Lynne A. Opperman

Cranial sutures as intramembranous bone growth sites

Intramembranous bone growth is achieved through bone formation within a periosteum or by bone formation at sutures. Sutures are formed during embryonic development at the sites of approximation of the membranous bones of the craniofacial skeleton. They serve as the major sites of bone expansion during postnatal craniofacial growth. For sutures to function as intramembranous bone growth sites, they need to remain in an unossified state, yet allow new bone to be formed at the edges of the overlapping bone fronts. This process relies on the production of sufficient new bone cells to be recruited into the bone fronts, while ensuring that the cells within the suture remain undifferentiated. Unlike endochondral growth plates, which expand through chondrocyte hypertrophy, sutures do not have intrinsic growth potential. Rather, they produce new bone at the sutural edges of the bone fronts in response to external stimuli, such as signals arising from the expanding neurocranium. This process allows growth of the cranial vault to be coordinated with growth of the neurocranium. Too little or delayed bone growth will result in wide‐open fontanels and suture agenesis, whereas too much or accelerated bone growth will result in osseous obliteration of the sutures or craniosynostosis. Craniosynostosis in humans, suture fusion in animals, and induced suture obliteration in vitro has been associated with mutations or alterations in expression of several transcription factors, growth factors, and their receptors. Much of the data concerning signaling within sutures has been garnered from research on cranial sutures; hence, only the cranial sutures will be discussed in detail in this review. This review synthesizes classic descriptions of suture growth and pathology with modern molecular analysis of genetics and cell function in normal and abnormal suture morphogenesis and growth in a unifying hypothesis. At the same time, the reader is reminded of the importance of the suture as an intramembranous bone growth site.

Histological and Scanning Electron Microscopy Assessment of Various Vital Pulp-Therapy Materials

Pulp capping and pulpotomy procedures were performed on 15 male mongrel dogs. Three materials were used: calcium hydroxide, acid-etched dentin bonding, and mineral trioxide aggregate. Six of the animals were killed at 50 days and nine were killed at 150 days. Samples from 11 dogs were used for histological evaluation, and the remaining dog samples were used for scanning electron microscopy evaluation. Each slide was graded histologically according to previously published criteria. Scanning electron microscopy analysis was performed, and the weight percentage of elements found in the dentin of a nontreated tooth versus the bridge formed in the exposed specimen was established. By evaluating pulp inflammation in vital pulp-therapy treatments, it was found that mineral trioxide aggregate was not significantly different from the untreated control group, both in pulp-capping procedures at 50 days (p = 0.357) or 150 days (p = 0.198) and pulpotomy procedures at 50 days (p = 0.357) or 150 days (p = 0.198). Moreover, histologically mineral trioxide aggregate was a considerably better material than calcium hydroxide or acid-etched dentin bonding in maintaining the integrity of the pulp.

Transforming growth factor‐β2 and TGF‐β3 regulate fetal rat cranial suture morphogenesis by regulating rates of cell proliferation and apoptosis

