Cell proliferation and viability of NIH3T3 in the conditioned media of scaffolds were assessed using a live-dead test conducted by the previously reported methodology34. Consequently, 5 mg/mL fluorescein diacetate (FDA) and 2 mg/mL propidium iodide (PI) were employed to identify living and dead cells, respectively. The stained NIH3T3 cells were examined using a fluorescence microscope (CKX53, Olympus, Japan).

In biomedical engineering, the regeneration and repair of nerve tissues can be significantly enhanced by the transplantation of biomaterial scaffolds loaded with stem cells. Utilizing composite scaffolds constructed from biodegradable materials enables the integration of cells into the central nervous system and brain at specified cellular densities, thereby facilitating the delivery of stem cells to the injury site and achieving superior therapeutic outcomes. A crucial characteristic of scaffolds is the existence of linked holes of suitable dimensions to enable cell infiltration, adhesion, and proliferation. Chitosan, a natural polysaccharide, exhibits effective shock-absorbing properties and can replace glycosaminoglycan in the extracellular matrix (ECM). Creating 3D porous scaffolds from chitosan, including interconnected pores and exhibiting superior biocompatibility and biodegradability, renders it an optimal selection for tissue engineering. Interestingly, chitosan enhances neuron attachment, proliferation, and neurite extension and exerts potent neuroprotective action.

The significant influence of mechanical signals on cellular behavior has been established in the regulation of stem cell differentiation. Nanomaterials generate mechanical stimuli that affect cells during their movement, thereby playing a crucial role in cellular processes. Numerous efforts have been undertaken to enhance the mechanical strength of chitosan scaffolds by integrating inorganic calcium phosphate particles. Studies indicate that the deposition of calcium phosphate on synthetic material surfaces enhances cellular proliferation. Furthermore, a hydroxyapatite-like layer on the scaffold can elicit a favorable biological response from host tissues, resulting in bioactive behavior following implantation. Numerous studies have emphasized the impact of several calcium phosphate phases, including beta-tricalcium phosphate and hydroxyapatite (HA), on fibroblast cell shape, adhesion, and proliferation. Research has demonstrated that HA promotes osteogenic differentiation of MSCs. Hence, it is likely that HA functions as an inorganic bioactive molecule to modulate the differentiation of NSCs.

In this study, as depicted in Fig. 2, spongy porous CS-Fe-HA and CS-Fe-HA-phyco scaffolds with pore diameters of approximately 30 μm were manufactured by a simple technique. This method sought to create a bio-integrable matrix with optimal geometries to facilitate cell adhesion, proliferation, and anchoring to the pore walls of the chitosan scaffold. Incorporating Fe ions and phycocyanin in the neuroinductive HA structure significantly enhanced the scaffolds biological activity and integration capability.

The capacity of scaffolds to retain water is a crucial criterion in assessing their effectiveness for tissue engineering. Scaffolds' swelling characteristics markedly affect cell adhesion, proliferation, and differentiation. The chitosan hydrophilicity is attributed to free amine groups in its structure. Some of the chitosan properties such as the molecular weight and deacetylation degree can indeed influence scaffold properties like porosity and degradation. The water uptake tests (Fig. 3) found diverse water absorption kinetics for 12 h. Compared to the other materials, CS-Fe-HA has a more gradual increase and sustains relatively steady water absorption. In contrast, CS-HA-phyco exhibits a notable decrease in water absorption following the initial peak.

All samples exhibited excellent water affinity, achieving maximum swelling within 1 h. The incorporation of HA into the chitosan scaffold and the doping of Fe ions into the apatite lattice structure seemingly did not influence the water absorption properties of the produced samples. Upon attaining the plateau, no statistically significant differences in SW% were seen for all samples over a 12-hour incubation period.

Incorporating phycocyanin into the CS-Fe-HA scaffold further enhanced its hydrophilicity, improving water uptake. The interaction of phycocyanin with the chitosan matrix increased the scaffold's ability to absorb and retain water, which is beneficial for maintaining a hydrated environment conducive to cell survival and function. This characteristic is particularly advantageous for applications in tissue engineering, where a hydrated matrix is essential for cell proliferation and differentiation.

