WO2017030900A1

WO2017030900A1 - Quantitative tool using impedance spectroscopy to monitor fracture healing

Info

Publication number: WO2017030900A1
Application number: PCT/US2016/046568
Authority: WO
Inventors: Monica LIN; Michel MAHARBIZ; Safa HERFAT; Chelsea Bahney; Meir MARMOR
Original assignee: The Regents Of The University Of California
Priority date: 2015-08-14
Filing date: 2016-08-11
Publication date: 2017-02-23

Abstract

A system for monitoring or observing a property or condition of a bone includes a first electrode and a second electrode configured to be implanted into the bone so as to be in electrical connection with each other through at least a portion of the bone or the bone fracture region, and a variable frequency power supply electrically connected to the first and second electrodes to provide a source signal. The system includes a detection system electrically connected between the first and second electrodes and configured to detect phase-sensitive current and voltage of a circuit formed by the variable frequency power supply, the first and second electrodes and the bone, and to provide detection signals for each of a plurality of frequencies. The system also includes a signal processor arranged to communicate with the detection system to receive the detection signals, the signal processor being configured to process the detection signals to calculate a complex impedance of the bone substrate corresponding to each of the plurality of frequencies to provide an output impedance spectrum.

Description

QUANTITATIVE TOOL USING IMPEDANCE SPECTROSCOPY

TO MONITOR FRACTURE HEALING

[0001] This application claims priority to U.S. Provisional Application No. 62/205,561 filed August 14, 2015, the entire content of which is hereby incorporated by reference.

[0002] This invention was made with Government support under Grant EFRI- 1240380 and Grant IIP-1361975, awarded by the National Science Foundation. The Government has certain rights in the invention.

BACKGROUND

1. Technical Field

[0003] The field of the currently claimed embodiments of this invention relates to monitoring fracture healing, and more particularly systems and methods for monitoring fracture healing using impedance spectroscopy.

2. Discussion of Related Art

[0004] An estimated 15 million fracture injuries occur each year in the United States alone, with up to 20% of patients experiencing some degree of impaired healing. [1], [2] Within this fracture population, 10% will fail to heal appropriately and result in delayed or non-union, [3] and incidence of non-union rises to 46% when the fractures occur in conjunction with vascular injury. [4] The risk of non-union is greater for certain patient populations such as smokers and diabetics, and open fractures of the tibia are especially susceptible. [5] Treatment of fractures costs the U.S. healthcare system $45 billion per year. In particular, multiple reoperations are often necessary to treat non-unions, and 51% of fracture patients do not return to work in 6 months. [2] This causes substantial disability to patients and represents a significant burden on the healthcare system.

[0005] Determining when a fracture is healed is crucial to making correct clinical decisions for patients, but there are currently no standardized methods of assessing fracture union. Current available tools for assessing fracture healing include radiographic methods, serologic markers, and clinical evaluation. [5] However, poor accuracy, unreliability, need for high doses of radiation, large expense, and/or subjectivity limit their clinical utility. [5], [6], [7], [8] SUMMARY

[0006] According to some embodiments of the invention, a system for monitoring or observing a property or condition of a bone, a bone fracture, or a bone graft substrate of a subject includes a first electrode and a second electrode configured to be implanted into the bone, the bone fracture, or the bone graft substrate so as to be in electrical connection with each other through at least a portion of the bone or the bone fracture region. The system also includes a variable frequency power supply electrically connected to the first and second electrodes to provide a source signal. The system also includes a detection system electrically connected between the first and second electrodes and configured to detect phase-sensitive current and voltage of a circuit formed by the variable frequency power supply, the first and second electrodes and the bone, the bone fracture, or the bone graft substrate, and to provide detection signals for each of a plurality of frequencies. The system also includes a signal processor arranged to communicate with the detection system to receive the detection signals, the signal processor being configured to process the detection signals to calculate a complex impedance of the bone, the bone fracture, or the bone graft substrate corresponding to each of the plurality of frequencies to provide an output impedance spectrum of the bone, the bone fracture, or the bone graft substrate.

[0007] According to some embodiments of the invention, a method of monitoring or characterizing a bone fracture region includes applying a first plurality of alternating electrical signals, each at a different frequency, to a pair of electrodes implanted into the bone fracture region on opposite sides of a bone fracture, and measuring a first set of complex impedances of the bone fracture region for each of the first plurality of alternating electrical signals to obtain a first impedance spectrum. The method further includes applying a second plurality of alternating electrical signals, each at a different frequency, to the pair of electrodes implanted into the bone fracture region on opposite sides of the bone fracture, and measuring a second set of complex impedances of the bone fracture region for each of the second plurality of alternating electrical signals to obtain a second impedance spectrum. The method further includes comparing the first and second impedance spectra to at least one of each other or to a reference spectrum to provide an indication of a degree of healing of the bone fracture. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Further objectives and advantages will become apparent from a consideration of the description, drawings, and examples.

[0009] Figure 1 is a schematic illustration of a system for monitoring or observing a property or condition of a bone, a bone fracture, or a bone graft substrate of a subject;

[0010] Figure 2 shows an overview of an impedance sensing system;

[0011] Figure 3 shows an example of sensor needles used in a murine model;

[0012] Figure 4A is a circuit model displaying the impedance measurement taken between two electrodes and what paths the signal may take;

[0013] Figure 4B shows the setup for taking measurements in a cadaveric model across

Kirschner wires in a tibia with a bone plate;

[0014] Figure 5 shows schematic representations of some embodiments of the invention involving a bone plate;

[0015] Figure 6 shows an example of bone screws according to some embodiments;

[0016] Figure 7 shows schematic representations of some embodiments of the invention involving a fixation rigid rod or plate disposed outside the body;

[0017] Figure 8 shows a schematic of an intramedullary nail according to some embodiments;

[0018] Figure 9 illustrates how electrodes can be integrated into a hip acetabular cup, a hip femoral stem, a femoral condyle replacement, and a tibial insert;

[0019] Figure 10 illustrates the integration of a circuit and/or sensor arrays on the surface of a spine cage and on a posterior stabilization rod;

[0020] Figure 11 is a schematic illustration of a system for diagnosing and/or monitoring bone healing according to some embodiments of the current invention [0021] Figure 12 is a schematic of a control board according to some embodiments of the current invention;

[0022] Figure 13 shows the layout of a control board used to perform the methods described herein;

[0023] Figure 14 shows a system for measuring intact fracture calluses dissected from mice;

[0024] Figure 15 shows multiple printed circuit board (PCB) designs for in vivo sensors in mice;

[0025] Figure 16 shows CAD diagrams to UV laser cut sensors for in vivo mouse studies.

[0026] Figure 17 shows Bode diagrams of impedance magnitude and phase versus frequency showing the effect of a bone plate and bone screws on impedance measurements;

[0027] Figure 18 shows impedance measurements of cadaveric tissues;

[0028] Figure 19 shows results from tracking impedance measurements of the fracture calluses in mice over time;

[0029] Figure 20 shows Bode diagrams of impedance magnitude and phase versus frequency showing the progression of impedance of a fracture callus over healing time;

[0030] Figure 21 shows Bode diagrams of impedance magnitude and phase versus frequency of muscle taken from multiple mice at the various timepoints;

[0031] Figure 22 shows representative histologic sections of fracture calluses at each of days 7, 14, and 21;

[0032] Figure 23 shows a regression analysis of phase angle (Θ) correlated to % volume fractions of cartilage for fracture calluses; and

[0033] Figure 24 shows a regression analysis of phase angle (Θ) correlated to % volume fractions of cartilage for trabecular bone. DETAILED DESCRIPTION

[0034] Some embodiments of the current invention are discussed in detail below. In describing embodiments, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. A person skilled in the relevant art will recognize that other equivalent components can be employed and other methods developed without departing from the broad concepts of the current invention. All references cited anywhere in this specification, including the Background and Detailed Description sections, are incorporated by reference as if each had been individually incorporated.

[0035] Determining when a fracture is healed is crucial to making correct clinical decisions for patients, but there are currently no standardized methods of assessing fracture union. This is especially important for early diagnosis and treatment of non-unions. A survey of over 400 orthopedic surgeons revealed that there is a lack of consensus in defining both clinical and radiographic criteria for delayed union and nonunion in tibial fractures.

