Synthetic hydrogels as scaffolds for manipulating endothelium cell behaviors

Synthetic hydrogels can be used as scaffolds that not only favor endothelial cells (ECs) proliferation but also manipulate the behaviors and functions of the ECs. In this review paper, the effect of chemical structure, Young's modulus (E) and zeta potential (ζ ) of synthetic hydrogel scaffolds on static cell behaviors, including cell morphology, proliferation, cytoskeleton structure and focal adhesion, and on dynamic cell behaviors, including migration velocity and morphology oscillation, as well as on EC function such as anti-platelet adhesion, are reported. It was found that negatively charged hydrogels, poly(2-acrylamido-2-methylpropanesulfonic sodium) (PNaAMPS) and poly(sodium p-styrene sulphonate) (PNaSS), can directly promote cell proliferation, with no need of surface modification by any cell-adhesive proteins or peptides at the environment of serum-containing medium. In addition, the Young's modulus (E) and Zeta potential (ζ ) of hydrogel scaffolds are quantitatively tuned by copolymer hydrogels, poly(NaAMPS-coDMAAm) and poly(NaSS-co-DMAAm), in which the two kinds of negatively charged monomers NaAMPS and NaSS are copolymerized with neutral monomer, N, N-dimethylacrylamide (DMAAm). It was found that the critical Zeta potential of hydrogels manipulating EC morphology, proliferation, and motility is critical ζ = 20.83 mV and critical ζ = 14.0 mV for poly(NaAMPS-co-DMAAm) and poly(NaSS-co-DMAAm), respectively. The above mentioned 2 EC behaviors well correlate with the adsorption of fibronectin, a kind of cell-adhesive protein, on the hydrogel surfaces. Furthermore, adhered platelets on the EC monolayers cultured on the hydrogel scaffolds obviously decreases with an increase of the Young's modulus (E) of the hydrogels, especially when E > 60 kPa. Glycocalyx assay and gene expression of ECs demonstrate that the anti-platelet adhesion well correlates with the EC-specific glycocalyx. The above investigation suggests that understanding the relationship between physic-chemical properties of synthetic hydrogels and cell responses is essential to design optimal soft & wet scaffolds for tissue engineering.


Introduction
In order to survive and proliferation, anchorage dependent cells must adhere to and spread on a scaffold. In vivo, multicellular organisms composed of many kinds of anchorage dependent cells, and the scaffolds supporting the cells are either other co-surviving cells or extracellular matrix (ECM) [1,2] with an elastic modulus of ca. 10 kPa [3]. For example, endothelial cell (EC) monolayers are supported by the ECM secreted by vascular smooth muscle cells (VSMCs), while the VSMCs are supported by type I collagen [4].
A hydrogel, comprising of a 3 well-defined chemical structure and high purity of synthetic hydrogels make it relatively easier for explaining the mechanism of interaction between cultured cell and scaffolds [5,6].
Even though the amazing physical-chemical properties of synthetic hydrogels that attract scientists to design hydrogels suitable for cell culture scaffolds, comparing with the commercially available naturally derived collagen gel and Matrigel TM (a mixture of murine sarcoma-derived ECM) [7], cell proliferation does not occur spontaneously on many neutral synthetic hydrogels, which is one of the significant disadvantages as cell cultivation scaffold.
Recently, we have reported that ECs from human, i.e., human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cell (HCAECs), as well as from bovine, i.e., bovine fetal aorta endothelial cells (BFAECs), could spread, proliferate, and reach confluent on the negatively charged hydrogels that are not modified by any cell adhesive proteins or peptides, such as poly(2-acrylamido-2-methylpropanesulfonic sodium) (PNaAMPS), poly(sodium p-styrene sulphonate) (PNaSS), and poly(acrylic acid) (PAA) [17]. EC is a kind of special cell covered on the inner wall of blood vessel as a monolayer, the cells play a wide variety of critical roles in the control of vascular function, such as blood vessel formation and inhibition of platelet adhesion [18][19][20][21][22]. Therefore, the relationship between the physic-chemical properties of the synthetic hydrogels and the behaviors of EC has been further investigated.
In this review, our recent progresses on manipulate EC behaviors and functions by the physic-chemical properties of the synthetic hydrogels, including the effect of chemical structure 4 Thus, it is easy to discuss the correlation between the properties of the synthetic hydrogels, including the chemical structure, Young's modulus and Zeta potential ( ζ ), and the cell properties and behaviors.

