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`MILLENIUM EXHIBIT 2016
`Baxter Healthcare Corp. et. al. v. Millenium Biologix, LLC
`IPR2013-00582,-00583,-00590,-00591
`
`

`

`Toxicity of silica-containing glass: M. Nagase et al.
`
`173
`
`taneously and were dosed intraperitoneally from the
`same single glass stock suspensions.
`
`IOO Ca (P03)2 to X Sio2
`x
`
`(pmoI/cma)
`
`Dissolved
`
`(.‘.a(P03)2
`
`0.|
`
`0.2
`Molar fraction
`
`Figure 2 Amount of dissolved ions in water at 37°C for 50 h
`versus. glass composition. The dissolution of ions into the
`water depended on the glass composition. (0) P5“;
`([1)
`Ca2+;(A) Si“.
`
`Effect of particle measurements
`
`The differences in grain sizes or surface areas among
`samples did not affect the amounts of dissolved ions from
`the samples. The diameters of grains of materials used in
`this experiment were 3.9—3.3 ,um [Table 1]. The surface areas
`of the materials were 2.3—3.2 mZ/g. except for the pure
`silica glass itself, which was 13.0 mzlg ['Rable 1).
`
`DISCUSSION
`
`Silicon is, next to oxygen, the most abundant element in
`the earth’s crust. Although only trace amounts appear in
`most animal tissues, silicon is an essential element in the
`chick‘5 and the rat7, required for normal growth and
`development. Silicon may function as a biological cross-
`linking agent and contribute to the architecture and
`resilience of connective tissue“. The ingestion of a small
`amount of amorphous silica (silicon dioxide] powder
`appears to be completely harmless, However, when
`
`Table 1 Grain sizes and surface areas
`
`
`
` Materials‘ Grain sizes (,u m) Surface areas (mZ/g)
`
`
`
`Quantitative determination of dissolved ions
`
`The amount of dissolved Si“, Ca2+ and P5+ from the
`glass specimen in water was quantitatively determined
`using an inductively coupled plasma [ICP] analyser
`[Shimadzu, [CPS-500). A plate—like specimen of a sample
`of glass [surface area = 2 cm2] was used. The dissolved
`ions were determined after immersing the specimen in
`300 ml of distilled water at 37°C for 50 h.
`
`RESULTS
`
`Mortality
`
`Intraperitoneal administration of calcium phosphate
`glass containing various concentrations of silica resulted
`in an increase in the mortality with increasing amounts
`of silica in the glass. The mortality was 0/10, 3/10, 9/10,
`10/10 and 10/10 at X = O, 5, 10, 15 and 40 mol of silica,
`respectively [Figure 1]. The mean time of death was 68.0,
`29.3. 19.2 and 6.0 h after treatment at x = 5, 10, 15 and
`40 mol of silica. respectively [Figure 1]. The mortality in
`mice given pure silica glass powder itself was 0/10. In
`contrast to the mortality with silica glass itself or calcium
`phosphate glass without silica, the silica in calcium
`phosphate glass decreased the mean time of death and
`increased the number of deaths within 30 days.
`
`Quantitative determination of dissolved ions
`The dissolved amount of Si“, Ca2+ and P5+ in the water
`from the calcium phosphate glasses increased with the
`increasing amount of silica in the glass (Figure 2). No
`dissolved Si“, Ca2+ or P5+ was detected by ICP after
`50 h of treatment of pure silica glass [SiOzz molar
`fraction = 1] in water.
`
`l
`
`
`
`010
`
`
`
`Mortality("/a)
`
`IOO
`
`l w
`
`24
`
`48
`
`72
`
`96
`
`a
`
`I20
`
`I44
`
`|68
`
`Time (h)
`
`Figure 1 The mortality in C57BL/6 mice given a single
`intraperitoneal dose of the glass powders. Administration of
`calcium phosphate glass containing increasing concentrations
`of silica increased the mortality depending on the amount of
`'0, 5, 10, 15 and 40 mol = calcium phosphate glasstCaO/P205 = 1)with the
`silica in the glass. (El) Omol; (4) 5mol; (A) 10 mol; (0)
`composition of 100 Ca(P0312 to x SlO2 in moles in which x = 0,5, 10, 15 or
`40 mol of slllca, and SIOZ = pure silica glass Itself.
`15 mol; (0)40 mol; (I) Sioz.
`
`
`0 mol
`5 mol
`10 mol
`15 mol
`40 mol
`Sio2
`
`5.9
`4.3
`3.9
`4.3
`8.3
`4.1
`
`3.2
`2.3
`3.0
`2.5
`3.1
`13.0
`
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`174
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`Toxicity of silica-containing glass: M. Nagase et aI.
