Periostin is essential for the integrity and function of the periodontal ligament during occlusal loading in mice

Abstract

Background: The ability of the periodontal ligament (PDL) to absorb and distribute forces is necessary for periodontal homeostasis. This adaptive response may be determined, in part, by a key molecule, periostin, which maintains the integrity of the PDL during occlusal function and inflammation. Periostin is primarily expressed in the PDL and is highly homologous to betaig-H3 (transforming growth factor-beta [TGF-beta] inducible gene). Cementum, alveolar bone, and the PDL of periostin-null mice dramatically deteriorate following tooth eruption. The purpose of this study was to determine the role of periostin in maintaining the functional integrity of the periodontium.

Methods: The periodontia from periostin-null mice were characterized followed by unloading the incisors. The effect of substrate stretching on periostin expression was evaluated using a murine PDL cell line. Real-time reverse transcription-polymerase chain reaction was used to quantify mRNA levels of periostin and TGF-beta. TGF-beta1 neutralizing antibodies were used to determine whether the effects of substrate stretching on periostin expression are mediated through TGF-beta.

Results: Severe periodontal defects were observed in the periostin-null mice after tooth eruption. The removal of masticatory forces in periostin-null mice rescue the periodontal defects. Periostin expression was increased in strained PDL cells by 9.2-fold at 48 hours and was preceded by a transient increase in TGF-beta mRNA in vitro. Elevation of periostin in response to mechanical stress was blocked by the addition of 2.5 ng/ml neutralizing antibody to TGF-beta1, suggesting that mechanical strain activates TGF-beta to have potential autocrine effects and to increase periostin expression.

Conclusion: Mechanical loading maintains sufficient periostin expression to ensure the integrity of the periodontium in response to occlusal load.

Conflict of interest statement

The authors report no conflicts of interest related to this study.

Figures

Figure 1

Periostin mRNA expressed by PDL fibroblasts. Periostin in situ hybridization showing its localization to PDL fibroblasts within the dental alveolar area (A). Note that the in situ hybridization signal (red color) is contained within the PDL spaces. B) Higher-magnification image clearly depicting the periostin message within the PDL fibroblasts but not in the osteoblasts or the cementoblasts. (Original magnification: A, ×4; B, ×20.)

Figure 2

Loss of periostin results in dramatic periodontal defects. A) Within the periodontal attachment apparatus, periostin localizes exclusively within the PDL as shown here by immunostaining (brown color). B) With normal periostin expression, as in the wild-type control mice, the periodontium can be described by a well-defined (a) gingival tissue, (b) cervical epithelial attachment, (c) intact crestal alveolar bone, (d) narrow and regular PDL thickness, and (e) appropriate thickness of root cementum. C) In the absence of periostin (periostin-null mice), several defects become evident after the tooth eruption. The null periodontium displays (a) enlarged gingival tissue, (b) attachment loss, (c) irregular PDL, (d) dramatic alveolar bone loss, and (e) external root resorption. D and E) The periodontium of the null and wild-type animals appears intact when the teeth are unerupted. F) The wild-type adults maintain an intact and functional periodontium. G) The adult null animal develops rapid alveolar bone loss and obvious enamel defects affecting the incisors. (Original magnification: A, ×20; B and C, ×4.)

Figure 3

Periodontal defects worsened with age in the periostin-null mouse. A) Normal periodontal development and homeostasis in the control mouse. B) In the periostin-null mouse, molar teeth develop normally as reflected in the 7-day image. However, significant periodontal changes occur after the teeth erupt and start to sustain a normal occlusal load. The periodontal abnormalities can be detected radiographically at 3, 5, and 7 months, demonstrating a tendency to worsen with age. C and D) Micro-CT visualization of the periodontal tissues confirms the presence, extent, and severity of these defects (red arrows) and clearly accentuates the dramatic periodontal difference that results from a complete lack of periostin expression. In addition, notice the complete obliteration of the incisor pulp chamber (green arrow).

Figure 4

Alveolar bone loss is significantly greater in the periostin-null mice compared to control mice. A) Micro-CT qualitative assessment of interradicular alveolar bone defects. B) Histology confirms the altered integrity of the periodontal structures (hematoxylin and eosin; original magnification, ×4.) C and D) Linear quantitation of the interproximal alveolar bone from the cemento-enamel junction (CEJ) to the most coronal aspect of the alveolar crest reflects significant differences in the absence of periostin protein. *P <0.05.

Figure 5

Cyst formation in the ameloblast layer and detached cementoblasts in the periostin-null peridontium. A) The wild-type adult mice present a well-organized ameloblast layer composed of polarized cylindrical simple epithelium. B) The periostin knock-out mice display a disorganized pseudostratified epithelial layer that is poorly attached and appears to produce an amorphous matrix that covers the dentin and that is also present ectopically within this matrix-producing organ. C and E) Normal attachment and distribution of cementoblasts at the root surface (arrow) in the wild-type control. D and F) Periostin-null periodontium. Notice the absence of cementoblasts and an apparent disengagement of the PDL from the root surface. Note the absence of cementoblasts attached to the root surface (arrow). (H&E staining: A and B; TRAP and hematoxylin staining: C, D, E, and F; original magnification: A and B, ×20; C and D, ×10; E and F, ×40.)

Figure 6

Incisal reduction of the lower incisor rescues the phenotype. A) The hypofunction condition was created by taking one mandibular incisor of the periostin-null mice out of occlusion by trimming the incisal edge weekly for 3 months starting at 10 days. B) The contralateral incisor was left intact to be used as an internal control. Arrows denote normal anatomy in A and altered anatomy in B.

Figure 7

Partial rescue of the ameloblast layer by removal of occlusal force. A) Trimmed incisor from the knock-out mouse. Note the fairly homogeneous enamel surface as well as the ameloblast layer. B) The untrimmed side showing increased cyst formation (arrow). C) Higher magnification of the ameloblast layer on the trimmed side showing one of the small defects. D) Higher magnification of the irregular and detached ameloblast layer on the untrimmed side. (H&E staining: A through D; original magnification: A and B, ×1; C and D, ×10).

Figure 8

Partial rescue of the PDL by removal of occlusal force. A) Wild-type PDL space: alveolar wall is intact and the root surface is covered by attached cementoblasts. In addition, the PDL fibers are well organized. B) Null PDL: untrimmed side. The alveolar wall is irregular and discontinuous, there is lack of attachment of cementoblasts to the root surface, the PDL space is widened, and the PDL fibers lack organization. C) Null PDL: trimmed side. Histologically, the PDL width has decreased close to that of the wild-type mouse, the orientation of the fibers appears mildly affected, and the adjacent alveolar bone wall recovers its continuity. D) PDL width calculated in microns from comparable areas in all three groups (*P <0.05). (Original magnification: A through C, ×10.) Arrows depict PDL width.

Figure 9

Mechanical strain modulates periostin mRNA expression in PDL fibroblasts through TGF-β1. A) Time course variation of periostin mRNA levels following 14% stretching regimen. Periostin mRNA increased by 3.8 and 9.2-fold at 24 and 48 hours (h), respectively. The letter P = unloaded culture time points. B) Changes in TGF-β mRNA levels are ahead of that of periostin after mechanical stretching. TGF-β mRNA increased by 6.9-, 16.7-, 2.5-, 3.4-, and 3.6-fold at 6, 12, 24, 36, and 48 hours, respectively. C) Neutralizing TGF-β antibody (2.5 μg/ml) sharply reduced periostin response to mechanical stretching in PDL cells. *P <0.05; **P <0.005.