ILC1 drive intestinal epithelial and matrix remodelling.

Organoids can shed light on the dynamic interplay between complex tissues and rare cell types within a controlled microenvironment. Here, we develop gut organoid cocultures with type-1 innate lymphoid cells (ILC1) to dissect the impact of their accumulation in inflamed intestines. We demonstrate that murine and human ILC1 secrete transforming growth factor ß1, driving expansion of CD44v6+ epithelial crypts. ILC1 additionally express MMP9 and drive gene signatures indicative of extracellular matrix remodelling. We therefore encapsulated human epithelial-mesenchymal intestinal organoids in MMP-sensitive, synthetic hydrogels designed to form efficient networks at low polymer concentrations. Harnessing this defined system, we demonstrate that ILC1 drive matrix softening and stiffening, which we suggest occurs through balanced matrix degradation and deposition. Our platform enabled us to elucidate previously undescribed interactions between ILC1 and their microenvironment, which suggest that they may exacerbate fibrosis and tumour growth when enriched in inflamed patient tissues.

Pluronic Micelle-Mediated Tissue Factor Silencing Enhances Hemocompatibility, Stemness, Differentiation Potential, and Paracrine Signaling of Mesenchymal Stem Cells.

Mesenchymal stem/stromal cells (MSCs) evoke great excitement for treating different human diseases due to their ability to home inflamed tissues, suppress inflammation, and promote tissue regeneration. Despite great promises, clinical trial results are disappointing as allotransplantation of MSCs trigger thrombotic activity and are damaged by the complement system, compromising their survival and function. To overcome this, a new strategy is presented by the silencing of tissue factor (TF), a transmembrane protein that mediates procoagulant activity. Novel Pluronic-based micelles are designed with the pendant pyridyl disulfide group, which are used to conjugate TF-targeting siRNA by the thiol-exchange reaction. This nanocarrier design effectively delivered the payload to MSCs resulting in ∼72% TF knockdown (KD) without significant cytotoxicity. Hematological evaluation of MSCs and TF-KD MSCs in an ex vivo human whole blood model revealed a significant reduction in an instant-blood-mediated-inflammatory reaction as evidenced by reduced platelet aggregation (93% of free platelets in the TF-KD group, compared to 22% in untreated bone marrow-derived MSCs) and thrombin-antithrombin complex formation. Effective TF silencing induced higher MSC differentiation in osteogenic and adipogenic media and showed stronger paracrine suppression of proinflammatory cytokines in macrophages and higher stimulation in the presence of endotoxins. Thus, TF silencing can produce functional cells with higher fidelity, efficacy, and functions.

Modulating Thiol p Ka Promotes Disulfide Formation at Physiological pH: An Elegant Strategy To Design Disulfide Cross-Linked Hyaluronic Acid Hydrogels.

The disulfide bond plays a crucial role in protein biology and has been exploited by scientists to develop antibody-drug conjugates, sensors, and for the immobilization other biomolecules to materials surfaces. In spite of its versatile use, the disulfide chemistry suffers from some inevitable limitations such as the need for basic conditions (pH > 8.5), strong oxidants, and long reaction times. We demonstrate here that thiol-substrates containing electron-withdrawing groups at the ß-position influence the deprotonation of the thiol group, which is the key reaction intermediate in the formation of disulfide bonds. Evaluation of reaction kinetics using small molecule substrate such as l-cysteine indicated disulfide formation at a 2.8-fold higher ( k1 = 5.04 × 10-4 min-1) reaction rate as compared to the conventional thiol substrate, namely 3-mercaptopropionic acid ( k1 = 1.80 × 10-4 min-1) at physiological pH (pH 7.4). Interestingly, the same effect could not be observed when N-acetyl-l-cysteine substrate ( k1 = 0.51 × 10-4 min-1) was used. We further grafted such thiol-containing molecules (cysteine, N-acetyl-cysteine, and 3-mercaptopropionic acid) to a biopolymer namely hyaluronic acid (HA) and determined the p Ka value of different thiol groups by spectrophotometric analysis. The electron-withdrawing group at the ß-position reduced the p Ka of the thiol group to 7.0 for HA-cysteine (HA-Cys); 7.4 for N-acetyl cysteine (HA-ActCys); and 8.1 for HA-thiol (HA-SH) derivatives, respectively. These experiments further confirmed that the concentration of thiolate (R-S-) ions could be increased with the presence of electron-withdrawing groups, which could facilitate disulfide cross-linked hydrogel formation at physiological pH. Indeed, HA grafted with cysteine or N-acetyl groups formed hydrogels within 3.5 min or 10 h, respectively, at pH 7.4. After completion of cross-linking reaction, both gels demonstrated a storage modulus G' ≈ 3300-3500 Pa, which indicated comparable levels of cross-linking. The HA-SH gel, on the other hand, did not form any gel at pH 7.4 even after 24 h. Finally, we demonstrated that the newly prepared hydrogels exhibited excellent hydrolytic stability but can be degraded by cell-directed processes (enzymatic and reductive degradation). We believe our study provides a valuable insight on the factors governing the disulfide formation and our results are useful to develop strategies that would facilitate generation of stable thiol functionalized biomolecules or promote fast thiol oxidation according to the biomedical needs.

