R399E, A Mutated Form of Growth and Differentiation Factor 5, for Disease Modification of Osteoarthritis.

Objective: To preclinically characterize a mutant form of growth and differentiation factor 5, R399E, with reduced osteogenic properties as a potential disease-modifying osteoarthritis (OA) drug.

Methods: Cartilage, synovium, and meniscus samples from patients with OA were used to evaluate anabolic and antiinflammatory properties of R399E. In the rabbit joint instability model, 65 rabbits underwent transection of the anterior cruciate ligament plus partial meniscectomy.

Early detection of osteoarthritis in the rat with an antibody specific to type II collagen modified by reactive oxygen species.

Background: Osteoarthritis (OA) is a disease of the whole joint, with articular cartilage breakdown as a major characteristic. Inflammatory mediators, proteases, and oxidants produced by chondrocytes are known to be responsible for driving cartilage degradation. Nevertheless, the early pathogenic events are still unclear.

Sprifermin (recombinant human FGF18) is internalized through clathrin- and dynamin-independent pathways and degraded in primary chondrocytes.

Sprifermin is a human recombinant fibroblast growth factor 18 (rhFGF18) in clinical development for knee osteoarthritis. Previously, we demonstrated that sprifermin exerts an anabolic effect on chondrocytes in 3D culture with cyclic but not permanent exposure. Here, we hypothesized that permanent exposure to sprifermin de-sensitizes the cells.

BMPR1A is necessary for chondrogenesis and osteogenesis, whereas BMPR1B prevents hypertrophic differentiation.

BMP2 stimulates bone formation and signals preferably through BMP receptor (BMPR) 1A, whereas GDF5 is a cartilage inducer and signals preferably through BMPR1B. Consequently, BMPR1A and BMPR1B are believed to be involved in bone and cartilage formation, respectively. However, their function is not yet fully clarified.

The GDF-5 mutant M1673 exerts robust anabolic and anti-catabolic effects in chondrocytes.

The growth and differentiation factor 5 (GDF-5) is known to play a key role in cartilage morphogenesis and homeostasis, and a single-nucleotide polymorphism in its promoter sequence was found to be associated with osteoarthritis (OA). In addition, GDF-5 was shown to promote extracellular matrix (ECM) production in healthy chondrocytes, to stimulate chondrogenesis of mesenchymal stem cells (MSCs) and to protect against OA progression in vivo. Therefore, GDF-5 appears to be a promising treatment for osteoarthritis.

Sprifermin (rhFGF18) versus vehicle induces a biphasic process of extracellular matrix remodeling in human knee OA articular cartilage ex vivo.

Sprifermin, recombinant human fibroblast growth factor 18 (rhFGF18), induces cartilage regeneration in knees of patients with osteoarthritis (OA). We hypothesized that a temporal multiphasic process of extracellular matrix (ECM) degradation and formation underlie this effect. We aimed to characterize the temporal ECM remodeling of human knee OA articular cartilage in response to sprifermin treatment.

Importance of Osmolarity and Oxygen Tension for Cartilage Tissue Engineering.

For cartilage repair or evaluation of new therapeutic approaches , the generation of functional cartilage tissue is of crucial importance and can only be achieved if the phenotype of the chondrocytes is preserved. Three-dimensional (3D) cell culture is broadly used for this purpose. However, adapting culture parameters like the oxygen tension or the osmolarity to their physiological values is often omitted.

Translational strategies in drug development for knee osteoarthritis.

Osteoarthritis (OA) is a common disease worldwide with large unmet medical needs. To bring innovative treatments to OA patients, we at Merck have implemented a comprehensive strategy for drug candidate evaluation. We have a clear framework for decision-making in our preclinical pipeline, to design our clinical proof-of-concept trials for OA patients.

Increasing the Medium Osmolarity Reduces the Inflammatory Status of Human OA Chondrocytes and Increases Their Responsiveness to GDF-5.

The environment surrounding chondrocytes changes drastically in osteoarthritis (OA). For instance, the osmolarity in cartilage (ranging from 350 to 460 mOsm in healthy tissue) decreases during the progression of OA, reaching 270 mOsm. The objective of this study was to evaluate how osmolarity influences human OA chondrocytes.

Effects of Sprifermin, IGF1, IGF2, BMP7, or CNP on Bovine Chondrocytes in Monolayer and 3D Culture.

