Cryobiology 92 (2020) 130–137

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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
Department of Orthopaedic Surgery, Kanazawa University School of Medicine, Kanazawa, Japan
b
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: The pedicle freezing procedure by liquid nitrogen is a method for the reconstruction of tumor-bearing bone after
Cryosurgery malignant tumor resection. However, the regenerative mechanism of bone after the pedicle freezing procedure is
Frozen autograft unclear. We investigated the complete process from devitalization to revitalization of bone after the pedicle
Revitalization
freezing procedure in 13 rabbits. After osteotomy the 5 mm distal femurs were immersed in liquid nitrogen, and
Liquid nitrogen
the specimens were divided into frozen area and sub-frozen area. The bilateral femurs were harvested for
Endochondral ossification
Bone regeneration evaluation of bone regeneration by histological and immunohistochemical examination (VEGF, CD31, BMP-2
Tumor-bearing bone 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.

long-term durability of limb function.

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
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: norinori@med.kanazawa-u.ac.jp (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 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
G. Xu et al. Cryobiology 92 (2020) 130–137

2.1. Surgical procedure

Abbreviations
All rabbits were given general anesthesia using intramuscular in­
LN Liquid nitrogen jection with 2 ml of Domitor and 2 ml of Dormicum, respectively. 1 ml of
PFP Pedicle freezing procedure lidocaine was intramuscularly injected into the surgical site. Surgery
HE Hematoxylin and eosin was carried out under aseptic condition, and each animal was placed in
EOLR Empty osseous lacunae rate the lateral position on the operating table. A 12 cm lateral longitudinal
IHC Immunohistochemistry skin incision was created in the right femur and soft tissues were sepa­
VEGF Vascular endothelial growth factor rated until the entire femur was exposed. Firstly, osteotomies were
CD31 Platelet endothelial cell adhesion molecule 1 performed in the middle of femoral shaft by osteotome. Secondly, soft
BMP-2 Bone morphogenetic protein-2 tissues were protected around the distal femur with sheets. Lastly, femur
RUNX-2 Runt-related transcription factor 2 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
bone and treatment with irradiation, pasteurization or freezing. fixation of the osteotomy site was performed by plate and 6 screws (LCP
Freezing bone with LN at 196 � C is one of approaches in recycling bone plate 2.0: VP4012-10, 10 holes, 69 mm; cortex screw: VS102-012, 12
reconstruction, which was firstly reported in large bone defects mm; Depuy Synthes) (Fig. 1 D). Soft tissues and skin were sutured using
following tumor excision by Tsuchiya in 1999 [28]. We reported in our nonabsorbable sutures. Cefmetazole (100 mg/kg) was intravenously
previous studies that osteosarcoma cells were inhibited by LN in vitro injected to prevent infection after the operation. Rabbits were kept in
and in vivo and the anti-cancer immunological response was remaining individual cages. All rabbits were euthanized by intravenously injection
[15,34]. To minimize complication related with osteotomy site, pedicle of 6 ml pentobarbital sodium at indicated weeks after surgery (Table 1).
freezing procedure (PFP) was developed [30]. Shimozaki et al. reported There were no complications such as fracture, infection or bone ab­
that complication rate was lower with PFP than the free frozen pro­ sorption around the femur until euthanization in all rabbits in our study.
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
Table 1
mechanism of PFP is not clear. In our work, the immunohistochemistry The osteocytes necrosis rate from 1 week to 52 weeks.
(IHC) assay was applied to detect angiogenetic and osteoinductive ca­ The necrotic range of sub-frozen area was gradually decreased from 12 weeks.
pacity after PFP. Investigation of regenerative mechanism from devi­ As EOLRs were only found in a few parts of sub-frozen area from 36 weeks, the
talization to revitalization in rabbit models was measured with necrotic range cannot be calculated.
histological examination. The following questions were addressed: How Sub-frozen of necrotic
does the bone change from necrosis to regeneration after PFP? How do Frozen area Mean � SD Sub-frozen area Mean � SD range
the osteoinductive capacity in process of revitalization? How does (%) (%)
osteogenesis occur after PFP? 100 � 0.52 84 � 9.51 1.80 cm
99 � 0.84 90 � 5.5 1.84 cm
2. Materials and methods 98 � 1.50 88 � 7.6 1.77 cm
99 � 0.95 92 � 4.22 1.83 cm
100 98 � 1.77 1.89 cm
Animal experiments were all approved by the Institutional Animal 98 � 1.94 98 � 1.51 1.82 cm
Care and Ethics Committee of Kanazawa University (AP-163808). All 92 � 6.09 88 � 7.27 1.75 cm
experiments were performed under the guidelines for animal experi­ 87 � 7.66 85 � 8.10 1.93 cm
ments at Kanazawa University of Medical Science. A total of 13 imma­ 78 � 9.93 65 � 12.98 1.30 cm
64 � 16.24 48 � 10.90 1.05 cm
ture female Japanese white rabbits (Kitayama Labes, Nagano, Japan)
42 � 12.88 24 � 7.73 0.60 cm
were involved in this study. The rabbits were aged from 15 to 17 weeks 28 � 11.70 6 � 3.77 N/A
and with body weight ranging from 3.0 to 3.5 kg. 8 � 8.98 2 � 1.58 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
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 zoomer-
XRC 12000; Hamamatsu Photonics. Hamamatsu, Japan).
2.3. Experimental groups
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).
empty ​ osseous ​ lacunae ​
Osteocytes ​ necrosis ​ rate ¼
empty ​ osseous ​ lacunae ​ ​ þ ​ ​ nucleated ​ osteocyte ​ ​
2.5. Immunohistochemical evaluation
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 runt-
related 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.
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Cryobiology 92 (2020) 130–137
2.6. Statistical analysis
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.
3. Results
3.1. Histological findings
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
� 100%
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).
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
G. Xu et al. Cryobiology 92 (2020) 130–137

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. 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).
3.4. Immunohistochemistry of BMP-2
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 sub-
frozen 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)
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Cryobiology 92 (2020) 130–137
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. 7. C).
4. Discussion
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
G. Xu et al. Cryobiology 92 (2020) 130–137

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

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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.
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.
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