Cutaneous wound healing through paradoxical MAPK activation by BRAF inhibitors

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Escuin-Ordinas H, Li S, Xie MW, Sun L, Hugo W, Huang RR, Jiao J, de-Faria FM, Realegeno S, Krystofinski P, others. Cutaneous wound healing through paradoxical MAPK activation by BRAF inhibitors. Nature Communications. Nature Research; 2016;7.

08.01.16Nature Communications Cutaneous wound healing through paradoxical MAPK activation by BRAF inhibitors

BRAF inhibitors are highly effective therapies for the treatment of BRAF(V600)-mutated melanoma, with the main toxicity being a variety of hyperproliferative skin conditions due to paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway in BRAF wild-type cells. Most of these hyperproliferative skin changes improve when a MEK inhibitor is co-administered, as it blocks paradoxical MAPK activation. Here we show how the BRAF inhibitor vemurafenib accelerates skin wound healing by inducing the proliferation and migration of human keratinocytes through extracellular signal-regulated kinase (ERK) phosphorylation and cell cycle progression. Topical treatment with vemurafenib in two wound-healing mice models accelerates cutaneous wound healing through paradoxical MAPK activation; addition of a mitogen-activated protein kinase kinase (MEK) inhibitor reverses the benefit of vemurafenib-accelerated wound healing. The same dosing regimen of topical BRAF inhibitor does not increase the incidence of cutaneous squamous cell carcinomas in mice. Therefore, topical BRAF inhibitors may have clinical applications in accelerating the healing of skin wounds.

Helena Escuin-Ordinas, Shuoran Li, Michael W Xie, Lu Sun, Willy Hugo, Rong Rong Huang, Jing Jiao, Felipe Meira de-Faria, Susan Realegeno, Paige Krystofinski, Ariel Azhdam, Sara Marie D Komenan, Mohammad Atefi, Begoña Comin-Anduix, Matteo Pellegrini, Alistair J Cochran, Robert L Modlin, Harvey R Herschman, Roger S Lo, William H McBride, Tatiana Segura, Antoni Ribas

Escuin-Ordinas H, Li S, Xie MW, Sun L, Hugo W, Huang RR, Jiao J, de-Faria FM, Realegeno S, Krystofinski P, Azhdam A, Komenan SMD, Atefi M, Comin-Anduix B, Pellegrini M, Cochran AJ, Modlin RL, Herschman HR, Lo RS, McBride WH, Segura T, Ribas A

Combining nanofiber vaccine and MAP scaffold technology to reduce inflammatory cytokines

From Pablo’s lab diary:

Did you know that putting MAP scaffolds into wounds causes elevated adaptive immune responses, which leads to enhanced regeneration of the skin? I learned about this in grad school, and it made me think – what if we could focus those immune responses to battle the inflammatory cytokines that drive chronic would healing? Chronic wounds that never heal are a big problem for a lot of people, so I’m working on modifying MAP scaffold particles with a nanofiber vaccine platform to see if I can reduce inflammatory cytokines and heal those wounds. My goal is to develop an active immunotherapy that can increase a special type of antibody – ones that are capable of downregulating dysregulated-cytokine activity at the wound site. Accomplishing this will not only avoid delayed healing in patients with underlying chronic conditions, but it will also prevent the recurrence of chronic wounds.

This collaborative project has been a challenging journey for sure, but we are on our way to exciting results! If you are a student interested in working at the intersection between hydrogels and active immunotherapy for enhanced tissue healing, contact us and add in the message that you are interested in my project! #pablo #mapfibers

Immune cloaking

From Sydney’s lab diary:

During a college research internship, I joined a team of scientists working to develop engineered organs for transplantation in order to expand the donor organ pool. I will never forget the first time I was up-close and personal with organs sustained outside of the body. Two large heaps of pink slowly popped open to reveal a pair of lungs rising and falling with the pressure waves from the ventilator. The heavy lobes of tissue became fragile, fluctuating balloons. Little did I know that a few years later, I would watch a lung transplant procedure and witness a patient take their first breath with their new lungs.

Every year, over 40,000 people receive an organ transplant in the US. In most cases, these organs come from genetically non-identical donors. This means that without immunosuppression, a transplant recipient’s immune system would mount a “stranger danger” response and attack the precious and life-saving transplanted graft. However, immunosuppressive therapies work broadly, and thus, lower the guard to other non-desirable invaders – such as infection or even malignancy. Managing this fine balance of over- or under-immunosuppression is a major challenge in the field of transplantation. But, what if we thought of this problem from the inside out, and rather than weakening the recipient immune forces, we altered the organ to mask foreign signals from the recipient’s immune system – in effect, presenting the organ as a trojan horse to be welcomed into the recipient while maintaining immune forces at the ready to face other undesirable invaders.

My research interest is to subvert solid organ rejection by masking donor antigen or even disguising foreign tissues as “self.” I am working to do this by utilizing novel, synthetic cell surfaces and by developing organ-specific drug delivery methods. Through this work, we hope to reduce the burden of system immunosuppression, enhance donor organ function, and prolong survival following organ transplantation.

If you’re a student who is intrigued by stealth-ing of antigens or tissue-targeting therapies, contact us and add in the message that you’re interested in my project! #sydney #immunecloacking

Trapping the pro-repair environment

From Shangjing’s lab diary:

I’m currently a postdoc in the lab, and my previous PhD project revolved around designing biomaterials (actually MAP gels) for bone tissue engineering. I delivered stem cells from the material to promote bone formation. I joined the Segura lab because I wanted to learn from the creators of MAP scaffolds and challenge myself to design materials for neural applications, which is completely new to me. The project Prof. Segura proposed sounded really interesting, and with some reading, we have a really cool approach.

The fact that brain injury may actually help you learn new things fascinates me. This occurs through a process called plasticity. When we are first born, our brain is very plastic, and we are able to learn many new things. However, as we age, our brains are less plastic, meaning neurons in the brain aren’t able to form as many new pathways and connections. On the one hand, having our brains settle on stable connections helps us become good at things, but unfortunately, it also hinders us from picking up new things easily. It turns out that after injury, our body turns on plasticity as a way to heal the injury.

My project is to leverage this plasticity window with a ‘repair soup’ to promote healing after stroke. In particular, I am working on strategies that would trap locally-secreted plasticity-proteins into our biomaterial in an effort to extend the window of cellular change. The end goal of course is to reduce physical impairment caused by stroke.