Effect of chemical structure of hydrogels on cell proliferation
The synthetic hydrogels used for the scaffolds of ECs are classified into 3 categories, that is, neutral, negatively charged weak polyelectrolyte, and strongly charged negative polyelectrolyte hydrogels [17]. The neutral hydrogels have no ionized group, including PVA, PDMAAm, and PAAm; the weakly charged hydrogels have carboxylic acid groups on the side chains of the polymers and they are pH-dependent, including PAA and PMAA; the strongly charged hydrogels have sulfonate groups on the side chains of the polymers, and they are fully dissociated, including PNaAMPS and PNaSS. The molecular structures of these hydrogels and the typical phase-contrast micrographs of BFAECs cultured for 120 h on these hydrogels with various cross-linker concentrations M (mol% in relative to the monomer concentration) , i.e., PAAm (2 mol%), PDMAAm (4 mol%), PVA (6 mol%), PAA (2 mol%), PMAA (1 mol%), PNaAMPS (6 mol%), and PNaSS (10 mol%), are shown in Figure 1.
After a prolonged culture time, the cell morphology and proliferation strongly depend on the chemical structure of the hydrogels (Figure 1 column V). Figure 2 shows the proliferation kinetics of BFAECs cultured on the various kinds of synthetic hydrogels, as well as on a control, On the strongly charged hydrgels, more than 90% adhered ECs spread with a fusiform or polygonal shape, and the ECs proliferate to confluent with a cell density of 1.32×10 5 and 1.12×10 5 on PNaAMPS (M = 2) and PNaSS (M = 4) hydrogels, respectively, after 144 h.
Although the proliferation rate of the PNaAMPS and PNaSS hydrogels are lower than that of type I collagen gel (Figure 2), the densities of the proliferated cells at confluent cultured on the two kinds of strongly charged hydrogels are comparable to those on collagen gel. The above results demonstrate that the synthetic hydrogels facilitate EC proliferation in the following order: strongly charged gels (PNaAMPS, PNaSS) > weakly charged gels (PAA, PMAA) > neutral gels (PVA, PAAm, PDMAAm).

Effect of Zeta potential on cell behaviors
The effect of chemical structure on EC proliferation indicates that the strongly charged hydrogels facilitate cell proliferation, and the charge density of hydrogel is an important parameter for controlling EC fate. Based on the above result, the effect of charge density of hydrogel on cell behaviors was systematically investigated by synthesizing copolymer hydrogels with different charge density. The copolymer hydrogels, poly(NaAMPS-co-DMAAm) and poly(NaSS-co-DMAAm), could be synthesized by combination of strongly charged moiety, PNaAMPS or PNaSS, on which ECs can proliferate very well, with a neutral moiety, PDMAAm, on which ECs cannot proliferate. The Zeta potential, ζ , of the poly(NaAMPS-co-DMAAm) and poly(NaSS-co-DMAAm) hydrogels was quantitatively tuned by adjusting the molar fraction (F) of the negatively charged NaAMPS and NaSS in the copolymer hydrogels. Because NaAMPS and NaSS are monomers with negative charge, the ζ of poly(NaAMPS-co-DMAAm) and poly(NaSS-co-DMAAm) copolymer hydrogels should be negative except F=0. Thus, a large absolute value of Zeta potential, ζ , denotes high charge density of the copolymer hydrogels. The static cell behaviors, including cell morphology, proliferation, cytoskeleton structure, focal adhesion, as well as dynamic cell behaviors, including migration velocity, migration distance and morphology oscillation, have been investigated on the poly(NaAMPS-co-DMAAm) and poly(NaSS-co-DMAAm) copolymer hydrogels. Furthermore, the correlation between the adsorption of fibronectin, a kind of typical cell-adhesive protein, on hydrogel surface and cell behavior was furthermore analyzed. Table 1 shows the degree of swelling (q) of HEPES buffer-equilibrated poly(NaAMPS-co-DMAAm) hydrogels as a function of F. It shows that q increases with an increase of F, due to the introduction of more sulfonate group of PNaAMPS in the copolymer hydrogels. Figure 3 shows the F as a function of ζ of HEPES buffer-equilibrated poly(NaAMPS-co-DMAAm) hydrogels. It shows that with the increase of F, the ζ of poly (NaAMPS-co-DMAAm) hydrogels increases and saturates to 30.0 mV at F = 1, confirming that the charge density of poly(NaAMPS-co-DMAAm) hydrogels increases with an increase of the amount of NaAMPS.

