Cytocompatibility Evaluation Research Articles

Comparative study of resazurin reduction and MTT assays for cytocompatibility evaluation of nanofibrous materials

REMA is a more reliable and accurate approach for cytocompatibility evaluation of nanofibrous materials than MTT assay.

3D printed Polylactid Acid based porous scaffold for bone tissue engineering: an in vitro study

The objective of this study was to fabricate PLA-based porous scaffold by 3D printing technology and to evaluate their cytotoxicity and biocompatibility under in vitro conditions in respect to bone tissue engineering. Pure PLA in filamentous form was processed via 3D printing technology of fused filament fabrication into porous scaffolds. The structure and porosity of scaffolds were measured by metrotomography. PLA scaffolds were pre-treated by human serum, foetal bovine serum and complete cell culture medium to enhance bio-attractivity of the scaffold's surface for the adherence of the cells. Cells were enzymatically isolated from the periosteum of the proximal tibia and then expanded in monolayer. Periosteum-derived osteoprogenitors (PDOs) were seeded on the pre-treated PLA scaffolds and subsequent cell proliferation was measured by commercially available cell proliferation assay. Adherence of PDOs on the PLA scaffold was confirmed by scanning electron microscopy (SEM). Prepared scaffolds had well-defined structure and were characterized by uniform distribution of pores. They were non-toxic and biocompatible with PDOs, however, PLA scaffold with the periosteum-derived progenitor cells was significantly better in the group of scaffolds pre-treated with normal human serum. The obtained PLA porous scaffolds favored attachment of periosteum derived progenitors and proliferation, furthermore, cells penetrated into the scaffold through the interstitial pores which was meaningful for cytocompatibility evaluation.

Preparation and characterization of folic acid functionalized bioactive glass for targeted delivery and sustained release of methotrexate.

We have successfully prepared a novel targeted drug delivery system, composed of folic acid (FA) as targeting molecule, methotrexate (MTX) as anticancer drug, and bioactive glass (BG) as carrier. The BG nanoparticles were synthesized by sol-gel method with the dodecylamine as template. The surface of BG nanoparticles was grafted with amino groups by (3-aminopropyl) triethoxysilane, then, FA and MTX were covalently conjugated to the surface of modified BG through amidation reaction, so FA functionalized BG (BG-FA) and targeted drug delivery system (MTX-BG-FA) were prepared. The physicochemical properties of BG, BG-NH2 , and BG-FA were characterized by various methods, such as X-ray diffraction, scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, ultraviolet-visible, and so on. Moreover, the drug release results showed that the MTX-BG-FA had sustained release property because of the peptide bond between MTX and BG-FA. And the cytocompatibility evaluation demonstrated that the BG and BG-FA were biocompatible and BG-FA even could promote cell proliferation, while the MTX-BG-FA had high cytotoxicity owning to the sustained release of anticancer drug. From the above, it could be concluded that MTX-BG-FA could kill tumor cells targetedly and sustainedly, which made it a good cancer targeted therapy material. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 319-329, 2019.

Polyester-based green renewable eco-composites by solar energy tube processing: characterization and assessment of properties

A solar energy tube was used for the processing of banana fiber (BF), which is employed as filler in polycaprolactone-based green composites. The composite was made by melt-blending acrylic acid-grafted polycaprolactone (PCL-g-AA) with a coupling agent-treated banana fiber (TBF). To improve the interface adhesion of the PCL/BF composites, acrylic acid was used as a stabilizer for the blending of PCL and TBF. The water resistance of PCL-g-AA/TBF was greater than that of PCL/BF and a cytocompatibility evaluation with human foreskin fibroblasts (FBs) indicated that both materials were nontoxic. The results from the FB proliferation assays indicated a higher cytocompatibility of the PCL/BF composites than of the PCL-g-AA/TBF composites. The cell-cycle test of FBs on PCL/BF and PCL-g-AA/TBF composites were not affected by the DNA content related to damage. Moreover, both BF and TBF enhanced the polysaccharide content and antioxidant properties of the composites, demonstrating the potential of PCL/BF and PCL-g-AA/TBF composites for biomedical material applications.

