3.5. Assessment of Cell Viability of Cryogels by MTT Assay, Trypan Blue Exclusion Assay and Live/Dead Staining
The cytotoxic impact of PVA/Gel, PVA/Gel/
P.emblica-0.5, PVA/Gel/
P.emblica-1, PVA/Gel/
P.emblica-1.5 and PVA/Gel/
P.emblica-2 cryogels on keratinocytes was investigated by the MTT assay at 24th, 48th and 72nd hour (
Figure 6). The cell viability in the control significantly increased by 40.7% at 48th hour compared to 24th hour, but decreased by 4.22% at 72nd hour as compared to the 48th hour (P< 0.0001, P= 0.0663). Also, the cell viability in PVA/Gel cryogel significantly increased by 29.78% and decreased by 6.99%, respectively (P< 0.0001, P= 0.0006). In PVA/Gel/
P.emblica-0.5 cryogel, it increased significantly by 46.98% and 15.53%, respectively. (P< 0.0001, P= 0.0006). In PVA/Gel/
P.emblica-1 cryogel it increased significantly by 48.26% and 17.73%, respectively (P< 0.0001, P= 0.0133). In PVA/Gel/
P.emblica-1.5 cryogel, it increased significantly as 52.33% and 14.47%, respectively (P< 0.0001, P= 0.0049). In PVA/Gel/
P.emblica-2 cryogel, it increased significantly as 34.32% and 6.92%, respectively (P< 0.0001, P= 0.0272) (
Figure 6a).
In comparision with the control, cell proliferation was decreased in the PVA/Gel cryogel by 3.77% ± 2.05%, 10.90% ± 0.75%, and 13.47% ± 1.86% at 24th, 48th and 72nd, respectively (P= 0.2530, P= 0.0049, P= 0.0001, respectively). On the other hand, cell proliferation in PVA/Gel/ P.emblica-0.5 showed an increase of 10.88% ± 2.20%, 10.88% ± 2.20%, and 40.25 ± 8.36%, respectively, compared to control (P= 0.0380, P= 0.0076, P< 0.0001, respectively). The cell proliferation was higher in PVA/Gel/P.emblica-1 cryogel by 13.39% ± 7.85%, 19.93% ± 3.10%, and 47.42 ± 6.16%, respectively, compared to control (P= 0.0185, P= 0.0008, P< 0.0001, respectively). The cell proliferation was increased in PVA/Gel/P.emblica-1.5 cryogel by 16.74% ± 6.10%, 26.87% ± 5.96% and 51.63 ± 7.74%, respectively, compared to control (P= 0.0171, P= 0.0013, P< 0.0001, respectively). The cell proliferation was significantly increased in PVA/Gel/P.emblica-2 cryogel by 9.83% ± 5.31%, 5.25% ± 3.89% and 17.50 ± 6.68%, respectively, compared to control (P= 0.0574, P= 0.2082, P= 0.0039, respectively).
According to MTT assay, cell proliferation in PVA/Gel/P.emblica-0.5 cryogel was 15.22%, 24.44% and 62.09% higher at 24, 48 and 72 h, respectively, compared to PVA/Gel (P= 0.0003, P< 0.0001, P< 0.0001). Moreover, the cell proliferation was increased in the PVA/Gel/P.emblica-1 cryogel compared to PVA/Gel by 17.83%, 34.60% and 70.37% (P= 0.0014, P< 0.0001, P< 0.0001). The cell proliferation was significantly increased in the PVA/Gel/P.emblica-1.5 cryogel compared to PVA/Gel by 21.30%, 42.38% and 75.24% (P= 0.0005, P< 0.0001, P< 0.000l). Also, the cell proliferation was 14.13%, 18.12% and 35.79% higher in the PVA/Gel/P.emblica-2 cryogel compared to PVA/Gel by (P= 0.0011, P= 0.0002, P= 0.0002, respectively).
