Calcification of Intraocular Implant Lens Surfaces - Langmuir (ACS

Calcification of octacalcium phosphate [Ca8H2(PO4)6·5H2O, OCP] on differently packaged “Ultem” and “Surefold” intraocular implant lens surfac...
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Calcification of Intraocular Implant Lens Surfaces Wenju Wu,† Xiangying Guan,† Ruikang Tang,† Daniel Hook,‡ Wenyan Yan,‡ George Grobe,‡ and George H. Nancollas*,† Chemistry Department, Natural Sciences Complex, University at Buffalo, The State University of New York, Buffalo, New York 14260, and Global Scientific Affairs, Bausch & Lomb, 1400 North Goodman Street, Rochester, New York 14609 Received August 28, 2003. In Final Form: December 3, 2003 Calcification of octacalcium phosphate [Ca8H2(PO4)6‚5H2O, OCP] on differently packaged “Ultem” and “Surefold” intraocular implant lens surfaces has been studied in vitro in solutions supersaturated with respect to OCP at pH ) 7.10 and 37 °C. No mineral deposition was observed on the lenses packaged in Ultem vials even after treatment with behenic acid, one of the fatty acids identified on explanted lenses. Following treatment with behenic acid, nucleation of OCP occurred on the lenses from Surefold vials, which incorporate silicone gaskets; induction periods preceding calcification were about 6 h. No mineralization was found on the lenses in vials with other gasket materials, including polytetrafluoroethylene, fluorocarbon elastomer, and polypropylene. The results of this study indicate that both silicone and fatty acids such as behenic acid play important roles in inducing the in vivo calcification of OCP on IOL lenses; all of the lens treatment steps were necessary for nucleation induction.

Introduction The calcification of medical devices fabricated from polyurethane, silicones, and hydrogels has been widely reported. These devices include bioprosthetic heart valves, cerebrospinal fluid shunts, mammary implants, nose implants, artificial finger joints, and contact lenses.1-6 Calcifications of ophthalmic devices have also been increasingly observed following implantation.7-14 Bucher,9 using energy-dispersive X-ray spectroscopy and X-ray diffraction, reported the presence of HAP in deposits inside a lens (IOGEL 1003) removed from a patient 10 months after implantation. These findings suggest that the intraocular environment may induce calcification, and this must be taken into consideration when evaluating the long-term biocompatibility of materials used for IOL fabrication. * Author to whom correspondence should be addressed: Professor George H. Nancollas. Tel.: (+1) 716-645-6800 ext. 2210. Fax: (+1) 716-645-6947. E-mail: [email protected]. † The State University of New York. ‡ Bausch & Lomb. (1) Johnston, T. P.; Schoen, F. J.; Levy, R. J. J. Pharm. Sci. 1988, 77, 740. (2) Jorge-Herrero, E.; Fernandez, P.; De la Torre, N.; Escudero, C.; Garcia-paez, J. M.; Bujan, J.; Castillo-Olivares, J. L. Biomaterials 1994, 15, 815. (3) Schoen, F. J.; Danielle, H.; Richard, W. B.; Levy, R. J. J. Thorac. Cardovasc. Surg. 1994, 108, 880. (4) Chanda, J. Artif. Organs 1993, 18, 408. (5) Chanda, J.; Bhaskara Rao, S.; Mohanty, M.; Lal, A. V.; Muraleedharan, C. V.; Bhuvaneshwar, G. S.; Valiathan, M. S. Artif. Organs 1994, 18, 752. (6) Peters, W.; Smith, D. Ann. Plast. Surg. 1995, 34, 8. (7) Bucher, P. J. M.; Buchi, E. R.; Daicker, B. C. Arch. Ophthalmol. 1995, 113, 1431. (8) Jensen, M. K.; Crandall, A. S.; Mamlis, N.; Olson, R. J. Arch. Ophthalmol. 1994, 112, 1037. (9) Bucher, P. Eur. J. Implant Refractive Surg. 1994, 6, 175. (10) Olson, R. J.; Caldwell, K. D.; Crandall, A. S.; Jensen, M. K.; Huang, S. C. Am. J. Ophhthalmol. 1998, 126, 177. (11) Werner, L.; Apple, D. J.; Kaskaloglu, M.; Pandey, S. K. J. Cataract Refractive Surg. 2001, 27, 1485. (12) Buchen, S. Y.; Cunanan, C. M.; Gwon, A. G.; Weinschenk, J. I.; Gruber, L.; Knight, P. M. J. Cataract Refractive Surg. 2001, 27, 1473. (13) Apple, D. J.; Werner, L.; Escobar-Gomez, M.; Pandey, S. K. J. Cataract Refractive Surg. 2000, 26, 796. (14) Werner, L.; Apple, D. J.; Escobar-Gomez, M.; Pandey, S. K. Ophthalmology 2000, 107, 2179.

