Preparation and application of PVA/Gelatin/laminin Scaffold in Neural Tissue Engineering

Preparation and application of PVA/Gelatin/laminin Scaffold in Neural Tissue Engineering
khadijeh zeinali,¹ Mohammad Taghi Khorasani,^2,* Alimorad Rashidi,³ Morteza Daliri Joupari,⁴
1. Department of Science and Research branch, Islamic Azad University
2. Biomaterials Department, Iran Polymer and Petrochemical Institute
3. Research Institute of Petroleum Industry (RIPI)
Introduction: An example of a complex phenomenon in biology is the regeneration of nerves. If any impairment occurs in the nervous system, the recovery process will be difficult. Plus, malfunctions may occur in other parts of the body due to the fact that mature neurons cannot perform cell division. In this study, a novel neural tissue scaffold is introduced based on PVA/Gelatin/laminin to provide an alternative solution for this problem. In this method, we cross linked the PVA/Gelatin scaffolds physically using the freezethaw technique and performed a follow-up coagulation bath treatment. The gelatin/PVA solution was freeze/thawed and pre-frozen at different temperatures (i.e. -4, -80, and -196°C). Then, the scaffold was constructed using the freeze-drying method. Afterward, samples were covered with laminin protein. Next, to evaluate the effects of various parameters in the gradient temperature on the scaffold’s morphology, Scanning Electron Microscope (SEM) was employed. To perform in vitro evaluation, the P19 mouse cells were differentiated into nerve cells on scaffolds. Finally, an immunofluorescence test was administered. As confirmed by the experimental results, this model can be applied as a suitable platform for neural tissue engineering.
Methods: 2.1. Materials For our study, gelatin was provided from Merck (Darmstadt, Germany), while MTT (3-[4.5-dimethylthiazol-2-yi]-2.5-diphenyltetrazolium bromide) and PVA (with molecular weight between 85000–124000 and alcoholysis∼99%) were obtained from Sigma-Aldrich. Cell lines L-929 (mouse fibroblast cells) and P19 (mouse embryo carcinoma) were obtained the National Institute of Genetic engineering and Biotechnology, Tehran-Iran. Alpha minimum essential medium, Trypsin-EDTA, and the Fetal Bovine Serum (FBS) were obtained from Gibco BRL laboratories, Germany. 2.2. The Porous Gelatin/PVA Scaffold Preparation Process To prepare the necessary Porous gelatin/PVA scaffold, we employed aqueous solutions with 10% PVA (by weight) and various gelatin amounts, (i.e., 3%, 5%, and 7%, by weight). The (10%w/v) PVA solution was produced at 90°C. Next, the produced PVA solution was combined with gelatin with a ratio of 1:10 V/V. Once the mixture was homogeneous at T=60 °C, the mixture was cross-linked physically in three freeze-thaw cycles, including freezing for 24h at -4°C, thawing for another 24h at 21°C (room temperature). After that, the freeze-thaw cycle is repeated (due to the significance of PVA and gelatin-bonding hydrogen in creating 3D structures during the nucleation and growth) so that the gelatin porous hydrogels are prepared. Finally, lyophilization was carried out on the frozen samples for 24 hours. During the freezing process, when the chain of the polymer was frozen at -4, -80, and -196°C, nucleation was taken place inside the polymer solution, as shown in Fig 1.[12, 13] . Fig. 1. The process for the Preparation of porous gelatin/PVA scaffold 2.3. Covering the scaffolds with laminin After the surface of the scaffolds is activated with plasma, to enhance the biocompatibility of the specimens with the cell, their surface is coated with laminin L2020. To this end, the samples were placed in a laminin solution with a concentration of 100 µg/ml for 4 h at 4 °C, immediately after activation with plasma. Once the surface of the scaffolds was bound with the laminin, they are washed several times using a PBS solution. Then, the samples were stored in the refrigerator for cellular culturing. 