Photocrosslinkable polysaccharides for in situ hydrogel formation

In situ photopolymerization is an exciting new technique for tissue engineering. Two photocrosslinkable polysaccharides composed of alginate and hyaluronan are described that upon photolysis form soft, flexible, and viscoelastic hydrogels. The degree of methacrylate modification and thus covalent affects mechanical properties such as swelling, compression, and creep compliance. Significant swelling is observed in aqueous solution; these hydrogels can swell up to 14 times their dry weight. Both hydrogels exhibit low phase angles and (G*) values indicative of viscoelastic materials. The hyaluronan based hydrogel is stronger and more resilient than the corresponding alginate gel. SEM and AFM studies on both hydrogels show smooth and uniform surfaces at the macroscopic level with salient features observed only on the nanometer scale. Rapid polymerization by an optical trigger allows for controlled in situ photopolymerization in a minimally invasive manner, indicating that these hydrogels are relevant for biomedical applications such as sealing wounds and reconstructing soft tissues.     

Photocrosslinkable polyvinyl alcohol hydrogels that can be modified with cell adhesion peptides for use in tissue engineering

Photoactive polyvinyl alcohol hydrogels (PVA) have been investigated for use as tissue engineering scaffolds. These materials allow in situ polymerization for minimally invasive implantation methods. The mechanical properties of these materials can be tailored for a variety of soft tissue applications. The Young's modulus and ultimate tensile strength of PVA hydrogels are increased with increasing polymer concentration, and highly elastic hydrogels can be formed by altering the number of crosslinkable groups per chain. Fibroblasts homogeneously seeded within 3 mm thick PVA hydrogels remained viable throughout 2 weeks in culture, with no differences in viability across the thickness of the hydrogel. Cells seeded within the PVA hydrogels also produce extracellular matrix proteins, as indicated by the production of hydroxyproline during culture. Intrinsically cell non-adhesive, these PVA hydrogels were functionalized with the cell-adhesive peptide RGDS and found to support the attachment and spreading of fibroblasts in a dose-dependent manner. These results suggest that photopolymerizable PVA hydrogels are promising for tissue engineering applications.     

Smooth muscle cell growth in photopolymerized hydrogels with cell adhesive and proteolytically degradable domains: synthetic ECM analogs for tissue engineering

Photopolymerizable polyethylene glycol (PEG) derivatives have been investigated as hydrogel tissue engineering scaffolds. These materials have been modified with bioactive peptides in order to create materials that mimic some of the properties of the natural extracellular matrix (ECM). The PEG derivatives with proteolytically degradable peptides in their backbone have been used to form hydrogels that are degraded by enzymes involved in cell migration, such as collagenase and elastase. Cell adhesive peptides, such as the peptide RGD, have been grafted into photopolymerized hydrogels to achieve biospecific cell adhesion. Cells seeded homogeneously in the hydrogels during photopolymerization remain viable, proliferate, and produce ECM proteins. Cells can also migrate through hydrogels that contain both proteolytically degradable and cell adhesive peptides. The biological activities of these materials can be tailored to meet the requirements of a given tissue engineering application by creating a mixture of various bioactive PEG derivatives prior to photopolymerization.     

Versatile click alginate hydrogels crosslinked via tetrazine–norbornene chemistry

Alginate hydrogels are well-characterized, biologically inert materials that are used in many biomedical applications for the delivery of drugs, proteins, and cells. Unfortunately, canonical covalently crosslinked alginate hydrogels are formed using chemical strategies that can be biologically harmful due to their lack of chemoselectivity. In this work we introduce tetrazine and norbornene groups to alginate polymer chains and subsequently form covalently crosslinked click alginate hydrogels capable of encapsulating cells without damaging them. The rapid, bioorthogonal, and specific click reaction is irreversible and allows for easy incorporation of cells with high post-encapsulation viability. The swelling and mechanical properties of the click alginate hydrogel can be tuned via the total polymer concentration and the stoichiometric ratio of the complementary click functional groups. The click alginate hydrogel can be modified after gelation to display cell adhesion peptides for 2D cell culture using thiol-ene chemistry. Furthermore, click alginate hydrogels are minimally inflammatory, maintain structural integrity over several months, and reject cell infiltration when injected subcutaneously in mice. Click alginate hydrogels combine the numerous benefits of alginate hydrogels with powerful bioorthogonal click chemistry for use in tissue engineering applications involving the stable encapsulation or delivery of cells or bioactive molecules.     

