HYADD®4 is a hexadecylamide derivative of Hyaluronan (HA) forming hydrogels with excellent lubricating and viscoelastic properties, widely used as viscosupplement in the treatment of knee OA. In this study, the effects of the intra-articular (i.a.) injection of FID-337, a ready-to-use formulation of HYADD®4 + Alendronate (ALN), and FID-338, a combined treatment based on HYADD®4 + Rapamycin (RAP), were evaluated on an OA model obtained by destabilization of the medial meniscus (DMM) performed in ovariectomized (OVX) female rats. Eight-week-old female Sprague Dawley rats were ovariectomized and after four weeks underwent DMM surgery on the right knee. Four weeks after, the animals were randomly divided into 4 groups of 9 rats each according to knee joint i.a. treatment, administered every 2 weeks (a total of 3 injections): FID-337, FID-338, HYADD®4 and saline; sham-operated rats (SHAM) were used as control. Mechanical allodynia was assessed weekly by the Von Frey test. Four weeks after the third i.a. injection rats were sacrificed. Microtomography (micro-CT) analysis was performed to assess bone volume, trabecular thickness and separation, and bone mineral density. Hematoxylin–eosin and safranin O/fast green staining were used to evaluate cartilage degradation, which was quantified through the OARSI score, and proteoglycan (PG) content. Withdrawal thresholds, which were low and unchanged over time in rats treated with saline, increased after the first injection of each treatment, reached levels of the SHAM group by the 10th week in the FID-338 group while continued to increase until the end of the study in the FID-337 group. The bone architecture was altered in NaCl-treated rats, and partially restored only after treatment with FID-337, FID-338 or HYADDÒ4. In the same groups, the synovial membrane was better preserved compared to the saline group. The OARSI (Fig.1) and PG loss scores were significantly lower in HYADD®4-treated joints and even further reduced significantly with FID-337 or FID-338 treatment. I.a. administration of HYADD®4 with ALN or RAP significantly impacts the OA progression, reducing pain and preventing cartilage damage in the OVX+DMM rat model, which describes a specific OA endotype. The local administration could maximize the local benefit while reducing the well known systemic side effects and this may lead to the development of a novel therapy with repurposed drugs combined with the HA-based viscosupplement. For any figures or tables, please contact the authors directly.

Photopolymerization is widely used in tissue engineering and biofabrication to pattern specific geometries and modulate physical properties. Commonly employed photochemistries rely on a photoinitiator that generates reactive free radicals when exposed to light, which can lead to cytotoxic effects due to interactions with biomolecules and cellular components. To mitigate these issues, we have developed hyaluronic acid and gelatin derivatives of umbelliferone, which can form dimers thanks to cyclobutene ring formation when exposed to long-wavelength UV light (365 nm). These reactions occur efficiently with reduced cytotoxicity and without the need of a photoinitiator. Ligation to the biopolymers was carried out with the incorporation of a triethylene glycol or n-octyl linker that enhances the conformational flexibility of umbelliferone and contributes to improve the rheological properties. By heat annealing these derivatives, we produced double network hydrogels with various compositions. We assessed their physical properties using rheological and uniaxial compression tests, evaluated their cytocompatibility by encapsulating articular chondrocytes, and conducted preliminary printability tests to determine their suitability for injection and extrusion-based biofabrication. The materials exhibited good cytocompatibility and cell adhesion, were successfully extrudable using a pneumatic bioprinter while maintaining cell viability and were compatible with regulatory-approved steam sterilization. Due to their unique properties, these umbelliferone derivatives are well-suited for tissue engineering and biofabrication applications, offering crucial advantages for future clinical translation. STATEMENT OF SIGNIFICANCE: This study introduces a method for preparing novel bioinks from coumarin derivatives of hyaluronic acid and gelatin, key biopolymers in tissue engineering. These derivatives enable photoinitiator-free photocrosslinking without generating free radicals, thereby reducing cytotoxic risks and facilitating easier clinical translation compared to existing approaches. They are compatible with steam sterilization and show promise for extrusion-based techniques like bioprinting and injectability. Through mechanical characterization and biological assessments, the interactions between the biopolymers at different ratios and their effect on encapsulated cells were studied, providing insights for optimizing future tissue engineering applications.