This data is crucial for understanding the absorption properties of the examined scaffolds, which have potential applications in neuroregeneration. The insights gained could inform the development of more effective and durable scaffolds.

The functional groups of pure HA, HA-Phyco, FeHA, and FeHA-Phyco powders were assessed after 1, 6, and 12 h of immersion using Fourier transform infrared spectroscopy (FTIR). The FTIR spectra of the materials displayed functional groups associated with hydroxyapatite. Figure 4 shows the hydroxyapatite sample's FTIR spectrum in the 400 to 3800 cm range. As can be seen, the broad bands from 560 to 604 cm, 1470 cm, and from 860 to 920 cm can be observed, and the broad bands from 1020 to 1100 cm are related to the vibrations of the phosphate group. Peaks at approximately 560-600, 960, and 1030 cm corresponded to the out-of-plane bending, symmetrical stretching, and asymmetrical stretching of phosphate groups, respectively. Carbonate peaks were detected at around 1650 and 1470 cm, whilst a broad band at 3566 cm was ascribed to the symmetrical stretching vibration of OH. In the phosphate network, P-O vibrations' bending and stretching modes appear as bands in the range of 1600 cm and 1047 cm. The wide band from 13,400 to 3569 cm is related to the stretching vibration of the OH group. The absorption band in the region of 13,400 cm can be attributed to the OH of water absorbed on the sample or the presence of this functional group on the surface of nanoparticles.

The presence of a peak in 1633 cm indicates the bending vibration of this functional group caused by water adsorbed on the sample's surface. The 1410 and 1460 cm peaks are related to the stretching vibration of the carbonate group.

The samples spectra showed no significant differences between HA (Fig. 4), HA-Fe (Fig. 5), HA-physio (Fig. 6), and HA-physio (Fig. 7) powders, indicating that the incorporation of Fe ions and phycocyanin did not alter the scaffold materials fundamental functional groups.

In the FTIR spectrum of hydroxyapatite modified with the iron in situ method, the absorption bands of hydroxyapatite functional groups are observed with a very small shift to a higher wave number and broadening.

Figure 6 shows the FTIR spectrum of the hydroxyapatite-phycocyanin sample in the range of 500 to 3800 cm. In addition to the hydroxyapatite indicator peaks explained, the peaks corresponding to the spectrum of phycocyanin are also known with a slight shift. The 2358 cm peak indicates carboxylic acid bands.

There is a broad peak in the 3000 to 3568 cm range in the in situ samples. This broad peak is related to the water groups present in the samples, which can be attributed to the method of preparing the samples, and it is also possible that the samples absorbed water from the surrounding environment or were not calcined. Also, in the prepared nanoparticle sample of hydroxyapatite modified with iron by the in situ method, the peak of the 470 cm regions, which is related to the vibration of the phosphate group, appeared with more displacement than the hydroxyapatite sample before modification with iron, which can confirm the formation of a bond between hydroxyapatite and iron. Compared to the IR spectrum of iron-free hydroxyapatite nanoparticles, a peak in the region of 500 to 580 indicates the bending vibrations of Fe-O bonds in the crystal structure.

The reduction of the band in the range of 450-650 cm occurred with the incorporation of Fe ions, corroborating prior findings. The strength of the hydroxyl peak at 3500 cm increased with the doping of Fe into the HA lattice structure, hence confirming the effective incorporation of Fe ions. As shown in Fig. 7, with the addition of phycocyanin to the hydroxyapatite and chitosan scaffold, the prominent peaks have slightly shifted to the right, and two peaks, 2924 and 2359, have appeared in the FTIR spectrum together with phycocyanin. Peak 2924 can be related to aliphatic N-C, and 2359 can be related to C = N. The presence of phycocyanin was confirmed by specific peaks in the FTIR spectra corresponding to its characteristic functional groups. These peaks indicated the successful integration of phycocyanin into the scaffold matrix, contributing to the scaffold's hydrophilicity and providing bioactive sites that could enhance cell attachment and proliferation (Figs. 8 and 9).

