Locally released dexamethasone and its effects on osteogenic activity at implant-tissue interface.

The osseointegration of titanium implants within the host tissue holds crucial importance. The introduction of functional coatings at tissue-implant interface enhances the bioactivity of titanium implants, improves their therapeutic outcomes, and enhances the effectiveness of treatments. In this study, we focused on enhancing the bioactivity of titanium-based implant materials by coating the titanium surfaces with chitosan microspheres, which are loaded with osseointegration-promoting agent dexamethasone (DEX). Initially, chitosan microspheres were successfully produced, followed by DEX loading through diffusion, resulting in a drug loading efficiency of around 50.2 (wt %). The subsequent drug release profile displayed a 24-hour duration, releasing approximately 32.6 (wt %) of the loaded DEX. In cell proliferation assays using human osteosarcoma (SAOS-2) cells, Ti surfaces coated with DEX-loaded chitosan microspheres initially exhibited lower cell numbers compared with DEX-free ones. This observation was attributed to transient osteogenic differentiation effects of DEX, since a notable increase in cell proliferation was observed on the 7th day. Von Kossa staining revealed mineralization beginning on the 14th day, particularly evident in DEX-loaded samples. Moreover, alkaline phosphatase (ALP) activity displayed a pattern of initial increase and subsequent decrease, with DEX release from chitosan microspheres showing a clear influence on the osteogenic differentiation, especially on the 7th day. These findings align with literature, highlighting DEX's potential to enhance osteogenic differentiation and cellular behavior on chitosan microsphere-coated titanium surfaces. This study emphasizes the promising implications for functionalizing surfaces of implant materials with DEX-loaded chitosan microspheres to improve their biocompatibility and bioactivity.

Surface analysis of (Ti,Mg)N coated bone fixation devices following the rabbit femur surgery.

BACKGROUND: Magnesium (Mg) enhances the bone regeneration, mineralization and attachment at the tissue/biomaterial interface. OBJECTIVE: In this study, the effect of Mg on mineralization/osseointegration was determined using (Ti,Mg)N thin film coated Ti6Al4V based plates and screws in vivo. METHODS: TiN and (Ti,Mg)N coated Ti6Al4V plates and screws were prepared using arc-PVD technique and used to fix rabbit femur fractures for 6 weeks. Then, mineralization/osseointegration was assessed by surface analysis including cell attachment, mineralization, and hydroxyapatite deposition on concave and convex sides of the plates along with the attachment between the screw and the bone. RESULTS: According to Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS) analyses; cell attachment and mineralization were higher on the concave sides of the plates from both groups in comparison to the convex sides. However, mineralization was significantly higher on Mg-containing ones. The mean gray value indicating mineralized area after von Kossa staining was found as 0.48 ± 0.01 and 0.41 ± 0.04 on Mg containing and free ones respectively. Similarly, Fourier Transform Infrared Spectroscopy (FTIR) and X-ray diffraction (XRD) analyses showed that hydroxyapatite growth was abundant on the Mg-containing and concave sides of the plates. Enhanced mineralization and strong attachment to bone were also detected in EDS and SEM analyses of Mg-containing screws. CONCLUSION: These findings indicated that (Ti,Mg)N coatings can be used to increase attachment at the implant tissue interface due to accelerated mineralization, cell attachment, and hydroxyapatite growth.

A functional coating to enhance antibacterial and bioactivity properties of titanium implants and its performance in vitro.

Bacterial infections and lack of osseointegration may negatively affect the success of titanium (Ti) implants. In the present study, a functional coating composed of chitosan (CS) microspheres and nano hydroxyapatite (nHA) was prepared to obtain antimicrobial Ti implants with enhanced bioactivity. First, the chitosan microspheres were fixed to Ti surfaces activated by alkali and heat treatment, then nHA coatings were precipitated onto these surfaces. Ciprofloxacin was loaded into the microspheres using two different procedures; encapsulation and diffusion. Scanning electron microscopy micrographs of the modified Ti surfaces showed that the coating was successfully deposited onto the Ti surfaces and stable for 30 days in PBS. The drug was completely released from free microspheres loaded by encapsulation in 21 days whereas only 89% release was observed after immobilization. The burst release also decreased from ca. 55% to ca. 35%. The release was further reduced following the nHA precipitation. The modified Ti surfaces showed antimicrobial activity based on the bacterial time-kill assay using S. aureus, but the efficiency was affected by both nHA precipitation and drug loading strategy. Highest antimicrobial activity was seen in the samples without nHA layer, and when the drug was loaded by diffusion. Fourier transform infrared spectroscopy and X-ray diffraction analyses revealed that nHA on the surface enhanced HA growth in simulated body fluid for 3 weeks, showing increased osseointegration potential. Therefore, the proposed coating may be used to prevent Ti implant failure originated from bacterial infection and/or low bioactivity.

