BMP signaling: A significant player and therapeutic target for osteoarthritis.

OBJECTIVE: To explore the significance of BMP signaling in osteoarthritis (OA) etiology, and thereafter propose a disease-modifying therapy for OA. METHODS: To examine the role of the BMP signaling in pathogenesis of OA, an Anterior Cruciate Ligament Transection (ACLT) surgery was performed to incite OA in C57BL/6J mouse line at postnatal day 120 (P120). Thereafter, to investigate whether activation of BMP signaling is necessary and sufficient to induce OA, we have used conditional gain- and loss-of-function mouse lines in which BMP signaling can be activated or depleted, respectively, upon intraperitoneal injection of tamoxifen. Finally, we locally inhibited BMP signaling through intra-articular injection of LDN-193189 pre- and post-onset surgically induced OA. The majority of the investigation has been conducted using micro-CT, histological staining, and immuno histochemistry to assess the disease etiology. RESULTS: Upon induction of OA, depletion of SMURF1-an intra-cellular BMP signaling inhibitor in articular cartilage coincided with the activation of BMP signaling, as measured by pSMAD1/5/9 expression. In mouse articular cartilage, the BMP gain-of-function mutation is sufficient to induce OA even without surgery. Further, genetic, or pharmacological BMP signaling suppression also prevented pathogenesis of OA. Interestingly, inflammatory indicators were also significantly reduced upon LDN-193189 intra-articular injection which inhibited BMP signaling and slowed OA progression post onset. CONCLUSION: Our findings showed that BMP signaling is crucial to the etiology of OA and inhibiting BMP signaling locally can be a potent strategy for alleviating OA.

Taxonomy of fibroblasts and progenitors in the synovial joint at single-cell resolution.

OBJECTIVES: Fibroblasts in synovium include fibroblast-like synoviocytes (FLS) in the lining and Thy1+ connective-tissue fibroblasts in the sublining. We aimed to investigate their developmental origin and relationship with adult progenitors. METHODS: To discriminate between Gdf5-lineage cells deriving from the embryonic joint interzone and other Pdgfrα-expressing fibroblasts and progenitors, adult Gdf5-Cre;Tom;Pdgfrα-H2BGFP mice were used and cartilage injury was induced to activate progenitors. Cells were isolated from knees, fibroblasts and progenitors were sorted by fluorescence-activated cell-sorting based on developmental origin, and analysed by single-cell RNA-sequencing. Flow cytometry and immunohistochemistry were used for validation. Clonal-lineage mapping was performed using Gdf5-Cre;Confetti mice. RESULTS: In steady state, Thy1+ sublining fibroblasts were of mixed ontogeny. In contrast, Thy1-Prg4+ lining fibroblasts predominantly derived from the embryonic joint interzone and included Prg4-expressing progenitors distinct from molecularly defined FLS. Clonal-lineage tracing revealed compartmentalisation of Gdf5-lineage fibroblasts between lining and sublining. Following injury, lining hyperplasia resulted from proliferation and differentiation of Prg4-expressing progenitors, with additional recruitment of non-Gdf5-lineage cells, into FLS. Consistent with this, a second population of proliferating cells, enriched near blood vessels in the sublining, supplied activated multipotent cells predicted to give rise to Thy1+ fibroblasts, and to feed into the FLS differentiation trajectory. Transcriptional programmes regulating fibroblast differentiation trajectories were uncovered, identifying Sox5 and Foxo1 as key FLS transcription factors in mice and humans. CONCLUSIONS: Our findings blueprint a cell atlas of mouse synovial fibroblasts and progenitors in healthy and injured knees, and provide novel insights into the cellular and molecular principles governing the organisation and maintenance of adult synovial joints.

Targeting the IL-6-Yap-Snail signalling axis in synovial fibroblasts ameliorates inflammatory arthritis.

OBJECTIVE: We aimed to understand the role of the transcriptional co-factor Yes-associated protein (Yap) in the molecular pathway underpinning the pathogenic transformation of synovial fibroblasts (SF) in rheumatoid arthritis (RA) to become invasive and cause joint destruction. METHODS: Synovium from patients with RA and mice with antigen-induced arthritis (AIA) was analysed by immunostaining and qRT-PCR. SF were targeted using Pdgfrα-CreER and Gdf5-Cre mice, crossed with fluorescent reporters for cell tracing and Yap-flox mice for conditional Yap ablation. Fibroblast phenotypes were analysed by flow cytometry, and arthritis severity was assessed by histology. Yap activation was detected using Yap-Tead reporter cells and Yap-Snail interaction by proximity ligation assay. SF invasiveness was analysed using matrigel-coated transwells. RESULTS: Yap, its binding partner Snail and downstream target connective tissue growth factor were upregulated in hyperplastic human RA and in mouse AIA synovium, with Yap detected in SF but not macrophages. Lineage tracing showed polyclonal expansion of Pdgfrα-expressing SF during AIA, with predominant expansion of the Gdf5-lineage SF subpopulation descending from the embryonic joint interzone. Gdf5-lineage SF showed increased expression of Yap and adopted an erosive phenotype (podoplanin+Thy-1 cell surface antigen-), invading cartilage and bone. Conditional ablation of Yap in Gdf5-lineage cells or Pdgfrα-expressing fibroblasts ameliorated AIA. Interleukin (IL)-6, but not tumour necrosis factor alpha (TNF-α) or IL-1ß, Jak-dependently activated Yap and induced Yap-Snail interaction. SF invasiveness induced by IL-6 stimulation or Snail overexpression was prevented by Yap knockdown, showing a critical role for Yap in SF transformation in RA. CONCLUSIONS: Our findings uncover the IL-6-Yap-Snail signalling axis in pathogenic SF in inflammatory arthritis.