Do these lipids make my brain look fat?

From Briana’s lab diary:

I’m a chemist, and I love the art of synthesis – creating new polymers from scratch with unique designs. I also have experience making nanoparticles, so Prof. Segura and I discussed using nanoparticles as drug carriers that would target the brain after stroke. Before joining the lab as a postdoc, I worked on developing dendritic polymers that have both a hydrophobic (‘fat-loving’) and a hydrophilic (‘water-loving’) part, so I’m incorporating this chemistry into my current project. By using these amphiphilic polymers to form nanocapsules, we could not only co-deliver both hydrophobic and hydrophilic drugs, but we could also leverage the lipid (fat) content of the nanocapsules to cross the blood-brain barrier. In this way, our material could reach the brain through IV injection into the arterial system as opposed to direct injection into the brain, making it even less invasive.

After talking with Prof. Segura, I also learned that the brain is 50% lipids by weight. Lipids do much more than make us fat – they’re active players in signaling and normal tissue function. I’ll be reading and learning about lipid biology, especially in the context of stroke, so that I can guide my synthesis to include brain-active materials. By using self-assembling lipids, I aim to make nanoparticles that are entirely bioactive.

Hydrogel microparticles (HMPs)

The first step is to design the particles that compose our MAP material. Each particle is actually a network of entangled polymers (like a microscopic hairball), and we typically work with hydrogel polymers that swell in water – hence the term HMPs (also referred to as µgels). The process starts with some good ‘ole polymer chemistry, where we customize our material to fit the therapeutic application. Once the polymers are just right, we form them into the desired shape, then lock the shape into place through a process called polymerization. Most of our MAP experiments use spherical HMPs produced through emulsion or microfluidic techniques, but we’ve been experimenting with other shapes as well. (I know we just dropped some technical terms, so if you’re unfamiliar and want to learn more about HMPs, click here, we won’t tell anyone 🤫)

Non-3D-pore descriptors

LOVAMAP analyzes more than just 3-D pores. For example, we study paths that course throughout the void space of a scaffold. (You can see a distinct path highlighted in red below.) The measurements taken for a path give rise to some of our non-3-D-pore descriptors.

Generate images & GIFs

Part of LOVAMAP’s power is its ability to offer visual insight into all that is void space. Visualization is an important aspect of science communication, and we put a lot of effort into making beautiful images and videos that help the scientific community see what we’re talking about. Below we’re highlighting a single 3-D pore that represents a pocket of empty space surrounded by particles.

Segment void space

Our goal is to isolate all of the distinct pockets of empty space throughout the scaffold – seems like the obvious way to break up the space, right? But doing this accurately is so much more complicated than you might think. LOVAMAP uses the spatial landmarks as a basis for segmenting the void space into these natural open pockets, which we called ‘3-D pores.’ Below you can get a taste for how crazy and diverse pore shapes can be!

Studying the effects of amino acid chirality on skin regeneration

From Elle’s lab diary:

Did you know that skin is actually the largest organ in our body? When we get skin injuries, the important barrier function of the skin is disrupted. The body tries to do a ‘quick fix’ to heal these wounds by depositing granulation tissue and building a scar rather than putting in the work to regenerate essential dermal structures (think: hair follicles, oil glands). But researchers aren’t satisfied with this type of wound healing and are actively working to regenerate better skin!

A previous postdoc in our lab, Dr. Don Griffin (who’s now running his lab at UVA), stumbled across an exciting and unexpected new way to modulate regeneration using our MAP scaffold platform. The best part? All we had to do was change the conformation of a few amino acids! The majority of the amino acids in the human body exist in the L-chiral configuration, but switching three amino acids in our crosslinking peptide to the D-chiral configuration caused a significant increase in regeneration. Check out that image below!

I was so intrigued by how such a small change in material composition could influence the regenerative properties of a material with that magnitude, so it’s become my goal to understand the mechanisms of this phenomenon and leverage this knowledge to improve wound healing.

If you’re interested in working on a skin project that studies chirality of biomaterials, regeneration, and the immune system, contact us and add in the message that you’re interested in my project! #elle #chiralityandregeneration

Neuron communication at the synapse

From Nhi’s lab diary:

Making connections is what I like to do: between friends, collaborators, or in this case – neurons. A synapse is when two different neurons almost touch. This junction is where chemicals, called neurotransmitters, are exchanged between them to communicate with each other. Fun fact: neurons can have 100’s to 1000’s of synapses! These synapses make up the circuits in your brain, which help you control movement, learn new skills, and create memories. Ischemic stroke destroys these connections, forcing patients to lose mobility, which impacts their day-to-day lives dramatically.

The brain does try to repair some of these connections after stroke but can only do so for a limited amount of time. My project investigates whether our MAP material can serve as a scaffold for synapse formation, and I’m incorporating different factors to help boost stable connections. That way, stroke patients will have more time to reform those neural circuits and eventually regain their mobility.

If you’re interested in connecting with me (pun intended) to study neural plasticity in the brain, contact us and add in the message that you’re interested in my project! #nhi #synapticmaterials

Extract spatial landmarks

LOVAMAP relies on accurately identifying key spatial landmarks within the void space of MAP scaffolds. These landmarks are subtypes of the medial axis, which are central locations in space, and they serve as a starting point to orient LOVAMAP to the space. Our custom algorithms are highly effective at locating these landmarks because we take advantage of information stored in the graphical representation of granular material. Once the information has been stored, we’re ready to start dissecting and analyzing the MAP scaffold.

3-D pore descriptors

3-D pores (or pockets of open void space) form such weird shapes!! How do you describe something that looks so weird? If someone asked you to come up with different ways to measure a cube, that’s easy – you could measure length, volume, corner angles, etc. 🥱 But for these 3-D pores, it’s more complicated. So we came up with 17 different measurements to describe them, like reporting their volume or the number of surrounding particles that form the space. We also distinguish between 3-D pores that are completely inside of the scaffold vs. those that are touching the edge (like the massive purple one below).

