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The process of bone regeneration from devitalization to revitalization after pedicle freezing with immunohistochemical and histological examination in rabbits
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The process of bone regeneration from devitalization to revitalization after pedicle freezing with immunohistochemical and histological examination in rabbits Gang Xu a, Norio Yamamoto a, *, Takayuki Nojima a, b, Katsuhiro Hayashi a, Akihiko Takeuchi a, Shinji Miwa a, Kentaro Igarashi a, Hiroyuki Tsuchiya a a b
Department of Orthopaedic Surgery, Kanazawa University School of Medicine, Kanazawa, Japan Section of Diagnostic Pathology, Kanazawa University Hospital, Kanazawa, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords: Cryosurgery Frozen autograft Revitalization Liquid nitrogen Endochondral ossification Bone regeneration Tumor-bearing bone
The pedicle freezing procedure by liquid nitrogen is a method for the reconstruction of tumor-bearing bone after malignant tumor resection. However, the regenerative mechanism of bone after the pedicle freezing procedure is unclear. We investigated the complete process from devitalization to revitalization of bone after the pedicle freezing procedure in 13 rabbits. After osteotomy the 5 mm distal femurs were immersed in liquid nitrogen, and the specimens were divided into frozen area and sub-frozen area. The bilateral femurs were harvested for evaluation of bone regeneration by histological and immunohistochemical examination (VEGF, CD31, BMP-2 and Runx2) from 1 week to 52 weeks. The diameter of operating femurs was compared with contralateral fe murs from 6 weeks to 52 weeks. No viable cells could be found from 1 to 8 weeks in the frozen area, and a mean 1.83 cm necrotic range were detected in the sub-frozen area. The periosteal reaction, massive fibrous tissue and immature bone matrix invaded from the normal area to the necrotic area from 12 weeks. Subsequently, the necrotic bone was gradually replaced by newly formed bone by creeping substitution, with endochondral and intramembrane bone forma tion. The diameter of frozen femurs was significantly larger than the contralateral femur at the same period from 8 weeks to 52 weeks (P < 0.01). All immunohistochemical factors were positively expressed in both areas at different time points. The active osteoblasts and microvessel migrated from marrow cavity and periosteum into dead bone. This study suggested that the frozen bone not only provides a scaffold but also possesses excellent osteoinductive properties.
1. Introduction Due to current imaging techniques, effective neoadjuvant or adju vant therapy, precise surgical techniques and multidisciplinary team cooperation, survival rates for malignant musculoskeletal tumors have been prolonged, and five-year survival rate for osteosarcoma has increased from 20% to 80% [6,9,20]. Currently, amputation is gradually replaced by megaprosthesis and biological reconstruction. However, long-term complications for megaprosthesis are inevitable owing to mechanical failures, although the design of megaprostheses has been continuously improved [7,12,16,35]. Therefore, how to bring more benefits for long-term limb function continues to be an issue for the surgeons. Promising biological reconstructions are expected to provide
long-term durability of limb function. Theoretically, purpose of biological reconstructions is that they achieve normal mechanical strength, integration with the host bone, anatomical remodeling and permanent rebuilding. In fact, various bio logical reconstruction methods have been widely used for limb salvage surgery, including allograft, autograft, distraction osteogenesis, fibula transplantation and recycling tumor-bearing bone. However, it is diffi cult to replace non-vascularized graft bone with living bone, especially in recycled tumor-bearing bone and allografts [11,31,33,35]. Liquid nitrogen (LN) has been widely used as a cryogenic agent to treat various benign and malignant bone tumors in the field of ortho pedics [28,32]. The rationale is that the use of low temperatures induces tissue necrosis by ice crystal formation and cell dehydration [8]. Tumor-bearing bone graft is frequently performed by excision of the
* Corresponding author. Department of Orthopaedic Surgery, Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa, Ishikawa, 920-8641, Japan. E-mail address: [email protected] (N. Yamamoto). https://doi.org/10.1016/j.cryobiol.2019.12.002 Received 25 September 2019; Received in revised form 13 November 2019; Accepted 20 December 2019 Available online 23 December 2019 0011-2240/© 2019 Published by Elsevier Inc.
Please cite this article as: Gang Xu, Cryobiology, https://doi.org/10.1016/j.cryobiol.2019.12.002
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2.1. Surgical procedure
Abbreviations LN PFP HE EOLR IHC VEGF CD31 BMP-2 RUNX-2
All rabbits were given general anesthesia using intramuscular in jection with 2 ml of Domitor and 2 ml of Dormicum, respectively. 1 ml of lidocaine was intramuscularly injected into the surgical site. Surgery was carried out under aseptic condition, and each animal was placed in the lateral position on the operating table. A 12 cm lateral longitudinal skin incision was created in the right femur and soft tissues were sepa rated until the entire femur was exposed. Firstly, osteotomies were performed in the middle of femoral shaft by osteotome. Secondly, soft tissues were protected around the distal femur with sheets. Lastly, femur with 5 mm distance from the osteotomy site was placed in LN at 196 � C for 20 min and then kept at room temperature until the frozen bone was completely thawed (Fig. 1 A, B, C). The freezing procedure is concor dant with the clinical pedicle freezing method [30]. Reduction and fixation of the osteotomy site was performed by plate and 6 screws (LCP plate 2.0: VP4012-10, 10 holes, 69 mm; cortex screw: VS102-012, 12 mm; Depuy Synthes) (Fig. 1 D). Soft tissues and skin were sutured using nonabsorbable sutures. Cefmetazole (100 mg/kg) was intravenously injected to prevent infection after the operation. Rabbits were kept in individual cages. All rabbits were euthanized by intravenously injection of 6 ml pentobarbital sodium at indicated weeks after surgery (Table 1). There were no complications such as fracture, infection or bone ab sorption around the femur until euthanization in all rabbits in our study.
Liquid nitrogen Pedicle freezing procedure Hematoxylin and eosin Empty osseous lacunae rate Immunohistochemistry Vascular endothelial growth factor Platelet endothelial cell adhesion molecule 1 Bone morphogenetic protein-2 Runt-related transcription factor 2
bone and treatment with irradiation, pasteurization or freezing. Freezing bone with LN at 196 � C is one of approaches in recycling bone reconstruction, which was firstly reported in large bone defects following tumor excision by Tsuchiya in 1999 [28]. We reported in our previous studies that osteosarcoma cells were inhibited by LN in vitro and in vivo and the anti-cancer immunological response was remaining [15,34]. To minimize complication related with osteotomy site, pedicle freezing procedure (PFP) was developed [30]. Shimozaki et al. reported that complication rate was lower with PFP than the free frozen pro cedure in long bone. It is thought that blood-flow recovery is faster in PFP, due to the preservation of bone integrity in other side [21]. How ever, there is no report of angiogenesis after PFP, and the regenerative mechanism of PFP is not clear. In our work, the immunohistochemistry (IHC) assay was applied to detect angiogenetic and osteoinductive ca pacity after PFP. Investigation of regenerative mechanism from devi talization to revitalization in rabbit models was measured with histological examination. The following questions were addressed: How does the bone change from necrosis to regeneration after PFP? How do the osteoinductive capacity in process of revitalization? How does osteogenesis occur after PFP?
