Preparation of 3D printing solution

One gram of MASF (Engineering for Life, EFL-SiLMA-001) was placed in a centrifuge tube, and 0.05% (w/v) ultraviolet absorber water soluble (UV-326, Milanchemical, 3896–11-5) was added to the centrifuge tube. Nanohydroxyapatite (Macklin, China, H875578) was weighed to achieve mass ratios of MASF: nHA of = 1:0, 1:0.5, and 1:1 and added to the centrifuge tube. The centrifuge tube was filled with photoinitiator standard solution composed of: Lithium Phenyl (2,4,6-trimethylbenzoyl) phosphinate (EFL-LAP, Engineering for Life, EFL-LAP-1). The solution was dissolved at ambient temperature for 0.5–1 h, during which the solution was stirred/shaken several times (to avoid severe ultrasound, high temperature and strong shear). The MASF solution was sterilized with a sterile 0.22 μm needle filter. The filtered solution was stored at low temperature about 4 °C.

Fabrication of scaffolds

To produce the scaffold, a digital light processing bioprinting (EFL Light Curing Biological 3D Printer, China, BP8600) with high accuracy about 25 μm was used.The solution prepared above was poured into the printer's feed tank, and the material was printed according to the following parameters: Setting the print height to 100 μm, light intensity to10 mW/cm2, and single-layer exposure time to15 s. This is the optimal parameter that has been tested and determined. Printing parameters have an impact on printing accuracy. Too much light intensity or too long a printing time will affect the molding accuracy of the printed scaffolds. The smaller the print height, the higher the molding accuracy. If the light intensity is too low or the printing time is too short, then the printed scaffolds are difficult to be shaped. After printing, the solution was removed. Three scaffolds with different proportions (Mass ratios of MASF: nHA of = 1:0, 1:0.5, and 1:1) were prepared with 5-mm diameter and 1-mm height, soaked and cleaned in PBS solution for several times, and then soaked in PBS for preservation. Design of a digital model of the 5-mm diameter and 1-mm height 3D bracket using the computer-aided design software SolidWorks. The layer resolution ranged from 400 to 600 μm for a high fidelity of the 3D-printed scaffolds structure.

Characterization of scaffolds

The apparent forms of the nHA/MASF composite scaffold were characterized by scanning electron microscopy (SEM, Sigma 300, ZEISS, Germany). The chemical components of the scaffolds were characterized using Fourier transform infrared spectroscopy. Compression measurements of the scaffolds were performed along the print axis (z-axis) with a universal testing machine (Shimazu, AG-2000A, Japan) using displacement control (4 mm/min) at ambient conditions and at least three specimens were measured for each sample for the above test.

Cell proliferation assay

The influence of the scaffolds on cell viability and proliferative activity was measured. Bone mesenchymal stem cells (BMSCs) were cultured in α-MEM medium (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA) at 37 °C and 5% CO2. The culture medium was replaced every 2 days, and once the cells achieved 80% confluency, they were treated with trypsin (Gibco, USA). Cells were utilized from the third to fifth generation. The cell inoculation involved 5.0 × 104 cells/cm2. In order to prepare scaffold extracts,in accordance with the international standard ISO 10993–12: 2021 ' Biological evaluation of medical devices—Part 12: Sample preparation and reference samples ',three scaffolds with different proportions (Mass ratios of MASF: nHA of = 1:0, 1:0.5, and 1:1) were cultured in 1 ml by the above α-MEM medium at 37 °C and 5% CO2 for 24 h. The effect of the extracts on BMSCs was tested using a medium containing 10% of the extracts. We designed four groups: the 1:0 group (SF group), 1:0.5 group (SFH group), 1:1 group (SFHH group) and blank group without extract (N group). Cells were seeded into 24-well tissue culture plates at a density of 5 × 104 cells/cm2. The cell viability was analyzed by a live/dead cell staining experiment (Beyotime, China). After a 24-h culture, 100 µl staining working solution was added to each well and incubated at 37 °C for 15 min. Live cells (green fluorescence, 490 nm) and dead cells (red fluorescence, 545 nm) were simultaneously detected under a fluorescence microscope.

Cell counting kit-8 (CCK-8) analyze cytotoxicity

The cell seeding and culture protocol was the same as detailed above. Briefly, 0.5 mL of fresh DMEM containing 10% CCK-8 solution (Biosharp, United States) was added to each well. After 2 h, 100 µL of the mixed medium was transferred to a 96-well plate. The solution absorbance was measured at a wavelength of 450 nm with a microplate reader (Thermo, United States).

Alkaline phosphatase activity

To determine the effect of the scaffold on the alkaline phosphatase (ALP) activity of BMSCs, osteogenic differentiation medium containing scaffold immersion solution was used to induce differentiation, and groups were treated as described previously. Medium for rat BMSCs osteogenic differentiation (Cyagen, China) was prepared as the product manual. Cultivation of medium for rat BMSCs osteogenic differentiation containing 10% V/V scaffold soaking solution. After 1 week and 2 weeks of osteogenic induction, the cells were digested, centrifuged and collected into centrifuge tubes. Ice lysis cells were processed with 100 μL RIPA cell lysis solution (without phosphatase inhibitors and protease inhibitors) for 30 min and centrifuged at 13,500 r in a 4 °C high speed centrifuge for 30 min, and then collected the supernatant. The ALP activity was detected by an ALP kit (Beyotime, China). A BCA protein detection kit (Beyotime, China) was used to measure the total protein content in the extracted samples and standardized alkaline phosphatase activity by the total protein content.

