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IMM Report Number 35: Nanomedicine


In conjunction with Foresight Update 50

Is Sapphire Biocompatible With Living Cells?

By Robert A. Freitas Jr.
Research Scientist, Zyvex Corp.

Robert A. Freitas  Jr.
Robert A. Freitas Jr.

Much of the discussion of medical nanorobotics building materials [1, 2] focuses on diamond because this form of carbon is expected to be convenient to synthesize mechanosynthetically and has high rigidity and strength, excellent chemical inertness, and good biocompatibility [1, 3]. However, in many proposed in vivo applications of medical nanorobots [4] and medical nanorobot aggregates [5], sapphire appears to be a useful material as well. Sapphire is a high-density monocrystalline alumina (Al2O3) already used in nanofluidics, dental implants ([1], Section 15.3.5.2), and other near-term nanomedical applications, and is being considered for use in the manufacture of prosthetic heart valves. Like diamond, sapphire [6, 7] is generally regarded as a nontoxic bioinert ceramic material that is likely noncarcinogenic [8].

The advantages of sapphire may be briefly summarized as follows. First, sapphire is almost as strong and hard as diamond ([2], Table 9.3), and only slightly more dense. Second, sapphire is already fully oxidized, so in particulate form (e.g., micron-size nanorobots) it cannot burn in air like diamond, and its crystalline structure remains stable to higher temperatures than diamond. Third, sapphire has more favorable bulk thermophysical characteristics. The thermal conductivity of sapphire is 100-1000 times less than for diamond, reducing the increase in the thermal conductivity of blood that is loaded with sapphire nanorobots, compared to blood containing pure-diamond nanorobots, especially at the highest loadings ([1], Section 15.3.8). Sapphire also has 60% greater heat capacity per unit volume than diamond. Fourth, sapphire offers designers an alternative hydrophilic surface chemistry as compared to hydrophobic diamond. Sapphire is amphoteric, absorbing H+ ions in very acidic environments (acquiring a positive charge) and absorbing OH ions in alkaline environments (acquiring a negative charge), while remaining isoelectric (electrically neutral) at intermediate pH values near human physiologic at ~7.4 ([1], Section 15.5.6.1). Fifth, sapphire can be manufactured in a full spectrum of colors ([2], Section 5.3.7) by replacing 0.01%-0.1% of the aluminum atoms with atoms of iron, titanium, or chromium, while producing only modest changes in the physical and chemical properties of the material.

Is sapphire, like diamond [3], a biocompatible material for living cells? A number of experiments have been performed to determine the response of fibroblasts to bulk alumina and sapphire surfaces. For example, alumina ceramic surface has shown excellent in vitro biocompatibility in a tissue culture of rabbit fibroblasts and cultured embryonic mouse fibroblasts [7]. Fibroblast-like mesenchymal cell populations cultured on solid alumina ceramic surfaces induce no cytotoxic or antiproliferative effects on monolayer populations in vitro. A scanning electron microscopic study [9] was conducted on the adhesion, spreading and formation of confluent cell monolayers from fibroblasts and epithelioid cells on Al2O3 ceramics. The study found that the cells adhered, spread, migrated and proliferated on the surfaces tested, leading to the conclusion that this implant material is compatible with cells [9]. In general, such cells adhere well to single-crystal or polycrystalline alumina. Other experiments found that human orbital fibroblasts grown on alumina bioceramic implant were free of debris and had the largest cell count, whereas cells grown on hydroxyapatite or porous polyethylene implants had cellular debris associated with them.

The response of bone cells to alumina ceramic has also been examined. For instance, the morphological responses of individual osteoblasts as they attached and spread on alumina surfaces in vitro have been examined by scanning electron microscopy [10]. The cells are round after 30 minutes, then spread radially during the next 1.5 hours until they are almost flat with a nuclear bulge on both rough and polished alumina [10]. Others have investigated the response of various oral cells to sapphire dental implant surfaces. In one study [6], the influence of single-crystal sapphire on the behavior of human epithelial cells and fibroblasts derived from biopsies of the oral mucosa was studied. Compared to control cultures, no effects on cell morphology and growth characteristics were observed. Another study [11] sought to elucidate the ultrastructure of peri-implant junctional epithelium (IJE) on single-crystal sapphire dental implants connected to adjacent teeth by a metal superstructure, by examining the peri-implant gingivae of ten monkeys using a transmission electron microscope at 3, 6 and 12 months after implant insertion. At the time of examination, the ultrastructural features of the IJE were almost identical to those of the natural junctional epithelium attached to natural teeth.

