**3. Biomedical applications**

**Figure 6.** chain of water consisting of hydrogen and oxygen atoms. The solid and dotted lines mark the covalent and

In simulations we considered simple chain consisting of two 1D water molecules. The initial velocity of the first atom was taken at *V* = 500 m/s. The Morse and Born-Mayer potentials were

Figure 7 represents the initial and final stabilized conditions of atom chains (after direct lowenergy ion impact to the first atom of the chain) calculated with Born-Mayer (Figure 7a) and

**Figure 7.** Initial and final positions of atoms of 1D water molecule after direct low-energy impact to the first atom

(oxygen): a) Born-Mayer potential, b) Morse potential. The right figures show the enlarged final chains.

hydrogen bonds respectively.

10 Advances in Biomaterials Science and Biomedical Applications

used for this investigation.

Morse (Figure 7b) potentials

Biological objects are known for their high sensitivity to weak external fields. The evidence that electromagnetic fields can have "non-thermal" biological effects is now overwhelming. When the production of heat shock proteins is triggered electromagnetically it needs 100 million times less energy than when triggered by heat [20]. Low-frequency weak magnetic fields may lead to the resonant change of the rate of biochemical reactions although the impact energy is by ten orders of magnitude less than *kBT* where *kB* is the Boltzmann constant and *T* is the temperature of the medium [21].

The therapeutic ability of the low intensity electromagnetic radiations is actively discussed [22]. The low-power millimeter wave irradiation and magnetic-resonance therapy are used in practical medicine already, which differ significantly from the drug treatment by the fact that they do not clog organism with the undesirable chemical compounds, i.e. xenobiotics. In this chapter we discuss the biomedical application of vacuum-plasma technologies.

#### **3.1. Activating and therapeutic properties of water processed in GDP**

To understand the above extreme sensitivity of living objects, investigations in influences of weak fields on water appear to be essential. Indeed, water plays a major role in biological processes. A man consumes about 2 l of drinking water a day. Water is the main component of human, animal, plant and generally every living being body. A new-born child body contains 97% of water, decreasing to 70–75% with aging. In particular, human brain consists of about 85% of water.

So, we performed experiments with water, crop seeds and baking yeast *S. cerevisiae*. The crop seeds and yeast were processed directly in GDP. Also, the untreated crop seeds and yeast were poured with the water processed in GDP. In all cases practically the same biotrophic effects were observed. Namely, the seed sprouts showed the growth in 3–4 times higher than the control samples. Both the processed yeast and the unprocessed one that immersed in the processed (by GDP) water showed greater metabolic activity compared to the control samples.

The obtained results allow suggesting that the discovered phenomena can be used for direct correction of pathological states. Therefore we processed water and physiological solution. The samples were exposed to low-energy ion irradiation in GDP of residual gases. The ion energy depends on the voltage in the plasma generator. The latter was kept at 1.2 keV while the current in the plasma generator was maintained at 70 mA. The temperature in the chamber was controlled during the irradiation process and did not exceed 298 K (25° C). The irradiation time was 60 minutes.

In test experiments, volunteers with different diseases either drunk the processed water or they were injected intravenously with the similarly processed physiological solution. The course of treatment included 3–5 sessions of 0.5 l physiological solution transfusion. The preliminary results appeared to be very promising. We were most interested in the therapeutic treatment of the global inflammatory processes such as cardio-vascular diseases and pancre‐ atic (insular) diabetes complicated by the acute and chronic forms of atherosclerosis. Also, different types of oncology, say, leukemia, etc., were under investigation.

The blood immune cells were taken for diagnostics. The immune system is known as one of the leading homeostatic systems in the organisms. It may serve as a mirror that reflects practically all adaptations and pathological rearrangements. The immunocompetent cells, lymphocytes and leukocytes, have a set of properties that may be used as an indicator of the organism state. In addition, the structural organization of blood lymphocytes and leukocytes makes possible a most efficient use of microspectral analysis and different fluorescent probes for their studies [23].

We used the dual-wavelength microfluorimetry analysis. The selected cell populations (monoand polynuclears of blood immunocytes) were mapped as clusters of points on the phase plane in coordinates of the red and green luminescence intensities, i.e. on the wavelengths *I530* (abscissa) and *I640* (ordinate). Figure 8 presents the above phase plane for an oncologic outpatient before and after the monthly course treatment. The black and grey pluses represent lymphocyte and leukocyte cells respectively. The white ellipses mark the distribution of fluorescent signals of lymphocytes (lower ellipse) and leukocytes (upper ellipse) in norm. As seen, the treatment results into significant normalization toward the homeostasis.

