Deficiency of the SMOC2 matricellular protein impairs bone healing and produces age-dependent bone loss

Abstract

Secreted extracellular matrix components which regulate craniofacial development could be reactivated and play roles in adult wound healing. We report a patient with a loss-of-function of the secreted matricellular protein SMOC2 (SPARC related modular calcium binding 2) presenting severe oligodontia, microdontia, tooth root deficiencies, alveolar bone hypoplasia, and a range of skeletal malformations. Turning to a mouse model, Smoc2-GFP reporter expression indicates SMOC2 dynamically marks a range of dental and bone progenitors. While germline Smoc2 homozygous mutants are viable, tooth number anomalies, reduced tooth size, altered enamel prism patterning, and spontaneous age-induced periodontal bone and root loss are observed in this mouse model. Whole-genome RNA-sequencing analysis of embryonic day (E) 14.5 cap stage molars revealed reductions in early expressed enamel matrix components (Odontogenic ameloblast-associated protein) and dentin dysplasia targets (Dentin matrix acidic phosphoprotein 1). We tested if like other matricellular proteins SMOC2 was required for regenerative repair. We found that the Smoc2-GFP reporter was reactivated in adjacent periodontal tissues 4 days after tooth avulsion injury. Following maxillary tooth injury, Smoc2-/- mutants had increased osteoclast activity and bone resorption surrounding the extracted molar. Interestingly, a 10-day treatment with the cyclooxygenase 2 (COX2) inhibitor ibuprofen (30 mg/kg body weight) blocked tooth injury-induced bone loss in Smoc2-/- mutants, reducing matrix metalloprotease (Mmp)9. Collectively, our results indicate that endogenous SMOC2 blocks injury-induced jaw bone osteonecrosis and offsets age-induced periodontal decay.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Radiographic and cone-beam computed tomography (CBCT) images of a 9 year-old girl with a homozygous SMOC2 mutation (also see Ref.). (A,B) Respective frontal and lateral radiographic views of the skull. Cranial bones and sutures, sella turcica, and orbit structures are normal except for the maxilla and mandible. (C) A full-frontal view of the dentition. The lower dentition displays more severe oligodontia and microdontia compared with the upper teeth. Affected teeth are typically 15–20% smaller than normal, except for the lower right first molar (red arrowhead), which is bigger. (D) Extracted CBCT images of full-lateral views. Defects include macrodontia (red arrowhead) and microdontia (yellow arrowhead). (E,F) Extracted frontal and lateral views with the lower dentition displaying more severe oligodontia and microdontia compared with the upper teeth. Image extraction of isolated teeth (using the Analyze 11.0 software) shows that a micro-root structure consistently accompanies microdontia. A false blue background was added using the Analyze 11.0 software (C–F), and the figure was labelled with Adobe Photoshop CS6.

Figure 2

Smoc2-driven GFP immunolocalization in E14.5 (A) or E18.5 (B,D) developing teeth, and in adult mouse teeth (C). Smoc2-driven GFP is detected in the molar dental follicle mesenchymal populations surrounding the outer dental epithelium (A, red arrowheads), and the mesenchyme surrounding labial cervical loop of the lower incisor at fetal (B) and adult stages (C). GFP expression in E18.5 first and second molar mesenchyme (D) is also in the mesenchyme surrounding molars. Twelve fetal E14.5 and E18.5 and six adult (7 week-old) samples were used for each analysis. Bu buccal side, Li lingual side. This, and all following figures were labelled with Adobe Photoshop CS6.

Figure 3

Dental alterations in Smoc2−/− mutant mice. Seven week-old WT (A) and Smoc2−/− mutant (C) lower incisors, the latter exhibiting slight size reductions. Scanning electron microscope (SEM) views of WT (B) and Smoc2−/− (D) incisors. (taken from the red boxed regions in panels (A,C)) show mutants have alterations in both outer and inner enamel prisms structure (red arrowheads). (E,F) μCT 3D renderings of the occlusal surface of all three lower molars to assess morphological defects. Mutants display a significant reduction in the 1st–3rd molar field length (compare green and red bars in F: WT vs. mutant overall molar field length). Additionally, an ectopic distal 4th molar is observed in a fraction of Smoc2 mutants (F, green arrowhead). (G,H) μCT-derived mid-sagittal virtual sectioning indicates thinner alveolar bone in Smoc2−/− mutant (H, yellow arrowhead) vs. WT (G, yellow arrowhead). (I,K) Standard histology (areas corresponding to those indicated by orange boxes in G,H) shows Smoc2−/− mutants have reduced alveolar ossification (yellow arrowheads). (J,L) SEM analysis (region comparable to the blue-boxed region in G,H) shows reduced enamel crystal compaction, with irregular alignments in Smoc2−/− mutant (blue arrowheads).

