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International Journal of Bioprinting G40T60@WNT5A promotes osteoblast differentiation

In this study, we investigated the application of 2.2. RNA extraction and sequencing
Masquelet technique and 3D printing technology in Total RNA was separated from peripheral blood samples
the context osteogenic differentiation and angiogenesis. using Trizol reagent (15596026, Invitrogen, CA, USA).
Preliminary findings suggest that utilizing the Masquelet RNA sample concentrations and purities were measured
technique and preparing a degradable WNT5A-loaded using a NanoDrop 2000 spectrophotometer (1011U;
TM
scaffold G40T60 through 3D printing could facilitate the NanoDrop, USA). The total RNA samples that meet the
formation of the induced membrane in rats with chronic following criteria are used for subsequent experiments:
tibial osteomyelitis and tibial bone defects (CTO&BD), RNA integrity number (RIN) ≥7.0 and 28S:18S ratio ≥1.5.
thus promoting osteoblast differentiation and angiogenesis.
The findings also underscored the potential of Masquelet The sequencing library was generated and sequenced
technique combined with 3D printing as a promising by CapitalBio Technology (Beijing, China). Each sample
treatment modality for CTO&BD, and showed that WNT5A used for sequencing contained 5 μg of RNA. The Ribo-
may be a potential therapeutic target in CTO&BD. The Zero™ Magnetic Kit (MRZE706, Epicentre Technologies,
purpose of this study is to explore and develop an advanced Madison, WI, USA) was used to remove ribosomal RNA
bone repair technique for the treatment of CTO&BD, (rRNA) from total RNA. NEB Next Ultra RNA Library
using a 3D-printed biodegradable WNT5A-loaded scaffold Prep Kit (#E7775, NEB, USA) was used for Illumina
constructed based on the Masquelet technique to promote sequencing library construction. Then, in NEB Next First
healing. This innovative technology has substantial clinical Strand Synthesis Reaction Buffer (5×), the RNA fragment
value in the arena of bone healing. Relative to other was broken into fragments of approximately 300 base pairs
3D-printed scaffolds, induced membranes created through (bp) in length. Reverse transcription primers and random
bioengineering approach combined with Masquelet primers were used to synthesize the first and second
technique have superior quality and are of favorable sizes, strands of cDNA in the reaction buffer of deoxyuridine
which are distinct technical and biomechanical attributes. triphosphate (dUTP) mix (10×) for the second strand
This technique can effectively treat orthopedic diseases, synthesis reaction. The ends of cDNA fragments were
such as CTO&BD, that pose challenges in clinical treatment. repaired through the addition of polyA tails and the ligation
Leveraging advanced technologies such as 3D printing and of sequencing adaptors. After ligating Illumina sequencing
bioengineering may offer new avenue for addressing critical adapters, the second strand of cDNA was digested using
issues in current orthopedic treatments, thereby helping to USER Enzyme (#M5508, NEB, USA) to construct a strand-
improve patient’s quality of life and life expectancy. specific library. The library DNA was amplified, purified,
and enriched through PCR. Then, the libraries were
2. Materials and methods assessed by Agilent 2100 and quantified using the KAPA
Library Quantification Kit (KK4844, KAPA Biosystems,
2.1. Clinical specimen collection USA). Finally, paired-end sequencing was performed on
Ten healthy individuals and 10 patients with chronic the NextSeq CN500 (Illumina, USA) sequencer. 41,42
osteomyelitis with bone defects were recruited in this study.
Peripheral blood samples were collected from the human 2.3. Quality control of sequencing data and
participants, under the approval of the Ethics Committee alignment to reference genome
of The Third Hospital of Hebei Medical University (Ethics Fast Quality Control (FastQC) software v0.11.8 was used
Committee number: Ke-2022-104-1). Peripheral blood to check the quality of the paired-end reads on the raw
mononuclear cells were isolated from the samples using sequencing data. Raw data were processed using Cutadapt
heparinized tubes. Fresh peripheral blood (2–5 ml) in software 1.18, which removed the Illumina sequencing
each tube was centrifuged at 400 × g at room temperature. adapters and poly(A) tail sequences. Reads with an N content
Afterward, 2 ml of the upper layer of plasma was discarded, exceeding 5% were removed using a Perl script. Reads with
followed by a top-up with an equal amount of phosphate- a 70% base quality above 20 were extracted using FASTX
buffered saline (PBS). Five milliliters of single-nucleus Toolkit software 0.0.13. The paired-end sequences were
isolation buffer (1:1) (bio-processing system, #25610) were repaired using BBMap software. Finally, the filtered high-
added to a 15-ml centrifuge tube. Then, 5 ml of diluted quality reads fragments were aligned to the mouse reference
whole blood was slowly added to the tube containing genome using hisat2 software (0.7.12). 41,42
the single-nucleus isolation buffer. The suspension was
centrifuged at room temperature for 20 min with a speed 2.4. Bioinformatics analysis
that ensures a smooth descent of the rotor. Specifically, the Differential gene expression analysis of mRNA was
deceleration was set to “no break” (DECEL: set to slow). based on mRNA read count using the “edgeR” package
The cell layer in which peripheral blood mononuclear cells in R language, with the criteria of |log2FC| >1 and P
(PBMCs) reside is white. 39,40 <0.05 for differential gene selection. The target genes for

Volume 10 Issue 2 (2024) 230 doi: 10.36922/ijb.1461