Vegera I., Gordienko A., Novik ., Rachuba D., Sidorenko A.
Journal “Medicine and Innovations” 2(72) 2009.
Authors’ comments: Now days the most reliable test to evaluate biocompatibility is considered tests with cell cultures: fibroblasts, macrophages and etc. Authors of this article conducted the experiment with two titanium alloys VT-1.0 and VT-6.This article is quite interesting for us because in our product Titanium Silk even purer alloy is used.
At present titanium and its alloys are widely used for implant and other medical articles production [1, 2]. The most important requirements for the implant materials are their biological compatibility (toxic, mutagenic, carcinogenic safety) and histological compatibility [1, 6].
Implant toxicity test in vitro with the help of cell cultures is a reliable method of preclinical evaluation of their biocompatibility. To conduct such a test fibroblasts, lymphocytes macrophages and epithelial cells cultures are used. In vitro tests with bacterial test systems have also been worked out [7-10]. Material influence is evaluated with a help of the following indexes: morphological properties changes, growth and cell population development inhibition, metabolic activity suppression etc [5, 11, 12].
Microorganisms’ usage as a subject- test has some advantages:
- Due to small size cells have larger contact surface with the environment.
- High growth and reproduction speed allows to follow up the material influence on several cell generations in a short period of time.
- There is a possibility to analyze factor influence on morphological, physiological and genetic properties.
- Those tests are cheaper than cells’ culture tests.
Any organism requiring no special conditions of cultivation may be taken as a test- organism for nonspecific toxicity evaluation.
Bifidobacterium type is a representative of normal intestinal micro flora of human and warm blood animals. They can be also detected in mouth cavity and urinary tract. Bifidobacteria have a leading role in intestinal microbiocenosis, nonspecific organism resistance, metabolism improvement, immune system stimulation and etc.
The choice of these bacteria as test-cultures was due to some reasons:
- Changes in qualitative or quantitative composition of bifida flora may contribute to the development of different disorders, that’s why it’s important to notify the implant influence on the cells of exactly those bacteria.
- Bifida bacteria are anaerobic organisms which gives the opportunity to conduct in vitro tests in the conditions of similarity to in vivo. It’s not possible with aerobic organisms.
The objective of this work is to study titanium alloys VT1-0 AND VT-6 biocompatibility in vitro under the conditions comparable with in vivo using Bifida bacteria as a subject-test.
Investigation material was comprised of samples of titanium alloy VT-6 scorched at 1000 °C with the further hardening in water (alloy №1) or in the air ( alloy№2), and VT1-0 scorched at 920 °C with further hardening in water (alloy №3) and in the air ( alloy№4). In our work we used strains of bifida bacteria Bifidobacterium adolescentis MS-42, B adolescentis GO -13, B. bifidum 1, B. longum V379 M, given by the Russian Scientific and Constructive Milk Institute and Moscow Scientific Institute of Epidemiology and Microbiology named after Gabrichevskiy, and Bifidobacterium adolescentis 94 BIM deposited as B. adolescentis BIM V-87 from Byelorussian collection of nonpathogenic organisms. Bacteria were grown at 37°C.in tripton lactose medium (trip-ton -1%, lactose-1%, yeast extract- 0.5%, sodium caseinat-0.1%, ascorbic acid-0.05%, NaH2PO4x12H2O-0.2%, K2HPO4-0.2%, MgSO4-0.1%, NaCl -0.1%, agar-agar-0.1%, pH 6.8).For the experiment 18- hours physiologically active cultures were used. Alloys’ plates of 10x1x5 mm size were polished with the grinding paper, sterilized in autoclave (30 minutes at 121°C) and placed into the 50 ml flasks. Flasks were filled with the sterile TL medium and 18-hours bifida bacterium cultures and they were incubated in thermostat at 37°C. Along side with it cultures were placed into the flasks with TL medium but without alloy (control) which were placed in the same condition for the same amount of time as the experimental ones. The evaluation of materials influence on the bacteria population development was carried out in 24 hours time. In order to study titanium alloys prolonged presence influence on the bifida bacteria viability, flasks with experimental and control groups were placed into the refrigerator for storage (+4C) after the pH correction in 6.8-7 diapason with the saturated sodium hydro carbonate solution. The evaluation of viability was conducted in 3 months of storage.
