Preparation of PCL/β-TCP/CS composite scaffold

A 3D printing method based on fused deposition molding technology was used to prepare the scaffold. Computer-aided design (CAD) software was used to design the 3D structure of the scaffold material: a hollow cylindrical structure with an outer diameter of 4.9 mm, an inner diameter of 2.0 mm, a length of 1.5 cm, a pore size of 400 μm, and a porosity of 60%. Then, the 3D model was converted to STL format and imported into a 3D printer (Model: Bio-Architect-Lite, Recongene Biomedical, Nanjing, China). Polycaprolactone (PCL, Mw = 60,000, eSUN New Material, Shenzhen, China), tricalcium β-phosphate (β-TCP, d50 = 0.2–5.0 μm 99% pure, EMPEROR Nanomaterials, Nanjing), and calcium sulfate (CS, AR, 97.0%, MACKLIN Biochemical, Shanghai, China) were weighed proportionally and slowly added to chloroform, and the ratios of PCL, β-TCP and CS were 60:20:20, 70:15:15, and 80:10:10:10 (abbreviated as P/T/S20, P/T/S15, and P/T/S10, respectively). The reagents were dissolved in a blender and stirred overnight. The material was dried and cut into granules, added to the extrusion head to test extrusion, and adjusted for stability. The specific parameters of the printer were nozzle diameter 200 μm and speed 2 mm/s. The prepared scaffolding program was run with a stainless-steel bar to fabricate the scaffolding and allow cooling between each layer, and the complete scaffold was allowed to cool and then removed for processing. The PCL/β-TCP/CS composite artificial bone was immersed in ethanol and sterilized by UV, making it ready for use.

Characterization of the PCL/β-TCP/CS composite scaffold

Microscopy images were captured using a scanning electron microscope (SEM, JSM-7900F, Nippon Electron Corporation JEOL, Japan) to observe the microstructure of the fabricated scaffolds. Four microliters of triple-distilled water was dripped vertically from the feeder to the surface of the scaffold at room temperature, and the static contact angle of the scaffold (LAUDA ScientificOCA20, Germany) was determined using a contact angle meter. The porosity and density of the scaffolds (BSD-PS1/2, BSD Instrument Technology, Beijing, China) were measured using a fully automatic specific surface and pore size analyzer. The measurement principle is the gas expansion displacement method, i.e., using gas molecules with a known molecular cross-sectional area as probes covering the whole surface of the scaffolds to be tested by adsorption and then multiplying the number of adsorbed molecules by the molecular cross-sectional area, which is regarded as the specific surface area of the sample, to derive the actual volume of the scaffold. Porosity = 1 − actual volume of the scaffold/appearance volume of the scaffold; density = mass of the scaffold/actual volume of the scaffold.

Preparation of PCL/β-TCP/CS composite scaffold infiltration solution

The scaffolds were infiltrated with DMEM (C11885500BT, Gibco, Grand Island, USA) supplemented with 10% (0.1 g/ml) fetal bovine serum (Gibco, 10,099-141C, Grand Island, USA). After incubation at 37 °C in a 5% CO2 incubator for 72 h, the culture solution was aspirated and centrifuged and filtered through a 0.22-μm filter to remove bacteria. Then, the extract was stored in a 4 °C refrigerator.

Measurement of the toxic effects of the PCL/β-TCP/CS composite scaffold on cells by the CCK8 assay

Trypsin digestion of logarithmically grown BMSCs (MUBMX-01001, Cyagen, Guangzhou, China) was performed, and the cells were counted by a cell counting plate and inoculated into 96-well plates at a concentration of 5 × 103 cells/well, 100 μl per well. The cells were incubated in a CO2 incubator for 6–8 h. After the cells were attached to the bottom of the culture plate, 100 μl of the extract was added to the experimental group, and an equal volume of 10% fetal bovine serum medium was added to the control group. 10% CCK-8 reagent (10%, CK04, Dojindo, Japan) was added on days 1, 3, 5, and 7, the OD value of the cells was determined, and the relative cell growth rate was calculated after continuing the incubation for 2 h.

