Literature Highlights

Growth factors used for tissue regeneration applications often do not perform well in later stages of clinical trials, betraying their early promise. One example is the large initial release of bone morphogenic protein (BMP)-2 from collagen powder/sponges over 21 days that is nonphysiological. A slower- and sustained-release profile may reduce the incidence of adverse effects such as heterotopic ossification, and more physiological recovery is highly desirable. Fracture healing involves different stages, involvement of BMP and vascular endothelial growth factors (VEGFs), and interaction between blood vessels and bone cells. VEGF expression peaks around day 5/10 before decreasing, whereas BMP-2 increases constantly until day 21. Novel engineering methods can be used to control the release of such growth factors and osteoinductive hydroxyapatite (HA) and laponite nanoparticles into regenerative implants for tight temporal control over growth factor release. In this study, VEGF and BMP-2 factors were tuned by printing functionalized alginate inks with distinct spatiotemporal release profiles to enhance the regeneration of critically sized bone defects. To produce printable biomaterial inks, various weights of methylcellulose were added to RGD alginates. The temporal release profile of growth factors (e.g., VEGF) were tuned by adding clay and hydroxyapatite nanoparticles (nHAs). While methylcellulose increased VEGF release, laponite (clay nanoparticles) slowed the release of VEGF. The alginate, methylcellulose, and nHA blend was then referred to as the vascular bioink, releasing VEGF for more than 10 days. The authors then demonstrated that spatial gradients of VEGF could be maintained for more than 14 days by printing VEGF at different spatial presentations. When gradient VEGF implants were compared with homogeneous and no VEGF conditions, vessels were observed following 2 weeks of subcutaneous implantation in mice. In the no-VEGF group, the blood vessels were absent. Unlike the homogeneous VEGF group, the gradient group showed blood vessels in the implant center. To modulate the slower release of BMP-2, laponite was added to alginate–methylcellulose bioinks. To assess the effect of slow-release BMP-2 bioinks, fast-release (alginate–methylcellulose) bioinks were bioprinted with bone marrow-derived mesenchymal stem cells (BMSCs) and compared with the slower-release candidates. Eight weeks after implantation in a segmental defect, greater amounts of mineralized tissue were observed in the slow-release compared with the fast-release group using micro-computed tomography (micro-CT) and histological analysis. Thereafter, VEGF and BMP-2 were co-released from printed constructs to heal a critically sized bone defect. Using micro-CT angiography, combined VEGF and BMP-2 (composite) groups led to a significant increase in vessel volume compared with the VEGF gradient group. Comparatively, there was also greater vessel connectivity and greater maturity (verified by α-SMA and vWF expression). In terms of cartilage and new bone deposition, the composite group also performed better (Safranin O staining, micro-CT imaging) with dense, cortical-like bone present, comparable to native bone. A greater quantity of bone marrow was similarly observed in the composite group. Thus, the authors’ approach to identifying suitable spatiotemporal presentation of growth factors to regenerate large bone defects is described herein. They demonstrated improved angiogenesis and neo-bone formation while reducing off-target effects and envision this approach in regenerating other tissue types. (Freeman, F.; et al. Sci. Adv. 2020, 6, eabb5093.) Cardiac tissue engineering integrates cardiac cells with biomaterials and is a promising approach for regenerating infarcted hearts. An obstruction to this approach is the selection of biomaterials with requisite properties, with biocompatible degradation products and minimal immune response. The Dvir group has demonstrated a personalizable approach for engineering autologous cardiac patches. A biopsy of fatty tissue is obtained from patients before the cells are reprogrammed into pluripotent stem cells, whereas the extracellular matrix (ECM) is processed into a hydrogel. One drawback about this approach is the lack of blood vessel networks that match patient vasculature. Other approaches in recent times were unable to print full, thick vascularized cardiac patches. In this study, advanced 3D printing methods were used to generate thick, vascularized, perfusable cardiac patches to match the patient’s immunological, biochemical, and anatomical properties. They further extend this to printing whole hearts with major blood vessels. From omental fatty tissue, patient cells were reprogrammed into iPSCs (OCT4+) and