Effects of lamellar microstructure of retinoic acid loaded-matrixes on physicochemical properties, migration, and neural differentiation of P19 embryonic carcinoma cells

1 Introduction

Nerve disorders usually caused by thermal injuries, mechanical strikes, and diseases relating to nervous system functions such as memory, cognition, language, and movement. So the lesions can have a critical effect on individual performance [1]. When nerves get damaged by severe nerve injury (neurotmesis or axonotmesis) or neurodegenerative diseases (peripheral neuropathy), medical interventions are needed [2]. Herein, the regeneration process is affected by patient age, injury mechanism, proximity to the nerve cell, other diseases, etc. [3]. Tissue engineering is one of the best ways to assist nerve regeneration procedures [4]. Scaffold design and material type are essential factors that affect cellular interactions, so the efforts are doing to simulate the natural tissue environment [5].

Gelatin is a biocompatible polymer that was partially derived from collagen [6]. This polymer contains an Arg-Gly-Asp (RGD)-like sequence which has a low antigenicity and improves cell adhesion and migrations [7]. Due to its favorable properties, gelatin is used in nerve tissue engineering widely. Baniasadi et al. [8] used gelatin-chitosan scaffolds for nerve tissue engineering. Their observation confirmed an excellent molecular compatibility and water absorption potential of constructs that are crucial factors in cell-scaffolds interactions. In another study, Kriebel et al. [9] used gelatin matrixes containing polycaprolactone (PCL) fibers for nerve tissue engineering. They showed that gelatin facilitated implant assembly. Koudehi et al. [10] used gelatin to increase the flexibility of the scaffolds, to benefit its low immunogenicity, and to reduce concern about pathogen transmission. According to the results, these achievements may associate with the chemical compositions of constructs and their similarity to natural tissue.

Although gelatin has excellent properties, it cannot provide good mechanical strength. Moreover, rapid degradation has been observed after soaking the gelatin-based structures in a physiological environment. Therefore, adding other compounds such as polylactic-co-glycolic acid (PLGA) is recommended to enhance mechanical properties and controllable degradation rate [11]. It is a copolymer of lactic acid and glycolic acid. Its degradation rate and mechanical behavior can be easily controlled by varying the copolymer composition [12]. PLGA is widely used in nerve regeneration due to its suitable properties, such as biodegradability, biocompatibility, and low toxicity [13]. In Liao et al. [14] study, PLGA scaffolds were used to neural differentiation of bone marrow mesenchymal stem cells. The cellular analysis confirmed cell supporting and strong adherence of differentiated cells to PLGA scaffolds. Besides, this study suggested that PLGA constructs can be useful in nerve tissue engineering. In another investigation, Hassan et al. [15] applied PLGA sheets for nerve regeneration. Prepared PLGA matrix showed proper interconnectivity, so nerve cell growth occurred. In Yucel et al. [16] work, PLGA conduit was used for culturing nerve stem cells and astrocytes. Results demonstrated that conduits guide the growth and proliferation of both nerve stem cells and astrocytes.

Composite of PLGA and gelatin is a famous composition in tissue engineering because of good cellular interactions and biocompatibility. Mehrasa et al. [17], [18] used PLGA-gelatin for nerve regeneration applications. According to the results, polymeric constructs with a suitable morphology, hydrophilicity, mechanical properties, and degradation rate were fabricated. In addition, the proposed composition could support cell proliferation for nerve regeneration. They exhibited that adding gelatin to PLGA improves bioactivity and cell attachments.

The presence of small molecules such as retinoic acid (RA) shows a significant impact of differentiation of the stem cells toward neural ones and regulates the regeneration process [19]. Investigation of Robertson et al. [20] mouse olfactory bulb pathway confirmed the effectiveness of RA on the promotion of cellular behavior.

