In vitro and in vivo evaluation of 3D constructs engineered with human iPSC-derived chondrocytes in gelatin methacryloyl hydrogel.

Articular cartilage defects have limited healing potential and, when left untreated, can lead to osteoarthritis. Tissue engineering focuses on regenerating the damaged joint surface, preferably in an early stage. Here, we investigate the regenerative potential of three-dimensional (3D) constructs consisting of human induced pluripotent stem cell (iPSC)-derived chondrocytes in gelatin methacryloyl (GelMA) hydrogel for stable hyaline cartilage production. iPSC-derived chondrocytes are encapsulated in GelMA hydrogel at low (1 × 107 ml-1 ) and high (2 × 107 ml-1 ) density. In a conventional medium, GelMA hydrogel supports the chondrocyte phenotype, as opposed to cells cultured in 3D in absence of hydrogel. Moreover, encapsulated iPSC-derived chondrocytes preserve their in vivo matrix formation capacity after 21 days in vitro. In differentiation medium, hyaline cartilage-like tissue forms after 21 days, demonstrated by highly sulfated glycosaminoglycans and collagen type II. Matrix deposition is delayed at low encapsulation density, corroborating with lower transcript levels of COL2A1. An ectopic assay in nude mice demonstrates further maturation of the matrix deposited in vitro. Direct ectopic implantation of iPSC-derived chondrocyte-laden GelMA, without in vitro priming, also generates hyaline cartilage-like tissue, albeit less mature. Since it is unclear what maturity upon implantation is desired for joint surface regeneration, this is an attractive technology to generate immature and more mature hyaline cartilage-like tissue.

Reduced knee extensor torque production at low to moderate velocities in postmenopausal women with knee osteoarthritis.

This study aimed to determine deficits in knee extensor muscle function through the torque-time and torque-velocity relationships and whether these deficits are associated with reduced functional performance in postmenopausal women with knee osteoarthritis (KOA). A clinical sample of postmenopausal women with established KOA (n = 18, ≥55 years) was compared to an age-matched healthy control sample (CON) (n = 26). The deficits in different parameters of the knee extensor torque-time (maximal isometric torque and rate of torque development) and torque-velocity relationship (maximum muscle power, maximal velocity and torque at 0-500°·s-1 ) were assessed through a protocol consisting of isometric, isotonic and isokinetic tests. Functional performance was evaluated with sit-to-stand and stair-climbing tasks using a sensor-based technology (ie, time- and power-based outcomes). Postmenopausal women with KOA showed reduced maximal isometric torque (Hedge's g effect size (g) = 1.05, p = 0.001) and rate of torque development (g = 0.77-1.17, all p ≤ 0.02), combined with impaired torque production at slow to moderate velocities (g = 0.92-1.70, p ≤ 0.004), but not at high or maximal velocities (g = 0.16, p > 0.05). KOA were slower (g = 0.81-0.92, p ≤ 0.011) and less powerful (g = 1.11-1.29, p ≤ 0.001) during functional tasks. Additionally, knee extensor deficits were moderately associated with power deficits in stair climbing (r = 0.492-0.659). To conclude, knee extensor muscle weakness was presented in postmenopausal women with KOA, not only as limited maximal and rapid torque development during isometric contractions, but also dynamically at low to moderate velocities. These deficits were related to impaired functional performance. The assessment of knee extensor muscle weakness through the torque-time and torque-velocity relationships might enable individual targets for tailored exercise interventions in KOA.

Mapping human serum-induced gene networks as a basis for the creation of biomimetic periosteum for bone repair.

