Effects of liquid nitrogen treatment on the proliferation ... - Springer Link

8 downloads 0 Views 362KB Size Report
Effects of liquid nitrogen treatment on the proliferation of osteosarcoma and the biomechanical properties of normal bone. Norio Yamamoto, Hiroyuki Tsuchiya, ...
J Orthop Sci (2003) 8:374–380

Effects of liquid nitrogen treatment on the proliferation of osteosarcoma and the biomechanical properties of normal bone Norio Yamamoto, Hiroyuki Tsuchiya, and Katsuro Tomita Department of Orthopaedic Surgery, School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan

Abstract To overcome problems of autografts for reconstruction in the presence of malignant bone and soft tissue tumors, we devised a method for treating autografts that utilizes the hypothermic effect of liquid nitrogen. We measured temperature changes inside the bone at each condition and established a one-cycle liquid nitrogen protocol that included 20 min in liquid nitrogen, 15 min in room air, and 15 min in physiological saline. The proliferation potential of the tumor cells treated with the liquid nitrogen method was examined by means of bromodeoxyuridine (BrdU) immunostaining. Tumor proliferation potential in vivo was examined in nude mice. Based on the results we concluded that the tumor cells died out as a result of the liquid nitrogen method. Regarding compression strength there was no significant difference between intact bone and liquid nitrogen-treated bone, whereas the strength of the autoclaved bone was decreased. Scanning electron microscopic examination of the fracture surface of the autoclaved bone after the compression test showed an irregular, uneven surface, whereas that of the liquid nitrogen-treated bone was smooth and fine-grained. This might be one of the reasons for the discrepancy in compression strength. Key words Liquid nitrogen · Malignant bone and soft tissue tumor · Cell viability · Bone strength · Autograft

Introduction Because of recent improvements in multimodality therapies for sarcomas, limb salvage surgery is now primarily performed to improve quality of life.7,23 Various methods are used for limb reconstruction, but none leads to permanent reconstruction. Even for a tumor prosthesis, Kawai et al.10 reported a 10-year survival rate of about 50%. Allografts are also used for reconstructive surgery but are potential carriers of Offprint requests to: H. Tsuchiya Received: June 3, 2002 / Accepted: December 2, 2002

transmitted diseases, such as acquired immunodeficiency disease (AIDS) or viral hepatitis.22 Moreover, in some countries, especially Japan, it is difficult to obtain allografts for socioreligious reasons. In view of these problems, many methods have been developed for reusing the resected bone for reconstruction, including irradiation,27 autoclaving,13,21 and pasteurization.6 These methods require special equipment or strict thermal control, however; and especially in the case of autoclaving they may cause weakness of the treated bone and loss of bone inductivity.26 Most of these methods manage autografts through heating. In contrast, we devised a new processing method that refrigerates the autograft with liquid nitrogen.

Materials and methods Animals and cell line Fresh metatarsal bones of cattle (4-year-old holsteins) were used that had been removed immediately after slaughtering. The shape of these bones resembles the distal part of the human femur. The osteosarcoma Takase (OST) cell line, which is derived from human osteosarcoma in our laboratory, as described previously by Yamazaki,28 was used. This cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with antibiotics and 10% decomplemented fetal bovine serum (FBS) in an incubator. Male nude mice (BALB C nu/nu; 10 weeks old) were used for the in vivo study. They were bred under germ-free and specific pathogen-free conditions. All mice were treated under anesthesia and killed by cervical dislocation. All animal studies were conducted in accordance with the principles and procedures outlined in the Kanazawa University’s guidelines for the care and use of laboratory animals and any national laws on the care and use of laboratory animals.

N. Yamamoto et al.: Liquid nitrogen-treated bone graft

Fig. 1. Measurement of temperature changes inside bone. The tip of one thermometer was inserted into the bone marrow and that of the other into the subchondral bone of the metatarsus. Temperature changes were measured under three incubation conditions: in liquid nitrogen at ⫺196°C, at room temperature of 20°C, and in physiological saline at 30°C

375

fragments and incubated in 0.1% BrdU and the culture solution for 1 h at room temperature. The tumor mass from the control group (not treated with the liquid nitrogen method) was processed similarly in the culture solution and incubated in 0.1% BrdU solution for 1 h. The specimens from the one-cycle processing group [submersion in liquid nitrogen (at ⫺196°C) for 20 min, followed by incubation in room air (at 20°C) for 15 min and then in physiological saline (at 30°C) for 15 min], the two-cycle processing group (repeat one cycle twice), and the control group were subjected to BrdU immunohistochemical staining. Immunostaining was performed with a BrdU staining kit (ZYMED Laboratories, San Francisco, CA, USA) in accordance with the manufacturer’s instructions. Positive control staining was performed with mouse intestines supplied with this kit; negative control staining did not use a primary antibody.

