Glycosaminoglycans Hyaluronic Acid Research Articles

Influence of Anticoagulants and Heparin Contaminants on the Suitability of MMP-9 as a Blood-Derived Biomarker.

In contrast to other common anticoagulants such as citrate and low-molecular-weight heparin (LMWH), high-molecular-weight heparin (HMWH) induces the expression of matrix metalloproteinase (MMP)-9, which is also measured as a biomarker for stroke in blood samples. Mechanistically, HMWH-stimulated T cells produce cytokines that induce monocytic MMP-9 expression. Here, the influence of further anticoagulants (Fondaparinux, Hirudin, and Alteplase) and the heparin-contaminating glycosaminoglycans (GAG) hyaluronic acid (HA), dermatan sulfate (DS), chondroitin sulfate (CS), and over-sulfated CS (OSCS) on MMP-9 was analyzed to assess its suitability as a biomarker under various conditions. Therefore, starved Jurkat T cells were stimulated with anticoagulants/contaminants. Subsequently, starved monocytic THP-1 cells were incubated with the conditioned Jurkat supernatant, and MMP-9 mRNA levels were monitored (quantitative (q)PCR). Jurkat-derived mediators secreted in response to anticoagulants/contaminants were also assessed (proteome profiler array). The supernatants of HMWH-, Hirudin-, CS-, and OSCS-treated Jurkat cells comprised combinations of activating mediators and led to a significant (in the case of OSCS, dramatic) MMP-9 induction in THP-1. HA induced MMP-9 only in high concentrations, while LMWH, Fondaparinux, Alteplase, and DS had no effect. This indicates that depending on molecular weight and charge (but independent of anticoagulant activity), anticoagulants/contaminants provoke the expression of T-cell-derived cytokines/chemokines that induce monocytic MMP-9 expression, thus potentially impairing the diagnostic validity of MMP-9.

WNT5B drives osteosarcoma stemness, chemoresistance and metastasis.

Treatment for osteosarcoma, a paediatric bone cancer with no therapeutic advances in over three decades, is limited by a lack of targeted therapies. Osteosarcoma frequently metastasises to the lungs, and only 20% of patients survive 5 years after the diagnosis of metastatic disease. We found that WNT5B is the most abundant WNT expressed in osteosarcoma tumours and its expression correlates with metastasis, histologic subtype and reduced survival. Using tumor-spheroids to model cancer stem-like cells, we performed qPCR, immunoblotting, and immunofluorescence to monitor changes in gene and protein expression. Additionally, we measured sphere size, migration and forming efficiency to monitor phenotypic changes. Therefore, we characterised WNT5B's relevance to cancer stem-like cells, metastasis, and chemoresistance and evaluated its potential as a therapeutic target. In osteosarcoma cell lines and patient-derived spheres, WNT5B is enriched in stem cells and induces the expression of the stemness gene SOX2. WNT5B promotes sphere size, sphere-forming efficiency, and cell proliferation, migration, and chemoresistance to methotrexate (but not cisplatin or doxorubicin) in spheres formed from conventional cell lines and patient-derived xenografts. In vivo, WNT5B increased osteosarcoma lung and liver metastasis and inhibited the glycosaminoglycan hyaluronic acid via upregulation of hyaluronidase 1 (HYAL1), leading to changes in the tumour microenvironment. Further, we identified that WNT5B mRNA and protein correlate with the receptor ROR1 in primary tumours. Targeting WNT5B through inhibition of WNT/ROR1 signalling with an antibody to ROR1 reduced stemness properties, including chemoresistance, sphere size and SOX2 expression. Together, these data define WNT5B's role in driving osteosarcoma cancer stem cell expansion and methotrexate resistance and provide evidence that the WNT5B pathway is a promising candidate for treating osteosarcoma patients. WNT5B expression is high in osteosarcoma stem cells leading to increased stem cell proliferation and migration through SOX2. WNT5B expression in stem cells increases rates of osteosarcoma metastasis to the lungs and liver in vivo. The hyaluronic acid degradation enzyme HYAL1 is regulated by WNT5B in osteosarcoma contributing to metastasis. Inhibition of WNT5B with a ROR1 antibody decreases osteosarcoma stemness.

