Exploration of Inactivated Bone Using High Hydrostatic Pressurization for Future Oncologic Application

Exploration of Inactivated Bone Using High Hydrostatic Pressurization for Future Oncologic Application | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Exploration of Inactivated Bone Using High Hydrostatic Pressurization for Future Oncologic Application Eiichi Sawaragi, Rie Akita, Hiroki Yamanaka, Michiharu Sakamoto, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7430280/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract High hydrostatic pressure (HHP) has emerged as a promising technique for inactivating tumor cells while preserving the structural integrity of biological tissues. This study investigated the potential of HHP-treated bone as a biologically compatible autograft material for oncological reconstruction. Bone morphogenetic protein-2 (BMP-2), a key osteoinductive factor, was subjected to various inactivation methods, including HHP, high-temperature heating, and liquid nitrogen, and its osteogenic activity was assessed through alkaline phosphatase (ALP) expression in MC3T3-E1 cells. BMP-2 activity was preserved following HHP and cryogenic treatment but was diminished by thermal methods. In a rat calvarial defect model, we compared the biological and structural performance of bone grafts processed using HHP, heating (Pasteur method), liquid nitrogen, and no treatment. Computed tomography and histological analyses demonstrated comparable bone healing and ossification in the HHP and liquid nitrogen-treated groups. Scanning electron microscopy and Vickers hardness testing revealed that the surface morphology and mechanical strength of the HHP-treated bone were similar to those of the untreated bone. These findings suggest that HHP treatment preserves both the osteogenic and biomechanical properties of autologous bone and should offer a clinically viable alternative to conventional tumor inactivation methods in bone reconstruction. Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Cancer Biological sciences/Cell biology Physical sciences/Materials science Health sciences/Medical research High hydrostatic pressure Cell inactivation Bone reconstruction Osteoinductive proteins Autologous grafts Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Soft-tissue tumors in the head and neck region present a reconstructive challenge, particularly when bone invasion necessitates en bloc resection with a bony margin. Autologous bone grafting is a well-established approach for reconstructing bone defects. In particular, vascularized free composite tissue grafts remain the gold standard for reconstructing extensive bone loss in the head and neck region, with various methods proposed to improve function and aesthetics; [1–4] however, replicating the complex three-dimensional anatomy of the facial skeleton and achieving satisfactory outcomes remains difficult [5, 6]. Additionally, reconstructive surgery is often highly invasive, requires prolonged procedures for tumor excision and reconstruction, and is associated with a considerable risk of complications [7]. As an alternative, alloplastic materials, including hydroxyapatite, β-tricalcium phosphate, titanium, and ceramics, have been utilized for bone reconstruction. However, these materials have notable limitations, including susceptibility to infection, insufficient mechanical strength, poor osseointegration, and long-term degradation [8–10]. Therefore, novel strategies that fulfill both the structural and biological requirements of bone reconstruction are needed. One promising strategy is to reuse the resected autologous tumor bone following complete inactivation of malignant cells. Several tumor inactivation techniques, including cryo-treatment (liquid nitrogen) [11–14], high-temperature treatment [15, 16], autoclaving [17], and irradiation [18], have been explored to allow the re-implantation of autologous bone while preserving the anatomical shape. However, these methods remain suboptimal because they often compromise bone matrix proteins, reduce mechanical integrity, prolong intraoperative preparation, and may even allow residual viable tumor cells to persist [14, 16–18]. To overcome these limitations, we focused on high hydrostatic pressure (HHP) treatment as a novel method for tumor cell inactivation. HHP involves exposing tissues to pressures exceeding 200 MPa for more than 10 min within a specialized chamber, effectively inactivating cells without the use of heat or radiation. We have previously demonstrated that HHP successfully eliminates malignant cells in melanoma [19], squamous cell carcinoma [20], and osteosarcoma [21]. Furthermore, clinical studies have demonstrated the feasibility and safety of HHP-treated nevus tissues for dermal skin regeneration [22, 23]. Based on these findings, we hypothesized that HHP-treated autologous bone could serve as a biologically compatible graft material for oncological bone reconstruction. In this study, we evaluated the biological and mechanical properties of HHP-treated bone in comparison with those of bone processed using conventional inactivation methods. Specifically, we examined (1) the preservation of osteogenic protein content, (2) the in vivo bone-healing capacity in animal models, and (3) biomechanical changes in inactivated bone. Our goal was to assess the potential of HHP-treated autologous tumor bone as a functional and structurally sound material for clinical reconstruction after oncological resection. Results cellular experiment BMP-2 was inactivated in the cellular experiment at 80°C for 30 min, which was not significantly different from that of the untreated group. In contrast, heating at 65°C for 30 min was more active than the untreated group, but significantly less active than the high hydrostatic pressure groups (200 MPa, 500 MPa), liquid nitrogen-treated group, untreated group, and osteoblast induction medium (osteoblast differentiation medium). Activity was significantly lower in the high hydrostatic pressure (200 MPa and 500 MPa), liquid-nitrogen-treated, untreated, and osteoblast differentiation medium groups. There was no significant difference between the high hydrostatic pressure and liquid nitrogen-treated groups, the untreated BMP-2 group, and the osteoblast differentiation medium group. The results are shown in Figure 1. Animal experiment One rat in the Pasteur-treated group (time point = 4 weeks) died intraoperatively during the bone transplantation procedure. Although standard surgical protocols were followed, the preparation of Pasteur-treated bone fragments required a longer handling time compared to the other groups. As a result, the depth of isoflurane anesthesia may have deepened, potentially contributing to the animal’s inability to recover. Additionally, some technical delays occurred during the transplantation procedure, which may have further prolonged the anesthesia duration. Although the exact cause of death could not be definitively identified, anesthesia-related complications were considered the most plausible explanation. No similar events occurred in other groups. To avoid potential batch-related variations resulting from delayed sample replacement, no additional animals were used. Consequently, this group consisted of three rats at the corresponding time point (n = 3), and the statistical analyses were adjusted accordingly. The results of each evaluation item are presented below. Manual Examination The findings of the manual examination are presented in Table 1. No statistically significant differences were detected in the mobility of the grafted bone fragments at any of the observation points. Computed tomography The results of the CT evaluation are shown in Fig. 2. No significant differences were observed in the bone volume in the ROI among the four groups. Evaluation of tissue sections The results of the evaluation of the interosseous space fusion, as observed in the HE-stained sections, are depicted in Fig. 3. All groups exhibited temporal progression toward fusion; however, no statistically significant differences were observed between the groups. Evaluation of MTC-stained sections yielded the following results: At 16 weeks, the untreated group exhibited significantly greater membranous ossification than the high hydrostatic pressure group. At 12 weeks, both the untreated and liquid nitrogen-treated groups demonstrated significantly higher bone mass than the Pasteur-treated and high hydrostatic pressure groups. Additionally, at 16 weeks, the remnants of the cortical bone were significantly more pronounced in the liquid nitrogen-treated group than in the Pasteur-treated group (Fig. 4). Angiogenesis was significantly higher in the liquid nitrogen-treated group than in the untreated and Pasteur-treated groups at 12 weeks. The results are shown in Fig. 5. Biomechanical change of the inactivated bone Evaluation of bone surface alterations due to cell inactivation techniques The SEM results are shown in Fig. 6. Although this was a qualitative evaluation, degeneration of the bone surface was observed in the group treated at 80°C for 30 min and in the autoclaved group. No significant structural changes were observed in the other four groups. Evaluation of changes in bone strength due to cell inactivation techniques The results of the Vickers hardness tests are presented in Fig. 7. No significant differences in hardness were observed among the four groups. Discussion Our previous investigations established that HHP treatment effectively induces cell death across various tumor types, including malignant melanoma [19], squamous cell carcinoma [20], and osteosarcoma [21], as well as in nevus cells during clinical studies of congenital giant melanocytic nevi [22, 23]. Building on these findings, we aimed to explore the potential of HHP as a viable cell inactivation technique for preparing autologous bone grafts for oncologic reconstruction, with an emphasis on preserving the biological and structural properties of the graft. To evaluate the preservation of osteogenic activity, we used BMP-2, a key member of the TGF-β superfamily, which has been widely used in clinical settings for bone regeneration, spinal fusion, and long bone repair [24]. Given its well-established osteoinductive properties and therapeutic relevance, BMP-2 serves as a representative marker for assessing the impact of inactivation methods on biologically active proteins. We assessed the differentiation potential of preosteoblast-like cells following BMP-2 exposure by using various inactivation methods. Our in vitro results demonstrated that BMP-2 activity was well preserved following HHP or liquid nitrogen treatment, exhibiting ALP expression comparable to that of the untreated controls. In contrast, high temperature treatment significantly diminished BMP-2 activity and suppressed osteoblast differentiation, suggesting greater denaturation of critical osteogenic signals. In vivo analysis using a rat