3D bioprinting platform development for high-throughput cancer organoid models construction and drug evaluation.

The evaluation of anti-tumor drugs is critical for their development and clinical guidance. Tumor organoid models are gaining increased attention due to their ability to better mimic real tumor tissues, as well as lower time and economic costs, which makes up for the shortcomings of cell lines and xenograft models. However, current tumor organoid cultures based on the Matrigel have limitations in matching with high-throughput engineering methods due to slow gelation and low mechanical strength. Here, we present a novel composite bioink for culturing colorectal cancer organoids that provides an environment close to real tissue growth conditions and exhibits excellent photocrosslinking properties for rapid gel formation. Most importantly, the tumor organoids viability in the composite bioink after printing was as high as 97%, which also kept multicellular polar structures consistent with traditional culture methods in the Matrigel. Using 3D bioprinting with this composite bioink loaded with organoids, we demonstrated the feasibility of this drug evaluation model by validating it with clinically used colorectal cancer treatment drugs. Our results suggested that the composite bioink could effectively cultivate tumor organoids using 3D bioprinting, which had the potential to replace less reliable manual operations in promoting the application of tumor organoids in drug development and clinical guidance.

Activating the healing process: three-dimensional culture of stem cells in Matrigel for tissue repair.

BACKGROUND: To establish a strategy for stem cell-related tissue regeneration therapy, human gingival mesenchymal stem cells (hGMSCs) were loaded with three-dimensional (3D) bioengineered Matrigel matrix scaffolds in high-cell density microtissues to promote local tissue restoration. METHODS: The biological performance and stemness of hGMSCs under 3D culture conditions were investigated by viability and multidirectional differentiation analyses. A Sprague‒Dawley (SD) rat full-thickness buccal mucosa wound model was established, and hGMSCs/Matrigel were injected into the submucosa of the wound. Autologous stem cell proliferation and wound repair in local tissue were assessed by histomorphometry and immunohistochemical staining. RESULTS: Three-dimensional suspension culture can provide a more natural environment for extensions and contacts between hGMSCs, and the viability and adipogenic differentiation capacity of hGMSCs were significantly enhanced. An animal study showed that hGMSCs/Matrigel significantly accelerated soft tissue repair by promoting autologous stem cell proliferation and enhancing the generation of collagen fibers in local tissue. CONCLUSION: Three-dimensional cell culture with hydrogel scaffolds, such as Matrigel, can effectively improve the biological function and maintain the stemness of stem cells. The therapeutic efficacy of hGMSCs/Matrigel was confirmed, as these cells could effectively stimulate soft tissue repair to promote the healing process by activating the host microenvironment and autologous stem cells.

Chondroitin sulfate proteoglycan 4: An attractive target for antibody-based immunotherapy.

Multifunctional molecules involved in tumor progression and metastasis have been identified as valuable targets for immunotherapy. Among these, chondroitin sulfate proteoglycan 4 (CSPG4), a significant tumor cell membrane-bound proteoglycan, has emerged as a promising target, especially in light of advances in chimeric antigen receptor (CAR) T-cell therapy. The profound bioactivity of CSPG4 and its role in pivotal processes such as tumor proliferation, migration, and neoangiogenesis underline its therapeutic potential. We reviewed the molecular intricacies of CSPG4, its functional attributes within tumor cells, and the latest clinical-translational advances targeting it. Strategies such as blocking monoclonal antibodies, conjugate therapies, bispecific antibodies, small-molecule inhibitors, CAR T-cell therapies, trispecific killer engagers, and ribonucleic acid vaccines against CSPG4 were assessed. CSPG4 overexpression in diverse tumors and its correlation with adverse prognostic outcomes emphasize its significance in cancer biology. These findings suggest that targeting CSPG4 offers a promising avenue for future cancer therapy, with potential synergistic effects when combined with existing treatments.

Decellularized Brain Extracellular Matrix Hydrogel Aids the Formation of Human Spinal-Cord Organoids Recapitulating the Complex Three-Dimensional Organization.

