Search

Bone Morphogenic Protein 2 (BMP2) can induce ectopic bone. This ability, which first motivated the widespread application of BMP2 in fracture healing and spinal arthrodesis has, more recently, been indicated as one of several serious adverse effects associated with the supra-physiological doses of BMP2 relied upon for clinical efficacy. Key to harnessing BMPs and other agents safely and effectively will be the ability to localize activity at a target site at substantially reduced doses. Clay (Laponite) nanoparticles can self assemble into gels under physiological conditions and bind growth factors for enhanced and localized efficacy. Here we show the ability to localize and enhance the activity of BMP2 to achieve ectopic bone formation at doses within the sub-microgram per ml range of concentrations sufficient to induce differentiation of responsive cell populations in vitro and at approximately 3000 fold lower than those employed in clinical practice. [ABSTRACT FROM AUTHOR]

Craniosynostosis is the premature fusion of cranial sutures, which can result in progressive cranial deformations, increased intracranial pressure, and restricted brain growth. Most cases of craniosynostosis require surgical reconstruction of the cranial vault with the goal of increasing the intracranial volume and correcting the craniofacial deformities. However, patients often experience rapid post-operative bone regrowth, known as re-synostosis, which necessitates additional surgical intervention. Bone morphogenetic protein (BMP) inhibitors have tremendous potential to treat re-synostosis, but the realization of a clinically viable inhibitor-based therapeutic requires the development of a delivery vehicle that can localize the release to the site of administration. Here, we present an in situ rapidly crosslinking injectable hydrogel that has the properties necessary to encapsulate co-administered proteins and demonstrate that the delivery of rmGremlin1 via our hydrogel system delays bone regrowth in a weanling mouse model of re-synostosis. Our hydrogel is composed of two mutually reactive poly(ethylene glycol) macromolecules, which when mixed crosslink via a bio-orthogonal Cu free click reaction. Hydrogels containing Gremlin caused a dose dependent inhibition of bone regrowth. In addition to craniofacial applications, our injectable click hydrogel has the potential to provide customizable protein, small molecule, and cell delivery to any site accessible via needle or catheter. [ABSTRACT FROM AUTHOR]

The development of pancreatic ductal adenocarcinoma (PDAC) is heavily influenced by local stromal tissues, or desmoplasia. Biomimetic hydrogels capable of mimicking tumor niches are particularly useful for discovering the role of independent matrix cues on cancer cell development. Here, we report a photo-curable and bio-orthogonal thiol-ene (i.e., cross-linked by mutually reactive norbornene and thiol groups via photoinitiation) hydrogel platform for studying the growth, morphogenesis, drug resistance, and cancer stem cell marker expression in PDAC cells cultured in 3D. The hydrogels were prepared from multi-arm poly(ethylene glycol)-norbornene cross-linked with protease-sensitive peptide to permit cell-mediated matrix remodeling. Collagen 1 fibrils were incorporated into the covalent network while cytokines (e.g., EGF and TGF-β1) were supplemented in the culture media for controlling cell fate. We found that the presence of collagen 1 enhanced cell proliferation and Yes-associated protein (YAP) translocation to cell nuclei. Cytokines and collagen 1 synergistically up-regulated MT1-MMP expression and induced cell spreading, suggestive of epithelial-mesenchymal transition (EMT) in the encapsulated cells. Furthermore, PDAC cells cultured in 3D developed chemo-resistance even in the absence of collagen 1 and cytokines. This phenotype is likely a consequence of the enrichment of pancreatic cancer stem cells that expressed high levels of CD24, sonic hedgehog (SHH), and vascular endothelial growth factor (VEGF). [ABSTRACT FROM AUTHOR]

Despite the recent expansion of peptide drugs, delivery remains a challenge due to poor localization and rapid clearance. Therefore, a hydrogel-based platform technology was developed to control and sustain peptide drug release via matrix metalloproteinase (MMP) activity. Specifically, hydrogels were composed of poly(ethylene glycol) and peptide drugs flanked by MMP substrates and terminal cysteine residues as crosslinkers. First, peptide drug bioactivity was investigated in expected released forms (e.g., with MMP substrate residues) in vitro prior to incorporation into hydrogels. Three peptides (Qk (from Vascular Endothelial Growth Factor), SPARC 113 , and SPARC 118 (from Secreted Protein Acidic and Rich in Cysteine)) retained bioactivity and were used as hydrogel crosslinkers in full MMP degradable forms. Upon treatment with MMP2, hydrogels containing Qk, SPARC 113 , and SPARC 118 degraded in 6.7, 6, and 1 days, and released 5, 8, and, 19% of peptide, respectively. Further investigation revealed peptide drug size controlled hydrogel swelling and degradation rate, while hydrophobicity impacted peptide release. Additionally, Qk, SPARC 113 , and SPARC 118 releasing hydrogels increased endothelial cell tube formation 3.1, 1.7, and 2.8-fold, respectively. While pro-angiogenic peptides were the focus of this study, the design parameters detailed allow for adaptation of hydrogels to control peptide release for a variety of therapeutic applications. [ABSTRACT FROM AUTHOR]

Appropriate mechanical properties and highly interconnected porosity are important properties for tissue engineering scaffolds. However, most existing hydrogel scaffolds suffer from poor mechanical properties limiting their application. Furthermore, it is relatively infrequent that precision control is achieved over pore size and structure of the scaffold because there are relatively few current technologies that allow such control and there is not a general appreciation that such control is important. To address these shortcomings, by combining double network polymerization and sphere-templating fabrication techniques, we developed a tough, intelligent scaffold based on poly(acrylic acid) and poly(N-isopropyl acrylamide) with a controllable, uniform, and interconnected porous structure. A mechanical assessment showed the toughness of the hydrogel and scaffold to be up to ~1.4 × 107 Jm-3 and ~1.5 × 106 Jm-3 respectively, as compared with 104-105 Jm-3 for most synthetic hydrogels. The thermosensitivity and pH-sensitivity were explored in a swelling study. In vitro testing demonstrated the scaffold matrices supported NIH-3T3 cell adhesion, proliferation and infiltration. An in vivorabbit study showed the scaffolds promote strong cellular integration by allowing cells to migrate into the porous structure from the surrounding tissues. These data suggest that the poly(acrylic acid)/poly(N-isopropyl acrylamide)-based scaffold could be an attractive candidate for tissue engineering. [ABSTRACT FROM AUTHOR]

Development of a xenogeneic biological liver support is important in providing a bridge to transplantation or liver regeneration, thus helping to overcome the chronic shortage of liver donors. Among the critical factors in developing biological liver support are the creation of in vivo mimetic micro liver tissue (mLT), especially mLTs containing liver-specific ultrastructure, and an encapsulation method that can package massive numbers of cells while providing immune-protection from the host immune system. We describe here the development of mLTs that include liver microarchitecture and their in situ encapsulation in hydrogel composites. Concave microwells and the tri-culture of three types of primary liver cells were applied for the construction of mLTs showing excellent liver functions and long-term (>1 month) viability in vitro. Large quantities of rat mLTs were encapsulated in collagen-alginate composites, implanted into hepatic failure mice and sustained their survival during regeneration of the remaining liver. The proposed liver support system offers xenogeneic hepatic assistance by mimicking native liver microarchitecture and providing immune-protection without the need for complicated devices or processes, and as such represents a promising system for recovery of organ function. [ABSTRACT FROM AUTHOR]

The present work investigated the use of biodegradable hydrogel composite scaffolds, based on the macromer oligo(poly(ethylene glycol) fumarate) (OPF), to deliver growth factors for the repair of osteochondral tissue in a rabbit model. In particular, bilayered OPF composites were used to mimic the structural layers of the osteochondral unit, and insulin-like growth factor-1 (IGF-1) and bone morphogenetic protein-2 (BMP-2) were loaded into gelatin microparticles and embedded within the OPF hydrogel matrix in a spatially controlled manner. Three different scaffold formulations were implanted in a medial femoral condyle osteochondral defect: 1) IGF-1 in the chondral layer, 2) BMP-2 in the subchondral layer, and 3) IGF-1 and BMP-2 in their respective separate layers. The quantity and quality of osteochondral repair was evaluated at 6 and 12 weeks with histological scoring and micro-computed tomography (micro-CT). While histological scoring results at 6 weeks showed no differences between experimental groups, micro-CT analysis revealed that the delivery of BMP-2 alone increased the number of bony trabecular islets formed, an indication of early bone formation, over that of IGF-1 delivery alone. At 12 weeks post-implantation, minimal differences were detected between the three groups for cartilage repair. However, the dual delivery of IGF-1 and BMP-2 had a higher proportion of subchondral bone repair, greater bone growth at the defect margins, and lower bone specific surface than the single delivery of IGF-1. These results suggest that the delivery of BMP-2 enhances subchondral bone formation and that, while the dual delivery of IGF-1 and BMP-2 in separate layers does not improve cartilage repair under the conditions studied, they may synergistically enhance the degree of subchondral bone formation. Overall, bilayered OPF hydrogel composites demonstrate potential as spatially-guided, multiple growth factor release vehicles for osteochondral tissue repair. [ABSTRACT FROM AUTHOR]

