Customization and tuning of the degradation rate of X–Ca–alginate aerogels in the presence of PC 12 cells

Abstract

Degradable substrates for nerve regeneration are of great interest due to the avoidance of a second surgery to remove non-degradable implants. Aerogels are a class of materials with great promise in the medical field as they have been proven to increase nerve regeneration. Therefore, degradable aerogels show promise as neuronal scaffolds. In this study, polyurea-crosslinked calcium alginate (X–Ca–Alg) aerogels were studied in vitro, showing a rapid change in area, especially within the first few hours. Stiffness changes were also observed to decrease over time. Previous studies have shown an increase in regeneration rate in nerve cells with an electrical bias. Therefore, X–Ca–Alg aerogels were monitored in vitro under the influence of a DC bias. PC12 neuronal cells adhered to and extended neurites on X–Ca–Alg aerogels and showed changes based on the timeline of aerogel area change. These findings show the promising medical applications of X–Ca–Alg aerogels as degradable nerve scaffolds.

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1. Introduction

Aerogels are a unique class of materials consisting of lightweight, open nanoporous networks with high surface areas, high porosities, and low bulk densities.^1–3 They are typically synthesized through sol–gel processes,^1,2,4–6 which can be tailored to produce aerogels with mechanical properties suited for a broad range of specific applications. This versatility is achieved by crosslinking various polymer backbones with different crosslinkers, such as polyurea and other organic or inorganic agents.^7–9 In addition to their unique physical characteristics, aerogels are also appealing because they are produced through environmentally friendly methods using abundant and low-cost precursors.

Aerogels have demonstrated significant promise for several biomedical applications such as drug delivery, regenerative medicine, and biosensing.^4,10–13 Our previous studies have shown that aerogels effectively support neurite outgrowth in vitro^5,14–20 and facilitate nerve repair in vivo.^4,21–24In vitro experiments have shown that multiple types of aerogels, when surface-coated with type I collagen, promote neuronal cell attachment and subsequent neurite extension^5,14–20,25 and that this is influenced by the pore size and Young's modulus of the aerogels. Imaging analyses have revealed that neuronal cell adhesion to aerogels is followed by vigorous neurite extension, which is a key step in the repair of peripheral nerve injuries.^{4,12,22,26,27} Interestingly, the outgrowth and the directionality of neurite extension by neuronal cells attached to collagen-coated aerogels is enhanced by electrical stimulation (DC bias).^14,19 The clinical application of aerogels for nerve repair is currently limited by their chemical stability, which may necessitate a surgical procedure to remove the aerogel implant once nerve repair is complete. Such an intervention may carry the risk of infection and inflammatory processes.¹⁹ Biodegradable materials have proven effective for drug delivery and have the advantage to naturally degrade without leaving harmful residue after they released the bioactive molecules^28–31 Similarly, the use of biodegradable aerogels may mitigate the need for surgical removal after nerve healing.

Alginates are polysaccharides extracted from various species of brown algae. The synthesis of calcium alginate aerogels is straightforward and cost-effective, with a lower environmental impact when compared to plastics. Such calcium alginate preformed gels can react wih triisocyanates to form polyurea-crosslinked calcium alginate (X–Ca–alginate) aerogels, which are mechanically stronger and more stable in natural waters and physiological environments compared to native calcium alginate aerogels.^7,32,33 Importantly, alginates are biodegradable suggesting that X–Ca–Alg aerogels could also be degradable, which has been confirmed by our recent studies.¹⁸

The aim of this study was to characterize the physical properties, degradability, and ability of X–Ca–Alg aerogels synthesized under different conditions to support the attachment and neurite outgrowth of PC12 neuronal cells. We also examined whether a DC electrical bias influences the degradation of X–Ca–Alg aerogel. Our results indicate that X–Ca–Alg aerogels represent a promising degradable and electrically responsive substrate for nerve repair.