Temperature changes inside the bone

Evaluation of tumor cell viability (in vivo)

To evaluate the temperature changes inside the bone, we used the distal part of the metatarsal bone (a 170 mm length from the distal end). The tip of a digital thermometer in the subchondral bone and the tip of another thermometer in the bone marrow. The metatarsal bone was soaked gently to prevent liquid nitrogen from entering the bone from the cut surface (Fig. 1). Temperature changes were measured under three conditions: in liquid nitrogen at ⫺196°C, at room temperature (20°C), and in physiological saline at 30°C. In the preliminary study, all three metatarsal bones developed fractures during physiological saline processing subsequent to liquid nitrogen processing. Later, we found that we could prevent all three bones from fracturing by adding room temperature air between the liquid nitrogen and physiological saline phases. Based on these results, we determined that the protocol comprised three procedures (liquid nitrogen, room air, physiological saline), not two (liquid nitrogen, physiological saline).

To examine the tumor proliferation potential, a piece of the tumor weighing 0.3 g was cut from the tumor mass, which had proliferated on the back of one nude mouse. It was transplanted to the back of another nude mouse after treatment with the liquid nitrogen method. We examined changes in tumor volume and histological findings for the liquid nitrogen treatment groups (oneand two-cycle processing groups) and the control group. Ten nude mice were used for each group. The estimated tumor volume (V) in cubic millimeters was obtained with the following equation.

Evaluation of tumor cell proliferation potential (in vitro) The proliferation potential of the tumor cells was measured with bromodeoxyuridine (BrdU) immunostaining. Cultured osteosarcoma cells were detached from the flask by means of 0.25% trypsin, and 5 ⫻ 106 cells with 0.2 ml of the culture solution were injected into the back of the nude mice to create a tumor mass. The tumor mass was then removed from the back and divided into pieces weighing 0.3 g each, which were treated with the liquid nitrogen method. After this treatment, the fragments were gently cut into small

V ⫽ 0.5 ⫻ (length) ⫻ (width)2 Examination of compression strength To evaluate the strength of the treated bone, we examined the compression strength of intact bone, bone processed with the one-cycle liquid nitrogen treatment, and autoclaved bone. For autoclaving, the bones were processed at 121°C for 15 min. Ten samples from each group were tested. The samples were taken from the middle part (diaphysis) of the metatarsal bones, which had the same diameter and thickness. We used the DSS5000 compression tester (Shimazu, Kyoto, Japan), and the compression load was directed vertical to the bone axis until fracture. The speed of the crosshead was set at 2 mm/min. Observation of fracture surface by scanning electron microscopy To evaluate the surface features of the fractured bones, specimens were obtained from the one-cycle liquid

376

N. Yamamoto et al.: Liquid nitrogen-treated bone graft

A,B

C Fig. 2. Temperature changes in the metatarsus. A During liquid nitrogen processing. B At room temperature (20°C) after liquid nitrogen processing. C In physiological saline at

30°C following room temperature processing. Temperatures at the medullary cavity ( filled circles) and subchondral bone (open circles) are indicated. Bars indicate standard deviation

nitrogen-processed bones and the autoclaved bones immediately after the compression test. The specimens were fixed to object tables by double-sided pressuresensitive tape and then sputter-coated with gold by an ion coater IB-3 (Eicho Engineering, Mito, Japan) to add electroconductivity. Subsequently, the fracture surface features were observed by a scanning electron microscope (SEM) (DS-130C; Topcon, Tokyo, Japan) at 7 kV acceleration voltage. Statistical estimation Values are expressed as means ⫾ standard deviation. The results were compared for statistical significance with analysis of variance (ANOVA) and the Scheffé test. A P value of ⬍0.05 was considered significant.