Sodium alginate hydrogel, stabilized with gelatin, is a suitable, biologically inert matrix that can be used for encapsulating and 3D bioprinting of bone-related SaOS-2 cells. However, the cells, embedded in this matrix, remain in a non-proliferating state. Here we show that addition of an overlay onto the bioprinted alginate/gelatine/SaOS-2 cell scaffold, consisting of agarose and the calcium salt of polyphosphate [polyP·Ca 2+ -complex], resulted in a marked increase in cell proliferation. In the presence of 100 µ m polyP·Ca 2+ -complex, the cells proliferate with a generation time of approximately 47-55 h. In addition, the hardness of the alginate/gelatin hydrogel substantially increases in the presence of the polymer. The reduced Young's modulus for the alginate/gelatin hydrogel is approximately 13-14 kPa, and this value drops to approximately 0.5 kPa after incubation of the cell containing scaffolds for 5 d. In the presence of 100 µ m polyP·Ca 2+ -complex, the reduced Young's modulus increases to about 22 kPa. The hardness of the polyP·Ca 2+ -complex containing hydrogel remains essentially constant if cells are absent in the matrix, but it drops to 3.2 kPa after a 5 d incubation period in the presence of SaOS-2 cells, indicating that polyP·Ca 2+ -complex becomes metabolized, degraded, by the cells. The alginate/gelatine-agarose system with polyP·Ca 2+ -complex cause a significant increase in the mineralization of the cells. SEM analyses revealed that the morphology of the mineral nodules formed on the surface of the cells embedded in the alginate/gelatin hydrogel do not significantly differ from the nodules on cells growing in monolayer cultures. The newly developed technique, using cells encapsulated into an alginate/gelatin hydrogel and a secondary layer containing the morphogenetically active, growth promoting polymer polyP·Ca 2+ -complex opens new possibilities for the application of 3D bioprinting in bone tissue engineering. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM), the most prevalent primary brain cancer, is characterized by diffuse infiltration of tumor cells into brain tissue, which severely complicates surgical resection and contributes to tumor recurrence. The most rapid mode of tissue infiltration occurs along blood vessels or white matter tracts, which represent topological interfaces thought to serve as "tracks" that speed cell migration. Despite this observation, the field lacks experimental paradigms that capture key features of these tissue interfaces and allow reductionist dissection of mechanisms of this interfacial motility. To address this need, we developed a culture system in which tumor cells are sandwiched between a fibronectin-coated ventral surface representing vascular basement membrane and a dorsal hyaluronic acid (HA) surface representing brain parenchyma. We find that inclusion of the dorsal HA surface induces formation of adhesive complexes and significantly slows cell migration relative to a free fibronectin-coated surface. This retardation is amplified by inclusion of integrin binding peptides in the dorsal layer and expression of CD44, suggesting that the dorsal surface slows migration through biochemically specific mechanisms rather than simple steric hindrance. Moreover, both the reduction in migration speed and assembly of dorsal adhesions depend on myosin activation and the stiffness of the ventral layer, implying that mechanochemical feedback directed by the ventral layer can influence adhesive signaling at the dorsal surface. [ABSTRACT FROM AUTHOR]

By providing a three-dimensional (3D) support to cells, hydrogels offer a more relevant in vivo tissue-like environment as compared to two-dimensional cell cultures. Hydrogels can be applied as screening platforms to investigate in 3D the role of biochemical and biophysical cues on cell behaviour using bioluminescent reporter cells. Gradients in oxygen concentration that result from the interplay between molecular transport and cell metabolism can however cause substantial variability in the observed bioluminescent reporter cell activity. To assess the influence of these oxygen gradients on the emitted bioluminescence for various hydrogel geometries, a combined experimental and modelling approach was implemented. We show that the applied model is able to predict oxygen gradient independent bioluminescent intensities which correlate better to the experimentally determined viable cell numbers, as compared to the experimentally measured bioluminescent intensities. By analysis of the bioluminescence reaction dynamics we obtained a quantitative description of cellular oxygen metabolism within the hydrogel, which was validated by direct measurements of oxygen concentration within the hydrogel. Bioluminescence peak intensities can therefore be used as a quantitative measurement of reporter cell activity within a hydrogel, but an unambiguous interpretation of these intensities requires a compensation for the influence of cell-induced oxygen gradients on the luciferase activity. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we investigated the in vitro and in vivo properties and performance of a celecoxib-loaded hydrogel based on a fully acetyl-capped PCLA-PEG-PCLA triblock copolymer. Blends of different compositions of celocoxib, a drug used for pain management in osteoarthritis, and the acetyl-capped PCLA-PEG-PCLA triblock copolymer were mixed with buffer to yield temperature-responsive gelling systems. These systems containing up to 50 mg celecoxib/g gel, were sols at room temperature and converted into immobile gels at 37 °C. In vitro, release of celecoxib started after a ∼10-day lag phase followed by a sustained release of ∼90 days. The release was proven to be mediated by polymer dissolution from the gels. In vivo (subcutaneous injection in rats) experiments showed an initial celecoxib release of ∼30% during the first 3 days followed by a sustained release of celecoxib for 4–8 weeks. The absence of a lag phase and the faster release seen in vivo were likely due to the enhanced celecoxib solubility in biological fluids and active degradation of the gel by macrophages. Finally, intra-articular biocompatibility of the 50 mg/g celecoxib-loaded gel was demonstrated using μCT-scanning and histology, where no cartilage or bone changes were observed following injection into the knee joints of healthy rats. In conclusion, this study shows that celecoxib-loaded acetyl-capped PCLA-PEG-PCLA hydrogels form a safe drug delivery platform for sustained intra-articular release. [Copyright &y& Elsevier]

Abstract: Cancer stem-like cells (CSCs) are rare subpopulations of cancer cells that are reported to be responsible for cancer resistance and metastasis associated with conventional cancer therapies. Therefore, effective enrichment/culture of CSCs is of importance to both the understanding and treatment of cancer. However, it usually takes approximately 10 days for the widely used conventional approach to enrich CSCs through the formation of CSC-containing aggregates. Here we report the time can be shortened to 2 days while obtaining prostate CSC-containing aggregates with better quality based on the expression of surface receptor markers, dye exclusion, gene and protein expression, and in vivo tumorigenicity. This is achieved by encapsulating and culturing human prostate cancer cells in the miniaturized 3D liquid core of microcapsules with an alginate hydrogel shell. The miniaturized 3D culture in core–shell microcapsules is an effective strategy for enriching/culturing CSCs in vitro to facilitate cancer research and therapy development. [Copyright &y& Elsevier]

Abstract: Hydrogels, microgels and nanogels have emerged as versatile and viable platforms for sustained protein release, targeted drug delivery, and tissue engineering due to excellent biocompatibility, a microporous structure with tunable porosity and pore size, and dimensions spanning from human organs, cells to viruses. In the past decade, remarkable advances in hydrogels, microgels and nanogels have been achieved with click chemistry. It is a most promising strategy to prepare gels with varying dimensions owing to its high reactivity, superb selectivity, and mild reaction conditions. In particular, the recent development of copper-free click chemistry such as strain-promoted azide-alkyne cycloaddition, radical mediated thiol-ene chemistry, Diels–Alder reaction, tetrazole-alkene photo-click chemistry, and oxime reaction renders it possible to form hydrogels, microgels and nanogels without the use of potentially toxic catalysts or immunogenic enzymes that are commonly required. Notably, unlike other chemical approaches, click chemistry owing to its unique bioorthogonal feature does not interfere with encapsulated bioactives such as living cells, proteins and drugs and furthermore allows versatile preparation of micropatterned biomimetic hydrogels, functional microgels and nanogels. In this review, recent exciting developments in click hydrogels, microgels and nanogels, as well as their biomedical applications such as controlled protein and drug release, tissue engineering, and regenerative medicine are presented and discussed. [Copyright &y& Elsevier]

