PCEC/Gel nanocomposite is one of the most commonly used scaffolds for bone tissue engineering as a result of biocompatibility and biodegradability. Herein, we present a proof-of-concept of creating nanocomposite scaffolds based on interconnection between PCEC copolymer and gelatin chains, which doped with hydroxyapatite and superparamagnetic iron oxide nanoparticles. Gelatin is denatured collagen; it has a structure and chemical composition that resembles the extracellular matrix. The studies have shown that HA and Fe3O4 can enhance the mineralization of the scaffold and have a vital role in the proliferation and differentiation of osteoblasts [31]. Although HA and Fe3O4 have been used as doping elements in scaffolds for Bone TE, the simultaneous incorporation of them in the PCEC/Gel system has not been reported yet. Here, we try to characterize the physicochemical properties of PCEC copolymer and PCEC/Gel scaffold as well as the feasibility of incorporating HA and Fe3O4 into the PCEC/Gel to yield the nanocomposite scaffolds for Bone TE. The construction route for the preparation of PCEC/Gel-HA& Fe3O4 is illustrated in Fig. 1A.

Fig. 1

A Ischemic illustration for the preparation of PCEC/Gel-Fe3O4&HA scaffold. FT-IR spectra of B HA and Fe3O4. C PCEC, PCEC/Fe3O4, PCEC/HA, PCEC/Fe3O4&HA. D 1HNMR spectroscopy belonging to synthesized PCEC polymer

FT-IR analysis

To evaluate the encapsulation of the HA and Fe3O4 into the PCEC copolymer FT-IR spectra of the PCEC, PCEC/Fe3O4, PCEC/HA, PCEC/Fe3O4&HA was conducted and compared with the spectra of HA and Fe3O4. As depicted in Fig. 1C the characteristic band at 1740 cm− 1 corresponded to C=O stretching of the ester groups. The results revealed that all spectra exhibited the same absorption band. The peaks at 1105 cm− 1 and 1169 cm− 1 were corresponding to the vibration bands of C-O-C. The stretching vibration of the OH group appeared at 3446 cm− 1. The peaks at 2800–2900 cm− 1 could be attributed to the aliphatic C-H stretching bonds. In Fig. 1B, in the spectrum of Fe3O4, the absorption band at 561 cm− 1 belonged to the Fe-O group, which was observed in the spectrum of the PCEC/Fe3O4 and PCEC/Fe3O4&HA, indicating the successful incorporation of magnetic nanoparticles with PCEC copolymer. Also, the peaks in 1620 cm− 1 attributed to the bending mode of OH groups of adsorbed water [32, 33]. In the spectrum of HA, the peaks located at 569 cm− 1 and 603 cm− 1 corresponded to the asymmetric and symmetric bending modes of the PO43− group. The appearance of phosphate stretching vibration in the spectrum of the PCEC/HA and PCEC/Fe3O4&HA also confirmed the successful incorporation of HA with PCEC copolymer. The peak at 1040 cm− 1 was attributed to the C-O stretching of the carbonate group which was substituted by the phosphate group [34]. The band at 3413 cm− 1 was corresponding to the stretching vibration of O-H groups in the apatite lattice [35].

1HNMR spectroscopy

The 1HNMR spectrum of the PCEC copolymer is depicted in Fig. 1D. The presence of a singlet peak at 3.63 ppm (Ha) was assigned to the methylene protons of the PEG block in the copolymer. Additional signals at 4 ppm (He), 2.35 ppm (Hb), 1.65 ppm (Hc), and 1.48 ppm (Hd) came from the PCL block in the copolymer chain [36].

Thermal gravimetric analysis

The thermal stability and degradation behavior of PCEC/Gel nanocomposites were examined by TGA and DTG with a heating rate of 10 °C per minute in the flow of N2 gas from 50 °C to 700 °C. As depicted in Fig. 2A, the degradation process of PCEC/Gel nanocomposites have two maximums around 400 °C and 500 °C. The lower degradation temperature, which referred to the greatest reduction in mass, was induced by disintegrating intermolecular interactions as well as the breakdown of the copolymer backbone [37], while the second maximum mostly referred to gelatin decomposition. As shown in the DTG curve, the initial degradation temperature (Ti) of the PCEC/Gel nanocomposites was around 200 °C and the main degradation prosses took place in the range of 200–450 °C and 450–550 °C, corresponding to 66.35 and 20.7% weight loss, respectively [38,39,40].