[0036] Current available tools for assessing fracture healing include radiographic methods, mechanical assessment, serologic markers, and clinical evaluation. [5] Plain X-ray radiographs are most commonly used to evaluate fractures, however studies have shown that these correlate poorly with bone strength, do not define union with enough accuracy, and are unreliable for determining the stage of fracture healing. Specifically, because they only detect mineralized tissues, we know that formation of a hematoma followed by a transition to a cartilage intermediate are critical steps of fracture healing that cannot be detected by x-ray. X- ray computed tomography (CT), especially quantitative computed tomography (qCT), has great accuracy in determining bone mineral density (BMD), however the cost and high radiation doses required of CT preclude it from being utilized clinically. Dual energy X-ray absorptiometry (DEXA) is often used to diagnosis osteoporosis due to its ability to measure BMD, but it has decreased accuracy when imaging fractures treated with implants so it is not widely used clinically. [17]

[0037] Ultrasound carries a number of advantages over other techniques, as it is less expensive, does not require exposure to ionizing radiation, and is noninvasive. While it is unable to penetrate cortical bone, there is evidence that it can detect callus formation before radiographic changes are evident. However, interpretation of findings are highly dependent on operator expertise and thick layers of soft tissue can obscure view of bones. [18]

[0038] There have been multiple studies correlating mechanical properties to bone strength, but soft tissue artifacts often limit the reliability of conclusions. Additional tests exist that require patients to be weight-bearing, so they have limited utility in understanding the early stages of healing. [19] Using serologic biomarkers as early predictors of fracture healing is gaining popularity, but there is still a lot of work to be done. Research is focused on identifying sensitive and specific markers of delayed or failed repair, but multiple patient factors like smoking status, age, gender, etc. make it difficult to present clinical recommendations.

[0039] In the clinical setting, physical examination by a physician is still the most relied upon technique to determine fracture union. Patients are examined for local signs of infection, ability to weight-bear, and extent of pain. However, relying on patient-reported questionnaires is very qualitative, and physician assessment is subjective and depends on experience. [5] The embodiments of the invention can fill the need for a quantitative, reliable method to monitor fracture healing and particularly to distinguish between the early stages of healing.

[0040] Clinically, fractures heal primarily through endochondral ossification, in which bone forms indirectly from a cartilage template. Healing occurs through four overlapping phases of repair, beginning with an inflammatory phase, followed by chondrogenesis of mesenchymal progenitors to form the early cartilage callus that matures into a hard callus of cancellous bone, and finally remodeling into healthy cortical bone. [1], [9], [10] These clearly defined stages of healing can be well characterized histologically, but they are not detectable by standard radiographic techniques.

[0041] Electrical impedance spectroscopy (EIS) measures the dielectric properties of tissue as a function of frequency, and has been used for decades to characterize biological tissues [11], [12], [13], [14] such as bone [15], [16]. Here, we apply this technique to monitoring fracture healing.

[0042] Figure 1 shows a system for monitoring or observing a property or condition of a bone, a bone fracture, or a bone graft substrate of a subject according to some embodiments of the invention. The system 100 includes a first electrode 102 and a second electrode 104 configured to be implanted into the bone, the bone fracture, or the bone graft substrate so as to be in electrical connection with each other through at least a portion of the bone or the bone fracture region. The system 100 also includes a variable frequency power supply 106 electrically connected to the first and second electrodes 102, 104 to provide a source signal. The system 100 also includes a detection system 108 electrically connected between the first and second electrodes 102, 104 and configured to detect phase-sensitive current and voltage of a circuit formed by the variable frequency power supply 106, the first and second electrodes 102, 104, and the bone, the bone fracture, or the bone graft substrate, and to provide detection signals for each of a plurality of frequencies. The system 100 also includes a signal processor 110 arranged to communicate with the detection system 108 to receive the detection signals. The signal processor 110 is configured to process the detection signals to calculate a complex impedance of the bone, the bone fracture, or the bone graft substrate corresponding to each of the plurality of frequencies to provide an output impedance spectrum of the bone, the bone fracture, or the bone graft substrate.

[0043] According to some embodiments, the system further includes a data storage device configured to communicate with the signal processor to receive and store the output impedance spectrum of the bone, the bone fracture, or the bone graft substrate for at least one of further processing or later retrieval. According to some embodiments, the system includes an RF transmitter configured to communicate with at least one of the signal processor or the data storage device to transmit the output impedance spectrum of the bone, the bone fracture, or the bone graft substrate.

[0044] According to some embodiments, the system further includes a display device configured to communicate with at least one of the signal processor, the data storage device or the RF transmitter to receive and display the output impedance spectrum of the bone, the bone fracture, or the bone graft substrate.

[0045] According to some embodiments, the first and second electrodes have an electrically conducting tip structured to make electrical connection with bone marrow while being insulated with respect to surrounding regions. However, the embodiments of the invention are not limited to the electrically conducting portion being located at the tip. For example, the electrically conductive part that acts as the exposed electrode could be, for example, a surface pad or the uninsulated center portion of a wire. [0046] According to some embodiments, the first and second electrodes are incorporated into at least one of bone screws, a bone plate, or an intramedullary nail. According to some embodiments, the variable frequency power supply is a type of power supply that is capable of providing a frequency of the source signal of at least 500 kHz. According to some embodiments, the variable frequency power supply is a type of power supply that is capable of providing a frequency of the source signal of at least 1 MHz. According to some embodiments, the variable frequency power supply is a type of power supply that is capable of providing a frequency of the source signal of about 1 KHz to about 1 MHz.

[0047] According to some embodiments, the system further includes a third electrode configured to be implanted into bone, and a multiplexer arranged in electrical connection with the variable frequency power supply and with the first, second and third electrodes. The multiplexer is configured to select pairs of electrodes between the first, second and third electrodes to provide position-sensitive impedance spectra.

[0048] According to some embodiments of the invention, a method of monitoring or characterizing a bone fracture region includes applying a first plurality of alternating electrical signals, each at a different frequency, to a pair of electrodes implanted into the bone fracture region on opposite sides of a bone fracture, and measuring a first set of complex impedances of the bone fracture region for each of the first plurality of alternating electrical signals to obtain a first impedance spectrum. The method further includes applying a second plurality of alternating electrical signals, each at a different frequency, to the pair of electrodes implanted into the bone fracture region on opposite sides of the bone fracture, and measuring a second set of complex impedances of the bone fracture region for each of the second plurality of alternating electrical signals to obtain a second impedance spectrum. The method further includes comparing the first and second impedance spectra to at least one of each other or to a reference spectrum to provide an indication of a degree of healing of the bone fracture.

[0049] According to some embodiments, the pair of electrodes penetrate into bone marrow on each side of the bone fracture. According to some embodiments, the pair of electrodes are incorporated into at least one of bone screws, a bone plate, or an intramedullary nail.

[0050] According to some embodiments, comparing comprises comparing at least one of the first and second impedance spectra to a reference spectrum to provide an indication of a stage of healing of the bone fracture. According to some embodiments, the stage of healing is one of a predefined number of healing stages.

[0051] According to some embodiments, each of the first plurality of alternating electrical signals and the second plurality of alternating electrical signals has a frequency between 20 Hz and 1 MHz. According to some embodiments, each of the first plurality of alternating electrical signals and the second plurality of alternating electrical signals has a frequency between 1 kHz and 500 kHz.

[0052] According to some embodiments, at least one of the first set of complex impedances and the second set of complex impedances is measured in a marrow canal or cortical bone of the bone fracture region. According to some embodiments, at least one of the first set of complex impedances and the second set of complex impedances is measured in a fracture gap of the bone fracture region.

[0053] According to some embodiments of the invention, impedance spectroscopy is utilized to understand bone tissue health and to distinguish between different tissue types present in fracture healing. According to some embodiments of the invention, the system consists of electrodes to contact the tissue of interest, which are routed to control hardware that interfaces with an LCR meter and laptop, allowing for automatic collection of impedance measurements across a range of frequencies. To optimize our system for use in the fracture healing environment, we have designed specialized electrodes and have run experiments to understand how impedance data differ between tissues present in healing fractures.

[0054] Sensors are placed at or near a fracture injury to gather as much information as possible about the tissues in the fracture gap. Multiple electrodes can be arrayed to probe the area at multiple locations to both spatially and temporally resolve the healing process. When used in vivo, impedance spectroscopy may detect subtle changes in the tissue, enabling objective assessment and providing a unique insight into the condition of an injury.

[0055] Measurements can be taken across electrodes in a 2-point or 4-point measurement configuration, for example. A microcontroller sends commands to multiplexers to determine which electrodes to measure between at any given time. The drive signal can be provided from an impedance analyzer or LCZ meter, which then calculates the complex impedance of the tissue of interest. The frequency response can be analyzed, in particular for any dispersions. Arrayed electrodes can allow for spatial information of tissue health in a given area of interest. According to some embodiments, for each pair of electrodes, 5 measurements are taken in about 2-3 seconds for each frequency.

