Self-Healing Polymeric Hydrogel Formed by Metal-Ligand Coordination Assembly: Design, Fabrication, and Biomedical Applications.

Self-healing hydrogels based on metal-ligand coordination chemistry provide new and exciting properties that improve injectability, rheological behaviors, and even biological functionalities. The inherent reversibility of coordination bonds improves on the covalent cross-linking employed previously, allowing for the preparation of completely self-healing hydrogels. In this article, recent advances in the development of this class of hydrogels are summarized and their applications in biology and medicine are discussed. Various chelating ligands such as bisphosphonate, catechol, histidine, thiolate, carboxylate, pyridines (including bipyridine and terpyridine), and iminodiacetate conjugated onto polymeric backbones, as well as the chelated metal ions and metal ions containing inorganic particles, which are used to form dynamic networks, are highlighted. This article provides general ideas and methods for the design of self-healing hydrogel biomaterials based on coordination chemistry.

Enzymatic degradation of hyaluronan hydrogels with different capacity for in situ bio-mineralization.

In situ cross-linked hyaluronan (HA) hydrogels with different capacities for biomineralization were prepared and their enzymatic degradation was monitored. Covalent incorporation of bisphosphonates (BPs) into HA hydrogel results in the increased stiffness of the hydrogel in comparison with the unmodified HA hydrogel of the same cross-linking density. The rate of enzymatic degradation of HABP hydrogel was significantly lower than the rate of degradation of control HA hydrogel in vitro. This effect is observed only in the presence of calcium ions that strongly bind to the matrix-anchored BP groups and promote further mineralization of the matrix. The degradation of the hydrogels was followed by noninvasive fluorescence measurements enabled after mild and chemoselective labeling of cross-linkable HA derivatives with a fluorescent tag.

A Modular Approach to Sensitized Two-Photon Patterning of Photodegradable Hydrogels.

Photodegradable hydrogels have emerged as useful platforms for research on cell function, tissue engineering, and cell delivery as their physical and chemical properties can be dynamically controlled by the use of light. The photo-induced degradation of such hydrogel systems is commonly based on the integration of photolabile o-nitrobenzyl derivatives to the hydrogel backbone, because such linkers can be cleaved by means of one- and two-photon absorption. Herein we describe a cytocompatible click-based hydrogel containing o-nitrobenzyl ester linkages between a hyaluronic acid backbone, which is photodegradable in the presence of cells. It is demonstrated for the first time that by using a cyclic benzylidene ketone-based small molecule as photosensitizer the efficiency of the two-photon degradation process can be improved significantly. Biocompatibility of both the improved two-photon micropatterning process as well as the hydrogel itself is confirmed by cell culture studies.

Hyaluronic Acid Hydrogels Formed in Situ by Transglutaminase-Catalyzed Reaction.

Enzymatically cross-linked hydrogels can be formed in situ and permit highly versatile and selective tethering of bioactive molecules, thereby allowing for a wealth of applications in cell biology and tissue engineering. While a number of studies have reported the bioconjugation of extracellular matrix (ECM) proteins and peptides into such matrices, the site-specific incorporation of biologically highly relevant polysaccharides such as hyaluronic acid (HA) has thus far not been reported, limiting our ability to reconstruct this key feature of the in vivo ECM. Here we demonstrate a novel strategy for transglutaminase-mediated covalent linking of HA moieties to a synthetic poly(ethylene glycol) (PEG) macromer resulting in the formation of hybrid HA-PEG hydrogels. We characterize the ensuing matrix properties and demonstrate how these cytocompatible gels can serve to modulate the cellular phenotype of human mammary cancer epithelial cells as well as mouse myoblasts. The use of HA as a novel building block in the increasingly varied library of synthetic PEG-based artificial ECMs should have applications as a structural as well as a signaling component and offers significant potential as an injectable matrix for regenerative medicine.

Exploiting Bisphosphonate-Bioactive-Glass Interactions for the Development of Self-Healing and Bioactive Composite Hydrogels.