Alizarin red S staining

Alizarin red S staining was used to detect the mineralized nodules of BMSCs in SD rats. The cells were inoculated at a density of 5 × 104/ml on a 24-well plate and incubated for 24 h, then medium for rat BMSCs osteogenic differentiation (Cyagen,Chian) containing 10% V/V scaffold immersion solution was used. Then BMSCs cultured in medium containing 10% V/V scaffold immersion solution for 14 and 21 days, and the groups were treated as described previously. At a predetermined time, the cells were fixed with a 4% paraformaldehyde solution. The immobilized cells were further washed in pure water to remove any salt residues, added to a 2% (WT/V) Alizarin red S (ARS, Solebo, China) solution and incubated at room temperature for 30 min. After the samples were dried, fluorescence microscopy was used to collect images. ImageJ was used to quantify the images taken, and detection was carried out in triplicate.

Western blot analysis

The protein expression of SD rat BMSCs was detected by Western Blotting. After 24 h, the medium was replaced with normal osteogenic induction medium and osteogenic induction medium containing 10% of different scaffold extracts. After culturing for 14 days, cell precipitation was collected; 100 µl of RIPAcell lysate (Beyotime, China) containing 1 mM PMSF, (Beyotime, China) was added to each 6-well plate, lysed, centrifuged, and collected the supernatant. Total cellular protein was quantified using a BCA protein assay kit. Then the samples were subjected to SDS electrophoresis and transferred to a polyvinylidene fluoride membrane. The membranes were first blocked with 5% skim milk for 1 h, and then incubated at 4 °C overnight with primary antibodies as follows: RUNX2 (AF5186, Affinity, 1:500), OCN (DF12303, Affinity, 1:500), ALP (DF6225, Affinity, 1:500), and Collagen I (AF7001, Affinity, 1:500). After being washed with TBST three times, the membranes were incubated with HRP-conjugated secondary antibodies (1:10,000). The antigen–antibody complex was visualized. Signal intensities were quantified using ImageJ software.

Quantitative real-time PCR analysis

The cells were seeded at a density in tissue culture plates and grouped as before. After 24 h, the osteogenic induction protocol was the same as before. After culturing for 14 days, total RNA was extracted from cells according to the manufacturer’s instructions. Complementary DNA was synthesized from total RNA using the PrimeScript RT Reagent Kit. After reverse transcription, real-time PCR was performed using a SYBR Green qPCR kit (Servicebio, China) and an ABI Step One Plus Real-Time PCR System. Each sample was analyzed in triplicate, and β-actin was used as a reference. The primer sequences used are described in Table 1.

Table 1 Information on the primers designed for each osteogenic protein genesEstablishment of the skull defect model

Female SD rats were used as the object of the study on osteogenic performance in vivo. Forty-eight SD rats were randomly divided into 4 groups with 12 rats in each group, and one group was the sham operation group. All the experimental procedures were carried out on animals with the formal approval of the Ethics and Animal Research Committee of Anhui Medical University, China (NO. YX2022-128). Clean environment feeding, free eating, drinking water, regular environmental disinfection. The rats were weighed by electronic balance, anesthetized by chloral hydrate, and fixed in the prone position on the operating table. The scalp hair of rats was shaved to expose the skin, the skin was disinfected with an iodine cotton ball, the sterile hole towel was covered, and the operation was carried out under strict aseptic conditions. First, the median line of the skull was determined, and then a skin incision of approximately 2 cm in length was made along its length. The skin and subcutaneous tissue were cut to the periosteum layer in turn, and the frontal bone and skull were fully exposed. The skull was drilled vertically at low speed using a surgical electric hollow trephine, approximately 4 mm in diameter. Normal saline was added regularly during drilling to avoid damage to brain tissue and blood vessels caused by the high temperature of the drill bit and friction. When a slight break was felt with the drill, the skull fraction along the circular hole was removed with tweezers. The nHA/MASF scaffold was implanted and sutured layer by layer. The incision was disinfected with iodophor for 3 consecutive days after the operation, and 200,000 U penicillin was injected subcutaneously once a day. The room temperature was maintained at (25 ± 2) °C.

Radiographic evaluation

At 4 and 8 weeks after stent implantation, 3 SD rats from each group were sacrificed by cervical dislocation at each time point, and the skulls were separated. The samples were fixed in paraformaldehyde solution and scanned using a micro-CT system (Skyscan1276, Bruker, Germany) to evaluate the new bone around the defect area. A cylindrical area of interest (ROI) with an appropriate diameter and depth was selected in the bone defect area and reconstructed by a CT analyzer. Bone volume/total volume (BV/TV), bone trabecular number (Tb.N) and bone surface area (BS) were calculated in the CTAn procedure (Skyscan, Germany) to evaluate new bone tissue.

Histological analysis

For analysis of bone histology, craniums were fixed in paraformaldehyde, and subsequently decalcified for four weeks before embedding in paraffin, and thick sections were used for hematoxylin and eosin (HE) staining and Masson’s trichrome staining.

Statistical analysis

Statistical analysis was performed using SPSS 19 (IBM, Armonk, IL, USA). All results are expressed as the mean ± SD. The results were analyzed by independent T test and one-way ANOVA. P < 0.05 was statistically significant. All quantization was performed on high-resolution images using ImageJ, followed by statistical analysis and mapping of the data using GraphPad Prism 8.0.1.