(50a) Heparin-Functionalized Peg Hydrogels Direct Three-Dimensional Human Mesenchymal Stem Cell Osteogenic Differentiation

The nature of cell adhesion to substrate materials has a tremendous effect on cell function and tissue development. Signaling via receptor-ligand interactions initiated by cell adhesion provides the cell with vital information about its extracellular environment, regulating a variety of events, including differentiation, tissue evolution, and apoptosis [1,2]. Understanding how cells interact with a substrate is crucial to the development of functional biomaterials.

Cell adhesion through cell surface receptors, such as integrins, selectins, and immunoglobulins, is mediated by interactions with proteins adsorbed on the material. However, a controlled environment in which individual proteins can be studied systematically is difficult to achieve when relying upon non-specific protein adsorption. Furthermore, modification of biomaterials with full proteins can be very complex; protein coupling to biomaterials requires mild reaction conditions, since the proteins are subject to both denaturation and degradation. As an alternative to inclusion of full proteins, significant interest has emerged in the design of cell scaffolds that incorporate moieties that can induce specific, controlled protein adsorption. Relative to integrins, interactions of proteoglycans and glycosaminoglycans (GAGs) with their cell surface ligands have not been widely employed in the design of biomaterials that regulate cell adhesion or function. A GAG of particular significance is heparin. Many proteins contain heparin-binding domains. Thus, immobilized heparin can be utilized as a sequestering molecule for heparin-binding signaling or adhesive proteins produced by or delivered to cells. Interestingly, heparin is capable of interacting with numerous proteins associated with osteoblast and osteoblast progenitor cell adhesion (e.g., fibronectin, vitronectin) and osteogenic differentiation (e.g., bone morphogenetic proteins, pleiotrophin) [3].

In our previous work [4], heparin-modified poly(ethylene glycol) (PEG) hydrogels were found to promote human mesenchymal stem cell (hMSC) adhesion, proliferation, and osteogenic differentiation in two-dimensional culture. In order to evaluate this system as a potential osteogenic, three-dimensional scaffold, as well to understand the osteogenic mechanism of heparin-functionalized PEG hydrogels, macromolecular photoreactive heparin monomers were synthesized and copolymerized with dimethacrylated PEG monomers in the presence of hMSCs to yield hydrogels. Prior to this work, viability of hMSCs encapsulated in PEG hydrogels was found to be limited [5] and improved dramatically by the incorporation of the adhesive peptide, Arg-Gly-Asp-Ser (RGDS). Therefore, to ensure viability and allow for comparison, hMSCs were encapsulated in RGDS-functionalized hydrogels.

hMSCs encapsulated in RGDS- , heparin-, and RGDS+heparin-functionalized PEG hydrogels were cultured in vitro and viability, alkaline phosphatase production, and gene expression of osteopontin and collagen type I were monitored as a function of time. After 35 days, viability of hMSCs in heparin-functionalized hydrogels was 94%, which is increased over unmodified PEGDM gels and RGDS-modified gels, which exhibited 63% and 87% viability over 5 weeks, respectively. As expected, the combination of heparin and RGDS lead to the greatest survivability (95%) over the 5 weeks. ALP production increased 4-fold and 3-fold, respectively, for hMSCs encapsulated in heparin-functionalized and heparin+RGDS-functionalized hydrogels. Cells encapsulated in RGDS-functionalized gels maintained a constant ALP production over the culture period, indicating that RGDS is not significantly affecting osteogenic differentiation. Osteopontin gene expression was at the same level for all treatments at day 4. A substantial increase in gene expression was found at day 10 for cells encapsulated in heparin-functionalized gels to nearly 3-fold over the RGDS- and heparin+RGDS-functionalized gels. OPN gene expression remained very high throughout the study for hMSCs encapsulated in heparin-functionalized gels and was greater than that of cells in other treatments. Collagen type I gene expression of hMSCs in heparin- and heparin+RGD-functionalized gels was greater than all treatments and steady over the culture days 4, 10, and 21 (1.6-fold, 2-fold, and 2-fold greater, respectively) than expression in RGDS-functionalized hydrogels. Cells in RGDS-functionalized gels exhibited the lowest COL I gene expression at all time points compared with the other treatments. OPN and COL I gene expression along with ALP production data supports the hypothesis that heparin-functionalized gels are inducing the osteogenic differentiation of hMSCs in three-dimensional in vitro culture.