LOcal Void Analysis of MAP scaffolds (LOVAMAP)

Straight from Lindsay’s dissertation abstract:

Our lab designs hydrogel microparticles (HMPs) that are interlinked to form microporous annealed particle (MAP) scaffolds for wound healing applications. The therapeutic effects of MAP are attributed, in part, to the void space between particles that creates inherent micro-porosity through which cells can infiltrate and migrate unhindered. Cell behavior is influenced by local geometry, and our goal is to design scaffolds that influence cells toward pro-healing behaviors. To accomplish this, we need a methodology for quantitatively characterizing the void space of MAP scaffolds in order to study the relationships between internal microarchitecture and therapeutic outcomes.

The work presented here is a visually-rich dissertation that covers our approach for analyzing the void space of packed particles. We use techniques from computational geometry and graph theory to develop a robust methodology for segmenting the void space into natural pockets of open space and outputting a set of descriptors that characterize the space. Our methods are developed using simulated MAP scaffolds covering a range of particle compositions, including mixed particle sizes, stiffnesses, and shapes. Our software, called LOcal Void Analysis of MAP scaffolds (LOVAMAP), has allowed us to study many aspects of void space, including global descriptors like void volume fraction, local ‘pore’ measurements of size and shape, and additional features like ligand availability, paths, isotropy/anisotropy, and available regions for unhindered migration based on size. LOVAMAP is an enabling technology that can be used for analyzing real scaffolds or studying simulated scaffolds to inform material design. It serves as a platform for void space analysis that can easily be built upon to encompass ever-growing innovations in scaffold characterization.

Read the full dissertation here: https://dukespace.lib.duke.edu/dspace/handle/10161/25268

Regenerating skin by reducing stress… on cells

From Aleja’s lab diary:

After I joined the lab, I read the work of a previous postdoc in the lab – Don Griffin – and learned how MAP scaffolds promote tissue regeneration in skin wound healing. Two questions stuck in my brain: How does MAP work to regenerate skin? And how can I improve our MAP technology? Prof. Segura and I had a lot of fun conversations trying to find the perfect focus for my project, and I remember the first time she pitched this great idea: maybe the unique mechanical properties of our granular scaffolds are the key to MAP’s superior ability to regenerate tissue!

We hypothesized that our microparticles may be dissipating the energy exerted by cells, which in turn influences cell behavior. Prof. Segura suggested studying fibroblasts since these cells are involved in skin wound healing and are very susceptible to mechanical cues. Once fibroblasts receive mechanical (and chemical) cues, they ‘activate’ into another cell type called myofibroblasts, which secrete collagen and other components of the extracellular matrix as well as contract the wound. This process is a feedback loop known as the fibroblast-myofibroblast transition, and it controls the balance between regeneration and scarring. My project is to study this process and investigate whether our material can modulate the communication between fibroblasts and myofibroblasts.

But if you’re interested in working on a skin project that studies my favorite cells, contact us and add in the message that you’re interested in my project! #alejandra #reducingstress

Synergizing glycotherapeutics and brain tissue engineering

From Yunxin’s lab diary:

Over the past decade, our interest and understanding of glycans (sugars) and their functions in the human body have expanded tremendously, thanks to the technological advances made towards studying these non-template-based, capricious molecules. Our lab and others are continuing to take deeper dives into the intricacies of glycan functions in various disease contexts, like brain cancer and ischemic stroke (take a look at our project ‘Sweet Brain’). Thanks to this explosion of glycan knowledge, glycan-based therapeutics are already showing promising results for treating specific diseases such as cancer and HIV. For me, this emerging field of glycotherapeutics is an exciting space to discover and develop more effective treatments.

In fact, the field of biomaterials-based tissue engineering is no stranger to glycans – many of us have been working with glycan materials such as hyaluronic acid and heparan sulfate for a long time! However, our current utilization of glycans in tissue engineering is only scratching the surface of the vast potential of glycans in regulating cellular- and tissue-level responses. In other words, there are both technological and clinical incentives to narrow the gap between glycotherapeutics and tissue engineering. Therefore, the overarching goal of my graduate research is to identify disease-relevant glycans and incorporate them into our biomaterial scaffolds to achieve tissue repair and regeneration.

My current disease-focus is on ischemic stroke, and I am trying to develop MAP-based materials that incorporate several glycans shown to be important in the pathophysiology of stroke. I hope to synergize the power of glycans with that of our MAP scaffolds in order to develop a platform that can dynamically address a multitude of stroke symptoms – leading to more comprehensive recovery in patients.

If you’re a student interested in working at the junction between glycotherapuetics and brain tissue engineering, contact us and add in the message that you’re interested in my project! #yunxin #sugarmap

Bioengineering Microporous Annealed Particle Scaffolds to Recruit Neural Progenitor Stem Cells and Promote Angiogenesis in the Stroke Core

Straight from Kat’s dissertation abstract:

There remains a significant gap in the need for regenerative therapies for stroke compared to what is currently available. An ideal therapy would be one that stimulates the formation of new tissue with the ability to regain any function previously lost due to stroke. Therefore, methods exploiting the plasticity of the brain and modulating endogenous cellular responses to promote repair in the stroke core after ischemia have become highly attractive. However, this process of neural regeneration is complex and requires a series of controlled biological events, such as recruitment and differentiation of neuron progenitor cells (NPC’s), angiogenesis, and axonogenesis. Biomaterials are now commonly used to research tissue regeneration and cellular mechanisms, both in vitro and in vivo.

We have designed a biocompatible biomaterial from macroporous annealed particles (MAP) hydrogels for injection into the stroke core five days after a photothrombotic stroke. Our hyaluronic acid-based material has been modified to dictate NPC fate in vitro through maintained stemness and the formation of neurospheres or towards Tuj1 positive NPCs, as well as enhance angiogenesis and the recruitment of endogenous NPCs after stroke. Within only 10 days after injection, we have observed the first case of NPC differentiation, enhanced recruitment, and angiogenesis after stroke, and at 30 days after injection we observe significant angiogenesis throughout the entire stroke core.

