Vincent B. Ho

Bioengineered dermal–epidermal composites (DECs) have demonstrated promise initiating skin regeneration and hair follicle neogenesis after injury. DECs in our work comprise a collagen matrix embedded with human dermal papilla cells (HDPCs) overlaid with human keratinocytes. HDPCs, as three-dimensional spheroids, enhance hair follicle formation, working in tandem with keratinocytes. Herein, 3D printed stamped PDMS microwell arrays were used as a strategy for spatially patterning dermal papilla spheroids in the dermal components of the DEC. DECs were transferred to cell culture media for 5 days followed by air–liquid interface culture for 2 days. Spheroid diameter, cell viability, and qPCR gene expression analyses were conducted. DECs were surgically grafted on immunocompromised mice, and healing was followed for 10 weeks. HDPCs cultured in the microwell arrays formed patterned viable spheroids and successfully transferred to the collagen dermal matrix. RNA analysis using qPCR showed upregulation of key HDPC markers (VCAN and BMP6) in DC microwell patterned HDPC spheroids compared to monolayers. This work represents a novel 3D printing strategy optimizing designing patterned HDPC spheroids in the extracellular matrix to regenerate functional human skin instead of scar tissue after injury.

Conducted in challenging environments such as disaster or conflict areas, operational medicine presents unique challenges for the delivery of efficient and quality healthcare. It exposes first responders and medical personnel to many unexpected health risks and dangerous situations. To tackle these issues, artificial intelligence (AI) has been progressively incorporated into operational medicine, both on the front lines and also more recently in support roles. The ability of AI to rapidly analyze high-dimensional data and make inferences has opened up a wide variety of opportunities and increased efficiency for its early adopters, notably for the United States military, for non-invasive medical imaging and for mental health applications. This review discusses the current state of AI and highlights its broad array of potential applications in operational medicine as developed for the United States military.

A phase-sensitive diffusion tensor magnetic resonance imaging (MRI) sequence is proposed with pulse timing optimization scheme to achieve velocity resolution of less than 20   μm s ⁻¹ and an integrated image reconstruction and velocity map generation pipeline. The application of ultra-slow flow relevant to neurofluids is enabled by the use of a recently developed, ultra-high-performance brain MRI gradient system. By simultaneously reconstructing magnitude and phase data, both metrics that characterize diffusive fluid motion and coherent velocity maps are calculated non-invasively in human subjects, time-resolved over the entire cardiac cycle. The resulting acquisition and reconstruction of velocity maps in brain parenchyma, enabled by high-performance brain imaging systems, promises to be an important approach to investigating ultra-slow neurofluid flow and glymphatic circulation.

A reliable, accessible, and high-quality blood supply is critical for the sustainment of any healthcare system. World events such as the COVID-19 pandemic have proven that maintaining the supply of blood presents a logistical challenge. The current blood supply is overseen by extensive donor programs around the world. In the United States, as in other countries, the need for blood has increased, with a decline in blood donations and increasing exclusions for blood donor qualification. While there is a need to improve blood donation participation, there is also need for new alternatives to traditional donation to ensure readiness to treat hemorrhagic shock common in the setting of trauma, as often occurs during a natural disaster or conflict. These operational medicine scenarios require significant blood availability which may tax the current blood supply chain. Aside from a walking blood bank (WBB) model for blood collection in suboptimal conditions, researchers have proposed alternatives for blood that include the manufacturing of blood from stem cell sources. Other alternatives include synthetic liquids that can carry oxygen such as Perfluoro-Chemicals (PFCs) and hemoglobin-based oxygen-carrying systems (HBCOs). Here, we review some of these alternatives to the traditional donor blood model. Researchers now have the technology that makes it feasible to develop blood alternatives that one day may supplement and help alleviate the limitations in blood supply.

Skin wounds often form scar tissue during healing. Early intervention with tissue-engineered materials and cell therapies may promote scar-free healing. Exosomes and extracellular vesicles (EV) secreted by mesenchymal stromal cells (MSC) are believed to have high regenerative capacity. EV bioactivity is preserved after lyophilization and storage to enable use in remote and typically resource-constrained environments. We developed a bioprinted bandage containing reconstituted EVs that can be fabricated at the point-of-need. An alginate/carboxymethyl cellulose (CMC) biomaterial ink was prepared, and printability and mechanical properties were assessed with rheology and compression testing. Three-dimensional printed constructs were evaluated for Young’s modulus relative to infill density and crosslinking to yield material with stiffness suitable for use as a wound dressing. We purified EVs from human MSC-conditioned media and characterized them with nanoparticle tracking analysis and mass spectroscopy, which gave a peak size of 118 nm and identification of known EV proteins. Fluorescently labeled EVs were mixed to form bio-ink and bioprinted to characterize EV release. EV bandages were bioprinted on both a commercial laboratory bioprinter and a custom ruggedized 3D printer with bioprinting capabilities, and lyophilized EVs, biomaterial ink, and thermoplastic filament were deployed to an austere Arctic environment and bioprinted. This work demonstrates that EVs can be bioprinted with an alginate/CMC hydrogel and released over time when in contact with a skin-like substitute. The technology is suitable for operational medical applications, notably in resource-limited locations, including large-scale natural disasters, humanitarian crises, and combat zones.

