Jon C. Daniel

Effects of long-term exposure of human periodontal ligament cells to milk and other solutions*

Periodontal ligament (PDL) cells cultured from healthy extracted human teeth were exposed to milk, Alcon Opti-Free contact lens solution, K-Mart contact lens solution, saline, and Hanks balanced salt solution. The appearance and rate of loss of the cells from the culture dishes were recorded over time at both room temperature (20 degrees C) and 4 degrees C. The results indicated that saline was superior to either of the contact lens solutions in its ability to maintain the vitality of the PDL cells. Milk at 4 degrees DC provided good short-term viability , but cells did not remain attached after 48 h. At 20 degrees C, however, milk resulted in a 24.4% retention of cells after 72 h. Hanks balanced salt solution was the best storage media, with 46.8% of sells remaining attached after 72 h of exposure. This study supports milk as a good short-term storage medium for maintaining the vitality of PDL cells in vitro.

Extracellular Matrix-mediated Tissue Remodeling Following Axial Movement of Teeth

Tooth eruption is a multifactorial process involving movement of existing tissues and formation of new tissues coordinated by a complex set of genetic events. We have used the model of the unopposed rodent molar to study morphological and genetic mechanisms involved in axial movement of teeth. Following extraction of opposing upper molars, lower molars supererupted by 0.13 mm. Labeled tissue sections revealed significant amounts of new bone and cementum apposition at the root apex of the unopposed side following supereruption for 12 days. Newly apposited cementum and alveolar bone layers were approximately 3-fold thicker in the experimental vs the control group, whereas periodontal ligament width was maintained. Tartrate-resistant acid phosphatase staining indicated bone resorption at the mesial alveolar walls of unopposed molars and provided in tandem with new bone formation at the distal alveolar walls an explanation for the distal drift of molars in this model. Microarray analysis and semiquantitative RT-PCR demonstrated a significant increase in collagen I, integrin β5, and SPARC gene expression as revealed by comparison between the unopposed molar group and the control group. Immunohistochemical verification revealed increased levels of integrin β5 and SPARC labeling in the periodontal ligament of the unopposed molar. Together our findings suggest that posteruptive axial movement of teeth was accomplished by significant formation of new root cementum and alveolar bone at the root apex in tandem with upregulation of collagen I, integrin β5, and SPARC gene expression.

An evaluation of two newly formulated calcium hydroxide cements: A leakage study

One-hundred extracted human single-rooted teethwere biomechanically instrumented. Sixty teeth were obturated with gutta-percha and three types of root canal cements, which were Roth Root Canal Cement, Calciobiotic Root Canal Sealer, and Sealapex. None of the remaining 40 teeth (controls) were obturated; however, 20 had their apical foramina sealed and the other 20 had patent foramina. Five microliters of [ 3 H]uridine were deposited into the prepared root canal of each tooth, and radioisotope counts were obtained at various intervals postoperatively (2 to 60 days). The results showed that there was no statistical difference in leakage between the obturated groups and the sealed control group; however, when compared with the control group with patent foramina there was a significant difference.

Development of the rabbit craniomandibular joint in association with tooth eruption

The adult rabbit craniomandibular joints (CMJs) are stress-bearing joints. The two CMJs and the teeth form an articular triad. In early fetal life the developing triad consists of the CMJ primordia, the tooth germs for the entire set of deciduous teeth, as well as the posterior extensions of the dental lamina, which will give rise to the permanent teeth with no deciduous predecessors. During postnatal life, before occlusion is established, there is a remodelling stage in which the CMJ builds up its matrix components such as collagenous and elastic fibres, proteoglycans and type II collagen. Remodelling gradually diminishes into the maintenance stage once occlusion is fully established and after eruption of the first and second molars. Chondrocytes first appear in the CMJ articular disc during the second week of postnatal development. These cells localize in the band areas of the disc and establish an extensive cartilaginous matrix 3-4 weeks postnatally. This study supports the concept that the full development of a fibrocartilaginous articular disc, rich in proteoglycans, occurs as adult occlusion is established.

Expression of type VI collagen during glioblastoma cell invasion in brain tissue cultures

Human glioblastoma cells, U-87 MG, were utilized in two separate rat brain tissue culture systems. In both cases, the glioblastoma cells deeply penetrated and formed tumor masses inside the brain tissues. Immunofluorescence technique, utilizing anti-type VI collagen antibodies demonstrated strong immunoreactivity of type VI collagen in the tumor masses, invading cells, and cell groups. We suggest that type VI collagen may be involved in tumor cells infiltration and invasion of healthy rat brain tissues. Furthermore, the brain tissue culture method may provide a rapid in vitro model with which cellular and extracellular determinants of invasiveness may be studied.

