The aim of this study was to examine the level and basic characteristics of cell-derived microparticles (MPs) in the cyst fluids of odontogenic keratocysts (OKCs). For this purpose, MPs from the cyst fluids (CFMPs) of OKCs were purified by a classic differential centrifugation method and characterized by a transmission electron microscope and fluorescence microscope. Flow cytometric analysis was used to determine the size, concentration and cellular origins of the CFMPs. Moreover, the expression level of receptor activator for nuclear factor-κB ligand in the OKCs was evaluated by immunohistochemical staining and then analyzed for its correlation with the concentration of CFMPs by Spearman's rank correlation test. In addition, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and tartaric-resistant acid phosphatase (TRAP) staining were performed to examine the osteoclastogenesis of mouse bone marrow-derived macrophages (BMMs) in response to CFMPs. The results revealed that the levels of total CFMPs were significantly elevated in OKCs compared with dentigerous cysts (DCs) and radicular cysts (RCs). In addition,
Odontogenic cysts of the jaws, which are space-occupying lesions, are generally classified into two groups: inflammatory cysts and developmental cysts (
Cell-derived microparticles (MPs) are small (100-1,000 nm in diameter) membrane-enclosed vesicles secreted from cells by direct budding from the plasma membrane (
Since odontogenic cysts are directly immersed in the milieu of cyst fluids, the epithelial cells or surrounding cells of odontogenic cysts may release MPs into the cavity. In the present study, the levels of total and subtype MPs derived from the cyst fluids (CFMPs) were examined and compared among patients with DCs, RCs and OKCs. In addition, the potential clinical significance and biological function of CFMPs were investigated.
This study was approved by the Review Board of the Medical Ethics Committee of the Hospital of Stomatology, Wuhan University, Wuhan, China and conducted from April, 2016 to February, 2017. This study included a total of 26 patients with OKCs, 20 patients with RCs and 13 patients with DCs (
Samples of cystic fluids were obtained from the cyst cavity by aspiration with a syringe attached to an 18G sterilized needle prior to surgery. The cyst fluids were immediately centrifuged at 3,000 × g for 20 min at 4°C using the Centrifuge 5810R (Eppendorf, Hamburg, Germany). Supernatants were collected and centrifuged at 3,000 × g for a further 20 min to obtain cell-free cyst fluids. Subsequently, an equal volume of Ca2+/Mg2+-free highly purified phosphate-buffered saline (PBS; Beyotime, Shanghai, China) was added to dilute the supernatants and the dilution was centrifuged at 10,000 × g for 40 min. This step was aimed to remove apoptotic bodies and larger vesicles. The supernatants were then centrifuged at 50,000 × g for 1 h at 4°C using the Avanti J-26 XP high-speed centrifuge (Beckman Coulter, Irving, TX, USA) to pellet the CFMPs as previously described (
TEM was performed at the Wuhan Institute of Virology, Chinese Academy of Sciences (Wuhan, China). Freshly-isolated CFMPs from patients with DCs, RCs and OKCs were placed on a copper grid. The grids were stained with 1% v/v uranyl acetate and the samples were examined on a HT7700 transmission electron microscope (Hitachi High-Tech; Hitachi, Tokyo, Japan) as previously described (
CFSE is a fluorescent dye used for the labeling of MPs (
Flow cytometric analysis was performed with a BD FACSAria II flow cytometer (BD Biosciences) as described in our previous studies (
Samples from 24 patients (2 cases were lacking the epithelial component in the IHC test) with OKCs were fixed in 4% paraformaldehyde and embedded in paraffin under the guidelines of the National Institutes of Health. The tissue samples and CFMPs used for the corollary analyses were collected from the same patient. In brief, the sections were dewaxed, rehydrated and antigen-retrieved by high pressure. Subsequently, the sections were incubated with 3% hydrogen peroxide for 20 min, goat serum for 20 min under room temperature, followed by incubation with the primary antibody [receptor activator for nuclear factor-κB ligand (RANKL), 1:200, Proteintech Group, Wuhan, China; 23408-1-AP] at 4°C overnight. For the staining, a diaminobenzidine substrate kit, and hematoxylin (both from Dako, Glostrup, Denmark) were used. For RANKL evaluation, the semi-quantitative analysis of immunohistochemical staining was performed using Image-Pro Plus 6.0 and the quantification was calculated as the mean density for each protein (IOD/area).