Purpose (the aim of the study): Therapeutic recombinant adeno-associated virus (rAAV) gene vectors have strong value to treat traumatic articular cartilage defects and osteoarthritic lesions, yet their safe application in patients remains challenging due to potential host immune responses against this class of viral vectors. In this regard, scaffold-guided rAAV gene transfer is an innovative strategy to overcome such a limitation by protecting the rAAV capsids from host humoral neutralization. Here, we examined the ability of a photopolymerizable biocompatible hyaluronic acid (HA) hydrogel to deliver rAAV vectors in human bone marrow-derived stromal cells (hMSCs) as a convenient, off-the-shelf system to modify such reparative cells for their safe implantation in cartilage lesions in patients in the future.

Articular cartilage defects represent an unsolved clinical challenge. Photopolymerizable hydrogels are attractive candidates supporting repair. This study investigates the short-term safety and efficacy of two novel hyaluronic acid (HA)-triethylene glycol (TEG)-coumarin hydrogels photocrosslinked in situ in a clinically relevant large animal model. It is hypothesized that HA-hydrogel-augmented microfracture (MFX) is superior to MFX in enhancing early cartilage repair, and that the molar degree of substitution and concentration of HA affects repair. Chondral full-thickness defects in the knees of adult minipigs are treated with either 1) debridement (No MFX), 2) debridement and MFX, 3) debridement, MFX, and HA hydrogel (30% molar derivatization, 30 mg mL(-1) HA; F3) (MFX+F3), and 4) debridement, MFX, and HA hydrogel (40% molar derivatization, 20 mg mL(-1) HA; F4) (MFX+F4). After 8 weeks postoperatively, MFX+F3 significantly improves total macroscopic and histological scores compared with all other groups without negative effects, besides significantly enhancing the individual repair parameters "defect architecture," "repair tissue surface" (compared with No MFX, MFX), and "subchondral bone" (compared with MFX). These data indicate that photopolymerizable HA hydrogels enable a favorable metastable microenvironment promoting early chondrogenesis in vivo. This work also uncovers a mechanism for effective HA-augmented cartilage repair by combining lower molar derivatization with higher concentrations.

Hyaluronic acid (HA) is frequently formulated in eye drops to improve the stability of the tear film by hydration and lubrication. Mucoadhesion is related to the ocular residence time and therefore to the effectiveness of the eye drops. The ocular residence time of the HA formulation is correlated with the ability of HA to create specific strong interactions in the ocular surface with the mucus layer, mainly composed of a mixture of secreted mucins (MUC; gel forming MUC5AC and MUC2) and shed membrane-bound soluble mucins (MUC1, MUC4, and MUC16). Dry eye disease (DED) is a multifactorial pathology of the preocular tear film with possible damage to the ocular surface classified in two types: (1) aqueous-deficient dry eye and (2) evaporative dry eye, caused by a decrease in goblet cell density that reduces MUC expression and/or by meibomian gland dysfunction, that results in a drop in the lipidic fraction of the tear film. In this work, the binding affinity between HA and MUC2 has been evaluated with three complementary approaches because the secreted MUCs play a pivotal role in the viscoelastic properties of the tear film: 1. Rheological analysis, measuring the mucoadhesive index and the complex viscosity in relation to MM (Molecular Mass) and concentration; 2. Fluorescence analysis, using a fluorescent hydrophobic probe, to investigate the conformational change of MUC2 during the interaction with the HA polymer; 3. Surface plasmon resonance analysis, used to measure the affinity between MUC2 (immobilized on the surface of a sensor chip) and the HA polymers that flowed on it at the molecular level. For all these tests, the mucoadhesive performance of the natural HA linearly increases with the MM, whereas cross-linked HA and other emollient and gelling agents (formulated in artificial tears) do not show the same mucoadhesive properties (with the exception of xanthan gum). The mucoadhesive performance of high MM HA has also been confirmed in conditions that simulate the pathological condition of the tear film during DED by decreasing the MUC2 or oleic acid concentration. Physico-chemical analysis of a series of marketed artificial tears confirms the linear correlation between the MM of the HA used in the products and the mucoadhesive index measured on the ocular surface model.