2. Experimental

Sodium alginate (PROTANAL LF 240 D; G 30–35%) was purchased from FMC. CaCl₂·2H₂O, acetone and MeCN (HPLC grade) were purchased from Fisher Scientific. Desmodur N3300 and Desmodur eco N7300 were generously provided by Covestro AG. All solvents and reagents were used as received. Supercritical fluid (SCF) drying was performed in an autoclave (Model E3100, Quorum Technologies, East Sussex, UK). The gels were placed in the autoclave at 12 °C and covered with acetone. Liquid CO₂ was then allowed in the autoclave; acetone was drained out as it was displaced by liquid CO₂ (5×; 1 per 30 min). The temperature of the autoclave was raised to 45 °C and maintained for 1 h. Finally, the pressure was slowly released, allowing CO₂ to escape as a gas.

N₂-sorption measurements were performed on a Micromeritics Tristar II 3020 surface area and porosity analyzer (Micromeritics, Norcross, GA, USA). Samples were degassed at 80 °C for 24 h using Micromeritics VacPrep 061. Skeletal densities (ρ_s) were determined by He pycnometry using a Micromeritics AccuPyc II 1340 pycnometer. Bulk densities (ρ_b) were calculated from the weight and dimensions of the material.

2.1 Preparation of polyurea-crosslinked calcium alginate micromeritic (X–Ca–Alg) aerogels

X–Ca–Alg aerogel types 2 and 5 were prepared as described before^7,18 (Table 1). To prepare X–Ca–Alg aerogel types 2HDI and 2PDI (Table 1), an aqueous solution of sodium alginate (2% w/w) was prepared by dissolving sodium alginate (2 g) in H₂O (98 g) at 25 °C. To induce gelation and aging, the solution was transferred into cellulose dialysis tubing (Sigma-Aldrich) and dialyzed for 24 h against 4 volumes of 0.2 M CaCl₂. After removal of the calcium alginate (Ca–Alg) gels from the dialysis tubing stepwise solvent-exchange was performed, first with MeCN/H₂O mixtures (30, 60, and 90% v/v) and then with dry MeCN (3×). The gels were then immersed in a solution of 0.6 M triisocyanate (Desmodur N3300 or Desmodur N7300) in dry MeCN for 24 h at room temperature and then for 72 h at 70 °C. The volume of the triisocyanate solution was 4× the volume of Ca–Alg gels. Afterward, the gels were solvent-exchanged with acetone (3×) and dried from supercritical CO₂ to yield the final aerogels. The exact formulations for the X–Ca–Alg aerogels used in this study are presented in Table 1.

Table 1 Formulations and selected material properties of X–Ca–Alg aerogel samples used in this study

Aerogel type	Na–alginate concentration (% w/w)	Triisocyanate used for X-linking	Triisocyanate concentration (g/100 mL MeCN) (M)	Bulk density ρb (g cm^−3)	Skeletal density ρs (g cm^−3)	Porosity Π^b (% v/v)	Avg. pore diameter d^c (nm)
a Values taken from ref. 7 and 18. b Porosity calculated according to formula: Π = [(ρs − ρb)/ρs] × 100. c Calculated by the 4V/σ method; V was set equal to the total pore volume (VTotal), which was calculated according to formula: VTotal = 1/ρb − 1/ρs.
2^a	0.9	N3300	11.9 (0.2)	0.071	1.34	95	224
5^a	1.35	N3300	24.2 (0.4)	0.088	1.35	93	190
2HDI	2.0	N3300	39.0 (0.6)	0.138	1.63	92	99
2PDI	2.0	N7300	43.5 (0.6)	0.146	1.53	90	110

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2.2 Aerogel properties

2.2.1 Surface roughness. High resolution 3D images of each aerogel's surface were obtained using the Profilm 3D (Filmetrics Inc, San Diego, CA) profilometer. These images were then used to measure the surface roughness of X–Ca–Alg aerogels. Prior to profilometry, the samples were sputter-coated with a 5 nm layer of AuPd using the Hummer 10.2 (Anatech, Hayward, CA) at 3–5 mA for 1–3 min.