calvarial defect model further corroborated these findings. In our evaluation system, no significant differences in bone healing or bone volume were observed among the treatment groups based on CT imaging. Histological analysis at 16 weeks showed that membrane ossification was more pronounced in the untreated group than in the high hydrostatic pressure group; however, there were no significant differences in the overall bone volume among the groups at that time point. Notably, at 12 weeks, the liquid nitrogen-treated group exhibited greater bone volume than the high-temperature and high hydrostatic pressure groups, indicating reduced resorption of the grafted bone. These observations are consistent with previous reports, suggesting that liquid-nitrogen-treated bone can serve as a scaffold for osteoblasts [25]. Nevertheless, by 16 weeks, volumetric analysis of the regions of interest (ROI) did not reveal any clear advantage of liquid nitrogen treatment over HHP, with both groups demonstrating similar outcomes. Temporary increases in angiogenesis were noted in the liquid nitrogen-treated group, but no significant differences were detected at the final evaluation point. Scanning electron microscopy (SEM) revealed no discernible differences in bone surface morphology among the untreated, high hydrostatic pressure, high-temperature, and liquid nitrogen-treated groups. Because of the difficulty of quantitative evaluation in this context, qualitative assessments were also performed. In the autoclave-treated group, which represented an extreme inactivation condition, the three-dimensional structure of collagen was no longer clearly visible. While previous studies have reported that both the mechanical properties and collagen architecture of bone can be preserved under heat treatment below 180 °C [26], our findings suggest that autoclaving 8-mm calvarial bone under the present conditions did not maintain structural integrity. Therefore, this processing method may be suboptimal for preserving the mechanical and morphological properties of the bone tissue. Importantly, consistent with the SEM findings, no notable morphological differences were observed among the four main experimental groups (untreated, high hydrostatic pressure, high-temperature, and liquid nitrogen-treated) in the animal model. Similarly, Vickers hardness testing showed no significant reduction in the mechanical strength across the treatment groups, indicating that neither HHP nor the other processing methods compromised the material properties of the bone. Although compression and three-point bending tests are commonly used to evaluate bone strength [27], this study specifically examined cranial vaults, which are characterized by their small size and intricate shape. Therefore, we selected the Vickers hardness test, which facilitates precise measurement of hardness. Collectively, these findings suggest that HHP-treated bone retains osteogenic activity, structural integrity, and mechanical properties comparable to bone processed using established inactivation techniques. From a clinical perspective, HHP offers additional advantages such as reduced processing time and minimal protein denaturation, making it a promising candidate for future applications in bone reconstruction following tumor resection. This study had several limitations. The primary constraint was the limited sample size, with only four animals per group for the in vivo bone regeneration experiments. This precludes formal power analyses and warrants validation in larger cohorts. Additionally, the variability in defect size due to the use of a trephine drill may have introduced inconsistencies in the bone graft preparation. Future animal models with standardized defect dimensions and improved reproducibility should be considered to minimize technical bias and enhance experimental reliability. Material and Methods Cellular experiment： the preservation of osteogenic protein content To evaluate the residual activity of the bioactive substances after inactivation treatment, BMP-2 was selected as a representative osteoinductive factor and added to the culture medium of mouse preosteoblast-like cells after treatment with HHP, heat, or liquid nitrogen. Osteoblast differentiation was evaluated based on ALP expression. Cell culture MC3T3-E1 cells (99072810; KAC Co., Ltd., Kyoto, Japan) were cultured in α-minimum Essential Medium devoid of ascorbic acid and nucleosides (A10490-01; Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (10270-106; Thermo-Fisher Scientific) and 1% antibiotic-antimycotic (15240062; Thermo Fisher Scientific). The culture medium was refreshed every 2-3 days. At the time of acquisition, the number of cell passages was +4. Two passages were cryopreserved in 5% dimethyl sulfoxide (13048-64; NACALAI TESQUE Inc.) and stored in liquid nitrogen. For the experimental procedures, cells were thawed, cultured in 100-mm dishes until they reached subconfluence, and subsequently resuspended in TrypLE Express (12605-028; Thermo-Fisher Scientific) for further experimentation. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. HHP treatment and the other cell inactivation treatment for rhBMP2 MC3T3-E1 cells were seeded in 24-well plates at a density of 1 × 10^4 cells per well and cultured for three days. On the fourth day, 1 ml of the medium in each well was replaced with the various treatments. Four wells were prepared for each group on days four and seven. The remainder of this paper is organized as follows: 1) Untreated group: A previously described medium was used. 2) 200 MPa high hydrostatic pressure treatment group: rhBMP-2 (026-14811; FUJIFILM Wako Pure Chemical) was diluted to 1000 ng/ml. Approximately 2 ml of this solution was placed in 1-ml cryovials (1-5479-01; Thermo Fisher Scientific) to eliminate air bubbles. A plastic bag containing 20 ml of phosphate-buffered saline (PBS) (diluted from 10×D-PBS (-)048-29805; FUJIFILM Wako Pure Chemical Corporation) was prepared. The cryovials were sealed and subjected to a pressure of 200 MPa for 10 min using a high-pressure hydrostatic treatment device (SERVO PRESSUER 500; Sugino Machine Limited, Toyama, Japan). BMP-2 was diluted to a concentration of 100 ng/ml. 3) 500 MPa high hydrostatic pressure group: rhBMP-2 was dissolved in distilled water to 100 μg/ml and then diluted with medium to 1000 ng/ml. Approximately 2 ml was injected into 1 ml cryovials to avoid air bubbles. The samples were placed in sealed plastic bags containing 20 ml of PBS. The tubes were then treated at 500 MPa for 10 min using a high-pressure hydrostatic apparatus. BMP-2 was subsequently diluted to 100 ng/ml. 4) Heat treatment at 65°C group: rhBMP-2 was prepared as described previously. Cryotubes containing the solution were sealed in a plastic bag with 20 ml of PBS and heated at 65°C for 10 min in a water bath (1-6111-11; AS ONE Corp., Osaka, Japan). BMP-2 was then diluted to 100 ng/ml with medium. 5) Heat treatment at 80 °C: This process mirrored that of the 65°C group, except that the sealed cryotubes were immersed in a water bath at 80°C for 10 min. Following this, BMP-2 was diluted to 100 ng/ml using medium. 6) The group was treated with liquid nitrogen; rhBMP-2 solution was created by dissolving the protein in distilled water to reach 100 μg/ml, followed by dilution with medium to 1000 ng/ml. Two tubes, each containing approximately 1 ml of liquid nitrogen, were prepared. The samples with slightly loosened caps were immersed in liquid nitrogen for 20 min. After removal, the tubes were placed in 15-ml Corning tubes containing cryotubes and allowed to thaw on a sterile bench at 25°C for at least 25 min. The BMP-2 solution was then further diluted with the medium to 100 ng/ml. 7) Untreated BMP-2 group: rhBMP-2 was dissolved in distilled water to achieve a concentration of 100 μg/ml and subsequently diluted with medium to 1000 ng/ml. For the preparation of osteoblast induction medium, 50 μg/ml of L(+)-Ascorbic Acid (012-04802; FUJIFILM Wako Pure Chemical Corporation), and 10 mM (0.01 mol/L) of β-glycerophosphate disodium (048-34332; FUJIFILM Wako Pure Chemical Corporation) were used. Estimation of differentiation of osteoblasts with ALP staining on day 4 and day 7 Alkaline phosphatase (ALP) was stained using the TRAP/ALP Stain Kit (294-67001; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) at 4 and 7 days post-treatment, following the manufacturer's protocol. The protocol used a 10% Formalin Neutral Buffer Solution (068-01663; FUJIFILM Wako Pure Chemical Corporation) for fixation. Acetone (016-00346; FUJIFILM Wako Pure Chemical Corporation) and ethanol (99.5) (14713-95; FUJIFILM Wako Pure Chemical Corporation) were used for permeabilization. Stained areas were quantified using a microscope (BZ-X810; Keyence Corp., Osaka, Japan). All experiments were conducted in duplicate. The ALP-positive area was normalized to the mean value of the osteoblast induction medium group in each experiment, and results from the two experiments were pooled for analysis. （２） Animal experiment ： in vivo bone healing capacity using animal models All animal experiments were performed in accordance with the ARRIVE guidelines, and the Animal Experimentation Committee of the Graduate School of Medicine, Kyoto University approved all experimental protocols for this study (Medkyo 24583). All methods were performed in compliance with the relevant guidelines and regulations. Healthy male Wistar rats (11 weeks old and weighing in 350–400 g range, were obtained from SLC Japan, Inc. The rats were individually caged and kept under specific pathogen-free conditions with a 12-hour light/dark cycle, had free access to food and water, and were allowed to acclimate to the laboratory for 1 week prior to the experiment. 