Despite several vaccines that are currently approved for human use to control the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an urgent medical need for therapeutic and prophylactic options. SARS-CoV-2 binding and entry in human cells involves interactions of its spike (S) protein with several host cell surface factors, including heparan sulfate proteoglycans (HSPGs), transmembrane protease serine 2 (TMPRSS2), and angiotensin-converting enzyme 2 (ACE2). In this paper we investigated the potential of sulphated Hyaluronic Acid (sHA), a HSPG mimicking polymer, to inhibit the binding of SARS-CoV-2 S protein to human ACE2 receptor. After the assessment of different sulfation degree of sHA backbone, a series of sHA functionalized with different hydrophobic side chains were synthesized and screened. The compound showing the highest binding affinity to the viral S protein was further characterized by surface plasmon resonance (SPR) towards ACE2 and viral S protein binding domain. Selected compounds were formulated as solutions for nebulization and, after being characterized in terms of aerosolization performance and droplet size distribution, their efficacy was assessed in vivo using the K18 human (h)ACE2 transgenic mouse model of SARS-CoV-2 infection.

Despite process similarities, distinctive manufacturing technologies offer hyaluronic acid dermal fillers with different in vitro physicochemical and rheological properties due to peculiar crosslinked hydrogel networks. A better understanding of dermal filler properties could provide specific clinical indications and expectations with more accurate performance correlations. In this study, with an emphasis on the degree of modification, hyaluronic acid concentration and molecular weight, these process parameters were able to modulate dermal filler properties, especially rheology. Moreover, an extensive characterization of commercial hyaluronic acid injectables of the Hyal System line was described to present product properties and help to elucidate related clinical effects. Standardized methodologies were applied to correlate in vitro parameters with feasible clinical indications. In view of an optimized dermal filler design, the results of the extrudability measurements allowed the quantification of the effect of hydrogel composition, rheological properties and needle size on injectability. Composition, dynamic viscosity and needle size showed an impactful influence on hydrogel extrudability. Finally, the positive influence of 200 KDa hyaluronic acid in comparison to fragments of ether-crosslinked hyaluronic acid on fibroblast recognition were shown with a migration assay.

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by pain and cartilage damage. Intra-articular (i.a) viscosupplementation with hyaluronic acid (HA) is frequently used for the management of OA. Preclinical studies have reported that bisphosphonates (BPs) may have a therapeutic potential to slow down or reverse the progression of OA. Among these, alendronate (ALN) has demonstrated chondroprotective effects in both in vitro and vivo experiments. This study evaluated the effects of a novel alendronate-hyaluronic acid (ALN-HA) conjugate on an OA in vivo model induced by medial meniscus destabilization (DMM). DMM surgery was performed on the knees of Sprague Dawley rats that received, after four weeks, one intra-articular (i.a.) injection of: (1) ALN-HA; (2) HA; (3) sodium chloride (NaCl). Sham-operated rats were used as control. Allodynia was assessed by Von Frey test. Joint degeneration was evaluated eight weeks after treatment by micro-computed tomography (micro-CT), histology, and immunohistochemistry. Collagen cross-linked C-telopeptides (CTX-I and CTX-II) serum levels were determined by ELISA. Paw withdrawal threshold increased in ALN-HA group when compared to rats treated with NaCl or HA. Micro-CT did not show differences between ALN-HA, HA and NaCl groups. ALN-HA injection produced significant improvements in articular cartilage degeneration showing an OARSI score lower than those of HA and NaCl, and reduced matrix metalloproteinase (MMP)-13, MMP-3, interleukin-6, vascular endothelial growth factor and Caspase-3 expression. CTX-I was reduced after ALN-HA treatment when compared to NaCl. Our results indicate that i.a. use of ALN after conjugation with HA limits OA development and progression in the rat DMM model, and may lead to the development of novel therapeutic strategies in OA management.

During osteoarthritis (OA) development, chondrocytes progressively decompensate, upregulating proteolytic enzymes and reducing the key growth factors involved in promoting chondrocyte anabolism. A combined therapeutic approach is needed to address this multifactorial pathology, which affects the whole joint. Based on the literature, three promising targets for OA treatment have been selected: MMP3 (matrix metallopepti-dase 3), TRPV4 (transient receptor potential cation channel subfamily V member 4) and mTOR (mammalian target of rapamycin). In this study, a novel water-soluble and biocompatible amphiphilic polymer named "sHA-oleylamide" was synthesized and screened from a series of hyaluronic acid derivatives for its anticata-bolic activity. This MMP inhibitor showed no cytotoxicity, and in an in vitro model of inflammatory OA, it reversed the inflammatory outcome at a concentration of 0.011 mg/mL. The ability of sHA-oleylamide to form 20-50 nm micelles in water with a critical micelle concentration of 0.27 +/- 0.1 mg/mL, was confirmed by TEM images and measured by Nile red staining. RN-1747 and rapamycin molecules were successfully loaded in sHA-oleylamide, previously prepared at 12 mg/mL in PBS; both formulations were stable, sterile and con-firmed in vitro to have mTOR inhibition by rapamycin and TRPV4 activation activity by RN-1747. The controlled release of RN-1747 from the micellar formulation with sHA-oleylamide showed that only approximately 60% of the total loaded RN-1747 was released within 7 days. These micellar formulations can potentially increase the bioavailability and pharmaceutical efficacy of the selected active molecules, combin-ing their anti-catabolic and pro-anabolic activities and making them suitable for i.a. administration as OA treatments.(c) 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.

