3D Printing of Materials and Printing Parameters with Animal Resources: A Review.

3D printing technology enables the production of creative and personalized food products that meet consumer needs, such as an attractive visual appearance, fortification of specific nutrients, and modified textures. To popularize and diversify 3D-printed foods, an evaluation of the printing feasibility of various food pastes, including materials that cannot be printed natively, is necessary. Most animal resources, such as meat, milk, and eggs, are not inherently printable; therefore, the rheological properties governing printability should be improved through pre-/post-processing or adding appropriate additives. This review provides the latest progress in extrusion-based 3D printing of animal resource-based inks. In addition, this review discusses the effects of ink composition, printing conditions, and post-processing on the printing performance and characteristics of printed constructs. Further research is required to enhance the sensory quality and nutritional and textural properties of animal resource-based printed foods.

Enhancing adoptive T-cell therapy with fucoidan-based IL-2 delivery microcapsules.

Adoptive cell therapy (ACT) with antigen-specific T cells is a promising treatment approach for solid cancers. Interleukin-2 (IL-2) has been utilized in boosting the efficacy of ACT. However, the clinical applications of IL-2 in combination with ACT is greatly limited by short exposure and high toxicities. Herein, a complex coacervate was designed to intratumorally deliver IL-2 in a sustained manner and protect against proteolysis. The complex coacervate consisted of fucoidan, a specific IL-2 binding glycosaminoglycan, and poly-l-lysine, a cationic counterpart (FPC2). IL-2-laden FPC2 exhibited a preferential bioactivity in ex vivo expansion of CD8+T cells over Treg cells. Additionally, FPC2 was embedded in pH modulating injectable gel (FPC2-IG) to endure the acidic tumor microenvironment. A single intratumoral administration of FPC2-IG-IL-2 increased expansion of tumor-infiltrating cytotoxic lymphocytes and reduced frequencies of myeloid populations. Notably, the activation and persistency of tumor-reactive T cells were observed only in the tumor site, not in the spleen, confirming a localized effect of FPC2-IG-IL-2. The immune-favorable tumor microenvironment induced by FPC2-IG-IL-2 enabled adoptively transferred TCR-engineered T cells to effectively eradicate tumors. FPC2-IG delivery system is a promising strategy for T-cell-based immunotherapies.

Engineering edgeless human skin with enhanced biomechanical properties.

Despite the advancements in skin bioengineering, 3D skin constructs are still produced as flat tissues with open edges, disregarding the fully enclosed geometry of human skin. Therefore, they do not effectively cover anatomically complex body sites, e.g., hands. Here, we challenge the prevailing paradigm by engineering the skin as a fully enclosed 3D tissue that can be shaped after a body part and seamlessly transplanted as a biological clothing. Our wearable edgeless skin constructs (WESCs) show enhanced dermal extracellular matrix (ECM) deposition and mechanical properties compared to conventional constructs. WESCs display region-specific cell/ECM alignment, as well as physiologic anisotropic mechanical properties. WESCs replace the skin in full-thickness wounds of challenging body sites (e.g., mouse hindlimbs) with minimal suturing and shorter surgery time. This study provides a compelling technology that may substantially improve wound care and suggests that the recapitulation of the tissue macroanatomy can lead to enhanced biological function.

Biomaterials and bioengineering to guide tissue morphogenesis in epithelial organoids.

Organoids are self-organized and miniatured in vitro models of organs and recapitulate key aspects of organ architecture and function, leading to rapid progress in understanding tissue development and disease. However, current organoid culture systems lack accurate spatiotemporal control over biochemical and physical cues that occur during in vivo organogenesis and fail to recapitulate the complexity of organ development, causing the generation of immature organoids partially resembling tissues in vivo. Recent advances in biomaterials and microengineering technologies paved the way for better recapitulation of organ morphogenesis and the generation of anatomically-relevant organoids. For this, understanding the native ECM components and organization of a target organ is essential in providing rational design of extracellular scaffolds that support organoid growth and maturation similarly to the in vivo microenvironment. In this review, we focus on epithelial organoids that resemble the spatial distinct structure and function of organs lined with epithelial cells including intestine, skin, lung, liver, and kidney. We first discuss the ECM diversity and organization found in epithelial organs and provide an overview of developing hydrogel systems for epithelial organoid culture emphasizing their key parameters to determine cell fates. Finally, we review the recent advances in tissue engineering and microfabrication technologies including bioprinting and microfluidics to overcome the limitations of traditional organoid cultures. The integration of engineering methodologies with the organoid systems provides a novel approach for instructing organoid morphogenesis via precise spatiotemporal modulation of bioactive cues and the establishment of high-throughput screening platforms.