According to MTT assay, the cell proliferation in the PVA/Gel/
P.emblica-1
, PVA/Gel/
P.emblica-1.5
and PVA/Gel/
P.emblica-2 cryogels showed increases of 2.26%, 5.28% and -0.94% at 24 hour, respectively, compared to PVA/Gel/
P.emblica-0.5 (P= 0.5198, P= 0.0958, P= 0.07148). In addition, the cell proliferation significantly increased at 48 h and 72 h by 8.17%, 14.42%, -5.07% and 5.11%, 8.11%, -16.22% (P= 0.0016 P= 0.0008, P= 0.0187 for 48th hour and P= 0.0252, P= 0.0066, P= 0.00297 for 72nd h). Moreover, the cell proliferation was increased in the PVA/Gel/
P.emblica-1.5
and P.emblica-2 cryogel compared to PVA/Gel/
P.emblica-1 cryogel by 2.95% and -3.14% respectively at 24th h (P= 0.5232, P= 0.3486). Also, the cell proliferation significantly increased at 48th and 72nd hours by 5.78%, -12.24% and 2.85%, -20.30% (P= 0.0479, P= 0.0001 for 48th hour and P= 0.0379, P= 0.0010 for 72nd hour, respectively). Also, the cell proliferation was decreased in the PVA/Gel/
P.emblica-2 cryogel compared to PVA/Gel/
P.emblica-1.5 by -5.91%, 17.04%, 22.51% respectively at 24, 48, 72 hours (P= 0.967, P= 0.0005, P= 0.0014, respectively) (
Figure 6b).
Trypan blue assay was performed to investigate the efficacy of PVA/Gel, PVA/Gel/
P.emblica-0.5, PVA/Gel/
P.emblica-1, PVA/Gel/
P.emblica-1.5 and PVA/Gel/
P.emblica-2 cryogels on keratinocytes viability at 24th, 48th and 72nd h (
Figure 7). In comparision with control, the cell number of PVA/Gel cryogel was declined by 7.23% ± 8.45%, 7.84% ± 3.81%, and 10.94% ± 3.47%, respectively (P= 0.0913, P= 0.0471, P= 0.0641 respectively). Moreover, the cell number in the PVA/Gel/
P.emblica-0.5 cryogel was significantly increased by 11.45% ± 7.69%, 20.41% ± 2.3%, and 39.29 ± 4.72%, respectively, compared to control (P= 0.0177, P< 0.0001, P= 0.0003, respectively). Also, the cell number in the PVA/Gel/
P.emblica-1 cryogel was increased by 31.93% ± 10.36%, 43.71% ± 3.94%, and 40.85 ± 2.31%, respectively, compared to control (P= 0.0002, P< 0.0001, P= 0.0001, respectively). The cell number in the PVA/Gel/
P.emblica-1.5 cryogel was significantly increased by 48.19% ± 4.88%, 56.08% ± 4.10% and 67.63 ± 3.83%, respectively, compared to control (P< 0.0001 for all). The cell number in the PVA/Gel/
P.emblica-2 cryogel was significantly increased by 1.41% ± 2.69%, 8.87% ± 2.07% and 10.71 ± 4.99%, respectively, compared to control (P= 0.0585, P= 0.0013, P= 0.0393, respectively).
According to trypan blue assay, the number of cells in the PVA/Gel/P.emblica-0.5 cryogel was increased by 20.13%, 30.65% and 56.39% at 24, 48 and 72 hours compared to PVA/Gel, respectively (P= 0.0061, P< 0.0001, P< 0.0001). The number of cells was 42.21%, 55.93% and 58.15% higher in PVA/Gel/P.emblica-1 cryogel compared to PVA/Gel (P= 0.0004, P< 0.0001, P< 0.0001). The cell number was significantly increased in the PVA/Gel/P.emblica-1.5 cryogel compared to PVA/Gel by 59.74%, 69.35% and 88.22% (P< 0.0001, for all). In addition, the number of cells was 9.31%, 18.12% and 24.31% higher in the PVA/Gel/P.emblica-2 cryogel compared to PVA/Gel (P= 0.0766, P= 0.0007, P= 0.0005, respectively).