Hyaluronan (HA) is a component of the extracellular matrix (ECM) it is the main non-sulfated glycosaminoglycan able to modulate cell behavior in the healthy and tumor context. Sulfated hyaluronan (sHA) is a biomaterial derived from chemical modifications of HA, since this molecule is not naturally sulfated. The HA sulfation modifies several properties of the native molecule, acquiring antitumor properties in different cancers. In this study, we evaluated the action of sHA of similar to 30-60 kDa with different degrees of sulfation (0.7 sHA1 and 2.5 sHA3) on tumor cells of a breast, lung, and colorectal cancer model and its action on other cells of the tumor microenvironment, such as endothelial and monocytes/macrophage cells. Our data showed that in breast and lung tumor cells, sHA3 is able to modulate cell viability, cytotoxicity, and proliferation, but no effects were observed on colorectal cancer cells. In 3D cultures of breast and lung cancer cells, sHA3 diminished the size of the tumorsphere and modulated total HA levels. In these tumor models, treatment of monocytes/ macrophages with sHA3 showed a downregulation of the expression of angiogenic factors. We also observed a decrease in endothelial cell migration and modulation of the hyaluronan-binding protein TSG-6. In the breast in vivo xenograft model, monocytes/macrophages preincubated with sHAl or sHA3 decreased tumor vasculature, TSG-6 and HA levels. Besides, in silico analysis showed an association of TSG-6, HAS2, and IL-8 with biological processes implicated in the progression of the tumor. Taken together, our data indicate that sHA in a breast and lung tumor context is able to induce an antiangiogenic action on tumor cells as well as in monocytes/macrophages (Mo/Mempty set) by modulation of endothelial migration, angiogenic factors, and vessel formation.

Purpose: Intra-articular injections of hyaluronic acid (HA) are widely used to treat osteoarthritis (OA). HYADD®4 (HS), a hexadecylamide derivative of HA, has demonstrated in preclinical studies greater beneficial effects to those of unmodified hyaluronans. Recently, in vivo experiments have demonstrated that bisphosphonates may have a therapeutic potential to attenuate the progression of OA. Among these, alendronate (ALN) has been shown to decrease MMP-13 and ADAMTS-5 expression and increase COL2A1 mRNA levels in in vitro OA models.

Objective:Osteoarthritis (OA) is a painful degenerative disease of the whole joint structure, including articular cartilage, synovial fluid, and subchondral bone. Hyaluronic acid (HA), an anionic non-sulfated glycosaminoglycan, is commonly used for intra-articular (IA) treatment in OA, while bisphosphonates (BPs) are anti-resorptive drugs that act on the bone. Here, a novel conjugate with a covalent and hydrolysable linker between HA and alendronate (ALD) was designed as an attractive therapeutic strategy for IA drug delivery.Design:The HA-ALD derivative was synthesized and tested in comparison with a simple mixture of HA and ALD for in vitro ALD release, rheological properties, cytotoxicity towards osteoblasts and chondrocytes and in an in vitro efficacy assay of OA inflammatory model on bovine cartilage explants.Results:The structure of HA-ALD was elucidated exhibiting no depolymerization and efficient drug incorporation. The controlled ALD release in vitro was slower compared to the simple mixture of HA and ALD; moreover, the derivative showed calcium-tuned rheological properties. The absence of cytotoxicity towards osteoblasts and chondrocytes was shown for up to 7 days, and the viability of chondrocytes was confirmed by fluorescence microscopy. Finally, a reduction in collagen release and MMP-13 expression was measured in the OA inflammatory model.Conclusion:This new HA-ALD derivative opens the door to a new approach for OA treatment, as it combines viscosupplementation and biological effects of HA with the pharmacological activity of BPs. Prolonged ALD release increased rheological properties and beneficial effect against cartilage degradation make it a promising IA therapy for OA.

