Magnetically powered microwheel thrombolysis of occlusive thrombi in zebrafish.

Tissue plasminogen activator (tPA) is the only FDA-approved treatment for ischemic stroke but carries significant risks, including major hemorrhage. Additional options are needed, especially in small vessel thrombi which account for ~25% of ischemic strokes. We have previously shown that tPA-functionalized colloidal microparticles can be assembled into microwheels (µwheels) and manipulated under the control of applied magnetic fields to enable rapid thrombolysis of fibrin gels in microfluidic models of thrombosis. Transparent zebrafish larvae have a highly conserved coagulation cascade that enables studies of hemostasis and thrombosis in the context of intact vasculature, clotting factors, and blood cells. Here, we show that tPA-functionalized µwheels can perform rapid and targeted recanalization in vivo. This effect requires both tPA and µwheels, as minimal to no recanalization is achieved with tPA alone, µwheels alone, or tPA-functionalized microparticles in the absence of a magnetic field. We evaluated tPA-functionalized µwheels in CRISPR-generated plasminogen (plg) heterozygous and homozygous mutants and confirmed that tPA-functionalized µwheels are dose-dependent on plasminogen for lysis. We have found that magnetically powered µwheels as a targeted tPA delivery system are dramatically more efficient at plasmin-mediated thrombolysis than systemic delivery in vivo. Further development of this system in fish and mammalian models could enable a less invasive strategy for alleviating ischemia that is safer than directed thrombectomy or systemic infusion of tPA.

Magnetically Powered Chitosan Milliwheels for Rapid Translation, Barrier Function Rescue, and Delivery of Therapeutic Proteins to the Inflamed Gut Epithelium.

Inflammatory bowel disease (IBD) is mediated by an overexpression of tumor necrosis factor-α (TNF) by mononuclear cells in the intestinal mucosa. Intravenous delivery of neutralizing anti-TNF antibodies can cause systemic immunosuppression, and up to one-third of people are non-responsive to treatment. Oral delivery of anti-TNF could reduce adverse effects; however, it is hampered by antibody degradation in the harsh gut environment during transit and poor bioavailability. To overcome these shortcomings, we demonstrate magnetically powered hydrogel particles that roll along mucosal surfaces, provide protection from degradation, and sustain the local release of anti-TNF. Iron oxide particles are embedded into a cross-linked chitosan hydrogel and sieved to produce 100-200 µm particles called milliwheels (m-wheels). Once loaded with anti-TNF, these m-wheels release 10 to 80% of their payload over 1 week at a rate that depends on the cross-linking density and pH. A rotating magnetic field induces a torque on the m-wheels that results in rolling velocities greater than 500 µm/s on glass and mucus-secreting cells. The permeability of the TNF-challenged gut epithelial cell monolayers was rescued in the presence of anti-TNF carrying m-wheels, which both neutralized the TNF and created an impermeable patch over leaky cell junctions. With the ability to translate over mucosal surfaces at high speed, provide sustained release directly to the inflamed epithelium, and provide barrier rescue, m-wheels demonstrate a potential strategy to deliver therapeutic proteins for the treatment of IBD.

Glycosaminoglycans Influence Extracellular Matrix of Human Trabecular Meshwork Cells Cultured on 3D Scaffolds.

Glaucoma is a multifactorial progressive optic neuropathy characterized by the loss of retinal ganglion cells leading to irreversible blindness. It is the leading cause of global irreversible blindness and is currently affecting over 70 million people. Elevated intraocular pressure (IOP) is considered the only modifiable risk factor and is a target of numerous treatment modalities. Researchers have assigned this elevation of IOP to accumulation of extracellular matrix (ECM) components in the aqueous humor (AH) outflow pathway. The major drainage structure for AH outflow is the trabecular meshwork (TM). The ECM of the TM is important in regulating IOP in both normal and glaucomatous eyes. In this work, we have studied the role of exogeneous glycosaminoglycans (GAGs), glucocorticoids, and culture conditions on the expression of the ECM gene and proteins by human TM (hTM) cells cultured on biomaterial scaffolds. Gene and protein expression levels of elastin, laminin, and matrix metalloproteinase-2 (MMP-2) were evaluated using quantitative PCR and immunohistochemistry. Pressure gradient changes in cell-laden scaffolds in perfusion cultures were also monitored. Our findings show that GAGs and dexamethasone play an influencing role in hTM ECM turnover at both transcriptional and translational levels by altering expression levels of elastin, laminin, and MMP-2. Understanding the role of exogeneous factors on hTM cell behavior is helpful in gaining insights on glaucoma pathogenesis and ultimately pivotal in development of novel therapeutics against the disease.

