# Patent

## Title
Radiopaque embolic particles

## Abstract

A radiopaque particulate material one or more of SiO2, TiO2, La2O3, Na2O and MgO and useful for embolization which optionally includes therapeutic components that are released in vivo.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the 35 USC §371 national stage entry of PCT/IB2012/000341 and claims the benefit of U.S. Provisional Application 61/437,566 filed on Jan. 28, 2011, which is hereby incorporated by reference in its entirety for all purposes.


FIELD OF THE INVENTION
The invention relates to particulate material that is radiopaque and suited for performing embolization.
BACKGROUND
Polymeric particles are frequently employed by interventional radiologists for the selective occlusion of blood vessels in the treatment of (but not limited to) hypervascular tumors such as leiomyoma uteri and vascular anomalies such as vascular malformations. However, current state of the art particles are beset with numerous drawbacks which limit market penetration for such products. The drawbacks include that the particles are not radiopaque. Thus, the clinician is not able to monitor placement of the particles radiographically. The result is that complications of embolization, such as reflux with non-target embolization and through embolization are not detectable. Current approaches to this problem include dispersing the particles in a medium that includes a contrast agent that is visible radiographically. This leads to its own complications however. Contrast-induced nephropathy occurs in about seven percent of patients exposed to contrast agents. Contrast-induced nephropathy is characterized by acute renal injury which can lead to renal failure. Exposure to contrast agents is the third most common cause of hospital acquired renal failure. Additionally, the degradation of the particles currently in use cannot be controlled. The ability to choose particles that are either non-degradable or that have differing degrees of degradability allows for more treatment options for the various lesions that are amenable to embolization. Use of particles that degrade with time may allow for revascularization of the occluded blood vessel after the treated lesion is gone resulting in recovery of blood flow to normal tissue. This would be desired in instances such as the treatment of gastrointestinal hemorrhage. In other instances, use of a non-degradable particle prevents revascularization of the underlying vessel. This would be desired in vascular lesions such as vascular malformations.
Thus, what are needed are improved embolic particles that limit the addition of a contrast agent for radiographic imaging and whose degradation may be controlled.
SUMMARY
A particulate material comprising one or more of TiO2, La2O3, Na2O and MgO or SrO is provided. In one embodiment, the particulate material comprises: 0.4-0.7 mole fraction SiO2; 0.04-0.7 mole fraction TiO2; 0.04-0.5 mole fraction La2O3; 0.03-0.3 mole fraction MgO; and 0.03-0.3 mole fraction Na2O. In one embodiment, MgO is present at 0.05 to 0.2 mole fraction. The particulate material may optionally comprise SrO. When SrO is included, the MgO and the SrO together can be present at 0.05-0.3 mole fraction. Alternatively, the MgO and the SrO together are present at 0.05-0.2 mole fraction. The La2O3 can be present at 0.04 to 0.4 mole fraction or 0.04 to 0.3 mole fraction. The TiO2 may be present at 0.04 to 0.3 mole fraction or 0.04 to 0.2 mole fraction. The SiO2 may be present at 0.4-0.6 mole fraction or 0.4-0.5 mole fraction. The Na2O is present at 0.03-0.2 mole fraction or 0.03-0.15 mole fraction.
In some aspects, the particulate material is radiopaque.
In some aspects, the particulate material is biocompatible.
The particulate material is optionally degradable in vivo. In one embodiment the particulate material degrades substantially in more than six months. Alternatively the particles are non-resorbable.
In some aspects, the particulate material releases therapeutic components under physiological conditions.
In some aspects, the particulate material has a Q-speciation of Q1-Q3 or about Q2.
In some aspects, the particles of the particulate material have an average diameter of 45-1180 μm, 200-1000 μm, 100-300 μm, 300-500 μm, 500-710 μm or 710-1000 μm.
In some aspects, the particulate material comprises no more than 0.1 mole fraction aluminosilicates, phosphates or a combination thereof.
The particulate material optionally includes a polymeric coating or is dispersed within a polymeric matrix. In one embodiment, the polymer comprises poly(lactic-co-glycolic acid). Additionally or alternatively, the polymer comprises a poloxamer. In one embodiment the poloxamer is Pluronic F127. The polymer optionally comprises therapeutic components which are released under physiological conditions.
In one aspect, the particulate material of any of the previous claims is used for vascular occlusion. The vascular occlusion may be for treatment of vascular malformations, organ ablation, chemoembolization, treatment of hemorrhage or uterine fibroid embolization.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a particle according to one embodiment.
FIG. 2 are DSC traces for eight particles.
FIG. 3 are XRD patterns for synthesized exemplary particles.
FIG. 4 are contour plots for synthesized exemplary particles.
FIG. 5 morphological data (SEM) for ORP5 and control sample from ContourTM.
FIG. 6 illustrates chemical (EDX) data for ORP5.
FIG. 7 illustrates 29Si MAS-NMR spectra for experimental embolic materials.
FIG. 8 illustrates 29Si MAS-NMR spectra for experimental embolic material ORP6.
FIG. 9 illustrate contour plots based on the 29Si MAS-NMR spectra of FIG. 7.
FIG. 10 illustrate additional contour plots based on the 29Si MAS-NMR spectra of FIG. 7.
FIG. 11 presents ion release profiles for Si (FIG. 11A) and Na (FIG. 11B) release of the eight embolic particulates.
FIG. 12 presents ion release profiles for Ca (FIG. 12A) and Sr (FIG. 12B) release of the eight embolic particulates.
FIG. 13 presents ion release profiles for Ti (FIG. 13A) and La (FIG. 13B) release of the eight embolic particulates.
FIG. 14 presents an ion release profile for Mg (FIG. 14A) and mean (±SD) release levels for Zn (FIG. 14B) of the eight embolic particulates.
FIG. 15 presents the effect of embolic material composition on release levels for Si (FIG. 15A) and Na (FIG. 15B) after 120 h incubation periods.
FIG. 16 presents the effect of embolic material composition on release levels for Ca (FIG. 16A) and Sr (FIG. 16B) after 120 h incubation periods.
FIG. 17 presents the effect of embolic material composition on release levels for Ti (FIG. 17A); La (FIG. 17B) and Mg (FIG. 17C) after 120 h incubation periods.
FIG. 18 presents cell viability and cell toxicity data.
FIG. 19 presents contour plots' showing the compositional-interaction effects on the experimental embolic materials cell viability (%) at four various additions of Ti.
FIG. 20 presents a comparison of radiopacity for ORP vs. PVA in various dilutions of saline/contrast agent.
FIG. 21 presents histological data, derived from one animal in each group, with respect to the performance of ORP5 and Contour.
FIG. 22 illustrates the intracutaneous irritation test design of NZW Rabbits.
FIG. 23 are images of the intradermal injection sites for the three NZW rabbits after 24, 48 and 72 h observations.
FIG. 24 illustrates changes in Fg conformation monitored by far-ultraviolet circular dichroism (CD) spectroscopy as a function of the ionic dissolution products with respect to time.
FIG. 25 illustrates results for platelet LDH assay (ORP5 vs. PVA) with (FIG. 25A) and without (FIG. 25B) outlier.



DETAILED DESCRIPTION
The particles of the particulate material comprise a core and an optional compressible shell/matrix. The core comprises one or more of SiO2, La2O3, Na2O, MgO and SrO. The optional compressible shell/matrix comprises a biocompatible polymer. Additional provided particulate materials have a core comprising one or more of SiO2, La2O3, TiO2, ZnO, MgO, Na2O, SrO, and CaO. In some embodiments, the particulate material is substantially free of phosphates and aluminosilicates. In some embodiments, the particulate material comprises no more than 0.1 mole fraction aluminosilicates, phosphates or a combination thereof. Surprisingly, the particulate materials are biocompatible and yet do not require phosphates or aluminosilicates.
Core
In one embodiment, the particulate material has the properties of a glass ceramic. In such an embodiment, the components make up a network, which can be amorphous or crystalline. Components include SiO2, La2O3, TiO2, ZnO, MgO, Na2O, SrO, and CaO. Modifying the amounts of the various core components (as well as the ratios of components to each other) allows for tuning the characteristics of the material to its intended use.
Network Components
La2O3 is present at a mole fraction of 0.04 to 0.5. In another embodiment, La2O3 is present at 0.04 to 0.4 mole fraction. In another embodiment, La2O3 is present at 0.04 to 0.3 mole fraction. La2O3 provides radiopacity to the particulate material. Lanthanum has been used for decades in dental applications as a radiopacifiying agent in glass ceramics and dental composites. The La2O3 provides superior radiopacity over current state of the art embolic agents. The release of La3+ as the material degrades has the additional potential benefit of providing therapeutic benefit. The La3+, for example, facilitates anoikis of certain cancers and is thus beneficial when the intended use of the particulate material is to initiate anoikis of those cancers. For those uses the amount of lanthanum is tuned for that purpose.
Another component of the particulate material is titanium. TiO2 is present in 0.04 to 0.7 mole fraction. In some embodiments, TiO2 is present at 0.04 to 0.3 mole fraction. In some embodiments, TiO2 is present at 0.04-0.2 mole fraction. Titanium is thrombogenic and its inclusion in the particulate material increases thrombogenicity around the particles in vivo. The thrombogenicity of the particulate material may be tuned by the amount of TiO2 present. Depending on the intended application, a greater or lesser amount of thrombogenicity is required.
A third component of the particulate materials is silicon. SiO2 is present in the particulate material at 0.4-0.7 mole fraction. In one embodiment, the SiO2 is present at 0.4-0.6 mole fraction. In yet another embodiment, the SiO2 is present at 0.4-0.5 mole fraction.
Magnesium is present in the particulate material in the form of MgO at a mole fraction of 0.03 to 0.3. In one embodiment, the MgO is present at 0.03 to 0.2 mole fraction. In alternative embodiments, MgO is present at 0.05 to 0.3 pr 0.05 to 0.2 mole fraction. Magnesium is a thrombogenic material and its inclusion in the particulate material increases thrombogenicity around the particles in vivo. Further, magnesium, a metallic element, enhances the imaging qualities of the particulate material and also provides additional control over the structure of the particulate material. In some embodiments, a second alkaline earth metal oxide is also used, SrO. The ratio of MgO to SrO allows tuning the biocompatibility of the particulate material as needed for different applications. For example, increased MgO results in a less stable glass network which would result in faster degradation which is desirable for some applications. Increasing SrO increases radiopacity and so the ratio of MgO to SrO also allows for tuning of this functional aspect of the particulate material.
Sodium imparts degradability to the particulate material. The more sodium present in the particulate material, the more quickly the particulate material degrades in vivo. Na2O is present in the particulate material at 0.03-0.3 mole fraction. In some embodiments, Na2O is present in the particulate material at 0.03-0.2 mole fraction. In yet other embodiments, Na2O is present in the particulate material at 0.03-0.15 mole fraction. In one embodiment, the particles take more than six months to degrade. Particles with this rate of degradation are useful when early recanalization of vessels is undesirable. Examples of such situations include organ ablation (i.e. embolization of native kidneys that are causing hypertension in a dialysis patient or a patient with a kidney transplant) or malignant tumor embolization (i.e hormonally active metastatic disease to the liver such as a carcinoid tumor, renal cell carcinoma, etc).
Additional Components
Additional components having therapeutic properties can be added to the particulate material. In one embodiment such therapeutic components release as a function of in vivo surface modifications to the particle. These components are then released from the particle in the body. The make-up of the network-modifying components is used to control the release of the therapeutic components. Examples of such components include therapeutic ions and chemotherapeutic agents. Therapeutic ions include, for example, Ca2+, which aids in coagulation. In one embodiment, calcium is provided in the particle in the form of CaO.
Nature of Core/Particle
In one embodiment, the particulate material is a ceramic. Ceramics are inorganic, non-metallic materials prepared by heating and subsequent cooling. Ceramics are usually formed between metallic and nonmetallic elements, such as, for example, aluminum and oxygen, calcium and oxygen and silicon and nitrogen. Ceramics can have crystalline, partly crystalline or amorphous structures and include ceramic glasses, also referred to as glasses. Other ceramics include enamels, glass-ceramics (glasses containing ceramic crystals), and inorganic cement-type materials (cement, plaster and lime). In one embodiment, the particulate material is a glass. Glass is any solid with a non-crystalline, or amorphous, structure and exhibits a glass transition upon heating.
Q-speciation is known in the art as a system for characterizing glass materials. It is a measure of how many of the oxygens surrounding silicon are bridging oxygens. The superscript, n, in Qn is higher the more bridging oxygens there are in the tetrahedral units of the network. The superscript n can be from 0 to 4. In one embodiment, the particulate material of the invention has a Q-speciation between Q1 and Q3 with a corresponding network connectivity of between 1 and 3. In one embodiment, the particulate material has a Q-speciation of Q2.
Compressible Shell/Matrix
FIG. 1 illustrates a particle according to one embodiment. The particle includes the core 101 described above and also a compressible shell/matrix 103 of a biocompatible polymer. Such an embodiment is illustrated in FIG. 1. As mentioned previously, the core can also be suspended in a polymer matrix. Possible polymers include poly(lactic-co-glycolic acids) (“PLGA”) and poloxamers. In one embodiment, the shell/matrix includes Pluronic F127 available from BASF. This shell/matrix allows for drug elution for therapeutic benefit. Examples include chemoembolization of malignant tumors. Specific examples include hepatocellular carcinoma of the liver. Additional drugs that can be eluted include, for example, thrombogenic agents that enhance the degree of vascular occlusion. The compressibility of the shell/matrix is useful in clinical deployment of the particulate material.
Size of Particulate Material
The individual particles of the particulate materials are between 45 and 1180 μm in average diameter. Alternatively the particles are between 200 and 1000 μm in average diameter. Various uses for the particles require different sizes of particles. Thus, in one embodiment, the particles have an average diameter of 100-300 μm which are useful for deep tumor necrosis as may be needed in malignant tumor embolization. In another embodiment, the particles have an average diameter of 300-500 μm and are used for treating gastrointestinal or traumatic bleeding. The 300-500 μm particles can also be used as the initial size of particle for uterine fibroid embolization and most other organ or tumor embolizations. In another alternative embodiment, the particles have an average diameter of 500-710 μm. The 500-710 μm particles are also used in embolization of uterine fibroids. Which size range, 300-500 μm or 500-710 μm, to use for embolization of uterine fibroids depends on other characteristics of the particle. In yet another alternative, the particles have an average diameter of 710-1000 μm and are useful for proximal embolization to reduce the “head pressure” to a site of hemorrhage such as in gastrointestinal hemorrhage or traumatic hemorrhage.
Additional Uses of Particulate Material
The particulate material of the invention may be used for vascular occlusion. In one embodiment, vascular occlusion is used in the treatment of uterine fibroids, vascular malformations and other vascular anomalies or lesions, organ ablation including portal vein embolization, chemoembolization and hemorrhage from any cause.
EXAMPLES
Synthesis of Glass Particulate Materials
Generally, appropriate amounts of analytical grade reagents are weighed out as appropriate and thoroughly mixed by shaking (30 mins) in a plastic container. Each batch of powder is fired at the appropriate temperature (initially 1520° C. for 1 hour) in a platinum crucible. The glass melt is then shock quenched into water. The resulting glass frit is dried in an oven (120° C., 1 day), ground and sieved to retrieve glass powder for subsequent analysis. Tables 1 and 2 show example compositions for particulate materials. Table 2 as appended at the end of the specification.