The effect of ascorbate on embryonic chick sternal chondrocytes cultured in agarose

Primary cultures of embryonic chick sternal chondrocytes were embedded in a three-dimensional matrix of 1% solid agarose which was overlaid with nutrient media. The chondrocytes divided and formed nests of spherically shaped cells which were surrounded by an extensive extracellular matrix containing high molecular weight proteoglycans. Using light and electron microscopy, condensation of proteoglycan was observed pericellularly, often forming septa between cells of a nest, and as part of the outer boundary of the cell nest. No cross-striated collagen fibers were observed in the extracellular matrix although proteoglycan appeared to decorate a network of fine strands. Upon the addition of ascorbate to the nutrient media high molecular weight proteoglycans were synthesized, but there was a marked decrease in the synthesis of proteoglycans after a 10 day exposure to ascorbate. Morphologically, the decrease in proteoglycan synthesis was manifested in the discontinuous arrangement of the pericellular matrix as well as the diffuse form of the cell-nest boundary. Both of these structures were clearly defined in control cultures and were enriched in proteoglycan as demonstrated by ruthenium red staining. This study demonstrates that embryonic chondrocytes remain differentiated when cultured in solid agarose for a period of up to 15 days. They continue to synthesize their tissue specific macromolecules and are phenotypically stable when exposed to ascorbate for extended periods of time.

Biosynthesis of type VI collagen by glioblastoma cells and possible function in cell invasion of three-dimensional matrices.

The biosynthesis of type VI collagen was studied in human glioblastoma cell line, U-87 MG. The effects of ascorbic acid on type VI collagen synthesis and secretion were investigated. After ascorbic acid treatment, type VI collagen in cell layers increased from 4.48% in control to 6.63% in the ascorbic acid treated cultures, an increase of 48%. The effect of ascorbic acid on type VI collagen synthesized by glioblastoma cells was lower than that reported for osteosarcoma cells (Engvall et al., 1986). The reason for these differences is still under investigation. The function of type VI collagen in glioblastoma cells is still unknown. We utilized the collagen gel system to elucidate the possible roles of type VI collagen in glioblastoma cells in vitro. Glioblastoma cells in collagen gels showed a stellate shape with long, branched processes in all directions. The strong positive reactivity of type VI collagen detected on cell bodies and cell processes by anti-type VI collagen antibody indicated that this specific collagen was associated with cell surfaces and processes, without releasing or diffusing into the gels. Type VI collagen was directly involved in the cell process extension. When living cells were treated with anti-type VI collagen antibody, a variation of cell morphology was observed. Instead of a stellate shape with processes, cells formed clusters without or with very short processes. These data suggest that type VI collagen, synthesized and secreted by glioblastoma cells, may play a role in tumor cell adhesion and spreading, and enhance cell process extension, penetration, and invasion into collagen gels.

Long-Term Sealing Efficacy of Four Root Surface Sealing Materials Used in Endodontic Leakage Studies

Fifty extracted human maxillary anterior teeth were biomechanically instrumented and divided into five equal groups, four experimental and a control. The teeth in the experimental groups had their root surface coated with one of four sealants; epoxy, casting resin, sticky wax, or nail polish. The roots of the remaining teeth were not coated and served as controls. All of the teeth were mounted in the caps of scintillation vials. Five microliters of [3H]uridine were deposited in the root canal space and disintegration counts were obtained over time periods of 1, 4, 8, 12, and 36 wk. At the conclusion of the experiment, sticky wax was demonstrated to provide a superior seal (p < 0.0001).

Immunofluorescence and biochemical studies of the type VI collagen expression by human glioblastoma cells in vitro

The human glioblastoma cell line U-87 MG was found to express a 140 kD polypeptide which was recognized on immunoblot analysis by a monoclonal antibody to type VI collagen. This polypeptide was digestible by a highly purified bacterial collagenase. After treatment of U-87 MG cells by pepsin, the protein profile revealed the two major pepsin-resistant fragments identical in Mr to those of collagen VI extracted from human placenta. The respective peptide maps from V8 protease one-dimensional gels of these two fragments were identical to those obtained with human collagen VI. Immunofluorescent staining by antibodies to type VI collagen was observed in the extracellular matrix. Moreover, U-87 MG cells were found to be positive for A2B5, a cell surface marker specific for O-2A type glial precursor cells. These data indicate that the human glioblastoma cell line U-87 MG exhibits the properties of glial precursor cells and expresses collagen type VI in vitro. This cell line therefore may prove valuable for comparative investigations of the regulation of type VI collagen synthesis, and may be useful as a model to study the function and pathological importance of type VI collagen in human brain tumours, both in vitro and in vivo.

Expression of Type VI Collagen in Psammoma Bodies

OBJECTIVE To demonstrate that psammoma bodies in human meningiomas contain type VI collagen and laminin. STUDY DESIGN Two fresh human meningiomas were obtained from surgery and the frozen sections examined for type VI collagen and laminin with specific antibodies. RESULTS Type VI collagen was found on the surface of the psammoma bodies. In addition, it was detected in the interstitial tissues surrounding the tumor cells and on the tumor cells themselves. Laminin was detected mainly in the walls of capillaries and large blood vessels. In contrast, in some psammoma bodies, laminin was detectable internally. Laminin was not found in the interstitial tissues or tumor cells. CONCLUSION This is the first report to describe the involvement of type VI collagen in psammoma bodies and whorl formations in meningiomas. This specific type of collagen may serve an anchoring function in psammoma body development.