Total RNA was isolated from the CFMPs and the supernatants of OKCs using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA was reverse transcribed into complementary DNA (cDNA) with Oligo(dT) and AMV reverse transcriptase (Fermentas/Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was then used for polymerase chain reaction (PCR) by amplifying 36 cycles for RANKL (94°C for 30 sec, 60°C for 1 min, 72°C for 1 min, and final elongation at 72°C for 10 min) on a 7900HT Real-time PCR System (Applied Biosystems, New York, NY, USA). The primer sequences were as follows: RANKL forward, 5′-TCAGAAGATGGCACTCACTG-3′ and reverse, 5′-AACATCTCCCACTGGCTGTA-3′. PCR products were separated in 1% agarose gels and stained with GelRed (Biotium, Fremont, CA, USA). The blots were imaged with a Syngene G-Box (Syngene, Cambridge, UK).
HIOECs were kindly provided by Professor San-Gang He from the School of Stomatology, Wuhan University. The HIOECs were plated in 6-well plates and cultured in defined keratinocyte serum-free medium (Gibco, Carlsbad, CA, USA). When the HIOECs were grown to 80% confluence, they were co-cultured with the CFMPs (5 and 10
This study was approved by the Medical Ethics Committee of Hospital of Stomatology, Wuhan University. To investigate the biological functions of CFMPs, cellular uptake assays were performed. For this purpose, 10 six-week-old male C57BL/6 mice were obtained from the Experimental Animal Centre of Wuhan University. The weight of these mice ranged from 15.2 to 18.4 g. The mice were injected intraperitoneally with a lethal dose of sodium pentobarbital (150 mg/kg) followed by cervical dislocation. The whole body of the mice were disinfected with 70% ethanol by soaking all the fur. The skin of the neck region was cut open, and the muscle attachments of the mandible were removed. Subsequently, the mandible was divided into 2 sections and pulled out. Mandibles were kept in α-minimal essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS) (both from Gibco), 1% antibiotics (100 U/ml penicillin and 100
Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The HIOECs and BMMs were seeded in a 96-well culture plate (3×103 cells/well) and incubated overnight at 37°C with a supply of 5% CO2. The cells were treated with or without various concentrations of CFMPs (5 or 10
Western blot analysis was performed using the CFMPs and CFMPs co-cultured with the HIOECs. The CFMPs and HIOECs were lysed in RIPA buffer, exposed to brief sonication, and proteins were quantified using the bicinchoninic acid assay (BCA). Subsequently, 40
The adherent BMMs were cultured for 5 days in α-MEM supplemented with FBS (10%), recombinant mouse M-CSF (20 ng/ml) (R&D Systems), mouse recombinant sRANKL (50 ng/ml; PeproTech, London, UK), anti-RANKL monoclonal antibody (1,000 ng/ml; R&D Systems) or CFMPs (5 and 10
The isolation of total RNA, synthesis of cDNA and RT-qPCR were performed. Total RNA was extracted from the BMMs using TRIzol reagent (Invitrogen). Subsequently, 2
Data are expressed as the means ± SEM. Differential levels (apart from the mean density of RANKL expression) were investigated using the Kruskal-Wallis test followed by the Dunn's Multiple Range test for post hoc comparisons. The mean density of RANKL expression levels in the patient samples were analyzed by the Student's t-test. Spearman's rank correlation analysis was used to investigate the correlation between CFMP levels and RANKL expression in the samples collected from the same patients. For the comparisons, a value of P<0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed using GraphPad Prism 6.0 software.