Medical response to military conflicts, natural disasters, and humanitarian crises are challenged by operational logistics with unreliable supply chains, delayed medical evacuation, and compatibility of the disparate medical equipment and consumables. In these environments, stocks of supplies will become more quickly depleted and the need for equipment parts increases secondary to their higher likelihood for failure from overuse. Additive Manufacturing (AM), or 3D printing, at or closer to the point-of-need provides potential solutions to mitigate these logistics challenges. AM’s ability to tailor the resultant product through computer design enables real-time modification of a product to meet a specific situation. In this study, we deployed two different 3D printers to an arctic locale to demonstrate the utility of 3D printing and bioprinting in austere environments. Deployment of AM solutions in austere environments will likely impact medical care following natural disasters and conflicts with contested logistics. The work presented here furthers the readiness status of AM for use in austere environments to manufacture medical equipment parts and demonstrates its potential use for tissue engineering and advanced medical treatments in remote environments.

Torn and damaged menisci resulting from trauma are very common knee injuries, which can cause pain and mobility limitations and lead to osteoarthritis. Meniscal injuries can require surgery to repair the tissue damage and restore mobility. Here we describe the biomechanical testing of a 3D-printed meniscus to illustrate methods to determine if it has the strength and durability to effectively repair meniscal tears and restore knee biomechanics. This work was designed to demonstrate the steps needed to test novel meniscus repair devices prior to moving toward animal testing. The first testing step determined the ability of the 3D-printed meniscus to withstand surgical fixation by measuring the suture pull-out force. We show that vertical 2/0 silk or Fiberwire sutures need an average of 1.4 or 1.8 N, respectively, to pull through the meniscus, while horizontal sutures need only 0.7 and 1.2 N, respectively. The next step measured the compressive strength of normal, damaged, and repaired porcine meniscus tissue. Here, we show that meniscectomy decreased the stiffness of meniscus tissue from 26.7 ± 0.85 N to 7.43 ± 0.81 N at 25% strain. Menisci repaired with the 3D-printed tissue restored 66% of the measured force at 25% strain. The final step measured the contact pressures and areas in an ex vivo porcine knee before and after meniscal repair was made with the 3D-printed meniscus tissue. The example 3D-printed meniscus was successfully sutured into the porcine knee joint but failed to restore normal knee contact pressures. This work demonstrates the need for an iterative biomechanical testing process of biomaterial development, 3D-printing optimization, and knee kinematics to develop a durable and functional meniscus repair device. In summary, the methods described here serve as a guide for the functional evaluation of novel meniscus repair devices.

Micro-computed tomography (microCT) is a common tool for the visualization of the internal composition of organic tissues. Collagen comprises approximately 25–35% of the whole-body protein content in mammals, and the structure and arrangement of collagen fibers contribute significantly to the integrity of tissues. Collagen type I is also frequently used as a key structural component in tissue-engineered and bioprinted tissues. However, the imaging of collagenous tissues is limited by their inherently low X-ray attenuation, which makes them indistinguishable from most other soft tissues. An imaging contrast agent that selectively alters X-ray attenuation is thus essential to properly visualize collagenous tissue using a standard X-ray tube microCT scanner. This review compares various contrast-enhanced techniques reported in the literature for MicroCT visualization of collagen-based tissues. An ideal microCT contrast agent would meet the following criteria: (1) it diffuses through the tissue quickly; (2) it does not deform or impair the object being imaged; and (3) it provides sufficient image contrast for reliable visualization of the orientation of individual fibers within the collagen network. The relative benefits and disadvantages of each method are discussed. Lugol’s solution (I₃K), phosphotungstic acid (H₃PW₁₂O₄₀), mercury(II) chloride (HgCl₂), and Wells–Dawson polyoxometalates came closest to fitting the criteria. While none of the contrast agents discussed in the literature met all criteria, each one has advantages to consider in the context of specific lab capabilities and imaging priorities.