Surface Modification Of Titanium Research Articles

Layer-by-layer self-assembly of PLL/CPP-ACP multilayer on SLA titanium surface: Enhancing osseointegration and antibacterial activity in vitro and in vivo

Dental Implants are expected to possess both excellent osteointegration and antibacterial activity because poor osseointegration and infection are two major causes of titanium implant failure. In this study, we constructed layer-by-layer self-assembly films consisting of anionic casein phosphopeptides-amorphous calcium phosphate (CPP-ACP) and cationic poly (L-lysine) (PLL) on sandblasted and acid etched (SLA) titanium surfaces and evaluated their osseointegration and antibacterial performance in vitro and in vivo. The surface properties were examined, including microstructure, elemental composition, wettability, and Ca2+ ion release. The impact the surfaces had on the adhesion, proliferation and differentiation abilities of MC3T3-E1 cells were investigated, as well as the material’s antibacterial performance after exposure to the oral microorganisms such as Porphyromonas gingivalis (P. g) and Actinobacillus actinomycetemcomitans (A. a). For the in vivo studies, SLA and Ti (PLL/CA-3.0)10 implants were inserted into the extraction socket immediately after extracting the rabbit mandibular anterior teeth with or without exposure to mixed bacteria solution (P. g & A. a). Three rabbits in each group were sacrificed to collect samples at 2, 4, and 6 weeks of post-implantation, respectively. Radiographic and histomorphometry examinations were performed to evaluate the implant osseointegration. The modified titanium surfaces were successfully prepared and appeared as a compact nano-structure with high hydrophilicity. In particular, the Ti (PLL/CA-3.0)10 surface was able to continuously release Ca2+ ions. From the in vitro and in vivo studies, the modified titanium surfaces expressed enhanced osteogenic and antibacterial properties. Hence, the PLL/CPP-ACP multilayer coating on titanium surfaces was constructed via a layer-by-layer self-assembly technology, possibly improving the biofunctionalization of Ti-based dental implants.

Fabrication and evaluation of Ti6Al4V /NiCrBSi bimetallic structure with Nb/Cu bilayer by laser melting deposition

The fabrication of high-quality bimetallic structures using titanium and nickel alloys is a desirable integration for fully leveraging their remarkable properties. This study is dedicated to the surface modification of titanium brake discs, using laser melting deposition to produce the Ti6Al4V/NiCrBSi bimetallic structure and inhibition of brittle phase formation by Nb/Cu bilayer. The results showed the generation of brittle intermetallic compound phases was effectively resisted using Nb/Cu bilayer, thus preventing cracking and peeling of the Ti6Al4V substrate and NiCrBSi coatings. Moreover, the bimetallic structure fractured at the weakest position, i.e. at the Nb/Cu interface, with a maximum strength of 243.20 ± 55.16 MPa during the tensile testing, exhibiting brittle-like failure behaviour. Additionally, the friction and wear properties of the Ti6Al4V substrate and NiCrBSi coatings were evaluated at both room temperature and elevated temperature (600 °C). The results reveal that the NiCrBSi coatings effectively improved the friction and wear properties of the Ti6Al4V.

Titanium Surface Modification Techniques to Enhance Osteoblasts and Bone Formation for Dental Implants: A Narrative Review on Current Advances

Titanium Surface Modification Techniques to Enhance Osteoblasts and Bone Formation for Dental Implants: A Narrative Review on Current Advances

Modified Titanium Surface with Nano Amorphous Calcium Phosphate@Chitosan Oligolactate as Ion Loading Platform with Multifunctional Properties for Potential Biomedical Application.