12-week-old rats underwent surgery. The rats were placed in a pre-anesthesia box and anesthetized by isoflurane inhalation (Viatris Pharmaceuticals, Inc., Tokyo, Japan). The pre-anesthesia conditions were as follows: isoflurane concentration was 3-4%, flow rate, 3 l/min, and recovery, 3.5 l/min. After the rats ceased to move, they were switched to maintenance anesthesia via mask inhalation. The conditions for maintenance anesthesia were an isoflurane concentration of 2-3%, a flow rate of 3 l/min, and a recovery rate of 3.5 l/min. The rat heads were shaved using an electric trimmer. The head was disinfected with isodine solution 10% (Shionogi, Osaka, Japan), and 0.5 cc of Xylocaine Injection 1% with epinephrine (Sand Pharma, Tokyo, Japan) was injected locally under the head skin for local anesthesia. A skin incision was made in the midline of the parietal region in the sagittal direction using scalpel No. 15 (KAI Industries, Inc., Tokyo, Japan). An incision was made in the periosteum and the cranial crown was clearly visible. The bone fragments were harvested from the center of the space using a trephine bar with an inner diameter of 8 mm. Depending on the method of cell-inactivation treatment of the bone fragments collected (treatment methods were randomly assigned), the rats were assigned to four groups, the details of which were as follows: (1) Group that received HHP treatment. The treatment was performed at 200 MPa for 10 min. (2) Group treated using the Pasteur method: High-temperature treatment at 65°C for 30 min. (3) Group treated with liquid nitrogen for 20 min. (4) No treatment (control). A detailed procedure is described in the Supplementary Fig. S1. Bone fragments that had undergone cell inactivation were reimplanted into cranial defects in rats. Following reimplantation, the periosteum and skin were closed using 4-0 nylon sutures (Bear Medic, Inc., Japan), and the animals were returned to their individual cages upon regaining consciousness. Each group consisted of 12 samples, and euthanasia was performed using gas at 4, 12, and 16 weeks after surgery (n = 4 per time point). Euthanasia was performed using CO2 gas inhalation without prior anesthesia in accordance with the institutional animal welfare guidelines. Death was confirmed by the cessation of heartbeat and respiration, and ocular decolorization. After euthanasia, the skin was incised to expose the cranial vault and the reimplanted bone. The cranial vault was then extracted as a single unit using a trephine bar, and the mobility of the reimplant was confirmed after manual testing. The extracted cranial vault was fixed in 10% formalin solution for 72 h and then transferred to 70% ethanol. The following techniques were used to assess outcomes. 1: Manual Examination The crania of the euthanized rats were incised to expose the calvaria while preserving the periosteum, ensuring that the grafted bone was clearly visible. Before sampling, the mobility of the grafted bone was assessed using forceps. This procedure is presented in Supplementary Video S2. 2: Computed Tomography A micro-focus X-ray system (TOSCANER-32300 μFD; Toshiba IT Control Systems Corporation, Tokyo, Japan) was employed to image the cranial vault volume. Additionally, three-dimensional image analysis was conducted using 3D Slicer (https://www.slicer.org), which is an open-source platform for medical image processing and informatics. Detailed information regarding the evaluation methodology is provided in Supplementary Movies S2-4. 3: Histological Evaluation The cranial vault was fixed in formalin, which was replaced with ethanol, decalcified with EDTA (7-14 days), and embedded in paraffin. Hematoxylin-eosin (HE) staining, Masson’s trichrome (MTC) staining, and CD31 staining were performed. After morphological confirmation by HE staining, the gap between the bone fragment and the skull was evaluated. The evaluation criteria were as follows: if both gaps were fused, it was considered completely fused; if only one gap was fused, it was considered partially fused; and if no fusion was observed, it was considered unfused. Next, membranous ossification and bone volume in the region of interest (ROI) were evaluated using MTC staining. Membranous ossification was identified as the blue areas in MTC staining, and bone volume was identified as the red area. For CD31 staining, the slides were deparaffinized, hydrated, treated for 20 min at 98°C in a pH 9 antigen retrieval solution (code: 415211), washed with distilled water, incubated at room temperature for 10 min in 3% hydrogen peroxide, and subsequently washed with distilled water and Tris-buffered saline with Tween 20 (TBST). The sections were then incubated for 60 min at room temperature with 3% bovine serum albumin, followed by incubation with Anti-CD31 antibody diluted 1:1000 (code: ab182981) at 4°C for 24 h. Thereafter, the sections were reacted with a polymer reagent (code: 714191) at room temperature for 30 min, washed with TBST, developed using DAB, washed with water, dehydrated, and mounted. The number and area of DAB-positive vascular endothelial cells in the ROI were evaluated for neovascularization. The details of the ROI settings are provided in the Supplementary Fig. S5. Observations were performed using the BZ-X800 Analyzer software (Keyence Corp., Osaka, Japan). (3) the biomechanical change of the inactivated bone Evaluation of bone surface alterations due to cell inactivation methods A dozen male Wistar rats aged 11 weeks were euthanized using gas, and circular bone samples (8 mm in diameter) were extracted from their cranial crowns. These samples were then randomly allocated into six groups, with two samples per group, and subjected to various cell inactivation methods: 1) untreated control, 2) liquid nitrogen exposure, 3) Pasteur method (65°C for 30 min), 4) high hydrostatic pressure, 5) elevated temperature (80°C for 30 min), and 6) autoclave sterilization (121°C for 20 min). The treated bone specimens were subsequently examined using scanning electron microscopy (SEM; JSM-7900F, JEOL Ltd. Tokyo, Japan) on both anterior and posterior surfaces. Evaluation of changes in bone strength due to cell inactivation methods A group of 20 male Wistar rats, aged 11 weeks, were euthanized using gas, and their cranial crowns were extracted as a single unit. The extracted crowns were randomly divided into four equal groups, each subjected to different cell inactivation techniques: (1) no treatment (control), (2) liquid nitrogen exposure, (3) high-temperature application, and (4) high-hydrostatic pressure treatment. To assess the bone strength, four random points on each sample were evaluated using the Vickers hardness test. The equipment used for the test was HMV2000AD (SHIMADZU, Kyoto, Japan). Statistical analysis All data are presented as mean ± standard deviation (SD). Statistical significance was set at P < 0.05. The analysis was conducted using JMP software (JMP Statistical Discovery LLC., Cary, NC, USA). The Tukey-Kramer test and Fisher's exact test were used. Declarations Funding This research was supported by JSPS KAKENHI (Grant Number 24K02606) and AMED (Grant Number JP24ym0126143). Acknowledgements We extend our sincere appreciation to Tsuboi Mizuki and Tanida Yukihiro of the Kyoto Prefectural Technology Center for Small and Medium Enterprises for their invaluable technical guidance in the execution of CT imaging and Vickers hardness testing. We are also grateful to Yoshitaka Tanaka for his dedicated assistance in the experimental procedures. Furthermore, this manuscript has been meticulously proofread by a native English speaker through Editage. Competing interest The authors declare no competing interests. Author contributions In the development of the study, E.S. and R.A. were responsible for the design, primary conceptualization, and the formulation of the proof's framework. Data collection was conducted by E.S. and R.A. The interpretation of the results and manuscript preparation were supported by H.Y., M.S., T.Y., S.D., and N.M. The manuscript was authored by E.S. and R.A. All authors engaged in discussions regarding the results and provided constructive feedback on the manuscript. Data availability statement The datasets generated and/or analyzed during the current study are available in the Figshare repository at https://doi.org/10.6084/m9.figshare.29964341.v1 References Hurley, C.M. et al. Current trends in craniofacial reconstruction. Surgeon 21 , e118-e125 (2023). Tsuge, I. et al. Central Mandibular Reconstruction by Semiopen Wedge Osteotomy Double-barrel Fibula Flap for a Slim Aesthetic Appearance. Plast Reconstr Surg Glob Open 10 , e4716 (2022). Tsuge, I. et al. Double-flap Mandibular Reconstruction around the Condylar Head Using Fibula and Anterolateral Thigh Flaps. Plast Reconstr Surg Glob Open 10 , e4607 (2022). Tsuge, I., Yamanaka, H., Katsube, M., Sakamoto, M. & Morimoto, N. Simultaneous Reconstruction of the Bilateral Maxillae and Nasal Hard Structure Using a Vascularized and Nonvascularized Fibula. Plast Reconstr Surg Glob Open 12 , e5936 (2024). Futran, N.D. & Mendez, E. Developments in reconstruction of midface and maxilla. Lancet Oncol 7 , 249-258 (2006). Chang, E.I. & Hanasono, M.M. State-of-the-art reconstruction of midface and facial deformities. J Surg Oncol 113 , 962-970 (2016). Lese, I., Biedermann, R., Constantinescu, M., Grobbelaar, A.O. & Olariu, R. Predicting risk factors that lead to free flap failure and vascular compromise: A single unit experience with 565 free tissue transfers. J Plast Reconstr Aesthet Surg 74 , 512-522 (2021). Koons, G.L., Diba, M. & Mikos, A.G. Materials design for bone-tissue engineering. Nature Reviews Materials 5 , 584-603 (2020). Zhang, J., Feng, Y., Zhou, X., Shi, Y. & Wang, L. Research status of artificial bone materials. International Journal of Polymeric Materials and Polymeric Biomaterials 70 , 37-53 (2021). Feng, P. et al. Structural and Functional Adaptive Artificial Bone: Materials, Fabrications, and Properties. Advanced Functional Materials 33 , 2214726 (2023). Yamamoto, N., Tsuchiya, H. & Tomita, K. Effects of liquid nitrogen treatment on the proliferation of osteosarcoma and the biomechanical properties of normal bone. J Orthop Sci 8 , 374-380 (2003). Tsuchiya, H. et al. Reconstruction using an autograft containing tumour treated by liquid nitrogen. J Bone Joint Surg Br 87 , 218-225 (2005). Tsuchiya, H. et al. Pedicle frozen autograft reconstruction in malignant bone tumors. J Orthop Sci 15 , 340-349 (2010). Errani, C., Tsukamoto, S., Almunhaisen, N., Mavrogenis, A. & Donati, D. Intercalary reconstruction following resection of diaphyseal bone tumors: A systematic review. J Clin Orthop Trauma 19 , 1-10 (2021). Ehara, S., Nishida, J., Shiraishi, H. & Tamakawa, Y. Pasteurized intercalary autogenous bone graft: radiographic and scintigraphic features. Skeletal Radiol 29 , 335-339 (2000). Sugiura, H. et al. Evaluation of long-term outcomes of pasteurized autografts in limb salvage surgeries for bone and soft tissue sarcomas. Arch Orthop Trauma Surg 132 , 1685-1695 (2012). Umer, M. et al. Autoclaved tumor bone for skeletal reconstruction in paediatric patients: a low cost alternative in developing countries. Biomed Res Int 2013 , 698461 (2013). Arpornchayanon, O., Leerapun, T., Pruksakorn, D. & Panichkul, P. Result of extracorporeal irradiation and re-implantation for malignant bone tumors: a review of 30 patients. Asia Pac J Clin Oncol 9 , 214-219 (2013). Jinno, C. et al. Extracorporeal high-pressure therapy (EHPT) for malignant melanoma consisting of simultaneous tumor eradication and autologous dermal substitute preparation. Regen Ther 15 , 187-194 (2020). Mitsui, T. et al. High Hydrostatic Pressure Therapy Annihilates Squamous Cell Carcinoma in a Murine Model. Biomed Res Int 2020 , 3074742 (2020). Li, Y. et al. Development of a novel regenerative therapy for malignant bone tumors using an autograft containing tumor inactivated by high hydrostatic pressurization (HHP). Regen Ther 22 , 224-231 (2023). Morimoto, N. et al. A Novel Treatment for Giant Congenital Melanocytic Nevi Combining Inactivated Autologous Nevus Tissue by High Hydrostatic Pressure and a