YAP promotes the early development of temporomandibular joint bony ankylosis by regulating mesenchymal stem cell … – Nature.com

Animal model and tissue processing

Twenty-six 3-month-old male small-tailed sheep with body weights ranging from 25 to 27kg were used in this study under a research protocol approved by the Ethics Committee of Tianjin Stomatological Hospital (approval code: Tjskq2013001). All experiments were performed in compliance with the Animal Management Regulations and Administrative Measures on Experimental Animals, and are reported in accordance with the ARRIVE guidelines. The animals were housed in a laboratory animal facility with adequate facility management services and specialised nursing care and husbandry practices, similar to our previous study8. The animals underwent unilateral TMJ surgery, involving the removal of two-thirds of the articular disc and severe damage to the articular fossa to induce bony ankylosis, following the protocol in our previous publication8. The anaesthesia, analgesia and euthanasia methods used were the same as those described in a previous study26.

Three sheep per time point were sacrificed for tissue specimen collection via euthanasia (120mg/kg of pentobarbitone sodium administered via intravenous injection) on Days 1, 4, 7, 9, 11, 14 and 28 postsurgery, and five were sacrificed on Day 14 after TMJ surgery for subsequent isolation and culture of MSCs. The TMJ complexes were removed en bloc with a band saw, and newly formed tissue within the joint space was bluntly dissected from the surrounding soft tissue using periosteum separators.

After fixation with 10% formalin, the collected tissue was routinely dehydrated and embedded and then cut into 5m-thick sections using a microtome for subsequent staining experiments. Successive slices were taken to ensure that the haematoxylin and eosin (HE), immunohistochemistry (IHC) and multiplex immunohistochemistry (mIHC) staining were performed on the same area.

To observe the histological manifestations of the early stages of traumatic bony ankylosis of the TMJ, paraffin sections of tissues collected on Days 1, 4, 7, 9, 11, 14 and 28 post -operation were stained with HE (Sigma, USA).

IHC was used to detect YAP and Runt-related transcription factor 2 (RUNX2) expression positions in TMJ ankylosis formation. Antibodies against RUNX2 was used to label the osteogenically active regions. After routine dewaxing, hydration, heat repair and antibody blocking of the tissue sections, a nonblocking kit (ZSGBBIO, China) was used to prepare the sections for staining with an anti-YAP antibody (rabbit monoclonal, 1:1000, ProteinTech Group, USA) and an anti-RUNX2 antibody (mouse monoclonal, 1:1000, Abcam, USA), to which a matching secondary antibody was then added, followed by incubation at 37C for 30min. Diaminobenzidine (DAB, 1:20, ZSGBBIO, China) was used for the final immunological colour development, and the nuclei were stained with haematoxylin (Sigma, USA). Image acquisition was performed using a microscope (Nikon, Japan).

We used mIHC to further analyse the expression level of YAP in the early stages of traumatic bony ankylosis of the TMJ. A specialised tyramine signal amplification-immunohistochemistry (TSA-IHC) multitarget immunofluorescence staining kit (Bruno, China) was used for this experiment. Different primary antibodies were applied sequentially, followed by horseradish peroxidase-coupled secondary antibody incubation and tyramine signal amplification (TSA). After each TSA, the slides were processed for antigen elution. After labelling YAP and RUNX2, cell nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI) (Bruno, China). The images were acquired using a panoramic microscope camera system (Jinan Tangier Electronics Co., China), and the digital scanning and viewing software used was CaseViewer 2.4 (3DHISTECH, Hungary).

All collected tissues were initially viewed at100 for each section, in which functioning YAP was stained red, RUNX2 was stained green, and nuclei were stained blue. Subsequently, 20micrographs were acquired and three fields of view were obtained for each specimen. The percentage of cells exhibiting positive coexpression of RUNX2+YAP (i.e., number of positive coexpression cellstotal number of cells) was calculated using the Halo (Indica Labs, USA) whole-slide image analysis platform. The fluorescence intensity measured as the integrated density (Int Den) and area were measured using ImageJ (National Institutes of Health, USA), and the mean grey value (mean) of each image was then calculated.