Read the full dissertation here: TBD

Modulating Macrophage Response with Microporous Annealed Particle Scaffolds

Straight from Yining’s dissertation abstract:

When designing biomaterials for clinical applications, the performance of these platforms hinges on their interaction with the host immune system. A failure in engaging and incorporating the correct immune response would lead to a foreign body response and subsequent rejection of the materials. To improve the biocompatibility of biomaterials and avoid undesired immune reactions, the key immunomodulatory cell type macrophage needs to be engaged and its phenotype modulated properly and in a timely manner. Therefore, the design parameters of biomaterials should be carefully considered in the context of macrophage modulation. Microporous annealed particle scaffolds (MAPS) are a new class of immunomodulatory granular materials generated through the interlinking of microgels. The modular nature of MAPS offers enormous tunability in not only the individual microgel design but also the homogenous or heterogenous microgel assembly into the bulk scaffold. We leveraged the plug-and-play feature of MAPS to study the effect of two design parameters, microgel crosslinking peptide (comprised of L- or D-amino acids) and spatial confinement (achieved through varying microgel size), on macrophage modulation and host responses.

We uncovered that a fine balance between pro-regenerative and pro-inflammatory macrophage phenotypes in MAPS with D-amino acid-based crosslinker was an indicator for regenerative scaffolds in a subcutaneous implantation model. We also discovered that scaffolds comprised of large microgels with pore sizes that can accommodate ~40 µm diameter spheres induced a more balanced pro-regenerative macrophage response and better wound healing outcomes with more mature collagen regeneration and reduced inflammation level. The role of spatial confinement on macrophage response was further explored in vitro, where we demonstrated that size-dependent macrophage response to M1/M2 cytokine stimulations was tied to the change in cell morphology and motility. This work offers valuable insights into the dynamic immune response to synthetic porous scaffolds with a specific focus on macrophages and establishes a foundation for further optimization of immunomodulatory pro-regenerative outcomes for wound healing and biomaterial implants.

Read the full dissertation here: TBD

Engineering biomaterials, culturing tolerance

From Holly’s lab diary:

Prior to joining the Segura Lab, my world was in the operating room. As a surgery resident at Duke, I’ve performed many procedures, and as you know, surgery requires cutting through the patient’s healthy tissues to gain access to internal structures. After we close them up, the wound healing process begins and usually a scar forms – but in some unfortunate cases, the wound never properly heals and takes much longer to close. We are trying to understand the molecular signals that lead to healthy wound healing and a return to normal function.

Before I was a surgeon, I spent my PhD investigating the roles of adult stem cells as well as their anti-inflammatory properties, and I later learned that biomaterials could be designed to mimic the pro-healing actions of stem cells. Since I was drawn to transplantation surgery during residency, I became interested in developing a biomaterials-based approach for improving the immune response to soft tissue transplants – and this brought me to the Segura lab. When I first approached Prof. Segura, I was struck by her welcoming attitude toward new applications of her MAP hydrogel technology. Although she was not familiar with the challenges of soft tissue transplantation, I explained what I wanted to do, and she was immediately on board. The plan is to use MAP scaffolds to promote immune tolerance. As you may have already read, MAP scaffolds promote blood and lymphatic vessel formation in healing tissues, both of which are critical processes in reconstructive and transplant surgeries like skin grafting and kidney transplantation, respectively.

Skin grafting (my current topic) is a cornerstone of reconstructive surgery. Over 160,000 skin grafts are performed annually for patients after burns, trauma or vascular disease (Serebrakian, et al. Plast Reconstr Surg Glob Open, 2018). Despite various FDA-approved dressings, up to half of skin grafts are rejected. Repeated skin grafting requires multiple surgeries, which increases risk of morbidity and mortality. Although a variety of cadaveric and synthetic alternatives exist, they are limited by allogeneic responses, dysregulated inflammatory reactions, worse scarring, poor functional outcome, and just overall lower quality of life. My research aims to improve the outcome of soft tissue reconstruction through the development of hydrogel biomaterials that can coordinate the interactions of immune cells to improve tolerance in the wound bed of allotransplantation.

If working on a biomaterials-based approach to promote tolerance in soft tissue transplants is of interest to you, contact us and add in the message that you’re interested in my project! #holly #culturingtolerance

Extending the window for cellular change

We’ve explored this approach the most. During the latter stages of wound healing, the microenvironment is in a state of endogenous plasticity, which means the tissue is still undergoing changes as it heals. The goal here is to place materials into wounds that extend the plastic time period by recruiting progenitor cells and other important factors. We primarily do this by designing materials that promote blood vessel formation. Blood vessels are a key component in tissue repair – they deliver important nutrients, regulate the immune response, and guide cell migration and tissue deposition. By maintaining and controlling the process of vessel formation with our materials, we’re thinking we can extend the window of plasticity and promote tissue regeneration. You can learn more about our approaches to promote vessel formation by reading the articles on Our Publications page that deal with therapeutic angiogenic material (filter by Topic: angiogenesis)!

Another way we’re extending endogenous plasticity is by designing materials that allow for rapid cellular infiltration while maintaining structural support. We do this by forming a microscopic scaffold made of packed hydrogel microparticles that are stuck together, which forms pockets of void space that are large enough for cells to fit. Alright, you guessed it – we’re describing MAP… you might as well read about it here.

Re-opening the window for cellular change

The most difficult wounds to treat are those that are chronic or well-past the phase of endogenous plasticity – when tissue is still being remodeled. For these wounds, we want to re-activate plasticity so that the body has another opportunity to heal itself properly. As you can imagine, this is harder to achieve than when wounds are still fresh in the acute or sub-acute phase. Our strategy is to first identify the molecules that are present in wounds during endogenous plasticity. We will then introduce materials that induce blood vessel formation as a source for replenishing the molecules that are present during plasticity. The goal is to determine the minimum molecular signals needed to re-open the window for cellular change.

Injectability

In the same way that it would be difficult to fit a sponge into a needle, it’s difficult for a non-granular matrix to be injected. And if something can’t be injected, then getting it to the right place might require some surgery. Luckily MAP is granular, so all of the tiny particles can fit inside a syringe. And – thanks to the concept of particle jamming – after injection, the particles reform into a scaffold that perfectly conforms to the shape of the wound. Once the material has been deposited, the last step is to perform some chemistry magic to interlink the particles so they don’t go anywhere. The injectability of MAP makes it much more convenient for physician’s to use, and patients can avoid undergoing unnecessary surgery – everyone wins!

