Regulated transcription of human matrix metalloproteinase 13 (MMP13) and interleukin-1ß (IL1B) genes in chondrocytes depends on methylation of specific proximal promoter CpG sites.

The role of DNA methylation in the regulation of catabolic genes such as MMP13 and IL1B, which have sparse CpG islands, is poorly understood in the context of musculoskeletal diseases. We report that demethylation of specific CpG sites at -110 bp and -299 bp of the proximal MMP13 and IL1B promoters, respectively, detected by in situ methylation analysis of chondrocytes obtained directly from human cartilage, strongly correlated with higher levels of gene expression. The methylation status of these sites had a significant impact on promoter activities in chondrocytes, as revealed in transfection experiments with site-directed CpG mutants in a CpG-free luciferase reporter. Methylation of the -110 and -299 CpG sites, which reside within a hypoxia-inducible factor (HIF) consensus motif in the respective MMP13 and IL1B promoters, produced the most marked suppression of their transcriptional activities. Methylation of the -110 bp CpG site in the MMP13 promoter inhibited its HIF-2α-driven transactivation and decreased HIF-2α binding to the MMP13 proximal promoter in chromatin immunoprecipitation assays. In contrast to HIF-2α, MMP13 transcriptional regulation by other positive (RUNX2, AP-1, ELF3) and negative (Sp1, GATA1, and USF1) factors was not affected by methylation status. However, unlike the MMP13 promoter, IL1B was not susceptible to HIF-2α transactivation, indicating that the -299 CpG site in the IL1B promoter must interact with other transcription factors to modulate IL1B transcriptional activity. Taken together, our data reveal that the methylation of different CpG sites in the proximal promoters of the human MMP13 and IL1B genes modulates their transcription by distinct mechanisms.

Loss of methylation in CpG sites in the NF-κB enhancer elements of inducible nitric oxide synthase is responsible for gene induction in human articular chondrocytes.

OBJECTIVE: To investigate whether the abnormal expression of inducible nitric oxide synthase (iNOS) by osteoarthritic (OA) human chondrocytes is associated with changes in the DNA methylation status in the promoter and/or enhancer elements of iNOS. METHODS: Expression of iNOS was quantified by quantitative reverse transcriptase-polymerase chain reaction. The DNA methylation status of the iNOS promoter and enhancer regions was determined by bisulfite sequencing or pyrosequencing. The effect of CpG methylation on iNOS promoter and enhancer activities was determined using a CpG-free luciferase vector and a CpG methyltransferase. Cotransfections with expression vectors encoding NF-κB subunits were carried out to analyze iNOS promoter and enhancer activities in response to changes in methylation status. RESULTS: The 1,000-bp iNOS promoter has only 7 CpG sites, 6 of which were highly methylated in both control and OA samples. The CpG site at -289 and the sites in the starting coding region were largely unmethylated in both groups. The NF-κB enhancer region at -5.8 kb was significantly demethylated in OA samples compared with control samples. This enhancer element was transactivated by cotransfection with the NF-κB subunit p65, alone or together with p50. Critically, methylation treatment of the iNOS enhancer element significantly decreased its activity in a reporter assay. CONCLUSION: These findings demonstrate the association between demethylation of specific NF-κB-responsive enhancer elements and the activation of iNOS transactivation in human OA chondrocytes, consistent with the differences in methylation status observed in vivo in normal and human OA cartilage and, importantly, show association with the OA process.

Epigenetic modifiers influence lineage commitment of human bone marrow stromal cells: Differential effects of 5-aza-deoxycytidine and trichostatin A.

Clinical imperatives for new bone to replace or restore the function of traumatized or bone lost as a consequence of age or disease has led to the need for therapies or procedures to generate bone for skeletal applications. However, current in vitro methods for the differentiation of human bone marrow stromal cells (HBMSCs) do not, to date, produce homogeneous cell populations of the osteogenic or chondrogenic lineages. As epigenetic modifiers are known to influence differentiation, we investigated the effects of the DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) or the histone deacetylase inhibitor trichostatin A (TSA) on osteogenic and chondrogenic differentiation. Monolayer cultures of HBMSCs were treated for 3 days with the 5-aza-dC or TSA, followed by culture in the absence of modifiers. Cells were subsequently grown in pellet culture to determine matrix production. 5-aza-dC stimulated osteogenic differentiation as evidenced by enhanced alkaline phosphatase activity, increased Runx-2 expression in monolayer, and increased osteoid formation in 3D cell pellets. In pellets cultured in chondrogenic media, TSA enhanced cartilage matrix formation and chondrogenic structure. These findings indicate the potential of epigenetic modifiers, as agents, possibly in combination with other factors, to enhance the ability of HBMSCs to form functional bone or cartilage with significant therapeutic implications therein.

