Scaffold Characterization

DHART-loaded PCL/Col NFs (5%, and 10% (wt/wt%)) were constructed through the electrospinning process under the optimal conditions. Any further addition in DHART amount resulted in the therapeutic compounds precipitate in the solution and the formation of electrospun beaded fibers.

According to FE-SEM results, the PCL/Col NFs exhibited smooth and free-bead surfaces with a 250 nm mean size (Fig. 1). Although comparable features to PCL/Col NFs were achieved by encapsulating DHART into PCL/Col NFs, the mean thickness was increased to 320 nm, demonstrating a homogenous distribution of the drug in the polymeric solution and a steady increase in the NF diameter as the drug and polymer are physically blended. These findings are in agreement with those of Hokmabad VR et al. They found that the manufactured PCL-PEG-PCL (PCEC) NFs were bead-free, and smooth fibers with a mean size of 138.851.7 nm while the average thickness of Elaeagnus angustifolia (EA)-loaded PCEC NFs incorporated with 5 wt-%, 10 wt-% and 15 wt-% EA were 180.856.3, 200.4768.5 and 237.2493.6 nm, respectively (1).

Fig. 1

Morphology and diameter distribution of electrospun NFs. FE-SEM images of A PCL/Col, B PCL/Col/DHART (5%), and C PCL/Col/DHART (10%)

The drug encapsulation process within NF carriers induced atypical shifts in FTIR spectroscopy. As a result, FTIR spectroscopic comparisons between pure DHART and PCL/Col/DHART NFs can be rather significant. Figure 2A displays the FTIR results of pure DHART, PCL/Col, and PCL/Col/DHART NFs. The therapeutic properties of DHART are attributed to its peroxide bridge, which can produce particularly potent oxidative stress [19]. The spectrum of pure DHART displayed typical bands at 3379 cm−1 (O–H stretching vibrations); 2947 cm−1 (C-H stretching); 1093 cm−1 (C-O stretching); 875 cm−1 (O–O-C stretching); 825 cm−1 (O–O stretching) presenting characters of O–O-C entityrespectively that characterizes 1,2,4-trioxanering [30]. In the PCL/Col NF spectra, the typical peaks at 1184 cm−1, 1220 cm−1, and 1727 cm−1 were assigned to the stretching band of C-O, C–O–C, and carboxylic acid functional groups of PCL, respectively. Col revealed the specific peaks at 1541 cm−1 (amide II) and 1647 cm−1 (amide I). As seen in Fig. 1, the presence of adsorption bands at 875, 1100 cm−1 in the pure DHART and PCL/Col/DHART NFs demonstrates the presence of DHART in its active state in PCL/Col/DHART NFs.

Fig. 2

A FTIR spectra of DHART, PCL/Col, PCL/Col/DHART (5%), and PCL/Col/DHART (10%), B TGA curves of DHART, PCL/Col PCL/Col/DHART (5%), and PCL/Col/DHART (10%) NFs from 0 to 600 °C, and C XRD patterns of DHART, physically mixed DHART@PCL/Col, PCL/Col/DHART (5%), and PCL/Col/DHART (10%) NFs

Thermal stability of DHART, PCL/Col, PCL/Col/DHART were evaluated through TGA. The detected initial weight loss for all samples up to 100 \(^\circ\) is due to the moisture evaporation of compounds. DHART was initiated to decompose at 110 \(^\circ\), monitored by loss of volatile substances of roughly 26% by weight to the second decomposition at 165 \(^\circ\) [31]. In comparison, thermal degradation of PCL/Col, and PCL/Col/DHART NFs was between 389.70 and 424.65 °C, respectively (Fig. 2B).

To further characterization the interaction between DHART and PCL/Col, the crystal properties of the PCL/Col/DHART were examined by X-ray diffraction (Fig. 2 C) The diffractogram for DHART displays distinct peaks in the initial state, indicating the presence of crystalline phase [31], whereas the PCL/Col/DHART revealed a characteristic amorphous pattern. For the elimination of the possibility that the lower efficacy is due to the reduced weight percentage of DHART in the drug-loaded NF apparatus, the XRD analysis was performed on the physical mixture of PCL/Col/DHART with the equal quantity proportion. As shown in Fig. 2C, the DHART peak intensity is still evident in the physical mixture, demonstrating that its crystalline configuration is still evident. As a result, it demonstrates that the absence of the typical signal of DHART in PCL/Col/DHART NFs is not owing to the weak signal but instead to the amorphous structure. The current findings indicate that scaffold NFs can prevent DHART from the re-crystallizing process.

Also, Fig. 3A shows the rate of degradation of the prepared NF scaffolds. The fiber weight reduction was not noticeable during the initial four days. After 28 days, more than 80% PCL/Col and PCL/Col/DHART NFs have degraded a relatively stable speed of weight reduction. Interestingly, no major differences in the removal percentage of PCL/Col and PLC/Col/DHART NFs were found. This was anticipated due to the small percentage of the total fiber weight that was related to DHART. After 28 days, the percentage of weight reduction for PCL/Col and PCL/Col/DHART NFs were 22.2% and 20.1%, respectively.