Abstract: Metastatic melanoma is highly resistant to drug treatment, and the underlying mechanisms of this resistance remain unclear. Increased tissue stiffness is correlated with tumor progression, but whether increased tissue stiffness contributes to treatment resistance in melanoma is not known. To investigate the effect of substrate stiffness on melanoma cell treatment responsiveness, PEG hydrogels were utilized as a cell culture system to precisely vary matrix elasticity and investigate melanoma cell responses to a commercially available pharmacological inhibitor (PLX4032). The tensile moduli were varied between 0.6 and 13.1 kPa (E) and the effects of PLX4032 on metabolic activity, apoptosis, and proliferation were evaluated on human cell lines derived from radial growth phase (WM35) and metastatic melanoma (A375). The A375 cells were found to be stiffness-independent; matrix elasticity did not alter cell morphology or apoptosis with PLX4032 treatment. The WM35 cells, however, were more dependent on substrate modulus, displaying increased apoptosis and smaller focal adhesions on compliant substrates. Culturing melanoma cells on PEG hydrogels revealed stage-dependent responses to PLX4032 that would have otherwise been masked if cultured strictly on TCPS. These findings demonstrate the utility of PEG hydrogels as a versatile in vitro culture platform with which to investigate the molecular mechanisms of melanoma biology and treatment responsiveness. [Copyright &y& Elsevier]

Abstract: Three-dimensional (3D) tissue-engineered tumor models have the potential to bridge the gap between monolayer cultures and patient-derived xenografts for the testing of nanoparticle (NP)-based cancer therapeutics. In this study, a hydrogel-derived prostate cancer (PCa) model was developed for the in vitro evaluation of doxorubicin (Dox)-loaded polymer NPs (Dox-NPs). The hydrogels were synthesized using chemically modified hyaluronic acid (HA) carrying acrylate groups (HA-AC) or reactive thiols (HA-SH). The crosslinked hydrogel networks exhibited an estimated pore size of 70–100 nm, similar to the spacing of the extracellular matrices (ECM) surrounding tumor tissues. LNCaP PCa cells entrapped in the HA matrices formed distinct tumor-like multicellular aggregates with an average diameter of 50 μm after 7 days of culture. Compared to cells grown on two-dimensional (2D) tissue culture plates, cells from the engineered tumoroids expressed significantly higher levels of multidrug resistance (MDR) proteins, including multidrug resistance protein 1 (MRP1) and lung resistance-related protein (LRP), both at the mRNA and the protein levels. Separately, Dox-NPs with an average diameter of 54 ± 1 nm were prepared from amphiphilic block copolymers based on poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) bearing pendant cyclic ketals. Dox-NPs were able to diffuse through the hydrogel matrices, penetrate into the tumoroid and be internalized by LNCaP PCa cells through caveolae-mediated endocytosis and macropinocytosis pathways. Compared to 2D cultures, LNCaP PCa cells cultured as multicellular aggregates in HA hydrogel were more resistant to Dox and Dox-NPs treatments. Moreover, the NP-based Dox formulation could bypass the drug efflux function of MRP1, thereby partially reversing the resistance to free Dox in 3D cultures. Overall, the engineered tumor model has the potential to provide predictable results on the efficacy of NP-based cancer therapeutics. [Copyright &y& Elsevier]

Abstract: Hydrogels have been widely used for 3-dimensional (3D) cell culture and tissue regeneration due to their tunable biochemical and physicochemical properties as well as their high water content, which resembles the aqueous microenvironment of the natural extracellular matrix. While many properties of natural hydrogel matrices are modifiable, their intrinsic isotropic structure limits the control over cellular organization, which is critical to restore tissue function. Here we report a generic approach to incorporate alignment topography inside the hydrogel matrix using a combination of electrical and mechanical stretching. Hydrogel fibres with uniaxial alignment were prepared from aqueous solutions of natural polymers such as alginate, fibrin, gelatin, and hyaluronic acid under ambient conditions. The unique internal alignment feature drastically enhances the mechanical properties of the hydrogel microfibres. Furthermore, the facile, organic solvent-free processing conditions are amenable to the incorporation of live cells within the hydrogel fibre or on the fibre surface; both approaches effectively induce cellular alignment. This work demonstrates a versatile and scalable strategy to create aligned hydrogel microfibres from various natural polymers. [Copyright &y& Elsevier]

Abstract: Regeneration of chronic myocardial infarction (CMI) is one of the challenging issues due to its limited regeneration activity compared to acute or sub-acute stage. In this study, we examined whether combination of stem cell homing factor (SDF-1) and angiogenic peptides (Ac-SDKP) injected with biomimetic hydrogels promote regeneration of cardiac function in a CMI model. We evaluated the regeneration of chronically infarcted myocardium using injectable biomimetic hydrogels containing two therapeutic factors; stromal-derived factor-1 (SDF-1) and Ac-SDKP for stem cell homing and angiogenesis, respectively. Injection of the two therapeutic factors into the infarct region of the left ventricle showed that the biomimetic hydrogels containing two therapeutic factor exhibited significantly improved left ventricle function, increased angiogenesis, decreased infarct size and greatest wall thickness within the infarct region at 4 weeks post-treatment. From these results, it is clear that hydrogels containing two therapeutic factors showed synergistic effects on regeneration in the chronic heart failure model. In conclusion, these results suggest that combination of stem cell homing factor with angiogenic peptides recruit stem cells to the microenvironments, increase the expression of angiogenic genes, enhance the matured vessel formation and improve the cardiac function in chronic MI. [Copyright &y& Elsevier]

Abstract: We developed an injectable hydrogel system to evaluate the effect of hydrogel stiffness on chondrocyte cellular functions in a three-dimensional (3D) environment and its subsequent influence on ectopic cartilage formation and early-stage osteochondral defect repair in a rabbit model. The hydrogels, composed of gelatin-hydroxyphenylpropionic acid (Gtn-HPA) conjugate, were formed using oxidative coupling of HPA moieties catalyzed by hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). The storage modulus (G′) of the hydrogels, which was tunable by changing the H2O2 and Gtn-HPA concentrations, ranged from 570 Pa to 2750 Pa. It was found that the cellular functions of chondrocytes encapsulated in hydrogels, including cell proliferation, biosynthesis of collagen and sulfated glycosaminoglycans (sGAG), as well as gene expression of type I (Col-I) and type II collagen (Col-II), were strongly affected by the stiffness of the hydrogels. Of note, chondrocytes cultured within the Gtn-HPA hydrogel of medium stiffness (G′ = 1000 Pa) produced highest level of sGAG production, as well as highest ratio of Col-II to Col-I gene expression among the Gtn-HPA hydrogels of different stiffness. Consistent with the results from in vitro and in vivo ectopic cartilage formation, osteochondral defect repair in a rabbit model showed stiffness-dependent tissue repair, with defects implanted with chondrocytes in hydrogels of medium stiffness having markedly more hyaline cartilage formation, smoother surface and better integration with adjacent cartilage, compared to defects treated with hydrogels of low or high stiffness. These results suggest that the tunable stiffness of Gtn-HPA hydrogels modulates chondrocyte cellular functions, and has a dramatic impact on cartilage tissue histogenesis and repair. [Copyright &y& Elsevier]

Abstract: Multilayered polymer hydrogels containing living cells were assembled for assessing the distance-dependent effects of soluble factors secreted by stroma cells on tumor cell cycle progression in vitro. A layer of tumor cells and a layer of stroma cells were separated with finely controlled spacing in a multilayered sandwich composed of a 2-methacryloyloxyethyl phosphorylcholine polymer and poly(vinyl alcohol) hydrogel. We demonstrated the utility of this tool for investigating intercellular communication between human cervical cancer HeLa cells and supportive stromal L929 fibroblast cells in co-culture. Time-lapse microscopic analyses of HeLa cells showed short distances (15 μm) between tumor cells and stroma cells induced a continuous increase in the percentage of HeLa cells in the S/G2/M phases of the cell cycle, while longer distances (70 μm) between the cells caused a slower increase followed by a sharp increase in the percentage of cells in S/G2/M phases. One possible explanation is gradient formation in the diffusion-dominant multilayer hydrogels by water-soluble factors such as those inducing growth, differentiation, and proliferation. This study provides insights into the potential effects of diffusion of soluble factors and related distance-dependent effects on cell behavior, which may contribute to the design of future co-culture systems. [Copyright &y& Elsevier]