Hydrogels are widely recognized as promising candidates for various biomedical applications, such as tissue engineering. Recently, extensive research efforts have been devoted to the improvement of the biological and mechanical performance of hydrogel systems by incorporation of functional groups and/or inorganic particles in their composition. Bisphosphonates are a class of drugs, commonly used for treatment of osteoporosis, which exhibit a strong binding affinity for hydroxyapatite. In this study, the binding affinity of a bisphosphonate-functionalized polymer, hyaluronan, toward a bioactive glass (i.e., 45S5 Bioglass) is evaluated using force-distance measurements with atomic force microscopy. The strong interaction between bisphosphonate and bioactive glass is then exploited to develop organic-inorganic composite hydrogels and the viscoelastic and self-healing ability of these materials are investigated. Finally, the stability and mineralization behavior of these hydrogels are evaluated in simulated body fluid. Following this approach, injectable, bioactive and self-healing organic-inorganic composite hydrogels are produced, which mineralize abundantly and rapidly in simulated body fluid. These properties render these composite gels suitable for applications in bone-tissue engineering.

Functionalization of hyaluronic acid with chemoselective groups via a disulfide-based protection strategy for in situ formation of mechanically stable hydrogels.

Functionalization of hyaluronic acid (HA) with chemoselective groups enables in situ (in vivo) formation of HA-based materials in minimally invasive injectable manner. Current methods of HA modification with such groups primarily rely on the use of a large excess of a reagent to introduce a unique reactive handle into HA and, therefore, are difficult to control. We have developed the new protective group strategy based on initial mild cleavage of a disulfide bond followed by elimination of the generated 2-thioethoxycarbonyl moiety ultimately affording free amine-type functionality, such as hydrazide, aminooxy, and carbazate. Specifically, new modifying homobifunctional reagents have been synthesized that contain a new divalent disulfide-based protecting group. Amidation of HA with these reagents gives rise to either one-end coupling product or to intra/intermolecular cross-linking of the HA chains. However, after subsequent treatment of the amidation reaction mixture with dithiothreitol (DTT), these cross-linkages are cleaved, ultimately exposing free amine-type groups. The same methodology was applied to graft serine residues to the HA backbone, which were subsequently oxidized into aldehyde groups. The strategy therefore encompasses a new approach for mild and highly controlled functionalization of HA with both nucleophilic and electrophilic chemoselective functionalities with the emphasis for the subsequent conjugation and in situ cross-linking. A series of new hydrogel materials were prepared by mixing the new HA-aldehyde derivative with different HA-nucleophile counterparts. Rheological properties of the formed hydrogels were determined and related to the structural characteristics of the gel networks. Human dermal fibroblasts remained viable while cultured with the hydrogels for 3 days, with no sign of cytotoxicity, suggesting that the gels described in this study are candidates for use as growth factors delivery vehicles for tissue engineering applications.

Bisphosphonate nanoclay edge-site interactions facilitate hydrogel self-assembly and sustained growth factor localization.

Nanoclays have generated interest in biomaterial design for their ability to enhance the mechanics of polymeric materials and impart biological function. As well as their utility as physical cross-linkers, clays have been explored for sustained localization of biomolecules to promote in vivo tissue regeneration. To date, both biomolecule-clay and polymer-clay nanocomposite strategies have utilised the negatively charged clay particle surface. As such, biomolecule-clay and polymer-clay interactions are set in competition, potentially limiting the functional enhancements achieved. Here, we apply specific bisphosphonate interactions with the positively charged clay particle edge to develop self-assembling hydrogels and functionalized clay nanoparticles with preserved surface exchange capacity. Low concentrations of nanoclay are applied to cross-link hyaluronic acid polymers derivatised with a pendant bisphosphonate to generate hydrogels with enhanced mechanical properties and preserved protein binding able to sustain, for over six weeks in vivo, the localized activity of the clinically licensed growth factor BMP-2.

In situ cross-linkable high molecular weight hyaluronan-bisphosphonate conjugate for localized delivery and cell-specific targeting: a hydrogel linked prodrug approach.