Results Temperature changes inside bone During liquid nitrogen processing the temperature approached stability in about 20 min (Fig. 2A). At room temperature (20°C) after the liquid nitrogen processing, recovery to about ⫺100°C took almost 15 min (Fig. 2B). In physiological saline at 30°C after room temperature processing, the temperature became nearly stable in about 15 min (Fig. 2C). Based on these results, we determined that one cycle of the liquid nitrogen treatment should consist of submersion in liquid nitrogen (at ⫺196°C) for 20 min followed by incubation in room air (at 20°C) for 15 min and then in physiological saline (at 30°C) for 15 min to obtain stable temperature changes without any fracture.

Fig. 3. Immunohistochemical staining of bromodeoxyuridine (BrdU). A Control. B Liquid nitrogen (one-cycle) processed tissue. In contrast to the control group, there were no positive cells in the liquid nitrogen in the one-cycle group. Bars 12.5 µm

liquid nitrogen group (Fig. 3B) and the two-cycle group (data not shown) were not.

Evaluation of tumor cell proliferation potential (in vitro)

Evaluation of tumor cell viability (in vivo)

The nuclei of the tumor cells in the control group were stained (Fig. 3A). In contrast, nuclei in the one-cycle

The volume of the control group tumor increased over time (Fig. 4). It was histologically demonstrated that the

N. Yamamoto et al.: Liquid nitrogen-treated bone graft

377

Fig. 4. Estimated volume of control-group tumor treated as described in the “Materials and methods” section. Estimated tumor volume was gradually increased as time passed. Bars indicate standard deviation

tumor cells permeated the spaces between the muscle fibers (Fig. 5). In contrast, in the one-cycle liquid nitrogen group the volume decreased over time (Fig. 6), and there was a similar decrease in tumor volume even in the two-cycle liquid nitrogen group (data not shown). Histological observations showed fibrous tissue around the transplanted mass (Fig. 7A). On the highly magnified image, necrosis and infiltration of inflammatory cells were identified (Fig. 7B). Examination of compression strength The mean value for the maximum load of the intact bone was 23.5 ⫾ 0.6 kN, that for the bone treated with the one-cycle liquid nitrogen process was 22.6 ⫾ 0.9 kN, and that for the autoclaved bone 14.8 ⫾ 2.5 kN. The Scheffé tests revealed that there was no significant difference in compression strength between the intact bone and the bone treated with the one-cycle liquid nitrogen process. However, the strength of the autoclaved bone was about two-thirds that of the intact bone, with the difference being significant (P ⬍ 0.05) (Fig. 8). Autoclaving thus clearly reduced the strength of the treated bone.

Fig. 5. A Low magnification image. B High magnification image. It was histologically demonstrated with high magnification that the tumor cells permeated the spaces between the muscle fibers. Bars 500 µm (A), 50 µm (B)

Observation of fracture surface by SEM The fracture surface of the autoclaved bone was irregular and uneven with many small holes (Fig. 9A). In contrast, the fracture surface of the liquid nitrogentreated bone was smooth and fine-grained (Fig. 9B).

Discussion We investigated cell viability in vitro and in vivo. For the in vitro experiment, we use BrdU, a thymidine

Fig. 6. Estimated volume of the liquid nitrogen method (one cycle processing) group tumor treated as described in the “Materials and methods” section. Estimated tumor volume was gradually decreased. Bars indicate standard deviation

378

Fig. 7. A Low magnification image. B High magnification image. Histologically fibrous tissue was observed around the transplanted mass. Necrosis and infiltration of inflammatory cells were observed on the highly magnified image. Bars 250 µm (A), 25 µm (B)

Fig. 8. Compression strengths. There was no statistical difference for compression strength between liquid nitrogentreated bone and intact bone. Autoclaved bone showed a statistically significant difference. Bars indicate standard deviation. *P ⬍ 0.05. **P ⬍ 0.05

N. Yamamoto et al.: Liquid nitrogen-treated bone graft

Fig. 9. Scanning electron micrographs of the fracture surface. A Autoclaved bone. B Liquid nitrogen-treated bone. The fracture surface of the autoclaved bone was irregular and uneven, whereas the liquid nitrogen-treated bone was smooth and fine-grained. Bars 3.33 µm

analogue, to detect the cell proliferation potential. BrdU is absorbed into the nucleus of the cell during the S phase, when the cell cycle changes. The nuclei of the tumor cells of the control group were stained, whereas those of the one-cycle liquid nitrogen group (liquid nitrogen for 20 min, room air for 15 min, physiological saline for 15 min) and the two-cycle group (repeating one cycle twice) were not. This finding suggests that either the tumor cells had died or the cell cycle was markedly slowed after treatment with liquid nitrogen. The in vivo experiment with nude mice demonstrated that the tumor volume increased over time in the control group, but that it decreased in the one- and two-cycle liquid nitrogen groups. The total tumor cell deaths could be determined histologically for the liquid nitrogen-treated groups. These results led us to conclude that the tumor cells had died as a result of the liquid nitrogen treatment. We also concluded that the one-cycle liquid nitrogen method is sufficient to achieve tumor cell extinction and produces the same results as those obtained with the two-cycle method.