Induction of osteogenic differentiation by PCL/β-TCP/CS composite scaffold extracts

BMSCs were inoculated into 12-well plates at a density of 1 × 104 cells/well, with 3 replicate wells per group. Cells were cultured until 60%–70% confluence, and then, the medium was replaced with PCL support extract. Half of the medium was changed every 3 days. The induction of early osteogenic differentiation of BMSCs was detected by alkaline phosphatase staining on day 7, and the induction of middle and late osteogenic differentiation was detected by alizarin red staining on day 21. Specifically, BMSCs were fixed with 4% formaldehyde solution for 30 min at room temperature on day 7 and then rinsed twice with PBS. They were then stained with 100 µl of alkaline phosphatase solution (C3206, Beyotime, China) for 2 h at 37 °C. On day 21, BMSCs were fixed with 4% paraformaldehyde solution for 30 min at room temperature, then washed twice with PBS, and stained with 100 µl alizarin red S (C0138, Beyotime, China) for 1 h. Photographs were taken under a microscope.

Real-time quantitative PCR analysis

To further elucidate the stimulatory effect of PCL/β-TCP/CS scaffolds on osteogenic differentiation,

the typical markers runt-related transcription factor 2 (RUNX2), osteopontin (OPN), and BMP2 were evaluated. BMSCs were inoculated in 12-well plates at a density of 1 × 104 cells/well with 3 replicate wells per group. Gene expression of typical markers of BMSCs was assessed after 7 days of culture. Total RNA was extracted using a nucleic acid kit (G3013, Servicebio, China) and converted to cDNA using a reverse transcription kit (G3337, Servicebio, China). SYBR Green master mix (G3320, Servicebio, China) was used for real-time PCR. β-Actin was used as an internal control. The primers used for real-time PCR are listed in Table 1.

Table 1 The primer sequences used for the RT-qPCR analysisHistocompatibility of the PCL/β-TCP/CS composite scaffold

Thirty healthy New Zealand Large White rabbits (Laboratory Animal Resource Center, Chongqing Medical University, Chongqing, China) weighing 2.5–3.0 kg, male and female, were selected. This experiment was approved by the Animal Research and Ethics Committee of the Children's Hospital of Chongqing Medical University. After intravenous anesthesia with sodium pentobarbital (30 mg/kg) was effective, a 2–3 cm skin incision was made along the long axis of the lateral radius, centered on the middle radius on the left side, and the skin, subcutaneous tissues, fascia, and muscular tissues were bluntly separated to the periosteum in sequence to fully expose the middle radius. A section of radius approximately 1.5 cm in length was excised by using midget electro-saw, and the severed end was rinsed with saline, filled with composite stent, and sutured layer by layer. The incision was sterilized with iodine and fixed with gauze. The rabbits were placed on warm pads and returned to the cage after awakening, and their physiological signs and recovery were observed. In the first postoperative month, rabbit liver and kidney tissues were removed, fixed in 10% formaldehyde, routinely dehydrated and fixed by paraffin embedding, and routinely sectioned before eosin (HE) staining for histological evaluation. Tissues 1 cm around the stent were removed at postoperative months 3, 6, and 12, fixed with formaldehyde, dehydrated and embedded in paraffin, and then stained with Goldner’s trichrome staining. Safranin O-fast green and Masson staining were performed according to routine protocols. Whole sections were scanned using a full paddle scanner (SQS-120P, Shengqiang Technology, Shenzhen, China), and images were acquired using Image Viewer software.

Statistical analysis

At least 3 replicates were performed in each group for all experiments. Experimental data are expressed using the mean ± standard deviation. Differences between the two samples were evaluated by the t test. One-way analysis of variance was used to assess between-group differences from GraphPad Prism 9.5 software. The findings were considered significantly different when a p value < 0.05 was obtained.