We present here a novel synthesis route to functionalize high molecular weight hyaluronan (HMW-HA) with a hydrazide group and a bioactive ligand, namely bisphosphonate (BP). For this purpose, a new symmetrical self-immolative biscarbazate linker has been devised. The hydrazide group was used to form hydrazone cross-linked hydrogel upon treating with previously described aldehyde modified hyaluronan. The 1:1 weight ratio of these two polymers gave hydrogel in less than 30 s. In this communication we present the first in vitro results showing that even though HA can target CD44 positive cancer cells (HCT-116), receptor mediated endocytosis could only occur by cleavage of high molecular weight HA with an ubiquitous enzyme, hyaluronidase (Hase). The cancer cells are known to overexpress CD44 receptors and also increase the hyaluronidase activity in vivo. Thus the pro-drug design, based on drug conjugation to HMW-HA, represents a new drug delivery platform where the drug potency is triggered by Hase mediated degradation of the HA-drug conjugate. We have successfully demonstrated that the cross-linkable HA-BP conjugate first undergoes Hase-mediated scission to the fragments of suitable sizes so as to be internalized by CD44 positive cells. The specificity of this targeting was proven by comparing the results with less CD44 positive HEK-293T cells. The localized delivery of such drugs at the surgical resection site opens up avenues to control tumor recurrence after removal of the tumor. In the form of hydrogel it would prevent systemic exposure of the drug and would allow its controlled release.

Injectable hyaluronic acid hydrogels with the capacity for magnetic resonance imaging.

Monitoring hydrogel degradation in real time using noninvasive imaging techniques is of great interest for designing a scaffold in tissue engineering. We report the preparation of gadolinium (Gd)-labeled and injectable hyaluronic acid (HA) hydrogels that can be visualized using T1- and T2-weighted magnetic resonance imaging (MRI). An HA derivative functionalized with thiol and hydrazide was labeled using a diethylenetriaminepentaacetate complex modified with "clickable" dithiopyridyl functionalities (degree of modification was 3.77% with respect to HA repeat units). The HA derivative modified with cross-linkable groups and Gd complex exhibited relaxivities r1 = 3.78 mM-1s-1 and r2 = 56.3 mM-1s-1. A hydrazone hydrogel network was obtained by mixing Gd-labeled HA-hydrazide and HA-aldehyde derivatives. Enzymatic hydrogel degradation could be followed using MRI because the MR images showed great correlation with the hydrogel mass loss. Ex vivo MRI of injected Gd-labeled hydrogels demonstrated that they show a significant contrast difference (SNRcoronal = 456; SNRaxial = 459) from the surrounding tissues. These results indicate that our Gd-labeled HA hydrogel has great potential as an injectable biocompatible hydrogel that can be used for longitudinal tracking in vivo using MRI.

A new synthetic granular calcium phosphate compound induces new bone in a sinus lift rabbit model.

OBJECTIVES: The aim of this study was to investigate if a synthetic granular calcium phosphate compound (CPC) and a composite bisphosphonate-linked hyaluronic acid-calcium phosphate hydrogel (HABP·CaP) induced similar or more amount of bone as bovine mineral in a modified sinus lift rabbit model. MATERIAL AND METHODS: Eighteen adult male New Zeeland White rabbits, received randomly one of the two test materials on a random side of the face, and bovine mineral as control on the contralateral side. In a sinus lift, the sinus mucosa was elevated and a titanium mini-implant was placed in the alveolar bone. Augmentation material (CPC, HABP·CaP or bovine bone) was applied in the space around the implant. The rabbits were euthanized three months after surgery and qualitative and histomorphometric evaluation were conducted. Histomorphometric evaluation included three different regions of interest (ROIs) and the bone to implant contact on each installed implant. RESULTS: Qualitative assessment (p = <.05), histomorphometric evaluations (p = < .01), and implant incorporation (p = <.05) showed that CPC and bovine mineral induced similar amount of bone and more than the HABP·CaP hydrogel. CONCLUSION: CPC induced similar amount of bone as bovine mineral and both materials induced more bone than HABP·CaP hydrogel. CLINICAL SIGNIFICANCE: The CPC is suggested as a synthetic alternative for augmentations in the maxillofacial area.