There are several methods to fabricate regenerative substitutes. Among them, freeze-casting (ice-templating), which was introduced by Deville et al. [21], has attracted a lot of attention owing to comfortable, simple, cost-effective, and environmentally friendly process. Freeze-casting technology has lots of adaptabilities so that by manipulating production factors, different characters, including pore structure, pore size, porosity rate, and pore orientation, can be controlled [22]. In this technique, first, the suspension or solution was frozen rapidly in a mold. The ice crystals were trapped in the polymer structure, and the porous structure was created via sublimation of the frozen solvent. Francis et al. [23] used this technique to fabricate the chitosan-alginate scaffolds for nerve tissue engineering. Since freeze-casting technology is not performed at high temperatures, it is possible to incorporate biomolecules in fabricated constructs. Their results revealed that this technology could create appropriate nerve scaffolds with sustained drug releasability. Linear aligned pores produced by gradient freezing can be beneficial for nerve tissue engineering. Accordingly, Francis et al. [24] used this method to fabricate a linearly oriented structure, which is achieved by directional solidification of the water-based solutions. The resultant scaffold was very porous and a linear aligned structure with continuous channels was fabricated. The direction of neuron growth are essential factors for nerve regenerations. The other investigation indicated that freeze-cast constructs can act as contact guidance in regeneration of neural lesions [25].

In this study, RA loaded-PLGA-gelatin scaffolds were fabricated with both freeze-casting and freeze-drying methods. Then, the potential of unidirectional pore channels as contact guidance on the release ratio and induction of cell adhesion, migration, and differentiation was evaluated.

2 Materials and methods

PLGA-gelatin solutions were prepared by dissolving the polymers in acetic acid with a concentration of 5% (w/v) and the ratio of 80:20 at ambient temperature under gentle stirring for 12 h. Hydrophilic functional groups were cross-linked by the addition of 0.5% (w/w) glutaraldehyde (based on gelatin concentration) to the polymeric solution. Drug-loaded solutions were prepared in a dark environment by the addition of 1% (w/w) RA to the stock solution [35].

Freeze-dried scaffolds were prepared through freezing the solution at −20 °C for 24 h. Freeze cast matrixes were fabricated via gradient freezing the solution with a rate of 1 °C/min, according to previous studies [26]. All the frozen constructs were lyophilized at −58 °C, 0.5 Torr, 48 h. The details about the synthesizing condition can be observed in table 1.

The morphology and microstructure of the scaffolds were observed by field emission scanning electron microscopy (FE-SEM, MIRA3, TESCAN Co., Brno, Czech Republic) at an accelerating voltage of 20 kV.

Fourier transforms infrared (FTIR, Nicolet Is10, USA) spectroscopy evaluated the chemical characterization of the constructs at 400–4000 cm⁻¹ wavenumber.

The absorption capacity of scaffolds was determined after incubation of samples in the PBS solution at 37 ± 0.5 °C. Accordingly, the dry weight (W₀) and wet weight (W) of constructs after 1, 3, 7, and 24 h was measured, and the swelling ratio was calculated using Eq. (1) [27]:

Biodegradation of scaffolds was calculated after incubation of samples in the PBS solution at 37 ± 0.5 °C. Accordingly, the initial dry weight (W₀) and secondary dry weight (W) of constructs during eight weeks were measured, and the degradation ratio was calculated using Eq. (2) [28]:

The release behavior was followed by immersion of 20 mg RA loaded-scaffolds in 4 mL PBS: ethanol (50:50) under shaking at 37 ± 0.5 °C and rotational speed 30 rpm. The release ratio was measured on days 1, 3, 7, 9, 14, and 21 using a UV–Vis spectrophotometer at a wavelength of 350 nm.

P19 embryonic carcinoma cell line (NCBI Code: ES7) was supplied by Pasteur Institute, Tehran, Iran, which was derived from embryonal carcinoma tissue of mice according to international ethical rules. P19 cells were preserved in DMEM supplemented with 2.5% fetal calf serum, 1% non-essential amino acids [28a], 25 mg/mL l-glutamine [28b], and 1% penicillin-streptomycin. Then, they were incubated in 37 °C, 5% CO₂ and 95% humidity by sub-culturing every 2 days.

