POLYMER BLEND PHANTOMS

CROSS-RELATED APPLICATIONS

[0001] The present application claims the benefit of the filing date of provisional application 60/926,648, filed on April 27, 2007, which is incorporated by reference in its entirety.

FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under NIBIB/NIH grant number 1 ROl EB004012-01A1. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates to the use of phantoms to calibrate imaging systems.

BACKGROUND

[0004] Patients with low bone mass due to osteomalacia are often misdiagnosed as only having osteoporosis, since radiographs and blood and urine measurements frequently fail to distinguish the two conditions. Osteomalacia is a disease in which patients have impaired bone mineralization, and may have a reduced amount of bone tissue. Patients with osteoporosis have a reduced amount of bone that is normally mineralized. To differentiate between osteomalacia and osteoporosis, information about the relative proportions of bone mineral and organic bone matrix in a given volume of bone substance is needed. Current X-ray based bone mineral density measurements, such as dual energy X-ray densitometry (DXA) and quantitative computed tomography (QCT), only provide a measurement of bone mineral content, which is lower than normal in both osteomalacia and osteoporosis, but do not provide information about bone matrix density. One of the most important parameters distinguishing osteoporosis from osteomalacia is the degree of bone mineralization, which may be closely defined as the ratio of bone mineral density divided by bone matrix density.

[0005] Magnetic resonance imaging (MRI) creates images of biological tissues using signals originating from the hydrogen (proton, or IH) content of the tissues. Conventional MRI, as performed on MR scanners designed for human use, can detect proton signals only from fluid substances such as free water and fat. Bone matrix exhibits intermediate molecular mobility.

This soft solid material exhibits intermediate spectral linewidths that may be on the order of only a few kHz, and beyond the detection limit of conventional MRI.

[0006] Calibration phantoms are essential for quantitative MRI measurements, since they provide stable, accurate and known references. While there are phantoms specifically designed for fluid state proton density measurements, there remains a need for phantoms designed for solid-state proton density measurements of soft solids, such as bones.

SUMMARY

[0007] Provided herein is a phantom, which may be used to calibrate imaging systems. The phantom may be a proton calibration phantom. The phantom may be designed for soft solid density measurement. The phantom may comprise one or more polymers and quartz sand. The phantom may comprise a first polymer, a second polymer, and quartz sand. The first polymer may be a poly(ethylene oxide). The second polymer may be a poly(methyl methacrylate). The soft solid may be a bone matrix. The first and second polymer may be in a blend. The poly(ethylene oxide) may be between 5 and 30% and poly(methyl methacrylate) may be between 70 and 95%. The phantom may have a single IH NMR peak. The phantom may have a line width of between 1000Hz and 4000Hz. The quartz may be silicon dioxide. [0008] Also provided herein is a method of calibrating an imaging system. The phantom may be inserted into an imaging system and the phantom imaged to calibrate the system. The method may allow for determining the bone matrix density. The density of a soft solid may be determined using an imaging system. The phantom may be used to calibrate the system. A calibration curve may then be generated. The composition of interest may be imaged on the calibrated system and the signal intensity plotted on the calibration curve. The soft solid is selected from the group consisting of bone matrix, wax, and protein. Also provide herein is a method for diagnosing osteomalacia in a subject. The method may comprise determining the bone matrix density of a tissue in a subject and comparing the bone matrix density to bone mineral density, wherein a ratio of bone mineral density to bone matrix density of lower than 1 is indicative of osteomalacia. Treatment for osteomalacia in a subject may be monitored by periodically determining bone matrix density of a tissue in a subject; and comparing the bone matrix density to bone mineral density.

[0009] Also provided herein is an imaging system calibrated by the phantoms as herein described.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 shows a proton spectrum (A) and SMRI image (B) of the 20%PEO/PMMA blend phantoms. The diameters of the cylindrical phantoms are 0.52cm and thickness is 0.2-

0.25cm.

[0011] Figure 2 shows proton spectra, SMRI and WASPI images of a piece of bovine cortical bone, 4 polymer phantoms and a tube of bone marrow. A. Single pulse IH spectrum. Water and fat peaks were at 0.00 and -3.50ppm, respectively. B. Water and fat suppressed spectrum. C.

Non-suppressed SMRI. D. WASPI images.

[0012] Figure 3 shows a calibration curve relating known phantom densities to the measure MRI signal intensity of the bovine cortical bone.

DETAILED DESCRIPTION

[0013] Magnetic resonance imaging (MRI) creates images of biological tissues using signals originating from the hydrogen (proton or IH) content of the tissues. One important application of MRI is measuring the density of soft tissues to determine the shape, status of any deterioration or damage of the tissue, or present of foreign cell types such as cancer cells. Conventional MRI, as performed on MR scanners designed for human use, can detect proton signals only from free water in soft tissue and fat.

[0014] Most MR imaging centers have some form of machine and image quality control that ensures that the acquired images are of sufficient quality for clinical evaluation. Test objects called phantoms are used for this purpose. Phantoms are non-living models of living tissue that attempt to recapitulate or mimic the optical behavior of tissue. They can be used to determine the accuracy of volumetric measurements, resolution, and relaxation time measurements for soft tissues. None of the prior art phantoms however have been useful for ensuring quality MRI images of bones. Bones are a class of tissue that proton MR spectrals cannot detect. [0015] The inventors have made the surprising discovery of a new type of phantom that have MR properties that allow for the desired values to image bones. Specifically, the phantom blend of PEO and PMMA in quartz provides a signal intensity that can be converted to a numerically

equivalent value of a bone density matrix. The use of this polymer blend phantom can be used in quantitative magnetic resonance imaging of bone density matrix and will now allow physicians to distinguish bone related diseases such as osteoporosis from osteomalacia.