TABLE 1







13 glass compositions (mol. fraction) formulated using design


of experiments. The Network Modifying (NM) components (Ca;


Mg; Sr and Na) are kept constant at 0.035 mol. fraction.

















Embolic

Design










Designation
NC
pointsa
SiO2
CaO
ZnO
MgO
La2O3
SrO
Ti2O
Na2O





ORP1
2
V
0.553
0.035
0.137
0.035
0.137
0.035
0.033
0.035


ORP2
2
V
0.570
0.035
0.240
0.035
0.000
0.035
0.050
0.035


ORP3
2
V
0.537
0.035
0.213
0.035
0.068
0.035
0.042
0.035


ORP4
2
V
0.570
0.035
0.000
0.035
0.240
0.035
0.050
0.035


ORP5
2
V
0.562
0.035
0.188
0.035
0.068
0.035
0.042
0.035


ORP6
2
V
0.562
0.035
0.068
0.035
0.188
0.035
0.042
0.035


ORP7
2
A-CB
0.562
0.035
0.213
0.035
0.068
0.035
0.017
0.035


ORP8
2
A-CB
0.570
0.035
0.000
0.035
0.290
0.035
0.000
0.035


ORP9
2
A-CB
0.520
0.035
0.290
0.035
0.000
0.035
0.050
0.035


ORP10
2
A-CB
0.520
0.035
0.000
0.035
0.290
0.035
0.050
0.035


ORP11
2
A-CB
0.570
0.035
0.290
0.035
0.000
0.035
0.000
0.035


ORP12
2
A-CB
0.562
0.035
0.068
0.035
0.213
0.035
0.017
0.035


ORP13
2
C
0.537
0.035
0.068
0.035
0.213
0.035
0.042
0.035









The optional compressible shell/matrix are added by a modified emulsification method. PLGA of various molecular weights are supplemented with glass particles at various wt %. PLGA are dissolved in methylene chloride, and then the glass will be added, at the preferred level, into the solution. The mixture is then added, drop-wise, into a stirred PVA solution. Micro-spheres are isolated by filtration, washed with deionized water air dried then vacuum dried. Table 3 provides the make-up of example particulate materials with a polymer coating.







TABLE 3







9 composite variations (wt %.) formulated using design of experiments.


The glass is expressed as a wt. % of the PLGA base.












Std.
Run.
PLGA
Glass
















ORP2
1
25.00
5.00



ORP8
2
25.00
60.00



ORP1
3
15.00
5.00



ORP9
4
25.00
60.00



ORP7
5
10.00
60.00



ORP6
6
10.00
60.00



ORP4
7
10.00
32.50



ORP3
8
25.0
5.00



ORP5
9
15.0
41.67










Example 1
Particle Compositions
Particle compositions (mol. fraction) (0.52-0.57)SiO2-0.035CaO-(0.00-0.29)-ZnO-0.035MgO-(0.00-0.188)La2O3-0.035SrO-(0.00-0.05)TiO2-0.035Na2O were synthesized for this work. Analytical grade reagents: silicon dioxide, calcium carbonate, zinc oxide, magnesium oxide, lanthanum (III) oxide, strontium carbonate, titanium dioxide and sodium carbonate (Sigma Aldrich, Canada) were weighed and homogeneously mixed in a plastic container (NalgeneTM, Sigma Aldrich, Canada) for 1 hour. Each batch of powder was placed in platinum crucibles (50 mL), then fired (1480° C., 1 hour) using a Bench-Top High Temperature Muffle Furnace (EQ-KSL, MTI Corporation. USA) and shock quenched into water. The resulting frit was dried in an oven (120° C., 1 day), pulverized in an agate planetary mill (Pulverisette 7; Laval Labs Inc., Canada) and sieved through various aperture to produce powder particulates in the following size ranges: <45 μm; 45-212 μm; 212-300 μm; 355-500 μm; >500 μm. All batches of particles produced in this work were subsequently stored in dry dessicators for further evaluation.
Characterization of Particulate Materials
Differential Scanning Calorimetry (DSC)
Generally, DSC is used to determine the onset of the glass transition temperature (Tg) for each glass using a differential scanning calorimeter (DSC). Temperature intervals of 258.15° C. (up to 725° C.) are employed in an air atmosphere with alumina (or other appropriate reference) in a matched platinum crucible. The tolerance of the DSC used is on the order of 2%.
Example 2
DSC analysis of the particles made in Example 1 was performed to obtain the values of the glass transition temperature (Tg). Samples ( ̃80 mg) were heated in a platinum crucible in a nitrogen environment alongside an empty reference crucible. A standard reference material is already pre-selected for a given temperature range. Tg was measured in a differential scanning calorimeter (DSC; TA Instruments-DQ200) between 25° C. and 725° C. at temperature intervals of 258.15° C. DSC traces for the eight particles (ORP1-3, ORP5-7, ORP9 and ORP11) are shown in FIG. 2. Table 4 is an abstracted ANOVA table for the Tg reduced linear mixture polynomial model. A tabulated comparison between observed and calculated Tg behaviors for each material composition, is provided for in Table 5, as based on the regression model developed in terms of L-Pseudo component coding. The final mathematical model in terms of L-Pseudo components is shown as follows in Equation 1:

Tg(° C.)=+634.82ZnO+833.28La2O3+677.195SiO2+699.01TiO2  Eqn. 1

















TABLE 4





Response











(sig./non


F
Prob. > F




Adq.


sig.)
SD
Mean
Value
Model
R2
Adj-R2
Pred-R2
CV %
Prec.







Tg (° C.)
5.05
662.27
31.54
0.0309
0.9793
0.9482
0.7089
0.76
10.728


significant
















TABLE 5







Residuals of the Tg study.













Embolic








Composition
ORP2
ORP3
ORP5
ORP7
ORP9
ORP11
















Calculated Tg
646.08
680.971
684.087
681.573
639.845
641.05


(° C.)


Experimental
643.65
680.20
688.94
677.49
640.23
643.09


Tg (° C.)


Residual
2.43
−0.78
4.85
−4.08
0.39
2.04


(Difference)









X-Ray Diffraction (XRD)

Generally, XRD is employed to validate the amorphous nature of the glasses. Powdered samples of each glass are pressed to form discs (Ø32 mm×3 mm). Diffraction patterns are collected using an X-ray Diffraction Unit with monochromated CuKα (λ=1.54060 A) radiation at 40 KV and 35 mA. The scanning angle range (2θ) is performed from 10° to 70° with a step size 0.033423° and step time of 59.69 s.
Example 3
X-ray diffraction (XRD) measurements for the particles were performed using an INEL CPS-120 diffractometer with a curved position sensitive detector coupled to an X-ray generator (40 kV; 35 mA) and equipped with a Cu target X-ray tube. Samples were prepared by pressing the selected particle particles (45-212 μm) into hollow square steel wafers. A monochromator in the incident beam path limits the wavelengths striking the sample to Cu Kα1,α2. The X-ray beam is incident upon the sample at approximately 6° and the curved position sensitive detector collects all scattered X-rays in the scan angle range 10°<2θ<110°. Collection time for the XRD spectra is 1800 seconds. The powder samples were placed on the INEL's x-y translating stage that allows measurement and move operations to be sequentially programmed.
FIG. 3 illustrates XRD patterns for each material synthesized (A) glasses ORP 2,3,5,7,9,11 and (B) ORP 1 and 6. The x axis denotes the scattering angle as measured in degree 2-theta and the y-axis in arbitrary units. Table 6 provides the legend for phase identification.











TABLE 6





Peak






Identi-
JCPDS Card

Chemical
Present in


fier
No.
Phase Identifier
Formula
Composition:







B
00-050-0522
Strontium-
Sr3La2Ti2O10
ORP1,6




Lanthanum-




Titanium oxide


C
00-042-0423
Perovskite,
CaTiO3
ORP6




Calcium




Titanium Oxide


D
00-049-1433
Titanium oxide
TiO2
ORP6


E
00-039-0190
Zinc-Titanium
ZnTiO3
ORP1




oxide









Surprisingly Tg for these complex multi-component systems is increased by increasing any of the four compositional variants, with its level of statistical significance for the compositional variants following the order: La2O3>TiO2>SiO2>ZnO. One might have expected that only increasing SiO2 content would lead to increases in Tg. Interestingly, with a decrease in Tg attributed to the formation of a weaker glass network, the strongest to weakest networks formed for the series of glasses herein therefore, follows the order: ORP5>ORP3>ORP7>ORP2>ORP11>ORP9.
Network Connectivity
Example 4
The network connectivity (NC) of each composition was calculated using Equation 2 and the molar compositions of the glass.1,2 The results are shown in Table 1.