Figure 8 shows FTIR analysis on an iron-modified nanohydroxyapatite scaffold presented in situ. The prominent peaks of hydroxyapatite related to the vibration of the P-O group (1020 to 1100 cm), the vibration of the C = O group (1410 to 1460 cm), and the vibration of the O-H group (3400 to 3569 cm) and the Fe-O bond peak at 563 cm can be observed.

As shown in Fig. 9, in the comparison of the FTIR spectrum of hydroxyapatite scaffold with three different percentages of phycocyanin, the peaks were repeated in the same order, with the difference that with the increase in the percentage of phycocyanin from the sample where the ratio of hydroxyapatite to phycocyanin was one to one. One is one to two and one to three; Peaks are broader and shifted to the right. Also, by increasing the percentage of phycocyanin, the IR spectrum becomes more like the spectrum of phycocyanin. The changes after adding phycocyanin in Fig. 7 are the same as explained in Fig. 9, with the difference that the peak related to iron binding is also observed in these two spectra.

As seen in Fig. 9, the peak related to the Fe-O bond in the region of 550 to 600 cm, the characteristic FTIR peak of phycocyanin for S-O stretching vibrations at 673.86 and 794.67 cm, the peak related to the group P-O in the range of 1083 to 1118 cm, C-H-O peak in the range of 1410 cm, the peak corresponding to C = C can be detected in the range of 1590 to 1633 cm and C-H and O-H peaks in the range of 2936 and 3400 cm.

In the Fe-doped HA, the phosphate vibration bands show a slight shift to higher frequency compared to pure HA, which suggests lattice substitution. Similarly, addition of phycocyanin introduces new peaks or shifts (around 2350-2360 cm) and a slight broadening of the OH band. A 5 cm shift to higher wavenumber was observed for the P-O stretch in Fe-HA compared to HA, indicating the hydroxyapatite's nanoscale and low-crystalline nature. In the Fe-HA samples, increased soaking time led to broader characteristic peaks of hydroxyapatite. No impurities were observed in the sample pattern. The broadness of the peaks is related to the low crystallinity of the formed hydroxyapatite, which is close to the natural bone structure.

The X-ray diffraction (XRD) patterns of Fe-doped hydroxyapatite (HA) with varying soaking durations (1, 6, and 12 h) (Fig. 10) and phyco-doped HA (Fig. 11) were contrasted with the patterns of pure HA (Fig. 10). The diffraction peaks in the XRD patterns were attributed to monophase, low-crystalline hydroxyapatite, by HA (JCPDS card No. 9-432). The peaks at 25.9°, 28.2°, 29°, 31.9°, 32.9°, 34.1°, 39.9°, 46.8°, 49.5°, and 53.1° correspond to the 002, 102, 210, 211, 300, 202, 310, 222, 213, and 004 planes of hydroxyapatite, respectively. No additional diffraction peaks were detected in any Fe-doped HA samples, and there were no notable shifts in peak locations or alterations in relative peak intensities following ion exchange, irrespective of soaking duration. This indicates that the ion exchange procedure did not alter the HA structure.

The X-ray diffraction pattern of iron-modified hydroxyapatite has been investigated in situ for sample identification (Fig. 11). As mentioned in Table 1, the sample prepared by the in situ method was prepared with Fe and Fe ions.

The Ca/P molar ratio of the sample was also determined according to the molar data of the spectra in Tables 2, 3 and 4.

The peak related to iron appeared in the region of 35.7°. In Figs. 10 and 11, the X-ray diffraction pattern of the iron-modified hydroxyapatite sample is compared with the hydroxyapatite pattern. This comparison indicates hydroxyapatite formation and confirms the presence of iron ions in the sample.

The preservation of the HA crystalline structure upon Fe substitution and phycocyanin incorporation is a positive outcome, as it means the scaffold retains the known bioactive properties of hydroxyapatite (such as osteoconductivity and biocompatibility). An unchanged HA lattice suggests that the modifications did not compromise the material's fundamental biofunctionality, which is important for its role in supporting cell growth. The inclusion of phycocyanin did not significantly alter the XRD patterns, suggesting that its incorporation did not affect the crystalline structure of HA. However, the presence of phycocyanin contributed to the overall stability and bioactivity of the scaffold, as evidenced by enhanced cell proliferation and attachment in subsequent biological tests. A highly crystalline HA like bone mineral is often favorable for cell responses, which is an advantage our scaffold keeps even with the added components.

