Laminin and hyaluronan supplementation of collagen hydrogels enhances endothelial function and tight junction expression on three-dimensional cylindrical microvessel-on-a-chip.

Vasculature-on-chip (VoC) models have become a prominent tool in the study of microvasculature functions because of their cost-effective and ethical production process. These models typically use a hydrogel in which the three-dimensional (3D) microvascular structure is embedded. Thus, VoCs are directly impacted by the physical and chemical cues of the supporting hydrogel. Endothelial cell (EC) response in VoCs is critical, especially in organ-specific vasculature models, in which ECs exhibit specific traits and behaviors that vary between organs. Many studies customize the stimuli ECs perceive in different ways; however, customizing the hydrogel composition accordingly to the target organ's extracellular matrix (ECM), which we believe has great potential, has been rarely investigated. We explored this approach to organ-specific VoCs by fabricating microvessels (MVs) with either human umbilical vein ECs or human brain microvascular ECs in a 3D cylindrical VoC using a collagen hydrogel alone or one supplemented with laminin and hyaluronan, components found in the brain ECM. We characterized the physical properties of these hydrogels and analyzed the barrier properties of the MVs. Barrier function and tight junction (ZO-1) expression improved with the addition of laminin and hyaluronan in the composite hydrogel.

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.

Laminin-Augmented Decellularized Extracellular Matrix Ameliorating Neural Differentiation and Neuroinflammation in Human Mini-Brains.

Non-neural extracellular matrix (ECM) has limited application in humanized physiological neural modeling due to insufficient brain-specificity and safety concerns. Although brain-derived ECM contains enriched neural components, certain essential components are partially lost during the decellularization process, necessitating augmentation. Here, it is demonstrated that the laminin-augmented porcine brain-decellularized ECM (P-BdECM) is xenogeneic factor-depleted as well as favorable for the regulation of human neurons, astrocytes, and microglia. P-BdECM composition is comparable to human BdECM regarding brain-specificity through the matrisome and gene ontology-biological process analysis. As augmenting strategy, laminin 111 supplement promotes neural function by synergic effect with laminin 521 in P-BdECM. Annexin A1(ANXA1) and Peroxiredoxin(PRDX) in P-BdECM stabilized microglial and astrocytic behavior under normal while promoting active neuroinflammation in response to neuropathological factors. Further, supplementation of the brain-specific molecule to non-neural matrix also ameliorated glial cell inflammation as in P-BdECM. In conclusion, P-BdECM-augmentation strategy can be used to recapitulate humanized pathophysiological cerebral environments for neurological study.

Exploring a Solvent Dependent Strategy to Control Self-Assembling Behavior and Cellular Interaction in Laminin-Mimetic Short Peptide based Supramolecular Hydrogels.

Self-assembled hydrogels, fabricated through diverse non-covalent interactions, have been extensively studied in regenerative medicines. Inspired from bioactive functional motifs of ECM protein, short peptide sequences have shown remarkable abilities to replicate the intrinsic features of the natural extracellular milieu. In this direction, we have fabricated two short hydrophobic bioactive sequences derived from the laminin protein i. e., IKVAV and YIGSR. Based on the substantial hydrophobicity of these peptides, we selected a co-solvent approach as a suitable gelation technique that included different concentrations of DMSO as an organic phase along with an aqueous solution containing 0.1 % TFA. These hydrophobic laminin-based bioactive peptides with limited solubility in aqueous physiological environment showed significantly enhanced solubility with higher DMSO content in water. The enhanced solubility resulted in extensive intermolecular interactions that led to the formation of hydrogels with a higher-order entangled network along with improved mechanical properties. Interestingly, by simply modulating DMSO content, highly tunable gels were accessed in the same gelator domain that displayed differential physicochemical properties. Further, the cellular studies substantiated the potential of these laminin-derived hydrogels in enhancing cell-matrix interactions, thereby reinforcing their applications in tissue engineering.