Thirteen patients with DCs (7 male, 6 female), 20 patients with RCs (12 male, 8 female) and 26 patients with OKCs (11 male, 15 female) were enrolled in this study (
CFMPs from patients with DCs, RCs and OKCs were purified by differential centrifugation. To directly visualize the CFMPs, TEM and CFSE fluorescence labeling were performed. As shown in
By the addition of one or more CD-specific monoclonal antibodies, we determined the cellular origin of the MPs present in the cyst fluid. Platelet-derived MPs (CD31+/CD41+), endothelium-derived MPs (CD144+/Annexin V+), erythrocyte-derived MPs (CD235a+/Annexin V+), leukocyte-derived MPs (CD45+/Annexin V+) and epithelium-derived MPs (EpCAM+/Annexin V+) were identified in the cyst fluid of DCs, RCs and OKCs. On the other hand, erythrocyte- and epithelium-derived MPs were the leading CFMPs from the OKCs and DCs, whereas platelet- and erythrocyte-derived MPs were the top two CFMP subsets from the RCs (
The results revealed that the levels of leukocyte- and platelet-derived MPs (CD45+/Annexin V+ and CD31+/CD41+, respectively) were significantly increased in the OKCs when compared with the DCs (P<0.05, P<0.01 respectively), whereas no significant differences were observed between the DCs and RCs (P>0.05), and between the OKCs and RCs (P>0.05) (
By applying flow-count beads, the concentrations of CFMPs were determined by flow cytometry (
To investigate the potential influence of CFMPs on the osteoclastic activity of odontogenic lesions, we detected the expression level of RANKL in the OKC samples and examined its correlation with the level of CFMPs. According to the mean concentration of CFMPs, the patients with OKCs were divided into 2 groups, with a high or low level of CFMPs. As shown in
The HIOECs and BMMs were co-cultured with or without various concentrations of CFMPs (5 or 10
To further determine the biological effects of CFMPs, a series of
To the best of our knowledge, this study demonstrates for the first time, the presence of MPs shed from platelets, endothelial cells, leukocytes, erythrocytes and epithelial cells into the cyst fluids of patients with DCs, RCs and OKCs, and demonstrates the elevation of CFMPs in patients with OKCs. The levels of CFMPs are closely associated with the diameters of lesions and RANKL expression levels in OKC tissues. In addition, we demonstrate that CFMPs isolated from patients with OKCs stimulate BMM differentiation and lead to the formation of osteoclasts, suggesting that CFMPs may contribute to the osteoclastogenesis of OKCs.
Cyst fluid is a crucial component of the microenvironment of odontogenic cysts. The levels of proteins and/or cytokines within the cyst fluid of OKCs have been shown to be quite different from those in DCs or RCs. For instance, it has been reported that the protein level of lactoferrin is significantly higher in OKCs compared with other cysts, due to the impermeability nature of the cyst wall of OKCs (
During the release of MPs, the asymmetric distribution of phospholipids in the two leaflets of the plasma membrane is lost, leading to phospholipid exposure (
For all the odontogenic cysts or tumors, bone resorption is one of the critical events (
Of note, we found that BMMs could easily uptake CFMPs which contained RANKL mRNA and protein at 2 h, indicating that CFMPs may exert biological effects on the recipient cells. To investigate the biological functions of CFMPs, CFMPs were added to the BMMs and the levels of osteoclastogenesis-related genes, such as TRAP and NFATc1 were found to be significantly elevated in the BMMs co-cultured with CFMPs. More importantly, we found that BMMs could successfully differentiate into osteoclasts in the presence of M-CSF and CFMPs, which may be a novel mechanism of osteoclastogenesis in OKCs. The osteoclasts absorb the adjacent bone to acquire the space of the cavity for the growth of the lesion. This may also imply the close association between the CFMP level and RANKL expression in the tissue. Taken together, our study demonstrates that the level of CFMPs may be an important indicator of the progression of OKCs.
In conclusion, the present study demonstrated that the level of CFMPs was significantly elevated in OKCs and was closely associated with cyst diameters.
The authors would like to thank the technician, Juan Min, from the Wuhan Institute of Virology, Chinese Academy of Sciences for supporting our flow cytometric analysis. The authors would also like to thank Professor San-Gang He from the School of Stomatology, Wuhan University for providing the HIOECs.
This study was supported by grants from the National Natural Science Foundation of China to GC (no. 81671816) and YFZ (no. 81570994).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
QWM contributed to the design of the study and wrote the manuscript. QWM, YYZ and JYL collected the clinical samples. QWM, JGR and WQZ performed the flow cytometry analysis and CFMP identification. RFL and BFN performed the cell experiments. YFZ, GC and BL performed the data analysis and revised the manuscript. All authors have read and approved this manuscript.
The use of human samples was approved by the Review Board of the Medical Ethics Committee of the Hospital of Stomatology, Wuhan University, Wuhan, China. All patients agreed to participate in the study and signed informed consent forms. The use of animals was approved by the Medical Ethics Committee of Hospital of Stomatology, Wuhan University.
Not applicable.
The authors declare that they have not competing interests.