An Unexpected Role of Hyaluronic Acid in Trafficking siRNA Across the Cellular Barrier: The First Biomimetic, Anionic, Non-Viral Transfection Method.

Circulating nucleic acids, such as short interfering RNA (siRNA), regulate many biological processes; however, the mechanism by which these molecules enter the cell is poorly understood. The role of extracellular-matrix-derived polymers in binding siRNAs and trafficking them across the plasma membrane is reported. Thermal melting, dynamic light scattering, scanning electron microscopy, and computational analysis indicate that hyaluronic acid can stabilize siRNA via hydrogen bonding and Van der Waals interactions. This stabilization facilitated HA size- and concentration-dependent gene silencing in a CD44-positive human osteosarcoma cell line (MG-63) and in human mesenchymal stromal cells (hMSCs). This native HA-based siRNA transfection represents the first report on an anionic, non-viral delivery method that resulted in approximately 60 % gene knockdown in both cell types tested, which correlated with a reduction in translation levels.

Insights into the mechanism and catalysis of oxime coupling chemistry at physiological pH.

The dynamic covalent-coupling reaction involving α-effect nucleophiles has revolutionized bioconjugation approaches, due to its ease and high efficiency. Key to its success is the discovery of aniline as a nucleophilic catalyst, which made this reaction feasible under physiological conditions. Aniline however, is not so effective for keto substrates. Here, we investigate the mechanism of aniline activation in the oxime reaction with aldehyde and keto substrates. We also present carboxylates as activating agents that can promote the oxime reaction with both aldehyde and keto substrates at physiological pH. This rate enhancement circumvents the influence of α-effect by forming H-bonds with the rate-limiting intermediate, which drives the reaction to completion. The combination of aniline and carboxylates had a synergistic effect, resulting in a ∼14-31-fold increase in reaction rate at pD 7.4 with keto substrates. The biocompatibility and efficiency of carboxylate as an activating agent is demonstrated by performing cell-surface oxime labeling at physiological pH using acetate, which showed promising results that were comparable with aniline.

Glycosaminoglycans' for brain health: Harnessing glycosaminoglycan based biomaterials for treating central nervous system diseases and in-vitro modeling.

Dysfunction of the central nervous system (CNS) following traumatic brain injuries (TBI), spinal cord injuries (SCI), or strokes remains challenging to address using existing medications and cell-based therapies. Although therapeutic cell administration, such as stem cells and neuronal progenitor cells (NPCs), have shown promise in regenerative properties, they have failed to provide substantial benefits. However, the development of living cortical tissue engineered grafts, created by encapsulating these cells within an extracellular matrix (ECM) mimetic hydrogel scaffold, presents a promising functional replacement for damaged cortex in cases of stroke, SCI, and TBI. These grafts facilitate neural network repair and regeneration following CNS injuries. Given that natural glycosaminoglycans (GAGs) are a major constituent of the CNS, GAG-based hydrogels hold potential for the next generation of CNS healing therapies and in vitro modeling of CNS diseases. Brain-specific GAGs not only offer structural and biochemical signaling support to encapsulated neural cells but also modulate the inflammatory response in lesioned brain tissue, facilitating host integration and regeneration. This review briefly discusses different roles of GAGs and their related proteoglycan counterparts in healthy and diseases brain and explores current trends and advancements in GAG-based biomaterials for treating CNS injuries and modeling diseases. Additionally, it examines injectable, 3D bioprintable, and conductive GAG-based scaffolds, highlighting their clinical potential for in vitro modeling of patient-specific neural dysfunction and their ability to enhance CNS regeneration and repair following CNS injury in vivo.

