La conceptualidad y su proceso

derived neuronal cells

Hydrogel compositions were first tested as a growth surface for the cells. The human neuronal cells were cultured on top of the hydrogel surfaces and analyzed after two weeks of culturing. All tested hydrogel compositions supported cell growth and no cytotoxicity was observed (data not shown). The best compositions were the HA-PVA-based hydrogels containing a high molecular weight HALD component (HP1, HP2), as well as the AP hydrogel. Both compositions supported cell growth and neurite outgrowth along the hydrogel surface (Fig. 5.15(a), (c), (e)), whereas the HP3 and HP4 hydrogels containing a low molecular weight HALD component were less supportive (Fig. 5.15(b), (d)). Neurite outgrowth from cell aggregates was the highest in the HP1 hydrogel. The increase in the DS% of the HALD component in HA-PVA-based hydrogels had a negative effect on neurite outgrowth when the HP1 and HP2 hydrogels were compared (Fig. 5.15(f)). The effect of increasing the DS% was not as dramatic on the cells as the change in molecular weight. The most prominent compositions, HP1 and AL, had the lowest stiffness of all the tested hydrogels.

The most prominent hydrogel compositions, HP1 and AP, in terms of cell growth and neurite outgrowth support were selected for the following 3D culturing experiments where the cells were cultured as encapsulated in the hydrogel. First, the viability of neuronal cells inside the HP1 and AP hydrogels was analyzed after two weeks in culture. The cell viability in 3D was at a similar level as viability in 2D control cultures on top of laminin (Fig. 5.16 (a-c)). This indicates that the hydrogel was well tolerated as a culturing environment. Next, the effect of the decreased polymer concentration of the HP1 hydrogel was studied in order to see how the cells behaved in even softer hydrogels. Neuronal cells were cultured for two weeks as encapsulated inside the four soft hydrogel compositions (HP1, HP1a, HP1b, and AP). Cell growth was seen inside all the studied compositions and the cells were positive for the neuronal markers MAP-2 and β-tubulin III (Fig. 5.16 (d-g)). Slight differences were seen in the cell growth. In HP1 and HP1a hydrogels, for example, more cells stayed in clusters (Fig. 5.16(d), (e)), whereas inside the softest HP1b and AP hydrogels the cell outgrowth from the aggregates into the hydrogel was more robust (Fig. 5.16(f), (g)).

DS 9 % DS 5 % HP (High Mw) HP (Low Mw) (a) HP1 (b) (e)AP (c) HP2 (d) HP4 (f) AP (Medium Mw) HP3 100 µm DS 7%

Neurite outgrowth on top of hydrogels

Neurite Length ( m m) HP1 HP2 HP3 HP4 AP 800 600 400 200 0

Figure 5.15: Representative images of neural cells growing on top of HA-PVA- and AL-PVA-

based hydrogel surfaces: the high molecular weight HA-containing hydrogels (a) HP1 and (b) HP2, the low molecular weight HA-containing hydrogels (c) HP3 and (d) HP4, and (e) the alginate-based AP hydrogel. (f) Neurite outgrowth measured from cultures on top of the hydrogels. Median with inter-quartile range of the measured neurites is shown as line and whiskers whereas individual values are presented as gray dots. The scale bar in all images is 100 µm.

50 µm

HP1 HP1a HP1b AP

DAPI/MAP-2/B-tubulin III

Modulus 2.8 ± 0.8 kPa Modulus 2.5 ± 0.8 kPa Modulus 0.9 ± 0.4 kPa Modulus 1.0 ± 0.5 kPa

(d)

(a) HP1 _(b) AP _(c) 2D laminin

Calcein-AM/EthD-1

(e) (f) (g)

100 µm

Figure 5.16: Live/dead labeling of the neurons cultured two weeks inside HP1 (a) and AP

(b) hydrogels, and on top of laminin coated plastic (c). Living cells are labeled with Calcein- AM (green) and dead cells with EthD-1 (red). Neurons grown for two weeks encapsulated inside the HP1 (d), HP1a (e), HP1b (f), and AP (g) hydrogels labeled with neuron specific immunocytochemical markers MAP-2 (green) and B-tubulin III (red), co-labeled with nuclear marker DAPI (blue). The second-order elastic constants of each hydrogel are shown under the images. The scale bars in images (a-c) is 100 µm and 50 µm in (d-g).