Titanium (Ti) is widely used in medical and dental implants. Calcium phosphate (CPs) coatings enhance Ti implants' osteoinductive properties, and additives further improve these coatings. Recently, a nano amorphous calcium phosphate (nACP) coating decorated with chitosan oligolactate (ChOL) and selenium (Se) showed immunomodulatory effects. This study investigates the surface morphology, composition, bioactivity, mechanical properties, and Se-release mechanism of the nACP@ChOL-Se hybrid coating on Ti substrates. Amorphous calcium phosphate (ACP) was synthesized, and the nACP@ChOL-Se hybrid coating was deposited on Ti substrates using in situ anaphoretic deposition. Physico-chemical characterization was used to analyze the surface of the coating (scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier Transform Infrared Spectroscopy). The distribution of Se within the coating was examined with energy-dispersive X-ray spectroscopy (EDS). Bioactivity was evaluated in simulated body fluid (SBF), and adhesion was tested using a scratch test method. In vitro testing determined the release mechanism of Se. SEM images illustrated the surface morphology, while AFM provided a detailed analysis of surface roughness. XRD analysis revealed structural and phase composition, and EDS confirmed Se distribution within the coating. The coating exhibited bioactivity in SBF and showed good adhesion according to the scratch test. In vitro testing uncovered the release mechanism of Se from the coating. This study successfully characterized the surface morphology, composition, bioactivity, and Se-release mechanism of the nACP@ChOL-Se hybrid coating on Ti substrates, offering insights for developing immunomodulatory coatings for medical and dental applications.

The effect of silver nanoparticles on biological and corrosion behavior of electrophoretically deposited hydroxyapatite film on Ti6Al4V

Surface modification of titanium and its alloys has been seriously considered by researchers to improve their biological behaviors, in the past few decades. In present research, hydroxyapatite (HA) based composite coatings with different concentrations of 0, 2, 4, and 6 wt% of silver (Ag) nanoparticles were electrophoretically deposited (EPD) on anodized and non-anodized Ti6Al4V, using a direct current at a voltage of 30 V for 10 min at room temperature. The specimens were then characterized by means of X-ray diffraction (XRD) analysis, Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) equipped with energy dispersive spectroscopy (EDS). The cell adhesion images and cell viability results showed that HA-Ag composite coatings significantly promoted the biocompatibility of samples compared with the non-anodized and anodized Ti6Al4V. The viabilities of Mg-63 cells on HA-4%Ag coating and bi-layer coating (HA-4%Ag on anodized specimen) were approximately 91% and they were considered as the best coatings in term of biocompatibility. On the other hand, the antibacterial assessments demonstrated that HA-6%Ag coating had the best antibacterial performance compared with other samples. Furthermore, Tafel polarization curves indicated that corrosion resistance of the bi-layer coating was higher than those of the other specimens. The polarization resistance of this coating was about 7 times more than that of theTi6Al4V alloy.Graphical

Effects of the application of low-temperature atmospheric plasma on titanium implants on wound healing in peri-implant connective tissue in rats

PurposeThis study aimed to clarify the effects of surface modification of titanium (Ti) implants by low-temperature atmospheric pressure plasma treatment on wound healing and cell attachment for biological sealing in peri-implant soft tissue.MethodsHydrophilization to a Ti disk using a handheld low-temperature atmospheric pressure plasma device was evaluated by a contact angle test and compared with an untreated group. In in vivo experiments, plasma-treated pure Ti implants using a handheld plasma device (experimental group: PL) and untreated implants (control group: Cont) were placed into the rat upper molar socket, and samples were harvested at 3, 7 and 14 days after surgery. Histological evaluation was performed to assess biological sealing, collagen- and cell adhesion-related gene expression by reverse transcription quantitative polymerase chain reaction, collagen fiber detection by Picrosirius Red staining, and immunohistochemistry for integrins.ResultsIn in vivo experiments, increased width of the peri-implant connective tissue (PICT) and suppression of epithelial down growth was observed in PL compared with Cont. In addition, high gene expression of types I and XII collagen at 7 days and acceleration of collagen maturation was recognized in PL. Strong immunoreaction of integrin α2, α5, and β1 was observed at the implant contact area of PICT in PL.ConclusionsThe handheld low-temperature atmospheric pressure plasma device provided hydrophilicity on the Ti surface and maintained the width of the contact area of PICT to the implant surface as a result of accelerated collagen maturation and fibroblast adhesion, compared to no plasma application.

