The Microdamage and Expression of Sclerostin in Peri-implant Bone under One-time Shock Force Generated by Impact
ScienTiFic REPORTS |
The Microdamage and Expression of Sclerostin in Peri-implant Bone under One-time Shock Force Generated by Impact
OPEN Osseointegration is the key to implant stability and occlusal support. Biomechanical response and remodeling of peri-implant bone occurs under impact loading. Sclerostin participates in bone formation and resorption through Wnt and RANKL pathways. However the mechanism of microdamage and expression of sclerostin in peri-implant bone under impact load is still unclear. In present study, specific impact forces were applied to the implants with favorable osseointegration in rabbits. The microdamage of peri-implant bone and the expression of sclerostin, β-catenin and RANKL during the process of bone damage and remodeling were investigated by micro-CT, histology, immunofluorescence and RT-qPCR analysis. Interface separation and trabecular fracture were found histologically, which were consistent with micro-CT analyses. Throughout remodeling, bone resorption was observed during the first 14 days after impact, and osseointegration and normal trabecular structure were found by 28 d. The expression of sclerostin and RANKL increased after impact and reached a maximum by 14 d, then decreased gradually to normal levels by 28 d. And β-catenin expression was opposite. Results indicated that sclerostin may involve in the peri-implant bone damage caused by impact and remodeling through Wnt/β-catenin and RANKL/RANK pathways. It will provide a new insight in the diagnosis and treatment for patients suffering impact.
Implant dentures are supported by dental implants, which are inserted into the alveolar bone and connect directly
to the bone without intervening soft tissue, following osseointegration1–3. Osseointegration is very important for
an implant to maintain its stability and provide occlusal support4. Occlusal force is transmitted from the implant
to the alveolar bone by functional loading during mastication. The biomechanical response of the bone to proper
occlusal force determines the osseointegration of the implant and bone remodeling after implantation5–8. A failed
osseointegration would decrease the stability of the implant and cause the failure of the restoration.
Implant dentures may suffer from impact, particularly during activities such as sports and physical training.
Impact is a complex phenomenon that occurs when two or more bodies undergo a collision9. Characteristics of
impact are very brief duration, high force levels reached, rapid dissipation of energy and large accelerations and
decelerations present9. When the pulse duration of the impact load is in the range of microseconds, the force is
transmitted and reflected, usually in the form of stress waves, through the composite structure consisting of the
implant, the interface and the alveolar bone, in which each has the different impedance. If the dental implant
and alveolar bone are unable to buffer the impact through deformation of their structure, the osseointegration
at the implant-bone interface and the bone microstructure around the implant would be damaged. However, the
mechanism of trauma is still unclear.
The bone is a functional remodeling biomaterial. Bone remodeling is usually defined as a process where
bone gradually alters its morphology when mechanical signals are sensed and conducted by osteocytes through
the lacunar-canalicular system to adapt to the biomechanical environment10–16. Bone remodeling includes two
opposing processes: resorption and deposition. Sclerostin secreted by osteocytes is a glycoprotein encoded by
the SOST gene17, 18. Several studies have reported that sclerostin plays important roles in the anabolic response
of bone to mechanical stimulation through the Wnt/β-catenin pathway and the catabolic response of bone to
mechanical stimulation through the RANK/RANKL pathway19–22. However, the expression of sclerostin in
peri-implant bone following impact is still unclear, and the correlation between the expression of sclerostin and
bone remodeling needs to be studied.
In order to address these questions, we established an animal model with implants under impact load. The
characteristics of the microdamage and the expression of sclerostin, β-catenin and RANKL were investigated to
explore the mechanism of impact damage and the subsequent repair of bone around the implant. These results
could help in the evaluation of alveolar bone trauma around implants and provide guidance for the management
of dental implants following impact.
Materials and Methods
Animal Studies. Animal Preparation. Thirty 20- to 22-week-old female New Zealand rabbits weighting
approximately 3.5–4.0 kg were purchased from the animal centre of the Fourth Military Medical University
(Shaanxi, China) and housed for 1 month. The animals were allowed access to water and pelleted commercial diet
ad libitum. The weights were monitored weekly in whole research.