Systematic analysis of inheritance pattern determination in genes that cause rare neurodevelopmental diseases.

Despite recent advancements in our understanding of genetic etiology and its molecular and physiological consequences, it is not yet clear what genetic features determine the inheritance pattern of a disease. To address this issue, we conducted whole exome sequencing analysis to characterize genetic variants in 1,180 Korean patients with neurological symptoms. The diagnostic yield for definitive pathogenic variant findings was 50.8%, after including 33 cases (5.9%) additionally diagnosed by reanalysis. Of diagnosed patients, 33.4% carried inherited variants. At the genetic level, autosomal recessive-inherited genes were characterized by enrichments in metabolic process, muscle organization and metal ion homeostasis pathways. Transcriptome and interactome profiling analyses revealed less brain-centered expression and fewer protein-protein interactions for recessive genes. The majority of autosomal recessive genes were more tolerant of variation, and functional prediction scores of recessively-inherited variants tended to be lower than those of dominantly-inherited variants. Additionally, we were able to predict the rates of carriers for recessive variants. Our results showed that genes responsible for neurodevelopmental disorders harbor different molecular mechanisms and expression patterns according to their inheritance patterns. Also, calculated frequency rates for recessive variants could be utilized to pre-screen rare neurodevelopmental disorder carriers.

Precisely Localized Bone Regeneration Mediated by Marine-Derived Microdroplets with Superior BMP-2 Binding Affinity.

Prompt and robust bone regeneration has been clinically achieved using supraphysiological doses of bone morphogenetic protein-2 (BMP-2) to overcome the short half-life and rapid clearance. However, uncontrolled burst release of exogenous BMP-2 causes severe complications such as heterotopic ossification and soft tissue inflammation. Therefore, numerous researches have focused on developing a new BMP-2 delivery system for a sustained release profile by immobilizing BMP-2 in various polymeric vehicles. Herein, to avoid denaturation of BMP-2 and enhance therapeutic action via localized delivery, a complex coacervate consisting of fucoidan, a marine-derived glycosaminoglycan, and poly-l-lysine (PLL) is fabricated. Superior BMP-2 binding ability and electrostatic interaction-driven engulfment enable facile and highly efficient microencapsulation of BMP-2. The microencapsulation ability of the coacervate significantly improves BMP-2 bioactivity and provides protection against antagonist and proteolysis, while allowing prolonged release. Moreover, BMP-2 containing coacervate is coated on conventional collagen sponges. The bioactivity and localized bone regenerating ability are confirmed through in vitro (human-derived stem cells), and in vivo (calvarial bone defect model) evaluations.

Development of a regenerative porous PLCL nerve guidance conduit with swellable hydrogel-based microgrooved surface pattern via 3D printing.

Peripheral nerve injury causes severe loss of motor and sensory functions, consequently increasing morbidity in affected patients. An autogenous nerve graft is considered the current gold standard for reconstructing nerve defects and recovering lost neurological functions; however, there are certain limitations to this method, such as limited donor nerve supply. With advances in regenerative medicine, recent research has focused on the fabrication of tissue-engineered nerve grafts as promising alternatives to the autogenous nerve grafts. In this study, we designed a nerve guidance conduit using an electrospun poly(lactide-co-ε-caprolactone) (PLCL) membrane with a visible light-crosslinked gelatin hydrogel. The PLCL nanoporous membrane with permeability served as a flexible and non-collapsible epineurium for the nerve conduit; the inner-aligned gelatin hydrogel paths were fabricated via 3D printing and a photocrosslinking system. The resultant gelatin hydrogel with microgrooved surface pattern was established as a conducting guidance path for the effective regeneration of axons and served as a reservoir that can incorporate and release bioactive molecules. From in vivo performance tests using a rat sciatic nerve defect model, our PLCL/gelatin conduit demonstrated successful axonal regeneration, remyelination capacities and facilitated functional recovery. Hence, the PLCL/gelatin conduit developed in this study is a promising substitute for autogenous nerve grafts. STATEMENT OF SIGNIFICANCE: Nerve guidance conduits (NGCs) are developed as promising recovery techniques for bridging peripheral nerve defects. However, there are still technological limitations including differences in the structures and components between natural peripheral nerve and NGCs. In this study, we designed a NGC composed of an electrospun poly(lactide-co-ε-caprolactone) (PLCL) membrane and 3D printed inner gelatin hydrogel to serve as a flexible and non-collapsible epineurium and a conducting guidance path, respectively, to mimic the fascicular structure of the peripheral nerve. In particular, in vitro cell tests clearly showed that gelatin hydrogel could guide the cells and function as a reservoir that incorporate and release nerve growth factor. From in vivo performance tests, our regenerative conduit successfully led to axonal regeneration with effective functional recovery.

