[show abstract][hide abstract] ABSTRACT: 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 de-methylation 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.
Journal of Biological Chemistry 02/2013; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
PLoS ONE 01/2013; 8(1):e54957. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: It is now well established that the onset of adult diseases such as heart disease, stroke, type 2 diabetes and hypertension
are linked to an adverse uterine growth environment, in particular through maternal nutrition, during development of the individual.
The geographical distribution of the incidence rate of heart disease is similar to that of osteoporosis. This may indicate
a link between maternal nutrition during pregnancy and the subsequent risk of developing osteoporosis in the offspring. This
review summarises what we know to date, from animal models, about maternal nutrition and the subsequent alterations in the
offspring’s skeletal structure.
Clinical Reviews in Bone and Mineral Metabolism 04/2012; 8(1):40-48.
[show abstract][hide abstract] ABSTRACT: 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-1β) 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-1β 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.
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-1β 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.
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-1β 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.
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.
Biochemical and Biophysical Research Communications 02/2011; 407(1):54-9. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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-1β, 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.
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-1β 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-1β, 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-1β, 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 IL1β and MMP13 proximal promoter were quantified by pyrosequencing.
IL1β expression was enhanced over 580-fold in articular chondrocytes treated with IL-1β and OSM. GlcN dramatically ameliorated the cytokine-induced expression by 4-fold. BAY alone increased IL1β expression by 3-fold. In the presence of BAY, IL-1β induced IL1B mRNA levels were decreased by 6-fold. The observed average percentage methylation of the -256 CpG site in the IL1β promoter was 65% in control cultures and decreased to 36% in the presence of IL-1β/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-1β/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.
We demonstrate for the first time that GlcN and BAY can prevent cytokine-induced demethylation of a specific CpG site in the IL1β promoter and this was associated with decreased expression of IL1β. 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.
Biochemical and Biophysical Research Communications 02/2011; 405(3):362-7. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
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.
[show abstract][hide abstract] ABSTRACT: Osteoarthritis (OA) is generally a disease of the elderly population, but can occur in young patients in exceptional cases. This study compares the cellular and epigenetic features of primary old-age OA with those of secondary OA in a 23-year-old patient with developmental dysplasia of the hip. In addition, control cartilage from a 14-year-old was compared with that from patients with a fracture of the neck of femur (#NOF) to establish to what extent the latter is a useful control for OA. Articular cartilage was obtained from discarded femoral heads after hip arthroplasty. MMP-3, MMP-9, MMP-13, and ADAMTS-4 were immunolocalized and the methylation status of specific promoter CpG sites was determined. Both primary and secondary OA were characterized by loss of aggrecan, formation of clones, and abnormal expression of the proteases that correlated with epigenetic DNA demethylation. The latter indicated that the abnormal expression of the cartilage-degrading proteases was not due to a short-term up-regulation, but a heritable, permanent alteration in gene expression. Comparing cell densities in young and old control cartilage estimated an age-related cell loss of approximately 1% per year. In aged #NOF cartilage, some superficial-zone chondrocytes expressed the proteases, but the majority of cells were immunonegative and their promoters were hypermethylated. The cellular and epigenetic features of the intermediate and deep zones of #NOF cartilage are thus similar to those of young healthy cartilage, justifying the use of #NOF cartilage as control cartilage for OA, providing the superficial zone is removed.