Injectable and long lasting delivery of genes to brain tissue

From Evan’s lab diary:

You all are probably very familiar now with the goals of non-viral gene delivery after the pandemic. If you are not, then great. I get to give you some new information. The goal of gene delivery is to use genetic information (like mRNA or DNA) as drugs. Our cells are designed to know what to do with genetic material, so if you can get this material to the right cellular compartment, the cell will use it as intended – generally to make a therapeutic protein. In the case of COVID vaccines, either mRNA or DNA was delivered depending on the manufacturer. But in all cases where genetic material was delivered, the vaccine expressed the spike protein of the SARS-CoV-2 virus, and this spike protein was able to give us immunity against COVID-19. The difficulty has been to deliver enough genetic material to the right cellular compartment so that enough therapeutic protein is expressed to reach the required dose.

My lab has worked for over a decade on approaches to deliver genetic material from hydrogel biomaterials (heck even my advisor’s thesis was on this topic, along with past lab members Leo, Quinn, Talar, Anandika, Cynthia, and Norman). These approaches have led to high levels of protein expression in vitro, but not in test animals. Also, previous approaches did not load genetic material into injectable gels…and just like a vaccine, if our gene therapy isn’t injectable, it limits applicability. My thesis work is focused on improving our delivery strategies to achieve effective genetic material delivery from injectable hydrogels into the brain. Once this is achieved, my goal is to express proteins that can regulate endogenous protein expression of pro-repair factors to promote recovery after stroke.

If you’re a student who recognizes that non-viral gene delivery is trending, contact us and add in the message that you’re interested in my project! #evan #genedelivery

Global descriptors

LOVAMAP computes 14 global descriptors for each scaffold. ‘Global’ refers to the fact that a single value is reported for the descriptor, which captures information spanning the entire scaffold. For example, the most common global descriptor reported in the literature for granular biomaterials is Void-Volume Fraction, where the top of the fraction is the volume of scaffold that’s occupied by empty space and the bottom of the fraction is the total volume of the scaffold.

Inner porosity

The picture below really says it all! MAP is designed so that the space between the particles is large enough to fit cells. Back in the day, researchers would create materials that resemble… Jello swiss cheese 🤔 The idea was to create a material that’s filled with interconnected holes (or ‘pores), which would allow cells to move around freely. The structure of MAP is essentially the opposite of that! The holes are now the particles, and the cheese(?) is now empty void space. Cells can still move around freely, and in fact, they seem to prefer the concave surface of the particles. We want to create materials that keep cells happy and push them toward healing wounds, so this is good news.

Outer porosity

Let’s first cover what MAP is not – MAP is not a solid hunk of material, like Jello. Imagine a cell approaching a giant wall of Jello. It would have to eat away at the material in order to get inside and hang out. We want to make our materials inviting to cells so that there’s minimal effort for them to get right in and start healing that wound. MAP is genius in that way because it’s made of a bunch of microscopic particles of Jello packed together, and the space between the particles is also on the micron-scale. Do you know what else is on the micron-scale? Cells. So with MAP, the micron-sized openings (or ‘pores) allow cells to easily crawl into the scaffold without wasting time and energy degrading the material first. This process of cell movement is called ‘cell infiltration,’ and the structure of MAP makes for some effortless cell infiltration. Do you see those brain cells stained in red making their way into the MAP stained in green?

Glycobiology of stroke

From Juhi’s lab diary:

Did you know that our body uses sugars to regulate its activity? In the brain, sugar molecules are everywhere – they’re attached to biomolecules and covering all of the cells. Sugars, which are called glycans in our world, can help cells communicate with each other and with their environment. And cells will display different glycans depending on their current state. As you may imagine, the glycans that get added and displayed during disease or injury are different to those that get displayed during a healthy state.

I am interested in understanding how glycosylation changes with aging and after a stroke, and I want to understand if part of the success of our previous materials are because of changes in the glycosylation profile. My ultimate goal is to design novel glycan-decorated materials to promote recovery after stroke.

If you’re a student who agrees that glycans > proteins and nucleic acids and you’re interested in their impact on stroke recovery, contact us and add in the message that you’re interested in my project! #juhi #sweetbrain

Microporous annealed particles (MAP) scaffolds

The magic of MAP comes from interlinking the HMPs in a granular scaffold so that the particles get stuck in place. There are many, many different techniques for doing this, but the very first MAP scaffold was designed to naturally crosslink in the body. This was great because it meant we could load a syringe with HMPs, inject into the body to form a granular scaffold, then wait for the body’s natural clotting factor to chemically link the particles together. Since then, we’ve also become fond of interlinking using UV light. The benefit of interlinking (or annealing) HMPs is that we create a stable structure that sticks around for longer and makes cells extra happy. We’ve seen MAP scaffolds promote aspects of wound healing in disease models like stroke, myocardial infarction, skin wounds, and spinal cord injury. And once the MAP scaffold has completed its job, it naturally gets degraded by the body.

Return descriptor data

LOVAMAP does some powerful computing to generate an ever-growing list of descriptors that quantitatively characterize the MAP scaffold. These measurements describe features of the void space between the particles, the pathways throughout the scaffold, integrin-binding hotspots, and more. By quantifying the microarchitecture of our scaffolds, researchers can study how scaffold geometry affects other measurable properties, like infiltrating cell behavior and material mechanics. Ultimately, these types of analyses will help us optimize our material for the best possible therapeutic effect.

Generate images and GIFs

Coming soon!

Granular scaffolds

The word ‘scaffold’ implies a supporting structure or framework, so at this step, we’re creating a framework using the HMPs as building blocks. The term ‘granular’ has a specific definition as well, but we’ll leave you to read this review for the nitty-gritty details. The take-home point is that granular materials transition from a ‘liquid-like’ state to a ‘solid-like’ state when particle jamming occurs – so to create a solid structure from HMPs, we pack them together. Now we have a granular scaffold with empty space in between the particles to support cell ingrowth. But remember, a granular scaffold is just a collection of particles jammed together, so it’s still a dynamic system where everything can slosh around.

Wound healing

We are inspired by our body’s ability to heal. This occurs through a process called endogenous regeneration, which takes advantage of the activation of ‘plasticity’ upon injury. Plasticity is the ability to transform local cells into tissue factories that produce extracellular matrix and other multicellular structures that got destroyed. The goal of plasticity is to produce brand-spanking-new tissue. As you may have guessed, these pathways are usually dormant and only awaken once the body recognizes that an injury has occurred. So this begs the question – if tissues have the ability to fix themselves back to working order, why do some injuries cause permanent damage? This is because there are two competing forces in healing: plasticity and scarring. Scarring is a pro-survival mechanism that evolved to ensure that we survive an injury. Regenerating tissue takes time and resources – time that we may not have if the injury starts inhibiting important bodily functions or introducing deadly bacterial infections. Thus, at the same time that pro-regenerative pathways are activated to promote functional recovery, there are also pro-survival mechanisms activated that favor a quick-fix. Usually, scarring wins, and scar tissue gets deposited that is not completely functional.

