A chitosan–glutathione based injectable hydrogel for suppression of oxidative stress damage in cardiomyocytes

Overproduction of reactive oxygen species (ROS) is closely associated with myocardial infarction. The oxidative stress damage caused by ROS in grafted cells and host cells presents a major obstacle for successful myocardial repairs in cardiac tissue engineering. Previous injectable biomaterials in use of myocardial repairs typically lack consideration of their antioxidant properties. In this work, a thermosensitive chitosan chloride–glutathione (CSCl–GSH) hydrogel was developed to suppress the oxidative stress injury in cardiomyocytes (CMs). Glutathione (GSH) was conjugated on the chitosan chloride (CSCl) chain via amide bonds between carboxylic acid moieties of GSH and amine groups of CSCl. Our data show that CSCl–GSH conjugates in vitro could effectively scavenge the superoxide anion, hydroxyl radical and DPPH radical even at high concentrations and its antioxidant capacity can be modulated via adjusting the grafted degree of CSCl–GSH conjugates. In addition, CSCl–GSH hydrogels have shown an excellent biocompatibility to support the adhesion and survival of CMs. Moreover, it can remove the excessive intracellular ROS and thus suppress the oxidative stress damage and apoptosis in CMs in the presence of high ROS. These results suggest CSCl–GSH hydrogels could effectively support the myocardial repair via attenuating the oxidative stress damage to cells.     

Sustained delivery of latanoprost by thermosensitive chitosan–gelatin-based hydrogel for controlling ocular hypertension

Glaucoma is an irreversible ocular disease that may lead to progressive visual field loss and eventually to blindness with inadequately controlled intraocular pressure (IOP). Latanoprost is one of the most potent ocular hypotensive compounds, the current first-line therapy in glaucoma. However, the daily instillation required for efficacy and undesirable side-effects are major causes of treatment adherence failure and persistence in glaucoma therapy. In the present study, we developed an injectable thermosensitive chitosan/gelatin/glycerol phosphate (C/G/GP) hydrogel as a sustained-release system of latanoprost for glaucoma treatment. The latanoprost-loaded C/G/GP hydrogel can gel within 1 min at 37  °C. The results show a sustained release of latanoprost from C/G/GP hydrogel in vitro and in vivo. The latanoprost-loaded C/G/GP hydrogel showed a good in vitro and in vivo biocompatibility. A rabbit model of glaucoma was established by intravitreal injection of triamcinolone acetonide. After a single subconjunctival injection of latanoprost-loaded C/G/GP hydrogel, IOP was significantly decreased within 8 days and then remained at a normal level. The results of the study suggest that latanoprost-loaded C/G/GP hydrogel may have a potential application in glaucoma therapy.     

Protein functionalized micro hydrogel features for cell–surface interaction

Cross-linked hydrogel features have been patterned using subtractive lift-off of polymerized hydrogel film. Projection lithography and oxygen plasma etch was used to pattern parylene C polymer film. Molecular self-assembly of polymerizable monolayer was obtained in solution-phase and acrylamide based hydrogel was polymerized using free-radical polymerization on this substrate. Parylene C film was mechanically lifted-off to remove the blanket hydrogel film and micro hydrogel features (muhf) were obtained attached to the predefined patterns in the range from 1 to 60 mum. The muhf were functionalized with aldehyde functional groups, and proteins were coupled to them using Schiff base chemistry followed by reductive amination. Interaction of mesenchymal stem cells with transforming growth factor-beta 1 (TGF-beta1) functionalized muhf was studied, and TGF-beta1 was found to retain its tumor suppression activity.     