6.1

Fabrication of fast gelling in situ formed hydrazone

crosslinked hydrogels from modified polymers

The choice of the fabrication method is important, especially when living cells are encapsulated. Mild reaction conditions, such as water-based methods, should be preferred, whereas any extreme conditions, such as extreme temperatures, free radicals, toxic components, or organic solvents, should be avoided. [69, 119] Injectable materials, on the other hand, offer a less invasive implantation process. Ideally, the cells would be premixed with the liquid precursors of the hydrogels, and then injected and gelated in situ. [27, 105] To meet these requirements, hydrazone crosslinking was chosen as a fabrication method for the hydrogels presented in this thesis. Hydrazone crosslinked (mainly HA-based) hydrogels have previously been studied, for example, for wound healing [57] and for cartilage and myocardial TE [26, 54 – 56]. However, for neural or corneal applications these hydrogel types have not really been studied before. Therefore, hydrazone crosslinked HA-PVA- (Study III), AL-PVA- (Study III), GG-HA- (Study I), and HA-HA(-col I) (Study II)-based hydrogels were fabricated and studied. HA, AL, GG, and PVA were chemically modified with complementary reactive aldehyde- and/or hydrazide-groups to allow their gelation. Overall, the DS% were kept as low as possible so as not to lose the original advantageous properties of the polymers.

Aldehyde-groups were introduced to HA, AL, and GG either through periodate oxidation by cleaving the C2-C3 bond from the polysaccharide chain (Studies I, III), or through selective oxidation of diol-modified polymer (only HA, Study II). Based on the1_H-NMR

and FTIR-analysis, the modifications were successful, although some things need to be considered with regard to the reaction used. For example, periodate oxidation can lead to the loss of native backbone structure and the molecular weight of a polymer can be reduced, which might have an effect on the ability of cells to recognize it. The benefit of using selective oxidation of diol-modified HA instead of periodate oxidation is that it keeps the ring-structure of HA intact, and therefore the cells can recognize the HA more easily. Thus, this modification method is usually preferred over the simple periodate oxidation method.

Mutually, hydrazide-groups were introduced to HA and PVA. Hydrazide-groups were introduced to PVA by using glycine ethyl ester and hydrazine as a source of hydrazide unit (Study III). HA, on the other hand, was modified either using homobifunctional ADH or CDH as a source of hydrazide unit (Studies I, II).1_{H-NMR and FTIR-analysis}

confirmed the success of these modifications. It should be noted, however, that when using ADH, it can act as a crosslinker if both of its hydrazide groups manage to react. Even when a large excess of ADH is used, the crosslinking effect can still be significant.

[158] The benefit of using CDH instead of ADH is discussed more deeply next. In the case of HA-HA-based hydrogels, the HH2 hydrogel was considered more stable than the HH1 hydrogel due to a resonance stabilization effect of CDH. As Oommen et al. [54] state, in the CDH-based hydrazone (C1_=N1_-N2_H-(C=O)N3_{H), the neighboring heteroatom (N}3₎

of CDH can provide resonance stabilization to the developing N2_{positive charge. Thus,}

due to its urea-type structure, the N2 _{positive charge can be delocalized. Instead, there}

is no stabilization effect with ADH. [54]

Successful gelation and hydrogels with variable properties were obtained by optimizing and varying the gel parameters, i.e., DS%, molecular weight and the ratio of polymer components, as well as the polymer concentration of hydrogel. The effect of these parameters on the properties of hydrogels is summarized in Section 6.8. The gelation time of the hydrogels varied from seconds to five minutes (Studies I-III), which should be enough for the proper mixing and injection of the cell-polymer solutions. However, it should be noted that injecting a large amount of fast-gelling material has the potential to damage the surrounding tissue. This might be avoided by injecting smaller amounts of gel over a longer period of time. The gelation time was not significantly affected by the gel parameters, only the decrease in polymer concentration of the hydrogel increased the gelation time (from seconds to minutes, Study III). The FTIR-based structure analysis confirmed the success of hydrazone crosslinking with all the gel types as the appearance of hydrazone C=N stretching signal was shown with all of them (Studies I-III).