Fig. 3

A Biodegradation profiles, B and typical tensile stress–strain curves of samples

Besides, mechanical features must be considered in the efficient constructing scaffolds for tissue engineering and regenerative medicine applications. The characteristic tensile strain–stress diagrams of PCL, PCL/Col, and PCL/Col/DHART NFs were presented in Fig. 3B. Tensile stress for PCL, PCL/Col, PCL/Col/DHART (5%), and PCL/Col/DHART (10%) NFs were 2.33, 6.2, and 6.14, 6.26 MPa and can bear a strain of 86, 66, 68 and 70%, respectively. The Young’s modulus value for PCL, PCL/Col, PCL/Col/DHART (5%), and PCL/Col/DHART (10%) NFs were 11.21, and 29.13, 30.2, 31.7 MPa. The result displayed greater tensile stress–strain values for PCL/Col than PCL NFs due to enhanced mechanical characteristics. The findings suggested that combining PCL with Col improves the mechanical characteristics of NF scaffolds [32]. Also, as shown in this diagram, the tensile mechanical and physical properties of DHART-loaded NFs and neat NFs were remarkably similar, demonstrating that incorporation of DHART molecules into the PCL/Col NFs could not significantly change the fiber's mechanical properties.

Drug release assay

Cumulative DHART release from PCL/Col/DHART (5%) NFs was evaluated for 21 days at 37 °C in PBS solution. The release profile of PCL/Col/DHART (5%) NFs is shown in Fig. 4. As shown in Fig. 4, PCL/Col/DHART (5%) NFs with the highest content of PCL demonstrated that fast drug release at first, then slower sustained release, which is typical of reservoir-type scaffolds. DHART burst release from membranes was first identified in the early days. PCL/Col/DHART NFs displayed a more constant release of DHART after 4 days. The burst release phase was due to the rapid release of amorphous DHART from the PCL/Col/DHART surfaces. The DHART encapsulated within the PCL/Col NFs dispersed slowly onto the NF surface and then into the medium solution, providing a gradual rate of diffusion. Free DHART control exhibited the typical rapid release, with > 85% DHART released within 4 h at pH 7.4. However, PCL/Col/DHART released about 87% of DHART after 2 weeks of incubation. This is explained by interactions between DHART and PCL/Col hybrid polymer, which resulted in a significant enhancement in the sustained release of drugs from the PCL/Col/DHART (5%) NFs.

Fig. 4

Cumulative DHART release in PBS (pH 7.4) at regular intervals. The data are presented as mean ± SD (n = 3)

These findings are in accordance with those from a study by Huo P and colleagues, demonstrating that the sustained release of Artemisinin (ART) from PCL/Col NFs. They reported that these molecules diffuse into the PBS medium from the surface of the PCL/Col NFs and form holes on the surface of the NFs. As the release progresses, pores are gradually formed inside the NFs, and the medium gradually penetrates the NFs. Subsequently, the ART embedded in the PCL/Col NFs gradually dissolves and eventually completely dissolves in the medium, leaving the NF scaffolds (19).

The viability and proliferation of hADSCs on PCL/Col/DHART NFs

hADSCs were seeded on the prepared NFs and their cytocompability and the viability and proliferation of these cells were evaluated using MTT assay at 1-, 7-, 14-, and 21- days (Fig. 5A). The results revealed that the viability and proliferation improved in all of the treated samples and there was no important difference in the viability of the PCL/Col treated group for different time intervals of the experiment. However, it is observed that the viability of PCL/Col/DHART treated groups was significantly higher on days 14 and 21 than for the neat PCL/Col treated groups. Importantly, it was found that the PCL/Col/DHART (5%) treated NFs had higher cell viability than PCL/Col/DHART (10%) treated NFs for different time intervals of the experiment, obviously on day 21 (p < 0.0001). The results demonstrated that PCL/Col/DHART (5%) NFs are nontoxic and could significantly improve the viability of hADSCs.

Fig. 5

The viability and proliferation of hADSCs on the NFs evaluated by MTT and PicoGreen assays, respectively. ****P < 0.0001 vs. control was considered significant. Results are mean ± SD (n = 3)

For the evaluation of the amount of cell proliferation, PicoGreen examine was used to determine the DNA quantity in the treated NF scaffolds (Fig. 5B). There was no considerable variance in the DNA quantity at periodic intervals for the control and PCL/Col treated groups. However, PCL/Col/DHART treated groups had a higher DNA value after 14 and 21 days. In comparison to the other groups, the DNA quantity in the PCL/Col/DHART (5%) treated NFs significantly increased from day 1 to day 21 (p < 0.0001). It is expected that the prolonged release of DHART from the NF scaffolds provides long-term attachment for the hADSCs viability and proliferation. These findings are in accordance with those from a study by Mashayekhi et al., demonstrating that the sustained release of DHART from NFs increase the adhesion, viability, and proliferation of hADSCs.