N. Yamamoto et al.: Liquid nitrogen-treated bone graft

We investigated bone strength after treatment, because one of the purposes of this study was to devise a new method for limb reconstruction. The strength of the autoclaved bone was found to be about two-thirds that of the intact bone, whereas liquid nitrogen-treated bone maintained its initial strength even after the treatment. SEM showed an irregular, uneven fracture surface of the autoclaved bone with holes that seemed to have been caused by the loss of protein and cell components due to thermal denaturation. We presumed that the weakness of the autoclaved bone was due to enlargement of these small holes under stress. In contrast, the fracture surface of the liquid nitrogentreated bone was smooth and fine-grained, leading to the hypothesis that preservation of protein and cell components is one of the reasons bone strength is maintained after liquid nitrogen treatment. We can therefore conclude that liquid nitrogen-treated bone has enough initial strength for limb reconstruction, comparable to that of allografts and pasteurized bones. However, in most clinical cases of malignant bone tumors, bone cortices were destroyed by tumor tissues and their strength decreased. In clinical cases, we may have to consider the use of internal structural support (e.g., bone cement) or external support (e.g., crutches) until bone substitution takes place. Surgical use of liquid nitrogen was first reported by Cooper3 in 1962. Since then, many tumors have been treated locally with liquid nitrogen.20,24 In the field of orthopedics, Gage et al.8 researched the effects of liquid nitrogen in 1966, and Marciani et al.16 reported on the good remodeling quality of liquid nitrogen-treated bone. Marcove and Miller17 reported the first clinical use of bone tumor in 1969. Orthopedically, liquid nitrogen is now used for benign or borderline malignant tumors, being applied directly to the lesions.1,14,15,18 Reimplantation of resected bone treated with liquid nitrogen has already been reported for oral maxillofacial surgery.2,4,5,19 Dong et al.4 reported their clinical results with reimplanted bone for patients with benign and malignant bone tumors, as well as their findings concerning the merits of liquid nitrogen treatment. The mechanisms of cell death caused by freezing are complex, but the main factors are ice crystal formation and dehydration of the cells.11 Tumor cells are considered more sensitive to low temperatures than normal cells.25 Helpap9 found that rapid freezing and slow thawing causes the most effective damage to tumor cells. Our process features rapid freezing and slow thawing steps, which can cause fatal damage to the treated cells; because only a 50 min period is needed for the bone treatment, it can be done during a single operation. The liquid nitrogen method does not pose any risk of a high frequency of recurrence or infection.14,15 In

379

clinical use for local treatment of benign bone tumors, no difference in the infection rate was found between the liquid nitrogen and phenol methods. In our in vivo study, none of the nude mice showed any sign of infection on their backs after implantation of the liquid nitrogen-treated mass. We can expect joints reconstructed with liquid nitrogen-treated bone to last much longer than those treated with allografts because of their perfect congruity. Kitaoka12 reported that in the case of allografts the reimplanted ligament was replaced with the recipient’s ligament, and that 24 weeks after reimplantation the strength of the ligament could be expected to reach 90% that of the control. Thus, histologically confirmed replacement can occur even in the case of allografts. In the case of autografts, we can therefore expect at least the same or better results. We conclude that the liquid nitrogen treatment method is one of the most effective for reconstruction in cases of malignant bone and soft tissue tumors.

