2.1. 3D printing of PLA scaffolds
First, a 3D computer-aided design (CAD) model of the PLA scaffold was designed in ABAQUS software. Subsequently, the "stl." File format of the model was imported to Simplify3D software to provide the g-codes for manufacturing. Poly(lactic) acid filament (diameter of 1.75 mm) was used to build the scaffolds with a conventional fused deposition modeling (FDM) printer. The PLA filament was heated above the PLA melting temperature (nozzle temperature was 210ºC). The melted PLA was extruded through a nozzle made up of stainless-steel on to a printing bed having temperature of 60ºC. The scaffolds were printed in a layer-by-layer manner having a 7.6 mm diameter and a 1.6 mm height. The strut thickness was 0.4 mm and the pore size was 800 μm. The fabricated PLA scaffolds were analyzed using X-ray diffraction (XRD) technique (Bruker, D8-advance) and Attenuated Total Reflection-Furrier Transform Infrared Spectroscopy (ATR-FTIR, Bruker’s Alpha FTIR Spectrometer, Germany). The XRD analysis was conducted at 35 kV and 30 mA using Cu Kα radiation (λ=1.5405980 Å). The scanning angle (2θ) was between 5°-80° at a step size of 0.06°. ATR-FTIR spectrum was obtained at the resolution of 2 cm-1 over the frequency range of 4000-600 cm−1.
2.2. BMSCs harvesting, culture and immunophenotype
The BMSCs harvesting and culture were done based on the previous study (Talebi et al. 2020). Briefly, after sacrificing an adult female rat, the femur and tibia were immediately removed. To kill the rat, it was first anesthetized by intraperitoneal injection of 80 mg/kg ketamine and 10 mg/kg xylazine, and it was followed by cervical dislocation. The bone marrow was flushed by 10 ml of Dulbecco's Modified Eagle Medium (DMEM, Gibco) with nutrient F12 Ham, supplemented with 10% fetal bovine serum (FBS, Gibco) in two T25 tissue culture flasks, and incubated in the culture medium containing 10% FBS and 1% penicillin/streptomycin at 37 °C, 95% humidity, and 5% CO2. After 48 hours, the culture medium was replaced. Adhesive cells were sub-cultured four times upon reaching 80–90% confluence.
To analyze the expression of BMSCs surface markers, more than 1 × 105 cells were incubated in fluorescently labeled monoclonal antibodies (BD Pharmingen) against CD29, CD34, CD44, CD45 and CD90 in a dark place. After 30 minutes of washing with PBS, the labeled cells were analyzed using flow cytometry (BD FACS Calibur). Furthermore, optical microscopy images were taken at different passages for morphology evaluation.
2.3. Sterilizing the PLA scaffolds
The 3D printed PLA scaffolds were first immersed in distilled water for 1 h. Then, they were washed and ultrasonically cleaned with distilled water for 5 minutes. Afterward, the cleaned samples were sterilized using ultra-violet (UV) light under a laminar flow bench; 10 min each side of the scaffolds. For the cell-free group, each sterilized PLA scaffold was individually put in a sterile petri dish and transferred for surgery. For the cell-seeded group, the sterilized PLA scaffolds were used to seed MSCs. After seeding (as explained in the following section), each cell-seeded scaffold was individually put in a sterile petri dish with a small amount of complete medium (500µL) and transferred for implantation.
2.4. BMSCs seeding and culturing on PLA scaffolds
Initially, the bottom of wells of a 24-well plate was evenly coated by 2% agarose (700 μL per well) having no defects including bubbles or scratches in the coatings. After ⁓30 minutes, the sterilized PLA scaffolds were placed on the agarose. For seeding, 230 µL of culture medium containing 106 BMSCs was added evenly on both sides of the scaffolds; 115 µL on each side. After seeding of the cells on each side of the scaffold, a time interval of about 20 minutes was considered for initial cell attachment. In the next step, 1 mL of additional culture medium was gently added to each well and the plate was placed in an incubator at 37 °C, with 95% humidity and 5% CO2 for 24h. Finally, the seeded scaffolds were transferred for surgery one-by-one.