One possible approach to treat osteoarthritis (OA) is to counteract cartilage degeneration with anabolic compounds that stimulate chondrocyte proliferation and/or extracellular matrix (ECM) production. Several molecules including sprifermin (recombinant human fibroblast growth factor [FGF18]), insulin-like growth factor-1 [IGF1] and -2 [IGF2], C-type natriuretic peptide [CNP], and bone metamorphic protein 7 [BMP7] have been shown to have these characteristics both in vitro and in vivo. However, it is not known how these molecules compare each other regarding their effect on phenotype and stimulation of ECM production in primary chondrocytes.

Sprifermin (rhFGF18) modulates extracellular matrix turnover in cartilage explants ex vivo.

Background: Sprifermin (recombinant human fibroblast growth factor 18) is in clinical development as a potential disease-modifying osteoarthritis drug (DMOAD). In vitro studies have shown that cartilage regenerative properties of sprifermin involve chondrocyte proliferation and extracellular matrix (ECM) production. To gain further insight into the process of sprifermin in the cartilage tissue, this study aimed at investigating the ECM turnover of articular cartilage explants in a longitudinal manner.

Sprifermin (rhFGF18) enables proliferation of chondrocytes producing a hyaline cartilage matrix.

Objective: Fibroblast growth factor (FGF) 18 has been shown to increase cartilage volume when injected intra-articularly in animal models of osteoarthritis (OA) and in patients with knee OA (during clinical development of the recombinant human FGF18, sprifermin). However, the exact nature of this effect is still unknown. In this study, we aimed to investigate the effects of sprifermin at the cellular level.

Nitrogen-rich plasma-polymerized coatings on PET and PTFE surfaces improve endothelial cell attachment and resistance to shear flow.

Low seeding efficiency and poor cell retention under flow-induced shear stress limit the effectiveness of in vitro endothelialization strategies for small-diameter vascular grafts. Primary-amine-rich plasma-polymerized coatings (PPE:N) deposited using low- and atmospheric-pressure plasma discharges on PET and PTFE are evaluated for their ability to improve endothelial cells' kinetics and strength of attachment. PPE:N coatings increase cell adhesion and adhesion rate, spreading, focal adhesion, and resistance to flow-induced shear compared with bare and gelatin-coated PET and PTFE.

CHO cells adhering to nitrogen-rich plasma-polymerised ethylene exhibit high production of a specific recombinant protein.

In many industrial applications, inadequate cell attachment can be a limitation, especially when serum-free media are used. Nitrogen-rich plasma-polymerised ethylene (PPE:N) exhibits high concentrations of polar groups that can help to promote the attachment of weakly adherent cell types. Tissue plasminogen activator-producing Chinese hamster ovary (CHO) cells, adapted to suspension, were grown in the presence PPE:N flakes and were found to adhere to them.

Chondrocytes cultured in stirred suspension with serum-free medium containing pluronic-68 aggregate and proliferate while maintaining their differentiated phenotype.

The study of chondrocyte biology requires culture conditions that maintain cell phenotype. Phenotype is rapidly lost in monolayer but is maintained in 3-dimensional scaffolds, which however, experience limited cell proliferation and limited mass transport. In this study, we cultured chondrocytes in aggregates in stirred spinner flask suspension cultures to control aggregate size and promote mass transport.

Chondrocyte aggregation in suspension culture is GFOGER-GPP- and beta1 integrin-dependent.

Isolated chondrocytes form aggregates in suspension culture that maintain chondrocyte phenotype in a physiological pericellular environment. The molecular mechanisms involved in chondrocyte aggregation have not been previously identified. Using this novel suspension culture system, we performed mRNA and protein expression analysis along with immunohistochemistry for potential cell adhesion molecules and extracellular matrix integrin ligands.

The fate of Pluronic F-68 in chondrocytes and CHO cells.

The surfactant Pluronic F-68 (PF-68) is widely used in large-scale mammalian cell culture to protect cells from shear stress that arises from agitation and gas sparging. Several studies suggested that PF-68 is incorporated into the cell plasma membrane and could enter the cells, but without providing any direct evidence. The current study has examined this question for two cell types, one of pharmaceutical interest (CHO cells) and the other of biomedical interest (chondrocytes or cartilage cells).

Low calcium levels in serum-free media maintain chondrocyte phenotype in monolayer culture and reduce chondrocyte aggregation in suspension culture.

Objective: Extracellular calcium influences chondrocyte differentiation and synthesis of extracellular matrix. Previously, calcium concentrations ranging from 0.1 mM to 2 mM have been used in vitro and these studies indicated that low calcium concentrations were generally favorable for chondrocyte culture.