Table 1 The osteocytes necrosis rate from 1 week to 52 weeks. The necrotic range of sub-frozen area was gradually decreased from 12 weeks. As EOLRs were only found in a few parts of sub-frozen area from 36 weeks, the necrotic range cannot be calculated.
2. Materials and methods Animal experiments were all approved by the Institutional Animal Care and Ethics Committee of Kanazawa University (AP-163808). All experiments were performed under the guidelines for animal experi ments at Kanazawa University of Medical Science. A total of 13 imma ture female Japanese white rabbits (Kitayama Labes, Nagano, Japan) were involved in this study. The rabbits were aged from 15 to 17 weeks and with body weight ranging from 3.0 to 3.5 kg.
Frozen area Mean � SD (%)
Sub-frozen area Mean � SD (%)
100 � 0.52 99 � 0.84 98 � 1.50 99 � 0.95 100 98 � 1.94 92 � 6.09 87 � 7.66 78 � 9.93 64 � 16.24 42 � 12.88 28 � 11.70 8 � 8.98
84 � 9.51 90 � 5.5 88 � 7.6 92 � 4.22 98 � 1.77 98 � 1.51 88 � 7.27 85 � 8.10 65 � 12.98 48 � 10.90 24 � 7.73 6 � 3.77 2 � 1.58
Sub-frozen of necrotic range 1.80 cm 1.84 cm 1.77 cm 1.83 cm 1.89 cm 1.82 cm 1.75 cm 1.93 cm 1.30 cm 1.05 cm 0.60 cm N/A N/A
Fig. 1. Pedicle freezing procedure.A B C D 5 mm distal femur inserted liquid nitrogen and fixed with plate. E. Blue area was defined as frozen area where no living cells were detected. Green area was defined as sub-frozen area with a length from 5 mm to detectable nucleated osteocytes, and only a few nucleated osteocytes were detected in this area. (Black circle: nucleated osteocyte; White circle: empty osseous lacunae). (For interpretation of the refer ences to colour in this figure legend, the reader is referred to the Web version of this article.)
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2.2. Histological evaluation
2.6. Statistical analysis
The bilateral femurs harvested from rabbits were fixed in 10% formalin solution, dehydrated in graded ethanol (80%, 90%, and 100%), decalcified in 10% formic sodium citrate solution, embedded in paraffin, and then cut into 2 μm-thick sagittal slides. The sections at each time point were stained with hematoxylin and eosin (HE) and observed under an optical microscope and NDP software (Biorevo BZ-9000; Keyence Co. Osaka, Japan; NDP. view 2 viewing software U12388-01; Nano zoomerXRC 12000; Hamamatsu Photonics. Hamamatsu, Japan).
All statistical analyses were performed with SPSS, version 24 (IBM Corp., Armonk, NY, USA). An analysis of variance test (ANOVA) was used to compare between-group differences in the different areas. A P value < 0.05 was considered statistically significant.
2.3. Experimental groups
In histological staining of the frozen area, no viable cells were found from 1 to 8 weeks (Fig. 2A, Fig. 3 A, B). Osteoblasts, chondrocytes, fibrous tissues were found around frozen bone from 8 weeks post operatively (Fig. 3B). From 12 weeks, new bone formation, fewer chondrocytes and fibrous tissues surrounding the necrotic bone was clearly observed. Some necrotic areas were replaced by new bone. From 24 weeks, the necrotic area became an irregular shape, although most lacunae osteocytes were still in situ (Fig. 2 B). At 52 weeks, only a little partial necrotic bone was not absorbed, and necrotic bone was completely wrapped by new bone (Fig. 2 C). In the sub-frozen area, from 1 to 8 weeks, most osteocytes remained empty of osseous lacuna (Fig. 2 D, G, Fig. 3 C, D). Periosteal reaction, massive fibrous tissues and immature bone matrix gradually migrated from normal bone on the surface of lateral bone cortex from 2 weeks, and original cortical border was uninterrupted (Fig. 2 G). From 8 weeks, massive immature bone matrix and osteoblasts were formed by intra membrane bone formation on the surface of lateral bone cortex and medial medullary. Necrotic bone was almost covered with woven bone and osteoblasts. The original cortical border was interrupted in few
3. Results 3.1. Histological findings
Surgical specimens were divided into two areas under microscope with HE staining. Frozen area: defined as 5 mm distal femur immersed in LN after osteotomy. Sub-frozen area: defined as a length from 5 mm to the detectable nucleated osteocyte (Fig. 1 E). Contralateral femur of non-operation was used as a control to the diameter with frozen bone at each timepoint. 2.4. Osteocytes necrosis rate analyses To quantify the process from devitalization to revitalization, the osteocytes necrosis rate (%) was defined as the empty osseous lacunae rate (EOLR), and calculated as follows: The sections were magnified 100 times under a microscope, 10 fields of view were randomly selected in each area, and empty osseous lacunae and nucleated osteocytes were counted at each timepoint [14,22]. All areas were quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Osteocytes necrosis rate ¼
empty osseous lacunae � 100% empty osseous lacunae þ nucleated osteocyte
2.5. Immunohistochemical evaluation
parts. Active osteocytes, osteoblasts and micro-vessels were gradually invaded into the sub-frozen bone (Fig. 3 C, D). From 24 weeks, massive fibrous tissue and woven bone matrix transformed into mature bone on the surface of the discontinuously original cortical border, and most necrotic areas were substituted. The number of osteoblasts were decreased around discontinuously original cortical border, and most newly formed bone and revitalized bone like the normal bone morphology (Fig. 2 E, H). Few parts remained with necrosis with empty osseous lacuna at 52 weeks, and the original cortical border was dis appeared (Fig. 2 F, I). From 8 weeks, the newly formed bone cause thickening of frozen femurs, and the diameter of original cortex is similar to contralateral femur (Fig. 4 A, B). In comparison, the diameter of frozen femurs was significantly larger than the contralateral femur at the same period from 8 week to 52 weeks (p < 0.01) (Fig. 4 C).
