Excess BMP Signaling in Heterotopic Cartilage Forming in Prg4-null TMJ Discs.

Heterotopic cartilage develops in certain pathologic conditions, including those affecting the human temporomandibular joint (TMJ), but the underlying molecular mechanisms remain obscure. This is in part due to the fact that a reliable animal model of such TMJ diseases is not available. Here, we show that aberrant chondrocyte differentiation and ectopic cartilage formation occur spontaneously in proteoglycan 4 (Prg4) mutant TMJ discs without further invasive procedure. By 2 mo of age, mutant disc cells displayed chondrocyte transdifferentiation, accompanied by strong expression of cartilage master gene Sox9 and matrix genes aggrecan and type II collagen. By 6 mo, heterotopic cartilage had formed in the discs and expressed cartilage hypertrophic markers Runx2 and ColX. The ectopic tissue grew in size over time and exhibited regional mineralization by 12 mo. Bone morphogenetic protein (BMP) signaling was activated with the ectopic chondrogenic cells and chondrocytes, as indicated by phosphorylated Smad 1/5/8 nuclear staining and by elevated expression of Bmp2, Bmpr1b, Bmpr2, and BMP signaling target genes. Likewise, we found that upon treatment with recombinant human BMP 2 in high-density micromass culture, mutant disc cells differentiated into chondrocytes and synthesized cartilage matrix more robustly than control cells. Importantly, a specific kinase inhibitor of BMP receptors drastically attenuated chondrogenesis in recombinant human BMP 2-treated mutant disc cultures. Unexpectedly, we found that Prg4 was expressed at joint-associated sites, including disc/muscle insertion and muscle/bone interface, and all these structures were abnormal in Prg4 mutants. Our data indicate that Prg4 is needed for TMJ disc integrity and function and that its absence leads to ectopic chondrogenesis and cartilage formation in conjunction with abnormal BMP signaling. Our findings imply that the BMP signaling pathway could be a potential therapeutic target for prevention or inhibition of ectopic cartilage formation in TMJ disease.

In vivo effectiveness of enamel sealants around orthodontic brackets.

OBJECTIVE: In this randomized study, the caries-protective effect on vestibular enamel of two fluoride-containing sealants (Protecto® and Light Bond®) during multibracket treatment was investigated. MATERIALS AND METHODS: In all, 40 orthodontic patients about to receive a multibracket appliance with the brackets bonded to the vestibular tooth surfaces were randomly included in this study. Each one was randomly assigned to one of four groups. A crossover design was selected in which a sealed quadrant was contralateral to an unsealed quadrant, then choosing the reverse configuration in the opposite jaw. Two sealants were, thus, tested on vestibular enamel on left and right anterior teeth and premolars in both jaws of each patient over 6 months of multibracket treatment. A DIAGNOdent® pen measuring laser fluorescence was used to analyze the relevant enamel surfaces both at baseline and after 6 months. RESULTS: Neither the incidence nor the characteristics of the demineralization we observed during the study differed between the 4 groups. CONCLUSION: Single application of smooth-surface sealants did not protect enamel around brackets from incipient carious lesions during the first 6 months of multibracket treatment.

Integration of a maxillary model into facial surface stereophotogrammetry.

AIMS: Three-dimensional (3D) integration of a maxillary model into a facial model has only been possible by a complex procedure using face bow transfer after taking impressions of certain maxillary and facial parts. In this study, we aimed to develop a method for integrating a scanned maxillary model into a scan-realized facial model. MATERIAL AND METHODS: A total of 19 patients with the medical indication for cone-beam computed tomography (CBCT) and orthodontic treatment were included in this study. Facial and maxillary scans were also taken. The construction of the integrated surface model required 10 steps. This integration procedure was evaluated by taking ten 3D dentofacial linear segment measurements in the integrated scan and the CBCT. These results were analyzed using descriptive statistics. RESULTS: All measurements demonstrated good intra-individual reliability. We observed almost perfect congruence between integrated scan and CBCT in vertical distances, while the sagittal measurements revealed more, yet clinically acceptable, deviations possibly caused by different error sources in either of the two methods. CONCLUSION: This new method is suitable for generating 3D integrated surface-scan models which can be used for growth and therapy control studies in orthodontics and other disciplines in the dentofacial fields. Since this method does not require ionizing radiation, it is highly recommendable as an application for children and adolescent patients.

Differential stem cell contributions to thymocyte succession during development of Xenopus laevis.