The biocompatibility of alumina and sapphire particles has also been investigated because it is important to understand the biological reaction to inhaled particles and to wear particles that might be produced by frictional forces in long-term prosthetic implants. Such studies generally involve micron-scale particles, roughly the size range of future medical nanorobots — which is why we’re interested in the results.

First, is alumina powder lethal? In one early experiment [12], massive administration of several bioactive <44-micron ceramic powders were lethal to Balb/c mice in 5 gm/kg doses when injected intraperitoneally, producing a swollen kidney with an ischemic color [12]. Alumina powder similarly injected as a control (equivalent to 90 trillion 1-micron3 nanorobots injected into the peritoneal cavity of a 70-kg human) was not lethal and elicited no changes in blood chemistry [12]. All powders had almost no systemic effects when injected intramuscularly or subcutaneously [12].

What about inflammation? Early studies [7] of 0.5-5 micron alumina particles implanted subcutaneously and intraarticularly (knee joint) for up to 5 months in mice revealed no persistent inflammatory or progressive fibrotic reactions around the powder deposits. After an initial acute (3-7 day) granulocytic inflammatory phase, the material was gradually contained within macrophages and deposited locally without significant fibrous tissue reaction. Some particles were transported via lymphatic vessels into regional lymph nodes [7]. Particles were found in the interstitium of the lung; in the reticuloendothelial cells of the liver, spleen, and bone marrow; and in one case in the meshwork of a renal glomerulum; but rarely in the bloodstream. Such crystal deposits caused no local cell necrosis, fibrosis, or granulomatous reaction in any of these organs [7].

Most cytocompatibility studies have examined the foreign-body reactions of macrophages to alumina particles. For example, it is known that rat alveolar macrophages readily ingest aluminum oxide particles [13]. Toxicity tests of alumina powder in vitro using rabbit alveolar macrophages and in vivo using direct intratracheal injection into rat lungs found that the powder had low toxicity for macrophages and minimal recruitment of airway cells and neutrophils in the rat lungs. Rat tissue responses to alumina powder administered at low doses were investigated by Di Silvestre et al. [14], who found that powdered alumina implantation in the subcutis, the muscle and the peritoneum of the rat produced the same intense acute inflammatory reaction in all implantation sites after 2 weeks. However, after 8 weeks the inflammatory reaction had regressed and there was a thin layer of connective tissue around the implanted material, completely isolating it from the surrounding tissues [14]. Examination of human biopsies from well-fixed human total hip prostheses showed that alumina particle deposits increase with time with only a low-grade macrophagic reaction. The amount of necrosis and fibrosis was lower for alumina implant wear debris than that associated with metal or polyethylene implants [15]. Intraperitoneal and intramuscular implantation of powdered alumina particles in rats showed an initial granulocytic reaction with some uptake by the reticuloendothelial system [13]. No antigenicity of alumina ceramic was found in another study that attempted induction of footpad swelling in ceramic-immunized mice.

Sapphire is generally biocompatible with macrophages. Harms and Mausle [13] tested the biocompatibility of alumina ceramic in macrophage cultures revealed no acute cytotoxicity. Pizzoferato et al. [16] found that saline-suspended 1-12.5 micron alumina particles were only slightly phagocytosed in vivo by mouse peritoneal macrophage cells lavaged 1 week post-injection. Christel [15] noted that an examination of human biopsies from failed total hip prostheses revealed a foreign-body reaction containing predominantly macrophages, loaded with alumina particles, that had no morphologic alteration and had not lost their chemotactic ability [15]. Nakashima et al. [17] reported that 1-, 100-, and 1000-micron alumina particles could induce the release of bone resorbing mediators (IL-6, TNF-α, IL1-α) by macrophages in a dose-dependent manner, but hydroxyapatite particles of equal size stimulated a greater release than the alumina. Alumina dust has no significant effect on the in vitro enzyme activity of alveolar macrophages in the rat.