According to trypan blue assay, the viable cell number was significantly increased in the other PVA/Gel/P.emblica-1, PVA/Gel/ P.emblica-1.5 and PVA/Gel/ P.emblica-2 cryogels compared to PVA/Gel/P.emblica-0.5 by 18.38%, 32.97% and -9.01%, respectively at 24 h (P= 0.0139, P= 0.0002, P= 0.0379). In addition, the cell number significantly increased at 48 and 72 hours by 19.35%, 29.62%, -9.59% and 1.12%, 20.35%, -20.51% (P< 0.0001, P< 0.0001, P= 0.0006 for 48 h and P= 0.4492, P= 0.0003, P= 0.0008 for 72 h). Furthermore, the cell number in the PVA/Gel/P.emblica-1.5 and PVA/Gel/P.emblica-2 cryogel showed significant increases of 12.33% and -23.14% compared to PVA/Gel/P.emblica-1 cryogels at 24 hour, respectively (P= 0.0047, P= 0.0004). Also, the cell number significantly increased at 48 and 72 hours by 8.61%, -24.25% and 19.02%, -21.39% (P= 0.0024, P< 0.0001 for 48 h and P< 0.0001, P< 0.0001 for 72 h). Also, the cell number was significantly decreased in the PVA/Gel/P.emblica-2 cryogel compared to PVA/Gel/P.emblica-1.5 by 31.57%, 30.25%, 33.95% at 24, 48, 72 h, respectively (P< 0.0001 for all).
A live/dead assay was performed to evaluate the cell viability of cells, where green fluorescence indicated viable cells and red fluorescence indicated dead cells. (
Figure 8). Cells were seeded on PVA/Gel, PVA/Gel/
P.emblica-0.5, PVA/Gel/
P.emblica-1, PVA/Gel/
P.emblica-1.5 and PVA/Gel/
P.emblica-2 cryogels for 24th, 48th and 72nd h. As seen in
Figure 8a–c relatively similar numbers were determined in live and dead cells cultured in control and PVA/Gel/
P.emblica-0.5, PVA/Gel/
P.emblica-1, PVA/Gel/
P.emblica-1.5 and PVA/Gel/
P.emblica-2 cryogels for all hours. The cells cultured on the PVA/Gel
P.emblica-0.5, PVA/Gel/
P.emblica-1, PVA/Gel/
P.emblica-1.5 and PVA/Gel/
P.emblica-2 cryogels exhibited a higher percentage of green fluorescence, which indicates viable cells compared to the PVA/Gel. The majority of cells cultured on the PVA/Gel/
P.emblica-0.5, PVA/Gel/
P.emblica-1, PVA/Gel/
P.emblica-1.5 and PVA/Gel/
P.emblica-2 cryogels emitted green fluorescence, which indicates a high level of cell viability and cell proliferation. As shown in 8a, 8b and 8c, the addition of
P. emblica extract to the PVA/Gel cryogels did not adversely affect cell viability relative to the live/dead assay. The percentage of viable (green) cells was higher in cultured in cryogels loaded with
P. emblica. Unlike controls, cells cultivated especially in PVA/Gel/
P.emblica-0.5, PVA/Gel/
P.emblica-1, PVA/Gel/
P.emblica-1.5 cryogels showed viable (green) cells with more rounded contours. In addition, compared to PVA/Gel cryogels, PVA/Gel/
P.emblica-0.5, PVA/Gel/
P.emblica-1, PVA/Gel/
P.emblica-1.5 and PVA/Gel/P.emblica-2 cryogels have more viable cells.
