Generation of Col2α1-Cre ERT2; Ihhfl/fl Mice
Col2α1-Cre ERT2; Ihhfl/fl mice (a gift from Professor Beate Lanske) were bred as previously described [22]. P0 Col2a1-Cre ERT2; Ihhfl/fl mice were randomly divided into two groups: TM (n = 24) and no TM (n = 24). Random numbers were generated using the standard = RAND ( ) function in Microsoft Excel. In the TM group, Col2a1-Cre ERT2; Ihhfl/fl mice were intraperitoneal injected with TM (5 μL of 20 mg/mL for 3 consecutive days) to delete Ihh. In the no TM group, mice were injected with 3 doses of solvent (corn oil) as a control. The experimenter could not be blinded to whether the animal was injected with TM or with corn oil due to severe phenotype. Animal were killed at different age and right hindlimbs were harvested immediately after the mice were killed. All study animals were bred at the Experimental Animal Center of Shanxi Medical University. Approval of the animal experiments was obtained from The Institutional Animal Care and Use Committee of Shanxi Medical University.
PCR Analysis for Genotyping
Total DNA were extracted from mouse tail and genotyping of mice was performed by conventional PCR. The quantification of DNA was performed by real-time PCR using the SYBR Premix Ex TaqTM kit (Takara, Dalian, China) with the IQ50 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Each reaction was performed in triplicate. The following primers were used: Cre allele; forward, 5′-ATCCGAAAAGAAAACGTTGA-3′, reverse, 5′-ATCCAGGGTACGGATATA GT-3′; Ihh allele; forward, 5′-AGCACCTTTTTTCTCGACTGCCTG-3′, reverse, 5′-TGTTAGGCCGAGAGGGATTTCGTG-3′.
Gross Appearance and Radiographical Analysis
8-week-old mice in each group were anesthetized by intraperitoneal injections of ketamine hydrochloride (100 mg/kg body weight) and plain radiographs were taken using a Faxitron X-ray apparatus (Faxitron, Tucson, AZ). After the animals were euthanized, hind limbs were harvested and the length of the femur and tibia were measured.
Micro-CT Analyses
8-week-old mice in each group were sacrificed by cervical dislocation, the tibias of mice were collected. Three-dimensional (3D) reconstructions of the tibial bone from each group were generated from images acquired on vivaCT 80 (SCANCO MEDICAL, Bassersdorf, Switzerland), following recommended guidelines from Bouxsein et al. [23]. For trabecular bone analysis, a region of interest (0.5 mm below the growth plate) were selected for analysis. The following morphometric parameters of bone volume to total volume (BV/TV), trabecular number (Tb. N), trabecularnseparation (Tb. Sp) and trabecular thickness (Tb. Th) were were calculated using builtin software.
Histology
After the animals were euthanized, the hind knee joints from each group were harvested and immersed in 10% formalin for 24 hours at postnatal days 6, 8, 10, 12, 14. The paraffin sections were prepared through normal procedures. Safranin O staining was performed using 0.5% Safranin-O solution and counterstaining of 0.2% fast green to assess glycosaminoglycan production. Von Kossa staining was performed using 4% silver nitrate solution with counterstaining of 1% fast red solution to evaluate the mineralization of bone [10]. Immunohistochemistry was used to detected PTHrP and BMP-6 using 6 μm thick section.
In Vivo Ectopic Bone Formation in Nude Mice
BALB/c nude mice at 3 weeks of age were purchased from Charles River (China). They were acclimated to our animal facilities for 1 week and then used in experiments. Murine costal chondrocytes were isolated from the ventral parts of the rib cages of 6-day-old mice and cultured in F-12 media with 10% fetal bovine serum (Thermo Fisher Scientifc, Inc., Waltham, MA) as previously described [24, 25]. A total of 1 × 106 Ihhfl/fl or Ihhd/d costal chondrocytes cells were seeded in 200 lL Matrigel (BD Biosciences, San Jose, CA, USA) and implanted subcutaneously into athymic nude mice at 4 weeks of age as previously described (n = 4 per group) [8]. The dynamic bone formation was monitored weekly by Faxitron X-ray (Faxitron, Tucson, AZ).
Quantitative Real-Time PCR Analysis
Total RNA was extracted from cartilage by using the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA) and reversely transcribed into complementary DNA by using the PrimeScript RT-PCR Kit (Takara, Dalian, China). Real-time PCR was conducted utilizing SYBR Premix Ex TaqTM (Takara, Dalian, China) according to the manufacturer’s instructions. Levels of gene expression were normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. The specific primers used for gene expression analysis are listed in Table 1. The cycle threshold value for target gene was measured and calculated by computer software IQ50 (Bio-Rad Laboratories, Hercules, CA, USA). Relative mRNA level was calculated as x = 2−ΔΔCt, in which ΔΔCt = ΔCt E − ΔCt C, and ΔCt E = Ctexp − Ct18S, and ΔCt C = CtC − Ct18S [26]. Each sample was analyzed in triplicate.
Table 1
Information of qPCR primers used in this study
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Forward (5’–3’)
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Reverse (5’–3’)
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Ihh
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F5’-CCACCTCCGGGCCACATTTG-3’
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R5’-GGCCACCACATCCTCCACCA-3’
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Cre
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F5’-ATCCGAAAAGAAAACGTTGA-3’
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F5’-ATCCAGGGTACGGATATAGT-3’
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PTHrP
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F 5’-TGGAGTGTCCTGGTATTCCTGCTC-3’
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R 5’-GCGGCGCAAGTCTTGGATGG-3’
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BMP-6
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F 5’-TCCTCTATCGGCGGCTCAAC-3’
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R 5’-GTCTGCTGCTGCTGCTGCTG-3’
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GAPDH
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F 5’-AGCAGTCCCGTACACTGGCAAAC-3’
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R 5’-TCTGTGGTGATGTAAATGTCCTT-3’
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Statistical Analysis
Data for each group were derived from at least three independent samples, and all sampling was repeated thrice in each study group. Data are presented as the mean ± standard deviation. Student’s t-test was used to compare the difference between Ihhfl/fl and Ihhd/d. The significance level was set at α = 0.05. The Graph Pad Prism 5 software was used for creating statistical graphs.