Petitioners – Baxter Healthcare Corp., Apatech, Inc., and Apatech, Ltd., Exhibit 1014, p. 1
`
`

`

`Silicon-Doped Hydroxyapatite
`
`'71
`
`Silicon-Doped Hydroxyapatite
`
`A.J. Ruys*
`
`* Senior Research Associate
`Centre for Biomedical Engineering
`University of New South Wales
`
`Abstract
`
`The role of many ions in biological systems is not fully understood owing to difficulties in
`microdetermination. Silicon is known to be essential in many biological processes, including
`skeletal development. Some evidence indicates that silicon acts as a calcifying agent rather
`than as a resident structural species. This suggests that silicon may be used for enhancement
`of bone ingrowth rates for bioactive prosthetic materials. Of particular interest is
`hydroxyapatite Ca 10(P04MOH)2 [HAp], which has a relatively low bioactivity . The purpose
`of the present work was to determine the feasibility of chemically doping HAp with silicon.
`This route is practical because apatites are well known for undergoing extensive isomorphous
`substitution at all of the cation lattice positions.
`
`HAp was synthesised by the metathesis method using Ca3(N03h, (NH4) 2HP04 , and NHPH.
`Silicon was added by a sol-gel route using tetraethyl orthosilicate [TEOS] and ethanol.
`Silicon was added at levels up to a Si:HAp molar ratio of 50, although most work was done
`at < 2. The samples were sintered at 11 006C for 1 h in air. Characterisation consisted only
`of X-ray diffraction for semiquantitative phase analysis and lattice parameter determination.
`
`At all silicon levels, Ca 10(P04MSi04h formed. At low silicon levels, {3-Ca3(P04) 2 [{3-TCP]
`[a-TCP]. A Si-P-0 glass also
`formed, while high silicon levels favoured a-Ca3(P04h
`formed at high silicon levels. Lattice parameter measurements indicated that silicon
`dissolved in the HAp structure up to a Si:HAp molar ratio of - 0.36 .
`Ionic radii
`considerations suggest that the most likely substitution site was that of phosphorus. Charge
`compensation requires substitution of pH by Si4+ to form holes on the oH- sites. If so, then
`the saturated silicon-substituted phase would have the formula Ca 10(P 1_xSix04MOH)2_6x. where
`X = 0.06, or Ca 10(P 1_xSix04MOHh6x- in the absence of charge compensation.
`
`Consequently, it is possible to dope HAp with silicon using a sol-gel route. Si:HAp and Si:P
`molar ratios ~0 . 36:1 and ~0.06:0.94, respectively, should be used in order to avoid
`formation of biodegradable TCP.
`
`Petitioners – Baxter Healthcare Corp., Apatech, Inc., and Apatech, Ltd., Exhibit 1014, p. 2
`
`