The effect of photopolymerization on stem cells embedded in hydrogels

Photopolymerizable hydrogels, formed by UV-exposure of photosensitive polymers in the presence of photoinitiators, are widely used materials in tissue engineering research employed for cellular entrapment and patterning. During photopolymerization, the entrapped cells are directly exposed to polymer and photoinitiator molecules. To develop strategies that prevent potential photoexposure-damage to osteoprogenitor cells, it is important to further characterize the effects of photopolymerization on the exposed cells. In this study we analyzed the viability, proliferation and osteogenic differentiation of multipotent stromal cell (MSC) monolayers after exposure to UV-light in the presence of Irgacure 2959, a frequently used photoinitiator in tissue engineering research. Cell cycle progression, apoptosis and osteogenic differentiation of encapsulated goat MSCs were studied in photopolymerized methacrylate-derivatized hyaluronic acid hydrogel and methacrylated hyperbranched polyglycerol gel. We demonstrate adverse effects of photopolymerization on viability, proliferation and reentry into the cell cycle of the exposed cells in monolayers, whereas the MSCs retain the ability to differentiate towards the osteogenic lineage. We further show that upon encapsulation in photopolymerizable hydrogels the viability of the embedded cells is unaffected by the photopolymerization conditions, while osteogenic differentiation depends on the type of hydrogel used.     

A review of photocrosslinked polyanhydrides: in situ forming degradable networks

Many orthopaedic injuries could benefit from a high-strength and degradable material with good tissue compatibility. In addition, there is a great clinical need for materials which are easily contoured or placed into complex-shaped defects by a surgeon. We have rationally designed a new class of photocrosslinkable polyanhydride monomers which in situ form high-strength and surface eroding networks of complex geometries. This paper highlights the advantages of these materials for orthopaedic applications and the technique of photopolymerization for reacting these monomers under physiological conditions. The rationale for the material design, photopolymerization kinetics, degradation behavior, and histology in subcutaneous tissue and a model bone defect are presented.     

Fibrous protein-based hydrogels for cell encapsulation

Tissue scaffolds play a vital role in tissue engineering by providing a native tissue-mimicking environment for cells, with the aim to promote cell proliferation, proper cell differentiation, and regeneration. To better mimic the microenvironment of native tissues, novel techniques and materials have emerged in recent years. Among them, hydrogels formed from self-assembled biopolymer networks are particularly interesting. This paper reviews the fabrication and use of fibrous protein-based hydrogels, with an emphasis on silk, keratin elastin and resilin proteins. Hydrogels formed by these proteins show close structural, chemical and mechanical similarities with the extracellular matrix, typically good biological compatibility, and they can trigger specific cellular responses. In addition, these hydrogels can be degraded in the body by proteolytic enzymes. For these reasons, fibrous protein hydrogels are one of the most versatile materials for tissue engineering.     

The host response to naturally-derived extracellular matrix biomaterials

Biomaterials based on natural materials including decellularized tissues and tissue-derived hydrogels are becoming more widely used for clinical applications. Because of their native composition and structure, these biomaterials induce a distinct form of the foreign body response that differs from that of non-native biomaterials. Differences include direct interactions with cells via preserved moieties as well as the ability to undergo remodeling. Moreover, these biomaterials could elicit adaptive immune responses due to the presence of modified native molecules. Therefore, these biomaterials present unique challenges in terms of understanding the progression of the foreign body response. This review covers this response to natural materials including natural polymers, decellularized tissues, cell-derived matrix, tissue derived hydrogels, and biohybrid materials. With the expansion of the fields of regenerative medicine and tissue engineering, the current repertoire of biomaterials has also expanded and requires continuous investigation of the responses they elicit.     

Microengineered hydrogels for tissue engineering

Hydrogels have been extensively used in various biomedical applications such as drug delivery and biosensing. More recently the ability to engineer the size and shape of biologically relevant hydrogels has generated new opportunities in addressing challenges in tissue engineering such as vascularization, tissue architecture and cell seeding. Here, we discuss the use of microengineered hydrogels for tissue engineering applications. We will initially provide an overview of the various approaches that can be used to synthesize hydrogels with controlled features and will subsequently discuss the emerging applications of these hydrogels.     