Wound dressing based on electrospun PVA/chitosan/starch nanofibrous mats: Fabrication, antibacterial and cytocompatibility evaluation and in vitro healing assay

Electrospun nanofibrous mats based on biopolymers have been widely investigated for tissue engineering in recent years, primarily due to remarkable morphological similarity to the natural extracellular matrix (ECM). In this research, electrospun PVA/Chitosan/Starch nanofibrous mats were fabricated using electrospinning method for wound dressing application. The prepared nanofibrous mats were then cross-linked to enhanced the water resistance and also optimize the biodegradation rate followed by characterization and evaluation of their properties as wound dressings. The morphological studies performed by SEM and AFM showed that uniform bead-free electrospun nanofibrous mats were formed. The structural properties of the fabricated mats were characterized by FTIR. The proper porosity and balanced water absorption and water vapor transmission rate (WVTR) of obtained dressings, demonstrate their ability in providing suitable moist environment for wound, result in the appropriate wound breathing and simultaneously efficient handling of wound exudates. Suitable mechanical properties of nanofibrous dressing in both dry and wet states confirm the capability of fabricated wound dressing to protect wound area against the external forces during the healing process. Antibacterial test revealed excellent antibacterial activity of nanofibrous mats against both gram negative and gram positive bacteria. Furthermore, the in vitro cytotoxicity evaluated by MTT assay, proved appropriate cytocompatibility and cell viability of the developed nanofibrous mats which were also verified with in vitro wound healing analysis performed by scratch assay, confirming the remarkable potential of the investigated nanofibrous mats for wound dressing application.

Cytocompatibility evaluation of nano-sintered Ti-15Zr-4Nb-2Ta-0.2Pd alloy produced by spark plasma sintering technique

Nano-sintered Ti-15Zr-4Nb-2Ta-0.2Pd alloy made up of non-toxic elements was produced by mechanical alloying using high energy ball mill and synthesized by spark plasma sintering (SPS) hot furnace with an objective to develop cytocompatible metallic implant. These were done to develop an alloy capable of avoiding adverse body reaction and increase the longevity of human life by replacing the lost or diseased organs to restore form and function. Microstructure obtained shows well defined grain boundary lines for sintered CP Ti with no surface porosity. Sintered Ti-6Al-4V microstructure showed well defined sinter-bonding of grain matrix with white rings on grain boundary lines and sintered Ti-15Zr-4Nb-2Ta-0.2Pd alloy showed no definite orientation of grains on the surface and had obvious visible grain boundary lines with a few observable surface pores across the area of the specimen. Cytocompatibility test was conducted by culturing CP Ti, Ti-6Al-4V and Ti-15Zr-4Nb2Ta-0.2Pd alloys separately with murine fibroblast L929 at a concentration of 70 cells/well, and incubated for 7 days at a temperature of 37 °C. Amongst CP Ti, Ti-6Al-4V and Ti-15Zr-4Nb-2Ta-0.2Pd specimen surfaces, the murine fibroblast L929 cells were well attached, grew and formed more colonies on CP Ti and Ti-15Zr-4Nb-2Ta-0.2Pd and least attached to the Ti-6Al-4V surface.

A high-strength mineralized collagen bone scaffold for large-sized cranial bone defect repair in sheep.

Large-sized cranial bone defect repair presents a great challenge in the clinic. The ideal cranioplasty materials to realize the functional and cosmetic recovery of the defect must have sufficient mechanical support, excellent biocompatibility, good osseointegration and biodegradability as well. In this study, a high-strength mineralized collagen (MC) bone scaffold was developed with biomimetic composition, microstructure and mechanical properties for the repair of sheep large-sized cranial bone defects in comparison with two traditional cranioplasty materials, polymethyl methacrylate and titanium mesh. The compact MC scaffold showed no distinct pore structure and therefore possessed good mechanical properties. The strength and elastic modulus of the scaffold were much higher than those of natural cancellous bone and slightly lower than those of natural compact bone. In vitro cytocompatibility evaluation revealed that the human bone marrow mesenchymal stem cells (hBMSC) had good viability, attachment and proliferation on the compact MC scaffold indicating its excellent biocompatibility. An adult sheep cranial bone defect model was constructed to evaluate the performances of these cranioplasty materials in repairing the cranial bone defects. The results were investigated by gross observation, computed tomography scanning as well as histological assessments. The in vivo evaluations indicated that compact MC scaffold showed notable osteoconductivity and osseointegration with surrounding cranial bone tissues by promoting bone regeneration. Our results suggested that the compact MC scaffold has a promising potential for large-sized cranial bone defect repair.