The images were analyzed with the web-based software from Filmetrics. The area roughness was determined using the arithmetic mean height (S_a) over an area of 200 × 200 µm². S_a is defined as the absolute average of the surface values above and below the mean plane within a selected region. Three independent trials (N = 3) were performed for each sample. Each trial consisted of 10 separate measurements (n = 10). Sputtered samples were also used to identify the nanomorphology of the aerogel samples by FE-SEM imaging (Hitachi, Schaumburg, IL).

2.2.2 Stiffness. The compressive modulus (Y) for each type of X–Ca–Alg aerogel was measured using the motorized test stand ESM303 (Mark-10, Copiague, NY, USA) equipped with a Series 5 (Mark-10) force gauge, set to deliver a compression rate of 11 mm min⁻¹. The Y values were calculated using eqn (1) and (2) where ΔL is the ‘travel’ and F the “load”,To evaluate the effect of in vitro conditions and of the degradation X–Ca–Alg aerogels on the compressive modulus, these measurements were repeated on a weekly basis for a total of 14 consecutive weeks. Aerogels were kept under physiological conditions (submerged in a solution of DI water, penicillin, and streptomycin) and at 37 °C between measurements. These conditions are here onwards referred to as in vitro conditions.

2.3 Kinetic of X–Ca–Alg aerogel degradation as a function of time

In this work, degradation is defined as the change of area as a function of time and is consistent with previous definitions.^18,34 X–Ca–Alg aerogels in vitro have been observed to undergo dimensional changes, namely a shrinkage in the surface area.²⁰ Understanding any changes in the superior surface of each type of X–Ca–Alg aerogel was critical for explaining the cells response since this is the surface that is in direct contact with the cells. X–Ca–Alg aerogel formulations 2HDI, 2PDI, 2 and 5 were sectioned and attached to the bottom of a tissue culture polystyrene (TCPS) petri dish to prevent them from floating to the surface using clear adhesive sealant (Silicone RTV, Permatex). Petri dishes were then filled with a physiologically relevant solution and kept at 37 °C in an incubator. Using the VHS970FN digital microscope (Keyence, Itasca, IL) the top face area (a) of each X–Ca–Alg aerogel sample was measured as a function of time (Fig. 1). The normalized area (A_n) was then calculated using the formulawere A_n is the normalized area change at a specific time, a_i is the initial area value, and a_t is the area value at a specific time t (Fig. 1b and c). Area measurements were performed over two-time frames and referred to as (1) short-term and (2) long-term studies.

Fig. 1 (a) Top-down view o X–Ca–Alg aerogel samples in vitro (b) schematic diagram of initial top area of aerogel coupon (a_i = L₁ × L₂). (c) Schematic diagram of area at a specific time .

Short-term studies. Short-term changes in the area of X–Ca–Alg aerogels were investigated with the aerogels immersed in a physiologically relevant solution and by acquiring an image every 5 min for a duration of 6 h. Image analysis was performed using the NIH open-source ImageJ (version 1.53t) software, and the scale of the image was calibrated using the scale bar.

Long-term studies. Long-term changes in the area of X–Ca–Alg aerogels were investigated similarly compared to short-term studies, but on a weekly basis, for a duration of 14 weeks. Samples were imaged before in vitro conditions (baseline), after the initial immersion of aerogels in a physiologically relevant solution, and weekly afterwards. Image analysis was performed using ImageJ and the scale of the image was set using the scale bar.

Effect of DC bias on aerogel degradation. A custom-built electro-stimulation chamber used in earlier studies^14,19 provided a constant DC electrical bias to X–Ca–Alg aerogels in vitro. To investigate the effect of DC bias on aerogel degradation, increasing voltages (0, 15, 30 and 60 V) were applied.

2.4 Cell culture

PC12 pheochromocytoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were grown following a previously published protocol.^{5,14,15,17–19,25} Before each experiment, PC12 cells were ‘primed’ for neural differentiation by being placed for 8 days in RPMI 1640 medium supplemented with GlutaMax, HEPES, horse serum, penicillin/streptomycin and 50 ng ml⁻¹ NGF. This medium was replaced every 2–3 days. After 8 days, the cells were harvested and cryopreserved with trypsin and stored in liquid nitrogen following standard protocols.