Heterogeneous nucleation, an almost ubiquitous phenomenon, is often initiated at impurity sites and foreign surfaces. This process is very important because potential nucleators such as implant surfaces, proteins, cells, and other tissue components are in constant contact with mineralizing solutions. In some cases, rapid heterogeneous nucleation of calcium phosphates may be desired as in many load-bearing dental and orthopedic implants to achieve fast bony adaptation, the absence of fibrous tissue seams, a reduced healing time, and an increased tolerance of surgical inaccuracies. In other situations, where calcification would destroy the utility of the implants, heterogeneous nucleation must be prevented or minimized. There are many direct factors controlling nucleation such as the thermodynamic driving force or relative supersaturation, temperature, pH, ionic strength, and interfacial free energy as well as other variables such as impurities and surgical adjuvant products (viscoelastic, irrigation liquids, myotics, etc.). Hydroxyapatite [Ca10(PO4)6(OH)2, HAP] is the most physiologically important calcium phosphate phase. Octacalcium phosphate [Ca8H2(PO4)6‚5H2O, OCP] has been proposed as a precursor phase in the formation of many biological apatites because the presence of the apatite layer in OCP may allow for the epitaxial overgrowth of HAP. Because the deposits from explanted IOLs, originally packaged with silicone gaskets, also appear to contain silicone and fatty acid contaminants such as behenic and aracadinic acids, the present in vitro study was aimed at determining the factors responsible for inducing the formation of OCP mineral on hydrogel IOLs with different packaging styles in the presence of a typical in vivo fatty acid. Experimental Section Intraocular lenses, optical implants for the replacement of human crystalline lenses, composed of a hydrogel (18% water) made from hydroxyethylmethacrylate, were provided by Bausch & Lomb. The foldable lenses had been lathe cut and polished from the central hydrogel portions of composites, which contained a bonded UV absorber. The haptics were formed from the outer poly(methyl methacrylate) portion. Ultem and Surefold lenses

10.1021/la035606q CCC: $27.50 © 2004 American Chemical Society Published on Web 01/23/2004

