Morphological characterization and elemental distribution of scaffolds

Since the morphological characteristics of the scaffolds affect their cellular, mechanical, and thermal behaviors, SEM images from surfaces of PGS/PCL, PGS/PCL/Gel, and PGS/PCL/Gel/HA with different amounts of nanoparticle (3% and 5%) were investigated and shown in Fig. 2. The results showed porous scaffolds with interconnected pores and irregular shapes. As shown in Fig. 2 HA nanoparticles show a monotonous dispersion and excellent adhesion inside the polymeric matrix, indicating a suitable synthesis method.

Fig. 2

Scanning electron microscopy images of scaffolds fabricated by salt leaching method in the surface, at 5kx magnification. a PGS/PCL b PGS/PCL/Gel c PGS/PCL/Gel/HA 3% d PGS/PCL/Gel/HA 5%. SEM images show porous scaffolds with irregular shapes, and HA nanoparticles

In addition, the mapping analysis shows the dispersion of calcium (Ca), phosphorus (P), and oxygen (O) atoms as the main elements in HA nanoparticles, so that the atomic percentage of Ca, P, and O in the samples increased with the increase in the percentage of HA nanoparticles. Figure 3A and B represent the dispersion of these atoms in PGS/PCL/Gel/HA 3% and 5%, respectively.

Fig. 3

SEM/EDX-Mapping of A) PGS/PCL/Gel/HA 3% sample and B) PGS/PCL/Gel/HA 5% sample a SEM image b calcium (Ca) atoms c phosphorus (P) atoms d oxygen (O) atoms and e The combination of elements

Hydrophilicity behavior of scaffolds

The hydrophilicity of five composite scaffolds, as an important factor in biocompatibility and biodegradability, was investigated using contact angle analysis [37,38,39]. The average contact angles for each sample after 20 s have been presented in Fig. 4A. According to the results, the PGS and PGS/PCL/Gel/HA 5% scaffolds as the most hydrophilic samples show a lower contact angle among others. PCL with hydrophobic nature due to aliphatic groups causes an increase in contact angles [37], while gelatin is a natural hydrophilic polymer consisting of a large number of glycine, proline, and 4-hydroxy proline residues, which provides a favorable physico-chemical microenvironment for cell adhesion and proliferation [40].

Fig. 4

A Results from contact angle analysis, B The absorption of scaffolds in PBS after 24 h, C Hydrolytic degradation of scaffolds in PBS at time interval of 1, 3, 7, 14, 28 and 40 days. The statistical significant analysis paired t-test compared to control; *p < 0.05, and **p < 0.01

As shown in Fig. 4A, the contact angle of the PGS/PCL/Gel sample is less than PGS/PCL sample, due to the hydrophilicity of gelatin.

On the other hand, the incorporation of hydroxyapatite nanoparticles with polar groups [41, 42], to the polymer mixture reduces the contact angle compared to the PGS/PCL/Gel scaffold. Among two nanocomposite scaffolds, the incorporation of 5 wt% of HA nanoparticle shows a more significant effect in increasing the hydrophilicity of PGS/PCL/Gel copolymers, so that the contact angle reaches a value lower than the contact angle of the PGS scaffold. These results showed the effect of gelatin and HA nanoparticles on enhancing the hydrophilic properties of composite scaffolds.

The swelling process is defined as an increase in the volume of a gel or solid material due to liquid absorbance. In polymers, swelling is the first step of interaction between liquid molecules and the polymeric network, which is usually followed by solving the polymer chains [37]. The swelling behavior of the scaffold is a crucial parameter that can influence cell adhesion and growth [43].

As shown in Fig. 4B, after 24 h, the PBS absorption of all composite scaffolds has significantly increased compared to the PGS scaffold as control. Results revealed that the PGS/PCL/Gel sample has the highest swelling percentage among others.

Hydrolytic degradation of scaffolds

The degradation behavior of polymeric scaffolds after tissue restoration is a critical feature that eliminates the need for secondary surgeries [43,44,45]. Since PGS has too fast degradation rate, PCL as a hydrophobic polymer with a low degradation rate, is a good candidate to control its degradation rate and mechanical stability in the body [46,47,48,49].

Figure 4C demonstrates the results of degradation during 40 days. Moreover, the combination of PCL with hydrophilic materials including Gelatin and HA nanoparticles increases the degradation rate [43]. Gelatin as a hydrophilic natural polymer enhances wettability, accelerates degradation, and improves cell recognition sites of PCL [12].

In this study, the degradation of pure PGS, PGS/PCL, PGS/PCL/Gel, and PGS/PCL/Gel/HA scaffolds with different contents of nanoparticles (3, 5 wt %) was investigated. As shown in Fig. 4C, PGS/PCL/Gel composite scaffolds show the highest weight losses among others because of their swelling behavior. As expected, scaffolds with higher HA nanoparticle content (5 wt%) showed a higher degradation rate compared to PGS/PCL/Gel/HA 3%, due to the hydrophilic nature of HA nanoparticles.

Mechanical properties of scaffolds

Cartilage is a composite load-bearing tissue found in animal and human joints [50]. Since ECM regeneration depends on the mechanical properties of the scaffold at both macroscopic and microscopic scales, evaluation of the mechanical properties is a critical factor for providing a stable structure and clinical application of the scaffold [43].

The mechanical characteristics including compression strength (Fig. 5A), elongation at break (Fig. 5B), and module (Table 3) of composite scaffolds were investigated to assess the effect of the combination of PCL and Gelatin as well as HA nanoparticles in PGS-based composites (Fig. 5). Considering dry scaffolds, the results revealed that compression strength of PGS/PCL/Gel and PGS/PCL/Gel HA3 wt % composites are higher than other scaffolds. On the contrary, the compression strength of PGS/PCL/Gel/HA 5% samples in the wet state shows a remarkable decrease (p-value ≤ 0.05) compared to PGS/PCL/Gel/HA 3% scaffolds, which can be attributed to the higher swelling in the scaffolds with 5% HA.