1. DEFINITIONS

[0016] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise.

[0017] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number

6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9, and 7.0 are explicitly contemplated. a. bone matrix

[0018] "Bone matrix" as used herein may mean an organic matrix made predominantly from the structural protein collagen. b. bone mineral

[0019] "Bone mineral" as used herein may mean calcium and phosphate as found almost exclusively as hydroxyapatite in the form of fine crystalline particles distributed selectively along collagen fibers of the bone matrix. The organic collagen matrix and the orientation of the proteins in that matrix may determine the architectural and structural integrity of bone. The normal lamellar pattern of the collagen fibrils may provide the tensile strength of bone. c. bone mineral density

[0020] "Bone mineral density" as used herein may mean an estimate of the total amount of mineral in a scanned area of whole bone. d. degree of bone mineralization

[0021] "Degree of bone mineralization" as used herein may be determined by the ratio of bone mineral density over bone matrix density. e. Fluid substances

[0022] "Fluid substances" as used herein may be free water and/or fat. The free water and/or fat may be dispersed within a rigid solid substance and soft solid.

f. gravimetric analysis

[0023] "Gravimetric analysis" as used herein may mean an analysis in which the amounts of the constituents are determined by weight; in distinction from volumetric analysis. g. rigid solid substance

[0024] "Rigid solid substance may be an organic or inorganic crystalline compound. The inorganic crystalline compound may be hydroxyapatite, which may include calcium phosphate, calcium carbonate, calcium fluoride, calcium hydroxide and citrate. h. solid state MRI

[0025] "Solid state MRF' as used herein may mean any magnetic resonance imaging system employed to generate data reflecting the spatial distribution of one or more isotopes carried in a solid-state specimen. i. soft solid

[0026] "Soft solid" as used herein may mean a protein aggregate, wax, polymer, or blend of polymers. The soft solid may be bone matrix comprising collagen and small amounts of other organic constituents and other extracellular, extravascular organic constituents. j. WASPI

[0027] "WASPF' as used herein may mean water- and fat-suppressed solid-state proton projection imaging. k. wet chemical analysis

[0028] "Wet chemical analysis" may involve dissolving a mineral into an acidic solution. In order for the dissolution to take place completely, the mineral may first be ground into a fine powder (to increase surface area) and the appropriate acid must be used. Wet chemical analysis may be classified into three different types: (1) gravimetric analyses, wherein the element of interest may be precipitated as a compound. The precipitate may then weighed to determine its proportion in the original sample; (2) volumetric analyses, wherein titration may be used to determine the amount of reagent that is added in order for a specific chemical reaction to occur that involves the element of interest. From the volume of reagent added, the concentration of the element may be calculated; and (3) colorometric analyses, wherein a reagent may be added to the solution that reacts with the element of interest to produce a color change in the solution. The intensity of the color may be proportional to the concentration of the element of interest, and thus when compared to standard solutions in which the concentration is known, the concentration of

the element in the unknown solution may be determined. All elements may not be able to be determined by the same wet chemical methods and different methods are more sensitive for different elements. Thus, a complete wet chemical analysis may involve a combination of methods. 2. Phantom

[0029] Provided herein is a test device for use in conjunction with medical imaging modalities including CT, ultrasound, x-ray, nuclear medicine, and magnetic resonance imaging (MRI) to ensure sufficient quality of images for clinical evaluation. The test device may be a phantom. The phantom may be a device or composition that simulates or mimics the resonance behavior of biological tissue or organs. Phantoms may be incorporated into a number of shapes and configurations depending on the purpose. Among these include cylinders, spheres, and irregular or deformable compartments. For example, the phantom may be configured into the shape of a particular bone of interest for imaging. a. Polymer Blend

[0030] The phantom may be a blend of one or more polymers and quartz. The polymer provides the proton signal and induces magnetic resonance properties (Tl, T2,and resonance line shape). The first polymer may be molecular softer polymer. The second polymer may be rigid glassy polymer.

[0031] The molecularly softer polymer may be high molecular weight polymers such as polyalkylene oxides such as polyethylene oxide, and cellulosic polymer derivatives including hydroxypropyl cellulose, hydroxypropyl methyl 5 cellulose, and hydroxyethyl cellulose. The rigid glassy polymers may include sodium and calcium polyacrylic acid, polyacrylic acid, polymethaacrylic acid, and poly (methyl methacrylate). The terms "cellulose" and "cellulosic" are used herein to denote a linear polymer of anhydroglucose. The alkyl-substituted cellulose derivatives may be those substituted with alkyl groups of 1 to 3 carbon atoms each such as methylcellulose, hydroxymethyl-cellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and carboxymethylcellulose. In terms of their viscosities, one class of alkyl-substituted celluloses may include those whose viscosity is within the range of about 100 to about 110,000 centipoise as a 2% aqueous solution at 20.degree. C. Another class may include those whose viscosity is within the range of about 1,000 to about 4,000 centipoise as a 1% aqueous solution at 20 ⁰ C. Alkyl-substituted celluloses may be hydroxyethylcellulose and