2.5. Analysis and Characterization 2.5.1. Scanning Electron Microscopy (SEM) The scanning electron microscope employed for capturing the images in this study was an AIS-2100, by Seron Technology, Korea. Plus, examinations were performed at room temperature. Sputter coating with gold was performed on samples before capturing the images to create conductive surfaces. For this aim, before performing the measurement, the samples were coated for 10 minutes with Au. Moreover, horizontal and/or vertical cross-sections were prepared via cutting the N2(l) to facilitate observation of the inner structures of the scaffolds from different directions. Furthermore, before performing SEM observation at 15 kV, the samples were freeze-dried and coated with Au. 2.5.2. Measurement Method for the Cavities’ Average Diameter An electron microscope was employed to perform the measurement for the average diameter of the cavities in the scaffold’s structure. To this aim, 30 cavities were randomly selected from the images obtained from each sample, and their diameters were determined using image-j software, while their average diameters were determined accordingly. Moreover, porosity was found fully bound that facilitated food transfer. 2.5.3. Swelling To determine the ratio of adsorption for the Phosphate Buffered Saline (PBS) caused by the scaffolds produced, the samples dried in freeze-dryer were initially weighed. Next, the weighted samples were placed for 24 h on a shaker in PBS with pH = 7.4 at room temperature, while the PBS solution was refreshed multiple times. Using a paper filter, the water on the surface of the samples was removed. Then, the samples with solution were weighed, while the sample’s absorbance ratio was derived according to the following equation: WPBS= (w2-w1)/w1*100 W1= The weight for the dry samples W2= The weight for the samples following immersion in PBS solution WPBS= PBS absorption percentage 2.5.4. Cellular attachment The reaction occurred between the scaffold and the cell culture was investigated during a number of in vitro cell culture trials. In these experiments, L929 mouse fibroblasts cells were cultured in culture medium (RPMI-1640) containing penicillin (100 µg /mL), streptomycin (100 µg/mL), and supplemented with fetal calf serum (10%). Prior to seeding, a cell suspension of 4×104 cells/mL was prepared. Samples were sterilized with 70% ethanol, followed by washing in the culture media. The samples were placed in a 6 well plate and cell suspension (5 mL) were added to each well keeping one well as control, plate was maintained in a CO2-controlled incubator for 48 hours at 37°C. When the incubation period was over, all samples were washed with phosphate-buffered saline solution. The cells were fixed using glutaraldehyde 2.5%. Finally, the cells were dehydrated by ascending grade of ethanol at 60, 70, 80 and 95%. 2.5.5. MTT Assay For the cytotoxicity test of the MTT, we used the L929 mouse fibroblast cells. First, a general examination was performed on the cells to ensure their proper condition. This task was carried out via observing the cellular proliferation, the morphologic structure of the flask containing the cell, and culture medium. Samples were sterilized with 70% ethanol and cell concentration was adjusted to 4×104 cell/ml. Scaffolds were placed in a 24 well plate with control well without sample and to each well added 1 ml of cell suspension. After incubating at 37 °C in a 5% CO2 and 95% humidity incubator for 48 hours, 100 μL of MTT solution (0.5 mg/ml) was added to each well and incubated at 37 °C for 4 h. Following the removal of culture medium, acidified isopropanol was added in order to dissolve the formazan crystals. The optical density of formazan was measured spectrophotometrically at 570 nm using an ELISA plate reader. This absorbance value is proportional to the number of viable cells. Each of the samples and control was plated in triplicate MTT assays 24 hours. 