CRYSTAL GROWTH & DESIGN 2005 VOL. 5, NO. 1 21-23
Communications Superadhesion: Attachment of Nanobacteria to Tissues - Model Simulation Andrei P. Sommer,*,† Murat Cehreli,‡ Kivanc Akca,‡ Tolga Sirin,# and Erhan Piskin# Central Institute of Biomedical Engineering, Department of Biomaterials, University of Ulm, 89081 Ulm, Germany, Hacettepe University, Faculty of Dentistry, Department of Prosthodontics, 06100 Sıhhiye, Ankara, Turkey, and Hacettepe University, Chemical Engineering Department, and Bioengineering Division, and TUBITAK-Center of Excellence-BIYOMUH, 06532, Beytepe, Ankara, Turkey Received June 12, 2004;
Revised Manuscript Received September 2, 2004
ABSTRACT: Adhesion is common in the bacterial world and is partially mediated by slime. Nanobacteria (NB) seem to attach by slime as well as by the protecting nanocrystalline apatite shell, securing their survival in hostile milieus. Identification of NB in the human heart has brought the particles into the focus of material scientists. In view of the size distribution of the apatite nanovesicles (80-300 nm), simulation of their interaction with tissues can be performed via nanosuspensions and mirror-polished titanium substrates. Due to its wide biocompatibility, titanium is an ideal body tissue substitute. Here we show in a model simulation that the apatite nanovesicles are likely to perform also an anchoring function, specific to the mineral apatite. Anchoring may prevent solitary NB from elimination from the body, and enforce existing toxic potentials. The capacity of the apatite nanoparticles to bind to tissues in aqueous liquids, and the pronounced tendency of NB to form mineralized biofilms, indicate that NB could affect intracardiac fluid forces. Adhesion and protection represent a unique combination, which may stimulate the design of novel drug release systems based on apatite nanovesicles. Nanobacteria (NB) are protected by a mineral shell of nearly spherical architecture, consisting of apatite. The diameter of NB isolated from the blood of humans is typically between 80 and 300 nm.1 Recent work indicates that their sizes could reach 60 nm.2 The pathogenic character of NB seems clear. In the body they can gain access to various organs via circulation in fluids. Preliminary observations of an ongoing experimental study in the rabbit have suggested that hydroxyapatite nanoparticles implanted in the tibia may be distributed in the body through circulation and even gain retention in the kidney. Hence, it is not surprising that NB nucleate kidney stones,2,3 are implicated in heart disease,4,5 and possibly also contribute to the realization of low bone mineral density (BMD) levels observed in HIV-infected patients.6-8 In case of kidney stones, models predict that the stones are nucleated by giant solitary NB immobilized within the kidney, where NB find favorable conditions for growth.9 In case of heart disease, their pathogenic quality seems to be primarily related to their capacity to block essential metabolic communication by attaching as colonies to tissues within the heart.10 Apatite biofilms in the heart are likely to change shear forces in the blood, and contribute in this way to nonspecific circulatory problems.11,12 A closer inspection of the SEM images showing NB in the heart of humans4,5 strongly supports this picture. * To whom correspondence should be addressed. † University of Ulm. ‡ Hacettepe University, Faculty of Dentistry, Department of Prosthodontics. # Hacettepe University, Chemical Engineering Department, and Bioengineering Division.
NB appear to possess at least three qualities, which may cause alone or cooperatively harm to living systems. These can be related to the mineral apatite itself,13 a selfsynthesized slime,10 and possibly a genetic material contained in the mineral cavity. Most recent studies indicated the presence of DNA.2,5 Cultured NB have been found to respond to environmental changes, corresponding to physiological and/or biomechanical stress, by producing slime.10 Collective slime release could interconnect individual NB, establishing formation of surface-covering biofilms on occupied substrates. Clearly, attachment and immobilization facilitate eventually the transfer of toxic agents from NB to cells. This intuitive picture receives relevance from the clinical side, in particular by the identification of deposition of apatite on the perineurium of diabetic patients with peripheral neuropathy.14-16 Notably, growth observed in cultured NB stimulated with low level light17,18 was associated with a reduced slime secretion.19,20 The successful treatment of severest peripheral neuropathy with lasers has led to the assumption that NB may play a central role in HIV-infected patients, as well as in diabetes mellitus,21 since both are frequently burdened by peripheral neuropathy. Thus, models allowing a better understanding of the mechanisms by which NB form biofilms on body tissues are of extreme interest, for the biomedical side as well as for nanotechnology. Nanotechnology offers powerful tools to virtually simulate interaction modalities between NB and tissues, and to design models, which can inspire novel therapeutic strategies. Attachment of NB to the perineurium has been simulated via 60 nm polystyrene nano-
10.1021/cg049812n CCC: $30.25 © 2005 American Chemical Society Published on Web 10/13/2004
22
Crystal Growth & Design, Vol. 5, No. 1, 2005
Communications
Figure 1. (Left) Light microscopy photograph of deposition pattern formed by hydroxyapatite particles (Ø ∼ 60 nm) on titanium disk, mirror polished to exclude surface roughness effects. (Right) Ring formed self-organization of 60 nm polystyrene nanospheres on same substrate. Drops were placed successively and allowed to evaporate together in a closed Petri dish. Diameter of ring: ∼ 5 mm.