`

`72
`
`J. Aust. Ceram. Soc., 29 [112] 71-80 (1993), A.J. Ruys
`
`INTRODUCTION
`
`Silicon and Bone Growth
`
`The role of silicon in biological systems is not yet fully understood since microdetermination
`of silicon in biological systems is very difficult [1] . However, silicon is now known to be
`essential for the growth and development of vertebrates, being involved in cell wall
`formation, cross-linking in connective tissues, nucleic acid synthesis, photosynthesis, and
`other biological processes, particularly with regard to ageing [2,3] . Silicon also has been
`found to perform a vital role in skeletal development. It is a constituent of collagen [4], and
`silicon deprivation has been found to retard bone development in chicks [5].
`
`Although the importance of silicon in bone matrix is now well established, its importance in
`bone mineral is not yet fully understood [1 ,5,6] . Dietary silicon has been found to increase
`the rate of bone calcification independent of vitamin D, and nodular ill-formed bone results
`from silicon deficiency [2]. Silicon has been localised in immature bone by both ion and
`electron microprobe studies [6, 7].
`
`Figure 1 shows an electron microprobe scan of calcium and silicon concentration across a
`bone mineralising front [6]. While phosphorous distribution is diffuse in calcifying regions
`[7], this scan revealed the calcium distribution to be preferentially localised to regions of
`mature bone, as expected, but the silicon distribution was quite the opposite [6]. The scan
`detected negligible silicon levels in the calcium-rich (mature bone) region and a maximum
`silicon level of -0.5 wt% in the calcium-deficient area- the region of active calcification.
`The figure of -0.5 wt% was a semiquantitative value since precise determination of the
`silicon level at a mineralisation site is difficult because these sites are localised and difficult
`to measure directly. A calcium level of only -2 wt% was detected in the silicon-rich
`region. This corresponded to a Ca:P ratio that was too low to form any of the known
`calcium phosphate phases, and so it was suggested by Carlisle [6] that the bone precursor
`must be an organic phase containing silicon. Silicon also was found in the metaphyseal blood
`vessels. These findings suggest that silicon is a calcifying agent rather than a resident
`structural species .
`
`As further evidence of the role of silicon as a calcification truttator or promoter, the
`nucleation and growth of bone mineral (hydroxyapatite crystals) have been found to be
`greatly enhanced on silicon-rich substrates, including Si02 [8,9], Si02-rich bioglass surfaces
`[10, 11], and polysilicic acid [8].
`
`If silicon is a calcifying agent, this offers the possibility of the use of silicon for the
`enhancement of bone ingrowth rates for bioactive prosthetic materials. There are only two
`prosthetic materials that are known to combine the desirable properties of biocompatibility,
`low to negligible resorption rates in-vivo, and the ability to bond chemically with bone.
`These are hydroxyapatite Ca10(P04M0Hh [HAp] and certain bioactive glass compositions .
`The bioactivities of these glass formulations are considerably higher than that of HAp [12] .
`Table 1 summarises the bioactivities of some of the important bioactive glasses and HAp.
`The bioactivity of these Si02-rich glass formulations has been found to be related to the
`formation of a Si02-rich surface layer, with bone-glass chemical bonding following three
`stages [10]:
`
`Petitioners – Baxter Healthcare Corp., Apatech, Inc., and Apatech, Ltd., Exhibit 1014, p. 3
`
`

`

`Silicon-Doped Hydroxyapatite
`
`73
`
`1. A silica-rich layer develops on the glass surface, with corresponding linear weight loss
`from the implant.
`2. A thin film of HAp crystals is deposited on the silica-rich layer.
`3. The rate of weight loss from the implant declines to the point of no further change.
`
`i
`~ c
`
`c
`0
`
`Q)
`0
`c
`0
`0
`c
`0
`,g
`i:i5
`
`0.6
`
`0.5
`
`0.4
`
`0.3
`
`0.2
`
`0.1
`
`0
`
`30
`
`25
`
`*
`20 1
`
`c
`0
`~
`c
`15 Q)
`0
`c
`0
`0
`E
`:I
`·o
`<ii
`0
`
`10
`
`5
`
`0
`
`Periosteum
`
`Osteoid
`
`Bone
`
`FIGURE 1. ELECTRON MICROPROBE SCANS OF SILICON AND CALCIUM ACROSS A BONE
`MINERALISING FRONT IN YOUNG RAT TmiA [6].
`
`TABLE 1. BIOACTIVITIES OF GLASS-BASED MATERIALS AND HYDROXYAPATITE [12].
`
`Bioactive Material
`
`System or Compound
`
`45S5-Bioglass
`
`KGS-Ceravital
`
`Na20-Ca0-Pz05-Si02
`Ca(P03)z-N az0-Ca0-Si02
`A-W Glass-Ceramic Ca 10(P04MO,F)z-Ca0-Mg0-Si02
`Ca 10(P04MOH}z
`Hydroxyapatite
`
`Wt% Si02 p;;]
`- 15 days
`- 45 days
`-100 days
`
`Bioac
`
`45
`
`46
`
`45
`
`--
`
`-110 days
`
`Since silicon may enhance the bioactivity of these glass-based materials, the relatively low
`bioactivity of HAp may be due to the absence of silicon from its structure. However, since
`A-W glass-ceramic has a peak bioactivity of the same scale as HAp, there is some question
`as to this suggestion. The purpose of the present work was to address the potential benefits
`and problems involved in the silicon doping of HAp, since HAp has the advantage over
`bioactive glasses and glass-ceramics of being chemically similar to bone mineral.
`
`Petitioners – Baxter Healthcare Corp., Apatech, Inc., and Apatech, Ltd., Exhibit 1014, p. 4
`
`

