Functionalization of tissue engineering scaffolds with in vitro–generated bone-like extracellular matrix (ECM) represents an effective biomimetic approach to promote osteogenic differentiation of stem cells in vitro. However, the bone-forming capacity of these constructs (seeded with or without cells) is so far not apparent. In this study, we aimed at developing a mineralizing culture condition to biofunctionalize three-dimensional (3D) porous scaffolds with highly mineralized ECM in order to produce devitalized, osteoinductive mineralized carriers for human periosteal-derived progenitors (hPDCs). For this, three medium formulations [i.e., growth medium only (BM1), with ascorbic acid (BM2), and with ascorbic acid and dexamethasone (BM3)] supplemented with calcium (Ca2+) and phosphate (PO43−) ions simultaneously as mineralizing source were investigated. The results showed that, besides the significant impacts on enhancing cell proliferation (the highest in BM3 condition), the formulated mineralizing media differentially regulated the osteochondro-related gene markers in a medium-dependent manner (e.g., significant upregulation of BMP2, bone sialoprotein, osteocalcin, and Wnt5a in BM2 condition). This has resulted in distinguished cell populations that were identifiable by specific gene signatures as demonstrated by the principle component analysis. Through devitalization, mineralized carriers with apatite crystal structures unique to each medium condition (by X-ray diffraction and SEM analysis) were obtained. Quantitatively, BM3 condition produced carriers with the highest mineral and collagen contents as well as human-specific VEGF proteins, followed by BM2 and BM1 conditions. Encouragingly, all mineralized carriers (after reseeded with hPDCs) induced bone formation after 8 weeks of subcutaneous implantation in nude mice models, with BM2-carriers inducing the highest bone volume, and the lowest in the BM3 condition (as quantitated by nano-computed tomography [nano-CT]). Histological analysis revealed different bone formation patterns, either bone ossicles containing bone marrow surrounding the scaffold struts (in BM2) or bone apposition directly on the struts' surface (in BM1 and BM3). In conclusion, we have presented experimental data on the feasibility to produce devitalized osteoinductive mineralized carriers by functionalizing 3D porous scaffolds with an in vitro cell-made mineralized matrix under the mineralizing culture conditions.
biomineralization; bone regeneration; devitalization; mineralized extracellular matrix; osteoinductive carriers
Generalized osteoporosis is common in patients with inflammatory diseases, possibly because of circulating inflammatory factors that affect osteoblast and osteoclast formation and activity. Serum levels of the inflammatory factors CXCL8 and CCL20 are elevated in rheumatoid arthritis, but whether these factors affect bone metabolism is unknown. We hypothesized that CXCL8 and CCL20 decrease osteoblast proliferation and differentiation, and enhance osteoblast-mediated osteoclast formation and activity. Human primary osteoblasts were cultured with or without CXCL8 (2–200 pg/ml) or CCL20 (5–500 pg/ml) for 14 days. Osteoblast proliferation and gene expression of matrix proteins and cytokines were analyzed. Osteoclast precursors were cultured with CXCL8 (200 pg/ml) and CCL20 (500 pg/ml), or with conditioned medium (CM) from CXCL8 and CCL20-treated osteoblasts with or without IL-6 inhibitor. After 3 weeks osteoclast formation and activity were determined. CXCL8 (200 pg/ml) and CCL20 (500 pg/ml) enhanced mRNA expression of KI67 (2.5–2.7-fold), ALP (1.6–1.7-fold), and IL-6 protein production (1.3–1.6-fold) by osteoblasts. CXCL8-CM enhanced the number of osteoclasts with 3–5 nuclei (1.7-fold), and with >5 nuclei (3-fold). CCL20-CM enhanced the number of osteoclasts with 3–5 nuclei (1.3-fold), and with >5 nuclei (2.8-fold). IL-6 inhibition reduced the stimulatory effect of CXCL8-CM and CCL20-CM on formation of osteoclasts. In conclusion, CXCL8 and CCL20 did not decrease osteoblast proliferation or gene expression of matrix proteins. CXCL8 and CCL20 did not directly affect osteoclastogenesis. However, CXCL8 and CCL20 enhanced osteoblast-mediated osteoclastogenesis, partly via IL-6 production, suggesting that CXCL8 and CCL20 may contribute to osteoporosis in rheumatoid arthritis by affecting bone cell communication.
