Breeding pairs of C57Bl/6 wild type (Cat# 000664) and ERβ KO mice (homozygous male, heterozygous female, Cat# 004745) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). For the studies with ERβ KO mice, only homozygous females were used. Seven-day-old to120-day-old female C57Bl/6 WT (total n = 42) and ERβ KO mice (total n = 43) were used for the study (see Table 1 for details). At either 3 or 16 hours prior to euthanasia, mice were injected intraperitoneally with 0.1 mg bromodeoxyuridine (BrdU) per gram body weight. All experiments were performed in accordance with animal welfare based on an approved IACUC protocol# AAAD0950 from Columbia University animal care committee.
Table 1. Number of Mice
|Mouse genotype||Total (n)||Age (days)||Real-time PCR (n)||Histology/µCT|
|3-hour BrdUa (n)||16-hour BrdU (n)|
RNA extraction and PCR amplification
For each mouse the mandibular condylar heads (left and right), containing both the mandibular condylar cartilage and subchondral bone, were carefully harvested and all the soft tissues were removed under a dissecting microscope. Total RNA was obtained from the condylar head and extracted with TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) following the manufacturer's protocol. Total RNA was reverse-transcribed into cDNA using the ABI High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer's protocol. Real-time PCR was performed, for expression of different genes, in separate wells (single-plex assay) of 96-well plates with reaction volume of 20 µL. Gapdh was used as an endogenous control. Three replicates of each sample were amplified using Assays-on-Demand Gene Expression (Applied Biosystems) for the particular gene of interest, with predesigned unlabeled gene-specific PCR primers and TaqMan MGB FAM dye-labeled probes. The PCR reaction mixture (including 2× TaqMan Universal PCR Master Mix, 20× Assays-on-Demand Gene Expression Assay Mix, 50 ng of cDNA) was run in an Applied Biosystems ABI Prism 7300 Sequence Detection System instrument using universal thermal cycling parameters. For the genes in which the efficiencies of target and endogenous control amplification were approximately equal, relative expression in a test sample was compared to a reference calibrator sample (ΔΔCt method) and used for data analysis. For the genes that were not amplified with the same efficiency as the endogenous control the Relative Standard Curve method, in which target quantity is determined from the standard curve and divided by the target quantity of the calibrator, was used. Gene expression was performed for proteoglycan 4 (Prg4), parathyroid hormone related protein (Pthrp), SRY-box containing gene 9 (Sox9), collagen type II (Col2a1), Indian hedgehog (Ihh), collagen type X (Col10a1), vascular endothelial growth factor (Vegf), insulin-like growth factor 1 (Igf-1), receptor activator for nuclear factor κ B ligand (Rankl), and osteoprotegerin (Opg).
Histology and immunohistochemistry
Whole mouse heads were sectioned into halves, fixed in 10% formalin for 4 days at room temperature and decalcified in 14% EDTA (pH 7.1) (Sigma, St. Louis, MO, USA) for 10 days and 14 days in 49-day and 120-day samples, respectively. Subsequently, the samples were processed through progressive concentrations of ethanol, cleared in xylene and embedded in paraffin. The TMJ was sagittally serially sectioned into 5-µm sections by Microm HM 355s microtome (Thermo Fisher Scientific, Waltham, MA, USA), and every fifth section was stained with hematoxylin and eosin (H&E).
Histomorphometry measurements were made in a blinded, nonbiased manner using the BioQuant computerized image analysis system (BioQuant, Nashville, TN, USA). Mandibular condylar cartilage thickness measurements were performed on H&E sagittal sections corresponding to the mid-coronal portion of the mandibular condylar head. The entire mandibular condylar cartilage area was divided into three layers, superficial layer (layer S), flattened layer (layer F), and hypertrophic layer (layer H) based on the cartilage cell size, shape, and staining (Fig. 2A).The entire anterior-posterior condylar cartilage area was measured for each section and the average of at least three sections was calculated as the cartilage thickness for that mouse. Measurements included cell number, thickness, and area of each layer. Six to eight mice from each age and genotype group were analyzed.
For immunohistochemistry, tissue sections were deparaffinized with xylene and rehydrated with decreasing concentrations of ethanol. Following rehydration, the sections were treated with 3% peroxide to block endogenous peroxidase activity and digested for 60 minutes with pepsin for unmasking (Lab Vision, Fremont, CA, USA; Cat# AP-9007-006). All sections were blocked with Protein Block Serum-Free (DakoCytomation, Carpinteria, CA, USA; Code# X0909). Immunohistochemical staining was performed using the LSAB + System-HRP Kit (DakoCytomation; Code# K0690) following the procedure recommended by the manufacturer. Primary antibodies used in this study were collagen type II antigen (Millipore; MAB8887, 1:200 dilution), Tieg1 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; sc-23159, 1:100 dilution), p57 (Santa Cruz; sc-8298, 1:200 dilution) and IGF-1 (Abcam; ab40657, 1:100 dilution). BrdU immunohistochemical analysis was completed using a BrdU staining kit following the manufacturer's instructions (Zymed Laboratories-Invitrogen Corporation, Carlsbad, CA, USA). Negative controls were prepared by omitting the primary antibody step and incubating with blocking solution. To quantify BrdU, Tieg1, and p57 staining, the labeling index (number of BrdU-, p57-, or Tieg1-positive cells divided by the total number of cells) was calculated. Three to five sections, corresponding to the same anatomical area (mid-sagittal), were counted for each animal and the average of these sections was used for the labeling index.
Osteoclastic cell measurement were performed on tartrate-resistant acid phosphatase (TRAP)-stained sections by TRAP kit (Sigma Aldrich, St. Louis, MO, USA). The measurements were made in the bone region beneath the mandibular condylar cartilage. The thickness of the measured region was designated to the approximate cartilage thickness of the correlated region. Osteoclast number, osteoclastic surface, and bone surface in this region were analyzed with BioQuant computerized image analysis system.