The epigenetic effect of glucosamine and a nuclear factor-kappa B (NF-kB) inhibitor on primary human chondrocytes--implications for osteoarthritis.

OBJECTIVE: Idiopathic osteoarthritis is the most common form of osteoarthritis (OA) world-wide and remains the leading cause of disability and the associated socio-economic burden in an increasing aging population. Traditionally, OA has been viewed as a degenerative joint disease characterized by progressive destruction of the articular cartilage and changes in the subchondral bone culminating in joint failure. However, the etiology of OA is multifactorial involving genetic, mechanical and environmental factors. Treatment modalities include analgesia, joint injection with steroids or hyaluronic acid, oral supplements including glucosamine and chondroitin sulfate, as well as physiotherapy. Thus, there is significant interest in the discovery of disease modifying agents. One such agent, glucosamine (GlcN) is commonly prescribed even though the therapeutic efficacy and mechanism of action remain controversial. Inflammatory cytokines, including IL-1ß, and proteinases such as MMP-13 have been implicated in the pathogenesis and progression of OA together with an associated CpG demethylation in their promoters. We have investigated the potential of GlcN to modulate NF-kB activity and cytokine-induced abnormal gene expression in articular chondrocytes and, critically, whether this is associated with an epigenetic process. METHOD: Human chondrocytes were isolated from the articular cartilage of femoral heads, obtained with ethical permission, following fractured neck of femur surgery. Chondrocytes were cultured for 5 weeks in six separate groups; (i) control culture, (ii) cultured with a mixture of 2.5 ng/ml IL-1ß and 2.5 ng/ml oncostatin M (OSM), (iii) cultured with 2mM N-acetyl GlcN (Sigma-Aldrich), (iv) cultured with a mixture of 2.5 ng/ml IL-1ß, 2.5 ng/ml OSM and 2mM GlcN, (v) cultured with 1.0 µM BAY 11-7082 (BAY; NF-kB inhibitor: Calbiochem, Darmstadt, Germany) and, (vi) cultured with a mixture of 2.5 ng/ml IL-1ß, 2.5 ng/ml OSM and 1.0 µM BAY. The levels of IL1B and MMP13 mRNA were examined using qRT-PCR. The percentage DNA methylation in the CpG sites of the IL1ß and MMP13 proximal promoter were quantified by pyrosequencing. RESULT: IL1ß expression was enhanced over 580-fold in articular chondrocytes treated with IL-1ß and OSM. GlcN dramatically ameliorated the cytokine-induced expression by 4-fold. BAY alone increased IL1ß expression by 3-fold. In the presence of BAY, IL-1ß induced IL1B mRNA levels were decreased by 6-fold. The observed average percentage methylation of the -256 CpG site in the IL1ß promoter was 65% in control cultures and decreased to 36% in the presence of IL-1ß/OSM. GlcN and BAY alone had a negligible effect on the methylation status of the IL1B promoter. The cytokine-induced loss of methylation status in the IL1B promoter was ameliorated by both GlcN and BAY to 44% and 53%, respectively. IL-1ß/OSM treatment increased MMP13 mRNA levels independently of either GlcN or BAY and no change in the methylation status of the MMP13 promoter was observed. CONCLUSION: We demonstrate for the first time that GlcN and BAY can prevent cytokine-induced demethylation of a specific CpG site in the IL1ß promoter and this was associated with decreased expression of IL1ß. These studies provide a potential mechanism of action for OA disease modifying agents via NF-kB and, critically, demonstrate the need for further studies to elucidate the role that NF-kB may play in DNA demethylation in human chondrocytes.

Suppressors of cytokine signalling (SOCS) are reduced in osteoarthritis.

