Antioxidant-Encapsulated Chondroitin-Sulfate Microparticles to Treat Low Back Pain
Alexandria Richardson
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08/04/2020
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Minimally invasive delivery system to treat disc-associated low back pain (Nebraska Virtual Summer Research Symposium 2020)
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- [00:00:01.104]Hello everyone.
- [00:00:02.000]My name is Alexandria Richardson
- [00:00:03.765]and I am an upcoming senior studying
- [00:00:05.868]Biological Systems Engineering here at
- [00:00:07.927]the University of Nebraska-Lincoln.
- [00:00:09.454]Today, I will be digitally presenting
- [00:00:10.741]our work on Antioxidant-Encapsulated,
- [00:00:12.923]Chondroitin-Sulfate microparticles
- [00:00:15.023]to treat low back pain.
- [00:00:16.640]This work would not have been possible
- [00:00:17.791]without the support and guidance from my
- [00:00:19.141]graduate mentor, Fei-San Lee and
- [00:00:21.162]lab PI, Dr. Rebecca Wachs.
- [00:00:24.823]I am an undergraduate member of the
- [00:00:26.830]Orthopedic and Neural Engineering Lab,
- [00:00:28.300]also known as ONE Lab.
- [00:00:29.754]ONE Lab aims to improve the lives of
- [00:00:31.920]patients with low back pain. We pursue
- [00:00:34.153]this goal by bringing together a diverse,
- [00:00:36.155]interdisciplinary team of engineers,
- [00:00:38.113]clinicians, and scientists to create
- [00:00:39.983]comprehensive and innovative approaches.
- [00:00:42.908]These are some of the research interests
- [00:00:45.108]encompassing ongoing projects in our lab.
- [00:00:46.928]While seemingly diverse at first glance,
- [00:00:48.844]these approaches are all fundamentally aimed
- [00:00:50.625]at better understanding and treating
- [00:00:51.789]low back pain. Several projects in the lab
- [00:00:54.128]involve elements of tissue engineering,
- [00:00:55.739]neural engineering, biomaterials, and
- [00:00:58.169]antioxidant and anti-inflammatory treatments.
- [00:01:00.905]Between 26-42% of chronic low back pain
- [00:01:04.434]is caused by pain originating from
- [00:01:06.445]the intervertebral disc [1].
- [00:01:07.644]As you can see on the diagram
- [00:01:09.114]on the right, the intervertebral disc
- [00:01:10.813]is the gelatinous shock absorber
- [00:01:12.473]sandwiched between adjacent vertebral
- [00:01:14.277]bodies in the spine.
- [00:01:16.219]The intervertebral disc consists of a
- [00:01:17.984]squishy, gelatinous core known as the
- [00:01:19.854]nucleus pulposus surrounded by
- [00:01:21.287]collagenous outer rings. Typically, the
- [00:01:23.732]matrix of the nucleus pulposus contains
- [00:01:25.937]a high concentration of the neuro-inhibitory
- [00:01:27.924]proteoglycan chondroitin sulfate.
- [00:01:30.194]This prevents painful innervation from
- [00:01:32.424]the nearby sensory neurons located
- [00:01:33.866]in the dorsal root ganglion.
- [00:01:35.672]However, many causes including
- [00:01:37.345]aging, injury, and smoking can lead to
- [00:01:39.558]breakdown of this disc matrix [2].
- [00:01:41.609]Therefore, this allows sprouting of
- [00:01:43.519]pain-sensing nerves into the disc core.
- [00:01:45.465]Matrix breakdown also produces a harsh
- [00:01:47.560]inflammatory environment that contains an
- [00:01:49.809]excess of reactive oxygen species (ROS),
- [00:01:52.581]and inflammatory cytokines [5] [6].
- [00:01:54.787]Invading nerves are stimulated by
- [00:01:56.615]inflammatory cytokines and ROS in this
- [00:01:58.311]environment, producing a painful sensation.
- [00:02:01.084]This imbalance of reactive oxygen species
- [00:02:03.403]and antioxidants is known as oxidative
- [00:02:05.312]stress. In response to tissue damage,
- [00:02:07.536]many inflammatory cytokines and ROS
- [00:02:09.640]are released in conjunction at the
- [00:02:10.947]injury site. The increase in inflammation
- [00:02:13.392]at the disc only releases more
- [00:02:14.862]inflammatory cytokines and ROS, creating
- [00:02:16.853]the positive feedback loop pictured on
- [00:02:18.531]the left [7]. This can kind of be thought
- [00:02:20.342]of as a quote on quote "inflammatory soup"
- [00:02:22.944]as shown in the diagram on the right,
- [00:02:24.258]where many things are working together
- [00:02:25.793]to create an inflamed environment.