Abstract: Multiplication of hematopoietic stem cells (HSCs) in vitro with current standard methods is limited and mostly insufficient for clinical applications of these cells. They quickly lose their multipotency in culture because of the fast onset of differentiation. In contrast, HSCs efficiently self-renew in their natural microenvironment (their niche) in the bone marrow. Therefore, engineering artificial bone marrow analogs is a promising biomaterial-based approach for culturing these cells. In the current study, a straight-forward, easy-to-use method for the production of biofunctionalized, macroporous hydrogel scaffolds that mimic the spongy architecture of trabecular bones was developed. As surrogates for cellular components of the niche, mesenchymal stem cells (MSCs) from different sources (bone marrow and umbilical cord) and osteoblast-like cells were tested. MSCs from bone marrow had the strongest pro-proliferative effect on freshly isolated human hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood. Co-culture in the pores of the three-dimensional hydrogel scaffold showed that the positive effect of MSCs on preservation of HSPC stemness was more pronounced in 3D than in standard 2D cell culture systems. Thus, the presented biomimetic scaffolds revealed to meet the basic requirements for creating artificial HSC niches. [Copyright &y& Elsevier]

Abstract: Temperature has been extensively explored as a trigger to control the delivery of a payload from environment-sensitive polymers. The need for an external heat source only allows limited spatiotemporal control over the delivery process. We propose a new approach by using the dissipative properties of a hydrogel matrix as an internal heat source when the material is mechanically loaded. The system is comprised of a highly dissipative hydrogel matrix and thermo-sensitive nanoparticles that shrink upon an increase in temperature. Exposing the hydrogel to a cyclic mechanical loading for a period of 5 min leads to an increase of temperature of the nanoparticles. The concomitant decrease in the volume of the nanoparticles increases the permeability of the hydrogel network facilitating the release of its payload. As a proof-of-concept, we showed that the payload of the hydrogel is released after 5–8 min following the initiation of the mechanical loading. This delivery method would be particularly suited for the release of growth factor as it has been shown that cell receptor to growth factor is activated 5–20 min following a mechanical loading. [Copyright &y& Elsevier]

Abstract: Thermosensitive hydrogels based on poly(γ-ethyl-l-glutamate)-poly(ethylene glycol)-poly(γ-ethyl-l-glutamate) triblock copolymers (PELG-PEG-PELG) were prepared for localized and sustained delivery of anticancer drugs. The polypeptide-based hydrogels showed much lower critical gelation concentration than the traditional polyester-based hydrogels. In vivo biocompatibility studies revealed that the in situ formed gels in the subcutaneous layer last for ∼21 days, and H&E staining study suggested acceptable biocompatibility of our materials in vivo. Then the hydrogels were tried as injectable implants to encapsulate antitumor drug, paclitaxel (PTX), to assess the in situ anti-tumoral activity using liver cancer xenograft model. The results demonstrated that the PTX-incorporated hydrogels could efficiently suppress the tumor growth, and did not result in obvious damage to normal organs. Therefore, the polypeptide-based thermosensitive hydrogels designed in the present study have great potential to serve as an effective platform for localized anti-cancer drug delivery. [Copyright &y& Elsevier]

Abstract: Current antimicrobial strategies have mostly been developed to manage infections due to planktonic cells. However, microbes in their nature state will tend to exist by attaching to and growing on living and inanimate surfaces that result in the formation of biofilms. Conventional therapies for treating biofilm-related infections are likely to be insufficient due to the lower susceptibility of microbes that are embedded in the biofilm matrix. In this study, we report the development of biodegradable hydrogels from vitamin E-functionalized polycarbonates for antimicrobial applications. These hydrogels were formed by incorporating positively-charged polycarbonates containing propyl and benzyl side chains with vitamin E moiety into physically cross-linked networks of “ABA”-type polycarbonate and poly(ethylene glycol) triblock copolymers. Investigations of the mechanical properties of the hydrogels showed that the G′ values ranged from 1400 to 1600 Pa and the presence of cationic polycarbonate did not affect the stiffness of the hydrogels. Shear-thinning behavior was observed as the hydrogels displayed high viscosity at low shear rates that dramatically decreased as the shear rate increased. In vitro antimicrobial studies revealed that the more hydrophobic VE/BnCl(1:30)-loaded hydrogels generally exhibited better antimicrobial/antifungal effects compared to the VE/PrBr(1:30) counterpart as lower minimum biocidal concentrations (MBC) were observed in Staphylococcus aureus (Gram-positive), Escherichia coli (Gram-negative) and Candida albicans (fungus) (156.2, 312.5, 312.5 mg/L for VE/BnCl(1:30) and 312.5, 2500 and 625 mg/L for VE/PrBr(1:30) respectively). Similar trends were observed for the treatment of biofilms where VE/BnCl(1:30)-loaded hydrogels displayed better efficiency with regards to eradication of biomass and reduction of microbe viability of the biofilms. Furthermore, a high degree of synergistic antimicrobial effects was also observed through the co-delivery of antimicrobial polycarbonates with a conventionally-used antifungal agent, fluconazole. These hydrogels also displayed excellent compatibility with human dermal fibroblasts with cell viability >80% after treatment with hydrogels loaded with cationic polymers and/or fluconazole at minimum biocidal concentrations (MBC). [Copyright &y& Elsevier]

Abstract: The aim of this study was to develop a magnetic thermosensitive hydrogel as intravesical Bacillus Calmette–Guérin (BCG) delivery system, which was formulated with chitosan (CS), β-glycerophosphate (GP) and Fe3O4 magnetic nanoparticle (Fe3O4-MNP). The gelation time and magnetic response of the gel system were investigated. The morphology of the gel was displayed by scanning electron microscope. Frozen section examination was creatively employed for exhibiting the structure of the gel and determining its intravesical residence time. The antitumor effect and local immune activity of BCG loaded magnetic gel were evaluated. The flowing solution of CS/GP under room temperature could gelate rapidly at body temperature both in vitro and in vivo. The magnetic injectable hydrogels significantly prolonged intravesical BCG residence time under an applied magnetic field. In comparison to traditional BCG therapy for superficial bladder tumor, BCG delivered by the gel system induced a stronger Th1 immune response and revealed higher antitumor efficacy. [Copyright &y& Elsevier]

Abstract: Non-viral gene delivery holds great promise for promoting tissue regeneration, and offers a potentially safer alternative than viral vectors. Great progress has been made to develop biodegradable polymeric vectors for non-viral gene delivery in 2D culture, which generally involves isolating and modifying cells in vitro, followed by subsequent transplantation in vivo. Scaffold-mediated gene delivery may eliminate the need for the multiple-step process in vitro, and allows sustained release of nucleic acids in situ. Hydrogels are widely used tissue engineering scaffolds given their tissue-like water content, injectability and tunable biochemical and biophysical properties. However, previous attempts on developing hydrogel-mediated non-viral gene delivery have generally resulted in low levels of transgene expression inside 3D hydrogels, and increasing hydrogel stiffness further decreased such transfection efficiency. Here we report the development of biodegradable polymeric vectors that led to efficient gene delivery inside poly(ethylene glycol) (PEG)-based hydrogels with tunable matrix stiffness. Photocrosslinkable gelatin was maintained constant in the hydrogel network to allow cell adhesion. We identified a lead biodegradable polymeric vector, E6, which resulted in increased polyplex stability, DNA protection and achieved sustained high levels of transgene expression inside 3D PEG-DMA hydrogels for at least 12 days. Furthermore, we demonstrated that E6-based polyplexes allowed efficient gene delivery inside hydrogels with tunable stiffness ranging from 2 to 175 kPa, with the peak transfection efficiency observed in hydrogels with intermediate stiffness (28 kPa). The reported hydrogel-mediated gene delivery platform using biodegradable polyplexes may serve as a local depot for sustained transgene expression in situ to enhance tissue engineering across broad tissue types. [Copyright &y& Elsevier]