Ultrathin chitosan–poly(ethylene glycol) hydrogel films for corneal tissue engineering

Due to the high demand for donor corneas and their low supply, autologous corneal endothelial cell (CEC) culture and transplantation for treatment of corneal endothelial dysfunction would be highly desirable. Many studies have shown the possibility of culturing CECs in vitro, but lack potential robust substrates for transplantation into the cornea. In this study, we investigate the properties of novel ultrathin chitosan–poly(ethylene glycol) (PEG) hydrogel films (CPHFs) for corneal tissue engineering applications. Cross-linking of chitosan films with diepoxy-PEG and cystamine was employed to prepare ～50 μm (hydrated) hydrogel films. Through variation of the PEG content (1.5–5.9 wt.%) it was possible to tailor the CPHFs to have tensile strains and ultimate stresses identical to or greater than those of human corneal tissue while retaining similar tensile moduli. Light transmission measurements in the visible spectrum (400–700 nm) revealed that the films were >95% optically transparent, above that of the human cornea (maximum ～90%), whilst in vitro degradation studies with lysozyme revealed that the CPHFs maintained the biodegradable characteristics of chitosan. Cell culture studies demonstrated the ability of the CPHFs to support the attachment and proliferation of sheep CECs. Ex vivo surgical trials on ovine eyes demonstrated that the CPHFs displayed excellent characteristics for physical manipulation and implantation purposes. The ultrathin CPHFs display desirable mechanical, optical and degradation properties whilst allowing attachment and proliferation of ovine CECs, and as such are attractive candidates for the regeneration and transplantation of CECs, as well as other corneal tissue engineering applications.     

A magnetic chitosan hydrogel for sustained and prolonged delivery of Bacillus Calmette–Guérin in the treatment of bladder cancer

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.     

Preparation and in vitro delivery performance of chitosan–alginate microcapsule for IgG

The study aimed to develop chitosan–alginate microcapsule as an oral delivery carrier for IgG. Bovine serum albumin (BSA) was used as the protein to be encapsulated while determining the microcapsule formula by evaluating encapsulation efficiency (EE), loading capacity (LC) and release profile. Results suggested that the optimal formula was composed of 0.1% chitosan, 0.5% CaCl₂, 2% sodium alginate and the loading rate of BSA reached 25%. IgG was substituted for BSA and an IgG microcapsule was prepared following the above formula. The EE and LC of the resulted IgG microcapsule reached 76.83% and 18.53%. After 2?h incubation in simulated gastric fluid, the activity of IgG in the microcapsule remained at 79.79% and the total release rate of IgG in a simulated intestinal fluid reached 82.52%. This encapsulation formula was proved to be an effective oral delivery system which would protect the IgG from severe gastric conditions and guarantee an efficient release in the intestinal tract.     

Characterization of PEG–iron oxide hydrogel nanocomposites for dual hyperthermia and paclitaxel delivery

Hyperthermia, the heating of tissue from 41 to 45?°C, has been shown to improve the efficacy of cancer therapy when used in conjunction with irradiation and/or chemotherapy. In this work, hydrogel nanocomposites have been developed that can control the delivery of both heat and a chemotherapeutic agent (e.g. paclitaxel). The nanocomposites studied involve a stealth, poly(ethylene glycol) (PEG)-based system comprised of PEG (n?=?1000) methyl ether methacrylate and PEG (n?=?400) dimethacrylate with iron oxide nanoparticles physically entrapped within the hydrogel matrices. The capability of the hydrogel nanocomposites to be heated in an alternating magnetic field was demonstrated. The heating of the hydrogel systems was dependent on the crosslinking of the hydrogel network where hydrogels with lower swelling ratios were found to heat to a greater extent than those with higher ratios. In addition, paclitaxel was shown to exhibit non-Fickian release from the hydrogel systems, with the amount of drug released dependent on the hydrogel network structure. Three cell lines: M059K (glioblastoma), MDA MB 231 (breast carcinoma), and A549 (lung adenocarcinoma) were exposed to paclitaxel only, hyperthermia only, and both paclitaxel and hyperthermia to determine if a synergistic cytotoxic effect was possible for these cell lines. The efficacy of paclitaxel was greater with hyperthermia for the A549 cells; however, the M059K and MDA MB 231 did not show the same response.     