Cultured Epidermal Autograft: First-in-Human, Open, Prospective Clinical Trial. Plastic and Reconstructive Surgery 148 , 71e-76e (2021). Yamanaka, H. et al. A high-hydrostatic pressure device for nevus tissue inactivation and dermal regeneration for reconstructing skin defects after giant congenital melanocytic nevus excision: a clinical trial. Regen Ther 24 , 167-173 (2023). Halloran, D., Durbano, H.W. & Nohe, A. Bone Morphogenetic Protein-2 in Development and Bone Homeostasis. J Dev Biol 8 (2020). Xu, G. et al. The process of bone regeneration from devitalization to revitalization after pedicle freezing with immunohistochemical and histological examination in rabbits. Cryobiology 92 , 130-137 (2020). Uniyal, P., Sihota, P. & Kumar, N. Effect of organic matrix alteration on strain rate dependent mechanical behaviour of cortical bone. Journal of the Mechanical Behavior of Biomedical Materials 125 , 104910 (2022). Singh, D. et al. Experimental assessment of biomechanical properties in human male elbow bone subjected to bending and compression loads. J Appl Biomater Funct Mater 17 , 2280800018793816 (2019). Tables Table 1. Flotation results from manual testing 4 week 12 week 16 week C 0% (0/4) 25% (1/4) 25% (1/4) LN 0% (0/4) 0% (0/4) 0%(0/4) P 0% (0/3) 25% (1/4) 50% (2/4) HHP 0% (0/4) 25% (1/4) 25% (1/4) The results of the manual test show the number of samples with flotation at each observation point. C, untreated (control) group; LN, liquid nitrogen group; P, the pasteurization group; HHP, high hydrostatic pressure treatment group. Additional Declarations No competing interests reported. Supplementary Files supplementaryinformationS16.pdf SupplementaryMovie.S2float.mp4 SupplementaryMovie.S2nofloat.mp4 SupplementaryMovie.S3.mp4 SupplementaryMovie.S4.mp4 SupplementaryMovie.S5.mp4 Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 25 Sep, 2025 Reviewers agreed at journal 24 Sep, 2025 Reviewers agreed at journal 23 Sep, 2025 Reviewers invited by journal 23 Sep, 2025 Editor assigned by journal 16 Sep, 2025 Editor invited by journal 02 Sep, 2025 Submission checks completed at journal 01 Sep, 2025 First submitted to journal 01 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7430280","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":522472865,"identity":"986418e8-b880-432a-a475-4142ce2792b1","order_by":0,"name":"Eiichi Sawaragi","email":"data:image/png;base64,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","orcid":"","institution":"Kyoto University","correspondingAuthor":true,"prefix":"","firstName":"Eiichi","middleName":"","lastName":"Sawaragi","suffix":""},{"id":522472867,"identity":"6eaad28e-e3f6-40bd-b42b-c6bd081bbd3a","order_by":1,"name":"Rie Akita","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Rie","middleName":"","lastName":"Akita","suffix":""},{"id":522472870,"identity":"3580c542-55b1-409b-8625-863bc6ec1a9e","order_by":2,"name":"Hiroki Yamanaka","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Hiroki","middleName":"","lastName":"Yamanaka","suffix":""},{"id":522472874,"identity":"1fd8482f-75f8-4a02-a11f-5bbfcd0a7000","order_by":3,"name":"Michiharu Sakamoto","email":"","orcid":"","institution":"Kagawa University","correspondingAuthor":false,"prefix":"","firstName":"Michiharu","middleName":"","lastName":"Sakamoto","suffix":""},{"id":522472876,"identity":"edc61c1b-2519-4142-930a-88eb5607b961","order_by":4,"name":"Shinji Miwa","email":"","orcid":"","institution":"Kanazawa University","correspondingAuthor":false,"prefix":"","firstName":"Shinji","middleName":"","lastName":"Miwa","suffix":""},{"id":522472877,"identity":"885b7818-4312-4889-81aa-a32510e429ff","order_by":5,"name":"Satoshi Kato","email":"","orcid":"","institution":"Kanazawa University","correspondingAuthor":false,"prefix":"","firstName":"Satoshi","middleName":"","lastName":"Kato","suffix":""},{"id":522472879,"identity":"438c58de-3ac6-4e72-a177-8b713d2a35f1","order_by":6,"name":"Yohei Yamada","email":"","orcid":"","institution":"Kanazawa University","correspondingAuthor":false,"prefix":"","firstName":"Yohei","middleName":"","lastName":"Yamada","suffix":""},{"id":522472880,"identity":"11c5cd60-073f-4359-a4dd-2b222af3ed83","order_by":7,"name":"Satoshi Nagatani","email":"","orcid":"","institution":"Kanazawa University","correspondingAuthor":false,"prefix":"","firstName":"Satoshi","middleName":"","lastName":"Nagatani","suffix":""},{"id":522472882,"identity":"f9bb254a-1765-4f8b-bddf-98e9beaf2ca5","order_by":8,"name":"Tetsuji Yamaoka","email":"","orcid":"","institution":"Komatsu University","correspondingAuthor":false,"prefix":"","firstName":"Tetsuji","middleName":"","lastName":"Yamaoka","suffix":""},{"id":522472884,"identity":"860364a0-00b5-4d6d-aa65-af2f37284dcb","order_by":9,"name":"Satoru Demura","email":"","orcid":"","institution":"Kanazawa University","correspondingAuthor":false,"prefix":"","firstName":"Satoru","middleName":"","lastName":"Demura","suffix":""},{"id":522472886,"identity":"52590170-e602-4a88-b3d5-16f3bf64d961","order_by":10,"name":"Naoki Morimoto","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Naoki","middleName":"","lastName":"Morimoto","suffix":""}],"badges":[],"createdAt":"2025-08-22 03:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7430280/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7430280/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":92894663,"identity":"c0f378d7-d1c7-4702-b778-d1d54b06ea19","added_by":"auto","created_at":"2025-10-06 18:53:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2226336,"visible":true,"origin":"","legend":"\u003cp\u003eALP staining following BMP-2 inactivation.\u003c/p\u003e\n\u003cp\u003eRepresentative images of ALP staining and quantification of ALP positive regions are shown. The HHP, LN, untreated BMP-2, and osteogenic medium groups exhibited significantly higher activity than the heat-treated groups (*P, #P＜0.05).\u003c/p\u003e","description":"","filename":"Figure113.png","url":"https://assets-eu.researchsquare.com/files/rs-7430280/v1/1a0445c5432a04a0891e2df5.png"},{"id":92894765,"identity":"24f73839-45d2-4723-9764-932c632c40b9","added_by":"auto","created_at":"2025-10-06 19:01:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3627253,"visible":true,"origin":"","legend":"\u003cp\u003eCT assessment of the bone volume.\u003c/p\u003e\n\u003cp\u003eCT-based ROI analysis revealed no significant differences in bone volume among the four groups at 4, 12, or 16 weeks.\u003c/p\u003e","description":"","filename":"Figure212.png","url":"https://assets-eu.researchsquare.com/files/rs-7430280/v1/7a1a52cf89686664ecf0d67d.png"},{"id":92894664,"identity":"44de7603-cc46-4208-a900-d3827ea1cb88","added_by":"auto","created_at":"2025-10-06 18:53:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1940365,"visible":true,"origin":"","legend":"\u003cp\u003eBone fusion status determined by HE staining.\u003c/p\u003e\n\u003cp\u003eHE-stained sections and bone fusion status classification indicated no significant differences in fusion among the four groups. Scale = 1mm.\u003c/p\u003e\n\u003cp\u003eC, untreated (control) group; LN, liquid nitrogen group; P, pasteurization group; HHP, high hydrostatic pressure treatment group.\u003c/p\u003e","description":"","filename":"Figure310.png","url":"https://assets-eu.researchsquare.com/files/rs-7430280/v1/6213d86334732fb11a440fe6.png"},{"id":92894668,"identity":"d7a343d6-38ed-4ce3-9cf8-50cd84a09d7c","added_by":"auto","created_at":"2025-10-06 18:53:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3579186,"visible":true,"origin":"","legend":"\u003cp\u003eOssification and bone volume were assessed by MTC staining.\u003c/p\u003e\n\u003cp\u003eAt 16 weeks, ossification was more pronounced in the control group than in the HHP group. At 12 weeks, the bone volume was greater in the control and LN groups than in the P and HHP groups (*P \u0026lt; 0.05). Scale = 500μm.\u003c/p\u003e\n\u003cp\u003eC, untreated (control) group; LN, nitrogen group, P, pasteurization group; HHP, high hydrostatic pressure treatment group.\u003c/p\u003e","description":"","filename":"Figure48.png","url":"https://assets-eu.researchsquare.com/files/rs-7430280/v1/c26fa90013b0e16814dd3bdb.png"},{"id":92894766,"identity":"7d1228cb-cc73-46f3-b943-5559333de31f","added_by":"auto","created_at":"2025-10-06 19:01:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2893901,"visible":true,"origin":"","legend":"\u003cp\u003eCD31 staining for angiogenesis.\u003c/p\u003e\n\u003cp\u003eRepresentative CD31-stained sections and quantification of the vascular area demonstrated significantly higher angiogenesis in the LN group than in the control and P groups at 12 weeks. No significant differences were observed at 16 weeks (P \u0026lt; 0.05). Scale = 500μm.\u003c/p\u003e\n\u003cp\u003eC, untreated (control) group; LN, liquid nitrogen group; P, pasteurization group; HHP, high hydrostatic pressure treatment group.\u003c/p\u003e","description":"","filename":"Figure58.png","url":"https://assets-eu.researchsquare.com/files/rs-7430280/v1/285f8279e89b748937dc81a0.png"},{"id":92895287,"identity":"f5071a4f-4da9-492c-a415-59843ad96eac","added_by":"auto","created_at":"2025-10-06 19:09:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":8399926,"visible":true,"origin":"","legend":"\u003cp\u003eSEM analysis of bone surface morphology.\u003c/p\u003e\n\u003cp\u003eSEM images revealed surface degeneration in the 80 °C and autoclave groups, whereas no morphological changes were observed in the untreated, LN, P, or HHP groups.\u003c/p\u003e","description":"","filename":"Figure68.png","url":"https://assets-eu.researchsquare.com/files/rs-7430280/v1/ff6ece245ec321aa63a2fc5f.png"},{"id":92894666,"identity":"7911467e-bd1f-4731-97a0-3b45d7dc3792","added_by":"auto","created_at":"2025-10-06 18:53:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":33339,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVickers hardness of bone post-treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVickers hardness tests indicated no significant differences in hardness among the four groups. C, untreated (control) group; LN, liquid nitrogen group; P, pasteurization group; and HHP, high hydrostatic pressure treatment group.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure75.png","url":"https://assets-eu.researchsquare.com/files/rs-7430280/v1/5ea5aa7b7d7dfabf4e4b5f80.png"},{"id":92895517,"identity":"e0eb1cc7-8f43-4bd4-be44-3d0c2f3fdb19","added_by":"auto","created_at":"2025-10-06 19:17:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":26177257,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7430280/v1/c9f4ac73-e072-4138-8d0f-85fc90fe8273.pdf"},{"id":92894667,"identity":"72bb0bf2-1298-407f-a976-f4eb0f85027d","added_by":"auto","created_at":"2025-10-06 18:53:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1410924,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryinformationS16.