The intricate electrophysiological functions and anatomical structures of spinal cord tissue render the establishment of in vitro models for spinal cord-related diseases highly challenging. Currently, both in vivo and in vitro models for spinal cord-related diseases are still underdeveloped, complicating the exploration and development of effective therapeutic drugs or strategies. Organoids cultured from human induced pluripotent stem cells (hiPSCs) hold promise as suitable in vitro models for spinal cord-related diseases. However, the cultivation of spinal cord organoids predominantly relies on Matrigel, a matrix derived from murine sarcoma tissue. Tissue-specific extracellular matrices are key drivers of complex organ development, thus underscoring the urgent need to research safer and more physiologically relevant organoid culture materials. Herein, we have prepared a rat decellularized brain extracellular matrix hydrogel (DBECMH), which supports the formation of hiPSC-derived spinal cord organoids. Compared with Matrigel, organoids cultured in DBECMH exhibited higher expression levels of markers from multiple compartments of the natural spinal cord, facilitating the development and maturation of spinal cord organoid tissues. Our study suggests that DBECMH holds potential to replace Matrigel as the standard culture medium for human spinal cord organoids, thereby advancing the development of spinal cord organoid culture protocols and their application in in vitro modeling of spinal cord-related diseases.

Optimising a self-assembling peptide hydrogel as a Matrigel alternative for 3-dimensional mammary epithelial cell culture.

Three-dimensional (3D) organoid models have been instrumental in understanding molecular mechanisms responsible for many cellular processes and diseases. However, established organic biomaterial scaffolds used for 3D hydrogel cultures, such as Matrigel, are biochemically complex and display significant batch variability, limiting reproducibility in experiments. Recently, there has been significant progress in the development of synthetic hydrogels for in vitro cell culture that are reproducible, mechanically tuneable, and biocompatible. Self-assembling peptide hydrogels (SAPHs) are synthetic biomaterials that can be engineered to be compatible with 3D cell culture. Here we investigate the ability of PeptiGel® SAPHs to model the mammary epithelial cell (MEC) microenvironment in vitro. The positively charged PeptiGel®Alpha4 supported MEC viability, but did not promote formation of polarised acini. Modifying the stiffness of PeptiGel® Alpha4 stimulated changes in MEC viability and changes in protein expression associated with altered MEC function, but did not fully recapitulate the morphologies of MECs grown in Matrigel. To supply the appropriate biochemical signals for MEC organoids, we supplemented PeptiGels® with laminin. Laminin was found to require negatively charged PeptiGel® Alpha7 for functionality, but was then able to provide appropriate signals for correct MEC polarisation and expression of characteristic proteins. Thus, optimisation of SAPH composition and mechanics allows tuning to support tissue-specific organoids.

Coarse-Grained Model of Glycosaminoglycans for Biomolecular Simulations.

Proteoglycans contain glycosaminoglycans (GAGs) which are negatively charged linear polymers made of repeating disaccharide units of uronic acid and hexosamine units. They play vital roles in numerous physiological and pathological processes, particularly in governing cellular communication and attachment. Depending on their sulfonation state, acetylation, and glycosidic linkages, GAGs belong to different families. The high molecular weight, heterogeneity, and flexibility of GAGs hamper their characterization at atomic resolution, but this may be circumvented via coarse-grained (CG) approaches. In this work, we report a CG model for a library of common GAG types in their isolated or proteoglycan-linked states compatible with version 2.2 (v2.2) of the widely popular CG Martini force field. The model reproduces conformational and thermodynamic properties for a wide variety of GAGs, as well as matching structural and binding data for selected proteoglycan test systems. The parameters developed here may thus be employed to study a range of GAG-containing biomolecular systems, thereby benefiting from the efficiency and broad applicability of the Martini framework.

Autoantigen-Dexamethasone Conjugate-Loaded Liposomes Halt Arthritis Development in Mice.