BACKGROUND: The periosteum is a highly vascularized, collagen-rich tissue that plays a crucial role in directing bone repair. This is orchestrated primarily by its resident progenitor cell population. Indeed, preservation of periosteum integrity is critical for bone healing. Cells extracted from the periosteum retain their osteochondrogenic properties and as such are a promising basis for tissue engineering strategies for the repair of bone defects. However, the culture expansion conditions and the way in which the cells are reintroduced to the defect site are critical aspects of successful translation. Indeed, expansion in human serum and implantation on biomimetic materials has previously been shown to improve in vivo bone formation. AIM: This study aimed to develop a protocol to allow for the expansion of human periosteum derived cells (hPDCs) in a biomimetic periosteal-like environment. METHODS: The expansion conditions were defined through the investigation of the bioactive cues involved in augmenting hPDC proliferative and multipotency characteristics, based on transcriptomic analysis of cells cultured in human serum. RESULTS: Master regulators of transcriptional networks were identified, and an optimized periosteum-derived growth factor cocktail (PD-GFC; containing ß-estradiol, FGF2, TNFα, TGFß, IGF-1 and PDGF-BB) was generated. Expansion of hPDCs in PD-GFC resulted in serum mimicry with regard to the cell morphology, proliferative capacity and chondrogenic differentiation. When incorporated into a three-dimensional collagen type 1 matrix and cultured in PD-GFC, the hPDCs migrated to the surface that represented the matrix topography of the periosteum cambium layer. Furthermore, gene expression analysis revealed a down-regulated WNT and TGFß signature and an up-regulation of CREB, which may indicate the hPDCs are recreating their progenitor cell signature. CONCLUSION: This study highlights the first stage in the development of a biomimetic periosteum, which may have applications in bone repair.

Predicting in vitro human mesenchymal stromal cell expansion based on individual donor characteristics using machine learning.

BACKGROUND: Human mesenchymal stromal cells (hMSCs) have become attractive candidates for advanced medical cell-based therapies. An in vitro expansion step is routinely used to reach the required clinical quantities. However, this is influenced by many variables including donor characteristics, such as age and gender, and culture conditions, such as cell seeding density and available culture surface area. Computational modeling in general and machine learning in particular could play a significant role in deciphering the relationship between the individual donor characteristics and their growth dynamics. METHODS: In this study, hMSCs obtained from 174 male and female donors, between 3 and 64 years of age with passage numbers ranging from 2 to 27, were studied. We applied a Random Forests (RF) technique to model the cell expansion procedure by predicting the population doubling time (PDT) for each passage, taking into account individual donor-related characteristics. RESULTS: Using the RF model, the mean absolute error between model predictions and experimental results for the PDT in passage 1 to 4 is significantly lower compared with the errors obtained with theoretical estimates or historical data. Moreover, statistical analysis indicate that the PD and PDT in different age categories are significantly different, especially in the youngest group (younger than 10 years of age) compared with the other age groups. DISCUSSION: In summary, we introduce a predictive computational model describing in vitro cell expansion dynamics based on individual donor characteristics, an approach that could greatly assist toward automation of a cell expansion culture process.

Neurofibromatosis type 1-related pseudarthrosis: Beyond the pseudarthrosis site.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder affecting approximately 1 in 2,000 newborns. Up to 5% of NF1 patients suffer from pseudarthrosis of a long bone (NF1-PA). Current treatments are often unsatisfactory, potentially leading to amputation. To gain more insight into the pathogenesis we cultured cells from PA tissue and normal-appearing periosteum of the affected bone for NF1 mutation analysis. PA cells were available from 13 individuals with NF1. Biallelic NF1 inactivation was identified in all investigated PA cells obtained during the first surgery. Three of five cases sampled during a later intervention showed biallelic NF1 inactivation. Also, in three individuals, we examined periosteum-derived cells from normal-appearing periosteum proximal and distal to the PA. We identified the same biallelic NF1 inactivation in the periosteal cells outside the PA region. These results indicate that NF1 inactivation is required but not sufficient for the development of NF1-PA. We observed that late-onset NF1-PA occurs and is not always preceded by congenital bowing. Furthermore, the failure to identify biallelic inactivation in two of five later interventions and one reintervention with a known somatic mutation indicates that NF1-PA can persist after the removal of most NF1 negative cells.