Implant Insertion. Femoral distal condyles were chosen as a standard site to insert implants as previously
described23, and the implantation surgeries were performed under general anesthesia. A skin incision was made
over the lateral femur-tibia joint, the femoral distal condyle was exposed after the muscle and fascia tissues were
peeled away. The implant, which was 2.3mm in diameter and 5 mm in length, was inserted into the prepared
hole with an implant placement torque of 5–10 N·cm. The incision was closed with sutures in a single layer. An
intramuscular injection of penicillin (200 thousand IU kg−1) was given to each animal once daily for 5 days
postoperatively to prevent infection, and the animals were allowed unrestricted activity.
Impact Loading. Three months later, impact experiments were performed after favorable osseointegration
occurred. The animals were randomly divided into two experimental groups suffering impact load and one
control group without impact. The impact protocol24, 25 is illustrated in the Supplementary Fig. S1. During impact
loading, a 25 g or 50 g impact mass was dropped from a height of 1 m onto a pressure sensor attached to an
implant. The masses were arrested immediately to prevent multiple impacts. The voltage signals received from the
pressure sensor were magnified by a charge amplifier (10N/V), and the impulse waveforms were captured with
an oscilloscope. Animals in experimental groups received a final impact load of 500 or 1000 N in 0.2 ms in order
to observe the trabecular microdamage around the implant, meanwhile, prevent the detachment of the implant
based on the results of our pre-experiments (Fig. 1). Then, animals were sacrificed on day 0, 7, 14, or 28, and the
implants and surrounding bone were harvested for micro-CT, histomorphometry, immunofluorescence (IF) and
RT-qPCR analysis. The sample sizes at each time point are illustrated in Table 1. In the experiment, one femur
was dissected for micro-CT, histomorphometry and immunofluorescence randomly, and another femur from the
same animal was used for RT-qPCR. So the total sample size was 60, and n (Table 1) indicates the numbers of the
femurs from different rabbits. In order to investigate the characteristics of microdamage, the samples of the two
experimental groups at 0d were only used for micro-CT and histomorphometry analysis. The 1000N group was
chosen for the study of peri-implant bone remodeling following impact due to its more typical microdamage.
The sample size of the control group for statistical analysis was the sum of that at every time point, for the control
group was not treated with an impact load.
Micro-computed Tomography Analysis. Bones containing implants were dissected, fixed in 4%
paraformaldehyde and scanned by micro-CT (Inveon Research Workplace 2.2, Siemens Inc., Germany) at a resolution
of 19.64 μm with an X-ray voltage of 80 kV and a current of 50 μA. The scanner software (Inveon Acquisition
Workplace 2.2, Siemens Inc., Germany) was applied for image reconstruction and analyses. The bone around
the implant with 0.5 mm thickness and 5.5 mm length (including 5 mm length of implant and 0.5 mm length of
bone under the implant) was chosen as the region of interest (ROI) in each sample for microstructure analyses26
Micro-CT & Histology & IF/RT-qPCR
Micro-CT & Histology & IF/RT-qPCR
Micro-CT & Histology & IF/RT-qPCR
Micro-CT & Histology & IF/RT-qPCR
(Fig. 2a), and parameters such as trabecular bone volume/total volume (Tb.BV/TV, %), trabecular number (Tb.N,
mm−1), trabecular thickness (Tb.Th, mm), trabecular separation (Tb.Sp, mm) and bone mineral density (BMD,
mg/cc) were measured.
Histomorphometry and Immunofluorescence. Undecalcified samples were prepared for subsequent
histomorphometry studies. Samples were cleaned of soft tissue, dehydrated in graded alcohols and embedded in
methyl methacrylate. Then, sections of 200μm in thickness were cut longitudinally, ground to 20 μm and stained
with Van Gieson (VG) and Hematoxylin-Eosin (H&E) for microscopic examination. Images were observed and
captured with a light microscope (DMI6000, Leica Inc., Germany).