OBJECTIVES: Suppressor of cytokine signalling (SOCS) proteins are inhibitors of cytokine signalling that function via the JAK/STAT pathway (Janus kinase/signal transducers and activators of transcription). Eight SOCS proteins, SOCS1-SOCS7 and CIS-1 (cytokine-inducible SH2-domain, with similar structure to the other SOCS proteins) have been identified, of which SOCS1, 2, and 3 and CIS-1 are the best characterised. A characteristic feature of osteoarthritis (OA) is increased production by articular chondrocytes of pro-inflammatory cytokines, such as interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNFα), which may be induced by mechanotransduction and contribute to cartilage destruction. In this study, we have compared the gene expression of SOCS1, 2, 3 and CIS-1 in healthy and OA human chondrocytes, and also analyzed the effects of IL-1ß and TNFα on the levels of mRNA encoding these SOCS family members. In addition, SOCS2 protein production was assessed and the CpG methylation status of the SOCS2 promoter was analyzed to determine the role of epigenetics in its regulation. METHODS: Femoral heads were obtained after joint replacement surgery for late stage OA and hemiarthroplasty following a fracture of the neck of femur (#NOF). Chondrocytes from the superficial layer of OA cartilage and the deep zone of #NOF cartilage were isolated by sequential treatment with trypsin, hyaluronidase and collagenase B. Total DNA and RNA were extracted from the same chondrocytes, and the levels of SOCS1, 2, 3 and CIS-1 mRNA were determined by qRT-PCR. The percentage of methylation in the CpG sites of the SOCS2 proximal promoter was quantified by pyrosequencing. Alternatively, healthy chondrocytes were isolated from #NOF cartilage and cultured with and without a mixture of IL-1ß and oncostatin M (OSM, both 2.5ng/ml) or TNFα (10ng/ml). The short-term cultures with single cytokine treatment were harvested 24 and 72h after treatment, and the long-term cultures were maintained for 4-5 weeks until confluent with periodical cytokine stimulation. Total RNA was extracted and mRNA levels were determined by qRT-PCR. RESULTS: The SOCS2 and CIS-1 mRNA levels were reduced by approximately 10-fold in OA samples compared to control samples, while SOCS1 and SOCS3 showed similar expression patterns in OA and control chondrocytes. The SOCS2 and CIS-1 mRNA levels declined by 6-fold and 3-fold with long-term treatment with IL-1ß and OSM in combination and TNFα, respectively. There was no significant difference in the CpG methylation status of the SOCS2 promoter between healthy and OA chondrocytes. Similarly, cytokine stimulation did not change the CpG methylation status of the SOCS2 promoter. CONCLUSIONS: This study demonstrates the reduced expression of SOCS2 and CIS-1 in OA, while SOCS1 and SOCS3 were unaffected. The observation that long-term treatment with inflammatory cytokines attenuated the expression of SOCS2 and CIS-1 suggests a potential positive feedback mechanism, and a role of SOCS in the pathology of OA.

Roles of inflammatory and anabolic cytokines in cartilage metabolism: signals and multiple effectors converge upon MMP-13 regulation in osteoarthritis.

Human cartilage is a complex tissue of matrix proteins that vary in amount and orientation from superficial to deep layers and from loaded to unloaded zones. A major challenge to efforts to repair cartilage by stem cell-based and other tissue engineering strategies is the inability of the resident chondrocytes to lay down new matrix with the same structural and resilient properties that it had upon its original formation. This is particularly true of the collagen network, which is susceptible to cleavage once proteoglycans are depleted. Thus, a thorough understanding of the similarities and particularly the marked differences in mechanisms of cartilage remodeling during development, osteoarthritis, and aging may lead to more effective strategies for preventing cartilage damage and promoting repair. To identify and characterize effectors or regulators of cartilage remodeling in these processes, we are using culture models of primary human and mouse chondrocytes and cell lines and mouse genetic models to manipulate gene expression programs leading to matrix remodeling and subsequent chondrocyte hypertrophic differentiation, pivotal processes which both go astray in OA disease. Matrix metalloproteinases (MMP)-13, the major type II collagen-degrading collagenase, is regulated by stress-, inflammation-, and differentiation-induced signals that not only contribute to irreversible joint damage (progression) in OA, but importantly, also to the initiation/onset phase, wherein chondrocytes in articular cartilage leave their natural growth- and differentiation-arrested state. Our work points to common mediators of these processes in human OA cartilage and in early through late stages of OA in surgical and genetic mouse models.

Synthesis of scaffold-free, three dimensional, osteogenic constructs following culture of skeletal osteoprogenitor cells on glass surfaces.