Synergy between Laminin-Derived Elastin-like Polypeptides (LELPs) Optimizes Cell Spreading.

A major component of the extracellular matrix (ECM), laminins, modulates cells via diverse receptors. Their fragments have emerging utility as components of "ECM-mimetics" optimized to promote cell-based therapies. Recently, we reported that a bioactive laminin peptide known as A99 enhanced cell binding and spreading via fusion to an elastin-like polypeptide (ELP). The ELP "handle" serves as a rapid, noncovalent strategy to concentrate bioactive peptide mixtures onto a surface. We now report that this strategy can be further generalized across an expanded panel of additional laminin-derived elastin-like polypeptides (LELPs). A99 (AGTFALRGDNPQG), A2G80 (VQLRNGFPYFSY), AG73 (RKRLQVQLSIRT), and EF1m (LQLQEGRLHFMFD) all promote cell spreading while showing morphologically distinct F-actin formation. Equimolar mixtures of A99:A2G80-LELPs have synergistic effects on adhesion and spreading. Finally, three of these ECM-mimetics promote the neurite outgrowth of PC-12 cells. The evidence presented here demonstrates the potential of ELPs to deposit ECM-mimetics with applications in regenerative medicine, cell therapy, and tissue engineering.

Biophysical phenotype mixtures reveal advantages for tumor muscle invasion in vivo.

Bladder, colon, gastric, prostate, and uterine cancers originate in organs surrounded by laminin-coated smooth muscle. In human prostate cancer, tumors that are organ confined, without extracapsular extension through muscle, have an overall cancer survival rate of up to 97% compared with 32% for metastatic disease. Our previous work modeling extracapsular extension reported the blocking of tumor invasion by mutation of a laminin-binding integrin called α6ß1. Expression of the α6AA mutant resulted in a biophysical switch from cell-ECM (extracellular matrix) to cell-cell adhesion with drug sensitivity properties and an inability to invade muscle. Here we used different admixtures of α6AA and α6WT cells to test the cell heterogeneity requirements for muscle invasion. Time-lapse video microscopy revealed that tumor mixtures self-assembled into invasive networks in vitro, whereas α6AA cells assembled only as cohesive clusters. Invasion of α6AA cells into and through live muscle occurred using a 1:1 mixture of α6AA and α6WT cells. Electric cell-substrate impedance sensing measurements revealed that compared with α6AA cells, invasion-competent α6WT cells were 2.5-fold faster at closing a cell-ECM or cell-cell wound, respectively. Cell-ECM rebuilding kinetics show that an increased response occurred in mixtures since the response was eightfold greater compared with populations containing only one cell type. A synthetic cell adhesion cyclic peptide called MTI-101 completely blocked electric cell-substrate impedance sensing cell-ECM wound recovery that persisted in vitro up to 20 h after the wound. Treatment of tumor-bearing animals with 10 mg/kg MTI-101 weekly resulted in a fourfold decrease of muscle invasion by tumor and a decrease of the depth of invasion into muscle comparable to the α6AA cells. Taken together, these data suggest that mixed biophysical phenotypes of tumor cells within a population can provide functional advantages for tumor invasion into and through muscle that can be potentially inhibited by a synthetic cell adhesion molecule.

A mutation affecting laminin alpha 5 polymerisation gives rise to a syndromic developmental disorder.

Laminin alpha 5 (LAMA5) is a member of a large family of proteins that trimerise and then polymerise to form a central component of all basement membranes. Consequently, the protein plays an instrumental role in shaping the normal development of the kidney, skin, neural tube, lung and limb, and many other organs and tissues. Pathogenic mutations in some laminins have been shown to cause a range of largely syndromic conditions affecting the competency of the basement membranes to which they contribute. We report the identification of a mutation in the polymerisation domain of LAMA5 in a patient with a complex syndromic disease characterised by defects in kidney, craniofacial and limb development, and by a range of other congenital defects. Using CRISPR-generated mouse models and biochemical assays, we demonstrate the pathogenicity of this variant, showing that the change results in a failure of the polymerisation of α/ß/γ laminin trimers. Comparing these in vivo phenotypes with those apparent upon gene deletion in mice provides insights into the specific functional importance of laminin polymerisation during development and tissue homeostasis.