2.5.6. Co-Culturing and Differentiation of Cells P19 mouse embryonic carcinoma cells were used in this study with the aim of confirming the scaffold’s suitability for the task of neural cell growth. Following the acquisition of the cells, they were maintained and cultured based on the procedure described in Robertson.[16] The P19 cells were obtained in a frozen package. Therefore, their package was melted rapidly at 37°C. Then, the maintenance of the cells was carried out in a standard environment containing α-MEM (GIBCO, Germany) and 10% FBS. Plus, the cells were supplemented by 100µl/ml Streptomycin and 100 IU/ml Penicillin and were incubated at 37°C with 5% CO2. While the cells were in the incubation, the culture medium was changed in an interval of 48 hours until the cell number of 5×104 cell/ml was reached. Then, we transferred the cells to 100 mm bacteriological containers. The cultural medium utilized in this stage included α MEM (GIBCO), 0.3 μM retinoic acid (Sigma), and 10% FBS in 100mm bacteriological Petri dish. Cell culturing continued for four days and the medium was changed regularly every 48 hours. Several cell aggregates formed during the culturing period. Aggregations formed in the cell were mechanically separated and were transferred into specific cell cultures that contained scaffolds in an acidic and non-retinoic environment. This process continued for fourteen days. The cell’s differentiation and growth during this period were observed using an optical microscope, while the negative control was considered the cultivation dishes in order to demonstrate the cell’s growth. The culturing medium was discharged slowly after the 14th day in order to stabilize the cells grown on the scaffold. Then, to remove the dead cells, the scaffolds were washed for 5 minutes using PBS and dehydrated via immersing for 5 min in a range of ethanol-water solutions with 90, 80, 70, 60, 50, and 100% v/s. Then, we washed the samples for another 5 minutes. For the final drying, we maintained the scaffolds for 24 h in the ambient temperature. Finally, after the culturing and once the P19 cells were differentiated into scaffolds, we investigated the samples under SEM and using optical light microscopy techniques.[17] 2.5.7. Immunofluorescence Staining The aim of performing immunohistochemistry was to demonstrate a distinction between nerve cells and P19 cells on scaffolds using monoclonal antibodies. Washing the samples was carried out using PBS in a four-step process with intervals of 5 minutes. For duration of 30 minutes, 2% normal chloride acid was added to the samples in order to recover the antigen. Moreover, a borate buffer was added to the samples to neutralize the acid for a duration of 5 minutes. Then, the cells were washed via PBS. Furthermore, 0.3% triton was utilized for a duration of 30 minutes to make the cell membranes penetrable. 10% goat serum was added to the sample to act as an extra background color in order to block the secondary antibody response for 30 minutes. To this aim, using PBS, the diluted antibodies (1 to 100) were first added to the sample. Then, after the wetting medium was prepared, the sample was maintained in a refrigerator overnight at a temperature between 2°C and 8°C to maintain the sample’s wetness. The sample was removed the next day and was washed using PBS 4 times for 5 minutes. The secondary antibody, with a dilution range between 1 and 150, was added to the sample afterward. The obtained sample was then maintained for one and a half hours in an incubator in a dark place at 37ºC and was transferred to a dark room afterward. Next, the sample was washed four times and DAPI was added to it. This DAPI was removed immediately and was replaced with PBS. Finally, in order to confirm the markers, the obtained samples were observed under a 400mm lens using an Olympus Fluorescent Microscope.