Effect of Zeta potential on static cell behaviors
It should be note that although PDMAAm is neutral, it shows a small negative ζ in HEPES buffer solution, perhaps due to its ionic adsorption in the buffer solution [25].
Typical phase-contrast micrographs of BFAECs and HUVECs cultured for 120 h on the poly (NaAMPS-co-DMAAm) hydrogels with various ζ are shown in Figure 4. To reveal the correlation between ζ and cell proliferation behavior, the cell density cultured for 120 h of two kinds of ECs, BFAEC and HUVEC, as a function of the ζ of the poly (NaAMPS-co-DMAAm) hydrogels, is plotted ( Figure 5).
In The similar critical ζ was also observed on the poly(NaSS-co-DMAAm) hydrogels with various ζ . Figure 6 shows the typical phase-contrast micrographs of BFAECs after 6 h and 96 h cultivation on the poly(NaSS-co-DMAAm) hydrogels with various absolute values of Zeta potential, ζ . The morphology of cells relates to the structures of actin fibers and focal adhesion.
Vinculin is a membrane cytoskeleton protein found in focal adhesion plaques that is involved in 8 the linkage of integrin adhesion molecules to the actin cytoskeleton. Figure  whereas the weak adhesion is formed on the hydrogels with low ζ value, i.e. low charge density [26]. hydrogel. It is considered that PNaSS with aromatic ring close to the ionizable group facilitates protein adsorption from serum containing culture medium than that of PNaAMPS. The present results suggest that not only Zeta potential, but also chemical structure affects cell proliferation.

Effect of Zeta potential on dynamic cell behaviors
Cell motility plays an important role in a wide variety of biological processes such as embryogenesis, inflammatory response, wound healing, and the metastasis of tumor cells [27][28][29][30][31]. In particular, the migration of vascular ECs, which form the inner lining of blood vessels, is essential for angiogenesis [32,33]. As described in 3.2.1, Zeta potential (ζ ) of the hydrogels affects the static behavior of ECs, such as cell morphology, proliferation, cytoskeleton structure and focal adhesion. Thus, effect of Zeta potential (ζ ) on dynamic behavior of ECs, such as migration velocity, migration distance, and morphology oscillation were further investigated.
It has been reported that the migration velocity of cells is affected by the Young′s modulus (E) of the PAAm gel modified by collagen [34]. Therefore, for studying the effect of ζ on cell dynamic behaviors, designing hydrogels with various ζ , and at the same time, maintaining an identical E are necessary. As shown in Figure 9, a series of poly(NaSS-co-DMAAm) copolymer and deposit a basement membrane to finally yield new operational blood vessels [34]. Therefore, studying on the motility of ECs that is not able to proliferate could reveal some information on angiogenesis.
We have found that an individual BFAEC repeatedly changes its morphology when 8.8 mV < ζ < 12.6 mV (F = 0.05-0.1). In this morphology oscillation process, a spreading cell shrink its extended morphology to a round shape only in the time less than 1 min, whereas a round shape cell changes its morphology to an extensively spreading cell need ca. 20 min (Figure 11a).
The large time difference between cell spreading and shrinkage implies that the polymerization of actin fibers take a longer time than their depolymerization. Figure 11b shows a typical example of a BFAECs with its morphology oscillating from a spread shape to a round shape when ζ = 9.4 mV (F = 0.06). The filopodia of an extensively spreading cell shrank (Figure 11b-2), indicating that the EC was released from the pre-existing sites of adhesion. As a result, the EC rapidly changed to a round shape and migrated to another location ( Figure 11b-3). This process was very fast and was completed within 1 min. The round EC subsequently remained at its new location for ca. 10 min (Figure 11b-11 It is well known that a spreading cell gradually reverts to a round shape in order to undergo mitosis. In the present study, however, the cells cultured on the hydrogels which 8.8 mV < ζ < 12.6 mV (F = 0.05-0.1) are not able to divide during the process of morphological change, indicating that the change in cell morphology is not associate with mitosis.
It has been reported that fibronectin affects certain cell behaviors, such as the rate and anisotropism of spread, traction force (the forces exerted by cells on its scaffold during spreading), and cell-scaffold adhesive force, as well as migration velocity [35,36]. Our study further found that fibronectin affects EC oscillation on the hydrogels with low charge density in a stick-slip mode.

