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Role of PTH1R signaling in Prx1+ mesenchymal progenitors during eruption
Chen Cui, Ruiye Bi, Weiqing Liu, Shuyuan Guan, Peiran Li, Dongzhe Song, Ruoshi
Xu, Liwei Zheng, Quan Yuan, Xuedong Zhou, Yi Fan
Appendix Material and Methods
Micro-computed tomographic (μCT) analyses
Mandibular bones from postnatal day (P) 10,P21 and P42 animals were used for
μCT with Scanco Medical μCT 35 system (Scanco), with an isotropic voxel size of 7
mm, according to guidelines (Dempster et al. 2013). The images were reconstructed
and perform three dimensional. Regions of interest were defined from alveolar bone
tissue nearby first molar. The selected regions were analyzed to determine the
trabecular bone volume fraction (BV/TV), trabecular thickness (Tb. Th), and trabecular
separation (Tb. Sp) at P10. Alveolar bone volume under first molar as well as the
corresponding enamel and dentin volume of the incisor was analyzed at P21.
Histology and Immunostaining
Tissues were dissected and fixed in 4% paraformaldehyde overnight. After
decalcification, samples were embedded in paraffin. 5μm sections were cut using
HM360 microtome (Microm). The sections were stained with hematoxylin (VWR) and
eosin (Sigma-Aldrich). Tartrate-resistant acid phosphatase (TRAP) (Sigma-Aldrich)
and Masson's trichrome (Solarbio) staining were performed according to the
manufacturers’ protocols, respectively. For immunostaining, slides were stained with
anti-Runx2 (1:200, Abcam, ab23981), anti-Osx (1:200, Abcam, ab22552), anti-ColαI
(1:200, Abcam, ab21286), anti-Periostin (1:200, Abcam, ab14041), anti-Red
Fluorescent Protein (1:100, Santa Cruz, sc-390909), anti-Sox9 (1:200, Abcam,
ab3697), anti-ColX (1:200, Abcam, ab58632), anti-OPN (1:400, R&D systems, AF808),
anti-PTH1R (1:200, Assay biotech, G220), anti-CD200 (1:200, Abcam, ab33734)
overnight at 4°C and stained with Alexa Fluor 488 (Invitrogen, 1:1000), Alexa Fluor
568 (Invitrogen, 1:1000) or a biotinylated secondary antibody, respectively. DAPI
(Vector) or hematoxylin (VWR) was used as counterstaining. Immunostaining images
were performed with Olympus confocal microscope FV3000 (Olympus). Quantitative
analysis including the numbers of Runx2+/tdTomato+ cells, Osx+/tdTomato+ cells and
tdTomato+ osteoblasts in ColαI-labeled alveolar bone region, were conducted by a
blinded observer with Image J software. At least three different sections were used
from each sample and three or more different samples were analyzed for each group.
Histomorphometry
Calcein mineral labels was performed to evaluate dynamic mineral apposition and
bone formation in mandibles of 3-week-old mice. Mice were injected with 20 mg of
calcein (Sigma-Aldrich) per kg of body weight in a 2% sodium bicarbonate solution at
5 and 2 days prior to sacrifice. Then, mandibles were collected and processed for
histologic sectioning and histomorphometry. 5μm frozen sections from undecalcified
samples were processed using Multipurpose Cryosection Preparation kit (Section-
LAB Co. Ltd, Japan) and cutting by a Cryomicrotome at -35°C (Leica Microsystems
KK, Germany). To determine the label, sections were directly viewed under fluorescent
light. Mineral surface/bone surface (MS/BS, %), mineral apposition rate (MAR, μm/day)
and bone formation rate/bone surface (BFR/BS, μm3/μm2/day) were measured.
Mouse Orofacial Bone-marrow-derived mesenchymal stem cells (OMSCs)
Culture
Isolation of OMSCs was as described previously (Yamaza et al. 2011). Briefly, after
removing attached soft tissues and teeth (incisors and molars), mice mandible was
digested with 3 mg/mL collagenase type I (Worthington Biochem) and 4 mg/mL
dispase II (Roche Diagnostic) for 60 min at 37°C. All nucleated cells were obtained
through 70-μm cell strainers (Corning) and seeded at 1-1.5 x 106 on a 100-mm dish
(Corning). Cells were cultured in α-MEM (Gibco) containing 1% penicillin/streptomycin
(HyClone) and 10% fetal bovine serum (FBS) (Gibco). Non-adherent cells were
removed by replacing the medium after 24 hours. The attached OMSCs were used for
experiments at passage 3 and plated at 3 x 104 cells/cm2. Osteogenic differentiation
was induced with osteogenic differentiation medium (α-MEM supplemented with 1%
PS, 10% FBS, 50 μg/ml ascorbic acid (Sigma-Aldrich), 10 mM β-glycerophosphate
(Sigma-Aldrich)). The medium was changed every other day. Alkaline phosphatase
(ALP) staining was performed using ALP assay kit (Beyotime) at 7 days and Alizarin
red S staining (Sigma-Aldrich) was performed at 14 days according to the
manufacturer’s instructions. For adipogenic differentiation, cells were induced with
adipocyte differentiation medium (α-MEM supplemented with 1% PS, 20% FBS, 500
μM IBMX, 1 μM Dexamethasone, 5 μg/ml Insulin and 1 μM Rosiglitazone) for first 2
days. Then the medium was changed to adipocyte differentiation base medium (α-
MEM supplemented with 1% PS, 20% FBS, 5 μg/ml Insulin and 1 μM Rosiglitazone)
for next 4 days, which was changed every other day. After 6 days, cells were washed
in PBS and collected for qPCR analysis or fixed with 10% formalin for oil red O staining.
