Researcher News:  Jin Hee Kwak, DDS, MS, UCLA Orthodontics

https://www.youtube.com/watch?v=PIgU95fL0qg&feature=youtu.be

Microgravity, or weightlessness, affects many observable phenomena and thus provides a unique environment for study. A successful launch of the SpaceX CRS-11 took place on June 3rd, 2017, carrying an experiment from the University of California, Los Angeles (UCLA) to NASA’s International Space Station (ISS) U.S. National Laboratory to test a new therapy that could prevent and treat bone loss in humans. This was one of more than 40 experiments that will showcase the breadth of research possible through the ISS National Laboratory, but the only one involving a mammalian model. This mission, called the Rodent Research-5 (RR-5), incorporates the first live-return of drug-treated rodents from space.

The Study

The UCLA study, “Systemic Therapy of NELL-1 for spaceflight-induced osteoporosis,” is designed to test a bone-forming molecule discovered by UCLA’s Dr. Kang Ting, NELL-1, which is a protein produced by the human body that is critical for normal bone and cartilage development. Understanding how NELL-1 functions in a microgravity environment will hopefully help in the development of human therapies that can prevent bone loss or regain bone that is lost from a combination of various factors, such as genetic predisposition, age, menopause in women, and lack of exercise. A microgravity environment provides distinct conditions for studying osteoporosis. For example, astronauts spending extended time in space experience rapid bone density loss that requires daily exercise as prevention, otherwise losing more than 1% of bone per month in space as compared to 2-3% of bone loss per year in senility on the ground. It was also found that skeletally mature rodents lose up to 30% of bone mass within three weeks in space (measured by micro-CT). The experiment description from the ISS website states that the “Systemic Therapy of NELL-1 for Osteoporosis (Rodent Research-5) investigation tests a new drug on rodents that can both rebuild bone and block further bone loss, improving health for crew members in orbit and people on Earth.”

The UCLA experiment sent 40 rodents to the space station to evaluate whether NELL-1 can prevent bone loss in microgravity. After the flight, live rodents returned to Earth and to UCLA on July 4th for further therapy, representing a first for American ISS research . To study the rodents pre- and post-flight, the UCLA lab will use a SkyScan 1172 micro-CT scanner. “MicroCT is definitely the gold standard now to evaluate the bone quantity and quality non-destructively,” states Jin Hee Kwak, DDS, MS, from the UCLA School of Dentistry and the Project Manager for the experiment. “We had/have three different ex vivo micro-CT scanners in UCLA Dentistry, and the SkyScan 1172 is definitely the best. We’ve published over 50 publications in the last ten years with the key data originating from the 1172. The software is also super easy to use and allows creative analysis and imaging.”

Ground and Flight Operations

The study comprised a Ground operation and a Flight operation. The Ground operation went on for two years prior to launch to optimize the drug delivery system. The UCLA lab generated many papers throughout this process, and micro-CT always provided the key data. The Ground operation consisted of nine trials of animals, all of which went through routine analysis, including fluorescent bone labeling, serum analysis, organ analyses for toxicity testing, DEXA, micro-CT, histology, and Immunohistochemistry (IHC). “MicroCT is definitely the key data in any in vivoanimal experiment , providing ‘cross-sectional’ data (post-mortem) that quantifies bone mineral density (BMD), BV/TV, trabecular spacing, thickness, and number, in addition to many more useful parameters” says Dr. Kwak. Other important data comes from DEXA, which assesses bone mineral density (BMD)BMD “longitudinally” (throughout the experiment).

The Flight operation began with the rocket launch on June 3rd. Prior to launch, all animals (a total of 200, accounting for launch scrubs) went through DEXA scanning for BMD screening, weight analysis, and oral swabs. They were then evenly distributed to cages based on BMD and body mass so that if the first launch attempt failed (which it actually did), the next set of cages to load would have the same average BMD and weight. The Flight group had 40 rodents housed in the International Space Station (ISS) and the Ground control had 40 rodents housed in Kennedy Space Center (KSC) in Florida. Among the Flight and Ground control groups, half of the animals were to be returned live to UCLA after spending about four weeks in space (see Figure 2). The live-return animals will beare housed and cared for in the UCLA Division of Laboratory and Animal Medicine (DLAM) vivarium for 4four additional weeks where they will continue to receive therapy. They willand they represent the return and recovery on Earth. Immediately upon their return to UCLA, they were tested for grip, gait, and other behavioral tests. They also had DEXA scans to assess dynamic BMD. They will continue to receive the same therapy for an additional four weeks at UCLA as will the remaining animals on the ISS and KSC. The total experiment duration is eight to nine weeks, at which point all animals will be sacrificed. The live-return animals will undergo live-tissue analysis, including RNA sequencing, in addition to post-mortem analysisanalyses, including micro-CT. The remaining animals in the ISS and KSC will be euthanized and returned to UCLA as frozen carcasses for micro-CT analysis as well.