Fig. 2

A TGA and DTG curves of nanocomposite scaffolds. B Thermal parameters derived from TGA and DTG data of nanocomposite scaffolds. Porosity and specific surface area of materials in nanocomposite scaffolds (PCEC/Gel), C Adsorption-desorption isotherm, D The BJH curve

Porosity measurement and BET surface area

The uptake property and adsorbent capacity of the prepared scaffold was predicted from the porosity and specific surface area. In this regard, the Brunauer-Emmett-Teller (BET) theory was used to determine the specific surface areas of PCEC/Gel and the corresponding N2 adsorption-desorption curve is shown in Fig. 2C. The obtained isotherm of PCEC/Gel corresponded to type III according to the IUPAC classification. The BET surface area of PCEC/Gel was 3.274 m2/g. The pore size distribution was determined by Barrett–Joyner–Hanlenda (BJH) theory and represented the nanoporous structure of PCEC/Gel with the average pore radius of 1.21 nm and a pore volume of 0.011 cm3/g. The corresponding pore size distribution is depicted in Fig. 2D, which represented the presence of mesopores and micropores.

X-ray diffraction (XRD) analysis

The X-ray diffraction (XRD) pattern of Fe3O4 NPs and HA, along with PCEC/Gel nanocomposite scaffolds and scaffolds containing 6% Fe3O4, and 6% HA, separately and in combination with together were shown in Fig. 3A. The characteristic diffraction peaks, which corresponded to the HA were observed at 2θ angle values of 25.7°, 31.65°, 32.89°, 39.62°, and 49.3° [34, 41]; and the peaks at 2θ of about 30.15°, 35.6°, 43.27°, 57.3°, and 63° were attributed to the reflection plane of Fe3O4 NPs [42]. The powder x-ray diffraction of the nanocomposite scaffold revealed that the structure is mostly amorphous. Compared to crystalline materials, amorphous materials are more prone to hydrolytic degradation. The high sensitivity of amorphous polymer to hydrolytic degradation is due to the easy transfer of water molecules to the inner region of the polymer that can prove the biodegradability of the copolymer. The peak at 2θ angle around 20° was attributed to the gelatin reflection plane [43], while the peak located at 2θ angle around 23.6° corresponded to the PCL block of the PCEC copolymer, which means that the crystallinity of the PEG block was restricted by the outer PCL block [44]. In the diffractogram of PCEC/Gel-Fe3O4, the peak at 2θ angle around 25° corresponded to the HA, which confirms the presence of HA in the nanocomposite scaffolds; however, the characteristic peaks of metal oxide nanoparticles were not observed in the diffractogram due to the utilizing negligible percentage for doping, which could be under the detection limit of the apparatus.

Fig. 3

A X-ray diffractograms belonging to magnetic nanoparticles, hydroxyapatite, and different groups of nanocomposite scaffolds. B Magnetic properties analysis for Fe3O4 nanoparticles and scaffolds containing magnetic nanoparticles (a, b). C Swelling behavior in gelatin and nanocomposite scaffolds. D Stress-strain curves in tensile tests of gelatin and scaffolds in dry condition. E Stress-strain curves in tensile tests of scaffolds in wet condition

Vibrating-sample magnetometer (VSM)

To measure the magnetic properties of the synthesized samples, the VSM was performed at 25 °C and the corresponding hysteresis curve was prepared by changing the magnetic field of H from − 15,000 to + 15,000 Oe. As shown in Fig. 3B, the saturation magnetization (Ms) value of Fe3O4 NPs was 61.54 emu.g− 1 at room temperature (300 K), which significantly reduced for the PCEC/Gel-Fe3O4 and PCEC/Gel-Fe3O4&HA. Reduction in saturation magnetization of scaffold nanocomposites was due to the use of a negligible amount (6 wt%) of magnetic nanoparticles, but the samples also represented paramagnetism. Hysteresis magnetization and negligible magnetic coercivity in the results indicated that the nanoparticles were superparamagnetic in both samples, and it was proven by converting the hysteresis loop into an S-shaped curve [45, 46].