Our main goals are to 1) scale-down scarring so that our tissues have a chance to regrow, and 2) deliver key signals that promote tissue regeneration. Luckily we know that our body actively works to repair itself after injury, so we only have to deliver the pro-healing components that are low or lacking in the wound environment. And the good news is that biomaterials can be designed to do just that – modulate the pathways that are responsible for scarring, while at the same time deliver key therapeutics to promote functional tissue deposition.

Heterogeneity

MAP is a collection of tiny particles, but nobody says we have to use the SAME particles throughout our scaffold. In fact, having the option to easily combine different types of particles into one scaffold is a big plus with our material. We can include particles of different shapes, sizes, and stiffnesses…made from different polymers…with different degradation rates…carrying different types of drugs… Really the world is our MAP-oyster! And not only can we precisely choose the proportion of these particle types, but we can also vary where in the scaffold they lie. Do you want everything to be mixed like rainbow Dippin’ Dots? Do you want particle types to be nicely layered like a cake? You choose. This is all to say, the heterogeneity offered by MAP allows for a highly personalized, versatile therapeutic material.

Stroke variation by age and sex

From Kevin’s lab diary:

As you have probably guessed by looking at our website, my lab is interested in coming up with new approaches to reduce the physical disability caused by stroke. However, before getting into material development, I became interested in the differences across sex and age when treating stroke. All of our previous work was studied in young male mice, so we don’t know how well our technology works in female mice or old mice. It’s important to study our materials in all contexts so that we can identify any differences among patient groups and develop therapies that work for everyone who needs them.

Aside from sex and age, my work focuses on further development of a dual-function angiogenic biomaterial, CLUVENA, that previous lab members developed (Sean, Shiva, and Lina). When injected intracranially into the stroke cavity, CLUVENA can both restore blood vessel density and decrease inflammation. This material was also able to reduce physical disability in a rodent model of stroke. My goal is to better understand the mechanism of action of CLUVENA in order to design a novel approach that further reduces physical disability. I also want to make sure our materials can be easily manufactured, shipped, and stored.

If you’re a student who wants to work on cutting-edge technology in the stroke recovery space, contact us and add in the message that you are interested in my project! #kevin #stroke

CLUVENA – Clustered VEGF Nanoparticle hydrogel (developed by our lab!)

Non-invasive therapeutics

We’re motived to develop non-invasive treatment options so that patients can avoid invasive surgical procedures whenever possible. Although surgery is oftentimes the best and only option for treating a condition, the process introduces secondary surgical wounds that both increase the risk for bacterial infections and also require additional energy and resources to heal. Technological advances are tending toward minimizing how much cutting has to be done, but even in the field of biomaterials, surgical procedures are often required to implant therapeutic materials. This is because many products on the market are bulk materials, such as sponges or mesh sheets, that have to be cut to the appropriate shape and physically placed into the wound site.

Our lab has been focused on a different approach – generating materials that are flowable so that they can be injected via needle or catheter to the target location without the need for extensive surgery. Even for sites that are easily accessible, such as skin wounds, our flowable materials offer a benefit because upon injection, they naturally fill the shape of the wound for a seamless interface between the material and surrounding tissue – even if the tissue is moving!

Aside from injectable hydrogel materials, we are also working on intravenous (IV) delivery and transdermal delivery of nanoparticles. IV delivery is a very common route of administration for many therapeutics today, so it makes sense to use this approach when delivering nanosized materials. Transdermal delivery across the skin barrier removes the need for needles and enables home therapy using patches. Of course, the skin has evolved to be a tough barrier to penetrate, so we need to outsmart evolution and find a side door.

Ninjabyte Computing

If you’ve ever watched a Marvel movie or played a video game like Witcher 3, you know graphics have come a long way. But you may not know how much serious math and research goes into 3D animation. Technologies in simulation and visual effects are incredibly powerful, yet most scientists don’t have access to them because software engineers in animation don’t often chill with scientists. Ninjabyte Computing was founded to connect the science community with high-performance animation tools used in industry.

The company is headed by Peter Cheng, PhD, who offers the perfect blend of expertise in mathematics, computer science, and animation. Ninjabyte Computing uses professional visual effects software, like SideFX Houdini, to generate physically-accurate simulations of randomly packed particles with varying shapes, sizes, stiffness, and textures. These simulations are fantastic mimics of a range of MAP scaffold types, which makes the data perfect to analyze with LOVAMAP in order to study our scaffolds. Ninjabyte Computing also renders beautiful images and videos of our material to help explain our work. One of their images was featured on the cover of Nature Materials!

While our collaboration focuses on MAP simulations, this only scratches the surface of animation technology. We’re happy to share the love for Ninjabyte, and we recommend reaching out to ninjabytecomputing@gmail.com for any inquiries.

Biomaterial scaffold

A biomaterial scaffold is exactly what it sounds like – a structure made from building blocks that provides support. In this case, the building blocks we use are nanoscopically small because our scaffolds interact with tissue at the microscopic level. The scaffolds act as a supplemental extracellular matrix that provides physical support in compromised tissue, and they also serve as a framework to help nearby cells move around and do their job. You can see below that a typical scaffold looks like a little slab of clear Jello.

The scaffold itself can also behave as a drug and influence biological processes in the tissue surrounding it. This is possible because cells in the tissue are mechanoresponsive, which means that they can sense the structural features around them and respond to any changes. We can engineer the structural features of our scaffold to influence cell behavior, which leads to therapeutic action.

When we make scaffolds out of hydrogel microparticles and link these particles together, we create MAP – but you can read more about that here.

Read in formatted data

LOVAMAP is a software designed for analyzing granular material. For this reason, LOVAMAP takes in a specific data format that highlights individual particles – basically each voxel (3D pixel) associated with a particular particle should be labeled with a unique identifying number. This ensures that our software is analyzing exactly what you intended. Alternatively, if you’re simulating spherical particles with a known center and radius, go ahead and input the sphere data – our code can handle it.