Injectable in situ forming biodegradable chitosan–hyaluronic acid based hydrogels for cartilage tissue engineering

Injectable, biodegradable scaffolds are important biomaterials for tissue engineering and drug delivery. Hydrogels derived from natural polysaccharides are ideal scaffolds as they resemble the extracellular matrices of tissues comprised of various glycosaminoglycans (GAGs). Here, we report a new class of biocompatible and biodegradable composite hydrogels derived from water-soluble chitosan and oxidized hyaluronic acid upon mixing, without the addition of a chemical crosslinking agent. The gelation is attributed to the Schiff base reaction between amino and aldehyde groups of polysaccharide derivatives. In the current work, N-succinyl-chitosan (S-CS) and aldehyde hyaluronic acid (A-HA) were synthesized for preparation of the composite hydrogels. The polysaccharide derivatives and composite hydrogels were characterized by FTIR spectroscopy. The effect of the ratio of S-CS and A-HA on the gelation time, microstructure, surface morphology, equilibrium swelling, compressive modulus, and in vitro degradation of composite hydrogels was examined. The potential of the composite hydrogel as an injectable scaffold was demonstrated by the encapsulation of bovine articular chondrocytes within the composite hydrogel matrix in vitro. The results demonstrated that the composite hydrogel supported cell survival and the cells retained chondrocytic morphology. These characteristics provide a potential opportunity to use the injectable, composite hydrogels in tissue engineering applications.     

A tunable silk–alginate hydrogel scaffold for stem cell culture and transplantation

One of the major challenges in regenerative medicine is the ability to recreate the stem cell niche, which is defined by its signaling molecules, the creation of cytokine gradients, and the modulation of matrix stiffness. A wide range of scaffolds has been developed in order to recapitulate the stem cell niche, among them hydrogels. This paper reports the development of a new silk–alginate based hydrogel with a focus on stem cell culture. This biocomposite allows to fine tune its elasticity during cell culture, addressing the importance of mechanotransduction during stem cell differentiation. The silk–alginate scaffold promotes adherence of mouse embryonic stem cells and cell survival upon transplantation. In addition, it has tunable stiffness as function of the silk–alginate ratio and the concentration of crosslinker – a characteristic that is very hard to accomplish in current hydrogels.     

A star-PEG–heparin hydrogel platform to aid cell replacement therapies for neurodegenerative diseases

Biofunctional matrices for in vivo tissue engineering strategies must be modifiable in both biomolecular composition and mechanical characteristics. To address this challenge, we present a modular system of biohybrid hydrogels based on covalently cross-linked heparin and star-shaped poly(ethylene glycols) (star-PEG) in which network characteristics can be gradually varied while heparin contents remain constant. Mesh size, swelling and elastic moduli were shown to correlate well with the degree of gel component cross-linking. Additionally, secondary conversion of heparin within the biohybrid gels allowed the covalent attachment of cell adhesion mediating RGD peptides and the non-covalent binding of soluble mitogens such as FGF-2. We applied the biohybrid gels to demonstrate the impact of mechanical and biomolecular cues on primary nerve cells and neural stem cells. The results demonstrate the cell type-specific interplay of synergistic signaling events and the potential of biohybrid materials to selectively stimulate cell fate decisions. These findings suggest important future uses for this material in cell replacement based-therapies for neurodegenerative diseases.     

Electrospun collagen–chitosan nanofiber: A biomimetic extracellular matrix for endothelial cell and smooth muscle cell

Electrospinning of collagen and chitosan blend solutions in a 1,1,1,3,3,3-hexafluoroisopropanol/trifluoroacetic acid (v/v, 90/10) mixture was investigated for the fabrication of a biocompatible and biomimetic nanostructure scaffold in tissue engineering. The morphology of the electrospun collagen–chitosan nanofibers was observed by scanning electron microscopy (SEM) and stabilized by glutaraldehyde (GTA) vapor via crosslinking. Fourier transform infrared spectra analysis showed that the collagen–chitosan nanofibers do not change significantly, except for enhanced stability after crosslinking by GTA vapor. X-ray diffraction analysis implied that both collagen and chitosan molecular chains could not be crystallized in the course of electrospinning and crosslinking, and gave an amorphous structure in the nanofibers. The thermal behavior and mechanical properties of electrospun collagen–chitosan fibers were also studied by differential scanning calorimetry and tensile testing, respectively. To assay the biocompatibility of electrospun fibers, cellular behavior on the nanofibrous scaffolds was also investigated by SEM and methylthiazol tetrazolium testing. The results show that both endothelial cells and smooth muscle cells proliferate well on or within the nanofiber. The results indicate that a collagen–chitosan nanofiber matrix may be a better candidate for tissue engineering in biomedical applications such as scaffolds.     