Convergent in situ assembly of injectable lipogel for enzymatically controlled and targeted delivery of hydrophilic molecules.

Applications of liposomes are limited due to their rapid blood clearance and non-specific biodistribution. Surface modification of liposomes could overcome these disadvantages. However, direct coating of liposome surface may cause disruption of liposomes. Herein we present a "top-down" method to coat liposomes in situ with tumor (CD44 receptor) targeting polymer, hyaluronan (HA), by taking advantages of "click" type chemistries and enzymatic degradation. Liposomes entrapped within HA gel were stable without leaking of small cargo molecules from the interior of the liposomes. This injectable liposome-in-hydrogel (lipogel) drug delivery system can achieve sequential two-step release: (1) liposomes release from lipogel after HA degradation; (2) small molecules release from liposomes after the liposomes disruption (either before or after cellular uptake). Similarly to HA coating, this strategy could be used for in situ "top-down" modification of liposomes with other targeting biopolymers. Additionally, it provides the possibility to deliver different types of molecules from two compartments of the lipogel, i.e. large biomacromolecules from the exterior of liposomes and small hydrophilic molecules from the interior of liposomes, locally and systemically.

Bisphosphonate-modified biomaterials for drug delivery and bone tissue engineering.

INTRODUCTION: Bisphosphonates (BPs) were introduced 45 years ago as anti-osteoporotic drugs and during the last decade have been utilized as bone-targeting groups in systemic treatment of bone diseases. Very recently, strategies of chemical immobilization of BPs in hydrogels and nanocomposites for bone tissue engineering emerged. These strategies opened new applications of BPs in bone tissue engineering. AREAS COVERED: Conjugates of BPs to different drug molecules, imaging agents, proteins and polymers are discussed in terms of specific targeting to bone and therapeutic affect induced by the resulting prodrugs in comparison with the parent drugs. Conversion of these conjugates into hydrogel scaffolds is also presented along with the application of the resulting materials for bone tissue engineering. EXPERT OPINION: Calcium-binding properties of BPs can be successfully extended via different conjugation strategies not only for purposes of bone targeting, but also in supramolecular assembly affording either new nanocarriers or bulk nanocomposite scaffolds. Interaction between carrier-linked BPs and drug molecules should also be considered for the control of release of these molecules and their optimized delivery. Bone-targeting properties of BP-functionalized nanomaterials should correspond to bone adhesive properties of their bulk analogs.

Hydrogel patterning by diffusion through the matrix and subsequent light-triggered chemical immobilization.

A novel approach to hyaluronic acid (HA) hydrogel with a chemical gradient of the matrix-linked bisphosphonate (BP) groups is presented. The method consists of two steps, including initial generation of physical gradient patterns of BPs by diffusion of BP acrylamide reagent into HA matrix carrying thiol groups and subsequent chemical immobilization of the BP groups by UV light-triggered thiol-ene addition reaction. This gradient hydrogel permits spatial three-dimensional regulation of secondary interactions of different molecules with the polymer matrix. In particular, graded amounts of cytochrome c (cyt c) were reversibly absorbed in the hydrogel, thus enabling the subsequent spatially controlled release of the therapeutic protein. The obtained patterned hydrogel acts also as a unique reactor in which peroxidase-catalyzed oxidation of a substrate is determined by spatial position of the enzyme (cyt c) in the matrix resulting in a range of product concentrations. As an example, matrix template-assisted oxidation of 3,3',5,5'-tetarmethylbenzydine (TMB) in the presence of H2O2 occurs simultaneously at different rates within the gradient hydrogel. Moreover, calcium binding to the gradient HABP hydrogel reflects the pattern of immobilized BP groups eventually leading to the graded biomineralization of the matrix. This approach opens new possibilities for use of hydrogels as dynamic models for biologic three-dimensional structures such as extracellular matrix.

In situ forming interpenetrating hydrogels of hyaluronic acid hybridized with iron oxide nanoparticles.