There is no curative treatment for chronic auto-inflammatory diseases including rheumatoid arthritis, and current treatments can induce off-target side effects due to systemic immune suppression. This work has previously shown that dexamethasone-pulsed tolerogenic dendritic cells loaded with the arthritis-specific antigen human proteoglycan can suppress arthritis development in a proteoglycan-induced arthritis mouse model. To circumvent ex vivo dendritic cell culture, and enhance antigen-specific effects, drug delivery vehicles, such as liposomes, provide an interesting approach. Here, this work uses anionic 1,2-distearoyl-sn-glycero-3-phosphoglycerol liposomes with enhanced loading of human proteoglycan-dexamethasone conjugates by cationic lysine tetramer addition. Antigen-pulsed tolerogenic dendritic cells induced by liposomal dexamethasone in vitro enhanced antigen-specific regulatory T cells to a similar extent as dexamethasone-induced tolerogenic dendritic cells. In an inflammatory adoptive transfer model, mice injected with antigen-dexamethasone liposomes have significantly higher antigen-specific type 1 regulatory T cells than mice injected with antigen only. The liposomes significantly inhibit the progression of arthritis compared to controls in preventative and therapeutic proteoglycan-induced arthritis mouse models. This coincides with systemic tolerance induction and an increase in IL10 expression in the paws of mice. In conclusion, a single administration of autoantigen and dexamethasone-loaded liposomes seems to be a promising antigen-specific treatment strategy for arthritis in mice.

Scale-Dependent Rheology of Synovial Fluid Lubricating Macromolecules.

Synovial fluid (SF) is the complex biofluid that facilitates the exceptional lubrication of articular cartilage in joints. Its primary lubricating macromolecules, the linear polysaccharide hyaluronic acid (HA) and the mucin-like glycoprotein proteoglycan 4 (PRG4 or lubricin), interact synergistically to reduce boundary friction. However, the precise manner in which these molecules influence the rheological properties of SF remains unclear. This study aimed to elucidate this by employing confocal microscopy and multiscale rheometry to examine the microstructure and rheology of solutions containing recombinant human PRG4 (rhPRG4) and HA. Contrary to previous assumptions of an extensive HA-rhPRG4 network, it is discovered that rhPRG4 primarily forms stiff, gel-like aggregates. The properties of these aggregates, including their size and stiffness, are found to be influenced by the viscoelastic characteristics of the surrounding HA matrix. Consequently, the rheology of this system is not governed by a single length scale, but instead responds as a disordered, hierarchical network with solid-like rhPRG4 aggregates distributed throughout the continuous HA phase. These findings provide new insights into the biomechanical function of PRG4 in cartilage lubrication and may have implications in the development of HA-based therapies for joint diseases like osteoarthritis.

Cartilage-like protein hydrogels engineered via entanglement.

Load-bearing tissues, such as muscle and cartilage, exhibit high elasticity, high toughness and fast recovery, but have different stiffness (with cartilage being significantly stiffer than muscle)1-8. Muscle achieves its toughness through finely controlled forced domain unfolding-refolding in the muscle protein titin, whereas articular cartilage achieves its high stiffness and toughness through an entangled network comprising collagen and proteoglycans. Advancements in protein mechanics and engineering have made it possible to engineer titin-mimetic elastomeric proteins and soft protein biomaterials thereof to mimic the passive elasticity of muscle9-11. However, it is more challenging to engineer highly stiff and tough protein biomaterials to mimic stiff tissues such as cartilage, or develop stiff synthetic matrices for cartilage stem and progenitor cell differentiation12. Here we report the use of chain entanglements to significantly stiffen protein-based hydrogels without compromising their toughness. By introducing chain entanglements13 into the hydrogel network made of folded elastomeric proteins, we are able to engineer highly stiff and tough protein hydrogels, which seamlessly combine mutually incompatible mechanical properties, including high stiffness, high toughness, fast recovery and ultrahigh compressive strength, effectively converting soft protein biomaterials into stiff and tough materials exhibiting mechanical properties close to those of cartilage. Our study provides a general route towards engineering protein-based, stiff and tough biomaterials, which will find applications in biomedical engineering, such as osteochondral defect repair, and material sciences and engineering.

Nanomechanics of Aggrecan: A New Perspective on Cartilage Biomechanics, Disease and Regeneration.