Fabrication of tannic acid/poly(N-vinylpyrrolidone) layer-by-layer coating on Mg-based metallic glass for nerve tissue regeneration application.

For improving recovery rates and functional outcomes in large nerve defects, a nerve guide conduit, in addition to topographic, physical and chemical cues should provide contact guidance and adequate mechanical support for cell migration and axon outgrowth. Among biomaterials, magnesium (Mg) metal has potential to support nerve regeneration owing to its electrical conductivity, biodegradation and ability to be formed into wires, filaments and ribbons. However, rapid degradation of magnesium can pose a challenge. Mg-based metallic glasses with desirable features including favorable biocompatibility, proper biodegradation and good mechanical properties are a good alternative to crystalline Mg alloys. This study investigates the biocorrosion and biocompatibility of Mg-Zn-Ca metallic glass ribbon with Mg70Zn26Ca4 composition. For controlling biocorrosion, layer-by-layer coating of tannic acid/ poly(N-vinylpyrrolidone) was applied on Mg70Zn26Ca4 ribbon and characterized by SEM and FTIR. Immersion and potentiodynamic polarization test results indicated that coating significantly improved the corrosion resistance of Mg70Zn26Ca4. Schwann cells were selected for the cytocompatibility evaluation of samples due to their key role in peripheral nerve regeneration and ability to repair spinal cord injuries. The MTT assay and cell morphology results showed good biocompatibility for Mg70Zn26Ca4 metallic glass as a promising candidate for nerve regeneration and implantable nervous prosthetic devices.

Validation of in vitro assays in three-dimensional human dermal constructs.

Three-dimensional cell culture systems are urgently needed for cytocompatibility testing of biomaterials. This work aimed at the development of three-dimensional in vitro dermal skin models and their optimization for cytocompatibility evaluation. Initially “murine in vitro dermal construct” based on L929 cells was generated, leading to the development of “human in vitro dermal construct” consisting of normal human dermal fibroblasts in rat tail tendon collagen type I. To assess the viability of the cells, different assays CellTiter-Blue®, RealTime-Glo™ MT, and CellTiter-Glo® (Promega) were evaluated to optimize the best-suited assay to the respective cell type and three-dimensional system. Z-stack imaging (Live/Dead and Phalloidin/DAPI-Promokine) was performed to visualize normal human dermal fibroblasts inside matrix revealing filopodia-like morphology and a uniform distribution of normal human dermal fibroblasts in matrix. CellTiter-Glo was found to be the optimal cell viability assay among those analyzed. CellTiter-Blue reagent affected the cell morphology of normal human dermal fibroblasts (unlike L929), suggesting an interference with cell biological activity, resulting in less reliable viability data. On the other hand, RealTime-Glo provided a linear signal only with a very low cell density, which made this assay unsuitable for this system. CellTiter-Glo adapted to three-dimensional dermal construct by optimizing the “shaking time” to enhance the reagent penetration and maximum adenosine triphosphate release, indicating 2.4 times higher viability value by shaking for 60 min than for 5 min. In addition, viability results showed that cells were viable inside the matrix. This model would be further advanced with more layers of skin to make a full thickness model.

Biomimetic mineralization and cytocompatibility of nanorod hydroxyapatite/graphene oxide composites

Nanorod hydroxyapatite (NRHA)/graphene oxide (GO) composites with weight ratios of 0.4, 1.5, and 5 have been fabricated by a facile ultrasonic-assisted method at room temperature and atmospheric pressure. The chemical structure properties and morphology of the composites were characterized by field emission source scanning electron microscope, X-ray diffraction, transmission electron microscopy, and high-resolution transmission electron microscopy. The results indicate that the NRHA/ GO composites have an irregular surface with different degree wrinkles and are stable, and NRHA are well combined with GO. In addition, the biomimetic mineralization mechanism of hydroxyapatite on the NRHA/GO composites in simulated body fluid (SBF) is presented. The presence of a bone-like apatite layer on the composite surface indicate that the NRHA/GO composites facilitate the nucleation and growth of hydroxyapatite crystals in SBF for biomimetic mineralization. Moreover, the NRHA- 1.5/GO composite and pure GO were cultured with MC3T3-E1 cells to investigate the proliferation and adhesion of cells. In vitro cytocompatibility evaluation demonstrated that the NRHA/GO composite can act as a good template for the growth and adhesion of cells. Therefore, the NRHA/GO composite could be applied as a GO-based, free-template, non-toxic, and bioactive composite to substitute for a damaged or defect bone.

