M180 amelogenin processed by MMP20 is sufficient for decussating murine enamel.

Amelogenin (AMELX) and matrix metalloproteinase-20 (MMP20) are essential for proper enamel development. Amelx and Mmp20 mutations cause amelogenesis imperfecta. MMP20, a protease secreted by ameloblasts, is responsible for processing enamel proteins, including AMELX, during the secretory stage of enamel formation. Of at least 16 different amelogenin splice products, the most abundant isoform found in murine ameloblasts and developing enamel is the M180 protein. To understand the role of MMP20 processing of M180 AMELX, we generated AmelxKO/Mmp20KO (DKO) mice with an amelogenin (M180Tg) transgene. We analyzed the enamel phenotype by SEM to determine enamel structure and thickness, µCT, and by nanoindentation to quantify enamel mechanical properties. M180Tg/DKO mouse enamel had 37% of the hardness of M180Tg/AmelxKO teeth and demonstrated a complete lack of normal prismatic architecture. Although molar enamel of M180Tg/AmelxKO mice was thinner than WT, it had similar mechanical properties and decussating enamel prisms, which were abolished by the loss of MMP20 in the M180Tg/DKO mice. Retention of the C-terminus or complete lack of this domain is unable to rescue amelogenin null enamel. We conclude that among amelogenins, M180 alone is sufficient for normal enamel mechanical properties and prism patterns, but that additional amelogenin splice products are required to restore enamel thickness.

Microgenomics of ameloblastoma.

Gene expression profiles of human ameloblastoma microdissected cells were characterized with the purpose of identifying genes and their protein products that could be targeted as diagnostic and prognostic markers as well as for potential therapeutic interventions. Five formalin-fixed, decalcified, paraffin-embedded samples of ameloblastoma were subjected to laser capture microdissection, linear mRNA amplification, and hybridization to oligonucleotide human 41,000 RNA arrays and compared with universal human reference RNA, to determine the gene expression signature. Assessment of the data by Significance Analysis of Microarrays (SAM) and cluster analysis showed that 38 genes were highly expressed (two-fold increase) in all samples, while 41 genes were underexpressed (two-fold reduction). Elements of the sonic hedgehog pathway and Wingless type MMTV integration site family were validated by immunohistochemistry. We have identified the expression of multiple genes and protein products that could serve as potential diagnostic, prognostic, and therapeutic targets.

The amelogenin C-terminus is required for enamel development.

The abundant amelogenin proteins are responsible for generating proper enamel thickness and structure, and most amelogenins include a conserved hydrophilic C-terminus. To evaluate the importance of the C-terminus, we generated transgenic mice that express an amelogenin lacking the C-terminal 13 amino acids (CTRNC). MicroCT analysis of TgCTRNC29 teeth (low transgene number) indicated that molar enamel density was similar to that of wild-type mice, but TgCTRNC18 molar enamel (high transgene number) was deficient, indicating that extra transgene copies were associated with a more severe phenotype. When amelogenin-null (KO) and TgCTRNC transgenic mice were mated, density and volume of molar enamel from TgCTRNCKO offspring were not different from those of KO mice, indicating that neither TgCTRNC18 nor TgCTRNC29 rescued enamel's physical characteristics. Because transgenic full-length amelogenin partially rescues both density and volume of KO molar enamel, it was concluded that the amelogenin C-terminus is essential for proper enamel density, volume, and organization.

Transgenic mice that express normal and mutated amelogenins.

Amelogenin proteins are secreted by ameloblasts within the enamel organ during tooth development. To better understand the function of the 180-amino-acid amelogenin (M180), and to test the hypothesis that a single proline-to-threonine (P70T) change would lead to an enamel defect similar to amelogenesis imperfecta (AI) in humans, we generated transgenic mice with expression of M180, or M180 with the proline-to-threonine (P70T) mutation, under control of the Amelx gene regulatory regions. M180 teeth had a relatively normal phenotype; however, P70T mineral was abnormally porous, with aprismatic regions similar to those in enamel of male amelogenesis imperfecta patients with an identical mutation. When Amelx null females were mated with P70T transgenic males, offspring developed structures similar to calcifying epithelial odontogenic tumors in humans. The phenotype argues for dominant-negative activity for the P70T amelogenin, and for the robust nature of the process of amelogenesis.

mRNA expression and phenotype of odontogenic tumours in the v-Ha-ras transgenic mouse.