Results: 3. Results and Discussion 3.1. Scanning Electron Microscopy (SEM) The freeze-drying method was utilized in this study to produce the PVA/gelatin scaffolds. As illustrated in Figure 2, the electron microscope utilized captured two types of vertical and horizontal images from the scaffold’s cross-sections. As can be seen, a wide range of porosity in the structure exists in the images. The existences of an expanded porosity spectrum on the scaffold is indicative of initial temperature conditions, while the porosity distribution and the pore size is determined using the heat transfer. The effect of the temperature of the polymer on the scaffold’s morphology is shown in Figure 2, where freezing at a constant concentration occurred in all samples. Moreover, since the polymeric chain density is in an inverse correlation with the temperature, it can be seen that increasing the wall’s thickness was contributed to the temperature decrease in the solution, along with a decrease in the cavities' diameter. Hence, the parts in which the solvent maintained its cavity form remained smaller. Alternatively, as can be seen in Fig. 2a, the scaffolds that were produced using the freeze-drying method demonstrated large pores following freezing at -4°C. These pores were visibly noticeable in the cross-section between the cylindrical structures of a number of samples and the orientation of the scaffolds in a direction where the temperature gradient is applied. SEM observations illustrated in Fig. 2a and b revealed heterogeneous pores in a network structure with average inner-diameters of 250 µm. On the other hand, the pores in the scaffold that was prepared via freezing at the temperature of -80C had inner diameters of 85 µm, as can be seen in Fig. 2c, d. However, following freezing in N2(1), highly elastic scaffolds were prepared in the dried state by freeze-drying. Moreover, as can be seen in Fig. 2 e, f, homogeneous polygonal pores were observed. . Fig. 2. (a) SEM image from the 5% gelatin/10% PVA vertical cross-section in -4°C, (b) SEM image from the 5% gelatin/10% PVA horizontal cross-section in -4°C, (c) SEM image from the 5% gelatin/10% PVA vertical cross-section in -80°C, (d) SEM image from the 5% gelatin/10% PVA horizontal cross-section in -80°C, (e) SEM images from the 5% gelatin/10% PVA vertical cross-section in -196°C, (f) SEM images from the 5% gelatin/10% PVA vertical cross-section in -196°C. As can be seen in Fig. 2, the freeze-dried scaffolds’ inner structure is affected by the freezing temperature via creating channels. The channel’s direction is most likely identical to the direction in which the crystals of ice were formed. If the ice were formed without applying external forces, crystals of ice will form with a random form and with no dominant direction. However, if an external stress is applied to the crystals, (e.g. rapid cooling) the resulting ice will be oriented according to the temperature gradient that is induced within the gelatin/PVA scaffold. Therefore, the size of the pore may be reduced only alongside the scaffold’s horizontal cross-section (i.e. in perpendicular to the gradient), while it will be enlarged towards the scaffold vertical cross-section (i.e. along its axis). The most internal structure in the scaffold freezes at -196°C. In this layer, very thin connections exist between the walls, which are mostly due to the fact that at -196°C, the environment provides crystal orientation. Moreover, in this temperature, ice crystallization occurs randomly. A hypothesis regarding the structure of the inner porous modified via variation in the freezing temperature is illustrated in Fig. 2e and f. The varying thickness between walls and interconnections in the scaffold that was produced at -196°C is the cause for the difference in orientations and times. The polygonal shapes are particular shapes of ice. In specific, marks of freezing at low temperatures are similar to the freezing of liquid nitrogen. However, it is unclear whether the individual crystals were shaped into a new alignment from their initial spherulitic association and thus, experienced a reduction in size, or they melted when the liquid nitrogen penetrated and therefore, were formed with new orientations.[12] 3.2. Swelling An important factor in the design of the scaffolds is swelling since nutrients should penetrate the scaffolds, while the produced wastes generated by the cellular metabolism should exit. Therefore, in designing the scaffolds, the utilization of hydrophilic scaffolds is more desirable. As can be seen in Table 1, the high amount of inflation in these scaffolds is in relation to their highly porous structure. The level of water absorption in a scaffold for tissue engineering not only is effective in its structure and morphology, but it is also effective in the growth of the cells. If the absorption of the water was occurred generally within the gelatin hydrogel walls, then it is expected that the gelatin scaffolds that are prepared using high concentrations of gelatin would be capable of demonstrating higher levels of water absorption, but the experimental result obtained showed a contrary outcome. Therefore, we believe that the storage of the water occurred mainly in the porous space in the gelatin scaffolds. On the other hand, gelatin scaffolds that were prepared using low concentrations