CLINICAL RESEARCH
e-ISSN 1643-3750
© Med Sci Monit, 2017; 23: 4278-4283
DOI: 10.12659/MSM.906293
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Accepted:
Published:
The Irisin Hormone Profile and Expression in
Human Bone Tissue in the Bone Healing Process
in Patients
2017.07.20
2017.08.09
2017.09.04
Authors’ Contribution:
Study Design A
Data Collection B
Statistical Analysis C
Data Interpretation D
Manuscript Preparation E
Literature Search F
Funds Collection G
ABCDEFG 1
ADE 1
BCEF 2
BE 3
Corresponding Author:
Source of support:
Background:
Material/Methods:
Results:
Conclusions:
MeSH Keywords:
Full-text PDF:
Sancar Serbest
Uğur Tiftikçi
Hacı Bayram Tosun
Üçler Kısa
1 Department of Orthopedics and Traumatology, Faculty of Medicine, Kirikkale
University, Kırıkkale, Turkey
2 Department of Orthopedics and Traumatology, Faculty of Medicine, Adıyaman
University, Adıyaman, Turkey
3 Department of Biochemistry, Faculty of Medicine, Kirikkale University, Kirikkale,
Turkey
Sancar Serbest, e-mail: dr.sancarserbest@hotmail.com
This study was funded by Kirikkale University Scientific Research Project (2015/001)
Whether or not there is a relationship between the newly-discovered irisin hormone and bone healing is not yet
known. The aim of this study was to investigate what effect irisin hormone has on the bone healing process.
The study included 21 adult patients with a diagnosed fracture of the lower extremity (femur or tibia). Informed
consent was obtained from all the patients. A total of four venous blood samples were taken from the patients:
before fracture stabilization, then postoperatively on days 1, 10, and 60. In patients with femoral neck fracture
who had hip prosthesis applied, bone tissue samples were taken from the removed femur head and irisin was
determined immunohistochemically in muscle biopsies taken from the same patients.
In analysis, it was revealed that the mean value of irisin 60 days after operation is significantly higher than the
values of irisin before operation, 1 day after operation, and 15 day after operation (p<0.001, p<0.001, p<0.001,
respectively). Intense staining was observed in compact bone tissue, muscle tissue, and in hypertrophic vascular endothelium within the Havers canal.
The level of irisin hormone increased in the bone union process and affects fracture healing due to irisin receptors in human bone tissue.
Bone and Bones • Bone Diseases, Endocrine • Fracture Healing
https://www.medscimonit.com/abstract/index/idArt/906293
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CLINICAL RESEARCH
Background
Bone healing occurs as a result of the complex events requiring sequential interaction between progenitor cells, osteoblasts, and mediators found in the region [1–3]. Approximately
7.9 million fractures occur per year in the USA, and in 5–20%
of these, severe morbidity is associated with delayed healing
and causes a reduction in productivity [4–6].
Irisin, which was discovered by Böstrom et al. in 2012, is formed
of molecular 112 amino acids and is synthesized primarily in
muscle tissue, and several other tissues such as the liver, pancreas saliva, and sweat glands [7]. Although many molecules
play a role in fracture healing, irisin hormone has recently been
reported to show an osteoblastic effect [8–11]. To the best of
our knowledge, there has been no study to date that has investigated the relationship between fracture union and the
recently discovered irisin hormone. The measurement of the
levels of this hormone in patients with a bone fracture could
provide an idea of the pathophysiology of fracture healing.
The aim of this prospective study was to determine the change
in the level of irisin hormone in the blood and to show irisin hormone in human bone and muscle tissue immunohistochemically.