Analysis of mechanical properties of nanocomposite scaffolds

As one of the most important biomaterials, gelatin has gained more attention in bone tissue engineering due to the induction of osteogenesis, angiogenesis, and wound healing [24]. However, compared to other polymers like metallic compounds, it has lower loading capacity and elastic moduli. To address the problem, the incorporation of the gelatine with a biocompatible polymer like PCEC could enhance the bioactivity and mechanical property of the resulting scaffold [24]. Determining the mechanical properties of scaffolds is a basic issue in tissue engineering as these properties can affect cell behavior during culturing, adhesion, proliferation, and signaling. Increasing the porosity of the scaffold reduces the mechanical properties [47]. Our results showed that Young’s Moduli increased from 29.44 MPa (pristine gelatine) to 78.79 MPa (PCEC/Gel nanocomposite scaffolds) and the tensile strength increased from 0.39 to 0.53, respectively. Improving the mechanical properties of the nanocomposite scaffold could be explained as a result of decreasing the porosity and increasing the wall thickness of the scaffold pores [48]. The mechanical strength values ​​obtained from hydrated nanocomposite scaffolds were also compared with dry nanocomposite scaffolds. In dry conditions, the values of Young’s modulus and tensile strength increased, but Young’s modulus and tensile stress decreased significantly due to the water absorption. As shown in Fig. 3E, Young’s modulus and tensile strength decreased to 1.29 and 0.11 MPa respectively, but the tensile at the fracture point increased by 45.22%. Hydration of scaffolds led to the plasticity effect, thereby reducing the mechanical properties [49]. Hence, the preparation of PCEC/Gel nanocomposite scaffolds can be considered as an effective scaffold with suitable mechanical and biological properties due to the intrinsic bioactivity of the nanocomposite scaffold, which, in turn could drive diferentation and mineralization of the hDPSCs in vitro.

Swelling behavior in nanocomposite scaffolds

Despite the advantages that come from the hydrophilic nature of the scaffolding hydrogels, the excess water uptake has been shown to interrupt cell migration and vascularization [50]. Therefore, the swelling ratio is usually used to assess the hydrophilicity, porosity, and pore size of the scaffold. In this regard, we evaluate the swellability of the gelatin, PCEC/Gel, PCEC/Gel-Fe3O4, PCEC/Gel-HA&Fe3O4, and PCEC/Gel-HA scaffolds in vitro. The results that were depicted in Fig. 3C, indicated that the gelatin scaffold had a higher water-binding capacity than other scaffolds. Generally, the results suggested that the collaboration of gelatin with PCEC copolymer increased the cross-linking density between polymer chains due to the use of glutaraldehyde cross-linker, which, in turn, decrease the in vitro swelling capacity of the nanocomposite scaffolds. The incorporation of the Fe3O4 NPs and HA into the PCEC/Gel scaffold decreased the swelling capacity of scaffolds due to the hydrophobic nature of Fe3O4 NPs and hydroxyapatite [51, 52]. It should be noted that the swelling rate initially increased for all samples. However, it decreased with the continued absorption of PBS molecules and ions. After the first rapid penetration of the solution into the porous structures of nanocomposite scaffolds, the osmotic pressure difference between the samples and the surrounding solution decreased, and the scaffolds began to absorb the solution at a slower rate until they reached equilibrium. Due to the general hydrophilic nature of nanocomposite scaffolds in tissue engineering applications, it is expected that the scaffold hydrophilicity will increase cell transplantation and proliferation at the bone-implant site.

Evaluation of structures and morphology of magnetic nanoparticles and hydroxyapatite nanoparticles.