Tunable chitosan-calcium phosphate composites as cell-instructive dental pulp capping agents.

Dental cavities are the most prevalent, preventable disease worldwide providing a need for robust treatment options to restore both the form and function of decaying teeth. Here is a presentation of a possible regenerative pulp capping agent that can serve to restore tooth function while regenerating healthy dentin tissue over a long period of time. To achieve this goal a material needs to crosslink quickly, be structurally rigid, and support the proliferation and differentiation of stem cells contained within the dental pulp. In this study, calcium phosphate nanoparticles were embedded in polymer hydrogels of carboxymethyl-chitosan and a diglycidyl ether. The particle size, surface, and mechanical properties of these materials were characterized. These composites have moduli up to 3 MPa, support the culture of dental pulp stem cells more than 3 weeks and exhibit osteogenic potential even without osteogenic media. These composites demonstrate a promising potential as the next generation of pulp capping materials.

Photoinduced Gelatin-Methacrylate Scaffolds to Examine the Impact of Extracellular Environment on Trabecular Meshwork Cells.

Glaucoma is the leading cause of irreversible blindness in the world, currently impacting 80 million people. Patients suffering from primary open-angle glaucoma experience aqueous humor accumulation within the eye causing an increase in intraocular pressure (IOP). The main cause of this rise in IOP is due to poor outflow of aqueous humor through the trabecular meshwork (TM), a tissue composed of collagen and glycosaminoglycans (GAGs) embedded with TM cells. The behavior of TM cells is impacted by their microenvironment, and studies conducted on two-dimensional plastic substrates do not necessarily reflect how TM cells would behave in their native setting. Here, we cultured human TM (hTM) cells on 3D biocompatible hydrogels composed of gelatin methacrylate (GelMA) incorporated with the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and hyaluronic acid (HA). Mechanical properties were quantified by storage moduli and viscosity data. Cellular response was measured by quantifying cellular proliferation and expression of an important extracellular matrix protein, fibronectin. We have shown substrate mechanical properties to impact hTM cell proliferation over 2 weeks. It was found that the incorporation of GAGs impacted cell proliferation and fibronectin expression in hTM cells. This work will help elucidate hTM cell response with changes in their microenvironment.

Human trabecular meshwork cell behavior is influenced by collagen scaffold pore architecture and glycosaminoglycan composition.

Glaucoma is a degenerative eye disease in which damage to the optic nerve leads to a characteristic loss of vision. The primary risk factor for glaucoma is an increased intraocular pressure that is caused by an imbalance of aqueous humor generation and subsequent drainage through the trabecular meshwork (TM) drainage system. The small size, donor tissue limitations, and high complexity of the TM make it difficult to research the relationship between the TM cells and their immediate environment. Thus, a biomaterial-based approach may be more appropriate for research manipulations and in vitro drug development platforms. In this work, human TM (hTM) cells were cultured on various collagen scaffolds containing different glycosaminoglycans (GAGs) and different pore architectures to better understand how hTM cells respond to changes in their extracellular environment. Cellular response was measured by quantifying cellular proliferation and expression of an important extracellular matrix protein, fibronectin. The pore architecture of the scaffolds was altered using freeze-casting technique to make both large and small pores that were aligned or with a non-aligned random structure. The composition of the scaffolds was altered with the addition of chondroitin sulfate and/or hyaluronic acid. It was found that the hTM cells grown on large pore scaffolds proliferate more than those grown on small pores. There was an increase in the fibronectin expression with the incorporation of GAGs, and its morphology was changed by the underlying pore architecture. This work will help provide an insight into the behavior of hTM cells when introducing changes in their microenvironment.

Injectable, self-healable zwitterionic cryogels with sustained microRNA - cerium oxide nanoparticle release promote accelerated wound healing.