Material and Methods
Approval for the study was granted by the Clinical Research
Ethics Committee of Kirikkale University Medical Faculty
(2015/10-14). Informed consent was obtained from all the
study participants. The study included a total of 21 adult patients who met the study inclusion criteria. All the patients
had a lower extremity long bone fracture (femur or tibia) that
was treated with closed intramedullary nailing. Patients were
excluded from the study if they had an open or pathological
fracture, required blood transfusion, had osteogenesis imperfecta, rickets, suspected malignancy, diabetes, chronic kidney
failure, osteomyelitis, a history of gastric or intestinal surgery, hepatic or hematological disorder, thyroid dysfunction,
or a hormonal disorder. All patients were operated on within 24 hours of presentation at the Emergency Department. A
detailed anamnesis was taken from all the cases and a thorough physical examination was done.
Hormonal and biochemical measurements
Venous blood samples of 5 mL were taken in the morning between 0800 and 1000 from the patients a total of four times;
before fracture stabilization and on postoperative days 1, 15,
and 60. As peptides are easily separated by proteases within the cell, a protease inhibitor containing aprotinin was added to the tubes with the blood samples to be able to correctly
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measure the amount of irisin. After centrifugation, 1 N HCl was
added to the obtained samples up to a volume of 1/10 and
this ensured that the samples could remain stable for up to
a year at –20°C. To determine the irisin levels in the collected
samples, Active Human Irisin ELISA kit was used (YH Biosearch
Laboratory Cat.No: YHB1765Hu).
Pathological examinations and irisin-specific
immunoperoxidase test
In patients with a femoral neck fracture where hip arthroplasty was applied, the level of irisin hormone expression was examined in tissues using bone tissue samples taken from the
femur head and muscle biopsy samples taken from the same
patients. Samples extirpated from the femoral head during the
operation were fixed in 4% buffered paraformaldehyde for 48
hours and were then decalcified with commercial EDTA disodium salt (Bio-optica, Italy W01030799) changed every other
day. After 15 days the samples were trimmed to a thickness of
1 cm, and without washing the decalcified bone tissue under
tap water, tissues of 3–5 mm were transferred to cassettes. For
routine follow-up procedure, the tissues were passed through
a series of alcohol (70%, 80%, 90%, 96% and 99.5%) and xylol,
then embedded in paraffin and sections of 4–5 µm thickness
were again cut from the paraffin blocks. The histology slices
were deparaffinized and dehydrated. Then, to inhibit endogenous peroxidase activity, they were incubated for five minutes with 3% hydrogen peroxidase solution. For the antigen
retrieval procedure, the sections were boiled for 30 minutes
in a pH 6.0 citrate buffer and to prevent non-specific binding,
they were incubated for seven minutes in 10% bovine serum
albumin. Then the sections were incubated for 60 minutes in
mouse anti-irisin (FNDC5) monoclonal primer antibody (Biorbyt,
orb229904, UK) diluted to a ratio of 1: 200.
According to the recommendations in the horse-radish peroxidase chromogen kit (Invitrogen, USA), the sections were
then processed first for 30 minutes with secondary biotin antibody, then for 30 minutes in an avidin-peroxidase solution.
When antigenic structures became apparent with AEC chromogen substrate, all the samples were counter-stained with
Mayer’s hematoxylin. The skeletal muscle biopsy samples taken during the operation were accepted as positive controls and
sections applied with phosphate buffered saline rather than
primer antibody were used as negative controls. Antigenic
structures stained red with AEC were evaluated as positive
and microphotographs were taken with an Olympus BX51
trinocular microscope.
Radiological evaluations
The Lane and Sandhu [12] radiological scoring system was
used to evaluate the formation of new bone in the fracture
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18
Mean values of irisin
14
12
10
8
6
4
2
0
16
Serum irisin levels μg/ml
14
12
10
8
6
4
Before operation
1 day after operation
15 day after operation
60 day after operation
15
20
25
2
0
0
5
10
6.38±1.05
6.35±1.01
6.87±0.97
Before operation
1 day after
operation
15 day after
operation
60 day after
operation
Figure 2. Serum irisin levels (mean ± standard error of the
mean) before operation and on postoperative days 1,
15, and 60.
