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Transparent mouse tibia / Courtesy of Science Translational Medicine, Greenbaum, Chan, et al; Gradinaru laboratory/Caltech

Transparent Bones!; New Biomechanics Testing Approach; IV Acetaminophen vs. Opioid

Elizabeth Hofheinz, M.P.H., M.Ed. • Mon, May 15th, 2017

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Transparent Bones: Now Available for Viewing Active Cells

Usually, when we say “get down to the bare bones” we can’t actually see inside the bones. But Caltech can (The California Institute of Technology).

A team of researchers working in the lab of Viviana Gradinaru, Ph.D., assistant professor of biology and biological engineering and a Heritage Medical Research Institute Investigator, expanded upon a technique called CLARITY. This technique, says the April 26, 2017 Caltech news release, was originally developed for clearing brain tissue during Gradinaru's postgraduate work at Stanford University.

The work, “Bone CLARITY: Clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow,” appears in the April 26 issue of Science Translational Medicine.

The PR department at Caltech described Dr. Gradinaru’s work this way: “CLARITY renders soft tissues, such as brain, transparent by removing opaque molecules called lipids from cells while also providing structural support by an infusion of a clear hydrogel mesh. Gradinaru's group at Caltech later expanded the method to make all of the soft tissue in a mouse's body transparent. The team next set out to develop a way to clear hard tissues, like the bone that makes up our skeleton.”

The authors wrote, “We used Bone CLARITY and a custom-built light-sheet fluorescence, microscope to detect the endogenous fluorescence of Sox9-tdTomato(+) osteoprogenitor cells in the tibia, femur, and vertebral column of adult transgenic mice.”

“To obtain a complete distribution map of these osteoprogenitor cells, we developed a computational pipeline that semi automatically detects individual Sox9-tdTomato(+) in their native 3D environment. Our computational method counted all labeled osteoprogenitor cells without relying on sampling techniques and displayed increased precision when compared with traditional stereology techniques for estimating the total number of these rare cells.”

The group collaborated with researchers at the biotechnology company Amgen Inc. to use Bone CLARITY to test a new osteoporosis drug.

"Our collaborators at Amgen sent us a new therapeutic that increases bone mass," said Ken Chan, graduate student and co-first author of the paper, in the news release. "However, the effect of these therapeutics on the stem cell population was unclear. We reasoned that they might be increasing the proliferation of stem cells."

So then the researchers gave one group of mice the treatment and, using Bone CLARITY, compared their vertebral columns with bones from a control group (no drug).

"We saw that indeed there was an increase in stem cells with this drug," he added. "Monitoring stem cell responses to these kinds of drugs is crucial because early increases in proliferation are expected while new bone is being built, but long-term proliferation can lead to cancer."

Dr. Gradinaru told OTW, “Clearing techniques were mostly developed and optimized toward clearing soft tissue such as the brain, and it achieves remarkable results with these types of tissues. However, bone is a unique histological sample, it has both hard (mineral) and soft (bone marrow) tissue. Therefore, it is challenging to tailor a clearing protocol that addresses these two very different types of tissue while at the same time maintaining the native fluorescence of reporter mouse lines.”

“Cell types like the osteoprogenitor population that we are trying to study are rare and non-uniformly distributed, therefore it is challenging to slice a few thin sections of the bone and accurately estimate their total numbers. Observing the bone in 3D allows us to detect all of the osteoprogenitor cells inside and detect minute changes in their population over the course of drug treatment.”

“In addition, the more traditional approach of studying the bones by serial sectioning is tedious and it may compromise the 3D morphology, which is especially important in applications of stem cell research where the cellular environment plays a large role in their regulation.”

The clearing process we developed uses gentle clearing reagents that preserves the native fluorescence of the reporter mouse, while enabling us to image regions of the bone from one end to the other.”

“Light-sheet microscopy was a big time saver and allowed us to shorten the imaging time to only [a] few hours per bone, while still maintaining single-cell resolution. Our computation pipeline helped speed up our analysis of the big datasets we generated.”

“To retain the integrity of the bone marrow, a fixative is perfused through the vasculature of the post-mortem mouse. The fixative prevents the tissue from decaying, and by using the animal vasculature we achieve uniform fixation and we were able to fix hard to access areas in the bone. Additionally, we use a hydrogel that locks in place the proteins and nucleic acids during removal of lipids. The hydrogel preserves the 3D architecture of the samples, allowing the gentle detergent flow throughout the bone to wash away the opaque lipids.”

Commenting on barriers that must be overcome to further improve upon the platform, Dr. Gradinaru told OTW, “In order to make the method more versatile, antibody staining is still an ongoing challenge with tissue clearing methods. Antibodies are large molecules that require time to diffuse into the tissue. Currently, the bones would need to be bisected in order to help penetration of the antibody. To overcome this barrier, systemic delivery of antibodies and the use of small molecule staining methods can help. Scalability to larger animals is a challenge, for example sample processing time, acquisition time, and data processing would scale linearly. Therefore, the development of stronger chelators, fast microscopes with very long working distances, and better ways to handle big data is required.”

New Testing Device for in vitro Knee Biomechanics

How to load the knee properly during testing, but not constrain it much? Six goats in the Netherlands are helping us determine just that.