Characterization of modified titanium surfaces by anodisation and immersion tests

This paper explores the features of both nanoporous and compact TiO2 films formed by titanium anodisation in two experimental conditions, given that they have completely different morphologies and properties than those found by our previous studies. After anodisation, samples have been subjected for 20 days to immersion tests in different media (H2O, H3PO4, and KOH). Surface morphology, phase composition and wettability of anodised films were investigated using FESEM, FTIR, Raman spectroscopy, contact angle measurements and XPS, and the hydrophilicity of modified surfaces was investigated by immersion tests. Nanoporous films exhibited hydrophobic surfaces, but contact angle values gradually decreased after immersing films in H2O, H3PO4 and KOH media, respectively. However, compact films produced superhydrophilic surfaces, both before and after immersion tests, with the exception of immersing the film in a H3PO4 medium due to film removal by acid attack. As for compact films, an unusual morphology revealed by the presence of cone-shaped particles might be responsible for the adsorption of –OH groups arranged so as to favour anatase phase formation.

Cold Atmospheric Plasma Improves the Colonization of Titanium with Primary Human Osteoblasts: An In Vitro Study.

Several studies have shown that cold atmospheric plasma (CAP) treatment can favourably modify titanium surfaces to promote osteoblast colonization. The aim of this study was to investigate the initial attachment of primary human osteoblasts to plasma-treated titanium. Micro-structured titanium discs were treated with cold atmospheric plasma followed by the application of primary human osteoblasts. The microwave plasma source used in this study uses helium as a carrier gas and was developed at the Leibniz Institute for Surface Modification in Leipzig, Germany. Primary human osteoblasts were analyzed by fluorescence and cell biological tests (alkaline phosphatase activity and cell proliferation using WST-1 assay). The tests were performed after 4, 12, and 24 h and showed statistically significant increased levels of cell activity after plasma treatment. The results of this study indicate that plasma treatment improves the initial attachment of primary human osteoblasts to titanium. For the first time, the positive effect of cold atmospheric plasma treatment of micro-structured titanium on the initial colonization with primary human osteoblasts has been demonstrated. Overall, this study demonstrates the excellent biocompatibility of micro-structured titanium. The results of this study support efforts to use cold atmospheric plasmas in implantology, both for preimplantation conditioning and for regeneration of lost attachment due to peri-implantitis.

Enhanced osteogenic response by collagen type I coating on surface-modified titanium bone implants

Surface modifications of titanium (Ti) bone implants can activate an osteogenic response and accelerate osseointegration after implantation. In this study, in vitro tests were performed to evaluate different titanium dioxide TiO2 modified surfaces, sandblasted, large grit, acid-etched implant surfaces and surfaces featuring nanotubular structures (nanopore diameters of 20 and 100 nm) produced through potentiostatic anodization before and after subjecting them to covalent treatment for functionalization and subsequent coating with collagen type I (Col I). The physicochemical and morphological properties were evaluated using spectroscopy and microscopy, which confirmed the structural and biochemical modifications of the surfaces after coating with Col I. Studies with pre-osteoblastic MC3T3-E1 cells indicate improved cell adhesion and proliferation on surfaces coated with Col I, whereas cell viability remained similar for all evaluated surfaces. The results of alkaline phosphatase expression and activity, as well as a mineralization assay, demonstrated that surfaces coated with Col I acted positively on the osteogenic differentiation of the cells in comparison with the non-coated surfaces. Our findings suggest that, regardless of the previous modification of the TiO2 surface, the addition of a Col I coating presents a promising modification for bone implant applications.

Penning ion source based surface modification of titanium by nitrogen ion implantation

The nitrogen ions of the Penning ion source are bombarded on commercially available pure titanium substrates in pulses of about 5.6 μs duration. A thin film (∼500 nm) of multiphase titanium nitride is produced without additional heating of the substrate. The surface modification is studied for various energies of implanted ions at a low flux density. The 2.4 × 109 ions of specific energy are bombarded on the sample in each single pulse of the ion. Each sample is exposed to one thousand such pulses at a repetition rate of 0.1 Hz. The corresponding energy flux was transferred to the sample, promoting the growth of a thin nitride layer. X-ray diffraction (XRD) analysis demonstrates the formation of a monocrystalline multiphase titanium nitride thin film. The XRD spectra show the multiphase reflections of Ti4N2 (111), Ti9N3.87 (009), and Ti12N7 (0012) that depend on the energy of the ion beam. Ti4N2 is observed to be the dominant phase in this ion implantation process. The morphological and compositional changes of ion implanted samples are investigated using field emission scanning electron microscopy (SEM) along with energy dispersive x-ray spectroscopy (EDX). Raman scattering analysis of treated samples verified the XRD results. The penetration depth of nitrogen ions inside the titanium is calculated using the SRIM code. Vickers hardness has improved three times compared to the original sample.