Patients
Figure 1. Serum irisin levels in patients with fracture before
fracture stabilization and on postoperative days 1, 15,
and 60.
line on anterior-posterior and lateral radiographs taken in the
first, third, and sixth month postoperatively.
Statistical Methods
Statistical analysis was applied using SPSS v.16.0 software
(SPSS Inc., Chicago, USA). Obtained data was assessed by a
confidence interval (CI) of 95% and a two-tailed p<0.05 were
determined to be statistically significant for all of the analyses. The numerical data was analyzed with Shapiro-Wilk test
in terms of assessment of data whether they were parametric.
The differences in the values of irisin before operation, 1 day
after operation, 15 days after operation and 60 days after operation were assessed with general linear model. If there would
be a significant difference in first assessment, Bonferroni test
was used to evaluate the pairwise comparisons. All of the numerical data were expressed as the mean SD values. The differences categorical variables were assessed using the c2 test.
Results
No statistically significant difference was determined in respect
of age, gender, treatment applied, and the affected extremity
(p>0.05). The mean values of irisin was before operation, 1 day
after operation, 15 days after operation and 60 days after operation were 6.38±1.05 µg/mL, 6.35±1.01 µg/mL, 6.87±0.97 µg/mL
and 12.87±1.66 µg/mL respectively (Figures 1, 2). In multivariate testing, it was found that there were significant differences in repeated measures of irisin (F=182.6, partial eta
squared=0.945, p<0.001). In post-hoc analysis, it was revealed
that the mean value of irisin 60 days after operation was significantly higher than the values of irisin before operation, 1
day after operation, and 15 days after operation (p<0.001,
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12.87±1.66
p<0.001, p<0.001). The mean value of irisin 15 days after operation was also significantly higher than the values of irisin
before operation, and 1 day after operation (p<0.001, p<0.001).
The mean values of irisin before operation and 1 day after operation were found to be similar (p=0.98).
According to the irisin antibody staining test with avidin-biotin peroxidase, cytoplasmic intense immunopositive staining was observed in the vascular cells in the vesicular nucleus
structure and extensive cytoplasm showing osteoblast morphology, especially in spongy bone tissue (Figure 3A, 3B). At
the same time, a mild level of irisin antigen immunopositive
reaction was seen in bone marrow cells (Figure 3C) between
the spongy tissue.
Although the osteoid matrix in compact bone tissue showed
a mild degree of irisin activity, most of the hypertrophic vascular endothelium in Havers canals showed intense staining
(Figure 3D). In the muscle tissue used as the positive control
group, irisin immunopositive staining generally showed an intense and diffuse nature within the sarcoplasm. While irisin immunoreactivity was observed in interstitial vascular endothelium and in some mid-diameter artery media layers, there was
no staining in any of the negative control sections (Figure 4).
According to the Lane and Sandhu radiological evaluation results, osseous union was seen to have started in all patients
in the radiological examinations made three months postoperatively. In the postoperative six-month examination, full osseous union was seen in all patients.
Discussion
The most important results of this study were the increase in
the amount of irisin hormone in the blood in the bone union
process and the finding that irisin receptors were seen in human
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B
A
C
D
Figure 3. Irisin immunoreactivities in the spongy and compact bone tissue. Avidin-biotin immunoperoxidase technique, anti-irisin
mouse antibody, Mayer’s hematoxylin counterstain. (A) Irisin activity in the capillary endothelia and osteoblasts (arrows),
bar=250 micrometer (B) Irisin positive immunostaining in the osteoblast cytoplasm (arrows), bar=60 micrometer (C) Compact
tissue irisin activity (arrows), bar=90 micrometer (D) Irisin immunopositive reactions in the bone marrow cells (arrows),
bar=120 micrometer.
bone cells. The mechanisms involved in the process of fracture
healing and the sequence of events continue to be a subject
attracting great attention in orthopedic science. Factors affecting fracture healing and research into the acceleration of
healing are popular topics of research. Many different studies
have been made on the subject of accelerating fracture healing in particular [13–16].