Figure 12a shows the SEM images of prepared HA nanopowder. The particles are uniform and spherical, and their size distribution is mostly in the range of 68 to 98 nanometers, with an average of 83 nanometers. Figure 12b shows the images of HA nanoparticles modified with iron using the in situ method. Scanning electron microscope images also show the distribution of non-spherical hydroxyapatite nanoparticles modified with iron.

One important point for cell growth is the need for a three-dimensional scaffold with appropriate porosity (Fig. 12) that creates a suitable place for cell migration and growth. For example, implants without porosity or an insufficient percentage of porosity are unsuitable for tissue engineering applications due to a lack of effective connection and tissue growth. Therefore, besides being biodegradable and biocompatible, chitosan resin provides a suitable substrate for ceramic nanoparticles such as nanohydroxyapatite in bone tissue engineering.

SEM images show the existence of internal communication (interconnection) between pore and pore, which plays an important role in cell nutrition and disposal of cell waste. Choosing or preparing the right material for the required applications can be done according to the SEM results. According to the SEM image in Fig. 12c, chitosan has a porous structure, most of which has open pores. In the figure, you can see the porosity created by the freeze-drying method with a distribution of 0.5 to 6.5 micrometers with an average size of 2.5 micrometers and hydroxyapatite nanoparticles in all scaffold areas. The SEM image of the nano-hydroxyapatite scaffold sample modified with iron on the chitosan substrate is shown in Fig. 12d. Examining the image of the sample shows the uniform distribution of iron-modified hydroxyapatite nanoparticles on the surface of the scaffold.

Figure 12e shows the morphology of a nanohydroxyapatite and phycocyanin scaffold on a chitosan substrate with 500 x magnification. The scaffold has a suitable porous structure with spherical holes, and hydroxyapatite and phycocyanin particles are scattered on its wall. The scaffolding has favorable characteristics.

As shown in Fig. 13, after increasing the concentration of phycocyanin powder to hydroxyapatite nanoparticles increases from 1:1 to 1:3, the particles placed in the chitosan scaffold bed become densely dispersed.

Figure 14 shows a nanohydroxyapatite scaffold modified with iron and phycocyanin on a chitosan substrate. The scaffolding has proper porosity, and the holes have changed from spherical to elongated and rod-shaped. Modified hydroxyapatite nanoparticles and phycocyanin can also be seen on the scaffold substrate.

Scaffolds with the higher phycocyanin content (1:3) showed compared to those with lower phycocyanin (1:1). From our results, one trend was that extremely high phycocyanin content might slightly decrease cell viability at 72 h, or that the distribution of particles changes (Figs. 15 and 19 in the original show denser particle coverage at 1:3.

Increasing phycocyanin concentration in the scaffold did not dramatically harm viability. This phenomenon is considered good since it shows that the phycocyanin is not overtly toxic at the studied doses. It may even provide more antioxidant protection up to an optimal concentration. However, if we observed any decrease at the highest content, we note that extremely high concentrations could potentially saturate the system or affect scaffold properties (like making it more brittle or altering surface chemistry). A systematic study of various phycocyanin concentrations would be valuable in the future to determine the optimal loading for maximal neural induction with minimal adverse effect.

In Fig. 15, the elongated structure of the scaffold holes and the dispersion of nanohydroxyapatite particles modified with iron and phycocyanin on the walls of the holes are clear in all three images. Comparing the three images, as the percentage of phycocyanin in the scaffold increases from 1:1 to 1:3, the density of the scattering of particles on the scaffold bed increases.

