A novel injectable hydrogel containing polyetheretherketone for bone regeneration in the craniofacial region

Materials

Silk cocoons from Bombyx mori (B. Mori) were obtained by the Silkworm Research Center (Gilan, Iran). Microcrystalline cellulose powder (MCC) was kindly donated from Zahravi Pharmaceutical Company (Iran). Sodium periodate (NaIO₄), Dimethyl sulfoxide (DMSO), lithium bromide (LiBr), sodium carbonate (Na₂CO₃), and dialysis bag (MWCO = 3000 and 12,000 Da) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). PEEK was also purchased from Merck (Mean particle size 80 microns, GF75065755).

Synthesis of injectable hydrogels

Silkworm cocoons were cut into pieces and then degummed for 30 min in boiling Na₂CO₃ solution to eliminate the sericin proteins. Then, the samples were rinsed thoroughly, deionized water several times and air-dried overnight. Next, the specimens were soaked in 9.3 M lithium bromide (1 g in 4 mL) for 4 h at 60 °C, then dialyzed (3 kDa MWCO) to remove LiBr for 4 days. The obtained solution was centrifuged to eliminate residual debris for further use.

The CNCs solution was prepared according to our previous work^69,70. After that, the reaction of the CNC solution with NaIO₄ solution (300 µg/mL) was performed in the dark condition for 8 h. To end the response, 600 µL ethylene glycol was added to the solution, and then the solution was dialyzed for further purification. The final product was freeze-dried to obtain the ADCNCs powder.

For this purpose, the prepared silk fibroin (~ 7%) and ADCNCs (~ 0.5% wt) solutions were transferred into two barrels and then injected into a mold, followed by keeping for 30 min to obtain the hydrogel (Fig. 11.A). The hydrogel was prepared using a double-barrel syringe with a gauge needle (21 G). For the hydrogel containing PEEK, the powder (10% wt) was added to the ADCNCs solution.

(A) Injectable ADCNCs/SF/PEEK hydrogel. (B) Critical size bone defect (8 mm) in rat cranial (C) ADCNCs/SF/PEEK hydrogel in created defect (D) Incision closure.

We calculated the aldehyde content to evaluate the oxidation degree of ADCNCs^71,72. First, 2 mL of hydroxylamine hydrochloride solution (0.35 M) was added to 1 mL ADCNCs solution (pH = 5.0) and then stirred for 8 h at 55 °C. The feeding values of NaOH solution (0.5 M) were recorded as Vc and Vb for the titration of ADCNCs and CNCs. The molecular weight of ADCNCs is about 162 g/moL. Then, the aldehyde content was estimated by the following Eq:

where m is the dry weight (g) of the ADCNCs sample.

Characterizations of developed hydrogels

This study used the TGA (TGA/SDTA 851/Mettlear Toledo, Spain) device under nitrogen atmosphere (20 mL/min). The temperature range was from 30 °C to 600 °C at a heating rate of 10 °C/min. In compressed KBr pellets, the FTIR spectra of hydrogels were recorded with a resolution (4.0 cm⁻¹) and 16 scans per minute by Bruker Tensor 27. The wavenumber range was set from 400–4000 cm^-1. The inverted tube test was considered to calculate the gelation time.

The analysis of the frequency sweep test was completed with a range of frequencies from 1 to 100 rad/s and a regular strain of ɤ = 0.01. The dynamic viscosity, storage modulus (G′), and loss modulus (G″) were assessed.

The hydrogels were soaked in 20 mL of phosphate-buffered saline (PBS, pH = 7.4). Then, the samples were placed in a shaking incubator at 37 °C. After predetermined time intervals (1, 2, 4, 6, and 8), pieces were removed from the medium and dried under a vacuum. Degradation was quantified using the following Eq:

where Wi is the initial dry polymer mass, and Wf is the dry polymer mass at a time.

To assess the swelling ability of hydrogels, the specified samples were dipped into deionized water at 37 °C. In summary, at first, the dry weight of the hydrogels was measured (Wd), and then the swollen hydrogels were weighed after 1, 6, 12, 24, 48, 72, and 96 h (Ws). The swelling degree (SD) was recorded according to the Eq:

Culture of hDPSCs on fabricated hydrogels

HDPSCs were purchased from Shahid Beheshti University and cultured on in High-content glucose Dulbecco’s Modified Eagle Medium (DMEM/HG; Cat No: 31600083; Gibco; USA) containing %10 fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco, USA). After reaching to 80% confluency, cells were trypsinized (Gibco, Singapore) and seeded on the sterilized scaffolds for in vitro tests.

Scanning electron microscopy (SEM) imaging

The surface morphology and structure of ADCNCS/SF/PEEK scaffolds with and without hDPSCs were evaluated by SEM three days after seeding. Before assessing, hDPSCs were fixed in 2.5% glutaraldehyde on the scaffolds as described recently⁷³. After fixation, the hydrogels containing hDPSCs were dehydrated using a graded series of alcohol concentrations (50, 70, 90, and 100%)⁷⁴. Afterward, scaffolds with and without stem cells were cut into three specimens and coated with a thick gold layer. To characterize these samples, FE-SEM 1430 vp (MIRA3 FEG-SEM—Tescan, Czech) was applied.