Abstract: Metastasis begins with the escape, or dissemination, of cancer cells from the primary tumor. We recently demonstrated that tumors preferentially disseminate into collagen I and not into basement membrane protein gels (Matrigel). In this study, we used synthetic polymer systems to define material properties that could induce dissemination into Matrigel. We first specifically varied rigidity by varying the crosslinking density of poly(ethylene glycol) (PEG) networks within Matrigel scaffolds. Increased microenvironmental rigidity limited epithelial growth but did not promote dissemination. We next incorporated adhesive signals into the PEG network using peptide-conjugated cyclodextrin (α-CDYRGDS) rings. The α-CDYRGDS rings threaded along the PEG polymers, enabling independent control of matrix mechanics, adhesive peptide composition, and adhesive density. Adhesive PEG networks induced dissemination of normal and malignant mammary epithelial cells at intermediate values of adhesion and rigidity. Our data reveal that microenvironmental signals can induce dissemination of normal and malignant epithelial cells without requiring the fibrillar structure of collagen I or containing collagen I-specific adhesion sequences. Finally, the nanobiomaterials and assays developed in this study are generally useful both in 3D culture of primary mammalian tissues and in the systematic evaluation of the specific role of mechanical and adhesive inputs on 3D tumor growth, invasion, and dissemination. [Copyright &y& Elsevier]

Abstract: Intervertebral disc (IVD) degeneration is one of the leading causes of lower back pain and a major health problem worldwide. Current surgical treatments include excision or immobilisation, with neither approach resulting in the repair of the degenerative disc. As such, a tissue engineering-based approach in which stem cells, coupled with an advanced delivery system, could overcome this deficiency and lead to a therapy that encourages functional fibrocartilage generation in the IVD. In this study, we have developed an injectable hydrogel system based on enzymatically-crosslinked polyethylene glycol and hyaluronic acid. We examined the effects of adding pentosan polysulphate (PPS), a synthetic glycosaminoglycan-like factor that has previously been shown (in vitro and in vivo) to this gel system in order to induce chondrogenesis in mesnchymal precursor cells (MPCs) when added as a soluble factor, even in the absence of additional growth factors such as TGF-β. We show that both the gelation rate and mechanical strength of the resulting hydrogels can be tuned in order to optimise the conditions required to produce gels with the desired combination of properties for an IVD scaffold. Human immunoselected STRO-1+ MPCs were then incorporated into the hydrogels. They were shown to retain good viability after both the initial formation of the gel and for longer-term culture periods in vitro. Furthermore, MPC/hydrogel composites formed cartilage-like tissue which was significantly enhanced by the incorporation of PPS into the hydrogels, particularly with respect to the deposition of type-II-collagen. Finally, using a wild-type rat subcutaneous implantation model, we examined the extent of any immune reaction and confirmed that this matrix is well tolerated by the host. Together these data provide evidence that such a system has significant potential as both a delivery vehicle for MPCs and as a matrix for fibrocartilage tissue engineering applications. [Copyright &y& Elsevier]

Abstract: A wide variety of hydrogels have been explored as 3D culture platforms and for applications in tissue engineering. Hydrogels formed from natural extracellular matrix (ECM) proteins readily support the formation of vasculature in vitro, but only a handful of hydrogels composed of synthetic materials have shown anything comparable. This relative lack of synthetic material options has hindered efforts to better understand how ECM cues direct vascularization. We developed a biosynthetic hydrogel consisting of polyethylene glycol diacrylamide conjugated to macromolecular type-I collagen. Through their acrylamide-based crosslinks, these materials allow for independent control of physical properties and bulk ligand concentration. These hydrogels exhibited hydrolytic stability, but the collagen component retained its susceptibility to enzymatic remodeling. Photoencapsulation of endothelial cells and fibroblasts within this hydrogel material and their subsequent co-culture led to the formation of capillary vessel-like networks with well-defined hollow lumens. Capillary formation was prevented by inhibiting matrix metalloproteinase (MMP) activity, recapitulating the MMP-dependence of vascularization observed in natural hydrogels. These findings validate the utility of this material platform to decipher how the ECM regulates capillary morphogenesis and to support the formation of vascularized tissue constructs for potential applications in regenerative medicine. [Copyright &y& Elsevier]

Abstract: Allografts remain the clinical “gold standard” for treatment of critical sized bone defects despite minimal engraftment and ∼60% long-term failure rates. Therefore, the development of strategies to improve allograft healing and integration are necessary. The periosteum and its associated stem cell population, which are lacking in allografts, coordinate autograft healing. Herein we utilized hydrolytically degradable hydrogels to transplant and localize mesenchymal stem cells (MSCs) to allograft surfaces, creating a periosteum mimetic, termed a ‘tissue engineered periosteum’. Our results demonstrated that this tissue engineering approach resulted in increased graft vascularization (∼2.4-fold), endochondral bone formation (∼2.8-fold), and biomechanical strength (1.8-fold), as compared to untreated allografts, over 16 weeks of healing. Despite this enhancement in healing, the process of endochondral ossification was delayed compared to autografts, requiring further modifications for this approach to be clinically acceptable. However, this bottom-up biomaterials approach, the engineered periosteum, can be augmented with alternative cell types, matrix cues, growth factors, and/or other small molecule drugs to expedite the process of ossification. [Copyright &y& Elsevier]

Abstract: Impairment of sphincter muscles or their neural and vascular support leads to stress urinary incontinence. The aim of this study was to determine the role of urine-derived stem cells (USCs) over-expressing vascular endothelial growth factor (VEGF) in collagen-I gel on angiogenesis, cell survival, cell growth, myogenic phenotype differentiation of the implanted cells and innervations following implantation in vivo. USCs were infected with adenovirus containing the human VEGF165 and green fluorescent protein genes. A total of 5 × 106 cells, USCs alone, or plus endothelial cells or human skeletal myoblasts (as control) suspended in collagen-I gel were subcutaneously implanted into nude mice. Extensive vascularization and more implanted cells was noted in VEGF-expressing USCs groups compared to the non-VEGF groups in vivo. Numbers of the cells displaying endothelial markers (CD 31 and von Willebrand's factor) and myogenic markers (myf-5, MyoD and desmin), and regenerated nerve fibers displaying neural markers (S-100, GFAP and neurofilament) significantly increased in the grafts of VEGF-expressing USCs. Improved angiogenesis by VEGF-expressing USCs enhanced grafted cell survival, recruited the resident cells and promoted myogenic phenotype differentiation of USCs and innervation. This approach has important clinical implications for the development of cell therapies for the correction of stress urinary incontinence. [Copyright &y& Elsevier]

Abstract: Peripheral nerve injuries requiring surgery can be repaired by autograft, the clinical “gold standard”, allograft, or nerve conduits. Most published clinical studies show the effectiveness of nerve conduits in small size defects in sensory nerves. Many preclinical studies suggest that peripheral nerve regeneration through conduits can be enhanced and repair lengths increased with the use of a biomaterial filler in the conduit lumen. We have previously shown that a luminal hydrogel filler derived from human hair keratin (HHK) can improve electrophysiological and histological outcomes in mouse, rabbit, and non-human primate nerve injury models, but insight into potential mechanisms has been lacking. Based on the premise that a keratin biomaterial (KOS) hydrogel provides an instantaneous structural matrix within the lumen, the current study compares the cellular behavior elicited by KOS hydrogel to Matrigel (MAT) and saline (SAL) conduit fillers in a 1 cm rat sciatic nerve injury model at early stages of regeneration. While there was little difference in initial cellular influx, the KOS group showed earlier migration of dedifferentiated Schwann cells (SC) from the proximal nerve end compared to the other groups. The KOS group also showed faster SC dedifferentiation and myelin debris clearance, and decreased macrophage infiltration during Wallerian degeneration of the distal nerve tissue. [Copyright &y& Elsevier]