then differentiated into cardiomyocytes (CMs) and endothelial cells (ECs). Computerized tomography (CT) was then used to visualize the 3D structure and the orientation of major blood vessels. Computer-aided design (CAD) was then used to model the smaller vessels using mathematical equations to simulate oxygen transport and consumption. The personalized hydrogels were then mixed with the iPSC-derived endothelial, cardiac, and fibroblast cells, together with gelatin as a sacrificial ink. A 3D printer was able to create 2 mm thick vascularized patches with high cell viability. Incubation at 37 °C led to gelatin liquefying to leave 300 µm lumens within the cardiac patches. These patches were physically robust, holding their shape, while liquid could be infused into the open lumens. These patches were positive for actinin (CM cells) and CD31 (EC cells), with the ECs and fibroblasts assembling into their native configuration. The printed vascularized cardiac patch was also contractile, exhibiting calcium transients to demonstrate its functionality. These were transplanted in rat omentum displaying elongated and aligned cells with massive striations, indicating their contractility and functionality. For organs or tissues with larger dimensions, an alternative strategy was used since these gels have unsuitable mechanical properties. To preserve the integrity of the delicate structures and sensitive cells, a gel support comprising alginate microparticles and xanthum gum is utilized for free-form printing and safe enzymatic or chemical degradation. The authors managed to generate thick, vascularized, perfusable tissue. Triaxial lumen structures allowed the perfusion of dye, indicating the potential for blood transfer. The authors then printed large (20 mm height, 14 mm diameter) volumetric tissue with anatomical architectures. These structures had similar mechanical properties to decellularized rat hearts. Sarcomeric actinin structures were also observed in the heart tissue internal compartment. In summary, the authors demonstrate a fully personalized method to print cardiac tissue and organs obtained from patient source tissue. These may be further tested in long-term in vitro and in vivo animal studies to evaluate the true therapeutic value of the printed cardiac tissue. (Noor, N.; et al. Adv. Sci. 2019, 6, 1900344.) Acute myeloid leukemia (AML) has a dismal prognosis and its standard of care, chemotherapy, is only partially successful with frequent disease relapse. Stem cell transplantation is another approach but suffers from a lack of donors and the risk of mortality. Since AML originates in the bone marrow, targeted drug delivery could improve efficacy and reduce collateral toxicity to nonhematopoietic tissue. AML cells exhibit marrow homing abilities; hence. the Gu lab repurposed them for drug delivery while neutralizing their disease-causing properties. They utilized cryo-shocking to render the AML cells (liquid nitrogen-treated [LNT] cells) nonviable before loading them with drugs (doxorubicin [DOX]). Following 12 h in liquid nitrogen, LNT cells were thawed at 37 °C. These shrank in size but maintained their nucleus and cytoskeleton, with a rougher cell surface. Calcein-AM/EthD-1 showed the lack of viability, while cell counting kit-8 (CCK8) and annexin V–propidium iodide (PI) showed that the cells were nonproliferative. In mouse models, the AML cells did not form a tumor and exhibited no circulating cancer cells. In LNT cells, most of the proteins expressed by live AML cells were present. Particularly, CXCR4 and CD44 were detected in both cells through confocal imaging and flow cytometry. LNT and viable AML cells exhibited similar accumulation efficiency in bone marrow following intravenous infusion. The intact nuclear and cytoplasmic structures allowed DOX intercalation up to 65 µg per 107 LNT cells. DOX was released in a sustained manner with 81% released over 10 h. While a higher amount of DOX from LNT was required for AML cytotoxicity compared with free DOX, LNT delivery led to longer detection of DOX in blood and bone marrow. DOX-loaded LNTs were able to reduce AML tumor growth. LNTs were then investigated for enhancing antigen uptake and mature antigen-presenting cells (APCs). LNTs upregulated CD40, CD80, CD86, and major histocompatibility complex II (MHC-II). CD4+ T cells and CD8+ T cells were also increased in peripheral blood. DC maturation and T-cell-activated cytokines—interferon-γ (IFN-γ), tumor necrosis factor–α (TNF-α), and interleukin-6 (IL-6)—were similarly detected following LNT and adjuvant treatment (monophosphoryl lipid A [MPLA]). The combination of LNT/DOX and adjuvant nearly eliminated AML tumors after 21 days. Increased inflammatory cytokines IFN-γ and TNF-α suggested boosted immunity from the combined therapeutic. The