Hyaluronic Acid‐based Cell‐free Composite Scaffold Promotes Osteochondral Repair In Vitro by Upregulating Osteogenic‐ and Chondrogenic‐Specific Gene Expressions

Osteochondral defects pose a significant challenge in clinical practice, leading to increased medical care, diminished quality of life for patients, and elevated economic burden. The restoration of osteochondral defects, particularly in cartilage, is limited by its finite repair capacity and complex architecture. Treatment based on regenerative therapies acclaims the favorable alternative to contemporary procedures. Studies reveal that engineered biomaterials hold the significant importance in stimulating tissue repair with minimal cost and risks. In this study, a porous composite scaffold is developed, where host cells infiltrate and create the microenvironment for cellular adhesion and enhanced differentiation. Thus, a composite scaffold composed of natural glycosaminoglycan hyaluronic acid (HA), the protein component fibrin, bioceramic nanohydroxyapatite (nHAP), and graphene oxide (GO) is fabricated. Scanning electron microscopy observation and physiochemical characterization reveal an interconnected pore structure, optimum swelling potential, high porosity (80.14%), controlled biodegradation, high mechanical properties, and mineralization. Evaluation of scaffold's biocompatibility using osteoblast – like MG‐63 cells – shows adequate viability, cell adhesion, and osteoinductive potential. The upregulated expressions of osteogenic and chondrogenic genes OCN, ALP, COL1A1, ACAN, SOX9, and COL2A1 promote mineralization and extracellular matrix formation. These findings suggest that the composite scaffold HA‐GO‐F‐nHAP exhibits promising potential for osteochondral repair.

TSG-6-Mediated Extracellular Matrix Modifications Regulate Hypoxic-Ischemic Brain Injury.

Proteoglycans containing link domains modify the extracellular matrix (ECM) to regulate cellular homeostasis and can also sensitize tissues/organs to injury and stress. Hypoxic-ischemic (H-I) injury disrupts cellular homeostasis by activating inflammation and attenuating regeneration and repair pathways. In the brain, the main component of the ECM is the glycosaminoglycan hyaluronic acid (HA), but whether HA modifications of the ECM regulate cellular homeostasis and response to H-I injury is not known. In this report, employing both male and female mice, we demonstrate that link-domain-containing proteoglycan, TNFα-stimulated gene-6 (TSG-6), is active in the brain from birth onward and differentially modifies ECM HA during discrete neurodevelopmental windows. ECM HA modification by TSG-6 enables it to serve as a developmental switch to regulate the activity of the Hippo pathway effector protein, yes-associated protein 1 (YAP1), in the maturing brain and in response to H-I injury. Mice that lack TSG-6 expression display dysregulated expression of YAP1 targets, excitatory amino acid transporter 1 (EAAT1; glutamate-aspartate transporter) and 2 (EAAT2; glutamate transporter-1). Dysregulation of YAP1 activation in TSG-6-/- mice coincides with age- and sex-dependent sensitization of the brain to H-I injury such that 1-week-old neonates display an anti-inflammatory response in contrast to an enhanced proinflammatory injury reaction in 3-month-old adult males but not females. Our findings thus support that a key regulator of age- and sex-dependent H-I injury response in the mouse brain is modulation of the Hippo-YAP1 pathway by TSG-6-dependent ECM modifications.

Extrusion bioprinting of elastin‐containing bioactive double‐network tough hydrogels for complex elastic tissue regeneration

AbstractDespite recent advances in extrusion bioprinting of cell‐laden hydrogels, using naturally derived bioinks to biofabricate complex elastic tissues with both satisfying biological functionalities and superior mechanical properties is hitherto an unmet challenge. Here, we address this challenge with precisely designed biological tough hydrogel bioinks featuring a double‐network structure. The tough hydrogels consisted of energy‐dissipative dynamically crosslinked glycosaminoglycan hyaluronic acid (o‐nitrobenzyl‐grafted hyaluronic acid) and elastin through Schiff's base reaction, and free‐radically polymerized gelatin methacryloyl. The incorporation of elastin further improved the elasticity, stretchability (∼170% strain), and toughness (∼45 kJ m−3) of the hydrogels due to the random coiling structure. We used this novel class of hydrogel bioinks to bioprint several complex elastic tissues with good shape retention. Furthermore, in vitro and in vivo experiments also demonstrated that the existence of elastin in the biocompatible bioinks facilitated improved cell behaviors and biological functions of bioprinted tissues, such as cell spreading and phenotype maintenance as well as tissue regeneration. The results confirmed the potential of the elastin‐containing tough hydrogel bioinks for bioprinting of 3D complex elastic tissues with biological functionalities, which may find widespread applications in elastic tissue regeneration.