  
    
      
        
          N
          ⁢
          
            
          
          ⁢
          C
        
        =
        
          
            
              
                No
                .
                
                  
                
                ⁢
                B
              
              ⁢
              
                
              
              ⁢
              Os
            
            -
            
              
                No
                .
                
                  
                
                ⁢
                N
              
              ⁢
              
                
              
              ⁢
              B
              ⁢
              
                
              
              ⁢
              Os
            
          
          
            Total
            ⁢
            
              
            
            ⁢
            
              No
              .
              
                
              
              ⁢
              Bridging
            
            ⁢
            
              
            
            ⁢
            species
          
        
      
    
    
      
        Eq
        .
        
          
        
        ⁢
        2
      
    
  




Wherein NC=Network Connectivity; BO=Bridging Oxygens and NBO=Non-Bridging Oxygens

Generation and Application of Mathematical Models Using a Design of Experiments (DoE) Approach

To estimate the coefficients of a second order canonical Scheffé polynomial3,4, a quadratic user-defined design with thirteen experiments representing different compositional variants (design points) within a defined domain (design space) was constructed using Design-Expert 8.0.4 software (Stat-Ease, Inc.). These design points were determined based on the constrained ranges for each composition: with six experiments set at the extreme vertices; a further six investigating axial check-blends and one overall centroid within the defined design space. These points are in clear agreement with Scheffé's proposal that the interesting points of a domain are at its tops, at the middle of the sides, at the middle of the faces and its centre of gravity (See Table 1). With the mixture design method, an equation is obtained. This formula connects Y, with the four compositional factors (ZnO, La2O3, SiO2 and TiO2, noted respectively as X1, X2, X3 and X4).
The ordinary second-order Scheffé (linear) polynomial equation fitted for Magic Angle Spinning-Nuclear Magnetic Resonance (MAS-NMR) spectroscopy and DSC responses (chemical shift and line width) is:

YA=β1X1+β2X2+β3X3+β4X4+e  Eq. 3

where X1 to X4 represent the compositional factors, β1-4 coefficients represent the effect of the individual compositional factors X1-4. e is called residual, this value is the difference between calculated and experimental values for each selected glass. It is equal to 0 if the number of retained glasses is the same as the number of coefficients in the formula. In the case of a number of chosen glasses higher than the number of coefficients, residual has a different value for each experiment.

The canonical Scheffé second-order polynomial equation fitted for density and cell viability responses is:

Y=β1X1+β2X2+β3X3+β4X4+β12X1X2+β13X1X3+β14X1X4+β23X2X3+β24X2X4+β123X1X2X3+β124X1X2X4+β134X1X3X4+β234X2X3X4+e   Eq. 4

where X1 to X4 represent the compositional factors, β1-4 coefficients represent the effect of the individual compositional factors X1-4; β12-24, are the coefficients of regression which represent the effects of two-way interactions between the compositional factors; β123-234, are the coefficients of regression which represent the effects of three-way interactions between the compositional factors and e is the residual.

From the estimated coefficients of a quadratic model presented in pseudo and actual values, the effect of each component can be derived. All mixture experiment models were developed relating the response variables to proportions of pseudo-components. Pseudo-component proportions (zi) are calculated as:

zi=(xi−Li)/(1−ΣL)  Eq. 5

where xi stands for the original component proportions, Li stands for the lower bound constraint (limit) for the ith component, L stands for the sum of all lower bound constraints (limits) for all components in the design, and 1 represents the mixture total.

The pseudo-components are combinations of the original (actual) components, which rescale the constrained composition region so that the minimum allowable proportion of each pseudo-component is zero. This transformation provides for estimating model coefficients more precisely compared to using the actual component system, and as such the coefficients derived based on the pseudo-component scaling is referred to in the context of the discussion to follow. Model validity, in terms of experimental versus calculated data points and graphical representation (contour plots) however, is presented in terms of actual component coding.
If several response characteristics y1, y2, . . . , yn have been modeled in the proportions of the same set of q components, where in the composition space the best overall set of properties are obtained by implementing the desirability function approach. Using the models, which are based on the response characteristics, compositions for particulate materials having desired properties for a particular purpose can be determined.
True Densities
Example 5
The true densities were measured using a helium pycnometer (AccuPyc 1340, Micromeritics) for all the particles synthesized herein and, also for the commercial control material: ContourTM (PVA particulates). Results are representative of an average of five recorded measurements. FIGS. 4A and 4B are resulting contour plots showing the compositional-interaction effects on the experimental particles density (g/cc) at four various additions of Ti (0.017, 0.033, 0.042 and 0.05 mol.) in actual component coding. (A) is Ti: 0.017 mol. fraction; (B) is Ti: 0.033 mol. fraction; (C) is Ti: 0.042 mol. fraction; and (D) is Ti: 0.050 mol. fraction.
Table 7 illustrates final regression equations in terms of L_Pseudo (1); Actual components (2) and R2 values; and summarized ANOVA for density. The ANOVA data demonstrates that the model adequately predicts the characteristics of the particulate material.









TABLE 7









Summarized ANOVA









Adeq.














Response
Regression Models
R2
R2adj.
R2pred.
Prob > F
CV (%)
Prec.


















Density
1
+3.39ZnO + 5.12La2O3 +
0.9989
0.9975
0.9859
<0.0001
0.55
69.809


(g/cc)

2.43SiO2 + 2.92TiO2 +




1.07ZnO * La2O3



2
+5.65559ZnO +




10.72595La2O3 +




2.82345SiO2 +




4.26905TiO2 +




9.211775ZnO * La2O3









Table 8 is a summary of the significant (positive and negative) main, interaction and quadratic compositional factors (ranked highest to lowest) and its effect on increasing density; where ↑ denotes an increase and ↓ a decrease. Based on actual component coding.








TABLE 8





Ranking of



Compositional


Factors
Density Response







1
↑La2O3


2
↑ZnO


3
↑TiO2


4
↑SiO2


5
↑ZnO*La2O3









As illustrated, density may be increased by increasing any of the four compositional variants in the order La2O3>ZnO>TiO2>SiO2. Interestingly, increasing the interaction of ZnO and La2O3 (fifth most influencing factor) may result in an increase in material density, a feature which would not have been predicted but which the disclosed models do predict for the disclosed compositions. The lowest levels of density achieved (3.2 g/cc (ORP2) and 3.3 (ORP11)) in the design matrix tested, demonstrate how a slight variation in compositional variants from ORP2 to ORP 11 (increased ZnO (0.05 mol. fraction) and reduced TiO2 (0.5 mol. fraction) content) may adversely affect the final cell viability obtainable; by reducing it from 84 to 61%, respectively. Interestingly, density in the range of 3.5 g/cc (±0.3) (for ORP2, 3 and 5) was also representative of the top three compositional variations in terms of cell viabilities (86% (±5) attainable.
Specific Surface Area Determination
N2 adsorption/desorption will be determined using the Brunauer-Emmett-Teller (BET) method to determine the specific surface area (SSA) and porosimetry of the prepared glass powders. Glass samples ( ̃0.15 g±0.05 g) will be placed under a nitrogen atmosphere at 77.35 K with an equilibration interval of 10 s.
Example 6
The theoretical external specific surface areas for the particle particulates and ContourTM were calculated according to the following equation5, under the assumption of spherical particulate morphology




  
    
      
        A
        =
        
          
            6
            
              ρ
              ⁢
              
                
              
              ⁢
              d
            
          
          .
        
      
    
    
      
        Eq
        .
        
          
        
        ⁢
        6
      
    
  




Table 9 provides specific surface area measurements (45 micron (Lower Level, LL) and 212 micron (Upper Level, UL).













TABLE 9







Sample
Density
SD
PD (LL)
PD (UL)
SSA (LL)
SSA (UL)


