Injectable Nanoengineered Adhesive Hydrogel for Treating Enterocutaneous Fistulas.

Enterocutaneous fistula (ECF) is a severe medical condition where an abnormal connection forms between the gastrointestinal tract and skin. ECFs are, in most cases, a result of surgical complications such as missed enterotomies or anastomotic leaks. The constant leakage of enteric and fecal contents from the fistula site leads to skin breakdown and increases the risk of infection. Despite advances in surgical techniques and postoperative management, ECF accounts for significant mortality rates, estimated between 15-20%, and causes debilitating morbidity. Therefore, there is a critical need for a simple and effective method to seal and heal ECF. Injectable hydrogels with combined properties of robust mechanical properties and cell infiltration/proliferation have the potential to block and heal ECF. Herein, we report the development of an injectable nanoengineered adhesive hydrogel (INAH) composed of a synthetic nanosilicate (Laponite®) and a gelatin-dopamine conjugate for treating ECF. The hydrogel undergoes fast cross-linking using a co-injection method, resulting in a matrix with improved mechanical and adhesive properties. INAH demonstrates appreciable blood clotting abilities and is cytocompatible with fibroblasts. The adhesive properties of the hydrogel are demonstrated in ex vivo adhesion models with skin and arteries, where the volume stability in the hydrated internal environment facilitates maintaining strong adhesion. In vivo assessments reveal that the INAH is biocompatible, supporting cell infiltration and extracellular matrix deposition while not forming fibrotic tissue. These findings suggest that this INAH holds promising translational potential for sealing and healing ECF.

Aerogel-Based Biomaterials for Biomedical Applications: From Fabrication Methods to Disease-Targeting Applications.

Aerogel-based biomaterials are increasingly being considered for biomedical applications due to their unique properties such as high porosity, hierarchical porous network, and large specific pore surface area. Depending on the pore size of the aerogel, biological effects such as cell adhesion, fluid absorption, oxygen permeability, and metabolite exchange can be altered. Based on the diverse potential of aerogels in biomedical applications, this paper provides a comprehensive review of fabrication processes including sol-gel, aging, drying, and self-assembly along with the materials that can be used to form aerogels. In addition to the technology utilizing aerogel itself, it also provides insight into the applicability of aerogel based on additive manufacturing technology. To this end, how microfluidic-based technologies and 3D printing can be combined with aerogel-based materials for biomedical applications is discussed. Furthermore, previously reported examples of aerogels for regenerative medicine and biomedical applications are thoroughly reviewed. A wide range of applications with aerogels including wound healing, drug delivery, tissue engineering, and diagnostics are demonstrated. Finally, the prospects for aerogel-based biomedical applications are presented. The understanding of the fabrication, modification, and applicability of aerogels through this study is expected to shed light on the biomedical utilization of aerogels.

pH-Responsive doxorubicin delivery using shear-thinning biomaterials for localized melanoma treatment.

Injectable shear-thinning biomaterials (STBs) have attracted significant attention because of their efficient and localized delivery of cells as well as various molecules ranging from growth factors to drugs. Recently, electrostatic interaction-based STBs, including gelatin/LAPONITE® nanocomposites, have been developed through a simple assembly process and show outstanding shear-thinning properties and injectability. However, the ability of different compositions of gelatin and LAPONITE® to modulate doxorubicin (DOX) delivery at different pH values to enhance the effectiveness of topical skin cancer treatment is still unclear. Here, we fabricated injectable STBs using gelatin and LAPONITE® to investigate the influence of LAPONITE®/gelatin ratio on mechanical characteristics, capacity for DOX release in response to different pH values, and cytotoxicity toward malignant melanoma. The release profile analysis of various compositions of DOX-loaded STBs under different pH conditions revealed that lower amounts of LAPONITE® (6NC25) led to higher pH-responsiveness capable of achieving a localized, controlled, and sustained release of DOX in an acidic tumor microenvironment. Moreover, we showed that 6NC25 had a lower storage modulus and required lower injection forces compared to those with higher LAPONITE® ratios. Furthermore, DOX delivery analysis in vitro and in vivo demonstrated that DOX-loaded 6NC25 could efficiently target subcutaneous malignant tumors via DOX-induced cell death and growth restriction.

Photoswitchable Microgels for Dynamic Macrophage Modulation.