Educational outreach visits to improve knee osteoarthritis management in primary care.

BACKGROUND: Knee osteoarthritis is a common problem, but often underdiagnosed and undertreated in primary care as compared to evidence-based guidelines. Educational outreach visits are an effective strategy to improve guideline adherence, but its contribution to knee osteoarthritis management is largely unknown. The aim of this study was to evaluate the overall effectiveness of educational outreach visits on process quality indicators for knee osteoarthritis management, more specifically on the referral for physical therapy. METHODS: An educational intervention study, non-randomized and controlled, was designed for general practitioners (GPs) in Belgium. During four months, 426 GPs were visited by academic detailers and allocated to the intervention group. The control group was selected from GPs not visited by academic detailers during the study period. Six months post-intervention, both groups received a questionnaire with two case-vignettes to measure the effectiveness of the educational outreach. Outcomes were assessed with a Belgian set of quality indicators for knee osteoarthritis management and focused on the number of prescriptions for appropriate physical therapy (i.e. muscle strengthening, aerobic, functional or range of motion exercises) and the adherence to eight additional quality indicators related to knee osteoarthritis management. For the analysis, multivariable logistic regression models were used and Generalized Estimating Equations to handle the correlation between the multiple results per GP. RESULTS: The intervention group showed a tendency to prescribe more frequently at least one appropriate physical therapy for a case (43.8%), compared to the control group (31.3%, p = 0.057). Muscle strengthening exercises were the most frequently prescribed therapy with 37.0% in the intervention versus 26.9% in the control group. The adherence to the other quality indicators showed no significant difference between the intervention and control group and varied between 8.9 and 100% in the intervention group. CONCLUSIONS: This intervention did not alter significantly the adherence to quality indicators and in particular the probability of prescribing physical therapy. To change general practitioners' prescription behavior, more extensive or combined interventional approaches seem warranted.

In Vitro Screening of Molecularly Engineered Polyethylene Glycol Hydrogels for Cartilage Tissue Engineering using Periosteum-Derived and ATDC5 Cells.

The rapidly growing field of tissue engineering and regenerative medicine has brought about an increase in demand for biomaterials that mimic closely the form and function of biological tissues. Therefore, understanding the cellular response to the changes in material composition moves research one step closer to a successful tissue-engineered product. With this in mind, polyethylene glycol (PEG) hydrogels comprised of different concentrations of polymer (2.5%, 4%, 6.5%, or 8% (w/v)); different protease sensitive, peptide cross-linkers (VPMSMRGG or GPQGIWGQ); and the incorporation or lack of a peptide cell adhesion ligand (RGD) were screened for their ability to support in vitro chondrogenesis. Human periosteum-derived cells (hPDCs), a mesenchymal stem cell (MSC)-like primary cell source, and ATDC5 cells, a murine carcinoma-derived chondrogenic cell line, were encapsulated within the various hydrogels to assess the effects of the different formulations on cellular viability, proliferation, and chondrogenic differentiation while receiving exogenous growth factor stimulation via the medium. Through the results of this screening process, the 6.5% (w/v) PEG constructs, cross-linked with the GPQGIWGQ peptide and containing the RGD cell binding molecule, demonstrated an environment that consistently supported cellular viability and proliferation as well as chondrogenic differentiation.

Immersed Boundary Models for Quantifying Flow-Induced Mechanical Stimuli on Stem Cells Seeded on 3D Scaffolds in Perfusion Bioreactors.