Thixotrophy property of hydrogels to evaluate the cell growing on the inside of the material bulk (Amber effect)

The realization of injectable hydrogels is one of the most challenging aims of biomedical research. Most injectable hydrogels are obtained by photopolymerization in situ. A hydrogel which is able to pass through a needle without losing its structure once crosslinked would be of great interest for several biomedical applications. In this work, hyaluronane and alginate-based 50% hydrogels were synthesized, their thixotrophic behavior was verified and their mechanical properties (G' and G') were determined before and after the passage through the needle. A morphological analysis by scanning electron microscopy (SEM) was also performed in order to evaluate the effect of the passage of the hydrogel through the needle on the morphological structure of the material. The thixotrophic property of these hydrogels is used to realize a cell-containing material that supports cell proliferation and growth permitting in vivo engineering of new tissues. In fact, thixotrophic hydrogel can be sucked together with cell suspension and in this way cells remain entrapped inside the hydrogel structure (Amber effect). A comparison of the cell growth using this technique with the classical seeding technique (cell seeding onto the hydrogel) was performed. A different behavior was found between hyaluronane and alginate-based hydrogels.     

Novel thermosensitive chitosan hydrogels: in vivo evaluation

Chitosan is an attractive biopolymer for the preparation of hydrogels. Its unique combination of biocompatibility, biodegradability, bioadhesivity, and tissue-promoting abilities allows pharmaceutical applications. We investigated novel thermosensitive hydrogels based on chitosan homogeneously reacetylated to a deacetylation degree of about 50%, combined with selected polyols or polyoses such as trehalose, a nontoxic polysaccharide. The latter, a gel-inducing and lyoprotective agent enabled the formulation to be lyophilized and rehydrated without affecting the thermosensitive behavior. This made possible long-term storage and promoted its use in a clinical setup. The thermally induced sol-gel transition allowed injectability and in situ setting. Rheological characterization revealed that storage moduli could be increased by one decade by increasing the chitosan concentration from 1.4 to 2.2% (w/w). Evaluation in vivo provided evidence of in situ implant formation in subcutaneous tissue of Sprague-Dawley rats and permanence for up to 3 months. Histopathological analysis demonstrated a mild, chronic, inflammatory reaction that disappeared with the complete absorption of the gel implant over a few months period. Such in situ forming hydrogels could be advantageous for specific applications in drug delivery and tissue engineering.     

Transparent, elastomeric and tough hydrogels from poly(ethylene glycol) and silicate nanoparticles

The structures and mechanical properties of both physically and covalently cross-linked nanocomposite hydrogels made from poly(ethylene glycol) (PEG) and silicate nanoparticles (Laponite RD) are investigated. Injectable nanocomposite precursor solutions can be covalently cross-linked via photopolymerization. The resulting hydrogels are transparent and have interconnected pores, high elongation and toughness. These properties depend on the hydrogel composition, polymer-nanoparticle interactions and degree of cross-linking (both physical and covalent). Covalent cross-linking of polymer chains leads to the formation of an elastic network, whereas physical cross-linking between nanoparticles and polymer chains induces viscoelastic properties. At high deformations covalent bonds may be broken but physical bonds rebuild and to some extent self-heal the overall network structure. Addition of silicate also enhances the bioactivity and adhesiveness of the hydrogel as these materials stick to soft tissue as well as to hard surfaces. In addition, MC3T3-E1 mouse preosteoblast cells readily adhere and spread on nanocomposite hydrogel surfaces. Collectively, the combinations of properties such as elasticity, stiffness, interconnected network, adhesiveness to surfaces and bio-adhesion to cells provide inspiration and opportunities to engineer mechanically strong and elastic tissue matrixes for orthopedic, craniofacial and dental applications.     

Synthesis,properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels

Gelatin methacryloyl (GelMA) hydrogels have been widely used for various biomedical applications due to their suitable biological properties and tunable physical characteristics. GelMA hydrogels closely resemble some essential properties of native extracellular matrix (ECM) due to the presence of cell-attaching and matrix metalloproteinase responsive peptide motifs, which allow cells to proliferate and spread in GelMA-based scaffolds. GelMA is also versatile from a processing perspective. It crosslinks when exposed to light irradiation to form hydrogels with tunable mechanical properties. It can also be microfabricated using different methodologies including micromolding, photomasking, bioprinting, self-assembly, and microfluidic techniques to generate constructs with controlled architectures. Hybrid hydrogel systems can also be formed by mixing GelMA with nanoparticles such as carbon nanotubes and graphene oxide, and other polymers to form networks with desired combined properties and characteristics for specific biological applications. Recent research has demonstrated the proficiency of GelMA-based hydrogels in a wide range of tissue engineering applications including engineering of bone, cartilage, cardiac, and vascular tissues, among others. Other applications of GelMA hydrogels, besides tissue engineering, include fundamental cell research, cell signaling, drug and gene delivery, and bio-sensing.     