Figure 2. Schematic representation of major system-immanent forces acting on nanoparticles suspended in a drop evaporating on a substrate. The principal forces are F1 and F2. F1 is active in the near-field of the substrate, corresponding to the nature of the van der Waals attraction. F2 stems from convective flow toward the liquid/solid/air-contact line. The interplay between F2 and F1 determines the deposition patterns on substrates: F2 > F1 f ring, F2 < F1 f no ring.
spheres deposited from an aqueous suspension on various cylindrical objects mimicking the perineurial tube. First experimental results have demonstrated that deposition is strongly controlled by the polarity of the surfaces.22 Here we proceed with the simulation by introducing an improved model system employing biocompatible substrates (mirrorpolished titanium disks with total surface roughness < 4 nm),23 and two aqueous suspensions, one containing 60 nm polystyrene nanospheres (Duke Scientific, Palo Alto, CA), the other hydroxyapatite nanoparticles with mean diameter of 60 nm, prepared in the Chemical Engineering Department (Bioengineering Division) of the Hacettepe University, Ankara, Turkey. The prime reason for using titanium as substrates is the wide biocompatibility of this
prominent biomaterial.24-27 Living cells are, in principle, not able to discriminate between a titanium implant and bone.28 Exploiting this unusual reciprocity, titanium advances to an ideal model material for mimicking body tissues. To test the attachment of the nanoparticles to the titanium surfaces, we prepared similarly concentrated suspensions from both systems, nanospheres and apatite. Drops of equal volume (∼10 µL) were placed on top of mirror-polished titanium disks and allowed to evaporate in a closed Petri dish (Ø 35 mm). The representative image on the right in Figure 1 shows a ring formed by slow evaporation of the drop containing the nanospheres. Similar rings have been described for drops of aqueous suspensions containing nanospheres with diameters of 60 nm, 200 nm, and a mix of them, slowly evaporating on titanium.29-31 Importantly, drops containing the apatite nanoparticles employed here to mimic NB did not form rings, as can be verified in the image on the left in Figure 1. The physical mechanism of the ring formation has been analyzed extensively.32-35 Rings are formed by a balanced interplay between at least four major processes: sedimentation controlled by gravitational force, drift to the periphery driven by a minimum in the liquid surface tension at the solid/liquid/air-interface, vertical movement driven by a temperature gradient, and attractive van der Waals interaction between suspended material and substrate. The interplay of the dominant forces, finally determining the nature of the pattern, is illustrated in Figure 2. Apparently, radial transport, the main actor in shaping rings, is not strong enough to carry the hydroxyapatite nanoparticles
Figure 3. Scanning electron microscopy (SEM) image of outer peripheral zone of the patterns presented in Figure 1. (Left) 60 nm apatite particles with irregular contour line and conglomerate formation. (Right) 60 nm polystyrene nanospheres with clear ordered contour line and minimal conglomerate formation. Obviously, attachment to the substrate prevents apatite nanoparticles to self-organize in a crystalline order (as observed in polystyrene nanospheres suspensions).31 The effect of the substrate material on pattern formation has been demonstrated on Cybernox (Sitram, France), a quasicrystalline material used for the production of nonstick fry pans; on it the employed apatite nanosuspension formed crystalline rings.42 Arrows point to individual nanoparticles. Bars ) 5 µm.
