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.

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.

Short-time pre-washing of brushite-forming calcium phosphate cement improves its in vitro cytocompatibility.

A pre-washing protocol was developed for resorbable, brushite-forming calcium phosphate cements (CPCs) to avoid harmful in vitro effects on cells. CPC discs (JectOS+, Kasios; self-developed CPC) were pre-washed with repeated changes of phosphate-buffered saline (PBS; 24h total). Unwashed or PBS-pre-washed discs were incubated in culture medium (5% fetal calf serum; up to 10days) and then tested for their influence on pH/calcium/phosphate levels in H2O extracts. Effects on pH/calcium/phosphate levels in culture supernatants, and morphology, adherence, number, and viability of ATDC5 cells and adipose-tissue derived stem cells were analyzed in co-culture. Pre-washing did not alter CPC surface morphology or Ca/P ratio (scanning electron microscopy; energy-dispersive X-ray spectroscopy). However, acidic pH of unwashed JectOS+ and self-developed CPC (5.82; 5.11), and high concentrations of Ca (2.17; 2.40mM) and PO4 (38.15; 49.28mM) in H2O extracts were significantly counteracted by PBS-pre-washing (pH: 7.92; 7.92; Ca: 0.64; 1.11mM; PO4: 5.39-5.97mM). Also, PBS-pre-washing led to physiological pH (approx. 7.5) and PO4 levels (max. 5mM), and sub-medium Ca levels (0.5-1mM) in supernatants and normalized cell morphology, adherence, number, and viability. This CPC pre-washing protocol improves in vitro co-culture conditions without influencing its structure or chemical composition.

Enhanced reconstruction of long bone architecture by a growth factor mutant combining positive features of GDF-5 and BMP-2.

Non healing bone defects remain a worldwide health problem and still only few osteoinductive growth factors are available for clinical use in bone regeneration. By introducing BMP-2 residues into growth and differentiation factor (GDF)-5 we recently produced a mutant GDF-5 protein BB-1 which enhanced heterotopic bone formation in mice. Designed to combine positive features of GDF-5 and BMP-2, we suspected that this new growth factor variant may improve long bone healing compared to the parent molecules and intended to unravel functional mechanisms behind its action. BB-1 acquired an increased binding affinity to the BMP-IA receptor, mediated enhanced osteogenic induction of human mesenchymal stem cells versus GDF-5 and higher VEGF secretion than BMP-2 in vitro. Rabbit radius defects treated with a BB-1-coated collagen carrier healed earlier and with increased bone volume compared to BMP-2 and GDF-5 according to in vivo micro-CT follow-up. While BMP-2 callus often remained spongy, BB-1 supported earlier corticalis and marrow cavity formation, showing no pseudojoint persistence like with GDF-5. Thus, by combining positive angiogenic and osteogenic features of GDF-5 and BMP-2, only BB-1 restored a natural bone architecture within 12 weeks, rendering this promising growth factor variant especially promising for long bone regeneration.

New insights into the molecular mechanism of multiple synostoses syndrome (SYNS): mutation within the GDF5 knuckle epitope causes noggin-resistance.

Growth and differentiation factor 5 (GDF5), a member of the bone morphogenetic protein (BMP) family, is essential for cartilage, bone, and joint formation. Antagonists such as noggin counteract BMP signaling by covering the ligand's BMP type I (BMPRI) and type II (BMPRII, ActRII, ActRIIB) interaction sites. The mutation GDF5-S94N is located within the BMPRII interaction site, the so-called knuckle epitope, and was identified in patients suffering from multiple synostoses syndrome (SYNS). SYNS is characterized by progressive symphalangism, carpal/tarsal fusions, deafness and mild facial dysmorphism. Here we present a novel molecular mechanism of a GDF5 mutation affecting chondrogenesis and osteogenesis. GDF5-S94N exhibits impaired binding to BMPRII causing alleviated Smad and non-Smad signaling and reduced chondrogenic differentiation of ATDC5 cells. Surprisingly, chondrogenesis in mouse micromass cultures was strongly enhanced by GDF5-S94N. By using quantitative techniques (SPR, reporter gene assay, ALP assay, qPCR), we uncovered that this gain of function is caused by strongly reduced affinity of GDF5-S94N to the BMP/GDF antagonist noggin and the consequential lack of noggin inhibition. Thus, since noggin is upregulated during chondrogenic differentiation, GDF5-S94N exceeds the GDF5 action, which results in the phenotypic outcome of SYNS. The detailed molecular characterization of GDF5-S94N as a noggin-resistant growth factor illustrates the potential of GDF5 mutants in applications with defined therapeutical needs.