According to live/dead assay, viable cells in the PVA/Gel cryogel decreased by 11.81% ± 1.38%, 12.86% ± 1.72%, and 25.77% ± 2.16% at 24, 48, and 72 hours compared to control, respectively (P= 0.0288, P= 0.0145, P= 0.0008). The viable cell ratio in PVA/Gel/P.emblica-0.5 was significantly increased by 10.48% ± 3.97%, 14.86% ± 2.74%, 34.05% ± 1.37% respectively cryogel compared to control (P= 0.0004, P= 0.0008, P< 0.0001). Also, the viable cell ratio in PVA/Gel/P.emblica-1 cryogel was significantly increased by 10.67% ± 1.03%, 17.07% ± 1.86% and 52.76% ± 0.75% respectively cryogel compared to control (P= 0.0002, P= 0.0034, P= 0.0001). Also, the viable cell ratio in PVA/Gel/P.emblica-1.5 cryogel was significantly increased by 9.33% ± 1.17%, 21.95% ± 1.79%, 66.87% ± 2.61% 10.06 respectively cryogel compared to control (P= 0.0060, P= 0.0004, P< 0.0001). Also, the viable cell ratio in PVA/Gel/P.emblica-2 cryogel was significantly enhanced by 1.52% ± 1.37%, 5.99% ± 1.03%, 15.03% ± 1.63%, respectively cryogel compared to control (P> 0.9999, 0.0342, P> 0.9999). Moreover, the viable cells in the PVA/Gel/P.emblica-0.5 cryogel increased significantly by 25.27%, 31.38% and 80.58%, respectively compared to PVA/Gel (P= 0.0042, P= 0.0017, P< 0.0001). Also, the viable cells in the PVA/Gel/P.emblica-1 cryogel increased by 25.49%, 34.35% and 105.79%, respectively compared to PVA/Gel (P= 0.0026, P= 0.0005, P< 0.0001). The viable cells in the PVA/Gel/P.emblica-1.5 cryogel increased by 23.97%, 39.95% and 129.79%, respectively compared to PVA/Gel (P= 0.0069, P= 0.0003, P< 0.0001). The viable cells in the PVA/Gel/P.emblica-2 cryogel increased significantly by 15.12%, 21.63% and 54.96%, respectively compared with PVA/Gel (P= 0.0361, 0.0036, P= 0.0002).
According to live/dead assay, dead cells in the PVA/Gel cryogel increased by 82.67% ± 1.38%, 38.93 % ± 1.72%, and 30.66 % ± 2.16% at 24, 48, and 72 hours, respectively, compared to control (P= 0.0288, P= 0. 0145, P= 0.0008). Furthermore, the dead cell ratio in PVA/Gel/
P.emblica-0.5 was decreased significantly by 73.33% ± 3.97%, 44.97% ± 2.74%, and 40.51% ± 1.37%, respectively cryogel compared to control (P= 0.0004, P= 0.0008, P< 0.0001). Also, the dead cell ratio in PVA/Gel/
P.emblica-1 was significantly decreased by 74.67% ± 1.17%, 51.68% ± 1.86%, and 62.77% ± 0.75%, respectively compared to control (P= 0.0002, P= 0.0034, P= 0.0001). Moreover, the dead cell ratio in PVA/Gel/
P.emblica-1.5 was significantly decreased by 65.33% ± 1.17%, 66.44% ± 1.79%, and 79.56% ± 2.61%, respectively cryogel compared to control (P= 0.0060, P= 0.0004, P< 0.0001). The dead cell ratio in PVA/Gel/
P.emblica-2 was decreased by 10.67% ± 1.37%, 18.12% ± 1.03%, and 17.88% ± 1.63%, respectively cryogel compared to control (P> 0.9999, P= 0.0342, P> 0.9999). The dead cells in the PVA/Gel/
P.emblica-0.5 cryogel decreased significantly by 85.40%, 60.39% and 54.47%, respectively compared to PVA/Gel (P= 0.0042, P= 0.0017, P< 0.0001). Also, the dead cells in the PVA/Gel/
P.emblica-1 cryogel declined by 86.13%, 65.22% and 71.51%, respectively compared to PVA/Gel (P= 0.0026, P= 0.0005, P< 0.0001). Moreover, the dead cells in the PVA/Gel/
P.emblica-1.5 cryogel decreased significantly by 81.02%, 75.85% and 84.36%, respectively compared to PVA/Gel (P= 0.0069, P= 0.0003, P< 0.0001). The dead cells in the PVA/Gel/
P.emblica-2 cryogel declined significantly by 51.09%, 41.06% and 37.15%, respectively compared with PVA/Gel (P= 0.0361, P= 0.0036, P= 0.0002) (
Figure 8d–f).