Cranial vault sutures are the major intramembranous bone growth sites during rapid expansion of the neurocranium. To function as bone growth sites, sutures need to remain patent, while allowing rapid bone formation at the edges of the bone fronts. Premature osseous obliteration of sutures (craniosynostosis) by fusion of bone fronts across the suture site prevents further bone formation at this site, often leading to severe facial dysmorphology. Although several growth factor receptor and transcription factor mutations have been implicated in craniosynostosis, the underlying mechanisms leading to sutural obliteration remain unclear. Previous studies have shown that dura secreted soluble factors responsible for maintaining suture patency and that suture fusion observed in the absence of dura was preceded by elevated levels of DNA synthesis and collagen production in the suture region. The use of neutralizing antibodies in a fetal calvarial culture model further demonstrated that removal of transforming growth factor (TGF) ‐β3 activity induced premature sutural obliteration, whereas removal of TGF‐β2 activity prevented sutural obliteration. Data presented here demonstrate that suture obliteration induced by removal of TGF‐β3 activity was preceded by elevated levels of DNA synthesis, similar to that seen upon removal of the dura. Addition of exogenous TGF‐β3 to calvaria cultured without dura both prevented suture obliteration and reduced DNA synthesis to levels comparable to those seen with intact dura. Addition of exogenous TGF‐β2 to calvarial cultures induced sutural fusion accompanied by elevated levels of cell proliferation. However, sutures rescued from obliteration by removal of TGF‐β2 activity did not have decreased levels of cell proliferation, but rather appeared to be due to inhibited differentiation. In all calvaria in which sutures remained patent in culture, numbers of apoptotic cells were high within the suture, whereas in sutures destined to fuse, numbers of apoptotic cells were low. Results indicate that one of the critical regulators of suture patency is cell number. Alterations in cell number can trigger premature differentiation of cells, resulting in sutural obliteration. Furthermore, a complex interplay between closely related molecules is required to maintain cranial vault sutures in an unossified state, while allowing new bone to be formed at the edges of the bone fronts.

The effects of bisphosphonates on osteoblasts in vitro

OBJECTIVE The purpose of this study was to test the effects of bisphosphonates on osteoblast viability and function. STUDY DESIGN Primary rat osteoblasts were cultured in normal media or media containing increasing concentrations of 2 bisphosphonates, alendronate and zoledronate. Enzyme-linked immunoabsorbent assays (ELISAs) were used to measure interleukin (IL)-6, transforming growth factor (TGF) beta1, and receptor activator of nuclear factor kappaB ligand (RANKL) expression in supernatants after 24, 48, and 72 h. Live and necrotic cell numbers were assessed with trypan blue assays. RESULTS As drug concentrations increased, cell viability decreased significantly. The ELISAs revealed significant increases in TGF-beta1 in treatment groups, but no significant change in RANKL or IL-6. CONCLUSIONS High concentrations of alendronate and zoledronate were cytotoxic, decreasing cell viability at 72 h. Transforming growth factor beta1 increased even as viability decreased, suggesting a mechanism for bisphosphonate action. These data suggest that lower concentrations of bisphosphonates may have therapeutic benefits without the cytotoxic effects which may result in osteonecrosis.

Regulation of cell proliferation in rat mandibular condylar cartilage in explant culture by insulin-like growth factor-1 and fibroblast growth factor-2

Insulin-like growth factor-1 (IGF-1) and fibroblast growth factor-2 (FGF-2) regulate the proliferation and differentiation of growth-plate chondrocytes, but surprisingly little is known of the mechanisms underlying growth regulation in secondary cartilages such as the mandibular condylar. The aims here were to investigate whether IGF-1 and FGF-2 receptors are present in mandibular condylar cartilage in vivo from 28-day-old male Sprague-Dawley rats (by immunohistochemistry), how proliferation in that cartilage responds to increasing concentrations of exogenous IGF-1 or FGF-2 in explant culture (by [3H]thymidine incorporation), and whether the expression of these growth factors and their receptors in the cartilage changes during the transition to puberty (quantitative reverse transcriptase-polymerase chain reaction). Immunoreactivity for receptors (R) for IGF-1 and FGF-2 (IGF-1R, FGFR1, and FGFR3) was most pronounced in chondroblasts and hypertrophic chondrocytes, while FGFR2 immunoreactivity was strongest in the articular and prechondroblastic zones. The proliferative response elicited by exogenous IGF-1 was considerably greater than that induced by FGF-2, although the threshold concentration for a significant response was lower for FGF-2. In the transition from prepuberty (31 days) to the beginning of late puberty (42 days), a pronounced trend of increasing IGF-1 and decreasing FGF-2 gene expression was evident. Of the receptors, only FGFR2 and FGFR3 expression increased. These data provide evidence that proliferation in the mandibular condylar cartilage might be regulated in part by IGF-1 and FGF-2, and that expression of these genes changes considerably at puberty. The data also suggest that mechanisms governing proliferation in mandibular condylar cartilage might have as much in common with those regulating cranial sutures as those regulating growth-plate.

