DOI 10.1007/s10527-017-9675-x Biomedical Engineering, Vol. 51, No. 1, May, 2017, pp. 1619. Translated from Meditsinskaya Tekhnika, Vol. 51, No. 1, Jan.Feb., 2017, pp. 1215. Original article submitted November 11, 2016.
Spectral Studies of Biodegradation and Hemolysis Caused by Contact of Bulk and Film Nanocomposites with Biological Fluids U. E. Kurilova1, N. N. Zhurbina1, M. V. Mezentseva2, L. I. Russu2, I. A. Suetina2, I. V. Pyanov1, D. V. Telyshev1, and A. Yu. Gerasimenko1*
The paper discusses the possibility of use of nanocomposites obtained by laser structuring of singlewalled (SWCNTs) and multiwalled (MWCNTs) carbon nanotubes in bovine serum albumin matrix as tissueengi neering scaffolds for blood vessel network sprouting in the process of biodegradation of the nanocomposite implant. The lowest rate of biodegradation was obtained in bulk and film nanocomposites based on SWCNTs immersed in NaCl solution: 2.45 and 9.77% weight loss after 14 days, respectively. Analysis of the dynamics of changes in the optical density of NaCl solution showed intense biodegradation of nanocomposites after six days of immersion. Spectral studies showed that contact of blood with bulk and film nanocomposites based on SWCNTs did not lead to hemolysis (hemolysis level ≤ 0.5%).
Introduction
Bulk nanocomposites can be used, for example, to fill largesize bone defects, while film nanocomposites allow, in particular, reconstruction of organ and vascular walls, as well as of connective tissue [3]. Carbon nanotubes (CNTs) are a promising filler material. Their properties are similar to those of compo nents of natural extracellular matrix. It has been shown that nanotubes can increase adhesion and proliferation of cells of connective, nerve and bone tissue [4, 5]. The goal of this work was to describe the results of studies of nanocomposites obtained by laser nanostruc turing of CNTs in bovine serum albumin (BSA) matrix [6]. Spectral methods were used to assess the possibility of applying nanocomposites as tissueengineering scaffolds for blood vessel network sprouting in the process of biore sorption of the nanocomposite implant. The rate of nanocomposite biodegradation in bloodimitating liquid medium and the level of blood hemolysis caused by con tact with nanocomposites were measured. In addition to being a promising material for tissue engineering, nanocomposites are shown in this work to offer considerable potential for development of biocom patible coatings for bloodcontacting implantable med ical devices used, for example, in cardiac surgery [7].
The problem of shortage of donor organs and tissues remains very urgent [1]. It has led to largescale research in the field of artificial organs and tissues that focuses on development of new materials for adequate replacement of human organs and tissues with artificial ones. The physical and chemical properties of these materials should be close to those of replaced biological tissue. They should also be biocompatible and provide cell growth at the site of tissue defect. Finding solutions for these problems is the task of tissue engineering, an actively developing discipline, which has as its pur pose the search for new scaffolds for human cell colonies [2]. Composite materials based on a biological matrix with nanosized filler particles (nanocomposites) are the most promising material for tissueengineering scaffolds. 1
National Research University of Electronic Technology, Zelenograd, Moscow, Russia; Email:
[email protected] 2 Gamaleya Federal Research Center for Epidemiology and Microbiology, Moscow, Russia. * To whom correspondence should be addressed.