We measured the pore dimensions from the SEM images to the extent possible. The chitosan scaffold (produced by freeze-drying) exhibits a pore size distribution roughly in the range of 0.5 to 6.5 μm, with an average pore diameter of about 2-3 μm. These values were obtained by analyzing the SEM micrographs (as shown in revised Fig. 12c). We also determined the pore interconnectivity: The SEM images (Fig. 12c and others) clearly show an interconnected porous network. The scaffold pores are largely open and connected, which is beneficial for cell migration and nutrient/waste exchange. While we did not use mercury porosimetry or micro-CT to quantify connectivity, we describe qualitatively that the structure contains numerous open pores that interlink (citing the SEM evidence). We acknowledge that we did not perform Brunauer-Emmett-Teller (BET) surface area analysis on these scaffolds. This is a limitation in this study. The future work should include BET surface area measurements to quantify the surface area available for cell attachment and to correlate with cell growth outcomes. According to the available literature, the higher surface area can enhance protein adsorption and cell responses, to rationalize why this would be important.

Energy Dispersive X-ray Analysis (EDX) conducted an elemental analysis of inorganic nanoparticles. The EDX spectra of HA (Fig. 16a) revealed the presence of Ca, P, and O, whereas Fe was identified in Fe-doped HA powders after 1 and 12 h of immersion (Fig. 16b). Fe ions, despite lacking thermodynamic stability in aqueous conditions at ambient temperature, were effectively incorporated into the hydroxyapatite lattice, signifying the substitution of Fe inside the HA structure.

The findings of the EDX analysis (Tables 2 and 3) also revealed that the Fe weight% was higher in FeHA-12 h compared to FeHA-1 h, confirming more significant Fe ion substitution with increased immersion time. The Ca/P atomic ratios were calculated as 1.7 for HA, 0.9 for FeHA-1 h, and 0.83 for FeHA-12 h. The decrease in the Ca/P ratio in FeHA samples compared to pure HA could be attributed to substituting divalent Ca ions with trivalent Fe ions, potentially leading to the formation of calcium vacancies to compensate for lattice charge imbalance. The in situ sample's molar ratio of calcium to phosphorus has decreased from 1.7 to 1.29 (Table 3).

Incorporating phycocyanin did not significantly alter the scaffold's elemental composition, as detected by EDX (Tables 4 and 5). However, it did contribute to its overall bioactivity, as evidenced by improved cell attachment and proliferation in subsequent biological tests.

Our goal in incorporating the iron in the scaffold was to introduce some magnetism without compromising biocompatibility, although the effect is modest. Future optimizations (for example, increasing the iron content or using magnetite nanoparticles in the scaffold) could be explored to achieve more pronounced magnetic responsiveness if the scaffold shows the requested appropriate characteristics.

Vibrating Sample Magnetometry (VSM) was used to assess the magnetic characteristics of FeHA at room temperature under a magnetic field of 10 kOe (Fig. 17). The hysteresis loop of ferromagnetic material depicts the relationship between magnetization and the applied magnetic field, showcasing a hysteresis loop characteristic of ferromagnetic materials. The x-axis represents the applied field (Oe) ranging from approximately - 12,000 to 12,000 Oe, while the y-axis indicates the magnetization (emu/g) ranging from approximately - 0.20 to 0.20 emu/g.

This hysteresis loop illustrates the materials behavior as an external magnetic field is applied and removed. The shape of the loop can infer key magnetic properties such as coercivity and remanence.

All FeHA samples exhibited paramagnetic properties, as seen by the positive gradient of their magnetization-magnetic field curves. Despite the introduction of magnetic properties through Fe doping in all FeHA samples, a definitive link between soaking duration and magnetic properties was absent, with FeHA-12 h showing lower saturation magnetization (Ms) than FeHA-6 h, contrary to the expectation that increased Fe substitution would lead to higher saturation magnetization. However, phycocyanin did not contribute to the magnetic properties of the scaffold.

Besides, the saturation magnetization is relatively negligible. We note that such a low Ms is unlikely to exert a strong magnetic force for techniques like guided cell alignment or targeted drug delivery. This provides a more realistic interpretation of our magnetic data.