Synthesis of the matriglycan hexasaccharide, -3Xylα1-3GlcAß1-trimer and its interaction with laminin.

Matriglycan, a polysaccharide that is a pivotal part of the core M3 O-mannosyl glycan composed of the repeating disaccharide -3Xylα1-3GlcAß1-, interacts with laminin to stabilize muscle tissue. We herein report the synthesis of matriglycan-repeating hexasaccharides equipped with an alkyne linker to form glycoconjugates. The key step in the formation of an α-linked xylosyl glycoside was resolved by solvent-specific separation from an anomeric mixture. Successful glycan elongation was regio- and stereoselectively performed to obtain (-3Xylα1-3GlcAß1)3-O(C2H4O)3CH2CCH and the biotin conjugate. We also investigated interactions between matriglycan hexasaccharides and laminin-G-like domains 4 and 5 of laminin-α2 using saturation transfer difference-NMR. The dissociation constant obtained from bio-layer interferometry was estimated to be 7.5 × 10-8 M. These results indicate that a chemical approach may be applied to the reconstruction of muscle tissue.

Effects of laminin-111 peptide coatings on rat neural stem/progenitor cell culture.

Neurons require adhesive scaffolds for their growth and differentiation. Laminins are a major cell adhesive component of basement membranes and have various biological activities in the peripheral and central nervous systems. Here, we evaluated the biological activities of 5 peptides derived from laminin-111 as a scaffold for mouse neuroblastoma Neuro2a cells and rat neural stem/progenitor cells (NPCs). The 5 peptides showed Neuro2a cell attachment activity similar to that of poly-d-lysine. However, when NPCs were cultured on the peptides, 2 syndecan-binding peptides, AG73 (RKRLQVQLSIRT, mouse laminin α1 chain 2719-2730) and C16 (KAFDITYVRLKF, laminin γ1 chain 139-150), demonstrated significantly higher cell attachment and neurite extension activities than other peptides including integrin-binding ones. Long-term cell culture experiments showed that both AG73 and C16 supported the growth of neurons and astrocytes that had differentiated from NPCs. Furthermore, C16 markedly promoted the expression of neuronal markers such as synaptosomal-associated protein-25 and syntaxin 1A. These results indicate that AG73 and C16 are useful for NPC cultures and that C16 can be applied to specialized research on synapses in differentiated neurons. These peptides have the potential for use as valuable biomaterials for NPC research.

Improvement of sciatic nerve regeneration by multichannel nanofibrous membrane-embedded electro-conductive conduits functionalized with laminin.

Multichannel structures in the design of nerve conduits offer potential advantages for regeneration of damaged nerves. However, lack of biochemical cues and electrical stimulation could hamper satisfactory nerve regeneration. The aim of this study was to simultaneously evaluate the effects of topographical, biological, and electrical cues on sciatic nerve regeneration. Accordingly, a series of multichannel nerve conduit was made using longitudinally-aligned laminin-coated poly (lactic-co-glycolic acid) (PLGA)/carbon nanotubes (CNT) nanofibers (NF, mean diameter: 455 ± 362 nm) in the lumen and randomly-oriented polycaprolactone (PCL) NF (mean diameter: 340 ± 200 nm) on the outer surface. In vitro studies revealed that the materials were nontoxic and able to promote cell attachment and proliferation on nanofibers and on fibrin gel. To determine the influence of laminin as biological and CNT as electrical cues on nerve regeneration, either of hollow PCL conduits, PLGA NF-embedded, PLGA/CNT NF-embedded or laminin-coated PLGA/CNT NF-embedded PCL conduits were implanted in rats. A new surgery method was utilized and results were compared with an autograft. The results of motor and sensory tests in addition to histopathological examination of the regenerated nerves demonstrated the formation of nerve fibers in laminin-coated PLGA/CNT NF-embedded PCL conduits. Results suggested that these conduits have the potential to improve sciatic nerve regeneration. Graphical abstract.