References 1. Biesecker JL, Marcove RC, Huvos AG, et al. Aneurysmal bone cysts: a clinicopathologic study of 66 cases. Cancer 1970;26:615– 25. 2. Bradley PF. A two-stage procedure for reimplantation of autogenous freeze-treated mandibular bone. J Oral Maxillofac Surg 1982;40:278–84. 3. Cooper IS. Cryogenic surgery of the basal ganglia. JAMA 1962; 181:600–4. 4. Dong YJ, Zhang GZ, Wang SP, et al. The use of immediately frozen autogenous mandible, for benign tumour mandibular reconstruction. Br J Oral Maxillofac Surg 1996;34:58–61. 5. Dougherty TP, Rafetto LK, Edwards RC, et al. Reimplantation of freeze-treated bone in immediate reconstruction of the mandible. Am J Surg 1982;144:463–5. 6. Ehara S, Nishida J, Shiraishi H, et al. Pasteurized intercalary autogenous bone graft: radiographic and scintigraphic features. Skeletal Radiol 2000;29:335–9. 7. Enneking WF. A system of staging musculoskeletal neoplasms. Clin Orthop 1986;204:9–24. 8. Gage AA, Greene GW Jr, Neiders ME, et al. Freezing bone without excision. JAMA 1966;196:770–4. 9. Helpap B. Cryosurgery techniques, morphology and immunology: an overview. Low Temp Med 1982;8:7–12. 10. Kawai A, Muschler GF, Lane JM, et al. Prosthetic knee replacement after resection of a malignant tumor of the distal part of the femur: medium to long-term results. J Bone Joint Surg Am 1998; 80:636–47. 11. Kimura T, Kojima Y, Nakagawara G. Current status of cryopreservation of pancreatic islets. Low Temp Med 1996;22:1–6 (in Japanese). 12. Kitaoka K. An experimental study regarding regeneration of the ligament and ligament-bone junction in the frozen allografts. J Juzen Med Soc 1994;103:96–107 (in Japanese). 13. Lauritzen C, Alberius P, Santanelli F, et al. Repositioning of craniofacial tumorous bone after autoclaving. Scand J Plast Reconstr Surg Hand Surg 1991;25:161–5. 14. Malawer MM, Dunham W. Cryosurgery and acrylic cementation as surgical adjuncts in the treatment of aggressive (benign) bone

380

15.

16.

17. 18. 19. 20. 21.

N. Yamamoto et al.: Liquid nitrogen-treated bone graft tumors: analysis of 25 patients below the age of 21. Clin Orthop 1991;262:42–57. Malawer MM, Bickels J, Meller I, et al. Cryosurgery in the treatment of giant cell tumor: a long-term followup study. Clin Orthop 1999;359:176–88. Marciani RD, Giansanti JS, Massey GB. Reimplantation of freeze-treated and saline-treated mandibular bone. J Oral Surg 1976;34:314–19. Marcove RC, Miller TR. Treatment of primary and metastatic bone tumors by cryosurgery. JAMA 1969;207:1890–4. Marcove RC, Sheth DS, Takemoto S, et al. The treatment of aneurysmal bone cyst. Clin Orthop 1995;311:157–63. Plezia RA, Smith DB, Weaver AW. Frozen autogenous mandible as an immediate replacement graft. J Oral Surg 1978;36:481–6. Shafir M, Shaprio R, Sung M, et al. Cryoablation of unresectable malignant liver tumors. Am J Surg 1996;171:27–31. Thompson VP, Steggall CT. Chondrosarcoma of the proximal portion of the femur treated by resection and bone replacement: a six-year result. J Bone Joint Surg Am 1956;33:357–67.

22. Tomford WW. Transmission of disease through transplantation of musculoskeletal allografts. J Bone Joint Surg Am 1995;77: 1742–54. 23. Tsuchiya H, Tomita K, Mori Y, et al. Caffeine-assisted chemotherapy and minimized tumor excision for nonmetastatic osteosarcoma. Anticancer Res 1998;18:657–66. 24. Uchida M, Imaide Y, Sugimoto K, et al. Percutaneous cryosurgery for renal tumours. Br J Urol 1995;75:132–7. 25. Uedaira H. Dynamic states of water in biological systems under low temperature. Low Temp Med 1977;3:87–9 (in Japanese). 26. Urist MR, Dawson E. Intertransverse process fusion with the aid of chemosterilized autolyzed antigen-extracted allogeneic (AAA) bone. Clin Orthop 1981;154:97–113. 27. Yamamuro T, Kotoura Y. Intraoperative radiation therapy for osteosarcoma. Cancer Treat Res 1993;62:177–83. 28. Yamazaki Y. Establishment of cell strain of human steogenic sarcoma cells and histological appearance of this strain in tissue culture. J Juzen Med Soc 1964;71:1–13 (in Japanese).