Self-assembling peptide nanofiber hydrogels for central nervous system regeneration

Central nervous system (CNS) presents a complex regeneration problem due to the inability of central neurons to regenerate correct axonal and dendritic connections. However, recent advances in developmental neurobiology, cell signaling, cell--matrix interaction, and biomaterials technologies have forced a reconsideration of CNS regeneration potentials from the viewpoint of tissue engineering and regenerative medicine. The applications of a novel tissue regeneration-inducing biomaterial and stem cells are thought to be critical for the mission. The use of peptide nanofiber hydrogels in cell therapy and tissue engineering offers promising perspectives for CNS regeneration. Self-assembling peptide undergo a rapid transformation from liquid to gel upon addition of counterions or pH adjustment, directly integrating with the host tissue. The peptide nanofiber hydrogels have mechanical properties that closely match the native central nervous extracellular matrix, which could enhance axonal growth. Such materials can provide an optimal three dimensional microenvironment for encapsulated cells. These materials can also be tailored with bioactive motifs to modulate the wound environment and enhance regeneration. This review intends to detail the recent status of self-assembling peptide nanofiber hydrogels for CNS regeneration.     

Macroporous hydrogels based on 2-hydroxyethyl methacrylate. Part 4: Growth of rat bone marrow stromal cells in three-dimensional hydrogels with positive and negative surface charges and in polyelectrolyte complexes

The growth of bone marrow stromal cells was assessed in vitro in macroporous hydrogels based on 2-hydro- xyethyl methacrylate (HEMA) copolymers with different electric charges. Copolymers of HEMA with sodium methacrylate (MA⁻) carried a negative electric charge, copolymers of HEMA with [2-(methacryloyloxy)ethyl] trimethylammonium chloride (MOETA⁻) carried a positive electric charge and terpolymers of HEMA, MA⁻ and MOETA⁺ carried both, positive and negative electric charges. The charges in the polyelectrolyte complexes were shielded by counter-ions. The hydrogels had similar porosities, based on a comparison of their diffusion parameters for small cations as measured by the real-time tetramethylammonium iontophoretic method of diffusion analysis. The cell growth was studied in the peripheral and central regions of the hydrogels at 2 hours and 2, 7, 14 and 28 days after cell seeding. Image analysis revealed the highest cellular density in the HEMA-MOETA⁺ copolymers; most of the cells were present in the peripheral region of the hydrogels. A lower density of cells but no difference between the peripheral and central regions was observed in the HEMA-MA⁻ copolymers and in polyelectrolyte complexes. This study showed that positively charged functional groups promote the adhesion of cells.     

Hyaluronic acid hydrogel immobilized with RGD peptides for brain tissue engineering

In this paper, hyaluronic acid hydrogels with open porous structure have been developed for scaffold of brain tissue engineering. A short peptide sequence of arginine–glycine–aspartic acid (RGD) was immobilized on the backbone of the hydrogels. Both unmodified hydrogels and those modified with RGD were implanted into the defects of cortex in rats and evaluated for their ability to improve tissue reconstruction. After 6 and 12 weeks, sections of brains were processed for DAB and Glees staining. They were also labeled with GFAP and ED1 antibodies, and observed under the SEM for ultrastructral examination. After implanting into the lesion of cortex, the porous hydrogels functioned as a scaffold to support cells infiltration and angiogenesis, simultaneously inhibitting the formation of glial scar. In addition, HA hydrogels modified with RGD were able to promote neurites extension. Our experiments showed that the hyaluronic acid-RGD hydrogel provided a structural, three-dimensional continuity across the defect and favoured reorganization of local wound-repair cells, angiogenesis and axonal growth into the hydrogel scaffold, while there was little evidence of axons regeneration in unmodified hydrogel.     