All immunohistochemical examinations were performed using uni form methods at each time point. Vascular endothelial growth factor, blood vessel endothelial cells, bone morphogenetic protein-2 and runtrelated transcription factor 2 were detected with anti-VEGF antibody (Abcam, ab1316; 1:200), anti-CD-31 antibody (Novus Biological, NB600-562; 1:250), anti-BMP-2 antibody (Abcam, ab6285; 1:200) and anti-RUNX2 antibody (Abcam, ab76956; 1:200), respectively. The sections were deparaffined in xylene, grade ethanol and Liberate Antibody Binding Solution (LAB solution; Polysciences, Philadelphia, PA, USA) at room temperature. They were blocked with hydrogen peroxide (Peroxidase-Blocking Solution, Dako, Glostrup, Denmark), phosphate buffer saline (PBS) and protein block serum-free (Dako, Glostrup, Denmark) for 10 min. Primary antibodies diluted in antibody diluent (Dako Antibody Diluent, Dako, Glostrup, Denmark) were drip ped onto slides and incubated at room temperature for 1 h. The speci mens were incubated with secondary antibody (Dako, REAL, Rabbit/ Mouse, Glostrup, Denmark) for 30 min at room temperature after washed with PBS for 15 min. Once positive signals were visible under microscope after DAB chromogen (Dako REAL DAB þ Chromogen, Glostrup, Denmark), the reactions were stopped with distilled water. Positive rate of osteoblasts was counted using above method of EOLR in the medullary cavity side of cortex and the lateral side of cortex from 6 weeks to 24 weeks, respectively. The immunohistochemical and his tological results were confirmed by blinded pathologists to make it accurate.
3.2. Necrosis rate of osteocyte analyses Mean necrotic range is 1.83 cm in sub-frozen area from 1 week to 8 weeks after surgery. The range of necrosis in sub-frozen area gradually decreased starting from 12 weeks. As EOLRs were only found in a few parts of sub-frozen area from 36 weeks, the necrotic range cannot be calculated (Table 1). 3.3. Immunohistochemistry of VEGF and CD31 No positive signals of CD31 and VEGF was observed in frozen area and sub-frozen area before 8 weeks. Expression of these two proteins 3
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Fig. 2. The process of revitalization with frozen bone in the different areas as revealed by HE staining (H&E staining; Scale ¼ 250 μm). A D G No living cells and vessels in frozen area and only few osteocytes in sub-frozen area, and massive fibrous tissues were migrated from normal bone to sub-frozen area on the surface of lateral cortex at two weeks. B E H Frozen area became an irregular shape, and osteoblasts invaded frozen bone. Osteoblasts covered necrosis bone in the medullary cavity of sub-frozen area, and massive new bone has formed in lateral side at 24 weeks.C F I Only few parts of necrotic bone were not absorbed, and necrotic bone was completely wrapped by vital osteocytes. Sub-frozen area almost restored normal morphology at 52 weeks. (triangle: original cortical border; square: newly formed bone; star: revitalized bone).
Fig. 3. HE staining showed the process of revitalization with frozen bone at 8 weeks. A. Massive fibrous tissue and new bone were formed around sub-frozen area (Scale ¼ 10 mm). A representative histological image at 8 weeks after surgery was presented, B. Massive chondrocytes on the surface of frozen area, and partial chondrocytes and osteoblasts invaded frozen bone. C. Few osteoblasts were detected in this part of medullary side, and original cortical border was interrupted. D. Massive new bone was formed, and osteoblasts invaded the frozen bone by creeping substitution and intra-membranous ossification in the lateral side (Scale ¼ 250 μm). (triangle: original cortical border; circle: chondrocyte; arrow: osteoblast; square: newly formed bone.).
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Fig. 4. Comparison of diameter of femoral shaft with contralateral femur, and the diameter of different regions at 24 weeks was shown by HE staining assay (Scale ¼ 10 mm). A B The histological results showed that diameter of metaphysis regions and original cortex were similar between the frozen and non-operative group, whereas the femoral shaft after freezing is larger than contralateral femur at 24 weeks after surgery (A: frozen femur; B: contralateral nonoperative femur; blue line: original cortex).C Comparison of diameter of femoral shaft after freezing is signifi cantly larger than contralateral femur from 8 weeks to 52 weeks (Mean � SD: group C: 5.46 mm � 0.83 mm; group F: 7.76 mm � 1.11 mm; *p < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 5. VEGF and CD31 expressed in the different areas. A B No positive reaction was detectable at 6 weeks. C D Positive expressions of VEGF were visible at 12 weeks in frozen area and sub-frozen area. E F The CD31 expression is positive in the different areas at 24 weeks. New vessel formation had penetrated from new bone to frozen bone, and living osteocyte was detected around the new vessel (Scale ¼ 100 μm).
started from 12 weeks in frozen area and from 8 weeks in sub-frozen area. Migration of microvessel into necrosis bone was found in both areas starting from 12 weeks (Fig. 5).
(Fig. 7. C).
3.4. Immunohistochemistry of BMP-2
In this study, we found that as necrotic frozen bone is gradually absorbed and substituted, the frozen bone not only provides a scaffold but also possesses excellent osteoinductive properties and earlier angiogenesis in PFP. Meanwhile, endochondral and intramembranous ossification participated in the process of revitalization. Osteoinduction, osteoconduction and earlier recovery of blood sup ply influence success or failure for bone transplantation, such as effec tive growth factors including BMP, VEGF and CD31 [17,18]. It is reported that abnormal expression of these proteins would lead to bone morphology alteration or osteogenesis disrupture [1,18,19,23,27]. Several clinical reports disclosed that reusing tumor-bearing bone induced loss of osteoinductive properties, which leads to increased incidence of complication, such as postoperative nonunion or absorption [2,24,30,33]. In our study, the expression of CD31, VEGF and BMP-2 were found
4. Discussion
Massive positive signals were detectable on the surface of newly formed bone from 8 weeks. Expression of BMP-2 in osteoblasts, active osteocyte and bone lacuna were observed in both areas from 12 weeks. After 36 weeks, no positive reactions were found in sub-frozen area (Fig. 6). 3.5. Immunohistochemistry of RUNX2 The Runx2 was expressed around the frozen bone and newly formed bone from 8 weeks in frozen area. Positive staining for osteoblast on the surface of cortical bone was detectable from 4 weeks to 24 weeks in subfrozen area (Fig. 7 A B). Percentage of positive osteoblast in the lateral side is higher than medullary side from 6 weeks to 24 weeks (P < 0.05) 5
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Fig. 6. BMP-2 expressed in the different areas. BMP-2 were expressed in active osteocyte and empty lacuna in the different areas at 12 weeks (A, B) and 24 weeks (C, D) (Scale ¼ 50 μm).