[0056] For clinical application according to some embodiments of the current invention, these impedance sensors can ultimately be integrated into the existing management techniques of surgically-treated fractures. In other embodiments, this could be used to monitor progression of de novo bone formation either through intramembranous bone or endochondral bone regeneration. One could incorporate the impedance sensing nail into bone grafts, demineralized bone matrix, and bone allografts that are commonly used to promote bone formation for spine fusion or in large segmental defects without departing from the broad concepts of the current invention. Target cases would include fractures stabilized by either internal or external fixation. Internally stabilized fractures involve a metal bone plate in which bone screws are drilled into healthy bone on either side of the fracture site to secure a plate in place and stabilize the fracture. Our sensors can be designed to mimic a bone screw and then drilled into the bone tissue at or immediately flanking the fracture site to measure impedance in or across the injury. External fixation is similar, except a metal fixator is left outside the body and only the bone screws pierce the skin and are fixed to the uninjured areas of bone.

[0057] Figure 2 shows an overview of an impedance sensing system according to some embodiments. Figure 3 show an example of sensor needles comprising electrodes that are used for a murine model. The electrodes can be made as a bone screw so the electrodes are exposed at the end or edge of the screw, and the traces (metal conducting lines) lead out the top of the screw for connection to another module to read out the signal. In some embodiments, the electrodes can lie either embedded in the center of the bone (in the marrow canal or the surrounding cortical bone) or within the fracture gap itself (centered in the cross-sectional area of the bone). In other embodiments, there can be an array of electrodes. If the electrodes are arrayed, it is possible that in one sensor there are some electrodes in the marrow canal and some in the cortical bone, for example. In an embodiment, a small RF chip can be placed at the top of the screw or on the corresponding bone plate and the system can be passively powered from outside the body to take a measurement and collect data. However, the general concepts of the current invention are not limited to this particular example. For example, in other embodiments the system could be powered by some form of piezoelectric mechanism (converting mechanical stress experienced by the bone/joint/etc. to an electric charge. In some embodiments, batteries and/or other energy storage devices could be used. [0058] This system also has the capability of providing electrical stimulation across the fracture gap to potentially increase the rate of healing.

[0059] According to some embodiments of the invention, the signal will travel through biological tissue at and around the fracture site so the impedance measurement will reflect the changing tissues as the fracture heals. However, in cases in which there is a metal bone plate pressed up against the bone, there is a possibility that the current will short through this highly conductive path rather than travel through the bone tissue. Figure 4a is a circuit model displaying the impedance measurement taken between two electrodes and possible paths the signal may take. We conducted measurements to determine how presence of a bone plate and bone screws would affect impedance measurements in a cadaveric model. Two Kirschner wires and a Syntheses 245.16 bone plate (stainless steel) were used in this study. Measurements were taken in intact cortical bone. Figure 4B shows the setup for taking measurements in a cadaveric model across Kirschner wires in a tibia with a bone plate.

[0060] With the electrodes spaced 12 mm apart, impedance measurements of the bone with and without a plate are virtually indistinguishable, signifying that there is something at the interface between the bone and the bone plate that makes it difficult for the current to pass from the bone to the plate. The bone plate can never be fully flush against the bone since bone is not entirely flat, so this poor electrical contact may prevent signal from traveling between these two structures. In other words, the impedance at the bone-plate interface, represented by Zb-p and Z'b-p, is high. Instead, the current should preferentially travel directly between the electrodes on the ends of the sensor screws, passing only through bone tissue and tissue filling the fracture gap.

[0061] As another test, the electrodes were placed 36 mm apart and bone was measured first without screws, and then with two bone screws inserted in between the electrodes. In this experiment, the impedance magnitude dropped after the screws were inserted. This result indicates that the bone screw is making good electrical contact to the bone, so current could travel through the bone screws and create a short circuit through the plate. This will only be an issue if the bone screws are placed too close to the sensors at the fracture site. However, in most clinical situations, bone screws are typically placed far from the site of injury to ensure they are secured in strong, healthy bone so the bone plate is adequately held in place against the fracture. Therefore, this is not a major concern, although this potential issue still needs to be considered in determining use cases for the device. [0062] This technology has the potential to fit a wide range of applications. Just in the monitoring of fracture healing, there are a number of ways in which embodiments of the invention can be integrated into existing management techniques. Impedance spectroscopy can also be applied widely to monitoring surface injuries as well as internal injuries.

[0063] For certain fracture cases, a surgical operation is performed to insert a bone plate at the fracture site to stabilize the two bone ends. In this scenario, extra sensor screws can be drilled into the bone tissue at or flanking the fracture site to measure the tissue in the fracture gap. The electrodes can be directly integrated into the bone screws and drilled in at the site of the fracture. This would allow for readings that reflect what tissue is in the gap, and thus what stage of healing the injury has progressed to. Figure 5 shows schematic representations of some embodiments of the invention. According to some embodiments, electrodes can be disposed on the underside of the bone plate. According to some embodiments, the electrodes can be disposed on sensor screws at or flanking the fracture site. According to some embodiments, a flexible array of electrodes is wrapped around the fracture beneath the bone plate. These embodiments can also be combined. Circuitry to collect the data/make measurements and send data can be integrated on the bone plate hardware. The underside of the bone plate can be coated with an insulating material, separating it from the bone.

[0064] Figure 6 shows an example of bone screws according to some embodiments.

An array of electrodes can be instrumented on a single screw, and the screw can include circuitry to collects and send data. The electrodes can be dispose along the side of the screw or at the tip of the screw. Any pairwise combination of electrodes can be measured. The electrodes may be disposed on the same screw or on different screws. The electrodes may also be included in a dedicated sensor screw that is not designed to secure sections of bone like a bone screw is. The sensor screw may have multiple electrodes at its tip that are design to be placed at the fracture cite. The electrodes can go into an empty fracture gap, a closed juncture between the reduced bone, or into bone grafts or other substrates. The bone screws can be coated with an insulating coating, as can the underside of the bone plate, separating it from the bone.

[0065] External fixators utilize a rigid rod or plate outside of the body with bone screws piercing the skin and going through the bone to stabilize a fracture in place. As illustrated in Figure 7, these bone screws can be utilized to take impedance measurements across the fracture gap. Circuitry to collect and send data can be included on the rigid rod or plate outside the body. In some embodiments, they could also be used to monitor healing progression for bone lengthening procedures that use external fixators for distraction osteogenesis, for example.

[0066] Technology has been developed to fabricate an array of electrodes on thin, flexible substrates that could be wrapped around, or embedded within, an intramedullary nail to instrument it for taking impedance measurements. A schematic of an intramedullary nail is shown in Figure 8. The nail comprises an electrode array long its length, and can include circuitry disposed, for example, at an end of the nail. This nail allows for readings directly through the center of the bone and fracture gap, revealing information about tissue health at and around the site of the injury. This method produces a map of electrical impedance that can reveal spatial information about the tissue health.

[0067] Electrode arrays can also be integrated into joint replacements. Figure 9 illustrates how electrodes can be integrated into a hip acetabular cup, a hip femoral stem, a femoral condyle replacement, and a tibial insert. Figure 10 illustrates the integration of a circuit and/or sensor arrays on the surface of a spine cage and on a posterior stabilization rod. The embodiments of the invention are not limited to these examples. Electrode arrays can be implemented into many types of replacement joints and other devices inserted into the body, as will be appreciated by one of ordinary skill in the art.

[0068] Some embodiments describe two-electrode systems. The general concepts of the current invention are not limited to only two-electrode systems. For example, some embodiments can take 3 -point measurements using 3 electrodes at one time or 4-point measurements using 4 electrodes. Furthermore, electrode arrays could be used in some embodiments of the current invention.

[0069] Micromotion at the site of internal fixation often leads to failure of a surgical implant used to stabilize a fracture. This motion would move the electrodes further apart, changing the distance between the electrodes and thus change how far the signal has to travel. These differences would manifest themselves in changes in impedance, which could be recorded to detect whether or not micromotion exists at a surgical site.

[0070] Compartment syndrome occurs as a result of increased pressure within a compartment of tissue that leads to insufficient blood supply to the muscles and nerves in the area. This may be detectable by impedance, so integrating sensors around the area that has suffered from a traumatic injury would enable objective data collection about the tissue health and reflect pressure in the area.

[0071] Wireless capability can be utilized for this device to function within the framework of internal monitoring. This can allow for remote monitoring, one of the major advantages of some embodiments of the invention. This would allow patient data to be collected on a more frequent basis and sent to the physician for analysis. The electrodes would be placed inside the body at or in the bone, and a control unit can be placed beside it using the bone plate or other surgical implant as a platform. This use case addresses a huge unmet clinical need, as there is no adequate alternative for monitoring internal healing.

[0072] Figure 11 is a schematic illustration of a system for diagnosing and/or monitoring bone healing according to some embodiments of the current invention. Figure 12 is a schematic of a control board according to some embodiments of the current invention. Figure 13 shows the layout of a control board used to perform the methods described herein. Figure 14 shows a system for measuring intact fracture calluses dissected from mice. Figure 15 shows multiple printed circuit board (PCB) designs for in vivo sensors in mice. Figure 16 shows CAD diagrams to UV laser cut sensors for in vivo mouse studies.