`
`silica enters the body in any other way than by ingestion,
`some type of toxicity almost invariably results". We
`showed that a calcium phosphate glass—ceramic con-
`taining 34.2 wt% of silica was lethal in C57BL/6 and A/I
`mice“. The LD50 of the silica containing glass—ceramic
`after intraperitoneal administration in C57BL/6 mice was
`750 mg/kg '(Ref. 4). The mice died from 2 to 6 days after
`administration of the glass—ceramic“. Significant footpad
`swelling occurred for a week after challenge in mice
`challenged with the glass—ceramic“. The lethality of the
`material administered weekly did not change during 5
`weeks‘. A special kind of inflammation, not an immuno—
`logical one“, plays an important
`role in the lethal
`reaction.
`
`The present study was undertaken to examine the
`intraperitoneal
`toxicity of calcium phosphate glass
`containing various concentrations of silica in C57BL/6
`mice. Calcium phosphate glasses containing large
`amounts of silica are extremely toxic. The Si“, Ca2+ and
`P5+ in water dissolved from the calcium phosphate glass
`depends on the amount of silica in the glasses, but the
`amounts of Ca2+ and P5+ were much lower than the
`amounts normally existing in serum. The dissolved silica
`(Si’Hl in this study may be monomeric or may not be
`highly polymerized“). It is suggested that the concentra-
`tion of monomeric or low molecular silicic anions
`determines the toxicity of the materials, but the total
`content of non-dissolved silica had no relation to its
`toxicity. In determining the biological consequences of
`dissolved or monomeric silica, what kind of reaction may
`play an important role in the lethal reaction will require
`further study.
`Under these circumstances it is interesting to speculate
`on the mechanism of the biological effects of dissolved or
`monomeric silica. There have been a great many studies
`to explore the pathogenesis of silicosisu'”. But almost
`all silicosis researchers studied the biological reactions
`of various forms of solid silica, e.g. crystalline silica
`(quartz, tridymite, cristobalite, coesite and stishovite)”,
`silica powder without an organophilic hydrophobic
`monolayer of hydrocarbon groups“, silica gel14 and the
`finest amorphous silica“. Nobody has investigated the
`toxicity of monomeric or low molecular silicic anions
`dissolved from composition materials. We showed in this
`study that there was no toxicity in pure silica (SiOz) glass
`or in calcium phosphate [Ca(P05)2] glass without silica.
`Monomeric silicic anions dissolved from silica-con-
`taining glasses may induce the toxicity. The difference in
`toxicity among the various forms of solid silica may
`depend on the amounts of monomeric silica dissolved
`from the silica. The difference in the mean time of death
`[6.0—68.0 h after administration in this study, 75.9 h after
`administration in our previous study‘] may depend on the
`different rates at which monomeric silica is dissolved
`from composition materials.
`Silica is also released from ‘bioglass‘, and the dissolved
`amount depends on the glass composition". It is hoped
`that future researchers and engineers will be mindful of
`this toxicity due to silica dissolved in calcium phosphate
`glass. Note also that we have not addressed any variation
`in sensitivity to silica with respect to age, sex, diet, route
`of exposure, or treatment regimen. These factors may also
`affect the 30-day mortality in mice.
`The marked physiological response at the high doses
`
`Biomaterials 1992, Vol. 13 No. 3
`
`of silica may well be symptomatic of the detrimental
`biochemical changes that follow silica exposure, even
`long after implantation, since glasses and ceramics are
`susceptible to both general corrosion and pitting attack in
`aqueous solutions, and silica is 20 times more soluble in
`organisms than in saline solution”. Several types of
`glass-ceramic in systems with calcium phosphate without
`silica have been developed” 19. That kind of glass—
`ceramic would be expected to be much better for implant
`systems than silica—containing glass—ceramics although
`the former have some disadvantages such as the weak
`mechanical
`strength in water and low chemical
`stability.
`
`ACKNOWLEDGEMENTS
`
`to Dr Hiroyasu Takeuchi,
`The authors are grateful
`Research and Development Center, Mitsubishi Material
`Co., Ltd for his assistance in measurements of grain size
`and surface area; and Mr Kiichi Oosawa, Department of
`Orthopaedic Surgery, Gunma University School of
`Medicine for his assistance in photography. This work
`was supported in part by Grant of Iapan Sports Medicine
`Foundation Inc. No. 1.
`
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