6.2

Swelling and deswelling kinetics of hydrogels

The swelling ratio (SR) of hydrogels is usually estimated by quantifying the water content as a function of time. Generally, the SR is determined by studying the water uptake of dry hydrogels, or the water uptake of wet hydrogels that are dehydrated after the swelling. The drawback with the first method is that the drying process can be harmful to the hydrogel structure and cause distortions to the swelling results (that was also noted in this thesis, data not shown). Moreover, these methods do not describe how an initially wet hydrogel behaves in studied conditions, for example, during the cell culture experiments, since the gel is not dehydrated at any point of the experiment. For these reasons, in this thesis the swelling kinetics of the wet hydrogels in deionized water and cell culture medium (DMEM/F12) were studied without drying the sample at any stage of the experiment.

The swelling of HA-PVA-, AL-PVA-, and HA-HA(-col I)-based hydrogels was shown to be dependent on the solvent and its ionic strength (Studies II, III). Unlike in water, in cell culture medium most hydrogels shrank instead of swelling. This was expected because in the presence of salts hydrogel networks lose their hydrophilic-hydrophobic balance and shrink due to ex-osmosis. As counter ions (Na+_{) condense around the fixed}

carboxylate ion charges of HA or AL, they cause a decrease in repulsive forces among the carboxylate groups. This leads to a decrease in swelling (internal network collapses). The cell culture medium and body fluids also contain divalent cations. [160] Therefore, it can be expected that when placed into such media or fluids, the AP hydrogel may also form ionic crosslinks that lead to a more crosslinked structure and reduced swelling, as was also reported, for example, by Kuo and Ma [161].

In water, the hydrogels swelled many times to that of cell culture medium (Studies II, III). The amount of hydrogel swelling depends on the degree of crosslinking and the chemical structure of the hydrogel. Hence, the more hydrophilic the structure, the stronger the

polymer-water interaction becomes. [61] Due to the large number of hydrophilic groups of HA-based hydrogels, they swelled more in water. It was also noted that the swelling ratio of HA-PVA-based hydrogels increased when the molecular weight of HA or the DS% of HALD-component decreased, whereas the swelling ratio decreased when the polymer concentration of the hydrogel decreased. Furthermore, as the crosslinking density increased, the swelling ratio decreased. The AP hydrogel was less stable in water and degraded in just a few hours. The addition of collagen into the HA-HA-based hydrogel decreased the swelling ratio by approximately 20% at each data point. One possible explanation for this is the imine formation between the aldehyde groups of HA and the amino groups of collagen that increases the crosslinking density. Collagen may also form gel itself. It should be also noted that there is a marked difference in the swelling behavior of the components themselves; HA is susceptible to swelling, whereas collagen is not. The swelling and deswelling behavior of GG-HA-based hydrogels was studied in deionized water and PBS. Similar to previous hydrogels, GG-HA-based hydrogels swelled in water, whereas in PBS they mainly shrank. The swelling ratio was shown to decrease when the DS% of HAADH-component was lower, but the ratio of gel components did not show any significant affect. The swelling ability of these hydrogels in water can be connected to the HA component, since the control GG-Ca gel without any HA (a control sample) hardly swelled in water, whereas it shrank the most in PBS. The shrinking of GG-based hydrogels in PBS can be explained by the ions of PBS being similar to cell culture medium, although PBS only contains monovalent cations. Due to these cations, PBS is able to form ionic crosslinks with GG (ions increase the formation of double helix and junction zones), and this leads to a more crosslinked structure [33]. The ionic nature of GG-HA-based hydrogel shrinking was confirmed by immersing the hydrogel by turns in water and PBS. Similar to Coutinho et al. [33], the results confirmed the ionic nature of the GG-HA-based hydrogels. Thus, by changing the solution environment, the control of the physical properties of these gels could be possible.