authors then evaluated LNT cells as a prophylactic cancer vaccine by immunization and adjuvant 1–3 weeks prior to inoculation of AML cells. The tumor bioluminescence intensity was significantly smaller compared with the control group. Whereas all control mice (without preimmunization) perished, 71% of mice were tumor-free 90 days following the challenge. Furthermore, blood serum levels of IFN-γ, TNF-α, IL-12, and IL-6, as well as CD3+ and CD8+ T cells, were significantly higher in LNT/adjuvant-treated mice. In summary, the authors used a simple liquid nitrogen treatment procedure to negate the tumorigenicity yet preserve cellular integrity. This renders the AML nonpathogenic and allows it to be exploited for drug delivery and antitumor immune responses. While a highly interesting approach, this will warrant further efforts in regard to manufacturing, quality control, and clinical application. (Ci, T.; et al. J. Sci. Adv. 2020, 6, eabc3013.) The COVID-19 pandemic has shown the importance of rapidly developing therapeutic strategies for ongoing or preparing for future disease outbreaks. The spike (S) protein (containing S1 and S2 subunits) has a vital role in viral infection. The S1 subunit engages with human angiotensin-converting enzyme II (ACE2). The infected host organism then mounts a sufficient immune response, with significantly increased inflammatory cytokines to eliminate pathogens and promote tissue repair. This in turn could spur the inflammatory state, leading to immune dysfunction—cytokine release syndrome (“cytokine storm”). While most patients display mild symptoms, ~20% progress to severe symptoms, including pneumonia, septic shock, and even multiple organ failure. Therefore, treatments that block SARS-CoV-2 progress through ACE2 could be highly promising. While antivirals such as remdesivir are showing encouraging signs for early intervention, few target late-stage infection-associated cytokine release syndrome (CRS). Monoclonal antibodies for IL (interleukin)-6 and GM-CSF (granulocyte–macrophage colony-stimulating factor) have been suggested as therapeutic candidates, but it is challenging to suppress CRS due to the complexity of cytokine targets and their interactions. From previous nanotechnology experience, the authors have shown that protein synthesis and display enabled the generation of nanovesicles expressing native proteins on their surface. They hypothesize that engineering and displaying ACE2 on nanovesicles can compete with and bind SARS-CoV-2 to create nanodecoys. Briefly, the engineered nanodecoys are created by transfecting ACE2 on 293T cells before fusing them with THP1 cells. The final product contains ACE2, IL-6R, and GM-CSF moieties, facilitating the neutralization of viruses and secreted inflammatory cytokines. Immunostaining and flow cytometry demonstrated positive ACE2 transfection in 293T cells. To extract cell membrane material, the intracellular material was removed by hypotonic lysis, mechanical disruption before gradient centrifugation. Both ACE2 and THP vesicles were then mixed, sonicated, and repeatedly extruded through nanopores to form nanodecoys. Confocal microscopy demonstrated successful fusion since ACE2 and THP1 were prelabeled with different fluorescence dyes. Western blots showed the successful combination and preservation of the different biological components (ACE2, CD130, CD116). Each 1 µg of nanodecoys was found to contain 140 pg of ACE2. In an in vitro infection model (huh-7 cells), pseudotyped SARS-CoV2 (PsV) was incubated with either nanodecoys or their component vesicles (293T-, THP1-, ACE2-Ves). Only ACE2-Ves and nanodecoys inhibited PsV infection, while 293T- and THP1-Ves were unable to do so. In addition to SARS-CoV-2, pseudotyped SARS-CoV and SARSr-CoV were also inhibited by the nanodecoys. This demonstrates the promising broad-spectrum antiviral properties of nanodecoys and their potential against emerging coronavirus outbreaks. The nanodecoys’ antiviral properties were further repeated on Vero-E6 monkey kidney cells and human Caco-2 cells using authentic SARS-CoV-2 with reduced levels of N protein and a reduction of viral copy number (using The nanodecoys further removed inflammatory µg removed pg of and pg of their potential for In and GM-CSF highly before nanodecoys significantly cytokine The nanodecoys were then tested on mouse and by while in the after h. The mice were then to by Following the mice were with before was obtained to assess cytokine the nanodecoys IL-6, and protein levels in showed a of the mice to a the nanodecoys did not to and long-term their Thus, the authors demonstrate a as a potential COVID-19 therapeutic. This can be further in to for ACE2 for the and may be to and of nanodecoys. et al. 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