In vitro cytocompatibility evaluation of poly(octamethylene citrate) monomers toward their use in orthopedic regenerative engineering

Citrate based polymer poly(octamethylene citrate) (POC) has shown promise when formulated into composite material containing up to 65 wt% hydroxylapatite (HA) for orthopedic applications. Despite significant research into POC, insufficient information about the biocompatibility of the monomers 1,8-Octanediol and Citrate used in its synthesis is available. Herein, we investigated the acute cytotoxicity, immune response, and long-term functionality of both monomers. Our results showed a cell-type dependent cytotoxicity of the two monomers: 1,8-Octanediol induced less acute toxicity to 3T3 fibroblasts than Citrate while presenting comparable cytotoxicity to MG63 osteoblast-like cells; however, Citrate demonstrated enhanced compatibility with hMSCs compared to 1,8-Octanediol. The critical cytotoxic concentration values EC30 and EC50, standard for comparing cytotoxicity of chemicals, were also provided. Additionally, Citrate showed slower and less inhibitory effects on long-term hMSC cell proliferation compared with 1,8-Octanediol. Furthermore, osteogenic differentiation of hMSCs exposure to Citrate resulted in less inhibitory effect on alkaline phosphatase (ALP) production. Neither monomer triggered undesired pro-inflammatory responses. In combination with diffusion model analysis of monomer release from cylindrical implants, based on which the maximum concentration of monomers in contact with bone tissue was estimated to be 2.2 × 10−4 mmol/L, far lower than the critical cytotoxic concentrations as well as the 1,8-Octanediol concentration (0.4 mg/mL or 2.7 mmol/L) affecting hMSCs differentiation, we provide strong evidence for the cytocompatibility of the two monomers degraded from citrate-based composites in the orthopedic setting.

In vitro and in vivo cytocompatibility evaluation of biodegradable magnesium-based stents: a review

Biodegradable magnesium (Mg)-based vascular stents have been designed as temporary scaffolds to treat angiostenotic lesions for the maintenance of normal blood flow. Numerous studies have presented in vitro and in vivo tests for the evaluation of the safety and feasibility of Mg-based vascular stents and the related materials. Therein the cytocompatibility is a basic and important parameter in the evaluation system. In this review, we summarize the applications and limitations of in vitro evaluation methods including basic characterization methods and direct and indirect cytotoxicity tests. We discuss the influencing factors on cytotoxicity, such as surface roughness, preconditioning of sample surface, cell type for the biocompatibility evaluation in direct contact as well as conditions for the formation of extracts/degradation products for indirect assays. Besides, we highlight the recent in vivo animal tests and clinical trials about Mg-based stents along with some associated results. The aim of this review is to provide a meaningful reference in the further developments and related evaluation methods of Mg-based stents.

Chitosan-Cellulose Multifunctional Hydrogel Beads: Design, Characterization and Evaluation of Cytocompatibility with Breast Adenocarcinoma and Osteoblast Cells.

Cytocompatible polysaccharide-based functional scaffolds are potential extracellular matrix candidates for soft and hard tissue engineering. This paper describes a facile approach to design cytocompatible, non-toxic, and multifunctional chitosan-cellulose based hydrogel beads utilising polysaccharide dissolution in sodium hydroxide-urea-water solvent system and coagulation under three different acidic conditions, namely 2 M acetic acid, 2 M hydrochloric acid, and 2 M sulfuric acid. The effect of coagulating medium on the final chemical composition of the hydrogel beads is investigated by spectroscopic techniques (ATR–FTIR, Raman, NMR), and elemental analysis. The beads coagulated in 2 M acetic acid displayed an unchanged chitosan composition with free amino groups, while the beads coagulated in 2 M hydrochloric and sulfuric acid showed protonation of amino groups and ionic interaction with the counterions. The ultrastructural morphological study of lyophilized beads showed that increased chitosan content enhanced the porosity of the hydrogel beads. Furthermore, cytocompatibility evaluation of the hydrogel beads with human breast adenocarcinoma cells (soft tissue) showed that the beads coagulated in 2 M acetic acid are the most suitable for this type of cells in comparison to other coagulating systems. The acetic acid fabricated hydrogel beads also support osteoblast growth and adhesion over 192 h. Thus, in future, these hydrogel beads can be tested in the in vitro studies related to breast cancer and for bone regeneration.