ID
(g/cc)
(g/cc)
cm
cm
cm2/g
cm2/g





ORP1
4.09
0.002
0.045
0.212
32.600
6.920


ORP2
3.17
0.001
0.045
0.212
42.048
8.925


ORP3
3.77
0.001
0.045
0.212
35.395
7.513


ORP5
3.70
0.002
0.045
0.212
36.075
7.657


ORP6
4.29
0.001
0.045
0.212
31.087
6.599


ORP7
3.73
0.002
0.045
0.212
35.737
7.586


ORP9
3.32
0.001
0.045
0.212
40.136
8.520


ORP11
3.25
0.003
0.045
0.212
41.038
8.711


Contour
1.77
0.003
0.045
0.212
75.406
16.006


















Sample
Density
Density SD
PD (LL)
PD (UL)
SSA (LL)
SSA (UL)
1 m2
1 cm2


ID
g/m3
g/m3
m
m
m2/g
m2/g
g
g





ORP1
4090000
2000
0.000045
0.000212
0.033
0.007
144.51
0.14


ORP2
3171000
1000
0.000045
0.000212
0.042
0.009
112.04
0.11


ORP3
3767000
1000
0.000045
0.000212
0.035
0.008
133.10
0.13


ORP5
3696000
2000
0.000045
0.000212
0.036
0.008
130.59
0.13


ORP6
4289000
1000
0.000045
0.000212
0.031
0.007
151.54
0.15


ORP7
3731000
2000
0.000045
0.000212
0.036
0.008
131.83
0.13


ORP9
3322000
1000
0.000045
0.000212
0.040
0.009
117.38
0.12


ORP11
3249000
3000
0.000045
0.000212
0.041
0.009
114.80
0.11


Contour TM
1768200
3000
0.000045
0.000212
0.075
0.016
62.48
0.06









Scanning Electron Microscopy

Example 7
For scanning electron microscopy (SEM) observations, powder samples (particle size range: 355-500 μm) were mounted onto 10 mm diameter×3 mm high A1 stubs and coated with  ̃27 nm platinum using a gold-sputter coater (SC7640, Fisons Instruments). The samples were subsequently transferred to the chamber of a Hitachi S-4700 FEG-SEM equipped with an Oxford Inca energy dispersive x-ray spectroscopy (EDS) system for morphological and chemical analysis. An accelerating voltage of 5.0 kV and a working distance of 11-12 mm were used with the lower secondary electron (SE) detector.
FIG. 5 presents morphological data (SEM) derived from one experimental composition (ORP5); whose morphology is representative of the full set of multi-component systems examined and compared to the commercially available predicate device, Contour. FIG. 5A is a control sample of ContourTM with particle size range of 355-510 μm. FIG. 5B is an experimental sample of ORP5 having particle size range of 355-510 μm.
Chemical (EDX) data for ORP5 is presented in FIG. 6 demonstrates that the synthesized composition is as reported in Table 1.
Magic Angle Spinning-Nuclear Magnetic Resonance (MAS-NMR) Spectroscopy for Determination of Non-Bridging Oxygens
Generally, the 29Si MAS-NMR spectra of the glasses are recorded at a spinning frequency of 5 kHz using a high power pulse (P1) acquisition of 1.5 μs for silicon. The 29Si MAS-NMR samples are spun with a recycle/delay time set to  ̃2 s. 29Si NMR chemical shifts are reported in ppm and recorded at an ambient probe temperature with 29Si referenced externally relative to 2,2-dimethyl-2 silapentane-5-sulfonate sodium salt (DSS) or other appropriate reference. For solid state NMR, shifts recorded using MAS are independent of the isotropic bulk magnetic susceptibility of the sample. The estimated error of chemical shift for data is ca. 0.1 ppm for an ideal material; small variations above this value may be observed.
Example 8
29Si magic angle spinning (MAS) NMR studies were carried out on a Bruker Avance NMR spectrometer with a 9.4 T magnet (79.51 MHz 29Si Larmor frequency) using a probe head for 7 mm rotor diameters. The specimens (<45 μm) were spun at 5.00 kHz. 200 scans were accumulated with single pulse excitation using a pulse length of eighty degrees at 28 kHz rf field strength. The recycle delays were chosen to be three times the spin lattice relaxation times as determined by inversion recovery sequences. Spin lattice relaxation times range between 15 and 26 seconds. The chemical shift scale was referenced externally against Kaolin as secondary chemical shift standard at −91.34 ppm. Error bars on the peak maxima and peak widths are ±1 ppm.
FIG. 7 illustrates 29Si MAS-NMR spectra for each experimental embolic material (peak maxima and peak width for each is recorded in Table 10, respectively) sorted by decreasing line width (a: ORP11, b: ORP2, c: ORP9, d: ORP7, e: ORP6, f: ORP5, g: ORP3, h: ORP1). To ensure that no crystal signals were missed due to too fast pulsing (50 s) an additional 29Si MAS NMR spectrum for ORP6 was acquired at longer repetition times (90 s). FIG. 8 illustrates those results—29Si MAS-NMR spectra for experimental embolic material ORP6 at (a) 90 s and (b) 50 s repetitions times and (c) their scaled difference; verifying that silica is not part of the crystals detected by XRD. In both spectra, no crystalline components were evident. The XRD diffractograms for ORP1 and ORP6 indicate the presence of some crystalline species which were identified in Table 6.
FIG. 9 illustrates contour plots' showing the compositional-interaction effects on the experimental embolic materials 29Si MAS-NMR chemical shift (in ppm) at four various additions of Ti ((FIG. 9A) 0.017, (FIG. 9B) 0.033, (FIG. 9C) 0.042 and (FIG. 9D) 0.05 mol.) in actual component coding.
FIG. 10 illustrates contour plots' showing the compositional-interaction effects on the experimental embolic materials 29Si MAS-NMR spectra line width (ppm) at four various additions of Ti ((FIG. 10A) 0.017, (FIG. 10B) 0.033, (FIG. 10C) 0.042 and (FIG. 10D) 0.05 mol.) in actual component coding.
Table 10 illustrates the total number of positive charges and associated NMR max. position and line width from the nominal compositions outlined in Table 1 (where SiO2, TiO2, ZnO and La2O3 are varied with equimolar concentrations of CaO, Na2O MgO and SrO).










TABLE 10









Maximum
















ORP
Si02
TiO2
ZnO
La2O3
#charges/
position
width













Sample
mol fraction
Si
±1 ppm
±1 ppm
Morphology


















11
0.570
0.000
0.290
0.000
1.51
−86.2
26.9
glassy


2
0.570
0.050
0.240
0.000
1.68
−89.8
26.9


9
0.520
0.050
0.290
0.000
2.04
−85.5
24.7


7
0.562
0.017
0.213
0.068
2.10
−83.6
19.7


5
0.562
0.042
0.188
0.068
2.19
−84.3
19.9


3
0.537
0.042
0.213
0.068
2.39
−82.2
18.8


1
0.553
0.033
0.137
0.137
2.73
−82.4
15.7
glassy + crystalline


6
0.562
0.042
0.068
0.188
3.05
−84.1
19.0


12
0.562
0.017
0.068
0.213
3.14


frit synthesis impossible


4
0.570
0.050
0.000
0.240
3.37


13
0.537
0.042
0.068
0.213
3.47


8
0.570
0.000
0.000
0.290
3.54


10
0.520
0.050
0.000
0.290
4.27









Table 11 illustrates regression equations in terms of L_Pseudo (1); Actual components (2) and R2 values; and summarized ANOVA for each response. The ANOVA data demonstrates that the model adequately predicts the characteristics of the particulate material for responses A and B.









TABLE 11









Summarized ANOVA














Response
Regression Models
R2
R2adj.
R2pred.
Prob > F
CV (%)
AP



















A
Chemical
1
−82.25ZnO − 63.59La2O3 −
0.9836
0.9589
0.7689
0.0246
0.62
16.512



Shift

111.10SiO2 − 103.60TiO2



(ppm)
2
−44.32971ZnO +





10.55810La2O3 −





129.18571SiO2 −





107.11371TiO2


B
Line
1
+24.65ZnO − 8.69La2O3 +
0.9997
0.9993
0.9958
0.0004
0.45
97.003



Width

39.61SiO2 + 25.19TiO2



(ppm)
2
+2.05426ZnO −





95.99452La2O3 +





46.05426SiO2 +





3.65426TiO2









Table 12 presents a summary of the significant (positive and negative) main, interaction and quadratic compositional factors (ranked highest to lowest) and its effect on producing a chemical shift/peak position towards a negative direction and increasing line width; where ↑ denotes an increase and ↓ a decrease. The data is based on actual component coding.









TABLE 12







Ranking of




Compositional
Responses









Factors
A
B





1
↑SiO2
↑ SiO2


2
↑TiO2
↑ TiO2


3
↑ZnO
↑ ZnO


4
↑La2O32
↓ La2O3


5









Table 13 presents residuals of the 29Si MAS-NMR chemical shift (a) and line width (b) study for all material compositions synthesized.










TABLE 13









A. 29Si MAS-NMR chemical shift (ppm)
B. 29Si MAS-NMR line width (ppm)














Experimental
Calculated
Residual
Experimental
Calculated
Residual


Glass
Data
Data
(Difference)
Data
Data
(Difference)
















ORP2
−89.803
−89.631
−0.172
26.900
26.927
−0.027


ORP3
−82.336
−82.528
0.192
18.800
18.747
0.053


ORP5
−84.305
−84.650
0.345
19.900
19.847
0.053


ORP7
−83.617
−83.080
−0.537
19.700
19.807
−0.107


ORP9
−85.484
−85.388
−0.096
24.700
24.727
−0.027


ORP11
−86.223
−86.491
0.268
26.900
26.847
0.053









As shown in the NMR spectra, both NMR peak shift may be increased by decreasing any of the four compositional variants in the order SiO2>TiO2>ZnO>La2O3, and that the line width follows the same order, except for La2O3 The NMR spectra (FIG. 7)) obtained for ORP2, 9 and 11 appear significantly broader than the other spectra obtained for all other compositions. The difference between the corresponding specimens is that ORP2, 9 and 11 do not contain Lanthanum, while the others do.
Specimen ORP2 is comprised of more TiO2 at the cost of ZnO compared to ORP11 and therefore has a higher charge-to-silica ratio, but, the Qn (nSi, 4-nTi) species resonate at lower ppm values than the Qn (nSi, 4-nZn) species. Based on the charge-to-silica ratio and assuming a roughly binary Q-species distribution, ORP11 and 2 should and experimentally do resonate in the Q3/Q2 range, specimens ORP9, 7, 5, 3, and 1 in the Q2/Q1 range (deconvolutions indicate the presence Q1 sites) and specimen ORP6 and below in the Q1/Q0 range, while specimen ORP10 should only consist of Q0 and separate metal oxide species. Interestingly, for specimen ORP1 and 6, (i.e. where the charge-to-silica ratio comes in the range of Q0 contributions), separation into glassy and crystalline components already occurs with a higher charge ratio and cannot be made into frits at all. This phase separation in specimen ORP6 is the reason, why its NMR properties fall in between those of the spectra for ORP7 and ORP5, due to the crystals withdrawing cation-oxides and to result in a glass of different composition studied by NMR. La2O3, even at modest concentrations causes a breakdown of the glasses. This is useful because it adds to the tunability of the degradation of the particulate material.
Quantification of Degradation Products from Glass and Composite
TRIS-HCl buffer and citric acid buffer solutions with a pH of 7.4±0.1 and pH of 3.0±0.2 respectively, are prepared to simulate normal and extreme physiological conditions (according to ISO10993-14). Both buffer solutions are used for the quantification of degradation products from the glasses. 100 mg of each particulate material are immersed in 10 ml of each solution (n=3) in polypropylene tubes maintained at 37° C. in a shaking waterbath, agitated at 2 Hz. Specimens are stored for various durations of time—for example, 1, 3, 7, or 30 days. After each time period, specimens are removed and filtered through Grade 5 Whatman filter paper, the filtrate retained for ionic content analysis. The degradation products from the glass are identified and quantified using Inductively Coupled Plasma-Mass Spectrometer. Analysis of each extract are performed in triplicate (n=3 (extracts per condition), 3 analyses performed on each extract).
Analysis of Ion Release Profiles
The ion release profiles from the embolic agents are described in terms of the ion release concentration (Y) over incubation time (X). Since the incubation time is not an input of the correlation function, the time dependent functions have been fitted to nonlinear regressive polynomial, gaussian, sine waves and exponential models using Prism 5.0 software (GraphPad software Inc.). The best fitting model for the four elements with respect to each embolic agent is the one phase-decay association model:

Y═Y0+(Plateau−Y0)*(1−exp(−K*X))  Eq. 7

Wherein:

    ‘Y’ and ‘X’ are the ion release concentrations in ppm and incubation time in hours, respectively;
    ‘Y0’ is the ion release concentration (ppm) at initial ion release; where Y value at X0=1;
    ‘Plateau’ is the ion release concentration at an infinite time (ppm), where Y value at X=120 h;
    ‘K’ is the rate constant, expressed in reciprocal of the ‘tau’ incubation time and unit is inverse days;
    ‘tau’ denotes the time necessary for ion release to reach 63% of the estimated ‘ymax’ (ppm);
    ‘t1/2’ denotes the half-life (time) to reach 50% of final ‘ymax’ value, ‘t1/2=tau*LN (2)’;
    ‘ts’ denotes the difference between Y0 and Plateau;
    ‘R2’ is the sum of the squares of the distances of the points from the best-fit of the exponential nonlinear regression as determined by Prism 5.0 (GraphPad Inc.) software. The value of R2 is a fraction between 0.0 and 1.0, with the best-fit line with a R2 equal to 1.0.

Cell Culture Testing with Mouse Fibroblast Cell Line L929



The established mouse fibroblast cell line L929 (American Type Culture Collection CCL 1 fibroblast, NCTC clone 929) is cultured in M199 media supplemented with 10% foetal bovine serum and 1% (2 mM) L-glutamine. Cells are grown in T-75 flasks at 37° C. in a 5% CO2 incubator. When the cells reach confluency, they are chemically removed using 0.25% trypsin, centrifuged and re-suspended in fresh culture media to create a new single cell suspension for further inoculation.
Cell Viability Assay
3T3 cells are seeded at a density of 1×104/ml in 24 well plates (Sarstedt, Ireland). M199 Culture media is used as a negative control and culture media plus cells used as a positive control. Plates are then incubated for 24 hrs in a cell culture incubator at 37° C. (5% CO2/95% air atmosphere). After 24 hrs, 100 μl of sterile tissue culture water are added to control wells. 100 μl of relevant experimental extracts (containing particles) are added to appropriate wells for testing. The plate is then incubated for 24 hrs in a cell culture incubator at 37° C. (5% CO2/95% air atmosphere). After 24 hrs incubation, each well is exposed to MTT (Sigma Aldrich, Ireland) at an amount equal to 10% of the culture media volume (100 μl). Plates are returned to the incubator for 3 hrs. After incubation, MTT solubilisation solution are added to each well at a volume equal to the original culture media volume (1 ml). Each well is titrated using a pipette in order to enhance dissolution of the crystals, after which the absorbance of each well is measured spectrophotometrically at a wavelength of 570 nm. Cell positive control wells were assumed to have metabolic activities of 100% and the percentage metabolic activity of the cells exposed to experimental extracts were calculated relative to this.
Example 9
Preparation of Particle Extracts for Degradation Studies
0.1 g of particles and ContourTM (commercial control) (Lot No.s: 13473927 and 13599201) were immersed in 10 ml6 of sterile tissue culture water (Sigma-Aldrich, Canada) for 12, 24, 48, 96 and 120 hours at 37° C. in a shaking waterbath (Stuart Sb40, Techne Inc., USA) vibrated rotationally at 2 Hz. After each storage period, samples were filtered using a sterile 0.20 μm filter (Sarstedt, Canada), and filtrates stored at 7° C. prior to in vitro evaluation.
The SO4+, Na+, Ca2+, Zn2+, Ti4+, La3+, Sr2+, and Mg2+ concentrations for each extract were analyzed using inductively coupled plasma atomic emission spectroscopy (ICP-AES, Perkin Elmer Optima 3000, MA, USA). The absorption wavelengths used for the determination of each element is reported in Table 14. Before each cycle of measurement, calibration curves were obtained by preparing standard solutions containing Ti4+, La3+, Sr2+, and Mg2+ and a separate set of standard solutions containing Si4+, Na+, Ca2+ and Zn2+ (as obtained from JVA Analytical Ltd, Ireland) at concentrations reported in Tables 15 and 16. Standard sample concentrations were measured periodically to ensure the accuracy of the calibration curve. Triplicates of each extract (from each incubated embolic agent) were measured for each element, with appropriate adjustments in outputs being deployed to balance dilutions of original extracts. The results demonstrate that the complex multi-component system provides for tailored rates of degradability.