Correlation between fibronectin adsorption and cell behaviors
The above results demonstrated that the ζ of the hydrogels has a profound effect on the static behaviors and dynamic behaviors of the ECs. It is a question why the EC behaviors can be affected by the ζ of synthetic hydrogels. Because the hydrogels that enhance cell proliferation are negatively charged, and the cell surface is negatively charged also, therefore, there is no direct electrostatic interaction between the hydrogel and the cultured ECs. We assumed that proteins contained in the FBS could adsorb on the hydrogel surface and act as bridges between cultured ECs and hydrogels, which affect static and dynamic behavior of the ECs. In other word, if ζ affects the amount of adsorbed proteins on the hydrogels, it should affect the behaviors of ECs. Thus, the correlation between adsorption total amount of protein and a typical cell-adhesive protein, fibronectin, from cell cultured medium on the hydrogel surface and cell behavior are furthermore analyzed.
The same as the relationship between the cell density and the absolute value of Zeta potential, ζ , the concentration of total adsorbed proteins ( p C ) and fluorescence intensity of fibronectin ( f F ) change with an increase of the ζ of the poly(NaAMPS-co-DMAAm) copolymer hydrogels could also be divided into three stages, i.e., slowly adsorbed stage, dramatically adsorbed stage and stable adsorption stage (Figure 12). indicates that the charge density of hydrogels adjusts protein adsorption, that is, more negative charge, more protein adsorption, which favors cell proliferation, and the migration velocity of the cells is low, on the other hand, a little negative charge, a little protein adsorption, which not suitable cell proliferation, and the migration velocity of the cells is high.
Effect of protein adsorption on cytoskeleton and focal adhesions could clearly visualize actin stress fibers and focal adhesions (Figure 7). When ζ = 9.4 mV, only a little protein adsorption, the spreading ECs deficient in actin stress fibers and focal adhesions, implying that the ECs are unable to form stable focal adhesions on the hydrogel. Consequently, the ECs undergo a dramatic oscillation in shape factor. On the other hand, when ζ = 20.5 mV, more protein adsorption, the spreading ECs has well-developed actin fibers and large focal adhesions, implying that the ECs are able to form stable focal adhesions on the hydrogel. Such ECs thus maintain a stable spreading shape factor.

Effect of hydrogel properties on EC function
The major problem of the artificial blood vessel is that blood clot occurs after a certain period of implantation, especially when the diameter of artificial blood vessel is smaller than 5mm [37].
The ideal artificial blood vessel should have the structure similar to in vivo blood vessel and take full advantages of cell functions. Therefore, hybrid artificial blood vessel with EC monolayer on its inner wall has been expected to inhibit thrombosis. Our investigation demonstrates that the platelet adhesion on the two kinds of human ECs, i.e., HUVECs and HCAEC, strongly dependents on the chemical structure and Young's modulus of synthetic hydrogels. Furthermore, the different platelet adhesion behaviors are attributed to the amount of glycocalyx secreted by the ECs cultured on different kinds of hydrogel scaffolds. The ECs cultured on the PNaSS hydrogel secrets more glycocalyx which contributes to inhibit platelet adhesion. The results imply that it is possible to fabricate hybrid artificial blood vessel with high blood compatibility from PNaSS hydrogel with ECs monolayer on its inner surfaces.