Double-layered adhesive microneedle bandage based on biofunctionalized mussel protein for cardiac tissue regeneration.

Heart failure following myocardial infarction (MI), the primary cause of mortality worldwide, is the consequence of cardiomyocyte death or dysfunction. Clinical efforts involving the delivery of growth factors (GFs) and stem cells with the aim of regenerating cardiomyocytes for the recovery of structural and functional integrity have largely failed to deliver, mainly due to short half-lives and rapid clearance in in vivo environments. In this work, we selected and genetically fused four biofunctional peptides possessing angiogenic potential, originating from extracellular matrix proteins and GFs, to bioengineered mussel adhesive protein (MAP). We found that MAPs fused with vascular endothelial growth factor (VEGF)-derived peptide and fibronectin-derived RGD peptide significantly promoted the proliferation and migration of endothelial cells in vitro. Based on these characteristics, we fabricated advanced double-layered adhesive microneedle bandages (DL-AMNBs) consisting of a biofunctional MAP-based root and a regenerated silk fibroin (SF)-based tip, allowing homogeneous distribution of the regenerative factor via swellable microneedles. Our developed DL-AMNB system clearly demonstrated better preservation of cardiac muscle and regenerative effects on heart remodeling in a rat MI model, which might be attributed to the prolonged retention of therapeutic peptides as well as secure adhesion between the patch and host myocardium by MAP-inherent strong underwater adhesiveness.

Adhesive protein-based angiogenesis-mimicking spatiotemporal sequential release of angiogenic factors for functional regenerative medicine.

Damaged vascular structures after critical diseases are difficult to completely restore to their original conditions without specific treatments. Thus, therapeutic angiogenesis has been spotlighted as an attractive strategy. However, effective strategies for mimicking angiogenic processes in the body have not yet been developed. In the present work, we developed a bioengineered mussel adhesive protein (MAP)-based novel therapeutic angiogenesis platform capable of spatiotemporally releasing angiogenic growth factors to target disease sites with high viscosity and strong adhesiveness in a mucus-containing environment with curvature. Polycationic MAP formed complex coacervate liquid microdroplets with polyanionic hyaluronic acid and subsequently gelated into microparticles. Platelet-derived growth factor (PDGF), which is a late-phase angiogenic factor, was efficiently encapsulated during the process of coacervate microparticle formation. These PDGF-loaded microparticles were blended with vascular endothelial growth factor (VEGF), which is the initial-phase angiogenic factor, in MAP-based pregel solution and finally crosslinked in situ into a hydrogel at the desired site. The microparticle-based angiogenic-molecule spatiotemporal sequential (MASS) release platform showed good adhesion and underwater durability, and its elasticity was close to that of target tissue. Using two in vivo critical models, i.e., full-thickness excisional wound and myocardial infarction models, the MASS release platform was evaluated for its in vivo feasibility as an angiogenesis-inducing platform and demonstrated effective angiogenesis as well as functional regenerative efficacy. Based on these superior physicochemical characteristics, the developed MASS release platform could be successfully applied in many biomedical practices as a waterproof bioadhesive with the capability for the spatiotemporal delivery of angiogenic molecules in the treatment of ischemic diseases.

