A total of 20 New Zealand rabbits (female; 4–5 months; weighing 2–3 kg; the Laboratory Animal Farm of Chunle, Tianjin, China) were used for this study. The environment was kept at 25°C and humidity 50%. A 12/12 h light/dark cycle was maintained. Access to food and water was restricted (twice per day). The new biopsy and bolt closure instruments and metallic casing are our patented products, which are available for purchase from the Affiliated Hospital of Chengde Medical College (Chengde, China) (Fig. 1). This study was performed in accordance with the regulations for the protection of animal and medical rights. The study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol has been reviewed and approved by the Institutional Animal Care and Use Committee of the Affiliated Hospital of Chengde Medical College (Chengde, Hebei, China).

The VX2 tumor cells (Jennio biotech company, Guangzhou, China) were injected into the left vastus lateralis of the rabbit for serial subcultivation, and Color Doppler ultrasonography (Medical Systems-LOGIQ9, GE Healthcare, Chicago, IL, USA) was used to assess the tumor. After 3 weeks, once the tumor size had reached a maximum of 2×2×2 cm, rabbits with VX2 muscle tumors were secured to the animal dissecting table subsequent to administration of anesthesia (sodium pentobarbital, 35 mg/kg) via the ear vein. An incision was made in the overlying skin and tissue, and then the VX2 tumor was resected. The tumor was resected carefully following elimination of necrotic and surrounding fibrous tissues, and then placed in phosphate-buffered saline (PBS) at 37°C. Next, the tumor was cut into 1×1×1-mm sections. Subsequent to the sections being centrifuged for 5 min at 3,960 × g at 20°C (centrifugal radius, 13.5 cm), the supernatant was removed. The pieces were again placed in PBS and centrifugation was repeated under the same conditions. A tumor cell suspension of 10⁶ cells/ml was ultimately prepared. All procedures were performed on a sterile bench. The tumor cells or tissue were stored in cryogenic refrigerator at −80°C until subsequent use. The size of any regrowth or metastasis was small, measuring ~0.5×0.5×0.5 cm.

A total of 20 rabbits were secured to the animal dissecting table after administering anesthesia (sodium pentobarbital, 35 mg/kg) via the ear vein. The hair on each lower extremity was removed, and then the surgical field was disinfected and outlined using sterile towels. An ~1-cm skin incision was made along the medial tibia stem epiphysis, and the muscle was separated to expose the tibial metaphyseal bone. A Kirschner wire (1.6 mm) was applied to puncture the tibial metaphyseal bone and then placed parallel to the long axis of the tibia. When the wire was passed through the marrow cavity at a distance of ~2 cm, the tumor cell suspension (0.3 ml) was transplanted into the tibia and the puncture hole was closed using bone wax, thus establishing the bone tumor model. This procedure was performed for each lower limb. Radiography (X-ray apparatus; model no. OEC9800; GE Healthcare) was performed on the tibias of each of the 20 rabbits every 7 days to assess tumor growth. Radiography performed 1 or 2 weeks after VX2 tumor cell transplantation showed that the tumors were growing slowly in the medullary space and that the cortices of the bones were not broken. Radiography performed in a preliminary experiment on additional rabbits revealed that in the third week the tumors grew rapidly and that the cortices of the bones were partially destroyed (Fig. 2). The rabbit model of bone tumors at the second week was therefore used for further study.

A total of 20 rabbits with bone tumors were secured to the animal dissecting table after administering anesthesia (sodium pentobarbital, 35 mg/kg) via the ear vein. The hair on the lower extremities was removed, and the surgical field was disinfected and outlined by sterile towels. All the left tibial tumors were punctured with the improved biopsy method (the experimental group). When the puncture needle reached the tumor, the stylet was pulled out and the casing was maintained in a stable position; the needle was then inserted across the casing, and several pathological samples of the tumor were obtained. Bolt closures were implanted into the puncture holes through the casing after biopsy. All the right tibias were punctured using the traditional biopsy technique (control group), and the puncture holes were not blocked (Fig. 3).

Color Doppler ultrasonography was performed to assess the exudation of the puncture holes in the 20 rabbits after 1 h. After 7 days, the 20 rabbits were sacrificed via air embolism, after administering anesthesia (sodium pentobarbital, 40 mg/kg) via intraperitoneal injection, and soft tissues, which were 0.5 cm away from the puncture holes, were obtained. The tissues were cut into 2×2×2 mm sections, incubated in 10% formalin (25°C) for 12 h, then the tissue sections were placed into increasing alcohol gradient (75, 80, 90%) for dehydration. After 5 h, the transparent sections were put into molten wax and embedded. When embedded successfully, the wax block was cut into 5 µm-thick slices and fixed on the glass slide. When the section was de-waxed, the section was heated for 20 min at 60°C prior to being de-waxed. Then sections were placed in the xylol (100%) for 20 min, the section was then placed into the xylol (50%) for 5 min (25°C). The sections were the rehydrated in a descending alcohol series (100, 95, 90, 80 and 70%) for 5 min at room temperature. Hematoxylin-eosin staining of the soft tissue was performed (eosin, 0.5% for 3 min; hematoxylin, 1% for 5 min) at room temperature, and the samples were examined using light microscopy (magnification, ×20; Olympus-BX53, Olympus Corporation, Tokyo, Japan). The microscopic results were investigated by a pathologist in a blinded manner to determine tumor cell spreading.

SPSS17.0 (SPSS, Inc., Chicago, IL, USA) was used for the statistical analysis. The exudation rate through the puncture hole in VX2 bone tumors and the tumor spread rate in the soft tissue near the puncture hole of the experimental and control groups were analyzed using χ² tests. Theoretical frequency (12) (T_RC=n_R × n_C/n, where T_RC represents the theoretical frequency from the R row and the C column, n_R and n_C represent the total number of the R row and the total number of the C column, n=40) was calculated as T>5, n=40, χ²=(ad-bc)²n/(a+b)(c+d)(a+c)(b+d); where a, b, c and d represent actual frequency with P<0.05 considered to indicate a statistically significant difference.

The success rate of the model was 100% for the 20 rabbits. After 1 h, color Doppler ultrasonography showed that all the closures covered the puncture holes; 5 cases in the experimental group showed a certain degree of exudation around the puncture holes, while 16 cases in the control group showed the same (Fig. 4).

After 2 weeks, pathological examinations showed that tumor cells were found in the soft tissue near the puncture holes in 7 cases in the experimental group and in 17 cases in the control group (Fig. 5).

The exudation rate through the puncture holes in the experimental group was significantly lower than that in the control group (25 vs. 80%; χ²=12.13; P<0.05; Table I). The rate of tumor spread in the experimental group was significantly lower than that in the control group (35 vs. 85%; χ²=10.41; P<0.05; Table II).