SC BioCRAFT

Project I

JEFFREY ANKER, Ph.D. Assistant Professor of Chemistry  Clemson UniversityDeveloping Luminescent Strain Sensors to Evaluate and Monitor Osteoinductive Therapies

Target Investigator

JEFFREY ANKER, Ph.D.
Assistant Professor of Chemistry

Clemson University
Email: janker@clemson.edu   
Phone: 864-656-1726

Developing Non-Invasive Luminescent Tension-Indicating Orthopedic Screws

Bone healing and maintenance is a consequence of proper fixation and there is a critical need to monitor strain in orthopedic screws in order to detect loosening early. Orthopedic screws apply stress and fix the position of implanted orthopedic devices such as fracture fixators and spinal grafts. Bone healing is accompanied by increased rigidity, while loosening is a major cause of implant failure. Loosening is often an indication of infection on the device surface, which is difficult to eradicate if not treated early and usually requires device removal with risk of mortality and morbidity from surgery and prolonged hospitalization. Approximately half of infections acquired in hospitals are associated with implanted or indwelling devices (one million in 2004) and of the two million fracture fixators implanted annually in the US, 5-10% become infected, and 40% for soldiers due to debris in blast injuries. Loosening is most commonly determined by radiography and is characterized by displacement of the screw in a time series of X-ray images or dark lines surrounding the screw due to bone degradation. However, these results are difficult to quantify, especially at early stages, and the X-ray images are ill-suited for studying dynamic motion. The objective of this pilot project is to provide the preliminary results and instrumentation to demonstrate that in situ changes in strain and spectrum can be monitored noninvasively through tissue. Optical tension-indicating bolts have been described in the patent literature since the 1970s; the novelty of our approach is that we use upconversion and radioluminescence to provide an essentially background-free near infrared signal that can propagate deeply through tissue for orthopedic applications. We will fabricate tension-indicating luminescent screws by modifying existing orthopedic screws. We will then characterize the modified screws both mechanically and optically. We will measure implant loosening after repeated strain cycling in bone mimics and ex vivo bovine bone. This pilot project will enable us to collect data for a future R01 proposal studying implant loosening and biofilm infection in rabbit models. The luminescent tension-indicating screws will enhance our ability to assess mechanical changes in vivo and enable quantitative assessment of implant loosening and bone healing.

SUMMARY:

The project has been highly productive on all fronts. In terms of research have fabricated mechanical test specimens, loaded them in an Instron Testing System, and are comparing optical strain measurements taken with a digital camera to the Instron strain data. We have also demonstrated that we can measure pH changes on implanted devices through tissue 6.5 mm of porcine tissue with minimal spectral distortion. Work is underway to combine these two aspects of the project for measuring strain via luminescence spectroscopy through tissue. On the publication front, the PI has published two articles relating to X-ray excited optical luminescence, has two articles in preparation regarding spectroscopic strain and chemical measurements through tissue, filed one provisional patent application on the luminescent strain sensors, and converted a provisional patent application on the X-ray luminescent chemical sensors into a full patent application. On the grant front, the PI was awarded a $216,227 NIH R15 grant, a $18,000 SC Space Grants award, and his $526,000 NSF CAREER proposal was recommended for funding.

HIGHLIGHTS / PROGRESS:

  • Developed protocol to validate the optical strain gauges by attaching the strain gauges to mechanical specimens being stressed with an Instron Testing System. We then acquire a series of photographs and compare the strain gauge color to the Instron displacement data and a nearby resistive strain sensor.