Perfusion bioreactors regulate flow conditions in order to provide cells with oxygen, nutrients and flow-associated mechanical stimuli. Locally, these flow conditions can vary depending on the scaffold geometry, cellular confluency and amount of extra cellular matrix deposition. In this study, a novel application of the immersed boundary method was introduced in order to represent a detailed deformable cell attached to a 3D scaffold inside a perfusion bioreactor and exposed to microscopic flow. The immersed boundary model permits the prediction of mechanical effects of the local flow conditions on the cell. Incorporating stiffness values measured with atomic force microscopy and micro-flow boundary conditions obtained from computational fluid dynamics simulations on the entire scaffold, we compared cell deformation, cortical tension, normal and shear pressure between different cell shapes and locations. We observed a large effect of the precise cell location on the local shear stress and we predicted flow-induced cortical tensions in the order of 5 pN/µm, at the lower end of the range reported in literature. The proposed method provides an interesting tool to study perfusion bioreactors processes down to the level of the individual cell's micro-environment, which can further aid in the achievement of robust bioprocess control for regenerative medicine applications.

Initiating human articular chondrocyte re-differentiation in a 3D system after 2D expansion.

Cartilage damage affects a large population via acute and chronic injury and disease. Since native cartilage does not self-renew, cartilage tissue engineering has gained traction as a potential treatment. However, a limiting factor is that the primary cell type in cartilage, the articular chondrocyte, tends to de-differentiate when grown on 2D surfaces for in vitro expansion. Thus, 3D systems are being developed and used to counter this loss of chondrogenic capabilities. We hypothesize that a 3D matrix that can be remodeled may be more supportive of the chondrogenic phenotype of encapsulated articular chondrocytes than a 2D surface and may allow for the re-differentiation of chondrocytes after 2D expansion. Hence, in this study, enzymatically degradable polyethylene glycol (PEG) hydrogels containing two different protease degradable peptide segments, with different degradation rates, were tested in combination with chondrogenic medium as a 3D in vitro culture system to better recapitulate the native environment of human articular chondrocytes (hACs). In addition, the effect of incorporation of the integrin binding ligand Arg-Gly-Asp (RGD) in the hydrogels was explored. Hydrogels crosslinked with a slower degrading crosslinker and not functionalized with RGD maintained hAC viability and led to increased GAG production and chondrogenic gene expression over time, suggesting that this system can initiate hAC re-differentiation after 2D expansion.

Do Psychosocial Factors Predict Muscle Strength, Pain, or Physical Performance in Patients With Knee Osteoarthritis?

OBJECTIVE: The aim of this study was to examine the relationship of psychosocial factors, namely, pain catastrophizing, kinesiophobia, and maladaptive coping strategies, with muscle strength, pain, and physical performance in patients with knee osteoarthritis (OA)-related symptoms. METHODS: A total of 109 women (64 with knee OA-related symptoms) with a mean age of 65.4 years (49-81 years) were recruited for this study. Psychosocial factors were quantified by the Pain Catastrophizing Scale, Tampa Scale for Kinesiophobia, and Pain Coping Inventory. Clinical features were assessed using isometric and isokinetic knee muscle strength measurements, visual analog scale, Western Ontario and McMaster Universities Osteoarthritis Index, and functional tests. Associations were examined using correlation and regression analysis. RESULTS: In knee OA patients, pain catastrophizing, kinesiophobia, and coping strategy explained a significant proportion of the variability in isometric knee extension and flexion strength (6.3%-9.2%), accounting for more overall variability than some demographic and medical status variables combined. Psychosocial factors were not significant independent predictors of isokinetic strength, knee pain, or physical performance. CONCLUSIONS: In understanding clinical features related to knee OA, such as muscle weakness, pain catastrophizing, kinesiophobia, and coping strategy might offer something additional beyond what might be explained by traditional factors, underscoring the importance of a biopsychosocial approach in knee OA management. Further research on individual patient characteristics that mediate the effects of psychosocial factors is, however, required in order to create opportunities for more targeted, personalized treatment for knee OA.

Large-scale progenitor cell expansion for multiple donors in a monitored hollow fibre bioreactor.

