Injectable Radiopaque Hyaluronic Acid Granular Hydrogels for Intervertebral Disc Repair.

Injectable hydrogels offer minimally-invasive treatment options for degenerative disc disease, a prevalent condition affecting millions annually. Many hydrogels explored for intervertebral disc (IVD) repair suffer from weak mechanical integrity, migration issues, and expulsion. To overcome these limitations, an injectable and radiopaque hyaluronic acid granular hydrogel is developed. The granular structure provides easy injectability and low extrusion forces, while the radiopacity enables direct visualization during injection into the disc and non-invasive monitoring after injection. The radiopaque granular hydrogel is injected into rabbit disc explants to investigate restoration of healthy disc mechanics following needle puncture injury ex vivo and then delivered in a minimally-invasive manner into the intradiscal space in a clinically-relevant in vivo large animal goat model of IVD degeneration initiated through degradation by chondroitinase. The radiopaque granular hydrogel successfully halted loss of disc height due to degeneration. Further, the hydrogel not only enhanced proteoglycan content and reduced collagen content in the nucleus pulposus (NP) region compared to degenerative discs, but also helped to maintain the structural integrity of the disc and promote healthy segregation of the NP and annulus fibrosus regions. Overall, this study demonstrates the great potential of an injectable radiopaque granular hydrogel for treatment of degenerative disc disease.

Mechanical crosstalk between the intervertebral disc, facet joints, and vertebral endplate following acute disc injury in a rabbit model.

Background: Vertebral endplate sclerosis and facet osteoarthritis have been documented in animals and humans. However, it is unclear how these adjacent pathologies engage in crosstalk with the intervertebral disc. This study sought to elucidate this crosstalk by assessing each compartment individually in response to acute disc injury. Methods: Eleven New Zealand White rabbits underwent annular disc puncture using a 16G or 21G needle. At 4 and 10 weeks, individual compartments of the motion segment were analyzed. Discs underwent T 1 relaxation mapping with MRI contrast agent gadodiamide as well T 2 mapping. Both discs and facets underwent mechanical testing via vertebra-disc-vertebra tension-compression creep testing and indentation testing, respectively. Endplate bone density was quantified via µCT. Discs and facets were sectioned and stained for histology scoring. Results: Intervertebral discs became more degenerative with increasing needle diameter and time post-puncture. Bone density also increased in endplates adjacent to both 21G and 16G punctured discs leading to reduced gadodiamide transport at 10 weeks. The facet joints, however, did not follow this same trend. Facets adjacent to 16G punctured discs were less degenerative than facets adjacent to 21G punctured discs at 10 weeks. 16G facets were more degenerative at 4 weeks than at 10, suggesting the cartilage had recovered. The formation of severe disc osteophytes in 16G punctured discs between 4 and 10 weeks likely offloaded the facet cartilage, leading to the recovery observed. Conclusions: Overall, this study supports that degeneration spans the whole spinal motion segment following disc injury. Vertebral endplate thickening occurred in response to disc injury, which limited the diffusion of small molecules into the disc. This work also suggests that altered disc mechanics can induce facet degeneration, and that extreme bony remodeling adjacent to the disc may promote facet cartilage recovery through offloading of the articular cartilage.

Influence of Microgel and Interstitial Matrix Compositions on Granular Hydrogel Composite Properties.

Granular hydrogels are an emerging class of biomaterials formed by jamming hydrogel microparticles (i.e., microgels). These materials have many advantageous properties that can be tailored through microgel design and extent of packing. To enhance the range of properties, granular composites can be formed with a hydrogel interstitial matrix between the packed microgels, allowing for material flow and then stabilization after crosslinking. This approach allows for distinct compartments (i.e., microgels and interstitial space) with varied properties to engineer complex material behaviors. However, a thorough investigation of how the compositions and ratios of microgels and interstitial matrices influence material properties has not been performed. Herein, granular hydrogel composites are fabricated by combining fragmented hyaluronic acid (HA) microgels with interstitial matrices consisting of photocrosslinkable HA. Microgels of varying compressive moduli (10-70 kPa) are combined with interstitial matrices (0-30 vol.%) with compressive moduli varying from 2-120 kPa. Granular composite structure (confocal imaging), mechanics (local and bulk), flow behavior (rheology), and printability are thoroughly assessed. Lastly, variations in the interstitial matrix chemistry (covalent vs guest-host) and microgel degradability are investigated. Overall, this study describes the influence of granular composite composition on structure and mechanical properties of granular hydrogels towards informed designs for future applications.