  • Preliminary results show that the optical strain gauge can detect elongation of stainless steel components during tension with a precision of ~50 µstrains using inkjet-printed 500 µm lines. Work is ongoing to improve the sensitivity using smaller photolithographically patterned lines. We are also developing multi-channel strain sensor that provide course and fine displacement measurement in order to resolve fine strain changes over a large range of strain (using the same principle as vernier scales and optical position encoders).
  • Developed upconversion luminescent films for measuring pH through tissue. These sensors comprise a pH indicating film with bromocresol green dye in silica placed over an upconversion luminescence film. Under 980 nm illumination, the upconversion particles luminescence in the red and NIR region. This luminescence passes through the pH indicator layer and the spectrum is modified by the dye absorption. We measure the ratio between closely spaced spectral peaks (669 nm and 661 nm) as a function of pH and develop a sensitive calibration curve. Because the peaks are closely spaced, the tissue scattering and absorption does not cause significant spectral distortion, and the calibration curves with and without passing through 6.5 mm of tissue essentially overlap. Tibial dynamic compression plates are generally placed 3-5 mm below skin surface, so the 6.5 mm depth would be sufficient for measurements on tibial fracture fixators. We next measured the spectrum through tissue for a film inoculated with S. epidermidis bacteria and a sterile control in buffer. We were able to track local acidosis of the film surface during bacterial growth in real time, with minimal change in pH for the control. We will use the same closely spaced upconversion luminescence approach for our strain measurements. In addition, these chemical measurements are complementary to the mechanical strain sensors for detecting implant infection.
  • Demonstrated proof-of-concept for magnetically and chemically modulating strain and measuring this with our optical strain sensors. The ability to both apply and measure strain non-invasively will be important for extending our strain concepts to “smart” sensors and actuators capable of measuring response to active stimuli.

GRANTS / PAPERS (PUBLISHED/SUBMITTED) / PATENT APPLICATIONS:

  • Awarded two small grants, an NIH R15, and recommended for funding for an NSF CAREER grant: 1) “Travel Scholarships for Frontiers in BioMagnetic Particles 2013,” Co-PI, $4,000 ($2000) (2013). 2) “Developing Optical Strain Gauges for Passive Remote Sensing.” SC Space Grant Consortium and NASA EPSCoR, PI, $18,000 direct, (6/1/13-5/31/13). 3) “Investigating Mechanism of Intracellular Rotational Transport with Optical Tracking (OPT-MTC),” NIH 1R15EB014560, PI, $150,148 direct ($216,277 total) 9/30/2012-8/31/2015. 4)“CAREER: High Resolution Spectrochemical Imaging Through Tissue,” NSF CHE1255535, PI, $526,216. 5/1/13-4/30/18. (Recommended for funding).
  • Published two papers, with two more in preparation: 1) Chen, H., Rogalski M., Anker, J.N.: “Advances in functional X-ray imaging techniques and contrast agents.” Physical Chemistry Chemical Physics, 14 13469-13486 (2012). Invited Review.  2) Chen H., Moore T., Qi B., Colvin D.C., Jelen E.K. Hitchcock D., He J., Mefford O.T., Alexis F., Gore J.C., Anker J.N.: “Monitoring pH-triggered Drug Release from Radioluminescent Nanocapsules with X-ray Excited Optical Luminescence.” ACS Nano, http://dx.doi.org/10.1021/nn304369m. (2013). Selected as the article of the month for the ACS Nano Podcast.
  • The COBRE pilot project was an important factor in the Clemson University Research Fund’s (CURF) decision to convert US Provisional patent application on X-ray excited optical nanosensors to a full patent application and to file a provisional application on luminescent strain gauges: 1) Anker J.N., Chen H., Alexis F. "X-ray excited optical materials & methods for high resolution chemical imaging.". US Patent Application 13/534,437 (2012). 2) Anker J.N., Rogalski M., Anderson D., Heath, J. “Optical strain gauges and orthopedic screws for imaging through tissue.” US Provisional Patent Application 61/680,419 (2012).