Engineering biomimetic scaffolds by combining silk protein nanofibrils and hyaluronic acid

Nanofibrous scaffolds mimicking important features of the native extracellular matrix (ECM) provide a promising strategy for tissue regeneration. However, 3D scaffolds mimicking natural protein nanofibers and bioactive glycosaminoglycans remain poorly developed. In this study, a biomimetic nanofibrous scaffold composed of natural silk protein nanofibers and glycosaminoglycan hyaluronic acid (HA) was developed. HA functionalization significantly improved the hydrophilicity and bioactivity of silk nanofibers (SNFs). SNFs can be assembled into nanofibrous aerogel scaffolds with low density and desirable shapes on a large scale. More importantly, with the assistance of HA, the silk nanofibrous aerogel scaffolds with ultra-high porosity, natural bioactivity, and structural stability in aqueous environment can be fabricated. In the protease/hyaluronidase solution, the SNF scaffolds with 10.0 % HA can maintain their monolithic shape for >3 weeks. The silk nanofibrous scaffolds not only imitate the composition of ECM but also mimic the hierarchical structure of ECM, providing a favorable microenvironment for cell adhesion and proliferation. These results indicate that this structurally and functionally biomimetic system is a promising tissue engineering scaffold.

Advances in the pharmacotherapeutic applications of hyaluronic acid: A comprehensive review

Natural non-sulfated glycosaminoglycan hyaluronic acid (HA), a biopolymer, has a variety of functions in controlling different biological processes. However, articular cartilage and synovial fluid contain more HA than other tissues and fluids. Over the past few decades, HA has been employed as a pharmacotherapeutic for a variety of conditions, such as osteoarthritis, cartilage regeneration, and wound healing. HA is used to create scaffold materials appropriate for tissue engineering because of its unique physicochemical characteristics. Recent cutting-edge research has shown that the molecular weight (Mw) of HA affects its efficacy. This paper provided a summary of recent developments in several pharmacotherapeutic applications and recommended research areas in order to improve HA activity.

Hyaluronic Acid in Periodontal Regeneration and Implant Dentistry-A Review

The glycosaminoglycan Hyaluronic Acid (HA) is present in the connective tissue of vertebrates. In the extracellular matrix of soft periodontal tissues, it is the most prevalent glycosaminoglycan with a higher molecular weight. In medical fields such as orthopaedics, dermatology, and ophthalmology, the use of HA in the treatment of inflammatory processes is well established. The extracellular matrix of various tissues, including connective tissue, synovial fluid, and other tissues, contains HA, a naturally occurring linear polysaccharide. Its efficacy in the treatment of inflammatory conditions has been proven. It has anti-inflammatory and anti-bacterial effects in the treatment of gingivitis and periodontitis in dentistry. It could be used as an adjunct to mechanical therapy in the treatment of periodontitis because of its tissue healing properties. Use of HA for implant surface modification has also been extensively studied. HA has proven to be effective in peri-implantitis. The purpose of this review paper is to explain HA’s involvement in periodontal therapy.

A Review of the Clinical Effectiveness and Safety of Hybrid Cooperative Complexes in Intra-articular Viscosupplementation.

Viscosupplementation by intra-articular (i.a.) injection of the non-sulfated glycosaminoglycan (GAG) hyaluronic acid (HA) is a conservative therapy widely accepted in clinical practice for the management of osteoarthritis (OA) and joint diseases. The aim of viscosupplementation is to restore the rheological properties of the synovial fluid to relieve joint inflammation and pain and improve joint function through a chondroprotective effect. However, there is a range of hyaluronic acid products for OA that differ in preparation, molecular weight, rheological characteristics and concentration, and different i.a. formulations are more suited to particular patient populations and clinical situations, in part because of anatomical differences between joints. This paper focuses on innovative hybrid cooperative complexes of high and low molecular weight hyaluronic acid (HA-HL) and hyaluronic acid plus sodium chondroitin (HA-SC) that have been developed. Both products are formulated with pharmaceutical-grade, highly purified hyaluronic acid obtained with a multi-step biofermentation process, with properties that make them suitable across a range of degenerative joint diseases. They represent progress in building on the symptomatic and functional benefits of viscosupplementation in joint disease, with the additional beneficial effect of treating the patient with a high concentration of GAGs by a low number of injections. Here, we review the clinical evidence for the efficacy of a hybrid cooperative compound of HA-HL in various degenerative joint diseases, which suggests a synergistic effect of the different molecular weight hyaluronans that together more closely mimic the physiological composition of synovial fluid. Similarly, the evidence shows that HA-SC is safe, effective, and well tolerated in hip OA, with rapid and clinically significant improvements in pain symptoms and functionality. Such innovations in viscosupplementation expand the usefulness of the modality in the management of OA and other joint diseases, complemented by a lack of systemic or local side effects that allow the concurrent use of other drugs if needed.

