Synthesis and characterization of silica-chitosan hybrid materials as antibacterial coatings for titanium implants

To avoid dental implant-related infections and to promote the osseointegration of titanium implants, the application of silicon and chitosan containing coatings is proposed. Silicon is a well-known osteogenic element and chitosan was selected to confer the antibacterial properties. The synthesis of hybrid silica-chitosan coatings using the sol-gel process is presented and the characterization using 29Si-NMR to verify the correct formation of the network is discussed. The 13C NMR spectroscopy was used to confirm the covalent union between chitosan and the silicon network. Hydrolytic degradation and silicon release studies showed the effective silicon release from the hybrids and, hence, the possibility to promote bone formation. The introduction of different amounts of chitosan and tetraethyl orthosilicate (TEOS) modulated the Si release. The analysis of cell cultures in vitro demonstrated that the hybrid coatings were not cytotoxic and promoted cell proliferation on their surfaces. The coatings containing 5%–10% chitosan had substantial antibacterial properties

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.This work was supported by MINECO [MAT2017-86043-R; RTC-2017-6147-1], Generalitat Valenciana [GRISOLIAP/2018/091, BEFPI/2021/043, PROMETEO/2020/069], Universitat Jaume I [UJI-B2017-37], and the University of the Basque Country [GIU18/189]. Andreia Cerqueira was supported by the Margarita Salas postdoctoral contract MGS/2022/10 (UP2022-024) financed by the European Union-NextGenerationEU. The University Medical Centre Hamburg-Eppendorf (Hamburg, Germany) and the Clinics for Gynecology AGAPLESION BETHESDA Hospital provided the blood and tissue for cell isolation. The authors would like to thank Raquel Oliver, Jose Ortega, Iraide Escobés, and Anke Borkam-Schuster for their valuable technical assistance and Antonio Coso (GMI-Ilerimplant) for producing the titanium discs

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.Funding for open access charge: CRUE-Universitat Jaume IThis work was supported by Ministerio Ciencia e Innovación [PID2020-113092RB-C21]; Generalitat Valenciana [GRISOLIAP/2018/091, APOSTD/2020/036, PROMETEO/2020/069]; Universitat Jaume I [UJI-B2021-25]; and Basque Government [PRE_2017_2_0044]. The authors would like to thank Raquel Oliver, José Ortega, José Miguel Pedra and Iraide Escobés for their valuable technical assistance and Antonio Coso (GMI-Ilerimplant) for making the titanium discs

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.This work was supported by Ministerio Ciencia e Innovación [PID2020-113092RB-C21], Generalitat Valenciana [APOSTD/2020/036, PROMETEO/2020/069], Universitat Jaume I [UJI-B2021-25] and Basque Government [MARSA21/07]. The authors would like to thank Raquel Oliver, José Ortega and Iraide Escobés for their valuable technical assistance and GMI-Ilerimplant for making the titanium discs

Protein adsorption/desorption dynamics on Ca-enriched titanium surfaces: biological implications

[EN] Calcium ions are used in the development of biomaterials for the promotion of coagulation, bone regeneration, and implant osseointegration. Upon implantation, the time-dependent release of calcium ions from titanium implant surfaces modifies the physicochemical characteristics at the implant-tissue interface and thus, the biological responses. The aim of this study is to examine how the dynamics of protein adsorption on these surfaces change over time. Titanium discs with and without Ca were incubated with human serum for 2 min, 180 min, and 960 min. The layer of proteins attached to the surface was characterised using nLC-MS/MS. The adsorption kinetics was different between materials, revealing an increased adsorption of proteins associated with coagulation and immune responses prior to Ca release. Implant-blood contact experiments confirmed the strong coagulatory effect for Ca surfaces. We employed primary human alveolar osteoblasts and THP-1 monocytes to study the osteogenic and inflammatory responses. In agreement with the proteomic results, Ca-enriched surfaces showed a significant initial inflammation that disappeared once the calcium was released. The distinct protein adsorption/desorption dynamics found in this work demonstrated to be useful to explain the differential biological responses between the titanium and Ca-ion modified implant surfaces.This work was supported by MINECO [MAT2017-86043-R; RTC-2017-6147-1], Generalitat Valenciana [GRISOLIAP/2018/091; APOSTD/2020/036, PROMETEO/2020/069], Universitat Jaume I under [ UJI-B2017-37], the University of the Basque Country under [GIU18/189] and Basque Government under [PRE_2017_2_0044]. The authors would like to thank Raquel Oliver, Jose Ortega and Iraide Escobes for their valuable technical assistance.Romero-Gavilán, F.; Cerqueira, A.; Anitua, E.; Tejero, R.; García-Arnáez, I.; Martínez-Ramos, C.; Ozturan, S.... (2021). Protein adsorption/desorption dynamics on Ca-enriched titanium surfaces: biological implications. JBIC Journal of Biological Inorganic Chemistry. 26(6):1-12. https://doi.org/10.1007/s00775-021-01886-4S11226

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.This work was supported by Ministerio de Ciencia e Innovación (PID2020-113092RB-C21/AEI/10.13039/501100011033), University of the Basque Country UPV/EHU (MARSA21/07), Diputación de Gipuzkoa (2021-CIEN-000072-01), Universitat Jaume I (UJI-B2021-25) and Generalitat Valenciana (APOSTD/2020/036, PROMETEO/2020/069). The authors would like to thank for technical and human support provided by SGIker (UPV/EHU/ERDF, EU), and the company GMI Dental Implantology SL for producing the titanium discs