TABLE 14






Absorption


Background


Element
Wavelength
Lower Limit
Upper Limit
Correction







Si4+
288.158
288.073
288.256
±0.026


Na+
330.237
330.136
330.348
±0.030


Ca2+
396.847
396.679
397.039
±0.072


Zn2+
334.501
334.400
334.614
±0.031


Ti4+
337.279
335.188
334.810
±0.031


La3+
407.735
407.971
407.596
±0.075


Mg2+
279.553
279.646
279.399
±0.026


Sr2+
421.552
421.759
421.371
±0.078









Table 15 provides the standard concentrations used for the ICP measurements (JVA Analytical, Ireland).











TABLE 15





Standard
Si4+ (mg/L)
Na+ (mg/L)
Ca2+ (mg/L)
Zn2+ (mg/L)



















1
2
1
0.5
1


2
4
2
1
2


3
10
4
3
4









Table 16 provides laboratory standard concentrations used for the ICP measurements.











TABLE 16





Standard
Ti4+ (mg/L)
La3+ (mg/L)
Mg2+ (mg/L)
Sr2+ (mg/L)



















4
0.1
0.1
0.1
0.1


5
1
1
1
1


6
10
10
10
10









FIG. 11 presents ion release profiles for Si (FIG. 11A) and Na (FIG. 11B) release of the eight embolic particulates with time dependency over 12, 24, 48, 96 and 120 h. Lines are drawn as visual guides. FIG. 12 presents ion release profiles for Ca (FIG. 12A) and Sr (FIG. 12B) release of the eight embolic particulates with time dependency over 12, 24, 48, 96 and 120 h. Lines are drawn as visual guides. FIG. 13 presents ion release profiles for Ti (FIG. 13A) and La (FIG. 13B) release of the eight embolic particulates with time dependency over 12, 24, 48, 96 and 120 h. Lines are drawn as visual guides. Lines are drawn as visual guides (excluding ambiguous data with R2<0.6). FIG. 14 presents an ion release profile for Mg (FIG. 14A) and mean (±SD) release levels for Zn (FIG. 14B) of the eight embolic particulates with time dependency over 12, 24, 48, 96 and 120 h (for Zn (FIG. 14B) only). Lines are drawn as visual guides. Lines are drawn as visual guides (excluding ambiguous data with R2<0.6). FIG. 15 presents the effect of embolic material composition on release levels for Si (FIG. 15A) and Na (FIG. 15B) after 120 h incubation periods. FIG. 16 presents the effect of embolic material composition on release levels for Ca (FIG. 16A) and Sr (FIG. 16B) after 120 h incubation periods. FIG. 17 presents the effect of embolic material composition on release levels for Ti (FIG. 17A); La (FIG. 17B) and Mg (FIG. 17C) after 120 h incubation periods.
Table 16 provides the goodness of fit (in terms of R2 values) for the non-linear one phase association model formed from Si4+ and Na+ release over 5 time points (12, 24, 48, 96, 120 h).











TABLE 16








Si4+
Na+



Embolic Composition
R2
R2









ORP1
0.8219
0.7752



ORP2
0.8115
0.7383



ORP3
0.7195
0.7174



ORP5
0.8691
0.8935



ORP6
0.8123
0.6263



ORP7
0.8672
0.7111



ORP9
0.7839
0.5419



ORP11
0.8697
0.6747










Table 17 provides the best-fit parameters for the non-linear one phase association model formed from Ca2+ release over 5 time points (12, 24, 48, 96, 120 h).












TABLE 17





Embolic Composition
t1/2 (h)
tau (h)
ts (h)
ymax (ppm)
R2







ORP1
4.938
7.123
7.948
7.953
0.9808


ORP2
4.003
5.776
3.932
3.936
0.9427


ORP3
4.840
6.982
2.860
2.868
0.9594


ORP5
4.523
6.525
2.663
2.669
0.9660


ORP6
3.926
5.664
2.650
2.653
0.9604


ORP7
3.771
5.440
2.605
2.607
0.9605


ORP9
1.379
1.989
5.515
5.515
0.6575


ORP11
4.044
5.835
3.189
3.193
0.9669









Table 18 provides the best-fit parameters for the non-linear one phase association model formed from Sr2+ release over 5 time points (12, 24, 48, 96, 120 h).












TABLE 18





Embolic Composition
t1/2 (h)
tau (h)
ts (h)
ymax (ppm)
R2




















ORP1
15.70
22.65
0.3583
0.3706
0.9550


ORP2
14.64
21.12
0.3446
0.3585
0.9181


ORP3
18.28
26.38
0.2980
0.3089
0.9239


ORP5
12.63
18.21
0.2772
0.2828
0.9713


ORP6
16.00
23.08
0.4494
0.4669
0.9645


ORP7
10.20
14.72
0.3201
0.3262
0.9666


ORP9
6.786
9.79
0.4188
0.4202
0.8559


ORP11
9.671
13.95
0.3096
0.3129
0.9749









Table 19 presents the best-fit parameters for the non-linear one phase association model formed from Ti4+ release over 5 time points (12, 24, 48, 96, 120 h). The asterisk (*) represents the models denoted as ambiguous.












TABLE 19





Embolic







Composition
t1/2 (h)
tau (h)
ts (h)
ymax (ppm)
R2




















ORP1*
~0.233
~0.336
0.1980
0.1980
0.9661


ORP2
2.563
3.698
0.1875
0.1875
0.9815


ORP3
4.300
6.203
0.1861
0.1862
0.9878


ORP5
2.885
4.162
0.1801
0.1801
0.9876


ORP6
2.885
4.162
0.1801
0.1801
0.9876


ORP7
2.885
4.162
0.1801
0.1801
0.9925


ORP9*
~0.000
~0.000
0.1920
0.1920
0.9867









Table 20 presents the best-fit parameters for the non-linear one phase association model formed from Mg2+ release over 5 time points (12, 24, 48, 96, 120 h). The asterisk (*) represents the models denoted as ambiguous.












TABLE 20





Embolic Composition
t1/2 (h)
tau (h)
ts (h)
ymax (ppm)
R2




















ORP1
57.52
82.98
0.11
0.1111
0.9639


ORP2
74.21
107.10
0.07
0.0721
0.7802


ORP3
9.50
13.70
0.03
0.0261
0.3176


ORP5*
~0.05
~0.01
0.05
0.0460
0.2356


ORP6
58.01
83.69
0.11
0.1058
0.9656


ORP7*
~0.00
~0.00
0.05
0.0460
0.1572


ORP9
7.71
11.12
0.08
0.0751
0.4458


OR11
3.91
5.64
0.04
0.0372
0.1779









Surprisingly, Zn2+ was only found to initially release after 120 h. All other ions were found to release continuously for all time periods. This is contrary to the expectation that Zn2+ would release ions with the immediate onset of its submersion into solution.
The ion release profiles for both Si4+ and Na2+ present stable time-dependent increases in ion release (denoted by the goodness of fit values represented by R2 values ranging from 0.81 to 0.87 with the exception of ORP3 and 9 yielding R2 values of 0.72 and 0.78, respectively for Si4+ and R2 values ranging from 0.71 to 0.90 with the exception of ORP6, 9 and 11 yielding R2 values of 0.63, 0.54 and 0.67, respectively for Na2+) up to 120 h, which potentially indicate the controllable nature for each composition. Full stabilization of ion release is not evident for both ions after 120 h.
An initial burst of Ca2+ released for ORP9 during the first few hours in solution was noted, ending abruptly to maintain a stable release level for the remainder of the study. ORP1 also exhibited an initial burst of Ca2+ release to gradually release Ca2+ at a stable rate past 24 h. The remaining compositions however, show more gradual initial Ca2+ release up to 24 h prior to reach full stabilization. This is likely due to the formation of a hydrogel like layer on the full surface area of the glass particulate as a result of initial Ca2+ release (from the particulate) in exchange for H3O+ in the incubation media, to precipitate onto the entire surface of the particulate; and further impact upon the rate of ion release from the particulates. Interestingly, the same release profiles were observed for Sr2+ and Ti4+.
Importantly, very slow release levels of Mg2+ into the solution were noted, to suggests that it is strongly chelated by the silicate network. The same is reported for La2+ with the exception of ORP1 (possibly due to an increase in crystalline species).
Preparation of Particle Extracts for MTT/Release of LDH Assays
For this protocol, all particles were sterilized by autoclaving (AMSCO Medallist) at 121°/25 bar for a period of 20 mins prior to incubation. Pre-sterilized batches of ContourTM (Lot No.s: 13473927 and 13599201) were used as a control. Subsequently, an equivalent amount (0.1 g) of each sterile glass (ORP1-3, ORP5-7, ORP9, ORP11) and ContourTM were immersed in 10 mL of sterile tissue culture water (Sigma-Aldrich, Canada) for 24 hours, placed on a roller (rotating at  ̃2 Hz.) positioned inside a 37° C. incubator. After each storage period, samples were filtered using a sterile 0.20 μm filter (Sarstedt, Canada), and filtrates stored at 7° C. prior to in vitro evaluation.
Fibroblast Cell Culture
Immortalized rat fibroblasts (NIH-3T3; American Type Tissue Collection, Manassas, Va.) at passages 15-20 were used for experiments. The cells were grown in 75-cm2 tissue culture flasks in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5% fetal calf serum (FCS; heat-inactivated at 56° C. for 60 min). Cells were passaged twice weekly at 70% confluence, using 2 mL of 0.25% trypsin-EDTA to detach cells, and re-suspended in 8 mL of DMEM. Flasks were maintained in a humidified atmosphere at 37° C. and 10% CO2. No antibiotics were used during routine subdivisions or for cell culture experiments to avoid altering cell metabolism. Fibroblasts for use in experiments were harvested at 70% confluence, detached using trypsin-EDTA, suspended at a concentration of 1×104 cells/mL, and verified using a Coulter counter.
Assessment of Cell Viability (MTT Assay)
NIH-3T3 cells (200 μL) were seeded at a density of 1×104 cells/mL in 96-nontissue culture-treated polystyrene plates (CoStar, Corning, Canada). DMEM+5% FCS culture media plus sterile tissue culture water only was used as a negative control and culture media plus cells plus sterile tissue culture water used as a positive control. Serial dilutions were performed on all samples (at 25, 50, 75 and 100%) and performed in triplicate. Hence, 4 plates (corresponding to each dilution) were incubated for 24 h in a cell culture incubator at 37° C. (10% CO2/95% air atmosphere). After 24 h, 5, 10 and 15 μL of sterile tissue culture water were added to each well in their respective plate (corresponding to 75, 50 and 25% dilutions). 20 μL of relevant experimental extracts were then added to appropriate wells for testing. The plate was incubated again for 24 h in a cell culture incubator at 37° C. (10% CO2/95% air atmosphere). A 5 mg/mL solution of MTT reagent (M2128, Sigma Aldrich Canada) was prepared in phosphate-buffered saline, vortexed and sterile-filtered (0.20 μm). Post 24 h incubation of the plates, each well was exposed to the prepared MTT at an amount equal to 10% of the culture media volume. The four plates representing each set of dilutions were then returned to the incubator for 3 h. After incubation, MTT solubilisation solution (Catalog Code: M8910) was added to each well at a volume equal to the original culture media volume. Each well was gently stirred on a rotating table to enhance dissolution of the formazan crystals. Subsequently, the absorbance of each well was spectrophotometrically measured at a wavelength of 570 nm on a multidetection microplate reader (Synergy HT, BIO-TEK). Cell positive control wells were assumed to have 100% metabolic activity corresponding to cellular viability of 100% and the percentage cell viability of the cells exposed to experimental extracts were calculated relative to this.
Assessment of Cell Cytotoxicity (Release of LDH Assay)
The lactate dehydrogenase (LDH) assay was measured by a colorimetric lactate dehydrogenase (LDH) assay (TOX-7 (Product Code: 050M6079), Sigma Aldrich, Canada), according to instructions from the supplier. The amount of LDH in the medium is proportional to the number of lysed/dead cells present; therefore, this assay can be used to estimate cell death. This assay measures membrane integrity as a function of the amount of cytoplasmic LDH released into the medium. Briefly, assay mixture was prepared by mixing equal amounts of LDH assay substrate (Catalog Number: L2402), cofactor (Catalog Number: L2527) and dye solutions (Catalog Number: L2277). For all cultures (70 μL), assay mixture was added to the medium in a proportion of two to one in 4×96-nontissue culture-treated polystyrene plates (CoStar, Corning, Canada). Each plate corresponds to medium dilutions of 25, 50, 75 and 100%, respectively. After incubation at room temperature in the dark (each plate covered with Al foil) and through gentle rotation on a roller, the color reaction was stopped by 1 N HCl. Similar to the MTT assay, DMEM+5% FCS culture media plus sterile tissue culture water only was used as a negative control and culture media plus cells plus sterile tissue culture water used as a positive control. Absorbance was determined at 490 nm using a multidetection microplate reader (Synergy HT, BIO-TEK), with the background correction performed at 650 nm.
Statistical Analysis
Each experiment was performed in triplicate and analysed using Prism 5.0 software (GraphPad software, Inc.) Results are expressed as mean±standard deviation of the triplicate determinations. One way analysis of variance (ANOVA) was carried out followed by a Tukey's post hoc test for comparisons between groups. The level of significance was set at p<0.05. FIG. 18A presents cell viability in the presence of ORP1-3, ORP5-7, ORP9 and ORP11 and ContourTM for 24 h embolic particulate extracts at 25, 50 and 100% serial dilutions. Results represent mean±SD (no significant statistical differences (p<0.05) were observed between any of the extracts tested). FIG. 18B presents cell toxicity in the presence of ORP1-3, ORP5-7, ORP9 and ORP11 and ContourTM for 24 h embolic particulate extracts at 25, 50, 75 and 100% serial dilutions. Results represent mean±SD (no significant statistical differences (p<0.05) were observed between any of the extracts tested). FIG. 19 presents contour plots' showing the compositional-interaction effects on the experimental embolic materials cell viability (%) at four various additions of Ti (0.017 (FIG. 19A), 0.033 (FIG. 19B), 0.042 (FIG. 19C) and 0.05 mol. (FIG. 19D)) in actual component coding.
Table 21 provides the final regression equations in terms of L_Pseudo (1); Actual components (2) and R2 values; and summarized ANOVA for cell viability. The ANOVA data demonstrates that the model adequately predicts the cell viability response of the particulate material.









