Cristina C. Teixeira

Phosphate ions mediate chondrocyte apoptosis through a plasma membrane transporter mechanism

In a previous investigation we showed that phosphate ions (Pi) induced apoptosis of terminally differentiated hypertrophic chondrocytes. To explore the mechanism by which Pi induces cell death, we asked the following two questions. First, can we prevent Pi-induced apoptosis by inhibiting plasma membrane Na-Pi cotransport? Second, which specific Na-Pi transporters are expressed in chondrocytes and are they developmentally regulated? Terminally differentiated hypertrophic chondrocytes were isolated from chick tibial cartilage and cell death was measured in the presence of 3-7 mmol/L Pi. To ascertain whether apoptosis was linked to a rise in cellular Pi loading, we examined the effect of phosphonoformic acid (PFA), a competitive inhibitor of Na-Pi cotransport on Pi-induced apoptosis in chondrocytes. We found that 1 mmol/L PFA blocked anion-induced cell death and prevented an increase in the cell Pi content. In a parallel study, we determined that the bisphosphonate, alendronate, also protected chondrocytes from death, albeit at a lower concentration than PFA. Using a DNA end-labeling procedure, we showed that the Pi-treated cells were apoptotic and, as might be predicted, the presence of PFA blocked induction of the death sequence. Next, we examined the expression of two Pi transporters in relation to chondrocyte maturation and anion treatment. We noted that there was expression of the constitutive transporter, Glvr-1, and a type II cotransporter in chick growth plate cells. Although these transport systems are active in terminally differentiated cells, it is probable that the initiation of apoptosis may require the induction of other Pi-transport systems. It is concluded that, at the mineralization front, cell death is linked directly to the elevation in environmental anion concentration and the concomitant rise in intracellular Pi levels.

Cytokine Expression and Accelerated Tooth Movement

It has been shown that inhibiting the expression of certain cytokines decreases the rate of tooth movement. Here, we hypothesized that stimulating the expression of inflammatory cytokines, through small perforations of cortical bone, increases the rate of bone remodeling and tooth movement. Forty-eight rats were divided into 4 groups: 50-cN force applied to the maxillary first molar (O), force application plus soft tissue flap (OF), force application plus flap plus 3 small perforations of the cortical plate (OFP), and a control group (C). From the 92 cytokines studied, the expression of 37 cytokines increased significantly in all experimental groups, with 21 cytokines showing the highest levels in the OFP group. After 28 days, micro-computed tomography, light and fluorescent microscopy, and immunohistochemistry demonstrated higher numbers of osteoclasts and bone remodeling activity in the OFP group, accompanied by generalized osteoporosity and increased rate of tooth movement.

Effect of micro-osteoperforations on the rate of tooth movement

INTRODUCTION Our objectives were to study the effect of micro-osteoperforations on the rate of tooth movement and the expression of inflammatory markers. METHODS Twenty adults with Class II Division 1 malocclusion were divided into control and experimental groups. The control group did not receive micro-osteoperforations, and the experimental group received micro-osteoperforations on 1 side of the maxilla. Both maxillary canines were retracted, and movement was measured after 28 days. The activity of inflammatory markers was measured in gingival crevicular fluid using an antibody-based protein assay. Pain and discomfort were monitored with a numeric rating scale. RESULTS Micro-osteoperforations significantly increased the rate of tooth movement by 2.3-fold; this was accompanied by a significant increase in the levels of inflammatory markers. The patients did not report significant pain or discomfort during or after the procedure, or any other complications. CONCLUSIONS Micro-osteoperforation is an effective, comfortable, and safe procedure to accelerate tooth movement and significantly reduce the duration of orthodontic treatment.

Engineering endochondral bone: in vitro studies.

Chitosan scaffolds have been shown to possess biological and mechanical properties suitable for tissue engineering and clinical applications. In the present work, chitosan sponges were evaluated regarding their ability to support cartilage cell proliferation and maturation, which are the first steps in endochondral bone formation. Chitosan sponges were seeded with chondrocytes isolated from chicken embryo sterna. Chondrocyte/chitosan constructs were cultured for 20 days, and treated with retinoic acid (RA) to induce chondrocyte maturation and matrix synthesis. At different time points, samples were collected for microscopic, histological, biochemical, and mechanical analyses. Results show chondrocyte attachment, proliferation, and abundant matrix synthesis, completely obliterating the pores of the sponges. RA treatment caused chondrocyte hypertrophy, characterized by the presence of type X collagen in the extracellular matrix and increased alkaline phosphatase activity. In addition, hypertrophy markedly changed the mechanical properties of the chondrocyte/chitosan constructs. In conclusion, we have developed chitosan sponges with adequate pore structure and mechanical properties to serve as a support for hypertrophic chondrocytes. In parallel studies, we have evaluated the ability of this mature cartilage scaffold to induce endochondral ossification.