Dynamic manipulation of supramolecular self-assembled structures is achieved irreversibly or under non-physiological conditions, thereby limiting their biomedical, environmental, and catalysis applicability. In this study, microgels composed of azobenzene derivatives stacked via π-cation and π-π interactions are developed that are electrostatically stabilized with Arg-Gly-Asp (RGD)-bearing anionic polymers. Lateral swelling of RGD-bearing microgels occurs via cis-azobenzene formation mediated by near-infrared-light-upconverted ultraviolet light, which disrupts intermolecular interactions on the visible-light-absorbing upconversion-nanoparticle-coated materials. Real-time imaging and molecular dynamics simulations demonstrate the deswelling of RGD-bearing microgels via visible-light-mediated trans-azobenzene formation. Near-infrared light can induce in situ swelling of RGD-bearing microgels to increase RGD availability and trigger release of loaded interleukin-4, which facilitates the adhesion structure assembly linked with pro-regenerative polarization of host macrophages. In contrast, visible light can induce deswelling of RGD-bearing microgels to decrease RGD availability that suppresses macrophage adhesion that yields pro-inflammatory polarization. These microgels exhibit high stability and non-toxicity. Versatile use of ligands and protein delivery can offer cytocompatible and photoswitchable manipulability of diverse host cells.

Polypseudorotaxane and polydopamine linkage-based hyaluronic acid hydrogel network with a single syringe injection for sustained drug delivery.

Polypseudorotaxane structure and polydopamine bond-based crosslinked hyaluronic acid (HA) hydrogels including donepezil-loaded microspheres were developed for subcutaneous injection. Both dopamine and polyethylene glycol (PEG) were covalently bonded to the HA polymer for catechol polymerization and inclusion complexation with alpha-cyclodextrin (α-CD), respectively. A PEG chain of HA-dopamine-PEG (HD-PEG) conjugate was threaded with α-CD to make a polypseudorotaxane structure and its pH was adjusted to 8.5 for dopamine polymerization. Poly(lactic-co-glycolic acid) (PLGA)/donepezil microsphere (PDM) was embedded into the HD-PEG network for its sustained release. The HD-PEG/α-CD/PDM 8.5 hydrogel system exhibited an immediate gelation pattern, injectability through single syringe, self-healing ability, and shear-thinning behavior. Donepezil was released from the HD-PEG/α-CD/PDM 8.5 hydrogel in a sustained pattern. Following subcutaneous injection, the weight of excised HD-PEG/α-CD/PDM 8.5 hydrogel was higher than the other groups on day 14. These findings support the clinical feasibility of the HD-PEG/α-CD/PDM 8.5 hydrogel for subcutaneous injection.

Recent developments in mussel-inspired materials for biomedical applications.

Over the decades, researchers have strived to synthesize and modify nature-inspired biomaterials, with the primary aim to address the challenges of designing functional biomaterials for regenerative medicine and tissue engineering. Among these challenges, biocompatibility and cellular interactions have been extensively investigated. Some of the most desirable characteristics for biomaterials in these applications are the loading of bioactive molecules, strong adhesion to moist areas, improvement of cellular adhesion, and self-healing properties. Mussel-inspired biomaterials have received growing interest mainly due to the changes in mechanical and biological functions of the scaffold due to catechol modification. Here, we summarize the chemical and biological principles and the latest advancements in production, as well as the use of mussel-inspired biomaterials. Our main focus is the polydopamine coating, the conjugation of catechol with other polymers, and the biomedical applications that polydopamine moieties are used for, such as matrices for drug delivery, tissue regeneration, and hemostatic control. We also present a critical conclusion and an inspired view on the prospects for the development and application of mussel-inspired materials.

Multi-layered cellulose nanocrystal system for CD44 receptor-positive tumor-targeted anticancer drug delivery.