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00063398/17/51010016 © 2017 Springer Science+Business Media New York
Biodegradation and Hemolysis Caused by Contact of Nanocomposites with Biofluids
Materials and Methods The following materials were used to prepare nanocomposite samples: distilled water, 99%pure lyophilized BSA, 99%pure singlewalled carbon nano tubes (SWCNTs), and 98%pure multiwalled carbon nanotubes (MWCNTs) [6]. At the first stage, water–BSA dispersion with addi tion of SWCNTs or MWCNTs was prepared. The BSA concentration was 250 g/L; CNT concentration, 0.1 g/L. To avoid CNT agglomeration and to uniformly distribute the components, the dispersion underwent ultrasound treatment in several modes. At the second stage, bulk nanocomposite samples were obtained by drying a given volume of dispersion until complete evaporation of water. Drying was performed by exposure to semiconductor laser radiation with a wave length of 810 nm and radiation power of 35 W during 20 60 min. Film nanocomposite samples were obtained by drying a thin dispersion layer applied uniformly to a sub strate made of polyethylene terephthalate (PET), a woven inert synthetic material. The drying time under irradia tion conditions identical to those described above was ~10 min. The electromagnetic field of laser radiation played a structuring role leading to selfassembly of nano tubes and giving rise to production of a rigid porous scaf fold. BSA served as a matrix that connects and envelopes the nanotubes. The rate of biodegradation of tissueengineering scaf folds should be controlled to provide the correct ratio between the cell sprouting time and the time of scaffold bioresorption in the process of tissue regeneration [8]. To assess the biodegradation rate, the samples were immersed in 0.9% NaCl solution for 14 days at 37°C. The rate was assessed from the change in sample weight before and after immersion. The obtained values were compared to the rates of biodegradation of samples obtained by drying the BSAbased CNT dispersions in a thermostat at 60°C. Quantitative assessment of the nanocomposite amount dissolved in NaCl was performed by measuring its optical density from the absorption spectra in the wavelength range of 300900 nm. The absorption spectra of NaCl solutions of nanocomposites allow the weight loss of the samples to be evaluated with more precision, taking into account the process of soaking. The absorp tion spectra also allow more detailed information about the products of biodegradation to be obtained. Tissue growth into implanted tissueengineering scaffolds begins with blood vessel sprouting. Thus, it becomes necessary to study the hemocompatibility of samples; namely, the intensity of hemolysis (rupturing of red blood cells and release of hemoglobin) caused by con
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tact of blood with the samples and products of their biodegradation. To study the process of hemolysis, the samples were decontaminated with UV radiation for 20 min on each side and placed into sterile test tubes. 1.5 mL of human blood was added to each test tube. Blood without added nanocomposites was used as negative control with natural hemolysis level. A positive control sample of completely hemolyzed blood was obtained by freezing and thawing a blood sample without added nanocomposites. After sedimentation of red blood cells, plasma from each test tube was studied by spectral methods using a Thermo Scientific Genesys 10S UVVis spectrophotome ter in the wavelength range λ = 300700 nm with resolu tion 0.2 nm. The hemolysis level (%) was calculated from the optical density of plasma samples at λ = 540 nm using the following formula: sam b
b
(1) b
where Dsam is the optical density of blood upon contact with nanocomposite sample; Db(–) is the optical density of blood with natural level of hemolysis (negative con trol); Db(+) is the optical density of completely hemolyzed blood (positive control).
Results The method described above provides production of bulk nanocomposite samples in the form of solid opaque black tablets with uniformly distributed components. Nanocomposite film samples deposited on a PET support were black; interwoven fibers of the support could be eas ily discerned through the film. Samples took their black color from carbon nanotubes. The weight loss values of the samples obtained using the laserbased and the thermostatbased drying tech niques are compared in Table 1. According to the obtained data, the CNTbased nanocomposite samples produced by laser structuring lost less weight after 14 days of drying than the samples dried in the thermostat. Gradual resorption of samples exposed to the NaCl solution led to an increase in the absorptance of the solution. The absorption spectra of the solutions were similar to the spectral curves of the initial BSA based dispersions of CNTs. Albumin is the main compo nent of the nanocomposites in terms of mass. Its absorp tion peak lies in the visible range at λ = 440 nm. Thus, the resorption characteristics of the samples can be evaluated from the optical density of the NaCl solution at λ =
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Kurilova et al.
TABLE 1. Mechanical Properties of Nanostructured SZD Crystals Production technique
Bulk sample weight loss, %
Film sample weight loss, %
BSA
Laser Thermostat
75.13 3.80
25.19 18.84
BSA, SWCNT
Laser Thermostat
2.45 2.52
8.43 19.44
BSA, MWCNT
Laser Thermostat
9.77 11.37
17.99 22.81
Sample composition
D, a.u.