[0073] There are also other embodiments for extracting the electrical impedance of a system without using variable frequency stimulation. In particular, one can apply a "white noise" or "flat spectrum" current or voltage and then measure the dual (voltage or current which arises). Since the input is flat spectrum and the output will not be, one can calculate the impedance that was required to generate the output spectrum. For example, the schematic figure could include either variable frequency or "white noise" signal generator. In addition, the input need not be perfect white noise (which, in practice, is not possible) just sufficiently flat in our frequency band of interest so as to allow for the impedance to be inferred from the output. The term "variable frequency power supply" is intended to have a broad meaning to include at least all types of power supplies discussed in relation to specific embodiments of the current invention. This is intended to include, but is not limited to, a white noise signal generator. It is intended to also include, but is not limited to, a power supply in which one can either manually or electronically select specific frequencies.

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Biomechanics, pp. 255-66, 1999.

[0094] [20] T. Yoshida, "Assessment of fracture and distraction callus by monitering bone electrical impedance in rabbits," in 54th Annual Meeting of the Orthopaedic Research Society, 2008.

[0095] [21] T. Yoshida, "Assessment of distraction callus in rabbits by monitoring of the electrical impedance of bone," Acta Orthopaedica, vol. 81, no. 5, pp. 628-33, 2010.

[0096] [22] N. Yamada, "Analysis of Increase in Bone Electrical Impedance in

Fracture Healing," in 55th Annual Meeting of the Orthopaedic Research Society, 2009. [0097] The following examples describe some embodiments in more detail. The broad concepts of the current invention are not intended to be limited to the particular examples. Further, concepts from each example are not limited to that example, but may be combined with other embodiments of the system.

[0098] EXAMPLES

[0099] Example 1 : Impedance Spectroscopy to Monitor Fracture Healing

[00100] Of the estimated 7.9 million fracture injuries that occur each year in the United

States alone, 10% will fail to heal appropriately and result in delayed or non-union [1], with incidence of non-union rising to 46% when the fractures occur in conjunction with vascular injury [2]. Treatment of fractures costs the U.S. healthcare system $45 billion per year. In particular, multiple reoperations are often necessary to treat non-unions, and 51% of fracture patients do not return to work in 6 months [3]. This causes substantial disability to patients and represents a significant burden on the healthcare system.

[00101] Determining when a fracture is healed is crucial to making correct clinical decisions for patients, but there are currently no standardized methods of assessing fracture union. Current available tools for assessing fracture healing include radiographic methods, serologic markers, and clinical evaluation [4]. However, poor accuracy, unreliability, need for high doses of radiation, large expense, and/or subjectivity limit their clinical utility [4-7].

[00102] Clinically, fractures heal primarily through endochondral ossification, in which bone forms indirectly from a cartilage template. Healing occurs through four overlapping phases of repair, beginning with an inflammatory phase, followed by chondrogenesis of mesenchymal progenitors to form the early cartilage callus that matures into a hard callus of cancellous bone, and finally remodeling into healthy cortical bone. [1, 8, 9] These clearly defined stages of healing can be well characterized histologically, but they are not detectable by standard radiographic techniques.

[00103] Electrical impedance spectroscopy (EIS) measures the dielectric properties of tissue as a function of frequency, and has been used for decades to characterize biological tissues [10-13] such as bone [14, 15]. Here, we apply this technique to monitoring fracture healing. First, we optimize a system to measure impedance in the fracture environment, then validate our system by measuring tissues present in healing fractures. We perform experiments to assess feasibility of our system to work within the current fracture treatment scheme, and present measurement data from a simulated fracture model created in a cadaver.

[00104] System Overview

[00105] Previous work has been done in our group to study skin health using impedance spectroscopy [16], and we are now applying this technique to study fracture healing. The system consists of electrodes to contact the tissue of interest, which are routed to control hardware that interfaces with an LCR meter and laptop, allowing for automatic collection of impedance measurements across a range of frequencies. We used a Keysight Technologies E4980AL-100 Precision LCR meter with a 100 mV voltage sine wave output signal at frequencies of 20 Hz to 1 MHz to measure impedance magnitude and phase. The control hardware currently runs off of four AA batteries, and a set of measurements between two electrodes that incorporates a frequency sweep takes a little over one minute. To optimize our system for use in the fracture healing environment, we are designing specialized electrodes and have run experiments to understand how impedance data differ between tissues present in healing fractures, which will be detailed in this paper.

[00106] Sensors will be placed at or near a fracture injury to gather as much information as possible about the tissues in the fracture gap. Multiple electrodes can be arrayed to probe the area at multiple locations to both spatially and temporally resolve the healing process.

[00107] Fracture Model

[00108] For clinical application, these impedance sensors will ultimately be integrated into the existing management techniques of surgically-treated fractures. Target cases would include fractures stabilized by either internal or external fixation. Internally stabilized fractures involve a metal bone plate in which bone screws are drilled into healthy bone on either side of the fracture site to secure a plate in place and stabilize the fracture. Our sensors would be designed to mimic a bone screw and then drilled into the bone tissue at or immediately flanking the fracture site to measure impedance in or across the injury. Fig. 1 shows how the sensors fit within this treatment regime and how the signal will travel between the electrodes. External fixation is similar, except the metal plate is left outside the body and only the bone screws pierce the skin and are fixed to the uninjured areas of bone. [00109] Ideally, the signal will travel through biological tissue at and around the fracture site so the impedance measurement will reflect the changing tissues as the fracture heals. However, since there is a metal bone plate pressed up against the bone, there is a possibility that the current will short through this highly conductive path rather than travel through the bone tissue. To understand this, we conducted an experiment to determine how presence of a bone plate and bone screws would affect impedance measurements in a cadaveric model, with results shown in Figure 17. Two Kirschner wires and a Syntheses 245.16 bone plate (stainless steel) were used in this study. Measurements were taken in intact cortical bone. The experimental setup is shown in Figure 4B. The setup includes a bone plate secured by two bone screws in between electrodes. Figure 4A is a fracture model depicting sensor integrated within the treatment scheme. The model shows how the signal can travel within the fracture environment.

[00110] The results obtained using the setup of Figure 4B are shown in Figure 17.

Figure 17 shows Bode diagrams of impedance magnitude and phase versus frequency showing the effect of a bone plate and bone screws on impedance measurements. The distance listed in the legend corresponds to the distance between the electrodes.

[00111] With the electrodes spaced 12mm apart, impedance measurements of the bone with and without a plate are virtually indistinguishable, signifying that there is something at the interface between the bone and the bone plate that makes it difficult for the current to pass from the bone to the plate. The bone plate can never be fully flush against the bone since bone is not entirely flat, so this poor electrical contact may prevent signal from traveling between these two structures. In other words, the impedance at the bone-plate interface, represented by Zb-p and Z'b-p, is high. Instead, the current should preferentially travel directly between the electrodes on the ends of the sensor screws, passing only through bone tissue and tissue filling the fracture gap. As another test, the electrodes were placed 36mm apart and bone was measured first without screws, and then with two bone screws inserted in between the electrodes. In this experiment, the impedance magnitude dropped after the screws were inserted. This result indicates that the bone screw is making good electrical contact to the bone, so current could travel through the bone screws and create a short circuit through the plate. This will only be an issue if the bone screws are placed too close to the sensors at the fracture site. However, in most clinical situations, bone screws are typically placed far from the site of injury to ensure they are secured in strong, healthy bone so the bone plate is adequately held in place against the fracture. Therefore, this is not a major concern, although this potential issue still needs to be considered in determining best use cases for our device.

[00112] Results and Discussion

[00113] A. Impedance Measurements of Fracture Tissues

[00114] Fracture healing occurs through four stages that are characterized by different tissue types. Stage one, the inflammatory phase, commences immediately after the injury with the formation of a hematoma. The hematoma is formed to stop the bleeding and contain the fracture debris after the bone break. The hematoma is a critical step in initiating healing and the tissue composition begins largely as coagulated blood that is remodeled into a fibrous scaffold. Stage two is characterized by a soft callus that bridges the fracture gap and is primarily composed of cartilage. During stage three, blood vessels invade the cartilage so the tissue becomes calcified and eventually converts into cancellous bone. Finally, the fracture heals fully in stage four by remodeling the cancellous bone into cortical bone that nearly perfectly resembles the original tissue in both form and function. [1,8,9]

[00115] To best replicate these four phases of fracture healing, impedance measurements were taken of blood, coagulated blood, cartilage, cancellous bone, and cortical bone to validate the ability of our system to distinguish between these various tissue types present in fracture healing using impedance spectroscopy. Gold-plated copper electrodes, 300 um in diameter, were used to measure these tissues, which were extracted from a cadaver. Each set of measurements consisted of 5 impedance readings at each of 18 frequencies spanning 20 Hz to 1 MHz. For each tissue type, 5 sets of measurements were taken, with mean and standard deviation calculated at each frequency and plotted in Figure 18. Figure 18 shows Bode diagrams of impedance magnitude and phase for various fracture tissue types plotted as a function of frequency. Despite the lack of water and other fluids in cadaveric tissue as compared to living tissue, these measurements can still demonstrate differences in impedance between the various tissue types.