X–Ca–Alg aerogel samples were attached to the bottom of a 24-well microplate (Kemtec, Hanover, PA) using a clear adhesive sealant (Silicone RTV, Permatex) which was allowed to cure overnight. X–Ca–Alg aerogel samples were sterilized and coated with rat tail type 1 collagen (Invitrogen, Carlsbad, CA) as previously described for other types of aerogels.^{5,14,15,17,19,25} Collagen solution was applied to the aerogel surface to reach a collagen density of 4 µg cm⁻². Primed PC12 cells were rapidly thawed as per the manufacturer instructions and plated on these substrates at a cell density of 1 × 10⁴ cells per cm² and maintained in RPMI 1640 medium supplemented with GlutaMax, HEPES (Life Technologies, CA), and 10% fetal calf serum. Cells were cultured in a 5% CO₂ incubator at 37 °C for 1 or 3 days. Three independent trials (N = 3) were conducted for each experiment. As a control, we used cells plated in the above described medium in the wells of tissue culture polystyrene (TCPS) dishes without X–Ca–Alg.

2.5 Fluorescence staining of PC12 cells with Alexa 488 phalloidin

Cells attached to X–Ca–Alg aerogels were stained with Alexa 488 phalloidin as previously described,²⁸ with minor modifications. Phalloidin is a molecule binding to actin, a protein which outlines cell shape, and especially cytoplasmic extensions such as neurites. The staining was performed through a series of incubations in which the wells containing the cells on X–Ca–Alg aerogels were filled with the different solutions used for the staining. First, cells on X–Ca–Alg aerogels were fixed for 15 min with 4% formaldehyde (Tousimis, Rockville, MD) in phosphate buffered saline (PBS). The fixation was followed by cell permeabilization with 0.1% NP40 in phosphate buffered saline (PBS) for 5 min. Next, the cells were washed 2 times 5 min with PBS and incubated for 1 h in Alexa 488 phalloidin diluted 1 : 200 in PBS (Cytoskeleton, Denver, CO). At the end of this incubation, the cells on the X–Ca–Alg aerogels were washed 2 × 5 min in PBS wash to remove unbound phalloidin. The X–Ca–Alg aerogels were then removed from the wells of the culture dish and mounted on 35 mm Petri dishes with bottom surface consisted of a #1 coverslip (MatTek, Ashland, MA) using Prolong Diamond mounting medium. The mounting medium was cured for 24 h at 4 °C. Imaging of cells stained with the fluorescent probe was performed with a Nikon A1 confocal scanning laser fluorescence microscope (Nikon, Tokyo, Japan).

Evaluation of neurite extension by PC12 neuronal cells. The length and branching shape of neurites extended by PC12 plated on X–Ca–Alg aerogels was assessed by the fluorescence microscopy of Alexa 488 phalloidin stained PC12 cells. Confocal fluorescence microscopy images were obtained using a 1 AU pinhole and a laser intensity and PMT gain appropriate for the intensity of fluorescence staining. The same settings were used for all observations. Z-stacks were obtained and sometime collated in maximum intensity projections images. Analysis of the length and branching of neurites was performed using the NIH open-source ImageJ (version 1.53t) software in conjunction with SNT, an ImageJ framework for tracing, visualizing, and quantifying neuronal morphology.^35,36 SNT allowed for the semi-automatic measurement of neurites length and branching for each cell. These measurements differ from the methodology used previously,²⁸ as the Z-stack provides a 3D view of the neurites. This is important because when neurite elongation takes place on a flat surface the neurites lay on the x–y axis. In contrast, when neurite elongation takes place on a contoured surface like that of aerogels, the neurites may travel in the x–y–z axis. Assessment of neurites branching should preferably be done on a z-stack as this collect's information in the x–y–z axes.