Communications to the peripheral zone of the drop. Instead the suspended material sticks to the titanium surface on contact (Figure 1). The associated scanning electron microscope (SEM) image in Figure 3 shows the peripheral zone from where ring formation is known to start.33 A closer examination of the SEM image could be very instructive: Compared to the ordered deposition line (on the right), by which the first nanospheres have pinned down the ring, there is no ordered deposition at the periphery of the pattern formed by the apatite nanoparticles (on the left). It may be argued that, in case of the apatite, immobilization of the suspended material did not start at the liquid/solid/air-contact line, but instead simultaneously, across the titanium surface wetted by the drop. Liquid deposition of appropriate nanoparticles allows simulation of the attachment scenarios by which NB adhere to tissues. There exist two principal modalities where NB can stick to tissues: via a protein-based slime and direct interfacial contact. The attachment mediated by slime operates preferably unspecificallysa vital key function for primordial biosystems.36 In case of direct interfacial contact with tissues, two adhesion modes seem now plausible: unspecific, promoted by the mineral apatite and the size of the nanovesicles, and/or specific, controlled by a surface protein (epitope).1 The distinct biological and physical aspects of the model system analyzed here could provide relevant information for the biomedical side as well as valuable concepts allowing to exploit potentials in nanotechnology, e.g., to design drug release systems adhering to body tissues, based on apatite nanovesicles.37,38 A more realistic simulation of attachment processes could be realized by the use apatite nanoparticles with a spherical geometry and a size below 300 nm. Their fabrication is, however, in no way trivial. As a first step, simulation could be performed by the use of NB, killed by γ-irradiation.36 Nanosuspensions containing spherical apatite particles are of special interest because of the possibility to produce densely packed crystalline patterns on substrates by selforganization, as has been exemplarily shown for 200 nm polystyrene nanospheres.31 Possible applications may motivate efforts to evaluate the conditions required to produce crystalline patterns by the use of apatite nanospheres. Such patterns may foster practicable routes toward enamel remineralization strategies in our teeth. It is certainly not merely a coincidence that nature has built our teeth from apatite: While apatite seems to play a major role in blocking nerve activities in peripheral neuropathy, an equivalent function might have a beneficial effect and could lower nerve activities within the root channels in out teeth, or even in nerves passing our bones. In contrast to hydroxyapatite, the mineral shells of the NB (predominantly carbonate apatite) present an extreme resistance against decomposition by cells and body fluids. The relationship between carbonate apatite and proteins is not unique for NB and has attracted much attention because of the phenomena related to the growth of calcium carbonate crystals in the presence of proteins, shown to be fruitful in understanding biomineralization processes occurring in the ear of the zebra fish.39 Analyzing these advanced natural materials promise to lead to unexpected progress in various fields, including nanotechnology, tissue engineering biomaterials and proteomics, and inspire novel routes for the design of materials and methods allowing to control the attachment of NB to tissues, including the definitive identification of the still disputed biological content of NB. Existing models,40,41 designed to simulate adhesion of cells to surfaces, may be adjusted to the NB problem. NB impose, in addition to the recognized pathogenic interactions with tissues, as solitary crystallization nuclei in the kidneys or collectively in the heart, also a
Crystal Growth & Design, Vol. 5, No. 1, 2005 23 challenge to the blood circulation. In particular, it seems that by anchoring to tissue in high flow regions of the heart, they presumably affect biomechanical forces. Correlated effects of tissue roughness on blood flow rate could be simulated in a rheological chamber model.