BACKGROUND: Efficient differentiation of stem cells into three-dimensional (3D) osteogenic construct is still an unmet challenge. These constructs can be crucial for patients with bone defects due to congenital or traumatic reasons. The modulation of cell fate and function as a consequence of interaction with the physical and chemical properties of materials is well known. METHODS: The current study has examined the osteogenic differentiation potential of human skeletal populations following culture on glass surfaces, as a monolayer, or in glass tubes as a pellet culture. The 3D prosperities were assessed morphometrically and the differentiation was evaluated through molecular characterization as well as matrix formation. RESULTS: Early temporal expression of alkaline phosphatase expression of skeletal populations was observed following culture on glass surfaces. Skeletal populations seeded on glass tubes, adhered as a monolayer to the tube base and subsequently formed 3D pellets at the air -media interface. The pellets cultured on glass displayed 4.9 ± 1.3 times the weight and 2.9 ± 0.1 the diameter of their counterpart cultured in plastic tubes and displayed enhanced production of osteogenic matrix proteins, such a collagen I and osteonectin. The size and weight of the pellets correlated with surface area in contrast to cell numbers seeded. Global DNA methylation level was decreased in pellets cultured on glass. In contrast, gene expression analysis confirmed upregulation extracellular matrix proteins and osteogenesis-related growth factors. CONCLUSION: This simple approach to the culture of skeletal cells on glass tubes provides a scaffold-free, 3D construct platform for generating pellets enabling analysis and evaluation of tissue development and integration of multiple constructs with implications for tissue repair and regenerative application on scale-up.

Expression of ADAMTS-4 by chondrocytes in the surface zone of human osteoarthritic cartilage is regulated by epigenetic DNA de-methylation.

The two major aggrecanases involved in osteoarthritis (OA) are ADAMTS-4 and ADAMTS-5. Knock-out studies suggested that ADAMTS-5, but not ADAMTS-4, is the major aggrecanase in murine OA. However, studies of human articular cartilage suggest that ADAMTS-4 also contributes to aggrecan degradation in human OA. This study investigated ADAMTS-4 in human OA. While ADAMTS-4 was virtually absent in control cartilage, numerous ADAMTS-4 immuno-positive chondrocytes were present in OA cartilage and their numbers increased with disease severity. RT-PCR confirmed expression, especially in the surface zone. DNA methylation was lost at specific CpG sites in the ADAMTS-4 promoter in OA chondrocytes, suggesting that the increased gene expression was more than a simple up-regulation, but involved loss of DNA methylation at specific CpG sites, resulting in a heritable and permanent expression of ADAMTS-4 in OA chondrocytes. These results suggest that ADAMTS-4 is epigenetically regulated and plays a role in aggrecan degradation in human OA.

Normal bone growth requires optimal estrogen levels: negative effects of both high and low dose estrogen on the number of growth plate chondrocytes.

Endochondral bone formation at epiphyseal growth plate consists of the synchronized processes of chondrogenesis and cartilage ossification. Estrogen, the major female sex hormone, plays an important role in this process, particularly during the pubertal growth spurt. However, its effects on the growth plate are not completely understood. The aims of this study were to clarify the effects of estrogen on the kinetics of chondrocytes in the growth plates of 10- to 25-week-old female rabbits by studying the effects of ovariectomy or high-dose administration of estrogen on the balance between cell proliferation and death. Forty-eight Japanese white rabbits were divided into three groups: sham operated, ovariectomized, or ovariectomized with subsequent weekly injection of high dose estrogen from 10 weeks. The chondrocyte kinetics was investigated by histomorphometry and immunohistochemistry, using antibodies for caspase-3, a marker of apoptosis, and for proliferating cell nuclear antigen. Both ovariectomized and estrogen-injected rabbits showed a declination of the chondrocyte number although the latter animals indicated a more dramatic effect. Estrogen-injected rabbits showed a decrease in the cell proliferating ability together with an increase in chondrocytes undergoing apoptosis while ovariectomy mainly reduced the cell proliferating ability. Given the known importance of estrogen for bone growth, one would expect that ovariectomy and high-dose administration of estrogen would have opposite effects. However, the present study indicated that both low and high concentration had a similar effect: a decrease in the chondrocyte number compared with control, suggesting that estrogen has to be maintained within a narrow range for optimal bone growth.

An ex vivo model for chondrogenesis and osteogenesis.

