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International Journal of Bioprinting Biomimetic scaffolds for mandibular repair

scaffold was manufactured using a digital laser process and at room temperature for 15 minutes. Before imaging,
was composed of 85% β-tricalcium phosphate and 15% cells were washed with DPBS. Subsequently, single-cell
hydroxyapatite. The 3D printing process parameters for morphological changes were captured under a confocal
the AUTOCERA-M (Beijing Shiwei Technology Co. Ltd, microscope at 60× magnification, and quantitative analysis
China) include a light wavelength of 405 nm, a spot size of was performed using ImageJ software (NIH, Germany).
50 µm, an exposure time of 6 seconds, an exposure power
of 30 mW/cm², and a layer thickness of 25 µm. After the 2.4. Analysis of differentially expressed genes
scaffold was cleaned with distilled water, it was sterilized in DESeq2 software was used to analyze differential gene
an autoclave at 121°C for 30 minutes, followed by drying at expression between two distinct groups (and edgeR was
37°C for 12 hours. used for two samples). Genes meeting the criteria of false
discovery rate (FDR) <0.05 and absolute fold change ≥2
To prepare SIT scaffolds, I-PRF was obtained by were identified as differentially expressed genes (DEGs).
collecting and centrifuging blood from the central artery Genes with varying expression levels were subsequently
of a rabbit’s ear. We mixed 500 μL of 200 ng/mL SDF- analyzed for Gene Ontology (GO) enrichment and
1 solution with 200 μL of I-PRF solution and used a Kyoto Encyclopedia of Genes and Genomes (KEGG)
pipette to repeatedly aspirate and dispense the mixture pathway enrichment.
onto the scaffold, ensuring even distribution throughout
the scaffold. 2.5. GO enrichment analysis
Gene Ontology is a globally recognized system for
2.2. Preparation of critical mandibular bone defect categorizing gene functions, providing a constantly
model of rabbits updated set of terms and clear definitions to fully
Nine male New Zealand white rabbits (Guangzhou Xinhua characterize the attributes of genes and their products
Animal Farm, China), all healthy and 6 months old, were across all organisms. GO consists of three ontologies:
chosen based on their weight of 3.0±0.5 kg. The participants molecular function, cellular component, and biological
were randomly divided into three groups: control, TPMS, process. GO term is the fundamental building block of
and SIT (n = 3 per group). Following the administration GO. Every GO term is associated with a specific ontology
of anesthesia, the skin in the right submandibular area category. First, all DEGs were mapped to the GO terms
was prepared in the usual manner, sterilized, covered with available in the Gene Ontology database (http://www.
sterile sheets, and then a 2–3 cm cut was made alongside geneontology.org/). Then, the number of genes associated
the lower border of the rabbit’s jaw from the back. An 8 with each term was calculated. To identify significantly
mm circular bone drill was utilized to create a defect in the enriched GO terms among the DEGs, a hypergeometric
center of the mandibular body. Cold physiological saline test was performed, comparing the DEG-associated terms
was then applied to lower the temperature, resulting in a against the genome background.
cylindrical bone defect measuring 8 mm in diameter and
4 mm in depth. Various scaffold materials with varying 2.6. KEGG pathway enrichment analysis
levels of porosity were inserted into the injury site based Genes typically collaborate with one another to fulfill
on pre-defined groups, followed by the sequential suturing specific biological functions. Analyzing pathways aids in
of the muscle membrane, fascia, and skin. Following gaining a deeper comprehension of the biological roles of
the procedure, a 30000-unit dose of penicillin was genes. The KEGG is a publicly available, primary pathway-
administered intramuscularly every three days, with the related database. Through the KEGG pathway enrichment
wound disinfected using iodophor to reduce the risk of analysis, metabolic pathways or signal transduction
infection after surgery. pathways that were significantly enriched in DEGs
2.3. Confocal microscopy and image analysis compared to the entire genome could be revealed.
Microscopy imaging was performed using Zeiss LSM 780 2.7. Protein-protein interaction analysis
and Olympus SD-OSR. Rabbit BMSCs were seeded onto the Protein-protein interactions were analyzed using the
SIT scaffolds. Cells were fixed with 4% paraformaldehyde STRING database (version 12.0) with a confidence
(PFA; 30525-89-4, Wako, Japan) at room temperature for threshold of 0.7. Interactions were limited to Homo sapiens
10 minutes, followed by washing, and permeabilization and visualized using Cytoscape (version 3.10.2). Key hub
with 0.1% Triton X-100 (Sigma, Germany) in phosphate- proteins were identified using plugin “cytoHubba.”
buffered saline (PBS) for 15 minutes. Cells were then
washed twice with Dulbecco’s phosphate-buffered saline 2.8. Gene set enrichment analysis
(DPBS) and incubated with ActinGreen (Alexa-Fluor- Gene set enrichment analysis (GSEA) was conducted
488-conjugated phalloidin; R37110, Invitrogen, USA) utilizing GSEA (v4.1.0) software and MSigDB. Enrichment

Volume 10 Issue 5 (2024) 528 doi: 10.36922/ijb.4147