References (1) Kajander, E. O.; Ciftcioglu, N. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 8274. (2) Khullar, M. et al. Urol. Res. Urol. Res. 2004, 32,190. (3) Ciftcioglu, N.; Bjo¨rklund, M.; Kuorikoski, K.; Bergstro¨m, K.; Kajander, E. O. Kidney Int. 1999, 56, 1893. (4) Jelic, T. M. et al. South. Med. J. 2004, 97, 194. (5) Miller, V. M. et al. Am. J. Physiol. Heart. Circ. Physiol. 2004, 287, H1115. (6) Glesby, M. J. Clin. Infect. Dis. 2003, 37, Suppl 2, S91. (7) Thomas, J.; Doherty, S. M. J. Acquired Immune Defic. Syndr. 2003, 33, 281. (8) Sommer, A. P. J. Proteome Res. 2004, 3, 670. (9) Sommer, A. P.; Kajander, E. O. Cryst. Growth Des. 2002, 2, 563. (10) Sommer, A. P.; Pretorius, A. M.; Kajander, E. O.; Oron, U. Cryst. Growth Des. 2004, 4, 45. (11) Hove, J. R. et al. Nature 2003, 421, 172. (12) Farzaneh-Far, A.; Proudfoot, D.; Shanahan, C.; Weissberg, P. L. Heart 2001, 85, 13. (13) Cehreli, M. C.; Onur, M. A.; Sahin, S. Clin. Oral Implants Res. 2003, 14, 269. (14) Paetau, A.; Haltia, M. Acta Neuropathol. 1976, 36, 185. (15) Kalimo, H.; Maki, J.; Paetau, A.; Haltia, M. Muscle Nerve 1981, 4, 228. (16) King, R. H.; Llewelyn, J. G.; Thomas, P. K.; Gilbey, S. G.; Watkins, P. J. Neuropathol. Appl. Neurobiol. 1988, 14, 105. (17) Sommer, A. P.; Pinheiro, A. L. B.; Mester, A. R.; Franke, R. P.; Whelan, H. T. J. Clin. Laser Med. Surg. 2001, 19, 29. (18) Sommer, A. P.; Oron, U.; Kajander, E. O.; Mester, A. R. J. Proteome Res. 2002, 1, 475. (19) Sommer, A. P.; Hassinen, H. I.; Kajander, E. O. J. Clin. Laser Med. Surg. 2002, 20, 241. (20) Sommer, A. P.; Oron, U.; Pretorius, A. M.; McKay, D. S.; Ciftcioglu, N.; Mester, A. R.; Kajander, E. O.; Whelan, H. T. J. Clin. Laser Med. Surg. 2003, 21, 231. (21) Sommer, A. P. J. Proteome Res. 2003, 2, 665. (22) Sommer A. P. J. Proteome Res. 2004, 3, 667. (23) Sommer, A. P.; Franke, R. P. J. Proteome Res. 2002, 1, 111. (24) Williams, D. F. In Biocompatibility of Clinical Implant Materials; Williams, D. F., Ed.; CRC Press: Boca Raton, FL, 1982; Vol. 1, p 10. (25) Mofid, M. M.; Thompson, R. C.; Pardo, C. A.; Manson, P. N.; Vander Kolk, C. A. Plast. Reconstr. Surg. 1997, 100, 14. (26) Albrektsson, T.; Brånemark, P. I.; Hansson, H. A.; Ivarsson, B.; Jo¨sson, U. Adv. Biomater. 1982, 4, 167. (27) Yanagisawa, T. et al. Neurosurg. 2004, 100, 146. (28) Brånemark, R.; Brånemark, P. I.; Rydevik, B.; Myers, R. R. J. Rehabil. Res. Dev. 2001, 38, 175. (29) Sommer, A. P.; Franke, R. P. Nano Lett. 2003, 3, 573. (30) Sommer, A. P.; Franke, R. P. Nano Lett. 2003, 3, 321. (31) Sommer, A. P.; Ben-Moshe, M.; Magdassi, S. J. Phys. Chem. B 2004, 108, 8. (32) Denkov, N. D.; Velev, O. D.; Kralchevsky, P. A.; Ivanov, I. B.; Yoshimura, H.; Nagayama, K. Nature 1993, 361, 26. (33) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Nature 1997, 389, 827. (34) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Phys. Rev. E. 2000, 62, 756. (35) Sommer, A. P. J. Phys. Chem. B 2004, 108, 8096. (36) Sommer, A. P.; McKay, D. S.; Ciftcioglu, N.; Oron, U.; Mester, A. R.; Kajander E. O. J. Proteome Res. 2003, 2, 441. (37) Sommer, A. P.; Pavla´th, A. E. J. Proteome Res. 2003, 2, 558. (38) Bell, S.; Morco, T.; He, Q. United States Patent 2002, Patent Nr. 6, 355, 271. (39) Nicolson, T. Cryst. Growth Des. 2004, 4, 667. (40) Ruckenstein, E.; Marmur, A.; Gill, W. N. J. Theor. Biol. 1977, 66, 147. (41) Ruckenstein, E.; Marmur, A.; Gill, W. N. J Theor. Biol. 1976, 58, 439. (42) Sommer, A. P. J. Proteome Res., in press.
CG049812N