Glycosaminoglycans immobilized core-shell gold mesoporous silica nanoparticles for synergetic chemo-photothermal therapy of cancer cells

In this study, glycosaminoglycans hyaluronic acid (HA), heparin sulfate (HS) and heparin (HP) functionalized core-shell gold@mesoporous silica nanoparticles (Au@MSN) were fabricated. Their cellular behaviors were evaluated by culturing with GSH overexpressed tumor cells, which can cut off the disulfide bond between glycosaminoglycans (GAGs) and particles. Notably, Au@MSN@HS, Au@MSN@HP NPs could be swallowed by cells faster than Au@MSN@HA. Additionally, these GAGs functionalized nanoparticles possessed favorable photothermal effect under NIR laser irrradiation, contributing to a synergistic chemophotothermal treatment of cancer cells.

Liposomes embedded in layer by layer constructs as simplistic extracellular vesicles transfer model

Extracellular vesicles (EVs) are particles originating from the exfoliation of the cellular membrane. They are involved in cell-to-cell and cell-to-matrix signaling, exchange of bioactive molecules, tumorigenesis and metastasis, among others. To mitigate the limited understanding of EVs transfer phenomena, we developed a simplistic model that mimics EVs and their interactions with cells and the extracellular matrix. The proposed model is a layer by layer (LbL) film built from the polycationic poly-l-lysine (PLL) and the glycosaminoglycan hyaluronic acid (HA) to provide ECM mimicry. Positively charged 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and N1,N1,N14,N14-tetramethyl-N1,N14-ditetradecyltetradecane-1,14-diaminium dibromide (GS14) liposomes were embedded in this construct to act as EVs analogs. To simulate EVs carrying substances, Nile Red was loaded as a model of lipophilic cargo molecules. The integration of each component was followed by quartz crystal microbalance measurements, which confirmed the immobilization of intact liposomes on the underlying (PLL/HA)3 soft film. The release of Nile Red from liposomes either embedded in the LbL construct or exposed at its surface revealed a fast first order release. This system was validated as a model for EV/cell interactions by incubation with breast cancer cells MDA-MB-231. We observed higher internalization for embedded liposomes when compared with surface-exposed ones, showcasing that the ECM mimic layers do not constitute a barrier to liposome/cell interactions but favor them.

Evaluation of Hyaluronic Acid to Modulate Oral Squamous Cell Carcinoma Growth In Vitro.

Introduction: Previous studies have demonstrated that glycosaminoglycan hyaluronic acid (HA) is capable of mediating oral tumor growth. Some clinical evidence has suggested reduced HA expression predicts poor cancer prognosis and that HA-chemotherapy conjugates may function synergistically to inhibit oral tumor growth. Other studies have found conflicting results that suggest enhanced CD44-HA-mediated growth and proliferation. Due to the lack of clarity regarding HA function, the primary goal of this study was to investigate the effects of HA using well-characterized oral cancer cell lines. Methods: Using several commercially available oral squamous cell carcinoma lines (and a normal non-cancerous control), 96-well growth and viability assays were conducted using HA (alone and in combination with chemotherapeutic agents paclitaxel and PD98059). Results: Different results were observed in each of the cell lines evaluated. HA induced small, non-significant changes in cellular viability among each of the cell lines within a narrow range (1–8%), p = 0.207. However, HA induced differing effects on growth, with minimal, non-significant changes among some cell lines, such as SCC4 (+1.7%), CCL-30 (−2.8%), and SCC15 (−2.5%), p = 0.211 and more robust inhibition among other cell lines, SCC9 (−24.4%), SCC25 (−36.6%), and CAL27 (−47.8%), p = 0.0001. Differing effects were also observed with growth and viability under concomitant administration of HA with PD98059 or paclitaxel. Further analysis of these data revealed strong inverse (Pearson’s) correlations between initial baseline growth rate and responsiveness to HA administration, ranging from R = −0.27 to R = −0.883. Conclusion: The results of this study revealed differing responses to HA, which may be inversely correlated with intrinsic characteristics, such as the baseline growth rate. This may suggest that the more rapidly growing cell lines are more responsive to combination therapy with hyaluronic acid; an important finding that may provide insights into the mechanisms responsible for these observations.

