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Orthopaedic
Article Surger y
Journal of Orthopaedic Surgery
25(2) 1–7
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conjugated linoleic acid in rats DOI: 10.1177/2309499017718910
journals.sagepub.com/home/osj

Zhongshu Shan1, Zong-Ping Luo2, Xiaozhong Shen1

and Liang Chen2

Abstract
Background: The ability of fracture healing in Tibetans is significantly superior to Chinese Hans, which may attribute to
factors from diet, altitude to physical conditions. Conjugated linoleic acid (CLA) is an important ingredient in Tibetan diet,
playing a role in antioxidation, antiatherosclerosis, and decrease in body fat accumulation. Methods: This study further
quantified CLA effect in fracture healing in rats using combined structural evaluation (X-ray and micro-computed
tomography), biomechanical test, and histological examination. Results: CLA could promote fracture healing with
quicker development of trabecular connection, network and thickening and were more active at the stage of bony union
and remodeling. The load to failure could reach 78.12 + 10.03 N, 41.4% stronger than the control by week 6 (p ¼
0.0209). Conclusions: CLA improved the quality and mechanical strength of fracture healing in rats callus. The infor-
mation may offer insight in development of new therapeutic strategies of fracture healing for general populations beyond
Tibetans.

Keywords
conjugated linoleic acid, fracture, fracture healing, rats

Introduction inducible factor, a pathway in bone development, increases

It has been observed clinically that fracture healing in Tibe-
tans is significantly better than Chinese Hans.1–3 Under-
standing the underlying mechanism may offer insight in
development of new therapeutic strategies for fracture heal-
ing in Hans and other non-Tibetans.
The possible reasons of the superior fracture healing in
Tibetans may be summarized as follows: stronger physical
conditions in both musculoskeletal and immune systems,
genetic factors, high elevation environment (extra ultraviolet
intake, hypoxia, and significant outdoor activities), and diet.
Muscle power of hand grip and legs has been shown to be
positively associated with the increase of bone mineral den-
sity (BMD) at the lumbar spine and hips.4 Studies have
reported that fracture healing potential is affected by the
patient genome, and variations in the IL1B and NOS2 genes
may contribute to delayed fracture-healing.5 Ultraviolet
radiation B produces significant alteration to vitamin D lev-
els via serum 25(OH)D, which in turn increases BMD and
reduces the risk of fracture.6,7 Activation of the hypoxia
bone modeling events through cell-nonautonomous mechan-
isms to coordinate the timing, direction, and degree of new
blood vessel formation in bone.8 Therapeutic exercise is
often recommended as a non-pharmacologic treatment of
vertebral fractures to reduce pain and restore functional
movement.9 To date, however, it remains undefined if the
diet can affect the fracture healing significantly.
The daily diet of Tibetans differs from that of Hans.
The primary differences include yak butter (73.0 g/day
1
Department of Orthopedic Surgery, People’s Hospital of Qinghai
Province, Xining, Qinghai, People’s Republic of China
2
Department of Orthopedic Surgery, The First Affiliated Hospital of
Soochow University, Suzhou, Jiangsu, People’s Republic of China
Corresponding author:
Liang Chen, Department of Orthopedic Surgery, The First Affiliated
Hospital of Soochow University, 188 Shizi St. Suzhou, Jiangsu 215006,
People’s Republic of China.
Email: chenliang1972@sina.com