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7430280/v1/3836ccbde38a76c0e611e510.pdf"},{"id":92894672,"identity":"13e9ccf5-b556-4199-85e1-1493e19f474a","added_by":"auto","created_at":"2025-10-06 18:53:36","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7822424,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMovie.S2float.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7430280/v1/558d6854dd17b11a56a9a95c.mp4"},{"id":92894669,"identity":"0c82e6d1-e4f2-4912-9303-05f253a5ccd6","added_by":"auto","created_at":"2025-10-06 18:53:36","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4323587,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMovie.S2nofloat.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7430280/v1/8723aa363cdd8f6e6272b103.mp4"},{"id":92894675,"identity":"090e6103-a703-4973-9bd1-6105fc914649","added_by":"auto","created_at":"2025-10-06 18:53:40","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":110706155,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMovie.S3.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7430280/v1/5bc5b170ae7b82dba32558d6.mp4"},{"id":92894676,"identity":"b4b8ffc1-bb93-46be-9449-4fddb6c27e01","added_by":"auto","created_at":"2025-10-06 18:53:46","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":264281159,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMovie.S4.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7430280/v1/d7e38dd74f7e4a08a82135bf.mp4"},{"id":92894674,"identity":"8a03f8a0-2565-47b4-a1c1-aa0e41c9e460","added_by":"auto","created_at":"2025-10-06 18:53:40","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":100552930,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMovie.S5.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7430280/v1/eee69a50dea9594e944080eb.mp4"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploration of Inactivated Bone Using High Hydrostatic Pressurization for Future Oncologic Application","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSoft-tissue tumors in the head and neck region present a reconstructive challenge, particularly when bone invasion necessitates en bloc resection with a bony margin. Autologous bone grafting is a well-established approach for reconstructing bone defects. In particular, vascularized free composite tissue grafts remain the gold standard for reconstructing extensive bone loss in the head and neck region, with various methods proposed to improve function and aesthetics; [1\u0026ndash;4] however, replicating the complex three-dimensional anatomy of the facial skeleton and achieving satisfactory outcomes remains difficult [5, 6]. Additionally, reconstructive surgery is often highly invasive, requires prolonged procedures for tumor excision and reconstruction, and is associated with a considerable risk of complications [7].\u003c/p\u003e\n\u003cp\u003eAs an alternative, alloplastic materials, including hydroxyapatite, \u0026beta;-tricalcium phosphate, titanium, and ceramics, have been utilized for bone reconstruction. However, these materials have notable limitations, including susceptibility to infection, insufficient mechanical strength, poor osseointegration, and long-term degradation [8\u0026ndash;10]. Therefore, novel strategies that fulfill both the structural and biological requirements of bone reconstruction are needed.\u003c/p\u003e\n\u003cp\u003eOne promising strategy is to reuse the resected autologous tumor bone following complete inactivation of malignant cells. Several tumor inactivation techniques, including cryo-treatment (liquid nitrogen) [11\u0026ndash;14], high-temperature treatment [15, 16], autoclaving [17], and irradiation [18], have been explored to allow the re-implantation of autologous bone while preserving the anatomical shape. However, these methods remain suboptimal because they often compromise bone matrix proteins, reduce mechanical integrity, prolong intraoperative preparation, and may even allow residual viable tumor cells to persist [14, 16\u0026ndash;18].\u003c/p\u003e\n\u003cp\u003eTo overcome these limitations, we focused on high hydrostatic pressure (HHP) treatment as a novel method for tumor cell inactivation. HHP involves exposing tissues to pressures exceeding 200 MPa for more than 10 min within a specialized chamber, effectively inactivating cells without the use of heat or radiation. We have previously demonstrated that HHP successfully eliminates malignant cells in melanoma [19], squamous cell carcinoma [20], and osteosarcoma [21]. Furthermore, clinical studies have demonstrated the feasibility and safety of HHP-treated nevus tissues for dermal skin regeneration [22, 23].\u003c/p\u003e\n\u003cp\u003eBased on these findings, we hypothesized that HHP-treated autologous bone could serve as a biologically compatible graft material for oncological bone reconstruction. In this study, we evaluated the biological and mechanical properties of HHP-treated bone in comparison with those of bone processed using conventional inactivation methods. Specifically, we examined (1) the preservation of osteogenic protein content, (2) the in vivo bone-healing capacity in animal models, and (3) biomechanical changes in inactivated bone. Our goal was to assess the potential of HHP-treated autologous tumor bone as a functional and structurally sound material for clinical reconstruction after oncological resection.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003ecellular experiment\u003c/h2\u003e\n\u003cp\u003eBMP-2 was inactivated in the cellular experiment at 80\u0026deg;C for 30 min, which was not significantly different from that of the untreated group. In contrast, heating at 65\u0026deg;C for 30 min was more active than the untreated group, but significantly less active than the high hydrostatic pressure groups (200 MPa, 500 MPa), liquid nitrogen-treated group, untreated group, and osteoblast induction medium (osteoblast differentiation medium). Activity was significantly lower in the high hydrostatic pressure (200 MPa and 500 MPa), liquid-nitrogen-treated, untreated, and osteoblast differentiation medium groups. There was no significant difference between the high hydrostatic pressure and liquid nitrogen-treated groups, the untreated BMP-2 group, and the osteoblast differentiation medium group. The results are shown in Figure 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne rat in the Pasteur-treated group (time point = 4 weeks) died intraoperatively during the bone transplantation procedure. Although standard surgical protocols were followed, the preparation of Pasteur-treated bone fragments required a longer handling time compared to the other groups. As a result, the depth of isoflurane anesthesia may have deepened, potentially contributing to the animal\u0026rsquo;s inability to recover. Additionally, some technical delays occurred during the transplantation procedure, which may have further prolonged the anesthesia duration. Although the exact cause of death could not be definitively identified, anesthesia-related complications were considered the most plausible explanation. No similar events occurred in other groups.\u003cbr\u003e\u0026nbsp;To avoid potential batch-related variations resulting from delayed sample replacement, no additional animals were used. Consequently, this group consisted of three rats at the corresponding time point (n = 3), and the statistical analyses were adjusted accordingly. The results of each evaluation item are presented below.\u003c/p\u003e\n\u003ch2\u003eManual Examination\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eThe findings of the manual examination\u003c/em\u003e\u003cem\u003e\u0026nbsp;are\u0026nbsp;\u003c/em\u003e\u003cem\u003epresented in Table 1. No statistically significant differences were detected in the mobility of the grafted bone fragments at any of the observation points.\u003c/em\u003e\u003c/p\u003e\n\u003ch2\u003eComputed tomography\u003c/h2\u003e\n\u003cp\u003eThe results of the CT evaluation are shown in Fig. 2. No significant differences were observed in the bone volume in the ROI among the four groups.\u003c/p\u003e\n\u003ch2\u003eEvaluation of tissue sections\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe results of the evaluation of the interosseous space fusion, as observed in the HE-stained sections, are depicted in Fig. 3. All groups exhibited temporal progression toward fusion; however, no statistically significant differences were observed between the groups. Evaluation of MTC-stained sections yielded the following results: At 16 weeks, the untreated group exhibited significantly greater membranous ossification than the high hydrostatic pressure group. At 12 weeks, both the untreated and liquid nitrogen-treated groups demonstrated significantly higher bone mass than the Pasteur-treated and high hydrostatic pressure groups. Additionally, at 16 weeks, the remnants of the cortical bone were significantly more pronounced in the liquid nitrogen-treated group than in the Pasteur-treated group (Fig. 4). Angiogenesis was significantly higher in the liquid nitrogen-treated group than in the untreated and Pasteur-treated groups at 12 weeks. The results are shown in Fig. 5.\u003c/p\u003e\n\u003ch2\u003eBiomechanical change of the inactivated bone\u003c/h2\u003e\n\u003ch2\u003eEvaluation of bone surface alterations due to cell inactivation techniques\u003c/h2\u003e\n\u003cp\u003eThe SEM results are shown in Fig. 6. Although this was a qualitative evaluation, degeneration of the bone surface was observed in the group treated at 80\u0026deg;C for 30 min and in the autoclaved group. No significant structural changes were observed in the other four groups.\u003c/p\u003e\n\u003ch2\u003eEvaluation of changes in bone strength due to cell inactivation techniques\u003c/h2\u003e\n\u003cp\u003eThe results of the Vickers hardness tests are presented in Fig. 7. No significant differences in hardness were observed among the four groups.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur previous investigations established that HHP treatment effectively induces cell death across various tumor types, including malignant melanoma [19], squamous cell carcinoma [20], and osteosarcoma [21], as well as in nevus cells during clinical studies of congenital giant melanocytic nevi [22, 23].\u003c/p\u003e\n\u003cp\u003eBuilding on these findings, we aimed to explore the potential of HHP as a viable cell inactivation technique for preparing autologous bone grafts for oncologic reconstruction, with an emphasis on preserving the biological and structural properties of the graft.\u003c/p\u003e\n\u003cp\u003eTo evaluate the preservation of osteogenic activity, we used BMP-2, a key member of the TGF-\u0026beta; superfamily, which has been widely used in clinical settings for bone regeneration, spinal fusion, and long bone repair [24]. Given its well-established osteoinductive properties and therapeutic relevance, BMP-2 serves as a representative marker for assessing the impact of inactivation methods on biologically active proteins. We assessed the differentiation potential of preosteoblast-like cells following BMP-2 exposure by using various inactivation methods. Our in vitro results demonstrated that BMP-2 activity was well preserved following HHP or liquid nitrogen treatment, exhibiting ALP expression comparable to that of the untreated controls. In contrast, high temperature treatment significantly diminished BMP-2 activity and suppressed osteoblast differentiation, suggesting greater denaturation of critical osteogenic signals.