3.1. Characterization of prepared scaffolds
The nanoparticles obtained from the mentioned manufacturing steps have dispersed in size, which is shown by DLS as a diagram in Fig. 2. As can be seen, the nanoparticles are in the range of 37.8 to 142 nm, among which the average size of nanoparticles is 61.4 nm.
Scaffold-related images were taken by scanning electron microscopy to examine the pore size, as well as the presence of nanoparticles, and their dispersion in the scaffold (Fig. 3(a, b)). The Image J software was used to evaluate the pore size of the scaffolds. At first, the samples are subjected to temperature decrease in stages and by having appropriate temperature fluctuations as well as the concentration of scaffold solution, the appropriate size and dispersion of porosity can be achieved. The average pore size of NP and NPRP, scaffolds were 160 ± 32, and 153.7 ± 38, respectively.
The size of the pores is one of the most important factors in determining the fate of cells. This size should be such that the cells can migrate between the pores and implant, and on the other hand, the culture medium can circulate in it and deliver nutrients and excrete waste products. For bone scaffolds, the porosity diameter of about 100 microns is suitable and for angiogenesis, there should be a distribution of 250–300 micrometers [34]. As can be seen, the pores are interconnected, and this is one of the important features of the structure from the perspective of biocompatibility because it causes migration and tissue formation and consequently angiogenesis.
In order to investigate the presence of nanoparticles loaded in the scaffold, FESEM images and their high resolution were used, the results of which can be seen in Fig. 3(c and d).
Figure 4. shows the FTIR results of nanoparticles used in gelatinous Nanobiomimetic scaffolds. The peaks observed in the range (1000 cm− 1) 1000 to 1100 are related to the symmetrical vibrations of the C-O bonds of the glycosidic bond in the starch polysaccharide. The peak in the 1739 cm− 1 region corresponds to the COOH groups formed in the nanoparticle structure. As can be seen, the intensity of this peak is reduced in PRP-carrying starch nanoparticles. Peak 3005 cm− 1 is also associated with tensile vibrations of C-H groups in starch polysaccharides [35]. The peaks of C = O observed in 1631 cm− 1 as well as N-H observed in 1532 cm− 1 in the NPRP sample are examples of protein adsorption on the surface of nanoparticles. This adsorption caused a decrease in citrated starch index peaks. Adsorption and protein layer formation and biological activation of nanoparticles are quantified by FTIR. The tensile vibration of the carboxyl group in the amino acid of this substance peaks at 1490, 1541, and 1645 cm− 1. As shown in the Fig. 4; The process of starch citrate increases the C = O groups, which is evident in the FTIR results [36].
On the other hand, to characterize the gelatinous scaffold (Fig. 5), we can refer to peaks 1631 cm− 1 related to the double bond of carbon and oxygen and 1532 cm− 1 related to the bond of hydrogen and nitrogen, as well as 1404 cm− 1 related to the bond of carbon and nitrogen. On the other hand, in samples with PRP, the intensity of these peaks has been reduced. After chemical cross-linking by EDC in the scaffold, a deeper peak of this carbon-nitrogen bond can be observed, which indicates an increase in the amide bond due to EDC cross-linking. The EDC reacts with the carboxyl functional group on the gelatin and protonates it, followed by a nucleophilic attack of the adjacent gelatin amine group, causing the gelatin cross-linking, followed by the release of EDC residues that can be washed away. The result of this amide bond is evident in the FTIR test [37].
Investigation of compressive strength is one of the most important indicators in evaluating the quality and suitability of scaffolds used in tissue engineering. The amount of elastic modulus and compressive strength of scaffolds are given in Table 2.
Table 2
Elastic modulus and compressive strength of the scaffolds
Samples | Compressive Modulus | Compressive Strength |
NP | 2.45 ± 0.327 | 0.764 ± 0.019 |
NPRP | 1.956 ± 0.201 | 0.711 ± 0.009 |
Table 2 shows that with the presence and use of PRP-containing scaffolds, the compressive strength decreases from 0.764 ± 0.019 MPa in the NP scaffold to 0.711 ± 0.005 MPa in the NPRP scaffold. The compression modulus has also changed in the same way and its value has decreased from 2.45 ± 0.327 MPa in the NP scaffold to 1.956 ± 0.201 MPa in the NPRP scaffold. According to these results, it seems that these composites are suitable for use in tissue engineering of bones with low stress. The inner ear, trabecular, or nose are the most commonly mentioned cases [38].