Evaluation of antibacterial, angiogenic, and osteogenic activities of green synthesized gap-bridging copper-doped nanocomposite coatings.

Titanium (Ti) and its alloys have been widely used in clinics for years. However, their bio-inert surface challenges application in patients with compromised surgical conditions. Numerous studies were conducted to modify the surface topography and chemical composition of Ti substrates, for the purpose of obtaining antibacterial, angiogenic, and osteogenic activities. In this study, using green electrophoretic deposition method, we fabricated gap-bridging chitosan-gelatin (CSG) nanocomposite coatings incorporated with different amounts of copper (Cu; 0.01, 0.1, 1, and 10 mM for Cu I, II, III, and IV groups, respectively) on the Ti substrates. Physicochemical characterization of these coatings confirmed that Cu ions were successfully deposited into the coatings in a metallic status. After rehydration, the coatings swelled by 850% in weight. Mechanical tests verified the excellent tensile bond strength between Ti substrates and deposited coatings. All Cu-containing CSG coatings showed antibacterial property against both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. The antibacterial property was positively correlated with the Cu concentration. In vitro cytocompatibility evaluation demonstrated that activities of bone marrow stromal cells were not impaired on Cu-doped coatings except for the Cu IV group. Moreover, enhanced angiogenic and osteogenic activities were observed on Cu II and Cu III groups. Overall, our results suggested that Cu-doped CSG nanocomposite coating is a promising candidate to functionalize Ti materials with antibacterial, angiogenic, and osteogenic properties.

A pro-angiogenic degradable Mg-poly(lactic-co-glycolic acid) implant combined with rhbFGF in a rat limb ischemia model.

Magnesium (Mg)-based implant has been already used in patients with critical limb ischemia. Site-specific controlled release of recombinant human basic fibroblast growth factor (rhbFGF), has become a promising approach to improve peripheral vascular disease. We report here on a novel combination implant composed of spiral magnesium and a coating made using poly(lactic-co-glycolic acid) (PLGA) with encapsulated rhbFGF (Mg-PLGA-rhbFGF). The preparation method does not involve any complex processes and results in a high encapsulation efficiency (approximately 100%). The degradation of metal Mg raise the microclimate pH in the PLGA polymer, which could well preserve the bioactivity of rhbFGF incorporated in the implant. Mg-PLGA-based, sustained local delivery of rhbFGF promotes post-ischemic angiogenesis and blood flow recovery in rat limb ischemic model. This work marks the first report for controlled release of rhbFGF in combination with metal Mg, and suggests potential therapeutic usefulness of Mg-PLGA-rhbFGF for tissue ischemia.

Biocompatibility, corrosion resistance and antibacterial activity of TiO2/CuO coating on titanium

In this work, TiO2/CuO coating was prepared on titanium (Ti) by combination of magnetron sputtering and annealing treatment. The microstructure, biocompatibility, corrosion resistance and antibacterial property of TiO2/CuO coating were investigated in comparison with pure Ti and TiO2 coating. The results show that TiO2/CuO coating is mainly composed of TiO2 and CuO. In vitro cytocompatibility evaluation suggests that no obvious toxicity appears on the TiO2/CuO coating, and the coating stimulates the osteoblast spreading and proliferation. Compared with Ti and TiO2 coating, TiO2/CuO coating exhibits improved corrosion resistance and antibacterial ability against S.aureus. This study is the first attempt to apply the combination of magnetron sputtering and annealing treatment to introduce the Cu into TiO2 coating for surface modification of Ti-based implant materials, which may provide a research foundation for further development of bioactive multifunctional coatings to meet the better clinical demand.