Fig. 7. Runx2 expressed in the different sides, and the percentage of positive osteoblast between lateral side and medullary side. A B Runx2 were positive expressed on the surface of medullary side and lateral side and at 16 weeks, respectively (Scale ¼ 50 μm). C The percentage of positive osteoblast in lateral side was significantly stronger than medullary side from 6 weeks to 24 weeks (Mean � SD: lateral side: 0.19 � 0.06; medullary side: 0.06 � 0.03; *P < 0.05).
positively expressed in the frozen bone and newly formed bone. This indicated that new vessels formed in necrotic bone, and a lot of fibro vascular tissues invaded into frozen bone from newly formed bone and medullary cavity. Meanwhile, massive osteoblasts and active osteocytes were found surrounding the fibrovascular in frozen bone. Although the osteoionductive capacity preserved by freezing was reported before, there was no evidence disclosed the expression of osteoinductive factors during frozen bone regeneration after PFP [4,25]. In our study, the angiogenesis and BMP-2 expressed in the earlier stage of bone revitali zation was found. It might be related to the potential advantage of PFP that preserving continuity of bone in one side. Massive bone growth factors and effective blood supply were provided from a continuous side, which can accelerate the process of revitalization. On the other hand, it is inevitable that subchondral collapse was resulted from cryosurgery [10]. However, PFP combined with prosthesis might avoid its
occurrence. Tanzawa et al. reported that osteoblasts were observed in the frozen bone 6 years after the freezing [26]. Shinimura et al. reported that the crushed bone graft following freezing can obtain bone fusion in the spinal reconstruction [22]. Tsuchiya et al. also reported that formation of the callus was induced with frozen bone during the bone transport [29]. These indicate the osteoconductive ability of frozen bone, although LN leads to additional infiltration depths of up to 1.83 cm, which might resemble a marginal or wide excision [32]. As massive fibrous tissues and periosteal reaction were gradually migrated from normal bone to necrotic areas, the necrotic areas were predominantly invaded by osteoblasts, fibroblasts and fibrovascular through creeping substitution. Keijser et al. reported periosteal osteogenesis and creeping substitution on the localized surface of necrosis bone after cryoprobe [13]. Remarkably, massive chondrocytes were observed around frozen 6
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area, eventually became mature bone. This finding leads us to hypoth esized that endochondral ossification also participated in the bone regeneration, like the process of normal bone healing. Furthermore, even if a few parts of cortex remained empty of osseous lacuna at the one-year model, newly formed bone had completely wrapped the necrotic bone, and EOLR gradually decreased (Table 1). It might indi cate a continuing revitalization potential, and necrotic bone will be completely revitalized. In order to prove that the process of revitaliza tion is not a bone-growing process, we compared the contralateral non-operative femur at the same time point. Our results showed that the region of femoral shaft after freezing is significantly larger than contralateral femur from 6 to 52 weeks (p < 0.01). In addition, expression of Runx2 is largely restricted to osteoblasts and forming bone [3]. In this study, Runx2 was positively expressed on the surface of the lateral cortical bone and the medullary side from 4 weeks, which gradually expressed replaced bone. Meanwhile, positive expression of Runx2 in later cortical side was significantly stronger than medullary side from 6 to 24 weeks (P < 0.05). In other words, most of the osteoblasts and new vessels migrated from the contiguous lateral cortical bone during bone revitalization. Enneking et al. reported that necrotic graft was repaired both in externally and internally, however, the process of internal repair was slow and incomplete, which occurred in the ends and the periphery of the cortex [5]. To our knowledge, this is the first study demonstrating the utilization of immunohistochemistry and histology to analyze the process of bone revitalization for PFP. This study has some limitations: firstly, we did not establish a control group with free frozen procedure, as previous studies already reported the regeneration in free frozen procedure. Secondly, only normal bone was used for the analysis of the process, as proving the regeneration ability and osteoinductive capacity after PFP is the aim of this study. However, in the clinic application many disadvantageous factors may affect the process of bone regeneration such as age, general, chemotherapy. In conclusion, even if cryosurgery results in bone necrosis, the bone regeneration properties are not interrupted. This study provides a new proof for bone revitalization after pedicle freezing. With early vascu larization and preservation of bone morphogenetic protein, the frozen bone can be regenerated by creeping substitution, endochondral and intramembrane bone formation.
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Trauma Surg. 132 (2012) 1685–1695. https://doi:10.1007/s00402-012-1606-4. [25] M. Takata, N. Sugimoto, N. Yamamoto, T. shirai, K. Hayashi, H. Nishida, Y. Tanzawa, H. Kimura, S. Miwa, A. Takeuchi, H. Tsuchiya, Activity of bone morphogenetic protein-7 after treatment at various temperatures: freezing vs.
Funding No funding or benefits were received from any specific grant in this research. Declaration of competing interest The authors declare that they have no conflict of interest. Acknowledgment The authors would like to thank professor Takayuki Nojima for the evaluation of the immunohistochemical and histological examination, and also thank Shinji Miwa and Akihiko Takeuchi for the review and comments on the manuscript. References [1] A. Bandyopadhyay, K. Tsuji, K. Cox, B.D. Harfe, V. Rosen, C.J. Tabin, Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis, PLoS Genet. 2 (2006) 216. https://doi:10.1371/journal.pge n.0020216. [2] P. Bohm, J. Fritz, S. Thiede, W. Budach, Reimplantation of extracorporeal irradiated bone segments in musculoskeletal tumor surgery: clinical experience in eight patients and review of the literature, Langenbeck’s Arch. Surg. 387 (2003) 355–365. https://doi:10.1007/s00423-002-0332-8.