Augmented peripheral nerve regeneration through elastic nerve guidance conduits prepared using a porous PLCL membrane with a 3D printed collagen hydrogel.

Peripheral nerve injury results in significant sensory and motor functional deficits. Although direct neurorrhaphy in the early phase may reduce its devastating effects, direct end-to-end neurorrhaphy is sometimes impossible owing to a defect at the injured site of the nerve. Autogenous nerve graft is a primary consideration for peripheral nerve defects; however, significant morbidity of the donor site is inevitable. Recently, the treatment using engineered synthetic nerve conduits has been regarded as a promising strategy to promote the regeneration of peripheral nerve defects. In this study, we developed longitudinally oriented collagen hydrogel-grafted elastic nerve guidance conduits (NGC) to reconstruct sciatic nerve defects. An elastic NGC was prepared by using poly(lactide-co-caprolactone) (PLCL), and electrospun PLCL was adopted to fabricate nanoporous structures with appropriate permeability for nerve regeneration. Oriented collagen hydrogels were prepared by the 3D printing method to achieve a microscale hydrogel pattern. Based on sciatic nerve injury models in rats, we confirmed the beneficial effects of the NGC with 3D printed collagen hydrogel on axonal regeneration and remyelination along with superior functional recovery in comparison with the NGC filled with the bulk collagen hydrogel. It is believed that the aligned collagen hydrogels provide a preferable environment for nerve regeneration, functioning as an oriented guidance path. In conclusion, the PLCL nerve guide conduit containing a 3D printed aligned collagen hydrogel can be useful for peripheral nerve regeneration.

Body temperature-activated protein-based injectable adhesive hydrogel incorporated with decellularized adipose extracellular matrix for tissue-specific regenerative stem cell therapy.

Adipose tissue engineering represents a valuable alternative for reconstructive and cosmetic applications to restore soft tissue loss. Herein, for the development of a tissue-engineered adipose substitute, we designed an injectable thermoresponsive tissue adhesive hydrogel by grafting bioengineered mussel adhesive protein (MAP) with poly(N-isopropylacrylamide) (PNIPAM) and incorporating decellularized adipose tissue (DAT) powder as a biochemical cue. The body temperature-activated PNIPAM-grafted MAP (MAP-PNIPAM) hydrogel showed 3.2-times higher water retention ability, high porosity, and 8.4-times stronger tissue adhesive properties compared to the PNIPAM gel alone with pore collapse. Moreover, we found that the introduction of 5 wt% DAT powder had adipo-inductive and adipo-conductive effects, which might be due to the provision of biochemical substrates enriched in collagen and laminin for cell-cell and cell-matrix interactions. In vivo subcutaneous injection of the adipose-derived stem cell-laden DAT-incorporated MAP-PNIPAM hydrogel further demonstrated better volume maintenance, angiogenesis, and lipid accumulation than control injectable alginate gel or DAT powder only. Collectively, our injectable body temperature-activated tissue adhesive MAP-PNIPAM hydrogel system with a decellularized extracellular matrix source can be utilized as a promising alternative for tissue-specific regenerative stem cell therapy. STATEMENT OF SIGNIFICANCE: For adipose tissue engineering, we designed an injectable body temperature-activated adhesive hydrogel by grafting bioengineered mussel adhesive protein (MAP) with poly(N-isopropylacrylamide) (PNIPAM) and incorporating adipose-derived stem cells (ASCs) and decellularized adipose tissue (DAT) powder as regenerative cell and ECM sources. PNIPAM has been widely used for cell sheet engineering, but not for cell carriers due to its dramatic thermal contractive properties. By conjugation with hydrophilic MAP, water retention ability and tissue adhesiveness of the scaffold increased by a factor of 3.2- and 8.4-fold, respectively, which are highly required for survival of the transplanted cells and interfacial integration with host tissues. In vivo performance demonstrated that ASCs/DAT powder-laden MAP-PNIPAM hydrogel achieved better volume maintenance, neovascularization, and adipogenesis than control injectable groups.

Genomic profiling of 553 uncharacterized neurodevelopment patients reveals a high proportion of recessive pathogenic variant carriers in an outbred population.