\u003c/p\u003e\n\u003cp\u003eIn vivo analysis using a rat calvarial defect model further corroborated these findings. In our evaluation system, no significant differences in bone healing or bone volume were observed among the treatment groups based on CT imaging. Histological analysis at 16 weeks showed that membrane ossification was more pronounced in the untreated group than in the high hydrostatic pressure group; however, there were no significant differences in the overall bone volume among the groups at that time point. Notably, at 12 weeks, the liquid nitrogen-treated group exhibited greater bone volume than the high-temperature and high hydrostatic pressure groups, indicating reduced resorption of the grafted bone. These observations are consistent with previous reports, suggesting that liquid-nitrogen-treated bone can serve as a scaffold for osteoblasts [25]. Nevertheless, by 16 weeks, volumetric analysis of the regions of interest (ROI) did not reveal any clear advantage of liquid nitrogen treatment over HHP, with both groups demonstrating similar outcomes. Temporary increases in angiogenesis were noted in the liquid nitrogen-treated group, but no significant differences were detected at the final evaluation point.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eScanning electron microscopy (SEM) revealed no discernible differences in bone surface morphology among the untreated, high hydrostatic pressure, high-temperature, and liquid nitrogen-treated groups. Because of the difficulty of quantitative evaluation in this context, qualitative assessments were also performed. In the autoclave-treated group, which represented an extreme inactivation condition, the three-dimensional structure of collagen was no longer clearly visible. While previous studies have reported that both the mechanical properties and collagen architecture of bone can be preserved under heat treatment below 180 \u0026deg;C [26], our findings suggest that autoclaving 8-mm calvarial bone under the present conditions did not maintain structural integrity. Therefore, this processing method may be suboptimal for preserving the mechanical and morphological properties of the bone tissue. Importantly, consistent with the SEM findings, no notable morphological differences were observed among the four main experimental groups (untreated, high hydrostatic pressure, high-temperature, and liquid nitrogen-treated) in the animal model. Similarly, Vickers hardness testing showed no significant reduction in the mechanical strength across the treatment groups, indicating that neither HHP nor the other processing methods compromised the material properties of the bone. Although compression and three-point bending tests are commonly used to evaluate bone strength\u0026nbsp;[27], this study specifically examined cranial vaults, which are characterized by their small size and intricate shape. Therefore, we selected the Vickers hardness test, which facilitates precise measurement of hardness.\u003c/p\u003e\n\u003cp\u003eCollectively, these findings suggest that HHP-treated bone retains osteogenic activity, structural integrity, and mechanical properties comparable to bone processed using established inactivation techniques. From a clinical perspective, HHP offers additional advantages such as reduced processing time and minimal protein denaturation, making it a promising candidate for future applications in bone reconstruction following tumor resection.\u003c/p\u003e\n\u003cp\u003eThis study had several limitations. The primary constraint was the limited sample size, with only four animals per group for the in vivo bone regeneration experiments. This precludes formal power analyses and warrants validation in larger cohorts. Additionally, the variability in defect size due to the use of a trephine drill may have introduced inconsistencies in the bone graft preparation. Future animal models with standardized defect dimensions and improved reproducibility should be considered to minimize technical bias and enhance experimental reliability.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003ch2\u003eCellular experiment： the preservation of osteogenic protein content\u003c/h2\u003e\n\u003cp\u003eTo evaluate the residual activity of the bioactive substances after inactivation treatment, BMP-2 was selected as a representative osteoinductive factor and added to the culture medium of mouse preosteoblast-like cells after treatment with HHP, heat, or liquid nitrogen. Osteoblast differentiation was evaluated based on ALP expression.\u003c/p\u003e\n\u003ch2\u003eCell culture\u003c/h2\u003e\n\u003cp\u003eMC3T3-E1 cells (99072810; KAC Co., Ltd., Kyoto, Japan) were cultured in \u0026alpha;-minimum Essential Medium devoid of ascorbic acid and nucleosides (A10490-01; Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (10270-106; Thermo-Fisher Scientific) and 1% antibiotic-antimycotic (15240062; Thermo Fisher Scientific). The culture medium was refreshed every 2-3 days. At the time of acquisition, the number of cell passages was +4. Two passages were cryopreserved in 5% dimethyl sulfoxide (13048-64; NACALAI TESQUE Inc.) and stored in liquid nitrogen. For the experimental procedures, cells were thawed, cultured in 100-mm dishes until they reached subconfluence, and subsequently resuspended in TrypLE Express (12605-028; Thermo-Fisher Scientific) for further experimentation. Cells were maintained at 37\u0026deg;C in a humidified atmosphere containing 5% CO2.\u003c/p\u003e\n\u003ch2\u003eHHP treatment and the other cell inactivation treatment for rhBMP2\u003c/h2\u003e\n\u003cp\u003eMC3T3-E1 cells were seeded in 24-well plates at a density of 1\u0026nbsp;\u0026times;\u0026nbsp;10^4 cells per well and cultured for three days. On the fourth day, 1 ml of the medium in each well was replaced with the various treatments. Four wells were prepared for each group on days four and seven. The remainder of this paper is organized as follows:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e1) Untreated group: A previously described medium was used.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2) 200 MPa high hydrostatic pressure treatment group: rhBMP-2 (026-14811; FUJIFILM Wako Pure Chemical) was diluted to 1000 ng/ml. Approximately 2 ml of this solution was placed in 1-ml cryovials (1-5479-01; Thermo Fisher Scientific) to eliminate air bubbles. A plastic bag containing 20 ml of phosphate-buffered saline (PBS) (diluted from 10\u0026times;D-PBS (-)048-29805; FUJIFILM Wako Pure Chemical Corporation) was prepared. The cryovials were sealed and subjected to a pressure of 200 MPa for 10 min using a high-pressure hydrostatic treatment device (SERVO PRESSUER 500; Sugino Machine Limited, Toyama, Japan). BMP-2 was diluted to a concentration of 100 ng/ml.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3) 500 MPa high hydrostatic pressure group: rhBMP-2 was dissolved in distilled water to 100 \u0026mu;g/ml and then diluted with medium to 1000 ng/ml. Approximately 2 ml was injected into 1 ml cryovials to avoid air bubbles. The samples were placed in sealed plastic bags containing 20 ml of PBS. The tubes were then treated at 500 MPa for 10 min using a high-pressure hydrostatic apparatus. BMP-2 was subsequently diluted to 100 ng/ml.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4) Heat treatment at 65\u0026deg;C group: rhBMP-2 was prepared as described previously. Cryotubes containing the solution were sealed in a plastic bag with 20 ml of PBS and heated at 65\u0026deg;C for 10 min in a water bath (1-6111-11; AS ONE Corp., Osaka, Japan). BMP-2 was then diluted to 100 ng/ml with medium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e5) Heat treatment at 80 \u0026deg;C: This process mirrored that of the 65\u0026deg;C group, except that the sealed cryotubes were immersed in a water bath at 80\u0026deg;C for 10 min. Following this, BMP-2 was diluted to 100 ng/ml using medium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e6) The group was treated with liquid nitrogen; rhBMP-2 solution was created by dissolving the protein in distilled water to reach 100 \u0026mu;g/ml, followed by dilution with medium to 1000 ng/ml. Two tubes, each containing approximately 1 ml of liquid nitrogen, were prepared. The samples with slightly loosened caps were immersed in liquid nitrogen for 20 min. After removal, the tubes were placed in 15-ml Corning tubes containing cryotubes and allowed to thaw on a sterile bench at 25\u0026deg;C for at least 25 min. The BMP-2 solution was then further diluted with the medium to 100 ng/ml.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e7) Untreated BMP-2 group: rhBMP-2 was dissolved in distilled water to achieve a concentration of 100 \u0026mu;g/ml and subsequently diluted with medium to 1000 ng/ml.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the preparation of osteoblast induction medium, 50 \u0026mu;g/ml of L(+)-Ascorbic Acid (012-04802; FUJIFILM Wako Pure Chemical Corporation), and 10 mM (0.01 mol/L) of \u0026beta;-glycerophosphate disodium (048-34332; FUJIFILM Wako Pure Chemical Corporation) were used.\u003c/p\u003e\n\u003ch2\u003eEstimation of differentiation of osteoblasts with ALP staining on day 4 and day 7\u003c/h2\u003e\n\u003cp\u003eAlkaline phosphatase (ALP) was stained using the TRAP/ALP Stain Kit (294-67001; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) at 4 and 7 days post-treatment, following the manufacturer\u0026apos;s protocol. The protocol used a 10% Formalin Neutral Buffer Solution (068-01663; FUJIFILM Wako Pure Chemical Corporation) for fixation. Acetone (016-00346; FUJIFILM Wako Pure Chemical Corporation) and ethanol (99.5) (14713-95; FUJIFILM Wako Pure Chemical Corporation) were used for permeabilization. Stained areas were quantified using a microscope (BZ-X810; Keyence Corp., Osaka, Japan). All experiments were conducted in duplicate. The ALP-positive area was normalized to the mean value of the osteoblast induction medium group in each experiment, and results from the two experiments were pooled for analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e（２）\u003c/strong\u003e\u003cstrong\u003eAnimal experiment\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003cstrong\u003ein vivo bone healing capacity using animal models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed in accordance with the ARRIVE guidelines, and the Animal Experimentation Committee of the Graduate School of Medicine, Kyoto University approved all experimental protocols for this study (Medkyo 24583). All methods were performed in compliance with the relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003eHealthy male Wistar rats (11 weeks old and weighing in 350\u0026ndash;400 g range, were obtained from SLC Japan, Inc. The rats were individually caged and kept under specific pathogen-free conditions with a 12-hour light/dark cycle, had free access to food and water, and were allowed to acclimate to the laboratory for 1 week prior to the experiment. 