[00116] The impedance magnitude measurements trend as expected, with readings steadily increasing from stage one through stage four. The largest spread in the data is found at frequencies between 5 and 15 kHz. Importantly, the shapes of these plots as a function of frequency vary amongst the tissues, with the dominant pole shifting to the right (higher frequency) as the tissues progress through the healing phases. The poles and zeros that describe the frequency responses of impedance of these tissues fall out of transfer function fits to their respective Bode plots. Ultimately, a parameter used to distinguish between the different tissue types may be a combination of information gathered from impedance magnitude and phase, as well as from transfer function fits. This will allow us to objectively classify a fracture within one of the four stages of healing, and track the progression of recovery over time.

[00117] B. Ex Vivo Simulated Fracture

[00118] We simulated a fracture ex vivo in a cadaveric tibia to understand our system in the context of a human injury. We first created a complete fracture in the center of a tibia bone extracted from a cadaver, and fixed it in place with an external fixator, resulting in a 5mm fracture gap. The external fixator pins, made of inert stainless steel, were screwed into the bone 28 mm apart and used as the electrodes in this study. Small amounts of cartilage and cancellous bone were individually stuffed into the fracture gap, and 5 sets of impedance measurements were taken for each of 18 frequencies from 20 Hz to 1 MHz. In addition, a heterogeneous mixture of cartilage and cancellous bone was stuffed into the fracture gap as a comparison. Figure 19 shows Bode diagrams of impedance magnitude and phase for measurements across a simulated fracture plotted as a function of frequency. Analysis of the data, shown in Figure 19, shows clear differences between the impedance across a fracture gap filled with cartilage and one filled with cancellous bone, with the graph for the mixture falling in between the two tissues as expected.

[00119] Cartilage and cancellous bone placed in a gap created in cortical bone represent stage two and stage three of the fracture healing process, respectively. Since cortical bone is of higher impedance than the other tissues present in the fracture gap, it is critical that impedance measurements taken across the fracture reflect the tissues in the gap and are not completely dominated by the cortical bone around the fracture. Data collected from this simulated fracture indicate that our system can at least distinguish between injuries at stage two versus stage three of the healing process. This will enable tracking of fracture healing over time, and allow physicians to spot when a fracture does not progress through the different stages of healing at the anticipated rate. This will allow for early intervention to prevent high risk fractures from failing to heal in an acceptable time frame.

[00120] Conclusion [00121] We have developed a system that utilizes impedance spectroscopy to study fractures and has shown promise in its ability detect the different tissue compositions that are expected to be at the fracture site during the progression of bone healing. Experiments have been conducted to ensure that presence of a bone plate and screws in the area of injury for fracture stabilization will not compromise our measurements. We have demonstrated that our system can distinguish between the different tissues present in healing fractures. Measurements taken across a simulated fracture further highlight the functionality of our sensors to classify fractures as being in one of the four stages of healing, which will provide physicians with additional quantitative information to help direct treatment. These experiments establish feasibility of our system to detect differences in electrical properties of the tissue at the fracture site as an injury heals, which can be validated in an in vivo model. Ultimately, understanding where a patient's injury is within the healing process can diagnose delayed healing at an earlier stage and allow for timely intervention to prevent problem fractures from progressing to nonunion.

[00122] References - Example 1

[00123] [1] M. L. Schenker, et al., "Fracture Repair and Bone Grafting,"

Orthopaedic Knowledge Update, pp. 15-25, 2014.

[00124] [2] K. F. Dickson, et al., "Importance of the blood supply in the healing of tibial fractures," Contemp Orthop, vol. 30, no. 6, pp. 489-493, 1995.

[00125] [3] American Academy of Orthopaedic Surgeons, "The Burden of

Musculoskeletal Diseases in the United States", Rosemont, IL, 2008.

[00126] [4] S. Morshed, "Current Options for Determining Fracture Union," Advances in Medicine, pp. 1-12, 2014.

[00127] [5] S. P. Whiley, et al., "Evaluating fracture healing using digital X-ray image analysis," CME, vol. 29, no. 3, pp. 102-106, 2011.

[00128] [6] V. C. Protopappas, et al., "Ultrasonic Monitoring of Bone Fracture Healing," Transactions on Ultrasonics, Ferroelectrics, and Frequency Control, vol. 55, no. 6, pp. 1243-1255, 2008. [00129] [7] L. E., Claes and C. A. Heigele, "Magnitudes of local stress and strain along bony surfaces predict the course and type of fracture healing," Journal of

Biomechanics, pp. 255-266, 1999.

[00130] [8] B. McKibbin, et al., "The Biology of Fracture Healing in Long Bones," Journal of Bone and Joint Surgery, pp. 150-162, 1978.

[00131] [9] H. M. Frost, et al., "The Biology of Fracture Healing: An Overview for Clinicians, Part I," Clinical Orthopaedics and Related Research, pp. 283-293, 1989.

[00132] [10] R. D. Stoy, K. R. Foster, and H. P. Schwan, "Dielectric properties of mammalian tissues from 0.1 to 100 MHz: a summary of recent data," Phys Med Biol, vol. 27, no. 4, pp. 501-513, 1982.

[00133] [11] H. Jongschaap, et al., "Electrical impedance tomography: a review of current literature," European Journal of Radiology, vol. 18, pp. 165-174, 1994.

[00134] [12] D. A. Dean, et al., "Electrical Impedance Spectroscopy Study of Biological Tissues," J Electrostat, vol. 66, no. 3-4, pp. 165-177, 2008.

[00135] [13] S. Gabriel, R. W. Lau, and C. Gabriel, "The dielectric properties of biological tissues: II," Phys Med Biol, vol. 41, pp. 2251-2269, 1996.

[00136] [14] I. V. Ciuchi, et al., "Impedance spectroscopy characterization of bone tissues," Journal of Advanced Research in Physics, vol. 1, no. 1, pp. 1-5, 2010.

[00137] [15] S. Schaur, B. Jakoby, and G. Kroureif, "Position-dependent

[00138] [16] S. L. Swisher, et al., "Impedance sensing device enables early detection of pressure ulcers in vivo," Nature Communications, vol. 6, 2015.

[00139] Example 2: Impedance Sensor to Monitor Fracture Healing

[00140] Fracture healing progresses via two pathways: intramembranous ossification and endochondral ossification. Intramembranous ossification features healing through direct osteogenesis, where mesenchymal cells directly differentiate into bone-forming osteoblasts. Endochondral ossification is a biphasic process involving chondrogenesis followed by osteogenesis. It occurs through four overlapping phases of repair, beginning with an inflammatory phase, followed by formation of an early callus that matures into a hard callus, and finally remodeling into healthy bone. The inflammatory phase begins with hematoma in response to the trauma, and inflammatory cells debride the wound while skeletal progenitor cells are recruited. As the progenitor cells differentiate into chondrocytes, the fracture enters the soft callus phase which is primarily made up of cartilage. The soft callus becomes a hard callus in the third stage as the matrix is vascularized and cancellous bone replaces the callus. In the final phase, the cancellous bone undergoes remodeling via organized osteoblast and osteoclast activity to form cortical bone. [1, 7, 8]

[00141] Clinically, fractures heal primarily through endochondral ossification, although intramembranous ossification occurs simultaneously to various degrees. For example, both processes may be occurring but in different spatial locations. Areas close to the bone ends sometimes heal through intramembranous ossification, while the center of the fracture site will first pass through a cartilage phase. The molecular mechanism for fracture repair is also heavily dependent on stability at the fracture site. Well stabilized fractures will tend to heal by direct osteogenesis, whereas micro-motion of the bone fragments will induce healing through the development of a callus. [1]

[00142] During fracture repair there are clearly defined stages of healing that can be well characterized histologically, but are not detectable by standard radiographic techniques.