For chondrogenic introduction, OMSCs were plated at 1.5mL microcentrifuge tube
with 3 × 106 cells/tube to form three-dimensional cartilage balls. Cells were cultured in
chondrogenic differentiation media (Cyagen) and were changed every other day. After
14 days, cartilage balls were fixed with 10% formalin and embedded in paraffin for
alcian blue staining and immunofluorescence staining.
Transfection
Recombinant adenoviruses CRE expression (Ad-CRE) were gotten from Dr. Dongzhe
Song which also express GFP as the marker for monitoring infection efficiency.
Adenovirus expressing only GFP (Ad-GFP) was used as a control. OMSCs were
transfected with Ad-CRE or Ad-GFP for 12 hours and then exchanged normal medium.
In order to enhance transgene transduction efficiency, polybrene (5 μg/ml) was added
to the culture medium for all adenovirus infections (Song et al. 2017; Wu et al. 2014).
Flow Cytometry
For preparation of flow cytometry analysis, OMSCs were digested with trypsin
(HyClone), and then suspended in αMEM and washed with 5% BSA. Cells were
incubated with PE anti-CD29 (BioLegend), PE anti-CD90 (BioLegend), FITC anti-
CD45 (BioLegend) and FITC anti-CD11b (BioLegend). Flow cytometry was carried out
using the FACSCalibur (FC500, Beckman Coulter). Each data set was analyzed by
FlowJo software (FLOWJO, LLC, OR, USA).
RNA Extraction and Quantitative Real-time PCR (qRT-PCR)
Total RNA from mice mandible at P0, P10 and P14 and in vitro cultured OMSCs were
extracted using Trizol (Invitrogen) according to the manufacturer’s protocol. RNA
concentration was determined with NanoDrop ND-1000 (Thermo Fisher Scientific).
cDNA was generated using PrimeScript RT reagent Kit (Takara) and qRT-PCR was
performed with SybrGreen Supermix (Bio-Rad Laboratories). Relative gene
expression levels were normalized by GAPDH (glyceraldehyde-3-phosphate
dehydrogenase) using the 2ΔΔCT method.
Protein Extraction and Western Blot
Protein from OMSCs was extracted by whole cell lysis assay (KeyGEN) and the
concentrations were evaluated by enhanced BCA protein assay kit (Beyotime). Equal
amount of protein samples was separated by NuPAGE 4-12% Bis-Tris SDS/PAGE
using precast gels (Invitrogen) and transferred onto polyvinylidene fluoride membrane
(Millipore). Following incubation in primary antibodies, we probed proteins by
enhanced chemiluminescence kit (Bio-Rad Laboratories, USA). Bands were
quantified by Image J software. Primary antibodies included anti-Osterix (abcam,
1:1000, ab22552), anti-Runx2 (abcam, 1:1000, ab23981), anti-Col αI (abcam, 1:1000,
ab21286), anti-Tubulin α (Signalway Antibody, 1:500, 41517). Horseradish
peroxidase-conjugated anti-rat or rabbit IgG was used as the secondary antibody
(Jackson ImmunoResearch Laboratories). Signal was detected by Amersham ECL
Prime Western Blotting Detection Reagent (GE Healthcare).
Statistics
For statistical analysis, GraphPad Prism 8.0 (GraphPad Software Inc.) was used.
Unpaired two-tailed Student’s t test was used between two groups. All values are
expressed as mean ± SEM. P values
Appendix Figure 1
Appendix Figure 1: Incisor eruption phenotype and distribution pattern of Prx1+
progenitors. (A) Gross phenotype of the incisors of PTH1Rfl/fl (upper) and
Prx1Cre;PTH1Rfl/lfl (lower) mice at postnatal days P7, P10, P14, P21 and P180 shows
arrested mandibular incisor eruption in mutants. Scale bars: 2mm. (B, C)
Quantification of mandibular incisor and molar eruption length or height measured in
mm at P10 and P21. n=4-7. *p
Appendix Figure 2
Appendix Figure 2: Characterization of incisor cervical loop and molar root. (A) H&E
staining of sagittal section of mandible at P0, P4, P10 and P14 indicate comparable
cervical loop histology in mutant when compared to age-matched control. n=4-6. Scale
bars: 200μm. (B) qRT-PCR analysis of gene expressions related to amelogenesis,
including Amelogenin (Amelx), Ameloblastin (Ambn) and Enamelin (Enam) in
mandibles of mutant and control littermates at P10 and P14. n=4-6. (C) 3D μCT
images of control and mutant at P42. Scale bars: 1mm. Individual slides of μCT images
at an interval of 6μm indicate the molar root are not dilacerated in mutants. (D) CD200
staining of P21 mice showed unchanged expression in root cementum. n=3. Scale
bars: 50μm.
Appendix Figure 3
Appendix Figure 3: Multilineage differentiation capacity of OMSCs and decreased
osteogenesis in PTH1R-deficient OMSCs after Ad-Cre transfection. (A,B) Alizarin red
staining (ARS) and quantitative analysis of osteogenic gene expressions of OMSCs
after 14-day osteogenic induction. (C,D) Oil red O staining and adipogenic-related
gene expressions of OMSCs after 7-day adipogenic induction. (E,F) Alcian blue and
immunofluorescence (Col X and Sox 9) staining of cartilage balls and gene expression
after chondrogenic induction of OMSCs. n=3. Scale bars: 100μm in C, E. *p
Appendix Figure 4
Appendix Figure 4: Alveolar bone resorption remained unchanged in
Prx1Cre;PTH1Rfl/fl mice (A) qRT-PCR analysis of osteoblast/osteoclast-relative gene
expressions (Rankl, Opg, Rank) in mandibles of mutant and control littermates at P10
and P14. n=4-6. *p
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