MTT assay

The effects of synthesized scaffolds on hDPSCs were assessed by MTT assay. Briefly, 5 × 10³ cells were seeded on ADCNCs/SF and ADCNCS/SF/PEEK scaffolds in the 96 well plates. hDPSCs were cultured without scaffolds on the polystyrene surface of three wells in the same plate and were considered as a control group. After 1, 3, and 5 days, 50 μL of 3-(4,5-Dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide) (Invitrogen, Carlsbad, CA, USA) solution (5 mg/mL) were added to the medium and after incubation for 4 h at 37 °C and 5% CO₂; the medium was replaced by 100 μL DMSO and the color change of the purple formazan crystals solution was measured by a microplate reader (BioTek, USA) in the wavelength of 570 nm.

Calcium deposition and alkaline phosphates activity

The calcium accumulation of hDPSCs seeded on ADCNCS/SF, and ADCNCS/SF/PEEK hydrogels were measured using Alizarin Red Staining to determine the mineralization of these cells. 5 × 10⁵ cells were seeded on the synthesized hydrogels, which were placed in 6 well plates as follows; three wells were filled with ADCNCS/SF hydrogels, three wells were filled with ADCNCS/SF/PEEK, and three wells contained hDPSCs without scaffolds, which was considered as the control group. After fourteen days, the cells were fixed with 2% paraformaldehyde (Sigma-Aldrich Co.) and washed with PBS. Then Cells were stained with 40 mM alizarin red (pH 4.2, Sigma-Aldrich Co.) for 40 min in the dark at room temperature. Finally, the wells were washed with distilled water three times and left to dry. The culture plates were photographed under an optical microscope to show mineralized nodules that appeared with a dark red center and light red peripheral area. For quantitative analysis, after adding 10% acetic acid solution for 30 min and shaking, 10% ammonium hydroxide solution was added to neutralize the reaction. The color intensity was determined using an ELISA reader (BioTek, USA) at 405 nm.

Alkaline phosphatase activity of seeded hDPSCs on synthesized hydrogels was determined based on manufacturer instruction (ALP assay kit, Pars Azmoon, Iran). Similar plates were prepared in the same condition as the ARS test. Seven days after cell seeding, the samples were washed twice with PBS and lysed in alkaline lysis buffer. After 45 min incubation, the concentration of P-nitrophenol was measured at 405 nm, and the results were reported as IU/mg protein.

Evaluation of osteogenic-related markers by real-time PCR and western blot

1 × 10⁶ hDPSCs were cultured on the synthesized scaffolds, which were placed on the six well plates. After fourteen days, total RNA was extracted according to the manufacture instruction by Ambion TRIzol buffer (Cat No: 15596–026, Invitrogen, USA), and the quality of obtained RNA was determined with Nanodrop (Thermo Scientific, Waltham, MA, USA). Then, 1 μg of total RNA was used for cDNA synthesizing by a cDNA synthesis kit (Cat No: YT4500). The expression level of three genes for osteogenic differentiation was evaluated by specific primers, including Runx2, OCN, and COL1A1 (Table1). The β-actin gene was used as a housekeeping gene for normalization. The expression was measured by an RT-PCR system (LightCycler 96). Each data was repeated in three separate experiments and three times. Data analysis was performed by the Pfaffl method⁷⁵.

The immunoblotting assay was conducted to evaluate the level of expression of osteogenic proteins in hDPSCs seeded on ADCNCS/SF and ADCNCS/SF/PEEK hydrogels. HDPSCs were seeded without scaffolds considered as a control group. This test was performed according to standard protocols described in our previous study⁷⁶. Briefly, after seven days, the cells were lysed in ice-cold cell lysis buffer solution (NaCl, NP-40, and Tris–HCl), including cocktail enzyme inhibitors. The solutions were sonicated and then centrifuged at 14,000 g for 20 min. The supernatant was analyzed for total protein contents by the Picodrop spectrophotometer system (Model No: PICOPET01, Serial No. 000212/1) and resolved by the SDS-PAGE method. The following primary antibody solution was added to samples and then was incubated overnight at 4 °C; Runx2 (RUNX2 (F-2), Cat No: sc-390351, Santa Cruz Biotechnology, Inc.), collagen type Ι alpha Ι (COL1α1 (3G3), Cat No: sc-293182, Santa Cruz Biotechnology, Inc.), osteocalcin (OCN (FL-100), Cat No: sc-30044, Santa Cruz Biotechnology, Inc.), and β-Actin (Cat No: sc-47778, Santa Cruz Biotechnology, Inc.). The samples were incubated with secondary HRP-conjugated anti-IgG antibody (Cat No: sc-2357, Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. ECL plus solution kit (BioRad) was used to detect the immunoreactive blots. Visualizing the reactive proteins on the blots process was performed according to the manufacturer’s instructions. This experiment was performed in triplicate.