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differed in the gasket materials used in their packaging. Behenic acid was purchased from Sigma Chemical Co. (lot 96F84151). To accelerate the possible interactions between IOLs and the gasket and holder materials of the vials, the polypropylene holder/ folders (15 units, lot 12483) and retainers (25 units, lot 12489) were Soxhlet-extracted with petroleum ether (AR grade, bp 3560 °C) for 48 h. The solvent was removed in vacuo affording 0.46 g of pale yellow oil. A solution of 9.4 mg of the extract was diluted to a total weight of 9.40 g with HPLC grade hexane. A total of 18 freshly manufactured, Ultem-packaged IOLs (H60M lot 44VD) were air-dried on a sheet of polytetrafluoroethylene (PTFE) overnight. Nine of them were dipped into this diluted extract before being air-dried. Each IOL was then replaced in its vial with the original water. In these in vitro tests, the original gasket material for the Ultem-packaged IOLs is referred to as the control standard. Solutions, prepared using triply distilled, carbon dioxide-free water, were filtered before use through washed 0.22-µm filters (Millipore, Bedford, MA). Calcium concentrations were determined either complexometrically by ethylenediaminetetraacetic acid titration with Eriochrome Black-T as the indicator or by atomic absorption (Perkin-Elmer, model 3100, Norwich, CT). Monobasic potassium phosphate and sodium chloride reagent grade salts were dried at 110 °C before use. Phosphate concentrations were determined spectrophotometrically (Varian Cary 210) as the phosphovanadomolybdate complex. Carbon dioxidefree potassium hydroxide solutions were prepared from reagent grade pellets washed in a nitrogen atmosphere. OCP nucleation and crystal growth experiments on the lens surfaces were made at a relative supersaturation, σOCP ) 2.28, pH ) 7.10, T ) 37.0 ( 0.05 °C, and ionic strength of 0.15 mol L-1, adjusted by the addition of sodium chloride. The reaction solutions contained CaCl2 ) 2.50 × 10-3 mol L-1, KH2PO4 ) 1.88 × 10-3 mol L-1, and NaCl ) 0.139 mol L-1. Behenic acid stock solutions contained 10 mg in 100 mL of 90% ethanol. Where pretreatment was required, IOL samples were washed with triply distilled water and immersed in diluted behenic acid solution (2 mL of 1:1 stock solution:triply distilled water) for 30 min. To exclude carbon dioxide, calcification tests were conducted in magnetically stirred water-jacketed Pyrex vessels, in an atmosphere of nitrogen gas presaturated with respect to the background electrolyte. Supersaturated solutions were prepared by introducing, in sequence, sodium chloride, calcium chloride, and potassium dihydrogen phosphate solutions. The pH was adjusted to the required value by the very slow addition of a 0.06 mol L-1 potassium hydroxide solution to avoid local concentration effects and unwanted spontaneous precipitation. Once equilibrium of the supersaturated solutions had been attained, as indicated by a constant glass electrode potential, the IOL test lenses were introduced. The initial consumption of crystal lattice ions, accompanying nucleation, was detected by a change in the hydrogen ion activity sensed by a glass electrode. The lowering of the pH was used to trigger the simultaneous addition of two titrant solutions from stepper-motor-driven burets that served to maintain constant the pH, the concentrations of calcium and phosphate, and the ionic strength of the reaction solutions. A glass electrode (Orion, model 9101), standardized using two N-bromosuccinimide buffer solutions at pH ) 7.386 and 4.028 at 37 °C,15 was used to control titrant addition through a potentiostat. The concentrations of the titrant solutions were calculated using mass balance and electroneutrality expressions. Titrant I consisted of calcium chloride and sodium chloride at concentrations given by eqs 1 and 2, respectively.

TCaCl2 ) 2WCaCl2 + 4Ceff

(1)

TNaCl ) 2WNaCl - 8Ceff

(2)

Titrant II contained potassium dihydrogen phosphate and potassium hydroxide with concentrations given by eqs 3 and 4, respectively. (15) Bates, R. G. pH Determination, Theory and Practice; Wiley: New York, 1973; p 73.

TKH2PO4 ) 2WKH2PO4 + 3Ceff

(3)

TKOH ) 2WKOH + 5Ceff

(4)

In eqs 1-4, T and W are the titrant and metastable supersaturated reaction solution concentrations, respectively, and Ceff is the effective concentration of added titrants with respect to OCP or the number of moles of OCP grown/L of mixed titrants. A suitable Ceff was chosen to provide convenient titrant volumes; the value of Ceff for this study was usually 2.00 × 10-4 mol L-1. During the reactions, solutions were periodically withdrawn, filtered (0.22-µm Millipore filters), and analyzed for calcium and phosphate to verify the constancy of the solution compositions. The IOL lenses were examined by scanning electron microscopy (SEM at 20 kV; JEOL JSM-5300, Noran Instrumental, Inc., Middleton, WI) and by diffuse reflectance infrared Fourier transform spectroscopy (Perkin-Elmer 1760X FT-IR spectrometer). Experiments were done at least three times. To determine whether hexane, used to dilute the silicone, may play a role in these calcification experiments, parallel experiments were made in which the IOLs were treated only with pure hexane and examined for nucleation potential following the same procedures. This possibility was eliminated because there was no induced calcification (Table 1). Relative supersaturations, σ, were calculated using eq 5