BACKGROUND AIMS: With the increasing scale in stem cell production, a robust and controlled cell expansion process becomes essential for the clinical application of cell-based therapies. The objective of this work was the assessment of a hollow fiber bioreactor (Quantum Cell Expansion System from Terumo BCT) as a cell production unit for the clinical-scale production of human periosteum derived stem cells (hPDCs). METHODS: We aimed to demonstrate comparability of bioreactor production to standard culture flask production based on a product characterization in line with the International Society of Cell Therapy in vitro benchmarks and supplemented with a compelling quantitative in vivo bone-forming potency assay. Multiple process read-outs were implemented to track process performance and deal with donor-to-donor-related variation in nutrient needs and harvest timing. RESULTS: The data show that the hollow fiber bioreactor is capable of robustly expanding autologous hPDCs on a clinical scale (yield between 316 million and 444 million cells starting from 20 million after ± 8 days of culture) while maintaining their in vitro quality attributes compared with the standard flask-based culture. The in vivo bone-forming assay on average resulted in 10.3 ± 3.7% and 11.0 ± 3.8% newly formed bone for the bioreactor and standard culture flask respectively. The analysis showed that the Quantum system provides a reproducible cell expansion process in terms of yields and culture conditions for multiple donors.

Systemic Inflammation Affects Human Osteocyte-Specific Protein and Cytokine Expression.

Bone remodeling can be disturbed in active rheumatoid arthritis (RA), possibly as a result of elevated levels of circulating inflammatory cytokines. Osteocyte-specific proteins and cytokines play a vital role in bone remodeling by orchestrating bone formation and/or bone resorption. Therefore, we aimed to investigate the effect of RA-serum or inflammatory cytokines on expression of human osteocyte-specific proteins and cytokines. Human trabecular bone chips were cultured with RA-serum or inflammatory cytokines for 7-days. Live-dead staining was performed to assess cell viability. Gene expression of osteocyte-specific proteins and cytokines was analyzed by qPCR. Immuno-staining was performed for osteocyte-specific markers. Approximately 60 % of the osteocytes on the bone chips were alive at day-7. Cells in or on the bone chips did express the gene for osteocyte markers SOST, FGF23, DMP1, and MEPE, and the cytokines IL-1ß, IL-6, and TNFα at day 0 and 7. Active RA-serum treatment enhanced IL-1ß, TNFα, SOST, and DKK1 gene expression. IL-1ß treatment enhanced IL-1ß, TNFα, IL-6, IL-8, FGF23, and SOST gene expression. TNFα treatment enhanced IL-1ß, TNFα, IL-6, IL-8, and FGF23 gene expression. IL-8 treatment enhanced TNFα, IL-8, and FGF23 gene expression. A combination of IL-1ß, IL-6, and TNFα treatment synergistically upregulated IL-1ß, IL-6, and IL-8 gene expression, as well as enhanced TNFα, OPG, SOST, and FGF23, and inhibited DKK1 gene expression. In conclusion, gene expression of human osteocyte-specific proteins and cytokines was affected by RA-serum, and exogenous recombinant cytokines treatment suggesting that osteocytes could provide a new target to prevent systemic inflammation-induced bone loss in RA.

Serum of patients with active rheumatoid arthritis inhibits differentiation of osteochondrogenic precursor cells.

Delayed fracture healing is frequently experienced in patients with systemic inflammation such as during rheumatoid arthritis (RA). The reasons for this are diverse, but could also be caused by inflammatory cytokines and/or growth factors in serum from patients with active disease. We hypothesized that serum from patients with active RA contains circulating inflammatory factors that inhibit differentiation of osteochondrogenic precursors. Serum was obtained from 15 patients with active RA (active RA-sera) and from the same patients in clinical remission 1 year later (remission RA-sera; controls). The effect of active RA-sera on osteochondrogenic differentiation of chondrogenic ATDC5 cells and primary human periosteum-derived progenitor cells (HPDC) was determined in micromass culture. In ATDC5 cells, active RA-sera reduced Ki67 transcription levels by 40% and cartilage matrix accumulation by 14% at day 14, and Alp transcription levels by 16%, and matrix mineralization by 17% at day 21 compared with remission RA-sera. In HPDCs, active RA-sera inhibited metabolic activity by 8%, SOX9 transcription levels by 14%, and cartilage matrix accumulation by 7% at day 7 compared with remission RA-sera. In conclusion, sera from patients with active RA negatively affect differentiation of osteochondrogenic precursors, and as a consequence may contribute to delayed fracture healing in these patients.

