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International Journal of Bioprinting 3D-printed bioceramic scaffolds for bone regeneration

2.2. Characterization of L-, S-, and LS-grafts guide block using a sagittal blade bone saw (Zimmer
The X-ray diffraction (XRD) patterns of L-, S-, and LS- Biomet Co., Ltd., Tokyo, Japan). The graft was implanted
grafts were recorded to observe the apatite crystalline phase into the defect and fixed with a stainless steel plate and
using a diffractometer (D8 Advance, Bruker AXS GmbH, four screws. A polyethylene terephthalate (PET) sheet was
Karlsruhe, Germany). Fourier-transform infrared (FTIR) placed between the graft and the radius. Finally, the graft
spectra of the L-, S-, and LS-grafts were recorded to evaluate was fixed to the plate using a single loop–4-0 nylon suture
the compositions using an FT/IR-6200 spectrometer loop (MANI Co., Ltd., Tochigi, Japan). After irrigation
(JASCO, Tokyo, Japan). The morphologies of the L-, S-, with saline, the muscles and skin were repaired using 4-0
and LS-grafts were confirmed using micro-computed nylon suture. The aforementioned surgery was performed
tomography (µ-CT; ScanXmate-L080T/L090T, Comscan on both forearms. The forearms were not immobilized
Techno Co., Ltd., Kanagawa, Japan) and scanning electron after surgery. At 4 and 12 weeks postoperative (PO), the
microscopy (SEM; S3400N, Hitachi High-Technologies rabbits were sacrificed using an overdose of ketamine and
Corporation, Tokyo, Japan). The lengths on the side of the xylazine (n = 4 per group). After sacrifice, both forearms
pore aperture and the strut thickness for each graft were were harvested and immersed in 10% formalin solution to
estimated from the SEM images. At least ten pores and fix the tissues.
struts in each graft were used to estimate the pore aperture
size and strut thickness, respectively. The porosities of the 2.4. Gross and radiographical analyses
macro-pores, in other words, macro-porosities, in L-, S-, The images of the specimens were obtained by photography,
and LS-grafts were analyzed using µ-CT software (TRI/3D- a radiographic system (HA-60, HITEX Co., Ltd, Osaka,
BON-FCS64, RATOC System Engineering Co., Ltd, Tokyo, Japan), and a µ-CT system (ScanXmate-L080T/L090T).
Japan). The total porosities of the L-, S-, and LS-grafts were Photographs were captured immediately after the surgery
measured using the theoretical density of HAp (3.16 g/cm , to demonstrate the orientation of the uniaxial pores in the
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five samples in each graft). The compressive strengths of defects and the types of tissue in contact with the pore
the L-, S-, and LS-grafts were measured using a universal apertures. Radiographs were obtained to observe temporal
testing machine (Autograph AGS-J; Shimadzu, Kyoto, changes in the exterior of the grafts. The µ-CT images were
Japan) by loading parallel to the long (10-mm length taken to observe temporal changes in the graft interior.
direction) and short (6-mm length direction) axes of the The µ-CT images were reconstructed using µ-CT software
graft at a crosshead speed of 1 mm/min until fracture. One (TRI/3D-BON-FCS64). The new bone volume-to-total
sample of each graft was used to measure the compressive volume (BV/TV) and material volume-to-total volume
strength in each direction. (MV/TV) ratios were measured according to previously
published methods. The total volume is defined as the
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2.3. Surgery volume enclosed by the graft perimeter.

The animal experiments were approved by the Animal Care 2.5. Histological analyses
and Use Committee of Kyushu University (Approval No. The hematoxylin–eosin (HE)-stained images were
A23-007-0). We used 12 Japanese white rabbits (18-week- prepared after scanning the photographs, radiographs, and
old, male, 2.9–3.4 kg, Japan SLC, Co., Ltd., Shizuoka, Japan) µ-CT images. The new bone area-to-total area (BA/TA)
in this study. A critical-sized (10 mm length) rabbit ulnar and material area-to-total area (MA/TA) were measured
segmental bone defect model was used to evaluate the using a BZ-X digital analyzer (Keyence Co., Ltd., Osaka,
effect of pore architecture on tissue ingrowth (Figure S1 Japan). The total area was defined as the area enclosed
in Supplementary File). To achieve analgesic and sedative by the graft perimeter. To clarify the effects of graft pore
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effects, ketamine (30 mg/kg) and xylazine (5.0 mg/kg) direction on vascularization, the thicknesses and angles
were administered intramuscularly to the thighs of rabbits of the blood vessels formed in each graft were analyzed
to achieve analgesic and sedative effects. Prior to skin using HE-stained sections. The angles of the blood vessels
incision, lidocaine (6 mg/kg) was injected subcutaneously. were estimated using an ImageJ plug-in, called FibrilTool.
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Gentamicin (4 mg/kg) was intraperitoneally administered The directions parallel and perpendicular to the bone axis
to prevent infection. The diaphyseal ulna was exposed by were defined as 0° and 90°, respectively. Twenty regions of
separating the intermuscular space between the extensor interest (ROIs) per section were used for the analysis. The
digitorum communis and extensor digitorum lateralis. The orientation of the new bone was analyzed from the HE-
muscles were treated carefully to minimize damage because stained images using the ImageJ plug-in FibrilTool. The
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excessive damage to the muscles can induce abnormal orientation of new bone was determined from that of the
accumulation of fibrotic tissue within the new bone. The collagen fibers in the new bone. Acidic eosin stains the
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ulna was amputated along both sides of a 10-mm width collagen fibers light pink and accentuates their structures.
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Volume 10 Issue 2 (2024) 447 doi: 10.36922/ijb.2323