σ)S-1)

( ) IP KSO

1/v

-1

(5)

in which v ()16) is the number of ions in a formula unit of the OCP growing phase, S is the supersaturation ratio, and IP and KSO are the ionic activity and solubility products, respectively. The solubility product of the OCP used in these calculations was taken as 2.51 × 10-99 mol16 L-16.16 The relative degrees of supersaturation and ionic activities were calculated from the experimental total concentrations using speciation the programs described previously17 based on expressions for ion-pair and complex formation constants, mass balance, and electroneutrality.18,19 Surfaces were characterized using a Phymetrics (PHI) Quantum 2000 scanning X-ray photoelectron spectroscopy (XPS) microprobe incorporating a monochromatic Al anode operated at 15 kV and 40 W in the standard power mode with dual beam neutralization (ions and electrons). Static point acquisitions were collected with a 200-µm2 analysis area. The base pressure of the instrument was 5 × 10-10 Torr, and during operation, the pressure was e1 × 10-7 Torr. This instrument made use of a hemispherical analyzer operated in the FAT mode. A gauze lens was coupled to a hemispherical analyzer to increase signal throughput. Data were collected with a SUN Ultra 5 workstation running Solaris 2.6 OS and COMPASS version 4.0A. The instrument utilized MultiPak version 6.0 software for data analysis. Scanned X-ray images (SXIs) were collected using a focused X-ray beam, which causes the emission, from the specimen surface, of photoelectrons, Auger electrons, and secondary electrons in competing processes. The analyzer was set (stage bias, gauze lens to +90 V) to accelerate the secondary electrons into the analyzer for imaging. The secondary electrons were spatially resolved by tracking the electron beam and gauze lens location through a two-dimensional image of the specimen. This image was then used to define the area for XPS analysis. For XPS examination, the IOL lenses were placed on a stainless steel mount and held in place with a molybdenum mask. Lens surfaces were analyzed utilizing low-resolution survey spectra (0-1100 eV) to identify the elements present. Quantification of elemental compositions was completed by integration of the photoelectron peak areas. Analyzer transmission, photoelectron (16) Shyu, L. J.; Perez, L.; Zawacki, S.; Heughebaert, J. C.; Nancollas, G. H. J. Dent. Res. 1983, 62, 398. (17) Wu, W.; Zhuang, H. Z.; Nancollas, G. H. J. Biomed. Mater. Res. 1996, 35, 93. (18) Koutsoukos, P.; Amjad, Z.; Tomson, M. B.; Nancollas, G. H. J. Am. Chem. Soc. 1980, 102, 1553. (19) Davies, C. W. Ion Association; Butterworth: London, 1962.

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Table 1. OCP Nucleation and Growth Tests of IOLs packaging style

gasket

number of tests

Ultem Ultem Ultem Ultem

standard standard standard standard

10 6 26 2

Ultem

standard

3

Ultem

standard

4

Surefold*

silicone silicone fluorocarbon clastomer, Kelrez extracted nitrile rubber PTFE standard

5 36 19 4 4 4

Surefold** Surefold Surefold Surefold Ultem

silicone pretreatment no none. treated only with hexane no lenses in 1:250 silicone in hexane solution lenses in 1:250 silicone in hexane solution solution containing silicone gasket cutting pieces no no no no no solution containing PTFE pieces

Figure 1. Plot of titrant addition as a function of time for calcification test of OCP on IOL lens (lot 44VD2) packaged in an Ultem vial after treatment with behenic acid. cross-sections, and source angle correction were taken into consideration to give accurate atomic concentration values.