Mild and efficient strategy for site-selective aldehyde modification of glycosaminoglycans: tailoring hydrogels with tunable release of growth factor.

Aldehydes have been used as an important bioorthogonal chemical reporter for conjugation of large polymers and bioactive substances. However, generating aldehyde functionality on carbohydrate-based biopolymers without changing its native chemical structure has always persisted as a challenging task. The common methods employed to achieve this require harsh reaction conditions, which often compromise the structural integrity and biological function of these sensitive molecules. Here we report a mild and simple method to graft aldehydes groups on glycosaminoglycans (GAGs) in a site-selective manner without compromising the structural integrity of the biopolymer. This regio-selective modification was achieved by conjugating the amino-glycerol moiety on the carboxylate residue of the polymer, which allowed selective cleavage of pendent diol groups without interfering with the C2-C3 diol groups of the native glucopyranose residue. Kinetic evaluation of this reaction demonstrated significant differences in second-order reaction rate for periodate oxidation (by four-orders of magnitude) between the two types of vicinal diols. We employed this chemistry to develop aldehyde modifications of sulfated and nonsulfated GAGs such as hyaluronic acid (HA), heparin (HP), and chondroitin sulfate (CS). We further utilized these aldehyde grafted GAGs to tailor extracellular matrix mimetic injectable hydrogels and evaluated its rheological properties. The composition of the hydrogels was also found to modulate release of therapeutic protein such as FGF-2, demonstrating controlled release (60%) for over 14 days. In short, our result clearly demonstrates a versatile strategy to graft aldehyde groups on sensitive biopolymers under mild conditions that could be applied for various bioconjugation and biomedical applications such as drug delivery and regenerative medicine.

Biomimetic polyelectrolyte coating of stem cells suppresses thrombotic activation and enhances its survival and function.

Mesenchymal stem cells (MSCs) therapy is a promising approach for treating inflammatory diseases due to their immunosuppressive and tissue repair characteristics. However, allogenic transplantation of MSCs induces thrombotic complications in some patients which limits its potential for clinical translation. To address this challenge, we have exploited the bioactivity of heparin, a well-known anticoagulant and immunosuppressive polysaccharide that is widely used in clinics. We have developed a smart layer-by-layer (LbL) coating strategy using gelatin and heparin polymers exploiting their overall positive and negative charges that enabled efficient complexation with the MSCs' glycocalyx. The stable coating of MSCs suppressed complement attack and mitigated thrombotic activation as demonstrated in human whole blood. Gratifyingly, the MSC coating retained its immunosuppressive properties and differentiation potential when exposed to inflammatory conditions and differentiation factors. We believe the simple coating procedure of MSCs will increase allogenic tolerance and circumvent the major challenge of MSCs transplantation.

Unveiling extracellular matrix assembly: Insights and approaches through bioorthogonal chemistry.

Visualizing cells, tissues, and their components specifically without interference with cellular functions, such as biochemical reactions, and cellular viability remains important for biomedical researchers worldwide. For an improved understanding of disease progression, tissue formation during development, and tissue regeneration, labeling extracellular matrix (ECM) components secreted by cells persists is required. Bioorthogonal chemistry approaches offer solutions to visualizing and labeling ECM constituents without interfering with other chemical or biological events. Although biorthogonal chemistry has been studied extensively for several applications, this review summarizes the recent advancements in using biorthogonal chemistry specifically for metabolic labeling and visualization of ECM proteins and glycosaminoglycans that are secreted by cells and living tissues. Challenges, limitations, and future directions surrounding biorthogonal chemistry involved in the labeling of ECM components are discussed. Finally, potential solutions for improvements to biorthogonal chemical approaches are suggested. This would provide theoretical guidance for labeling and visualization of de novo proteins and polysaccharides present in ECM that are cell-secreted for example during tissue remodeling or in vitro differentiation of stem cells.