The developed magnetic scaffold in this study might have potential future applications. Although the magnetization of the current scaffolds is low, the concept of a magnetic scaffold opens possibilities for magnetically guided cell delivery or alignment. In principle, if the magnetic response were enhanced in future iterations, one could envision guiding neural stem cells to injury sites or orienting neurite growth by applying external magnetic fields. The magnetic scaffolds have been explored for controlled drug delivery, where an external magnet can concentrate a drug-laden scaffold at a target site.

Figure 18 shows that cell viability after 24 h was highest in the HA/CH and HA/Fe groups, showing a significant difference from the control group (p < 0.05). Additionally, the survival rate in groups without Fe was higher than that of those with Fe, with a notable difference between the 1.1 and 1.1/Fe groups.

After 48 h, cell viability and proliferation increased, showing significant differences between the control group and other groups, except for the 1.3/Fe and HA/Fe groups (p < 0.05).

However, by 72 h, cell viability in the treatment groups decreased compared to the 48-hour mark, with the highest survival rate observed in the control group, which was significantly different (p < 0.05). A significant difference was also observed between the 1.1 and 1.1/Fe groups and within the groups between 1.1 and 1.3. Moreover, the treatment groups containing Fe exhibited the lowest survival rates compared to their corresponding groups.

Overall, the highest survival and proliferation were seen under conditions without Fe and with a low percentage of phycocyanin. Additionally, survival rates also improved as the Fe content and phycocyanin percentage increased.

The qualitative assessment of the NIH3T3 using the Live-Dead assay revealed that the cells underwent morphological changes, became spindle-shaped, and showed cytoplasmic extensions like neurites, which could indicate the cells mobility and tendency to establish new connections with their surroundings (Fig. 19). These changes were more pronounced in the Fe-deprived groups. The cells' confluence is also consistent with MTT results.

The observed pore size range in SEM and XRD analyses are explicitly connected to the neural tissue engineering relevance. It should be noted that our scaffold's pores (on the order of a few micrometers, roughly 0.5-6.5 μm with an average ~ 2.5 μm as measured) are within a scale that can be conducive to neural cell interaction. Specifically, the small pores in the low micron range can support neurite extension and guidance, as well as allow diffusion of nutrients in a dense neural network. The neuronal processes (neurites) can penetrate pores of a few microns and that having a microporous structure can encourage tissue ingrowth. This alignment of pore size with neuroregenerative needs was implicit before; now we have made it explicit. The interconnected nature of the pores (as seen in SEM) is beneficial for nerve tissue integration because it could allow nerve fiber penetration throughout the scaffold. The scaffold's physical structure was intentionally designed via freeze-drying of chitosan to yield such pore sizes, which indeed match well with the requirements of neural tissue scaffolds.

In this study, to synthesize hydroxyapatite from quaternary calcium nitrate and diammonium hydrogen phosphate, to make iron-modified hydroxyapatite from iron dichloride and trichloride, and to make scaffolds from different powder samples, chitosan polymer was selected. The size of the particles, proper dispersion of hydroxyapatite in the chitosan scaffold substrate, and the favorable biological characteristics showed the potential of this scaffold. The appropriate porosity of the hydroxyapatite and chitosan scaffold, evident in this study's SEM images, can create a suitable place for the migration and growth of neural cells.

By examining the EDX patterns, the peaks have become more expensive with the increase in the percentage of iron in the samples. With the increase of iron and its entry into the hydroxyapatite network, the number of calcium atoms decreases, the structure loses its ideal order, and its degree of crystallinity decreases. The reason for this is the small radius of the iron ion (64 angstroms) compared to the calcium ion (99 angstroms), which causes contraction in the structure. On the other hand, in addition to the presence of a doubly positive iron ion in the in situ method, there is also a triply positive iron ion, whereas calcium ion is divalent. This load imbalance leads to the formation of faults in the network.

Also, the width of the peaks related to hydroxyapatite compared to the sample modified with iron in the XRD diagrams indicates the low crystallinity of the hydroxyapatite formed in the iron substrate, which is close to the natural bone structure.

Interpreting the EDX results of the scaffold modified with iron reveals that less calcium phosphate mineral phase is deposited on the sample's surface. This indicates the existence of a suitable substrate for the formation and growth of the hydroxyapatite mineral phase and confirms the scaffolds' bioactivity.