[00143] Electrical Impedance Spectroscopy

[00144] Electrical impedance spectroscopy (EIS) measures the dielectric properties of tissue as a function of frequency. It has been used to characterize biological tissues [9-12], with some studies being done specifically on bone [13, 14]. In addition, a small body of literature exists where impedance spectroscopy has been used to monitor fracture healing and has tracked differences in impedance over healing time between fracture groups and control groups [15- 18]. While this data is promising, these studies are limited to fractures treated via external fixation, using long pins as electrodes. This may constrain the dynamic range of the signal, and simple two point measurements result in low spatial resolution. More work needs to be done to optimize impedance spectroscopy for use in the fracture environment and to expand its utility to study healing. [00145] To determine sensitivity of the impedance measurement system to study fracture healing, multiple tissue types present in healing fractures are measured in an ex vivo study. The goal of this study is to validate the measurement system's ability to utilize impedance spectroscopy to distinguish between different tissue types. Impedance magnitude and phase as well as frequency response are studied to draw conclusions. Specifically, this experiment elucidates what frequency range will show the greatest spread in impedance between tissues and whether impedance magnitude or phase provides more information.

[00146] The electrodes can be integrated in or flanking the fracture site with a bone plate over the fracture gap. The measurement path of interest goes from one electrode through bone to the fracture gap, then through bone again to the other electrode. A confounding parallel path exists through a short in the bone plate. Bone plates are typically made of titanium, stainless steel, or cobalt chrome, so this provides a highly conductive path through which the signal can travel. An experiment is conducted to determine the effect of this parallel path on the impedance of the system. This study tests the sensitivity of the impedance measurement system in a simulated fracture environment and to inform device design for translation.

[00147] To validate the utility of an impedance measurement system to track fracture healing, we measure intact fracture calluses from mice at days 8, 14, and 21. These represent the progressive phases of fracture healing, as the tissue in the fracture gap develops from a soft callus primarily comprised of cartilage to a hard callus primarily composed of trabecular bone. As the types of tissue at each phase change, we observe changes in the impedance measurement.

[00148] By processing the samples for histology, we can determine the volume fractions of the different types of tissue in each intact fracture callus that was measured. These can then be correlated with the impedance measurements.

[00149] Methods

[00150] We collaborate with an Electrical Engineering group at UC Berkeley who have developed an impedance measurement system to study skin health [19]. The system consists of electrodes to contact the tissue of interest, which are routed to control hardware that interfaces with an LCR meter and laptop, allowing for automatic collection of impedance measurements across a range of frequencies. We used a Keysight Technologies E4980AL-100 Precision LCR meter with a 1 V voltage sine wave output signal at frequencies of 20 Hz to 1 MHz to measure impedance magnitude and phase. The control hardware currently runs off of four AA batteries, and a set of measurements between two electrodes that incorporates a frequency sweep takes a little over one minute. To optimize our system for use in the fracture healing environment, we designed specialized electrodes that meet the device specifications shown in Table 1.

[00151] Table 1 : Device specifications of electrodes to measure impedance in bone.

[00152] Cadaveric samples of blood, coagulated blood, cartilage, cancellous bone, cortical bone, muscle, and fat are obtained and two point measurements taken across each of these tissues in a frequency sweep from 20 Hz - 1 MHz. For each set of measurements for a given tissue type, five readings are collected at each frequency, and then the electrodes are removed, cleaned, and reapplied five times for repeated measurements. To analyze the data, the five readings per trial are first averaged, then these five numbers are averaged across trials to produce graphs of impedance magnitude and phase as functions of frequency.

[00153] Two Kirschner wires are first drilled into a healthy, cadaveric long bone to use as electrodes, and impedance is measured. Then a Syntheses 245.16 bone plate (stainless steel) is screwed into the set-up and impedance is measured again. The spacing between the electrodes and the placement of the bone screws are adjusted to isolate their effects.

[00154] We first created a complete fracture in the center of a tibia bone extracted from a cadaver, and fixed it in place with an external fixator, resulting in a 5 mm fracture gap. The external fixator pins, made of inert stainless steel, were screwed into the bone 28 mm apart and used as the electrodes in this study. Small amounts of cartilage and cancellous bone were individually stuffed into the fracture gap, and 5 sets of impedance measurements were taken for each of 18 frequencies from 20 Hz to 1 MHz. In addition, a heterogeneous mixture of cartilage and cancellous bone was stuffed into the fracture gap as a comparison.

[00155] Our Laboratory for Skeletal Regeneration has developed and validated fracture models in a murine model. Adult, male mice (10 weeks old) are anesthetized and a standardized, closed non-stable fracture will be created in the mid-diaphysis of the tibia using a custom-built apparatus. The apparatus creates a reproducible three-point bending fracture by controlling the impact force with a weight of 460g and drop distance of 14cm. Fractures are not stabilized to promote endochondral repair, and animals are provided analgesics as needed. A 2 mm segmental defect is created by an osteotomy in the mid-diaphysis of the right tibia. We take impedance measurements of 5 mice at each of 3 timepoints (days 8, 14, and 21). An intact fracture callus is dissected out of each mouse, and pressed up against specially designed electrodes (gold-plated, 150um diameter). The fracture callus is removed and re-measured in the same orientation (relative to the electrode) 5 times, and then placed in a different orientation twice more, with 5 trials per orientation. In each trial, 5 measurements are taken at each of 18 frequencies from 20 Hz to 1 MHz.

[00156] After measurement, the samples are fixed immediately and processed for histology by the Laboratory for Skeletal Regeneration. After sectioning, they are stained by classic Trichrome or Direct Red histology techniques for visualization, and the amount of each tissue type (i.e. cartilage, cancellous bone, etc.) is quantified.

[00157] Results and Discussion

[00158] Measurements of different types of fracture tissues produced impedance magnitude measurements that trend as expected, with readings steadily increasing from stage one through stage four, as plotted in Figure 18. The largest spread in the data is found at frequencies between 5 and 15 kHz. Importantly, the shapes of these plots as a function of frequency vary amongst the tissues, with the dominant pole shifting to the right (higher frequency) as the tissues progress through the healing phases. The poles and zeros that describe the frequency responses of impedance of these tissues fall out of transfer function fits to their respective Bode plots. Ultimately, a parameter used to distinguish between the different tissue types may be a combination of information gathered from impedance magnitude and phase, as well as from transfer function fits. This will allow us to objectively classify a fracture within one of the four stages of healing, and track the progression of recovery over time.

[00159] To understand the impedance measurement system for the fracture environment, we developed a circuit model to understand electrical and geometric constraints on the sensors, shown in Figure 4A. The experimental set-up is shown in Figure 4B, and experimental results are shown in Figure 17.

[00160] With the electrodes spaced 12 mm apart, impedance measurements of the bone with and without a plate are virtually indistinguishable, signifying that there is something at the interface between the bone and the bone plate that makes it difficult for the current to pass from the bone to the plate. The bone plate can never be fully flush against the bone since bone is not entirely flat, so this poor electrical contact may prevent signal from traveling between these two structures. In other words, the impedance at the bone-plate interface, represented by Zb-p and Z'b-p, is high. Instead, the current should preferentially travel directly between the electrodes on the ends of the sensor screws, passing only through bone tissue and tissue filling the fracture gap. As another test, the electrodes were placed 36mm apart and bone was measured first without screws, and then with two bone screws inserted in between the electrodes. In this experiment, the impedance magnitude dropped after the screws were inserted. This result indicates that the bone screw is making good electrical contact to the bone, so current could travel through the bone screws and create a short circuit through the plate. This will only be an issue if the bone screws are placed too close to the sensors at the fracture site. However, in most clinical situations, bone screws are typically placed far from the site of injury to ensure they are secured in strong, healthy bone so the bone plate is adequately held in place against the fracture. Therefore, this is not a major concern, although this potential issue still needs to be considered in determining use cases for the device.

[00161] Cartilage and cancellous bone placed in a gap created in cortical bone represent stage two and stage three of the fracture healing process, respectively. Since cortical bone is of higher impedance than the other tissues present in the fracture gap, it is critical that impedance measurements taken across the fracture reflect the tissues in the gap and are not completely dominated by the cortical bone around the fracture. Data collected from this simulated fracture, shown in Figure 19, indicate that our system can at least distinguish between injuries at stage two versus stage three of the healing process. This will enable tracking of fracture healing over time, and allow physicians to spot when a fracture does not progress through the different stages of healing at the anticipated rate. This will allow for early intervention to prevent high risk fractures from failing to heal in an acceptable time frame.

[00162] Results from tracking impedance measurements of the fracture callus in mice over time are shown in Figure 20. Figure 20 shows Bode diagrams of impedance magnitude and phase versus frequency showing the progression of impedance of a fracture callus over healing time. The magnitude of impedance is clearly distinguishable between days 8, 14, and 21, particularly between 10³ and 10⁴ Hz. Furthermore, the impedance values trend upward as expected as the mouse is healing over time. This is due to the fact that cartilage prevalent in the early stages of healing is of lower impedance than cancellous bone prevalent in the later stages, as we found in Figure 18. As a reference, muscle measurements were also taken in mice, plotted in Figure 21. Figure 21 shows Bode diagrams of impedance magnitude and phase versus frequency of muscle taken from multiple mice at the various timepoints. It is clear that the muscle measurements do not change across mice and between the different timepoints, so they serve as a good reference point for measurement.