Figure 4

Increased bone loss and osteoclast activation in post-extraction Smoc2 mutants. Six-week post-extraction μCT images of two months-old WT (A, D) and Smoc2−/− (B, E) males show delayed healing and an extensive resorption around the extracted first molar (yellow arrowhead) and around 2nd molar (red arrowhead) in the Smoc2−/− mutant. Bone loss defects in Smoc2−/− mutant are rescued by short-term ibuprofen treatment (30 mg/kg for 10 days following surgery) (C,F). (G–I) Histological images near resorbed roots (corresponding to purple-boxed regions in D–F, respectively) show multinucleated cells (potentially osteoclasts), more pronounced in Smoc2−/− mutant (H, blue arrowheads), and smaller in the Smoc2−/− ibuprofen-treated specimen (I, green arrowheads). Three independent samples were analyzed for each group. (J) RT-PCR analysis of M1 or M2 molars and adjacent periodontal tissue shows relative expression levels of Cox2, Mmp9, and Tnfa normalized to Gapdh. Cox2 and Mmp9 mRNA are increased, while Tnfa is decreased 7 days post-extraction in Smoc2−/− mutant in both M1 and M2, in comparison to WT. Both Cox2 and Mmp9 are drastically reduced in Smoc2−/− mutants treated with ibuprofen. Each sample was obtained from 7 mice and done 3 times repeatedly. Statistical analysis was performed using Student’s t test: *p value < 0.05; **p value < 0.01; ***p value < 0.001.

Figure 5

Smoc2-GFP reporter reveals Smoc2 activation following injury. (A–C) In Smoc2-GFP reporter mice, Smoc2-driven GFP immunolocalization, weakly detected in the non-injured first molar, appears increased 4 days after molar extraction injury in adults. Smoc2-driven GFP is detected in periodontal tissue between root and bone (A), and is increased at 4 days after tooth injury (B). The increase in GFP was quantitated by RT-PCR, showing 40% higher GFP levels at day 4 (relative to Gapdh). (D–H) TRAP staining shows an increase of osteoclast cells 7 days after tooth injury in Smoc2−/− mutants (E,G), especially around the M2 roots. The resorption of M2 root is observed in a Smoc2−/− mutant with adjacent osteoclasts (G, red arrowhead). In the periodontal area, TRAP-positive cells were counted around the root-bone interface, five similar sections from each sample (magnification ×100) were captured and assessed using a HC2500 image analysis system (Fuji Photo Film, Tokyo, Japan) and the number of TRAP positive cells in each area was counted. Six independent samples were analyzed for each group.

Figure 6

Periodontal bone loss in aging Smoc2−/− mutants. One year-old Smoc2−/− mutant mice have extensive spontaneous alveolar bone resorption. μCT 3D renderings of a 1 year-old WT (A) and an age-matched Smoc2−/− mutant (B) show the resorption of alveolar bone and 2nd and 3rd maxillary molar roots in the Smoc2−/− mutant (A–D, red arrowheads). The same resorptions are also found in mandibular 4th molars posteriorly to 3rd molars of Smoc2−/− mutant (E–H, yellow arrowheads). Standard histology (of the regions indicated by purple boxes in C,D, respectively) shows that the Smoc2−/− mutant has multinucleated cells infiltrated inside of the resorbed roots (I,J, blue arrowhead).

Figure 7

A model for the role of SMOC2 following tooth injury. In the normal (WT) situation (A), SMOC2 is induced 4 days after injury. The presence of SMOC2 reduces Mmp9, regulating over-activation of osteoclasts, and allowing reparative bone remodeling in a controlled effective manner. Smoc2−/− mutants (B) display increased Mmp9 and COX2 expression, promoting osteoclast activation, leading to bone and root destruction during healing. When Smoc2−/− mutants received a post-injury ibuprofen treatment (C), drug anti-inflammatory actions reduce Mmp9 and COX2, a potential explanation for why Smoc2−/− mutants display less bone and root deterioration after treatment. Figure from scanned hand-drawing, labelled using Adobe Photoshop CS6.