A biomimetic extracellular matrix for cartilage tissue engineering centered on photocurable gelatin,hyaluronic acid and chondroitin sulfate

The development of hydrogels tailored for cartilage tissue engineering has been a research and clinical goal for over a decade. Directing cells towards a chondrogenic phenotype and promoting new matrix formation are significant challenges that must be overcome for the successful application of hydrogels in cartilage tissue therapies. Gelatin–methacrylamide (Gel-MA) hydrogels have shown promise for the repair of some tissues, but have not been extensively investigated for cartilage tissue engineering. We encapsulated human chondrocytes in Gel-MA-based hydrogels, and show that with the incorporation of small quantities of photocrosslinkable hyaluronic acid methacrylate (HA-MA), and to a lesser extent chondroitin sulfate methacrylate (CS-MA), chondrogenesis and mechanical properties can be enhanced. The addition of HA-MA to Gel-MA constructs resulted in more rounded cell morphologies, enhanced chondrogenesis as assessed by gene expression and immunofluorescence, and increased quantity and distribution of the newly synthesized extracellular matrix (ECM) throughout the construct. Consequently, while the compressive moduli of control Gel-MA constructs increased by 26 kPa after 8 weeks culture, constructs with HA-MA and CS-MA increased by 114 kPa. The enhanced chondrogenic differentiation, distribution of ECM, and improved mechanical properties make these materials potential candidates for cartilage tissue engineering applications.     

Cell encapsulation in biodegradable hydrogels for tissue engineering applications

Encapsulating cells in biodegradable hydrogels offers numerous attractive features for tissue engineering, including ease of handling, a highly hydrated tissue-like environment for cell and tissue growth, and the ability to form in vivo. Many properties important to the design of a hydrogel scaffold, such as swelling, mechanical properties, degradation, and diffusion, are closely linked to the crosslinked structure of the hydrogel, which is controlled through a variety of different processing conditions. Degradation may be tuned by incorporating hydrolytically or enzymatically labile segments into the hydrogel or by using natural biopolymers that are susceptible to enzymatic degradation. Because cells are present during the gelation process, the number of suitable chemistries and formulations are limited. In this review, we describe important considerations for designing biodegradable hydrogels for cell encapsulation and highlight recent advances in material design and their applications in tissue engineering.     

Visible light crosslinkable chitosan hydrogels for tissue engineering

In situ gelling constructs, which form a hydrogel at the site of injection, offer the advantage of delivering cells and growth factors to the complex structure of the defect area for tissue engineering. In the present study, visible light crosslinkable hydrogel systems were presented using methacrylated glycol chitosan (MeGC) and three blue light initiators: camphorquinone (CQ), fluorescein (FR) and riboflavin (RF). A minimal irradiation time of 120 s was required to produce MeGC gels able to encapsulate cells with CQ or FR. Although prolonged irradiation up to 600 s improved the mechanical strength of CQ- or FR-initiated gels (compressive modulus 2.8 or 4.4 kPa, respectively), these conditions drastically reduced encapsulated chondrocyte viability to 5% and 25% for CQ and FR, respectively. Stable gels with 80-90% cell viability could be constructed using radiofrequency (RF) with only 40s irradiation time. Increasing irradiation time up to 300s significantly improved the compressive modulus of the RF-initiated MeGC gels up to 8.5 kPa without reducing cell viability. The swelling ratio and degradation rate were smaller at higher irradiation time. RF-photoinitiated hydrogels supported proliferation of encapsulated chondrocytes and extracellular matrix deposition. The feasibility of this photoinitiating system as in situ gelling hydrogels was further demonstrated in osteochondral and chondral defect models for potential cartilage tissue engineering. The MeGC hydrogels using RF offer a promising photoinitiating system in tissue engineering applications.     