Transforming growth factor-beta 3(Tgf-β3) in a collagen gel delays fusion of the rat posterior interfrontal suture in vivo

Postnatal expansion of the intramembranous bones of the craniofacial skeleton occurs as bone growth at sutures. Loss of the bone growth site occurs when the suture fails to form, or when the newly formed sutures become ossified, resulting in premature obliteration. Previous experiments demonstrated that removal of dura mater from fetal rat coronal sutures, or neutralizing transforming growth factor‐beta 2 (Tgf‐β2) activity using antibodies resulted in premature obliteration of the suture in vitro. Conversely, addition of Tgf‐β3 to coronal sutures in vitro rescued them from osseous obliteration. To examine whether Tgf‐β3 rescues sutures from obliteration in vivo, a collagen gel was used as a vehicle to deliver Tgf‐β3 to the normally fusing rat posterior interfrontal (IF) suture. Surgery was done on postnatal day 9 (P9) rats, in which collagen gels containing 0, 3, or 30 ng Tgf‐β3 were placed above the IF suture, underneath the periosteum for 2 weeks. By P24, 75–100% of animals in control unoperated, sham‐operated, and collagen gel‐only groups had fused IF sutures. In contrast, 40% of sutures exposed to 3 ng Tgf‐β3 remained open, while sutures exposed to 30 ng Tgf‐β were similar to controls. By immunohistochemistry, sutures rescued from obliteration by Tgf‐β3 had the same Tgf‐β receptor type II (Tβr‐II) distribution as controls. However, Tgf‐β3‐treated sutures had altered Tgf‐β2 and Tβr‐I distribution compared to controls. Anat Rec 267:120–130, 2002.

Transforming Growth Factor-β Isoform Expression in the Perisutural Tissues of Craniosynostotic Rabbits

Objective To describe the expression patterns of the various transforming growth factor-β (Tgf-β) isoforms, known to be involved in suture development, in the perisutural tissues of rabbits with naturally occurring craniosynostosis and relate such differential expression to the pathogenesis of premature suture fusion. Method Twenty-one coronal sutures were harvested from six wild-type control New Zealand White rabbits and five rabbits with familial coronal suture synostosis at 25 days of age for histomorphometric and immunohistochemical analyses. Tgf-β isoform immunoreactivity was assessed using indirect immunoperoxidase procedures with specific antibodies. Results Synostosed sutures had significantly (p < .01) greater bone area and relatively more osteoblasts and osteocytes in the osteogenic fronts, compared with wild-type sutures. Tgf-β isoform immunoreactivity showed differential staining patterns between wild-type and synostosed perisutural tissues. In wild-type sutures, Tgf-β1 and Tgf-β3 immunoreactivity was significantly (p < .001) greater than Tgf-β2 staining in all perisutural tissues. In synostosed sutures, the opposite pattern was observed, with Tgf-β2 immunoreactivity significantly (p < .001) greater than Tgf-β1 and Tgf-β3 in the osteogenic fronts, dura mater, and periosteum. Conclusions Findings from this study suggest that an overexpression of Tgf-β2, either in isolation or in association with an underexpression of Tgf-β1 and Tgf-β3, may be related to premature suture fusion (craniosynostosis) in this pathological rabbit model. These abnormal expression patterns may be involved in premature suture fusion either through increased cell proliferation, decreased apoptosis of the osteoblasts or both at the osteogenic fronts.