hydroxypropylmethylcellulose. Hydroxyethylcellulose may be NATRAS OL.RTM. 250HX NF (National Formulary), available from Aqualon Company, Wilmington, Del., USA. [0032] Polyalkylene oxides may have those properties described above for alkyl- substituted cellulose polymers. A polyakylene oxide polymer may be poly(ethylene oxide), which term is used herein to denote a linear polymer of unsubstituted ethylene oxide. Poly(ethylene oxide) polymers may have a molecular weights of about 4,000,000 and higher. PEO may also have molecular weights within the range of about 4,500,000 to about 10,000,000, or with molecular weights within the range of about 5,000,000 to about 8,000,000. Poly(ethylene oxide)s may be in the weight- average molecular weight within the range of about 1 x 10 ⁵ to about 1 x 10 ⁷ , or within the range of about 9 x 10 ⁵ to about 8 x 10 ⁶ .

[0033] Poly(ethylene oxide)s may also be characterized by their viscosity in solution. The viscosity range of PEO may be about 50 to about 2,000,000 centipoise for a 2% aqueous solution at 20 ⁰ C. Two presently preferred polyethylene oxide)s are POLYOX.RTM. NF, grade WSR Coagulant, molecular weight 5 million, and grade WSR 303, molecular weight 7 million, both products of Union Carbide Chemicals and Plastics Company Inc. of Danbury, Conn., USA. [0034] Crosslinked polyacrylic acids may be those properties as described above for alkyl- substituted cellulose and polyalkylene oxide polymers. Polyacrylic acids may be those with a viscosity ranging from about 4,000 to about 40,000 centipoise for a 1% aqueous solution at 25°C. Polyakylene oxide polymers may be CARBOPOL®. NF grades 97 IP, 974P and 934P (BFGoodrich Co., Specialty Polymers and Chemicals Div., Cleveland, Ohio, USA). Polyakylene oxide polymers may also be WATER LOCK®, which are starch/acrylates/acrylamide copolymers available from Grain Processing Corporation, Muscatine, Iowa, USA. [0035] The polymer may be a blend of poly(ethylene oxide) and poly(methyl methacrylate) (PMMA). The PEO and PMMA blend may comprise between 5% and 30% PEO and between 70-95% PMMA. The blend may comprise between 10 and 25% PEO and between 75 and 90% PMMA. The blend may comprise 20% PEO and 80% PMMA. b. Quartz

[0036] The quartz may be a substance that contributes no proton signal and has minimal artifact- inducing magnetic properties. The quartz may be natural or synthetic. Natural quartz may be smoky, bubble, coontail, cubic, phantom, rainbow, rose, amethyst, milky, chalcedony, agate, onyx, jasper, aventurine, tiger's eye, rock crystal, citrine, prasiolite, rutilated, morion, or

carnelian. Prasiolite may also be synthetically derived. Quartz may be crushed silica dioxide

(SiO ₂ ). The quartz may be quartz sand.

3. Combination of Phantom with Contrast Agent

[0037] The phantom of the invention may include a contrast enhancing agent. The phantom of the invention may include one or more contrast enhancing agents selected from an X-ray contrast agent, a CAT contrast agent, an ultrasound contrast agent and a magnetic resonance imaging contrast agent. A phantom of the invention may include more than one type of contrast agent for the same or different imaging modalities.

[0038] Contrast agents are useful adjuncts in radiological imaging, making it possible to determine the location, size and conformation of organs or other structures of the body in the context of their surrounding tissues. Exemplary X-ray contrast agents may include insoluble inorganic barium salts, which may enhance X-ray attenuation in the body zones into which they distribute. Other X-ray contrast agents may include soluble iodine containing compounds such as those marketed by Nycomed AS under the trade names Omnipaque® and Amipaque®. Recent work on X-ray contrast agents has concentrated on aminopolycarboxylic acid (APCA) chelates of heavy metal ions.

[0039] Contrast agents for MR imaging may be based on paramagnetic metal chelates or ferri- or ferro -magnetic particles. Chelates with high thermodynamic and kinetic stabilities may be used because their ability to remain stable in vivo offers a distinct benefit to MR imaging. Chelating agents include 1,4,7, 10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA). Further examples are 1,4,7, 10-tetraazacyclododecane-N,N',N"-triacetic acid (D03A), diethylene- triaminepentaacetic acid (DTPA) and various analogs and derivatives of both ligands. [0040] Chelated or unchelated paramagnetic metal ions may be of use in the phantom of the invention. Paramagnetic metals of a wide range are suitable for complexation with these ligands. Suitable metals are those having atomic numbers of 22-29 (inclusive), 42, 44 and 58 70 (inclusive), and have oxidations states of 2 or 3. Those having atomic numbers of 22-29 (inclusive) and 58-70 (inclusive) are preferred, and those having atomic numbers of 24-29 (inclusive) and 64-68 (inclusive) are more preferred. Examples of such metals are chromium (III), manganese (II), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III),

erbium (III) and ytterbium (III). Manganese (II), iron (III) and gadolinium (III) are particularly preferred, with gadolinium (III) the most preferred. 4. Use of Phantom

[0041] The phantom may be used to simulate a body of tissue or organ in conjunction with medical imaging modalities including CT, MRI, solid state MRI, WASPI, ultrasound, x-ray, and nuclear medicine. The phantom may be used marking, calibrating, and aligning soft-solid images from CAT and MRI systems. The phantom may be used to determine the accuracy of volumetric arrangements, resolution, and relaxation time measurements of soft-solids. The phantom may also be used to determine longitudinal and inter-site stability of measuring soft- solids. The phantom may also be used in image quality control to compensate for noise, assess spatial resolution, sensitivity, slice, thickness, focal zone, system sensitivity, gray scale, dynamic range, penetration, dead zone, and dose.

