<0.05) between the two CD44BP and s-CD44BP groups within the same form of peptide addition (Dis or Conj). Values are expressed as mean ± SD (n = 3).</p

Forward and reverse sequence of the PCR primers.

<p>Forward and reverse sequence of the PCR primers.</p

Effect of CD44BP conjugated to the gel on tumor formation <i>in vivo</i>.

<p>The gel without cell (negative control, light blue), 4T1 tumospheres in suspension (positive control, red), 4T1 cells encapsulated in the gel without CD44BP (green), and 4T1 cells encapsulated in the gel with CD44BP (light blue) were inoculated subcutaneously in syngeneic Balb/C mice. Tumor sizes were measured daily from post-inoculation day 11 (n = 6/group). Tumor growth was not observed in the negative control group (the gel without cell) and the group with 4T1 cells in the gel with CD44BP (the lines for these two groups are overlapped in the figure).</p

CSC population in cells encapsulated in PEGDA gel conjugated with CD44BP, IBP, or FHBP.

<p>4T1 cells were encapsulated in PEGDA gels with 5 kPa modulus and cultured in stem cell culture medium. Cells before encapsulation (a), 9 days after encapsulation in the gel without peptide (b), 9 days in the gel conjugated with FHBP (c), 9 days in the gel conjugated with IBP (d), and 9 days in the gel conjugated with CD44BP were stained with CD44-FITC and CD24-PE antibodies. The population of CD24+, CD44+ and CD44+/CD24− cells was determined by flow cytometry. Flow cytometry was repeated multiple times on each sample to ascertain reproducibility of the results.</p

CSC population in the cells encapsulated in PEGDA gel.

<p>MCF7 cells were encapsulated in PEGDA gels with 5 kPa modulus and cultured in stem cell culture medium. Cells before encapsulation (a), 3 days (b), 8 days (c) and 11 days (d) after encapsulation were stained with CD44−FITC and CD24-PE antibodies. The population of CD24+, CD44+ and CD44+/CD24− cells was determined by flow cytometry. Flow cytometry was repeated multiple times on each sample to ascertain reproducibility of the results.</p

Expression of the markers related to CSC maintenance in cells grown in the gel conjugated with CD44BP, IBP, or FHBP.

<p>Effect of cell binding peptide on N-Cadherin (a), E-Cadherin, (b), integrin α_V (c), and integrin β₃ (D) mRNA marker expression for 4T1 tumor cells encapsulated in PEGDA hydrogel and incubated in the stem cell culture medium for 9 days. Effect of cell binding peptide on vimentin (e) and VEGF (f) protein expression. The protein expression was determined by western blot and quantified with imageJ. Actin was used as the internal control and the protein expressions were normalized to those at time zero. RNA levels of the cells were normalized to those at time zero. A star indicates a statistically significant difference (p<0.05) between the test group and “Ctrl”. Values are expressed as mean ± SD (n = 3).</p

Sphere formation and the effect of cell type encapsulated in PEGDA gel on the expression of CSC markers.

<p>Representative fluorescent images of the tumorsphere size and distribution for 4T1 (a), MCF7 (b), and MCF10a (c) cells encapsulated in PEGDA gels (1.4×10⁵ cells/ml), and cultured in stem cell culture medium. Encapsulated cells were stained with phalloidin for cytoskeleton (red) and DAPI for nucleus (blue). Effect of cell type on cell number density (d) and tumorsphere diameter (e) for tumor cells encapsulated in PEGDA hydrogel and incubated in stem cell culture medium for 6 or 9 days. The sphere size distribution (f) was determined 9 days after encapsulation. Effect of cell type on CD44 (g), CD24 (h) and ABCG2 (i) mRNA marker expression for tumor cells encapsulated in PEGDA hydrogel and incubated in stem cell culture medium for 6 or 9 days. RNA levels of the cells were normalized to those at time zero. A star indicates a statistically significant difference (p<0.05) between the test group and the groups with different cell type in the same time point (the same diameter range in f). Values are expressed as mean ± SD (n = 3).</p

Peptides and their scrambled sequence.

<p>Peptides and their scrambled sequence.</p