Combining sandblasting and pink anodisation of Ti implants as a promising method for improving fibroblast adhesion and immune response.

This study examined the effect of combining the sandblasting and anodising of titanium alloys used in implants on the cell response and protein adsorption patterns. The titanium samples were divided into four groups depending on the surface treatment: machining (MC), pink anodisation (PA), sandblasting (MC04) and a combination of the last two (MC04 + PA). Their physicochemical properties were analysed by SEM/EDX, Raman, contact angle measurements and profilometry. In vitro responses were examined using human gingival fibroblastic (HGF) cells and THP-1 macrophages. Cytokine secretion, macrophage adhesion and gene expression were measured by ELISA, confocal microscopy and RT-PCR. Cell adhesion and collagen secretion were evaluated in HGF cultures. The adsorption of immune and regenerative proteins onto the surfaces was assessed employing nLC-MS/MS. MC04 + PA surfaces exhibited a change in the roughness, chemical composition and hydrophilicity of the material, showing more elongated HGF cells and a considerable increase in the area of cells exposed to the MC04 + PA surfaces. Moreover, cells cultured on MC04 + PA generally showed a reduction in the expression of proinflammatory genes (TNF-α, MCP-1, C5, NF-kB and ICAM-1) and an increase in the secretion of anti-inflammatory cytokines, such as IL-4. These results correlated with the proteomic data; we found preferential adsorption of proteins favouring cell adhesion, such as DSC1 and PCOC1. A considerable reduction in the adsorption of immunoglobulins and proteins associated with acute inflammatory response (including SAA4) was also observed. The study highlights the potential advantages of MC04 + PA surface treatment to modify dental implant abutments; it enhances their compatibility with soft tissues and reduces the inflammatory response.

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.

Enhancing the correlation between in vitro and in vivo experiments in dental implant osseointegration: investigating the role of Ca ions.

This study delves into the osteogenic potential of a calcium-ion modified titanium implant surface, unicCa, employing state-of-the-art proteomics techniques both in vitro (utilizing osteoblasts and macrophage cell cultures) and in vivo (in a rabbit condyle model). When human osteoblasts (Hobs) were cultured on unicCa surfaces, they displayed a marked improvement in cell adhesion and differentiation compared to their unmodified counterparts. The proteomic analysis also revealed enrichment in functions associated with cell migration, adhesion, extracellular matrix organization, and proliferation. The analysis also underscored the involvement of key signalling pathways such as PI3K-Akt and mTOR. In the presence of macrophages, unicCa initially exhibited improvement in immune-related functions and calcium channel activities at the outset (1 day), gradually tapering off over time (3 days). Following a 5-day implantation in rabbits, unicCa demonstrated distinctive protein expression profiles compared to unmodified surfaces. The proteomic analysis highlighted shifts in adhesion, immune response, and bone healing-related proteins. unicCa appeared to influence the coagulation cascade and immune regulatory proteins within the implant site. In summary, this study provides a comprehensive proteomic analysis of the unicCa surface, drawing correlations between in vitro and in vivo results. It emphasizes the considerable potential of unicCa surfaces in enhancing osteogenic behavior and immunomodulation. These findings significantly contribute to our understanding of the intricate molecular mechanisms governing the interplay between biomaterials and bone cells, thereby facilitating the development of improved implant surfaces for applications in bone tissue engineering.

Using osteogenic medium in the in vitro evaluation of bone biomaterials: Artefacts due to a synergistic effect.

In vitro tests using bone cells to evaluate the osteogenic potential of biomaterials usually employ the osteogenic medium (OM). The lack of correlation frequently reported between in vitro and in vivo studies in bone biomaterials, makes necessary the evaluation of the impact of osteogenic supplements on these results. This study analysed the proteomic profiles of human osteoblasts (HOb) cultured in the media with and without osteogenic agents (ascorbic acid and ß-glycerol phosphate). The cells were incubated for 1 and 7 days, on their own or in contact with Ti. The comparative Perseus analysis identified 2544 proteins whose expression was affected by osteogenic agents. We observed that the OM strongly alters protein expression profiles with a complex impact on multiple pathways associated with adhesion, immunity, oxidative stress, coagulation, angiogenesis and osteogenesis. OM-triggered changes in the HOb intracellular energy production mechanisms, with key roles in osteoblast maturation. HOb cultured with and without Ti showed enrichment in the skeletal system development function due to the OM. However, differentially expressed proteins with key regenerative functions were associated with a synergistic effect of OM and Ti. This synergy, caused by the Ti-OM interaction, could complicate the interpretation of in vitro results, highlighting the need to analyse this phenomenon in biomaterial testing.

Adverse Childhood Experiences: Relationship with Empathy and Alexithymia.

Several studies showed that adults who have experienced childhood adversity are more likely to develop alexithymia and low empathy. Therefore, this research aims to analyze the relationship between childhood adversity and alexithymia and empathy in adulthood and verify a predictive explanatory model of alexithymia. The sample comprised 92 adults who responded to the sociodemographic questionnaire, the Childhood History Questionnaire, the Interpersonal Reactivity Index, and the Alexithymia Scale of Toronto. Childhood adversity showed a positive relationship with alexithymia and a negative relationship with empathy. Predictive validity showed that marital status, adverse childhood experiences (ACEs), and empathic concern predicted higher alexithymia scores. These results show the impact of these childhood experiences on adult life, underlining the importance of developing intervention programs in this field.