Tweaking the immune system

The goal with this approach is to design materials that communicate with our body’s immune system and urge it to generate a lasting pro-regenerative response in the wound. The immune system is most commonly known for helping us fight disease. When external objects enter our body, specialized cells will recognize them as foreign and trigger a specific immune response depending on the type of invader. Viruses, for example, often trigger a danger! danger! response that causes the immune system to produce antibodies against the virus for long-lasting defense. This means you’re protected from getting sick if you get infected with the same virus again. Materials made in lab, however, don’t always elicit the same immune response, which means our materials can be explicitly engineered to tweak the immune system in a certain direction.

It turns out that in addition to fighting foreign invaders, immune cells are also primary players in healing the body. Inflammation, for example, is an early-stage part of wound healing that relies on the hustle and bustle of immune cells. As you probably know, too much inflammation can be a bad thing that actually delays healing. This is why RICE (Rest, Ice, Compression, Elevation) is used to treat twisted ankles and sprained knees. RICE helps keep inflammation down and is often accompanied by the administration of anti-inflammatory drugs such as ibuprofen, which helps our body heal faster. I’m sure it comes as no surprise that modulating inflammation is one of the key features of our materials. In fact, it has recently been discovered that the same cells involved in creating immunity against viruses could be used to promote healing, so we’re learning how our materials can transition from controlling inflammation to generating long-lasting immune responses.

Using pro-repair matrices to fight cancer

From Alex’s lab diary:

My lab primarily works on designing novel biomaterials to help heal our body after injury or disease. So, when I first approached Prof. Segura about wanting to work on cancer, she was not super receptive. She eventually said that if I could figure out how to use our pro-repair matrices for cancer, then she would be ok with it. It turns out that using pro-repair matrices to fight cancer is a brand-new approach that has only begun in the last decade! This approach treats cancer as a wound that won’t heal and uses materials to change the tumor microenvironment towards a healing rather than inflammatory state. The tumor microenvironment has evolved to evade the body’s defenses so that it can grow without containment – meaning if one could normalize the tumor microenvironment so that the body can recognize it, then the body could prevent its growth and kill the cancerous cells. Our plan is to inject our material either inside the tumor core or in a resected-tumor cavity and use the material to promote tumor microenvironment normalization. In particular, I am focused on designing materials to promote normal vasculature so that therapeutics and host immune cells can penetrate the tumor core.

Since MAP scaffolds are a highly versatile biomaterial that have been shown to support cell growth, we are hoping they can be used to combat cancer as well. So far, we have experimented with different polymer types, as well as varying material stiffness, and our custom MAP scaffolds are already showing promising results – with more functionality on the way.

Our focus is on glioblastoma multiform, an aggressive brain cancer with particularly low survival rates. If you’re a student with a passion to fight cancer using pro-repair matrices, contact us and add in the message that you are interested in my project! #alex #healingcancer

Engineering components of the amniotic membrane for wound healing

From Drew’s lab diary:

Our lab uses hyaluronic acid (HA) as a base for many of our materials because it is naturally found in the human body and because it can be recombinantly produced, which makes it easier for translation. Finding natural ways to modify HA to introduce novel functionality would make it an even better material. One day Prof. Segura asked me to look into natural covalent modifications of HA. She knew that most modifications of hyaluronic acid are non-covalent, but she had come across a covalent modification. Although our original plan to use this chemistry to modify HA was not feasible (it was more complicated than we expected…), it turns out that the covalent modification of HA with proteins is involved in the scarless healing of the amniotic membrane. I was super intrigued with amniotic membrane biology, and I am basing my thesis on the design on synthetic placenta biomaterials. My goal is to use these matrices to promote scarless healing of skin wounds.

Since the amniotic membrane is used to promote healing of non-healing wounds, I will incorporate components of the amniotic membrane – which I will produce recombinantly – into HA hydrogels.

If you’re a student who’s excited about scarless healing and synthetic placenta biomaterials, contact us and add in the message that you’re interested in my project! #drew #syntheticplacenta

Modified building blocks

Our starting materials are hydrogel polymers, such as

hyaluronic acid
polyethylene glycol
poly(propylene sulfide)
heparin
poly(allyl mercaptan)

We chemically engineer these polymers to precisely fit our needs, like adding handles that allow the strands to link to one another and form a gel. Our polymer modifications include end-group modifications, side-chain modifications, and even attachment of biologically relevant ligands that make our materials more biocompatible and useful for therapeutic applications. We also use these polymers to create higher-level building blocks called hydrogel microparticles, which you can read more about on our MAP Scaffolds page.

In vivo work

In vivo studies are performed in a living organism and are an essential and exciting part of the research we do. These types of experiments are invaluable because they not only teach us about biocompatibility, but we also get the full effect of a living tissue. This means we gain access to the complicated signaling pathways that would be impossible to recreate in a test tube, and therefore, we get a complete picture of the healing process. Understanding our in vivo results bring us one step closer to developing a product that can help real patients.

During an in vivo study above, we injected one of our biomaterial scaffolds into a controlled skin wound.

In vitro work

In vitro studies are performed in the lab outside of a living organism – think test tubes and petri dishes. These types of experiments allow us to characterize important properties of our scaffolds, like how much they deform or how well the molecules we’ve attached get released. It also offers us the luxury of studying the effects of our material on specific phenomenon, like cell behavior and vessel growth, in a controlled environment. It’s basically Step 1 in understanding what we’ve created.

Look how our gel squishes during compression testing to measure its stiffness!

Ischemic Stroke Project

Ischemic strokes are characterized by a sudden stop of blood flow to the brain due to a clot. The lack of blood flow rapidly kills brain tissue leaving > 40% of patients with a physical disability. There are currently no therapies to decrease stroke related disabilities. Although all current therapeutic treatments for stroke are administered immediately after stroke onset and focus on preventing tissue death, our goal is to develop materials to treat stroke related disability that can be administered days to months after the stroke occurs. Our materials are delivered intracranially, directly into the stroke core, to promote and encourage the brain plasticity needed to form new brain tissue and reduce disability.

Although their name would imply that NPCs become neural cells (e.g., nerves), I am interested in how these cells also play a role in blood vessel formation. There is a synergistic relationship between growing blood vessels in the brain and NPCs. Low blood vessel growth results in low NPC migration, but also NPCs secrete factors that are essential for blood vessel sprouting and maturation. My studies revolve around the use of MAP scaffolds as well as our VEGF delivery technology CLUVENA to promote NPC migration and vascularization. In addition, I am changing the physical and biochemical cues of the scaffold to modulate NPC phenotype and mature vessel formation.