Articular cartilage is a hydrated macromolecular composite mainly composed of type II collagen fibrils and the large proteoglycan, aggrecan. Aggrecan is a key determinant of the load bearing and energy dissipation functions of cartilage. Previously, studies of cartilage biomechanics have been primarily focusing on the macroscopic, tissue-level properties, which failed to elucidate the molecular-level activities that govern cartilage development, function, and disease. This chapter provides a brief summary of Dr. Alan J. Grodzinsky's seminal contribution to the understanding of aggrecan molecular mechanics at the nanoscopic level. By developing and applying a series of atomic force microscopy (AFM)-based nanomechanical tools, Grodzinsky and colleagues revealed the unique structural and mechanical characteristics of aggrecan at unprecedented resolutions. In this body of work, the "bottle-brush"-like ultrastructure of aggrecan was directly visualized for the first time. Meanwhile, molecular mechanics of aggrecan was studied using a physiological-like 2D biomimetic assembly of aggrecan on multiple fronts, including compression, dynamic loading, shear, and adhesion. These studies not only generated new insights into the development, aging, and disease of cartilage, but established a foundation for designing and evaluating novel cartilage regeneration strategies. For example, building on the scientific foundation and methodology infrastructure established by Dr. Grodzinsky, recent studies have elucidated the roles of other proteoglycans in mediating cartilage integrity, such as decorin and perlecan, and evaluated the therapeutic potential of biomimetic proteoglycans in improving cartilage regeneration.

Introduction to the Complexity of Cell Surface and Tissue Matrix Glycoconjugates.

This chapter provides an overview of structures and functions of complex carbohydrates (commonly called glycans) that are covalently linked to proteins or lipids to form glycoconjugates known as glycoproteins, glycolipids, and proteoglycans. To understand the complexity of the glycan structures, the nature of their monosaccharide building blocks, how the monomeric units are covalently linked to each other, and how the resulting glycans are attached to proteins or lipids are discussed. Then, the classification, nomenclature, structural features, and functions of the glycan moieties of animal glycoconjugates are briefly described. All three classes of glycoconjugates are constituents of plasma membranes of all animal cells, including those of the nervous system. Glycoproteins and proteoglycans are also found abundantly as constituents of tissue matrices. Additionally, glycan-rich mucin glycoproteins are the major constituents of mucus secretions of epithelia of various organs. Furthermore, the chapter draws attention to the incredible structural complexity and diversity of the glycan moieties of cell surface and extracellular glycoconjugates. Finally, the involvement of glycans as informational molecules in a wide range of essential functions in almost all known biological processes, which are crucial for development, differentiation, and normal functioning of animals, is discussed.

Identification and Structural Characterization of Novel Chondroitin/Dermatan Sulfate Hexassacharide Domains in Human Decorin by Ion Mobility Tandem Mass Spectrometry.

Chondroitin sulfate (CS) and dermatan sulfate (DS) are found in nature linked to proteoglycans, most often as hybrid CS/DS chains. In the extracellular matrix, where they are highly expressed, CS/DS are involved in fundamental processes and various pathologies. The structural diversity of CS/DS domains gave rise to efforts for the development of efficient analytical methods, among which is mass spectrometry (MS), one of the most resourceful techniques for the identification of novel species and their structure elucidation. In this context, we report here on the introduction of a fast, sensitive, and reliable approach based on ion mobility separation (IMS) MS and MS/MS by collision-induced dissociation (CID), for the profiling and structural analysis of CS/DS hexasaccharide domains in human embryonic kidney HEK293 cells decorin (DCN), obtained after CS/DS chain releasing by ß-elimination, depolymerization using chondroitin AC I lyase, and fractionation by size-exclusion chromatography. By IMS MS, we were able to find novel CS/DS species, i.e., under- and oversulfated hexasaccharide domains in the released CS/DS chain. In the last stage of analysis, the optimized IMS CID MS/MS provided a series of diagnostic fragment ions crucial for the characterization of the misregulations, which occurred in the sulfation code of the trisulfated-4,5-Δ-GlcAGalNAc[IdoAGalNAc]2 sequence, due to the unusual sulfation sites.

Regulation of biomineralization by proteoglycans: From mechanisms to application.

Proteoglycans consist of core proteins and one or more covalently-linked glycosaminoglycan chains. They are structurally complex and heterogeneous. Proteoglycans bind to cell surface receptors, cytokines, growth factors and have strong affinity for collagen fibrils. Together with their complex spatial structures and different charge densities, proteoglycans are directly or indirectly involved in biomineralization. The present review focused on the potential mechanisms of proteoglycans-mediated biomineralization. Topics covered include the ability of proteoglycans to influence the proliferation and differentiation of odontoblasts and osteoblasts through complex signaling pathways, as well as regulate the aggregation of collagen fibrils and mineral deposition. The functions of proteoglycans in mineralization regulation and biomimetic properties render them important components in bone tissue engineering. Hence, the integrated impact of proteoglycans on bone formation was also succinctly deliberated. The potential of proteoglycans to function therapeutic targets for relieving the symptoms of ectopic mineralization and mineralization defects was also comprehensively addressed.