`

`74
`
`J. A ust. Ceram. Soc., 29 [112] 71-80 (1993), A.J. Ruys
`
`Silicon in Hydroxyapatite
`
`It is well known that the bone ingrowth capacity of HAp can be enhanced only by increasing
`the porosity, but the low strength of porous HAp limits its use to monolithic implants in
`nonload-bearing sites or bioactive coatings. If silicon is a bone calcifying agent [6], it may
`be possible 'to enhance the bone ingrowth rate of dense HAp by silicon impregnation, thereby
`increasing the bioactivity without compromising strength. Silicon doping of HAp is known
`to occur in geological systems since apatites are capable of undergoing extensive isomorphous
`substitution [13]. The existence of naturally occurring calcium silicophosphate apatite
`minerals has been documented in various studies.
`
`Apatites have a hexagonal crystal structure [14] . Molecular models of fluorapatite are shown
`in Figures 2 and 3. The fluorapatite structure represents the generalised apatite formula
`range [13]:
`
`In the case of HAp, with the formula Ca10(P04MOH)2, calcium, phosphorous, and hydroxyl
`groups occupy the A, X, and Z sites, respectively. Isomorphous substitution at each of these
`sites is governed by the upper and lower ionic radii limits, as listed by Cockbain [13]:
`
`A site
`X site
`Z site
`
`0.069 - 0.174 nm
`0.026 - 0.056 nm
`0.131 -0.216 nm
`
`The Z sites are large channels parallel to the c axis, analogous to the channels in a zeolite,
`so they can be vacant. Thus, the occupants of the Z site are weakly held, and many apatites,
`such as tricalcium phosphate ,6-Ca3(P04h [,6-TCP], have empty Z sites [13].
`
`The present work involved the development of techniques for the doping of HAp by silicon
`and subsequent investigation of the effects of silicon on the crystal structure of HAp, with
`the aim of establishing whether or not such a material would be suited to clinical trials.
`
`Methods and Materials
`
`HAp Synthesis
`
`The HAp used in the present work was synthesised by the metathesis method of Jarcho et al.
`[1 5]. The manufacturer's specifications of the Ca3(N03) 2 , (NH4hHP04 , and NH40H used
`in the synthesis are compiled in Table 2. The Ca3(P04) 2 precipitate was simultaneously
`stirred and boiled. The stir/boil method was necessary in order to eliminate TCP from the
`calcined product. This could not be achieved reliably with cold-stirring for the recommended
`time of 24 h nor for longer periods up to 48 h. The resulting precipitates of - 20 nm
`diameter (assumed size [15]) HAp crystallites were washed twice to remove the NH4N03 by
`filtering through a Buchner funnel and resuspending in demineralised water by means of
`high-speed stirring. After the second washing, the wet filter cake was resuspended in ethanol
`in preparation for the silicon addition process. The filter cake was not allowed to dry
`between the final washing stage and resuspension in ethanol in order to prevent aggregation.
`
`Petitioners – Baxter Healthcare Corp., Apatech, Inc., and Apatech, Ltd., Exhibit 1014, p. 5
`
`

`

`Silicon-Doped Hydroxyapatite
`
`75
`
`FIGURE 2.
`
`FLUORAPATITE STRUCfURE WITH C AxiS ORIENTED HORIZONTALLY WITHIN
`PAGE (BLACK = CA, WHITE = P, GREY = 0) [11].
`
`FIGURE 3.
`
`FLUORAPATITE STRUCfURE WITH C AxiS ORIENTED NORMAL TO PAGE
`(BLACK = CA, WHITE = P, GREY = 0) [11].
`
`Petitioners – Baxter Healthcare Corp., Apatech, Inc., and Apatech, Ltd., Exhibit 1014, p. 6
`
`

