Osteoarthritis (OA) is a joint degenerative disease leading to chronic pain and disability, thus resulting in a major socioeconomic health burden. OA, which has long been believed to be a cartilage disease, is now considered a whole-joint disorder affecting various anatomical structures, including subchondral bone.

Hyaluronic Acid (HA) is commonly used as intra-articular viscosupplementation therapy for its mechanical features and biological effects. Bisphosphonates (BPs) are antiresorptive agents inhibiting recruitment and maturation of osteoclast precursors and activity of mature osteoclasts in the bone. Pre-clinical evidences in the literature, show that intra-articular BPs could impact on OA progression, slowing down or reversing it. The combination of HA biological and mechanical role and Alendronate (ALD) antiresorptive effect could be an interesting strategy for OA treatment. This study describes the synthesis and characterization of FID-134, a new chemical derivative of HA conjugated with ALD by means of a covalent bond, cleavable in physiological condition.

FID-134 was synthesized starting from 500 kDa HA: chemical structure and functionalization degree with ALD were investigated by NMR and ICP-OES. Kinetics of ALD release from FID-134 was determined in TRIS buffer at 37°C and compared to a simple mixture of HA+ALD. 20mg/mL formulations of FID-134 and HA+ALD were investigated for viscoelastic properties, in absence and presence of Ca²⁺ ions. The cytotoxicity of FID-134 and free ALD were tested on Saos-2 osteoblasts (ATCC HTB-85) and on primary bovine chondrocytes (PBC) at day 1, 3 and 7. The efficacy of FID-134 was assessed in an inflammatory arthritis in vitro model, where bovine cartilage biopsies were exposed to IL-1β/OSM (10ng/mL) for 3 weeks; at the same time, cartilage explants were treated with FID-134. Collagen release in the surnatants was quantified and compared to controls.

FID-134 structure was confirmed by NMR and the 20% mol/mol functionalization degree was determined by ICP-OES. Only about 50% of total bound ALD was released from FID-134 within 7 days, resulting slower compared to HA+ALD mixture. In presence of Ca²⁺ ions, viscoelastic properties of FID-134 dramatically improved, while HA+ALD formulation remained unaffected. The cytotoxicity of ALD was evident at 100 μM on Saos-2 and PBC after 3 days, while no cytotoxicity was observed at 7 days with FID-134. In the cartilage explant model, a strong collagen release was detected in inflammatory conditions after 3 weeks; this tendency was reversed, and collagen release halved when FID-134 was added to the biopsies.

The synthesized HA-ALD adduct, FID-134, opens the door for a new approach for OA treatment. The results suggest that FID-134 could be beneficial in cartilage degradation and in restoration of subchondral bone function. Finally, local administration and controlled BP release would likely overcome the drawbacks of ALD oral administration, such as unspecific features and long-term toxic side effects.

Abstract

Background

Hyaluronic acid (HA) hydrogels are becoming an increasingly attractive choice for the creation of new biomaterials useful in wound care, tissue engineering and regenerative medicine, because of their high level of biocompatibility and biodegradability, and for their ability to imitate the environment of the extracellular matrix (ECM). Due to the poor biomechanical properties of native hyaluronan, a variety of chemical modifications have been devised to provide mechanically and chemically stiffer materials.

Introduction: Pathological conditions, which determine human atrophy, are numerouses and heterogeneous.

Experimental studies prove that these different pathological conditions use common enzymatic pathways leading muscle atrophy. In every catabolic conditions where there is proteolyses’s increase, this one happens in association with up-regulation of two specific genes of skeletal muscle atrophy. These genes, MuRf1 (muscle ring finger-1) and MAFbx (muscle atrophy F-box), encode ubiquitin ligases. These ligases bind and mediate ubiquitination of myofibrillar proteins for subsequent degradation during muscle atrophy.

The aim of our study is to obtain a better understanding of human muscle physiopathology in atrophy by use of histochemistry and immunolocalisation of MuRF-1 and MAFbx.

Patients and Methods: 15 patients, amputated at third distal or proximal leg because of different acute or chronic pathology, were divided in two group. Group A: 12 elderly patients (mean age 72 years) amputated for vascular diseases (8) and complication of a diabetic foot (4). Group B: 3 young patients involved in car accident (mean age 25) amputated for limb’s acute arterial insufficiency. Gastrocnemius muscle biopsy specimens were obtained for all the patients, after that the informed consent was obtain, for histochemical (haematossilineosin), and immunohistochemical (anti- MuRf1, anti- MAFbx) analysis.

Results: Histochemistry: Group A: skeletal muscle showed a decrease in fiber size in cross-sectional area and fiber length with adipose tissue. Group B: light skeletal muscle structural alteration. Immuno-histochemistry: in group A, in muscular drawings, polyclonal antibodies direct against MuRf1 and MAFbx had stained muscle biopsy specimens. Muscle fiber cells showed MuRf1 and MAFbx subsarcolemmatic immunoreactivity and weakly immunoreactivity of the extracellular matrix. We noticed no positivity to anti- MuRf1 and anti- MAFbx less in sections from group B muscle biopsy specimens and in sections in which were present tissue muscle degeneration with replacement of adipose tissue.

Conclusion: The pathological results supported the concept that MuRf1 and MAFbx appeared to be regulatory peptide in cellular pathology that conduce to muscular atrophy. Our data support the hypothesis that different pathological conditions use common enzymatic pathways leading muscle atrophy.

The demonstration that the muscle-specific proteins MAFbx and MuRF1 are upregulated in multiple pathological conditions of skeletal muscle atrophy it is critical to continue studying the cellular pathways to discover promising targets for the development of effective new treatments for skeletal muscle disease.

Stimulation of bone healing and bone formation through local application of growth factors may improve the clinical outcome in high tibial osteotomy in varus knee. The goal of the present study is to evaluate the effectiveness of autologous growth factors asscociated with or without granular coralline hydroxyapatite used to fill open wedge defects after tibial osteotomy for valgisation in 13 patients with medial femorotibial degeneration.

The integration of the coralline hydroxyapatite was complete in every case; we did not encounter any general or local problems, nor were there any intra-operative, peri-operative or post-operative complications. We conclude that local application of autologous growth factors associated with coralline hydroxyapatite is a good solution to stimulate callus formation and ossification in the early phase of bone healing and can substitute bone graft to fill bone defects.