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2 Journal of Orthopaedic Surgery 25(2)
in Tibetans vs. 3.7 g/day in Hans), beef and lamb (226.2
g/day in Tibetans vs. 115.7 g/day in Hans), vegetables
and fruits (217.8 g/day in Tibetans vs. 514.3 g/day in
Hans).10 As the main portion of daily food intake of
Tibetans, yak butter, beef and lamb are all high in the
content of multifunctional fatty acids.11 For example,
yak butter consists of conjugated linoleic acid (CLA,
17.2 mg/g), linolenic acid and arachidonic acid (9.07
mg/g), eicosapentaenoic acid (0.56 mg/g), and docasa-
hexaenoic acid (0.28 mg/g).12
It has been known that CLA functions include anticancer,
antioxidation, antiatherosclerosis, lessening markers of cata-
bolism, enhancement of the immune system, and decrease in
body fat accumulation.13 Studies suggested that dietary CLA
suppressed the production of prostaglandin E2 (PGE2) in
serum and spleen,14 CLA might reduce PGE2 level by limit-
ing arachidonic acid.15 Anticarcinogenic effects of CLA indi-
cated a modifying role in peroxisome proliferator–activated
receptor a (PPARa) action.16 Moreover, CLA actions on fat
and energy metabolism could, in part, be directed through
changes in both PPARa and PPARg.17,18 Recent studies fur-
ther indicated the role that CLA played in osteoblast-like
cells. CLA increased levels of osteocalcin and activity of
alkaline phosphatase (markers of bone formation) in murine
MC3T3-E1 osteoblast-like cells in culture, providing the
involvement in osteoblast-like cells.15,19–21
Fracture healing is a complicated biological repairing
process involving the coordination of a variety of differ-
ent processes. Healing is typically characterized by four
overlapping stages: the initial inflammatory response,
soft callus formation, hard callus formation and initial
bony union, and remodeling.22 Studies have shown that
PGs are multifunctional regulators of bone metabolism
stimulating both bone resorption and formation.23 Evi-
dences indicate an important role of PPARg in bone
metabolism. Heterozygous PPARg-deficient (PPARg
þ/–) mice exhibit enhanced bone formation with
increased osteoblastogenesis.24
As CLA is closely related to PGs, PPARg, and
osteoblast-like cells, we hypothesized that CLA can signif-
icantly regulate fracture healing. In this study, we devel-
oped a rat fracture model to examine this hypothesis, and
used structural, biomechanical and histological techniques
to evaluate the outcomes of fracture healing.
Materials and methods
Animal model
The experiment was performed on Sprague-Dawley rats (n
¼ 30, 200 + 15 g in weight; approved by the institutional
animal committee). The specimens were divided randomly
into two groups equally: A. CLA (10 g CLA/kg diet) with
the basal diet, and B. control with the basal diet only. The
rats were harbored individually in cages at 20  C with
access of water and foods.
Figure 1. Illustration of the rat fracture model (the arrow
pointed to the fracture site).
Surgical technique
The tibial fracture was created following a standard proce-
dure.25,26 In brief, after anesthesia via the intramuscular
injection of 10% chloral hydrate (0.3 ml/100 g), rats were
placed in supine position. The left lower extremity was
shaved, cleaned, and then properly rotated to abduction
position to ensure superior migration of fibula. Infrapatellar
tibia was palpated and accessed via anteromedial incision.
After the skin and cutaneous maximus muscle were dis-
sected, the tibia was reached through the tibialis cranialis,
peroneus longus, and brevis muscles. The tibial tuberositas
caudal to the patellar joint was palpated and a 0.8-mm
K-wire advanced until the tarsal joint was delivered. The
osteotomy line was performed manually using 2-mm osteo-
tome from 15 mm caudal to the patellar joint resulting in
fracture (Figure 1). After surgery, rats were assessed with
respect to wound site infection and dehiscence daily.
Structural evaluation
Fracture healing process was first evaluated radiologically
at weeks 2, 4, and 6. The degree of union was scored using
a five-point system: 0—no callus, 1—little-to-moderate
callus, 2—profuse callus tissue, 3—bridging periosteal cal-
lus, 4—mature callus with interfragmentary bridging, and
5—callus resorption after solid union.27 The scores were