Bcl11a is required for neuronal morphogenesis and sensory circuit formation in dorsal spinal cord development.

Dorsal spinal cord neurons receive and integrate somatosensory information provided by neurons located in dorsal root ganglia. Here we demonstrate that dorsal spinal neurons require the Krüppel-C(2)H(2) zinc-finger transcription factor Bcl11a for terminal differentiation and morphogenesis. The disrupted differentiation of dorsal spinal neurons observed in Bcl11a mutant mice interferes with their correct innervation by cutaneous sensory neurons. To understand the mechanism underlying the innervation deficit, we characterized changes in gene expression in the dorsal horn of Bcl11a mutants and identified dysregulated expression of the gene encoding secreted frizzled-related protein 3 (sFRP3, or Frzb). Frzb mutant mice show a deficit in the innervation of the spinal cord, suggesting that the dysregulated expression of Frzb can account in part for the phenotype of Bcl11a mutants. Thus, our genetic analysis of Bcl11a reveals essential functions of this transcription factor in neuronal morphogenesis and sensory wiring of the dorsal spinal cord and identifies Frzb, a component of the Wnt pathway, as a downstream acting molecule involved in this process.

A homeostatic function of CXCR2 signalling in articular cartilage.

OBJECTIVE: ELR+ CXC chemokines are heparin-binding cytokines signalling through the CXCR1 and CXCR2 receptors. ELR+ CXC chemokines have been associated with inflammatory arthritis due to their capacity to attract inflammatory cells. Here, we describe an unsuspected physiological function of these molecules in articular cartilage homeostasis. METHODS: Chemokine receptors and ligands were detected by immunohistochemistry, western blotting and RT-PCR. Osteoarthritis was induced in wild-type and CXCR2(-/-) mice by destabilisation of the medial meniscus (DMM). CXCR1/2 signalling was inhibited in vitro using blocking antibodies or siRNA. Chondrocyte phenotype was analysed using Alcian blue staining, RT-PCR and western blotting. AKT phosphorylation and SOX9 expression were upregulated using constitutively active AKT or SOX9 plasmids. Apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. RESULTS: CXCL6 was expressed in healthy cartilage and was retained through binding to heparan sulfate proteoglycans. CXCR2(-/-) mice developed more severe osteoarthritis than wild types following DMM, with increased chondrocyte apoptosis. Disruption of CXCR1/2 in human and CXCR2 signalling in mouse chondrocytes led to a decrease in extracellular matrix production, reduced expression of chondrocyte differentiation markers and increased chondrocyte apoptosis. CXCR2-dependent chondrocyte homeostasis was mediated by AKT signalling since forced expression of constitutively active AKT rescued the expression of phenotypic markers and the apoptosis induced by CXCR2 blockade. CONCLUSIONS: Our study demonstrates an important physiological role for CXCR1/2 signalling in maintaining cartilage homeostasis and suggests that the loss of ELR+ CXC chemokines during cartilage breakdown in osteoarthritis contributes to the characteristic loss of chondrocyte phenotypic stability.

Expansion of murine periosteal progenitor cells with fibroblast growth factor 2 reveals an intrinsic endochondral ossification program mediated by bone morphogenetic protein 2.