[0042] The phantom may be used to determine the accuracy of volumetric arrangements, resolution, and relaxation time measurements of solid bone matrix. The phantom may also be used to determine the longitudinal and inter-site stability of a solid bone matrix. The phantom includes an offset configuration that produces three-dimensional spaces that models critical aspects of the solid bone matrix. The phantom may be used mimic the collagen fibrils and other immobile intracellular and extracellular molecules such as tightly bound water within the solid bone matrix.

[0043] When the phantom of the invention is used in conjunction with a nuclear medicine procedure, the phantom is useful to assess the performance of gamma cameras (single photon emission computed tomography and positron emission tomography) for field uniformity, volume sensitivity, spatial resolution, lesion detectability, etc.

[0044] The phantom of the invention is useful for quality control, calibration and testing of radiographic, fluoroscopic, tomographic and angiographic equipment. The phantom is used to evaluate the system for contrast, resolution, image quality, image, intensifier performance, and exposure.

[0045] The phantom of the invention may also be used in conjunction with radiotherapy. The phantom is utilized to measure radiation dose, dose distributions, and other treatment parameters. [0046] The phantom may be imaged at regular intervals over the entire time period (e.g., months to years) that a given patient or group of patients is to be studied. The images and measurements

derived from the phantom may be used to check the accuracy and longitudinal precision of corresponding images and measurements derived from patients and correct them as necessary. Accurate measurements of known precision may be essential in tracking small changes in disease state and response to therapy.

[0047] The phantom may be used as a training tool for simulations of operation of the system under real conditions. For example, rather than having a technician practice using a new imaging system (e.g., for imaging breast tissue) on living patients, often at considerable discomfort to the patient, technicians can use the new tissue-like phantoms.

[0048] For surgeons, the new tissue-like phantoms may permit surgeons to "operate" (physically cut and dissect) on them to learn the functions of the imaging system and to become proficient in using, e.g., the NIR fluorescence imaging and pseudo-colored overlay of a given system to find target tumors and perform surgery (e.g., removal of a tumor mass). It may be far better and cheaper training for the surgeon than to use an animal, human cadaver, or living patient for training purposes. Anything that decreases the learning curve for surgeons may ultimately save thousands of dollars and decrease risk to patients. a. Method of Determining Bone Matrix Density

[0049] The phantom described herein may be used to convert MRI intensity of a bone. The bone of a subject may contain both fluid and soft solid constituents. The phantom may be used to determine the true density (in g/cm ³ ) of the soft solid constituent of the bone, and/or of the subject. The polymers of the phantom may be utilized with MRI properties similar to those of solid bone matrix. The MRI properties may be Tl, T2, and reasonance line shape.

(1) Calibration Curve

[0050] The phantom may be used in solid state MRI according to the methods described in Wu et ah, Proc. Natl. Acad. ScL USA, 96:1574-1578 (1999). The phantom may be used in WASPI. The solid state MRI and/or WASPI may be described by the level of line widths of the phantom and a T2* value of the phantom. The phantom may be created at different densities (g/cm ³ ) in order to establish a calibration curve of MRI signal intensity vs. phantom density. Phantom densities may be between 0.1 g/cm ³ to 10 g/cm ³ or 0.10 g/cm ³ , 0.20 g/cm ³ , 0.30 g/cm ³ , 0.40 g/cm ³ , 0.50 g/cm ³ , 0.60 g/cm ³ , 0.70 g/cm ³ , 0.80 g/cm ³ , 0.90 g/cm ³ , 1.0 g/cm ³ , 1.1 g/cm ³ , 1.2 g/cm ³ , 1.3 g/cm ³ , 1.4 g/cm ³ , 1.5 g/cm ³ , 1.6 g/cm ³ , 1.7 g/cm ³ , 1.8 g/cm ³ , 1.9 g/cm ³ , 2.0 g/cm ³ , 2.1 g/cm ³ , 2.2 g/cm ³ , 2.3 g/cm ³ , etc.

[0051] The phantom may be used to minimize the need to correct differences in Tl and/or T2 relaxation between the phantom and the subject. The differences in Tl and T2 time constrants involved in relaxation processes that establish equilibrium following resonance frequency excitation. As the high-energy nuclei relax and realign, they emit energy at rates which are recorded to provide information about the material they are in. The realignment of nuclear spins with the magnetic field is termed longitudinal relaxation and the time required for a certain percentage of the tissue's nuclei to realign is termed "Time 1" or Tl, which is typically about 1 second at 1.5 tesla main field strength. T2-weighted imaging relies upon local dephasing of spins following the application of the tranvsere energy pulse; the transverse relaxation time is termed "Time 2" or T2, typically < 100 ms for tissue at 1.5 tesla main field strength. A subtle but important variant of the T2 technique is called T2* imaging. T2 imaging employs a spin echo technique, in which spins are refocused to compensate for local magnetic field inhomogeneities. T2* imaging is performed without refocusing. This sacrifices some image integrity (resolution) but provides additional sensitivity to relaxation processes that cause incoherence of transverse magnetization. The phantom may have a single T2* value that is comparable to that of solid bone matrix. The T2* value may be within the range of 10μs-300 μs, 50-250 μs, or 75-200 μs, 100 μs-150 μs.