Injectable thermosensitive PEG–PCL–PEG hydrogel/acellular bone matrix composite for bone regeneration in cranial defects

We have certified that the injectable thermosensitive ABM/PECE composite presented promising potential in bone regeneration benefited from the incorporation of the intrinsic osteoinductive acellular bone matrix (ABM) granules into the poly(ethylene glycol)–poly(ε-capro-lactone)–poly(ethylene glycol) (PEG–PCL–PEG, PECE) hydrogel. In this study, the 12 mm × 8 mm × 2 mm cranial defects of the New Zealand white rabbits were fabricated to evaluate the bone regeneration effect. The ABM/PECE composite was injected into the defect while the pure PECE as control, and the bone regeneration was evaluated at 4, 12 and 20 weeks post-surgery by X-radiological examination, micro-computed tomography examination and histological analysis. In ABM/PECE composite treated group, the new bone formed originally from both the margin and the center of the defect, and the defect region had healed up to 20 weeks. Furthermore, the shadow density of the newly formed bone eventually approximated to host cortical bone. In comparison, the control group was filled with sparing new bone with low-density only from the periphery of the defect. Meanwhile, the quantitative determination of new bone by histomorphometry confirmed the excellent bone regeneration of ABM/PECE composite. All the results suggested that the ABM/PECE composite presented enhanced bone regeneration guidance in rabbit cranial defects.     

Acoustic droplet–hydrogel composites for spatial and temporal control of growth factor delivery and scaffold stiffness

Wound healing is regulated by temporally and spatially restricted patterns of growth factor signaling, but there are few delivery vehicles capable of the “on-demand” release necessary for recapitulating these patterns. Recently we described a perfluorocarbon double emulsion that selectively releases a protein payload upon exposure to ultrasound through a process known as acoustic droplet vaporization (ADV). In this study, we describe a delivery system composed of fibrin hydrogels doped with growth factor-loaded double emulsion for applications in tissue regeneration. Release of immunoreactive basic fibroblast growth factor (bFGF) from the composites increased up to 5-fold following ADV and delayed release was achieved by delaying exposure to ultrasound. Releasates of ultrasound-treated materials significantly increased the proliferation of endothelial cells compared to sham controls, indicating that the released bFGF was bioactive. ADV also triggered changes in the ultrastructure and mechanical properties of the fibrin as bubble formation and consolidation of the fibrin in ultrasound-treated composites were accompanied by up to a 22-fold increase in shear stiffness. ADV did not reduce the viability of cells suspended in composite scaffolds. These results demonstrate that an acoustic droplet–hydrogel composite could have broad utility in promoting wound healing through on-demand control of growth factor release and/or scaffold architecture.     

Core–shell microspheres by dispersion polymerization as promising delivery systems for proteins

Functional poly(methyl methacrylate) core–shell microspheres were prepared by dispersion polymerization. An appropriate selection of experimental parameters and in particular of the initiator and stabilizer amount and of the medium solvency power allowed a monodisperse sample as large as 600 nm to be prepared. To this purpose, low initiator concentration, high steric stabilizer amount and a low solvency power medium were employed. The microspheres present a core–shell structure in which the outer shell is constituted by the steric stabilizer which affords carboxylic groups able to interact with basic proteins, such as trypsin, whose adsorption is essentially driven by the carboxylic group density in the microsphere shell. Finally, fluorescent microspheres were prepared for biodistribution studies and shown to be readily taken up by the cells both in vitro and in vivo. These results suggest that these microspheres are promising delivery systems for the development of novel protein-based vaccines.     