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pasteurization vs. allograft, Cryobiology 63 (2011) 235–239. https://doi:10.101 6/j.cryobiol.2011.09.001. Y. Tanzawa, H. Tsuchiya, T. Shira, K. Hayashi, Z. Yo, K. Tomita, Histological examination of frozen autograft treated by liquid nitrogen removed 6 years after implantatio, J. Orthop. Sci. 13 (2008) 259–264. https://doi:10.1007/s00776-00 9-1392-1. K. Tatsuyama, Y. Maezawa, H. Baba, Y. Imamura, M. Fukuda, Expression of various growth factors for cell proliferation and cytodifferentiation during fracture repair of bone, Eur. J. Histochem. 44 (2000) 269–278. H. Tsuchiya, S.L. Wan, K. Sakatama, N. Yamamoto, H. Nishida, K. Tomita, Reconstruction using an autograft containing tumour treated by liquid nitrogen, J. Bone. Joint Surg. Br. 87 (2005) 218–225. H. Tsuchiya, K. Sakurakichi, N. Yamashiro, K. Wantanabe, Y. Inoue, N. Yamamoto, K. Tomita, Bone transport with frozen devitalized bone: an experimental study using rabbits and a clinical application, J. Orthop. Sci. 9 (2004) 619–624. https:// doi:10.1007/s00776-004-0836-x. H. Tsuchiya, H. Nishida, P. Srisawat, T. shirai, K. Hayashi, A. Takeuchi, N. Yamamoto, K. Tomita, Pedicle frozen autograft reconstruction in malignant
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Cryobiology journal homepage: http://www.elsevier.com/locate/cryo
The process of bone regeneration from devitalization to revitalization after pedicle freezing with immunohistochemical and histological examination in rabbits Gang Xu a, Norio Yamamoto a, *, Takayuki Nojima a, b, Katsuhiro Hayashi a, Akihiko Takeuchi a, Shinji Miwa a, Kentaro Igarashi a, Hiroyuki Tsuchiya a a b
Department of Orthopaedic Surgery, Kanazawa University School of Medicine, Kanazawa, Japan Section of Diagnostic Pathology, Kanazawa University Hospital, Kanazawa, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords: Cryosurgery Frozen autograft Revitalization Liquid nitrogen Endochondral ossification Bone regeneration Tumor-bearing bone
The pedicle freezing procedure by liquid nitrogen is a method for the reconstruction of tumor-bearing bone after malignant tumor resection. However, the regenerative mechanism of bone after the pedicle freezing procedure is unclear. We investigated the complete process from devitalization to revitalization of bone after the pedicle freezing procedure in 13 rabbits. After osteotomy the 5 mm distal femurs were immersed in liquid nitrogen, and the specimens were divided into frozen area and sub-frozen area. The bilateral femurs were harvested for evaluation of bone regeneration by histological and immunohistochemical examination (VEGF, CD31, BMP-2 and Runx2) from 1 week to 52 weeks. The diameter of operating femurs was compared with contralateral fe murs from 6 weeks to 52 weeks. No viable cells could be found from 1 to 8 weeks in the frozen area, and a mean 1.83 cm necrotic range were detected in the sub-frozen area. The periosteal reaction, massive fibrous tissue and immature bone matrix invaded from the normal area to the necrotic area from 12 weeks. Subsequently, the necrotic bone was gradually replaced by newly formed bone by creeping substitution, with endochondral and intramembrane bone forma tion. The diameter of frozen femurs was significantly larger than the contralateral femur at the same period from 8 weeks to 52 weeks (P < 0.01). All immunohistochemical factors were positively expressed in both areas at different time points. The active osteoblasts and microvessel migrated from marrow cavity and periosteum into dead bone. This study suggested that the frozen bone not only provides a scaffold but also possesses excellent osteoinductive properties.
1. Introduction Due to current imaging techniques, effective neoadjuvant or adju vant therapy, precise surgical techniques and multidisciplinary team cooperation, survival rates for malignant musculoskeletal tumors have been prolonged, and five-year survival rate for osteosarcoma has increased from 20% to 80% [6,9,20]. Currently, amputation is gradually replaced by megaprosthesis and biological reconstruction. However, long-term complications for megaprosthesis are inevitable owing to mechanical failures, although the design of megaprostheses has been continuously improved [7,12,16,35]. Therefore, how to bring more benefits for long-term limb function continues to be an issue for the surgeons. Promising biological reconstructions are expected to provide
long-term durability of limb function. Theoretically, purpose of biological reconstructions is that they achieve normal mechanical strength, integration with the host bone, anatomical remodeling and permanent rebuilding. In fact, various bio logical reconstruction methods have been widely used for limb salvage surgery, including allograft, autograft, distraction osteogenesis, fibula transplantation and recycling tumor-bearing bone. However, it is diffi cult to replace non-vascularized graft bone with living bone, especially in recycled tumor-bearing bone and allografts [11,31,33,35]. Liquid nitrogen (LN) has been widely used as a cryogenic agent to treat various benign and malignant bone tumors in the field of ortho pedics [28,32]. The rationale is that the use of low temperatures induces tissue necrosis by ice crystal formation and cell dehydration [8]. Tumor-bearing bone graft is frequently performed by excision of the
* Corresponding author. Department of Orthopaedic Surgery, Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa, Ishikawa, 920-8641, Japan. E-mail address: [email protected] (N. Yamamoto). https://doi.org/10.1016/j.cryobiol.2019.12.002 Received 25 September 2019; Received in revised form 13 November 2019; Accepted 20 December 2019 Available online 23 December 2019 0011-2240/© 2019 Published by Elsevier Inc.
Please cite this article as: Gang Xu, Cryobiology, https://doi.org/10.1016/j.cryobiol.2019.12.002
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2.1. Surgical procedure
Abbreviations LN PFP HE EOLR IHC VEGF CD31 BMP-2 RUNX-2
All rabbits were given general anesthesia using intramuscular in jection with 2 ml of Domitor and 2 ml of Dormicum, respectively. 1 ml of lidocaine was intramuscularly injected into the surgical site. Surgery was carried out under aseptic condition, and each animal was placed in the lateral position on the operating table. A 12 cm lateral longitudinal skin incision was created in the right femur and soft tissues were sepa rated until the entire femur was exposed. Firstly, osteotomies were performed in the middle of femoral shaft by osteotome. Secondly, soft tissues were protected around the distal femur with sheets. Lastly, femur with 5 mm distance from the osteotomy site was placed in LN at 196 � C for 20 min and then kept at room temperature until the frozen bone was completely thawed (Fig. 1 A, B, C). The freezing procedure is concor dant with the clinical pedicle freezing method [30]. Reduction and fixation of the osteotomy site was performed by plate and 6 screws (LCP plate 2.0: VP4012-10, 10 holes, 69 mm; cortex screw: VS102-012, 12 mm; Depuy Synthes) (Fig. 1 D). Soft tissues and skin were sutured using nonabsorbable sutures. Cefmetazole (100 mg/kg) was intravenously injected to prevent infection after the operation. Rabbits were kept in individual cages. All rabbits were euthanized by intravenously injection of 6 ml pentobarbital sodium at indicated weeks after surgery (Table 1). There were no complications such as fracture, infection or bone ab sorption around the femur until euthanization in all rabbits in our study.
