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. In this study, GDF5 mutants with a decreased affinity for BMPR1A were generated. These mutants, and wild-type GDF5 and BMP2, were tested for their ability to induce dimerization of BMPR1A or BMPR1B with BMPR2, and for their chondrogenic, hypertrophic and osteogenic properties in chondrocytes, in the multipotent mesenchymal precursor cell line C3H10T1/2 and the human osteosarcoma cell line Saos-2. Mutants with the lowest potency for inducing BMPR1A-BMPR2 dimerization exhibited minimal chondrogenic and osteogenic activities, indicating that BMPR1A is necessary for chondrogenic and osteogenic differentiation. BMP2, GDF5 and the GDF5 R399E mutant stimulated expression of chondrogenic and hypertrophy markers in C3H10T1/2 cells and chondrocytes. However, GDF5 R399E, which induces the dimerization of BMPR1B and BMPR2 more potently than GDF5 or BMP2, displayed reduced hypertrophic activity. Therefore, we postulate that stronger BMPR1B signaling, compared to BMPR1A signaling, prevents chondrocyte hypertrophy and acts as a cartilage stabilizer during joint morphogenesis.This article has an associated First Person interview with the first author of the paper.

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. To test this, a combination of Western-blot and cell staining methods was used. We demonstrate that sprifermin is transiently internalized in chondrocytes along with a transient increase in ERK1/2 activation. We also show that sprifermin is intracellularly degraded, probably together with its receptor FGFR3, thus preventing further stimulation. However, incubation without sprifermin re-sensitizes the cells. Finally, we show that sprifermin endocytosis is clathrin- and dynamin-independent and that receptor activation is not necessary for sprifermin's endocytosis. In this study, we link the role of endocytosis to the cell response and elucidate for the first time a de-sensitization phenomenon to a FGF.

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. However, GDF-5 also promotes osteogenesis and hypertrophy, limiting its therapeutic utility. To circumvent this, a GDF-5 mutant with lower hypertrophic and osteogenic properties was engineered: M1673. The present study aimed to evaluate and compare the effects of GDF-5 and M1673 on primary porcine and human OA chondrocytes. We found that both GDF-5 and M1673 can robustly stimulate ECM accumulation, type II collagen and aggrecan expression in porcine and human OA chondrocytes in 3D culture. In addition, both molecules also down-regulated MMP13 and ADAMTS5 expression. These results suggest that M1673 retained the anabolic and anti-catabolic effects of GDF-5 on chondrocytes and is an alternative to GDF-5 for osteoarthritis.

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. For this purpose, the osmolarity of the culture medium (340 mOsm) was increased to 380, 420 or 460 mOsm and its effect on the phenotype, matrix production, protease expression, cytokine release and growth and differentiation factor-5 (GDF-5) receptor expression in human OA chondrocytes was evaluated in a monolayer. Afterwards, the same parameters, as well as the responsiveness to GDF-5, were evaluated in 3D culture at 340 and 380 mOsm. Our results revealed that increasing the medium osmolarity increased matrix production but also reduced cytokine release, type I collagen and protease expression. It was also demonstrated that at 380 mOsm, the response to GDF-5 in 3D culture was more robust than at 340 mOsm. For the first time, it was established that a decreased osmolarity plays a role in sustaining inflammation and catabolic activities in OA chondrocytes and decreases their responsiveness to GDF-5. This indicates that osmolarity is a critical aspect of OA pathobiology.

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. METHODS: Bovine full-depth articular cartilage explants were stimulated with sprifermin or placebo at weekly intervals, similar to the dosing regimen used in clinical trials. Pre-culturing with oncostatin M and tumour necrosis factor-α, was also used to induce an inflammatory state before treatment. Metabolic activity was measured using AlamarBlue, and chondrocyte proliferation was visualized by immuno-histochemical detection of proliferating cell nuclear antigen. ECM turnover was quantified by biomarker ELISAs; ProC2 reflecting type II collagen formation, CS846 reflecting aggrecan formation, active MMP9, C2M and AGNx2 reflecting matrix metalloproteinase activity, and AGNx1 reflecting aggrecanase activity. RESULTS: Sprifermin was able to reach the chondrocytes through the extracellular matrix, as it increased cell proliferation and metabolic activity of explants. ProC2 and CS846 was dose-dependently increased (P < 0.05) by sprifermin compared to placebo, while C2M and AGNx2 were unaffected, active MMP9 was slightly decreased, and AGNx1 was slightly increased. Over the course of treatment, the temporal order of ECM turnover responses was AGNx1, then ProC2, followed by CS846 and MMP9. Pro-inflammatory activation of the explants diminished the ECM turnover responses otherwise observed under non-inflammatory conditions. CONCLUSIONS: The data suggest that sprifermin has chondrogenic effects on articular cartilage ex vivo, exerted through a sequential process of ECM turnover; aggrecan degradation seems to occur first, while type II collagen and aggrecan production increased at a later time point. In addition, it was observed that these chondrogenic effects are dependent on the inflammatory status of the cartilage prior to treatment.