To progress the fields of tissue engineering (TE) and regenerative medicine, development of quantitative methods for non-invasive three dimensional characterization of engineered constructs (i.e. cells/tissue combined with scaffolds) becomes essential. In this study, we have defined the most optimal staining conditions for contrast-enhanced nanofocus computed tomography for three dimensional visualization and quantitative analysis of in vitro engineered neo-tissue (i.e. extracellular matrix containing cells) in perfusion bioreactor-developed Ti6Al4V constructs. A fractional factorial ‘design of experiments’ approach was used to elucidate the influence of the staining time and concentration of two contrast agents (Hexabrix and phosphotungstic acid) and the neo-tissue volume on the image contrast and dataset quality. Additionally, the neo-tissue shrinkage that was induced by phosphotungstic acid staining was quantified to determine the operating window within which this contrast agent can be accurately applied. For Hexabrix the staining concentration was the main parameter influencing image contrast and dataset quality. Using phosphotungstic acid the staining concentration had a significant influence on the image contrast while both staining concentration and neo-tissue volume had an influence on the dataset quality. The use of high concentrations of phosphotungstic acid did however introduce significant shrinkage of the neo-tissue indicating that, despite sub-optimal image contrast, low concentrations of this staining agent should be used to enable quantitative analysis. To conclude, design of experiments allowed us to define the most optimal staining conditions for contrast-enhanced nanofocus computed tomography to be used as a routine screening tool of neo-tissue formation in Ti6Al4V constructs, transforming it into a robust three dimensional quality control methodology.
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.
Rheumatoid arthritis; Generalized osteoporosis; Inflammatory cytokines; Pulsating fluid flow; Osteoclastogenesis
To successfully implement tissue-engineered (TE) constructs as part of a clinical therapy, it is necessary to develop quality control tools that will ensure accurate and consistent TE construct release specifications. Hence, advanced methods to monitor TE construct properties need to be further developed. In this study, we showed proof of concept for contrast-enhanced nanofocus computed tomography (CE-nano-CT) as a whole-construct imaging technique with a noninvasive potential that enables three-dimensional (3D) visualization and quantification of in vitro engineered extracellular matrix (ECM) in TE constructs. In particular, we performed a 3D qualitative and quantitative structural and spatial assessment of the in vitro engineered ECM, formed during static and perfusion bioreactor cell culture in 3D TE scaffolds, using two contrast agents, namely, Hexabrix® and phosphotungstic acid (PTA). To evaluate the potential of CE-nano-CT, a comparison was made to standardly used techniques such as Live/Dead viability/cytotoxicity, Picrosirius Red staining, and to net dry weight measurements of the TE constructs. When using Hexabrix as the contrast agent, the ECM volume fitted linearly with the net dry ECM weight independent from the flow rate used, thus suggesting that it stains most of the ECM. When using PTA as the contrast agent, comparing to net weight measurements showed that PTA only stains a part of the ECM. This was attributed to the binding specificity of this contrast agent. In addition, the PTA-stained CE-nano-CT data showed pronounced distinction between flow conditions when compared to Hexabrix, indicating culture-specific structural ECM differences. This novel type of information can contribute to optimize bioreactor culture conditions and potentially critical quality characteristics of TE constructs such as ECM quantity and homogeneity, facilitating the gradual transformation of TE constructs in well-characterized TE products.