Then, 10⁵ cells/mL P19 embryonic cell line was cultured on the surface of samples for 10 days in neurobasal medium with 2% B27. Then, the media was removed, and the cell loaded samples were fixed and dehydrated, respectively. After drying the specimens in air, the morphology of the adhered and migrated cells was observed by FE-SEM. The gene expression as a function of microstructure was followed after the differentiation of P19 cells. Accordingly, after 10 days, the total RNA was isolated from the cells through the usage of NucleoSpin RNA II Kit. Then, to remove contaminating genomic DNA, DNase I was used to digesting RNA samples. Additionally, extracted RNA was treated by DNase I to avoid cross-contamination of RNA by genomic DNA. Finally, extracted RNA converted to cDNA using reverse transcriptase (RT) and oligo (dT) primer. Polymerase chain reaction (PCR) was done in three steps, including denaturation at 94 °C for 30 s, annealing at 58 °C for 45 s, and extension at 72 °C for 45 s. The below genes with determined oligonucleotide sequences were used for the determination of differentiation. The separated products by electrophoresis were observed by ethidium bromide staining. Scaffold-free wells are considered as a control group.

β-tubulin V (housekeeping gene): (forward) 5′-GGA ACA TAG CCG TAA ACT GC-3′ and (reverse) 5′-TCA CTG TGC CTG AAC TTA CC-3′ (288 bp)

β-tubulin III: (forward) 5′-GGA ACA TAG CCG TAA ACT GC-3′ and (reverse) 5′-TCA CTG TGC CTG AAC TTA CC-3′ (288 bp)

Pax-6: (forward) 5′-GAG AGG ACC CAT TAT CCA GAT G-3′ and (reverse) 5′-GCT GAC TGT TCA TGT TTT G-3′ (435 bp)

Nestin: (forward) 5′- TCG AGC AGG AAG TGG TAGG-3′ and (reverse) 5′- TTG GGA CCA GGA CCA GGG ACT GTT A-3′ (288 bp).

3 Results and discussion

Topography and structure of the matrixes are critical factors for favorable nerve regeneration [29]. According to Chiono et al. [30] investigation, suitable nerve scaffolds should simulate a natural ECM environment and should not prohibit tissue formation. They stated that the high degree of porosity, the interconnectivity of the pores, biodegradability, and absorption capacity are important factors that affect the in-vitro performance of scaffolds, vascularization, and tissue regeneration. Notably, in the case of neurite growth, directional guidance for nerve reconstruction is necessary. The topography and surface properties of fabricated scaffold affect tissue ingrowth and aligned microstructure are crucial for the directional guidance of axons [30].

Although freeze-drying is a useful and straightforward fabrication method with several advantages, there is no control over the microstructure of the pores [31]. The freeze-casting method somewhat overcame this problem. In the freeze-casting technology, prepared solution pours into a PTFE mold and freezing happens according to the predetermined rate. Then, the frozen solvent sublimates and removed ice crystals lead to the formation of pores so that the pattern of solidification affects pore structure. In the case of unidirectional freezing, the resulting scaffolds have unidirectional pores [32].

Figure 1A–D demonstrates the morphology of PGD and PGC scaffolds. As can be seen, both scaffolds have a porous structure with open pores, which is an essential parameter in cell adhesion and proliferation. In the parallel direction compared with solidification, the pores are distributed in the range of 50–250 µm and 50–350 µm for PGD constructs and 50–250 µm and 50– 550 µm for PGC scaffolds in horizontal and vertical measurements, respectively. The measured values and pore size distribution indicated the formation of spread pores according to the freezing direction and proved the unidirectional microstructure of PGC matrixes. Highly porous scaffolds provide more space for nutrition transfer, oxygen supply, and extraction of waste by-products between cells and the microenvironment [25]. The directional pores in the freeze-casting method facilitate the fluid flow, provide a more favorable environment for cell growth compared with the freeze-drying one, and may lead to longitudinal outgrowth of axons, in-vitro uniform alignment of Schwan cells, and better tissue regeneration [33].

Figure 1:

FE-SEM micrographs of freeze-dried (A, C) and freeze-cast (B, D) PLGA-gelatin-RA scaffolds with different magnification. FTIR spectra of raw materials and drug-loaded composite matrixes (E).