Measurements performed with ImageJ included neurite length, neurite density (number of neurites per cell), number of branches per neurite, and branch length. Each of these variables was measured for approximately 50 cells (n = 50) in three trials (N = 3) for control and for each type of X–Ca–Alg aerogel (X–Ca–Alg aerogel 2, X–Ca–Alg aerogel 2HDI, X–Ca–Alg aerogel 2PDI, X–Ca–Alg aerogel 5) at time points of 1 and 3 days after plating on aerogel.

2.6 Statistical analysis

Statistical analysis of the data was performed using Student's t-test to calculate a p-value and determine whether the average of two sample groups were statistically different or not. The difference was considered significant when p < 0.05. Statistical significance is represented with (*) on the graphs. The error bars in the graphs represent the standard error of the mean of three independent trials (N = 3) and OriginLab9 was used for preparing all the graphs.

3. Results and discussion

3.1 Physical properties of X–Ca–Alg aerogels

In previous studies⁵ we showed that the length and branching of neurites extended by PC12 neuronal cells grown on aerogels critically depend on the average pore diameter (d), surface roughness (S_a), and stiffness (Y) of the aerogels. This prompted us to determine the values of these three parameters for the different types of X–Ca–Alg aerogels in this study. This provides baseline values to understand the length and shapes of neurites extended by PC12 cells on X–Ca–Alg aerogels. The nanomorphology of the different types of X–Ca–Alg aerogels examined here was determined by SEM (Fig. 2). The observations show that the different types of X–Ca–Alg aerogels were fibrous and porous but that the porosity was variable, with the pores of aerogels 2 and 5 (Fig. 2a and b) being larger than those of aerogels 2HDI and 2PDI (Fig. 2c and d). Thus, the aerogels synthesized with the lowest alginate concentrations (Tables 1 and 2) had larger pores than aerogels synthesized with higher alginate concentrations.

Fig. 2 Scanning electron microscope (SEM) images of the surface of each aerogel type (a) X–Ca–Alg aerogel 2, (b) X–Ca–Alg aerogel 5, (c) X–Ca–Alg aerogel 2HDI, and (d) X–Ca–Alg aerogel 2PDI.

Table 2 Average pore diameter, surface roughness and stiffness of X–Ca–Alg aerogel samples used for this study

Aerogel sample	Triisocyanate concentration (M)	Triisocyanate used for X-linking	Avg. pore diameter d (nm)	Surface roughness Sa (nm)	Stiffness Y (MPa)
2	0.2	N3300	224	582.9	4.85
5	0.4	N3300	190	311.3	3.85
2HDI	0.6	N3300	99	200.6	14.37
2PDI	0.6	N7300	110	221.3	1.69

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The stiffness of the different X–Ca–Alg aerogels ranged from 1.69 to 14.37 MPa (Fig. 3a). These differences are attributed to the precursor types and the concentrations used for synthesis. The aerogel 2HDI had the largest Y value (Y = 14.37 MPa) and was synthesized with the highest concentration of triisocyanate N3300 and Na–alginate. The Y value decreased with decreasing concentrations of N3300 and Na–alginate and this decrease was statistically significant. In contrast, for aerogels 2 and 5 which were synthesized under similar conditions, the Y values (4.85 MPa for aerogel 2 and 3.85 MPa for aerogel 5) were not statistically different. On the other hand, aerogel 2PDI had the lowest Y value (1.69 Pa), although the concentrations of triisocyanate and Na–alginate were the same as for aerogel 2HDI. The different triisocyanates used for crosslinking (N7300 vs. N3300) have a tremendous effect on the stiffness of the aerogels, despite their very similar structure. The structures of these two aerogels have been provided and is available in the supplemental section. There is a clear correlation between the average pore diameter (d) and surface roughness (S_a) since an increase in d results in an increase in S_a.⁵ Summary of Y, S_a, and d measurements are shown in Table 2.

Fig. 3 Stress over strain graphs of X–Ca–Alg aerogel samples, as indicated. (a) Stiffness differences between the aerogel samples before in vitro conditions (baseline). (b) Stiffness comparison of aerogel samples 2HDI (top) and 2PDI (bottom) between baseline, day 1, and day 2. (c) Small change in stiffness of aerogel sample 2HDI over time while immersed (week 0–14). (d) Increase of stiffness of aerogel sample 2PDI over time (week 0–14).