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications.

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., "microgels"). In the field of biomaterials, granular hydrogels have many advantageous properties, including injectability, microscale porosity, and tunability by mixing multiple microgel populations. Methods to fabricate microgels often rely on water-in-oil emulsions (e.g., microfluidics, batch emulsions, electrospraying) or photolithography, which may present high demands in terms of resources and costs, and may not be compatible with many hydrogels. This work details simple yet highly effective methods to fabricate microgels using extrusion fragmentation and to process them into granular hydrogels useful for biomedical applications (e.g., 3D printing inks). First, bulk hydrogels (using photocrosslinkable hyaluronic acid (HA) as an example) are extruded through a series of needles with sequentially smaller diameters to form fragmented microgels. This microgel fabrication technique is rapid, low-cost, and highly scalable. Methods to jam microgels into granular hydrogels by centrifugation and vacuum-driven filtration are described, with optional post-crosslinking for hydrogel stabilization. Lastly, granular hydrogels fabricated from fragmented microgels are demonstrated as extrusion printing inks. While the examples described herein use photocrosslinkable HA for 3D printing, the methods are easily adaptable for a wide variety of hydrogel types and biomedical applications.

Sticking Together: Injectable Granular Hydrogels with Increased Functionality via Dynamic Covalent Inter-Particle Crosslinking.

Granular hydrogels are an exciting class of microporous and injectable biomaterials that are being explored for many biomedical applications, including regenerative medicine, 3D printing, and drug delivery. Granular hydrogels often possess low mechanical moduli and lack structural integrity due to weak physical interactions between microgels. This has been addressed through covalent inter-particle crosslinking; however, covalent crosslinking often occurs through temporal enzymatic methods or photoinitiated reactions, which may limit injectability and material processing. To address this, a hyaluronic acid (HA) granular hydrogel is developed with dynamic covalent (hydrazone) inter-particle crosslinks. Extrusion fragmentation is used to fabricate microgels from photocrosslinkable norbornene-modified HA, additionally modified with either aldehyde or hydrazide groups. Aldehyde and hydrazide-containing microgels are mixed and jammed to form adhesive granular hydrogels. These granular hydrogels possess enhanced mechanical integrity and shape stability over controls due to the covalent inter-particle bonds, while maintaining injectability due to the dynamic hydrazone bonds. The adhesive granular hydrogels are applied to 3D printing, which allows the printing of structures that are stable without any further post-processing. Additionally, the authors demonstrate that adhesive granular hydrogels allow for cell invasion in vitro. Overall, this work demonstrates the use of dynamic covalent inter-particle crosslinking to enhance injectable granular hydrogels.

Methods to Characterize Granular Hydrogel Rheological Properties, Porosity, and Cell Invasion.

Granular hydrogels are formed through the packing of hydrogel microparticles and are emerging for various biomedical applications, including as inks for 3D printing, substrates to study cell-matrix interactions, and injectable scaffolds for tissue repair. Granular hydrogels are suited for these applications because of their unique properties including inherent porosity, shear-thinning and self-healing behavior, and tunable design. The characterization of their material properties and biological response involves technical considerations that are unique to modular systems like granular hydrogels. Here, we describe detailed methods that can be used to quantitatively characterize the rheological behavior and porosity of granular hydrogels using reagents, tools, and equipment that are typically available in biomedical engineering laboratories. In addition, we detail methods for 3D cell invasion assays using multicellular spheroids embedded within granular hydrogels and describe steps to quantify features of cell outgrowth (e.g., endothelial cell sprouting) using standard image processing software. To illustrate these methods, we provide examples where features of granular hydrogels such as the size of hydrogel microparticles and their extent of packing during granular hydrogel formation are modulated. Our intent with this resource is to increase accessibility to granular hydrogel technology and to facilitate the investigation of granular hydrogels for biomedical applications.