- [00:02:27.508]When nerves invade the disc core in
- [00:02:28.965]response to tissue damage, they are
- [00:02:30.330]painfully stimulated be these excess
- [00:02:32.069]ROS and inflammatory factors.
- [00:02:34.586]So, how can you prevent this painful
- [00:02:36.254]stimulation? We theorize that targeting
- [00:02:38.332]the excess ROS with an antioxidant
- [00:02:40.469]treatment could reduce the inflammation
- [00:02:42.023]present after disc matrix breakdown.
- [00:02:44.299]Excess ROS can be neutralized through
- [00:02:46.328]antioxidant treatment, reducing high
- [00:02:48.164]levels of oxidative stress and potentially
- [00:02:50.110]returning the disc back to a healthy state [6].
- [00:02:53.172]This could prevent pain by
- [00:02:54.467]preventing stimulation of invading
- [00:02:55.706]sensory neurons by the inflammatory environment.
- [00:02:59.597]We are currently developing a minimally
- [00:03:01.275]invasive, injectable delivery system to
- [00:03:03.418]treat low back pain [8]. Designing
- [00:03:05.046]neuro-inhibitory methacrylated
- [00:03:07.201]chondroitin sulfate microparticles,
- [00:03:08.963]MACS-A microparticles, with loaded
- [00:03:11.753]antioxidants has the potential to target
- [00:03:13.823]both undesired disc innervation and
- [00:03:15.561]excess ROS that contribute to many
- [00:03:17.491]cases of disc-associated low back pain.
- [00:03:20.291]A schematic of this process is pictured below.
- [00:03:23.149]As you can see, stressed NP cells
- [00:03:24.775]would theoretically be targeted by
- [00:03:26.457]our loaded MACS-A microparticles and
- [00:03:28.334]treated with antioxidants to reduce
- [00:03:29.852]oxidative stress, and therefore
- [00:03:31.621]reducing inflammation in the disc.
- [00:03:34.106]The neuro-inhibitory chondroitin sulfate
- [00:03:35.822]comprising our particles could
- [00:03:37.284]potentially prevent further innervation of
- [00:03:39.306]sensory neurons into the disc.
- [00:03:41.056]Taken together, this could be a
- [00:03:42.557]one-two punch to treat two major sources
- [00:03:44.628]of inflammation and pain in
- [00:03:46.348]disc-associated low back pain.
- [00:03:49.200]So, how do you actually make a microparticle?
- [00:03:51.706]I will break the process for these
- [00:03:53.786]microparticles at a relatively high level,
- [00:03:55.674]step-by-step [8].
- [00:03:57.497]First, chondroitin-sulfate A undergoes
- [00:03:59.664]a modification process that adds a
- [00:04:01.508]methacrylate group forming MACS-A.
- [00:04:04.016]This allows for the later formation of
- [00:04:05.629]a polymer during cross-linking.
- [00:04:07.685]This MACS-A is added to cold 1xPBS
- [00:04:10.523]along with reaction catalysts and the
- [00:04:12.243]antioxidant of choice.
- [00:04:15.776]Next, this solution is added dropwise
- [00:04:17.766]into a cold corn oil solution.
- [00:04:20.616]This corn oil mixture is homogenized
- [00:04:22.777]at 3000 rpm for 5 minutes on ice,
- [00:04:25.729]dispersing the aqueous mixture containing
- [00:04:27.327]the reaction catalysts, antioxdants, and MACS-A
- [00:04:30.167]throughout the corn oil, forming a
- [00:04:32.017]single water-in-oil emulsion.
- [00:04:34.555]Next, the emulsion is polymerized to form
- [00:04:36.759]the actual microparticles.
- [00:04:39.783]This is done over heat in a 50 degree Celsius
- [00:04:41.984]water bath and continuous stirring,
- [00:04:43.541]inducing thermal cross-linking of the
- [00:04:45.488]MACS-A polymer around antioxidants
- [00:04:48.094]in the aqueous solution. Additionally,
- [00:04:50.135]this process occurs under an inert
- [00:04:52.140]environment with Nitrogen gas bubbling
- [00:04:53.900]to ensure the presence of oxygen in the
- [00:04:55.900]atmosphere doesn't affect cross-linking.
- [00:04:58.432]Lastly, the mixture undergoes a series
- [00:05:00.381]of washes to separate and purify the
- [00:05:02.330]microparticles from the surrounding corn oil.