Abstract: Sentinel lymph nodes (SLNs) are the first lymph nodes which cancer cells reach after traveling through lymphatic vessels from the primary tumor. Evaluating the nodal status is crucial in accurate staging of human cancers and accordingly determines prognosis and the most appropriate treatment. The commonly used methods for SLN identification in clinics are based on employment of a colloid of radionuclide or injection of a small dye. Although these methods have certainly contributed to improve surgical practice, new imaging materials are still required to overcome drawbacks of the techniques such as inconvenience of handling radioactive materials and short retention time of small dyes in SLNs. Here, we prepare a fluorescence-labeled DNA tetrahedron and perform SLN imaging by using the DNA nanoconstruct. With a successful identification of SLNs by the DNA nanoconstruct, we suggest that DNA tetrahedron hold great promises for clinical applications. [Copyright &y& Elsevier]

Abstract: Islet transplantation offers a promising treatment for type 1 diabetes (T1D). However, a major hurdle in this treatment is the rapid loss of functional islets during culture and after transplantation. The liver site, currently utilized for transplantation, is suboptimal for achieving long-term insulin independence due to a rapid islet loss followed by a chronic decline in islet function after transplantation. Herein, we report a synthetic saccharide–peptide (SP) hydrogel that allows suspending islets in liquid and injecting for in situ polymerization without forming islet clumps, indicating its potential in extrahepatic islet transplantation. In vitro, rat islets in SP hydrogel maintained a 3D structure and high glucose-stimulated insulin release similar to that observed in freshly isolated islets for 4 weeks, while control islets cultured in suspension lost their 3D structure and insulin release responses by 2 weeks. Biocompatibility of SP hydrogel was shown by the absence of cytokine mRNA activation in peripheral blood mononuclear cells (PBMCs) exposed to hydrogel in vitro and by the absence of cellular infiltrates in and around the hydrogel implanted subcutaneously. Syngeneic Lewis rat islets transplanted in SP hydrogel in various extrahepatic sites stained strongly for insulin, and more effectively reversed diabetes than unencapsulated islets when transplanted in an omental pocket. In conclusion, the SP hydrogel is non-cytotoxic and supports normal islet structure and function both in vitro and in vivo. Specifically, the ability of the hydrogel to separate individual islets after transplantation is important for maintaining their function in vivo. This important property, combined with the versatility and biocompatibility, makes our SP hydrogel a promising synthetic scaffold that can facilitate transplantation of organized heterogeneous cells to preserve their micro-structure and function. [Copyright &y& Elsevier]

Abstract: This work reports the self-assembly of a sparingly soluble antibiotic (ciprofloxacin) and a hydrophobic tripeptide (DLeu–Phe–Phe) into supramolecular nanostructures that yield a macroscopic hydrogel at physiological pH. Drug incorporation results in modified morphology and rheological properties of the self-assembled hydrogel. These changes can be correlated with intermolecular interactions between the drug and the peptide, as confirmed by spectroscopic analysis (fluorescence, circular dichroism, IR). The drug appears bound within the hydrogel by non-covalent interactions, and retains its activity over a prolonged release timescale. Antimicrobial activity of the ciprofloxacin-peptide self-assembled hydrogel was evaluated against Staphylococcus aureus, Escherichia coli, and a clinical strain of Klebsiella pneumoniae. Interestingly, the peptide hydrogel alone exhibited a mild anti-bacterial activity against Gram-negative bacteria. While toxic to bacteria, no major cytotoxicity was seen in haemolysis assays of human red blood cells or in mouse fibroblast cell cultures. This new approach of drug incorporation into the nanostructure of a simple tripeptide hydrogel by self-assembly may have important applications for cost-effective wound dressings and novel antimicrobial formulations. [Copyright &y& Elsevier]

Abstract: This study is aimed to develop a long-acting injectable formulation in treatment of type II diabetes. A glucoregulatory polypeptide, exenatide (EXT), was chosen as the model drug, and an aqueous block copolymer system with a sol–gel transition upon the increase of temperature was selected as the delivery matrix of EXT. The thermoreversible hydrogel composed of poly(lactic acid-co-glycolic acid)-poly(ethylene glycol)-poly(lactic acid-co-glycolic acid) (PLGA-PEG-PLGA) triblock copolymers was found to slower the degradation of the polypeptide to a large extent. However, the initial formulation in this study exhibited a significant drug burst effect, which is a common problem to load a hydrophilic small or medium-size polypeptide into a hydrogel. Zinc acetate was then introduced to slow down the EXT release by formation of insoluble Zn-EXT complexes in the thermogel matrix. Yet an incomplete release became another crucial problem, which is also common for peptide and protein delivery. The synergistic effect of three excipients (zinc acetate, PEG, and sucrose) under an appropriate condition overcame these two problems simultaneously, and the sustained release of drug lasted for 1 week. In vivo experiments via mice oral glucose tolerance tests demonstrated an improved glucose tolerance for 1 week after a single subcutaneous injection of the optimal EXT formulation. As a result, a formulation of antidiabetic drugs was set up, and meanwhile a strategy using synergistic excipients to adjust release profiles of peptides from hydrogels was put forward. [Copyright &y& Elsevier]

Abstract: Nanoparticles with pH-sensitive behavior may enhance the success of chemotherapy in many cancers by efficient intracellular drug delivery. Here, we investigated the effect of a bioactive surfactant with pH-sensitive properties on the antitumor activity and intracellular behavior of methotrexate-loaded chitosan nanoparticles (MTX–CS–NPs). NPs were prepared using a modified ionotropic complexation process, in which was included the surfactant derived from Nα,Nε-dioctanoyl lysine with an inorganic lithium counterion. The pH-sensitive behavior of NPs allowed accelerated release of MTX in an acidic medium, as well as membrane-lytic pH-dependent activity, which facilitated the cytosolic delivery of endocytosed materials. Moreover, our results clearly proved that MTX–CS–NPs were more active against the tumor HeLa and MCF-7 cell lines than the free drug. The feasibilty of using NPs to target acidic tumor extracellular pH was also shown, as cytotoxicity against cancer cells was greater in a mildly acidic environment. Finally, the combined physicochemical and pH-sensitive properties of NPs generally allowed the entrapped drug to induce greater cell cycle arrest and apoptotic effects. Therefore, our overall results suggest that pH-sensitive MTX–CS–NPs could be potentially useful as a carrier system for tumor and intracellular drug delivery in cancer therapy. [Copyright &y& Elsevier]

Abstract: The effectiveness of systemically administered anticancer treatments is limited by difficulties in achieving therapeutic doses within tumors, a problem that is complicated by dose-limiting side effects to normal tissue. To increase the efficacy and reduce the toxicity of systemically administered anticancer 5-fluorouracil (5-Fu) treatments in patients, intratumoral administration of an injectable hydrogel has been evaluated in the current work. The MPEG-b-(PCL-ran-PLLA) diblock copolymer (MCL) containing 5-Fu existed in an emulsion-sol state at room temperature and rapidly gelled in vivo at the body temperature. MCL acted as in vivo biodegradable drug depot over a defined experimental period. A single injection of 5-Fu-loaded MCL solution resulted in significant suppression of tumor growth, compared with repeated injection of free 5-Fu as well as saline and MCL alone. For both repeated injections of free 5-Fu and single injection of 5-Fu-loaded MCL, most of the 5-Fu was found in the tumor, indicating the maintenance of therapeutic concentrations of 5-Fu within the target tumor tissue and the prevention of systemic toxicity associated with 5-Fu in healthy normal tissues. In conclusion, this work demonstrated that intratumoral injection of 5-Fu-loaded MCL may induce significant suppression of tumor growth through effective accumulation of 5-Fu in the tumor. [Copyright &y& Elsevier]