The incorporation of Fe into HA could have a dual effect: on one hand, providing magnetic responsiveness, even if small, which could be harnessed in the future for magnetically steering cells, and on the other hand, possibly affecting cell behavior. Considering our viability data an interesting trend was comprehended. The scaffolds without iron (those with HA and phyco but no Fe) tended to show slightly higher cell viability than those with Fe. It could be suggested that excess iron can sometimes have cytotoxic effects or alter the local environment (for example, through reactive oxygen species generation if Fe catalyzes Fenton reactions). The presence of Fe, while endowing magnetic properties, must be balanced against potential cytocompatibility trade-offs. The Fe-doped scaffolds still supported good cell viability overall, but the slightly lower viability compared to Fe-free scaffolds is noted and is consistent with the idea that Fe substitution should be kept at a moderate level.

Additionally, we emphasize some positive aspects of Fe presence in the HA lattice. It could potentially stimulate certain cellular pathways or improve the mechanical properties of the scaffold which can influence cell behavior.

In the experience of hydroxyapatite synthesis, divalent ions with the same diameter as calcium can be used to modify the powder's physical and chemical characteristics, including wearability and creating the appropriate ratio of calcium to phosphorus.

The SEM results show that the hydroxyapatite crystals have become smaller with the addition of iron in the sample prepared by the in situ method. Since the ionic radius of Fe is smaller than the ionic radius of Ca, this substitution decreases the size of the lattice parameter, and the crystals become smaller. Also, the SEM result of this study shows the presence of iron ions in all the selected points of the scaffold surface and the dispersion on the hydroxyapatite surface.

In this way, ions such as iron and titanium with a similar diameter sit instead of calcium in the hydroxyapatite structure. By creating a suitable molar ratio of calcium to phosphorus, they improve the physic powder's physical and chemical characteristics.

To evaluate the efficiency of a scaffold for tissue engineering purposes; the first stage of product development is to evaluate its possible toxicity on selected cell lines of the human body. The MTT assay method was used on the NIH3T3 cell line to evaluate the possible toxicity of different synthesized scaffolds. The study was conducted in 1-3 days on eight groups with three concentrations of phycocyanin and the effect of different hydroxyapatite, iron, and phycocyanin components on cell viability. An important next step will be to evaluate the scaffold with actual neural cells (such as neural stem cells or neurons) and in relevant in vivo models to confirm the scaffold's efficacy in a physiological environment for neuro regenerative purposes.

The cell line survival percentage was studied with three readings (n = 3). The results of cell viability in 24, 48, and 72 h were presented in Fig. 18. The survival of hydroxyapatite groups with different percentages of phycocyanin was better on the second day than on the third day and better on the third day than on the first day. This order of days also applied to the survival percentage for the hydroxyapatite-iron groups with different percentages of phycocyanin. Regarding the groups without phycocyanin, the survival percentage was higher on day 3 than day 2, and day 2 was higher than day 1, respectively. The results of the intervention groups with phycocyanin on the second day were better than the first and third days.

In the 24-hour group, No significant change was seen in scaffolds with different phycocyanin concentrations. In the 48-hour study of the survival rate of three hydroxyapatite-iron groups with different concentrations of phycocyanin, the survival rate increased significantly with the increase in the percentage of phycocyanin. In the 72-hour study, the hydroxyapatite group with a lower ratio of phycocyanin had a significantly higher survival rate than the same group with a higher phycocyanin concentration. This comparison showed that in the groups modified with iron, the increase in the amount of phycocyanin compared to iron and in the hydroxyapatite-phycocyanin groups, the higher percentage of hydroxyapatite than phycocyanin is more tolerable for the cell.

The cell's lower tolerance to iron was probably related to its concentration. Results of a study showed the non-toxic effects of iron oxide nanoparticles in concentrations less than 1000 µg/mL.

When comparing the effect of scaffold components on cell survival, it should be noted that the survival of groups without phycocyanin was higher than that of other groups in all three days. This amount was significantly higher in the CS-HA-Fe group than the CS-HA and control groups on the second day and significantly lower than that of the control group on the third day.