Effect of chemical structure on platelet adhesion
HUVECs could proliferate to sub-confluent (PAA M =1, 2 mol%) or confluent on negatively charged hydrogels (PNaAMPS M =2, 10 mol%, PNaSS M =4, 10 mol%) which is not sensitive to the M of the hydrogels. Figure 13 shows the typical phase-contrast micrographs of the

Effect of Young's modulus on platelet adhesion
It was reported that heparan sulfate proteoglycans (HSPGs), which is a main component of proteoglycans contained in glycocalyx, exhibit antithrombin activity [39,40]. Combining with the above described results, we hypothesized that the glycocalyx secreted the ECs can be affected by the physic-chemical properties of the hydrogel scaffolds because platelet adhesion closely correlates to the glycocalyx. To verify this, the correlation between the platelet adhesion and the amount of glycocalyx of ECs cultured on the PNaSS hydrogels with various levels of Young's modulus (E) rang in 3 ~ 263 kPa was further studied.
Same as HUVECs, HCAECs could proliferate to confluence on the PNaSS hydrogels with various E. In addition, real-time polymerase chain reaction (PCR) and glycosaminoglycan assay showed that the amount of EC-specific glycocalyx, glypican, syndecan-4, and perlecan, secreted by the cultured HCAECs increases with an increase of the E of PNaSS hydrogel, which obviously higher than that cultured on TCPS. Furthermore, the HCAECs cultured on PNaSS hydrogels showed excellent property against platelet adhesion. The largest amount of EC-specific glycocalyx and excellent blood compatibility were observed when E > 60 kPa. . Furthermore, the cell morphology is more homogenous on the hydrogel scaffolds than that on TCPS [41].
HSPGs of ECs consist of a core protein and heparan sulfate-type glycosaminoglycans (GAGs).
There are five distinct HSPG core proteins in ECs: syndecan-1, syndecan-2, syndecan-4, glypican and prelecan. It was reported that prelecan exhibit antithrombin activity by activing antithrombin III though heparin-like sequences in the heparin sulfate chains [42]. For investigating the effect of the Young's modulus (E) of hydrogel on the amount of glycocalyx secreted by HCAECs, the relative RNA levels of core proteins and the amount of GAG were analyzed.
The relative RNA expressions of the specific core proteins in glycocalyx, glypican, syndecan-4, and perlecan, and the relative amount of total GAG, are also shown in Figure 14. The results demonstrate that HCAECs cultured on hydrogels secret a larger amount of glycocalyx than those cultured on TCPS scaffold. Furthermore, the amount of glycocalyx is influenced by the elasticity of the PNaSS hydrogel scaffolds, harder PNaSS hydrgels promote the secretion of glycocalyx.
The density of platelets that adhered on the HCAEC monolayer cultured on PNaSS hydrogels decreases with an increase of E. The density of platelets that adhered to the HCAEC monolayer cultured on the PNaSS hydrogels with E = 60, 100 kPa is 1.2 × 10 5 platelets/µm 2 and 3 × 10 4 platelets/μm 2 , respectively, which is c.a. 8 and 30 times lower than that on the PNaSS hydrogel with E = 3 kPa. The result is consistent with the result of the relative RNA levels of glypican, syndecan-4, and perlecan, as well as the amount of GAG assay described above.
The results suggested that the glycocalyx of cultured ECs modulates platelet compatibility, and the amount of glycocalyx secreted by ECs dependents on the chemical structure and Young's modulus (E) of hydrogel scaffolds. This result should be applied to make the hybrid artificial blood vessel composes of hydrogels and ECs with high platelet compatibility.

. Conclusions
We have found that some negatively charged synthetic hydrogels, such as PNaAMPS and     Reproduced with permission from the literature [26]. Revised with permission from the literature [26].   Reproduced with permission from the literature [26]. Reproduced with permission from the literature [26]. permission from the literature [25]. Figure 13 The typical phase-contrast micrographs of the HUVECs cultured on the various kinds of hydrogels at 144 h (Column I), as well as the SEM images (Column II) and the density of adhered platelets ( p D ) on the corresponding HUVECs. Revised with permission from the literature [39]. Figure 14 The typical phase-contrast micrographs of HCAECs cultured for 120 h on PNaSS hydrogels with E = 3, 60, 100 kPa, corresponding M =4, 10, 13 mol%, as well as the relative RNA expressions of the specific core proteins in glycocalyx, glypican, syndecan-4, and perlecan, the relative amount of total GAG. TCPS was used as a control. Revised with permission from the literature [41].