Bidirectional cell-matrix interaction dictates neuronal network formation in a brain-mimetic 3D scaffold.

Human pluripotent stem cells (hPSC) derived neurons are emerging as a powerful tool for studying neurobiology, disease pathology, and modeling. Due to the lack of platforms available for housing and growing hPSC-derived neurons, a pressing need exists to tailor a brain-mimetic 3D scaffold that recapitulates tissue composition and favourably regulates neuronal network formation. Despite the progress in engineering biomimetic scaffolds, an ideal brain-mimetic scaffold is still elusive. We bioengineered a physiologically relevant 3D scaffold by integrating brain-like extracellular matrix (ECM) components and chemical cues. Culturing hPSCs-neurons in hyaluronic acid (HA) gels and HA-chondroitin sulfate (HA-CS) composite gels showed that the CS component prevails as the predominant factor for the growth of neuronal cells, albeit to modest efficacy. Covalent grafting of dopamine (DA) moieties to the HA-CS gel (HADA-CS) enhanced the scaffold stability and stimulated the gel's remodeling properties by entrapping cell-secreted laminin, and binding brain-derived neurotrophic factor (BDNF). Neurons cultured in the scaffold expressed Col1, Col11, and ITGB4; important for cell adhesion and cell-ECM signaling. Thus, the HA-CS scaffold with integrated chemical cues (DA) supported neuronal growth and network formation. This scaffold offers a valuable tool for tissue engineering and disease modeling and helps in bridging the gap between animal models and human diseases by providing biomimetic neurophysiology. STATEMENT OF SIGNIFICANCE: Developing a brain mimetic 3D scaffold that supports neuronal growth could potentially be useful to study neurobiology, disease pathology, and disease modeling. However, culturing human induced pluripotent stem cells (hiPSC) and human embryonic stem cells (ESCs) derived neurons in a 3D matrix is extremely challenging as neurons are very sensitive cells and require tailored composition, viscoelasticity, and chemical cues. This article identified the key chemical cues necessary for designing neuronal matrix that trap the cell-produced ECM and neurotrophic factors and remodel the matrix and supports neurite outgrowth. The tailored injectable scaffold possesses self-healing/shear-thinning property which is useful to design injectable gels for regenerative medicine and disease modeling that provides biomimetic neurophysiology.

Dynamic covalent crosslinked hyaluronic acid hydrogels and nanomaterials for biomedical applications.

Hyaluronic acid (HA), one of the main components of the extracellular matrix (ECM), is extensively used in the design of hydrogels and nanoparticles for different biomedical applications due to its critical role in vivo, degradability by endogenous enzymes, and absence of immunogenicity. HA-based hydrogels and nanoparticles have been developed by utilizing different crosslinking chemistries. The development of such crosslinking chemistries indicates that even subtle differences in the structure of reactive groups or the procedure of crosslinking may have a profound impact on the intended mechanical, physical and biological outcomes. There are widespread examples of modified HA polymers that can form either covalently or physically crosslinked biomaterials. More recently, studies have been focused on dynamic covalent crosslinked HA-based biomaterials since these types of crosslinking allow the preparation of dynamic structures with the ability to form in situ, be injectable, and have self-healing properties. In this review, HA-based hydrogels and nanomaterials that are crosslinked by dynamic-covalent coupling (DCC) chemistry have been critically assessed.

Interpenetrating gallol functionalized tissue adhesive hyaluronic acid hydrogel polarizes macrophages to an immunosuppressive phenotype.