Fourier Transform Infrared Spectroscopy (FTIR) was performed on the raw materials and PLGA-gelatin composite to identify chemical interactions. The obtained spectra are shown in Figure 1E. According to spectra, the broad peak in 3200–3600 cm⁻¹ is related to N-H and O-H stretching vibration. The detected peaks at 1633 and 1525 cm⁻¹ are assigned to C=O and N-H stretching vibration of amide I and amide II, respectively. In addition, the existence of amide III leads to a peak detection at 1230 cm⁻¹[34]. In PLGA spectra, the observed peaks at 1725 and 1130 cm⁻¹ are related to the C-O bond, and the detected peak at 1452 cm⁻¹ can be attributed to the C-H bond. The hydroxyl group of the PLGA peak is observed in 3433 cm⁻¹[35]. The observed peaks at 1255 and 1690 cm⁻¹ are assigned to C-O stretch vibration and carbonyl functional groups in the chemical structure of RA [36]. In the composite FTIR spectra, the broad peak at approximately 3400 cm⁻¹ is related to the intramolecular hydrogen bonding between gelatin and PGLA substrate. On the other hand, the detected peaks approximately between 1470 and 1570 cm⁻¹ are assigned to cross-linking reactions of gelatin by glutaraldehyde.

The potential of scaffolds to interact with water molecules was determined with a swelling experiment, as indicated in Figure 2A. Accordingly, the scaffolds could absorb a high level of PBS within the first hour, and after that, the swelling ratio increased with a steady gradient; however, the enhancement in the absorption capacity of PGC matrixes followed less slope until 24 h compared with PGD ones. Besides, the PGC samples indicated a higher potential to absorb PBS that can arise from a unidirectional pore structure. A similar report was published previously [37]. It should be considered that a high level of swelling in the PGC constructs can provide a suitable condition for cell interactions and promote regeneration.

Figure 2:

Absorption capacity (A) and biodegradation ratio (B) of PGD and PGC matrixes at 37 ± 0.5 °C in the PBS solution.

The biodegradation ratio of scaffolds as a function of incubation time in the PBS solution has been presented in Figure 2B. According to observations, the microstructure of pores is an effective parameter on long-lasting the constructs since the applied materials and cross-linking procedure are similar in both structures. Herein, the PGC scaffolds with unidirectional pore channels lost lower weight within eight weeks, and the degradation process occurred by a slower gradient compared with PGD constructs. Although PGC matrixes showed a higher absorption rate than PGD ones, lower biodegradation may arise from higher stability and integrity of scaffolds that prevent sudden collapsing. Other investigations demonstrated that regeneration of the injured nerve starts after 1–3 months of implantation [38]; accordingly, the biodegradation rate in PGC samples can supply required stability and provide a balance between regeneration and biodegradation.

The in-vitro retinoic acid release mechanism from PLGA-gelatin scaffolds, which are prepared with both freeze-casting and freeze-drying techniques, were investigated based on thermodynamics and kinetics viewpoint. The mechanism of release depends on the fabrication method since it can occur via various mechanisms such as diffusion, erosion, desorption and degradation, swelling, or different contributions simultaneously [39–41]. A simultaneous examination of thermodynamics and kinetics viewpoint can provide useful information about the release mechanism [42]. The Gibbs free energy is the most important thermodynamic parameters for describing the release mechanism since it can describe chemical events in the drug transfer process. Also, it can be evidence of structural resistance against drug release. Hence, the Gibbs free energy transfer values of retinoic acid, ΔGtr0, from PGD and PGC matrixes were calculated by following equation [43] (Eq. (3)):

where R and T are the gas constant and the temperature, respectively. Also, CT and Ct represent the total concentration of retinoic acid and the concentration of retinoic acid during release time, respectively [40, 43], respectively. As shown in Figure 3A, the values of Gibbs free energy transfer for retinoic acid from freeze-cast PLGA-gelatin scaffolds are more spontaneous than the freeze-drying ones at 37 ± 0.5 °C. The difference arises from the structural difference in applied methods. Directional porous structures in PGC constructs can be responsible for the observed phenomenon. The values of Gibbs free energy transfer showed that PGD matrixes are rigid so that they resist structural changes. In other words, the properties of directional porous structures allow more retinoic acid release. Thus, according to the thermodynamic point of view, it can be concluded that the PGC scaffolds have a convenient release because of unidirectionally oriented pores.

Figure 3:

The plot of Gibbs free energy changes of retinoic acid transfer (A) and the release profile of retinoic acid (B) from PGD and PGC matrixes at 37 ± 0.5 °C.

The release profile of retinoic acid from PGD and PGC scaffolds is shown in Figure 3. The results revealed that the maximum release occurred in prepared constructs with freeze-casting. The release data were fitted in mathematical models to indicate the release kinetics.