Diabetics are prone to chronic wounds that have slower healing, and methods of accelerating the wound closure and to ensure protection from infections are critically needed. MicroRNA-146a gets dysregulated in diabetic wounds and injection of this microRNA combined with reactive oxygen species-scavenging cerium oxide nanoparticles (CNPs) can reduce inflammation and improve wound healing; however, a better delivery method than intradermal injections is needed. Here we demonstrate a biomaterial system of zwitterionic cryogels (gels formed below freezing temperatures) laden with CNP-miR146a that are topically applicable, injectable, self-healable, and provide sustained release of the therapeutic molecules. These cryogels are comprised of CBMA or SBMA and HEMA, and do not contain chemical crosslinkers. Properties of the gels can be manipulated by changing monomer type and ratio. These materials have demonstrated efficacy and viability in vivo with a diabetic mouse wound healing model. Overall, these materials have a high potential for application in wound treatments due to their ease of production, antifouling characteristics, durability, topical application, and sustained release mechanics. STATEMENT OF SIGNIFICANCE: This work presents the development of zwitterionic cryogels with unique physical properties including injectability and self-healing, that also offer highly sustained release of nanoparticles over time to improve wound healing in a diabetic mouse model. The nanoparticles are made of cerium oxide, which is known to scavenge reactive oxygen species and reduce oxidative stress, and these particles have been further tagged with a microRNA146a that has been shown to reduce inflammation. Zwitterionic materials are known for their superior antifouling properties and good biocompatibility and ability to incorporate bioactive factors. Given these properties, the use of these materials as wound healing dressings would be exciting, yet to date it has been difficult to prolong the release of bioactive factors from them due to their hydrophilicity. Previously we developed zwitterionic cyrogels with very sustained protein release over time, but those materials were quite brittle and difficult to handle. Here, we demonstrate for the first time that by removing the crosslinker molecule from our reaction and polymerizing gels under cryo-conditions, we are able to form zwitterionic cryogels that are injectable, self-healing, and with sustained release profiles. The sustained release of miRNA146a-tagged cerium oxide nanoparticles from these gels is demonstrated to speed up diabetic wound healing time and significantly reduce inflammation.

* Engineering Osteoinductive Biomaterials by Bioinspired Synthesis of Apatite Coatings on Collagen Hydrogels with Varied Pore Microarchitectures.

Biomaterial controlled osteoinduction is influenced by the porous microenvironment and the composition of incorporated calcium orthophosphate (CaPi) polymorphs, however, for the design of materials that rival the efficacies of natural grafts a systematic approach to assessing the physicochemical properties that affects cellular differentiation is needed. In this research, we introduce a bioinspired synthetic approach to the mineralization of preformed porous collagen hydrogel scaffolds with tunable apatite coatings. Initially, collagen scaffolds are mineralized with dicalcium phosphate dihydrate (DCPD) by alternate immersion in Ca2+ and HPO42- salt solutions. Utilizing classic DCPD conversion chemistry, the surface coatings are selectively transformed to apatite by immersion of the DCPD-collagen substrate in Tris buffer at pH 7.4, 37°C, for 5 days. The composition and morphology of the deposited mineral coatings are characterized by XRD, SEM, and AFM. Variations in the porous microarchitecture of the collagen hydrogel substrate, pore size (9.5 ± 5 µm, 165 ± 50 µm) and pore alignment altered the morphology of the deposited apatite particles. Intrafibrillar and extrafibrillar mineralization of the collagen templates were observed for both investigated pore sizes. However, templates with aligned pores of both sizes were observed to restrict intrafibrillar mineralization resulting in the exclusive deposition of surface coatings. The osteoinductive activity of the apatite-collagen materials with varied pore microarchitectures was evaluated by in vitro culture of human mesenchymal stem cells for 28 days based on cellular proliferation, alkaline phosphatase activity, and the expression of RUNX2. The combined effects of apatite coatings, reduced pore size, and pore alignment contributed to reductions in cellular proliferation. However, the apatite mineral coating was determined to induce high levels of RUNX2 expression in the absence of additional osteoinductive agents, indicative of biomaterial-induced osteogenesis. This work establishes a versatile synthetic platform for the preparation of bone-like apatite collagen materials with osteoinductive activity.