If you’re interested in working on strategies to promote recovery after stroke focusing on NPCs and vascularization, contact us and add in the message that you’re interested in my project! #kat #brainheroes

VEGF – vascular endothelial growth factor
CLUVENA – Clustered VEGF Nanoparticle hydrogel (developed by our lab!)

Stem cell delivery

Tbd

We injected MAP into a skin wound, and you can see cells infiltrating between the particles.

Stroke-induced disability

A stroke is like a heart attack of the brain – it occurs when oxygen can’t get delivered to the organ. But unlike a heart attack, a stroke will leave a patient with substantially increased risk for lifelong disabilities [1]. In fact, stroke is one of the leading causes of serious long-term disability in the United States [2], and in 2018 alone, over 7.8 million individuals suffered from a stroke [3]. Sadly, although thrombolytic drugs are used to minimize damage within the first few hours of stroke onset, there are no therapeutic drugs available to treat damaged brain tissue after stroke. Patients must therefore rely on rehabilitation efforts like physical therapy and video-assisted training to reduce their disability and regain independence.

After a stroke, the brain tissue that was starved of oxygen is irreversibly injured, and the body attempts to repair the injury by forming a scar – but scar tissue is no longer functional. Patient disability is the direct result of non-functional scar tissue at the stroke site. Rehabilitation efforts attempt to recover function by training the brain to rebuild neuronal connections that bypass the stroke site. The brain is an adaptive organ, so figuring out new ways to form connections without the original tissue is challenging, but not always impossible.

Instead of working around scar tissue, we’re motivated to develop a material that can be placed directly into the stroke cavity to promote endogenous tissue regeneration, which is the body’s way of rebuilding functional tissue after injury. Theoretically, the body has all of the tools necessary to replace injured tissue with healthy tissue, and our material will offer physical and chemical cues to help guide this process in the stroke cavity. The end-goal is minimal scar tissue and maximal functional recovery.

[1] Dhamoon MS, et al. (2017) Disability Trajectories Before and After Stroke and Myocardial Infarction The Cardiovascular Health Study. JAMA Neurology. 74(12): 1439-1445.

[2] Centers for Disease Control and Prevention (CDC). (2009) Prevalence and most common causes of disability among adults: United States, 2005. MMWR Morb Mortal Wkly Rep. 58: 421–426.

[3] NCHS, National Health Interview Survey, 2018. Table A-1a: 1-9.

Poorly healing wounds

We all know that tearing your skin creates a wound. And even though skin wounds are a pain (literally), most heal right up – possibly with the help of some stitches. But the bigger and deeper the wound, the harder it is for the body to heal. What’s worse – if a patient has an underlying disease that stunts wound closure, like diabetes, wounds are more susceptible to chronic inflammation, infection, and poor healing. For some perspective, in 2014 there were 8.4 million claims for wound healing related diagnosis codes in Medicare patients alone [1]. Although there is a long history of therapies that have been developed to improve wound healing (check out reference [2] below), there is no universal approach to treat the ones that fail to close up. This means that despite the thousands of products available, many individuals continue to suffer from painful, long-lasting wounds that severely impact their quality of life.

The challenge of fixing a disrupted natural process in the body motivates us to develop a biomaterials-based approach that can break the inflammatory cycle and turn a non-healing environment into a healing one.

[1] Nussbaum SR, et al. An Economic Evaluation of the Impact, Cost, and Medicare Policy Implications of Chronic Nonhealing Wounds. Value in Health. (2018) 27 – 32.

[2] Han, G and Ceilley, R, 2017. Chronic wound healing: a review of current management and treatments. Advances in therapy, 34(3), 599 – 610.

Scarless healing

You could call this the Holy Grail of wound healing – everyone dreams of applying a topical cream that miraculously prevents scarring. It’s no wonder scar treatments are a $17.94 billion market [1]. But we’re motivated by more than cosmetic benefits. Many people hear ‘scar’ and think of visible scars that tell an interesting story. But scarring is a physiological process that can occur at any site of injured tissue, including internal organs. Think of scarring like the body filling up the injured site with cement. On the one hand, the cement (or scar tissue) has prevented further damage locally…but on the other hand, the region that contains the cement is no longer normal tissue, and it can’t function properly. Depending on the size and location of the scar, this can lead to compromised organ function and serious health issues.

The medical field is in need of a material that can prevent scarring and recapitulate* the native tissue environment. We are developing biomaterials that are intended to be placed in a wound prior to the formation of a scar. The goal is for our material to modulate the wound environment in a way that promotes normal tissue deposition while reducing scar formation. This means less cement and more healthy, functioning tissue.

[1] Grand View Research: Market Analysis Report. Scar Treatment Market Size, Share & Trends Analysis Report By Scar Type (Atrophic, Hypertrophic & Keloid Scars, Stretch Marks), By Product (Injectables, Topical, Laser), By End Use, And Segment Forecasts, 2019 – 2026.

* Fun fact: Lindsay in our lab was responsible for changing the official Merriam-Webster definition of the word ‘recapitulate’ to include the way scientists have been using it for years:

Wound healing

Tbd

We injected MAP into a skin wound, and you can see cells infiltrating between the particles.

Bells and whistles

We hate to be boring, so we’re always exploring new features to incorporate into our materials. Most of our scaffolds include additional bioactive signals that help make our material more effective at promoting healing, like:

peptides / proteins
nucleic acids
small molecules
cells

These additions can be used to attract and influence cells that make their way into the scaffold, or the material can be designed as a drug depot that releases factors at desired time points.

Above you can see DNA nanoparticles (highlighted in green) that have been loaded into a scaffold.

Sept 2015 – June 2019

Elias Sideris

Clearview Healthcare Partners

Elias obtained his PhD from the Segura Lab in 2019 with a thesis titled “Hydrogel scaffolds and their influence on the brain.” He is now a consultant at Clearview Healthcare Partners based in San Francisco.

Sept 2015 – June 2019

Elias Sideris

Clearview Healthcare Partners

Impact Factor:

02.02.20
Nature Materials

Hydrogel micopraticles for
Biomedical aplications

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