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International Journal of Bioprinting 3D-Printed scaffolds for diabetic bone defects

peptides onto the surface of the PCL porous scaffolds were analyzed using scanning electron microscopy (SEM;
through chemical bonding for functional modification. Evo 50 XVP, Zeiss, Germany). The elemental composition
In vitro, experiments were performed to investigate of the surface of the porous scaffold was determined using
any improvements in physicochemical properties, an Al Kα X-ray spectrometer (ESCALAB250 XPS, Thermo
biocompatibility, cell migration, and cell respiration in Fisher, England) for analysis of the changes in elemental
a high-glucose environment, as well as the osteogenic content before and after modification. The materials’
differentiation of BMSCs achieved with the 3D-printed hydrophilicity was examined using a water contact angle
porous scaffold. In vivo experiments verified the efficacy tester (SEO Phoenix 300, Korea) to photograph and observe
of the scaffolds in promoting bone defect repair in diabetic the changes in surface hydrophilicity of the four porous
rats. The results of this study are expected to provide new scaffolds. All experiments were repeated three times.
methods and approaches to overcome the clinical challenge To assess the rate and amount of peptide retardation,
of diabetic bone defects (Figure 1). the porous scaffolds were immersed in PBS (pH 7.4,
37°C) according to an area:volume ratio of 1.25 cm /mL
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2. Materials and shaken slowly and uniformly. The supernatant was
2.1. Preparation and chemical modification of PCL collected regularly from day 1 to day 21 and replenished
porous scaffolds with an equal amount of PBS. The absorbance values of
First, 3D scaffolds with porous structure were prepared 0, 0.125, 0.25, 0.5, 0.75, and 1.0 mg/mL peptide solutions
by importing the Solidworks digital model into a bio-3D were determined using a ultraviolet spectrophotometer,
printer (Novaprint, Novaprint Regenerative Medicine Co., and a standard curve was plotted; the absorbance values
Ltd., Suzhou, China) and 3D-printing the PCL material measured from the supernatants collected at each time
using FDM technology. The pore geometry was a square, point were then converted by substituting the measured
with a pore diameter of 600 μm. Then, 400 mg of heparin values into the standard curve to assess the amount and
(Aladdin), 190.6 mg of EDAC (Aladdin), and 115 mg of rate of peptide release. The experiment was repeated
N-hydroxysuccinimide (Aladdin) were dissolved in 10 mL three times.
of 2-(N-morpholino) ethanesulfonic acid (MES, Aladdin) 2.3. In vitro experiments
buffer (pH 4.5) and reacted for 10 min at room temperature,
and then dopamine hydrochloride (Aladdin) was dissolved 2.3.1. Culture of BMSCs
in MES buffer (pH 8.5) at 102.2 mg/mL. The PCL porous BMSCs for this study were purchased from Procell
scaffold material was immersed in the solution, and the Biologicals Ltd (Suzhou, China). BMSCs at passages 3 to
reaction was carried out overnight away from light at 25°C. 5 were used for subsequent experiments. To establish in
On the second day, the PCL scaffolds were washed three vitro diabetes model, we cultured BMSCs in high-glucose
times using buffer 1 (0.1 M phosphate-buffered saline [PBS], medium, with cells cultured in non-high-glucose medium
0.15 M NaCl, pH 7.2), submerged in sulfo-SMCC (Aladin) used as a control. The porous scaffolds were cut into 10 × 10
solution (2 mg/mL), and reacted for 1 h at room temperature, mm thin slices, sterilized with ethylene oxide, and placed
followed by reaction with buffer 2 (0.1 M PBS, 0.15 M NaCl, in six-well plates. Then, 1 × 10 BMSCs were inoculated
5
0.1 M EDTA, pH 7.0) three times. Finally, the peptide was in Dulbecco’s modified Eagle medium (DMEM) complete
dissolved in buffer 2 at 2 mg/mL and placed in a PCL holder, medium (containing 1% double antibody and 10% fetal
and the reaction was sealed overnight at 4°C away from light. bovine serum) and incubated at 37°C and 5% CO to allow
2
At the end of the reaction, the peptide was washed repeatedly cells to attach to the scaffolds. Cell viability, proliferation,
with double-distilled water until colorless, lyophilized, and adhesion, and osteogenic differentiation were assessed.
stored at -20°C away from the light. Fluorescence microscopy
was used to observe the peptide branching efficiency and to 2.3.2. Viability and proliferative capacity of scaffolds
confirm peptide activity, and all scaffolds were sterilized with for BMSCs
ethylene oxide before use. Cell proliferative capacity was assessed using CCK-8 (Cell
Counting Kit-8, Sigma, USA). The porous scaffolds were
2.2. Material characteristics of composite scaffolds added to DMEM complete medium at an area:volume ratio
The chemical composition of the four scaffolds (PCL, of 1.25 cm /mL and immersed for 72 h at 37°C. BMSCs
2
PCL@SS31, PCL@E7, and PCL@SS31@E7) was analyzed at passage 4 were then inoculated into 96-well plates at 5
by Fourier transform infrared spectroscopy using an × 10 /well, and 200 μL of the extract was added to each
3
attenuated total reflectance spectrometer (Perkin Elmer, well, followed by incubation in an incubator set at 37℃
USA). The microstructural morphology, pore shape, size, and infused with 5% CO . The specimens were prepared
2
and distribution of peptide particles of the four scaffolds according to the CCK-8 instruction manual on days 1, 2,

Volume 10 Issue 4 (2024) 206 doi: 10.36922/ijb.2379