PEG-based bioresponsive hydrogels with redox-mediated formation and degradation

A hydrogel which will undergo macroscopic transition responding to redox stimuli is prepared. Mercapto precursors are prepared from 4-armed polyethylene glycol and after deprotection of thiolate anions, they can transform into disulfide crosslinked hydrogels within 3 min by responding to oxidant H₂O₂. Desirable elasticity is exhibited with a wide range of storage modulus from 50 Pa to 14 kPa through rheological investigation. In addition, the hydrogels are found to be hydrolytically stable but degrade within 75 days when exposed to reductant such as glutathione (GSH). So gelation time and degradation behavior can be regulated by concentrations of precursor, oxidant, reductant, temperature, and pH value. Notably, interest arises from the long-period degradation under low GSH concentration of 0.01 mM that is similar to extracellular level, but not the fast disintegration under high concentration intracellular, providing the possibility of “smart” degradation responding to those cell-secreted biomacromolecules during the process of tissue regeneration. Furthermore, both hydrogels and their degradation products show cell viability above 90% culturing with C2C12 cells, representing nontoxic properties. Such a stimuli-responsive degradation strategy will give promising application in tissue repair and regeneration; especially enable the achievement of matching the degradation kinetics with physiological environment.     

Surface-modified hyaluronic acid hydrogels to capture endothelial progenitor cells

A major challenge to the effective treatment of injured cardiovascular tissues is the promotion of endothelialization of damaged tissues and implanted devices. For this reason, there is a need for new biomaterials that promote endothelialization to enhance vascular repair. The goal of this work was to develop antibody-modified polysaccharide-based hydrogels that could selectively capture endothelial progenitor cells (EPCs). We showed that CD34 antibody immobilization on hyaluronic acid (HA) hydrogels provides a suitable surface to capture EPCs. The effect of CD34 antibody immobilization on EPC adhesion was found to be dependent on antibody concentration. The highest level of EPC attachment was found to be 52.2 cells per mm(2) on 1% HA gels modified with 25 μg mL(-1) antibody concentration. Macrophages did not exhibit significant attachment on these modified hydrogel surfaces compared to the EPCs, demonstrating the selectivity of the system. Hydrogels containing only HA, with or without immobilized CD34, did not allow for spreading of EPCs 48 h after cell seeding, even though the cells were adhered to the hydrogel surface. To promote spreading of EPCs, 2% (w/v) gelatin methacrylate (GelMA) containing HA hydrogels were synthesized and shown to improve cell spreading and elongation. This strategy could potentially be useful to enhance the biocompatibility of implants such as artificial heart valves or in other tissue engineering applications where formation of vascular structures is required.     

Hyaluronic acid hydrogels for biomedical applications

Hyaluronic acid (HA), an immunoneutral polysaccharide that is ubiquitous in the human body, is crucial for many cellular and tissue functions and has been in clinical use for over thirty years. When chemically modified, HA can be transformed into many physical forms-viscoelastic solutions, soft or stiff hydrogels, electrospun fibers, non-woven meshes, macroporous and fibrillar sponges, flexible sheets, and nanoparticulate fluids-for use in a range of preclinical and clinical settings. Many of these forms are derived from the chemical crosslinking of pendant reactive groups by addition/condensation chemistry or by radical polymerization. Clinical products for cell therapy and regenerative medicine require crosslinking chemistry that is compatible with the encapsulation of cells and injection into tissues. Moreover, an injectable clinical biomaterial must meet marketing, regulatory, and financial constraints to provide affordable products that can be approved, deployed to the clinic, and used by physicians. Many HA-derived hydrogels meet these criteria, and can deliver cells and therapeutic agents for tissue repair and regeneration. This progress report covers both basic concepts and recent advances in the development of HA-based hydrogels for biomedical applications.     