The film formation on the titanium alloys’ surfaces was studied in the following way: in the center of Petri cups with agar medium holes of 15 mm diameters were cut. The plates of one of the alloys were immersed into those holes. Cups were inoculated with 10 ml of bacterial suspense, were placed into the station for microaerofilic organisms’ BUG BOX M growth and incubated at 37°C. in gas mixture during 21 days ( 10% CO2, 10%H2, 80%N2). Bio film formation was inspected visually. Microorganism growth and profusion on the agar- alloy and material surface border was taken into consideration. In 21 days alloys’ plates were taken from the holes and placed into 10 ml of liquid TL medium. The viability of bacteria forming films was evaluated by the growth presence in 24-72 hours of cultivation at 37°C. The quality of violable cells was notified by the method of dilution with the further seeding into the semi liquid tioglicolic medium. Counting of colonies formed was taken in 48-72 hours cultivation at 37°C. Physiological activity of bacteria was assessed according to the level of biomass accumulation and acids formation activity 24 hours after cultivation. Morphological properties assessment was done with light microscopy of preparations colored by Gramm method and phase –contrast microscopy with Nicon Eclipse E 200 (Nicon corporation, Japan). Statistical data assessment was made with programs of “Microsoft Excel XP. Results of influence evaluation of titanium alloy on the bifida bacteria population development are presented in picture 1-2 and table #1. It’s seen that experimental materials had no negative effect on the growth and acid formation activity of the experimental organisms. In all cases indexes of biomass accumulation and active acidity of the media in control and experimental samples were not significantly different. Titer of bifida bacteria grown in the titanium alloys samples presence corresponded to the control samples. At the same time an individual sensitivity of cultures to those alloys was observed. In some cases an inhibition or stimulating effects can be seen. For example with the present of alloys VT-6 and VT1-0 in the media a decrease of cell titer and biomass accumulation B bifidum № 1were noted. For the rest of bifidum bacteria strains experimental materials presence led to biomass accumulation growth. The most prominent stimulating effect for B adolescentis GO -13 and B adolescentis MS -42 was seen in the presence of alloy VT-6. In cases of B adolescentis 94 BIM and B. longum B379M- alloy VT1-0 independently of treatment.
Visual differences in colony morphology after seeding from control and experimental variants was not found: they had a typical form of nails or seeds with 1-3 mm in diameter. Microscopic examination showed that titanium alloys didn’t bring light optic visual morphological changes of bifida bacteria, which were presented as gram positive straight or slightly curved sticks with thickened ends. There was not formation of forms characteristic for cultures grown in unfavorable conditions or in the presence of toxic compounds.
Data received confirm good biocompatibility and non toxicity of titanium alloys on the growing populations of bifidum bacteria.
It’s defined that titers of B adolescentis 94 BIM, B adolescentis MS -42, and B adolescentis GO -13 after 3 months of storage with alloy №1 and B. longum B379M with alloy №4 had no difference with control variants. At the same time there was a full lack of vitality of B adolescentis GO -13 and B bifidum № 1 with alloy№ 2 and B. longum B379M with alloy№ 1. In general better conditions of bacteria independently of strains were in media with alloy№ 3. At the same time individual sensitivity of test-cultures to titanium alloys was noted. In all variants of experiment there was seen a significant reduction of titer B bifidum № 1, and the survival of B adolescentis 94 BIM, B adolescentis MS -42 was 80-100%. According to the data achieved strains of B adolescentis 94 BIM, B adolescentis MS -42 were found to be perspective for in vitro experiments.