**Figure 8.** Dual-wavelength microfluorimetry analysis (abscissa and ordinate represent the luminescent intensity *I530* and *I640* respectively) of blood immunocytes of oncologic patient (second stage breast cancer). The state before (a) and after (b) the water treatment course (see the text). The black and grey pluses represent lymphocytes and leukocytes respectively. The white ellipses mark the distribution of fluorescent signals of lymphocytes (lower ellipse) and leuko‐ cytes (upper ellipse) in norm.

#### **3.2. Biocompatibility of titanium alloys and stainless steel processed in GDP**

atic (insular) diabetes complicated by the acute and chronic forms of atherosclerosis. Also,

The blood immune cells were taken for diagnostics. The immune system is known as one of the leading homeostatic systems in the organisms. It may serve as a mirror that reflects practically all adaptations and pathological rearrangements. The immunocompetent cells, lymphocytes and leukocytes, have a set of properties that may be used as an indicator of the organism state. In addition, the structural organization of blood lymphocytes and leukocytes makes possible a most efficient use of microspectral analysis and different fluorescent probes

We used the dual-wavelength microfluorimetry analysis. The selected cell populations (monoand polynuclears of blood immunocytes) were mapped as clusters of points on the phase plane in coordinates of the red and green luminescence intensities, i.e. on the wavelengths *I530* (abscissa) and *I640* (ordinate). Figure 8 presents the above phase plane for an oncologic outpatient before and after the monthly course treatment. The black and grey pluses represent lymphocyte and leukocyte cells respectively. The white ellipses mark the distribution of fluorescent signals of lymphocytes (lower ellipse) and leukocytes (upper ellipse) in norm. As

**Figure 8.** Dual-wavelength microfluorimetry analysis (abscissa and ordinate represent the luminescent intensity *I530* and *I640* respectively) of blood immunocytes of oncologic patient (second stage breast cancer). The state before (a) and after (b) the water treatment course (see the text). The black and grey pluses represent lymphocytes and leukocytes respectively. The white ellipses mark the distribution of fluorescent signals of lymphocytes (lower ellipse) and leuko‐

seen, the treatment results into significant normalization toward the homeostasis.

different types of oncology, say, leukemia, etc., were under investigation.

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for their studies [23].

cytes (upper ellipse) in norm.

Stainless steel, titanium and its alloys are among the most utilized biomaterials and are still the materials of choice for many structural implantable device applications [24, 25]. We processed both the titanium and stainless steel samples and investigated changing in their properties caused by GDP.

Current titanium implants face long-term failure problems due to poor bonding to juxtaposed bone, severe stress shielding and generation of debris that may lead to bone cell death and perhaps eventual necrotic bone [26–28]. Improving the bioactivity of titanium implants, especially with respect to cells, is a major concern in the near and intermediate future. Surface properties such as wettability, chemical composition and topography govern the biocompat‐ ibility of titanium. Conventionally processed titanium currently used in the orthopedic and dental applications exhibits a micro-rough surface and is smooth at the nanoscale. Surface smoothness on the nanoscale has been shown to favor fibrous tissue encapsulation [27–29]. An approach to design the next-generation of implants has recently focused on creating unique nanotopography (or roughness) on the implant surface, considering that natural bone consists of nanostructured materials like collagen and hydroxyapatite. Some researchers have achieved nano-roughness in titanium substrates by compacting small (nanometer) constituent particles and/or fibers [30]. However, nanometer metal particles can be expensive and unsafe to fabricate. For this reason, alternative methods of titanium surface treatment are desirable.

For the investigation of biocompatibility of implanted materials the tests *in vitro* with the cultures of different cells (fibroblasts, lymphocytes, macrophages, epithelial cells, *etc.*) are used. The influence of material is typically evaluated according to such indicators as adhesion, change in the morphological properties, inhibition of an increase in the cellular population, oppression of metabolic activity and others.

The adhesion of cells, as is known, plays exceptionally important role in the biological process‐ es,suchasformationoftissuesandorgansduringembryogenesis,reparativeprocesses,immune and inflammatory reactions, *etc*. Capability for movement is the characteristic property of fibroblasts, cells of immune system and cells, which participate in the inflammation. More‐ over, in immunocytes and leukocytes it consists not only in the free recirculation in the blood stream or lymph but also in the penetration into vascular walls and active migration into the surrounding tissues. Adhesion and flattening of cells to the base layer always precede their locomotion.Thedegreeofflatteningis importantpreparatorysteptothecellamoeboidmobility. We concentrate our attention on the above components in experiments with titanium alloys.