Directed cell growth and alignment on protein-patterned 3D hydrogels with stereolithography

The stereolithography apparatus (SLA) is a computer-assisted, three-dimensional (3D) printing system that is gaining attention in the medical field for the fabrication of patient-specific prosthetics and implants. An attractive class of implantable biomaterials for the SLA is photopolymerisable hydrogels because of their resemblance to soft tissues and intrinsic support of living cells. However, most laser-based SLA machines lack the minimum feature size required to imitate cell growth and alignment patterns in complex tissue architecture. In this study, we demonstrate a simple method for aligning cells on 3D hydrogels by combining the micro-contact printing (µCP) technique with the stereolithographic process. Fibronectin modified with acrylate groups was printed on glass coverslips with unpatterned, 10, 50, and 100 µm wide line patterns, which were then transferred to hydrogels through chemical linkages during photopolymerisation. Fibroblasts cultured on protein-printed 3D hydrogels aligned in the direction of the patterns, as confirmed by fast Fourier transform and cell morphometrics.     

Morphological evaluation of bioartificial hydrogels as potential tissue engineering scaffolds

Poly(vinyl alcohol) hydrogels prepared by freeze-thawing procedure represent synthetic systems widely investigated as non-biodegradable scaffolds for tissue regeneration. In order to improve the biocompatibility properties of pure poly(vinyl alcohol) (PVA) hydrogels, blends of PVA with different biological macromolecules, such hyaluronic acid, dextran, and gelatin were prepared and used to produce bioartificial hydrogels. The porosity characteristics of these hydrogels were investigated by scanning electron microscopy and mercury intrusion porosimetry. The morphology of bioartificial hydrogels was evaluated and compared with that of pure PVA hydrogels. In particular the effect exerted by each biological component on pore size and distribution was investigated. The obtained results indicate that when a natural macromolecule is added to PVA the internal structure of the material changes. A small amount of biopolymer induces the structural elements of PVA matrix to take on a well evident lamellar appearance and an apparent preferential orientation. Comparing the results of SEM and mercury intrusion porosimetry it was concluded that hydrogels containing 20% of biological component have the most regular structure and at the same time the lowest total porosity. On the contrary samples with the highest content of natural polymer (40%) show the less regular structure and the highest total porosity.     

Effect of composition on gelation kinetics of unfilled and nanoapatite-filled poly(lactide-ethylene oxide-fumarate) hydrogels

Oscillatory shear rheometry at small strains was used to investigate the effects of composition (concentration of macromer, crosslinker, initiator/accelerator, and filler) on the gel point and ultimate storage modulus of poly(l-lactide-co-ethylene oxide-co-fumarate) (PLEOF) hydrogels. PLEOF in situ polymerizing hydrogels can be used as biodegradable scaffolds for cell encapsulation and tissue regeneration. The time evolution of the viscoelastic properties of the polymerizing mixture during the sol-gel transition was monitored using mechanical rheometry. According to the results, while the ultimate storage modulus linearly increased with the concentration of methylene bisacrylamide (BISAM) crosslinker, the values of the storage and loss moduli at the gel point were insensitive to the concentration of BISAM. Similar behavior was observed for PLEOF hydrogels reinforced with hydroxyapatite nanoparticles.     

Adhesive Hemostatic Conducting Injectable Composite Hydrogels with Sustained Drug Release and Photothermal Antibacterial Activity to Promote Full‐Thickness Skin Regeneration During Wound Healing

Developing injectable nanocomposite conductive hydrogel dressings with multifunctions including adhesiveness, antibacterial, and radical scavenging ability and good mechanical property to enhance full‐thickness skin wound regeneration is highly desirable in clinical application. Herein, a series of adhesive hemostatic antioxidant conductive photothermal antibacterial hydrogels based on hyaluronic acid‐graft‐dopamine and reduced graphene oxide (rGO) using a H₂O₂/HPR (horseradish peroxidase) system are prepared for wound dressing. These hydrogels exhibit high swelling, degradability, tunable rheological property, and similar or superior mechanical properties to human skin. The polydopamine endowed antioxidant activity, tissue adhesiveness and hemostatic ability, self‐healing ability, conductivity, and NIR irradiation enhanced in vivo antibacterial behavior of the hydrogels are investigated. Moreover, drug release and zone of inhibition tests confirm sustained drug release capacity of the hydrogels. Furthermore, the hydrogel dressings significantly enhance vascularization by upregulating growth factor expression of CD31 and improve the granulation tissue thickness and collagen deposition, all of which promote wound closure and contribute to a better therapeutic effect than the commercial Tegaderm films group in a mouse full‐thickness wounds model. In summary, these adhesive hemostatic antioxidative conductive hydrogels with sustained drug release property to promote complete skin regeneration are an excellent wound dressing for full‐thickness skin repair.     