FUTURE DIRECTIONS:

  • Perform a NZW rabbit study to demonstrate in vivo optical imaging of implanted X-ray excited optical luminescence (XEOL) pH sensing films with and without bacterial infection. We will surgically implant a modified pH sensing film and glue to the tibia. We will then measure the pH for several days at two locations in six New Zealand White rabbits. Two will be controls, two will be innoculated with S. epidermidis at the proximal screw, and two will be innoculated at the distal screw. We will exposue the animal to either 980 nm near infrared laser or a ~9 cGy local X-ray beam while acquiring a luminescence image at two wavelengths in order to calculate a spectral ratio an measure pH. The beam will be moved relative to the animal to acquire pH measurements at multiple locations. We will also measure liver and kidney enzymes to determine if the implants create toxic reactions. Our hypotheses are that 1) the pH will decrease in the case of infection, 2) that we will be able to quantify this pH decrease in time and determine whether the distal or priximal screws were innoculated, and 3) that the titanium implant surfaces, coated with silica will not cause toxic reactions. This research could have great impact in development of "smart" orthopedic devices which can detect and monitor infection and in research on methods to reduce infection.
  • Submit R21 “Non-Invasive Luminescent Strain Indicators,” Anker, DesJardins, Tzeng. (Feb 16, 2013).
  • Submit R01 “Luminescent pH sensor films for non-invasive detection of implant Infection.” Anker, DesJardins, Tzeng. (June 12, 2013).

SPECIFIC AIMS FOR RENEWAL

Developing Luminescent Stain Gauges for Non-Invasive Evaluation of Bone Fracture Healing and Pathologies
Proper fixation is critical for healing bone fractures and changes in load sharing and strain in orthopedic devices during healing can be used to assess healing rates and detect pathologies. Although most orthopedic implant surgeries are successful, failures occur due to non-union, aseptic implant loosening, infection, and implant fracture; such failures usually require revision surgery.1, 2 The risk of morbidity/mortality from this surgical intervention and hospitalization makes early diagnosis critical.3 Although most of these failures can be assessed via radiographic images, they do not provide early quantitative warnings. Currently available strain sensors (e.g. resistive and capacitive strain gauges,4-6 ultrasound of liquid-filled cavities,7-9 X-ray diffraction,10 optical moiré pattern analysis,11, 12 and video tracking)13 are either unsuitable for non-invasive transdermal measurements or require relatively large and complex devices for wireless detection or telemetry. Herein, we will develop a strain-indicating luminescent sensor that can be read via a simple fiber-coupled laser and spectrometer setup. The challenge involves developing a sensor that emits enough luminescence at wavelengths that penetrate through the tissue with minimal spectral distortion and background tissue autofluorescence. In our preliminary results, we used dye absorption to modulate the upconversion luminescence spectra, an effect that we measured through at least 7 mm of porcine muscle with minimal spectral distortion, even for 650 nm luminescence. We will build upon those findings to improve the optical collection efficiency, improve the strain indicator design, and test it in bone mimics and small animal models.

Our target applications will be to A) Design strain indicators that can be applied to the disused screw holes in a dynamic compression plate around a fracture site. B) Design a tension-indicating orthopedic screw to directly measure tension in load bearing screws. C) Apply the indicators to magnetically actuated orthopedic devices in order to measure in situ strain under controlled loads. These applications are chosen because of their medical importance and in order to highlight the critical advantages of the technique such as its simplicity, the small size of the sensor, and its flexibility for integration into more complex systems. We also expect that the devices will find use in strain measurements in ligaments and soft tissue. The strain measurements will also be complementary to the PI’s high resolution chemical measurements on implanted medical devices with X-ray addressed optical luminescence spectroscopy. The project fits well with the COBRE mission by providing mentorship and support to develop a highly innovative idea for NIH R01 funding. Our three specific aims are:

Aim 1: Sensor Fabrication and Characterization: We will fabricate luminescent comprising a bottom indicator plate patterned with a series of luminescent lines, and a top transparent analyzer plate with an opaque mask pattern positioned so that only a portion of the indicator plate is visible through the mask. Displacement of the analyzer plate with respect to the indicator plate changes the region of the indicator that is visible through the mask, thereby altering the luminescence spectrum.