`

`76
`
`J. Aust. Ceram. Soc., 29 [112] 71-80 (1993}, A.J. Ruys
`
`TABLE 2. RAW MATERIALS.
`
`II
`
`' I
`
`. I
`
`CaiN03h
`(NH4) 2HP04
`NH40H
`(C2H5) 4Si04
`C2H50H
`Al
`
`*Minimum
`
`Purity
`
`98.5 wt%
`
`98.0 wt%
`
`99.4 wt%
`
`Supplier
`
`II
`
`Ajax Chemicals
`
`Ajax Chemicals
`
`Ajax Chemicals
`
`99.0 wt%*
`
`Union Carbide
`
`95.8 vol%
`
`CSR Chemical
`
`99.5 wt%
`
`Cerac Inc.
`
`Silicon Additions
`
`A sol-gel method was used to dope the HAp with silicon. This method utilised tetraethyl
`orthosilicate (TEOS), which decomposes into ethanol and colloidal silica in the presence of
`water in accordance with the reaction:
`
`A solution of TEOS was added in measured amounts by burette to the HAp/ethanol
`suspension, and the suspension was subjected to high-speed stirring for 10 min. Excess
`water then was added to hydrolyse the ethyl silicate, followed by another high-speed stirring
`for 10 min. After this, the ethanol was removed by evaporation. The resulting filter cake
`then was crushed and pelletised at 200 MPa into 12.5 mm 0 x 2.5 mm thickness samples.
`These pellets were sintered at 1100°C for 1 h in air, using a heating rate of 60°C/h.
`
`Silicon addition levels were defined as atoms of silicon per HAp unit cell, which is the
`Si:HAp molar ratio:
`
`Si:HAp = ___ M_o_le_s_S_i_0..:..2 __
`Moles Ca 10(P04MOH)2
`The silicon addition level was varied from 0 to 50, with the majority of the samples being
`below 2.
`
`Sample Characterisation
`
`For each sample, the semiquantitative phase composition and the lattice parameters of the
`HAp phase of the samples were measured by X-ray powder diffraction (Philips, Type PW
`1140/00 powder diffractometer) using CuKa radiation. Powdered aluminium was used as
`an internal standard for the lattice parameter measurements. The lattice parameters were
`calculated using the computer program PARA 1 [16], which uses the sin2/cos2 extrapolation
`model for the hexagonal crystal system.
`
`Petitioners – Baxter Healthcare Corp., Apatech, Inc., and Apatech, Ltd., Exhibit 1014, p. 7
`
`

`

`Silicon-Doped Hydroxyapatite
`
`7'7
`
`Results and Discussion
`
`Phase Analysis
`
`The X-ray diffraction scans revealed that the HAp gradually decomposed to tricalcium
`phosphate Ca3(P04)z in the presence of increasing silicon concentrations according to the
`reaction:
`
`Previous studies have shown that Si02 can induce the HAp~ TCP decomposition [17-19],
`although none has addressed the case of silicon as the sole additive in an air atmosphere.
`These studies have investigated the presence of three additives - Si02, Al20 3 , and C in
`combination- in air [19] or under hydrothermal conditions [17, 18]. TCP is an undesirable
`phase since it is biodegradable in-vivo [20].
`
`In the present work, both a- and /1-TCP were formed, although /1-TCP was favoured at low
`silicon levels and a-TCP was favoured at higher silicon levels. Further, at higher silicon
`concentrations, a broad X-ray diffraction peak with ad spacing of 0.16-0.26 nm formed.
`Since both silicon and phosphorus are oxide glass f9rmers, this peak is likely to result from
`the presence of a Si-P-0 glass. For progressively higher silicon levels, the glass became the
`dominant phase. At very high dopant levels, approximate area ratios of the main diffraction
`peaks of HAp and TCP suggested that the TCP content was slightly greater than the HAp
`content.
`
`Traces of the calcium silicophosphate phase Ca10(P04MSi04)z appeared at the lowest silicon
`addition level, and the amount of this phase increased with increasing silicon levels up to the
`saturation Si:HAp molar ratio of -0.36. At this point, the main diffraction peak area ratios
`indicated that the concentration of this phase was similar to that of HAp. This ratio
`underwent little change at higher silicon addition levels. The appearance of this phase
`corresponds to the reaction:
`
`A recent EDS study of SiOiHAp mixtures has confirmed that the dissolution of silicon in
`HAp occurs at a temperature of 1000°C or greater [21].
`
`Variation in the lattice parameters of the HAp with respect to silicon content was measured
`up to a Si:HAp substitution ratio of 6.94. Beyond this, the HAp diffraction peaks were too
`depleted by the excessive silicon levels to be measured with any degree of certainty.
`However, the silicon saturation level, after which the increase in lattice parameters levelled
`off to a large degree, was graphically estimated to occur at a Si:HAp ratio of -0.36. Over
`the range 0-0.36, the following linear lattice expansions were observed:
`
`a = 0.939 to 0.955 nm
`c = 0.686 to 0.703 nm
`
`The data for the corresponding to the unit cell volume expansion are shown in Figure 4.
`
`Petitioners – Baxter Healthcare Corp., Apatech, Inc., and Apatech, Ltd., Exhibit 1014, p. 8
`
`

