Synthetic cementum protein 1–derived peptide regulates mineralization in vitro and promotes bone regeneration in vivo

Abstract

The use of recombinant proteins has revolutionized the development of biologic pharmaceuticals; however, they are not free of complications. Some have very high molecular weight, some demonstrate in vivo instability, and the high cost of producing them remains a major problem. On the other hand, it has been shown that peptides derived from active domains keep their biologic activity and can trigger events, such as osteogenesis and bone regeneration. Small peptides are advantageous because of their ease of synthesis and handling and their low immunogenic activity. The purpose of this study was to investigate the functions of a synthetic peptide, cementum protein 1-peptide1 (CEMP-1-p1), both in vitro and in vivo. Our results show that CEMP-1-p1 significantly enhanced the proliferation and differentiation of human periodontal ligament cells toward a mineralizing-like phenotype, as evidenced by increasing alkaline phosphatase (ALP)-specific activity and osterix, runt-related transcription factor (RUNX)-2, integrin binding sialoprotein, bone morphogenetic protein-2, osteocalcin, and cementum protein (CEMP)-1 expression at mRNA and protein levels. In vivo assays performed through standardized critical-size calvarial defects in rats treated with CEMP-1-p1 resulted in newly formed bone after 30 and 60 d. These data demonstrate that CEMP-1-p1 is an effective bioactive peptide for bone tissue regeneration. The application of this bioactive peptide may lead to implementing new strategies for the regeneration of bone and other mineralized tissues.-Correa, R., Arenas, J., Montoya, G., Hoz, L., López, S., Salgado, F., Arroyo, R., Salmeron, N., Romo, E., Zeichner-David, M., Arzate, H. Synthetic cementum protein 1-derived peptide regulates mineralization in vitro and promotes bone regeneration in vivo.

Cementum protein 1‐derived peptide (CEMP 1‐p1) modulates hydroxyapatite crystal formation in vitro

“…Lyophilized peptide was dissolved in distilled deionized water at room temperature (RT). All peptide solutions were filtered (0.22‐μm filter) and stored at 4°C before use …”

“…CEMP1 may be considered as an intrinsically disordered protein (IDP), since its sequence contains high percentage of random coil secondary structures . Recently, we have demonstrated that CEMP1's N‐terminus ‐derived peptide promotes differentiation of periodontal ligament cells toward a “mineralizing‐like” phenotype, and more importantly, the bioactive peptide shows osteoinductive and osteogenic properties, which enhanced the physiological deposition and maturation of newly formed bone in critical‐sized calvarial defects in Wistar rats . However, the characterization of undefined protein regions anticipates the disclosure of innovative functions into current biological processes; therefore, this study was designed to show that CEMP1‐p1 nucleates, has affinity, and promotes the growth of HA crystals in vitro.…”

Carboxy-Terminal Cementum Protein 1-Derived Peptide 4 (cemp1-p4) Promotes Mineralization through wnt/β-catenin Signaling in Human Oral Mucosa Stem Cells

“…HOMSCs were plated at 5 × 10 4 density in 6-well culture plates and cultured for 5, 10 and 15 days in the conditions as previously described [12]. Alkaline phosphatase (ALP)-specific activity was performed as described by Lowry et al [76].…”

“…Cultures were stained with 2% Alizarin Red S at pH 4.2 (ARS; Millipore Sigma, Burlington, MA, USA) for 10 min. Calcium deposits were documented and analyzed with ImageJ software (National Institutes of Health (NIH), Bethesda, MD, USA) Mineralization was quantified by the cetylpyridinium chloride (CPC) method [12]. Briefly, alizarin red was diluted in a 2 mL/well with 10% CPC, and incubated for 1 h at 37 • C. Then, the solution was diluted five times in CPC, and the absorbance reading was at 570 nm (Filter Max F5; Molecular Devices, LLC.…”

“…These two proteins have shown to promote the proliferation and differentiation of periodontal ligament cells toward a "mineralizing-like" phenotype [9][10][11]. Preclinical studies have shown that CEMP1 s peptide sequences can be utilized in therapeutic interventions such as critical-sized calvarial defects, mimicking hrCEMP1 s (human recombinant Cementum Protein 1) biological and therapeutic capabilities [12]. Therefore, CEMP1 s short peptide sequences are possible regulators of stem cell differentiation toward a "mineralizing-like" cell phenotype [12].…”