In Vivo Imaging of the Buccal Mucosa Shows Loss of the Endothelial Glycocalyx and Perivascular Hemorrhages in Pediatric Plasmodium falciparum Malaria.

Severe malaria is mostly caused by Plasmodium falciparum, resulting in considerable, systemic inflammation and pronounced endothelial activation. The endothelium forms an interface between blood and tissue, and vasculopathy has previously been linked with malaria severity. We studied the extent to which the endothelial glycocalyx that normally maintains endothelial function is involved in falciparum malaria pathogenesis by using incident dark-field imaging in the buccal mucosa. This enabled calculation of the perfused boundary region, which indicates to what extent erythrocytes can permeate the endothelial glycocalyx. The perfused boundary region was significantly increased in severe malaria patients and mirrored by an increase of soluble glycocalyx components in plasma. This is suggestive of a substantial endothelial glycocalyx loss. Patients with severe malaria had significantly higher plasma levels of sulfated glycosaminoglycans than patients with uncomplicated malaria, whereas other measured glycocalyx markers were raised to a comparable extent in both groups. In severe malaria, the plasma level of the glycosaminoglycan hyaluronic acid was positively correlated with the perfused boundary region in the buccal cavity. Plasma hyaluronic acid and heparan sulfate were particularly high in severe malaria patients with a low Blantyre coma score, suggesting involvement in its pathogenesis. In vivo imaging also detected perivascular hemorrhages and sequestering late-stage parasites. In line with this, plasma angiopoietin-1 was decreased while angiopoietin-2 was increased, suggesting vascular instability. The density of hemorrhages correlated negatively with plasma levels of angiopoietin-1. Our findings indicate that as with experimental malaria, the loss of endothelial glycocalyx is associated with vascular dysfunction in human malaria and is related to severity.

TMIC-25. MODIFICATION OF EXTRACELLULAR MATRIX ENHANCES ONCOLYTIC ADENOVIRUS IMMUNOTHERAPY IN GLIOBLASTOMA

Abstract Complex interplay between heterogeneous cells and extracellular matrix (ECM) in the tumor microenvironment drives the pathogenesis of glioblastoma (GBM). Glycosaminoglycan hyaluronic acid (HA) is a major component of GBM ECM, and is associated with tumor growth, invasion, and resistance to treatments. Delta24-RGD oncolytic adenovirus is a powerful cancer therapeutic and is currently under clinical evaluation for GBM. We hypothesized that degradation of HA would enhance oncolytic adenovirus immunotherapy of GBM by overcoming the immunosuppressive functions of GBM ECM. Intratumoral injections of ICOVIR17, a delta24-RGD oncolytic adenovirus carrying hyaluronidase PH20 cDNA, increased animal survival, in comparison to its parental virus ICOVIR15, in an orthotopic GBM model generated with murine 005 stem-like GBM cells in immunocompetent C57/Bl6 mice. ICOVIR17 promoted the degradation of HA, and induced an increase in tumor infiltrating CD8+ T cells and macrophages, compared with ICOVIR15. This led to the upregulation of immune checkpoint PD-L1 on GBM cells and macrophages, while it downregulated immune checkpoints TIM3 and CTLA-4 on CD8+ T cells. The combination therapy of ICOVIR17 and systemic administration of anti-PD-1 antibody significantly prolonged the survival of mice bearing intracerebral GBM compared to no treatment control group and monotherapy groups, and achieved long-term remission in 21% of treated animals. Flow cytometry analysis showed that the combination treatment significantly increased the ratio of effector/regulatory (CD8/Treg) T cells in the brain, compared with all the other three groups. Furthermore, antibody and clodronate-mediated in vivo depletion of immune subsets revealed that CD4+, CD8+ T cells and macrophages were all necessary for combination therapy to be effective as any of these depletions abrogated efficacy. Our findings reveal that ICOVIR17-mediated degradation of HA within GBM dramatically alters the immune landscape of the tumor microenvironment, and offer a mechanistically rationale combination immunotherapy with PD-L1/PD-1 blockade that results in superior anti-GBM efficacy.