Human Mesenchymal Stromal Cells Enhance Cartilage Healing in a Murine Joint Surface Injury Model.

Human umbilical cord (hUC)- or bone marrow (hBM)-derived mesenchymal stromal cells (MSCs) were evaluated as an allogeneic source of cells for cartilage repair. We aimed to determine if they could enhance healing of chondral defects with or without the recruitment of endogenous cells. hMSCs were applied into a focal joint surface injury in knees of adult mice expressing tdTomato fluorescent protein in cells descending from Gdf5-expressing embryonic joint interzone cells. Three experimental groups were used: (i) hUC-MSCs, (ii) hBM-MSCs and (iii) PBS (vehicle) without cells. Cartilage repair was assessed after 8 weeks and tdTomato-expressing cells were detected by immunostaining. Plasma levels of pro-inflammatory mediators and other markers were measured by electrochemiluminescence. Both hUC-MSC (n = 14, p = 0.009) and hBM-MSC (n = 13, p = 0.006) treatment groups had significantly improved cartilage repair compared to controls (n = 18). While hMSCs were not detectable in the repair tissue at 8 weeks post-implantation, increased endogenous Gdf5-lineage cells were detected in repair tissue of hUC-MSC-treated mice. This xenogeneic study indicates that hMSCs enhance intrinsic cartilage repair mechanisms in mice. Hence, hMSCs, particularly the more proliferative hUC-MSCs, could represent an attractive allogeneic cell population for treating patients with chondral defects and perhaps prevent the onset and progression of osteoarthritis.

Regulation of Gdf5 expression in joint remodelling, repair and osteoarthritis.

Growth and Differentiation Factor 5 (GDF5) is a key risk locus for osteoarthritis (OA). However, little is known regarding regulation of Gdf5 expression following joint tissue damage. Here, we employed Gdf5-LacZ reporter mouse lines to assess the spatiotemporal activity of Gdf5 regulatory sequences in experimental OA following destabilisation of the medial meniscus (DMM) and after acute cartilage injury and repair. Gdf5 expression was upregulated in articular cartilage post-DMM, and was increased in human OA cartilage as determined by immunohistochemistry and microarray analysis. Gdf5 expression was also upregulated during cartilage repair in mice and was switched on in injured synovium in prospective areas of cartilage formation, where it inversely correlated with expression of the transcriptional co-factor Yes-associated protein (Yap). Indeed, overexpression of Yap suppressed Gdf5 expression in chondroprogenitors in vitro. Gdf5 expression in both mouse injury models required regulatory sequence downstream of Gdf5 coding exons. Our findings suggest that Gdf5 upregulation in articular cartilage and synovium is a generic response to knee injury that is dependent on downstream regulatory sequence and in progenitors is associated with chondrogenic specification. We propose a role for Gdf5 in tissue remodelling and repair after injury, which may partly underpin its association with OA risk.

Joint morphogenetic cells in the adult mammalian synovium.

The stem cells that safeguard synovial joints in adulthood are undefined. Studies on mesenchymal stromal/stem cells (MSCs) have mainly focused on bone marrow. Here we show that lineage tracing of Gdf5-expressing joint interzone cells identifies in adult mouse synovium an MSC population largely negative for the skeletal stem cell markers Nestin-GFP, Leptin receptor and Gremlin1. Following cartilage injury, Gdf5-lineage cells underpin synovial hyperplasia through proliferation, are recruited to a Nestin-GFPhigh perivascular population, and contribute to cartilage repair. The transcriptional co-factor Yap is upregulated after injury, and its conditional ablation in Gdf5-lineage cells prevents synovial lining hyperplasia and decreases contribution of Gdf5-lineage cells to cartilage repair. Cultured Gdf5-lineage cells exhibit progenitor activity for stable chondrocytes and are able to self-organize three-dimensionally to form a synovial lining-like layer. Finally, human synovial MSCs transduced with Bmp7 display morphogenetic properties by patterning a joint-like organ in vivo. Our findings further the understanding of the skeletal stem/progenitor cells in adult life.

The multidisciplinary management of hip fractures in older patients.