The Effect of Titanium Anodization on the Bond Strength of 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP) Resin Cement.

To examine the effects of anodization and different surface modifications of titanium on bond strength to 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP) resin cement. Grade 5 titanium alloy disks (n = 160) were assigned to one of five prebonding surface treatment study groups (polished; polished and anodized; polished, etched, and anodized; sandblasted; sandblasted and anodized). Disks were adhesively bonded with 10-MDP resin cement (Panavia 21; bonding area: 3.3 mm in diameter) to composite resin cylinders. In each study group, tensile bond strength tests were conducted after 24 ± 1 hours and after 6 months (180 ± 2 days) of water storage (n = 16 specimens per water storage subgroup). Debonded specimens were stereomicroscopically analyzed to determine their fracture mode. Statistics included one-way and multifactorial ANOVA and Tukey post hoc tests (α = .05). Anodization and water storage did not significantly (P ≥ .389) affect tensile bond strength. Although subgroups of polished samples had significantly (P ≤ .031) lower bond strengths (subgroup mean values: 20 to 26 MPa) than etched or sandblasted samples (subgroup mean values: 29 to 33 MPa), they nonetheless exceeded the empirical threshold of 10 MPa used as a criterion for clinical recommendation. The fracture mode of all test specimens was predominantly cohesive. Anodization of titanium surfaces can be performed without weakening the adhesion of 10-MDP-based resin cement to titanium.

A review of functionalizing plasma electrolytic oxidation (PEO) coatings on titanium substrates with laser surface treatments

Nowadays, hybrid surface modification techniques are desirable approaches that are widely used to enhance the corrosion and wear behavior and biocompatibility of titanium dental and orthopedic implants. Laser processing is an effective, facile method with high flexibility, accuracy, and controllability for surface modification of titanium implant materials. Plasma electrolytic oxidation (PEO) is a cost-effective and eco-friendly process that can enhance the surface of valve metals like titanium, zirconium, magnesium, and aluminum, as well as non-valve metals such as zinc and niobium. This is achieved by creating thick, hard coatings that are resistant to both corrosion and wear, similar to ceramics. This review focuses on the improvement of anti-corrosion and anti-wear behavior, as well as the biocompatibility of titanium and its alloys through laser surface pre- or post-treatments involving laser surface melting (LSM) and laser surface texturing (LST), combined with PEO technology.

Surface Modification of Titanium by Oxidation Followed by Electrospark Alloying with a Graphite Electrode

Surface Modification of Titanium by Oxidation Followed by Electrospark Alloying with a Graphite Electrode

Effect of Electrocautery and Laser Treatment on the Composition and Morphology of Surface-Modified Titanium Implants.

The purpose of this study was to investigate the effects of different peri-implantitis treatment methods (Er,Cr:YSGG laser, diode laser, and electrocautery) on various titanium implant surfaces: machined; sandblasted, large-grit, and acid-etched; and femtosecond laser-treated surfaces. Grade 4 titanium (Ti) disks, with a diameter of 10 mm and a thickness of 1 mm, were fabricated and treated using the aforementioned techniques. Subsequently, each treated group of disks underwent different peri-implantitis treatment methods: Er,Cr:YSGG laser (Biolase, Inc., Foothill Ranch, CA, USA), diode laser (Biolase, Inc., Foothill Ranch, CA, USA), and electrocautery (Ellman, Hicksville, NY, USA). Scanning electron microscopy, energy-dispersive X-ray spectroscopy, and wettability were used to characterize the chemical compositions and surfaces of the treated titanium surfaces. Significant changes in surface roughness were observed in both the electrocautery (Sa value of machined surface = 0.469, SLA surface = 1.569, femtosecond laser surface = 1.741, and p = 0.025) and Er,Cr:YSGG laser (Ra value of machined surface = 1.034, SLA surface = 1.380, femtosecond laser surface = 1.437, and p = 0.025) groups. On femtosecond laser-treated titanium implant surfaces, all three treatment methods significantly reduced the surface contact angle (control = 82.2°, diode laser = 74.3°, Er,Cr:YSGG laser = 73.8°, electrocautery = 76.2°, and p = 0.039). Overall, Er,Cr:YSGG laser and electrocautery treatments significantly altered the surface roughness of titanium implant surfaces. As a result of surface composition after different peri-implantitis treatment methods, relative to the diode laser and electrocautery, the Er,Cr:YSGG laser increased oxygen concentration. The most dramatic change was observed after Er:Cr;YSGG laser treatment, urging caution for clinical applications. Changes in surface composition and wettability were observed but were not statistically significant. Further research is needed to understand the biological implications of these peri-implantitis treatment methods.

