Zambaga Khorolsuren1,2, Orsolya Lang1, Eva Pallinger1, Anna Foldes3, Gyulai-Gaál Szabolcs4, Gabor Varga3, Gabor Mezo5, Janos Vag2, Laszlo Kohidai1. 1. Department of Genetics, Cell and Immunobiology, Semmelweis University, Budapest, Hungary. 2. Department of Conservative Dentistry, Semmelweis University, Budapest, Hungary. 3. Department of Oral Biology, Semmelweis University, Budapest, Hungary. 4. Department of Oral Diagnostics, Faculty of Dentistry, Semmelweis University, Budapest, Hungary. 5. MTA-ELTE Research Group of Peptide Chemistry, Budapest, Hungary.
Abstract
BACKGROUND AND OBJECTIVE: Periodontal ligament cells (PDLCs) are an important source for periodontal tissue healing and regeneration. Proper cell adhesion is a key for survival of anchorage-dependent cells and also initiates further intracellular signals for essential cellular functions. We aimed to test 3 different synthetic conjugates with integrin-binding RGD sequence (SAK-c[RGDfC], AK-c[RGDfC], and SAK-opn on the adhesion of human PDLCs and subsequent events including proliferation, migration, behavior of cell surface molecules, and osteogenic differentiation. MATERIALS AND METHODS: Synthetic peptides were synthesized by solid-phase technique and attached to branched chain polymeric polypeptides via thioether linkage. Simple adsorption method was used to coat tissue culture plastic or electric arrays. PDLCs were isolated from 24 surgically extracted human third molars. Cell adhesion and proliferation were measured with real-time impedimetric xCELLigence SP system. Cell migration assay was performed with Ibidi® Culture inserts. Cell surface antigens were detected using flow cytometry analysis. Osteogenic differentiation was assessed with alkaline phosphatase (ALP) assay and Alizarin Red S staining, and real-time qPCR was performed to analyze the osteoblast-related gene expression. Osteogenic differentiation and adipogenic differentiation of PDLCs were monitored by real-time Electrical Cell-Substrate Impedance Spectroscopy (ECIS). RESULTS: Primary outcome of this study relies on that all three synthetic RGD peptides improved PDLC adhesion (P < .05). When animal serum is absent in culture medium, SAK-c[RGDfC] and AK-c[RGDfC] elevated cell adhesion (P < .05). Cell migration was enhanced by SAK-c[RGDfC] and AK-c[RGDfC] (P < .05). After 1-week treatment, all synthetic peptides elevated CD105 (1.7- to 2.2-fold) and CD146 (1.3- to 1.5-fold) markers and caused different integrin patterns. ALP activity (1.4-fold) and ARS (1.8- and 2.0-fold) were increased by SAK-c[RGDfC] and AK-c[RGDfC] in absence of osteogenic supplements, and all the peptides supported the mineralization under osteogenic condition (P < .05). RT-qPCR revealed the upregulation of bone sialoprotein (5.0- to 7.8-fold), osteocalcin (2.3- to 2.7-fold), and ALP (1.9- to 2.3-fold) gene expression in osteogenesis-induced PDLCs. ECIS monitoring showed that higher impedance was generated by the osteogenic induction compared with the adipogenic or the non-induced (P < .05). CONCLUSIONS: Our study demonstrates that SAK-c[RGDfC] and AK-c[RGDfC] improved adhesion and migration of PDLCs and supported osteogenic differentiation of PDLCs. These cyclic RGD peptides proved to be applicable biocompatible material in regenerative medicine.
BACKGROUND AND OBJECTIVE: Periodontal ligament cells (PDLCs) are an important source for periodontal tissue healing and regeneration. Proper cell adhesion is a key for survival of anchorage-dependent cells and also initiates further intracellular signals for essential cellular functions. We aimed to test 3 different synthetic conjugates with integrin-binding RGD sequence (SAK-c[RGDfC], AK-c[RGDfC], and SAK-opn on the adhesion of human PDLCs and subsequent events including proliferation, migration, behavior of cell surface molecules, and osteogenic differentiation. MATERIALS AND METHODS: Synthetic peptides were synthesized by solid-phase technique and attached to branched chain polymeric polypeptides via thioether linkage. Simple adsorption method was used to coat tissue culture plastic or electric arrays. PDLCs were isolated from 24 surgically extracted human third molars. Cell adhesion and proliferation were measured with real-time impedimetric xCELLigence SP system. Cell migration assay was performed with Ibidi® Culture inserts. Cell surface antigens were detected using flow cytometry analysis. Osteogenic differentiation was assessed with alkaline phosphatase (ALP) assay and Alizarin Red S staining, and real-time qPCR was performed to analyze the osteoblast-related gene expression. Osteogenic differentiation and adipogenic differentiation of PDLCs were monitored by real-time Electrical Cell-Substrate Impedance Spectroscopy (ECIS). RESULTS: Primary outcome of this study relies on that all three synthetic RGD peptides improved PDLC adhesion (P < .05). When animal serum is absent in culture medium, SAK-c[RGDfC] and AK-c[RGDfC] elevated cell adhesion (P < .05). Cell migration was enhanced by SAK-c[RGDfC] and AK-c[RGDfC] (P < .05). After 1-week treatment, all synthetic peptides elevated CD105 (1.7- to 2.2-fold) and CD146 (1.3- to 1.5-fold) markers and caused different integrin patterns. ALP activity (1.4-fold) and ARS (1.8- and 2.0-fold) were increased by SAK-c[RGDfC] and AK-c[RGDfC] in absence of osteogenic supplements, and all the peptides supported the mineralization under osteogenic condition (P < .05). RT-qPCR revealed the upregulation of bone sialoprotein (5.0- to 7.8-fold), osteocalcin (2.3- to 2.7-fold), and ALP (1.9- to 2.3-fold) gene expression in osteogenesis-induced PDLCs. ECIS monitoring showed that higher impedance was generated by the osteogenic induction compared with the adipogenic or the non-induced (P < .05). CONCLUSIONS: Our study demonstrates that SAK-c[RGDfC] and AK-c[RGDfC] improved adhesion and migration of PDLCs and supported osteogenic differentiation of PDLCs. These cyclic RGD peptides proved to be applicable biocompatible material in regenerative medicine.