Primary cells were isolated using the tissue attachment method27. The specific isolation, incubation, culture and purification processes applied to the MSCs were the same as described in a previous study13. Third-passage cells were harvested for subsequent experimental procedures.

The third-passage cells were digested with 0.25% trypsin (Solarbio, China) to obtain a single-cell suspension. The cells were then resuspended with phosphate buffered saline (PBS) solution supplemented with 1% foetal bovine serum (FBS) after centrifugation. For 30min at 4C in the dark, 1106 cells were incubated with the corresponding commercial monoclonal antibodies (CD44, Immunostep, clone 25.32, 1:10; CD29, Biolegend, clone TS2/16, 1:20; CD31, AbD Serotec, clone CO.3E1D4, 1:10; CD45, AbD Serotec, clone 1.11.32, 1:10), and then measurements were made using a FACSCanto (BD Biosciences, USA) flow cytometer. Flow cytometric analyses were performed using FACSDiva (BD Biosciences, USA) and FlowJo software (TreeStar, Ashland, Oregon).

Third-passage cells were seeded at a density of 1105/ml in a six-well plate (Corning, USA). When the cells reached 6070% confluence, the medium was removed, and the cells were washed with PBS three times. Osteogenic induction (OI) medium (the formula used for this osteogenic induction medium is described in an earlier study13) was added to the OI group, while normal control (NC) group was still added complete medium (-minimal essential medium (-MEM) plus 10% foetal bovine serum, 100 U/ml penicillin, 100g/ml streptomycin and 2.5g/ml amphotericin B).

The cells were stained with 1% Alizarin red (pH=4.3, Solarbio, China) after undergoing OI for 7days and, subsequently, 14days. Mineralised nodules were then dissolved in 10% cetylpyridinium chloride (Solarbio, China) for semiquantitative analysis by examining the absorbance at 562nm. Alkaline phosphatase (ALP) activity was tested using an ALP colour development kit (Beyotime, China) after the cells underwent OI for 7days and, subsequently, 14days. The mRNA expression levels of YAP, RUNX2, the Sp7 transcription factor (Osterix) and osteocalcin (OCN) were measured via real-time polymerase chain reaction (PCR) after induction for 7 and 14days.

To detect the intracellular localisation of YAP during the OI of MSCs, third-passage cells were inoculated into confocal culture dishes (NEST, China) at a density of 1103 cells/ml, the OI medium was cultured for three days, and immunofluorescence staining was performed as described in a previous study13. The monoclonal antibody used was anti-mouse YAP1 (clone: 3A7A9, ProteinTech Group, 1:800) with 594-conjugated goat anti-mouse lgG. Fluorescently labelled cells were photographed using an inverted fluorescence microscope (Nikon, Japan).

Total cellular RNA was extracted using a universal RNA extraction kit (TaKaRa, Japan), and the RNA concentration was determined. cDNA was extracted via reverse transcription using a synthesis kit (Promega, USA), and quantitative real-time PCR was performed with FastStart Universal SYBR Green Master Mix (Roche, Switzerland, Cat. #04,913,850,001) using a LightCycler 480 II (Roche, Switzerland). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference. The reaction system and PCR cycling parameters were the same as those described in a previous study28. The sequences of the primers used for PCR amplification are listed in Table 1.

A YAP-silenced MSC model was constructed using shRNA plasmid transfection technology (RuiboBio, Guangzhou, China). The target sequences are presented in Table 2. Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, USA) was used to transfect the MSCs, according to the manufacturers instructions. Third-passage cells were briefly inoculated into 35mm dishes (Corning, USA) at a density of 1105 cells/ml after transient transfection with shYAP1, shYAP2, shYAP3 and shRNA plasmid empty vectors. The transfection efficiency was tested after 24h of culture to determine the highest transfection efficiency of the shRNA plasmid. The MSCs were then reinoculated in 35mm dishes following the previously described density and method, and divided into three groups: The blank group (the blank control group, in which the shRNA plasmid was not transfected), the shRNA-YAP-NC group (the negative control group, in which the shRNA plasmid was transfected with an empty vector), and the shRNA-YAP group (the silencing group, in which the shYAP plasmid was transfected with the highest efficiency). After one day of transfection, the stock solutions of the three groups were discarded, after the following experimental procedures: (1) The stock medium was replaced with OI medium to detect the effect of YAP silencing on the osteogenic ability of the MSCs; the procedures for Alizarin red staining, ALP activity detection and real-time PCR were the same as those described earlier. (2) Continue to use complete medium to detect the effects of YAP silencing on the proliferation and migration ability of MSCs.