Anisotropic Rod-Shaped Particles Influence Injectable Granular Hydrogel Properties and Cell Invasion.

Granular hydrogels have emerged as a new class of injectable and porous biomaterials that improve integration with host tissue when compared to solid hydrogels. Granular hydrogels are typically prepared using spherical particles and this study considers whether particle shape (i.e., isotropic spheres vs anisotropic rods) influences granular hydrogel properties and cellular invasion. Simulations predict that anisotropic rods influence pore shape and interconnectivity, as well as bead transport through granular assemblies. Photo-cross-linkable norbornene-modified hyaluronic acid is used to produce spherical and rod-shaped particles using microfluidic droplet generators and formed into shear-thinning and self-healing granular hydrogels, with particle shape influencing mechanics and injectability. Rod-shaped particles form granular hydrogels that have anisotropic and interconnected pores, with pore size and number influenced by particle shape and degree of packing. Robust in vitro sprouting of endothelial cells from embedded cellular spheroids is observed with rod-shaped particles, including higher sprouting densities and sprout lengths when compared to hydrogels with spherical particles. Cell and vessel invasion into granular hydrogels when injected subcutaneously in vivo are significantly greater with rod-shaped particles, whereas a gradient of cellularity is observed with spherical particles. Overall, this work demonstrates potentially superior functional properties of granular hydrogels with rod-shaped particles for tissue repair.

Influence of Microgel Fabrication Technique on Granular Hydrogel Properties.

Bulk hydrogels traditionally used for tissue engineering and drug delivery have numerous limitations, such as restricted injectability and a nanoscale porosity that reduces cell invasion and mass transport. An evolving approach to address these limitations is the fabrication of hydrogel microparticles (i.e., "microgels") that can be assembled into granular hydrogels. There are numerous methods to fabricate microgels; however, the influence of the fabrication technique on granular hydrogel properties is unexplored. Herein, we investigated the influence of three microgel fabrication techniques (microfluidic devices (MD), batch emulsions (BE), and mechanical fragmentation by extrusion (EF)) on the resulting granular hydrogel properties (e.g., mechanics, porosity, and injectability). Hyaluronic acid (HA) modified with various reactive groups (i.e., norbornenes (NorHA), pentenoates (HA-PA), and methacrylates (MeHA)) were used to form microgels with an average diameter of ∼100 µm. The MD method resulted in homogeneous spherical microgels, the BE method resulted in heterogeneous spherical microgels, and the EF method resulted in heterogeneous polygonal microgels. Across the various reactive groups, microgels fabricated with the MD and BE methods had lower functional group consumption when compared to microgels fabricated with the EF method. When microgels were jammed into granular hydrogels, the storage modulus (G') of EF granular hydrogels (∼1000-3000 Pa) was consistently an order of magnitude higher than G' for MD and BE granular hydrogels (∼50-200 Pa). Void space was comparable across all groups, although EF granular hydrogels exhibited an increased number of pores and decreased average pore size when compared to MD and BE granular hydrogels. Furthermore, granular hydrogel properties were tuned by varying the amount of cross-linker used during microgel fabrication. Lastly, granular hydrogels were injectable across formulations due to their general shear-thinning and self-healing properties. Taken together, this work thoroughly characterizes the influence of the microgel fabrication technique on granular hydrogel properties to inform the design of future systems for biomedical applications.

Chemically Modified Biopolymers for the Formation of Biomedical Hydrogels.