For sclerostin immunofluorescence, sections were washed in phosphate buffered saline (PBS) and blocked in
goat serum for 10 min at room temperature. Endogenous peroxidases were quenched with 3% H2O2, and sections
were incubated in rabbit sclerostin polyclonal antibody (Bioss Inc., China) for 24 h at 4 °C. After washing in PBS,
sections were incubated with secondary antibody labeled with FITC. Images were captured with a confocal laser
scanning microscope (CLSM) (Fluo View FV-1000, Olympus Inc., Japan). Fluorescence intensity was measured
in five areas of each section using the CLSM software, and the average was calculated for statistical analyses.
RNA Extraction and RT-qPCR. Bone tissue at the base of the implant was snap frozen and crushed in
liquid N2. According to the manufacturer’s instructions, total RNA was extracted using Trizol reagent and was
dissolved in DEPC H2O. cDNA was synthesized using a 10 μl reverse transcription reaction mixture composed
of 0.5 μg total RNA, 2 μl 5× PrimeScriptTM Buffer, 0.5 μl PrimeScriptTM RT Enzyme Mix I and 0.5 μl random
primer. Partial sequences of SOST, β-catenin, RANKL and β-actin in the reverse transcribed cDNA were
amplified using a fluorescence RT-qPCR instrument (RG-3000, Gene Inc., Australia). The forward and reverse primer
sequences used for amplification are listed in Supplementary Table S2. Relative mRNA expression levels were
standardized to β-actin expression for quantified analyses using the ∆Ct method.
Ethics. This study was carried out in strict accordance with the recommendations in the Guide for the Care
and Use of Laboratory Animals of the Animal Center of the Fourth Military Medical University. The protocol
was approved by the Laboratory Animal Care & Welfare Committee, Fourth Military Medical University (No:
20150201). All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to
Statistical Analysis. The data were presented as mean± standard deviation and analyzed using SPSS19.0
(IBM Inc., USA) for one-way ANOVA statistical analyses. Statistical significance was considered as p < 0.05.
Bone-implant Osseointegration. Implants inserted into the rabbit femoral distal condyle were stable by
3 months after implantation. Micro-CT scan was performed to study the osseointegration at this time. The image
showed that the areas around the implant were full of trabeculae. The intact and thick trabeculae were arranged
regularly and distributed around and at the bottom of the implant. A good interface was observed between the
implant and the bone (Fig. 2a).
The Microdamage of Bone around the Implant and the Failure of the Implant-bone
Interface. Histological Study. No obvious change in the cortical bone around the implant was observed after
impact (Fig. 3a). However, histological study showed that the osseointegration failed at the interface between the
bone and the implant. Fractured trabeculae were observed around and at the bottom of the implant.
The implant and bone stained with VG are shown in Fig. 3b. In this figure, new bone appeared around the
implant threads and at the bottom of implant by 3 months after insertion, and trabeculae were regularly arranged
in the control group, which indicated that the osseointegration was favorable. After impact loading, bone tissues
in some areas were broken away from the implant, and the trabeculae at the bottom of implant were fractured.
This demonstrated that the interface between the implant and the surrounding tissues was incomplete in the
Similar microdamage in peri-implant bone is shown in Fig. 3c with H&E staining. Trabeculae around the
implant were fractured and disordered under impact loading, while trabeculae in the control group were
regularly arranged. Damage photography of interface and surrounding bone was more severe in group 1000 N than
in group 500 N.
Micro-CT Analyses. The image of micro-CT shows similar failed osseointegration and fractured trabeculae
around and at the bottom of the implant (Fig. 2b). To describe the microdamage quantitatively, micro-CT analyses
were performed in this study. The results demonstrated that BV/TV and Tb.Th decreased, while Tb.Sp and BMD
increased compared with the control group (p < 0.05). The results indicated that the bone mass decreased after
impact damage, but BMD increased when the trabeculae were fractured and compressed. There were statistically
significant differences in BV/TV and Tb.Sp between the test groups (p< 0.05), which indicated that the damages
were different under impact load (Fig. 2c).
The Peri-implant Bone Healing Process: From Impact Damage to Remodeling. To investigate the
remodeling of peri-implant bone after impact damage, bone structures along the interface between the implant
and the bone were studied at different time points. Cancellous bones around the implant are shown in Fig. 4.