Foxo1, a Novel Regulator of Osteoblast Differentiation and Skeletogenesis

Skeletogenesis depends on the activity of bone-forming cells derived from mesenchymal cells. The pathways that control mesenchymal cell differentiation are not well understood. We propose that Foxo1 is an early molecular regulator during mesenchymal cell differentiation into osteoblasts. In mouse embryos, Foxo1 expression is higher in skeletal tissues, while Foxo1 silencing has a drastic impact on skeletogenesis and craniofacial development, specially affecting pre-maxilla, nasal bone, mandible, tibia, and clavicle. Similarly, Foxo1 activity and expression increase in mouse mesenchymal cells under the influence of osteogenic stimulants. In addition, silencing Foxo1 blocks the expression of osteogenic markers such as Runx2, alkaline phosphatase, and osteocalcin and results in decreased culture calcification even in the presence of strong osteogenic stimulants. Conversely, the expression of these markers increases significantly in response to Foxo1 overexpression. One mechanism through which Foxo1 affects mesenchymal cell differentiation into osteoblasts is through regulation of a key osteogenic transcription factor, Runx2. Indeed, our results show that Foxo1 directly interacts with the promoter of Runx2 and regulates its expression. Using a tibia organ culture model, we confirmed that silencing Foxo1 decreases the expression of Runx2 and impairs bone formation. Furthermore, our data reveals that Runx2 and Foxo1 interact with each other and cooperate in the transcriptional regulation of osteoblast markers. In conclusion, our in vitro, ex vivo, and in vivo results strongly support the notion that Foxo1 is an early molecular regulator in the differentiation of mesenchymal cells into osteoblast.

Nitric oxide, C-type natriuretic peptide and cGMP as regulators of endochondral ossification

Coordinated proliferation and differentiation of growth plate chondrocytes is required for endochondral bone growth, but the mechanisms and pathways that control these processes are not completely understood. Recent data demonstrate important roles for nitric oxide (NO) and C-type natriuretic peptide (CNP) in the regulation of cartilage development. Both NO and CNP stimulate the synthesis of cGMP and thus the activation of common downstream pathways. One of these downstream mediators, cGMP-dependent kinase II (cGKII), has itself been shown to be essential for normal endochondral bone formation. This review summarizes our knowledge of the roles and mechanisms of NO, CNP and cGKII signaling in cartilage and endochondral bone development.

A rapid and ultrasensitive method for measurement of DNA, calcium and protein content, and alkaline phosphatase activity of chondrocyte cultures

Most investigators are cognizant of the problems inherent in counting cells embedded in a complex and abundant extracellular matrix. To overcome these obstacles, we developed a new method of isolating nucleic acids from chondrocytes which facilitates measurement of cell number by DNA analysis. Chondrocytes were isolated from chick embryo sterna and grown continuously without subculturing for 2–3 weeks in monolayer. The cells were treated with triton X-100 and the nucleic acid content of the extract was determined by measuring DNA fluorescence in the presence of Hoechst dye 33258. To minimize background fluorescence due to the triton, we precipitated the DNA with alcohol and then solubilized the nucleic acids in EDTA. This simple procedure removed the detergent and substantially increased the sensitivity of the method. Thus, we could measure with high precision and high recovery, the DNA content of cultures of 10,000–50,000 cells. In a single well containing 0.5–1.0 million cells, sufficient material remained for subsequent measurements of alkaline phosphatase activity and protein and calcium content. As the mineral present in the triton-treated samples was soluble in EDTA, we experienced no problems in measuring the calcium content of the culture. In addition, as triton X-100 is a nonionic detergent, we were able to measure cell and matrix proteins; moreover, the presence of the triton maintained the catalytic state of alkaline phosphatase. We conclude that this procedure provides a simple and rapid approach to measuring major indicators of chondrocyte maturation and function.