Osteogenic properties of bioactive titanium in inflammatory environment.

It is very important that the effects of surface modified titanium on osteogenic differentiation of bone marrow mesenchymal stem cells in the process of bone regeneration. The bio-function of modified titanium could be affected by the inflammatory micro-environment. The aim of this study was to investigate the effects of modified titanium on osteogenic differentiation in the inflammatory conditions and the osteogenic properties of the modified titanium dental implant in vivo. The medical pure titanium metals (PT-Ti) subjected to Anodic Oxidation (AO-Ti), Sand Blasting/acid etching (SLA-Ti) and Plasma-sprayed HA coating (HA coating-Ti) were used for regulating the osteogenic properties of MSCs in the normal and inflammatory conditions. The amount of the MSCs in the inflammatory environment were more similar to that in the non-inflammatory environment after cultured on AO-Ti samples for 7D. However, the proliferation of the MSCs was obviously inhibited on the other groups in the inflammatory condition. The morphology of MSC cells on the modified titanium surface was affected in the inflammatory conditions and the AO-Ti was more conducive to maintain the skeletal morphology of MSCs. The results of osteogenic related proteins expression showed that the amount of BMP-2 on AO-Ti group was the highest in the inflammatory conditions, and followed the order of AO-Ti >HA coating-Ti >SLA-Ti >PT-Ti. What's more, the AO-Ti samples were more beneficial to promote the expression of osteogenic genes ALP, OCN, COL-I and Runx2 in the inflammatory conditions. The results of osteogenic properties in vivo showed that the gingival depth of the AO-Ti group was smaller than that on the other groups. Some new bone could be observed around the AO-Ti implant at two weeks. The bone binding rates on AO-Ti group was the highest of 81.3% after implanted for one year. The AO-Ti was beneficial to osteogenic differentiation than other modified titanium metals in inflammatory condition. The anodic oxidation is an effective surface modification method on titanium to promote bone regeneration.

Surface Modification of Porous Titanium and Titanium Alloy Implants Manufactured by Selective Laser Melting: A Review

Titanium (Ti) and its alloy implants with porous structures manufactured by selective laser melting (SLM) can match the elastic modulus of human bone to reduce the stress‐shielding effect and satisfy the personalized requirement in orthopedic surgery. Compared with conventional casting and forging Ti and its alloy implants, SLM implants possess unique microstructural features and excellent comprehensive mechanical properties. However, the unmelted powder particles inevitably adhere to the surfaces of SLM implants, which may result in excessive surface roughness and potential health risks. Moreover, there are significant issues encountered, such as bioactivity, toxicity, antibacterial activity, corrosion, and wear resistance. Consequently, surface modification methods are essential to remove the unmelted powder particles and improve biological and mechanical properties of SLM implants. Herein, the research efforts focus exclusively on chemical (acid treatment, alkali treatment, sol–gel, chemical vapor deposition, and atomic layer deposition) and electrochemical methods (anodization and microarc oxidation) for SLM Ti and its alloy implants, especially for porous structures. Particularly, the characteristics of these methods are summarized, and their commonly used pre‐ and post‐treatment methods are introduced. In addition, the development trends and challenges in surface modification of SLM Ti and its alloy implants are discussed.

Improving Pure Titanium's Biological and Mechanical Characteristics through ECAP and Micro-Arc Oxidation Processes.