Results and Discussion Nucleation Tests. Experimental conditions and induction times, τin, preceding nucleation, are summarized in Table 1. The reaction solution was highly supersaturated with respect to OCP (σOCP ) 2.28) but stable for more than 10 h, after which OCP crystals precipitated on the walls of the reaction cell or the electrode. A typical titrant volume curve as a function of time for OCP nucleation on an IOL surface is shown in Figure 1. The induction time, τin, needed to reach steady-state nucleation was measured as the time from the introduction of IOL surfaces to the intersection of a volume/time tangent (dV/ dt) with the time axis. Although there is no precise method to draw the tangent, for consistency it was obtained from the linear regression of titrant addition (0.5-1.5 mL) volumes as a function time (see Figure 2). The reproducibility of the induction periods depends both upon their magnitude and the thermodynamic driving force. In all the tests, three stages are clearly distinguished (Figure 2). First, in the induction region, the solution concentrations and pH were unchanged and the volume, dV, of added titrant remained essentially 0, confirming the absence of mineral nucleation or growth.20,21 In second stage, titrant addition commenced as heterogeneous nucleation set in. This stage reflected the commencement of a stable nucleus

behenic acid induction confirmed observed pretreatment time (min) by CC by SEM no no yes no

>600 >600 >600 >600

no no no no

no no no no

yes

230-300

yes

yes

yes

400-480

yes

yes

no yes yes yes yes yes

>960 290-330 >600 >600 >600 >600

no yes no no no no

no yes no no no no

Figure 2. Plot of titrant addition as a function of time for the calcification test of OCP on the IOL lens (lot 131-82-2) packaged in an Ultem vial after the lens was precoated with 1:250 dilution cyclic silicone and then treated with behenic acid.

formation with an approximately linear titrant addition; this line was extrapolated to zero titrant to estimate the induction time.22,23 In the third region, a more rapid addition reflected both heterogeneous nucleation and the exponential growth of OCP crystals on the nuclei formed in the second stage.22,23 A total of 117 IOL lenses packaged in either Ultem or Surefold vials were examined. No calcification could be detected on Ultem IOLs, even when tests were extended for as long as 3 days. Twenty-six IOL lenses in Ultem packaging and treated only with behenic acid also showed no evidence of heterogeneous nucleation when tested for OCP calcification. SEM micrographs (Figure 3) also confirmed the absence of OCP crystals on the IOL surfaces. Similarly, no OCP crystallization was observed on the IOLs, which were dip-coated in a 1:250 dilution of the cyclic silicone hexane extract prior to the calcification tests. In contrast, OCP consistently nucleated on lenses treated first with silicone and then with behenic acid with titrant plots similar to Figure 2 and SEM shown in Figure 4. In (20) Nielsen, A. E. Kinetics of Precipitation; The Macmillan Co.: New York, 1964. (21) Walton, A. G. In Nucleation; Zettlemoyer, A. C., Ed.; Marcel Dekker: New York, 1969. (22) Ohara, M.; Reid, R. C. Modeling Crystal Growth Rates from Solution; Prentice Hall: Englewood Cliffs, NJ, 1973. (23) Hartman, P. Crystal Growth: An introduction; North-Holland: Amsterdam, 1975.

Intraocular Implant Lens Surfaces

Figure 3. SEM micrograph, following calcification test, of OCP on IOL lens (lot VP-1) packaged in an Ultem vial after treatment with behenic acid.

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Figure 5. Plot of titrant addition as a function of time for calcification test of OCP on IOL lens (lot 45B713) packaged in a silicone Surefold vial after the lens was treated with behenic acid.

Figure 6. SEM micrograph, following calcification test, of OCP on IOL lens (lot 375 × 07) packaged in a silicone Surefold vial after treatment with behenic acid.