12-week-old rats underwent surgery. The rats were placed in a pre-anesthesia box and anesthetized by isoflurane inhalation (Viatris Pharmaceuticals, Inc., Tokyo, Japan). The pre-anesthesia conditions were as follows: isoflurane concentration was 3-4%, flow rate, 3 l/min, and recovery, 3.5 l/min. After the rats ceased to move, they were switched to maintenance anesthesia via mask inhalation. The conditions for maintenance anesthesia were an isoflurane concentration of 2-3%, a flow rate of 3 l/min, and a recovery rate of 3.5 l/min. The rat heads were shaved using an electric trimmer. The head was disinfected with isodine solution 10% (Shionogi, Osaka, Japan), and 0.5 cc of Xylocaine Injection 1% with epinephrine (Sand Pharma, Tokyo, Japan) was injected locally under the head skin for local anesthesia. A skin incision was made in the midline of the parietal region in the sagittal direction using scalpel No. 15 (KAI Industries, Inc., Tokyo, Japan). An incision was made in the periosteum and the cranial crown was clearly visible. The bone fragments were harvested from the center of the space using a trephine bar with an inner diameter of 8 mm.\u003c/p\u003e\n\u003cp\u003eDepending on the method of cell-inactivation treatment of the bone fragments collected (treatment methods were randomly assigned), the rats were assigned to four groups, the details of which were as follows: (1) Group that received HHP treatment. The treatment was performed at 200 MPa for 10 min. (2) Group treated using the Pasteur method: High-temperature treatment at 65\u0026deg;C for 30 min. (3) Group treated with liquid nitrogen for 20 min. (4) No treatment (control).\u003c/p\u003e\n\u003cp\u003eA detailed procedure is described in the Supplementary Fig. S1.\u003c/p\u003e\n\u003cp\u003eBone fragments that had undergone cell inactivation were reimplanted into cranial defects in rats. Following reimplantation, the periosteum and skin were closed using 4-0 nylon sutures (Bear Medic, Inc., Japan), and the animals were returned to their individual cages upon regaining consciousness. Each group consisted of 12 samples, and euthanasia was performed using gas at 4, 12, and 16 weeks after surgery (n = 4 per time point).\u0026nbsp;Euthanasia was performed using CO2 gas inhalation without prior anesthesia in accordance with the institutional animal welfare guidelines. Death was confirmed by the cessation of heartbeat and respiration, and ocular decolorization. After euthanasia, the skin was incised to expose the cranial vault and the reimplanted bone. The cranial vault was then extracted as a single unit using a trephine bar, and the mobility of the reimplant was confirmed after manual testing. The extracted cranial vault was fixed in 10% formalin solution\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003efor 72 h and then transferred to 70% ethanol. The following techniques were used to assess outcomes.\u003c/p\u003e\n\u003ch2\u003e1: Manual Examination\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe crania of the euthanized rats were incised to expose the calvaria while preserving the periosteum, ensuring that the grafted bone was clearly visible. Before sampling, the mobility of the grafted bone was assessed using forceps. This procedure is presented in Supplementary Video S2.\u003c/p\u003e\n\u003ch2\u003e2: Computed Tomography\u003c/h2\u003e\n\u003cp\u003eA micro-focus X-ray system (TOSCANER-32300 \u0026mu;FD; Toshiba IT Control Systems Corporation, Tokyo, Japan) was employed to image the cranial vault volume. Additionally, three-dimensional image analysis was conducted using 3D Slicer (https://www.slicer.org), which is an open-source platform for medical image processing and informatics. Detailed information regarding the evaluation methodology is provided in Supplementary Movies S2-4.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3: Histological Evaluation\u003c/h2\u003e\n\u003cp\u003eThe cranial vault was fixed in formalin, which was replaced with ethanol, decalcified with EDTA (7-14 days), and embedded in paraffin. Hematoxylin-eosin (HE) staining, Masson\u0026rsquo;s trichrome (MTC) staining, and CD31 staining were performed. After morphological confirmation by HE staining, the gap between the bone fragment and the skull was evaluated. The evaluation criteria were as follows: if both gaps were fused, it was considered completely fused; if only one gap was fused, it was considered partially fused; and if no fusion was observed, it was considered unfused. Next, membranous ossification and bone volume in the region of interest (ROI) were evaluated using MTC staining. Membranous ossification was identified as the blue areas in MTC staining, and bone volume was identified as the red area. For CD31 staining, the slides were deparaffinized, hydrated, treated for 20 min at 98\u0026deg;C in a pH 9 antigen retrieval solution (code: 415211), washed with distilled water, incubated at room temperature for 10 min in 3% hydrogen peroxide, and subsequently washed with distilled water and Tris-buffered saline with Tween 20 (TBST). The sections were then incubated for 60 min at room temperature with 3% bovine serum albumin, followed by incubation with Anti-CD31 antibody diluted 1:1000 (code: ab182981) at 4\u0026deg;C for 24 h. Thereafter, the sections were reacted with a polymer reagent (code: 714191) at room temperature for 30 min, washed with TBST, developed using DAB, washed with water, dehydrated, and mounted. The number and area of DAB-positive vascular endothelial cells in the ROI were evaluated for neovascularization. The details of the ROI settings are provided in the Supplementary Fig. S5. Observations were performed using the BZ-X800 Analyzer software (Keyence Corp., Osaka, Japan).\u003c/p\u003e\n\u003ch2\u003e(3) the biomechanical change of the inactivated bone\u003c/h2\u003e\n\u003ch2\u003eEvaluation of bone surface alterations due to cell inactivation methods\u003c/h2\u003e\n\u003cp\u003eA dozen male Wistar rats aged 11 weeks were euthanized using gas, and circular bone samples (8 mm in diameter) were extracted from their cranial crowns. These samples were then randomly allocated into six groups, with two samples per group, and subjected to various cell inactivation methods: 1) untreated control, 2) liquid nitrogen exposure, 3) Pasteur method (65\u0026deg;C for 30 min), 4) high hydrostatic pressure, 5) elevated temperature (80\u0026deg;C for 30 min), and 6) autoclave sterilization (121\u0026deg;C for 20 min). The treated bone specimens were subsequently examined using scanning electron microscopy (SEM; JSM-7900F, JEOL Ltd. Tokyo, Japan) on both anterior and posterior surfaces.\u003c/p\u003e\n\u003ch2\u003eEvaluation of changes in bone strength due to cell inactivation methods\u003c/h2\u003e\n\u003cp\u003eA group of 20 male Wistar rats, aged 11 weeks, were euthanized using gas, and their cranial crowns were extracted as a single unit. The extracted crowns were randomly divided into four equal groups, each subjected to different cell inactivation techniques: (1) no treatment (control), (2) liquid nitrogen exposure, (3) high-temperature application, and (4) high-hydrostatic pressure treatment. To assess the bone strength, four random points on each sample were evaluated using the Vickers hardness test. The equipment used for the test was HMV2000AD (SHIMADZU, Kyoto, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are presented as mean \u0026plusmn; standard deviation (SD). Statistical significance was set at P \u0026lt; 0.05. The analysis was conducted using JMP software (JMP Statistical Discovery LLC., Cary, NC, USA). The Tukey-Kramer test and Fisher\u0026apos;s exact test were used.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by JSPS KAKENHI (Grant Number 24K02606) and AMED (Grant Number JP24ym0126143).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our sincere appreciation to Tsuboi Mizuki and Tanida Yukihiro of the Kyoto Prefectural Technology Center for Small and Medium Enterprises for their invaluable technical guidance in the execution of CT imaging and Vickers hardness testing. We are also grateful to Yoshitaka Tanaka for his dedicated assistance in the experimental procedures. Furthermore, this manuscript has been meticulously proofread by a native English speaker through Editage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the development of the study, E.S. and R.A. were responsible for the design, primary conceptualization, and the formulation of the proof\u0026apos;s framework. Data collection was conducted by E.S. and R.A. The interpretation of the results and manuscript preparation were supported by H.Y., M.S., T.Y., S.D., and N.M. The manuscript was authored by E.S. and R.A. All authors engaged in discussions regarding the results and provided constructive feedback on the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available in the Figshare repository at https://doi.org/10.6084/m9.figshare.29964341.v1\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHurley, C.M. et al. Current trends in craniofacial reconstruction. \u003cem\u003eSurgeon\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, e118-e125 (2023).\u003c/li\u003e\n \u003cli\u003eTsuge, I. et al. Central Mandibular Reconstruction by Semiopen Wedge Osteotomy Double-barrel Fibula Flap for a Slim Aesthetic Appearance. \u003cem\u003ePlast Reconstr Surg Glob Open\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, e4716 (2022).\u003c/li\u003e\n \u003cli\u003eTsuge, I. et al. Double-flap Mandibular Reconstruction around the Condylar Head Using Fibula and Anterolateral Thigh Flaps. \u003cem\u003ePlast Reconstr Surg Glob Open\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, e4607 (2022).\u003c/li\u003e\n \u003cli\u003eTsuge, I., Yamanaka, H., Katsube, M., Sakamoto, M. \u0026amp; Morimoto, N. Simultaneous Reconstruction of the Bilateral Maxillae and Nasal Hard Structure Using a Vascularized and Nonvascularized Fibula. \u003cem\u003ePlast Reconstr Surg Glob Open\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, e5936 (2024).\u003c/li\u003e\n \u003cli\u003eFutran, N.D. \u0026amp; Mendez, E. Developments in reconstruction of midface and maxilla. \u003cem\u003eLancet Oncol\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 249-258 (2006).\u003c/li\u003e\n \u003cli\u003eChang, E.I. \u0026amp; Hanasono, M.M. State-of-the-art reconstruction of midface and facial deformities. \u003cem\u003eJ Surg Oncol\u003c/em\u003e \u003cstrong\u003e113\u003c/strong\u003e, 962-970 (2016).