`

`78
`
`J. Aust. Ceram. Soc., 29 [112] 71-80 (1993), A.J. Ruys
`
`0.56
`
`c:>
`E .s
`Q) 0.55
`E
`:::J
`0 >
`Qi 0.54
`()
`:t=
`c
`::J
`
`0.53
`
`2
`
`5
`4
`3
`Si Atoms per HAp Unit Cell
`
`6 '
`
`7
`
`8
`
`FIGURE 4. UNIT CELL VOLUME EXPANSION OF HAP OWING TO SILICON DISSOLUTION.
`
`The lattice parameters of the undoped HAp measured in the present work are in good
`agreement with the published literature values, which are a = 0.941 nm [14] or 0.942 nm
`[22]; c = 0.688 nm [14,22] .
`
`The HAp lattice underwent silicon substitution to a saturation level of -0.36 atoms per unit
`cell. The substitution site in the A 10(X04)ch apatite structure probably was the X site since
`this site accepts ions within the radius range 0.026-0.056 nm in fourfold coordination [13].
`The ionic radius of ps+, the original ion in the X site, is 0.031 nm in fourfold coordination,
`while Si4+ is 0.040 nm in fourfold coordination [23].
`
`The effect on the lattice parameters of substituting a larger ion into the X site would be an
`increase in the a and c axes, with oa/lk = 2 [24] .
`In the present work, oa!oc was
`significantly lower at - 1. This probably was due to hole formation and partial loss of oR(cid:173)
`ions from the Z sites owing to charge compensation requirements. This would serve to
`reduce the unit cell volume. The effect on the lattice parameters of depletion of the Z sites
`would be the opposite of the effect of substitution of larger ions on the Z sites, as reported
`by Simpson [25].
`In the former case, there would be a large decrease in a and a small
`increase in c.
`Combination of the effects of expansion in the X site, giving oa!oc = 2, and contraction in
`the Z site, giving a large and negative oa but a small and positive oc, should result in
`significant oa and oc values. However, the oa!oc value 'would be very difficult to predict
`reliably. Thus, the present finding of oa!oc = 0.8 does not confirm the literature value [24],
`nor does it contradict it.
`
`Petitioners – Baxter Healthcare Corp., Apatech, Inc., and Apatech, Ltd., Exhibit 1014, p. 9
`
`

`

`Silicon-Doped Hydroxyapatite
`
`79
`
`The reasons for the relatively low saturation level of silicon in HAp of - 0.36, probably
`stem from the lattice destabilising effect of the charge imbalance induced by replacing
`pentavalent p5+ with tetravalent Si4+. This charge imbalance was probably compensated by
`the loss of the weakly held OH- ions in the Z site. If a sufficient number OH- ions are lost,
`structural collapse and the concomitant HAp ~ TCP transition can result. The Si:HAp
`substitution limit of - 0. 36 may have been a kinetic effect resulting from the relatively short
`heat treatment time of 1 h, resulting in the retention of the phase
`Ca 10(P0.94Sio.060 4MOH) 164 [Ca 10(P 1.xSix04MOH)2.6x, where X = 0.06], in the case of charge
`compensation. If no charge compensation by oH- loss occurred, then the phase could be
`described as Ca 10(P 1.xSix04MOH)z6x-, where X = 0.06. The limit of - 0.36 also may have
`been a result of structural stability limitations.
`
`Conclusions
`
`Doping of HAp by silicon using a sol-gel process resulted in isomorphous substitution of
`silicon, which probably entered the phosphorous sites in HAp, producing four products:
`
`Calcium Silicophosphate:
`
`Tricalcium Phosphate:
`
`Silicon-Doped HAp:
`
`Ca3(P04) 2 (significant for Si:HAp > 0.36)
`a-Ca3(P04) 2 at higher Si levels
`(1-Ca3(P04)z at lower Si levels
`
`Ca 10(P 1.xSix0 4MOH)2•6x or
`6x-, where X = 0.06
`Ca 10(P 1.xSix0 4MOH)2
`
`Si-P-0 Glass:
`
`Present at higher Si levels
`
`Therefore, it has been found to be feasible to dope HAp by silicon using a sol-gel route,
`thereby allowing the future assessment of the effects of silicon on the bioactivity of HAp
`through clinical trials . However, only low dopant levels should be used in order to maintain
`the TCP content to a minimum and so eliminate the possibility of biodegradability in-vivo .
`These molar ratio levels are Si:HAp = 0.36:1 and Si:P = 0.06 :0 .94.
`
`Acknowledgements
`
`The author gratefully acknowledges the assistance of Dr B.J. Baggaley in the quantitative
`X-ray diffraction analyses and the late Assoc . Prof. E.R. McCartney for helpful discussions .
`
`References
`
`[1] E.M. Carlisle, Nutr. Rev ., 40 (1982) 193 .
`[2] E.M. Carlisle, Calc if. Tissue Int., 33 (1981) 27.
`[3] F.H . Nielsen, Ann. Rev . Nutr., 4 (1984) 21.
`[4] R.E. Olson and A.A. Doisy, Nutr. Rev ., 38 (1980) 194.
`[5] E.M. Carlisle, J. Nutr., 110 (1980) 352.
`
`Petitioners – Baxter Healthcare Corp., Apatech, Inc., and Apatech, Ltd., Exhibit 1014, p. 10
`
`