Abstract: Colloidal gels have recently emerged as a promising new class of materials for regenerative medicine by employing micro- and nanospheres as building blocks to assemble into integral scaffolds. To this end, physically crosslinked particulate networks are formed that are injectable yet cohesive. By varying the physicochemical properties of different particle populations, the suitability of colloidal gels for programmed delivery of multiple therapeutic proteins is superior over conventional monolithic gels that lack this strong capacity for controlled drug release. Colloidal gels made of biodegradable polymer micro- or nanospheres have been widely investigated over the past few years, but a direct comparison between micro- vs. nanostructured colloidal gels has not been made yet. Therefore, the current study has compared the viscoelastic properties and capacity for drug release of colloidal gels made of oppositely charged gelatin microspheres vs. nanospheres. Viscoelastic properties of the colloidal gelatin gels were characterized by rheology and simple injectability tests, and in vitro release of two selected osteogenic proteins (i.e. bone morphogenetic protein-2 (BMP-2) and alkaline phosphatase (ALP)) from the colloidal gelatin gels was evaluated using radiolabeled BMP-2 and ALP. Nanostructured colloidal gelatin gels displayed superior viscoelastic properties over microsphere-based gels in terms of elasticity, injectability, structural integrity, and self-healing behavior upon severe network destruction. In contrast, microstructured colloidal gelatin gels exhibited poor gel strength and integrity, unfavorable injectability, and did not recover after shearing, resulting from the poor gel cohesion due to insufficiently strong interparticle forces. Regarding the capacity for drug delivery, sustained growth factor (BMP-2) release was obtained for both micro- and nanosphere-based gels, the kinetics of which were mainly depending on the particle size of gelatin spheres with the same crosslinking density. Therefore, the optimal gelatin carrier for drug delivery in terms of particle size and crosslinking density still needs to be established for specific clinical indications that require either short-term or long-term release. It can be concluded that nanostructured colloidal gelatin gels show great potential for sustained delivery of therapeutic proteins, whereas microstructured colloidal gelatin gels are not sufficiently cohesive as injectables for biomedical applications. [Copyright &y& Elsevier]

Abstract: Increasing evidence suggests that the improper synaptic reconnection of regenerating axons is a significant cause of incomplete functional recovery following peripheral nerve injury. In this study, we evaluate the use of collagen hydrogels functionalized with two peptide glycomimetics of naturally occurring carbohydrates—polysialic acid (PSA) and human natural killer cell epitope epitope (HNK-1)—that have been independently shown to encourage nerve regeneration and axonal targeting. Our novel biomaterial was used to bridge a critical gap size (5 mm) in a mouse femoral nerve injury model. Functional recovery was assessed using gait and hind limb extension, and was significantly better in all glycomimetic peptide-coupled collagen conditions versus non-functional scrambled peptide-coupled collagen, native collagen, and saline controls. Analysis of cross-sections of the regenerated nerve demonstrated that hydrogels coupled with the PSA glycomimetic, but not HNK, had significant increases in the number of myelinated axons over controls. Conversely, hydrogels coupled with HNK, but not PSA, showed improvement in myelination. Additionally, significantly more correctly projecting motoneurons were observed in groups containing coupled HNK-1 mimicking peptide, but not PSA mimicking peptide. Given the distinct morphological outcomes between the two glycomimetics, our study indicates that the enhancement of recovery following peripheral nerve injury induced by PSA- and HNK-functionalized collagen hydrogels likely occurs through distinct mechanisms. [Copyright &y& Elsevier]

Abstract: We have developed a hydrogel-based cell cultivation platform for forming 3D restiform hepatic micro-organoids consisting of primary rat hepatocytes and feeder cells (Swiss 3T3 cells). Sodium alginate solutions containing hepatocytes/3T3 cells were continuously introduced into a microfluidic channel to produce cell-incorporating anisotropic Ba-alginate hydrogel microfibers, where hepatocytes at the center were closely sandwiched by 3T3 cells. Hydrogel fiber-based cultivation under high oxygen tension enabled the formation of heterotypic micro-organoids with a length of up to 1 mm and a diameter of ∼50 μm, mimicking the hepatic cord structures found in the liver, while maintaining a high hepatocyte viability (∼80%) over 30 days. Long-term observation of up to 90 days revealed a significant enhancement of hepatic functions because of heterotypic and homotypic cell–cell interactions, including albumin secretion and urea synthesis as well as expression of hepatocyte-specific genes, compared with conventional monolayer culture and single cultivation in the hydrogel fibers. The encapsulated hepatic constructs were recovered as scaffold-free micro-organoids by enzymatically digesting the hydrogel matrices using alginate lyase. This technique for creating heterotypic micro-organoids with precisely ordered multiple cell types will be useful for the development of a new liver tissue engineering approach and may be applicable to the fabrication of extracorporeal bioartificial liver (BAL) devices and assessment tools for drug development and testing. [Copyright &y& Elsevier]

Abstract: Cell migration is controlled by the integration of numerous distinct components. Consequently, the analysis of cell migration is advancing towards comprehensive, multifaceted in vitro models. To accurately evaluate the contribution of an underlying substrate to cell motility in complex cellular environments we developed a migration assay using magnetically attachable stencils (MAts). When attached to a culture surface, MAts create a defined void in the cell monolayer without disrupting the cells or damaging the underlying substrate. Quantitative analysis of migration into this void reveals the substrate''s contribution to migration. The magnetically-guided placement of a microfabricated stencil allows for full experimental control of the substrate on which migration is analyzed. MAts enable the evaluation of intact, defined matrix, and make it possible to analyze migration on unique surfaces such as micropatterned proteins, nano-textured surfaces, and pliable hydrogels. These studies also revealed that mechanical disruption, including the damage that occurs during scratch assays, diminishes migration and confounds the analysis of individual cell behavior. Analysis of migration on increasingly complex biomaterials reveals that the contribution of the underlying matrix depends not only on its molecular composition but also its organization and the context in which it is presented. [Copyright &y& Elsevier]

Abstract: Tissue engineering-based approaches have the potential to improve stem cell engraftment by increasing cell delivery to the myocardium. Our objective was to develop and characterize a naturally-derived, autologous, biodegradable hydrogel in order to improve acute stem cell retention in the myocardium. HA-blood hydrogels (HA-BL) were synthesized by mixing in a 1:1(v/v) ratio, lysed whole blood and hyaluronic acid (HA), whose carboxyl groups were functionalized with N-hydroxysuccinimide (NHS) to yield HA succinimidyl succinate (HA–NHS). We performed physical characterization and measured survival/proliferation of cardiosphere-derived cells (CDCs) encapsulated in the hydrogels. Hydrogels were injected intra-myocardially or applied epicardially in rats. NHS-activated carboxyl groups in HA react with primary amines present in blood and myocardium to form amide bonds, resulting in a 3D hydrogel bound to tissue. HA-blood hydrogels had a gelation time of 58 ± 12 s, swelling ratio of 10 ± 0.5, compressive and elastic modulus of 14 ± 3 and 1.75 ± 0.6 kPa respectively. These hydrogels were not degraded at 4wks by hydrolysis alone. CDC encapsulation promoted their survival and proliferation. Intra-myocardial injection of CDCs encapsulated in these hydrogels greatly increased acute myocardial retention (p = 0.001). Epicardial application of HA-blood hydrogels improved left ventricular ejection fraction following myocardial infarction (p = 0.01). HA-blood hydrogels are highly adhesive, biodegradable, promote CDC survival and increase cardiac function following epicardial application after myocardial infarction. [Copyright &y& Elsevier]

Abstract: Induced pluripotent stem cells (iPSCs) have promising potential in regenerative medicine, but whether iPSCs can promote corneal reconstruction remains undetermined. In this study, we successfully reprogrammed human corneal keratocytes into iPSCs. To prevent feeder cell contamination, these iPSCs were cultured onto a serum- and feeder-free system in which they remained stable through 30 passages and showed ESC-like pluripotent property. To investigate the availability of iPSCs as bioengineered substitutes in corneal repair, we developed a thermo-gelling injectable amphiphatic carboxymethyl-hexanoyl chitosan (CHC) nanoscale hydrogel and found that such gel increased the viability and CD44 + proportion of iPSCs, and maintained their stem-cell like gene expression, in the presence of culture media. Combined treatment of iPSC with CHC hydrogel (iPSC/CHC hydrogel) facilitated wound healing in surgical abrasion-injured corneas. In severe corneal damage induced by alkaline, iPSC/CHC hydrogel enhanced corneal reconstruction by downregulating oxidative stress and recruiting endogenous epithelial cells to restore corneal epithelial thickness. Therefore, we demonstrated that these human keratocyte-reprogrammed iPSCs, when combined with CHC hydrogel, can be used as a rapid delivery system to efficiently enhance corneal wound healing. In addition, iPSCs reprogrammed from corneal surgical residues may serve as an alternative cell source for personalized therapies for human corneal damage. [Copyright &y& Elsevier]