Osteoarthritis is a chronic degenerative disorder of the joint and represents one of the most common diseases worldwide. Its prevalence and severity are increasing owing to aging of the population, but treatment options remain largely limited to painkillers and anti-inflammatory drugs, which only provide symptomatic relief. In the late stages of the disease, surgical interventions are often necessary to partially restore joint function. Although the focus of osteoarthritis research has been originally on the articular cartilage, novel findings are now pointing to osteoarthritis as a disease of the whole joint, in which failure of different joint components can occur. In this Review, we summarize recent progress in the field, including data from novel ‘omics’ technologies and from a number of preclinical and clinical trials. We describe different in vitro and in vivo systems that can be used to study molecules, pathways and cells that are involved in osteoarthritis. We illustrate that a comprehensive and multisystem approach is necessary to understand the complexity and heterogeneity of the disease and to better guide the development of novel therapeutic strategies for osteoarthritis.
Osteoarthritis; Cartilage; Bone; Animal models
GPR22 is an orphan G protein-coupled receptor (GPCR). Since the ligand of the receptor is currently unknown, its biological function has not been investigated in depth. Many GPCRs and their intracellular effectors are targeted to cilia. Cilia are highly conserved eukaryotic microtubule-based organelles that protrude from the membrane of most mammalian cells. They are involved in a large variety of physiological processes and diseases. However, the details of the downstream pathways and mechanisms that maintain cilia length and structure are poorly understood. We show that morpholino knock down or overexpression of gpr22 led to defective left-right (LR) axis formation in the zebrafish embryo. Specifically, defective LR patterning included randomization of the left-specific lateral plate mesodermal genes (LPM) (lefty1, lefty2, southpaw and pitx2a), resulting in randomized cardiac looping. Furthermore, gpr22 inactivation in the Kupffer’s vesicle (KV) alone was still able to generate the phenotype, indicating that Gpr22 mainly regulates LR asymmetry through the KV. Analysis of the KV cilia by immunofluorescence and transmission electron microscopy (TEM), revealed that gpr22 knock down or overexpression resulted in changes of cilia length and structure. Further, we found that Gpr22 does not act upstream of the two cilia master regulators, Foxj1a and Rfx2. To conclude, our study characterized a novel player in the field of ciliogenesis.
The pathogenesis of pulmonary fibrosis remains poorly understood. The Wnt signaling pathway regulates fibrogenesis in different organs. Here, we studied the role of two extracellular Wnt antagonists, secreted frizzled-related protein-1 (SFRP1) and frizzled-related protein (FRZB) on lung fibrosis in vitro and in vivo. For this purpose, we used an alveolar epithelial cell line and a lung fibroblast cell line, and the bleomycin-induced lung fibrosis model, respectively.
During the course of bleomycin-induced lung fibrosis, Sfrp1 and Frzb expression are upregulated. Expression of Sfrp1 appears much higher than that of Frzb. In vitro, recombinant SFRP1, but not FRZB, counteracts the transforming growth factor β1 (TGFβ1)-induced upregulation of type I collagen expression both in pulmonary epithelial cells and fibroblasts. Both SFRP1 and FRZB inhibit the TGFβ1-induced increase of active β-catenin, but do not influence the TGFβ1-induced phosphorylation levels of SMAD3, positioning Wnt signaling activity downstream of the active TGFβ signal in lung fibroblasts, but not in alveolar epithelial cells. In vivo, Sfrp1
mice showed identical responses to bleomycin in the lung compared to wild-type controls.
Although SFRP1 counteracts the effect of TGFβ1 in pulmonary cells in vitro; loss of neither SFRP1 nor FRZB alters fibrotic outcomes in the lungs in vivo. The lack of in vivo effect in the absence of specific SFRPs suggests functional redundancy within this family of Wnt antagonists.
Transforming growth factor-beta; WNT signaling; Secreted frizzled related proteins; Bleomycin; Pulmonary fibrosis; Skin fibrosis
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-C2H2 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.
