Animals and diet

The construction process of homozygote Enpp1flox/flox/EIIa-Cre mice is shown in Fig. 1A. Enpp1CKI/+ conditional targeted mice were generated as described in Fig. 1B by Cyagen Biosciences Inc. (Guangzhou, China). The mouse Enpp1 gene (NCBI Reference Sequence: NM_008813.4) is located on mouse chromosome 10. For the KI model, the “loxP-Endogenous SA of intron 17-Exon 18–25 CDS-3*SV40 pA-loxP” will be inserted into intron 17, and the p.G568* (GGA to TGA) mutation will be introduced into Exon 18 through gRNA cleavage. A silent mutation will also be introduced into the “Exon 18–25 CDS” cassette and Exon 18 to prevent the binding and re-cutting of the sequence by gRNA after homology-directed repair. To engineer the targeting vector, homology arms will be generated by PCR using BAC clone RP23-85B3 as a template. Cas9 and gRNA will be co-injected into fertilized eggs using a targeting vector for mice production. A PCR test will be performed on the pups, followed by sequencing analysis. EIIa-Cre tool mice were obtained from Cyagen Biosciences Inc. (Guangzhou, China). EIIa-cre mice carry a cre transgene under the control of the adenovirus EIIa promoter that targets Cre recombinase expression in the early mouse embryo and is useful for germline deletion of loxP-flanked genes.

Fig. 1

Gene mouse construction process. A Mouse hybridization process. B The construction process of Enpp1CKI /+ mice and Enpp1flox/flox/EIIa-Cre, the schematic of gene mouse identification. The p.G568*(GGA to TGA) mutation will be introduced into Exon 18 through gRNA cleavage. C Results of gene identification in the tail of WT, Enpp1CKI/+/EIIa-Cre, and Enpp1 flox/flox/EIIa-Cre mice. D Sequencing results of tail DNA sequence of Enpp1 flox/flox/EIIa-Cre mice

The genetically modified mice were raised in-house and weaned at 3–4 weeks of age, and the mice were maintained on a normal laboratory diet. All behavioral experiments were performed during the same circadian period, and all mice were maintained on a 12 h:12 h natural light: dark cycle (8:00 a.m to 8:00 p.m). Our experiments were conducted with only male mice because it is necessary to breed the appropriate number of female mice to obtain the desired genotypes for genetic experiments. Genotyping was performed by standard PCR-based procedures using tail genomic DNA. The sample size was estimated based on previous work and published literature. All procedures were in accordance with protocols approved by the Institutional Animal Care and Use Committee, Sun Yat-Sen University.

Mouse tail genotype identification

Mouse genotyping was performed using standard protocols followed by sequencing analysis. In brief, 2 mm tail fragments were clipped when the mice were 2 weeks old, digested using a test mouse genotyping kit (MG500, Genecopoeia), inactivated, and subjected to DNA amplification. PCR was performed using Pro Taq Master Mix, dye plus (AG11112, Accurate Biology AG, CHN) on a Real-Time System (Bio-Rad). Each reaction mixture was 25 μl and contained 1 μl of the cleaved product, 12.5 μl of 2 × Master Mix, 10.5 μl of nuclease-free water, and 0.5 μl each of 10 μM. The following cycle conditions were applied: 94 °C for 30 s, 55 °C for 30 s and 72 °C for 2 min for 34 cycles; 72 °C for 7 min; and finally kept at 4 °C. The specific primers used in this study were designed using Primer 6.0 software (Applied Biosystems, Foster City, CA, USA), and the sequences of the forward and reverse primers were as follows: F4, 5′-TCAGGAATCTGGGTGGCATAGC-3′, R4, 5′-TTGATTGGCACAATCCAAGGGT-3′; F5, 5′-GACAAGTGGACTTTGGCTTCTGT T-3′, R5, 5′-TTCTCCACCCCAAATGCGCTG-3′; EIIa-M-F2, 5′-TGGCCGCTGGAG ATGAC-GTAGTTT-3′, EIIa-M-R, 5′-GAACATCTTCAGGTTCTGCGGG-3′; and R6, 5′-TTAAGGCTGCCATGCGATCAAT-3′. To further confirm the successful location of the mutation, mouse tail DNA was amplified with the primer F4R6 and subjected to DNA sequencing (Tsingke Biotechnology).

Assessment of body weight and behavioral function

All animals underwent body weight measurements once a week until 28W of age. Three unbiased observers (ZY.H, ZY.Z, T.T) analyzed neurological and hindlimb performance using the BMS. Three observers performed ROM measurements and anesthetized the mice with isoflurane low-flow inhalation and placed them on a horizontal tabletop. A full extension of the ankle joints of the mice was limited to the horizontal plane, and the hindlimb movement was restricted to the horizontal plane. The ankle ROM was measured using a goniometer, which measures the angle between the tibial long axis and the plantar aspect of the hind foot.

Micro-CT analyses

The HOTL process was observed using micro-CT analyses performed on mice of different ages (Scanco medical, μCT100, Bassersdorf, Zurich, Switzerland). To detect ectopically mineralized components, the data were analyzed at a threshold of 255. CTvol, CTAn, and DataViewer software was used to analyze the reconstructed images.

Isolation of ligament fibroblasts from mice

Drawing from prior literature, novel methods were formulated for the acquisition and cultivation of primary ligament fibroblasts [33]. Under a dissection microscope, the PLL of mice was carefully excised from a non-ossified site to avoid the possibility of contamination with osteogenic cells. The ligaments were minced into approximately 1–3 mm pieces after being rinsed with phosphate-buffered saline (PBS) to remove blood and debris. Following ligament fragment dissociation, collagenase I was used to extract cells from ligament fragments (Gibco, USA, 17100017). The cells were harvested every half hour and cultured in Dulbecco’s modified Eagle’s medium (Dmem; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum and antibiotics at 37 °C in 5% CO2.