Pure titanium is limited to be used in biomedical applications due to its lower mechanical strength compared to its alloy counterpart. To enhance its properties and improve medical implants feasibility, advancements in titanium processing technologies are necessary. One such technique is equal-channel angular pressing (ECAP) for its severe plastic deformation (SPD). This study aims to surface modify commercially pure titanium using micro-arc oxidation (MAO) or plasma electrolytic oxidation (PEO) technologies, and mineral solutions containing Ca and P. The composition, metallography, and shape of the changed surface were characterized using X-ray diffraction (XRD), digital optical microscopy (OM), and scanning electron microscope (SEM), respectively. A microhardness test is conducted to assess each sample's mechanical strength. The weight % of Ca and P in the coating was determined using energy dispersive spectroscopy (EDS), and the corrosion resistance was evaluated through potentiodynamic measurement. The behavior of human dental pulp cell and periodontal cell behavior was also studied through a biomedical experiment over a period of 1-, 3-, and 7-days using culture medium, and the cell death and viability can be inferred with the help of enzyme-linked immunosorbent assay (ELISA) since it can detect proteins or biomarkers secreted by cells undergoing apoptosis or necrosis. This study shows that the mechanical grain refinement method and surface modification might improve the mechanical and biomechanical properties of commercially pure (CP) titanium. According to the results of the corrosion loss measurements, 2PassMAO had the lowest corrosion rate, which is determined to be 0.495 mmpy. The electrode potentials for the 1-pass and 2-pass coated samples are 1.44 V and 1.47 V, respectively. This suggests that the coating is highly effective in reducing the corrosion rate of the metallic CP Ti sample. Changes in the grain size and the presence of a high number of grain boundaries have a significant impact on the corrosion resistance of CP Ti. For ECAPED and surface-modified titanium samples in a 3.6% NaCl electrolyte solution, electrochemical impedance spectroscopy (EIS) properties are similar to Nyquist and Bode plot fitting. In light of ISO 10993-5 guidelines for assessing in vitro cytotoxicity, this study contributes valuable insights into pulp and periodontal cell behavior, focusing specifically on material cytotoxicity, a critical factor determined by a 30% decrease in cell viability.

A facile fabrication method of wettability modification of TiAl6V4 based on alkali solution treatment: Superhydrophilic, superhydrophobic and slippery surface

The rapid development of nanoscale surface modification technology has led to the development of superhydrophilic, superhydrophobic, and slippery liquid-infused porous surfaces (SLIPS) in recent decades. These surfaces have excellent self-cleaning and anti-contamination performance, and many studies have reported their industrial applications. Industrial and modern medical applications of titanium are rapidly expanding owing to its advantages of chemical stability, high mechanical strength, stable corrosion resistance, and biocompatibility; however, performing surface modification of titanium remains challenging. In this study, superhydrophilic, superhydrophobic, and SLIPS were fabricated on titanium using a simple surface fabrication method, and their wettabilities were analyzed and compared. Furthermore, we succeeded in applying the method used in this study to bulky and complex objects. Chemical and physical analyses were performed to confirm the fabrication, wettability properties, and nanostructures of the modified surfaces. The simple fabrication method presented a significant advancement as there are no limitations in its application to large areas, complex shapes, or mass production, unlike existing titanium wettability modification methods.

Study on the Mechanical Characteristics, Heat Resistance, and Corrosion Resistance of Unsaturated Polyester Resin Composite

In this study, the researchers aimed to broaden the applications of unsaturated polyester resin (UPR) composites in the building field by incorporating surface-modified titanium dioxide (TiO2) nanoparticles and reinforced chopped glass fibers within the UPR matrix. Specifically, the composites containing 4 wt.% TiO2 nanoparticles and 30 wt.% chopped glass fiber exhibited remarkable properties of 46.1 kJ/m2 in impact strength, 90.1 MPa in flexural strength, 78.8 MPa in tensile strength, and 121.5 °C in heat deflection temperature, which improve 207.5%, 80.3%, 62.5%, and 73.6%, respectively, when compared with the blank sample. Furthermore, the results of the corrosion resistance test showed that the composites performed poorly against polar organic and alkaline solutions, presented good corrosion resistance against acidic media, and behaved relatively stable in the salt solution.

Аnti-corrosion coatings based on modified epoxy compositions

The influence of surface-modified titanium dioxide on the electrochemical features of epoxy compositions was researched. The raise of epoxy primer protective properties by the presence of surface-modified fillers in its composition was determined. It is shown that the mechanism of the modified filler protective effect in epoxy coating applied on the steel is similar to the effect of solid zinc phosphate which effectiveness corresponds to the level of the known inhibitory pigment.