Tooth movements in foxhounds after one or two alveolar corticotomies

The aim of this split-mouth experimental study was to determine (1) whether corticotomy procedures increase tooth movement and (2) the effects of a second corticotomy procedure after 4 weeks on the rate of tooth movement. The mandibular third and maxillary second premolars of five skeletally mature male foxhounds, approximately 2 years of age, were extracted. One randomly selected mandibular quadrant had buccal and lingual flaps and corticotomies performed around the second premolar; the other quadrant served as the control. Both maxillary quadrants had initial buccal flaps and corticotomies; one randomly selected quadrant had a second buccal flap surgery and corticotomy after 28 days. Coil springs (200 g force), along with a 0.045 mm diameter tube on a 0.040 mm diameter guiding wire, were used to move the mandibular second and maxillary third premolars. Records, including digital calliper measurements and radiographs, were taken on days 0, 10, 14, 28, 42, and 56. Multilevel statistical procedures were used to model longitudinal tooth movements. The radiographic measurements initially showed increasing mandibular tooth movement rates, peaking between 22 and 25 days, and then decelerating. Total mandibular tooth movements were significantly (P < 0.05) greater on the experimental (2.4 mm) than on the control (1.3 mm) side. The rates of maxillary tooth movement slowed over time, with significantly (P < 0.05) more overall tooth movement on the side that had two (2.3 mm) than one (2.0 mm) corticotomy procedure. Alveolar corticotomy significantly increases orthodontic tooth movement. Performing a second corticotomy procedure after 4 weeks maintained higher rates of tooth movement over a longer duration and produced greater overall tooth movement than performing just one initial corticotomy, but the difference was small.

Effect of storage media on human periodontal ligament cell apoptosis

The ability of storage media to preserve periodontal ligament (PDL) cell vitality has been previously evaluated. However, the mechanisms by which different storage conditions alter the functional status of PDL cells have not been determined. The purpose of the present study was to investigate, in vitro, the level of programed cell death or apoptosis in a population of PDL cells following storage under different conditions. Primary human PDL cells were plated into 24-well-culture plates and allowed to attach for 24 h. Cells were then exposed for 1 h to milk, Hanks balanced salt solution (HBSS), Soft Wear contact lens solution or Gatorade at room temperature or on ice. Culture medium was used as a negative control. Apoptosis was evaluated at 24, 48, and 72 h after treatment on quadruplicate samples by using the ST 160 ApopTag Fluorescein Direct In Situ Detection Kit. The total number of cells and the total number of apoptotic cells were counted. The results indicated that at 24 and 72 h, PDL treated with Gatorade and the contact lens solution displayed the highest percentages of apoptotic cells when compared with the other treatment groups at room temperature. Overall, cells treated on ice showed significantly lower levels of apoptosis when compared with treatments at room temperature. In conclusion, the results indicated that apoptosis plays a major role in cell death in cells treated with Gatorade and contact lens solutions in comparison to other storage solutions and that storage on ice can inhibit programed cell death.

In Vitro Evaluation of Dentinal Tubule Penetration and Biomineralization Ability of a New Root-end Filling Material

INTRODUCTION Capasio is being developed as a new generation of endodontic material with potential use as a root-end filling material. The aim of this study was to compare the ability of Capasio and mineral trioxide aggregate (MTA) to penetrate human dentinal tubules and examine the interaction of Capasio and MTA with a synthetic tissue fluid (STF) and root canal walls in extracted human teeth. METHODS Root-end preparations were filled with Capasio or MTA, allowed to set for 4 weeks in STF, and then sectioned at 1, 2, and 3 mm from resected surface. Depth of penetration was evaluated by using scanning electron microscopy (SEM). Next, Capasio and MTA samples were prepared both in 1-g pellets and in root-end preparations. Samples were placed in STF, allowed to set, and then characterized by using SEM, energy dispersive x-ray analysis (EDXA), and x-ray diffraction (XRD) techniques. RESULTS Penetration of Capasio into dentinal tubules was observed at all levels. No penetration of MTA into dentinal tubules was observed at any level. Both Capasio and MTA formed apatite crystals in the supernatant, on their exposed surfaces, and in the interfacial layers that were similar in structure and elemental composition when evaluated by using SEM and EDXA. XRD analysis of these crystals corresponds with those reported for hydroxyapatite. CONCLUSIONS When used as a root-end filling material, Capasio is more likely to penetrate dentinal tubules. Both Capasio and MTA promote apatite deposition when exposed to STF.