Journal of Orthopaedic Research 12/2008; 27(5):593-601. · 2.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: The relative paucity of techniques currently available to repair bone tissue necessitates the development of innovative and more effective clinical strategies. [1,2] Of these, the combined integration of macroporous scaffolds, primed human-cell populations, and growth factors to organize and promote tissue formation is a particularly attractive approach. [3,4] However, bone tissue engi-neering is currently compromised by its inability to produce load-bearing scaffolds. For example, collagen scaffolds are used for a range of osteogenic applications, [5–7] but exhibit a compressive strength of ca. 0.034 MPa,  approximately three orders of magnitude lower than that of cancellous bone with values between 10 and 50 MPa. On the other hand, pure calcium phosphate mineral-based scaffolds, which currently dominate the commercial bone substitute materials market, lack a fibrillar protein component and are correspondingly brittle, typically failing catastrophically at compressive loads of less than 5 MPa. Recent studies have explored the possibility of replacing collagen with silk-based resorbable implants. [9–12] Silk is a fibrillar protein with excellent biocompatibility and mechanical strength,  and methodologies exist to convert silk fibers into regenerated silk fibroin solutions that can be subsequently reconstituted into macroporous 3D architectures with b-sheet secondary structure by salt leaching, gas foaming, extrusion layering or freeze drying. [9,13–18] Although these scaffolds are potentially suitable for tissue engineering, similarly to collagen-based biomaterials the reconstituted silks are compromised by low mechanical strength, due in part to partial degradation of the native protein structure during fibroin dissolution. Moreover, attempts to impart significant mechanical reinforcement by calcium phosphate mineralization have not been very successful, due to poor adhesion and integration at the protein/mineral interface. [19,20] As a consequence, more advanced uses of silk are currently focused on novel delivery devices for morphogens, cytokines, and cell populations in models of bone defects. [21–25] In contrast, we present herein the first example of a silk/calcium phosphate macroporous scaffold that is load-bearing with mechanical properties comparable to cancellous bone. The mechanical strength is far in excess of other materials previously produced, and is achieved through the use of high-quality silk fibroins  and an integrated procedure for gelation, freezing, and mineralization. Moreover, we demonstrate the effectiveness of these load-bearing materials as nonpyrogenic osteoregenerative scaffolds by in vitro and in vivo testing, and suggest that such materials represent a new class of potentially implantable alternatives to the use of allograft and autograft procedures, for example in surgical applications, where immediate load bearing is required. Silk/calcium phosphate macroporous scaffolds were prepared by freezing phosphate-containing aqueous gels of nondegraded silk fibroin, followed by slow infiltration of the frozen gel at 37 8C with an aqueous calcium chloride solution at pH 9, treatment with ethanol, and cross-linking of the dried miner-alized scaffold with hexamethylene diisocyanate (HMI, see Experimental). Various aspects of this protocol were critical for the fabrication of high-strength macroporous silk/calcium phosphate scaffolds. First, addition of phosphate to the fibroin solution resulted in the formation of an opaque gel (Fig. 1A) with sufficient viscosity to support fabrication of a self-supporting macroporous architecture by ice crystal templating. [27,28] Second, uniform calcium phosphate mineralization of the silk scaffold depended on the addition of phosphate before freezing, as this established a homogeneous distribution of the anion within the matrix prior to reaction with Ca 2þ ions. Third, formation of macropores required the formation of relatively large ice crystals, and this was achieved using an optimum freezing temperature of À12 8C (Fig. 1B). Fourth, although freezing of the fibroin gel resulted in dehydration and aggregation of the silk proteins, the extent of b-sheet formation and concomitant hydrophobicity of the matrix were limited under these conditions, such that the COMMUNICATION
[show abstract][hide abstract] ABSTRACT: 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.
The Tohoku Journal of Experimental Medicine 04/2008; 214(3):269-80. · 1.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Heterogeneity of cells with respect to the DNA methylation status at a specific CpG site is a problem when assessing methylation status. We have developed a simple two-step method for the quantification of the percent of cells that display methylation at a specific CpG site in the promoter of a specific gene. The first step is overnight digestion of genomic DNA (optimal conc. 20 ng/5 microl) with a relevant methylation-sensitive restriction enzyme (optimal 2 units). This is followed by real time PCR, using the SYBR Green method, with primers that bracket the site cleaved by the enzyme. By including fully methylated and fully non-methylated DNA in each PCR plate, the errors caused by non-specific digestion or incomplete digestion can be measured and used to adjust the raw results and thus increase specificity. The method can detect differences in methylation status if these are more than 10%. No specialized equipment is required beyond the real-time PCR system and the method can be adapted for any of the 53 commercially available methylation-sensitive restriction enzymes.
Epigenetics: official journal of the DNA Methylation Society 01/2007; 2(2):86-91. · 4.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
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.
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.
The Journal of Bone and Joint Surgery 12/2006; 88 Suppl 3:155-61. · 3.23 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: In the last few years there has been an explosion of research into epigenetics and, in particular, the roles of DNA methylation in the normal functioning of the mammalian organism as well as whether changes in methylation status contribute to or cause aberrant gene expression in diseases. While abnormal patterns of DNA methylation in cancer cells have been intensively investigated, little attention has so far been paid to the role of DNA methylation in cartilage and cartilage degeneration. This review summarizes the current knowledge of the mechanism of methylation, its association with transcriptional silencing, possible mechanisms of hyper- and hypomethylation as well as age- and disease related changes in methylation pattern. We discuss the possible involvement of DNA methylation in chondrogenesis as well as its potential importance for cartilage degradation. Overall, epigenetic gene regulation has largely been neglected in cartilage research, but is likely to be an important issue in future. There is increasing evidence that besides cytokines, growth factors and changes in matrix composition, variations in the genetic methylation pattern might also be important determinators of the complex gene expression pattern pathognomically observed in osteoarthritic cartilage tissue.
Current Rheumatology Reviews 07/2006; 2(3):221-232.