- [00:05:05.263]These microparticles are then stained
- [00:05:06.644]with a chondroitin sulfate dye, and imaged
- [00:05:08.812]for later size quantification.
- [00:05:11.264]Here are sample results from recent encapsulation
- [00:05:13.629]experiments. A sample image of encapsulated
- [00:05:15.854]microparticles is shown on the upper right.
- [00:05:17.898]When particles are empty and unstained,
- [00:05:20.471]they are completely clear. The antioxidant
- [00:05:22.831]used in this experiment is a dark brown
- [00:05:24.618]color. This image clearly shows that the
- [00:05:27.244]antioxidant was successfully encapsulated
- [00:05:29.014]into the microparticle. Moving to the left,
- [00:05:31.432]the mean size, standard deviation, and
- [00:05:33.362]number of particles are calculated
- [00:05:35.182]for each experimental group using
- [00:05:36.562]ZEN Blue image analysis software.
- [00:05:39.017]As shown on the bottom right,
- [00:05:40.282]this data can be used to generate histograms
- [00:05:42.139]displaying the size distribution of particles.
- [00:05:45.073]This size distribution will be
- [00:05:46.173]important for future work looking
- [00:05:47.580]at nucleus cell uptake of particles.
- [00:05:50.355]Particles greater than 10 microns
- [00:05:52.085]theoretically remain extracellular, while
- [00:05:54.066]particles smaller than 5 microns
- [00:05:55.797]are theoretically endocytosed.
- [00:05:57.794]Therefore, separating microparticles by
- [00:05:59.832]size may allow for specific extracellular or
- [00:06:02.221]intracellular targeting upon injection into
- [00:06:04.649]the disc. We are currently developing
- [00:06:06.655]a filtration method to separate
- [00:06:08.363]particles by size for this purpose.
- [00:06:10.408]Results from a recent filtration
- [00:06:11.853]experiment are shown below,
- [00:06:13.384]indicating that we are successfully
- [00:06:15.033]moving toward the separation of
- [00:06:16.123]particles into distinct size
- [00:06:17.616]populations after filtering. More work
- [00:06:20.555]needs to be done in this area to improve
- [00:06:22.299]accuracy and retain microparticle yield
- [00:06:24.471]through subsequent filtrations.
- [00:06:26.598]A large portion of my summer was spent
- [00:06:28.518]improving size analysis for microparticles.
- [00:06:30.918]In the past, diameter was calculated by
- [00:06:33.090]manually drawing straight line across
- [00:06:35.082]particles in ImageJ software. A total of
- [00:06:37.228]40 microparticles were randomly selected
- [00:06:39.348]for each experimental condition. The
- [00:06:41.172]mean and standard deviation were then
- [00:06:42.547]calculated based upon these measurements.
- [00:06:45.680]We are attempting to improve this
- [00:06:47.285]size analysis using more intelligent
- [00:06:49.005]software. By writing an image analysis
- [00:06:51.007]program in ZEN Blue, it will
- [00:06:52.564]theoretically be able to automatically
- [00:06:54.086]detect particles and calculate the resultant
- [00:06:56.210]diameter. A screenshot of this has been
- [00:06:58.424]shown below, where detected particles
- [00:07:00.494]are highlighted in bright red.
- [00:07:02.428]This automation will hopefully save
- [00:07:03.625]us a lot of time and give us much higher
- [00:07:05.630]output for our data analysis in the future.
- [00:07:08.623]In upcoming experiments, we will be
- [00:07:10.370]analyzing the effectiveness of our
- [00:07:11.865]microparticles at scavenging ROS using
- [00:07:14.261]a super-oxide dismutase assay.
- [00:07:17.009]We will also be looking further at in vitro
- [00:07:19.127]characterization of nucleus pulposus cells
- [00:07:21.338]when treated with loaded and unloaded
- [00:07:23.008]microparticles. Lastly, we will
- [00:07:25.018]characterize, analyze, and evaluate the
- [00:07:27.170]neuro-inhibitory properties of our
- [00:07:28.687]microparticles to see if they are
- [00:07:29.957]sufficient a stopping further
- [00:07:31.489]DRG sensory neuron innervation
- [00:07:33.519]into the disc core.
- [00:07:35.299]I would like to thank all member of ONE Lab
- [00:07:37.259]for support through this project.
- [00:07:38.349]I would also like to thank UCARE for
- [00:07:39.592]helping to fund my summer research experience.
- [00:07:42.657]My references are listed below
- [00:07:44.507]on this slide, if you are interested.
- [00:07:46.748]And last but not least,
- [00:07:47.548]thank you all for watching!
- [00:07:48.969]I hope you have a great day.
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