Spinal cord; Transcription factor; Neuronal differentiation; Bcl11a (CTIP1); Mouse
The current White paper summarizes the discussions and exchange of experiences during the first European Interdisciplinary Summit on Cell-Based ATMPs held in Vienna, Austria, May 02–03, 2013. The meeting was supported by the Research Networking Programme REMEDIC (regenerative medicine) funded by the European Science Foundation and by the British Medical Research Council. To improve the competitiveness of Europe in the field of cell-based Advanced Medicinal Therapy Products (ATMPs), the following key issues were identified during the meeting: removal of national hurdles in the European Union, harmonization of national and subnational differences in Hospital Exemption rules, improved treatment algorithms for reimbursement, better knowledge on the mode of action, predictive preclinical efficacy and safety testing, need for innovative systems for preclinical testing, appropriate product characterization, manufacturing with cost of goods in mind, and appropriate design of clinical trials.
Joint ankylosis is a major cause of disability in the human spondyloarthropathies. Here we report that this process partially recapitulates embryonic endochondral bone formation in a spontaneous model of arthritis in DBA/1 mice. Bone morphogenetic protein (BMP) signaling appears to be a key molecular pathway involved in this pathological cascade. Systemic gene transfer of noggin, a BMP antagonist, is effective both as a preventive and a therapeutic strategy in the mouse model, mechanistically interfering with enthesial progenitor cell proliferation in early stages of the disease process. Immunohistochemical staining for phosphorylated smad1/5 in enthesial biopsies of patients with spondyloarthropathy reveals active BMP signaling in similar target cells. Our data suggest that BMP signaling is an attractive therapeutic target for interfering with structural changes in spondyloarthropathy either as an alternative or complementary approach to current antiinflammatory treatments.
We have demonstrated previously that adult human synovial membrane-derived mesenchymal stem cells (hSM-MSCs) have myogenic potential in vitro (De Bari, C., F. Dell'Accio, P. Tylzanowski, and F.P. Luyten. 2001. Arthritis Rheum. 44:1928–1942). In the present study, we have characterized their myogenic differentiation in a nude mouse model of skeletal muscle regeneration and provide proof of principle of their potential use for muscle repair in the mdx mouse model of Duchenne muscular dystrophy. When implanted into regenerating nude mouse muscle, hSM-MSCs contributed to myofibers and to long term persisting functional satellite cells. No nuclear fusion hybrids were observed between donor human cells and host mouse muscle cells. Myogenic differentiation proceeded through a molecular cascade resembling embryonic muscle development. Differentiation was sensitive to environmental cues, since hSM-MSCs injected into the bloodstream engrafted in several tissues, but acquired the muscle phenotype only within skeletal muscle. When administered into dystrophic muscles of immunosuppressed mdx mice, hSM-MSCs restored sarcolemmal expression of dystrophin, reduced central nucleation, and rescued the expression of mouse mechano growth factor.
Duchenne muscular dystrophy; dystrophin; cell transplantation; muscle development; insulin-like growth factor I
The osteoarthritic diseases are common disorders characterized by progressive destruction of the articular cartilage in the joints, and associated with remodeling of the subchondral bone, synovitis and the formation of bone outgrowths at the joint margins, osteophytes. From the clinical perspective, osteoarthritis leads to joint pain and loss of function. Osteoarthritis is the leading cause of progressive disability. New data from genetic, translational and basic research have demonstrated that pathways with essential roles in joint and bone development also contribute to the postnatal homeostasis of the articular cartilage and are involved in osteoarthritis, making these potential therapeutic targets. Other systems of interest are the tissue-destructive enzymes that break down the extracellular matrix of the cartilage as well as mediators of inflammation that contribute to synovitis. However, the perspective of a durable treatment over years to decades highlights the need for a personalized medicine approach encompassing a global view on the disease and its management, thereby including nonpharmaceutical approaches such as physiotherapy and advanced surgical methods. Integration of novel strategies based on their efficacy and safety with the identification of individuals at risk and optimal individual rehabilitation management remains a major challenge for the medical community in particular, as the incidence of osteoarthritis is likely to further increase with the overall aging of the population.