evaluated by three surgeons independently and averaged.
The healing process was also examined by micro-CT
scanning of 17.51 mm resolution at 4 and 6 weeks post-
operation (Skyscan 1176, Bruker, Kontich, Belgium).
Acquired tomographic images were at the energy settings
of 65 kV and 385 mA, integration time of 250 ms, with 0.7
rotation between frames .The fracture line was identified
after reconstruction by simultaneously viewing multiple
orthogonal slices. The analysis determined the average
cross-sectional area (CSA) of the callus and the corre-
sponding BMD. The bone strength index (BSI) of the frac-
ture callus was then calculated from 3.5 mm proximal to
3.5 mm distal of the fracture line as CSA  BMD.28
Shan et al.
Figure 2. Radiographic evaluation at weeks 2, 4, and 6. (a) A typical radiographic image showing the fracture healing with CLA
intervention at week 6 (the arrow pointed to the fracture site). (b) The radiographic grade showing significant difference between the
CLA and control groups at weeks 4 and 6 (p < 0.05). CLA: conjugated linoleic acid.
Biomechanical test
29
Fracture healing strength was tested at week 6. The rats
were killed using high-dose anesthesia of 10% chloral
hydrate, and the k-wire in each tibia was kept to protect
the callus tissue. The entire tibia was removed by being
detached from its surrounding soft tissue. Between steps,
the tibia was soaked in saline. The mechanical properties of
the tibia were measured using a three-point bending test
and the load to failure was documented.30
Histological examination
Fracture healing was further examined at week 6 histolo-
gically. Fracture calluses were decalcified and embedded in
paraffin as described previously.31 Sagittal sections (5 mm)
were cut in standardized plane using a microtome (Eclipse
80i, Nikon, Japan) and stained with hematoxylin and eosin
for basic morphology, or alcian blue and nuclear fast red for
analysis. A scoring system was used for quantification. The
system scored zero for nonunion, one for healing with com-
plete fibrous callus, two for some calcification with fibrous
tissue, and three for completely calcified callus or new
bone formation.32 The scores were evaluated by a pathol-
ogist independently.
Statistical analysis
Mean and standard deviation in each group were calcu-
lated. The Wilcoxon signed-rank test was performed with
a significant difference among two groups (p < 0.05).
Results
Structural evaluation
Radiological results indicated that fracture lines were
clearly visible and fracture was not bridged in both CLA
3
and control groups at week 2 with the evaluation scores
of 1.3 + 0.9 and 1.0 + 0.5, respectively (p ¼ 0.4058;
Figure 2). At week 4, the fracture line became obscure in
both groups. The CLA group appeared to have more callus
formation with connection than the control, they were
different statistically in evaluation scores (3.7 + 0.7 in
CLA and 3.0 + 0.5 in the control, p ¼ 0.0197). At week 6,
the fracture line in the CLA group vanished, and 40%
specimens had complete callus absorption. The evaluation
score was 4.4 + 0.6. In contrast, the control group still
had the vague fracture lines with matured callus and sig-
nificant callus bridges. The evaluation score was 3.4 +
0.5 (p < 0.001).
The micro-CT images indicated that at week 4, both
groups had no significant difference in CSA (p ¼
0.1172), BMD (p ¼ 0.6015) except for BSI (p ¼ 0.0472;
Figure 3). However, at week 6, the CLA group had 14.05
+ 3.13 mm2 in CSA, 0.98 + 0.04 mg/mm3 in BMD and
13.73 + 2.67 mg/mm in BSI, which were significantly
greater than the control (9.38 + 1.87 mm2 in CSA, p ¼
0.0163, 0.86 + 0.78 mg/mm3 in BMD, p ¼ 0.0163, and
8.07 + 1.66 mg/mm in BSI, p ¼ 0.0039).
Biomechanical test
The three-point bending test indicated that the load to fail-
ure of the CLA and the control groups was 78.12 + 10.03
N and 32.34 + 8.99 N, respectively. The difference was
statistically significant (p ¼ 0.0209; (Figure 4)).
Histological examination
Histological results demonstrated the evidence of fracture
healing in the CLA group at week 6 (Figure 5). Under 4
microscope, trabeculae were densely reconnected and
medullary canal was redeveloped. The control group still
showed disorganized and loose trabeculae. Under 10
4 Journal of Orthopaedic Surgery 25(2)