Biocompatibility of chemically cross-linked gelatin hydrogels for ophthalmic use

Biocompatibility is a major requirement for the development of functional biomaterials for ophthalmic applications. In this study, we investigated the effect of cross-linker functionality on ocular biocompatibility of chemically modified gelatin hydrogels. The test materials were cross-linked with glutaraldehyde (GTA) or 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC), and were analyzed using in vitro and in vivo assays. Primary rat iris pigment epithelial cultures were incubated with various gelatin discs for 2 days, and the cellular responses were monitored by cell proliferation, viability, and pro-inflammatory gene and cytokine expression. The results demonstrated that the cells exposed to EDC cross-linked gelatins had relatively lower lactate dehydrogenase activity, cytotoxicity, and interleukin-1β and tumor necrosis factor-α levels than did those to GTA treated samples. In addition, the gelatin implants were inserted in the anterior chamber of rabbit eyes for 12 weeks and characterized by clinical observations and scanning electron microscopy studies. The EDC cross-linked gelatin hydrogels exhibited good biocompatibility and were well tolerated without causing toxicity and adverse effects. However, a significant inflammatory reaction was elicited by the presence of GTA treated materials. It was noted that, despite its biocompatibility, the potential application of non-cross-linked gelatin for local delivery of cell and drug therapeutics would be limited due to rapid dissolution in aqueous environments. In conclusion, these findings suggest ocular cell/tissue response to changes in cross-linker properties. In comparison to GTA treatment, the EDC cross-linking is more suitable for preparation of chemically modified gelatin hydrogels for ophthalmic use.     

Synthesis and characterization of matrix metalloprotease sensitive-low molecular weight hyaluronic acid based hydrogels

Hyaluronic acid is a naturally derived glycosaminoglycan (GAG) involved in biological processes. A low molecular weight hyaluronic acid (50 kDa)-based hydrogel was synthesized using acrylated hyaluronic acid. Matrix metalloproteinase (MMP) sensitive hyaluronic acid-based hydrogels were prepared by conjugation with two different peptides: cell adhesion peptides containing integrin binding domains (Arg-Gly-Asp: RGD) and a cross-linker with MMP degradable peptides to mimic the remodeling characteristics of natural extracellular matrices (ECMs) by cell-derived MMPs. Mechanical properties of these hydrogels were evaluated with different molecular weights of acrylated hyaluronic acid (10 kDa and 50 kDa) cross-linked by MMP sensitive peptides by measuring elastic modulus, viscous modulus, swelling ratio and degradation rate. The MMP sensitive hydrogel based on the 50 kDa hyaluronic acid showed a 31.5-fold shorter gelation time, 4.7-fold higher storage modulus and 0.51-fold smaller swelling ratio than those of the hydrogel based on the 10 kDa. Degradation rate was dependent on MMP sensitivity of the peptide cross-linker. MMP sensitive hyaluronic acid based hydrogels were degraded faster than MMP insensitive-hyaluronic acid-based hydrogels. Human mesenchymal stem cells (MSCs) were cultured in MMP-sensitive or insensitive hyaluronic acid-based hydrogels (50 kDa hyaluronic acid) and/or immobilized cell adhesive RGD peptides. Cells cultured in the MMP-sensitive hydrogel with RGD peptides showed dramatic cell spreading compared with that of the control, which remained round. This MMP-sensitive low molecular weight hyaluronic acid-based hydrogel could be useful in tissue engineering by improving tissue defect regeneration and tissue remodeling.     