Identification of cyst fluid microparticles (CFMPs) derived from patients with dentigerous cysts (DCs), radicular cysts (RCs) and odontogenic keratocysts (OKCs). (A) Transmission electron microscopy (TEM) images of CFMPs purified from DCs, RCs and OKCs. (B) Fluorescence images showing the successful labeling of CFMPs by the fluorescent dye, carboxyfluorescein succinimidyl ester (CFSE). (C) The size distribution of CFMPs derived from DCs, RCs and OKCs was characterized by flow cytometry based on Nile Red fluorescent particles (red) with known diameters ranging from 0.7–0.9
Cellular origin of cyst fluid microparticles (CFMPs). To determine the cellular origin, antibodies were used directly against CD31+/CD41+ (platelets), CD235a+/Annexin V+ (erythrocyte), CD144+/Annexin V+ (endothelium), CD45+/Annexin V+ (leukocyte) and EpCAM+/Annexin V+ (epithelium). Data are expressed and sorted by the number of microparticles per microlitre.
Quantification of the subtypes of cyst fluid microparticles (CFMPs). (A–E) Quantitative analysis of the levels of CD31+/CD41+ (platelets) CFMPs, CD235a+/Annexin V+ (erythrocytes) CFMPs, CD144+/Annexin V+ (endothelium) CFMPs, CD45+/Annexin V+ (leukocytes) CFMPs and EpCAM+/Annexin V+ (epithelium) CFMPs in patients with dentigerous cysts (DCs), radicular cysts (RCs) and odontogenic keratocysts (OKCs). Data are expressed as the means ± SEM. *P<0.05 vs. the control group.
Quantification of the level of cyst fluid microparticles (CFMPs) and the correlation between the level of CFMPs and the clinical characteristics of patients with dentigerous cysts (DCs), radicular cysts (RCs) and odontogenic keratocysts (OKCs). (A) Representative flow cytometry dot-plots showing gated CFMPs (green dots) and the fluorescent beads for counting (red dots). (B) Quantitative analysis of the level of CFMPs derived from patients with DCs, RCs and OKCs. (C) Representative immunohistochemical staining of receptor activator for nuclear factor-κB ligand (RANKL) in OKCs with different level of CFMPs. (D) Quantitative analysis of staining intensity of RANKL in OKCs with different levels of CFMPs. (E) Spearman's rank correlation test was performed to determine the correlation between the level of CFMPs and the mean density of RANKL in OKCs. The data are expressed as the means ± SEM. *P<0.05 vs. the control group; N. S, not significant.
Effect of cyst fluid microparticles (CFMPs) isolated from two patients with odontogenic keratocysts (OKCs) on human immortalized oral epithelial cells (HIOECs). (A) Uptake assays for CFSE-labeled CFMPs (green) were performed by using HIOECs (red) as the recipient cells. (B) DNA electrophoresis indicated that CFMPs from OKCs expressed receptor activator for nuclear factor-κB ligand (RANKL) mRNA. (C) RANKL expression was examined by western blot analysis in CFMPs co-cultured with HIOECs. The CFMPs were selected from two patients with OKCs.
Cells were treated with or without CFMPs (5 or 10
Receptor activator for nuclear factor-κB ligand (RANKL) expression in cyst fluid microparticles (CFMPs) of jaw cysts. (A) Statistic analysis showing the RANKL7Annexin V+ microparticles in patients with dentigerous cysts (DCs), radicular cysts (RCs) and odontogenic keratocysts (OKCs). (B) The protein level of RANKL was examined by western blot analysis of the CFMPs from 4 patients with OKCs.
Effect of cyst fluid microparticles (CFMPs) isolated from 2 patients with odontogenic keratocysts (OKCs) on the osteoclastogenesis of bone marrow-derived macrophages (BMMs). (A) Uptake assays for CFSE-labeled CFMPs (green) were performed by using BMMs (labeled with CellMask) as the recipient cells. (B) Tartaric-resistant acid phosphatase (TRAP) staining showed that multinucleated osteoclasts were observed in the presence of macrophage colony-stimulating factor (M-CSF) (20 ng/ml) and CFMPs (5 or 10
Summary of clinical characteristics of patients with odontogenic keratocysts (OKCs).