Osteoarthritis (OA) is the most prevalent form of arthritis and accounts for substantial morbidity and disability, particularly in the elderly. It is characterized by changes in joint structure including degeneration of the articular cartilage and its etiology is multifactorial with a strong postulated genetic component. We performed a meta-analysis of four genome-wide association (GWA) studies of 2,371 knee OA cases and 35,909 controls in Caucasian populations. Replication of the top hits was attempted with data from additional ten replication datasets. With a cumulative sample size of 6,709 cases and 44,439 controls, we identified one genome-wide significant locus on chromosome 7q22 for knee OA (rs4730250, p-value=9.2×10−9), thereby confirming its role as a susceptibility locus for OA. The associated signal is located within a large (500kb) linkage disequilibrium (LD) block that contains six genes; PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, beta), HPB1 (HMG-box transcription factor 1), COG5 (component of oligomeric golgi complex 5), GPR22 (G protein-coupled receptor 22), DUS4L (dihydrouridine synthase 4-like), and BCAP29 (the B-cell receptor-associated protein 29). Gene expression analyses of the (six) genes in primary cells derived from different joint tissues confirmed expression of all the genes in the joint environment.
In vivo high-resolution micro-computed tomography allows for longitudinal image-based measurements in animal models of lung disease. The combination of repetitive high resolution imaging with fully automated quantitative image analysis in mouse models of lung fibrosis lung benefits preclinical research. This study aimed to develop and validate such an automated micro-computed tomography analysis algorithm for quantification of aerated lung volume in mice; an indicator of pulmonary fibrosis and emphysema severity.
Mice received an intratracheal instillation of bleomycin (n = 8), elastase (0.25U elastase n = 9, 0.5U elastase n = 8) or saline control (n = 6 for fibrosis, n = 5 for emphysema). A subset of mice was scanned without intervention, to evaluate potential radiation-induced toxicity (n = 4). Some bleomycin-instilled mice were treated with imatinib for proof of concept (n = 8). Mice were scanned weekly, until four weeks after induction, when they underwent pulmonary function testing, lung histology and collagen quantification. Aerated lung volumes were calculated with our automated algorithm.
Our automated image-based aerated lung volume quantification method is reproducible with low intra-subject variability. Bleomycin-treated mice had significantly lower scan-derived aerated lung volumes, compared to controls. Aerated lung volume correlated with the histopathological fibrosis score and total lung collagen content. Inversely, a dose-dependent increase in lung volume was observed in elastase-treated mice. Serial scanning of individual mice is feasible and visualized dynamic disease progression. No radiation-induced toxicity was observed. Three-dimensional images provided critical topographical information.
We report on a high resolution in vivo micro-computed tomography image analysis algorithm that runs fully automated and allows quantification of aerated lung volume in mice. This method is reproducible with low inherent measurement variability. We show that it is a reliable quantitative tool to investigate experimental lung fibrosis and emphysema in mice. Its non-invasive nature has the unique benefit to allow dynamic 4D evaluation of disease processes and therapeutic interventions.
To identify genes involved in osteoarthritis (OA), the most prevalent form of joint disease, we performed a genome-wide association study (GWAS) in which we tested 500,510 Single Nucelotide Polymorphisms (SNPs) in 1341 OA cases and 3496 Dutch Caucasian controls. SNPs associated with at least two OA-phenotypes were analysed in 14,938 OA cases and approximately 39,000 controls. The C-allele of rs3815148 on chromosome 7q22 (MAF 23%, 172 kb upstream of the GPR22 gene) was consistently associated with a 1.14-fold increased risk (95%CI: 1.09–1.19) for knee- and/or hand-OA (p=8×10−8), and also with a 30% increased risk for knee-OA progression (95%CI: 1.03–1.64, p=0.03). This SNP is in almost complete linkage disequilibrium with rs3757713 (located 68 kb upstream of GPR22) which is associated with GPR22 expression levels in lymphoblast cell lines (p=4×10−12). GPR22 encodes an G-protein coupled receptor with unkown ligand (orphan receptor). Immunohistochemistry experiments showed absence of GPR22 in normal mouse articular cartilage or synovium. However, GPR22 positive chondrocytes were found in the upper layers of the articular cartilage of mouse knee joints that were challenged by in vivo papain treatment or in the presence of interleukin-1 driven inflammation. GRP22 positive chondrocyte-like cells were also found in osteophytes in instability-induced OA. In addition, GPR22 is also present in areas of the brain involved in locomotor function. Our findings reveal a novel common variant on chromosome 7q22 to influence susceptibility for prevalence and progression of OA.