Surface modification of titanium substrates for enhanced osteogenetic and antibacterial properties

The insufficient osseointegration and bacterial infection of titanium and its alloys remain the key challenges in their clinic applications, which may result in failure implantation. To improve osteogenetic and antibacterial properties, TiO2 nanotube arrays were fabricated on titanium substrates for loading of antibacterial drug. Then, TiO2 nanotube arrays were covered with chitosan/sodium alginate multilayer films. The successful construction of this system was verified via scanning electron microscopy and contact angle measurement. The cytocompatibility evaluation in vitro, including cytoskeleton observation, cell viability measurement, and alkaline phosphatase activity assay, confirmed that the present system was capable of accelerating the growth of osteoblasts. In addition, bacterial adhesion and viability assay verified that treated Ti substrates were capable of reducing the adhesion of bacteria. This study may provide an alternative to develop titanium-based implants for enhanced bone osseointegration and reduced bacterial infection.

Effects of copper nanoparticles in porous TiO2 coatings on bacterial resistance and cytocompatibility of osteoblasts and endothelial cells

Copper (Cu) has garnered increasing interest due to its excellent antimicrobial activity and important roles in human metabolism. Although the biological effects of Cu have been studied, the effects of Cu nanoparticles (NPs) on cell behavior are not well understood. In this study, porous TiO2 coatings doped with different amounts of Cu NPs (designated as 0 Cu, 0.3 Cu, and 3.0 Cu) are deposited on titanium by micro-arc oxidation (MAO). The Cu NPs coated samples exhibit excellent antibacterial activity against Staphylococcus aureus (S. aureus). In vitro cytocompatibility evaluation discloses that 0 Cu and 0.3 Cu have no toxicity to osteoblasts but 3.0 Cu shows cytotoxicity. 0.3 Cu promotes proliferation and adhesion of osteoblasts and enhances extracellular matrix mineralization (ECM), but has little effects on the alkaline phosphatase activity (ALP) and collagen secretion. Surprisingly, the Cu NPs coated samples show a different behavior with endothelial cells. Both 0.3 Cu and 3.0 Cu show no cytotoxicity on endothelial cells and promote cell proliferation. Production of nitric oxide (NO) and secretion of vascular endothelial growth factor (VEGF) by the endothelial cells are observed from the Cu NPs doped TiO2 coatings.

Fabrication, characterization, cytocompatibility, and biological activity of lemon fiber-filled polyester composites

ABSTRACTThe structure, cytocompatibility, and biological activity of composites prepared from polycaprolactone (PCL) and lemon fiber (LF) blends (PCL/LF) were evaluated. Acrylic acid-grafted PCL (PCL-g-AA) was used to enhance the desirable characteristics of these composites, which had better tensile properties than the corresponding PCL/LF composites. A cytocompatibility evaluation with normal human foreskin fibroblasts of the water released from the PCL/LF and PCL-g-AA/LF composites indicated that both materials were nontoxic. Moreover, LF enhanced the flavonoid and polyphenol contents and the anti-inflammatory and antioxidant properties of the PCL-g-AA/LF and PCL/LF composites. We analyzed the effects of these composites in mushroom tyrosinase activity and B16-F10 melanoma cells; the PCL/LF and PCL-g-AA/LF membranes demonstrated reduced tyrosinase activity. Composites of PCL-g-AA or PCL containing LF had better antibacterial activity.

Evaluation of antibacterial activity and cytocompatibility of α-TCP based bone cements with silver-doped hydroxyapatite and CaCO3

Abstract The implant-related infections are a severe problem in orthopedic surgery. In this work, bone cements based on α-TCP, silver doped hydroxyapatite and calcite were examined. Developed bioactive materials revealed good surgical handiness and appropriate setting times allowing for their application. In order to assess antibacterial properties, two independent experiments (test imitating in vivo conditions and test of cumulative extraction) were used. It was found that bone cement matrix do not impede the Ag + release from the Ag-HA agglomerates. Materials showed mild antibacterial activity which depended on the kind of bacterial strain and the type of test. Presence of silver in the tested materials increased the antibacterial activity in the extracts only for E. coli strain and the effect was transient. In the case of Gram-positive strains ( S. aureus and S. epidermidis ), antibacterial activity was most probably connected with the properties of cement material, not with the content of silver. Observed reduction of osteoblast viability in vitro resulted from the synergic effect: decrease in concentrations of Ca 2+ , Mg 2+ , and PO 4 3- ions in the culture medium and the presence of silver ions.