Assessment of bone healing using (Ti,Mg)N thin film coated plates and screws: Rabbit femur model.

Magnesium (Mg) based implants such as plates and screws are often preferred to treat bone defects because of the positive effects of magnesium in bone growth and healing. Their low corrosion resistance, however, leads to fast degradation and consequently failure before healing was completed. Previously, we developed Mg doped titanium nitrate (TiN) thin film coatings to address these limitations and demonstrated that <10 at% Mg doping led to enhanced mineralization in vitro. In the present study, in vivo performance of (Ti,Mg)N coated Ti6Al4V based plates and screws were studied in the rabbit model. Bone fractures were formed on femurs of 16 rabbits and then fixed with either (Ti,Mg)N coated (n = 8) or standard TiN coated (n = 8) plates and screws. X-ray imaging and µCT analyses showed enhanced bone regeneration on fracture sites fixed with (Ti,Mg)N coated plates in comparison with the Mg free ones. Bone mineral density, bone volume, and callus volume were also found to be 11.4, 23.4, and 42.8% higher, respectively, in accordance with µCT results. Furthermore, while TiN coatings promoted only primary bone regeneration, (Ti,Mg)N led to secondary bone regeneration in 6 weeks. These results indicated that Mg presence in the coatings accelerated bone regeneration in the fracture site. (Ti,Mg)N coating can be used as a practical method to increase the efficiency of existing bone fixation devices of varying geometry.

Magnesium doping on TiN coatings affects mesenchymal stem cell differentiation and proliferation positively in a dose-dependent manner.

BACKGROUND: In vitro evaluation of cell-surface interactions for hard tissue implants have mostly been done using osteoblasts. However, when an implant is placed in the body, mesenchymal stem cells (MSCs) play a major role in new bone formation. Therefore, using MSCs in cell-surface investigations may provide more reliable information on the prediction of in vivo behavior of implants. OBJECTIVE: In this study, Mg doped TiN coatings ((Ti,Mg)N) were prepared and tested for their effect on MSC differentiation and mineralization. METHODS: MSCs were isolated from rat bone marrow (rBMSCs) and seeded onto bare Ti, TiN and Mg containing (Ti,Mg)N surfaces. Cell proliferation, osteogenic differentiation (collagen type 1, alkaline phosphatase activity), calcium phosphate deposition (von Kossa staining, Scanning Electron Microscopy) analysis were conducted. RESULTS: Differentiation towards osteoblast lineage was significantly improved with the increment in Mg presence. Collagen type I deposition, mineralization, and the ALP activity were higher on high Mg containing (>10 at% Mg) surfaces but differentiation of rBMSCs were found to be delayed. CONCLUSIONS: Mg presence affected rBMSCs proliferation and differentiation positively in a dose-dependent manner. However, high Mg amounts delayed both proliferation and differentiation.

Behavior of mammalian cells on magnesium substituted bare and hydroxyapatite deposited (Ti,Mg)N coatings.

TiN and (Ti,Mg)N thin film coatings were deposited on titanium substrates by using cathodic arc physical vapor deposition (arc-PVD) technique with magnesium contents of 0, 4.24 at% (low Mg) and 10.42 at% (high Mg). The presence of magnesium on both normal (hFOB) and cancer (SaOS-2) osteoblast cell behavior was investigated in (Ti,Mg)N surfaces with or without prior hydroxyapatite (HA) deposition (in simulated body fluid, SBF). Mg incorporation on TiN films was found to have no apparent effect on the cell proliferation in bare surfaces but cell spreading was better on low Mg content surface for hFOB cells. SaOS-2 cells, on the other hand, showed an increased extra cellular matrix (ECM) deposition on low Mg surfaces but ECM deposition almost disappeared when Mg content was increased above 10 at%. HA deposited surfaces with high Mg content was shown to cause a significant decrease in cell viability. While the cells were flattened, elongated and spread over the surface in contact with each other via cellular extensions on unmodified and low Mg doped surfaces, unhealthy morphologies of cells with round shape with a limited number of extended arms was visualized on high Mg containing samples. In summary, Mg incorporation into the TiN coatings by arc-PVD technique and successive HA deposition led to promising cell responses on low Mg content surfaces for a better osteointegration performance.

Magnesium substituted hydroxyapatite formation on (Ti,Mg)N coatings produced by cathodic arc PVD technique.