In exploration of specific proteins affecting hMSC function during this study, hMSCs were seeded on two-dimensional heparin gels and cultured in the presence of media with defined exogenous proteins. Specifically, fibronectin (FN) and BMP2 were selected. To isolate the individual effects of fibronectin and BMP2, media was depleted of all heparin binding proteins. The individual effects of FN and/or BMP2 on ALP production and OPN gene expression of hMSCs cultured on two-dimensional heparin-functionalized gels was assessed by adding in both FN or BMP2 alone or in combination. As a control, media containing all available heparin-binding proteins was utilized. ALP production by hMSCs increased in all treatments from day 2 to day 14 as compared to levels on unmodified PEGDM. The greatest increases, however, were found by hMSCs cultured in osteogenic media (2.5-fold increase) and in FN+BMP2 media (2.5-fold increase). Both FN and BMP2 media alone had a strong impact on ALP production, where FN and BMP2 media, individually, caused a 2-fold increase and 1.6-fold increase, respectively, of ALP production by hMSCs. Even media without any heparin-binding proteins caused a significant, yet small (1.2-fold), increase in ALP production, indicating that heparin, by itself, could be initiating signal transduction pathways involved with cell differentiation.

OPN gene expression was greatest at days 2 and 14 by cells cultured in media with all heparin-binding proteins. Without any heparin-binding proteins, however, OPN gene expression was low at day 2 and decreased 2-fold by day 14. Cells cultured in BMP2 media exhibited the greatest increase in OPN gene expression (4-fold) over the culture period. In the presence of FN, hMSC OPN gene expression was statistically the same at all time points as that of cells in the presence of all heparin-binding proteins but did not change over the time course of the study. Interestingly, although BMP2 media increased OPN gene expression and FN media resulted in high and consistent OPN gene expression over the study, the combination of the two proteins resulted in the lowest gene expression at both time points and expression reduced 2-fold from day 2 to 14. Fibronectin and BMP2, therefore, do not act in combination to increase OPN gene expression but do have individual effects. Therefore, the temporal availability of these proteins may play a role in heparin-facilitated hMSC osteogenic differentiation.

In addition to the availability and interaction with signaling and adhesion proteins, integrin expression and interactions with extracellular molecules mediate the expression of genes controlling survival, differentiation, and matrix production [6-8]. The distribution of integrins alpha5beta1 and alphavbeta3 were analyzed in cells encapsulated in hydrogels at day 35. The greatest alpha5beta1 expression was found by cells encapsulated in heparin-functionalized gels, with 88% positive cells. Heparin+RGDS-functionalized hydrogels elicited the second-greatest alpha5beta1 expression (48% positive cells), followed by RGDS-functionalized gels (37% positive cells), and PEGDM hydrogels (12% positive cells). When examining alphavbeta3 integrin expression, RGDS-functionalized hydrogels elicited the most expression, with 46% positive cells. Heparin+RGDS-functionalized gels induced 23% positive cells, followed by heparin-functionalized gels (15% positive cells), and PEGDM hydrogels (5% positive cells). Based on these data, integrin expression is linked to viability, as the hydrogels presenting RGDS and heparin showed the greatest survivability of hMSCs and the greatest number of cells expressing integrins alpha5beta1 and alphavbeta3. In addition, based on data that shows heparin, specifically, is augmenting ALP production and OPN and Col I gene expression while also expressing the highest percentage of α5β1-positive cells, it can be deduced that α5β1 expression is positively correlated with the osteogenic differentiation of hMSCs.

In sum, hMSCs, multipotent precursor cells, were encapsulated in heparin-functionalized poly(ethylene glycol) hydrogels and analyzed for viability and osteogenic differentiation. Heparin-functionalized hydrogels supported hMSC viability and induced osteogenic differentiation, likely through cell-material interactions established by heparin-binding proteins fibronectin and bone morphogenetic protein 2. Furthermore, the viability and differentiation were differentially affected by integrin production, where both α5β1 and αvβ3 integrins-ligand interactions supported viability while only the α5β1 integrin played a role in hMSC osteogenic differentiation.

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