In this section, the morphology of the synthesized Fe3O4 and HA nanoparticles, dopped into the polymer scaffold as a reinforcing mineral phase, was evaluated. According to Fig. 4A, the average size of magnetic particles was about 56.84, which was calculated using image j software (Fig. 4a). The nanoparticles, which were prepared by the chemical coprecipitation method, were spherical. In Fig. 4B, the SEM micrograph related to the synthesized HA nanoparticles, and represented the average size distribution of about 85.88 nm according to the image j software (Fig. 4b). Figure 4C and D showed the images of magnetic nanoparticles and hydroxyapatite on nanocomposite scaffolds. These nanoparticles were well placed in the scaffolding and spread on the surface of the composites.

Fig. 4

A, a) Structures and morphology of magnetic nanoparticles and diameter distribution of magnetic nanoparticles. B, b) Structures and morphology of HA nanoparticles and diameter distribution of hydroxyapatite nanoparticles. C, D) Structures and morphology of scaffolds containing HA and Fe3O4

Research on cell compatibility of nanocomposite scaffolds

MTT assay was used to evaluate the biocompatibility of the nanocomposite scaffolds. For this, several groups of scaffolds including gelatin, PCEC/Gel, PCEC/Gel-Fe3O4, PCEC/Gel-HA, PCEC/Gel-HA&Fe3O4 were prepared and seeded with the hDPSC cells and cultured for 3, 7, and 14 days. The hDPSCs without scaffold were used as the control group. Cell viability and proliferation of the hDPSCs were determined via the absorption amount of the produced formazan due to the mitochondrial activity of the living cells. Our results represented that nanocomposite scaffolds could support the proliferation of the hDPSC cells as can be seen in Fig. 6A. Also, the results showed that viability and proliferation of the cells increased during 14 days of culture for all groups. By comparing the result of different groups we can see the positive effect of HA and Fe3O4 NPs incorporated with nanocomposite scaffolds, especially at day 14. The results indicated that hydrophilicity of the mineral nanoparticles facilitates adhesion and then proliferation of the cells over time. The highest viability of the cells incorporated with PCEC/Gel-HA, PCEC/Gel-Fe3O4, PCEC/Gel-HA&Fe3O4 scaffolds at day 14 confirmed the effective cell attachment and proliferation during this time. Taken together, the PCEC/Gel-HA&Fe3O4 scaffolds were non-toxic and presented excellent supports for cell proliferation; this means they can be an ideal candidate for tissue engineering.

The morphology study of cells cultured on nanocomposite scaffolds

One of the important issues expected from the scaffolding biomaterials is mimicking the porosity and structure of the native bone, which could, in turn, develop cell adhesion along with in vivo tissue ingrowth and vascularization [53]. First, nanocomposite scaffolds were formed and freeze-dried. Then. hDPSCs with the size of about 7–11 μm were cultured on nanocomposite scaffolds for 14 days, and the morphology of attached cells on the scaffolds was observed using SEM images. Figure 5 represented the SEM micrograph of PCEC/Gel-HA, PCEC/Gel-Fe3O4, PCEC/Gel-HA&Fe3O4 and confirmed the formation of microstructures and interconnected pores for all samples before and after cell culture. As shown in Fig. 5A, PCEC/Gel had porous structures before cell implantation. We used the freeze dryer method in the formation of porous nanocomposite scaffolds [41]. Porosity is an important factor in cell growth because it provides a proper interaction of the cell with the scaffold. Large pores in the scaffold can support the transformation of nutrients and elimination of metabolic wastes, and thus they are essential for effective cell growth, but they can reduce cell adhesion. Small pores can improve cell adhesion, despite reducing the transfer of nutrients and gas [42, 54]. Hence, the size of a scaffold pore is an important factor. It must be large enough for nutrients to be released and for cells to migrate, and it must also be small enough for cells to have the right area to attach [55]. Scaffolds in a size of 325–100 μm are suitable for tissue engineering [56, 57]. The micro-pores in the prepared porous scaffolds have a size in the range of 192.14 ± 70.33 μm, indicating the best adhesion. Figure 5B, C, and D show the attachment of cells and their random distribution on the surfaces of scaffolds. The cells could disperse and distribute properly and sufficiently on the surface of the scaffolds and fill the pores. Furthermore, there was no delay or inhibition of dental cell proliferation by scaffolds after 14 days due to the non-toxicity of scaffold components and their favorable conditions for cell attachment and growth [58]. It also seems that nanoparticles provide better space for cells and better interaction of scaffolds with cells due to having a larger surface area to volume ratio [56, 58].