of gelatin possessed high porosity and large pore size and thus, offered more water storage capacity and yielded higher absorption. As confirmed by the experimental results obtained, absorption of water in the scaffolds is in relation to the scaffold’s porosity that is adjusted via modifying the concentration of gelatin during the freezing process. -196 c -80 c -4 c Sample 3 Sample2 Sample1 PVA/gel 3% 848 727 414 1h 881 805 545 3h 968 914 748 6h 1547 1231 1120 24h Sample6 Sample5 Sample4 PVA/gel 5% 568 547 492 1h 807 684 589 3h 904 821 698 6h 1606 1556 1380 24h Sample9 Sample8 Sample7 PVA/gel 7% 371 344 302 1h 608 530 468 3h 855 789 741 6h 1363 1326 1331 24h 3.3. MTT Assay The results from the MTT test on scaffolds following 48 hours of culturing of the cells are demonstrated in Fig. 3. In specific, the cell’s survival ratio on the scaffolds was measured using the formazan absorption rate. The cell’s growth ratio can be seen at 570nm wavelength in the diagrams illustrated. Cell viability of 73% is a standard value for the nontoxicity of the biomaterial samples. All the samples produced in this study were biocompatible after 48 hours. This biocompatibility implies a lack of toxicity in L-929. The scaffold’s cell viability and the L-929 cell contact were 85%. Fig. 3. MTT assay for a scaffold 3.4. Cell Culture The images obtained using the SEM can display the cell’s overall interaction, structure, and adhesion to the samples. In the images obtained from the scaffold, the cell’s interconnection is illustrated clearly. However, due to the porous structure of the scaffolds and their respective cell penetration, obtaining appropriate images is difficult. The penetration of cells to the porous structure is demonstrated in Fig. 4. However, it is worth mentioning that due to the non-uniform surface of the structure, distinguishing the cells from the roughness over the surface is difficult. In any case, images with high quality were utilized in this article. Fig. 4. (a) The images captured using the optical microscope from the interaction between negative control and the fibroblast cells after 48 hours culture, (b) The sample from the scaffold after 48 hours culture. 3.5. P19 Cells Culturing and Differentiation The images captured using an optical microscope for the control samples (i.e. the samples that did not have scaffold) and the scaffolds after 8 and 14 days of culturing are demonstrated in Fig. 5. In this figure, cell aggregation is seen around the scaffolds. Moreover, it can be seen in the images for the 14th day that cellular connections were generally disconnected from the cells and made a connection to other cells. According to the images, over a course of 14 days, there was a complete change in the P19 cells and there was the emergence of the appendixes in the form of axons and neuronal dendrites. In addition, there were differences in terms of cellular expansion, growth, and cellular network expansion between the samples with scaffolds and the control. It can be concluded that no detrimental effects were seen from the presence of the scaffold on the activity of P19 cells of the mice. Finally, as confirmed from the results of the study, scaffolds are a biocompatible option for P19 cells. Fig. 5. (a) Interaction between P19 mouse cells and positive control captured by the optical microscope following 8-day culture, (b) scaffold sample following 8-day culture, (c) positive control following 14-day culture, (d) scaffold sample following 14-day culture. 3.6. Staining of Immunofluorescence and DAPI To this aim, the primary antibody was applied to the microtubule-associated Protein-2 (MAP2). This protein is a specific protein that is encoded in the nerve cells. In this study, the major section under consideration was the characteristics of the differentiation process of the P19 cells. In specific, the acquisition of a neuronal phenotype was evaluated by expressing the microtubule-associated protein 2 (map-2) as a dendritic indicator in order to identify axon. The cells that were marked using MAP-2 antibodies were visible when seen under the microscope, as is demonstrated in Fig.6 In this figure, a positive reaction between green phosphorous areas and their respective antibodies was observed due to the presence of MAP-2 proteins. Since this protein represents neuronal cells, its presence confirms successful differentiation of P19 cells into neurons following the culturing process. Furthermore, the tubular morphology provided by the method for separating the phase is a proper structure for the growth and penetration of the neural cells.[18] Fig. 6. Fluorescence imaging of the cells marked with Map-2 antibody on the scaffold after 14 days of culturing, a) control, b) 5% gelatin/10% PVA in -196° Overall, the suitability of the obtained scaffolds for adhesion, differentiation, and cellular aggregation of P19 cells into nerve cells is confirmed using the immunofluorescence and the cell-culture experiments. As can be seen in the results obtained, the technique proposed in this paper is an excellent method for the construction and fabrication of scaffolds. This technique can also be utilized for applications in neuron repair. Furthermore, it facilitates porous, free-standing and flexible 3D fabrication to support neural and glial cell growth, adhesion, and differentiation for up to two weeks.