At day 7 after impact, the gap between the implant and the bone was observed in VG stained sections and the
structure of the trabeculae stained with H&E was broken (Fig. 4). The gaps attributed to impact damage were
extended, and the trabeculae were scattered at day 14, which indicated that bone absorption may have occurred
in this area. By the last time point, the gaps disappeared, and regularly arranged trabeculae were observed. There
was no significant difference in the microstructure compared with the control group, which demonstrated that
the osseointegration had completely reformed by day 28.
Micro-CT scanning and analyses were applied to quantify the changes in trabecular microstructure (Fig. 5).
It could be observed that BV/TV, Tb.N decreased while Tb.Sp increased 14 days after impact (p < 0.05). And
BMD decreased at day 7 and 14 after temporary increasing at day 0. There was no significant difference in these
parameters between the experimental and control group by day 28. These results were consistent with the
Immunofluorescence Staining of Sclerostin in Trabeculae after Impact. Immunofluorescence
staining was used to study the expression of sclerostin during the process of remodeling following bone impact
damage. Immunofluorescence images are shown in Fig. 6a. In this figure, the sclerostin protein staining is
indicated by the green fluorescence points, and sclerostin protein expression is measured as fluorescence intensity
in Fig. 6d. From the results (Fig. 6d), it can be observed that the expression of sclerostin was higher than that
in the control group on days 7 and 14 after impact (p < 0.05). During the process of remodeling after damage,
it increased significantly from day 7 to 14 (p < 0.05). However, there was no obvious difference in expression of
sclerostin between the experimental and control group by day 28.
The Expression of SOST, β-catenin and RANKL mRNA in Bone Tissue around the Implant after
Impact. RT-qPCR was used to quantify the expression of SOST and to correlate sclerostin expression with
β-catenin and RANKL. Figure 6b shows the extracted total RNA in agarose gel electrophoresis and Fig. 6c
illustrates the SOST PCR amplification curve. The expression of SOST, β-catenin and RANKL mRNA was quantified
using the value of 2−∆Ct in Fig. 6e. In the figure, the expression of SOST and RANKL mRNA increased rapidly
after impact compared with the control group, and the values reached a maximum at day 14 (p < 0.05). Then, the
expression of SOST and RANKL mRNA decreased gradually to the control group levels at day 28. The expression
of SOST mRNA was similar to sclerostin protein expression based on the immunofluorescence staining results.
The expression of β-catenin mRNA was opposite to that of SOST and RANKL mRNA, and it did not return to the
level of the control group completely until day 28 (p < 0.05).
When an implant denture experiences an impact load, there will be microdamages in the peri-implant bone and
failure of the implant-bone interface. In addition, the expression of proteins will be changed to modify the bone
structure correspondingly. In this study, implants were inserted into the femoral distal condyles of New Zealand
white rabbit, and an impact load was applied to the implants after osseointegration occurred. The features of bone
damage around the implant and the remodeling of bone were studied through micro-CT analyses and hard tissue
slicing with VG and H&E staining. Further, the expression of sclerostin, β-catenin and RANKL were analyzed by
immunofluorescence and RT-qPCR during the process of remodeling following bone damage. The results showed
that there was no significant change in the cortical bone around the implant, but debonding at the interface and
impaired osseointegration in specific areas around the implant were observed. Microdamage in cancellous bone
was also observed around the implant. The expression of sclerostin, β-catenin and RANKL correlated with the
bone damage and process of remodeling. These data indicate that sclerostin may be involved in bone formation
and resorption caused by impact through regulating the Wnt/β-catenin and RANKL/RANK pathways. The results
of this study reveal the characteristics of the impact damage to the bone around the implant, and provide a
reference for damage assessment and clinical treatment of patients with impact loading.