In vivo biocompatibility of radiation induced acrylamide and acrylamide/maleic acid hydrogels

In vitro swelling and in vivo biocompatibility of radiation induced acrylamide (AAm) and acrylamide/maleic acid (AAm/MA) hydrogels were investigated. The swelling kinetics of AAm and AAm/MA hydrogels of are investigated in distilled water, human serum and some simulated physiological fluids such as phosphate buffer at pH 7.4, glycine-HCl buffer at pH 1.1, physiological saline solution and, some swelling and diffusion parameters have been calculated. AAm and AAm/MA hydrogels were subcutaneously implanted in rats for up to 10 weeks and the tissue response to these implants was studied. Histological analysis indicated that tissue reaction at the implant site progressed from an initial acute inflammatory response characterized. No necrosis, tumorigenesis or infection was observed at the implant site up to 10 week. In vivo studies indicated that the radiation induced acrylamide and acrylamide/maleic acid hydrogels were found to be well-tolerated, non-toxic and highly biocompatible.     

Composite hydrogels for intervertebral disc prostheses

Different PHEMA/PCL semi-IPNs hydrogels and their relative composite systems reinforced with PET fibres have been investigated for potential use as intervertebral disc prostheses. Compression properties and water absorption were evaluated. Uniaxial compression tests on the swollen samples showed an increase of the modulus and maximum stress with increasing content of PCL and PET fibres. In particular, the composite PHEMA/PCL hydrogels showed compression properties similar to those expressed by the canine intervertebral discs in different spinal locations. The equilibrium water content of modified semi-IPNs decreased as function of the PCL and PET fibres. These tests indicate that the use of composite hydrogels as disc prostheses is very promising because it is possible to combine transport and mechanical properties which are crucial for the performance of the intervertebral disc.This paper was accepted for publication after the 1995 Conference of the European Society of Biomaterials, Oporto, Portugal, 10–13 September.     

Modulating the mechanical properties of photopolymerised polyethylene glycol–polypropylene glycol hydrogels for bone regeneration

Hydrogels formulated from single polymers are often insufficient in terms of their mechanical properties for use as bone substitute materials. Hence, hydrogels synthesised from combinations of polymers have been investigated to optimise the performance of such materials. In the current study, polypropylene glycol dimethacrylate was added to polyethylene glycol dimethacrylate of a variety of molecular weights and photopolymerised to form a series of hydrogels. Polyethylene glycol and polypropylene glycol have the same chemical structure with the exception of a methyl group on the later. Herein, the influence of the methyl group of polypropylene glycol on the mechanical properties of hydrogels for bone regeneration applications is reported. For both unconfined and cyclic compression testing, results demonstrated that the incorporation of PEGDMA into the precursor improves the compression strength of the hydrogels. For example, in unconfined compression tests the Young’s modulus varied between 6.62?±?0.31?MPa and 8.08?±?0.81?MPa with the incorporation of PEGDMA 400.     

Stiff gelatin hydrogels can be photo-chemically synthesized from low viscous gelatin solutions using molecularly functionalized gelatin with a high degree of methacrylation

Gelatin is a very promising matrix material for in vitro cell culture and tissue engineering, e.g. due to its native RGD content. For the generation of medical soft tissue implants chemical modification of gelatin improves the mechanical properties of gelatin hydrogels and the viscous behavior of gelatin solutions for liquid handling. We present a systematic study on the influence of high degrees of methacrylation on the properties of gelatin solutions and photo-chemically crosslinked hydrogels. Changes from shear thinning to shear thickening behavior of gelatin solutions were observed depending on mass fraction and degree of methacrylation. Degrees of swelling of crosslinked hydrogels ranged from 194 to 770?% and storage moduli G′ from 368 to 5?kPa, comparable to various natural tissues including several types of cartilage. Crosslinked gels proofed to be cytocompatible according to extract testings based on DIN ISO 10933-5 and in contact with porcine chondrocytes.     