Four derivatives of hyaluronic acid (HA) bearing thiol (HA-SH), hydrazide (HA-hy), 2-dithiopyridyl (HA-SSPy), and aldehyde groups (HA-al) respectively were synthesized. Thiol and 2-dithiopyridyl as well as hydrazide and aldehyde make up two chemically orthogonal pairs of chemo-selective functionalities that allow in situ formation of interpenetrating (IPN) disulfide and hydrazone networks simultaneously upon the mixing of the above derivatives at once. The formation of IPN was demonstrated by comparing it with the formulations of the same total HA concentration but lacking one of the reactive components. The hydrogel composed of all four components was characterized by a larger elastic modulus than those of the control single networks (either disulfide or hydrazone) and the three component formulations gave the softest hydrogels. Moreover, a hydrazone cross-linkage was designed to contain a 1,2-diol fragment. This allowed us to partially disassemble one type of network in the IPN leaving another one unaffected. In particular, treatment of the IPN with either sodium periodate or dithiothreitol resulted in disassembly of the hydrazone and disulfide networks respectively and thus softening of the hydrogel. Contrarily, the single network hydrogels completely dissolved under the corresponding conditions. In corroboration with this, enzymatic degradation of the IPN by hyaluronidase was also substantially slower than the degradation of the single networks. In order to further improve the mechanical properties of the elaborated injectable IPN, it has been in situ hybridized with iron oxide nanoparticles (IONPs). The mesh size of the IPN was smaller than the size of the IONPs resulting in the retention of nanoparticles in the matrix under equilibrium swelling conditions. However, these nanoparticles were released upon enzymatic degradation suggesting their use as MRI tags for non-invasive tracking of the hydrogel material in vivo. Additionally, this injectable hybridized hydrogel with encapsulated IONPs can be used in hyperthermia cancer therapy.

Bisphosphonate-functionalized hyaluronic acid showing selective affinity for osteoclasts as a potential treatment for osteoporosis.

Current treatments for osteoporosis involve the administration of high doses of bisphosphonates (BPs) over a number of years. However, the efficiency of the absorption of these drugs and specificity towards targeted osteoclastic cells is still suboptimal. In this study, we have exploited the natural affinity of high (H) and low (L) molecular-weight hyaluronic acid (HA) towards a cluster of differentiation 44 (CD44) receptors on osteoclasts to use it as a biodegradable targeting vehicle. We covalently bonded BP to functionalised HA (HA-BP) and found that HA-BP conjugates were highly specific to osteoclastic cells and reduced mature osteoclast numbers significantly more than free BP. To study the uptake of HA-BP, we fluorescently derivatised the polymer-drug with fluorescein B isothiocyanate (FITC) and found that L-HA-BP could seamlessly enter osteoclastic cells. Alternatively, we tested polyvinyl alcohol (PVA) as a synthetic polymer delivery vehicle using similar chemistry to link BP and found that osteoclast numbers did not reduce in the same way. These findings could pave the way for biodegradable polymers to be used as vehicles for targeted delivery of anti-osteoporotic drugs.

Rapid screening of mineralization capacity of biomaterials by means of quantification of enzymatically deposited calcium phosphate.

The current study focused on the development of a rapid, straightforward quantification method based on the use of enzymatic decomposition of urea using urease to assess the mineralization capacity of a wide range of biomaterials for bone regeneration. Urea-containing mineralizing solutions (MSs) (containing: Na2HPO4, CaCl2, and NaCl at 37°C and pH 6.0) were used in the mineralization experiments. Urease was added to these solutions to induce enzymatic decomposition of urea resulting in increased pH and deposition of calcium phosphate. By optimizing the ionic and urease concentrations in these MSs, it was shown that the proposed system could mineralize titanium substrates with six different pretreatments, as opposed to normal simulated body fluid that mineralized only two of them. It was possible to rank the mineralization capacity of these substrates by measuring the amount of calcium deposited. Furthermore, the ranking of (i) various polymeric substrates and (ii) hydrogels with and without functionalization with calcium-binding bisphosphonate groups was also possible. These results confirm that the proposed testing system has a broad applicability in the field of biomaterials due to its inherent versatility and discriminative power.

