Massimo Sabatini

Cartilage and Osteoarthritis

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Culture of Chondrocytes in Alginate Beads

A classic method for the encapsulation and culture of chondrocytes in alginate beads is described. Chondrocytes are released from cartilage matrix by collagenase/dispase digestion and mixed with a solution of 1.25% alginic acid until a homogenous suspension is obtained. The suspension is drawn into a syringe and pushed gently through a needle, so that drops fall into a solution of calcium chloride. Beads form instantaneously and further polymerize after 5 min in the calcium chloride solution. Chondrocytes from any species, including human osteoarthritic chondrocytes, can be cultured with this technique. Under these conditions, chondrocytes maintain a high degree of differentiation. Beads can be dissolved by chelation of calcium with EDTA. In this way, chondrocytes can be recovered and further separated from the matrix by centrifugation. Almost all molecular and biochemical techniques, as well as a number of biological assays, are compatible with the culture of chondrocytes in alginate.

Recent progress toward biomarker identification in osteoarthritis.

Osteoarthritis (OA), the most common and disabling form of arthritic disease, is characterized by a slow and progressive degeneration of articular cartilage. Its etiology is multifactorial and includes genetic predisposition, obesity and aging. In addition to the cartilage itself, OA also involves the surrounding tissues, including the synovium and the subchondral bone. This clinical heterogeneity complicates the identification of biomarkers that are crucial for prompt pharmacological intervention at the early stages of the disease and for monitoring treatment efficacy with higher sensitivity than existing imaging methods. In this review, we highlight the difficulties associated with OA diagnosis and discuss the most recent research efforts and successes for the identification of reliable OA biomarkers.

Solid-phase synthesis of α-substituted 3-bisarylthio N-Hydroxy propionamides as Specific MMP Inhibitors

A novel series of potent and specific MMP-2,3,9,13 inhibitors has been obtained by modulation on solid phase by alpha and aryl substitutions on 3-arylthio-N-hydroxy-propionamides starting from itaconic acid.

General synthesis of α-substituted 3-bisaryloxy propionic acid derivatives as specific mmp inhibitors

Modulations of alpha and aryl substitutions on 3-aryloxy propionic acid hydroxamates led to novel and potent inhibitors of MMP-2,3,9 and 13, and selectivity versus MMP-1.

Assays of Proteoglycan and Collagen Degradation in Cultures of Rabbit Cartilage Explants

Cultures of cartilage explants have long been used to study the effects of modulators of extracellular matrix degradation. We present a simple and rapid assay system, based on culture of rabbit cartilage explants, which permits study of the effects of protease inhibitors on proteoglycan degradation (caused by either aggrecanases or matrix metalloproteinases [MMPs]), and on collagen degradation. The assay is based on the ability of interleukin-1 to stimulate both aggrecanase activity and synthesis of inactive MMPs, which are then activated by p-aminophenylmercuric acetate for the study of MMP-mediated proteoglycan degradation or by plasmin for the study of collagen degradation. Proteoglycan degradation is quantified as percent release of radioactivity from cartilage explants previously labeled with (35)SO4(2-). Collagen degradation is calculated as percent release of collagen, measured by colorimetric assay of hydroxyproline.

Divergent effects of strontium and calcium‐sensing receptor positive allosteric modulators (calcimimetics) on human osteoclast activity

Strontium ranelate, a drug approved and until recently used for the treatment of osteoporosis, mediates its effects on bone at least in part via the calcium‐sensing (CaS) receptor. However, it is not known whether bone‐targeted CaS receptor positive allosteric modulators (PAMs; calcimimetics) represent an alternative (or adjunctive) therapy to strontium (Sr2+o).

G protein-coupled receptors as anabolic drug targets in osteoporosis

ABSTRACT Osteoporosis is a progressive bone disorder characterised by imbalance between bone building (anabolism) and resorption (catabolism). Most therapeutics target inhibition of osteoclast‐mediated bone resorption, but more recent attention in early drug discovery has focussed on anabolic targets in osteoblasts or their precursors. Two marketed agents that display anabolic properties, strontium ranelate and teriparatide, mediate their actions via the G protein‐coupled calcium‐sensing and parathyroid hormone‐1 receptors, respectively. This review explores their activity, the potential for improved therapeutics targeting these receptors and other putative anabolic GPCR targets, including Smoothened, Wnt/Frizzled, relaxin family peptide, adenosine, cannabinoid, prostaglandin and sphingosine‐1‐phosphate receptors.