A substantial portion of Mendelian disease patients suffers from genetic variants that are inherited in a recessive manner. A precise understanding of pathogenic recessive variants in a population would assist in pre-screening births of such patients. However, a systematic understanding of the contribution of recessive variants to Mendelian diseases is still lacking. Therefore, genetic diagnosis and variant discovery of 553 undiagnosed Korean patients with complex neurodevelopmental problems (KND for Korean NeuroDevelopmental cohort) were performed using whole exome sequencing of patients and their parents. Disease-causing variants, including newly discovered variants, were identified in 57.5% of the probands of the KND cohort. Among the patients with the previous reported pathogenic variants, 35.1% inherited these variants in a recessive manner. Genes that cause recessive disorders in our cohort tend to be less constrained by loss-of-function variants and were enriched in lipid metabolism and mitochondrial functions. This observation was applied to an estimation that approximately 1 in 17 healthy Korean individuals carry at least one of these pathogenic variants that develop severe neurodevelopmental problems in a recessive manner. Furthermore, the feasibility of these genes for carrier screening was evaluated. Our results will serve as a foundation for recessive variant screening to reduce occurrences of rare Mendelian disease patients. Additionally, our results highlight the utility and necessity of whole exome sequencing-based diagnostics for improving patient care in a country with a centralized medical system.

Bio-inspired swellable hydrogel-forming double-layered adhesive microneedle protein patch for regenerative internal/external surgical closure.

Significant tissue damage, scarring, and an intense inflammatory response remain the greatest concerns for conventional wound closure options, including sutures and staples. In particular, wound closure in internal organs poses major clinical challenges due to air/fluid leakage, local ischemia, and subsequent impairment of healing. Herein, to overcome these limitations, inspired by endoparasites that swell their proboscis to anchor to host's intestines, we developed a hydrogel-forming double-layered adhesive microneedle (MN) patch consisting of a swellable mussel adhesive protein (MAP)-based shell and a non-swellable silk fibroin (SF)-based core. By possessing tissue insertion capability (7-times greater than the force for porcine skin penetration), MAP-derived surface adhesion, and selective swelling-mediated physical entanglement, our hydrogel-forming adhesive MN patch achieved ex vivo superior wound sealing capacity against luminal leaks (139.7 ±â€¯14.1 mmHg), which was comparable to suture (151.0 ±â€¯23.3 mmHg), as well as in vivo excellent performance for wet and/or dynamic external and internal tissues. Collectively, our bioinspired adhesive MN patch can be successfully used in diverse practical applications ranging from vascular and gastrointestinal wound healing to transdermal delivery for pro-regenerative or anti-inflammatory agents to target tissues.

Indoxyl Sulfate-Induced Extracellular Vesicles Released from Endothelial Cells Stimulate Vascular Smooth Muscle Cell Proliferation by Inducing Transforming Growth Factor-Beta Production.

Vascular access stenosis predominantly occurs as a result of neointimal hyperplasia (NH) formation at the anastomosis. Moreover, in the presence of NH, transforming growth factor-beta (TGF-ß) promotes vascular smooth muscle cell (VSMC) proliferation. Extracellular vesicles (EVs) released by endothelial cells are closely associated with vascular dysfunction. Here, we investigated the effects of EVs on TGF-ß signaling and VSMC proliferation. Specifically, EVs were collected from the culture medium of indoxyl sulfate (IS)-treated human umbilical vein endothelial cells and used (2 × 106) to stimulate human aortic smooth muscle cells (SMCs) (1 × 106). Western blotting was performed to assess the levels of Akt, ERK1/2, p38 MAPK, and Smad3. BrdU proliferation assays, quantitative PCR, and ELISA assays were performed to evaluate SMC proliferation and TGF-ß production. The IS-induced EVs stimulated the proliferation of aortic SMCs in a concentration-dependent manner. The EVs both contained TGF-ß and promoted TGF-ß production by SMCs by phosphorylating Akt, ERK1/2, p38 MAPK, and Smad3, which was significantly inhibited by an anti-TGF-ß antibody. SMC proliferation was suppressed by both an anti-TGF-ß antibody and inhibitors of the downstream factors. These results suggest that EVs are involved in the pathogenesis of vascular access stenosis by modulating TGF-ß signaling in VSMCs under uremic conditions.