Proteomic Analysis of Mesenchymal Stem Cells and Monocyte Co-Cultures Exposed to a Bioactive Silica-Based Sol–Gel Coating

New methodologies capable of extensively analyzing the cell-material interactions are necessary to improve current in vitro characterization methods, and proteomics is a viable alternative. Also, many studies are focused on monocultures, even though co-cultures model better the natural tissue. For instance, human mesenchymal stem cells (MSCs) modulate immune responses and promote bone repair through interaction with other cell types. Here, label-free liquid chromatography tandem mass spectroscopy proteomic methods were applied for the first time to characterize HUCPV (MSC) and CD14+ monocytes co-cultures exposed to a bioactive sol–gel coating (MT). PANTHER, DAVID, and STRING were employed for data integration. Fluorescence microscopy, enzyme-linked immunosorbent assay, and ALP activity were measured for further characterization. Regarding the HUCPV response, MT mainly affected cell adhesion by decreasing integrins, RHOC, and CAD13 expression. In contrast, MT augmented CD14+ cell areas and integrins, Rho family GTPases, actins, myosins, and 14-3-3 expression. Also, anti-inflammatory (APOE, LEG9, LEG3, and LEG1) and antioxidant (peroxiredoxins, GSTO1, GPX1, GSHR, CATA, and SODM) proteins were overexpressed. On co-cultures, collagens (CO5A1, CO3A1, CO6A1, CO6A2, CO1A2, CO1A1, and CO6A3), cell adhesion, and pro-inflammatory proteins were downregulated. Thus, cell adhesion appears to be mainly regulated by the material, while inflammation is impacted by both cellular cross-talk and the material. Altogether, we conclude that applied proteomic approaches show its potential in biomaterial characterization, even in complex systems

Roughness affects the response of human fibroblasts and macrophages to sandblasted abutments

Background A strong seal of soft-tissue around dental implants is essential to block pathogens from entering the peri-implant interface and prevent infections. Therefore, the integration of soft-tissue poses a challenge in implant-prosthetic procedures, prompting a focus on the interface between peri-implant soft-tissues and the transmucosal component. The aim of this study was to analyse the effects of sandblasted roughness levels on in vitro soft-tissue healing around dental implant abutments. In parallel, proteomic techniques were applied to study the interaction of these surfaces with human serum proteins to evaluate their potential to promote soft-tissue regeneration. Results Grade-5 machined titanium discs (MC) underwent sandblasting with alumina particles of two sizes (4 and 8 μm), resulting in two different surface types: MC04 and MC08. Surface morphology and roughness were characterised employing scanning electron microscopy and optical profilometry. Cell adhesion and collagen synthesis, as well as immune responses, were assessed using human gingival fibroblasts (hGF) and macrophages (THP-1), respectively. The profiles of protein adsorption to the surfaces were characterised using proteomics; samples were incubated with human serum, and the adsorbed proteins analysed employing nLC–MS/MS. hGFs exposed to MC04 showed decreased cell area compared to MC, while no differences were found for MC08. hGF collagen synthesis increased after 7 days for MC08. THP-1 macrophages cultured on MC04 and MC08 showed a reduced TNF-α and increased IL-4 secretion. Thus, the sandblasted topography led a reduction in the immune/inflammatory response. One hundred seventy-six distinct proteins adsorbed on the surfaces were identified. Differentially adsorbed proteins were associated with immune response, blood coagulation, angiogenesis, fibrinolysis and tissue regeneration. Conclusions Increased roughness through MC08 treatment resulted in increased collagen synthesis in hGF and resulted in a reduction in the surface immune response in human macrophages. These results correlate with the changes in protein adsorption on the surfaces observed through proteomics.This work was supported by Nueva Galimplant S.L. and by Ministerio Ciencia e Innovación [PDC2021-120924-I00/ AEI/https://doi.org/10.13039/501100011033/ Unión Europea NextGenerationEU/PRTR], Universitat Jaume I [UJI-B2021-25]. Andreia Cerqueira was supported by the Margarita Salas postdoctoral contract MGS/2022/10 (UP2022-024), funded by the European Union-NextGenerationEU