Theranostics 2019; 9(15):4265-4286. doi:10.7150/thno.35914 This issue
1. State Key Laboratory of Military Stomatology and National Clinical Research Center for Oral Diseases and Shaanxi Clinical Research Center for Oral Diseases, Department of Orthodontics, School of Stomatology, the Fourth Military Medical University, Xi'an, 710032, China.
2. State Key Laboratory of Military Stomatology, National Clinical Research Center for Oral Diseases, Shaanxi International Joint Research Center for Oral Diseases, Department of Oral Anatomy and Physiology and TMD, School of Stomatology, the Fourth Military Medical University, Xi'an, 710032, China.
3. State Key Laboratory of Oral Diseases, Department of Orthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
4. Medical College, Xijing University, Xi'an, 710123, China.
5. Division of Research, Department of Comprehensive Dentistry, the University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
6. State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, Xi'an, China.
*These authors contributed equally to this work.
Periodontal ligament stem cells (PDLSCs) can repair alveolar bone defects in periodontitis in a microenvironment context-dependent manner. This study aimed to determine whether different extracellular matrices (ECMs) exert diverse effects on osteogenic differentiation of PDLSCs and accurately control alveolar bone defect repair.
Methods: The characteristics of PDLSCs and bone marrow mesenchymal stem cells (BMSCs) with respect to surface markers and multi-differentiation ability were determined. Then, we prepared periodontal ligament cells (PDLCs)-derived and bone marrow cells (BMCs)-derived ECMs (P-ECM and B-ECM) and the related decellularized ECMs (dECMs). Transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), and protein mass spectrometry were used to distinguish the ECMs. The expression of Type IV collagen A2 (COL4A2) in the ECMs was inhibited by siRNA or activated by lentiviral transduction of relevant cells. The stemness, proliferation, and differentiation of PDLSCs were determined in vitro in different dECMs. For the in vivo analysis, different dECMs under the regulation of COL4A2 mixed with PDLSCs and Bio-Oss bone powder were subcutaneously implanted into immunocompromised mice or in defects in rat alveolar bone. The repair effects were identified by histological or immunohistochemical staining and micro-CT.
Results: B-dECM exhibited more compact fibers than P-dECM, as revealed by TEM, SEM, and AFM. Protein mass spectrometry showed that COL4A2 was significantly increased in B-dECM compared with P-dECM. PDLSCs displayed stronger proliferation, stemness, and osteogenic differentiation ability when cultured on B-dECM than P-dECM. Interestingly, B-dECM enhanced the osteogenic differentiation of PDLSCs to a greater extent than P-dECM both in vitro and in vivo, whereas downregulation of COL4A2 in B-dECM showed the opposite results. Furthermore, the classical Wnt/β-catenin pathway was found to play an important role in the negative regulation of osteogenesis through COL4A2, confirmed by experiments with the Wnt inhibitor DKK-1 and the Wnt activator Wnt3a.
Conclusion: These findings indicate that COL4A2 in the ECM promotes osteogenic differentiation of PDLSCs through negative regulation of the Wnt/β-catenin pathway, which can be used as a potential therapeutic strategy to repair bone defects.
Keywords: bone defect, extracellular matrix, osteogenic differentiation, periodontal ligament stem cells, type IV collagen A2