The best model for retinoic acid release was selected based on the correlation coefficient (R²) value. The mathematical kinetics models used for fitting data were zero-order, first-order [44], Weibull [45], Higuchi [46], Hixson–Crowell [47], and Korsmeyer–Peppas [48]. (Eqs. (4–9)):

where t represents the release time; M₀, M_t, and M_∞ represent the amount of drug released at time zero, t, and infinity, respectively. The parameters w₀ and w_t in the Hixson-Crowell model represent the drug weight at time zero and t, respectively. The parameters k₀, k₁, k_H, k_HC, and k_KP are the release kinetic constants in zero-order, first-order, the Higuchi, Hixson–Crowell, and Korsmeyer–Peppas models, respectively. Also, variables a and b are constants in the Weibull model. Besides, variable n in the Korsmeyer-Peppas is the release exponent, so that indicates the release mechanism of the drug. The fitting results are summarized in Table 2.

The fitting results explored that release data were well fitted to the Korsmeyer–Peppas model. Accordingly, the release exponent in Korsmeyer–Peppas model was calculated. Hence, the release exponent value determines the release mechanism. Herein, n=0.5, 0.5 < n < 1, n=1 and n > 1 represent Fickian diffusion, anomalous (non-Fickian) diffusion (i.e. by both diffusion and erosion [49]), case II transport (zero-order (time-independent) release), and super case II transport, respectively. So, the mechanism of retinoic acid release from PGD and PGC scaffolds has an anomalous (non-Fickian) diffusion because the release of exponent values are located between 0.5 and one in both conditions. The results are summarized in Table 3. According to the diffusion and erosion processes that co-occur for retinoic acid release, the Kopcha release model was used to indicate the exact contribution of diffusion and erosion using the following equation [41] (Eq. (10)):

Where A and B represent the diffusion and erosion terms, respectively, and t represents the release time. The A to B ratio (A/B) indicates the diffusion and erosion contributions in the release mechanism. The contribution is represented in three forms of A/B = 1, A/B > 1, and A/B < 1 so that equal contribution between diffusion and erosion is described. Herein, the diffusion predominates over erosion, and the erosion predominates over diffusion, respectively [47–50]. The Kopcha release model parameters are summarized in Table 2. The release data were well fitted in the Kopacha model. Thus, the non-Fickian process occurs in the retinoic acid release from PGD and PGC constructs. There is no lag time and explosion in the release mechanism since the values of A parameter are not large negative or positive. Also, the A/B parameter demonstrates that the erosion is predominant over diffusion. The kinetics results are well in agreement with the obtained results from thermodynamic studies. In other words, the results illustrated that the erosion of the structure affected by the fabrication method determines the release mechanism. As a result, PGD scaffolds do not show adequate erosion due to the rigid structure. Instead, PGC matrixes have more favorable erosion due to better accessibility. The difference in erosion only caused by unidirectionally oriented pores as a result of the preparation technique. Consequently, the fabrication method affects the amount of released retinoic acid from PLGA-gelatin scaffolds.

Figure 4A and B shows the FE-SEM images of cultured P19 embryonic carcinoma cells on the surface of PGD and PGC scaffolds, respectively. Another study demonstrated that PLGA-gelatin composition has a hydrophilic characterization, which is a critical requirement for cell adhesion and growth [51]. FE-SEM images of cultured cells on the PGC and PGD scaffolds presented that a higher density of cells could attach well on the freeze-cast matrixes, and the spreading has been improved significantly so that the adhered and spread cells could cover the surface completely. Note that pore shape, size, and interconnectivity are affecting important parameters on cell adhesion and spreading. Accordingly, it can be concluded that the unidirectional pore channels in PGC matrixes promote cell-scaffold interactions through facilitating nutrition and cell by-products flow and may provide a contact guidance for cultured cells.

Figure 4:

Adhesion and differentiation of P19 embryonic carcinoma cells on the PLGA-gelatin-RA scaffolds. FE-SEM images of P19 embryonic carcinoma cells, cultured on the surface of the PGD (A) and PGC (B) scaffolds. Expression of neural genes including, β-tubulin III, Pax-6, and Nestin after differentiation of P19 embryonic carcinoma cells on the control group (C1, D1, E1, and F1), PGD (C2, D2, E2, and F2), and PGC (C3, D3, E3, F3) matrixes. [β-Tubulin V (C1-C3), β-tubulin III (D1-D3), Pax-6 (E1-E3), and Nestin (F1-F3)].