Liquid nitrogen Pedicle freezing procedure Hematoxylin and eosin Empty osseous lacunae rate Immunohistochemistry Vascular endothelial growth factor Platelet endothelial cell adhesion molecule 1 Bone morphogenetic protein-2 Runt-related transcription factor 2
bone and treatment with irradiation, pasteurization or freezing. Freezing bone with LN at 196 � C is one of approaches in recycling bone reconstruction, which was firstly reported in large bone defects following tumor excision by Tsuchiya in 1999 [28]. We reported in our previous studies that osteosarcoma cells were inhibited by LN in vitro and in vivo and the anti-cancer immunological response was remaining [15,34]. To minimize complication related with osteotomy site, pedicle freezing procedure (PFP) was developed [30]. Shimozaki et al. reported that complication rate was lower with PFP than the free frozen pro cedure in long bone. It is thought that blood-flow recovery is faster in PFP, due to the preservation of bone integrity in other side [21]. How ever, there is no report of angiogenesis after PFP, and the regenerative mechanism of PFP is not clear. In our work, the immunohistochemistry (IHC) assay was applied to detect angiogenetic and osteoinductive ca pacity after PFP. Investigation of regenerative mechanism from devi talization to revitalization in rabbit models was measured with histological examination. The following questions were addressed: How does the bone change from necrosis to regeneration after PFP? How do the osteoinductive capacity in process of revitalization? How does osteogenesis occur after PFP?
Table 1 The osteocytes necrosis rate from 1 week to 52 weeks. The necrotic range of sub-frozen area was gradually decreased from 12 weeks. As EOLRs were only found in a few parts of sub-frozen area from 36 weeks, the necrotic range cannot be calculated.
2. Materials and methods Animal experiments were all approved by the Institutional Animal Care and Ethics Committee of Kanazawa University (AP-163808). All experiments were performed under the guidelines for animal experi ments at Kanazawa University of Medical Science. A total of 13 imma ture female Japanese white rabbits (Kitayama Labes, Nagano, Japan) were involved in this study. The rabbits were aged from 15 to 17 weeks and with body weight ranging from 3.0 to 3.5 kg.
Frozen area Mean � SD (%)
Sub-frozen area Mean � SD (%)
100 � 0.52 99 � 0.84 98 � 1.50 99 � 0.95 100 98 � 1.94 92 � 6.09 87 � 7.66 78 � 9.93 64 � 16.24 42 � 12.88 28 � 11.70 8 � 8.98
84 � 9.51 90 � 5.5 88 � 7.6 92 � 4.22 98 � 1.77 98 � 1.51 88 � 7.27 85 � 8.10 65 � 12.98 48 � 10.90 24 � 7.73 6 � 3.77 2 � 1.58
Sub-frozen of necrotic range 1.80 cm 1.84 cm 1.77 cm 1.83 cm 1.89 cm 1.82 cm 1.75 cm 1.93 cm 1.30 cm 1.05 cm 0.60 cm N/A N/A
Fig. 1. Pedicle freezing procedure.A B C D 5 mm distal femur inserted liquid nitrogen and fixed with plate. E. Blue area was defined as frozen area where no living cells were detected. Green area was defined as sub-frozen area with a length from 5 mm to detectable nucleated osteocytes, and only a few nucleated osteocytes were detected in this area. (Black circle: nucleated osteocyte; White circle: empty osseous lacunae). (For interpretation of the refer ences to colour in this figure legend, the reader is referred to the Web version of this article.)
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2.2. Histological evaluation
2.6. Statistical analysis
The bilateral femurs harvested from rabbits were fixed in 10% formalin solution, dehydrated in graded ethanol (80%, 90%, and 100%), decalcified in 10% formic sodium citrate solution, embedded in paraffin, and then cut into 2 μm-thick sagittal slides. The sections at each time point were stained with hematoxylin and eosin (HE) and observed under an optical microscope and NDP software (Biorevo BZ-9000; Keyence Co. Osaka, Japan; NDP. view 2 viewing software U12388-01; Nano zoomerXRC 12000; Hamamatsu Photonics. Hamamatsu, Japan).
All statistical analyses were performed with SPSS, version 24 (IBM Corp., Armonk, NY, USA). An analysis of variance test (ANOVA) was used to compare between-group differences in the different areas. A P value < 0.05 was considered statistically significant.
2.3. Experimental groups
In histological staining of the frozen area, no viable cells were found from 1 to 8 weeks (Fig. 2A, Fig. 3 A, B). Osteoblasts, chondrocytes, fibrous tissues were found around frozen bone from 8 weeks post operatively (Fig. 3B). From 12 weeks, new bone formation, fewer chondrocytes and fibrous tissues surrounding the necrotic bone was clearly observed. Some necrotic areas were replaced by new bone. From 24 weeks, the necrotic area became an irregular shape, although most lacunae osteocytes were still in situ (Fig. 2 B). At 52 weeks, only a little partial necrotic bone was not absorbed, and necrotic bone was completely wrapped by new bone (Fig. 2 C). In the sub-frozen area, from 1 to 8 weeks, most osteocytes remained empty of osseous lacuna (Fig. 2 D, G, Fig. 3 C, D). Periosteal reaction, massive fibrous tissues and immature bone matrix gradually migrated from normal bone on the surface of lateral bone cortex from 2 weeks, and original cortical border was uninterrupted (Fig. 2 G). From 8 weeks, massive immature bone matrix and osteoblasts were formed by intra membrane bone formation on the surface of lateral bone cortex and medial medullary. Necrotic bone was almost covered with woven bone and osteoblasts. The original cortical border was interrupted in few
3. Results 3.1. Histological findings
Surgical specimens were divided into two areas under microscope with HE staining. Frozen area: defined as 5 mm distal femur immersed in LN after osteotomy. Sub-frozen area: defined as a length from 5 mm to the detectable nucleated osteocyte (Fig. 1 E). Contralateral femur of non-operation was used as a control to the diameter with frozen bone at each timepoint. 2.4. Osteocytes necrosis rate analyses To quantify the process from devitalization to revitalization, the osteocytes necrosis rate (%) was defined as the empty osseous lacunae rate (EOLR), and calculated as follows: The sections were magnified 100 times under a microscope, 10 fields of view were randomly selected in each area, and empty osseous lacunae and nucleated osteocytes were counted at each timepoint [14,22]. All areas were quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Osteocytes necrosis rate ¼
empty osseous lacunae � 100% empty osseous lacunae þ nucleated osteocyte
2.5. Immunohistochemical evaluation
parts. Active osteocytes, osteoblasts and micro-vessels were gradually invaded into the sub-frozen bone (Fig. 3 C, D). From 24 weeks, massive fibrous tissue and woven bone matrix transformed into mature bone on the surface of the discontinuously original cortical border, and most necrotic areas were substituted. The number of osteoblasts were decreased around discontinuously original cortical border, and most newly formed bone and revitalized bone like the normal bone morphology (Fig. 2 E, H). Few parts remained with necrosis with empty osseous lacuna at 52 weeks, and the original cortical border was dis appeared (Fig. 2 F, I). From 8 weeks, the newly formed bone cause thickening of frozen femurs, and the diameter of original cortex is similar to contralateral femur (Fig. 4 A, B). In comparison, the diameter of frozen femurs was significantly larger than the contralateral femur at the same period from 8 week to 52 weeks (p < 0.01) (Fig. 4 C).