Loss of bone and cartilage are major healthcare issues. At present, there is a paucity of therapies for effectively repairing these tissues sustainably in the long term. A tissue engineering approach using advanced functional scaffolds may provide a clinically acceptable alternative. In this study, an innovative mineralized alginate/chitosan scaffold was used to provide tailored microenvironments for driving chondrogenesis and osteogenesis from single and mixed populations of human articular chondrocytes and human bone marrow stromal cells. Polysaccharide capsules were prepared with combinations of these cell types with the addition of type I or type II collagen to augment cell-matrix interactions and promote the formation of phenotypically distinct tissues and placed in a rotating (Synthecon) bioreactor or held in static 2D culture conditions for up to 28 days. Significant cell-generated matrix synthesis was observed in human bone marrow bioreactor samples containing type I collagen after 21-28 days, with increased cell proliferation, cell activity and osteocalcin synthesis. The cell-generated matrix was immuno-positive for types I and II collagen, bone sialoprotein and type X collagen, a marker of chondrogenic hypertrophy, demonstrating the formation of a mature chondrogenic phenotype with areas of new osteoid tissue formation. We present a unique approach using alginate/collagen capsules encapsulated in chitosan to promote chondrogenic and osteogenic differentiation and extracellular matrix formation and the potential for tissue-specific differentiation. This has significant implications for skeletal regeneration and application.

Strategies to promote chondrogenesis and osteogenesis from human bone marrow cells and articular chondrocytes encapsulated in polysaccharide templates.

The aim of this study was to synthesize functional in vitro and in vivo 3-dimensional (3D) constructs using a mix of human mesenchymal populations and articular chondrocytes encapsulated in biomineralized polysaccharide templates. Single-cell-type populations or mixtures of both cell types were encapsulated in alginate/chitosan and cultured within a rotating-bioreactor, perfused bioreactor system, or static conditions for 28 days. Within single cell-type populations, type II collagen immunopositive cells were present within lacunae in rotating-bioreactor capsules, with an increased proportion of metabolically active cells compared with perfused and static constructs. Biochemical analysis indicated significantly increased ( p < 0.05) DNA and protein in rotating-bioreactor conditions compared with perfused or static. However, in coculture samples, DNA and protein was significantly increased in static cultures owing to the formation of large regions of partially mineralized osteoid. This osteoid was found only in static cultures and when the ratio of human bone marrow cells to chondrocytes was 2:1 or, to a lesser extent, 5:1 ratio capsules. Subcutaneous implantation of capsules into immunocompromised mice also showed optimal osteoid formation when the ratio was 2:1. The current studies demonstrate the pivotal role of robust 3D biomimetic microenvironments and indicate the potential to harness the interactions between different cell types to create specific tissues.

Matrix metalloproteinase-9 induces the formation of cartilage canals in the chondroepiphysis of the neonatal rabbit.

BACKGROUND: An investigation of matrix metalloproteinase-9 (MMP-9) and its influence on vascular invasion in the secondary ossification center at the chondroepiphysis of developing long bones was undertaken. The effect of MMP-9 was compared with that of basic fibroblast growth factor (b-FGF), a potent angiogenic factor, and we assessed the chorioallantoic membrane (CAM) culture as a model for angiogenesis in osteochondral tissue. METHODS: Seventy-two femoral and seventy-two humeral heads of thirty-six four-day postnatal rabbits were dissected immediately after each animal was killed. Solutions of MMP-9, b-FGF, and phosphate-buffered saline solution were applied, and the femoral and humeral chondroepiphyseal explants were incubated for ten days in CAM culture. This was used as an in vivo model to investigate the growth of blood vessels into the femoral and humeral heads of the neonatal rabbit. The explants were harvested from the CAM culture and analyzed histologically. A three-day incubation was also performed to look for early signs of vascular ingrowth into the cartilage matrix. RESULTS: One hundred and twenty epiphyses from thirty rabbits were placed onto CAM culture successfully; of these, two were harvested at three days to assess early changes and 118 were harvested at ten days. Forty of the 118 cultures were still viable when harvested after ten days, giving a 33% yield. Both MMP-9 and b-FGF caused an increased vascular invasion into the chondroepiphysis. New blood vessels derived from the chorioallantoic membrane within cartilage canals were more numerous in MMP-9 treated epiphyses, and larger canals were more commonly seen when compared with a control group. CONCLUSIONS: These findings confirmed that b-FGF is angiogenic at the chondroepiphysis. Matrix metalloproteinase-9 appears to be implicated in vascular invasion and induces the formation of new cartilage canals at the chondroepiphysis. The CAM culture model was a useful model for investigating angiogenesis in osteochondral tissue. CLINICAL RELEVANCE: This study adds to the understanding of the complex biochemical interaction that occurs in cartilage when the advancing vasculature begins growing into the chondroepiphysis. A better knowledge of this angiogenic process will enable a better understanding of the pathological failure or disturbance of vasculogenesis, which results in dysplastic growth disorders and osteonecrosis.

