Abstract
Spheroids reproduce the tissue structure that is found in vivo more accurately than classic two-dimensional (2D) monolayer cultures. We cultured human periodontal ligament stem cells (HPLSCs) as spheroids that were embedded in collagen gel to examine whether their cementogenic differentiation could be enhanced by treatment with recombinant human plasminogen activator inhibitor-1 (rhPAI-1). The upregulated expression of cementum protein 1 (CEMP1) and cementum attachment protein (CAP), established cementoblast markers, was observed in the 2D monolayer HPLSCs that were treated with rhPAI-1 for 3 weeks compared with that in the control and osteogenic-induction medium groups. In the embedded HPLSC spheroids, rhPAI-1 treatment induced interplay between the spheroids and collagenous extracellular matrix (ECM), indicating that disaggregated HPLSCs migrated and spread into the surrounding ECM 72 h after three-dimensional (3D) culture. Western blot and immunocytochemistry analyses showed that the CEMP1 expression levels were significantly upregulated in the rhPAI-1-treated embedded HPLSC spheroids compared with all the 2D monolayer HPLSCs groups and the 3D spheroid groups. Therefore, 3D collagen-embedded spheroid culture in combination with rhPAI-1 treatment may be useful for facilitating cementogenic differentiation of HPLSCs.
Highlights
The periodontium supports and houses the dentition as a periodontal complex that consists of tooth root cementum, periodontal ligament (PDL), and alveolar bone [1]
These findings suggest that plasminogen activator-inhibitor type 1 (PAI-1) treatment can enhance cementogenic differentiation in the human periodontal ligament stem cells (HPLSCs) spheroids that were embedded in collagen gel and5can of 13 generate embedded cementoblastic spheroids after 3D culture for 2 days
There were significant differences in the percentage of cementum protein 1 (CEMP1)-positive cells between the PAI-1 and the control and the osteogenic inducing medium (OIM) groups (PAI-1 vs. control and OIM; p = 0.00123) (Figure 2E). These findings suggest that PAI-1 treatment can enhance cementogenic differentiation in the HPLSC spheroids that were embedded in collagen gel and can generate embedded cementoblastic spheroids after 3D culture for 2 days
Summary
The periodontium supports and houses the dentition as a periodontal complex that consists of tooth root cementum, periodontal ligament (PDL), and alveolar bone [1]. Mesenchymal stem/stromal cells (MSCs) are multipotent somatic stem cells that can differentiate into mesodermal cells, such as osteoblasts, chondrocytes, myocytes, and adipocytes [5,6]. Previous studies have examined the use of dental mesenchymal stem cells for periodontal tissue formation [7,8]. Among the tooth-derived stem cells, human PDL stem cells (HPLSCs) express various stromal cell markers, such as STRO-1, CD13, CD29, CD44, CD59, CD90, CD146, and CD105, that have similar morphological, phenotypic, and proliferation characteristics to adult MSCs [9,10]. Adult MSCs are derived from adult human tissues and provide a potentially powerful candidate for cell-based tissue engineering and regeneration [11]. HPLSCs are considered the most promising source of stem cells for periodontal regenerative therapies, including cementum formation
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