Layer-by-layer approach based on the electrostatic interactions has been introduced to make multi-layered targeting ligand-chemotherapeutics-cellulose nanocrystal (CNC) structure for tumor-targeted drug delivery. Negatively charged CNC was covered with cationic doxorubicin (DOX) molecule (as a chemotherapeutic agent) to fabricate DOX@CNC and sequentially wrapped with anionic hyaluronic acid (HA) polymer (as a CD44 receptor targeting ligand). Rod-shaped HA-coated DOX@CNC (HA@DOX@CNC) has been successfully fabricated and it exhibited 327 nm length, 12 nm width, -38 mV zeta potential, and 3% DOX content. HA@DOX@CNC displayed higher cellular accumulation efficiency and antiproliferation potentials in CD44 receptor-positive lung adenocarcinoma (A549) cells compared to DOX and DOX-wrapped CNC (DOX@CNC). In A549 spheroid model, HA@DOX@CNC group exhibited superior tumor penetration capability, reactive oxygen species (ROS) production level, and cancer cell killing capacity rather than DOX and DOX@CNC group. In A549 tumor implanted mouse model, Cy5.5-labeled HA@DOX@CNC group exhibited higher tumor accumulation efficiency rather than free Cy5.5 after intravenous injection. All these findings suggest that designed HA@DOX@CNC can be one of promising biocompatible tumor-targeted nano-size drug delivery systems.

Monopotassium phosphate-reinforced in situ forming injectable hyaluronic acid hydrogels for subcutaneous injection.

Monopotassium phosphate and pH modulation-reinforced hydrogel based on hyaluronic acid (HA) grafted with dopamine (dopa) was fabricated as one of subcutaneous injection formulations of donepezil (DPZ). Both incorporation of KH2PO4 and pH adjustment finally attributed to tuning viscoelastic and biodegradable properties of hydrogel system. Appropriate gelation time for in situ gel formation, single syringe injectability, self-healing capability, and viscoelastic features were accomplished with the optimization of KH2PO4 concentration in hydrogel systems. DPZ base (as a poorly water soluble drug) was encapsulated in poly(lactic-co-glycolic acid) (PLGA) microsphere (MS) and it was further embedded in the hydrogel structure for sustained drug release. Biodegradability of designed KH2PO4-incorporated HA-dopa/DPZ MS hydrogel system was assessed by optical imaging and the remained gel weight of crosslinked HA-dopa hydrogel group was 3.4-fold higher than that of unmodified HA-dopa mixture group on day 14 (p < 0.05). Subcutaneous injection of KH2PO4-incorporated HA-dopa/DPZ MS hydrogel did not induce any severe systemic toxicities. All these data suggest that designed HA-dopa/DPZ MS hydrogel structure crosslinked by KH2PO4 incorporation and pH adjustment can be one of promising subcutaneous injection formulations for sustained drug delivery.

Ferrous sulfate-directed dual-cross-linked hyaluronic acid hydrogels with long-term delivery of donepezil.

Ferrous sulfate (FeSO4)-directed dual-cross-linked hydrogels were designed for application in single-syringe injections. The use of FeSO4, rather than other iron salts, can modulate the gelation time and make it available for subcutaneous injection with a single syringe. These hydrogels are based on hyaluronic acid-dopamine (HA-dp) that contain donepezil (DPZ)-entrapping poly(lactic-co-glycolic acid) (PLGA) microsphere (MS). Although DPZ has been administered orally, its sustained release formulation via subcutaneous injection may reduce the dosing frequency for patients with Alzheimer's disease. The HA-dp conjugate was synthesized via an amide bond reaction for coordination of dp with a metal ion (Fe2+ or Fe3+) and self-polymerization of dp. The HA-dp/DPZ-loaded PLGA MS (PD MS)/FeSO4 gel system was considerably hardened via both the coordination of the metal ion with HA-dp and covalent bonding of dp. In addition, a quick restoration of the collapsed gel structure and sustained DPZ release from the HA-dp/PD MS/FeSO4 structure were achieved. The pharmacokinetic parameters after its subcutaneous injection in a rat indicate the sustained release and absorption of DPZ from the HA-dp/PD MS/FeSO4 system. The proposed system can be prepared by a simple method and can be efficiently and safely used for the long-term delivery of DPZ after the subcutaneous injection.

Non-transdermal microneedles for advanced drug delivery.