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The effect of irisin on bone metabolism is one of the important subjects still under discussion [17]. Despite the association of reduced muscle strength with vitamin D deficiency and
the risk of falling, no correlation has been shown between irisin and 25 (OH) D levels [9,18,19]. However, it has been shown
that recombinant irisin (r-irisin) applied at a low dose to young
mice increased cortical bone mineral density and changed bone
geometry in a positive way. Furthermore, with an increase in
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cells [24]. These findings suggest that irisin could be a bone
anabolic factor marker.
In another study, it was shown that irisin partially supported
osteoblast differentiation by the bone morphogenic protein
(BMP) pathway, and inhibited osteoclast differentiation by suppressing the RANKL-Akt1/MITF/PU1-NFATc1 pathway [25]. In
this way, irisin can be a mediator of the positive effects of exercise on the skeleton and could constitute a therapeutic target for bone diseases, including osteoporosis [26,27].
Conclusions
Figure 4. Irisin immunoreactivities in the skeletal muscle tissue.
Avidin-biotin immunoperoxidase technique, anti-irisin
mouse antibody, Mayer’s hematoxylin counterstain,
bar=120 micrometer.
irisin activating transcription factor 4 expression there was
seen to be a positive effect on osteoblasts by an increase in
the activity and differentiation of bone building cells [20–22].
Anastasilakis et al. reported a relationship between previous
osteoporotic fractures and low irisin levels in the circulation of
postmenopausal females with low bone mass [9]. In an in vitro
study by Colaianni et al., it was shown that irisin increased osteoblast differentiation via the Wnt-b-catenin signaling pathway and osteoclast differentiation was prevented by the suppression of the RANK/RANKL pathway [23]. In another study,
Zhang et al. reported that irisin facilitated osteoblast differentiation in osteoblastic cells and inhibited osteoclast differentiation by suppressing the c1 nuclear factor kappa B ligand
(RANKL)/receptor activator of active T cells (NFAT) in Raw264.7
The results of this study showed that irisin levels in the blood
increased in the fracture union process, and as irisin receptors
were in human bone tissue, fracture union was affected. This
study could form the basis of future research. As the presence
of irisin has been shown in fracture union, perhaps in future
studies, non-union could be eliminated with compensatory
irisin hormone applied to bones with non-union of fractures.
Conflict of interests
None.
Acknowledgments
We thank Professor Dr. Oğuz Kul from Kirikkale University,
Faculty of Veterinary, Department of Pathology, who provided
assistance in immunohistochemically evaluation in this study.
References:
1. Cruess RL, Dumont J: Fracture healing. Can J Surg, 1975; 18: 403–13
2. Simmons DJ: Fracture healing perspectives. Clin Orthop Relat Res, 1985;
200: 100–13
11. Guo B, Zhang ZK, Liang C et al: Molecular communication from skeletal muscle to bone: A review for muscle-derived myokines regulating bone metabolism. Calcif Tissue Int, 2017; 100(2): 184–92