Integration of hydrogels with hard and soft microstructures

Hydrogels, i.e., water-swollen polymer networks, have been studied and utilized for decades. These materials can either passively support mass transport, or can actively respond in their swelling properties, enabling modulation of mass and fluid transport, and chemomechanical actuation. Response rates increase with decreasing hydrogel dimension. In this paper, we present three examples where incorporation of hydrogels into solid microstructures permits acceleration of their response, and also provides novel functional capabilities. In the first example, a hydrogel is immobilized inside microfabricated pores within a thin silicon membrane. This hydrogel does not have a swelling response under the conditions investigated, but under proper conditions it can be utilized as a part of an electrolytic diode. In the second example, hydrogels are polymerized under microcantilever beams, and their swelling response to pH or glucose concentration causes variable deflection of the beam, observable under a microscope. In the third example, swelling and shrinking of a hydrogel embedded in a microfabricated valve structure leads to chemical gating of fluid motion through that valve. In all cases, the small size of the system enhances its response rate.     

Design of novel 3D gene activated PEG scaffolds with ordered pore structure

The ability to genetically modify cells seeded inside synthetic hydrogel scaffolds offers a suitable approach to induce and control tissue repair and regeneration guiding cell fate. In fact the transfected cells can act as local in vivo bioreactor, secreting plasmid encoded proteins that augment tissue regeneration processes. We have realized a DNA bioactivated high porous poly(ethylene glycol) (PEG) matrix by polyethyleneimine (PEI)/DNA complexes adsorption. As the design of the microarchitectural features of a scaffold also contributes to promote and influence cell fate, we appropriately designed the inner structure of gene activated PEG hydrogels by gelatine microparticles templating. Microarchitectural properties of the scaffold were analysed by scanning electron microscopy. 3D cell migration and transfection were monitored through time-lapse videomicroscopy and confocal laser scanning microscopy.     

Multifunctional Nanoengineered Hydrogels Consisting of Black Phosphorus Nanosheets Upregulate Bone Formation

Tissue‐engineered hydrogels have received extensive attention as their mechanical properties, chemical compositions, and biological signals can be dynamically modified for mimicking extracellular matrices (ECM). Herein, the synthesis of novel double network (DN) hydrogels with tunable mechanical properties using combinatorial screening methods is reported. Furthermore, nanoengineered (NE) hydrogels are constructed by addition of ultrathin 2D black phosphorus (BP) nanosheets to the DN hydrogels with multiple functions for mimicking the ECM microenvironment to induce tissue regeneration. Notably, it is found that the BP nanosheets exhibit intrinsic properties for induced CaP crystal particle formation and therefore improve the mineralization ability of NE hydrogels. Finally, in vitro and in vivo data demonstrate that the BP nanosheets, mineralized CaP crystal nanoparticles, and excellent mechanical properties provide a favorable ECM microenvironment to mediate greater osteogenic cell differentiation and bone regeneration. Consequently, the combination of bioactive chemical materials and excellent mechanical stimuli of NE hydrogels inspire novel engineering strategies for bone‐tissue regeneration.     

可降解水凝胶作为关节软骨修复材料的研究进展

可降解水凝胶因其良好的生物相容性和生物降解性被广泛用于关节软骨的修复和再生。本文以可降解水凝胶在软骨组织工程中的三类应用策略为主线,概述了用于原位成型可注射水凝胶的蛋白多糖类材料及纳米复合类材料;系统总结了传统工艺制造组织工程支架的优缺点及多种工艺结合的制备方法;重点归纳了近年来3D打印组织工程支架从纯软骨到骨/软骨一体化、从单层到多层的研究进展;最后分析了可降解水凝胶作为关节软骨支架材料在微观定向结构和生物活性功能化方面的局限性,并作出展望：未来开展多材料、多尺度、多诱导的高仿生梯度支架是关节软骨组织工程的一个重要研究方向。