3.2 Effect of incubation time on the stiffness of X–Ca–Alg aerogels

The different types of X–Ca–Alg aerogels were stored at 37 °C for 14 consecutive weeks, and the stiffness (Y) of each coupon was measured periodically. Overall, Y values increased for all types X–Ca–Alg aerogels as a function of incubation time. A more pronounced change was observed for aerogel sample 2PDI, which can be correlated to the different triisocyanate type used when compared to aerogel samples 2HDI, 2, and 5. Results shown in Fig. 3b present the change of Y in aerogels 2HDI (top) and 2PDI (bottom) within the first 24 h. Fig. 3c and d show the stiffness change for 2HDI and 2PDI, respectively, during the 14 weeks. Stiffness changes for aerogels 2HDI, 2 and 5 were similar, with an increase in Y over the 14 week-period of 1.46, 1.4 and 1.48, respectively. On the other hand, aerogel 2PDI experienced a 1.4-fold increase after one day of incubation and a 2-fold increase after 14 weeks of incubation.

3.3 Degradation X–Ca–Alg aerogels

Without DC bias. The time dependent degradation of X–Ca–Alg aerogels was observed while they were immersed in a physiologically relevant solution. This involved imaging the top area in 5 min intervals for a total of 6 h. During this time, aerogels outgassed and their area decreased gradually. The area change was normalized and then compared to previously tested X–Ca–Alg aerogels¹⁸ and presented in Fig. 4a and b. Only X–Ca–Alg aerogel 2 and 5 from previously published studies were used in this study for comparison. Sample 2HDI was included in this study as it was synthesized using higher concentrations of Na–alginate and triisocyanate compared to earlier formulations. On the other hand, sample 2PDI is a direct comparison from 2HDI as both share the same preparation conditions but differ in the triisocyanate used (N3300 or N7300). The two triisocyanates have very similar structures, differing only in the length of the carbon chains (6 carbons for N3300 and 5 carbons for N7300) between the isocyanurate ring and the –NCO groups. N7300 is a bio-based triisocyanate, containing a significant portion (∼70%) of renewable carbon derived from biomass. The bulk of the area change occurred within the first 2 h as shown in Fig. 4b. Aerogels 2HDI and 2PDI had a 52% and 36%, respectively, change in area. Aerogel types 1–5 show a decrease of less than 30%. Further investigation was conducted over a 14-week period as shown in Fig. 4c and d showing similar results.

Fig. 4 (a) and (b) Short term and (c) and (d) long term degradation of X–Ca–Alg aerogels depicted as normalized area in each time frame. Greater degradation rates were observed in the first hours of in vitro conditions. The degradation rate slowed down gradually in subsequent weeks.

In the presence of DC bias. The new formulations of X–Ca–Alg aerogels (2HDI and 2PDI) were further investigated by monitoring the degradation rate of the samples in the presence of DC bias. Aerogel 2PDI showed a degradation kinetic that differed from that observed in the absence of an applied DC bias (baseline kinetic). Baseline behavior showed a 19% area decrease at the 15 min mark, compared to 23%, 24% and 38% with an increasing voltage of 15 V, 30 V, and 60 V as shown in Fig. 5a. On the other hand, aerogel 2HDI showed little change in degradation regardless of the presence or absence of DC bias as shown in Fig. 6b. Therefore, X–Ca–Alg 2PDI, the aerogel sample with the lowest stiffness among the four X–Ca–Alg aerogel samples and higher change in stiffness over time while in vitro, shows an increase in degradation rate with increasing DC bias.

Fig. 5 Degradation of aerogel (a) 2PDI and (b) 2HDI under in vitro conditions with increasing DC electrical stimulation (0–60 V). 2PDI shows an increase of degradation with increasing DC bias.

Fig. 6 Degradation of X–Ca–Alg 2PDI aerogel in the presence of a DC bias on and off every 30 min. Increase in slope of area are observed with DC bias, with a higher decrease in the first 30 min due to in vitro incubation of X–Ca–Alg aerogels. However, an increase in area decrease is observed between 1 h and 1.5 h.