Biopolymers are natural polymers sourced from plants and animals, which include a variety of polysaccharides and polypeptides. The inclusion of biopolymers into biomedical hydrogels is of great interest because of their inherent biochemical and biophysical properties, such as cellular adhesion, degradation, and viscoelasticity. The objective of this Review is to provide a detailed overview of the design and development of biopolymer hydrogels for biomedical applications, with an emphasis on biopolymer chemical modifications and cross-linking methods. First, the fundamentals of biopolymers and chemical conjugation methods to introduce cross-linking groups are described. Cross-linking methods to form biopolymer networks are then discussed in detail, including (i) covalent cross-linking (e.g., free radical chain polymerization, click cross-linking, cross-linking due to oxidation of phenolic groups), (ii) dynamic covalent cross-linking (e.g., Schiff base formation, disulfide formation, reversible Diels-Alder reactions), and (iii) physical cross-linking (e.g., guest-host interactions, hydrogen bonding, metal-ligand coordination, grafted biopolymers). Finally, recent advances in the use of chemically modified biopolymer hydrogels for the biofabrication of tissue scaffolds, therapeutic delivery, tissue adhesives and sealants, as well as the formation of interpenetrating network biopolymer hydrogels, are highlighted.

Correction to: Cladribine Tablets: In Relapsing-Remitting Multiple Sclerosis.

During the creation of the HTML version of the article, incorrect body text (including all tables and figures) and an incorrect Additional Information section were uploaded; the PDF version was correct. The HTML version has now been corrected.

Efficient tuning of siRNA dose response by combining mixed polymer nanocarriers with simple kinetic modeling.

Two of the most prominent challenges that limit the clinical success of siRNA therapies are a lack of control over cargo release from the delivery vehicle and an incomplete understanding of the link between gene silencing dynamics and siRNA dosing. Herein, we address these challenges through the formulation of siRNA polyplexes containing light-responsive polymer mixtures, whose varied compositions and triggered release behavior provide enhanced gene silencing and controlled dose responses that can be predicted by simple kinetic models. Through the straightforward mixing of two block copolymers, the level of gene knockdown was easily optimized to achieve the maximum level of GAPDH protein silencing in NIH/3T3 cells (~70%) using a single siRNA dose. The kinetic model was used to describe the dynamic changes in mRNA and protein concentrations in response to siRNA treatment. These predictions enabled the application of a second dose of siRNA to maximally suppress gene expression over multiple days, leading to a further 50% reduction in protein levels relative to those measured following a single dose. Furthermore, polyplexes remained dormant in cells until exposed to the photo-stimulus, demonstrating the complete control over siRNA activity as well as the stability of the nanocarriers. Thus, this work demonstrates that pairing advances in biomaterials design with simple kinetic modeling provides new insight into gene silencing dynamics and presents a powerful strategy to control gene expression through siRNA delivery. STATEMENT OF SIGNIFICANCE: Our manuscript describes two noteworthy impacts: (1) we designed mixed polymer formulations to enhance gene silencing, and (2) we simultaneously developed a simple kinetic model for determining optimal siRNA dose responses to maintain silencing over several days. These advances address critical challenges in siRNA delivery and provide new opportunities in therapeutics development. The structure-function relationships prevalent in these formulations were established to enable tuning and forecasting of nanocarrier efficiency a priori, leading to siRNA dosing regimens able to maximally suppress gene expression. Our advances are significant because the mixed polymer formulations provide a straightforward and scalable approach to tailor siRNA delivery regimens. Moreover, the implementation of accurate dosing frameworks addresses a major knowledge gap that has hindered clinical implementation of siRNA.

Olmesartan medoxomil in children and adolescents with hypertension†: profile report.

†Adapted and reproduced from the original article published in Drugs 2010; 70 (18): 2439-47.

Steroids in villous atrophy: opening a can of worms?

A 60 year old man presented to the emergency department with septic shock and respiratory distress. He noted significant weight loss over the last 6 months with associated postprandial bloating. Endoscopy demonstrated partial villous atrophy. He had been on a gluten free diet, received empirical treatment for giardiasis and was receiving treatment with high dose systemic steroids for retroperitoneal fibrosis. This case discusses histology findings, differentials and reason for this acute presentation.

Botulinum toxin A (Dysport®): in dystonias and focal spasticity.