Synthesis, Self-Assembly, and Cell Responses of Aromatic IKVAV Peptide Amphiphiles.

Synthetic bioactive aromatic peptide amphiphiles have been recognized as key elements of emerging biomedical strategies due to their biocompatibility, design flexibility, and functionality. Inspired by natural proteins, we synthesized two supramolecular materials of phenyl-capped Ile-Lys-Val-Ala-Val (Ben-IKVAV) and perfluorophenyl-capped Ile-Lys-Val-Ala-Val (PFB-IKVAV). We employed UV-vis absorption, fluorescence, circular dichroism, and Fourier-transform infrared spectroscopy to examine the driving force in the self-assembly of the newly discovered materials. It was found that both compounds exhibited ordered π-π interactions and secondary structures, especially PFB-IKVAV. The cytotoxicity of human mesenchymal stem cells (hMSCs) and cell differentiation studies was also performed. In addition, the immunofluorescent staining for neuronal-specific markers of MAP2 was 4.6 times (neural induction medium in the presence of PFB-IKVAV) that of the neural induction medium (control) on day 7. From analyzing the expression of neuronal-specific markers in hMSCs, it can be concluded that PFB-IKVAV may be a potential supramolecular biomaterial for biomedical applications.

Curvature-dependent constraints drive remodeling of epithelia.

Epithelial tissues function as barriers that separate the organism from the environment. They usually have highly curved shapes, such as tubules or cysts. However, the processes by which the geometry of the environment and the cell's mechanical properties set the epithelium shape are not yet known. In this study, we encapsulated two epithelial cell lines, MDCK and J3B1A, into hollow alginate tubes and grew them under cylindrical confinement forming a complete monolayer. MDCK monolayers detached from the alginate shell at a constant rate, whereas J3B1A monolayers detached at a low rate unless the tube radius was reduced. We showed that this detachment is driven by contractile stresses in the epithelium and can be enhanced by local curvature. This allows us to conclude that J3B1A cells exhibit smaller contractility than MDCK cells. Monolayers inside curved tubes detach at a higher rate on the outside of a curve, confirming that detachment is driven by contraction.

Establishment of a novel monoclonal antibody against truncated glycoforms of α-dystroglycan lacking matriglycans.

α-Dystroglycan (α-DG) is a glycoprotein specifically modified with O-mannosyl glycans bearing long polysaccharides, termed matriglycans, which comprise repeating units of glucuronic acid and xylose. The matriglycan is linked to the O-mannosyl glycan core through two ribitol phosphate units that can be replaced with glycerol phosphate (GroP) units synthesized by fukutin and fukutin-related protein that transfer GroP from CDP-Gro. Here, we found that forced expression of the bacterial CDP-Gro synthase, TagD, from Bacillus subtilis could result in the overproduction of CDP-Gro in human colon carcinoma HCT116 cells. Western blot and liquid chromatography-tandem mass spectrometry analyses indicated that α-DG prepared from the TagD-expressing HCT116 cells contained abundant GroP and lacked matriglycans. Using the GroP-containing recombinant α-DG-Fc, we developed a novel monoclonal antibody, termed DG2, that reacts with several truncated glycoforms of α-DG, including GroP-terminated glycoforms lacking matriglycans; we verified the reactivity of DG2 against various types of knockout cells deficient in the biosynthesis of matriglycans. Accordingly, forced expression of TagD in HCT116 cells resulted in the reduction of matriglycans and an increase in DG2 reactivity. Collectively, our results indicate that DG2 could serve as a useful tool to determine tissue distribution and function of α-DG lacking matriglycans under physiological and pathophysiological conditions.