Figure 6 shows the results of the equilibrium water uptake test after 24 hours for NP and NPRP samples. According to the Fig, the amount of water uptake is higher in the NPRP sample, which is related to the presence of protein and hydrophilic groups in it and the higher size and porosity percentage of the scaffold [39].
3.2. In vitro release study
3.2.1. Releasing of VEGF and PDGF growth factors
One of the most important purposes of using PRP is to use a cocktail of growth factors. By loading PRP into the scaffold as well as into the nanoparticles, a more controlled release is expected. To examine this factor, two growth factors, VEGF and PDGF, have been studied.
Figure 7 shows the in vitro cumulative release profile of two growth factors, PDGF and VEGF. The initial explosive release was observed in both growth factors, with about 50% of the total growth factors measured during the experiment released during the first day. Release kinetics of both growth factors follow the same pattern, and of course, PDGF has a higher concentration than VEGF, which is consistent with the fact that the amount of VEGF in PRP is lower than in PDGF [40]. The important point is that in the scaffold preparation stages, steps such as sterilization and cross-linking, the initial explosive release is partially removed from the system and a more stable system is achieved.
3.2.2. Releasing of protein
Since the burst release is not a desirable consequence, it is intended to minimize this effect in the scaffolds. Continuous release over time helps scaffolds and migrating cells to enter the next stages of tissue differentiation and growth. As can be seen, the release of VEGF is faster while the release of PDGF is gradual and its concentration did not reach zero on day 21. This phenomenon has been studied in previous research and in fact, the release of VEGF causes vascular formation and consequently, the continuous release of PDGF causes vascular growth and repair of bone tissue [41].
The diagram of protein release from gelatinous scaffold is shown in Fig. 8. The amount of released protein in this scaffold depends on the amount of chemical crosslink as well as the porosity of the scaffold, and further affects the mechanical and biological properties of the scaffold. The continuous release rate and degradation rate are commensurate with tissue growth without loss of premature mechanical properties; It is one of the cases that can be considered by observing this rate of continuous release of protein from the scaffold [42]. It is observed that the rate of scaffold degradation and protein release is slow, which can indicate the appropriate strength and density of the crosslinked scaffold. It should be noted that although the biology of platelet-rich plasma remains somewhat unknown; it can be easily used in clinical applications and benefit from its properties.
3.3. Biological characterization
3.3.1. Cell viability test
The MTT assay is a colorimetric method based on the reduction and refraction of yellow tetrazolium (3- (4,5-Dimethylthiazol-2-Yl) -2,5-Diphenyltetrazolium Bromide) crystals by the enzyme succinate dehydrogenase and makes of Insoluble dyed crystals. During incubation, MTT is regenerated by the succinate dehydrogenase system, one of the enzymes of the respiratory cycle of mitochondria. The regeneration and breakage of this ring produce blue formazan crystals that are easily detectable under a microscope. The amount of dye produced is directly related to the number of cells that are metabolically active. Formazan crystals are insoluble in water and must be soluble in solvents such as DMSO before colorimetry. Finally, the optical absorption of the resulting solution can be read at a wavelength of 490 nm and cell viability can be compared.
Figure 9 shows the survival rate of cells in the scaffold based on the MTT test. It can be seen that scaffolds containing starch nanoparticles have always had a higher survival rate than the control. However; Statistically only a significant difference occurred on the first day. This may be due to the explosive release of growth factors during the first day of culture. Generally; By adding PRP to the scaffold; Cell adhesion increases and this increases the initial adhesion and consequently the growth of cells in the structure. But the phenomenon of inhibition of contact between cells, explosive release, and also lower growth factors used in the scaffold, has caused that despite the growth of cells in the sample with starch nanoparticles more than samples without PRP, this difference was not statistically significant (p < 0.05) [43].
3.3.2. Cell attachment
The morphology, adhesion, and distribution of differentiated cells to osteoblasts cells can be observed and interpreted using SEM images at different times (Fig. 10). As expected, the cells would eventually attach to the outer surface of the scaffold through the pores that appear on their outer membranes, indicating that their biological conditions were favorable. Scaffolds containing PRP have earlier and more attachment, which can be due to the release of growth factors and other suitable materials for improving the microenvironment of cells.