All immunohistochemical examinations were performed using uni form methods at each time point. Vascular endothelial growth factor, blood vessel endothelial cells, bone morphogenetic protein-2 and runtrelated transcription factor 2 were detected with anti-VEGF antibody (Abcam, ab1316; 1:200), anti-CD-31 antibody (Novus Biological, NB600-562; 1:250), anti-BMP-2 antibody (Abcam, ab6285; 1:200) and anti-RUNX2 antibody (Abcam, ab76956; 1:200), respectively. The sections were deparaffined in xylene, grade ethanol and Liberate Antibody Binding Solution (LAB solution; Polysciences, Philadelphia, PA, USA) at room temperature. They were blocked with hydrogen peroxide (Peroxidase-Blocking Solution, Dako, Glostrup, Denmark), phosphate buffer saline (PBS) and protein block serum-free (Dako, Glostrup, Denmark) for 10 min. Primary antibodies diluted in antibody diluent (Dako Antibody Diluent, Dako, Glostrup, Denmark) were drip ped onto slides and incubated at room temperature for 1 h. The speci mens were incubated with secondary antibody (Dako, REAL, Rabbit/ Mouse, Glostrup, Denmark) for 30 min at room temperature after washed with PBS for 15 min. Once positive signals were visible under microscope after DAB chromogen (Dako REAL DAB þ Chromogen, Glostrup, Denmark), the reactions were stopped with distilled water. Positive rate of osteoblasts was counted using above method of EOLR in the medullary cavity side of cortex and the lateral side of cortex from 6 weeks to 24 weeks, respectively. The immunohistochemical and his tological results were confirmed by blinded pathologists to make it accurate.
3.2. Necrosis rate of osteocyte analyses Mean necrotic range is 1.83 cm in sub-frozen area from 1 week to 8 weeks after surgery. The range of necrosis in sub-frozen area gradually decreased starting from 12 weeks. As EOLRs were only found in a few parts of sub-frozen area from 36 weeks, the necrotic range cannot be calculated (Table 1). 3.3. Immunohistochemistry of VEGF and CD31 No positive signals of CD31 and VEGF was observed in frozen area and sub-frozen area before 8 weeks. Expression of these two proteins 3
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Fig. 2. The process of revitalization with frozen bone in the different areas as revealed by HE staining (H&E staining; Scale ¼ 250 μm). A D G No living cells and vessels in frozen area and only few osteocytes in sub-frozen area, and massive fibrous tissues were migrated from normal bone to sub-frozen area on the surface of lateral cortex at two weeks. B E H Frozen area became an irregular shape, and osteoblasts invaded frozen bone. Osteoblasts covered necrosis bone in the medullary cavity of sub-frozen area, and massive new bone has formed in lateral side at 24 weeks.C F I Only few parts of necrotic bone were not absorbed, and necrotic bone was completely wrapped by vital osteocytes. Sub-frozen area almost restored normal morphology at 52 weeks. (triangle: original cortical border; square: newly formed bone; star: revitalized bone).
Fig. 3. HE staining showed the process of revitalization with frozen bone at 8 weeks. A. Massive fibrous tissue and new bone were formed around sub-frozen area (Scale ¼ 10 mm). A representative histological image at 8 weeks after surgery was presented, B. Massive chondrocytes on the surface of frozen area, and partial chondrocytes and osteoblasts invaded frozen bone. C. Few osteoblasts were detected in this part of medullary side, and original cortical border was interrupted. D. Massive new bone was formed, and osteoblasts invaded the frozen bone by creeping substitution and intra-membranous ossification in the lateral side (Scale ¼ 250 μm). (triangle: original cortical border; circle: chondrocyte; arrow: osteoblast; square: newly formed bone.).
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Fig. 4. Comparison of diameter of femoral shaft with contralateral femur, and the diameter of different regions at 24 weeks was shown by HE staining assay (Scale ¼ 10 mm). A B The histological results showed that diameter of metaphysis regions and original cortex were similar between the frozen and non-operative group, whereas the femoral shaft after freezing is larger than contralateral femur at 24 weeks after surgery (A: frozen femur; B: contralateral nonoperative femur; blue line: original cortex).C Comparison of diameter of femoral shaft after freezing is signifi cantly larger than contralateral femur from 8 weeks to 52 weeks (Mean � SD: group C: 5.46 mm � 0.83 mm; group F: 7.76 mm � 1.11 mm; *p < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 5. VEGF and CD31 expressed in the different areas. A B No positive reaction was detectable at 6 weeks. C D Positive expressions of VEGF were visible at 12 weeks in frozen area and sub-frozen area. E F The CD31 expression is positive in the different areas at 24 weeks. New vessel formation had penetrated from new bone to frozen bone, and living osteocyte was detected around the new vessel (Scale ¼ 100 μm).
started from 12 weeks in frozen area and from 8 weeks in sub-frozen area. Migration of microvessel into necrosis bone was found in both areas starting from 12 weeks (Fig. 5).
(Fig. 7. C).