In this study, formation of magnesium substituted hydroxyapatite (Ca10-xMgx(PO4)6(OH)2) on (Ti,Mg)N and TiN coating surfaces were investigated. The (Ti1-x,Mgx)N (x=0.064) coatings were deposited on titanium substrates by using cathodic arc physical vapor deposition technique. TiN coated grade 2 titanium substrates were used as reference to understand the role of magnesium on hydroxyapatite (HA) formation. The HA formation experiments was carried out in simulated body fluids (SBF) with three different concentrations (1X SBF, 5X SBF and 5X SBF without magnesium ions) at 37 °C. The coatings and hydroxyapatite films formed were characterized by scanning electron microscope (SEM), X-ray diffraction (XRD) and FTIR Spectroscopy techniques. The energy dispersive X-ray spectroscopy (EDS) analyses and XRD investigations of the coatings indicated that magnesium was incorporated in the TiN structure rather than forming a separate phase. The comparison between the TiN and (Ti, Mg)N coatings showed that the presence of magnesium in TiN structure facilitated magnesium substituted HA formation on the surface. The (Ti,Mg)N coatings can potentially be used to accelerate the HA formation in vivo conditions without any prior hydroxyapatite coating procedure.

Immobilization of laccase on polymer grafted polytetrafluoroethylene membranes for biosensor construction.

In this study, Trametes versicolor laccase was immobilized on polytetrafluoroethylene (PTFE) membranes using two different techniques, entrapment to gelatin and covalent immobilization to the surface. For surface immobilization, functional groups were formed on PTFE surface by radiofrequency (RF) plasma treatment followed by polymer grafting. Two different polymers, polyacrylamide (pAAm) and polyacrylic acid (pAAc) were tried. For polyacrylamide grafted PTFE, a two-step polymerization process was used. The membranes were first treated with hydrogen plasma and pAAm grafted PTFE (pAAm-g-PTFE) was then formed by argon plasma treatment. To produce pAAc grafted PTFE (pAAc-g-PTFE), the surface was first treated with argon plasma and AAc was then attached to the surface by heat treatment (70°C, 6h). For both cases, an optimized carbodiimide coupling reaction was used for laccase immobilization. Enzyme activity was measured by an oxygen electrode using guaiacol as substrate. All three biosensing membranes were characterized and compared in terms of optimum working conditions, storage stability and reusability. Our study concluded that although a higher activity was obtained by gelatin entrapped laccase, its mechanical instability and poor storage life makes the gelatin biosensor unattractive for multiple usages and for field measurements. pAAc-g-PTFE biosensor was found to be more stable and highly reusable (ca. 50 times) when compared with the other two biosensors. In addition, its sensitivity was suitable for field applications. Therefore, the pAAc-g-PTFE biosensor could be proposed as an alternative on-site detection tool for phenolic compound monitoring.

Alteration of PTFE surface to increase its blood compatibility.

The aim of this study is to increase the blood compatibility of polytetrafluoroethylene (PTFE), one of the preferred materials for soft-tissue application, by a two-step procedure: first, the surface was activated by hydrogen plasma followed by acrylamide attachment and, secondly, hirudin, a potent antithrombogenic protein from leeches, was immobilized to the surface. Plasma treatment conditions were optimized and different surfaces were characterized by water contact angle measurements, ATR-FT-IR and X-ray photoelectron spectroscopy (XPS). It was seen that the contact angle of the PTFE decreased from 126° to 55° in optimum conditions. Acrylamide (25% (w/v) in ethanol/acetone (50%, v/v)) was grafted to the surface by the help of argon plasma treatment (1 min, 50 W, 13 Pa). The water contact angle was further decreased to 33° with acrylamide grafting and amide groups, which were subsequently used in protein immobilization, and could be detected both by ATR-FT-IR and XPS analysis. In the second part, hirudin was attached to these amide groups on PTFE surface by an optimized EDC/NHS activation procedure. Then a thrombogenicity test was done to detect hirudin activity. The results showed that there is a significant decrease in the clot formation compared with the untreated PTFE samples and ca. 0.3-0.4 ATU/cm(2) (22-29 ng/cm(2)) of hirudin was enough to prevent the clot formation. A preliminary study showed that the hirudin immobilized membranes keep their antithrombogenic activity for at least 40 days in 37°C in PBS (0.1 M, pH 7.4). As a result, the blood compatibility of PTFE surfaces was ameliorated by plasma-induced monomer grafting and hirudin immobilization, and an alternative material was obtained to be used in medical applications such as vascular grafts, catheters, etc.