Excavating proteoglycan structure-function relationships: modern approaches to capture the interactions of ancient biomolecules.

Proteoglycans are now well regarded as key facilitators of cell biology. Although a majority of their interactions and functions are attributed to the decorating glycosaminoglycan chains, there is a growing appreciation for the roles of the proteoglycan core protein and for considering proteoglycans as replete protein-glycan conjugates. This appreciation, seeded by early work in proteoglycan biology, is now being advanced and exalted by modern approaches in chemical glycobiology. In this review, we discuss up-and-coming methods to unearth the fine-scale architecture of proteoglycans that modulate their functions and interactions. Crucial to these efforts is the production of chemically defined materials, including semisynthetic proteoglycans and the in situ capture of interacting proteins. Together, the integration of chemical biology approaches promises to expedite the dissection of the structural heterogeneity of proteoglycans and deliver refined insight into their functions.

Biologically Derived Neoproteoglycans for Profiling Protein-Glycosaminoglycan Interactions.

Glycosaminoglycans (GAGs) are a class of highly negatively charged membrane-associated and extracellular matrix polysaccharides involved in the regulation of myriad biological functions, including cell adhesion, migration, signaling, and differentiation, among others. GAGs are typically attached to core proteins, termed proteoglycans (PGs), and can engage >500 binding proteins, making them prominent relays for sensing external stimuli and transducing cellular responses. However, their unique substructural protein-recognition domains that confer their binding specificity remain elusive. While the emergence of glycan arrays has rapidly enabled the profiling of ligand specificities of a range of glycan-binding proteins, their adaptation for the analysis of GAG-binding proteins has been considerably more challenging. Current GAG microarrays primarily employ synthetically defined oligosaccharides, which capture only a fraction of the structural diversity of native GAG polysaccharides. Augmenting existing array platforms to include GAG structures purified from tissues or produced in cells with engineered glycan biosynthetic pathways may significantly advance the understanding of structure-activity relationships in GAG-protein interactions. Here, we demonstrate an efficient and tunable strategy to mimic cellular proteoglycan architectures by conjugating biologically derived GAG chains to a protein scaffold, defined as neoproteoglycans (neoPGs). The use of a reactive fluorogenic linker enabled real-time monitoring of the conjugation reaction efficiency and tuning of the neoPG valency. Immobilization of the reagents on a 96-well array platform allowed for efficient probing of ligand binding and enzyme-substrate specificity, including growth factors and the human sulfatase 1. The neoPGs can also be used directly as soluble probes to evaluate GAG-dependent growth factor signaling in cells.

Proteoglycans and glycosaminoglycans in central nervous system injury.

The brain and spinal cord constitute the central nervous system (CNS), which when injured, can be exceedingly devastating. The mechanistic roles of proteoglycans (PGs) and their glycosaminoglycan (GAG) side chains in such injuries have been extensively studied. CNS injury immediately alters endothelial and extracellular matrix (ECM) PGs and GAGs. Subsequently, these alterations contribute to acute injury, postinjury fibrosis, and postinjury repair. These effects are central to the pathophysiology of CNS injury. This review focuses on the importance of PGs and GAGs in multiple forms of injury including traumatic brain injury, spinal cord injury, and stroke. We highlight the causes and consequences of degradation of the PG and GAG-enriched endothelial glycocalyx in early injury and discuss the pleiotropic roles of PGs in neuroinflammation. We subsequently evaluate the dualistic effects of PGs on recovery: both PG/GAG-mediated inhibition and facilitation of repair. We then report promising therapeutic strategies that may prove effective for repair of CNS injury including PG receptor inhibition, delivery of endogenous, pro-repair PGs and GAGs, and direct degradation of pathological GAGs. Finally, we discuss the importance of two PG- and GAG-containing ECM structures (synapses and perineuronal nets) in CNS injury and recovery.