During endochondral ossification, chondrocyte growth and differentiation is controlled by many local signalling pathways. Due to crosstalks and feedback mechanisms, these interwoven pathways display a network like structure. In this study, a large-scale literature based logical model of the growth plate network was developed. The network is able to capture the different states (resting, proliferating and hypertrophic) that chondrocytes go through as they progress within the growth plate. In a first corroboration step, the effect of mutations in various signalling pathways of the growth plate network was investigated.
Studies in the spontaneous ankylosis model in aging male DBA/1 mice and in patients with ankylosing spondylitis provide evidence that inflammation and new tissue formation leading to joint or spine ankylosis are likely linked but largely uncoupled processes. We previously proposed the 'entheseal stress' hypothesis that defines microdamage or cell stress in the enthesis as a trigger for these disease processes. Here, we further investigated the relationship between inflammation and ankylosis by focusing on the early phase of the spontaneous arthritis model.
Aging male DBA/1 mice from different litters were caged together at the age of ten weeks and studied for signs of arthritis. A group of DBA/1 mice were treated daily with dexamethasone (0.5 μg/g body weight). Severity of disease was assessed by histomorphology and by positron emission tomography (PET) using 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) as a tracer. Bone loss in dexamethasone-treated or control mice was determined by in vivo dual-energy X-ray absorptiometry. Chemokine gene expression was studied ex vivo in dissected paws and in vitro in mesenchymal cells (periosteal and bone marrow stromal cells) by quantitative real-time PCR in the presence or absence of bone morphogenetic protein 2 (BMP2) and dexamethasone.
Dexamethasone treatment did not affect incidence or severity of ankylosis, but led to an expected reduction in inflammation in the paws at week 15 as measured by PET tracer uptake. Treatment with dexamethasone negatively affected bone mineral density. Chemokines attracting neutrophils and lymphocytes were expressed in affected paws. In vitro, BMP2 stimulation upregulated chemokines in different mesenchymal joint-associated cell types, an effect that was inhibited by dexamethasone.
BMP signaling may be a trigger for both inflammation and ankylosis in the spontaneous model of ankylosing enthesitis. The lack of inhibition by glucocorticoids on new bone formation while causing systemic bone loss highlights the paradoxical simultaneous loss and gain of bone in patients with ankylosing spondylitis.
The aim of this research was to study molecular changes in the articular cartilage and subchondral bone of the tibial plateau from mice deficient in frizzled-related protein (Frzb) compared to wild-type mice by transcriptome analysis.
Gene-expression analysis of the articular cartilage and subchondral bone of three wild-type and three Frzb-/- mice was performed by microarray. Data from three wild-type and two Frzb-/- samples could be used for pathway analysis of differentially expressed genes and were explored with PANTHER, DAVID and GSEA bioinformatics tools. Activation of the wingless-type (WNT) pathway was analysed using Western blot. The effects of Frzb gain and loss of function on chondrogenesis and cell proliferation was examined using ATDC5 micro-masses and mouse ribcage chondrocytes.