Conclusion: 4. Conclusion In this study, morphological examinations were carried out using images from the scaffolds obtained using a scanning electron microscope (SEM). The gelatin scaffold’s inner structure along with the difference in the size of the pores demonstrated the differences in the ratio of heat transfer during the scaffold’s freezing. The number of nuclei of the crystallization of ice may be fewer at higher freezing temperatures, which increases the final size of the crystals. Since larger crystals tend to expand the gelatin chains, the scaffold’s pore size will increase, leading to the destruction of the structure. On the other hand, rapid cooling may form many nuclei of ice crystals and create pores with smaller size. The fast freezing procedure in N2 (l) for a duration of 20 minutes created sponges that have smaller pores (compared to what was obtained through a slow freezing process that is carried out in a freezer and includes maintaining in -4°C for 24 hours). In other words, the difference in the pores' inner structure and their size demonstrates the differences in the ratio of heat transfer while the swollen scaffolds are being frozen. Hence, a sufficiently high cooling ratio, as in N2 (l), will be capable of extracting the crystallization heat and thus, preventing the formation of larger crystals of ice. Finally, frozen substances are dried by subliming the crystals of ice under vacuum at a temperature lower than the freezing temperature. Therefore, the freeze-dried scaffolds pore size can be exclusively controlled using the size of the crystals of ice formed in the freezing process. Formation of larger porous structures that have brittle and walls with more rapture is probably the result of larger crystals of ice that formed at lower freezing ratios. On the other hand, the N2 (l) cooling process yields a larger number of crystals of ice with smaller sizes. In this study, similar effects of the ratio of freezing on the mechanism in which scaffold’s porous microstructure was formed were studied. The temperature in which freezing occurred is effective in the freeze-dried scaffold’s inner structure through constructing channels, as shown in Fig. 5. In these channels, the direction is most likely identical to the direction of the formation of the crystals of ice. If the ice is formed with no external force, the crystals will most likely form randomly with no dominant direction. However, in the presence of an external stress, e.g. rapid cooling, crystals are oriented according to a temperature gradient that is induced in the gelatin scaffold.[12] Providing priority over the channel pores’ alignment may provide exceptional advantages in certain applications in medical engineering, including nerve regeneration. In order to provide sufficient space for extracellular matrix production and growth of the cells, most scaffolds employed in tissue engineering are highly porous. Regarding this point of view in tissue regeneration, the proposed method provides a desirable method to obtain biodegradable scaffolds that have interconnected pores to be utilized in mass diffusion control. L-929 fibroblast cells, mediated by the MTT test, were utilized to determine the scaffold’s suitability and interaction of cells. According to in vitro analysis, a significant increase in the metabolic activity was observed in contrast with the control, which resulted in the differentiation of the P19 cells over the surface of the scaffold. Through employing biocompatible procedure, a highly consistent, interrelated, and viable neural network was formed on scaffolds over a course of 14 days. Adapting these scaffolds to the necessary roughness and architecture requires a more comprehensive study on P19 cells' growth control, along with predicting the potential applications of these substances in the construction of neural repairers.
Keywords: neural tissue engineering, gelatin, PVA, laminin.