Pleiotrophin/Osteoblast-stimulating factor 1: dissecting its diverse functions in bone formation.

OSF-1, more commonly known as pleiotrophin (PTN) or heparin-binding growth-associated molecule (HB-GAM), belongs to a new family of secreted HB proteins, which are structurally unrelated to any other growth factor family. The aims of this study were to dissect the diverse functions of PTN in bone formation. The study showed that PTN was synthesized by osteoblasts at an early stage of osteogenic differentiation and was present at sites of new bone formation, where PTN was stored in the new bone matrix. Low concentrations (10 pg/ml) of PTN stimulated osteogenic differentiation of mouse bone marrow cells and had a modest effect on their proliferation, whereas higher concentrations (ng/ml) had no effect. However, PTN did not have the osteoinductive potential of bone morphogenetic proteins (BMPs) because it failed to convert C2C12 cells, a premyoblastic cell line, to the osteogenic phenotype, whereas recombinant human BMP-2 (rhBMP-2) was able to do so. When PTN was present together with rhBMP-2 during the osteoinductive phase, PTN inhibited the BMP-mediated osteoinduction in C2C12 cells at concentrations between 0.05 pg/ml and 100 ng/ml. However, when added after osteoinduction had been achieved, PTN enhanced further osteogenic differentiation. An unusual effect of PTN (50 ng/ml) was the induction of type I collagen synthesis by chondrocytes in organ cultures of chick nasal cartilage and rat growth plates. Thus, PTN had multiple effects on bone formation and the effects were dependent on the concentration of PTN and the timing of its presence. To explain these multiple effects, we propose that PTN is an accessory signaling molecule, which is involved in a variety of processes in bone formation. PTN enhances or inhibits primary responses depending on the prevailing concentrations, the primary stimulus, and the availability of appropriate receptors.

Induction of human osteoprogenitor chemotaxis, proliferation, differentiation, and bone formation by osteoblast stimulating factor-1/pleiotrophin: osteoconductive biomimetic scaffolds for tissue engineering.

The process of bone growth, regeneration, and remodeling is mediated, in part, by the immediate cell-matrix environment. Osteoblast stimulating factor-1 (OSF-1), more commonly known as pleiotrophin (PTN), is an extracellular matrix-associated protein, present in matrices, which act as targets for the deposition of new bone. However, the actions of PTN on human bone progenitor cells remain unknown. We examined the effects of PTN on primary human bone marrow stromal cells chemotaxis, differentiation, and colony formation (colony forming unit-fibroblastic) in vitro, and in particular, growth and differentiation on three-dimensional biodegradable porous scaffolds adsorbed with PTN in vivo. Primary human bone marrow cells were cultured on tissue culture plastic or poly(DL-lactic acid-co-glycolic acid) (PLGA; 75:25) porous scaffolds with or without addition of recombinant human PTN (1 pg-50 ng/ml) in basal and osteogenic conditions. Negligible cellular growth was observed on PLGA scaffold alone, generated using a super-critical fluid mixing method. PTN (50 microg/ml) was chemotactic to human osteoprogenitors and stimulated total colony formation, alkaline phosphatase-positive colony formation, and alkaline phosphatase-specific activity at concentrations as low as 10 pg/ml compared with control cultures. The effects were time-dependent. On three-dimensional scaffolds adsorbed with PTN, alkaline phosphatase activity, type I collagen formation, and synthesis of cbfa-1, osteocalcin, and PTN were observed by immunocytochemistry and PTN expression by in situ hybridization. PTN-adsorbed constructs showed morphologic evidence of new bone matrix and cartilage formation after subcutaneous implantation as well as within diffusion chambers implanted into athymic mice. In summary, PTN has the ability to promote adhesion, migration, expansion, and differentiation of human osteoprogenitor cells, and these results indicate the potential to develop protocols for de novo bone formation for skeletal repair that exploit cell-matrix interactions.

Maternal protein deficiency affects mesenchymal stem cell activity in the developing offspring.