The role of versican in the skin ECM and its interaction with hyaluronic acid

The extracellular matrix (ECM) is a structural network that comprises the bulk of the tissues. It acts as a supporting scaffold for the cells to develop their function and plays an active role in many processes such as proliferation and migration. Therefore, the ECM is an interesting object of study for regenerative medicine. In this article we make an extensive review of two key components of the ECM: the glycosaminoglycan Hyaluronic Acid (HA) and the proteoglycan Versican (Ver). These two molecules are present in the skin ECM and play active roles in processes such as differentiation, wound healing, hair follicle cycle and development.

Impact of a Hyaluronic Acid-Grafted Layer on the Surface Properties of Model Silicone Hydrogel Contact Lenses

The introduction of high oxygen transmissibility silicone hydrogel lenses ameliorated hypoxia-related complications, making them the most prescribed type of contact lens (CL). Despite the progress made over the last 2 decades to improve their clinical performance, symptoms of ocular dryness and discomfort and a variety of adverse clinical events are still reported. Consequently, the rate of CL wear discontinuation has not been appreciably diminished by their introduction. Aiming to improve the interfacial interactions of silicone hydrogel CLs with the ocular surface, a biomimetic layer of hydrophilic glycosaminoglycan hyaluronic acid (HA) (100 kDa) was covalently attached to the surface of model poly(2-hydroxyethyl methacrylate- co-3-methacryloxypropyl-tris-(trimethylsiloxy)silane) (pHEMA- co-TRIS) silicone hydrogel materials via UV-induced thiol-ene "click" chemistry. The surface structural changes after each modification step were studied by Fourier transform infrared spectroscopy-attenuated total reflectance and X-ray photoelectron spectroscopy (XPS). Successful grafting of a homogeneous HA layer to the surface of the model silicone hydrogels was confirmed by the consistent appearance of N (1s) and the significant decrease of the Si (2p) peaks, as determined by low-resolution angle-resolved XPS. The HA-grafted surfaces demonstrated reduced contact angles, dehydration rate, and nonspecific deposition of lysozyme and albumin, while maintaining their optical transparency (>90%). In vitro studies demonstrated that the HA-grafted pHEMA- co-TRIS materials did not show any toxicity to human corneal epithelial cells. These results suggest that surface immobilization of HA via thiol-ene "click" chemistry can be used as a promising surface treatment for silicone hydrogel CLs.

The role of glycosaminoglycanes in the development of intimal hyperplasia in the shunting of coronary arteries

The article is devoted to the study of the role of glycosaminoglycans (hyaluronic acid, heparan sulfate derivatives, dermatan sulfate) in the genesis of intimal arterial hyperplasia. It is shown that these substances play important role in the structural reorganization of vessels, including the development of intimal hyperplasia of the arteries. Scientific works on the role of GAG in the genesis of intimal hyperplasia are based on experimental data. The authors draw attention to GAG involvement in cell-intercell nteractions of artery walls, including proliferation, migration, signal transduction of smooth muscle cells, endotheliocytes, platelets. Undoubtedly, attempts to create a 3D shunt with hyaluronate, atorvastatin can be used in the clinic for full and longterm use, which can prevent the development of restenosis of the arteries.

3D bioprinting of methacrylated hyaluronic acid (MeHA) hydrogel with intrinsic osteogenicity.

In bone regenerative medicine there is a need for suitable bone substitutes. Hydrogels have excellent biocompatible and biodegradable characteristics, but their visco-elastic properties limit their applicability, especially with respect to 3D bioprinting. In this study, we modified the naturally occurring extracellular matrix glycosaminoglycan hyaluronic acid (HA), in order to yield photo-crosslinkable hydrogels with increased mechanical stiffness and long-term stability, and with minimal decrease in cytocompatibility. Application of these tailor-made methacrylated hyaluronic acid (MeHA) gels for bone tissue engineering and 3D bioprinting was the subject of investigation. Visco-elastic properties of MeHA gels, measured by rheology and dynamic mechanical analysis, showed that irradiation of the hydrogels with UV light led to increased storage moduli and elastic moduli, indicating increasing gel rigidity. Subsequently, human bone marrow derived mesenchymal stromal cells (MSCs) were incorporated into MeHA hydrogels, and cell viability remained 64.4% after 21 days of culture. Osteogenic differentiation of MSCs occurred spontaneously in hydrogels with high concentrations of MeHA polymer, in absence of additional osteogenic stimuli. Addition of bone morphogenetic protein-2 (BMP-2) to the culture medium further increased osteogenic differentiation, as evidenced by increased matrix mineralisation. MeHA hydrogels demonstrated to be suitable for 3D bioprinting, and were printed into porous and anatomically shaped scaffolds. Taken together, photosensitive MeHA-based hydrogels fulfilled our criteria for cellular bioprinted bone constructs within a narrow window of concentration.