TABLE 21









Summarized ANOVA









Adeq.














Response
Regression Models
R2
R2adj.
R2pred.
Prob > F
CV (%)
Prec.


















Cell
1
+66.63ZnO − 145.51La2O3 +
0.9999
0.9994
0.9817
0.0179
0.36
123.770


Viability

338.30SiO2 + 815.98TiO2 +


(%)

265.33ZnO * La2O3 −




365.03ZnO * SiO2 −




913.28ZnO * TiO2



2
+1236.37607ZnO −




1029.59161La2O3 +




393.36925SiO2 +




1798.32577TiO2 +




2295.21826ZnO * La2O3 −




3157.72134ZnO * SiO2 −




7900.33004ZnO * TiO2









Table 22 provides the residuals of the cell viability study for all material compositions synthesized.









TABLE 22









Cell Viability (%)














Experimental

Calculated
Residual



Glass
Data
SD
Data
(Difference)

















ORP1
73.790
°19.77
73.954
−0.164



ORP2
84.120
°17.23
84.087
−0.033



ORP3
81.270
°16.15
81.153
0.117



ORP5
91.790
°18.74
91.885
−0.095



ORP6
72.370
°17.05
72.301
0.069



ORP7
71.440
°10.90
71.323
0.117



ORP9
62.240
°22.03
62.278
−0.038



ORP11
60.760
°03.49
60.798
−0.038










Table 23 provides the summary of the significant (positive and negative) main, interaction and quadratic compositional factors (ranked highest to lowest) and its effect on increasing cytocompatibility; where ↑ denotes an increase and ↓ a decrease. The data is based on actual component coding.








TABLE 23





Ranking of



Compositional
Cell Viability


Factors
Response







1
↓ZnO*TiO2


2
↑TiO2


3
↓ZnO*SiO2


4
↑SiO2


5
↑ZnO*La2O3









The data demonstrates that the most significant ‘main’ compositional variants which impact upon cell viability at 75% are on the order TiO2>SiO2. An interesting feature is that, counter to what would be expected, ZnO in its own right does not contribute to a reduction in cell viability. Rather, ZnO is dependant on interactions with TiO2 (Table 23 G; ZnO*TiO2 is the 1st most significant factor with respect to the MTT assay) to reduce cell viability; whereby a decrease in ZnO for an increase in TiO2 may enhance cell viability. Similarly, the interactions between ZnO and SiO2 indicates that decreased ratio of ZnO:SiO2 will allow for enhanced cytocompatibility. Other surprising relationships shown in the data are described below.
It also would not have been predicted that increasing the ratio of ZnO:La2O3 provides for increased levels of cell viabilities. This interaction effect deems La2O3 more cytotoxic than ZnO. To the contrary, it would have been expected that the Zn containing materials described herein would demonstrate a cytotoxicity profile counter to requirements. However, it is clearly demonstrated that the complex multi-component system provides for tailored (equivalent or superior) cytocompatibility versus conventional embolic materials (Contour).
Evaluation of Radiopacity
Radiopacity of OccluRad versus PVA (Contour) was determined using computed tomography scans of Gammex Tissue Characterization phantom inserts filled with each product. FIG. 20 is a comparison of radiopacity for ORP vs. PVA in various dilutions of saline/contrast agent. Results are provided in terms of the materials Hounsfield Unit (HU) values.
Due to the complexity of the multi-component systems (compositions) disclosed, one could not predict the response for each, with respect to its inherent radiopacity. It is noted that the HU values for ORP5 representative for all sets of compositions disclosed) demonstrates far superior levels to Contour in the absence of any contrast agent.
In Vivo Evaluation of Safety and Efficacy
Animal units (New Zealand White Rabbits) were used for a pilot examination of local effects after implantation in the uterine artery. Material (ORP 5) was delivered to the relevant vasculature, using a 25G butterfly cannulae, in a suspension of saline (8 mg/mL). 8 animals were utilized; 4 treated with ORP5 and 4 with Contour. Animals were euthanized after 21 days, using Isofluorane and KCl (the latter administered via IV at 2 mg/kg). Laparotomy was performed, and the uterus, including both ovaries, was removed. The left and right uterine horns were dissected, fixed in 10% formaldehyde solution and sectioned appropriately for histological evaluation. Histological samples were taken at four to six different levels (depending on size) along the uterine horn; routine hematoxylin and eosin staining was performed thereafter.
FIG. 21 presents histological data, derived from one animal in each group, with respect to the performance of Contour (FIG. 21A), ORP5 (FIG. 21B) and Control (FIG. 21C).
That ORP5 worked as well as and in some measures, out performed, the Contour could not have been predicted. Gross examination of the pathology specimens from all 8 rabbits showed a necrotic uterus at the embolization site, with no obvious changes in any of the untreated uterine horns. No adverse reactions were observed for any of the H&E stained tissues examined (in both experimental and commercial groups) with comparable data to untreated (normal) uterine tissues. In the intracutaneous irritation test, there was no evidence of irritation in both the polar (normal saline) and non-polar (sesame oil) vehicles (the mean score difference less than 1 in all observations), to demonstrate equivalency for ORP5 to the embolic predicate Contour.
Intracutaneous Irritation Test
This study was conducted according to ISO 10993-10. The potential of the test article (extract of particle ORP5) producing irritation was evaluated after intracutaneous injection of the test article. Three New Zealand White rabbits were used in this study. Each rabbit was injected intracutaneously with 0.2 mL of test article or vehicles. The polar vehicle was normal saline, and the nonpolar vehicle was sesame oil. The treatment designs of each animal are presented in FIG. 22. Table 24 provides the Draize grading system for the experiment.








TABLE 24






Numerical


Reaction
Grading







1. Erythema and eschar formation



No erythema
0


Very slight erythema (barely perceptible)
1


Well-defined erythema
2


Moderate erythema
3


Severe erythema (beet-redness) to escher formation preventing
4


grading of erythema


2. Edema formation


No edema
0


Very slight edema (barely perceptible)
1


Well-defined edema (edges of area well-defined by definite
2


raising)


Moderate edema (raised approximately 1 mm)
3


Severe erythema (raised approximately 1 mm and extending
4


beyond exposure area)