The preservation of the bone-forming potential of skeletal progenitor cells during their ex vivo expansion remains one of the major challenges for cell-based bone regeneration strategies. We report that expansion of murine periosteal cells in the presence of FGF2, a signal present during the early stages of fracture healing, is necessary and sufficient to maintain their ability to organize in vivo into a cartilage template which gives rise to mature bone. Implantation of FGF2-primed cells in a large bone defect in mice resulted in complete healing, demonstrating the feasibility of using this approach for bone tissue engineering purposes. Mechanistically, the enhanced endochondral ossification potential of FGF2-expanded periosteal cells is predominantly driven by an increased production of BMP2 and is additionally linked to an improved preservation of skeletal progenitor cells in the cultures. This characteristic is unique for periosteal cells, as FGF2-primed bone marrow stromal cells formed significantly less bone and progressed exclusively through the intramembranous pathway, revealing essential differences between both cell pools. Taken together, our findings provide insight in the molecular regulation of fracture repair by identifying a unique interaction between periosteal cells and FGF2. These insights may promote the development of cell-based therapeutic strategies for bone regeneration which are independent of the in vivo use of growth factors, thus limiting undesired side effects.

Enhanced endogenous bone morphogenetic protein signaling protects against bleomycin induced pulmonary fibrosis.

BACKGROUND: Effective treatments for fibrotic diseases such as idiopathic pulmonary fibrosis are largely lacking. Transforming growth factor beta (TGFß) plays a central role in the pathophysiology of fibrosis. We hypothesized that bone morphogenetic proteins (BMP), another family within the TGFß superfamily of growth factors, modulate fibrogenesis driven by TGFß. We therefore studied the role of endogenous BMP signaling in bleomycin induced lung fibrosis. METHODS: Lung fibrosis was induced in wild-type or noggin haploinsufficient (Nog +/LacZ ) mice by intratracheal instillation of bleomycin, or phosphate buffered saline as a control. Invasive pulmonary function tests were performed using the flexiVent® SCIREQ system. The mice were sacrificed and lung tissue was collected for analysis using histopathology, collagen quantification, immunohistochemistry and gene expression analysis. RESULTS: Nog +/LacZ mice are a known model of increased BMP signaling and were partially protected from bleomycin-induced lung fibrosis with reduced Ashcroft score, reduced collagen content and preservation of pulmonary compliance. In bleomycin-induced lung fibrosis, TGFß and BMP signaling followed an inverse course, with dynamic activation of TGFß signaling and repression of BMP signaling activity. CONCLUSIONS: Upon bleomycin exposure, active BMP signaling is decreased. Derepression of BMP signaling in Nog +/LacZ mice protects against bleomycin-induced pulmonary fibrosis. Modulating the balance between BMP and TGFß, in particular increasing endogenous BMP signals, may therefore be a therapeutic target in fibrotic lung disease.

Mechanical loading reduces inflammation-induced human osteocyte-to-osteoclast communication.

Multiple factors contribute to bone loss in inflammatory diseases such as rheumatoid arthritis (RA), but circulating inflammatory factors and immobilization play a crucial role. Mechanical loading prevents bone loss in the general population, but the effects of mechanical loading in patients with RA are less clear. Therefore, we aimed to investigate whether mechanical stimuli reverse the stimulatory effect of RA serum on osteocyte-to-osteoclast communication. Human primary osteocytes were pretreated with 10 % RA serum or healthy control serum for 7 days, followed by 1 h ± mechanical loading by pulsating fluid flow (PFF). Nitric oxide (NO) and prostaglandin E2 were measured in the medium. Receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), interleukin-6 (IL-6), cyclooxygenase-2 (COX2), matrix-extracellular phosphoglycoprotein (MEPE), cysteine-rich protein 61 (CYR61), and SOST gene expression was quantified by qPCR. Osteoclast precursors were cultured with PFF-conditioned medium (PFF-CM) or static-conditioned medium (stat-CM), and osteoclast formation was assessed. RA serum alone did not affect IL-6, CYR61, COX2, MEPE, or SOST gene expression in osteocytes. However, RA serum enhanced the RANKL/OPG expression ratio by 3.4-fold, while PFF nullified this effect. PFF enhanced NO production to the same extent in control serum (2.6-3.5-fold) and RA serum-pretreated (2.7-3.6-fold) osteocytes. Stat-CM from RA serum-pretreated osteocytes enhanced osteoclastogenesis compared with stat-CM from control serum-pretreated osteocytes, while PFF nullified this effect. In conclusion, RA serum, containing inflammatory factors, did not alter the intrinsic capacity of osteocytes to sense mechanical stimuli, but upregulated osteocyte-to-osteoclast communication. Mechanical loading nullified this upregulation, suggesting that mechanical stimuli could contribute to the prevention of osteoporosis in inflammatory disease.