This result shows that the CS-HA-Fe samples have provided a favorable surface to support the adhesion, expansion, and proliferation of fibroblast cells compared with the CS-HA sample, and the effect of the amount of hydroxyapatite-modified with iron on the cell response is positive.

Also, comparing the survival rates of different components showed that the group without phycocyanin had a higher survival rate than the group that intervened with phycocyanin. Due to phycocyanin's favorable characteristics, it is likely that this issue is related to the concentration of phycocyanin, which is predicted by using lower concentrations of phycocyanin. In future studies, more favorable results of phycocyanin should be revealed.

In the comparison of the second day, the survival percentage of the HA group without phycocyanin compared to the group with phycocyanin was 1.03, and the same comparison for the iron-modified group without phycocyanin compared to the group with phycocyanin was 1.13. In general, the use of phycocyanin in the scaffold in these concentrations does not affect the cell viability very much. So, the results of this study can be scaled up for effectiveness studies.

Future studies will focus on direct neural stem cell culture on these scaffolds and assessment of neural differentiation markers to conclusively establish neuroinductive capacity. Cell differentiation is a critical indicator for understanding the mechanisms of action of genes, proteins, and pathways related to cell survival or death after exposure to toxic agents. Cell differentiation assays are generally used to screen test samples to determine whether they affect cell proliferation and differentiation or exhibit direct cytotoxic effects.

Various assay methods based on different cell functions include enzyme activity, cell membrane permeability, cell adhesion, ATP production, coenzyme production, and nucleotide absorption activity. These methods can be classified into different categories: (1) dye removal methods such as trypan blue dye removal assay, (2) methods based on metabolic activity, (3) ATP assay, (4) sulforhodamine B colorimetric assay, (5) protease viability marker assay, (6) clonogenic cell survival assay, (7) DNA synthesis cell proliferation assay and (8) Raman spectroscopy.

Fluorescein diacetate/propidium iodide (FDA/PI) staining is a combination of fluorescent dyes to assess the viability of various cell types, including mammalian cells, yeast, and pollen grains. FDA is a non-fluorescent dye that penetrates the cell membrane and is hydrolyzed by the esterase activity of living cells to produce the green fluorescent compound fluorescein. FDA is retained in living cells with intact membranes and acts as a viable cell marker. PI, a membrane-impermeable red dye, selectively penetrates dead cells or cells with damaged membranes. PI is a dead cell marker that binds to DNA and RNA through interstitial junctions.

The qualitative results from the fluorescent microscope images confirmed the quantitative results obtained from the cytotoxicity test. The fluorescence microscope images showed that the confluency of the cells in the groups without iron was higher compared to the groups with iron on all three days. Also, due to the growth of cells after 48 and 72 h, this index was higher on the second day than on the first day and the third day than on the second day.

A study investigated the effect of different amounts of polystyrene microplastics on the differentiation and transformation of human lung cells. Phase contrast imaging of live cells at 72 h revealed significant changes in the morphology of cells exposed to microplastics. This study is the first report of exposure of human cells to an environmental pollutant that results in the effects of inhibiting cell differentiation and significant changes in cell morphology. Likewise, this study investigated the effect of three different amounts of phycocyanin on the differentiation and morphological change of neural stem cells in this study.

Changing the morphology of the cells from round to spindle shapes and creating axon-like appendages was the first evidence to prove the differentiation of cells into the nervous system. The results of the fluorescent images showed that the change in cell morphology and their differentiation was more in groups without iron than in iron-containing groups with phycocyanin. Also, this marker was more visible for groups with lower amounts of phycocyanin.

This study did not include direct measurements of neural differentiation such as immunostaining for neural markers like MAP2, GFAP, β-III tubulin, etc. While the Live/Dead assay revealed some interesting cell morphology changes, this is not definitive proof of differentiation. This as a limitation of the current study. To confirm neural differentiation, future studies should evaluate specific neural marker expression in cells cultured on these scaffolds. Additionally, seeding actual neural stem cells or progenitor cells onto the scaffold and checking for neuronal or glial marker upregulation should be considered as a follow-up experiment.