Wnt1 Promotes Cementum and Alveolar Bone Growth in a Time-Dependent Manner

The WNT/β-catenin signaling pathway plays a central role in the biology of the periodontium, yet the function of specific extracellular WNT ligands remains poorly understood. By using a Wnt1-inducible transgenic mouse model targeting Col1a1-expressing alveolar osteoblasts, odontoblasts, and cementoblasts, we demonstrate that the WNT ligand WNT1 is a strong promoter of cementum and alveolar bone formation in vivo. We induced Wnt1 expression for 1, 3, or 9 wk in Wnt1Tg mice and analyzed them at the age of 6 wk and 12 wk. Micro–computed tomography (CT) analyses of the mandibles revealed a 1.8-fold increased bone volume after 1 and 3 wk of Wnt1 expression and a 3-fold increased bone volume after 9 wk of Wnt1 expression compared to controls. In addition, the alveolar ridges were higher in Wnt1Tg mice as compared to controls. Nondecalcified histology demonstrated increased acellular cementum thickness and cellular cementum volume after 3 and 9 wk of Wnt1 expression. However, 9 wk of Wnt1 expression was also associated with periodontal breakdown and ectopic mineralization of the pulp. The composition of this ectopic matrix was comparable to those of cellular cementum as demonstrated by quantitative backscattered electron imaging and immunohistochemistry for noncollagenous proteins. Our analyses of 52-wk-old mice after 9 wk of Wnt1 expression revealed that Wnt1 expression affects mandibular bone and growing incisors but not molar teeth, indicating that Wnt1 influences only growing tissues. To further investigate the effect of Wnt1 on cementoblasts, we stably transfected the cementoblast cell line (OCCM-30) with a vector expressing Wnt1-HA and performed proliferation as well as differentiation experiments. These experiments demonstrated that Wnt1 promotes proliferation but not differentiation of cementoblasts. Taken together, our findings identify, for the first time, Wnt1 as a critical regulator of alveolar bone and cementum formation, as well as provide important insights for harnessing the WNT signal pathway in regenerative dentistry.


Histology 27
For decalcified histology, jaws were fixed in 3.5% PBS-buffered formaldehyde, dehydrated in 28 ascending alcohol solution, and embedded in methylmethacrylate. 4-μm-thick sections were 29 cut with a Microtec rotation microtome (Techno-Med, Germany) and stained by von Kossa/van 30 Gieson, toluidine blue, and Masson's Trichrome staining according to standard protocols. 31 Calcein double labels (injected 10 and 3 days before euthanization) were visualized with a 32 fluorescence microscope (Axioscope, Carl Zeiss Microscopy GmbH, Germany). 1 Histomorphometry of cementum was performed using the OsteoMeasure system (Osteometrics 2 Inc., USA) as previously described (Koehne et al. 2016). Briefly: Histomorphometric 3 measurements were performed on toluidineblue-stained sagittal sections of the mandibular 4 molars. Acellular cementum was quantified along its full length (500 to 700 µm) on the distal 5 root surface of the first mandibular molar. Cellular cementum was measured at the distal root 6 apex of the first mandibular molar. Calcein measurements were performed using the 7 OsteoMeasure system (Osteometrics Inc., USA). Measurements of osteoid, cementoid, and the 8 non-mineralized organic tissue within the ectopic calcified matrix were performed using 9 OsteoidHisto according to the protocol (van 't Hof et al. 2017). Blood vessel areas were 10 measured using ZEN Blue software and compared to the total pulp volume. 11

Immunohistochemistry 12
For immunohistochemistry, animals were deeply anesthetized by a mixture of ketanest and 13 rompun and perfused through the heart with 4% paraformaldehyde (w/v) and 0.1% 14 The jaws were dissected sagittally and cryoprotected in 20% sucrose overnight. Thereafter, the 16 tissue was embedded in optimal cutting temperature OCT compound (Tissue Tek O.C.T. 17 Compound, Sakura) and frozen onto specimen holders at -20 °C. 16µm cryosections were 18 prepared at -20 °C with and collected on super frost glass slides (CM 3050S from Leica). 19 After washing in phosphate-buffered saline (PBS) (pH 7,2), the sections were treated with 0.3% 20  Wnt1Tg and WT mouse skulls were fixed in 4% PFA (pH 7.4) overnight. Next, they were either 4 decalcified in 10% EDTA for two weeks and dehydrated in 30% sucrose for 24 hours or 5 embedded directly. Embedding was performed with OCT (Tissue Tek O.C.T. Compound, 6 Sakura) and 10µm frozen tissue sections were cut using a Kryostat (CM 3050S from Leica). 7 The fresh samples were washed with PBST 0,1% for 3x5min. The staining was performed, 8 using the Molecular Instruments HCR v.3.0 protocol for "generic sample on slide". The used 9 probes (Wnt1, Col1a1) were designed and purchased from Molecular instruments (Los Angeles, 10 CA 90041). 11

Stable transfection and selection 17
OCCM cells were transfected with the pLNCX control vector and the Wnt1-HA (Schulze et al. 18

Cell number determination 23
The OCCM cells were diluted to a suitable concentration. 10 µL OCCM cell suspension was 24 pipetted in a Neubauer chamber. In order to accurately count the number, two out of 4 corner 25 squares were employed to take the average cell count from each of the sets. The counted number 26 was multiplied by 10,000 (10 4 ) and the dilution ratio. The unit is per milliliter. 27

Mineralization analysis 28
After 2 to 12 days of differentiation (induced with 50µg/mL Ascorbic acid and 10mM -29 Glycerophosphate), cells were washed with 1x PBS, fixed with cold 90% ethanol for 10 30 minutes, washed twice with water, incubated with 40mM alizarin red staining solution at room 31 temperature for 10 minutes, and washed 3 times with water. After the cells were photographed 1 on the lightbox, quantification was performed. For this, the stained cultures were incubated for 2 30 minutes in 800 µL 10 % acetic acid at room temperature, scraped off and pipetted into a 1.5 3 mL reaction tube, incubated for 5 minutes at 85°C and 2 minutes at 4 °C, and then centrifuged 4 for 10 minutes at 13000xg. From the supernatant, 400 µL were carefully transferred to a new 5 reaction tube and mixed with 50 µL 10% ammonium hydroxide solution. The absorbance was 6 measured at 450nm. 7

Protein isolation 8
The transfected OCCM cells were lysed with 400 µL radio-immunoprecipitation assay buffer 9 (RIPA buffer) per cell culture dish (⌀10), including protease and phosphatase inhibitors on day 10 5 of differentiation, scraped off and transferred to 1.5 mL reaction tube, incubated at 4 °C for 11 15 minutes, and then centrifuged for 15 minutes at 13000xg and 4 °C. The supernatant was 12 pipetted into a new reaction tube, frozen in liquid nitrogen, and stored at -80°C. 13

Western blot 14
Cell lysis was transferred from a SDS-PAGE gel (10%) onto a nitrocellulose membrane. The 15 transfer process was performed using electroblotting at 4°C and 15V overnight. To examine the 16 success of the transfer step, the total protein Ponceau S staining was performed. Subsequently, 17 the nitrocellulose membrane was put into blotting buffer (5% BSA + 95% TBST) for 60 minutes (Wnt1Tg) mice. Wnt1 was induced for 1 week (1W Wnt1) and 3 weeks (3W Wnt1) in 6-weeks-4 old mice and 9 weeks (9W Wnt1) in 12-weeks-old mice. The incisors appear chalky white after 5