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 β-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.This work was supported by Ministerio Ciencia e Innovación [PID2020-113092RB-C21/AEI/10.13039/501100011033], Generalitat Valenciana [APOSTD/2020/036, PROMETEO/2020/069], Universitat Jaume I [UJI-B2021-25] and Basque Government [MARSA21/07]. The authors would like to thank Raquel Oliver, José Ortega and Iraide Escobés for their valuable technical assistance, and GMI-Ilerimplant for making the titanium discs

The effect of calcium–magnesium mixtures in sol–gel coatings on bone tissue regeneration

Calcium and magnesium are two elements essential for bone structure and metabolism. However, their synergistic or competitive effects on bone regeneration are often overlooked during biomaterial development. We examined the interactions between Ca and Mg in sol–gel coatings doped with mixtures of CaCl2 (0.5%) and MgCl2 (0.5, 1, and 1.5%). After physicochemical characterisation, the materials were incubated in vitro with MC3T3-E1 osteoblastic cells and RAW264.7 macrophages, and the protein adsorption was analysed using nLC-MS/MS. The incorporation of the ions did not lead to the formation of crystalline structures and did not affect the sol–gel network cross-linking. The release of the ions did not cause cytotoxic effects at any tested concentration. The proteomic analysis showed that adding the Ca and Mg ions elevated the adsorption of proteins associated with inflammatory response regulation (e.g., ALBU, CLUS, HPT, HPTR, A1AG1 and A1AG2) but decreased the adsorption of immunoglobulins. The CaMg coatings had reduced affinity to proteins associated with coagulation (e.g., FA9, FA10, FA11, FA12) but increased the adsorption of proteins involved in cell adhesion (DSG1, DESP, FBLN1, ZA2G). In vitro assays revealed that the cellular response was affected by changing the concentration of Mg. Moreover, our results show that these differences reflect the changes in the concentrations of both ions in the mix but are not a simple additive effect.The work was supported by Ministerio Ciencia e Innovación [PID2020-113092RB-C21; RTC-2017-6147-1], Generalitat Valenciana [GRISOLIAP/2018/091, APOSTD/2020/036, PROMETEO/2020/069], Universitat Jaume I [GACUJI/2021/14] and Basque Government [PRE_2017_2_0044]. The authors would like to thank Raquel Oliver, José Ortega, José Miguel Pedra and Iraide Escobés for their valuable technical assistance, and Antonio Coso (GMI-Ilerimplant) for making the titanium discs

Antibacterial and osteogenic ­Cu-­doped sol-­gel coatings for Ti implant functionalization

Póster presentado en 30th Annual Scientific Meeting of the European Association for Osseointegration September 28th – October 30th 2023

Characterization of magnesium doped sol-gel biomaterial for bone tissue regeneration: the effect of Mg ion in protein adsorption

[EN] Magnesium is the fourth most abundant element in the human body with a wide battery of functions in the maintenance of normal cell homeostasis. In the bone, this element incorporates in the hydroxyapatite structure and it takes part in mineral metabolism and regulates osteoclast functions. In this study, sol-gel materials with increasing concentrations of MgCl2 (0.5, 1, and 1.5%) were synthesized and applied onto Ti surfaces as coatings. The materials were first physicochemically characterized. In vitro responses were examined using the MC3T3-E1 osteoblastic cells and RAW264.7 macrophages. Human serum protein adsorption was evaluated employing nLC-MS/MS. The incorporation of Mg did not affect the crosslinking of the sol-gel network, and a controlled release of Mg was observed; it was not cytotoxic at any of the tested concentrations. The cytoskeleton arrangement of MC3T3-E1 cells cultured on the Mg-doped materials changed in comparison with controls; the cells became more elongated, with protruded lamellipodia and increased cell surface. The expression of integrins (ITGA5 and ITGB1) was boosted by Mg-coatings. The ALP activity and expression of TGF-beta, OSX and RUNX2 genes were also increased. In RAW264.7 cells, TNF-alpha secretion was reduced, while TGF-beta and IL-4 expression rose. These changes correlated with the altered protein adsorption patterns. The Mg-doped coatings showed increased adsorption of anti-inflammatory (CLUS, IC1, CFAH, and VTNC), cell adhesion (DSG1, FILA2, and DESP) and tissue regeneration (VTNC and CYTA) proteins. This integrated approach to biomaterial characterization revealed the potential of Mg in bone tissue regeneration.This work was supported by MINECO [MAT2017-86043-R; RTC2017-6147-1], Generalitat Valenciana [GRISOLIAP/2018/091, APOSTD/2020/036, PROMETEO/2020/069], Universitat Jaume I under [UJI-B2017-37, Posdoc/2019/28], the University of the Basque Country under [GIU18/189] and Basque Government under [PRE_2017_2_0044]. The authors would like to thank Raquel Oliver, Jose Ortega, Jos ' e Miguel Pedra and Iraide Escob ' es for their valuable technical assistance and Antonio Coso (GMI-Ilerimplant) for producing the titanium discs.Cerqueira, A.; Romero-Gavilán, F.; García-Arnáez, I.; Martínez-Ramos, C.; Ozturan, S.; Izquierdo, R.; Azkargorta, M.... (2021). Characterization of magnesium doped sol-gel biomaterial for bone tissue regeneration: the effect of Mg ion in protein adsorption. Materials Science and Engineering C: Materials for Biological Applications (Online). 125:1-11. https://doi.org/10.1016/j.msec.2021.112114S11112