Cell scaffolds must be mechanically stable, biocompatible, biodegradable, but also porous with good interconnectivity for cell viability, proliferation, migration and other metabolic needs [
51]. PVA is a synthetic polymer widely used to produce macroporous, spongy matrices in tissue engineering applications due to its biocompatibility, non-carcinogenic and non-toxic properties [
54]. Gel is frequently preferred in biomaterial synthesis as an attractive natural polymer with its chemical structure, biocompatibility, and biodegradability properties that increase cell adhesion [
55,
56]. However, studies have revealed that biocompatible PVA and Gel-based scaffolds maintain the functionality of cells metabolically thanks to their strong, elastic and porous 3D structure, at the same time they support cell viability, adhesion, proliferation, mobility and allow the absorption of the necessary substrate for cell nutrition. For these reasons, it has been reported to be a promising platform in tissue engineering. Pterostilbene-loaded PVA/Gel cryogels have been reported as a potential dressing material in wound therapy, promoting cell viability and proliferation [
57,
58,
59]. However, it has been reported that PVA/Gel cryogel promotes the viability and proliferation of endothelial cells and is particularly suitable for vascular tissue engineering applications [
59]. In addition, it was determined that there was no cytotoxic effect for human skin cells cultured in PVA/Gelatin cryogel and the scaffold exhibited good biocompatibility [
43]. The antioxidant, antidiabetic, anti-inflammatory, antiaging, wound healing, cryoprotective and hepatoprotective activities of
P. emblica extracts used in traditional medicine were studied [
25,
26,
27]. In in vitro studies, it has been reported that
P. emblica extract promotes wound healing by supporting endothelial cells, fibroblast, and keratinocyte vitality, proliferation, migration, and angiogenesis through its high bioactive component content [
28,
29,
30]. In addition, it has been shown in an in vivo animal model that
P. emblica treatment increases tissue regeneration by reducing oxidative stress in the wound area, inducing collagen expression and angiogenesis [
31,
60]. In a different study, the potential of using the spray solution prepared with polyvinylpyrrolidone solution containing silver nanoparticles and
P. emblica extract as an antibacterial, antioxidant and biocompatible wound dressing for fibroblasts and keratinocytes was demonstrated [
61]. In addition, green tea, ginger, and
P. emblica extracts loaded hydrgel have been reported to treat acne and accelerate wound healing in humans [
62]. However, to the best of our knowledge, the effect of
P. emblica binding to cryogel on cytotoxicity and its potential in tissue regeneration as a wound dressing are not yet known. Evaluation of cell viability and proliferation in the microenvironment as an indicator of biocompatibility in synthesized scaffold structures is very important in tissue engineering applications [
62,
63]. Therefore, in our study, for the first time in the literature, cell viability, and proliferation were evaluated after different incubation times with MTT and trypan blue exclusion assay in cells cultured in PVA/Gel cryogels to which different concentrations of
P. emblica were added. In addition, live/dead assay was applied to visualize live and dead cells in culture medium to understand the effect of scaffolds on cell behavior [
40,
63].
In our study, the MTT test showed that cells were viable in PVA/Gel and PVA/Gel/
P.emblica cryogels. Furthermore, a highly increased cellular metabolic activity was observed over time in cryogels with different concentrations of
P.emblica. However, in the control, although the cell viability increased until the 2nd day of the cell culture, the cell viability decreased. This may be due to the rapid confluence of cells in the flat culture system compared to the 3D culture system, as the doubling time of the HaCaT cell is about 26.4-48 h [
64,
65]. Cells in monolayer culture with limited substrate area proliferate rapidly until they are confluent, but when the limit is reached, cell viability decreases due to contact inhibition [
66]. In agreement with the characterization study data, the 3D macroporous structure, high swelling rate, pore sizes, the large surface area of the PVA/Gel/
P.emblica cryogels were sufficient to facilitate the diffusion of macromolecules necessary for the metabolic activity of the cells and the removal of wastes. In this way, unlike the 2D culture medium, cell viability and proliferation continuously increased in PVA/Gel/
P.emblica cryogels until the end of the 3rd day of culture, so that the cells filled the pores of the matrix [
67]. Also, the increase in cell viability and proliferation was very limited in PVA/Gel cryogels compared to containing
P. emblica cryogels. It has been reported in the literature that high antioxidant activity originating from bioactive secondary compounds reduces intracellular oxidative stress and increases cell viability [
28,
30,
68]. It is known that
P. emblica has strong antioxidant activity mainly due to the presence of phenolics, flavonoids and various gallic acid derivatives [
27,
29,
69]. This high bioactive capacity of
P. emblica may have promoted the viability and proliferation of cells cultured in cryogels. Similarly, in agreement with the MTT assay data, the trypan blue exclusion test confirmed that PVA/Gel/
P. emblica cryogels provide a biosafe environment that promotes cell viability and increase in number. More importantly, the live/dead assay results confirmed other results from direct cytotoxicity studies by providing visible evidence that the
P. emblica extract, together with the polymers used in the manufacture of cryogels, promotes cell viability by a functionally significant difference. These results demonstrate the high potential of the macroporous scaffold synthesized with natural