[0052] MRI density values of phantoms may be calculated by summing all the pixel values above a threshold value in a rectangular volume of the 3D image and divided by that volume. The line widths of the phantom may be between 500 Hz and 4500 Hz or 500 Hz, 600 Hz, 700 Hz, 800 Hz, 900 Hz, 1000 Hz, 11000 Hz, 12000 Hz, 1300 Hz, 1400 Hz, 1500 Hz, 1600 Hz, 1700 Hz, 1800 Hz, 1900 Hz, 2000 Hz, 2100 Hz, 2200 Hz, 2300 Hz, 2400 Hz, 2500 Hz, 2600 Hz, 2700 Hz, 2800 Hz, 2900 Hz, 3000 Hz, 3100 Hz, 3200 Hz, 3300 Hz, 3400 Hz, 3500 Hz, 3600 Hz, 3700 Hz, 3800 Hz, 3900 Hz, 4000 Hz, 4100 Hz, 4200 Hz, 4300 Hz, 4400 Hz, and 4500 Hz, which comparable that solid bone matrix. The MR intensity of a bone specimen can be converted to density information as g/cm3 by comparing the MR intensity of bone to the calibration curve generated by the phantoms of varying densities.

[0053] The bone matrix density may be derived from a phantom generated calibration curve; bone specimens may be compared with the calibration curve to determine the matrix density of the specimens.

(2) Soft Solid Comparison

[0054] The phantom may have magnetic resonance properties similar to those of the soft solid. The phantom may have linewidth properties similar to those of the soft solid. The soft solid may have a magnetic resonance ("MR") spectral linewidth of between 150 Hz and 25kHz. The soft solid may have a MR spectral linewidth of between 500 Hz and 10 kHz, 750 Hz and 7 kHz, 1 kHz and 5 kHz, 1 kHz and 4 kHz, 1 kHz and 3 kHz, 1.5 kHz and 3 kHz , 2 kHz and 4 kHz , or 2 kHz and 3 kHz. The density of bone matrix may be used to determine the degree of bone mineralization ("DM"), as described above.

(3) Fluid Comparison

[0055] The soft solid may be present in a specimen that also comprises fluid, and/or a rigid solid substance. The fluid may have MR spectral linewidths of less than 150 Hz, less than 125 Hz, less than 100 Hz, less than 75 Hz, or less than 50 Hz. The fluid may have spin-spin relaxation times T ₂ or T ₂ * of 3 milliseconds ("ms"), 4 ms, 5 ms, 10 ms, 20 ms, or longer. The fluid may be free water and/or fat. The rigid solid substance may have MR spectral linewidths of 25 kHz or more. The rigid solid substance may have a MR spectral linewidth of 30 kHz, 40 kHz, 50 kHz, 60 kHz, 70 kHz, 80 kHz, 90 kHz, 100 kHz, or more. The rigid solid substance may have spin-spin relaxation times T2 of between 5 and 25 microseconds ("μs"). The rigid solid substance may have a spin-spin relaxation time T2 of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 μs. The rigid solid substance may be an organic or inorganic crystalline compound. The inorganic crystalline compound may be hydroxyapatite, which may include calcium phosphate, calcium carbonate, calcium fluoride, calcium hydroxide and citrate. [0056] The imaging system may be a water and fat suppressed ¹ H projection MRI ("WASPI") system. The phantom may be used in conjunction with any imaging system to quantitatively measure bone matrix density.

[0057] The phantom of the solid bone matrix may be used to provide a signal of fluid constituents, primarily the molecularly mobile water and lipids that are largely suppressed in solid bone matrix. The phantom may provide a calibration line width of WASPl IH NMR signals in the range of several thousand Hz, corresponding to T2*s on the order of lOOμs. The phantom may have a single IH NMR peak corresponding to a single T2*, and a line width ranging from 500Hz to 3000 Hz, or 1000Hz to 2000 Hz, corresponding to the T2*s on the order

of lOOμs. The solid state MRI images of the phantom may be used to show the linearity of the

MRI intensities with different phantom densities.

[0058] Various methods may be employed as controls to obtain bone matrix density of a specimen. Such methods may include wet chemistry analysis techniques. Such techniques may include gravimetric analysis.

5. Measurement of Bone Mineral Content

[0059] Several noninvasive techniques may be used for estimating skeletal mass or density.