Enzymatically crosslinked carboxymethylcellulose–tyramine conjugate hydrogel: Cellular adhesiveness and feasibility for cell sheet technology

An aqueous solution of carboxylmethylcellulose with phenolic hydroxyl groups (CMC-Ph) is gellable within 1 min via a peroxidase-catalyzed oxidative reaction under mild conditions suitable for mammalian cells. In this research, we evaluated cellular adhesion and proliferation on the resultant hydrogel, and the feasibility of the hydrogel as a substrate for cell sheet technology. Within 4 h of seeding, 76.9% of L929 fibroblast cells adhered to the gel and showed similar morphology of spreading to that on cell culture dish. Subsequently, the adherent cells proliferated on the gel and formed a confluent monolayer after 168 h of culture. From the confluent monolayer we could harvest a cell sheet after about 5 min of digestion of the gel using cellulase dissolved in medium at 5 U ml^?1. The cells in the cell sheet showed well-preserved morphology similar to that shown before they were detached from the gel. In addition, the harvested cell sheet readhered and proliferated after being transferred to another culture dish. These results demonstrate that CMC-Ph gel is a good candidate material for obtaining cell sheets.     

Continuous gradient scaffolds for rapid screening of cell–material interactions and interfacial tissue regeneration

In tissue engineering, the physical and chemical properties of the scaffold mediates cell behavior, including regeneration. Thus a strategy that permits rapid screening of cell–scaffold interactions is critical. Herein, we have prepared eight “hybrid” hydrogel scaffolds in the form of continuous gradients such that a single scaffold contains spatially varied properties. These scaffolds are based on combining an inorganic macromer (methacrylated star polydimethylsiloxane, PDMS_star-MA) and organic macromer (poly(ethylene glycol)diacrylate, PEG-DA) as well as both aqueous and organic fabrication solvents. Having previously demonstrated its bioactivity and osteoinductivity, PDMS_star-MA is a particularly powerful component to incorporate into instructive gradient scaffolds based on PEG-DA. The following parameters were varied to produce the different gradients or gradual transitions in: (1) the wt.% ratio of PDMS_star-MA to PEG-DA macromers, (2) the total wt.% macromer concentration, (3) the number average molecular weight (M_n) of PEG-DA and (4) the M_n of PDMS_star-MA. Upon dividing each scaffold into four “zones” perpendicular to the gradient, we were able to demonstrate the spatial variation in morphology, bioactivity, swelling and modulus. Among these gradient scaffolds are those in which swelling and modulus are conveniently decoupled. In addition to rapid screening of cell–material interactions, these scaffolds are well suited for regeneration of interfacial tissues (e.g. osteochondral tissues) that transition from one tissue type to another.     

Enzymatic conjugation of a bioactive peptide into an injectable hyaluronic acid–tyramine hydrogel system to promote the formation of functional vasculature

In this study, one-step enzyme-mediated preparation of a multi-functional injectable hyaluronic-acid-based hydrogel system is reported. Hydrogel was formed through the in situ coupling of phenol moieties by horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂), and bioactive peptides were simultaneously conjugated into the hydrogel during the gel formation process. The preparation of this multi-functional hydrogel was made possible by synthesizing peptides containing phenols which could couple with the phenol moieties of hyaluronic-acid–tyramine (HA–Tyr) during the HRP-mediated crosslinking reaction. Preliminary studies demonstrated that two phenol moieties per molecule resulted in a consistently high degree of conjugation into the HA–Tyr hydrogel network, unlike the one modified with one phenol moiety per molecule. Therefore, an Arg–Gly–Asp (RGD) peptide bearing two phenol moieties (phenol₂–poly(ethylene glycol)–RGD) was designed for conjugation to endow the HA–Tyr hydrogel with adhesion signals and enhance its bioactivities. Human umbilical vein endothelial cells (HUVECs) cultured on or within the RGD-modified hydrogels showed significantly different adhesion behavior, from non-adherence on the HA–Tyr hydrogel to strong adhesion on hydrogels modified with phenol₂–poly(ethylene glycol)–RGD. This altered cell adhesion behavior led to improved cell proliferation, migration and formation of capillary-like network in the hydrogel in vitro. More importantly, when HUVECs and human fibroblasts (HFF1) were encapsulated together in the RGD-modified HA–Tyr hydrogel, functional vasculature was observed inside the cell-laden gel after 2 weeks in the subcutaneous tissue. Taken together, the in situ conjugation of phenol₂–poly(ethylene glycol)–RGD into HA–Tyr hydrogel system, coupled with the ease of incorporating cells, offers a simple and effective means to introduce biological signals for preparation of multi-functional injectable hydrogels for tissue engineering application.     