Microneedles (MNs) have been used to deliver drugs for over two decades. These platforms have been proven to increase transdermal drug delivery efficiency dramatically by penetrating restrictive tissue barriers in a minimally invasive manner. While much of the early development of MNs focused on transdermal drug delivery, this technology can be applied to a variety of other non-transdermal biomedical applications. Several variations, such as multi-layer or hollow MNs, have been developed to cater to the needs of specific applications. The heterogeneity in the design of MNs has demanded similar variety in their fabrication methods; the most common methods include micromolding and drawing lithography. Numerous materials have been explored for MN fabrication which range from biocompatible ceramics and metals to natural and synthetic biodegradable polymers. Recent advances in MN engineering have diversified MNs to include unique shapes, materials, and mechanical properties that can be tailored for organ-specific applications. In this review, we discuss the design and creation of modern MNs that aim to surpass the biological barriers of non-transdermal drug delivery in ocular, vascular, oral, and mucosal tissue.

Combinatorial screening of biochemical and physical signals for phenotypic regulation of stem cell-based cartilage tissue engineering.

Despite great progress in biomaterial design strategies for replacing damaged articular cartilage, prevention of stem cell-derived chondrocyte hypertrophy and resulting inferior tissue formation is still a critical challenge. Here, by using engineered biomaterials and a high-throughput system for screening of combinatorial cues in cartilage microenvironments, we demonstrate that biomaterial cross-linking density that regulates matrix degradation and stiffness-together with defined presentation of growth factors, mechanical stimulation, and arginine-glycine-aspartic acid (RGD) peptides-can guide human mesenchymal stem cell (hMSC) differentiation into articular or hypertrophic cartilage phenotypes. Faster-degrading, soft matrices promoted articular cartilage tissue formation of hMSCs by inducing their proliferation and maturation, while slower-degrading, stiff matrices promoted cells to differentiate into hypertrophic chondrocytes through Yes-associated protein (YAP)-dependent mechanotransduction. in vitro and in vivo chondrogenesis studies also suggest that down-regulation of the Wingless and INT-1 (WNT) signaling pathway is required for better quality articular cartilage-like tissue production.

Synthesis of Injectable Shear-Thinning Biomaterials of Various Compositions of Gelatin and Synthetic Silicate Nanoplatelet.

Injectable shear-thinning biomaterials (iSTBs) have great potential for in situ tissue regeneration through minimally invasive therapeutics. Previously, an iSTB was developed by combining gelatin with synthetic silicate nanoplatelets (SNPs) for potential application to hemostasis and endovascular embolization. Hence, iSTBs are synthesized by varying compositions of gelatin and SNPs to navigate their material, mechanical, rheological, and bioactive properties. All compositions (each component percentage; 1.5-4.5%/total solid ranges; 3-9%) tested are injectable through both 5 Fr general catheter and 2.4 Fr microcatheter by manual pressure. In the results, an increase in gelatin contents causes decrease in swellability, increase in freeze-dried hydrogel scaffold porosity, increase in degradability and injection force during iSTB fabrication. Meanwhile, the amount of SNPs in composite hydrogels is mainly required to decrease degradability and increase shear thinning properties of iSTB. Finally, in vitro and in vivo biocompatibility tests show that the 1.5-4.5% range gelatin-SNP iSTBs are not toxic to the cells and animals. All results demonstrate that the iSTB can be modulated with specific properties for unmet clinical needs. Understanding of mechanical and biological consequences of the changing gelatin-SNP ratios through this study will shed light on the biomedical applications of iSTB on specific diseases.

Minimally Invasive and Regenerative Therapeutics.

Advances in biomaterial synthesis and fabrication, stem cell biology, bioimaging, microsurgery procedures, and microscale technologies have made minimally invasive therapeutics a viable tool in regenerative medicine. Therapeutics, herein defined as cells, biomaterials, biomolecules, and their combinations, can be delivered in a minimally invasive way to regenerate different tissues in the body, such as bone, cartilage, pancreas, cardiac, skeletal muscle, liver, skin, and neural tissues. Sophisticated methods of tracking, sensing, and stimulation of therapeutics in vivo using nano-biomaterials and soft bioelectronic devices provide great opportunities to further develop minimally invasive and regenerative therapeutics (MIRET). In general, minimally invasive delivery methods offer high yield with low risk of complications and reduced costs compared to conventional delivery methods. Here, minimally invasive approaches for delivering regenerative therapeutics into the body are reviewed. The use of MIRET to treat different tissues and organs is described. Although some clinical trials have been performed using MIRET, it is hoped that such therapeutics find wider applications to treat patients. Finally, some future perspective and challenges for this emerging field are highlighted.