The approach is related to traditional moiré phase analysis, except that the structured illumination pattern is created by the rigid analyzer plate and thus does not depend upon the angle or precise placement of the strain gauge. We will use photolithography to fabricate the device and study the strain sensitivity and luminescence intensity as a function of incident laser power, ex vivo porcine tissue thickness, and collection optics. To improve sensitivity while maintain a large dynamic range, we will fabricate multichannel gauges with distinguishable luminescent spectra for fine and course measurement, similar to vernier scale measurements and optical encoders.

Aim 2: Mechanical Testing and Validation: We will validate the optical strain gauges by attaching the strain gauges to mechanical specimens being stressed with an Instron Testing System and compare the optical measurements to the displacement of the grips and an electronic strain gauge adjacent to the optical strain gauge. For early device design work in the absence of tissue we will employ reflective strain sensors, printed with an inkjet printer for viewing by camera and eye. Preliminary results show that the optical strain gauge can detect elongation of a SS316L specimen during tension with a precision of ~50 µstrains using inkjet-printed 500 µm lines. We expect that employing lithographically patterned surfaces and using a low background luminescence spectrometer will further improve sensitivity even for measurements through tissue. We will then validate the technique for measuring loosening in bone mimics and ex vivo bovine tibiae during physiologic stress cycling. We will compare loosening with normal bones, fractured bones and bones weakened by decalcification.

Aim 3: Measurement in Animal Models. We will use our strain gauges to measure strain during bone healing in a tibial fixation NZW rabbit study. We will measure the time course of strain in healthy animals control and compare to populations innoculated with S. epidermidis at the proximal or distal screws. We expect to distinguish between the healthy and diseased animals based upon the rate of bone healing and load sharing.

References

  1. Zimmerli, W., Prosthetic-joint-associated infections. Best Pract. Res. Cl. Rh. 2006, 20, 1045.
  2. Bryers, J. D., Medical biofilms. Biotechnology and Bioengineering 2008, 100, 1.
  3. Esposito, S.; Leone, S., Prosthetic joint infections: microbiology, diagnosis, management and prevention. International Journal of Antimicrobial Agents 2008, 32, 287.
  4. Fleming, B. C.; Beynnon, B. D., In vivo measurement of ligament/tendon strains and forces: a review. Annals of biomedical engineering 2004, 32, 318.
  5. Straty, G.; Adams, E., Highly sensitive capacitive pressure gauge. Review of Scientific Instruments 1969, 40, 1393.
  6. French, P.; Evans, A., Piezoresistance in polysilicon and its applications to strain gauges. Solid-State Electronics 1989, 32, 1.
  7. Joshi, S.; Pathare, R., Ultrasonic instrument for measuring bolt stress. Ultrasonics 1984, 22, 261.
  8. Heyman, J. S., A CW ultrasonic bolt-strain monitor. Experimental Mechanics 1977, 17, 183.
  9. Allison, S.; Heyman, J., Nondestructive ultrasonic measurement of bolt preload using the pulsed-phase locked-loop interferometer. Welding, Bonding and Fastening, 1984 p 197-209(SEE N 86-11227 02-23) 1985.
  10. Noyan, I.; Huang, T.; York, B., Residual stress/strain analysis in thin films by X-ray diffraction. Critical Reviews in Solid State and Material Sciences 1995, 20, 125
  11. McKelvie, J., Moire strain analysis: an introduction, review and critique, including related techniques and future potential. The Journal of Strain Analysis for Engineering Design 1998, 33, 137.
  12. He, X.; Zou, D.; Liu, S.; Guo, Y., Phase-shifting analysis in moiré interferometry and its applications in electronic packaging. Optical engineering 1998, 37, 1410.
  13. Downs, J.; Halperin, H. R.; Humphrey, J.; Yin, F., An improved video-based computer tracking systems for soft biomaterials testing. Biomedical Engineering, IEEE Transactions on 1990, 37, 903.