Epidemiological studies suggest that environmental influences such as maternal nutrition, programme skeletal growth during intrauterine and early postnatal life. However, the mechanism whereby the skeletal growth trajectory is modified remains unclear. We have addressed this using a rat model of maternal protein insufficiency to investigate the cellular mechanisms involved in the programming of bone development. The aims of this study were to determine whether colony formation (colony forming unit-fibroblastic, CFU-F), proliferation, and differentiation of bone marrow stromal cells from offspring of female rats maintained on normal (18% casein) or low (9% casein) protein was altered and, whether their responses to growth hormone (GH), 1,25(OH)(2)D3, and IGF-1 differed. Dams were fed an 18% casein (control) diet or 9% casein (low protein) diet from conception until the end of pregnancy. Offspring were then fed a normal protein diet until harvest at 8, 12, and 16 weeks after birth. At 8 weeks, total CFU-F and alkaline phosphatase-positive CFU-F were significantly (P < 0.01) reduced in the low protein group compared to controls. At 12 weeks, no significant differences were observed in colony formation. Modulation of osteoblast proliferation and differentiation by IGF-1 and GH was observed (P < 0.01) in the control group at 8 weeks and the low protein group at 12 weeks. Alkaline phosphatase specific activity was significantly decreased at 8 weeks (P < 0.001) in the low protein group. At 12 and 16 weeks this was reversed, with significantly increased specific activity in the low protein group. These results suggest that normal proliferation and differentiation of mesenchymal stem cells were delayed by maternal protein restriction during early life. Furthermore, these results suggest that, with skeletal maturity, "catch-up" or a physiological shift in bone cell activity was present in the low protein group. These alterations in mesenchymal stem cell function by the early environment may represent an important candidate mechanism for the programming of osteoporosis and associated consequences in later life.

Temporal analysis of rat growth plates: cessation of growth with age despite presence of a physis.

Despite the continued presence of growth plates in aged rats, longitudinal growth no longer occurs. The aims of this study were to understand the reasons for the cessation of growth. We studied the growth plates of femurs and tibiae in Wistar rats aged 62-80 weeks and compared these with the corresponding growth plates from rats aged 2-16 weeks. During skeletal growth, the heights of the plates, especially that of the hypertrophic zone, reflected the rate of bone growth. During the period of decelerating growth, it was the loss of large hydrated chondrocytes that contributed most to the overall decrease in the heights of the growth plates. In the old rats we identified four categories of growth plate morphology that were not present in the growth plates of younger rats: (a). formation of a bone band parallel to the metaphyseal edge of the growth plate, which effectively sealed that edge; (b). extensive areas of acellularity, which were resistant to resorption and/or remodeling; (c). extensive remodeling and bone formation within cellular regions of the growth plate; and (d). direct bone formation by former growth plate chondrocytes. These processes, together with a loss of synchrony across the plate, would prevent further longitudinal expansion of the growth plate despite continued sporadic proliferation of chondrocytes.

Apoptosis in osteoarthritis.

Many studies have shown that apoptotic cell death occurs at an increased rate in osteoarthritic cartilage. Whichever type of cell death takes places in articular cartilage, it is important to prevent, because it is detrimental to articular cartilage maintenance. Thus, it is important to characterize events going on during cellular degeneration in more detail. Overall, physicians have reached a reasonable level of understanding of the extent of cell death occurring in the disease process, but they are still at an early stage in the understanding of the mechanisms underlying this process and the means of intervening in this facet of cartilage destruction.

Association of reduced type IX collagen gene expression in human osteoarthritic chondrocytes with epigenetic silencing by DNA hypermethylation.

OBJECTIVE: To investigate whether the changes in collagen gene expression in osteoarthritic (OA) human chondrocytes are associated with changes in the DNA methylation status in the COL2A1 enhancer and COL9A1 promoter. METHODS: Expression levels were determined using quantitative reverse transcription-polymerase chain reaction, and the percentage of DNA methylation was quantified by pyrosequencing. The effect of CpG methylation on COL9A1 promoter activity was determined using a CpG-free vector; cotransfections with expression vectors encoding SOX9, hypoxia-inducible factor 1α (HIF-1α), and HIF-2α were carried out to analyze COL9A1 promoter activities in response to changes in the methylation status. Chromatin immunoprecipitation assays were carried out to validate SOX9 binding to the COL9A1 promoter and the influence of DNA methylation. RESULTS: Although COL2A1 messenger RNA (mRNA) levels in OA chondrocytes were 19-fold higher than those in the controls, all of the CpG sites in the COL2A1 enhancer were totally demethylated in both samples. The levels of COL9A1 mRNA in OA chondrocytes were 6,000-fold lower than those in controls; 6 CpG sites of the COL9A1 promoter were significantly hypermethylated in OA patients as compared with controls. Treatment with 5-azadeoxycitidine enhanced COL9A1 gene expression and prevented culture-induced hypermethylation. In vitro methylation decreased COL9A1 promoter activity. Mutations in the 5 CpG sites proximal to the transcription start site decreased COL9A1 promoter activity. Cotransfection with SOX9 enhanced COL9A1 promoter activity; CpG methylation attenuated SOX9 binding to the COL9A1 promoter. CONCLUSION: This first demonstration that hypermethylation is associated with down-regulation of COL9A1 expression in OA cartilage highlights the pivotal role of epigenetics in OA, involving not only hypomethylation, but also hypermethylation, with important therapeutic implications for OA treatment.

