Clopidogrel Enhances Mesenchymal Stem Cell Proliferation Following Periodontitis

Abstract

Bone formation is dependent on the differentiation of osteoblasts from mesenchymal stem cells (MSCs). In addition to serving as progenitors, MSCs reduce inflammation and produce factors that stimulate tissue formation. Upon injury, MSCs migrate to the periodontium, where they contribute to regeneration. We examined the effect of clopidogrel and aspirin on MSCs following induction of periodontitis in rats by placement of ligatures. We showed that after the removal of ligatures, which induces resolution of periodontal inflammation, clopidogrel had a significant effect on reducing the inflammatory infiltrate. It also increased the number of osteoblasts and MSCs. Mechanistically, the latter was linked to increased proliferation of MSCs in vivo and in vitro. When given prior to inducing periodontitis, clopidogrel had little effect on MSC or osteoblasts numbers. Applying aspirin before or after induction of periodontitis did not have a significant effect on the parameters measured. These results suggest that clopidogrel may have a positive effect on MSCs in conditions where a reparative process has been initiated.

Keywords: aspirin; gingiva; inflammation; osteoblast; periodontal diseases; regeneration.

Figure 1.

Clopidogrel (clop) increases the number of osteoblasts and mesenchymal stem cells (CD271⁺ and CD146⁺) during resolution of inflammation. Serial paraffin sections were obtained from buccal-lingual aspects from animals treated with saline solution, aspirin (asp), and clop at baseline and during resolution of inflammation. Osteoblast number was counted as cuboidal bone–lining cells in areas of bone remodeling in hematoxylin and eosin–stained sections, and mesenchymal stem cells were identified by immunohistochemistry and immunofluorescence with antibody specific for CD271 and CD146. (A) Number of osteoblasts per unit length in areas of bone remodeling. (B) Percentage of mesenchymal stem cells (CD271⁺) from the connective tissue. (C) Percentage of mesenchymal stem cells (CD146⁺) from the connective tissue. Results are from 10 animals/group. Values are expressed as mean ± SEM. *P < 0.05.

Figure 2.

Clopidogrel (clop) increases proliferation of mesenchymal stem cells (MSCs). Serial paraffin sections were obtained from buccal-lingual aspects from animals treated with saline solution, aspirin (asp), and clop. The effect on MSC proliferation was measured in the connective tissue by double immunofluorescence assay using CD271- and Ki-67-specific antibodies. (A) Percentage of cells in proliferation (Ki-67⁺). (B) The percent total cells that are double positive for CD271 and Ki67. (C) The percent CD271⁺ cells that are Ki67⁺. Results are from 10 animals/group. Values are expressed as mean ± SEM. Significant difference (P < 0.05) among groups at *baseline and ^#experimental groups.

Figure 3.

Clopidogrel (clop) reduces the inflammatory infiltrate during resolution of inflammation. Double immunofluorescence assay using an specific antibody to CD271 and CD45 was performed to rule out the possibility that hematopoietic stem cells were included in the analysis of mesenchymal stem cells. (A) Percentage of CD45⁺ cells. (B) Percentage of mesenchymal stem cells that are also CD45⁺. (C) The percentage CD271-positive cells that are CD271/CD45 double positive. Results are from 10 animals/group. Values are expressed as mean ± SEM. *P < 0.001. ^#P < 0.05. asp, aspirin.

Figure 4.

Clopidogrel (clop) increases proliferation of human primary mesenchymal stem cells (MSCs) and murine MSCs C3H10T1/2. In vitro experiments were carried out to examine the direct effect of clop and aspirin (asp) on proliferation of human (A) and murine MSCs C3H10T1/2 (B). Five fields per well were examined in triplicate at 600×. Values are expressed as mean ± SEM. *P < 0.001.