3.3.3. Alkaline phosphatase activity (ALP activity)
ALP is an enzyme that has the most suitable activity at alkaline pH. It comes in different forms in the blood and is found in large amounts in the liver and bones. But it is also found in other tissues such as the kidney, placenta, intestinal wall, and thymus gland. Alkaline phosphatase contains several isoenzymes, including bone marrow (ALP2) and liver (ALPI). In bone disorders, ALP levels increase due to abnormal osteoblastic activity. Alkaline phosphatase is an early marker for the detection of osteoblasts, which is an enzyme in the cell membrane that hydrolyzes phosphate ions and allows the formation of hydroxyapatite crystals and the onset of mineralization.
The activity of the alkaline phosphatase enzyme is shown in Fig. 11(a) in which it can be seen that the activity of this enzyme in all samples containing PRP is up to day 14 and then decreases with increasing calcium deposition. But this is not the case in the sample without PRP.
Dolder et al reported that PRP could affect early matrix growth and mineralization of mouse bone marrow stem cells. As can be seen, scaffolds containing platelet-rich plasma show alkaline phosphatase activity peak earlier than other samples. On day 14, the trend is reversed and the unloaded sample shows higher enzyme activity. After day 14, enzyme activity decreases for both samples. These results indicate that release from PRP accelerates the process of bone differentiation.
3.3.4. Calcium assay
In this assay, calcium in an alkaline environment forms a purple complex with the cresol-phthalein complex. The intensity of the color created is proportional to the amount of calcium in the sample. During the osteogenesis process, after the highest level of alkaline phosphatase activity, a decrease in the activity of this enzyme occurs and mineralization improves. Peak alkaline phosphatase activity was observed earlier in PRP samples and then calcium production was expected to rise. Figure 11(b) shows a significant difference in the amount of calcium in the protein-carrying sample with the unloaded sample on day 14.
The process of transforming stem cells into osteoblasts that make up the functional matrix is briefly divided into three stages: (a) cell growth and proliferation and extracellular matrix biosynthesis; (b) cell maturation, spreading, and regulation of the extracellular matrix; and C) Mineralization. At each of these stages, changes in gene expression occur. In the first stage, which lays the foundation for the development of bone phenotypes, genes involved in the production of extracellular matrix materials (such as collagen type I and fibronectin and growth factor-beta) are expressed, after which this gene expression is gradually reduced. Also, the mRNA of collagen remains a low level at the later stages of differentiation. Thymidine [3H] and MTT assay showed increased cell divisions and the accuracy of the first phase of cell differentiation. Immediately after the expression of genes involved in cell growth and proliferation decreases, proteins that are related to the bone phenotype of the cells are identified. For example, the activity of the enzyme alkaline phosphatase and its mRNA is 10 times higher. At this time, changes occur in the structure of chromatin that transcribes the genes that are responsible for the functional and structural proteins of differentiated osteoblasts. In the post-proliferation period, the extracellular matrix and its components are arranged and changed in the direction of mineralization. In the mineralization medium, mRNA levels of the alkaline phosphatase decreased and the expression of other genes responsible for proteins such as sialoprotein, osteopontin, and osteocalcin increased with matrix mineralization [44].
The effect of alkaline phosphatase activity on the initiation of the mineralization process can be sought in the balance between mineralization promoting factors such as inorganic phosphate (Pi) and inhibitors such as extracellular inorganic pyrophosphate (ePPi). ePPi prevents Pi from binding to form crystals with calcium ions and produce hydroxyapatite. The role of TNAP and the phosphodiesterase pyrophosphatase (NPP1-PC) nucleotide (NPP1-PC) in the mechanism of ankylosing proteins, which are actually intermembrane proteins responsible for the transport of inorganic pyrophosphate, is complementary. Inhibition of mineralization, therefore, TNAP improves and promotes mineralization by converting ePPi produced by NPP1 and hydrolyzing them to inorganic phosphate. Finally, with the advancement of mineralization; Osteoblasts reduce the activity of this enzyme by a suitable Pi / PPi response mechanism [45].