Synthetic Biomaterials to Rival Nature's Complexity-a Path Forward with Combinatorics, High-Throughput Discovery, and High-Content Analysis.

Cells in tissue receive a host of soluble and insoluble signals in a context-dependent fashion, where integration of these cues through a complex network of signal transduction cascades will define a particular outcome. Biomaterials scientists and engineers are tasked with designing materials that can at least partially recreate this complex signaling milieu towards new materials for biomedical applications. In this progress report, recent advances in high throughput techniques and high content imaging approaches that are facilitating the discovery of efficacious biomaterials are described. From microarrays of synthetic polymers, peptides and full-length proteins, to designer cell culture systems that present multiple biophysical and biochemical cues in tandem, it is discussed how the integration of combinatorics with high content imaging and analysis is essential to extracting biologically meaningful information from large scale cellular screens to inform the design of next generation biomaterials.

Matrix directed adipogenesis and neurogenesis of mesenchymal stem cells derived from adipose tissue and bone marrow.

UNLABELLED: Mesenchymal stem cells (MSCs) can differentiate into multiple lineages through guidance from the biophysical and biochemical properties of the extracellular matrix. In this work we conduct a combinatorial study of matrix properties that influence adipogenesis and neurogenesis including: adhesion proteins, stiffness, and cell geometry, for mesenchymal stem cells derived from adipose tissue (AT-MSCs) and bone marrow (BM-MSCs). We uncover distinct differences in integrin expression, the magnitude of traction stress, and lineage specification to adipocytes and neuron-like cells between cell sources. In the absence of media supplements, adipogenesis in AT-MSCs is not significantly influenced by matrix properties, while the converse is true in BM-MSCs. Both cell types show changes in the expression of neurogenesis markers as matrix cues are varied. When cultured on laminin conjugated microislands of the same adhesive area, BM-MSCs display elevated adipogenesis markers, while AT-MSCs display elevated neurogenesis markers; integrin analysis suggests neurogenesis in AT-MSCs is guided by adhesion through integrin αvß3. Overall, the properties of the extracellular matrix guides MSC adhesion and lineage specification to different degrees and outcomes, in spite of their similarities in general characteristics. This work will help guide the selection of MSCs and matrix components for applications where high fidelity of differentiation outcome is desired. STATEMENT OF SIGNIFICANCE: Mesenchymal stem cells (MSCs) are an attractive cell type for stem cell therapies; however, in order for these cells to be useful in medicine, we need to understand how they respond to the physical and chemical environments of tissue. Here, we explore how two promising sources of MSCs-those derived from bone marrow and from adipose tissue-respond to the compliance and composition of tissue using model extracellular matrices. Our results demonstrate a source-specific propensity to undergo adipogenesis and neurogenesis, and uncover a role for adhesion, and the degree of traction force exerted on the substrate in guiding these lineage outcomes.

Temporal Modulation of Stem Cell Activity Using Magnetoactive Hydrogels.

Cell activity is coordinated by dynamic interactions with the extracellular matrix, often through stimuli-mediated spatiotemporal stiffening and softening. Dynamic changes in mechanics occur in vivo through enzymatic or chemical means, processes which are challenging to reconstruct in cell culture materials. Here a magnetoactive hydrogel material formed by embedding magnetic particles in a hydrogel matrix is presented whereby elasticity can be modulated reversibly by attenuation of a magnetic field. Orders of magnitude change in elasticity using low magnetic fields are shown and reversibility of stiffening with simple permanent magnets is demonstrated. The broad applicability of this technique is demonstrated with two therapeutically relevant bioactivities in mesenchymal stem cells: secretion of proangiogenic molecules, and dynamic control of osteogenesis. The ability to reversibly stiffen cell culture materials across the full spectrum of soft tissue mechanics, using simple materials and commercially available permanent magnets, makes this approach viable for a broad range of laboratory environments.

Cell shape and the presentation of adhesion ligands guide smooth muscle myogenesis.