Extracellular matrix-associated integrin and cadherin pathways, as well as WNT pathway genes were up-regulated in Frzb-/- samples. Several WNT receptors, target genes and other antagonists were up-regulated, but no difference in active β-catenin was found. Analysis of ATDC5 cell micro-masses overexpressing FRZB indicated an up-regulation of aggrecan and Col2a1, and down-regulation of molecules related to damage and repair in cartilage, Col3a1 and Col5a1. Silencing of Frzb resulted in down-regulation of aggrecan and Col2a1. Pathways associated with cell cycle were down-regulated in this transcriptome analysis. Ribcage chondrocytes derived from Frzb-/- mice showed decreased proliferation compared to wild-type cells.
Our analysis provides evidence for tight regulation of WNT signalling, shifts in extracellular matrix components and effects on cell proliferation and differentiation in the articular cartilage - subchondral bone unit in Frzb-/- mice. These data further support an important role for FRZB in joint homeostasis and highlight the complex biology of WNT signaling in the joint.
To address the need for standardization of osteoarthritis (OA) phenotypes by examining the effect of heterogeneity among symptomatic (SOA) and radiographic osteoarthritis (ROA) phenotypes.
Descriptions of OA phenotypes of the 28 studies involved in the TREAT-OA consortium were collected. To investigate whether different OA definitions result in different association results, we created hip OA definitions used within the consortium in the Rotterdam Study-I and tested the association of hip OA with gender, age and BMI using one-way ANOVA. For radiographic OA, we standardized the hip, knee and hand ROA definitions and calculated prevalence's of ROA before and after standardization in 9 cohort studies. This procedure could only be performed in cohort studies and standardization of SOA definitions was not feasible at this moment.
In this consortium, all studies with symptomatic OA phenotypes (knee, hip and hand) used a different definition and/or assessment of OA status. For knee, hip and hand radiographic OA 5, 4 and 7 different definitions were used, respectively. Different hip OA definitions do lead to different association results. For example, we showed in the Rotterdam Study-I that hip OA defined as “at least definite JSN and one definite osteophyte” was not associated with gender (p=0.22), but defined as “at least one definite osteophyte” was significantly associated with gender (p=3×10−9). Therefore, a standardization process was undertaken for radiographic OA definitions. Before standardization a wide range of ROA prevalence's was observed in the 9 cohorts studied. After standardization the range in prevalence of knee and hip ROA was small. Standardization of SOA phenotypes was not possible due to the case-control design of the studies.
Phenotype definitions influence the prevalence of OA and association with clinical variables. ROA phenotypes within the TREAT-OA consortium were standardized to reduce heterogeneity and improve power in future genetics studies.
Regenerative medicine seeks to repair or replace damaged tissues or organs, with the goal to fully restore structure and function without the formation of scar tissue. Cell based therapies are promising new therapeutic approaches in regenerative medicine. By using mesenchymal stem cells, good results have been reported for bone engineering in a number of clinical studies, most of them investigator initiated trials with limited scope with respect to controls and outcome. With the implementation of a new regulatory framework for advanced therapeutic medicinal products, the stage is set to improve both the characterization of the cells and combination products, and pave the way for improved controlled and well-designed clinical trials. The incorporation of more personalized medicine approaches, including the use of biomarkers to identify the proper patients and the responders to treatment, will be contributing to progress in the field. Both translational and clinical research will move the boundaries in the field of regenerative medicine, and a coordinated effort will provide the clinical breakthroughs, particularly in the many applications of bone engineering.
regenerative medicine; stem cell therapy; bone regeneration; clinical studies; ethics
Hedgehog protein IHH expression is regulated by transcription factor δ-EF1 to control endochondral bone formation
Indian hedgehog (Ihh) regulates proliferation and differentiation of chondrocytes in the growth plate. Although the biology of Ihh is currently well documented, its transcriptional regulation is poorly understood. δ-EF1 is a two-handed zinc finger/homeodomain transcriptional repressor. Targeted inactivation of mouse δ-EF1 leads to skeletal abnormalities including disorganized growth plates, shortening of long bones, and joint fusions, which are reminiscent of defects associated with deregulation of Ihh signaling. Here, we show that the absence of δ-EF1 results in delayed hypertrophic differentiation of chondrocytes and increased cell proliferation in the growth plate. Further, we demonstrate that δ-EF1 binds to the putative regulatory elements in intron 1 of Ihh in vitro and in vivo, resulting in down-regulation of Ihh expression. Finally, we show that δ-EF1 haploinsufficiency leads to a postnatal increase in trabecular bone mass associated with enhanced Ihh expression. In summary, we have identified δ-EF1 as an in vivo negative regulator of Ihh expression in the growth plate.