In this truly multicenter project, Dr. Kwak’s role is to take lead and draft all paperwork (proposals; workflow; worksheets—including one that astronauts follow as well; publications and abstracts), and oversee all procedures done by the groups involved. She is also the point of contact at UCLA for communications with NASA and many other parties involved in this project, namely: NASA; the Center for the Advancement of Science in Space (CASIS), which is the funding agency and manager of ISS US National Lab; BioServe (implementation partner); Taconic (animal vendor); Space Solutions; World Courier, and other collaborating universities. Within UCLA, Dr. Kwak also arranges collaborative work. For example, protein production is primarily done in Dr. Ting’s lab, while animal surgeries are done in Dr. Soo’s lab. Engineering of the drug delivery system was in Dr. Wu’s lab. Analysis is done in all three labs, also involving postdocs and students from all labs. Regulation of funds, compliance, and ownership of material all fall under Dr. Kwak as well.

Spaceflight-induced Osteoporosis and NELL-1

Spaceflight-induced osteoporosis (OP) is extreme and almost impossible to replicate with any single OP model on Earth. Dr. Kwak explains that, “to put this into perspective, middle-aged women lose around 2-3% of their bone per year. Space-travelers—young and with superior physiques—lose more than 1% of bone mass in just one month spent in space.” Micro-CT and DXA data from previous rodent missions (RR-1 and RR-2) provided some good reference data. The RR-1 mission had skeletally mature 32-week-old rodents that lost 15% of BMD and up to 30% of bone volume over total volume (BV/TV) in just 3 weeks in space, which is four times faster than by ovariectomized (OVX) rodents on the ground. In the RR-2 mission, 12-week-old rodents lost up to 20% of BMD in 8 weeks in space, twice as much as by OVX on the ground.

“This data shows that spaceflight-induced OP is super extreme and animal age matters,” says Dr. Kwak.  “If the NELL-1 therapy proves to be successful in treating spaceflight-induced OP in this project, we will know for sure this could be used in any extreme OP case on the ground, especially those in elderly populations and those complicated by fracture-associated disuse atrophy (extended bed rest).” Although the mechanics of OP that develop from age, menopause, or disuse all vary, most drugs have common therapeutic effects. This means that the implications of this therapy could be huge.

Other therapies don’t have this stunning efficacy. Similar to Anti-sclerostin and Wnt signaling pathway therapies, NELL-1 is both osteogenic (forms bone) and anti-resorptive (prevents bone loss). NELL-1 is therefore categorized as a new type of therapy that targets both functions. As an extension of the Ground Operation and part of Dr. Ting’s R01 grant from NIH/NIAMS, the UCLA lab is testing the same NELL-1 therapy in a large animal model (ovine), which is a step before preclinical trials.

Special Challenges and a Look to the Future

Dr. Kwak discussed some special challenges that the team encountered during the scope of this project in scanning bones that contain fluorescent bone labels. They found that when a sample wasn’t secured properly within the tube, the slight movement during the scanning resulted in blurry images that required re-scanning. This sometimes caused micro-breakage of small bones, which appeared on the histology. There were a few solutions the team put in place to protect valuable samples. First, they secured the samples in the tube using styrofoam. Secondly, because the University relies on students to handle the scanning, extra training using dummy samples was put in place. This ensures that the valuable samples are scanned by well-trained students and the re-scan frequency is limited to none to a maximum of one.

Credits

Special and heartfelt thanks to Jin Hee Kwak, DDS, MS, Assistant Professor and Clinic Director, UCLA Section of Orthodontics, UCLA Division of Associated Clinical Specialties, for her time and generous help with this article.

Many thanks also to Hsin Chuan (Dan) Pan, Orthodontic Resident, UCLA School of Dentistry, for sharing his visuals for this article.

 The research on this and related experiments is by collaboration of Drs. Chia Soo (UCLA School of Medicine), Kang Ting (UCLA School of Dentistry and the discoverer of NELL-1 osteogenic protein), and Ben Wu (UCLA School of Dentistry and School of Engineering). Dr. Kwak (UCLA School of Dentistry) is the Project Manager for this space mission, and has provided invaluable help to Micro Photonics for this article. The UCLA DLAM is the animal care facility, with staff and vets on standby to receive the animals and look after the invaluable space mice closely until the end of experiment. This research is funded by multiple grants, including CASIS, the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) and the National Institute of Dental and Craniofacial Research (NIDCR) within the National Institutes of Health (NIH), and the American Association of Orthodontists Foundation (AAOF). This experiment builds on those previous research investigations.

RR-5 mission overview video created by NASA: https://www.youtube.com/watch?v=ht9zTT4qPeI&feature=share&app=desktop

Link to UCLA:  https://www.dentistry.ucla.edu/directory/profile/jin-hee-kwak-dds-ms

Figure 1 (at top of page): Micro-CT of an adult mouse scanned on a SkyScan 1176. Courtesy of Hsin Chuan  Dan Pan, Orthodontic Resident,  UCLA School of Dentistry.