P. emblica extract as a suitable dressing material with its good biocompatibility.
3.6. Investigation of Cell Morphology, Adhesion, Viability, Cell-Cell and Cell-Matrix Interactions on Cryogels via Phase Contrast Microscopy, Giemsa Staining, Immunofluorescence Staining and SEM
Cells secrete ECM proteins in response to chemical-mechanical signals through their receptors and exhibit a characteristic morphology [
70]. Cell-cell and cell-cryogel interaction can be investigated by using microscopic imaging of features such as focal adhesion of the cell, cytoskeleton with fluorophores, and nuclear distribution. [
71]. Microscopic examination, giemsa, F-Actin/DAPI and Calcein-AM/Eth-1 staining was performed to assess the proliferation and morphology of cells cultured on control, PVA/Gel, PVA/Gel/
P.emblica-0.5, PVA/Gel/
P.emblica-1, PVA/Gel/
P.emblica-1.5 and PVA/Gel/
P.emblica-2 cryogels at 72nd h [
72] (
Figure 9). Phase contrast microscopy and giemsa staining showed that the cells adhered to the surface of the cryogels and penetrated the pores. Moreover, especially in the PVA/Gel/
P.emblica cryogels, cells proliferated extensively, forming large-sized contiguous colonies/spheroids. In addition, cells in PVA/Gel/
P.emblica cryogels were dose-dependently affected by
P. emblica in terms of proliferation and distrubition. In contrast, cells in PVA/Gel grew in scattered colonies and in fewer numbers. Unlike control cells, which grew in a monolayer in polystyrene, adherence and infiltration of cells into the cryogel were observed and their 3D structure was clear (
Figure 9a,b). Cells show different structural conformations from typical images of cells adhering to 2D surfaces when in contact with 3D scaffolds. [
73]. Cells in PVA/Gel and PVA/Gel/
P.emblica cryogels retained a tightly arranged cubic/spherical morphology. F-Actin/DAPI staining showed peripheral accumulation of strongly organized F-actin following cell contours (
Figure 9c) [
74]. In this paper, it was observed that loading
P. emblica on cryogel effectively enhanced cell number and adhesion in spherical morphology. However, morphological studies should be confirmed molecularly and biochemically for known morphological differentiation markers such as involucrin, cytokeratin, keratin and vimentin [
75,
76]. According to C-AM/Eth-1 staining, the compounds used in the synthesis of PVA/Gel and PVA/Gel/
P.emblica cryogels did not affect cell viability. It was observed as live (green) and dead (red) cells with cubic boundaries inside the scaffold (
Figure 9d). Our microscopic imaging data confirmed the cell viability and proliferation data, demonstrating good compatibility of PVA/Gel/
P.emblica cryogels to keep a high number of cells viable in healthy morphology. Our data showed that PVA/Gel/
P.emblica cryogels are suitable for use in skin tissue engineering applications and can be developed with advanced studies.