Epigenetic regulation during fetal femur development: DNA methylation matters.

Epigenetic modifications are heritable changes in gene expression without changes in DNA sequence. DNA methylation has been implicated in the control of several cellular processes including differentiation, gene regulation, development, genomic imprinting and X-chromosome inactivation. Methylated cytosine residues at CpG dinucleotides are commonly associated with gene repression; conversely, strategic loss of methylation during development could lead to activation of lineage-specific genes. Evidence is emerging that bone development and growth are programmed; although, interestingly, bone is constantly remodelled throughout life. Using human embryonic stem cells, human fetal bone cells (HFBCs), adult chondrocytes and STRO-1(+) marrow stromal cells from human bone marrow, we have examined a spectrum of developmental stages of femur development and the role of DNA methylation therein. Using pyrosequencing methodology we analysed the status of methylation of genes implicated in bone biology; furthermore, we correlated these methylation levels with gene expression levels using qRT-PCR and protein distribution during fetal development evaluated using immunohistochemistry. We found that during fetal femur development DNA methylation inversely correlates with expression of genes including iNOS (NOS2) and COL9A1, but not catabolic genes including MMP13 and IL1B. Furthermore, significant demethylation was evident in the osteocalcin promoter between the fetal and adult developmental stages. Increased TET1 expression and decreased expression of DNA (cytosine-5-)-methyltransferase 1 (DNMT1) in adult chondrocytes compared to HFBCs could contribute to the loss of methylation observed during fetal development. HFBC multipotency confirms these cells to be an ideal developmental system for investigation of DNA methylation regulation. In conclusion, these findings demonstrate the role of epigenetic regulation, specifically DNA methylation, in bone development, informing and opening new possibilities in development of strategies for bone repair/tissue engineering.

DNA demethylation at specific CpG sites in the IL1B promoter in response to inflammatory cytokines in human articular chondrocytes.

OBJECTIVE: To determine whether changes in the DNA methylation status in the promoter region of the gene encoding interleukin-1beta (IL-1beta) account for expression of IL1B messenger RNA (mRNA) after long-term treatment of human articular chondrocytes with inflammatory cytokines. METHODS: IL-1beta, tumor necrosis factor alpha (TNFalpha) plus oncostatin M (OSM), or 5-azadeoxycytidine (5-aza-dC) was added twice weekly for 4-5 weeks to primary cultures of normal human articular chondrocytes derived from the femoral head cartilage of patients with a fracture of the femoral neck. Expression of MMP13, IL1B, TNFA, and DNMT1 was determined by SYBR Green-based quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of genomic DNA and total RNA extracted from the same sample before and after culture. Bisulfite modification was used to identify which CpG sites in the IL1B promoter showed differential methylation between IL1B-expressing and IL1B-nonexpressing cells. The percentages of cells that were methylated at that critical CpG site (-299 bp) were quantified by a method that depended on methylation-sensitive restriction enzymes and real-time RT-PCR. Secretion of IL-1beta into the culture media was assessed by enzyme-linked immunosorbent assay. RESULTS: Healthy chondrocytes did not express IL1B mRNA, but the levels were increased 5-fold by treatment with 5-aza-dC and were increased 100-1,000-fold by treatment with TNFalpha/OSM. The percentage CpG methylation was decreased by 5-aza-dC treatment but was reduced considerably more by IL-1beta and was almost abolished by TNFalpha/OSM. The mRNA was translated into protein in cytokine-treated chondrocytes. CONCLUSION: These novel findings indicate that inflammatory cytokines can change the DNA methylation status at key CpG sites, resulting in long-term induction of IL1B in human articular chondrocytes.