3. Marsell R, Einhorn TA: The biology of fracture healing. Injury, 2011; 42(6):
551–55
12. Lane JM, Sandhu HS: Current approaches to experimental bone grafting.
Orthop Clin North Am, 1987; 18: 213–25
4. Einhorn TA, Lane JM: Significant advances have been made in the way surgeons treat fractures. Clin Orthop Relat Res, 1998; 355: 2–3
13. Serbest S, Tiftikci U, Tosun HB et al: Is there a relationship between fracture healing and mean platelet volume? Ther Clin Risk Manag, 2016; 12:
1095–99
5. Calori GM, Albisetti W, Agus A et al: Risk factors contributing to fracture
non-unions. Injury, 2007; 38: 11–18
6. Zeckey C, Mommsen P, Andruszkow H et al: The aseptic femoral and tibial shaft non-union in healthy patients – an analysis of the health-related quality of life and the socioeconomic outcome. Open Orthop J, 2011; 5:
193–97
14. Calori GM, Albisetti W, Agus A et al: Risk factors contributing to fracture
non-unions. Injury, 2007; 38: 11–18
15. Einhorn TA: The science of fracture healing. J Orthop Trauma, 2005; 19: 4–6
16. Liu P, Cai M: Leptin influences healing in the sprague dawley rat fracture
model. Med Sci Monit, 2017; 23: 258–65
7. Bostrom P, Wu J, Jedrychowski MP et al: A PGC1-alpha-dependent myokine that drives brown-fat-likedevelopment of white fat and thermogenesis. Nature, 2012; 481(7382): 463–68
17. Sanchis-Gomar F, Alis R, Pareja-Galeano H et al: Circulating irisin levels are
not correlated with BMI, age, and other biological parameters in obese and
diabetic patients. Endocrine, 2014; 46(3): 674–77
8. Timmons JA, Baar K, Davidsen PK, Atherton PJ: Is irisin a human exercise
gene? Nature, 2012; 488: E9–10
18. Ensrud KE, Ewing SK, Fredman L et al: Circulating 25-hydroxyvitamin D levels and frailty status in older women. J Clin Endocrinol Metab, 2010; 95:
5266–73
9. Anastasilakis AD, Polyzos SA, Makras P et al: Circulating irisin is associated with osteoporotic fractures in postmenopausal women with low bone
mass but is not affected by either teriparatide or denosumab treatment
for 3 months. Osteoporos Int, 2014; 25: 1633–42
10. Yan J, Liu H-J, GuoW-C, Yang J: Low serum concentrations of irisin are associated with increased risk of hip fracture in Chinese older women. Joint
Bone Spine, 2017 [Epub ahead of print]
This work is licensed under Creative Common AttributionNonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)
19. Murad MH, Elamin KB, Abu Elnour NO et al: Clinical review: The effect of
vitamin D on falls: A systematic review and metaanalysis. J Clin Endocrinol
Metab, 2011; 96(10): 2997–3006
20. Colaianni G, Grano M: Role of Irisin on the bone–muscle functional unit.
Bonekey Rep, 2015; 4: 765
4282
Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System]
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Serbest S. et al.:
The irisin hormone profile and expression in human bone tissue…
© Med Sci Monit, 2017; 23: 4278-4283
CLINICAL RESEARCH
21. Colaianni G, Mongelli T, Colucci S et al: Crosstalk between muscle and bone
via the muscle-myokine irisin. Curr Osteoporos Rep, 2016; 14: 132–37
24. Zhang J, Cheng J, Tu Q, Chen JJ: Effects of irisin on bone metabolism and
its signal mechanism. J Bone Miner Res, 2013; 28 (Suppl. 1): SA0185
22. Colaianni G, Cuscito C, Mongelli T et al: The myokine irisin increases cortical bone mass. Proc Natl Acad Sci USA, 2015; 112(39): 12157–62
25. Zhang J, Wu Y, Yu L et al: Exercise strengthens bone through myokine irisin. J Bone Miner Res, 2012; 27 (Suppl. 1): 1222
23. Colaianni G, Cuscito C, Mongelli T et al: Irisin enhances osteoblast differentiation in vitro. Int J Endocrinol, 2014; 2014: 902186
26. DiVasta AD, Gordon CM: Exercise and bone: where do we stand? Metabolism,
2013; 62: 1714–17
27. Qiao YQ, Nie Y, Ma YX et al: Irisin promotes osteoblast proliferation and
differentiation via activating the MAP kinase signaling pathways. Sci Rep,
2016; 6: 18732
This work is licensed under Creative Common AttributionNonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)
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