These include dual-energy x-ray absorptiometry (DXA), single-energy x-ray absorptiometry

(SXA), quantitative computed tomography (CT), and ultrasound. These x-ray based bone mineral density measurements, including quantitative computed tomography (QCT), may only provide a measurement of bone mineral content, which may be lower than normal in both osteomalacia and osteoporosis, but do not provide information about bone matrix density. a. DXA

[0060] DXA is a highly accurate x-ray technique that has become the standard for measuring bone density in most centers. Though it can be used for measurements of any skeletal site, clinical determinations are usually made of the lumbar spine and hip. Portable DXA machines have been developed that measure the heel (calcaneus), forearm (radius and ulna), or finger (phalanges), and DXA can also be used to measure body composition. In the DXA technique, two x-ray energies are used to estimate the area of mineralized tissue, and the mineral content is divided by the area, which partially corrects for body size. However, this correction is only partial since DXA is a two-dimensional scanning technique and cannot estimate the depths or posteroanterior length of the bone. Thus, small people tend to have lower-than-average bone mineral density (BMD). Newer DXA techniques that measure information BMD are currently under evaluation. Bone spurs, which are frequent in osteoarthritis, tend to falsely increase bone density of the spine. Because DXA instrumentation is provided by several different manufacturers, the output varies in absolute terms. Consequently, it has become standard practice to relate the results to "normal" values using T-scores, which compare individual results to those in a young population that is matched for race and gender. Alternatively, Z-scores compare individual results to those of an age-matched population that is also matched for race and gender. Thus, a 60-year-old woman with a Z-score of -1 (1 SD below mean for age) could have a T-score of -2.5 (2.5 SD below mean for a young control group).

b. CT

[0061] CT is used primarily to measure the spine, and peripheral CT is used to measure bone in the forearm or tibia. Research into the use of CT for measurement of the hip is ongoing. CT has the added advantage of studying bone density in subtypes of bone, e.g., trabecular vs. cortical. The results obtained from CT are different from all others currently available since this technique specifically analyzes trabecular bone and can provide a true density (mass of bone per unit volume) measurement. However, CT remains expensive, involves greater radiation exposure, and is less reproducible. c. Ultrasound

[0062] Ultrasound is used to measure bone mass by calculating the attenuation of the signal as it passes through bone or the speed with which it traverses the bone. It is unclear whether ultrasound assesses bone quality, but this may be an advantage of the technique. Because of its relatively low cost and mobility, ultrasound is amenable for use as a screening procedure. [0063] All of these techniques for measuring BMD have been approved by the U.S. Food and Drug Administration (FDA) based upon their capacity to predict fracture risk. The hip may be the preferred site of measurement in most individuals, since it directly assesses bone mass at an important fracture site. When hip measurements are performed by DXA, the spine may be measured at the same time. In younger individuals, such as perimenopausal women, spine measurements may be the most sensitive indicator of bone loss. 6. Degree of Bone Mineralization

[0064] Bone is made in two stages; matrix is formed first, and about two weeks later (in children) it begins to mineralize. The process of adding mineral to bone is often referred to as bone mineralization. In the absence of rickets or osteomalacia, the degree of mineralization of matrix varies between fairly narrow limits, so that for the most part mineral mass is a reasonable estimate of bone mass. The degree of bone mineralization may be defined as the ratio of bone mineral density divided by bone matrix density. Quantitative bone mineral density may be obtained by solid-state 3 IP magnetic resonance imaging (3 IP SMRI) as described in Wu et al., Calcif. Tissue Int (62):512-518 (1998), which is herein incorporated by reference. As described herein, the bone matrix density may be derived from a phantom generated calibration curve; bone specimens may be compared with the calibration curve to determine the matrix density of the specimens. MRI intensity of the bone specimens may be converted to density information as

g/cm ³ by comparing the MR intensity of bone to the calibration curve generated by the phantoms. As described herein, use of the phantom may allow the conversion of imaging measurements of bone matrix into density information. Therefore, upon obtaining the bone mineral density and the bone matrix density, one may determine the degree of bone mineralization.

7. Diagnosis of Diseases and Disorders of the Bone

[0065] Provided herein is a method for diagnosing disease and/or disorders of the bone. The method for diagnosing a bone disease and/or disorder in a subject may comprise (a) determining bone matrix density of a tissue in a subject; and (b) comparing the bone matrix density to bone mineral density. A ratio of bone matrix density to bone mineral density of greater than X may be indicative of osteomalacia.

[0066] Several types of bone disorders may occur from an imbalance of the growth and breakdown cycles of bone. a. Osteoporosis

[0067] The most common, osteoporosis, is a metabolic bone disease characterized by a low bone mass and microarchitectural deterioration of bone tissue leading to progressive decrease in the density of bones, causing them to weaken. A distinguishing characteristic of osteoporosis is the normal mineral/collagen ration in affected tissues, in contrast to the disease osteomalacia in which a mineral deficiency relative to collagen is observed. Osteoporosis occurs in several different types and is seen more often in older women. Postmenopausal osteoporosis is generally found in women between the ages 51 and 75 and is caused by the lack of estrogen. Senile osteoporosis results not only from the imbalance between growth and breakdown but also from the calcium deficiency associated with age. Secondary osteoporosis is caused by secondary effects of another medical condition (e.g., chronic renal failure, hormonal disorders) or by drugs (e.g., barbiturates, anticonvulsants). Idiopathic juvenile osteoporosis is a rare form that occurs in children and young adults who, for no obvious reason, have weak bones. Treatment for osteoporosis may be aimed at increasing bone density (e.g., estrogen intake, bisphosphonates, fluoride supplements). b. Osteomalacia

[0068] Osteomalacia may be a condition marked by softening of the bones, with pain, tenderness, muscular weakness, anorexia and loss of weight, resulting from deficiency of vitamin

D and calcium. The softening of bones may be due to impaired mineralization, with excess accumulation of osteoid. Osteomalacia may be caused by vitamin D deficiency or by a digestive tract or kidney disorder. These disorders may interfere with the body's use of vitamin D. Osteomalacia may be caused by a low phosphate level.