[00163] The Skeletal Regeneration Laboratory is currently processing the samples for histology, and will have quantitative volume fraction analysis for each of the timepoints in a few weeks. These can then be correlated with the impedance measurements to determine if the impedance measurement is sensitive enough to detect differences at a single timepoint depending on volume fraction of various tissues of the callus. This is important because there can be variation between mice at the same timepoint; some mice inherently heal faster than others. For example, one mouse may have significantly more cancellous bone in its callus at day 21 than another mouse that has a slower healing response and thus has a callus with proportionally more cartilage at the same timepoint. More rigorous statistical tests need to be performed on the study data as well as correlations made between the impedance measurements and the individual tissue make up of each callus.

[00164] Conclusion

[00165] We have developed a system that utilizes impedance spectroscopy to study fractures and has shown promise in its ability to detect the different tissue compositions that are expected to be at the fracture site during the progression of bone healing. Experiments have been conducted to ensure that presence of a bone plate and screws in the area of injury for fracture stabilization will not compromise our measurements. We have demonstrated that our system can distinguish between the different tissues present in healing fractures. Measurements taken of fracture calluses in mice on days 8, 14, and 21 after tibia fracture further highlight the functionality of our sensors to track the progression of healing. If we can classify fractures as being in one of the four stages of healing, we can provide physicians with additional quantitative information to help direct treatment. These experiments establish feasibility of our system to detect differences in electrical properties of the tissue at the fracture site as an injury heals, which can be validated in an in vivo model. Ultimately, understanding where a patient's injury is within the healing process can diagnose delayed healing at an earlier stage and allow for timely intervention to prevent problem fractures from progressing to non-union.

[00166] References - Example 2

[00167] [1] M. L. Schenker, et al., "Fracture Repair and Bone Grafting," Orthopaedic Knowledge Update, pp. 15-25, 2014.

[00168] [2] American Academy of Orthopaedic Surgeons, "The Burden of

Musculoskeletal Diseases in the United States", Rosemont, IL, 2008.

[00169] [3] K. F. Dickson, et al., "Importance of the blood supply in the healing of tibial fractures," Contemp Orthop, vol. 30, no. 6, pp. 489-493, 1995.

[00170] [4] S. Morshed, "Current Options for Determining Fracture Union," Advances in Medicine, pp. 1-12, 2014.

[00171] [5] S. P. Whiley, et al., "Evaluating fracture healing using digital X-ray image analysis," CME, vol. 29, no. 3, pp. 102-106, 2011.

[00172] [6] V. C. Protopappas, et al., "Ultrasonic Monitoring of Bone Fracture

Healing," Transactions on Ultrasonics, Ferroelectrics, and Frequency Control, vol. 55, no. 6, pp. 1243-1255, 2008.

[00173] [7] B. McKibbin, et al., "The Biology of Fracture Healing in Long Bones,"

Journal of Bone and Joint Surgery, pp. 150-162, 1978.

[00174] [8] H. M. Frost, et al., "The Biology of Fracture Healing: An Overview for

Clinicians, Part I," Clinical Orthopaedics and Related Research, pp. 283-293, 1989. [00175] [9] R. D. Stoy, K. R. Foster, and H. P. Schwan, "Dielectric properties of mammalian tissues from 0.1 to 100 MHz: a summary of recent data," Phys Med Biol, vol. 27, no. 4, pp. 501-513, 1982.

[00176] [10] H. Jongschaap, et al., "Electrical impedance tomography: a review of current literature," European Journal of Radiology, vol. 18, pp. 165-174, 1994.

[00177] [11] D. A. Dean, et al., "Electrical Impedance Spectroscopy Study of

Biological Tissues," J Electrostat, vol. 66, no. 3-4, pp. 165-177, 2008.

[00178] [12] S. Gabriel, R. W. Lau, and C. Gabriel, "The dielectric properties of biological tissues: II," Phys Med Biol, vol. 41, pp. 2251-2269, 1996.

[00179] [13] I. V. Ciuchi, et al., "Impedance spectroscopy characterization of bone tissues," Journal of Advanced Research in Physics, vol. 1, no. 1, pp. 1-5, 2010.

[00180] [14] S. Schaur, B. Jakoby, and G. Kroureif, "Position-dependent

[00181] [15] T. Yoshida, et al., "Assessment of fracture and distraction callus by monitoring bone electrical impedance in rabbits," 54th Annual Meeting of the Orthopaedic Research Society, 2008.

[00182] [16] T. Hirashima, et al., "Evaluating Bone Union of Distal Radius Fractures by Measuring Impedance Values", Orthopedics, vol. 32, no. 1, 2009.

[00183] [17] N. Yamada, et al., "Analysis of Increase in Bone Electrical Impedance in Fracture Healing", 55th Annual Meeting of the Orthopaedic Research Society, 2009.

[00184] [18] T. Yoshida, et al., "Assessment of distraction callus in rabbits by monitoring the electrical impedance of bone", Acta Orthopaedica, vol. 81, no. 5, pp. 628-633, 2010.

[00185] [19] S. L. Swisher, et al., "Impedance sensing device enables early detection of pressure ulcers in vivo," Nature Communications, vol. 6, 2015

[00186] Example 3 : Impedance Spectroscopy Device Detects Fracture Progression in Mice [00187] An estimated 15 million fracture injuries occur each year in the United States alone, with up to 20% of patients experiencing some degree of impaired healing.

Radiography remains the standard technique to monitor healing, but because it relies on detection of mineralized tissue, it can only diagnose delayed healing at the late stages of fracture repair. We are developing a device that utilizes impedance spectroscopy to monitor progression of fracture healing. Importantly, impedance spectroscopy can distinguish non- mineralized tissues, such as fibrous and cartilage tissues, from the mineralized bone tissue, which enables us to monitor the process of fracture repair. Non-unions fail to progress to mineralized tissue and the composition of the fracture callus in these cases is critical in directing clinical management of the fracture. Our novel sensor system that utilizes impedance spectroscopy will allow clinicians to monitor fracture healing and detect delays in union at a very early stage, thus enabling earlier intervention in poor bone healing.

[00188] Various cadaveric tissues representing fracture calluses were measured to determine the ability of our system to differentiate tissues types. Fracture healing was measured in an established murine model of endochondral repair where a standardized, closed fracture is made in the mid-diaphysis of the tibia. Days 8, 14 and 21 post-fracture mice were euthanized and their fracture calluses dissected out for measurement (N=5 per time point). The intact callus was placed on custom-made gold electrodes (150um diameter) and 590g of weight placed on top to allow for consistent contact. 2-point impedance measurements were taken across the electrodes over a range of frequencies (20 Hz to 1 MHz). Statistical significance was computed by running single-factor ANOVA tests comparing the 3 time points at each frequency, and then 2-sample t tests (assuming unequal variances) were used to test each pair of means for significant differences. After measurement, the samples were fixed immediately, processed for histology, and stained by Milligan's Trichrome.

[00189] Impedance measurements of cadaveric tissues are shown in Figure 18, demonstrating that the various tissue types are distinguishable between 10³ and 10⁵ Hz. Results from tracking impedance measurements of the fracture calluses in mice over time are shown in Figure 19. The magnitude of impedance trends upward as healing time increases, and values between days 8, 14, and 21 are particularly spread out between 10³ and 10⁵ Hz. With N=5 for each time point, ANOVA and 2-sample t tests revealed phase angles that were significantly different at 500 Hz and 1000 Hz (p<0.04). As a reference, muscle measurements were also taken in mice at each time point, and there were no discernable differences across mice and between the various time points, as expected. Representative histologic sections of fracture calluses at each of days 7, 14, and 21 are presented in Figure 22. In the histologic sections shown in Figure 22, the light grey dashed line indicates the tibia diaphysis, and the dark grey dashed line outline is an approximation of the dissected callus that was measured.

[00190] Initial impedance measurements of cadaveric tissues highlight the ability of our device to distinguish between tissues present in healing fractures. In the fracture callus, the upward trend of impedance magnitude over time was expected, since cartilage has lower impedance than cancellous bone. This was confirmed by histology, which shows the expected change in tissue composition as the fracture heals over time. Being able to distinguish between the different time points indicates that we can use impedance to track the progression of healing and may be able to detect when fracture healing is plateauing. However, since this study involved sacrificing mice at the time of measurement, some noise may be introduced with the insertion of electrodes in the set-up. Work is being done to develop custom sensors that can be implanted in the fracture site and measure impedance in vivo in mice over time.