Purpose: Hyaluronan(HA)-based viscosupplements, although resorbable, present different in vivo residence times, which are mainly related to structural modifications of the native polymer. The residence time of unmodified HA generally does not exceed 5 days, while chemically crosslinked HA can be detected in the joint up to one month. Artificial synovial fluid (ASF) have been described in the literature to mimic rheology of or drug release in human SF (SF). These models suffer from a limitation from the biological point of view: the absence of hyaluronidases, HA degrading enzymes expressed by the chondrocytes in contact with SF, well described in literature. A novel in vitro preparation of ASF with hyaluronidase is hereby reported and tested with three marketed viscosupplements: Sinovial® HL (SHL, IBSA Farmaceutici), which contains native HA of both low and high MW, Hyalubrix® (HBX, Fidia Farmaceutici S.p.A.), which contains unmodified high MW HA and Hymovis® (HYM, Fidia Farmaceutici S.p.A.), composed of HA modified with hexadecyl alkyl chains via amidic bonds (MW comprised between 500 and 700 kDa). The effect of this ASF towards HA molecular weight and rheological properties was reported and compared with the degradation profile in centrifuged human SF. Methods: ASF was formulated with the following composition: NaCl 8 g/L, KCl 0.2 g/L, Na2HPO4 1.7 g/L, Bovine Serum Albumin (BSA) 19 g/L, Bovine Testes Hyaluronidase (BTH) 6 U/mL, Gentamicin 0.01 mg/mL (pH 7.4). The salt and BSA composition and concentration in SF are well reported in the literature. At the best of our knowledge, the quantification of hyaluronidase activity in human SF is not reported. Considering that Hyaluronidase (Hyal) activity titrated in serum is at least 6 U/mL in BTH equivalent, and that Hyal (MW 55-60 kDa) can permeate synovial membrane, the same concentration of BTH was added to artificial SF. Centrifuged (cell free) human SF collected from OA patients was used as “degradation media”. Sinovial® HL, Hyalubrix® and Hymovis® were separately incubated either in ASF or SF at 37 °C for 14 days at a w/w ratio of approximately 1:1, under mild shaking. At different time points (0, 1, 7 and 14 days) the viscoelastic moduli and dynamic viscosity of the mixtures were measured with an Anton Paar MCR 92 rheometer. Moreover, at each time point the mixtures were diluted in water and filtered through Merck Vivaspin® 2 centrifugal concentrators (MWCO = 100 kDa, comparable to the synovial membrane cut-off) in order to quantitate the permeated and retained HA fractions. PBS+SF at a w/w ratio of 1:1 was used as blank for HA measurement. The percent recovery of the HA fraction with MW ≤ 100 kDa was determined by SEC-TDA (Omnisec Resolve/Reveal, Malvern instruments). Results: The degradation profile of HYM, HBX and SHL in SF or ASF is shown in the graph below: after 14 days the viscoelasticity (expressed as storage modulus: G’) of HYM/SF, HBX/SF and SHL/SF is slightly decreased; however, the elasticity of all viscosupplements in ASF is completely impaired after only few hours. After 14 days, the percent recovery of high MW HA (>100 kDa) confirms the rheological data: in SF, HYM, HBX and SHL were not depolymerized to low MW polymers while, in ASF, the percent recovery in higher MW HA was only about 45%, 10% and 13% for HYM, SHL and HBX respectively. The strong depolymerizing activity of ASF was confirmed for all the viscosupplements tested; nonetheless, HYM showed higher resistance to degradation in ASF compared to SHL. Conclusions: In this work, two different in vitro and ex vivo models were compared in order to predict the in vivo residence time of three HA-based viscosupplements in the intra-articular environment. Based on the literature, after a single intra-articular administration of high MW HA (2000 kDa), the measured residence time was as short as about 5 days. The ex vivo model with centrifuged human SF bears the limitation of the absence of hyaluronidases (a cell surface glycosylphosphatidylinositol-anchored protein, removed with the chondrocytes during the centrifugation process), while the assay in ASF added with BTH turned out to be more reliable and can therefore be employed during viscosupplement development to predict the intra-articular residence time of HA-based products