[0069] Osteomalacia causes fatigue and pain in the back, ribs, and hips. Muscles in the upper arms and thighs become weak. People with osteomalacia may have trouble getting up from a chair or climbing steps. Like osteoporosis, osteomalacia may lead to bone fractures. [0070] Doctors diagnose osteomalacia with blood tests, x-rays, and sometimes a biopsy. Osteomalacia is treated with vitamin D or phosphate supplements depending on the cause. c. Paget's Disease

[0071] Paget's Disease also results from an imbalance of the growth and breakdown of bone. The turnover rate is areas affected by Paget's Disease increases tremendously; resulting in abnormal, enlarged bone that is soft and weak. Although no specific genetic pattern has been determined, Paget's Disease tends to appear in family lineages. There may be no direct treatment for Paget's Disease, rather treatment may be given only alleviate pain and discomfort. d. Infection

[0072] Bone disorders can also result from infection. Bone can be infected through three routes: bloodstream, direct invasion, and adjacent soft tissue infections. Osteomyelitis is a bone infection usually caused by bacteria (e.g., Staphylococcus aureus) which results in swelling of the soft bone marrow tissue, compression of the blood vessels, and possibly death of parts of bone. Pott's disease is an infection of the vertebrae by the bacteria that cause tuberculosis (e.g., Mycobacterium tuberculosis, M. bovis, or M. africanum.) For acute infections, antibiotics may be the most effective treatment for this disease. However, if the infection is severe or chronic, surgery may also be required to remove the infected tissue and replaced with healthy bone, muscle, or skin. e. Cancer

[0073] Most bone carcinomas are benign. The most common type of benign bone tumor, usually occurring in people aged 10 to 20, is osteochrondroma. Osteochrondromas are growths on the surface of a bone that protrude as hard lumps. Benign chondromas, usually occurring in people aged 10 to 30, develop in the central part of the bone. Chrondroblastomas, usually occurring in

people aged 10 to 20, are rare, painful tumors that grow in the ends of bones. Osteoid osteomas are very small tumors that commonly develop in the arms or legs but can occur in any bone. Giant cell tumors, usually occurring in people aged 20-40, most commonly originate in the ends of the bones and may extend into adjacent tissue. Treatment of these tumors may involve pain management and/or surgery to remove the tumor.

[0074] Although rare, malignant bone tumors may be primary or metastatic. In children, most malignant bone tumors are primary; in adults, most are metastatic. The most common type of malignant primary tumor, multiple myeloma, originates in the red bone marrow cells and most commonly occurs in older people. Osteosarcoma, usually occurring in people aged 10 20, commonly occurs in or around the knee and cause pain and swelling. These tumors tend to spread to the lungs. Chrondrosarcomas are slow-growing tumors composed of cancerous cartilage cells. Ewing's sarcoma, occurring most commonly in males aged 10 to 20, develop most often in arms and legs. These tumors can become large and can affect the entire length of a bone. Metastatic bone tumors most often originate from breast, lung, prostate, kidney and thyroid cancers.

[0075] Treatment for bone tumors depends on the type of cancer. Most treatments are complex and involve a combination of chemotherapy, radiotherapy, and surgery. Prompt treatment is especially important for malignant bone tumors.

8. Method of Monitoring Treatment of Diseases or Disorders of the Bone [0076] Also provided herein is a method of monitoring a subject for effective treatment of disease and/or disorders of the bone. The subject may have been determined to have a predisposition for a disease or disorder of the bone. The subject may already have a disease or disorder of the bone. It may be desirable to measure the effects of treatment on the bone disease or disorder by treating the patient using a method comprising monitoring the bone disease or disorder. Monitoring for bone disease or bone disorder, or progression of bone disease or disorder, may include any test that measures bone matrix density and/or bone mineral density. A method for monitoring a treatment of a bone disease and/or disorder in a subject may comprise (a) periodically determining bone matrix density of a tissue in a subject; and (b) comparing the bone matrix density to bone mineral density. The bone mineral density may also be periodically determined. A ratio of bone mineral density to bone matrix density of lower than 5, 4, 3, 2, 1, 0.5, or 0.25 may be indicative of a bone disorder or disease. A ratio of bone mineral density to

bone matrix density may correspond to bone mineral loss of between 1% and 40%, between 1% and 20%, between 5% and 15%, between 5% and 10%, between 2% and 7%, or between 3% and 6%. The phantom may be imaged at regular intervals over the entire time period (e.g., months to years) that a given patient or group of patients is to be studied.

9. Method of Calibrating an Imaging System

[0077] Also provided herein is a method of calibrating an imaging system. The inserting the phantom into an imaging system and imaging the phantom to calibrate the system. The imaging system may be any imaging system. The imaging system may be a CAT scan (CT) system, positron emission tomography (PET), fluoroscopy, MRI system, ultrasound system, x-ray system, WASPI system, and/or NMR system. The MRI system may be a solid-state MRI system as described in Wu. et al, Proc. Natl. Acad. Sci. USA, Vol. 96, pp. 1574-1578 (1999) and PCT/US2003/015801, which are herein incorporated by reference.