EdU assay, cell formation assays and cell counting kit-8 (CCK-8) assays were used to investigate whether YAP silencing affects the proliferative capacity of MSCs.

A BeyoClick EdU-488 Cell Proliferation Assay Kit (Beyotime, China) was used to determine the cell proliferative capacity. Third-passage MSCs were seeded in 24-well plates (Corning, USA) at a density of 2,000 cells /well. After transfection using the preceding method, the three groups of cells (Blank, shRNA-YAP-NC and shRNA-YAP) were stained following the protocol of the kit. Fluorescence detection was performed on each well at a 495nm wavelength (10) using an inverted fluorescence microscope (Nikon, Japan). Quantitative analysis of EdU proliferation was performed using ImageJ; the percentage of EdU-positive=cells was calculated as (number of EdU-positive cells divided by the total number of cells)100%.

Third-passage cells were inoculated in 6-well plates (Corning, USA) at a density of 500 cells/well. After transfection using the method described earlier, the three groups of cells (Blank, shRNA-YAP-NC and shRNA-YAP) were fixed with 4% paraformaldehyde and stained with 5% Giemsa (Hydrogen, China) for 40min on Day 7 of culture, and colonies containing>50 cells were counted under the microscope (Nikon, Japan). To clearly observe cell proliferation under a microscope, the number of colonies was analysed using ImageJ.

Third-passage cells were seeded in four 96-well plates (Corning, USA) at a density of 2,000 cells/well. After transfection following the method described earlier, cell proliferation tests were performed on the three groups of cells (Blank, shRNA-YAP-NC and shRNA-YAP) using a CCK-8 kit (Beyotime, China) as described in a previous study13, and the detection time points were 1day, 3days, 5days and 7days.

Wound healing and Transwell migration assays were used to investigate whether YAP silencing affects the migratory capacity of MSCs.

Third-passage MSCs were inoculated in 6-well plates (Corning, USA) at a density of 1105 cells /well. When the cells reached to 90% confluence after transfection, a straight line was drawn in the cell layer with a sterile 200l pipet tip. Images were acquired under a microscope (Nikon, Japan) at 0h, 6h, 24h and 48h. The distance migrated by the cells was analysed using ImageJ. The relative wound closure rate was calculated as the ratio of the wound distance at 6h, 24h and 48h to the wound distance at 0h.

A 24well plate with Transwell chambers (Beyotime, China) with an 8.0m pore size was used for this experiment. Serumfree -MEM was added to the upper chambers, and a medium containing 20% FBS was added to the lower chamber. After transfection, the cells of the three groups (Blank, shRNA-YAP-NC, shRNA-YAP) were inoculated in the upper chamber at a cell density of 3104/well. After 24h of incubation at 37C, the migratory cells on the membrane were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet (Beyotime, China) for 30min. The membrane was air-dried and then photographed under a light microscope (Nikon, Japan) at 10magnification. The number of migrated cells was quantified using ImageJ.

The results of the experiments were analysed using unpaired t tests between two groups, while comparisons among three or more groups were performed using one-way ANOVA combined with Bonferronis multiple comparisons test (GraphPad Prism 9.0, USA); P<0.05 was considered to indicate statistical significance. The DAgostino-Pearson test was used to assess normality. Lines and error bars in all figures denote of the mean and standard error mean (SEM), respectively.

Read the original:
YAP promotes the early development of temporomandibular joint bony ankylosis by regulating mesenchymal stem cell ... - Nature.com