Innovative scaffold designs that modulate the local inflammatory microenvironment through favorable macrophage polarization and suppressing oxidative stress are needed for successful clinical translation of regenerative cell therapies and graft integration. We herein report derivation of a hydrazone-crosslinked gallol functionalized hyaluronic acid (HA-GA)-based hydrogel that displayed outstanding viscoelastic properties and immunomodulatory characteristics. Grafting of 6% gallol (GA) to a HA-backbone formed an interpenetrative network by promoting an additional crosslink between the gallol groups in addition to hydrazone crosslinking. This significantly enhanced the mechanical stability and displayed shear-thinning/self-healing characteristics, facilitated tissue adhesive properties to porcine tissue and also displayed radical scavenging properties, protecting encapsulated fibroblasts from peroxide challenge. The THP-1 human macrophage cell line or primary bone-marrow-derived murine macrophages cultured within HA-GA gels displayed selective polarization to a predominantly anti-inflammatory phenotype by upregulating IL4ra, IL-10, TGF-ß, and TGF-ßR1 expression when compared with HA-HA gels. Conversely, culturing of pro-inflammatory activated primary murine macrophages in HA-GA gels resulted in a significant reduction of pro-inflammatory TNF-α, IL-1ß, SOCS3 and IL-6 marker expression, and upregulated expression of anti-inflammatory cytokines including TGF-ß. Finally, when the gels were implanted subcutaneously into healthy mice, we observed infiltration of pro-inflammatory myeloid cells in HA-HA gels, while immunosuppressive phenotypes were observed within the HA-GA gels. Taken together these data suggest that HA-GA gels are an ideal injectable scaffold for viable immunotherapeutic interventions. STATEMENT OF SIGNIFICANCE: Host immune response against the implanted scaffolds that are designed to deliver stem cells or therapeutic proteins in vivo significantly limits the functional outcome. For this reason, we have designed immunomodulatory injectable scaffolds that can favorably polarize the recruited macrophages and impart antioxidant properties to suppress oxidative stress. Specifically, we have tailored a hyaluronic acid-based extracellular matrix mimetic injectable scaffold that is grafted with immunomodulatory gallol moiety. Gallol functionalization of hydrogel not only enhanced the mechanical properties of the scaffold by forming an interpenetrating network but also induced antioxidant properties, tissue adhesive properties, and polarized primary murine macrophages to immunosuppressive phenotype. We believe such immunoresponsive implants will pave the way for developing the next-generation of biomaterials for regenerative medicine applications.

Discrepancies on the Role of Oxygen Gradient and Culture Condition on Mesenchymal Stem Cell Fate.

Over the past few years, mesenchymal stem (or stromal) cells (MSCs) have garnered enormous interest due to their therapeutic value especially for their multilineage differentiation potential leading to regenerative medicine applications. MSCs undergo physiological changes upon in vitro expansion resulting in expression of different receptors, thereby inducing high variabilities in therapeutic efficacy. Therefore, understanding the biochemical cues that influence the native local signals on differentiation or proliferation of these cells is very important. There have been several reports that in vitro culture of MSCs in low oxygen gradient (or hypoxic conditions) upregulates the stemness markers and promotes cell proliferation in an undifferentiated state, as hypoxia mimics the conditions the progenitor cells experience within the tissue. However, different studies report different oxygen gradients and culture conditions causing ambiguity in their interpretation of the results. In this progress report, it is aimed to summarize recent studies in the field with specific focus on conflicting results reported during the application of hypoxic conditions for improving the proliferation or differentiation of MSCs. Further, it is tried to decipher the factors that can affect characteristics of MSC under hypoxia and suggest a few techniques that could be combined with hypoxic cell culture to better recapitulate the MSC tissue niche.

Redox responsive Pluronic micelle mediated delivery of functional siRNA: a modular nano-assembly for targeted delivery.

There is an unmet need to develop strategies that allow site-specific delivery of short interfering RNA (siRNA) without any associated toxicity. To address this challenge, we have developed a novel siRNA delivery platform using chemically modified pluronic F108 as an amphiphilic polymer with a releasable bioactive disulfide functionality. The micelles exhibited thermoresponsive properties and showed a hydrodynamic size of ∼291 nm in DLS and ∼200-250 nm in SEM at 37 °C. The grafting of free disulfide pyridyl groups enhanced the transfection efficiency and was successfully demonstrated in human colon carcinoma (HCT116; 88%) and glioma cell lines (U87; 90%), non-cancerous human dermal fibroblast (HDF; 90%) cells as well as in mouse embryonic stem (mES; 54%) cells. To demonstrate the versatility of our modular nanocarrier design, we conjugated the MDGI receptor targeting COOP peptide on the particle surface that allowed the targeted delivery of the cargo molecules to human patent-derived primary BT-13 gliospheres. Transfection experiments with this design resulted in ∼65% silencing of STAT3 mRNA in BT-13 gliospheres, while only ∼20% of gene silencing was observed in the absence of the peptide. We believe that our delivery method solves current problems related to the targeted delivery of RNAi drugs for potential in vivo applications.