Since the degradation of 2PDI was influenced by DC bias, a further study monitoring the change in area of the aerogel with the DC bias switched on and then off is shown in Fig. 6. A change in slope can be observed between all periods of times with a higher decrease in the first stage which is consistent with the rapid shrinkage of area observed when placing X–Ca–Alg aerogels in vitro. Any effect of DC bias is obscured by this rapid change. Once the DC bias was turned off, the change in area decreased at a lower rate compared to the second time that the aerogel was exposed to DC bias. This rate decreased again once the DC bias was terminated.

The observed increase in the rate of degradation (area reduction) of submerged aerogel coupons under DC bias can be attributed to electroosmosis-enhanced wetting of the aerogels rather than electrochemically-driven (e.g., migration) or solid-state field effects. X–Ca–Alg aerogels have a charged solid–liquid interface, and an external electric field can induce electro-osmotic flow of the surrounding solution into the porous network. This accelerates liquid infiltration, displaces trapped gas (air) faster, and triggers earlier capillary stresses, leading to enhanced physical contraction. The absence of this effect under dry conditions, combined with the use of insulated electrodes, argues against direct electrochemical or dielectric-driven mechanisms. The stronger response of the mechanically compliant 2PDI aerogels aligns with their greater sensitivity to capillary-induced network rearrangement.

3.4 Investigation of cell distribution on degradable alginate aerogels

For precise evaluation of cell behavior on the 3D contoured aerogel surface, Z-stacking and fluorescence microscopy was essential. Fluorescence images of PC12 cells cultured on X–Ca–Alg aerogel types 2, 5, 2HDI, and 2PDI are presented in Fig. 7. Fig. 7a, c, and e show representative fluorescence images after day 1 and Fig. 7b, d, and f show cell behavior after day 3 where a significant number of processes can be observed, at much longer lengths compared to day 1.

Fig. 7 Confocal scanning microscopy fluorescence images of PC12 cells cultured onto X–Ca–Alg aerogels and stained with Alexa 488 phalloidin to reveal the outlines of the neurites extending from the cell body. PC12 cells plated on 2HDI aerogel (a) and (b), 2PDI aerogel (c) and (d), and control substrate (e) and (f) for 1 (a), (c), (e) and 3 (b), (d), (f) days. Note that after 3 days the neurites of cells plated on 2HDI (b) are longer than those of cells plated on 2PDI and control substrate. 2HDI (a) and (b) aerogels display some autofluorescence which is observable even in the absence of staining with Alexa 488 phalloidin (data not shown).

Neurites were observed for all substrates and for all time frames. At the 3-day marker, cells showed longer neurites. Comparison of cell behavior based on neurite length, neurite density, branch density, and branch length for X–Ca–Alg aerogels 2HDI, 2PDI, 5, and control (tissue culture polystyrene) are shown in Fig. 8a, b, c, and d, respectively. PC12 cells grown on X–Ca–Alg aerogel 2 were not fully imaged and analyzed due to excessive substrate warping.

Fig. 8 Cell behavior of PC12 cells on X–Ca–Alg aerogel 2HDI, 2PDI and 5 as well as control (tissue culture polystyrene) based on (a) neurite length, (b) neurite density, (c) branch length, and (d) branch density.

Cell behavior was analyzed by measuring neurite length, neurite density, branch length, and branch density as a function of time, and shown in Fig. 8. When compared to the control (tissue culture polystyrene) and, to previous studies^{5,14–17,19,20} neurite length at day 1 was lower in all the alginate aerogels by a two-fold difference (Fig. 8a). It is important to point out that previous investigations have consistently demonstrated that aerogels enhance the regeneration rate of neurites.^{5,14–17,19,20} For the first time, it is reported that change in the physical environment of neurons has led to temporary disruption and suppression of the outgrowth rate, caused by the drastic change in interface area, which in turn leads to substrate stiffness change. After the initial change stabilized, the outgrowth rate recovered and was consistent with previous observations.