Catelas et al. [18] measured the effects of size (0.6-4.5 micron), concentration (5-1250 particles/macrophage), and composition (e.g., alumina) of ceramic particles on phagocytosis and cell mortality in the J774 mouse macrophage cell line. Kinetic studies (from 5 min to 24 hours) revealed that phagocytosis of the particles begins very early after cell exposure, increasing with time and particle concentration and reaching a plateau after ~15 hours. Phagocytosis increases with concentration for particles up to 2 microns. For larger particles up to 4.5 microns, phagocytosis reaches a plateau independent of particle size and concentration, suggesting a saturation effect most likely dependent on the total volume ingested [18]. Cytotoxicity studies revealed that macrophage mortality increases with particle size and concentration for sizes greater than 2 microns (to >30% cell mortality). Smaller particles (0.6 microns) cause cell mortality only at higher concentrations, and the mortality is still very low (~10%) [18]. Related studies by Catelas using the same cell model investigated the induction of apoptotic cell death in macrophages by alumina ceramic and other powders of different sizes and concentrations. Of some concern, Catelas found that the apoptotic effect of ceramic particles on nuclear morphology was size- and concentration-dependent, but that alumina ceramic particles induce apoptosis more effectively than polyethylene particles at concentrations of 125-250 particles/macrophage for ~2 hours. More work is needed to resolve this issue.

The responses of a few other cell types to alumina ceramic powders have also been investigated. For example, cultured human fibroblasts exposed to 1-500 micrograms/cm3 of alumina powder showed no cytotoxic effects with cell viability at different exposure times measured by colony formation efficiency, neutral red uptake and colorimetric tetrazolium reduction [19]. No cytotoxic or antiproliferative effects were induced in fibroblast-like mesenchymal cell monolayer populations cultured in vitro on powdery alumina ceramic. Alumina powders generally induce no cytotoxicity in cell cultures [20] of human gingival fibroblasts or osteoblastlike cells. Rodrigo et al. [21] found some change in osteoblast function from 10-micron α-alumina particles in human bone cell cultures, and that while both polyethylene and a-alumina increase the expression and secretion of IL-6 in human osteoblastic cells, the stimulation is weaker from α-alumina at the same particle dose. Oonishi et al. [22] observed no inflammation or cell infiltration for 10- and 100-micron alumina particles implanted in holes drilled in the femoral condyles of rabbits. Dion et al. [23] found that the hemolysis eventually initiated in vitro by alumina powder is almost zero.

As for inhalation toxicity, human experience with alumina powders strongly suggests that they are not associated with major specific pulmonary hazards under typical 20th century conditions of occupational inhalation exposure, though rodent experiments suggest that clearance of alumina particles from the lung is slow [24]. OSHA occupational exposure limits for alumina dust are 10 mg/m3 (total fraction) and 5 mg/m3 (respirable fraction), respectively, according to the official Material Safety Data Sheets. (10 mg/m3 of sapphire particles equates to ~3 billion/m3 airborne 1-micron3 nanorobots.) α-alumina 100-700 nm particles have only minimal or no fibrogenic reactivity, and only at doses instilled intratracheally that are massive compared to the amount which could reasonably be inhaled in any one breath.

We can be guardedly optimistic that monocrystalline sapphire should prove to have very good biocompatibility with living cells.

Copyright 2002 Robert A. Freitas Jr. All Rights Reserved

References

1. Robert A. Freitas Jr., Nanomedicine, Volume IIA: Biocompatibility, Landes Bioscience, Georgetown, TX, 2003; http://www.nanomedicine.com

2. Robert A. Freitas Jr., Nanomedicine, Volume I: Basic Capabilities, Landes Bioscience, Georgetown, TX, 1999; http://www.nanomedicine.com

3. Robert A. Freitas Jr., “Is Diamond Biocompatible With Living Cells?” Foresight Update No. 39, 30 December 1999, pp. 7-9; http://www.imm.org/Reports/Rep012.php

4. Robert A. Freitas Jr., “Exploratory Design in Medical Nanotechnology: A
Mechanical Artificial Red Cell,” Artificial Cells, Blood Substitutes, and
Immobil. Biotech.
26(1998):411-430; http://www.foresight.org/Nanomedicine/Respirocytes.html;
Robert A. Freitas Jr., “Microbivores: Artificial Mechanical Phagocytes using
Digest and Discharge Protocol,” Zyvex preprint, March 2001; http://www.rfreitas.com/Nano/Microbivores.htm.