2.5. Animals
Wistar female (n=24) adult rats, weighing 250 ± 20 g were used in this study. Animals were kept in a controlled temperature (22 ± 2℃) place and a 12-h regular light/dark cycle (light on from 07:00 to 19:00), housed 2 to 4 per cage with free access to food and water (Safakhah et al. 2017). The experimental protocol was approved by the Ethical Review Board of Semnan University of Medical Sciences (Ethic code: IR.SEMUMS.REC.1400.048). All experiments were conducted in agreement with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
2.6. Implantation
The scaffold implantation was performed through a method which was described by Sadeghi et al. (Sadeghi et al. 2016). After anesthetizing of the rats by intraperitoneal (IP) injection of a mixture containing ketamine hydrochloride and xylazine hydrochloride with the volume ratio of 8:2 at 1mL/kg (Bahraminasab et al. 2021), the head of the rat was completely fixed in a stereotaxic apparatus, and the hair was shaved and the skull was disinfected using povidone iodine solution. To expose the full extent of the calvaria, subperiosteal dissection was done bilaterally in a non-infectious manner and the subcutaneous muscles were completely pushed away. The skull was drilled to the size of the scaffold using a surgical trephine bur. One calvaria through-and-through osteotomy was made in the dorsal portion of the parietal bone midsagittal suture (Figure 1a) under irrigation with sterile normal saline. After preparing the transplant conditions, the scaffold was placed into the whole and finally, the scalp was sutured. The bone repair was analyzed after 8 and 12 weeks, postoperatively. The details of animal groups are given in Table 1.
Table 1: Details of studied animal groups
Group number
|
Group name
|
Time (week)
|
Number of implanted rats for each implantation time
|
1
|
defect
|
8
|
2
|
12
|
2
|
2
|
PLA
|
8
|
5
|
12
|
5
|
3
|
PLA+Cell
|
8
|
5
|
12
|
5
|
2.7. Histological analysis
The histological analysis was conducted after 8 and 12 weeks. First, the rats were sacrificed and then the defect sites were judiciously dissected. These samples were fixed in neutral-buffered formalin (10%), and decalcified in formic acid (10%), sequentially. The standard dehydration was then conducted on the decalcified samples in serially increasing alcohol (ethanol) solutions. The dehydrated samples were embedded in paraffin and subsequently 5µm sections were provided. The prepared sections were stained with hematoxylin and eosin (H&E). The analyzed area in histology is shown in Figure 1b. Some sections were also stained by toluidine blue. A light microscope was employed to analyze the histology slides. The percentages of the new bone area were measured from H&E images using ImageJ software. Furthermore, immediately after harvesting the defect sites, photos were taken and the macroscopically filled area by new bone was calculated in percent using ImageJ software. Figure 2, shows the whole procedure used in this study.
2.8. Serum biochemistry and osteocalcin detection
To assess the systemic toxicity of the scaffolds, the level of liver and muscle enzymes was measured by serum biochemistry. At the time of sacrifice (8 or 12 weeks postoperatively), about 5 mL of blood was collected from the heart of each rat and centrifuged at 3000 rpm for 10 min to obtain blood serum. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed using the commercial kits (Paadco, Golestan Technology Park, Iran). Furthermore, the osteocalcin level (a bone formation marker) was also measured in the serum, using a sandwich ELISA method (Rat Osteocalcin/Bone Gamma-Carboxyglutamic Acid Containing Protein (OT/BGLAP) EISA Kit; ZellBio, Germany) according to the manufacturer instruction.
2.9. Statistical analysis
Analysis of Variance (ANOVA) was done for statistical analyses using Minitab V17 software. The confidence level was set to be 95% (α=0.05) in all analyses. Moreover, the post-hoc pairwise comparisons were conducted using Tukey test.