In vitro study and characterization
Preparation of CPC-containing polymer complex
Tetracalcium phosphate (TTCP) powder was synthesized as follows. A calcium hydroxide solution (Nacalai Tesque Inc., Japan) was added dropwise into a phosphoric acid solution (Wako Pure Chemical Industries, Ltd., Japan) that was kept below 10 °C, and the solution was stirred at room temperature for 24 h. The precipitate was filtered, dried, and heated at 1500 °C for 5 h, ground in an agate mortar, and then sieved under 75 μm to obtain the TTCP powder (average particle size: 21.5 µm). Commercial dicalcium phosphate anhydrous (DCPA) powder (Taihei Chemical Industrial Co., Ltd., Japan) was ground in a ball mill with pure water and zirconia balls for 48 h to obtain the DCPA powder (average particle size: 0.52 µm). Equal molars of TTCP powder and DCPA powder were mixed in a blender and with an agate mortar to form the CPC powder.
A mixed solution of dipotassium hydrogen phosphate and potassium dihydrogen phosphate (0.2 and 0.1 g, respectively, both from Kishida Chemical Co., Ltd., Japan) was prepared with 10 mL of Milli-Q water. Low viscosity sodium alginate (UIV-L3, viscosity 20–50 mPa · s, KIMICA Co., Japan) was used as a biocompatible polysaccharide and gelation agent to develop the nonrigid CPC. Four liquids (Liquid 0 to Liquid 3) were prepared with 0, 5, 10, and 20 wt% low viscosity sodium alginate, respectively. The CPC powder was mixed with each curing liquid at a powder/liquid ratio of 3, and the mixture was placed in a cylinder (Table 1). The corresponding hardened specimens were denoted CPC0–CPC3.
The pH of the prepared curing liquid was measured using a pH meter (D-51, HORIBA, Ltd., Japan). The pH meter was first calibrated using three points measured in phosphate pH standard solution (pH 9.18), neutral phosphate pH standard solution (pH 6.86), and phthalate pH standard solution (pH 4.01), all from Wako Pure Chemical Industries, Ltd., Japan. Five replicates were measured for each liquid, and the average value is reported.
The setting time was measured according to JIS (Japanese Industrial Standards) T0330-4. Kneaded CPC paste was placed in a cylindrical Teflon mold (inner diameter: 6 mm, height: 12 mm) within 90 s after the start of kneading. The mold was placed in a container that also held a sponge soaked in Milli-Q water in an incubator held at 37 °C. Setting of the material was monitored using a Vicat needle (A-004, MECC Co., Ltd, Japan) with a 1 mm2 tip at 300 g load. Every 30 s, the Vicat needle was placed on the surface of the specimen, and the specimen was considered set when the needle failed to make a perceptible circle on its surface. The test was performed in triplicates for each condition, and the average value was reported as the setting time.
The compressive strength was determined in accordance with JIS T0330-4. The mixture of CPC powder and liquid was placed in the hole of a Teflon mold (diameter: 6 mm, height: 12 mm). The mold was stored in an incubator at 37 °C and 100% humidity for 1 h, and then immersed in Milli-Q water for 1 day. The diameter of each specimen was measured using a micrometer (M300, Mitsutoyo, Japan). Then, the specimen was individually placed on the stage of a universal tester (Autograph AGI-20kN, Shimadzu Co., Japan). Measurement was carried out at a head speed of 0.500 mm s–1 and a sampling interval of 100 ms. Five specimens from each type of CPC were used, and each specimen was measured ten times. Data analysis was performed using control/analysis software (Trapezium, Shimadzu Co., Japan), and the average value was used as the compressive strength.
Scanning electron microscopy (SEM) analysis employed an S-4700 (Hitachi Ltd., Japan) instrument at an accelerating voltage of 5.0 kV under a pressure of 1.00 × 10−3 Pa or less, on samples coated with 12 nm of Pt–Pd using an ion sputter (E-1030, Hitachi Ltd., Japan). The mixture of CPC powder and liquid was placed in a cylindrical Teflon mold (diameter: 6 mm, height: 12 mm). Each mold was stored in an incubator at 37 °C and 100% humidity for 1 h, and then immersed in acetic acid–sodium acetate buffer (see details below) for 7 days. The diameter of each specimen was measured using a micrometer (M300, Mitsutoyo, Japan). For the buffer, first two solutions were prepared using Milli-Q water: one containing 0.08 mol L–1 acetic acid (Kishida Chemical Co., Ltd. Japan) and the other containing 0.08 mol L–1 sodium acetate (Kishida Chemical Co., Ltd., Japan). Then, the two solutions were mixed at a ratio of 1:7 to prepare the buffer, which had a pH of 5.50 ± 0.02.
In vivo studies
The animal protocol was approved by the Animal Ethics Committee of Osaka City University. Ten retired female New Zealand white rabbits weighting 4–4.5 kg were used for implantation of CPC0 (control), CPC1, CPC2, and CPC3. Five femurs were used in each group. The rabbits were anesthetized with ketamine hydrochloride, and a cylindrical defect (diameter: 4 mm, depth: 10 mm) was drilled on both femur condyles using a trephine drill. A CPC sample was injected into the defect site, which was then sutured (Fig. 1). After 6 weeks, the rabbits were sacrificed, and the femur condyles were obtained for analysis. The surgery was performed by two authors (AS and KO).
X-ray and micro-CT analysis
X-ray images were taken for each femur condyle immediately after surgery as well as 3 and 6 weeks later. The extracted rabbit femur condyles were fixed in 10% neutral buffered formation solution at room temperature. Micro-CT was done using inspeXio SMX-90CT Plus (Shimadzu Corporation, Japan). The scan data were reconstructed using 3D image processing software (ExFact VR, Nihon Visual Science, Inc., Japan).
After the micro-CT analysis, each sample was decalcified with Morse solution (Wako Pure Chemical Industries, Ltd., Japan) and dehydrated using an alcohol series. Residual alcohol was removed by immersion in xylene. After embedding the tissue sample in paraffin block, 4-µm-thick slices were cut using a microtome and treated with hematoxylin and eosin (H&E) and Masson’s trichrome stains. The sections were observed using a model BX53F microscope (Olympus, Japan) and photographed with an Olympus DP74 camera. The images were analyzed using Cellsens software (Olympus, Japan). The area of new bone mass was measured using ImageJ based on Masson’s trichrome staining. The total area of new bone was measured in a 100-fold visual field at three locations: on the cortical bone side, the central side, and the cancellous bone side of the bone defects. Image analysis and measurement of the total area of new bone were conducted by two authors (HT and HN) in a blinded situation.
Statistical analysis was performed using the Excel Statistics software for Windows (version 2019; SSRI Co. Ltd., Tokyo, Japan). Data were expressed as mean ± standard deviation. The data analysis used one-way analysis of variance followed by a multiple comparison using the Tukey test. P values < 0.05 were considered statistically significant.