Abstract: Cells have been extensively studied for their uses in various therapies because of their capacities to produce therapeutic proteins and recreate new tissues. It has often been suggested that the efficacy of cell therapies can greatly be improved through the ability to localize and regulate cellular activities at a transplantation site; however, the technologies for this control are lacking. Therefore, this study reports a cell-Laden hydrogel patch engineered to support the proliferation and angiogenic growth factor expression of cells adhered to their surfaces, and to further promote neovascularization. Hydrogels consisting of alginate chemically linked with pyrrole units, termed alginate-g-pyrrole, were prepared through an oxidative cross-linking reaction between pyrrole units. Fibroblasts adhered to the alginate-g-pyrrole hydrogels, and exhibited increased proliferation and overall vascular endothelial growth factor (VEGF) expression, compared to those on pyrrole-free hydrogels. Furthermore, the alginate-g-pyrrole hydrogel surfaces were modified to present microposts, subsequently increasing the amount of pyrrole units on their surfaces. Cells adhered to the microfabricated gel surfaces exhibited increased proliferation and overall VEGF expression proportional to the density of the microposts. The resulting micropatterned alginate-g-pyrrole hydrogels exhibited increases in the size and density of mature blood vessels when implanted on chick chorioallantoic membranes (CAMs). The hydrogel system developed in this study will be broadly useful for improving the efficacy of a wide array of cell-based wound healing and tissue regenerative therapies. [Copyright &y& Elsevier]

Abstract: The extracellular matrix is continually remodelled by the action of various enzymes such as heparanase, which specifically targets heparan sulfate (HS) and is found in human platelets at high levels. The activity of heparin-containing hydrogels following incubation with platelet extract (PE) was investigated in order to simulate the responses that might occur when the hydrogels, as tissue engineered scaffolds, come in contact with blood products at the site of an injury. The heparanase activity of PE on heparin, used as a model of HS, was confirmed by the decrease in molecular weight. PE treatment diminished heparin''s anticoagulation property but increased its FGF-2 signalling activity, suggesting that the PE''s heparanase activity cleaves at the 3-O-sulfated glucosamine to produce large fragments that can signal cell receptors. The dual effect observed when poly(vinyl alcohol)/heparin co-hydrogels were incubated with PE supports the hypothesis of platelets having the capacity to limit anticoagulation and thus promote blood clot formation, which may be critical in the process of tissue repair. [Copyright &y& Elsevier]

Abstract: To overcome the unresolved issues of conventional therapeutic approaches such as radiation therapy, chemotherapy, combinational chemotherapy, and surgical treatment, we designed an injectable ‘MRI-monitored long-term therapeutic hydrogel (MLTH)’ system as an alternative/adjuvant approach for brain tumors. The MLTH system consists of a thermosensitive/magnetic poly(organophosphazene) hydrogel (the magnetic hydrogel) as a biodegradable imaging platform and an anticancer drug as a therapeutic agent via a simple physical mixing. The MLTH system has adequate properties for the MRI-monitored long-term therapy as follows: injectability, localizability due to fast gelation at body temperature, biocompatibility, biodegradability, sustained drug release, and MR imaging function. Since the MLTH system only requires a very small-sized pin hole injected into the area of brain tumors stereotactically, we suggest that the MLTH system can be an alternative/adjuvant approach to treat the malignant brain tumors without any surgical resection. Furthermore, we expect that the MLTH system can minimize the side effects from either an intravenous injection or surgical operation because one of the aims of MLTH is to focus on the sustained local delivery of anticancer drugs via a one- or two-time intratumoral injections. Thus, we assessed successfully the MRI-monitored long-term therapeutic potentialities of the MLTH system for brain tumors and estimated the inhibition efficacy of tumor growth via an MRI-monitored long-term therapy in this study. [Copyright &y& Elsevier]

Abstract: A novel three-component biomimetic hydrogel composite was successfully prepared in this study, which was composed of triblock PEG-PCL-PEG copolymer (PECE), collagen and nano-hydroxyapatite (n-HA). The microstructure and thermo-responsibility of the obtained PECE/Collagen/n-HA hydrogel composite were characterized. Scanning electronic microscopy (SEM) showed that the composite exhibited an interconnected porous structure. The rheological analysis revealed that the composite existed good thermo-sensitivity. In vivo biocompatibility and biodegradability was investigated by implanting the hydrogel composite in muscle pouches of rats for 3, 7, and 14 days. Moreover, the osteogenic capacity was evaluated by means of implanting the composite material in cranial defects of New Zealand White rabbits for 4, 12 and 20 weeks. In vivo performances confirmed that the biodegradable PECE/Collagen/n-HA hydrogel composite had good biocompatibility and better performance in guided bone regeneration than the self-healing process. Thus the thermal-response PECE/Collagen/n-HA hydrogel composite had the great potential in bone tissue engineering. [Copyright &y& Elsevier]

Abstract: Enrichment of progenitor cells in ischemic tissue has become a promising therapeutic strategy in the treatment of myocardial infarction. Towards this aim, we report a biology-inspired concept using sulfated glycosaminoglycans to sustainably generate chemokine gradients for the localized accumulation of early endothelial progenitor cells (eEPCs). StarPEG-heparin hydrogels, which have been previously demonstrated to support angiogenesis, were functionalized with SDF-1α, a potent chemoattractant known to act on EPCs. The gels were quantitatively shown to release the chemokine in amounts that are adjustable by the choice of loading concentrations and by matrix metalloprotease (MMP) mediated hydrogel cleavage. Transwell assays confirmed significantly enhanced migration of early EPCs towards concentration gradients of hydrogel-delivered SDF-1αin vitro. Subcutaneous implantation of SDF-1α-releasing gels in mice resulted in massive infiltration of early EPCs and subsequently improved vascularization. In conclusion, sustained delivery of SDF-1α from pro-angiogenic starPEG-heparin hydrogels can effectively attract early EPCs, offering a powerful means to trigger endogenous mechanisms of cardiac regeneration. [Copyright &y& Elsevier]

Abstract: Fibrillar collagen gels, which are used extensively in vitro to study tumor–microenvironment interactions, are composed of a cell-instructive network of interconnected fibers and pores whose organization is sensitive to polymerization conditions such as bulk concentration, pH, and temperature. Using confocal reflectance microscopy and image autocorrelation analysis to quantitatively assess gel microarchitecture, we show that additional polymerization parameters including culture media formulation and gel thickness significantly affect the dimensions and organization of fibers and pores in collagen gels. These findings enabled the development of a three-dimensional culture system in which cell-scale gel microarchitecture was decoupled from bulk gel collagen concentration. Interestingly, morphology and migration characteristics of embedded MDA-MB-231 cells were sensitive to gel microarchitecture independently of collagen gel concentration. Cells adopted a polarized, motile phenotype in gels with larger fibers and pores and a rounded or stellate, less motile phenotype in gels with small fibers and pores regardless of bulk gel density. Conversely, cell proliferation was sensitive to gel concentration but not microarchitecture. These results indicate that cell-scale gel microarchitecture may trump bulk-scale gel density in controlling specific cell behaviors, underscoring the biophysical role of gel microarchitecture in influencing cell behavior. [Copyright &y& Elsevier]

Abstract: Immune cells are present in the blood and in resident tissues, and the nature of their reaction towards biomaterials is decisive for materials success or failure. Macrophages may for example be classically activated to trigger inflammation (M1), or alternatively activated which supports healing and vascularisation (M2). Here, we have generated 3D nanofibrous meshes in different porosities and precisely controlled surface chemistries comprising PLGA, hydrogel-coated protein repellant and protein repellant endowed with the bioactive peptide sequences GRGDS or GLF. We also prepared 2D substrates with corresponding surface chemistry for a systematic evaluation of primary human macrophage adhesion, migration, transcriptome expression, cytokine release and surface marker expression. Our data show that material morphology is a powerful means in biomaterial design to influence immune cell response. Flat substrates lead to an increased number of M2 classified CD163+ macrophages. However, these M2 cells released large amounts of pro-inflammatory cytokines. In contrast, 3D nanofibres with corresponding surface chemistry yielded M1 classified 27E10+ macrophages with a significantly increased release of pro-angiogenic chemokines and angiogenesis related molecules and a strong decrease of pro-inflammatory cytokines. We thus suggest that, for macrophages in contact with biomaterials, cytokine release is taken as main criterion instead of surface-markers for macrophage classifications. [Copyright &y& Elsevier]