Overall, for all aerogel types, neurite length increased with time and exceeded the lengths reported for the controls. Neurite length increased 75%, 90%, 217%, and 320% when cultured on control, 2HDI, 2PDI, and 5, respectively. The optimal surface roughness (S_a) for maximum regrowth rate was previously established to be 0.5 µm.⁵ 2HDI, 2PDI, and 5 have S_a values of 0.2, 0.22, and 0.3 µm respectively. Type 5, with a S_a value of 0.3 µm resulted in the fastest outgrowth and regeneration rate confirming our previous observations.

Neurite density represents the average number of neurites per cell body. For the control sample, there was no significant change in the neurite density between days 1 and 3. However, aerogels 5, 2HDI, and 2PDI showed an increase of 38%, 75%, and 96% respectively (see Fig. 8b). It is hypothesized that the rapid initial geometry change negatively impacts the extension of neurites. This change stabilizes by day 3 which leads to a recovery of regeneration rate and neurite density that can be seen in Fig. 8a and b. A similar trend was observed when evaluating branch length and density and comparing day 1 behavior with day 3 (Fig. 8c and d).

A significant increase in the number of branches per cell and branch length was observed for aerogels 2HDI and 5 on day 3 compared to day 1. This increase was greater for those alginate-based aerogels with N3300 triisocyanate (2HDI and 5) than those containing N7300 triisocyanates (2PDI). This was followed by the control showing minimal increase.

4. Conclusions

This work reports on the physical properties and degradability of different X–Ca–Alg aerogels and on the effect of these aerogels on the extension of neurites by PC-12 cells. The physical properties and kinetics of degradation of X–Ca–Alg aerogel formulations strongly correlate with the type of triisocyanate used for crosslinking. From the data, it is evident that the small structural difference between the two triisocyanates (N3300 vs. N7300) was very important and has a significant impact on the stiffness and biodegradability of the resulting aerogel. This highlights that X–Ca–Alg alginate aerogels derived from N7300, a bio-based triisocyanate, contribute to the sustainable materials development for biomedical applications. Moreover, results show that chemical composition, specifically the type of triisocyanate, can be an effective tool for tuning biodegradability, offering greater control in biomaterial design.

Under cell culture conditions tested here, all alginate-based aerogels experienced a significant area change (reduction) within the first 1–2 hours after incubation. The degradation rate further increased significantly in the presence of a DC bias, for all the voltages (15, 30, and 60 V) for X–Ca–Alg 2PDI containing N7300. Therefore, the behavior of alginate-based aerogels can be further tuned by combining a DC bias with the choice of triisocyanates.

PC12 cells were successfully cultured on all alginate-based aerogels and developed neurites indicating cell viability. However, the significant area change that occurred in the initial incubation window delayed neurite outgrowth and regeneration rate. Once the change in area stabilized, the rate increased to previously observed behavior.

Author contributions

Martina Rodriguez Sala: writing – review & editing, writing – original draft, data curation, methodology, investigation, formal analysis, conceptualization. Grigorios Raptopoulos: writing – review & editing, writing – original draft, data curation, conceptualization, methodology. Patrina Paraskevopoulou: writing – review & editing, resources, data curation, methodology, funding. Omar Skalli: writing – review & editing, writing – original draft, resources. F. Sabri: writing – review & editing, writing – original draft, validation, supervision, methodology, investigation, formal analysis, data curation, conceptualization, resources, project administration, funding.

Conflicts of interest

There are no conflicts to declare.

Data availability

Supplementary information (SI) is available. See DOI: https://doi.org/10.1039/d5sm01269d.

The data that support the findings of this study are provided in the files uploaded to the system. Any other data are available from the authors upon reasonable request.

Acknowledgements

The authors are grateful to Covestro AG for their kind supply of Desmodur Ultra N3300 and Desmodur eco N7300. We also thank Prof. Nicholas Leventis for insightful discussions on the accelerated osmotic infiltration of solvent in the gels. The Special Account for Research Grants of the National and Kapodistrian University of Athens is also acknowledged for partial support.

Notes and references

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