5. Robert A. Freitas Jr., Christopher J. Phoenix, “Vasculoid: A personal nanomedical appliance to replace human blood,” Journal of Evolution and Technology 11(April 2002); http://www.jetpress.org/volume11/vasculoid.html

6. K. Arvidson et al., “In vitro and in vivo experimental studies on single crystal sapphire dental implants,” Clin. Oral Implants Res. 2(April-June 1991):47-55.

7. Peter Griss, Gunther Heimke, “Biocompatibility of High Density Alumina and its Applications in Orthopedic Surgery,” in David F. Williams, ed., Biocompatibility of Clinical Implant Materials, Volume I, CRC Press, Boca Raton, FL, 1981, pp. 155-198.

8. Robert A. Freitas Jr., “Could Medical Nanorobots Be Carcinogenic?” Foresight Update No. 49, August 2002, pp. 8-9; http://www.imm.org/Reports/Rep033.php

9. G. Neupert et al., “Significance of scanning electron microscopy observations on cellular reactions in the biological evaluation of endoprosthetic materials,” Z. Exp. Chir. Transplant Kunstliche Organe 20(1987):139-143. In German.

10. M.A. Malik et al., “Osteoblasts on hydroxyapatite, alumina and bone surfaces in vitro: morphology during the first 2 h of attachment,” Biomaterials 13(1992):123-128.

11. M. Hashimoto et al., “Ultrastructure of the peri-implant junctional epithelium on single-crystal sapphire endosseous dental implant loaded with functional stress,” J. Oral Rehabil. 16(May 1989):261-270.

12. Keiichi Kawanabe et al., “Effects of injecting massive amounts of bioactive ceramics in mice,” J. Biomed. Mater. Res. 25(January 1991):117-128.

13. J. Harms, E. Mausle, “Tissue reaction to ceramic implant material,” J. Biomed. Mater. Res. 13(January 1979):67-87.

14. M. Di Silvestre et al., “Powdered alumina implants in the experimental animal: a histological study conducted in the rat,” Chir. degli Organi. di Mov. 76(April-June 1991):167-172.

15. P.S. Christel, “Biocompatibility of surgical-grade dense polycrystalline alumina,” Clin. Orthop. 282(September 1992):10-18.

16. A. Pizzoferrato et al., “The effect of injection of powdered biomaterials on mouse peritoneal cell populations,” J. Biomed. Mater. Res. 21(April 1987):419-428.

17. Y. Nakashima et al., “The stimulatory effects of ceramic particles on the production of the bone-resorbing mediators in vitro,” Trans. Orthop. Res. Soc. 20(1995):780.

18. Isabelle Catelas et al., “Cytotoxicity and macrophage cytokine release induced by ceramic and polyethylene particles in vitro,” J. Bone Joint Surg. Br. 81(May 1999):516-521.

19. J. Li et al., “Evaluation of biocompatibility of various ceramic powders with human fibroblasts in vitro,” Clin. Mater. 12(1993):197-201.

20. I. Dion et al., “Physicochemistry and Cytotoxicity of ceramics Part II: Cytotoxicity of ceramics,” J. Mater. Sci.: Materials in Medicine 5(1994):18-24.

21. A.M. Rodrigo et al., “Effects of polyethylene and alpha-alumina particles on IL-6 expression and secretion in primary cultures of human osteoblastic cells,” Biomaterials 23(February 2002):901-908.

22. H. Oonishi et al., “Comparative bone growth behavior in granules of bioceramic materials of various sizes, ” J. Biomed. Mater. Res. 44(January 1999):31-43.

23. I. Dion et al., “Blood haemolysis by ceramics,” Biomaterials 14(1993):107-110.

24. R.B. Schlesinger et al., “Clearance and translocation of aluminum oxide (alumina) from the lungs,” Inhal. Toxicol. 12(October 2000):927-939.

More on the medical applications of nanotechnology at:
http://www.foresight.org/Nanomedicine/index.html

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