In vitro osteogenic induction

The cells at P1 were seeded in 6-well plates at the density recommended previously in the culture medium. The osteogenic medium was applied after the cells had reached 70–80% confluence, consisting of 50 μg/ml ascorbic acid (A8960, Sigma), 10 mm β-minimal medium replacement with glycerophosphate (G9422, Sigma), and 100 nM dexamethasone (D4902). To facilitate osteogenic differentiation, the osteogenic medium was replaced every 2 days for a period of 14 and 28 days [34].

Histochemistry and immunofluorescence

After the mice were systemically perfused with paraformaldehyde at 4W, 8W, and 28W time points, the spine, knee joints, and Achilles tendons were harvested and washed three times for half an hour each time in PBS at 4 °C. The tissues were decalcified with 12.5% EDTA and embedded in OCT. Frozen sections were sliced to a thickness of 10 mm. Non-decalcified Achilles tendons were used for A For staining, non-decalcified Achilles ten-dons were subjected to Alizarin Red staining, while other stains used decalcified tissues. Cells were fixed and incubated in 1% Alizarin Red S for 10 min, followed by rinsing with distilled water. Images were then captured. Standard protocols were employed for HE, ALP, and Alcian Blue & Nuclear Fast Red Staining, and images were captured using the Leica DM4B system and Digital Pathology Section Scanner (KF-PRO-005, KFBIO technology). Immunofluorescent staining was carried out by incubating samples overnight at 4 °C with antibodies such as OCN, OPN, IBSP, RUNX2, COL2A1, SOX9, SHH, PTCH, GLI1, SUFU, and Vimentin. Subsequently, the samples were incubated with secondary antibodies, donkey anti-mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594, and donkey anti-rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594, for 1 h. Following this, the samples were stained with DAPI. Images were captured using the Leica DM6B system and Zeiss LSM 880 confocal microscope.

Measurement of mouse serum PPi and phosphorus (Pi)

To measure mouse serum PPi and Pi, we collected blood and let it clot at room temperature for 30 min. Centrifuge the clotted blood at 3000g for 15 min to separate the serum, then carefully remove the serum and store it at − 80 °C. Detection of PPi using Pyrophosphate Assay Kit (ab112155, Abcam), when ready to assay, equilibrates all reagents to room temperature and prepares the reaction mix as per the kit instructions. Add 50 µL each of standards, samples, and reaction mix to a 96-well plate, incubate for 30 min, monitor fluorescence at Ex/Em = 316/456 nm, and calculate PPi activity using the standard curve. The detection of Pi uses a serum inorganic phosphorus detection kit (BB-47426, Bestbio), which combines inorganic phosphorus with ammonium molybdate to generate ammonium phosphomolybdate. The absorbance at 325–340 nm is detected by an ELISA reader, and the inorganic phosphorus content is calculated according to the formula.

RNA extraction and RT-qPCR

Cells were harvested for gene expression analysis at 14 and 28 days of osteogenic induction. For RNA extraction, the RNeasy Animal RNA Extraction Kit (Beyotime, Shanghai, CHN) was used, followed by the Primescript™ RT Master Mix (perfect real-time) cDNA Synthesis Kit (Takara Biomedical Technology, Beijing, CHN), which converted 400 ng of RNA to cDNA. qRT-PCR was performed using PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific, USA) on a Real-Time System (Bio-Rad). Each reaction mixture was 10 μl and contained 2 μl of 5 ng/μl cDNA, 5 μl of 2× PowerUp SYBR Green Master Mix, 2 μl of nuclease-free water, and 0.5 μl each of 10 μM forward and reverse primers. The following cycle conditions were applied: 50 °C for 2 min and 95 °C for 2 min followed by 44 cycles of 15 s at 95 °C and 1 min at 60 °C. The specific primers used in this study were designed using Primer 6.0 software (Applied Biosystems, Foster City, CA, USA), and the sequences are provided in the supplementary table. The data were analyzed using the 2−ΔΔCt algorithm.

Protein extraction and WB analysis

Following treatment, cells were promptly placed on ice and then washed thrice with ice-cold PBS. Cells were lysed with RIPA lysis buffer (Boster, Wuhan, China), containing 1% HaltTM Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA) and 1% PMSF (Boster). Subsequently, the cells were sonicated for 30 s using the Sonics CVX130 (Newtown, MA, USA). After centrifugation at 12000g for 10 min at 4 °C, protein isolation was carried out. Protein extracts were separated using NuPAGETM 4–12% Bis–Tris gels (Invitrogen), followed by electroblotting to PVDF membranes (Invitrogen). The membranes were water-rinsed and blocked with 5% nonfat dry milk in TBST (Leagene, Beijing, China) for 1 h at room temperature with continuous agitation. Thereafter, the membranes were incubated overnight at 4 °C in TBST with various antibodies including ENPP1, RUNX2, SP7, COL2A1, SOX9, SHH, PTCH, GLI1, p21, p16, and GAPDH. Post-incubation, the membranes were washed thrice with TBST. Goat anti-rabbit HRP (1:4000, ab205718, Abcam) was applied and incubated at room temperature for 1 h. After washing three times with TBST, immunoblots were visualized utilizing an ECL reagent (Beyotime). This experiment was replicated three times.

Statistical analysis

The results were quantified based on at least three independent experimental groups and presented as the mean ± standard deviation (SD). This study was statistically analyzed using GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA). Two-tailed Student's t-test or one-way analysis of variance (ANOVA) was used for comparing groups, followed by Bonferroni's multiple comparison test. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001), and P < 0.05 was considered significant.