Patient No. | Sex | Age (years) | Location | Cyst fluid drained (ml) |
---|---|---|---|---|
1 | F | 35 | Left mandible | 1 |
2 | F | 26 | Right maxilla | 3 |
3 | F | 39 | Mandible | 4.5 |
4 | F | 47 | Left mandible | 6 |
5 | F | 24 | Left mandible | 2.5 |
6 | F | 24 | Right mandible | 2 |
7 | M | 32 | Left mandible | 4.5 |
8 | M | 52 | Left mandible | 1 |
9 | M | 79 | Right mandible | 5 |
10 | M | 24 | Right mandible | 2 |
11 | F | 11 | Right mandible | 5 |
12 | F | 52 | Right maxilla | 2 |
13 | F | 45 | Right mandible | 3 |
14 | M | 34 | Left mandible | 0.8 |
15 | F | 28 | Right mandible | 3 |
16 | M | 37 | Left maxilla | 7 |
17 | M | 26 | Right maxilla | 4 |
18 | M | 24 | Left mandible | 1 |
19 | F | 32 | Left mandible | 1 |
20 | M | 16 | Right mandible | 2 |
21 | F | 30 | Left maxilla | 2 |
22 | F | 18 | Right mandible | 1 |
23 | F | 27 | Maxilla | 1 |
24 | F | 47 | Right maxilla | 2 |
25 | M | 23 | Right mandible | 2 |
26 | M | 17 | Left maxilla | 1 |
M, male; F, female.
Summary of clinical characteristics of patients with dentigerous cysts (DCs).
Patient No. | Sex | Age (years) | Location | Cyst fluid drained (ml) |
---|---|---|---|---|
1 | F | 30 | Left maxilla | 5 |
2 | F | 18 | Right mandible | 2 |
3 | M | 18 | Maxilla | 3 |
4 | F | 33 | Right maxilla | 2 |
5 | M | 51 | Right mandible | 9 |
6 | M | 42 | Left maxilla | 2 |
7 | F | 50 | Left mandible | 11 |
8 | M | 34 | Right mandible | 3.5 |
9 | M | 34 | Right mandible | 2 |
10 | M | 56 | Right mandible | 4 |
11 | F | 28 | Left maxilla | 5 |
12 | M | 36 | Right maxilla | 4 |
13 | F | 33 | Left mandible | 5 |
M, male; F, female.
Summary of clinical characteristics of patients with radicular cysts (RCs).
Patient No. | Sex | Age (years) | Location | Cyst fluid drained (ml) |
---|---|---|---|---|
1 | M | 14 | Left mandible | 4 |
2 | F | 45 | Right maxilla | 0.8 |
3 | F | 44 | Left mandible | 7.5 |
4 | M | 54 | Right maxilla | 10 |
5 | F | 34 | Maxilla | 2 |
6 | F | 63 | Right mandible | 4 |
7 | F | 32 | Right maxilla | 3 |
8 | M | 7 | Left maxilla | 2 |
9 | M | 43 | Left mandible | 4 |
10 | M | 32 | Mandible | 7 |
11 | F | 44 | Left mandible | 7.5 |
12 | M | 66 | Left maxilla | 3 |
13 | M | 29 | Left maxilla | 2 |
14 | F | 73 | Right maxilla | 5 |
15 | F | 51 | Right maxilla | 3 |
16 | M | 8 | Right maxilla | 5 |
17 | M | 17 | Left mandible | 3 |
18 | M | 24 | Left mandible | 6 |
19 | M | 65 | Right mandible | 5 |
20 | M | 41 | Right mandible | 4 |
M, male; F, female.
Cyst fluid microparticle levels in patients with dentigerous cysts (DCs), radicular cysts (RCs) and odontogenic keratocysts (OKCs).
Variables | OKCs | DCs | P<0.05 OKCs/DCs | RCs | P<0.05 OKCs/RCs |
---|---|---|---|---|---|
n | 26 | 13 | ND | 20 | ND |
Age |
33±15 | 36±12 | ND | 39±20 | ND |
Sex (male/female) | 11/15 | 7/6 | ND | 12/8 | ND |
Total MPs |
6,751±5,800 | 2,755±2,471 | Y | 3,477±3,346 | Y |
CD31+/CD41+MPs |
1,559±1,435 | 533±582 | Y | 891±870 | N |
CD235a+ MPs |
3,212±3,675 | 1,464±1,201 | Y | 2,523±2,863 | Y |
CD45+ MPs |
1,319±1,214 | 527±591 | Y | 639±601 | N |
CD144+ MPs |
996±1,144 | 357±417 | Y | 411±449 | Y |
EpCAM+ MPs |
1,867±2,072 | 586±534 | Y | 719±634 | Y |
Results are expressed as the mean number ± SEM of years;
Results are expressed as the mean number ± SEM of MPs per cyst fluid (