440 nm. The obtained data were used to construct a time curve of the optical density at λ = 440 nm for the period of sample immersion in the NaCl solution (Fig. 1). Figure 1 shows the dynamics of variation of the opti cal density of NaCl solutions with immersed SWCNT and MWCNTbased nanocomposite samples dried in thermostat (curves 1 and 2, respectively) and SWCNT and MWCNTbased samples obtained by laser structuring (curves 3 and 4, respectively). The spectral curves of the solutions are flat and virtually linear for the first 6 days of immersion; then, the process of weight loss is intensified. The optical density curve of the NaCl solution with immersed SWCNTbased sample dried in thermostat dif fers considerably from the other curves. In this sample, the intensity of degradation was significantly higher during the first 6 days of immersion. Higher levels of the optical den sity for solutions with immersed thermostatdried samples correlate with the results of measurement of the weight loss in different samples. The optical density variation was less pronounced for the samples with smaller weight losses, i.e., the nanocomposites obtained by laser nanostructuring.
Time of sample immersion in NaCl solution
Fig. 1. Dynamics of changes in optical density of NaCl solution with immersed nanocomposite samples.
TABLE 2. Optical Density and Hemolysis Level in Experimental and Control Samples Sample composition
Bulk samples
Film samples
D, a.u.
δ, %
D, a.u.
δ, %
BSA
0.19
0
0.11
0
BSA, SWCNT
0.20
0.25
0.12
0
BSA, MWCNT
0.22
9.49
0.13
1.20
−
−
0.11
0
Negative control
0.19
0
0.11
0
Positive control
0.61
100
1.05
100
PET
Absorption spectra of blood plasma after 24h con tact with samples are shown in Fig. 2. These spectra were obtained during hemolysis studies. The curves in Fig. 2, ac have no absorption peaks in the range of 400600 nm that could be interpreted as characteristic absorption bands of hemoglobin compounds. Such peaks can be seen in Fig. 2d (complete hemolysis) [9]. The measured optical density D at the hemoglobin absorption wavelength λ = 540 nm are given in Table 1 together with the hemolysis levels in the experimental and control samples calculated using Eq. (1) . The obtained results demonstrate rather high hemo compatibility of SWCNTbased bulk and film nanocom posites (0.25 and 0%, respectively). Levels of hemolysis observed for these samples are comparable to those for pure BSAbased samples and in the negative control. The hemolysis level for MWCNTbased film nanocomposites is rather low (1.20%), while the contact of a MWCNT based bulk sample with blood leads to 9.49% destruction of red blood cells and hemoglobin release. The most probable explanation is that this type of hemolysis is caused by the form factor of the nanotubes constituting the BSA matrix scaffold.
Biodegradation and Hemolysis Caused by Contact of Nanocomposites with Biofluids
b D, a.u.
D, a.u.
a
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d D, a.u.
D, a.u.
c
Fig. 2. Absorption spectra of blood plasma after contact with film samples: a) SWCNTbased sample; b) MWCNTbased sample; c) negative control; d) positive control.
Conclusion The possibility of use of nanocomposites obtained by laser structuring of SWCNTs and MWCNTs in BSA matrix as tissueengineering scaffolds for blood vessel network sprouting in the process of biodegradation of a nanocomposite implant was evaluated. The rate of biodegradation in SWCNTbased bulk nanocomposites immersed in NaCl solution was lower than in otherwise identical film nanocomposites: 2.45 and 9.77% weight loss after 14 days, respectively. Extended analysis of the dynamics of changes in the opti cal density of NaCl solution showed intense biodegrada tion of nanocomposites after six days of immersion. Spectral studies showed that contact of blood with bulk and film nanocomposites based on SWCNTs did not lead to hemolysis (0.25 and 0%, respectively). However, the hemolysis level for MWCNTbased bulk and film nanocomposites was 9.49 and 1.20%, respectively. Hemolysis caused by contact of blood with SWCNT based bulk and film nanocomposites does not exceed the permissible hemolysis level for medical devices contact ing with blood [10]. Thus, it can be concluded that BSAbased CNT dis persions can be effectively used in bioresorbable implants, for example, for regeneration of bone and connective tis sue, or in temporary biocompatible coatings for implant able medical devices.
This work was supported by the Russian Science Foundation (Project No. 143900044).
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