Dysport®, a formulation of botulinum toxin A, blocks acetylcholine release at neuromuscular junctions causing denervation and temporary muscle paralysis. It is used to treat several medical conditions, including dystonias and focal spasticity. Subcutaneous Dysport® was effective in improving functional disability in adults with blepharospasm in a placebo-controlled trial with 16 weeks' follow-up, and in adults with hemifacial spasm in case series. Similarly, intramuscular Dysport® was effective in improving symptoms of cervical dystonia in adults, focal spasticity in adults with post-stroke upper limb spasticity and dynamic equinus spasticity in paediatric patients with cerebral palsy in placebo-controlled trials with up to 20 weeks' follow-up. However, in two 12-week, placebo-controlled trials in adults with focal lower limb spasticity (spastic equinovarus deformity after stroke and hip adductor spasticity associated with multiple sclerosis) intramuscular Dysport® had limited efficacy. Available longer-term data indicated that Dysport® treatment was effective over several treatment cycles in patients with cervical dystonia or upper limb spasticity. Dysport® was generally well tolerated in patients with dystonias or focal spasticity. Most adverse events were mild to moderate and transient.

Denosumab: in the prevention of skeletal-related events in patients with bone metastases from solid tumours.

Denosumab, a fully human monoclonal antibody, binds to the receptor activator of nuclear factor-κB ligand (RANKL) and thereby inhibits RANKL-mediated bone resorption. In various individual countries, subcutaneous denosumab is indicated for the prevention of skeletal-related events in patients with bone metastases from solid tumours (featured indication), and/or for the treatment of postmenopausal osteoporosis and/or of cancer treatment-induced bone loss in prostate or breast cancer patients. In three, pivotal, double-blind, multinational trials in adult patients with cancer-related bone metastases (total n > 5700), including trials in patients with advanced breast or prostate cancer, subcutaneous denosumab (120 mg every 4 weeks) was shown to be noninferior to intravenous zoledronic acid (4 mg every 4 weeks), as determined by the median time to first on-study skeletal-related event (primary endpoint) at the time of the primary analysis (≈34 or 41 months). Denosumab treatment was superior to zoledronic acid in terms of the primary endpoint in two trials in patients with breast cancer or prostate cancer, based on secondary superiority analyses. In a third trial in patients with solid tumours excluding breast or prostate cancer, superiority of denosumab treatment versus zoledronic acid treatment was not demonstrated. The tolerability profile of denosumab was manageable in patients with bone metastases from solid tumours. Osteonecrosis of the jaw occurred in 1.8% and 1.3% of patients in the denosumab and zoledronic acid groups during the primary treatment phase; the incidence after approximately 4 additional months of denosumab treatment was 2.2%.

Erlotinib: as maintenance monotherapy in non-small-cell lung cancer.

Erlotinib is a low molecular weight, orally active, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. Inhibition of EGFR tyrosine kinase results in the disruption of processes involved in cancer growth and development, including cell migration, proliferation, angiogenesis, and apoptosis. In the well designed, phase III SATURN study, oral erlotinib 150 mg/day as maintenance treatment prolonged progression-free survival (PFS) in patients with non-small-cell lung cancer (NSCLC) who had not progressed after four cycles of first-line platinum doublet chemotherapy. PFS was significantly longer with erlotinib than with placebo in patients who were analyzable for PFS and in the subgroup of these patients with EGFR immunohistochemistry-positive tumors (co-primary endpoints). The improvement in PFS was independent of several baseline and clinical characteristics, including histology, smoking status, and EGFR mutation status, although a greater treatment benefit was observed in patients with tumors bearing EGFR-activating mutations than in those with wild-type EGFR tumors. Overall survival in the SATURN study was significantly longer with erlotinib than with placebo in the intent-to-treat population, in patients with EGFR immunohistochemistry-positive tumors, and in patients with wild-type EGFR tumors. Median overall survival had not yet been reached in patients with tumors bearing EGFR-activating mutations. Oral erlotinib as maintenance therapy was generally well tolerated in patients with NSCLC in the SATURN study and had a tolerability profile generally similar to that observed in a trial of erlotinib monotherapy as second-line treatment in patients with NSCLC.