Radionuclide Esophageal Transit Scintigraphy in Primary Hypothyroidism.

Background/AimsEsophageal dysmotility is associated with gastrointestinal dysmotility in various systemic and neuroregulatory disorders. Hypothyroidism has been reported to be associated with impaired motor function in esophagus due to accumulation of glycosaminoglycan hyaluronic acid in its soft tissues, leading to changes in various contraction and relaxation parameters of esophagus, particularly in the lower esophageal sphincter. In this study we evaluated esophageal transit times in patients of primary hypothyroidism using the technique of radionuclide esophageal transit scintigraphy.MethodsThirty-one patients of primary hypothyroidism and 15 euthyroid healthy controls were evaluated for esophageal transit time using 15–20 MBq of Technetium-99m sulfur colloid diluted in 10–15 mL of drinking water. Time activity curve was generated for each study and esophageal transit time was calculated as time taken for clearance of 90% radioactive bolus from the region of interest encompassing the esophagus. Esophageal transit time of more than 10 seconds was considered as prolonged.ResultsPatients of primary hypothyroidism had a significantly increased mean esophageal transit time of 19.35 ± 20.02 seconds in comparison to the mean time of 8.25 ± 1.71 seconds in healthy controls (P < 0.05). Esophageal transit time improved and in some patients even normalized after treatment with thyroxine. A positive correlation (r = 0.39, P < 0.05) albeit weak existed between the serum thyroid stimulating hormone and the observed esophageal transit time.ConclusionsA significant number of patients with primary hypothyroidism may have subclinical esophageal dysmotility with prolonged esophageal transit time which can be reversible by thyroxine treatment. Prolonged esophageal transit time in primary hypothyroidism may correlate with serum thyroid stimulating hormone levels.

Monocytes/Macrophages Upregulate the Hyaluronidase HYAL1 and Adapt Its Subcellular Trafficking to Promote Extracellular Residency upon Differentiation into Osteoclasts.

Osteoclasts are giant bone-resorbing cells originating from monocytes/macrophages. During their differentiation, they overexpress two lysosomal enzymes, cathepsin K and TRAP, which are secreted into the resorption lacuna, an acidified sealed area in contact with bone matrix where bone degradation takes place. Here we report that the acid hydrolase HYAL1, a hyaluronidase able to degrade the glycosaminoglycans hyaluronic acid (HA) and chondroitin sulfate, is also upregulated upon osteoclastogenesis. The mRNA expression and protein level of HYAL1 are markedly increased in osteoclasts differentiated from RAW264.7 mouse macrophages or primary mouse bone marrow monocytes compared to these precursor cells. As a result, the HYAL1-mediated HA hydrolysis ability of osteoclasts is strongly enhanced. Using subcellular fractionation, we demonstrate that HYAL1 proteins are sorted to the osteoclast lysosomes even though, in contrast to cathepsin K and TRAP, HYAL1 is poorly mannose 6-phosphorylated. We reported previously that macrophages secrete HYAL1 proforms by constitutive secretion, and that these are recaptured by the cell surface mannose receptor, processed in endosomes and sorted to lysosomes. Present work highlights that osteoclasts secrete HYAL1 in two ways, through lysosomal exocytosis and constitutive secretion, and that these cells promote the extracellular residency of HYAL1 through downregulation of the mannose receptor. Interestingly, the expression of the other main hyaluronidase, HYAL2, and of lysosomal exoglycosidases involved in HA degradation, does not increase similarly to HYAL1 upon osteoclastogenesis. Taken together, these findings point out the predominant involvement of HYAL1 in bone HA metabolism and perhaps bone remodeling via the resorption lacuna.