The effect of microstructure on the expression of neural genes and cell differentiation has been indicated in Figure 4 (C1–C3, D1–D3, and E1–E3) and has been proved by a reverse transcriptase-polymerase chain reaction. Herein, the effect of RA on the expression of β-tubulin III, Pax-6, and Nestin should not be ignored. Other literature reported similar observations [52, 53]. However, since both scaffolds have similar composition, the improved differentiation of the cells and expression of neural genes may arise from unidirectional pore channels of freeze-cast scaffolds that act as a guide in the cell differentiation process. In addition to channel-like structures, a higher release rate in PGC scaffolds can accelerate cell access to required differentiation factors and promote the expression of genes.

In order to investigate the cellular migration inside the freeze-dried and freeze-cast scaffolds, the cultured scaffolds with P19 embryonic carcinoma cells were cut, and FE-SEM observation was performed on the cross-section side. Figure 5 demonstrates the FE-SEM images of migrated cells inside the PGD and PGC scaffolds. Accordingly, there is no sign of cell diffusion in the FE-SEM images of the freeze-dried scaffold, and the adhesion is restricted to the surface. The observation indicated that the randomly oriented pores could not support migration and penetration of the cells inside the scaffold. In contrast, the cross-sectioned FE-SEM images of the freeze-cast scaffolds showed that the cells well migrated, adhered, and formed filopodia that suggested suitable microenvironment for the cells. This migration can provide 3D tissue formation and may enhance nerve regeneration.

Figure 5:

FE-SEM micrographs of PGD (A) and PGC (B1, B2) cross-sectioned-scaffolds after culturing the P19 embryonic carcinoma cells. PGC scaffolds act as contact guidance, and migrated cells inside the constructs are obvious.

Contact or topographic guidance is the tendency of cell mobility to be guided physically under topographical features such as grooves, fiber, edges, etc. [54]. This mechanism is performed by physical/structural cues from the cell, tissue, and ECM components at both nano and microscale [55]. Oliveira et al. [56] study demonstrated that the different morphological features affect cellular behavior, and aligned porosity in the micrometer range can positively regulate cell growth. Besides, Gomez et al. [57] compared the influence of topographical and chemical ligand on axon formation and growth. They found that when both chemical and topographical cues were applied, the cultured cells preferred topographical cues more than chemical ones. This phenomenon demonstrated that topography is a more potent stimulus for cell guidance. So topography and pore structure of fabricated scaffold is a critical factor that should be considered.

Furthermore, in Riblett et al. [58] study, it was concluded that the freeze-casting method has the capability of creating an aligned and rigid porous structure that can guide Schwann neural cell. The contact guidance of created grooves without any chemical or electrical stimuli provides a suitable condition for neuron growth. Johnson et al. [59] study demonstrated that aligned neurons express aligned adhesive ligands, such as fibronectin, laminin, and neural cell adhesion molecule (NCAM). The aligned channels of substrate lead to neurite growth and neurites migration neurites according to the pattern of structure.

Although the exact effect of topography on the cells is not investigated thoroughly, it seems that topographical features improve the focal adhesion complexes (FAC) reorganization. This phenomenon can regulate cytoskeletal tension, activate signal transduction, and express genes [60]. Integrins are the main physical and topographical reorganized molecules that provide the assembly of FACs (scaffolding proteins such as vinculin, talin, etc.). FACs link integrins in the actin filaments of the cytoskeleton and lead to cell shape deformations [61].

4 Conclusion

In conclusion, the present study determines the role of unidirectionally oriented pores on migration and differentiation of the cultured cells on the PLGA-gelatin-RA scaffolds. It was concluded that the freeze-casting method has the capability of creating a porous directional structure that is an effective factor in cell adhesion and spreading. Directional contact guidance provides better micro-environment for easy access of the cells to biological factors and promotes cell migration and penetration. Also, promoted expression of the neural genes in lamellar microstructures confirmed the potential of constructs for peripheral nerve repair. Further preclinical and clinical studies are under evaluation and will be presented in the future.