The article, “A novel physiological testing device to study knee biomechanics in vitro,” was published online April 25, 2017 in The Knee.

Fabian van de Bunt, Ph.D., with the Department of Orthopaedics at the VU University Medical Center in Amsterdam, The Netherlands and a co-author on the study, told OTW, “Osteoarthritis (OA) is a major health care problem in Western societies and there is growing awareness that kinetics and mechanical loading play important roles in this process. In our group, we gained ample experience with intervertebral disc degeneration—a type of OA—using a loaded disc culture system.”

“More specifically, we found that overloading led to katabolic and inflammatory responses by the cells. When we turned to study OA in the knee, we found no device that could accurately reproduce both movements and loading at the pace of a natural gait pattern. Therefore, we set ourselves to design and build such a device.”

“The interesting feature of our dynamic biomechanical knee system (BKS) is that it can produce regular knee kinetics by solely applying flexion–extension and normal joint loading: all other movements were unconstrained, so that the femoral part of the knee could find its natural pathway. To achieve that, we made sure that the outline of the center of rotation of the condyles was in line with the center of rotation of the femur shackle, while the femur shackle itself kept full freedom of movement.”

The authors wrote, “Six goat knees were used in the current study. Flexion and extension simulating gait was imposed by a servo-motor, while normal joint load was applied by two artificial muscles. Intra-class correlation coefficients (ICCs) were assessed for inter-test measures, while paired t-tests were performed for comparison between intact knees and knees with ACL [anterior cruciate ligament]-lesion.”

Dr. van de Bunt commented to OTW, “Unconstrained motion is essential for the physiological assessment of the healthy and pathological knee. Therefore, our most important finding is that we succeeded in achieving a stable but unconstrained movement of the knee under full physiological loading during normal gait. The movement patterns were reproducible after complete removal and remounting of the knee from and into the system. Finally, we found substantial but reproducible deviations of knee kinematics after cutting the ACL.”

The main surprise for us was that the motion of the knee was so stable and reproducible, despite the fact that we only applied one motion (flexion-extension) and a joint load of physiological magnitude. This shows that the knee is an inherently stable joint, as long as the ligaments and the meniscus are intact.”

“The biomechanical knee system has huge potential for the evaluation of implants and surgical techniques, such as ACL repair, meniscus repair and unloading devices. While the device described was designed for goat knees, we now have an upgrade to evaluate surgical innovations on human knees under physiological biomechanical conditions. This way we can be more efficient and at the same time reduce the need for animal experiments.”

IV Acetaminophen Reduces Opioid Need After Hip Surgery

Pain isn’t just a pain…it's an obstacle to staying on track with postop recovery.

Surgeons at Hospital for Special Surgery (HSS) in New York wanted to know if using intravenous (IV) acetaminophen might reduce the amount of opioids patients need after hip replacement surgery (at present, these patients typically receive opioids and Tylenol). “While there are numerous studies on this topic in other surgical subspecialties, there is nothing recent in the hip and knee literature,” says Geoffrey Westrich, M.D., to OTW.

Dr. Westrich, Director of Research for the Adult Reconstruction and Joint Replacement Service at HSS, and the principal investigator, has worked with colleagues to launch a randomized, double-blinded study on this topic. “We are constantly striving to enhance the joint replacement experience for patients. One of our primary goals is to reduce the amount of postoperative pain and the side effects of narcotic pain medication, so that patients can fully participate in physical therapy and are thus discharged in a timely manner. If we can reduce opioid use then that can get us closer to those goals. The fact is that once patients are on narcotics they may have side effects such as nausea/vomiting and constipation and also become lethargic and don’t fully engage in physical therapy…which may affect their discharge.”

“We regularly collaborate with HSS anesthesia researchers, looking at the efficacy of different drugs and injections in hips and knees. Not long ago IV acetaminophen was introduced and it soon became clear that it is much more effective than the oral version of the medication (i.e., Tylenol). The peak plasma concentration is much higher with the IV medication, and the time it takes to reach the maximum dose is much earlier—at least two hours quicker than with oral acetaminophen. All of this means that the drug is passing through the blood-brain barrier more quickly and in higher concentrations…which results in better, and more immediate, pain relief.”

This study, which is aiming for 150 individuals, is enrolling new participants each week. “I am very pleased with the rigor of our methodology. Since it is a double blind study, patients who enroll receive IV acetaminophen and acetaminophen tablets—but only the pharmacy knows which patients are getting the real acetaminophen in the IV and placebo tablets versus the placebo IV infusion and the real tablets.”

“There is also a strict outcome assessment protocol; we are measuring amount of narcotics needed, sedation effects, whether patients meet their physical therapy milestones, and length of stay. Not only have there been no complications as of yet, but we have had several patients go home the day after surgery. I think that there is a good chance that these are individuals who received IV acetaminophen.”

Dr. Westrich adds, “Our ultimate goal is to have a greater understanding of which multimodal pain protocol is the best for a given patient (i.e., spinal anesthesia, periarticular injections, anti-inflammatories, IV acetaminophen, and narcotics) and try to minimize the amount of narcotics to limit the side effects associated with such medications. We hope to optimize this in future research as well.”

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