\u003c/li\u003e\n \u003cli\u003eLese, I., Biedermann, R., Constantinescu, M., Grobbelaar, A.O. \u0026amp; Olariu, R. Predicting risk factors that lead to free flap failure and vascular compromise: A single unit experience with 565 free tissue transfers. \u003cem\u003eJ Plast Reconstr Aesthet Surg\u003c/em\u003e \u003cstrong\u003e74\u003c/strong\u003e, 512-522 (2021).\u003c/li\u003e\n \u003cli\u003eKoons, G.L., Diba, M. \u0026amp; Mikos, A.G. Materials design for bone-tissue engineering. \u003cem\u003eNature Reviews Materials\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 584-603 (2020).\u003c/li\u003e\n \u003cli\u003eZhang, J., Feng, Y., Zhou, X., Shi, Y. \u0026amp; Wang, L. Research status of artificial bone materials. \u003cem\u003eInternational Journal of Polymeric Materials and Polymeric Biomaterials\u003c/em\u003e \u003cstrong\u003e70\u003c/strong\u003e, 37-53 (2021).\u003c/li\u003e\n \u003cli\u003eFeng, P. et al. Structural and Functional Adaptive Artificial Bone: Materials, Fabrications, and Properties. \u003cem\u003eAdvanced Functional Materials\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 2214726 (2023).\u003c/li\u003e\n \u003cli\u003eYamamoto, N., Tsuchiya, H. \u0026amp; Tomita, K. Effects of liquid nitrogen treatment on the proliferation of osteosarcoma and the biomechanical properties of normal bone. \u003cem\u003eJ Orthop Sci\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 374-380 (2003).\u003c/li\u003e\n \u003cli\u003eTsuchiya, H. et al. Reconstruction using an autograft containing tumour treated by liquid nitrogen. \u003cem\u003eJ Bone Joint Surg Br\u003c/em\u003e \u003cstrong\u003e87\u003c/strong\u003e, 218-225 (2005).\u003c/li\u003e\n \u003cli\u003eTsuchiya, H. et al. Pedicle frozen autograft reconstruction in malignant bone tumors. \u003cem\u003eJ Orthop Sci\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 340-349 (2010).\u003c/li\u003e\n \u003cli\u003eErrani, C., Tsukamoto, S., Almunhaisen, N., Mavrogenis, A. \u0026amp; Donati, D. Intercalary reconstruction following resection of diaphyseal bone tumors: A systematic review. \u003cem\u003eJ Clin Orthop Trauma\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 1-10 (2021).\u003c/li\u003e\n \u003cli\u003eEhara, S., Nishida, J., Shiraishi, H. \u0026amp; Tamakawa, Y. Pasteurized intercalary autogenous bone graft: radiographic and scintigraphic features. \u003cem\u003eSkeletal Radiol\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 335-339 (2000).\u003c/li\u003e\n \u003cli\u003eSugiura, H. et al. Evaluation of long-term outcomes of pasteurized autografts in limb salvage surgeries for bone and soft tissue sarcomas. \u003cem\u003eArch Orthop Trauma Surg\u003c/em\u003e \u003cstrong\u003e132\u003c/strong\u003e, 1685-1695 (2012).\u003c/li\u003e\n \u003cli\u003eUmer, M. et al. Autoclaved tumor bone for skeletal reconstruction in paediatric patients: a low cost alternative in developing countries. \u003cem\u003eBiomed Res Int\u003c/em\u003e \u003cstrong\u003e2013\u003c/strong\u003e, 698461 (2013).\u003c/li\u003e\n \u003cli\u003eArpornchayanon, O., Leerapun, T., Pruksakorn, D. \u0026amp; Panichkul, P. Result of extracorporeal irradiation and re-implantation for malignant bone tumors: a review of 30 patients. \u003cem\u003eAsia Pac J Clin Oncol\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 214-219 (2013).\u003c/li\u003e\n \u003cli\u003eJinno, C. et al. Extracorporeal high-pressure therapy (EHPT) for malignant melanoma consisting of simultaneous tumor eradication and autologous dermal substitute preparation. \u003cem\u003eRegen Ther\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 187-194 (2020).\u003c/li\u003e\n \u003cli\u003eMitsui, T. et al. High Hydrostatic Pressure Therapy Annihilates Squamous Cell Carcinoma in a Murine Model. \u003cem\u003eBiomed Res Int\u003c/em\u003e \u003cstrong\u003e2020\u003c/strong\u003e, 3074742 (2020).\u003c/li\u003e\n \u003cli\u003eLi, Y. et al. Development of a novel regenerative therapy for malignant bone tumors using an autograft containing tumor inactivated by high hydrostatic pressurization (HHP). \u003cem\u003eRegen Ther\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 224-231 (2023).\u003c/li\u003e\n \u003cli\u003eMorimoto, N. et al. A Novel Treatment for Giant Congenital Melanocytic Nevi Combining Inactivated Autologous Nevus Tissue by High Hydrostatic Pressure and a Cultured Epidermal Autograft: First-in-Human, Open, Prospective Clinical Trial. \u003cem\u003ePlastic and Reconstructive Surgery\u003c/em\u003e \u003cstrong\u003e148\u003c/strong\u003e, 71e-76e (2021).\u003c/li\u003e\n \u003cli\u003eYamanaka, H. et al. A high-hydrostatic pressure device for nevus tissue inactivation and dermal regeneration for reconstructing skin defects after giant congenital melanocytic nevus excision: a clinical trial. \u003cem\u003eRegen Ther\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 167-173 (2023).\u003c/li\u003e\n \u003cli\u003eHalloran, D., Durbano, H.W. \u0026amp; Nohe, A. Bone Morphogenetic Protein-2 in Development and Bone Homeostasis. \u003cem\u003eJ Dev Biol\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e (2020).\u003c/li\u003e\n \u003cli\u003eXu, G. et al. The process of bone regeneration from devitalization to revitalization after pedicle freezing with immunohistochemical and histological examination in rabbits. \u003cem\u003eCryobiology\u003c/em\u003e \u003cstrong\u003e92\u003c/strong\u003e, 130-137 (2020).\u003c/li\u003e\n \u003cli\u003eUniyal, P., Sihota, P. \u0026amp; Kumar, N. Effect of organic matrix alteration on strain rate dependent mechanical behaviour of cortical bone. \u003cem\u003eJournal of the Mechanical Behavior of Biomedical Materials\u003c/em\u003e \u003cstrong\u003e125\u003c/strong\u003e, 104910 (2022).\u003c/li\u003e\n \u003cli\u003eSingh, D. et al. Experimental assessment of biomechanical properties in human male elbow bone subjected to bending and compression loads. \u003cem\u003eJ Appl Biomater Funct Mater\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 2280800018793816 (2019).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Flotation results from manual testing\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"622\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4 week\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e12 week\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e16 week\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0% (0/4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e25% (1/4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e25% (1/4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0% (0/4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0% (0/4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0%(0/4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0% (0/3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e25% (1/4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e50% (2/4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHHP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0% (0/4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e25% (1/4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e25% (1/4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe results of the manual test show the number of samples with flotation at each observation point.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC, untreated (control) group; LN, liquid nitrogen group; P, the pasteurization group; HHP, high hydrostatic pressure treatment group.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"High hydrostatic pressure, Cell inactivation, Bone reconstruction, Osteoinductive proteins, Autologous grafts","lastPublishedDoi":"10.21203/rs.3.rs-7430280/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7430280/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"High hydrostatic pressure (HHP) has emerged as a promising technique for inactivating tumor cells while preserving the structural integrity of biological tissues. This study investigated the potential of HHP-treated bone as a biologically compatible autograft material for oncological reconstruction. Bone morphogenetic protein-2 (BMP-2), a key osteoinductive factor, was subjected to various inactivation methods, including HHP, high-temperature heating, and liquid nitrogen, and its osteogenic activity was assessed through alkaline phosphatase (ALP) expression in MC3T3-E1 cells. BMP-2 activity was preserved following HHP and cryogenic treatment but was diminished by thermal methods. In a rat calvarial defect model, we compared the biological and structural performance of bone grafts processed using HHP, heating (Pasteur method), liquid nitrogen, and no treatment. Computed tomography and histological analyses demonstrated comparable bone healing and ossification in the HHP and liquid nitrogen-treated groups. Scanning electron microscopy and Vickers hardness testing revealed that the surface morphology and mechanical strength of the HHP-treated bone were similar to those of the untreated bone. These findings suggest that HHP treatment preserves both the osteogenic and biomechanical properties of autologous bone and should offer a clinically viable alternative to conventional tumor inactivation methods in bone reconstruction.","manuscriptTitle":"Exploration of Inactivated Bone Using High Hydrostatic Pressurization for Future Oncologic Application","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-06 18:53:32","doi":"10.21203/rs.3.rs-7430280/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"336681051321931051945539705802704541742","date":"2025-09-25T20:08:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"132923225998161076573121067394827121247","date":"2025-09-24T07:41:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"17369673406096171570304493928754475874","date":"2025-09-23T17:01:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-23T16:41:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-16T19:26:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-02T13:23:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-01T11:43:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-01T11:40:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b66aa168-9097-43bc-a299-299eb0f0fc8d","owner":[],"postedDate":"October 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":55533084,"name":"Biological sciences/Biological techniques"},{"id":55533086,"name":"Biological sciences/Biotechnology"},{"id":55533088,"name":"Biological sciences/Cancer"},{"id":55533089,"name":"Biological sciences/Cell biology"},{"id":55533090,"name":"Physical sciences/Materials science"},{"id":55533091,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2025-10-06T18:53:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-06 18:53:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7430280","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7430280","identity":"rs-7430280","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}