In a skin injury, migration and proliferation of keratinocytes are triggered first due to the inflammatory response, then they interact with the ECM and their adhesion changes [
77]. Activation of keratinocyte migration is closely related to intercellular transmission and signal transduction regulated by Connexins, (Cx) proteins, which form an intercellular space junction [
78]. In particular, gap junction protein Cx43 is involved in inducing signaling pathways including growth factors and ECM deposition that are key in wound closure [
79]. Cx43 distribution in cells cultured on control, PVA/Gel, PVA/Gel/
P.emblica-0.5, PVA/Gel/
P.emblica-1, PVA/Gel/
P.emblica-1.5 and PVA/Gel/
P.emblica-2 cryogels was visualized under the microscope at 72nd h by immunofluorescence staining (
Figure 10). Cx43 emits intense and bright fluorescent light in direct proportion to the increase in cell-cell interaction [
71]. An overall increase in gap junction density was observed for cells cultured on PVA/Gel/
P.emblica cryogels compared to control and PVA/Gel (
Figure 10a–c). According to the quantitative analysis of immunofluorescent staining, Cx43 antibody fluorescence intensity in PVA/Gel/
P.emblica-0.5, PVA/Gel/
P.emblica-1, PVA/Gel/
P.emblica-1.5 and PVA/Gel/
P.emblica-2 cryogels was increased by 38.41% ± 8.02%, 131.03% ± 7.80%, 186.87% ± 10.62%, 311.93% ± 14.42% and 63.21% ± 8.35%, respectively, compared to control (P< 0.0001 for all). Moreover, gap junction density increased by 275.12%, 365.77%, 568.84% and 165.51% in PVA/Gel/
P.emblica-0.5, PVA/Gel/
P.emblica-1, PVA/Gel/
P.emblica-1.5 and PVA/Gel/
P.emblica-2 compared to PVA/Gel cryogel (P< 0.0001 for all). In addition, the fluorescence intensity of Cx43 increased by 56.09%, 69.64% and 39.70% in PVA/Gel/P.emblica-1.5 compared to other PVA/Gel/
P.emblica cryogels, respectively (
Figure 10d) (P< 0.0001 for all). This data generally indicates enhanced cell-to-cell interaction in cryogels loaded with
P. emblica (P= 0.0007 for
P.emblica-0.5/
P.emblica-1; P< 0.0001 for
P.emblica-0.5/
P.emblica-2 and P< 0.0001 for
P.emblica-1/
P.emblica-2).
The purpose of biomaterial production is to form good cellular adhesion areas for controlling cell migration, proliferation, differentiation and ECM synthesis [
80]. Cells were visualized 72 h after incubation to evaluate the effect of control, PVA/Gel, PVA/Gel/
P.emblica cryogels on cell proliferation, morphology, localization and cell-matrix interaction by SEM. SEM can visualize cell attachment, spread, and morphology within the porous structure of the cryogel, revealing important information about the distribution of cells and their interaction with the surrounding matrix [
81]. Cells with indistinct borders located in the pores of the scaffold and inside the polymeric walls grew on the cryogels and the presence of ECM was observed. There was also strong contact between cells and formed colonies/spheroids. It was observed that cells also encompass the interpore spaces, as evidenced by the spherical morphology covering the surface [
74]. Notably, in the PVA/Gel/
P.emblica cryogel compared to PVA/Gel, cells settled over the entire surface of the scaffold, forming diffuse, proliferating and contiguous colonies/spheroids with ECM formation. Cell number and density were affected by the
P. emblica concentration in the cryogels (
Figure 11).
P. emblica contains bioactive secondary metabolites such as gallic acid, ascorbic ecid, quercetin, punigluconin, apigenin 7-glucoside, kaempferol and so on [
29].
P. emblica loaded cryogels possess bioactive components with multiple hydroxyl functional groups that promote cell attachment as well as cell proliferation. Macroporosity in biomaterials allows nutrient supply and metabolic waste diffusion for cell-cell network formation [
82]. The ECM is vital for cell adhesion, receiving-transmitting physical external stimuli, and has a significant impact on cellular behavior [
70]. SEM data revealed that the macroporous structure of PVA/Gel/
P.emblica cryogels promotes cell adhesion and growth to simulate epithelialization by synthesizing proinflammatory and growth factors from keratinocytes for wound healing [
83]. In vitro results showed that the PVA/Gel/
P.emblica cryogel, which provides versatile support for cell-material interaction with its 3D structure, has the potential to pave the way for the design of more efficient next generation tissue engineering materials and implants.