Self-healing hybrid nanocomposites consisting of bisphosphonated hyaluronan and calcium phosphate nanoparticles.

Non-covalent interactions are often regarded as insufficient to construct macroscopic materials of substantial integrity and cohesion. However, the low binding energy of such reversible interactions can be compensated by increasing their number to work in concert to create strong materials. Here we present the successful development of an injectable, cohesive nanocomposite hydrogel based on reversible bonds between calcium phosphate nanoparticles and bisphosphonate-functionalized hyaluronic acid. These nanocomposites display a capacity for self-healing as well as adhesiveness to mineral surfaces such as enamel and hydroxyapatite. Most importantly, these non-covalently cross-linked composites are surprisingly robust yet biodegradable upon extensive in vitro and in vivo testing and show bone interactive capacity evidenced by bone ingrowth into material remnants. The herein presented method provides a new methodology for constructing nanoscale composites for biomedical applications, which owe their integrity to reversible bonds.

Improving the osteogenic potential of BMP-2 with hyaluronic acid hydrogel modified with integrin-specific fibronectin fragment.

While human bone morphogenetic protein-2 (rhBMP-2) is a promising growth factor for bone regeneration, its clinical efficacy has recently shown to be below expectation. In order to improve the clinical translation of rhBMP-2, there exists strong motivation to engineer better delivery systems. Hyaluronic acid (HA) hydrogel is a suitable carrier for the delivery of rhBMP-2, but a major limitation of this scaffold is its low cell adhesive properties. In this study, we have determined whether covalent grafting of an integrin-specific ligands into HA hydrogel could improve cell attachment and further enhance the osteogenic potential of rhBMP-2. A structurally stabilized fibronectin (FN) fragment containing the major integrin-binding domain of full-length FN (FN III9*-10) was engineered, in order to be incorporated into HA hydrogel. Compared to non-functionalized HA hydrogel, HA-FN hydrogel remarkably improved the capacity of the material to support mesenchymal stem cell attachment and spreading. In an ectopic bone formation model in the rat, delivery of rhBMP-2 with HA-FN hydrogel resulted in the formation of twice as much bone with better organization of collagen fibers compared to delivering the growth factor in non-functionalized HA hydrogel. This engineered hydrogel carrier for rhBMP-2 can be relevant in clinical bone repair.

Calcium-mediated secondary cross-linking of bisphosphonated oligo(poly(ethylene glycol) fumarate) hydrogels.

This study demonstrates, for the first time, that synthetic PEG-based hydrogels can be cross-linked reversibly by calcium upon functionalization of the polymer backbone with bisphosphonate groups (BPs) that allow for the formation of strong coordination bonds with divalent metal ions such as Mg(2+) and Ca(2+). More specifically, it is shown that BP-functionalized hydrogels can be shaped by providing calcium ions as reversible physical cross-linkers.

Minimally invasive mandibular bone augmentation using injectable hydrogels.

Hyaluronic acid-based hydrogels are proven biocompatible materials and excellent carriers of bone morphogenetic protein-2 (BMP-2) that have been successfully tested for bone generation in vivo. Different formulations, with or without nanohydroxyapatite, have shown promise for craniofacial applications. In this study, 28 rats were used to investigate whether it is possible to achieve mandibular bone augmentation upon injection of novel hyaluronic acid-based hydrogels containing nanohydroxyapatite and different concentrations of BMP-2 (0, 5 and 150 µg/ml). The biomaterials were injected subperiosteally through fine needles into the innate mandibular diastema, imitating a clinical procedure for resorbed mandibles. No incisions, flaps or sutures were necessary. After 8 weeks the mandibles were evaluated by peripheral quantitative computed tomography (pQCT), micro-computed tomography (µCT), histology, immunohistochemistry and fluorochrome labelling. As a result, engineered bone was observed in all treated mandibles, with a statistically significant increase in mandibular bone volume correlated with the amount of BMP-2 loaded in the hydrogel formula. We therefore demonstrated that minimally invasive mandibular bone augmentation is possible upon injection in rats, when using the appropriate injectable scaffolds. This represents an attractive clinical alternative for oral implantology patients.