An improved Collagen Scaffold for Skeletal Regeneration

Bone repair and regeneration is one of the most extensively studied areas in the field of tissue engineering. All of the current tissue engineering approaches to create bone focus on intramembranous ossification, ignoring the other mechanism of bone formation, endochondral ossification. We propose to create a transient cartilage template in vitro, which could serve as an intermediate for bone formation by the endochondral mechanism once implanted in vivo. The goals of the study are (1) to prepare and characterize type I collagen sponges as a scaffold for the cartilage template, and (2) to establish a method of culturing chondrocytes in type I collagen sponges and induce cell maturation. Collagen sponges were generated from a 1% solution of type I collagen using a freeze/dry technique followed by UV light crosslinking. Chondrocytes isolated from two locations in chick embryo sterna were cultured in these sponges and treated with retinoic acid to induce chondrocyte maturation and extracellular matrix deposition. Material strength testing as well as microscopic and biochemical analyzes were conducted to evaluate the properties of sponges and cell behavior during the culture period. We found that our collagen sponges presented improved stiffness and supported chondrocyte attachment and proliferation. Cells underwent maturation, depositing an abundant extracellular matrix throughout the scaffold, expressing high levels of type X collagen, type I collagen and alkaline phosphatase. These results demonstrate that we have created a transient cartilage template with potential to direct endochondral bone formation after implantation.

F-spondin, a neuroregulatory protein, is up-regulated in osteoarthritis and regulates cartilage metabolism via TGF-β activation

In osteoarthritis (OA) articular chondrocytes undergo phenotypic changes culminating in the progressive loss of cartilage from the joint surface. The molecular mechanisms underlying these changes are poorly understood. Here we report enhanced (‐7‐fold) expression of F‐spondin, a neuronal extracellular ma‐trix glycoprotein, in human OA cartilage (P<0.005). OA‐specific up‐regulation of F‐spondin was also dem‐onstrated in rat knee cartilage following surgical meni‐sectomy. F‐spondin treatment of OA cartilage explants caused a 2‐fold increase in levels of the active form of TGF‐β1(P<0.01) and a 10‐fold induction of PGE2 (P< 0.005) in culture supernatants. PGE2 induction was found to be dependent on TGF‐β and the throm‐bospondin domain of the F‐spondin molecule. F‐spondin addition to cartilage explant cultures also caused a 4‐fold increase in collagen degradation (P< 0.05) and a modest reduction in proteoglycan synthesis (~20%;P<0.05), which were both TGF‐β and PGE2 dependent. F‐spondin treatment also led to increased secretion and activation of MMP‐13 (P<0.05). Together these studies identify F‐spondin as a novel protein in OAcartilage, where it may act in situ at lesional areas to activate latent TGF‐β and induce cartilage degradation via pathways that involve production of PGE2.—Attur, M. G., Palmer, G. D., Al‐Mussawir, H. E., Dave, M., Teixeira, C. C., Rifkin, D. B., Appleton, C. T. G., Beier, F., Abramson, S. B. F‐spondin, a neuroregulatory protein, is up‐regulated in osteoarthritis and regulates cartilage metabolism via TGF‐β activation. FASEB J. 23, 79‐89 (2009)

Chondrogenic ATDC5 cells: An optimised model for rapid and physiological matrix mineralisation

The development of chondrogenic cell lines has led to major advances in the understanding of how chondrocyte differentiation is regulated, and has uncovered many signalling pathways and gene regulatory mechanisms required to maintain normal function. ATDC5 cells are a well established in vitro model of endochondral ossification; however, current methods are limited for mineralisation studies. In this study we demonstrate that culturing cells in the presence of ascorbic acid and 10 mM β-glycerophosphate (βGP) significantly increases the rate of extracellular matrix (ECM) synthesis and reduces the time required for mineral deposition to occur to 15 days of culture. Furthermore, the specific expression patterns of Col2a1 and Col10a1 are indicative of ATDC5 chondrogenic differentiation. Fourier transform-infrared spectroscopy analysis and transmission electron microscopy (TEM) showed that the mineral formed by ATDC5 cultures is similar to physiological hydroxyapatite. Additionally, we demonstrated that in cultures with βGP, the presence of alkaline phosphatase (ALP) is required for this mineralisation to occur, further indicating that chondrogenic differentiation is required for ECM mineralisation. Together, these results demonstrate that when cultured in the presence of ascorbic acid and 10 mM βGP, ATDC5 cells undergo chondrogenic differentiation and produce a physiological mineralised ECM from Day 15 of culture onwards. The rapid and novel method for ATDC5 culture described in this study is a major improvement compared with currently published methods and this will prove vital in the pursuit of underpinning the molecular mechanisms responsible for poor linear bone growth observed in a number of chronic diseases such as cystic fibrosis, chronic kidney disease, rheumatological conditions and inflammatory bowel disease.