The adhesion and proliferation of hADSCs on PCL/Col/DHART NFs

The FE-SEM analysis showed the morphology and distribution of the treated NF groups after 21 days of incubation (Fig. 6). These results indicated that cell lines cover the surface of all the NFs and can adhere to them. After 21 days of culture, the PCL/Col/DHART treated groups revealed better attachment and proliferation compared to PCL/Col treated NFs. The most distribution and covering of hADSCs were found on PCL/Col/DHART (5%) treated group, demonstrating high biocompatibility and non-toxicity of PCL/Col/ DHART (5%) for hADSCs. These results were compatible with the detected results of the viability and proliferation of hADSCs on PCL/Col/DHART NFs.

Fig. 6

FE-SEM images of adhesion of hADSCs on the A PCL/Col, B PCL/Col/DHART (5%), C PCL/Col/DHART (10%) groups after 21 days of cell seeding

The expression levels of osteogenic-specific genes of hADSCs on PCL/Col/DHART NFs

For the evaluation of the proliferation and differentiation of treatment groups, the comparative mRNA expression of major osteoblast differentiation factors such as OSX, BMP-2, RUNX-2, Col I, and OCN were assessed by real-time PCR analysis (Fig. 7).

Fig. 7

mRNA expression levels of OSX, BMP-2, OCN, Col I, and RUNX-2, in hADSCs seeded on the NFs after 7-, 14-, and 21- days of culture. The GAPDH gene was used as an internal control. P < 0. 05 vs. control was considered significant. Results are mean ± SD (n = 3). *** represents p ≤ 0.001, **** represents p ≤ 0.0001

Col I is an initial osteoblast differentiation factor that helps to generate the external collagen network, which is necessary for the formation of the osteoblastic appearance [33]. The Col I expression peak was higher in DHART-loaded NFs on day 7, but it was reduced on day 14. Also, previous studies have revealed that Col I is up-regulated during the osteoblastic differentiation early phases and down-regulated during the later phases. Also, Runx2 is another initial key factor for osteoblastic differentiation, which up-regulates OCN fundamental bone matrix factor [34]. The expression level of Runx2 mRNA in the treated scaffolds increased until the 14 days and then decreased in all of the treated groups (Fig. 7). The expression level of Runx2 mRNA was rather higher in the DHART-loaded NF (5%) groups compared with the other treated groups.

OSX, BMP-2, and OCN are the most prominent non-collagenous bone formation factors that show a critical role in the early stages of bone formation calcification and are widely used as the late factor in bone formation differentiation [35]. As anticipated, the expression mRNA level of these proteins increased dramatically over time, with the greatest value expression observed on day 21 for the DHART and PCL/Col/DHART (5%) (Fig. 6). Previous studies have suggested that DHART plays an unknown mechanism in the control of human mesenchymal stem cells (hMSCs) osteogenic differentiation and proliferation. Licheng Ni, et al. reported DHART’s effect on hMSCs’ osteogenic differentiation and proliferation, together with its fundamental metabolic pathways. They found that DHART had no effect on the hMSC proliferation but improved osteogenic differentiation. It most possibly carried out its activity via the ERK1/2 and Wnt/β signaling pathways [23].

Osteoblastic differentiation on PCL/Col/DHART NFs

The most common procedures for assessing stem cell osteoblastic development on platforms are the evaluation of total calcium concentration and ALP activity. Alizarin Red staining was performed to visualize calcium deposition in osteoblasts at 7-, 14- and 21- days following the induction of osteogenesis. (Fig. 8A and B). It was found that calcium deposition in osteoblasts was significantly increased, and these promoting effects were time-dependent. These results are in accordance with those from a study by Xia T et al. [36]. After 24-days incubation, the results of mineralization demonstrated that the treated NF scaffolds had increased calcium deposition compared to the control-treated group. The calcium deposited quantity on the DHART-loaded NFs was found to be much higher than that on control, and neat NF treated scaffolds due to the biofunctionality of released DHART which stimulated the osteogenic differentiation. Moreover, the osteoblast phenotype was evaluated by determining ALP activity. The results revealed that ALP activity was also increased in a time-dependent manner (Fig. 8C). In comparison to the control-treated group, PCL/Col, and PCL/Col/DHART NF scaffolds considerably improved ALP activity on the 21 days. Particularly, it was found that PCL/Col/DHART (5%) treated groups considerably enhanced the cell ALP activity.

Fig. 8

A Alizarin red staining of hADSCs seeded on the treatment groups of a) control, b) PCL/Col, c) PCL/Col/DHART (5%) and d) PCL/Col/DHART (10%) at 7-, 14- and 21- days after osteogenic differentiation, and B in quantitatively colorimetric data of alizarin red staining. Results are mean ± SD (n = 3). C The ALP activity of hADSCs seeded on the treatment groups of control, PCL/Col, PCL/Col/DHART (5%) and PCL/Col/DHART (10%) at 7-, 14- and 21- days after osteogenic differentiation. P < 0. 05 vs. control was considered significant. Results are mean ± SD (n = 3). * represents p ≤ 0.05, *** represents p ≤ 0.001, **** represents p ≤ 0.0001