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. Three intraarticular (IA) R399E doses were administered biweekly 6 times, and static incapacitance was determined to assess joint pain. OA was evaluated 13 weeks after surgery. In sheep, medial meniscus transection was performed to induce OA, dynamic weight bearing was measured in-life, and OA was assessed after 13 weeks. RESULTS: Intermittent exposure to R399E (1 week per month) was sufficient to induce cell proliferation and release of anabolic markers in 3-dimensional chondrocyte cultures. R399E also inhibited the release of interleukin-1ß (IL-1ß), IL-6, and prostaglandin E2 from cartilage with synovium, meniscal cell, and synoviocyte cultures. In rabbits, the mean difference (95% confidence interval [95% CI]) in weight bearing for R399E compared to vehicle was -5.8 (95% confidence interval [95% CI] -9.54, -2.15), -7.2 (95% CI -10.93, -3.54), and -7.7 (95% CI -11.49, -3.84) for the 0.6, 6, and 60 µg doses, respectively, 6 hours after the first IA injection, and was statistically significant through the entire study for all doses. Cartilage surface structure improved with the 6-µg dose. Structural and symptomatic improvement with the same dose was confirmed in the sheep model of OA. CONCLUSION: R399E influences several pathologic processes contributing to OA, highlighting its potential as a disease-modifying therapy.

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. To investigate this, we employed an antibody that is specific to oxidative post-translationally modified collagen type II (anti-oxPTM-CII) to detect early cartilage pathogenic changes in two rat models of OA. METHODS: The animals underwent surgery for destabilization of the medial meniscus (DMM) and were sacrificed after 3, 5, 7, 14, and 28 days. Alternatively, anterior cruciate ligament transection with partial meniscectomy (ACLT+pMx) was performed and animals were sacrificed after 1, 3, 5, 7, and 14 days. Joints were stained with toluidine blue and saffron du Gatinais for histological scoring, anti-oxPTM-CII, and anti-collagen type X antibodies (anti-CX). RESULTS: We observed positive oxPTM-CII staining as early as 1 or 3 days after ACLT+pMx or DMM surgeries, respectively, before overt cartilage lesions were visible. oxPTM-CII was located mostly in the deep zone of the medial tibial cartilage, in the pericellular and territorial matrix of hypertrophic chondrocytes, and co-localized with CX staining. Staining was weak or absent for the lateral compartment or the contralateral knees except at later time points. CONCLUSION: The results demonstrate that oxidant production and chondrocyte hypertrophy occur very early in the onset of OA, possibly initiating the pathogenic events of OA. We propose to use anti-oxPTM-CII as an early biomarker for OA ahead of radiographic changes.

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. The effects of sprifermin, IGF1, IGF2, CNP, and BMP7 were evaluated on bovine articular chondrocytes, first in monolayer to determine their effective concentrations, and then in three-dimensional (3D) culture at concentrations of 100 ng/ml for sprifermin; 300 ng/ml for IGF1, IGF2, and BMP7; and 10 nM for CNP. In 3D culture, the effects of a permanent exposure or a cyclic exposure consisting of 24 h incubation per week with the compounds were evaluated. All growth factors increased ECM production and cell proliferation to a similar extent but CNP had almost no effect on bovine chondrocytes. Sprifermin was more effective with cyclic exposure, IGF1, and IGF2 with permanent exposure, and BMP7 showed similar results with both exposures. Regarding the cell phenotype, sprifermin appeared to be the only compound favoring the chondrocyte phenotype; it decreased type I collagen expression and had no hypertrophic effect. Together, these results confirmed that sprifermin is a promising disease-modifying OA drug. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 38:653-662, 2020.

Importance of Osmolarity and Oxygen Tension for Cartilage Tissue Engineering.