Figure 4. SEM micrograph, following calcification test, of OCP on IOL lens (lot 131-82-2) packaged in an Ultem vial after the lens was precoated with 1:250 dilution cyclic silicone and then treated with behenic acid (titrant curve shown in Figure 1).

these cases, the induction periods ranged from 5 to 6 h. Moreover, IOL lenses packaged in Ultem vials but with added silicone gasket fragments and subsequently treated with behenic acid induced calcification in all cases (Table 1). As can be see in Table 1, IOL lenses packaged in Surefold vials having silicone gaskets but untreated with behenic acid did not induce OCP nucleation. The tests could be extended in supersaturated solutions for periods of 16 h to 3 days without mineral deposition. However, after treatment with behenic acid, calcification was initiated in less than 6 h (Table 1). A typical plot of titrant addition as a function time for Surefold IOLs (Hydroview, sample lot 45B713) is shown in Figure 5. The SEM micrograph of the well-grown OCP crystals on the lens surfaces is shown in Figure 6; OCP crystals cover the entire IOL surface.

Other gasket materials were tested for potential calcification including PTFE, fluorocarbon elastomer, and polypropylene. After storage in vials containing these materials, the IOLs were treated with behenic acid for 30 min and no nucleation of OCP was detected. Moreover, no OCP nucleation was observed, following treatment with behenic acid, on IOLs in Surefold vials in which the silicone gaskets had been replaced by Kelrez (fluorocarbon elastomer) gaskets. XPS Data. XPS surface elemental analysis (10-100 Å) and short-range chemical bonding showed the surface composition of control lenses to be 72% carbon and 28% oxygen. The silicon in the Ultem-packaged IOL lenses (148 samples) was in the form of a silicone (102.4 eV) and ranged from 0 to about 1.9% with an average of 0.2% ((0.4%); the average elemental composition was 73% carbon, 0.5% nitrogen, 26% oxygen, and 0.2% silicon. The silicon in the Surefold-packaged IOL lenses (227 samples) was also present as a silicone ranging from 0 to 9.1% (average of 2.3 ( 1.6%) with an average elemental composition of 70.6% carbon, 0.2% nitrogen, 27.1% oxygen, and 2.1% silicon. Surefold control lenses (Table 1) were analyzed by SXIs prior to calcification testing; the silicon elemental map

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Figure 7. SXIs of IOL lens of pre- (a) and post- (b) calcification. Note that the shape of the silicone contamination island in part a is the same as that of the calcification in part b.

Figure 8. XPS post-calcification elemental maps of IOL lens, which show clearly that the calcium phosphates crystallized on those parts of the lens surfaces contaminated by silicone.

for a typical control Surefold surface (Surefold* in Table 1) is shown in Figure 7. In Figure 7, areas light in intensity represent silicon atoms; more intense light areas represent a greater density of silicon atoms than that of the darker areas. The individual lens images were registered such that the same areas could be imaged by XPS before and after placement in the calcification model. The XPS image results for the same lens that had undergone behenic acid

exposure prior to introduction into the OCP supersaturated solutions (Surefold** in Table 1) are shown in Figure 8. In this figure, the individual XPS intensity maps can be seen for calcium, phosphorus, oxygen, and carbon together with the total intensity map or the SXI. Comparison of the silicon map in Figure 7 with the calcium, phosphorus, or oxygen maps in Figure 8 shows that the calcified areas correspond to the position of the islands of

Intraocular Implant Lens Surfaces

silicon contamination prior to testing. Thus, the calcified areas correspond to the areas previously contaminated by silicone. Conclusions The results of this study show that both silicone and fatty acids such as behenic acid play important roles in inducing OCP crystallization or calcification of IOL lenses. The reaction sequence probably involves silicone molecular adsorption at the IOL lens surfaces, creating a hydrophobic interface, followed by the adsorption of the hydrophobic

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tail of the behenic acid, thereby exposing hydrophilic carboxyl groups to the supersaturated solutions. It is interesting to note that all of the treatment steps were necessary for nucleation induction. Direct observations have shown that calcification occurred only on the areas that had been shown to contain silicon atoms. Acknowledgment. We thank Bausch & Lomb Company for support this work and the National Institutes of Health for partial support (NIDCR 03223). LA035606Q