[00191] Currently, delayed or non-union is determined using a combination of physical examination and radiography typically requiring months to diagnose. There is a clinical need for physicians to better understand the non-mineralized stages of healing so intervention of mal -unions can occur earlier and thus reduce both patient morbidity and healthcare cost burden. In this study, we show that impedance spectroscopy holds promise for early diagnosis of nonunion by understanding electrical properties of tissue that reflect tissue health and composition.

[00192] Example 4: Impedance Measurements Correlate to Callus Maturation of Mice Tibia Fractures

[00193] Approximately 15 million fracture injuries occur in the United States each year.

Accurate monitoring of fracture healing can determine timing of return to function or the need for early intervention in case of a fracture nonunion. Fracture healing is currently monitored by radiographic methods, which rely on mineralization of tissue that only occurs in the later stages of fracture healing, and other monitoring techniques are either subjective or inaccurate. Electrical impedance spectroscopy (EIS) provides a measure of the dielectric properties of a medium and has been used to differentiate between different tissue types. We hypothesized that EIS can be used to monitor fracture healing by tracking the changing tissue composition of a fracture callus as it progresses through the various stages of healing. [00194] Standardized, closed fractures were created in the mid-diaphyses of mice tibia according to an established murine model of endochondral repair. Mice were euthanized and their fracture callus tissues dissected out at days 8, 14, and 21 post-fracture for measurement (N = 11). Each intact callus was pressed onto custom-made sensors with a 590g weight, and 2- point impedance measurements were taken across two gold electrodes (150um diameter) over a range of frequencies (20 Hz to 1 MHz). Samples were also fixed in 4% paraformaldehyde overnight, decalcified in 19% EDTA (pH 7.4) for 14 days at 4°C, and embedded in paraffin. Serial 10 um longitudinal sections throughout the entire callus tissue were collected and stained with modified Milligan's Trichrome. To quantify tissue volume fractions, histomorphometric analyses of total callus, cartilage, trabecular bone, cortical bone, muscle, fibrous tissue, and bone marrow space volumes were performed using an Olympus CAST system and Visiopharm software. The total tissue volumes were calculated in cubic millimeters (mm³) using the equation for a conical frustum and Cavalieri's principle. Univariate linear regression analysis was performed to assess correlative relationships between impedance measurements and volume fraction percentages of the various tissues present in the fracture calluses, and two- tailed t-tests were used to determine whether regression slopes were significantly different than zero. Significance was set at p<0.05 and trends were defined as 0.05<p<0.1.

[00195] Figure 23 shows a regression analysis of phase angle (Θ) correlated to % volume fractions of cartilage for fracture calluses. Figure 24 shows a regression analysis of phase angle (Θ) correlated to % volume fractions of cartilage for trabecular bone. Linear regression analyses indicated negative relationships between impedance magnitude (|Z|) and % trabecular bone as well as % marrow space, and positive relationships between |Z| and % cartilage as well as % fibrous tissue. The opposite trends were found when comparing phase angle (Θ) to these same volume fractions of tissues. These correlations were as expected - as healing time increases, % cartilage decreases and % trabecular bone increases as the spongy bone replaces the early soft callus. As a result, |Z| rises over the course of healing as more conductive tissue (cartilage) is remodeled into more resistive tissue (bone). % fibrous tissue decreases with healing time as it is replaced by trabecular bone or marrow space, and consequently % marrow space increases. Specifically at 500 kHz, |Z| and Θ both showed significant correlation with % cartilage and % trabecular bone (R²>0.40, p<0.05). At 1 MHz, Θ became less negative with greater % cartilage and % fibrous tissue (R²>0.54, p<0.01) and more negative with greater % trabecular bone (R²=0.58, p=0.007). In addition, Θ became less negative significantly with % trabecular bone at 5 kHz (R²=0.39, p=0.04), and this trend was maintained for frequencies less than 5 kHz (p<0.1).

[00196] Impedance magnitude and phase angle have significant correlations with volume fractions of cartilage, trabecular bone, fibrous tissue, and marrow space at multiple frequencies, particularly below 5 kHz and above 500 kHz. These findings support use of electrical impedance spectroscopy for monitoring fracture healing.

[00197] The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art how to make and use the invention. In describing embodiments of the invention, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. The above-described embodiments of the invention may be modified or varied, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.

Claims

WE CLAIM:

1. A system for monitoring or observing a property or condition of a bone, a bone fracture, or a bone graft substrate of a subject, comprising: a first electrode and a second electrode configured to be implanted into said bone, said bone fracture, or said bone graft substrate so as to be in electrical connection with each other through at least a portion of said bone or said bone fracture region; a variable frequency power supply electrically connected to said first and second electrodes to provide a source signal; a detection system electrically connected between said first and second electrodes and configured to detect phase-sensitive current and voltage of a circuit formed by said variable frequency power supply, said first and second electrodes and said bone, said bone fracture, or said bone graft substrate, and to provide detection signals for each of a plurality of frequencies; and a signal processor arranged to communicate with said detection system to receive said detection signals, said signal processor being configured to process said detection signals to calculate a complex impedance of said bone, said bone fracture, or said bone graft substrate corresponding to each of said plurality of frequencies to provide an output impedance spectrum of said bone, said bone fracture, or said bone graft substrate.

2. The system according to claim 1, further comprising a data storage device configured to communicate with said signal processor to receive and store said output impedance spectrum of said bone, said bone fracture, or said bone graft substrate for at least one of further processing or later retrieval.

3. The system according to claim 1 or 2, further comprising an RF transmitter configured to communicate with at least one of said signal processor or said data storage device to transmit said output impedance spectrum of said bone, said bone fracture, or said bone graft substrate.

4. The system according to any one of claims 1 to 3, further comprising a display device configured to communicate with at least one of said signal processor, said data storage device or said RF transmitter to receive and display said output impedance spectrum of said bone, said bone fracture, or said bone graft substrate.

5. The system according to any one of claims 1 to 4, wherein said first and second electrodes have an electrically conducting tip structured to make electrical connection with bone marrow while being insulated with respect to surrounding regions.

6. The system according to any one of claims 1 to 5, wherein said first and second electrodes are incorporated into at least one of bone screws, a bone plate, or an intramedullary nail.

7. The system according to any one of claims 1 to 6, wherein said variable frequency power supply is a type of power supply that is capable of providing a frequency of the source signal of at least 500 kHz.

8. The system according to any one of claims 1 to 7, wherein said variable frequency power supply is a type of power supply that is capable of providing a frequency of the source signal of at least 1 MHz.

9. The system according to any one of claims 1 to 8, further comprising: a third electrode configured to be implanted into bone; and a multiplexer arranged in electrical connection with said variable frequency power supply and with said first, second and third electrodes, wherein said multiplexer is configured to select pairs of electrodes between said first, second and third electrodes to provide position-sensitive impedance spectra.

10. A method of monitoring or characterizing a bone fracture region, comprising: applying a first plurality of alternating electrical signals, each at a different frequency, to a pair of electrodes implanted into said bone fracture region on opposite sides of a bone fracture;

measuring a first set of complex impedances of said bone fracture region for each of said first plurality of alternating electrical signals to obtain a first impedance spectrum;

applying a second plurality of alternating electrical signals, each at a different frequency, to said pair of electrodes implanted into said bone fracture region on opposite sides of said bone fracture;

measuring a second set of complex impedances of said bone fracture region for each of said second plurality of alternating electrical signals to obtain a second impedance spectrum; and

comparing said first and second impedance spectra to at least one of each other or to a reference spectrum to provide an indication of a degree of healing of said bone fracture.

11. The method according to claim 10, wherein said pair of electrodes penetrate into bone marrow on each side of the bone fracture.

12. The method according to claim 10 or 11, wherein said pair of electrodes are incorporated into at least one of bone screws, a bone plate, or an intramedullary nail.

13. The method according to any one of claims 10 to 12, wherein said comparing comprises comparing at least one of said first and second impedance spectra to a reference spectrum to provide an indication of a stage of healing of said bone fracture.

14. The method according to claim 13, wherein said stage of healing is one of a predefined number of healing stages.

15. The method according to any one of claims 10-14, wherein each of the first plurality of alternating electrical signals and the second plurality of alternating electrical signals has a frequency between 20 Hz and 1 MHz.

16. The method according to any one of claims 10-15, wherein each of the first plurality of alternating electrical signals and the second plurality of alternating electrical signals has a frequency between 1 kHz and 500 kHz.

17. The method according to any one of claims 10-16, where at least one of the first set of complex impedances and the second set of complex impedances is measured in a marrow canal or cortical bone of the bone fracture region.

18. The method according to any one of claims 10-17, where at least one of the first set of complex impedances and the second set of complex impedances is measured in a fracture gap of the bone fracture region.