The surgical procedure in rat calvarial bone defects

Twelve mature Wistar rats, 8 weeks old and weighing 3500–400 g, were entered into the current study and randomly divided into three groups. Each group contained four rats, which were kept single in pathogen-free boxes for seven days to be adapted to the condition of the animal house. This condition included the temperature of 22 ± 5 °C, the humidity of 50–60%, and a cycle of dark/light for 12 h. All the rats had access to standard rat chow and water in the same amount and condition.

To evaluate the osteogenesis effect of synthesized hydrogels, one defect of 8 mm in diameter was created in the calvarial of each rat (Fig. 11B). For anesthesia, a ketamine (Rotexmedica, Trittau, Germany) /xylazine (Alfasan, Netherland) mixture (45/10 mg/kg) was injected intramuscularly. The calvarial area was shaved and disinfected with povidone-iodine. After that, an approximately 25 mm sharp incision was made by surgical blade #13. Prichard Periosteal Elevator was used to retract the tissues. Then an eight mm defect was created by the trephine bur of the dental implant kit (DASK, Dentium Advanced Sinus Kit, South Korea) while the surgical site was cooled by sterile saline. The created defects in four rats were filled by ADCNCS/SF, while the other four rates received ADCNCS/SF/PEEK hydrogels (Fig. 11C). The defect sites in remained four rats did not fill with any material and were considered the control group. The incisions were sutured using nonabsorbable 3/0 USP surgical black braided silk (HURTEB Medical Devices, Tehran, Iran) (Fig. 11D). For postoperative pain, the subcutaneous injection of Piroxicam (Exir, Tehran, Iran) was performed for each rat immediately after surgery and 24 h later. After rats became active, they were transferred to their boxes and received food and water, as mentioned. The surgery sites were covered with Gentamicin skin ointment to prevent infection. The sutures were removed seven days after surgery.

After eight weeks, rats were sacrificed by overdosing on pentobarbital (100 mg/kg), and the defect areas were removed from the calvarial of each rat with 2 mm extra safe margins. After the collection of bone samples, the carcasses were discarded by burial.

The bone pieces were washed with a phosphate buffer saline (PBS) to remove the attached surrounding tissue and fixed in 10% neutral buffered formalin (Pars Chemie, Tehran, Iran). New bone formation was analyzed using a radiology (CBCT) and histology (H&E) evaluation.

CBCT assay

After sacrificing rats and harvesting the bony segments, the samples were scanned with Cone Beam Computed Scan (CBCT, NewTom VGi, Verona, Italy). The direction of the cone was placed parallel to the coronal surface of bone defects as described previously⁷⁷. To create 3D reconstruction, the analysis was performed with Mimics Medical 21.0 (Materialise, Leuven, Belgium), and the total volume of bone formation was measured. Briefly, DICOM files were uploaded to the software, and for reconstruction, the lower and upper thresholds ranged between 0 and 700 Hounsfield units. The total bone volume was measured in the cylindrical region (8 mm × 1 mm). Four defect models were calculated for each group, and data were reported as mean ± SD.

Histological examinations

Following the CBCT analysis, all samples were decalcified. In this process, 3% nitric acid (7697-37-2, Sigma, USA) was used for decalcification⁷⁸. After decalcification, the specimens were bisected and dehydrated by the gradient of ethanol solutions in a tissue processor (MeyMed, DS 2080/H, Tehran, Iran). The dehydrated samples were embedded in paraffin wax blocks (HistoWax, SCILAB, UK). The samples were sectioned into 5 μm histological slides using a rotary microtome (DID SABZ, DS 4055, Urmia, Iran), then transferred to glass slides and glued with mounting medium (05-BMHM100, Bio Mount HM, Milano, Italy). Hematoxylin (PadtanTeb, Tehran, Iran) and Eosin (CARLO ERBA), were carried out to observe new bone formation under the light microscope (Olympus).

Statistical analysis

Statistical analyses were performed using Prism software (version 8.0, GraphPad, San Diego, CA, USA). The Kolmogorov–Smirnov test analyzed the normality and homogeneity of the data distribution. The continuous values with normally distributed were reported as mean ± SD and analyzed by Student’s t-test, one-way ANOVA, and Tukey post hoc analysis. P-value < 0.05 was considered statistically significant. All experiments were carried out in triplicates.

Ethical approval

All experiments of the current study were affirmed by the published guideline of The Care and Use of Laboratory Animals (NIH Publication No. 85–23, revised 1996) and reported in accordance with ARRIVE guidelines, and approved by the Ethics committee of Tabriz University of Medical Sciences (IR.TBZMED.REC.1400.103) that complied with the Helsinki declaration.