Harnessing hyaluronic acid-based nanoparticles for combination therapy: A novel approach for suppressing systemic inflammation and to promote antitumor macrophage polarization.

Anti-inflammatory drugs such as dexamethasone (DEX) are commonly administered to cancer patients along with anticancer drugs, however, the effect of DEX on human cancers is poorly understood. In this article, we have tailored self-assembled nanoparticles derived from hyaluronic acid (HA) wherein, anti-inflammatory DEX was used as a hydrophobic moiety for inducing amphiphilicity. The HA-DEX micelles were subsequently loaded with chemotherapeutic agent, doxorubicin (DOX) (HA-DEX-DOX) and was utilized to deliver drug cargo to human cancer cells expressing different levels of CD44 receptors. We found that DEX suppressed the cytotoxicity of DOX in HCT116, while it synergistically enhanced cytotoxicity in MCF-7 cells. When we tested DOX and HA-DEX-DOX in an ex-vivo human whole blood, we found activation of complement and the coagulation cascade in one group of donors. Encapsulation of DOX within the nanoparticle core eliminated such deleterious side-effects. The HA-DEX-DOX also polarized bone-marrow-derived anti-inflammatory M2 macrophages, to pro-inflammatory M1 phenotype with the upregulation of the cytokines TNF-α, iNOS and IL-1ß.

Hyaluronic Acid-Functionalized Nanomicelles Enhance SAHA Efficacy in 3D Endometrial Cancer Models.

Histone Deacetylase (HDAC) enzymes are upregulated in cancer leading to the development of HDAC inhibiting compounds, several of which are currently in clinical trials. Side effects associated with toxicity and non-specific targeting indicate the need for efficient drug delivery approaches and tumor specific targeting to enhance HDAC efficacy in solid tumor cancers. SAHA encapsulation within F127 micelles functionalized with a surface hyaluronic acid moiety, was developed to target endometrial cancer cells expressing elevated levels of CD44. In vitro viability and morphology analyses was conducted in both 2D and 3D models to assess the translational potential of this approach. Encapsulation enhanced SAHA delivery and activity, demonstrating increased cytotoxic efficacy in 2D and 3D endometrial cancer models. High-content imaging showed improved nanoparticle internalization in 2D and CD44 enhanced penetration in 3D models. In addition, the nano-delivery system enhanced spheroid penetration resulting in cell growth suppression, p21 associated cell cycle arrest, as well as overcoming the formation of an EMT associated phenotype observed in free drug treated type II endometrial cancer cells. This study demonstrates that targeted nanoparticle delivery of SAHA could provide the basis for improving its efficacy in endometrial cancer. Using 3D models for endometrial cancer allows the elucidation of nanoparticle performance and CD44 targeting, likely through penetration and retention within the tumor model.

Streptococcus pneumoniae pneumolysin and neuraminidase A convert high-density lipoproteins into pro-atherogenic particles.

High-density lipoproteins (HDLs) are a group of different subpopulations of sialylated particles that have an essential role in the reverse cholesterol transport (RCT) pathway. Importantly, changes in the protein and lipid composition of HDLs may lead to the formation of particles with reduced atheroprotective properties. Here, we show that Streptococcus pneumoniae pneumolysin (PLY) and neuraminidase A (NanA) impair HDL function by causing chemical and structural modifications of HDLs. The proteomic, lipidomic, cellular, and biochemical analysis revealed that PLY and NanA induce significant changes in sialic acid, protein, and lipid compositions of HDL. The modified HDL particles have reduced cholesterol acceptor potential from activated macrophages, elevated levels of malondialdehyde adducts, and show significantly increased complement activating capacity. These results suggest that accumulation of these modified HDL particles in the arterial intima may present a trigger for complement activation, inflammatory response, and thereby promote atherogenic disease progression.