Emerging proteoglycans and proteoglycan-targeted therapies in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a common autoimmune disease that causes inflammation of the joints and damage to the cartilage and bone. The pathogenesis of RA is characterized in many patients by the presence of antibodies against citrullinated proteins. Proteoglycans are key structural elements of extracellular matrix in the joint articular cartilage and synovium and are secreted as lubricants in the synovial fluid. Alterations of proteoglycans contribute to RA pathogenesis. Proteoglycans such as aggrecan can be citrullinated and become potential targets of the rheumatoid autoimmune response. Proteoglycans are also upregulated in RA joints and/or undergo alterations of their regulatory functions over cytokines and chemokines, which promotes inflammation and bone damage. Recent studies have aimed to not only clarify these mechanisms but also develop novel proteoglycan-modulating therapeutics. These include agents altering the function and signaling of proteoglycans as well as tolerizing agents targeting citrullinated aggrecan. This mini-review summarizes the most recent findings regarding the dysregulation of proteoglycans that contributes to RA pathogenesis and the potential for proteoglycan-modulating agents to improve upon current RA therapy.

The function of glypicans in the mammalian embryo.

Glypicans are proteoglycans that are bound to the outer surface of the plasma membrane by a glycosylphosphatidylinositol anchor. The mammalian genome contains six members of the glypican family (GPC1 to GPC6). Although the degree of sequence homology within the family is rather low, the three-dimensional structure of these proteoglycans is highly conserved. Glypicans are predominantly expressed during embryonic development. Genetic and biochemical studies have shown that glypicans can stimulate or inhibit the signaling pathways triggered by Wnts, hedgehogs, fibroblast growth factors, and bone morphogenetic proteins. The study of mutant mouse strains demonstrated that glypicans have important functions in the developmental morphogenesis of various organs. In addition, a role of glypicans in synapsis formation has been established. Notably, glypican loss-of-function mutations are the cause of three human inherited syndromes. Recent analysis of glypican compound mutant mice has demonstrated that members of this protein family display redundant functions during embryonic development.

Comprehensive analysis of chondroitin sulfate and aggrecan in the head cartilage of bony fishes: Identification of proteoglycans in the head cartilage of sturgeon.

Cartilage in the head of sturgeon or salmon has been gaining attention as a rich source of functional chondroitin sulfate (CS) or proteoglycans. Although the cartilage was found in the heads of other bony fishes, the structure of CS and its core protein, especially aggrecan, was not fully investigated. In this study, comprehensive analysis of CS and aggrecan in the head cartilage of 10 bony fishes including sturgeon and salmon was performed. The 4-O-sulfation to 6-O-sulfation ratio (4S/6S ratio; S: sulfate residue) of CS in Perciformes was ≧1.0, while the 4S/6S ratios of CS from sturgeons and salmon were less than 0.5. Dot blotting and proteomic analysis revealed that aggrecan was a major core protein in head cartilage of all bony fishes. These results suggest that the head cartilage of bony fishes is a promising source for the preparation of CS or proteoglycans as a health food ingredient.

YY-11, a camel milk-derived peptide, inhibits TGF-ß-mediated atherogenic signaling in human vascular smooth muscle cells.

Atherosclerosis, the major underlying pathology of cardiovascular disease, commences with the binding and trapping of lipids on modified proteoglycans, with hyperelongated glycosaminoglycan chains. Transforming growth factor (TGF)-ß stimulates glycosaminoglycan elongation in vascular smooth muscle cells. We have recently shown that this TGF-ß signaling pathway involves reactive oxygen species (ROS). YY-11 is a dodecapeptide derived from camel milk and it has antioxidant activity. We have investigated the role of YY-11 in blocking ROS signaling and downstream atherogenic responses. YY-11 inhibited TGF-ß stimulated ROS production and inhibited the expression of genes for glycosaminoglycan chain elongation as a component of an in vitro model of atherosclerosis. This study provides a biochemical mechanism for the role of camel milk as a potential nutritional product to contribute to the worldwide amelioration of cardiovascular disease. PRACTICAL APPLICATIONS: The identification of readily accessible foods with antioxidant properties would provide a convenient and cost-effective approach community wide reducing oxidative stress induced pathologies such as atherosclerosis. We demonstrate that camel milk-derived peptide is an antioxidant that can inhibit growth factor-mediated proteoglycan modification in vitro. As proteoglycan modification is being recognized as one of the earliest atherogenic responses, these data support the notion of camel milk as a suitable nutritional product to contribute to the prevention of early stage of atherosclerosis development.