[show abstract][hide abstract] ABSTRACT: To date, the plasticity, multipotentiality, and characteristics of progenitor cells from fetal skeletal tissue remain poorly defined. This study has examined cell populations from human fetal femurs in comparison with adult-derived mesenchymal cell populations. Real-time quantitative polymerase chain reaction demonstrated expression of mesenchymal progenitor cell markers by fetal-derived cells in comparison with unselected adult-derived and immunoselected STRO-1-enriched adult populations. Multipotentiality was examined using cells derived from femurs and single-cell clones, culture-expanded from explants, and maintained in basal medium prior to exposure to adipogenic, osteogenic, and chondrogenic conditions. Adipocyte formation was confirmed by Oil Red O lipid staining and aP2 immunocytochemistry, with expression of peroxisome proliferation-activated receptor-gamma detected only in adipogenic conditions. In chondrogenic pellets, chondrocytes lodged within lacunae and embedded within dense proteoglycan matrix were observed using Alcian blue/Sirius red staining and type II collagen immunocytochemistry. Osteogenic differentiation was confirmed by alkaline phosphatase staining and type I collagen immunocytochemistry as well as by gene expression of osteopontin and osteocalcin. Single-cell clonal analysis was used to demonstrate multipotentiality of the fetal-derived populations with the formation of adipogenic, chondrogenic, and osteogenic populations. Mineralization and osteoid formation were observed after culture on biomimetic scaffolds with extensive matrix accumulation both in vitro and in vivo after subcutaneous implantation in severely compromised immunodeficient mice. These studies demonstrate the proliferative and multipotential properties of fetal femur-derived cells in comparison with adult-derived cells. Selective differentiation and immunophenotyping will determine the potential of these fetal cells as a unique alternative model and cell source in the restoration of damaged tissue.
[show abstract][hide abstract] ABSTRACT: To investigate whether the abnormal expression of matrix metalloproteinases (MMPs) 3, 9, and 13 and ADAMTS-4 by human osteoarthritic (OA) chondrocytes is associated with epigenetic "unsilencing."
Cartilage was obtained from the femoral heads of 16 patients with OA and 10 control patients with femoral neck fracture. Chondrocytes with abnormal enzyme expression were immunolocalized. DNA was extracted, and the methylation status of the promoter regions of MMPs 3, 9, and 13 and ADAMTS-4 was analyzed with methylation-sensitive restriction enzymes, followed by polymerase chain reaction amplification.
Very few chondrocytes from control cartilage expressed the degrading enzymes, whereas all clonal chondrocytes from late-stage OA cartilage were immunopositive. The overall percentage of non-methylated sites was increased in OA patients (48.6%) compared with controls (20.1%): 20% versus 4% for MMP-13, 81% versus 47% for MMP-9, 57% versus 30% for MMP-3, and 48% versus 0% for ADAMTS-4. Not all CpG sites were equally susceptible to loss of methylation. Some sites were uniformly methylated, whereas in others, methylation was generally absent. For each enzyme, there was 1 specific CpG site where the demethylation in OA patients was significantly higher than that in controls: at -110 for MMP-13, -36 for MMP-9, -635 for MMP-3, and -753 for ADAMTS-4.
This study provides the first evidence that altered synthesis of cartilage-degrading enzymes by late-stage OA chondrocytes may have resulted from epigenetic changes in the methylation status of CpG sites in the promoter regions of these enzymes. These changes, which are clonally transmitted to daughter cells, may contribute to the development of OA.
[show abstract][hide abstract] ABSTRACT: Utilizing ATDC5 murine chondrogenic cells and human articular chondrocytes, this study sought to develop facile, reproducible three-dimensional models of cartilage generation with the application of tissue engineering strategies, involving biodegradable poly(glycolic acid) scaffolds and rotating wall bioreactors, and micromass pellet cultures. Chondrogenic differentiation, assessed by histology, immunohistochemistry, and gene expression analysis, in ATDC5 and articular chondrocyte pellets was evident by the presence of distinct chondrocytes, expressing Sox-9, aggrecan, and type II collagen, in lacunae embedded in a cartilaginous matrix of type II collagen and proteoglycans. Tissue engineered explants of ATDC5 cells were reminiscent of cartilaginous structures composed of numerous chondrocytes, staining for typical chondrocytic proteins, in lacunae embedded in a matrix of type II collagen and proteoglycans. In comparison, articular chondrocyte explants exhibited areas of Sox-9, aggrecan, and type II collagen-expressing cells growing on fleece, and discrete islands of chondrocytic cells embedded in a cartilaginous matrix.
Biochemical and Biophysical Research Communications 08/2005; 333(2):609-21. · 2.41 Impact Factor