Titanium samples were cut into pieces (1 cm × 0.5 cm) and placed in a specially constructed plasma generator. They were exposed to glow discharge plasma by ions of the residual gases of the vacuum. The ion energy depended on the voltage in the plasmatron and did not exceed 1– 10 keV. Irradiated fluence was 1017 ion∙cm-2. The temperature of the specimens was controlled during the irradiation process and did not exceed 343 K while the irradiation time varied from 5 to 60 minutes. Rutherford Backscattering Spectrometry (RBS) was used to study the changes after the irradiation. Cell adhesion to titanium samples was tested with L929 mouse connec‐ tive tissue (fibroblasts-like cells). L929 cells were cultured in Dulbecco's modified Eagle's

medium with 10% fetal bovine serum. Initial cell density was 5∙10<sup>5</sup> cells/ml. The samples were placed into the sterile disposable 9 cm diameter tissue culture Petri dishes. 2 ml growth medium with cells were distributed into each Petri then incubated in the 5% CO2 at 37º C for 2 hours. After that period, cultures were prepared for scanning electron microscopy (SEM).

RBS data for the irradiated sample show the presence of iron on the surface that occurred from high-carbon steel cathode as a result of secondary emission process (Figure. 9). Percentage of iron and thickness of the layer were calculated using RUMP, the program for simulation and analysis based on RBS and Elastic Recoil Detection techniques.

**Figure 9.** RBS spectrum of the titanium sample irradiated for 5 min at 10 kV.


The obtained data for different voltage and time of the irradiation are presented in Table 2.

**Table 2.** Data obtained from the experiments with titanium samples exposed to GDP.

medium with 10% fetal bovine serum. Initial cell density was 5∙10<sup>5</sup>

14 Advances in Biomaterials Science and Biomedical Applications

analysis based on RBS and Elastic Recoil Detection techniques.

**Figure 9.** RBS spectrum of the titanium sample irradiated for 5 min at 10 kV.

that period, cultures were prepared for scanning electron microscopy (SEM).

placed into the sterile disposable 9 cm diameter tissue culture Petri dishes. 2 ml growth medium with cells were distributed into each Petri then incubated in the 5% CO2 at 37º C for 2 hours. After

RBS data for the irradiated sample show the presence of iron on the surface that occurred from high-carbon steel cathode as a result of secondary emission process (Figure. 9). Percentage of iron and thickness of the layer were calculated using RUMP, the program for simulation and

cells/ml. The samples were

Calculated data indicate an increase in the density of flattened cells as well as in the cell amount in comparison with the control sample. According to Table 2 one conclude that best adhesion (column 4) and most prolific cell attachment (column 5) correspond to the samples that were exposed to GDP for maximum time at minimum voltage. For this sample we observed less percentage of iron and thickness of the iron layer in comparison with others that were exposed to higher voltage plasma irradiation.

Figure 10 demonstrates SEM images of control and irradiated samples. In comparison with the control sample, analysis of cell attachment for the irradiated samples shows high conflu‐ ence (attachment ratio) and better spreading.

We also performed experiments on the adhesion of immune-competent cells of human blood to the stainless steel samples. Figure 11 represents the microphotography of the healthy person lymphocytes and leukocytes adhered to the irradiated and non-irradiated plates. As can be seen from photographs, cells, which are located on the different samples, are essentially different. The morphology of leukocytes and lymphocytes, which were adhered to the irradiated material, indicates the expressed amoeboid mobility.

In the majority of the cases endoprosthetics is conducted not in the healthiest people. This fact is very important and it must be considered. Figure 12 displays the results of similar study of the blood nucleus of person who suffers from second stage hypertonia, coronary artery disease and atherosclerosis. From the above data one can conclude that the nature of adhesion of cells to the base layer depends on both the physico-chemical state of this base layer and the state of organism, the owner of cells.

(a) (b)

**Figure 10.** SEM images of cell attachment on (a) the control sample and (b) the titanium sample that was irradiated for 5 min at 10 kV.

**Figure 11.** Luminescent microscopy (1000×) of lymphocytes and granulocytes of the blood of healthy donor adhered to (a) non-irradiated and (b) irradiated in GDP surface of the stainless steel samples. The cell nucleus fluorochromiza‐ tion is performed by propidium iodide (λfl = 615 nm).

**Figure 12.** Luminescent microscopy (1000×) *of* lymphocytes and granulocytes of the blood of donor suffering from second stage hypertonia, coronary artery disease and atherosclerosis. The cell nuclei are adhered to (a) non-irradiated and (b) irradiated in GDP surface of the stainless steel samples. The fluorochromization is performed by propidium iodide (λfl = 615 nm).

(a) (b)

**Figure 10.** SEM images of cell attachment on (a) the control sample and (b) the titanium sample that was irradiated

for 5 min at 10 kV.

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