First Aldol Cross-Linked Hyaluronic Acid Hydrogel: Fast and Hydrolytically Stable Hydrogel with Tissue Adhesive Properties.

Currently, there are limited approaches to tailor 3D scaffolds cross-linked with a stable covalent C-C bond that does not require any catalysts or initiators. We present here the first hydrogels employing aldol condensation chemistry that exhibit exceptional physicochemical properties. We investigated the aldol-cross-linking chemistry using two types of aldehyde-modified hyaluronic acid (HA) derivatives, namely, an enolizable HA-aldehyde (HA-Eal) and a non-enolizable HA-aldehyde (HA-Nal). Hydrogels formed using HA-Eal demonstrate inferior cross-linking efficiency (due to intramolecular loop formation), when compared with hydrogels formed by mixing HA-Eal and HA-NaI leading to a cross-aldol product. The change in mechanical properties as a result of cross-linking at different pH values is determined using rheological measurements and is interpreted in terms of molecular weight between cross-links (Mc). The novel HA cross-aldol hydrogel demonstrate excellent hydrolytic stability and favorable mechanical properties but allow hyaluronidase-mediated enzymatic degradation. Interestingly, residual aldehyde functionality within the aldol product rendered the tissue-adhesive properties by bonding two bone tissues. The aldehyde functionality also facilitated facile post-synthetic modifications with nucleophilic reagents. Finally, we demonstrate that the novel hydrogel is biocompatible with encapsulated stem cells that show a linear rate of expansion in our 3-6 days of study.

Tissue adhesive hyaluronic acid hydrogels for sutureless stem cell delivery and regeneration of corneal epithelium and stroma.

Regeneration of a severely damaged cornea necessitates the delivery of both epithelium-renewing limbal epithelial stem cells (LESCs) and stroma-repairing cells, such as human adipose-derived stem cells (hASCs). Currently, limited strategies exist for the delivery of these therapeutic cells with tissue-like cellular organization. With the added risks related to suturing of corneal implants, there is a pressing need to develop new tissue adhesive biomaterials for corneal regeneration. To address these issues, we grafted dopamine moieties into hydrazone-crosslinked hyaluronic acid (HA-DOPA) hydrogels to impart tissue adhesive properties and facilitate covalent surface modification of the gels with basement membrane proteins or peptides. We achieved tissue-like cellular compartmentalization in the implants by encapsulating hASCs inside the hydrogels, with subsequent conjugation of thiolated collagen IV or laminin peptides and LESC seeding on the hydrogel surface. The encapsulated hASCs in HA-DOPA gels exhibited good proliferation and cell elongation, while the LESCs expressed typical limbal epithelial progenitor markers. Importantly, the compartmentalized HA-DOPA implants displayed excellent tissue adhesion upon implantation in a porcine corneal organ culture model. These results encourage sutureless implantation of functional stem cells as the next generation of corneal regeneration.

Carbon nanotube doped pericardial matrix derived electroconductive biohybrid hydrogel for cardiac tissue engineering.

Cardiovascular diseases represent a major socio-economic burden. In recent years, considerable effort has been invested in optimizing cell delivery strategies to advance cell transplantation therapies to restore heart function for example after an infarct. A particular issue is that the implantation of cells using a non-electroconductive matrix potentially causes arrhythmia. Here, we demonstrate that our hydrazide-functionalized nanotubes-pericardial matrix-derived electroconductive biohybrid hydrogel provides a suitable environment for maturation of human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. hiPSC-derived cardiomyocytes exhibited an improved contraction amplitude (>500%) on conductive hydrogels compared to cells cultured on Matrigel®. This was accompanied by increased cellular alignment, enhanced connexin 43 expression, and improved sarcomere organization suggesting maturation of the hiPSC-derived cardiomyocytes. Sarcomeric length of these cells increased from 1.3 to 1.7 µm. Moreover, 3D cell-laden engineered tissues exhibited enhanced calcium handling as well as positive response to external electrical and pharmaceutical stimulation. Collectively, our data indicate that our biohybrid hydrogels consisting of solubilized nanostructured pericardial matrix and electroconductive positively charged hydrazide-conjugated carbon nanotubes provide a promising material for stem cell-based cardiac tissue engineering.