The contribution of two embryonic stem cell compartments to the developing thymus in the amphibian Xenopus was examined throughout the larval, postmetamorphic, and adult periods. Hematopoietic chimeras were produced by transplanting either the ventral blood islands (VBI) or the dorsal stem cell compartment (DSC) from diploid donors onto triploid hosts. The DNA content of isolated nuclei harvested from the thymus and circulating E populations was analyzed using propidium iodide staining and flow cytometry. The DNA content of mitotic figures derived from PHA reactive splenocytes was analyzed using the Feulgen reaction and microdensitometry. These data suggested that both the VBI and DSC contribute to the thymocyte populations from the earliest developmental stages examined. Moreover, the contribution of both stem cell compartments was cyclic. However, the periods of these cycles were different. Both VBI- and DSC-derived cells entered the thymus 4 days postfertilization. VBI-derived thymocytes were at a minimum at 28 days postfertilization, reached a maximum at 35 days postfertilization and a second minimum at 42 days postfertilization. However, DSC-derived cells reached a maximum at 28 days, a minimum at 35 days, and a second maximum at 42 days. The PHA-reactive splenocyte population followed a similar temporal pattern. In contrast, the VBI-derived E population was at a maximum during early development and steadily declined throughout the larval period. DSC-derived E were undetectable during early development but steadily increased throughout the larval period. Both VBI- and DSC-derived hematopoietic cells persisted after metamorphosis and contributed to all populations examined in adult frogs. Because of temporal differences in the VBI and DSC contributions to the developing thymus, these data suggest heterogeneity within the thymocyte population associated with the embryonic origin of the colonizing stem cells.

Construction of T cell hybridomas secreting allogeneic effect factor.

T cell hybridoma lines were constructed by fusion of DBA/2 alloantigen-activated T cell blasts with the AKR thymoma line BW5147. Certain of the hybridomas prepared in this manner secreted spontaneously into their culture supernates biologically active molecules that displayed B cell- and T cell-activating properties characteristic of allogeneic effect factor (AEF). Cell surface phenotype analysis documented that the hybridomas were, indeed, somatic cell hybrids between the two respective partner cells used for fusion. The B cell-activating properties of these hybridoma supernates was demonstrated by their capacity to stimulate T cell-depleted spleen cells to respond in vitro to T-dependent antigens. The T cell-activating properties of these hybridoma supernates was verified by their capacity to stimulate autonomous development of self-specific cytotoxic T lymphocytes and by their capacity to exert mitogenic effects on unprimed T cells. The biologically active molecules secreted by these hybridomas were, like conventional AEF, inhibitable by specific anti-Ia antibodies thus indicating the presence of Ia determinants on the relevant hybridoma products. Finally, these AEF-secreting hybridomas could be stimulated to proliferate and to secrete increased quantities of AEF when exposed to the specific alloantigen-bearing target cells to which the T cell blasts had been originally sensitized.

The biologic effects of allogeneic effect factor on T lymphocytes. I. The mitogenic activity and the autonomous induction of cytotoxic T lymphocytes by AEF.

Studies presented herein illustrate the capacity of the soluble mediator, allogeneic effect factor (AEF), which is derived from histoincompatible cell interactions, to induce the in vitro differentiation of normal murine splenic lymphocytes into mature cytotoxic cells capable of exerting activity on H-2-identical target cells. This process requires the presence of T lymphocytes during the sensitization phase, and the lytic activity on tumor cells is mediated by cytotoxic T lymphocytes (CTL). The capacity of AEF to induce differentiation of such CTL does not require the presence of stimulating target cells in the sensitization phase. The induction of CTL requires the presence of AEF at the initiation of culture, although exposure to AEF as brief as 1 hr is sufficient to induce fresh spleen cells to differentiate into CTL during the subsequent 5 days in culture. In addition to its ability to induce CTL, AEF is highly mitogenic for T lymphocytes. However, the mitogenic and the CTL-inducing activities of AEF can be experimentally dissociated, indicating that different subpopulations of T lymphocytes may be involved in the response to AEF. In contrast to similar soluble helper factors derived from allogeneic cell interactions, AEF appears to be unique in its ability to autonomously induce a primary CTL response in vitro.

Biological effects of allogeneic effect factor on T lymphocytes: in vitro induction of cytotoxic T lymphocytes manifesting preferential lytic activity against H-2-identical tumor cells.

The studies reported herein were designed to determine the effects of allogeneic effect factor (AEF), a soluble mediator generated in the course of allogeneic cell interactions, on the differentiation of cytotoxic T lymphocytes in vitro. Normal, unprimed spleen cells from various strains of mice cultured with AEF for 5 days, in the absence of any stimulator cells, developed into cytotoxic lymphocytes capable of lysing target cells in a short-term 51Cr release assay. T lymphocyte-depleted spleen cells did not become cytotoxic when cultured with AEF, and the cytotoxic cells themselves were found to be T lymphocytes. AEF-induced cytotoxic T lymphocytes preferentially lysed H-2-identical target cells. Thus, AEF, as opposed to similar "helper" factors derived from mixed lymphocyte cultures, appears to be unique in its ability to trigger normal, unprimed T lymphocytes to differentiate into cytotoxic T lymphocytes in the absence of specific antigenic stimulation.