Fig. 5

Mechanical properties A Compression strength, B Elongation at break of the scaffold; at dry (left) and wet (right) conditions; The values are expressed as means (± SEM; n = 3), (*p < 0.05), (** p < 0.01)

Table 3 Calculated modulus of fabricated scaffolds

No significant changes (p-value > 0.05) were observed in compression strength between PGS, PGS/PCL, and PGS/PCL/Gel samples in dry condition, however as shown in Fig. 5A in wet state, the compression strength of PGS/PCL/Gel scaffolds showed a significant decrease (p-value ≤ 0.05) compared with PGS, and PGS/PCL samples. Additionally, there is considerable difference (p ≤ 0.05) between PGS/PCL/Gel, PGS/PCL/Gel/HA 3%, and PGS/PCL/Gel/HA 5% samples, in the wet state.

As shown in Fig. 5B, the addition of PCL to PGS is associated with a significant decrease (p-value ≤ 0.01) in elongation in the dry state, due to the toughness of the PCL, while the addition of gelatin increases elongation (p-value ≤ 0.05). Meanwhile, by introducing HA nanoparticles, the elongation represents a significant increase (p-value ≤ 0.01), which shows a higher value than the elongation of pure PGS sample by the increase of HA percentage. However, in the wet state, the elongation of the samples in PGS, PGS/PCL, PGS/PCL/Gel, and PGS/PCL/Gel/HA 3%/ scaffolds is not significantly different (p-value > 0.05), while with the increase of HA nanoparticles to 5%, a sharp decrease in elongation is observed (p-value ≤ 0.01).

Cell viability and proliferation

To determine the effect of nanocomposite scaffolds on cytocompatibility and cell proliferation, the optical absorbance of ADSCs at 570 nm has been evaluated at the time intervals of 1, 7, and 14 days. According to MTT results, shown in Fig. 6, after 24 h, the engineered scaffolds in PGS/PCL group had inhibitory effects on cell proliferation. After that, on day 7, a significant increase in cell proliferation was observed in the PGS/PCL/Gel and PGS/PCL/Gel/HA 5% groups compared to the control samples (PGS scaffolds). Finally, after 14 days of treatment, all groups except PGS/PCL/Gel/HA 5% showed higher cell proliferation, especially in the presence of 3% HA. Based on these results, PGS-PCL-Gel and PGS-PCL-Gel-HA 3% scaffolds can support more cell survival and proliferation in vitro over time.

Fig. 6

In vitro evaluation of human mesenchymal stem cells survival and proliferation on 1, 3, and 14 days after cell seeding. The statistically significant analysis paired t-test compared to control cells. *p < 0.05, and **p < 0.01

Cell attachment

The cell attachment and behavior of PGS, PGS/PCL, PGS/PCL/Gel, and PGS/PCL/Gel/HA (3 and 5%) scaffolds three days after cell seeding have shown in Fig. 7, revealing the interaction of cells with scaffolds as well as cell attachment and spreading of them inside the pores. Since hydrophilicity and functional groups at the scaffold surface are the major factors in cell adhesion [51, 52], more cell-scaffold interaction can be observed at PGS/PCL/Gel/HA 3%( g, h) and PGS/PCL/Gel/HA 5%,( i, j) due to hydroxyapatite nanoparticles with polar groups. In addition, PGS/PCL/Gel scaffolds (e, f) have a very similar trend in this property, due to the hydrophilicity of Gelatin. These findings are confirmed by contact angle analysis in "Hydrophilicity behavior of scaffolds" section.

Fig. 7

Scanning electron microscopy images of human adipose derived mesenchymal stem cells seeded on the scaffolds 3 days after cell seeding (a&b) PGS, (c&d) PGS/PCL, (e&f) PGS/PCL/Gel, (g&h) PGS/PCL/Gel/HA 3%, (i&j) PGS/PCL/Gel/HA 5%

Cell differentiation

To evaluate the chondroconductive capability of the scaffolds, mRNA levels of Aggrecan, Sox9, Collagen II,as well as Osteocalcin were investigated after 21 days of cell seeding and results are depicted in Fig. 8.

Fig. 8

Gene expression profile of chondrogenic markers A Aggrecan, B Sox9, and C Col2 D Osteocalcin on PGS and PGS/PCL/Gel, PGS/PCL/Gel/HA 3 & 5%; the results are averages of three independent experiments,* p-value ≤ 0.05

The increase of Aggrecan expression as a marker of chondrogenic differentiation was observed after 21 days of seeding for scaffolds with 3 and 5 wt% of HA nanoparticles as compared to PGS and PGS/PCL/Gel samples (P < 0.05) (Fig. 8A).

The results showed an increase in the Sox9 gene expression level for samples with 3 and 5 wt% of HA nanoparticles as compared to PGS and PGS/PCL/Gel scaffolds revealing mineralization effect of nHA (P < 0.05) (Fig. 8B). As a result, mRNA analysis revealed significant cartilage-related gene expression for samples containing HA, which was consistent with other studies [18,19,20, 53], indicating the chondroconductive properties of HA. In addition, normalized data in Fig. 8C show the highest increase in Col2 expression in PGS/PCL/Gel/HA 3% and 5% scaffolds compared to other groups (P < 0.05), indicating the role of nHA in chondrogenic differentiation. The mRNA expression of Sox9 was also in accordance with the results observed for Aggrecan and Col2 genes. As shown in Fig. 8D,the result of gene expression for Osteocalcin as a bone-related gene showed down-regulation (P > 0.05).