3.4. Immunohistochemistry of BMP-2
In this study, we found that as necrotic frozen bone is gradually absorbed and substituted, the frozen bone not only provides a scaffold but also possesses excellent osteoinductive properties and earlier angiogenesis in PFP. Meanwhile, endochondral and intramembranous ossification participated in the process of revitalization. Osteoinduction, osteoconduction and earlier recovery of blood sup ply influence success or failure for bone transplantation, such as effec tive growth factors including BMP, VEGF and CD31 [17,18]. It is reported that abnormal expression of these proteins would lead to bone morphology alteration or osteogenesis disrupture [1,18,19,23,27]. Several clinical reports disclosed that reusing tumor-bearing bone induced loss of osteoinductive properties, which leads to increased incidence of complication, such as postoperative nonunion or absorption [2,24,30,33]. In our study, the expression of CD31, VEGF and BMP-2 were found
4. Discussion
Massive positive signals were detectable on the surface of newly formed bone from 8 weeks. Expression of BMP-2 in osteoblasts, active osteocyte and bone lacuna were observed in both areas from 12 weeks. After 36 weeks, no positive reactions were found in sub-frozen area (Fig. 6). 3.5. Immunohistochemistry of RUNX2 The Runx2 was expressed around the frozen bone and newly formed bone from 8 weeks in frozen area. Positive staining for osteoblast on the surface of cortical bone was detectable from 4 weeks to 24 weeks in subfrozen area (Fig. 7 A B). Percentage of positive osteoblast in the lateral side is higher than medullary side from 6 weeks to 24 weeks (P < 0.05) 5
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Fig. 6. BMP-2 expressed in the different areas. BMP-2 were expressed in active osteocyte and empty lacuna in the different areas at 12 weeks (A, B) and 24 weeks (C, D) (Scale ¼ 50 μm).
Fig. 7. Runx2 expressed in the different sides, and the percentage of positive osteoblast between lateral side and medullary side. A B Runx2 were positive expressed on the surface of medullary side and lateral side and at 16 weeks, respectively (Scale ¼ 50 μm). C The percentage of positive osteoblast in lateral side was significantly stronger than medullary side from 6 weeks to 24 weeks (Mean � SD: lateral side: 0.19 � 0.06; medullary side: 0.06 � 0.03; *P < 0.05).
positively expressed in the frozen bone and newly formed bone. This indicated that new vessels formed in necrotic bone, and a lot of fibro vascular tissues invaded into frozen bone from newly formed bone and medullary cavity. Meanwhile, massive osteoblasts and active osteocytes were found surrounding the fibrovascular in frozen bone. Although the osteoionductive capacity preserved by freezing was reported before, there was no evidence disclosed the expression of osteoinductive factors during frozen bone regeneration after PFP [4,25]. In our study, the angiogenesis and BMP-2 expressed in the earlier stage of bone revitali zation was found. It might be related to the potential advantage of PFP that preserving continuity of bone in one side. Massive bone growth factors and effective blood supply were provided from a continuous side, which can accelerate the process of revitalization. On the other hand, it is inevitable that subchondral collapse was resulted from cryosurgery [10]. However, PFP combined with prosthesis might avoid its
occurrence. Tanzawa et al. reported that osteoblasts were observed in the frozen bone 6 years after the freezing [26]. Shinimura et al. reported that the crushed bone graft following freezing can obtain bone fusion in the spinal reconstruction [22]. Tsuchiya et al. also reported that formation of the callus was induced with frozen bone during the bone transport [29]. These indicate the osteoconductive ability of frozen bone, although LN leads to additional infiltration depths of up to 1.83 cm, which might resemble a marginal or wide excision [32]. As massive fibrous tissues and periosteal reaction were gradually migrated from normal bone to necrotic areas, the necrotic areas were predominantly invaded by osteoblasts, fibroblasts and fibrovascular through creeping substitution. Keijser et al. reported periosteal osteogenesis and creeping substitution on the localized surface of necrosis bone after cryoprobe [13]. Remarkably, massive chondrocytes were observed around frozen 6
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area, eventually became mature bone. This finding leads us to hypoth esized that endochondral ossification also participated in the bone regeneration, like the process of normal bone healing. Furthermore, even if a few parts of cortex remained empty of osseous lacuna at the one-year model, newly formed bone had completely wrapped the necrotic bone, and EOLR gradually decreased (Table 1). It might indi cate a continuing revitalization potential, and necrotic bone will be completely revitalized. In order to prove that the process of revitaliza tion is not a bone-growing process, we compared the contralateral non-operative femur at the same time point. Our results showed that the region of femoral shaft after freezing is significantly larger than contralateral femur from 6 to 52 weeks (p < 0.01). In addition, expression of Runx2 is largely restricted to osteoblasts and forming bone [3]. In this study, Runx2 was positively expressed on the surface of the lateral cortical bone and the medullary side from 4 weeks, which gradually expressed replaced bone. Meanwhile, positive expression of Runx2 in later cortical side was significantly stronger than medullary side from 6 to 24 weeks (P < 0.05). In other words, most of the osteoblasts and new vessels migrated from the contiguous lateral cortical bone during bone revitalization. Enneking et al. reported that necrotic graft was repaired both in externally and internally, however, the process of internal repair was slow and incomplete, which occurred in the ends and the periphery of the cortex [5]. To our knowledge, this is the first study demonstrating the utilization of immunohistochemistry and histology to analyze the process of bone revitalization for PFP. This study has some limitations: firstly, we did not establish a control group with free frozen procedure, as previous studies already reported the regeneration in free frozen procedure. Secondly, only normal bone was used for the analysis of the process, as proving the regeneration ability and osteoinductive capacity after PFP is the aim of this study. However, in the clinic application many disadvantageous factors may affect the process of bone regeneration such as age, general, chemotherapy. In conclusion, even if cryosurgery results in bone necrosis, the bone regeneration properties are not interrupted. This study provides a new proof for bone revitalization after pedicle freezing. With early vascu larization and preservation of bone morphogenetic protein, the frozen bone can be regenerated by creeping substitution, endochondral and intramembrane bone formation.
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Funding No funding or benefits were received from any specific grant in this research. Declaration of competing interest The authors declare that they have no conflict of interest. Acknowledgment The authors would like to thank professor Takayuki Nojima for the evaluation of the immunohistochemical and histological examination, and also thank Shinji Miwa and Akihiko Takeuchi for the review and comments on the manuscript. References [1] A. Bandyopadhyay, K. Tsuji, K. Cox, B.D. Harfe, V. Rosen, C.J. Tabin, Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis, PLoS Genet. 2 (2006) 216. https://doi:10.1371/journal.pge n.0020216. [2] P. Bohm, J. Fritz, S. Thiede, W. Budach, Reimplantation of extracorporeal irradiated bone segments in musculoskeletal tumor surgery: clinical experience in eight patients and review of the literature, Langenbeck’s Arch. Surg. 387 (2003) 355–365. https://doi:10.1007/s00423-002-0332-8.
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