Laminins and interaction partners in the architecture of the basement membrane at the dermal-epidermal junction.

The basement membrane at the dermal-epidermal junction keeps the epidermis attached to the dermis. This anatomical barrier is made up of four categories of extracellular matrix proteins: collagen IV, laminin, nidogen and perlecan. These proteins are precisely arranged in a well-defined architecture through specific interactions between the structural domains of the individual components. Some of the molecular constituents are provided by both fibroblasts and keratinocytes, while others are synthesized exclusively by fibroblasts or keratinocytes. It remains to be determined how the components from the fibroblasts are targeted to the dermal-epidermal junction and correctly organized and integrated with the proteins from the adjacent keratinocytes to form the basement membrane.

Cell contraction induces long-ranged stress stiffening in the extracellular matrix.

Animal cells in tissues are supported by biopolymer matrices, which typically exhibit highly nonlinear mechanical properties. While the linear elasticity of the matrix can significantly impact cell mechanics and functionality, it remains largely unknown how cells, in turn, affect the nonlinear mechanics of their surrounding matrix. Here, we show that living contractile cells are able to generate a massive stiffness gradient in three distinct 3D extracellular matrix model systems: collagen, fibrin, and Matrigel. We decipher this remarkable behavior by introducing nonlinear stress inference microscopy (NSIM), a technique to infer stress fields in a 3D matrix from nonlinear microrheology measurements with optical tweezers. Using NSIM and simulations, we reveal large long-ranged cell-generated stresses capable of buckling filaments in the matrix. These stresses give rise to the large spatial extent of the observed cell-induced matrix stiffness gradient, which can provide a mechanism for mechanical communication between cells.

Potent laminin-inspired antioxidant regenerative dressing accelerates wound healing in diabetes.

The successful treatment of chronic dermal wounds, such as diabetic foot ulcers (DFU), depends on the development of safe, effective, and affordable regenerative tools that the surgeon can rely on to promote wound closure. Although promising, strategies that involve cell-based therapies and the local release of exogenous growth factors are costly, require very long development times, and result in modest improvements in patient outcome. We describe the development of an antioxidant shape-conforming regenerative wound dressing that uses the laminin-derived dodecapeptide A5G81 as a potent tethered cell adhesion-, proliferation-, and haptokinesis-inducing ligand to locally promote wound closure. A5G81 immobilized within a thermoresponsive citrate-based hydrogel facilitates integrin-mediated spreading, migration, and proliferation of dermal and epidermal cells, resulting in faster tissue regeneration in diabetic wounds. This peptide-hydrogel system represents a paradigm shift in dermoconductive and dermoinductive strategies for treating DFU without the need for soluble biological or pharmacological factors.

Grip and slip of L1-CAM on adhesive substrates direct growth cone haptotaxis.

Chemical cues presented on the adhesive substrate direct cell migration, a process termed haptotaxis. To migrate, cells must generate traction forces upon the substrate. However, how cells probe substrate-bound cues and generate directional forces for migration remains unclear. Here, we show that the cell adhesion molecule (CAM) L1-CAM is involved in laminin-induced haptotaxis of axonal growth cones. L1-CAM underwent grip and slip on the substrate. The ratio of the grip state was higher on laminin than on the control substrate polylysine; this was accompanied by an increase in the traction force upon laminin. Our data suggest that the directional force for laminin-induced growth cone haptotaxis is generated by the grip and slip of L1-CAM on the substrates, which occur asymmetrically under the growth cone. This mechanism is distinct from the conventional cell signaling models for directional cell migration. We further show that this mechanism is disrupted in a human patient with L1-CAM syndrome, suffering corpus callosum agenesis and corticospinal tract hypoplasia.