For cartilage repair in vivo or evaluation of new therapeutic approaches in vitro, 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. Indeed, articular cartilage is an avascular tissue subjected to reduced oxygen tension and presenting and increased osmolarity compared with most other tissues. In this study, we aimed at evaluating the effect of a physiological oxygen tension (3% instead of 21%) and physiological osmolarity (430 vs. 330 mOsm in nonadjusted DMEM) and the combination of both on the cell proliferation, matrix production, and the phenotype of porcine chondrocytes in a scaffold-free 3D culture system. We observed that a physiological osmolarity had no effect on cell proliferation and matrix production but positively influences the chondrocyte phenotype. A physiological oxygen level prevented cell proliferation but resulted in an increased matrix content/million cells and had a positive influence on the chondrocyte phenotype as well. The strongest benefit was reached with the combination of both physiological osmolarity and oxygen levels; with these conditions, type I collagen expression became undetectable. In addition, at 3% O2 the chondrocytes-matrix constructs were found to more closely resemble native cartilage regarding the matrix-to-cell ratio. In conclusion, this study clearly demonstrates the benefit of using physiological oxygen tension and osmolarity in cartilage tissue engineering with the combination of both showing the strongest benefit on the chondrocyte phenotype.

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. We have qualified our strategy to define and refine dose and dosing regimen, for treatments administered either systemically or intra-articularly (IA). We do this through preclinical in vitro and in vivo studies, and by back-translating results from clinical studies in OA patients.

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). A fluorescent derivative of PF-68 was synthesized to detect and localize internalized Pluronic with culture time. PF-68 uptake by the cells was quantified and characterized. We clearly demonstrate that PF-68 enters the cells, and possibly accumulates in the endocytic pathway. CHO cells showed an average uptake of 11.7 +/- 6.7 (SEM) microg PF-68/10(6) cells while the uptake of chondrocytes was 56.0 +/- 10.9 (SEM) microg PF-68/10(6) cells, independently of the initial PF-68 concentration (between 0.01 and 0.2%, w/v) and of cell concentration (from 1 x 10(6) to 4 x 10(6) cells/mL). These uptake values were identical for both static and agitated culture conditions. Finally, we found that CHO cells are able to eliminate intracellular fluorescent PF-68 but chondrocytes are not. These results show that the uptake of PF-68 by the cells can severely affect PF-68 concentration in the culture medium and thus shear protection effect.

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. In particular, about 90% of the cells remain on coated surfaces after 1 h exposure to shear. These coatings, therefore, appear as a promising versatile approach to improve cell seeding strategies for vascular grafts.

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. A previously optimized serum-free medium, containing the following growth factors (GFs), epidermal growth factor, platelet-derived growth factor-BB, and basic fibroblast growth factor, all at 2 ng/mL, was used as a control medium. In addition, two modified media were tested: one containing Pluronic F-68 (PF-68) and the other containing PF-68 with 10 times greater GF concentration (20 ng/mL, medium PF-68/10 x GF). Chondrocytes formed limited-size aggregates within 24 h and exhibited high viability (>95%), and cell concentration doubled in 7 days in the presence of PF-68. Low or no collagen I expression was found for any of the three media, whereas collagen II accumulated between cells, as revealed by a dense immunostaining. Integrin alpha10, a marker of differentiated chondrocytes and chondrogenic cells, was also found to be highly expressed. Aggregates resulting from spinner culture were found to be relevant in vitro models and their use for cartilage repair to be also conceivable.

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. The growth rate was reduced, but cell viability was enhanced and their metabolism was more efficient, with generally higher recombinant protein productivity. Finally, cell adhesion on PPE:N surfaces was found to be independent of integrins, and was probably mediated by certain non-specific interactions with the PPE:N surface.

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. Inhibition of aggregation assays were performed using specific blocking agents. We found that: (i) direct cell-cell interactions were not involved in chondrocyte aggregation, (ii) chondrocytes in aggregates were surrounded by a matrix rich in collagen II and cartilage oligomeric protein (COMP), (iii) aggregation depends on a beta1-integrin, which binds a triple helical GFOGER sequence found in collagens, (iv) integrin alpha10-subunit is the most highly expressed alpha-subunit among those tested, including alpha5, in aggregating chondrocytes. Taken together, this body of evidence suggests that the main molecular interaction involved in aggregation of phenotypically stable chondrocytes is the alpha10beta1-collagen II interaction.