Cladribine tablets: in relapsing-remitting multiple sclerosis.

Cladribine, an immunosuppressant that selectively reduces peripheral lymphocyte levels, has potential as an oral therapy for relapsing-remitting multiple sclerosis. An oral (tablet) formulation is being investigated in clinical trials. In the large, well designed, phase III CLARITY trial, short-course treatment with oral cladribine (cumulative dose of 3.5 or 5.25 mg/kg) resulted in a significantly greater reduction in annualized relapse rates at 96 weeks compared with placebo in patients with relapsing-remitting multiple sclerosis. Improvements in the annualized relapse rate with oral cladribine were independent of key baseline patient characteristics which included age, sex, previous treatment with disease-modifying drugs and the number of relapses in the previous 12 months. In addition, a significantly higher proportion of patients were relapse-free at 96 weeks and there were significant reductions in the risk of 3-month sustained progression of disability in cladribine recipients compared with placebo recipients. The mean numbers of brain lesions on magnetic resonance imaging were also significantly reduced with cladribine compared with placebo in the CLARITY trial. Lymphocytopenia, herpes zoster infections and neoplasms (including malignancies) were more common in cladribine than placebo recipients.

Olmesartan medoxomil: in children and adolescents with hypertension.

Olmesartan medoxomil is an orally administered angiotensin II receptor antagonist, selective for the angiotensin II type 1 receptor, which has established antihypertensive efficacy in adults. In children and adolescents with hypertension (n = 302), oral olmesartan medoxomil significantly and dose-dependently reduced seated systolic blood pressure (BP) and seated dystolic BP from baseline (the primary endpoint) in a 3-week, dose-response period in a well designed phase II/III clinical trial. Patients received olmesartan medoxomil high dose (20 or 40 mg once daily depending on bodyweight) or low dose (2.5 or 5.0 mg once daily depending on bodyweight). The response was significant for both cohorts, which were stratified by race (cohort A was mixed race [62% White] and cohort B was 100% Black). In addition, BP control was maintained in olmesartan recipients relative to placebo recipients in cohort A and the combined cohort A + B, but not for patients in cohort B, during a placebo-controlled withdrawal period of this trial. Oral olmesartan medoxomil was generally well tolerated in children and adolescents with hypertension. The majority of adverse events were of mild to moderate intensity.

Denosumab: in cancer treatment-induced bone loss.

Denosumab is a fully human monoclonal IgG(2) antibody that binds to receptor activator of nuclear factor-κB ligand (RANKL) and inhibits bone resorption due to RANKL-mediated osteoclastogenesis. In Europe, subcutaneous denosumab is indicated for cancer treatment-induced bone loss in men with prostate cancer and in postmenopausal women with breast cancer. In a large (n= 1468), well designed, multinational, phase III trial in adult patients with prostate cancer who were receiving androgen-deprivation therapy, bone mineral density (BMD) at the lumbar spine was significantly improved from baseline after 24 (primary endpoint) and 36 months of treatment with subcutaneous denosumab (60 mg once every 6 months), relative to that with placebo. Moreover, the risk of new vertebral fracture was significantly reduced by 62% in the denosumab group compared with the placebo group. In breast cancer patients receiving aromatase inhibitor therapy (n =252), subcutaneous denosumab (60 mg once every 6 months) significantly improved BMD at the lumbar spine from baseline after 12 (primary endpoint) and 24 months of treatment relative to placebo in a pivotal phase III trial. There were significant improvements in BMD at all skeletal sites, including the lumbar spine, total hip, and femoral neck, after 24 and 36 months' denosumab treatment in prostate cancer patients and after 12 and 24 months' treatment in breast cancer patients. In general, these improvements occurred irrespective of baseline characteristics, including age, duration of hormone ablation therapy, and baseline BMD. Denosumab treatment was generally well tolerated for up to 24 months in breast cancer patients and for up to 36 months in prostate cancer patients.