Modified Hyaluronic Acid-Laminin-Hydrogel as Luminal Filler for Clinically Approved Hollow Nerve Guides in a Rat Critical Defect Size Model.

Hollow nerve guidance conduits are approved for clinical use for defect lengths of up to 3 cm. This is because also in pre-clinical evaluation they are less effective in the support of nerve regeneration over critical defect lengths. Hydrogel luminal fillers are thought to improve the regeneration outcome by providing an optimized matrix inside bioartificial nerve grafts. We evaluated here a modified hyaluronic acid-laminin-hydrogel (M-HAL) as luminal filler for two clinically approved hollow nerve guides. Collagen-based and chitosan-based nerve guides were filled with M-HAL in two different concentrations and the regeneration outcome comprehensively studied in the acute repair rat sciatic nerve 15 mm critical defect size model. Autologous nerve graft (ANG) repair served as gold-standard control. At 120 days post-surgery, all ANG rats demonstrated electrodiagnostically detectable motor recovery. Both concentrations of the hydrogel luminal filler induced improved regeneration outcome over empty nerve guides. However, neither combination with collagen- nor chitosan-based nerve guides resulted in functional recovery comparable to the ANG repair. In contrast to our previous studies, we demonstrate here that M-HAL slightly improved the overall performance of either empty nerve guide type in the critical defect size model.

Proteins Binding to the Carbohydrate HNK-1: Common Origins?

The human natural killer (HNK-1) carbohydrate plays important roles during nervous system development, regeneration after trauma and synaptic plasticity. Four proteins have been identified as receptors for HNK-1: the laminin adhesion molecule, high-mobility group box 1 and 2 (also called amphoterin) and cadherin 2 (also called N-cadherin). Because of HNK-1's importance, we asked whether additional receptors for HNK-1 exist and whether the four identified proteins share any similarity in their primary structures. A set of 40,000 sequences homologous to the known HNK-1 receptors was selected and used for large-scale sequence alignments and motif searches. Although there are conserved regions and highly conserved sites within each of these protein families, there was no sequence similarity or conserved sequence motifs found to be shared by all families. Since HNK-1 receptors have not been compared regarding binding constants and since it is not known whether the sulfated or non-sulfated part of HKN-1 represents the structurally crucial ligand, the receptors are more heterogeneous in primary structure than anticipated, possibly involving different receptor or ligand regions. We thus conclude that the primary protein structure may not be the sole determinant for a bona fide HNK-1 receptor, rendering receptor structure more complex than originally assumed.

A Rho Kinase (ROCK) Inhibitor, Y-27632, Inhibits the Dissociation-Induced Cell Death of Salivary Gland Stem Cells.

Salivary gland stem cells (SGSCs) are potential cell sources for the treatment of salivary gland diseases. The control of cell survival is an essential factor for applying stem cells to regenerative medicine or stem cell-based research. The purpose of this study was to investigate the effects of the ROCK inhibitor Y-27632 on the survival of SGSCs and its underlying mechanisms. SGSCs were isolated from mouse submandibular glands and cultured in suspension. Treatment with Y-27632 restored the viability of SGSCs that was significantly decreased during isolation and the subsequent culture. Y-27632 upregulated the expression of anti-apoptotic protein BCL-2 in SGSCs and, in the apoptosis assay, significantly reduced apoptotic and necrotic cell populations. Matrigel was used to mimic the extracellular environment of an intact salivary gland. The expression of genes regulating apoptosis and the ROCK signaling pathway was significantly reduced when SGSCs were embedded in Matrigel. SGSCs cultured in Matrigel and treated with Y-27632 showed no difference in the total numbers of spheroids and expression levels of apoptosis-regulating genes. Matrigel-embedded SGSCs treated with Y-27632 increased the number of spheroids with budding structures and the expression of acinar cell-specific marker AQP5. We demonstrate the protective effects of Y-27632 against dissociation-induced apoptosis of SGSCs during their culture in vitro.