`

`80
`
`J. Aust. Ceram. Soc., 29 [112] 71-80 (1993), A.J. Ruys
`
`[6] E.M. Carlisle, p. 69 in Silicon and Siliceous Structures in Biological Systems . Eds
`T.L. Simpson and B.E. Volcani. Springer-Verlag, New York, 1981.
`[7] W.J. Landis, D.D. Lee, J.T. Brenna, S. Chandra, and G.H. Morrison, Calcif. Tissue
`Int., 38 (1986) 52.
`[8] J.J. Damen and J.M. Ten Cate, J. Dent. Res., 68 (1989) 1355 .
`[9] J.J. Damen and J.M. Ten Cate, J. Dent. Res., 71 (1992) 453 .
`[10] P. Li and F. Zhang, Boli Yu Tangci, 16 (1988) 8.
`[11] R. Li, Diss. Abs. Int., B52 (1992) 233.
`[12] L.L. Hench, J. Amer. Ceram. Soc., 74 (1991) 1487.
`[13] A.G. Cockbain, Miner. Mag., 36 (1968) 654.
`[14] C.A. Beevers and D.B. Mcintyre, Miner. Mag., 27 (1945) 254.
`[15] M. Jarcho, C.H. Bolen, M.B. Thomas, J. Bobick, J.F. Kay , and R.H. Doremus, J.
`Mater. Sci., 11 (1976) 2027.
`[16] B.K. Damkroger, University of New South Wales, School of Materials Science and
`Engineering, Private Communication.
`[17] M.A. Veiderma, Tr. Tallinsk. Politekhn. Inst., Ser. A, 228 (1965) 41.
`[18] H. Monma and T. Kanazawa, Bull. Chern. Soc. Japan, 48 (1975) 1816.
`[19] E.E. Pomoshchnikov, G.l. Gordeeva, and Y.P. Nikolskaya, Sib . Chern. J., [6] (1973)
`109.
`[20] S.R. Radin and P. Ducheyne, J. Mater. Sci. Mater. Med., 3 ·~(1992) 33.
`[21] K.A. Zeigler, A.J. Ruys, C.C. Sorrell and B.K. Milthorpe, p. 623 in Ceramics:
`Adding the Value, Volume 2. Ed. M.J. Bannister. CSIRO, Melbourne, 1992.
`[22] K. Ioku, Y. Masahiro, and S. Somiya, p. 1308 in Sintering '87, Volume 2. Eds S.
`Sorniya, M. Shimada, M. Yoshimura, and R. Watanabe. Elsevier, London, 1988.
`[23] 0 . Muller and R. Roy, The Major Ternary Structural Families. Springer-Verlag,
`Berlin, 1974.
`[24] D. McConnell, Amer. Mineralog., 22 (1937) 977.
`[25] D.R. Simpson, 53 (1968) 432.
`
`Petitioners – Baxter Healthcare Corp., Apatech, Inc., and Apatech, Ltd., Exhibit 1014, p. 11
`
`