Impact load is a transient load that transmits through or reflects between an implant and bone in stress waves
when the pulse duration of the load is in the microsecond range. The stress waves not only spread through the
implant-bone interface but also reflect at the interface when they pass through the anisotropic composite
structure of the implant and bone9, 27. The impact energy is spread and dissipated quickly along the interface, which
may cause the failure of the implant-bone interface and surrounding bone when the energy cannot be absorbed
and buffered by this composite structure. However, the characteristics of impact damage in the bone around the
implant have not been reported. In this study, microdamage of peri-implant bone under impact was investigated.
The results showed that the bone tissue was broken away from the implant and fractured trabeculae around
significant difference in the bone mass and BMD compared with the control group. The microstructure of the
peri-implant bone returned to normal morphology, and a favorable osseointegration reformed. These results
indicate that bone formation is dominated in the process of bone remodeling from day 14 to 28. From these results, it
could be observed that the characteristics of the impact damage of bone around the implant were different than
that of clinically normal surgical insertion, but the processes of bone remodeling were similar for the formation
of the osseointegration around the implant. Therefore, the principles for treatment after insertion surgery could
be used as a reference for the management of impact-loaded patients.
Sclerostin encoded by the SOST gene is a glycoprotein secreted by osteocytes28–30. Previous studies have
reported that there are alterations in its expression in animal models of bone defects or fractures31–34. Changes
in the microstructure could cause fluid flow in the bone matrix, which would change the mechanical
environment surrounding the osteocytes11. This mechanical signal is sensed and conducted by osteocytes though the
lacunar-canalicular system15, 35, 36, and then sclerostin is secreted by osteocytes to adapt to the biomechanical
environment37–39. In this study, the expression of sclerostin continued to increase after impact and reached a
maximum at day 14. Then, it decreased gradually to normal levels at day 28. From the results above, it can be observed
that the expression of sclerostin is related with the process of bone damage and remodeling.
Other studies have reported that sclerostin is an effective antagonist of bone formation40–42. The nodular
cystine domain in sclerostin allows the protein to competitively bind to the Wnt co-receptor LRP5/6 of the
osteoblast. This reduces β-catenin nuclear translocation. As a result, osteoblast activity is decreased, and new bone
formation and mineralization are inhibited43, 44. Meanwhile, sclerostin could promote the secretion of RANKL
from osteoblasts, which would stimulate the differentiation of osteoclasts from precursors and accelerate bone
resorption22. The relationship of sclerostin with the Wnt/β-catenin and RANKL/RANK pathways is illustrated in
In this study, the expression of RANKL mRNA continued to increase with the increase of SOST, while
β-catenin decreased over the first 14 days after impact. Then, the expression of RANKL and SOST mRNA
decreased gradually to the level of the control group, while, in contrast, β-catenin increased by day 28. The
aforementioned changes in protein expression and mRNA levels were consistent with the behavior of peri-implant
bone absorption and formation in the micro-CT and histological results. These results suggest that sclerostin
may be involved in both bone anabolism and catabolism in response to mechanical stimulation by regulating the
Wnt/β-catenin and RANKL/RANK pathways, respectively. The lower expression of β-catenin than that of control
group at day 28 in this study indicated that it didn’t reach the normal value. The main reason might be that it
will take longer for the cancellous bone to return to normal completely, and β-catenin may continue to increase
during this process.
In future experiments, the expression of β-catenin and RANKL should be investigated when the expression of
sclerostin is down-regulated, and the change in bone microstructure should also be analyzed to determine that
whether sclerostin serves as a potential target to regulate bone remodeling under damage through Wnt/β-catenin
and RANKL/RANK pathways, which would provide a new therapeutic target for patients with dental implant to
improve the osseointegration by regulating the expression of sclerostin.
This study was supported by National Natural Science Foundation of China (Contract no. 11672327).
D.X. and X.H. contributed to the design of the study. D.X., L.Z., A.B., F.F. and D.C. contributed to the acquisition,
collection and assembly of data. D.X. and X.H. contributed to the statistical analyses of data and wrote the main
manuscript text. D.X., X.H., W.Y. and L.K. contributed in revising the manuscript. All authors reviewed the
manuscript and approved the final version to be submitted.
Competing Interests: The authors declare that they have no competing interests.
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Additional Information Supplementary information accompanies this paper at doi:10.1038/s41598-017-06867-9