Amorphous titanium oxide (aTiO2) thin films biofunctionalized with CAP-p15 induce mineralized-like differentiation of human oral mucosal stem cells (hOMSCs).

Insufficient osseointegration of titanium-based implants is a factor conditioning their long-term success. Therefore, different surface modifications, such as multifunctional oxide coatings, calcium phosphates, and the addition of molecules such as peptides, have been developed to improve the bioactivity of titanium-based biomaterials. In this work, we investigate the behavior of human oral mucosal stem cells (hOMSCs) cultured on amorphous titanium oxide (aTiO2), surfaces designed to simulate titanium (Ti) surfaces, biofunctionalized with a novel sequence derived from cementum attachment protein (CAP-p15), exploring its impact on guiding hOMSCs towards an osteogenic phenotype. We carried out cell attachment and viability assays. Next, hOMSCs differentiation was assessed by red alizarin stain, ALP activity, and western blot analysis by evaluating the expression of RUNX2, BSP, BMP2, and OCN at the protein level. Our results showed that functionalized surfaces with CAP-p15 (1 µg ml-1) displayed a synergistic effect increasing cell proliferation and cell attachment, ALP activity, and expression of osteogenic-related markers. These data demonstrate that CAP-p15 and its interaction with aTiO2surfaces promote osteoblastic differentiation and enhanced mineralization of hOMSCs when compared to pristine samples. Therefore, CAP-p15 shows the potential to be used as a therapeutical molecule capable of inducing mineralized tissue regeneration onto titanium-based implants.

Characterization of aTiO2 surfaces functionalized with CAP-p15 peptide.

Functionalization of Titanium implants using adequate organic molecules is a proposed method to accelerate the osteointegration process, which relates to topographical, chemical, mechanical, and physical features. This study aimed to assess the potential of a peptide derived from cementum attachment protein (CAP-p15) adsorbed onto aTiO2 surfaces to promote the deposition of calcium phosphate (CaP) minerals and its impact on the adhesion and viability of human periodontal ligament cells (hPDLCs). aTiO2 surfaces were synthesized by magnetron sputtering technique. The CAP-p15 peptide was physically attached to aTiO2 surfaces and characterized by atomic force microscopy, fluorescence microscopy, and water contact angle measurement. We performed in vitro calcium phosphate nucleation assays using an artificial saliva solution (pH 7.4) to simulate the oral environment. morphological and chemical characterization of the deposits were evaluated by scanning electronic microscopy (SEM) and spectroscopy molecular techniques (Raman Spectroscopy, ATR-FTIR). The aTiO2 surfaces biofunctionalized with CAP-p15 were also analyzed for hPDLCs attachment, proliferation, and in vitro scratch-healing assay. The results let us see that the homogeneous amorphous titanium oxide coating was 70 nanometers thick. The CAP-p15 (1 µg/mL) displayed the ability to adsorb onto the aTiO2 surface, increasing the roughness and maintaining the hydrophilicity of the aTiO2 surfaces. The physical adsorption of CAP-p15 onto the aTiO2 surfaces promoted the precipitation of a uniform layer of crystals with a flake-like morphology and a Ca/P ratio of 1.79. According to spectroscopy molecular analysis, these crystalline deposits correspond to carbonated hydroxyapatite. Regarding cell behavior, the biofunctionalized aTiO2 surfaces improved the adhesion of hPDLCs after 24 h of cell culture, achieving 3.4-fold when compared to pristine surfaces. Moreover, there was an increase in cell proliferation and cell migration processes. Physical adsorption of CAP-p15 onto aTiO2 surfaces enhanced the formation of carbonate hydroxyapatite crystals and promoted the proliferation and migration of human periodontal ligament-derived cells in in vitro studies. This experimental model using the novel bioactive peptide CAP-p15 could be used as an alternative to increasing the osseointegration process of implants.