Fig. 5

FE-SEM images relating to nanocomposite scaffolds. A, a) nanocomposite scaffolds before cell implantation, and diagram of the pore size distribution of nanocomposite scaffolds. B) hDPSCs cultivated on nanocomposite scaffolds after three days. C) hDPSCs cultivated on nanocomposite scaffolds after 14 days

Alizarin red S test

Histological assessment for several groups of nanocomposite scaffolds using Alizarin Red S staining was shown in Fig. 6B. Differentiation of hDPSCs to the osteoblast can be evaluated by the calcium deposition. In the presence of calcium, Alizarin Red S binds to it and forms a pigment that is in orange to red [59]. The hDPSCs were cultured for 14 days for quantification with Alizarin Red S staining. The Quantitative measurements indicated that cells treated with PCEC/Gel-HA, PCEC/Gel-Fe3O4, PCEC/Gel-HA&Fe3O4 exhibited a higher level of calcium deposition in comparison with pristine gelatine, PCEC/Gel, and control group. Also based on the above-mentioned morphological study of the scaffolds, we can explain that the weaker structural integrity and larger pore size of the pristine gelatine caused minimal calcium deposition, while smaller pore size of the PCEC/Gel-HA induced significant calcium deposition as demonstrated in Fig. 6B. In addition, the observation of intensified staining in these groups confirmed the positive effect of HA and magnetic nanoparticles on osteogenic differentiation of human dental pulp stem cells. Mineralization refers to the extracellular deposition of calcium and phosphate ions, which ultimately leads to calcification and is important for bone regeneration. Therefore, hydroxyapatite and magnetic nanoparticles in nanocomposite scaffolds may play an important role in stimulating mineralization.

Fig. 6

A) MTT assay of hDPSCs cultivated on Gelatin, PCEC/Gel, PCEC/Gel-Fe3O4, PCEC/Gel-HA, PCEC/Gel-HA&F3O4 scaffolds. B, b) Alizarin Red staining was performed on day 14 to observe calcium and mineral deposits in nanocomposite scaffolds. The amount of Alizarin Red staining in different compounds was drawn with Prism software. The groups were compared by the one-way ANOVA, and they were then analyzed using the Tukey’s HSD, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

qRT-PCR analysis

To evaluate the effect of HA and Fe3O4 on the osteogenesis differentiation level of hDPSCs cultured from the scaffolds, we performed qRT-PCR after 21 days under the osteogenic conditions. As representative osteogenic markers, the gene expression of RUNX2 (the main transcription factor for bone formation), BGLAP (bone mineralization factor), BMP2 (skeletal development and extracellular matrix maturation factor), and SPARC (osteonectin marker) was chosen, which are capable of bone formation and mineralization at the same time. As depicted in Fig. 7 there was no significant difference between the control and gelatin groups, however, the scaffolds containing HA and Fe3O4 exhibited the highest expression compared to the other type of scaffold and genes, suggesting the optimized concentration of HA and Fe3O4 incorporated into the nanocomposite scaffolds. In other words, the doping process enhanced the bioactivity for osteogenesis and bone regeneration despite being used in negligible amounts. Considering the aforementioned results, we can speculate that the mineral nanoparticles inside the scaffolds could affect the gene expression of cultured cells compared to the bare PCEC/Gel and gelatin.

Fig. 7

The expression of osteogenic-related genes. Relative expression of A BMP2, B BGLAP, C RUNX2 and D SPARC by hDPSCs seeded on gelatin, PCEC/Gel, PCEC/Gel-Fe3O4, PCEC/Gel-HA, PCEC/Gel-Fe3O4&HA scaffolds after 21 days by real-time PCR analysis. The values were normalized to GAPDH. (*p < 0.05, **p < 0.01, and ***p < 0.001)