The reliable generation of smooth muscle cells is important for a number of tissue engineering applications. Human mesenchymal stem cells (MSCs) are a promising progenitor of smooth muscle, with high expression of smooth muscle markers observed in a fraction of isolated cells, which can be increased by introduction of soluble supplements that direct differentiation. Here we demonstrate a new micropatterning technique, where peptides of different ligand affinity can be microcontact printed onto an inert background, to explore MSC differentiation to smooth muscle through controlled biochemical and biophysical cues alone. Using copper-catalyzed alkyne-azide cycloaddition (CuAAC), we patterned our surfaces with RGD peptide ligands-both a linear peptide with low integrin affinity and a cyclic version with high integrin affinity-for the culture of MSCs in shapes with various aspect ratios. At low aspect ratio, ligand affinity is a prime determinant for smooth muscle differentiation, while at high aspect ratio, ligand affinity has less of an effect. Pathway analysis reveals a role for focal adhesion turnover, Rac1, RhoA/ROCK, and calpain during smooth muscle differentiation of MSCs in response to cell shape and the affinity of the cell adhesion interface. Controlling integrin-ligand affinity at the biomaterials interface is important for mediating adhesion but may also prove useful for directing smooth muscle myogenesis. Peptide patterning enables the systematic investigation of single to multiple peptides derived from any protein, at different densities across a biomaterials surface, which has the potential to direct multiple MSC differentiation outcomes without the need for soluble supplements.

Geometric guidance of integrin mediated traction stress during stem cell differentiation.

Cells sense and transduce the chemical and mechanical properties of their microenvironment through cell surface integrin receptors. Traction stress exerted by cells on the extracellular matrix mediates focal adhesion stabilization and regulation of the cytoskeleton for directing biological activity. Understanding how stem cells integrate biomaterials properties through focal adhesions during differentiation is important for the design of soft materials for regenerative medicine. In this paper we use micropatterned hydrogels containing fluorescent beads to explore force transmission through integrins from single mesenchymal stem cells (MSCs) during differentiation. When cultured on polyacrylamide gels, MSCs will express markers associated with osteogenesis and myogenesis in a stiffness dependent manner. The shape of single cells and the composition of tethered matrix protein both influence the magnitude of traction stress applied and the resultant differentiation outcome. We show how geometry guides the spatial positioning of focal adhesions to maximize interaction with the matrix, and uncover a relationship between αvß3, α5ß1 and mechanochemical regulation of osteogenesis.

Controlling cell geometry on substrates of variable stiffness can tune the degree of osteogenesis in human mesenchymal stem cells.

The physical properties of the extracellular matrix (ECM) play an important role in regulating tissue-specific human mesenchymal stem cell (MSC) differentiation. Protein-coated hydrogels with tunable stiffness have been shown to influence lineage specific gene expression in MSCs. In addition, the control of cell shape - either through changing substrate stiffness or restricting spreading with micropatterning - has proved to be important in guiding the differentiation of MSCs. However, few studies have explored the interplay between these physical cues during MSC lineage specification. Here, we demonstrate geometric control of osteogenesis in MSCs cultured on micropatterned polyacrylamide gels. Cells cultured on fibronectin-coated gels express markers associated with osteogenesis in a stiffness dependent fashion with a maximum at ~30kPa. Controlling the geometry of single cells across the substrate demonstrates elevated osteogenesis when cells are confined to shapes that promote increased cytoskeletal tension. Patterning MSCs across hydrogels of variable stiffness will enable the exploration of the interplay between these physical cues and their relationship with the mechanochemical signals that guide stem cell fate decisions.

Directing stem cell fate on hydrogel substrates by controlling cell geometry, matrix mechanics and adhesion ligand composition.

There is a dynamic relationship between physical and biochemical signals presented in the stem cell microenvironment to guide cell fate determination. Model systems that modulate cell geometry, substrate stiffness or matrix composition have proved useful in exploring how these signals influence stem cell fate. However, the interplay between these physical and biochemical cues during differentiation remains unclear. Here, we demonstrate a microengineering strategy to vary single cell geometry and the composition of adhesion ligands - on substrates that approximate the mechanical properties of soft tissues - to study adipogenesis and neurogenesis in adherent mesenchymal stem cells. Cells cultured in small circular islands show elevated expression of adipogenesis markers while cells that spread in anisotropic geometries tend to express elevated neurogenic markers. Arraying different combinations of matrix protein in a myriad of 2D and pseudo-3D geometries reveals optimal microenvironments for controlling the differentiation of stem cells to these "soft" lineages without the use of media supplements.