The appearance of each injection site was noted and marked immediately after injection. Observation of the tissue reaction at 0 hours (immediately after injection), 24 hours, 48 hours, and 72 hours after injection were graded. Any reaction at the injection site was recorded accordingly. FIG. 23 provides images of the intradermal injection sites for the three NZW rabbits after 24, 48 and 72 h observations.
Conformation of Fibrinogen in Embolic Extracts Containing Degradation Products
Far-ultraviolet (UV) Circular Dichroism (CD) spectra were recorded using a Jasco J-810 spectropolarimeter (Easton, Md.) with temperature control capability. A 6.4 mg/mL solution of fibrinogen from human plasma (F4883, plasminogen free, containing 58% protein, 96% clottable protein; Sigma Aldrich, ON) in 25 mM sodium phosphate buffer adjusted to pH 7.4±0.05 using NaOH and H2SO4 was diluted to a concentration of 0.2 mg/mL with the solutions of embolic extracts (containing the ion degradation products, as prepared in Example 9 at n=3 for each extract)). The concentration of the stock fibrinogen solution was determined by UV spectroscopy at 280 nm using the manufacturer's molar extinction coefficient. All spectra were collected at 37° C. (controlled with a NESLAB RTE-111 bath, Thermo Scientific, Newington, N.H.) in three repetitions (260-190 nm, 1 nm steps, 50 nm/min) in a 0.5 mm pathlength quartz cuvette (Hellma, Müllheim, Germany). The triplicate spectra for each ion degradation solution were averaged, blank subtracted with phosphate buffer (25 mM; pH 7.4±0.05), and converted to mean residue ellipticity ([θ]). Relative fibrinogen conformation between samples was monitored by comparing [θ] at the minima corresponding to the α-helical bands at  ̃208 and 222 nm. FIG. 24 illustrates changes in Fg conformation monitored by far-ultraviolet circular dichroism (CD) spectroscopy as a function of the ionic dissolution products with respect to time. FIG. 24A is an example of CD spectra for ORP5 over time. FIGS. 24B and C are intensity (mean of 3 replicates, blank subtracted; average deviation shown) at the minima at  ̃208 and 222 nm of Fg CD spectra for each ionic dissolution product. All data is reported in mean residue ellipticity [θ]. The provided legend applies for all three panels.
The effect of the degradation by-products from such complex multi-component systems on the secondary structure of fibrinogen, Fg (an essential precursor protein for clot formation) could not have been predicted. Interestingly, no significant conformational changes to Fg structure were observed by CD (FIG. 24), despite previous literature reports of this possibility. The response of platelets in contact with ORP5 and Contour using LDH assays could not have been predicted.
Platelet Lactate Dehydrogenase (LDH) Assay Studies
All protocols pertaining to the use of whole blood and platelets were approved by the Capital Health Research Ethics Board. The blood (31.5 mL) was collected via venipuncture from healthy, aspirin-free human volunteers at the Laboratory Blood Collection facility at the Victoria General Hospital in seven 4.5 mL glass BD Vacutainer tubes (Catalog No. 364606, Becton-Dickinson, Franklin Lakes, N.J.) containing an acid-citrated dextrose (ACD) anticoagulant. It is important to note that the first tube (4.5 mL) of blood was discarded, as it is rich in clotting factors, and then the remaining 27 mL was collected. Platelet rich plasma (PRP) was generated by centrifuging the ACD-anticoagulated blood (1500 rpm, 8 min, 25° C.) using an Eppendorf 5702 centrifuge. Careful transfer of the PRP to individual centrifuge tubes was completed using sterile plastic pasteur pipettes.
Platelet concentration was measured using a LH 785 CBC analyzer. The platelet concentration was recorded for each patient but not adjusted. The platelet suspension was then added to the preferred particles (ORP5 and ContourTM) at a final concentration of 0.1 cc per mL of platelet suspension (3 mL of patient PRP for each embolic agent with the exception of patient 1006 which only had enough PRP to add 2.5 ml of PRP per embolic agent) and allowed to adhere for 1 h at 37° C. under static conditions.
At the end of this step, the suspension was aspirated from each well, and the non-adherent platelets were rinsed away by filling and aspirating the wells ten times with 2.5 mL of PBS. The entire duration from blood collection to the conclusion of this step was less than 4 h. Platelet LDH was quantified by measuring the lactate dehydrogenase (LDH) released when the adherent platelets were lysed with a Triton-PSB buffer. A calibration curve was constructed using a known number of platelets and the platelet adhesion on the embolic agents was determined from this calibration curve. FIG. 25 illustrates results for platelet LDH assay (ORP5 vs. PVA) with (FIG. 25A) and without (FIG. 25B) outlier.
Given the complexity of the disclosed compositions, one could not have predicted the response of platelets to the material using the LDH assay. It is noted that performance of the multi-component systems in this assay demonstrate equivalent compatibility to Contour.
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the invention as defined in the appended claims.
REFERENCES


    1. Zhang X F, Kehoe S, Adhi S K, Ajithkumar T G, Moane S, O'Shea H, Boyd D, Composition-structure-property (Zn2+ and Ca2+ ion release) evaluation of Si—Na—Ca—Zn—Ce glasses: Potential components for nerve guidance conduits. Materials Science and Engineering C 2011; doi:10.1016/j.msec.2010.12.016
    2. Murphy S, Boyd D, Moane S, and Bennett M. The effect of composition on ion release from Ca—Sr—Na—Zn—Si glass bone grafts. J Mater Sci Mater Med 2009; 20: 2207-2214.
    3. Duee, C., et al., Mixture designs applied to glass bioactivity evaluation in the Si—Ca—Na system. Journal of Non-Crystalline Solids, 2009. 355(16-17): p. 943-950.
    4. Akalin, O., et al., Optimization of chemical admixture for concrete on mortar performance tests using mixture experiments. Chemometrics and Intelligent Laboratory Systems, 2010. 104(2): p. 233-242.
    5. Papelis C, Um W, Russell C E, and Chapman J B. Measuring the specific surface area of natural and manmade glasses: effects of formation process, morphology, and particle size. Colloids and Surfaces a-Physicochemical and Engineering Aspects 2003; 215: 221-239.
    6. Ning J, Yao A, Wang D P, Huang W H, Fu H L, Liu X, Jiang X Q, and Zhang X L. Synthesis and in vitro bioactivity of a borate-based bioglass. Materials Letters 2007; 61: 5223-5226.
    7. Cheung S, Gauthier M, Lefebvre L P, Dunbar M, and Filiaggi M. Fibroblastic interactions with high-porosity Ti-6Al-4V metal foam. Journal of Biomedical Materials Research Part B-Applied Biomaterials 2007; 82B: 440-449.









TABLE 2







177 glass compositions (mol. fraction) formulated


using design of experiments. The NM components


are varied between 0 to 0.140 mol. fraction.
















Std.