[0078] The phantom may be used to compare performance among different imaging systems in terms of sensitivity, reconstruction algorithms, and other system features. For commercial companies, the phantom may provide a valuable standardization tool to compare performance of any one imaging system against another type of system.

10. Imaging System Calibrated by the Phantom

[0079] Provided herein is an imaging system calibrated by the phantom. The imaging system may be any imaging system. The imaging system may be a CAT scan (CT) system, positron emission tomography (PET), fluoroscopy, MRI system, ultrasound system, x-ray system, WASPI system, and/or NMR system. The MRI system may be a solid-state MRI system as described in Wu. et al., Proc. Natl. Acad. Sci. USA, Vol. 96, pp. 1574-1578 (1999), which is herein incorporated by reference.

[0080] The phantom of the invention may be configured to model various regions and tissues of the body. . In a preferred example, the phantom is configured to model articular cartilage of a mammalian joint, preferably a knee joint.

[0081] The phantom can be utilized to model any property of a constituent or array of constituents of the mammalian joint. For example, properties of articular cartilage that are modeled using a phantom of the invention include the thickness of the cartilage, the curvature of the cartilage and a combination thereof.

EXAMPLES

Example 1 Polymer Blend Phantom

[0082] 20% polyethylene oxide) (PEO)/80% poly(methyl methacrylate (PMMA) blends were prepared by solvent casting from a chloroform solution. Clear and transparent blend film samples were obtained and ground into powder under liquid nitrogen. A fine uniform polymer powder was obtained by sieving through a 200-mesh screen. Cylindrical pellets of the polymer blend powder diluted with silicon dioxide (Fisher Scientific, 40-100 mesh) were made under high pressure (2000-4000 Ib) in a hydraulic press and used as an MRI calibration phantom. The polymer phantom densities were 1.17, 0.80, 0.56, and 0.40 g/cm ³ .

Example 2 MRI Values for Bone Specimens and Phantoms

[0083] Bovine bone specimens of 1.5cm (L) x 1.5cm (W) x 0.2cm (thick) were cut from the midshaft cortices of fresh bovine femora from a local slaughterhouse. Bone marrow was extracted from the same bovine cortical bone as reference for water and fat suppression in WASPI experiments. Volumes of bone specimens were measured by the water displacement method.

[0084] Four-month- old virgin female NIHRNU rats (Charles River Laboratories, Charlestown, MA) were used in this study. Following sacrifice the femurs were disarticulated from the hip and knee for MRI scanning. Solid state MRI and WASPI data were acquired and processed according to the previous description (See Wu et al., Calcif. Tissue Int (62):512-518 (1998), and Wu et al., Magn. Reson. Med. (2003) 50:59-68, which are herein incorporated by reference) with a Bruker 4.7T 33cm scanner equipped with a 400mT/m gradient system (Bruker Biospin, Billerica, MA, USA). The IH larmor frequency was 200.13 MHz. MRI density values of bone specimens and phantoms were calculated by summing all the pixel values above a threshold value in a rectangular volume of the 3D image containing each object to be analyzed and dividing by that volume. After MRI, gravimetric analyses (2) were performed on the same bone specimens to obtain bone matrix density for comparison.

[0085] The observable WASPI IH MRI signal of bone arises from solid matrix, which is dominated by collagen fibrils and other immobile intracellular and extracellular molecules such as tightly bound water. The signal of fluid constituents, primarily molecularly mobile water and lipid were largely suppressed. The line widths of WASPI IH NMR signals are in the range of several thousand Hz, corresponding to T2*s on the order of lOOμs. Therefore, the calibration phantom may have a single T2* which is comparable to that of solid bone matrix. As shown in figure IA, the polymer phantoms have a single IH NMR peak, corresponding to a single T2*, and the line width is around 2000Hz, corresponding to the T2*s on the order of lOOμs. The SMRI images of the polymer phantoms also show the linearity of the MRI intensities with different phantom densities (figure IB).

[0086] Figure 2A shows the proton spectrum of a piece of bovine cortical bone, four polymer phantoms and a tube of fresh bovine bone marrow held in a 1.5cm diameter sample holder. The receiver dead time was 100 ms, largely eliminating the solid signals. The water and fat peaks were at 0.00 and -3.50ppm respectively. Figure 2B shows the water and fat suppressed spectrum of same sample acquired with the WASPI sequence without projection gradient, in which water and fat peaks were suppressed to the baseline. Figures 2C and D show the SMRI and WASPI images of the samples. The signal of bone marrow was suppressed in the WASPI images. [0087] The linear least squares fit of the phantom calibration data is shown in Figure 3, along with the MRI-derived bone matrix density results. The bone matrix densities determined by MRI and gravimetric analysis are listed in Table 1. The ratio of MRI density/gravimetric density is 0.86 and 0.83 for bovine and rat bone specimens, respectively.

Table 1 Bone matrix densities determined by MRI and gravimetric analysis

[0088] Data related to bovine and rat bone specimens shows that bone matrix density values measured by MRI and gold standard gravimetric methods are linearly correlated. This study demonstrates that using a polymer blend phantom to quantitatively measure bone matrix density is feasible and may be implemented on clinical scanners.