The cementum, a mineralized structure surrounding the tooth root, is essential for anchoring the tooth inside the alveolar bone with collagenous fibers of the periodontal ligament (PDL).

It contains two major non-collagenous proteins: Osteopontin (OPN) and bone sialoprotein (BSP) (Grzesik and Narayanan, 2002). Bone sialoprotein (gene: Ibsp, protein: BSP) is part of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family and is involved in cell signaling, collagen binding and hydroxyapatite nucleation (Hunter and Goldberg, 1993).

BSP is involved in cell attachment and signaling through an arginine-glycine-aspartate (RGD) integrin-binding region, and acts as a positive regulator for mineral precipitation by nucleating hydroxyapatite (Ao et al., 2017, Fisher Larry W., 2008, Fisher et al., 2001, Foster et al., 2013). It is multifunctional through the C-terminal integrin-binding (RGD) domain and mediates cell attachment and migration via integrins. (Bellahcène et al., 2000, Karadag and Fisher, 2006, Rapuano et al., 2004) Additionally, the RGD domain of BSP is known for osteoblast and osteoclast formation and activation (Gordon et al., 2009, Valverde et al., 2005)

Mice with a BSP knockout genotype were found to have a nonfunctional hypo-mineralized cementum and a disconnected and disorganized PDL. Additionally, periodontal disarray and increased root resorption were detected for BSP knockout mice (Foster et al. (2013)).

To provide essential information for the molecular understanding of Ibsp in cementoblasts, this study investigates three cementoblast cell lines: I. OCCM.30-WT: The well-established cementoblast cell line (D’Errico et al., 2000, D’Errico et al., 1999),which express Bsp (Azraq et al., 2021) II. IbspΔNterm: Immortalized Ibsp knockout murine cementoblasts (Ao et al., 2017); III. IbspKAE: An Immortalized homozygous murine cementoblasts featuring a knock-in KAE mutation to replace and inactivate the RGD integrin-binding motif for a functional inhibition of BSP sequence in OCCM.30-WT cementoblasts. Knock-in inactivation was achieved by a KAE point mutation in the mouse Ibsp gene using the CRISPR/Cas9 system (Nagasaki et al., 2022).

Several established cementoblast markers have been shown to play an essential role in functional cement. Basal gene expression of these markers in the previously listed cementoblast cell lines will be investigated to quantify the role of lbsp in cementum functionality.

Tissue nonspecific alkaline phosphatase (Alpl) has a dramatic effect on acellular cement growth (Ao et al., 2017). Osteocalcin (Ocn) is a key regulator of mineralization (Chun et al., 2005). Runt related transcription factors 2 (Runx2) and Collagen type 1 alpha 1 (Col1a1) have been shown to be essential for the mineralization of the periodontium, too (Chun et al., 2005, Dalla-Bona et al., 2008, D’Errico et al., 2000, Zhang et al., 2013).

Furthermore, osteoclast markers, like Carbonic anhydrase 2 (Car2) and Cathepsin K (Ctsk), play a substantial role during tooth development and degradation of cementum (Xue et al., 2015).

Ctsk is known to degrade collagen type I and Ocn (Hou et al., 1999, Schilling et al., 2007) which are correlated with Cementum composite. Therefore, Ctsk may play a role in cementoblasts as well. Additionally, osteoclastic cells are formed by serial fusion of monocytes and are involved in process of cementum degradation.

Mechanical stress has been shown to up-regulate BSP mRNA expression in bovine cementoblasts in vitro (Yua et al., 2008). Further, the relation between gene expression regulation on mechanical force in murine cementoblasts has been presented in previous studies (Azraq et al., 2021, Niederau et al., 2020). Since the BSP knockout mice study confirmed the mentioned disorganizations of cementum and PDL, investigation of these markers in cementoblast cell lines might provide effective information about the influence of BSP on the periodontium.

The aim of this study is to analyze the basal gene expression of cementum associated markers in OCCM.30-WT, IbspΔNterm and IbspKAE cell lines, with and without compression force. In particular, possible changes in gene expression were analyzed when BSP was absent only in cementoblast cells and not in the whole tissues. This can contribute to better understanding the role of cementoblasts and BSP in functional organization of PDL and in cementum remodeling. Furthermore, a monocyte adhesion assay was performed to investigate the ability of cementoblasts with and without mechanical stimulation to trigger osteoclast-differentiation of mononuclear cells THP1.