The synovium is a major target tissue in chronic arthritis and is intensively studied at the cellular and molecular level. The aim of this study was to develop flow cytometry for the quantitative analysis of synovial cell populations pre and post culture and to characterize mesenchymal cell populations residing in the inflammatory synovium.
Knee synovium biopsies from 39 patients with chronic arthritis and from 15 controls were treated in a short, standardized tissue digestion procedure. Stored, thawed digests were routinely analyzed with flow cytometry including live-dead staining and use of the markers CD45, CD3, CD14, CD20, CD34, CD73, CD105, CD90, CD146, CD163 and HLA-DR to distinguish inflammatory and stromal cells. The influence of the digestion method on the detection of the different surface markers was studied separately. In addition, we studied the presence of a specific cell population hypothesized to be mesenchymal stem cells (MSC) based on the CD271 marker. Cell expansion cultures were set up and a MSC-related surface marker profile in passages 3 and 6 was obtained. Immunohistochemistry for CD34 and von Willebrand factor (vWF) was done to obtain additional data on synovium vascularity.
The cell yield and viability normalized to tissue weight were significantly higher in inflammatory arthritis than in controls. Within the hematopoietic CD45-positive populations, we found no differences in relative amounts of macrophages, T-lymphocytes and B-lymphocytes between patient groups. Within the CD45-negative cells, more CD34-positive cells were seen in controls than in arthritis patients. In arthritis samples, a small CD271 positive population was detected. Culture expanded cells were found to fulfill the multipotent mesenchymal stromal cell marker profile, except for CD34 negativity. Detection of peripheral blood macrophage and B-cell markers was decreased after enzymatic exposure and mechanical forces, respectively, but stromal markers were not affected.
Flow cytometry can distinguish synovial cell populations in tissue digests. The preparation method can influence the detection levels of macrophage and B-cell populations. However, stromal cell markers seem not affected and quantification is possible, supporting flow cytometry tissue analysis as a tool to study these cell populations in arthritis.
We present a patient with longstanding ankylosing spondylitis complicated with cauda equina syndrome. The patient suffered from increasing pain in the leg with reduced sensitivity and extremely cold feet associated with incontinence. Diagnostic workup revealed dural ectasia, arachnoiditis and a spinal inflammatory mass leading to extensive vertebral bone destruction. Of interest, this was not only found in the lumbar spine region (which is typical in cases of cauda equina syndrome associated with ankylosing spondylitis) but also in the lower cervical spine (C7) and upper dorsal spine. Moreover, the bone destructive phenotype of this complication of long-standing AS contrasts with the usual characteristics of new bone formation and ankylosis. As initial treatment with anti-inflammatory drugs was not sufficiently successful, infliximab therapy was started which resulted in manifest clinical improvement as chronic pain, incontinence and laboratory signs of inflammation progressively disappeared.
Ankylosing spondylitis; cauda equina syndrome; bone destruction; anti-TNF.
Targeted therapies that neutralize tumour necrosis factor are often able to control the signs and symptoms of spondyloarthritis. However, recent animal model data and clinical observations indicate that control of inflammation may not be sufficient to impede disease progression toward ankylosis in these patients. Bone morphogenetic proteins and WNTs (wingless-type like) are likely to play an important role in ankylosis and could be therapeutic targets. The relationship between inflammation and new bone formation is still unclear. This review summarizes progress made in our understanding of ankylosis and offers an alternative view of the relationship between inflammation and ankylosis.