Film formation on the titanium materials showed that all strains used in the experiment formed films 24-48 hours after cultivating. Those films grew and became thicker along the whole incubation period. Cultures grew similarly well on the thick nutritional media, agar-alloy border and material surface immersed into liquid media TL. There wasn’t noted any proliferation and bifidum bacteria properties change. In 21 days of incubation under modeled conditions titanium alloys’ surface was covered with multi layers film composed of alive microorganisms. Study of morphological properties of bacteria isolated from this film showed that cells are of small size and coccid form. This effect can be explained by the microorganisms’ reaction to increasing population pressure and lack of nutritional substrates in the media because of long time of incubation. Involutionary or abnormal morphology forms were not noted. The results prove good biocompatibility of titanium alloys with Bifidobacterium cells. The best material for implants and other medical items is considered to be titanium and its’ alloys having the whole complex of properties satisfactory for medicine: high corrosive stability, enough mechanical and cyclic durability, wear resistance, relatively low cost. High corrosive stability is due to quick formation of passive oxide film interfering with ion exit from implant into organism’s media. Titanium and its’ alloys stability in passive condition is higher than in iron, chrome, nickel and stainless steel. More over those materials are able to be passive in water solution containing ion of chlorine together with oxygen in ay concentrations. Sterilization of titanium alloys with boiling, autoclaving, formalin gases, burning, and immersion in alcohol doesn’t lead to metal surface damage. Longitudinal testing of titanium and its’ alloys showed that they are durable. The most successful application of those materials is in dental practice – alloys prosthesis durability is 10-15 years [1, 2].
One of the most important criteria for implant choice is biocompatibility – which means absence of negative influence on organism. Microorganism usage for the assessment of implant biocompatibility is now days conducted only to find out mutagenic effect and toxicity, although there is a tendency for a wider application of test-systems [7-10]. There is a number of papers devoted to the question of titanium influence on microphlora of mouth cavity in dental prosthesis [14, 15]. But we think that not enough attention is paid to the implant materials influence on the natural micro flora representatives. It’s known that after implant insertion its’ component come in contact both with surrounding tissue cells and the whole inner organism’s environment . Thus they will influence micro flora directly (dental implants) or indirectly through oxidation products diffusing into the blood, the process which can lead to significant changes of autochthonic flora. It has been established that changes in normal flora composition can lead to development of a number of diseases such as gastritis, ulcers, diarrhea colitis hypo and hyper tension, ischemia, hypo and hyper cholesterinimia, arthritis, caries, bile ducts disorders, dermatitis, allergic reactions and so on. It was also shown the presence of correlated interaction between the degree of gastric microbiocenosis and the rate of purulent inflammatory processes in patients in postoperative period . That’s why studying the materials biocompatibility it’s important both their toxicity towards the eukaryotic cells and their influence on representative of normal flora. It has been established that many compounds have the same toxicity towards the microorganisms and cultures of the human being: materials with high antibacterial toxicity usually have prominent cytotoxic and mutagenic properties [6, 9]. According to those factors we have worked out scheme of biocompatibility assessment of titanium alloys with Bifidobacterium in the modeled experiments in vitro. The test for non specific toxicity of alloys VT 1-0 and VT-6 has been also conducted. The absence of inhibiting and/or toxic action of titanium alloys on the growing population of Bifidobacterium has been shown. It ‘s been established that Bifidobacterium viability after 3 month of storage with titanium alloys VT1-0 and VT -6 was a bit lower than in control groups, but cultures’ morphological properties didn’t change significantly. In 24-48 hours period of cultivation Bifidobacteria are able to form multi layers films composed of viable bacteria with characteristic morphology.
Thus absence of inhibiting and/or toxic action of titanium alloys on the growing population of Bifidobacterium and good compatibility of VT 1-0 and VT -6 alloys with microorganisms of Bifidobacterium type have been proved. The data received can be necessary to study biocompatibility of implant materials and to work out tests for non specific toxicity evaluation of new medical alloys. Experiments were conducted in the frame of State complex scientific investigations program “Physics of crystal and atom –molecule structures” under the name “Creation of new materials on the base of titanium and cobalt alloys with biologically active coverings for their application in implants’ production”.