Older patients presenting with hip fractures are some of the frailest and sickest patients in hospital. In addition to complex medical problems and comorbidities, they have to overcome the additional physiological challenges posed by the hip fracture itself, and subsequent surgery. Hip fracture associated morbidity and mortality at one year remains high. Published guidelines stress the need for a multidisciplinary approach and the importance of the care environment for good outcomes. A combined management approach identifies and addresses not only the surgical but also the complex analgesic, medical, cognitive, nutritional, social and rehabilitation needs of our patients, thereby improving outcome for our patients.

Bone marrow contribution to synovial hyperplasia following joint surface injury.

BACKGROUND: Joint surface injury, a known risk factor for osteoarthritis, triggers synovial hyperplasia, which involves proliferation of mesenchymal stromal/stem cells (MSCs). Whether these proliferative MSCs are resident synovial cells or move into the tissue from elsewhere is not known. The aim of this study was to determine the contribution of bone marrow-derived cells to synovial hyperplasia following joint surface injury. METHODS: Lethally irradiated mice were transplanted with green fluorescent protein (GFP)-labelled bone marrow, and MSC chimerism was determined by the colony-forming unit fibroblast (CFU-F) assay and phenotypic analysis. To label host slow-cycling cells prior to bone marrow transplant, mice received iododeoxyuridine for 3 weeks. Mice then were subjected to GFP(+) bone marrow transplant, underwent joint surface injury and received chlorodeoxyuridine (CldU) for 7 days to label cells proliferating after injury. GFP- and nucleoside-labelled cells in normal and injured knee joint synovium were quantified in situ by immunofluorescence staining of paraffin-embedded tissue sections. The phenotype of GFP-labelled cells was determined by co-staining for the haematopoietic marker CD16/CD32 and the MSC/fibroblast marker platelet-derived growth factor receptor α (Pdgfrα). RESULTS: CFU-F assay and phenotypic analysis demonstrated successful bone marrow mesenchymal lineage chimerism in mice that underwent transplants. Bone marrow reconstitution preceded the detection of GFP-labelled cells in synovium. The percentage of GFP(+) cells in synovium increased significantly in response to injury, while the proportion of GFP(+) cells that were labelled with the proliferation marker CldU did not increase, suggesting that the expansion of the GFP(+) cell population in synovium was due mainly to bone marrow cell infiltration. In contrast, proliferation of host slow-cycling cells was significantly increased in the hyperplastic synovium. In both control and injured knee joints, the majority of marrow-derived GFP(+) cells in the synovium were haematopoietic (CD16/32(+)), while a minority of cells expressed the pan-fibroblast/MSC marker Pdgfrα. CONCLUSIONS: Our findings indicate that synovial hyperplasia following joint surface injury involves proliferation of resident slow-cycling cells, with a contribution from infiltrating bone marrow-derived cells. Understanding the process of synovial hyperplasia may reveal ways to restore homeostasis in injured joints and prevent secondary osteoarthritis.

Two cases of neurofibromatosis presenting with a foot deformity.

Two children presented with an isolated foot and ankle deformity. Examination in each suggested a plexiform neurofibroma although this diagnosis had not been considered before referral. Diagnosis of neurofibromatosis type 1 was confirmed by MRI scanning and on investigation both patients were proved to have widespread disease. One had a plexiform neurofibroma encasing the aorta and oesophagus. Both cases remain under observation and have not undergone surgery for their disease. Neurofibromatosis can present with isolated foot and ankle deformity and when such a diagnosis is suspected thorough investigation is important in a condition in which unsuspected widespread disease may exist.

Crystal structure and binding properties of the Serratia marcescens chitin-binding protein CBP21.

Chitin proteins are commonly found in bacteria that utilize chitin as a source of energy. CBP21 is a chitin-binding protein from Serratia marcescens, a Gram-negative soil bacterium capable of efficient chitin degradation. When grown on chitin, S. marcescens secretes large amounts of CBP21, along with chitin-degrading enzymes. In an attempt to understand the molecular mechanism of CBP21 action, we have determined its crystal structure at 1.55 angstroms resolution. This is the first structure to be solved of a family 33 carbohydrate-binding module. The structure reveals a "budded" fibronectin type III fold consisting of two beta-sheets, arranged as a beta-sheet sandwich, with a 65-residue "bud" consisting of three short helices, located between beta-strands 1 and 2. Remarkably, conserved aromatic residues that have been suggested previously to play a role in chitin binding were mainly found in the interior of the protein, seemingly incapable of interacting with chitin, whereas the structure revealed a surface patch of highly conserved, mainly hydrophilic residues. The roles of six of these conserved surface-exposed residues (Tyr-54, Glu-55, Glu-60, His-114, Asp-182, and Asn-185) were probed by site-directed mutagenesis and subsequent binding studies. All single point mutations lowered the affinity of CBP21 for beta-chitin, as shown by 3-8-fold increases in the apparent binding constant. Thus, binding of CBP21 to chitin seems to be mediated primarily by conserved, solvent-exposed, polar side chains.