In situ tissue engineering using magnetically guided three-dimensional cell patterning

Manipulation of cell patterns in three dimensions in a manner that mimics natural tissue organization and function is critical for cell biological studies and likely essential for successfully regenerating tissues--especially cells with high physiological demands, such as those of the heart, liver, lungs, and articular cartilage.(1, 2) In the present study, we report on the feasibility of arranging iron oxide-labeled cells in three-dimensional hydrogels using magnetic fields. By manipulating the strength, shape, and orientation of the magnetic field and using crosslinking gradients in hydrogels, multi-directional cell arrangements can be produced in vitro and even directly in situ. We show that these ferromagnetic particles are nontoxic between 0.1 and 10 mg/mL; certain species of particles can permit or even enhance tissue formation, and these particles can be tracked using magnetic resonance imaging. Taken together, this approach can be adapted for studying basic biological processes in vitro, for general tissue engineering approaches, and for producing organized repair tissues directly in situ.     

Production of heparin-containing hydrogels for modulating cell responses

Successful tissue regeneration requires that biomaterials have optimal bioactivity and mechanical properties. Heparin-containing hydrogels that can be crosslinked in situ were designed to contain tunable amounts of biological components (e.g. heparin, arginine–glycine–aspartate (RGD)) as well as to exhibit controlled mechanical properties (e.g. shear modulus). These gel parameters can also be tuned to provide controlled delivery of proteins, such as growth factors, for regulating cellular behavior. Maleimide-functionalized low-molecular-weight heparin (LWMH) was conjugated to a poly(ethylene glycol) (PEG) hydrogel. The elastic shear modulus, as assessed via oscillatory rheology experiments, could be tuned by the concentration of polymer in the hydrogel, and by the end group functionality of PEG. Hydrogels of two different moduli (2.8 and 0.4 kPa) were used to study differences in the response of human aortic adventitial fibroblasts (AoAF) in two-dimensional cell culture experiments. These experiments indicated that the AoAFs show improved adhesion to materials with the higher modulus. Evaluation of cell responses to hydrogels with RGD linked to the hydrogels via conjugation to PEG or to LMWH indicated improved cellular responses to these materials when the bioactive ligands were chemically attached through linkage to the PEG rather than to the LMWH. These results highlight important design considerations in the tailoring of these materials for cardiovascular tissue engineering applications.     

Engineering alginate as bioink for bioprinting

Recent advances in three-dimensional (3-D) printing offer an excellent opportunity to address critical challenges faced by current tissue engineering approaches. Alginate hydrogels have been used extensively as bioinks for 3-D bioprinting. However, most previous research has focused on native alginates with limited degradation. The application of oxidized alginates with controlled degradation in bioprinting has not been explored. Here, a collection of 30 different alginate hydrogels with varied oxidation percentages and concentrations was prepared to develop a bioink platform that can be applied to a multitude of tissue engineering applications. The authors systematically investigated the effects of two key material properties (i.e. viscosity and density) of alginate solutions on their printabilities to identify a suitable range of material properties of alginates to be applied to bioprinting. Further, four alginate solutions with varied biodegradability were printed with human adipose-derived stem cells (hADSCs) into lattice-structured, cell-laden hydrogels with high accuracy. Notably, these alginate-based bioinks were shown to be capable of modulating proliferation and spreading of hADSCs without affecting the structure integrity of the lattice structures (except the highly degradable one) after 8 days in culture. This research lays a foundation for the development of alginate-based bioink for tissue-specific tissue engineering applications.     

Synthesis of 3,4-dihydroxyphenylalanine (DOPA) containing monomers and their co-polymerization with PEG-diacrylate to form hydrogels

L-3,4-Dihydroxyphenylalanine (DOPA) is an unusual amino acid found in mussel adhesive proteins (MAPs) that is believed to lend adhesive characteristics to these proteins. Most previous efforts to incorporate DOPA into hydrogels have utilized oxidative cross-linking, which is hypothesized to reduce the adhesive properties of DOPA and requires reagents that are harmful to biological tissues. In this paper, we describe the synthesis of N-methacrylated DOPA monomers and their copolymerization with poly(ethylene glycol) diacrylate (PEG-DA) using either ultraviolet (UV) or visible light. The effect of DOPA containing monomers on gelation time, gel conversion and elastic modulus of the photocured hydrogels was investigated. Despite a retarding effect of DOPA on photopolymerization, DOPA was successfully incorporated into hydrogels with elastic moduli suitable for many biomedical applications. The incorporation of DOPA into hydrogels by photopolymerization may lead to new adhesive hydrogels for medical applications.