Figure 3. Micro-CT examination at weeks 4 and 6. (a) A typical micro-CT image revealing the fracture healing with CLA intervention at
week 6 (the arrow pointed to the fracture site). (b) The average CSA of callus. (c) The BMD of callus. (d) The BSI of callus. CLA:
conjugated linoleic acid; CSA: cross-sectional area; BMD: bone mineral density; BSI: bone strength index.

Figure 4. Biomechanical strength of fractured tibia 6 from three-

point bending test at week 6.

microscope, the CLA group had matured callus, and woven

bone spicules were observed predominantly with trabecu-
lae significantly thicker than the control. The averaged
score was 2.2 + 0.75 in the CLA group and 0.8 + 0.75
in the control group (p ¼ 0.041).
Figure 5. Histological evaluation at week 6. (a) and (b) were with
the CLA intervention, and (c) and (d) were the control. CLA:
conjugated linoleic acid.

the control at week 6, suggesting the improvement of

Discussion
In the present study, we demonstrated that dietary supple-
mentation with CLA benefited to fracture healing in rats.
Structural evaluation of X-ray indicated that fracture was
healed in 6 weeks with the CLA intervention; and the
evaluation scores were all between 4 and 5, significantly
higher than those in the control (p ¼ 0.002). Micro-CT
examination further showed fracture healing at the micro-
structural level; and the BSI was significantly greater than
overall strength being achieved via reestablishing trabe-
cular connections. Biomechanical test of three-point
bending at week 6 also revealed the improvement of bend-
ing strength after CLA intervention. Histologically, the
CLA group, in comparison with the control, showed the
maturity of healing with solid network connection and
thickening of trabeculae.
The rationale of this study needs to be discussed: 1. The
animal model. The fracture model of rats has been used
Shan et al.
routinely in the laboratory.25 As reported here, the model
served the purpose of showing CLA role in promoting
fracture healing. 2. CLA dose. We used 10 g CLA/kg diet
for intervention. This dose was similar to the typical daily
intake dose of Tibetans.11 CLA intake was confirmed on
one rat with higher serum high density lipoprotein choles-
terol and triacylglycerol levels (0.55 and 0.94 mmol/L)
comparing to the control (0.38 and 0.53 mmol/L) following
the protocol in the literature.33 3. Measurement timing.
During fracture healing of rats, the inflammatory response
takes 2 weeks, callus formation another 2 weeks, and bony
union and remodeling additional 2 weeks.22 Our measure-
ment was therefore chosen at weeks 2, 4, and 6.
CLA is a collective term describing a mixture of posi-
tional and geometric isomers of linoleic acid with conju-
gated double bonds, which may be of cis or trans
configuration at positions 9 and 11 or 10 and 12.34,35 The
cis 9-trans 11 and trans 10-cis 12 isomers of CLA are
routinely found in animal tissues.36 CLA isomers occur
naturally in ruminant food products (beef, lamb, and dairy)
during the process of bacterial biohydrogenation of linoleic
acid in the rumen.34,37,38 The cis 9-trans 11 and trans 10-cis
12 isomers of CLA have been shown to possess biological
activity.39,40 The trans 10-cis 12 isomer of CLA reduces
adipose mass in mice and humans, while the cis 9-trans 11
isomer has anti-inflammatory properties. All of the studies
conducted to date on the functional effects of CLA have
used mixed preparations of CLA with approximately equal
amounts of the bioactive 9, 11 and 10, 12 isomers. In our
study, this mixture of CLA was therefore adopted.
Fracture healing is a process that recapitulates certain
aspects of skeletal development and growth involving a
complex interplay of cells, growth factors, and extracellular
matrix. Repair is typically characterized by four overlap-
ping stages. The initial inflammatory response is coordi-
nated by and involves the secretion of a range of cytokines
and growth factors including platelet-derived growth fac-
tor, fibroblast growth factor-2, vascular endothelial growth
factor, macrophage colony stimulating factor, interleukins-
1 and -639–43 in the initial inflammatory response. These
factors facilitate the migration and invasion of multipotent
mesenchymal stem cells.44,45 The soft callus formation
stage is dominated at the cellular level by chondrocytes
and fibroblasts although the relative proportions of the dif-
ferent cell types can vary between fractures. The hard cal-
lus formation is characterized by high levels of osteoblast
activity and the formation of mineralized bone matrix,
which arises directly in the peripheral callus in areas of
stability, it represents most active period of osteogenesis.
In the final stage of fracture reunion remodeling, the key
cell type involved with resorption of mineralized bone is
the osteoclast. Cytokines that are secreted by osteoblasts,
growth factors, and cytokines are critical for the induction,
survival, and competency of osteoclasts. Our structural
evaluation suggested that the CLA intervention was the
most effective in the stage of bony union and remodeling
5
from week 4 to 6, followed by the (soft and hard) callus
formation stage from week 2 to 4 and the initial inflamma-
tory stage up to 2 weeks (Figure 2). In addition, CLA have
various effects besides fracture healing. A diet containing
CLA reduced body weight and body fat mass in the animal
model as well as in human trials.46,47 CLA administration
with 1% dietary calcium significantly improved total ash
percentage in femurs, suggesting the CLA potential to
improve bone mass.48
As the first step toward systematic investigation of CLA
regulation on fracture healing, this study was limited to the
animal model study. CLA may accelerate healing through
osteoblast differentiation and function, promotion of cal-
cium and phosphate absorption to improve bone strength.
These questions need to be clarified in the future.
Conclusions
The unusual ability of fracture healing in Tibetans stems
from multiple intrinsic and extrinsic factors. This study
suggested the involvement of CLA, a major dietary ingre-
dient in Tibetans, in promoting the fracture healing in rats.
The heal was achieved by quicker development of trabe-
cular connection, network, and thickening. The informa-
tion offered insight in development of new therapeutic
strategies of fracture healing for general populations
beyond Tibetans.
Acknowledgement
This work is partly supported by the Natural Science Foundation
of China under grants of 81320108018, 31270995, 31570943, and
81071450, and the Priority Academic Program Development of
Jiangsu Higher Education Institutions. Authors also thank Dr. Lei
Wang for histological analysis.
Author’s Note
Zhongshu Shan and Zong-Ping Luo—these authors contribute
equally.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
Funding
The author(s) received no financial support for the research,
authorship, and/or publication of this article.
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