Genome-wide association and functional studies identify the DOT1L gene to be involved in cartilage thickness and hip osteoarthritis.

Hip osteoarthritis (HOA) is one of the most disabling and common joint disorders with a large genetic component that is, however, still ill-defined. To date, genome-wide association studies (GWAS) in osteoarthritis (OA) and specifically in HOA have yielded only few loci, which is partly explained by heterogeneity in the OA definition. Therefore, we here focused on radiographically measured joint-space width (JSW), a proxy for cartilage thickness and an important underlying intermediate trait for HOA. In a GWAS of 6,523 individuals on hip-JSW, we identified the G allele of rs12982744 on chromosome 19p13.3 to be associated with a 5% larger JSW (P = 4.8 × 10(-10)). The association was replicated in 4,442 individuals from three United Kingdom cohorts with an overall meta-analysis P value of 1.1 × 10(-11). The SNP was also strongly associated with a 12% reduced risk for HOA (P = 1 × 10(-4)). The SNP is located in the DOT1L gene, which is an evolutionarily conserved histone methyltransferase, recently identified as a potentially dedicated enzyme for Wnt target-gene activation in leukemia. Immunohistochemical staining of the DOT1L protein in mouse limbs supports a role for DOT1L in chondrogenic differentiation and adult articular cartilage. DOT1L is also expressed in OA articular chondrocytes. Silencing of Dot1l inhibited chondrogenesis in vitro. Dot1l knockdown reduces proteoglycan and collagen content, and mineralization during chondrogenesis. In the ATDC5 chondrogenesis model system, DOT1L interacts with TCF and Wnt signaling. These data are a further step to better understand the role of Wnt-signaling during chondrogenesis and cartilage homeostasis. DOT1L may represent a therapeutic target for OA.

Smoc2 modulates embryonic myelopoiesis during zebrafish development.

BACKGROUND: SMOC2 is a member of the BM-40 (SPARC) family of matricellular proteins, reported to influence signaling in the extracellular compartment. In mice, Smoc2 is expressed in many different tissues and was shown to enhance the response to angiogenic growth factors, mediate cell adhesion, keratinocyte migration, and metastasis. Additionally, SMOC2 is associated with vitiligo and craniofacial and dental defects. The function of Smoc2 during early zebrafish development has not been determined to date. RESULTS: In pregastrula zebrafish embryos, smoc2 is expressed ubiquitously. As development progresses, the expression pattern becomes more anteriorly restricted. At the onset of blood cell circulation, smoc2 morphants presented a mild ventralization of posterior structures. Molecular analysis of the smoc2 morphants indicated myelopoietic defects in the rostral blood islands during segmentation stages. Hemangioblast development and further specification of the myeloid progenitor cells were shown to be impaired. Additional experiments indicated that Bmp target genes were down-regulated in smoc2 morphants. CONCLUSIONS: Our findings reveal that Smoc2 is an essential player in the development of myeloid cells of the anterior lateral plate mesoderm during embryonic zebrafish development. Furthermore, our data show that Smoc2 affects the transcription of Bmp target genes without affecting initial dorsoventral patterning or mesoderm development.