ORP#
Run
Si
Ca
Zn
Mg
La
Sr
Ti
Na



















78
1
0.000
0.150
0.520
0.050
0.140
0.000
0.140
0.000


4
2
0.290
0.000
0.570
0.000
0.000
0.140
0.000
0.000


17
3
0.290
0.140
0.520
0.050
0.000
0.000
0.000
0.000


132
4
0.000
0.000
0.570
0.050
0.100
0.140
0.000
0.140


150
5
0.000
0.150
0.520
0.050
0.000
0.000
0.140
0.140


73
6
0.200
0.000
0.520
0.000
0.140
0.000
0.140
0.000


6
7
0.290
0.000
0.570
0.000
0.000
0.000
0.000
0.140


43
8
0.290
0.000
0.520
0.000
0.000
0.140
0.050
0.000


10
9
0.000
0.290
0.570
0.000
0.000
0.000
0.140
0.000


125
10
0.000
0.200
0.520
0.000
0.000
0.140
0.000
0.140


14
11
0.290
0.000
0.520
0.050
0.000
0.140
0.000
0.000


142
12
0.000
0.010
0.520
0.050
0.140
0.140
0.000
0.140


121
13
0.000
0.100
0.570
0.050
0.140
0.000
0.000
0.140


20
14
0.000
0.290
0.520
0.050
0.000
0.000
0.140
0.000


144
15
0.000
0.000
0.520
0.050
0.140
0.140
0.010
0.140


171
16
0.000
0.000
0.570
0.010
0.000
0.140
0.140
0.140


62
17
0.000
0.100
0.570
0.050
0.140
0.140
0.000
0.000


152
18
0.000
0.100
0.570
0.050
0.000
0.000
0.140
0.140


151
19
0.100
0.000
0.570
0.050
0.000
0.000
0.140
0.140


15
20
0.290
0.000
0.520
0.050
0.000
0.000
0.140
0.000


153
21
0.000
0.000
0.570
0.050
0.100
0.000
0.140
0.140


27
22
0.290
0.090
0.570
0.050
0.000
0.000
0.000
0.000


8
23
0.000
0.290
0.570
0.000
0.140
0.000
0.000
0.000


145
24
0.200
0.000
0.520
0.000
0.000
0.000
0.140
0.140


63
25
0.290
0.000
0.520
0.000
0.000
0.000
0.140
0.050


134
26
0.060
0.000
0.520
0.000
0.140
0.140
0.000
0.140


107
27
0.290
0.000
0.520
0.000
0.000
0.000
0.050
0.140


148
28
0.000
0.150
0.570
0.000
0.000
0.000
0.140
0.140


160
29
0.000
0.000
0.570
0.010
0.140
0.000
0.140
0.140


120
30
0.100
0.000
0.570
0.050
0.140
0.000
0.000
0.140


75
31
0.150
0.000
0.570
0.000
0.140
0.000
0.140
0.000


128
32
0.150
0.000
0.520
0.050
0.000
0.140
0.000
0.140


108
33
0.050
0.290
0.520
0.000
0.000
0.000
0.000
0.140


159
34
0.000
0.010
0.570
0.000
0.140
0.000
0.140
0.140


24
35
0.290
0.000
0.570
0.050
0.000
0.090
0.000
0.000


164
36
0.000
0.000
0.530
0.050
0.140
0.000
0.140
0.140


9
37
0.000
0.290
0.570
0.000
0.000
0.140
0.000
0.000


124
38
0.200
0.000
0.520
0.000
0.000
0.140
0.000
0.140


90
39
0.100
0.000
0.570
0.050
0.000
0.140
0.140
0.000


126
40
0.150
0.000
0.570
0.000
0.000
0.140
0.000
0.140


174
41
0.000
0.010
0.520
0.050
0.000
0.140
0.140
0.140


141
42
0.010
0.000
0.520
0.050
0.140
0.140
0.000
0.140


7
43
0.290
0.140
0.570
0.000
0.000
0.000
0.000
0.000


92
44
0.000
0.000
0.570
0.050
0.100
0.140
0.140
0.000


74
45
0.000
0.200
0.520
0.000
0.140
0.000
0.140
0.000


122
46
0.000
0.000
0.570
0.050
0.140
0.100
0.000
0.140


135
47
0.000
0.060
0.520
0.000
0.140
0.140
0.000
0.140


64
48
0.290
0.050
0.520
0.000
0.000
0.000
0.140
0.000


93
49
0.000
0.000
0.520
0.000
0.140
0.140
0.140
0.060


12
50
0.140
0.290
0.570
0.000
0.000
0.000
0.000
0.000


95
51
0.000
0.060
0.520
0.000
0.140
0.140
0.140
0.000


119
52
0.000
0.150
0.520
0.050
0.140
0.000
0.000
0.140


162
53
0.010
0.000
0.520
0.050
0.140
0.000
0.140
0.140


69
54
0.000
0.290
0.520
0.000
0.050
0.000
0.140
0.000


80
55
0.100
0.000
0.570
0.050
0.140
0.000
0.140
0.000


112
56
0.240
0.000
0.570
0.050
0.000
0.000
0.000
0.140


49
57
0.050
0.290
0.520
0.000
0.000
0.140
0.000
0.000


157
58
0.000
0.000
0.520
0.000
0.140
0.060
0.140
0.140


133
59
0.000
0.000
0.570
0.050
0.000
0.140
0.100
0.140


23
60
0.290
0.000
0.570
0.050
0.090
0.000
0.000
0.000


86
61
0.000
0.150
0.570
0.000
0.000
0.140
0.140
0.000


47
62
0.000
0.290
0.520
0.000
0.000
0.140
0.050
0.000


21
63
0.000
0.290
0.520
0.050
0.000
0.000
0.000
0.140


19
64
0.000
0.290
0.520
0.050
0.000
0.140
0.000
0.000


71
65
0.240
0.000
0.570
0.050
0.000
0.000
0.140
0.000


56
66
0.000
0.150
0.570
0.000
0.140
0.140
0.000
0.000


176
67
0.000
0.000
0.520
0.050
0.010
0.140
0.140
0.140


102
68
0.000
0.000
0.520
0.050
0.140
0.140
0.140
0.010


67
69
0.000
0.290
0.520
0.000
0.000
0.000
0.140
0.050


76
70
0.000
0.150
0.570
0.000
0.140
0.000
0.140
0.000


99
71
0.000
0.000
0.570
0.010
0.140
0.140
0.140
0.000


115
72
0.000
0.200
0.520
0.000
0.140
0.000
0.000
0.140


58
73
0.000
0.150
0.520
0.050
0.140
0.140
0.000
0.000


34
74
0.290
0.000
0.520
0.000
0.140
0.000
0.050
0.000


29
75
0.000
0.290
0.570
0.050
0.000
0.090
0.000
0.000


41
76
0.240
0.000
0.570
0.050
0.140
0.000
0.000
0.000


163
77
0.000
0.010
0.520
0.050
0.140
0.000
0.140
0.140


87
78
0.150
0.000
0.520
0.050
0.000
0.140
0.140
0.000


139
79
0.000
0.000
0.570
0.010
0.140
0.140
0.000
0.140


167
80
0.000
0.060
0.520
0.000
0.000
0.140
0.140
0.140


48
81
0.000
0.290
0.520
0.000
0.000
0.140
0.000
0.050


65
82
0.290
0.000
0.520
0.000
0.050
0.000
0.140
0.000


5
83
0.290
0.000
0.570
0.000
0.000
0.000
0.140
0.000


156
84
0.000
0.060
0.520
0.000
0.140
0.000
0.140
0.140


88
85
0.000
0.150
0.520
0.050
0.000
0.140
0.140
0.000


45
86
0.290
0.050
0.520
0.000
0.000
0.140
0.000
0.000


83
87
0.200
0.000
0.520
0.000
0.000
0.140
0.140
0.000


40
88
0.050
0.290
0.520
0.000
0.140
0.000
0.000
0.000


173
89
0.010
0.000
0.520
0.050
0.000
0.140
0.140
0.140


51
90
0.240
0.000
0.570
0.050
0.000
0.140
0.000
0.000


165
91
0.000
0.000
0.520
0.050
0.140
0.010
0.140
0.140


28
92
0.000
0.290
0.570
0.050
0.090
0.000
0.000
0.000


113
93
0.000
0.240
0.570
0.050
0.000
0.000
0.000
0.140


44
94
0.290
0.000
0.520
0.000
0.000
0.140
0.000
0.050


118
95
0.150
0.000
0.520
0.050
0.140
0.000
0.000
0.140


131
96
0.000
0.100
0.570
0.050
0.000
0.140
0.000
0.140


66
97
0.290
0.000
0.520
0.000
0.000
0.050
0.140
0.000


26
98
0.290
0.000
0.570
0.050
0.000
0.000
0.000
0.090


170
99
0.000
0.010
0.570
0.000
0.000
0.140
0.140
0.140


52
100
0.000
0.240
0.570
0.050
0.000
0.140
0.000
0.000


104
101
0.290
0.050
0.520
0.000
0.000
0.000
0.000
0.140


46
102
0.290
0.000
0.520
0.000
0.050
0.140
0.000
0.000


37
103
0.000
0.290
0.520
0.000
0.140
0.050
0.000
0.000


36
104
0.290
0.050
0.520
0.000
0.140
0.000
0.000
0.000


146
105
0.000
0.200
0.520
0.000
0.000
0.000
0.140
0.140


158
106
0.010
0.000
0.570
0.000
0.140
0.000
0.140
0.140


89
107
0.000
0.000
0.570
0.050
0.000
0.140
0.140
0.100


109
108
0.000
0.290
0.520
0.000
0.050
0.000
0.000
0.140


96
109
0.010
0.000
0.570
0.000
0.140
0.140
0.140
0.000


18
110
0.000
0.290
0.520
0.050
0.140
0.000
0.000
0.000


25
111
0.290
0.000
0.570
0.050
0.000
0.000
0.090
0.000


42
112
0.000
0.240
0.570
0.050
0.140
0.000
0.000
0.000


32
113
0.090
0.290
0.570
0.050
0.000
0.000
0.000
0.000


116
114
0.150
0.000
0.570
0.000
0.140
0.000
0.000
0.140


149
115
0.150
0.000
0.520
0.050
0.000
0.000
0.140
0.140


129
116
0.000
0.150
0.520
0.050
0.000
0.140
0.000
0.140


117
117
0.000
0.150
0.570
0.000
0.140
0.000
0.000
0.140


101
118
0.000
0.010
0.520
0.050
0.140
0.140
0.140
0.000


82
119
0.000
0.000
0.570
0.050
0.140
0.100
0.140
0.000


84
120
0.000
0.200
0.520
0.000
0.000
0.140
0.140
0.000


2
121
0.190
0.290
0.520
0.000
0.000
0.000
0.000
0.000


61
122
0.100
0.000
0.570
0.050
0.140
0.140
0.000
0.000


175
123
0.000
0.000
0.530
0.050
0.000
0.140
0.140
0.140


166
124
0.060
0.000
0.520
0.000
0.000
0.140
0.140
0.140


11
125
0.000
0.290
0.570
0.000
0.000
0.000
0.000
0.140


97
126
0.000
0.010
0.570
0.000
0.140
0.140
0.140
0.000


168
127
0.000
0.000
0.520
0.000
0.060
0.140
0.140
0.140


13
128
0.290
0.000
0.520
0.050
0.140
0.000
0.000
0.000


22
129
0.140
0.290
0.520
0.050
0.000
0.000
0.000
0.000


147
130
0.150
0.000
0.570
0.000
0.000
0.000
0.140
0.140


68
131
0.050
0.290
0.520
0.000
0.000
0.000
0.140
0.000


39
132
0.000
0.290
0.520
0.000
0.140
0.000
0.000
0.050


1
133
0.290
0.190
0.520
0.000
0.000
0.000
0.000
0.000


81
134
0.000
0.100
0.570
0.050
0.140
0.000
0.140
0.000


54
135
0.000
0.200
0.520
0.000
0.140
0.140
0.000
0.000


94
136
0.060
0.000
0.520
0.000
0.140
0.140
0.140
0.000


31
137
0.000
0.290
0.570
0.050
0.000
0.000
0.000
0.090


50
138
0.000
0.290
0.520
0.000
0.050
0.140
0.000
0.000


53
139
0.200
0.000
0.520
0.000
0.140
0.140
0.000
0.000


138
140
0.000
0.010
0.570
0.000
0.140
0.140
0.000
0.140


114
141
0.200
0.000
0.520
0.000
0.140
0.000
0.000
0.140


127
142
0.000
0.150
0.570
0.000
0.000
0.140
0.000
0.140


143
143
0.000
0.000
0.530
0.050
0.140
0.140
0.000
0.140


91
144
0.000
0.100
0.570
0.050
0.000
0.140
0.140
0.000


38
145
0.000
0.290
0.520
0.000
0.140
0.000
0.050
0.000


123
146
0.000
0.000
0.570
0.050
0.140
0.000
0.100
0.140


155
147
0.060
0.000
0.520
0.000
0.140
0.000
0.140
0.140


16
148
0.290
0.000
0.520
0.050
0.000
0.000
0.000
0.140


55
149
0.150
0.000
0.570
0.000
0.140
0.140
0.000
0.000


161
150
0.000
0.000
0.570
0.000
0.140
0.010
0.140
0.140


105
151
0.290
0.000
0.520
0.000
0.050
0.000
0.000
0.140


59
152
0.000
0.000
0.570
0.050
0.140
0.140
0.100
0.000


33
153
0.290
0.000
0.520
0.000
0.140
0.050
0.000
0.000


172
154
0.000
0.000
0.570
0.000
0.010
0.140
0.140
0.140


72
155
0.000
0.240
0.570
0.050
0.000
0.000
0.140
0.000


3
156
0.290
0.000
0.570
0.000
0.140
0.000
0.000
0.000


77
157
0.150
0.000
0.520
0.050
0.140
0.000
0.140
0.000


177
158
0.085
0.085
0.543
0.023
0.066
0.066
0.066
0.066


35
159
0.290
0.000
0.520
0.000
0.140
0.000
0.000
0.050


57
160
0.150
0.000
0.520
0.050
0.140
0.140
0.000
0.000


100
161
0.010
0.000
0.520
0.050
0.140
0.140
0.140
0.000


169
162
0.010
0.000
0.570
0.000
0.000
0.140
0.140
0.140


60
163
0.000
0.000
0.570
0.050
0.140
0.140
0.000
0.100


111
164
0.000
0.290
0.520
0.000
0.000
0.000
0.050
0.140


70
165
0.000
0.290
0.520
0.000
0.000
0.050
0.140
0.000


140
166
0.000
0.000
0.570
0.000
0.140
0.140
0.010
0.140


154
167
0.000
0.000
0.570
0.050
0.000
0.100
0.140
0.140


137
168
0.010
0.000
0.570
0.000
0.140
0.140
0.000
0.140


106
169
0.290
0.000
0.520
0.000
0.000
0.050
0.000
0.140


98
170
0.000
0.000
0.570
0.000
0.140
0.140
0.140
0.010


79
171
0.000
0.000
0.570
0.050
0.140
0.000
0.140
0.100


30
172
0.000
0.290
0.570
0.050
0.000
0.000
0.090
0.000


85
173
0.150
0.000
0.570
0.000
0.000
0.140
0.140
0.000


103
174
0.000
0.000
0.530
0.050
0.140
0.140
0.140
0.000


110
175
0.000
0.290
0.520
0.000
0.000
0.050
0.000
0.140


130
176
0.100
0.000
0.570
0.050
0.000
0.140
0.000
0.140


136
177
0.000
0.000
0.520
0.000
0.140
0.140
0.060
0.140

## Claims

1. A particulate material comprising:
0.4-0.6 mole fraction SiO2;
0.00-0.1 mole fraction TiO2;
0.04-0.188 mole fraction La2O3;
0.008-0.290 mole fraction ZnO;
about 0.035 mote fraction Na3O;
about 0.035 mole fraction MgO;
about 0.035 mole fraction SrO; and
about 0.035 mole fraction CaO.

2. The particulate material of claim 1 wherein La2O3 is present at 0.04 to 0.1 mole fraction.

3. The particulate material of claim 2 wherein La2O3 is present at 0.04 to 0.07 mole fraction.

4. The particulate material of claim 1 wherein TiO2 is present at 0.017 to 0.05 mole fraction.

5. The particulate material of claim 1 wherein SiO2 present at 0.5-0.57 mole fraction.

6. The particulate material of claim 1 wherein the particulate material is radiopaque.

7. The particulate material claim 1 wherein the particulate material is biocompatible.

8. The particulate material of claim 1 wherein said particulate material is degradable in vivo.

9. The particulate material of claim 8 wherein said particulate material degrades substantially in more than six months.

10. The particulate material of claim 1 further comprising a therapeutic component.

11. The particulate material of claim 10 wherein said therapeutic component is released under physiological conditions.

12. The particulate material of claim 1 having a Q-speciation of Q1-Q3.

13. The particulate material of claim 12 having a Q-speciation of Q1.

14. The particulate material of claim 1 having an average diameter of 45-1180 μm.

15. The particulate material of claim 14 having an average diameter of 200-1000 μm.

16. The particulate material of claim 15 having an average diameter of 300-400 μm.

17. The particulate material of claim 15 having an average diameter of 500-710 μm.

18. The particulate material of claim 1 further comprising a polymeric coating.

19. The particulate material of claim 18 wherein said polymeric coating comprises poly(lactic-co-glycolic acid).

20. The particulate material of claim 18 wherein said polymeric coating comprises a poloxamer.

21. The particulate material of claim 20 wherein said polymeric coating comprises Pluronic F-127.

22. The particulate material of claim 18 further comprising a therapeutic component.

23. The particulate material of claim 22 wherein said therapeutic component is released under physiological conditions.

24. The particular material of claim 1 comprising no more than 0.1 mole fraction aluminosilicates, phosphates or a combination thereof.

25. The particulate material according to claim 1, wherein the particulate material consists of:
0.562 mole fraction SiO2;
0.042 mole fraction TiO2;
0.068 mole fraction La2O3;
0.035 mule fraction MgO;
0.035 mole fraction Na2O;
0.188 mole fraction ZnO;
0.035 mole fraction CaO; and
0.035 mole fraction SrO.

26. The particulate material of claim 1, wherein the particulate material consists essentially of:
0.4-0.6 mote fraction SiO2;
0.00-0.1 mole fraction TiO2;
0.04-0.188 mole fraction La2O3;
0.008-0.290 mole fraction ZnO;
about 0.035 mole fraction Na2O;
about 0.035 mole fraction MgO;
about 0.035 mole fraction SrO; and
about 0.035 mole fraction CaO.

27. The particulate material of claim 1, wherein the particulate material consists of:
0.4-0.6 mole fraction SiO2;
0.00-0.1 mole fraction TiO2;
0.04-0.188 mole fraction La2O3;
0.068-0.290 mole fraction ZnO; and
about 0.0435 mole fraction Na2O;
about 0.035 mole fraction MgO;
about 0.035 mole fraction SrO; and
about 0.035 mole fraction CaO.