The development of a nanocrystalline apatite reinforced crosslinked hyaluronic acid–tyramine composite as an injectable bone cement

We have developed an injectable bone cement composed of nanocrystalline apatite and crosslinked hyaluronic acid–tyramine conjugates (HA–Tyr). This bone cement was formed via the oxidative coupling of tyramine moieties catalyzed by hydrogen peroxide (H₂O₂) and horseradish peroxidise (HRP). The bone cement set within 60 s after H₂O₂ and HRP were added to the apatite/HA–Tyr pastes. The mechanical strength of the apatite/HA–Tyr cement was tuned by varying the apatite loading and H₂O₂ concentration. This rapid enzyme-mediated setting of our bone cement results in minimal heat release (ΔH = ?11.39 J/g) as compared to conventional bone cements. The crystalline phase and crystallite size (20 nm) of the apatitic phase in our bone cement matched that of trabecular bone. The storage modulus (G′), yield stress (σ_y), and compressive stiffness (E_c) of our bone cement prepared with different apatite loadings and H₂O₂ concentrations were measured, and optimized at G′ = 40 MPa, σ_y = 0.308 MPa and E_c = 2.270 MPa when the cement was formed with 0.4 g/ml of apatite, 0.61 units/ml of HRP and 6.8 mm of H₂O₂. Our biocompatible bone cement also successfully healed small bone and joint defects in mice within 8 weeks.     

PEG–PCL based micelle hydrogels as oral docetaxel delivery systems for breast cancer therapy

In this study, a composite drug delivery system was developed and evaluated for oral delivery of docetaxel: docetaxel-loaded micelles in pH-responsive hydrogel (DTX-micelle–hydrogel). Docetaxel was successfully loaded in micelles with small particle size of 20 nm and high drug loading of 7.76%, which contributed to the drug absorption in the intestinal tract. The experiments of cytotoxicity on 4T1 cells demonstrated the effective antitumor activity of DTX micelles. Meanwhile, a pH-responsive hydrogel was synthesized and optimized for incorporating the docetaxel micelles. The pH-responsiveness and reversibility of the hydrogel were investigated under the pH conditions of the gastrointestinal tract. Furthermore, the DTX-micelle–hydrogel system showed much quicker diffusion of micelles in simulated intestinal fluid than in simulated gastric fluid, which was mainly caused by the change of pH value. The docetaxel released from the micelle–hydrogel system quite slowly, so it had little influence on the absorption of DTX micelles in small intestine. More important, the pharmacokinetic study revealed that the DTX-micelle–hydrogel significantly improved the oral bioavailability of docetaxel (75.6%) about 10 times compared to DTX micelles, and this increase in bioavailability was probably due to the small intestine targeting release of the pH-responsive hydrogel. Consequently, the oral DTX-micelle–hydrogel system was effective in inhibiting tumor growth in subcutaneous 4T1 breast cancer model, and decreased systemic toxicity compared with intravenous treatment. The apoptosis cells in the immunofluorescent studies and the proliferation-positive cells in the immunohistochemical studies were also consistent with the results. Therefore, the DTX-micelle–hydrogel system might be a promising candidate oral drug for breast cancer therapy.