Proteomics as a tool to study the osteoimmunomodulatory role of metallic ions in a sol-gel coating.

The success of bone implants depends on the osteoimmunomodulatory (OIM) activity of the biomaterials in the interactions with the periimplantary tissues. Many in vitro tests have been conducted to evaluate the osteoimmunology effects of biomaterials. However, results of these tests have often been inconclusive. This study examines the properties of newly developed sol-gel coatings doped with two metal ions associated with bone regeneration, Ca and Zn. The study uses both proteomic methods and traditional in vitro assays. The results demonstrate that proteomics is an effective tool to scrutinize the OIM properties of the materials. Moreover, sol-gel coatings offer excellent base materials to evaluate the effects of metal ions on these properties. The obtained data highlight the highly tunable nature of sol-gel materials; studying the materials with different doping levels supplies valuable information on the interactions between the immune and bone-forming processes.

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.

Proteomic evaluation of human osteoblast responses to titanium implants over time.

Titanium is widely used in bone prostheses due to its excellent biocompatibility and osseointegration capacity. To understand the effect of sandblasted acid-etched (SAE) Ti implants on the biological responses of human osteoblast (HOb), their proteomic profiles were analyzed using nLC-MS/MS. The cells were cultured with the implant materials, and 2544 distinct proteins were detected in samples taken after 1, 3, and 7 days. Comparative analyses of proteomic data were performed using Perseus software. The expression of proteins related to EIF2, mTOR, insulin-secretion and IGF pathways showed marked differences in cells grown with SAE-Ti in comparison with cells cultured without Ti. Moreover, the proteomic profiles obtained with SAE-Ti were compared over time. The affected proteins were related to adhesion, immunity, oxidative stress, coagulation, angiogenesis, osteogenesis, and extracellular matrix formation functions. The proliferation, mineralization and osteogenic gene expression in HObs cultured with SAE-Ti were characterized in vitro. The results showed that the osteoblasts exposed to this material increase their mineralization rate and expression of COLI, RUNX2, SP7, CTNNB1, CAD13, IGF2, MAPK2, and mTOR. Overall, the observed proteomic profiles can explain the SAE-Ti osteogenic properties, widening our knowledge of key signaling pathways taking part in the early stages of the osseointegration process in this type of implantations.

Insight into the antibacterial mechanism of Cu-enriched sol-gel coatings employing proteomics.

Advanced antibacterial biomaterials can help reduce the severe consequences of infections. Using copper compounds is an excellent option to achieve this goal; they offer a combination of regenerative and antimicrobial functions. In this study, new CuCl2-doped sol-gel coatings were developed and physicochemically characterised. Their osteogenic and inflammatory responses were tested in vitro using human osteoblasts and THP-1 macrophages. Their antibacterial effect was evaluated using Escherichia coli and Staphylococcus aureus. The Cu influence on the adsorption of human serum proteins was analysed employing proteomics. The materials released Cu2+ and were not cytotoxic. The osteoblasts in contact with these materials showed an increased ALP, BMP2 and OCN gene expression. THP-1 showed an increase in pro-inflammatory markers related to M1 polarization. Moreover, Cu-doped coatings displayed a potent antibacterial behaviour against E. coli and S. aureus. The copper ions affected the adsorption of proteins related to immunity, coagulation, angiogenesis, fibrinolysis, and osteogenesis. Interestingly, the coatings had increased affinity to proteins with antibacterial functions and proteins linked to the complement system activation that can lead to direct bacterial killing via large pore-forming complexes. These results contribute to our understanding of the antibacterial mechanisms of Cu-biomaterials and their interaction with biological systems.

Complex effects of Mg-biomaterials on the osteoblast cell machinery: A proteomic study.

The cell-biomaterial interface is highly complex; thousands of molecules and many processes participate in its formation. Growing demand for improved biomaterials has highlighted the need to understand the structure and functions of this interface. Proteomic methods offer a viable alternative to the traditional in vitro techniques for analyzing such systems. Magnesium is a promoter of cell adhesion and osteogenesis. Here, we used the LC-MS/MS to compare the protein expression profiles of human osteoblasts (HOb) exposed to sol-gel coatings without (MT) and with Mg (MT1.5Mg) for 1, 3, and 7 days. PANTHER, DAVID, and IPA databases were employed for protein identification and data analysis. Confocal microscopy and gene expression analysis were used for further characterization. Exposure to MT1.5Mg increased the HOb cell area and the expression of SP7, RUNX2, IBP3, COL3A1, MXRA8, and FBN1 genes. Proteomic analysis showed that MT1.5Mg affected the early osteoblast maturation (PI3/AKT, mTOR, ERK/MAPK), insulin metabolism, cell adhesion (integrin, FAK, actin cytoskeleton regulation) and oxidative stress pathways. Thus, the effects of Mg on cell adhesion and osteogenesis are rather complex, affecting several pathways rather than single processes. Our analysis also confirms the potential of proteomics in biomaterial characterization, showing a good correlation with in vitro results.