UNLABELLED: Ameloblastomas are the most common odontogenic neoplasia in humans, and although typically considered locally invasive and benign, frequently recur subsequent to surgical resection. The Tg.AC transgenic mouse carrying the v-Ha-ras oncogene has been found to spontaneously develop ameloblastoma-like tumours (35% by 1 year of age) that are rare in the wild type FVB background strain. OBJECTIVE: The purpose of this study was to characterise the mRNA expression of genes in the mouse tumours that are either expressed in human ameloblastomas or essential for normal odontogenesis and to correlate the expression to the histological phenotype. STUDY METHODS: Histological, immunohistochemical and RT-PCR studies were used to evaluate clinically demonstrable odontogenic tumours occurring spontaneously in seven Tg.AC v-Ha-ras transgenic mice (homozygous, at 7 months of age or heterozygous at 11 months of age). RESULTS: Most genes profiled were expressed in all tumour samples, however three (amelogenin, matrix metalloproteinase-20 (MMP-20) and Dlx7) displayed differential expression. In addition, only the most highly differentiated tumour stained positively for collagen. In most cases, the variable expression could be explained by reference to the histological phenotype, although differences in gene expression were apparent within the Type 2 and the mixed phenotype tumours. CONCLUSIONS: These data confirm that many of the genes thought to be important in odontogenesis and odontogenic tumour formation in humans are also expressed in these murine ameloblastoma-like tumours however genes associated with terminal differentiation of ameloblasts demonstrate differential expression between the tumour phenotypes.

Characterization and mRNA expression in an unusual odontogenic lesion in a patient with tricho-dento-osseous syndrome.

UNLABELLED: Odontogenic lesions are rare, but can be associated with significant morbidity. While their molecular determinants are unknown, they likely express many genes common to normal odontogenesis. This study evaluated the histology and mRNA expression of an unusual odontogenic lesion in a patient with a confirmed history of tricho-dento-osseous syndrome. METHODS: Decalcified, frozen 8 micro m sections of the lesion were cut and mounted on glass slides and stained with hematoxylin/eosin for analysis. The expression of multiple genes associated with normal odontogenesis and related pathologies were evaluated by RT-PCR, where possible in samples of the hard and soft tissue components of the lesion. RESULTS: Histological examination showed the lesion had large areas of irregular, dentine-like material, enamel matrix, areas of woven immature bone and multiple fully mineralised tooth crowns. Although most of the gene transcripts were amplified from both samples, some, including DLX3/7 and Collagen I demonstrated differential expression. CONCLUSIONS: This study shows the gene expression profile of aberrant odontogenesis with associated odontoma formation is similar to that of normal tooth and the genes expressed in other odontogenic lesions. While the role of altered gene expression in the development of such lesions has previously been postulated from transgenic models, this is the only report of an odontogenic lesion in a patient with TDO, and begins to elucidate possible gene interactions key to its development.

A dose-escalating study of weekly bolus topotecan in previously treated ovarian cancer patients.

OBJECTIVE: Topotecan is an established topoisomerase I inhibitor for the treatment of relapsed ovarian cancer. Myelotoxicity and suboptimal patient convenience associated with daily topotecan, however, have prompted investigators to explore alternate regimens, including a weekly regimen of topotecan. The objective of this study was to determine the maximum tolerated dose (MTD) of topotecan given as a weekly bolus in previously treated ovarian cancer patients. METHODS: Second- and third-line ovarian cancer patients with measurable disease or elevated cancer antigen 125 received weekly bolus topotecan intravenously starting at 1.5 mg/m(2). Topotecan was escalated in dose increments of 0.5 mg/m(2) every 21 days as tolerability allowed. Dose-limiting toxicity was defined as grade 3/4 neutropenia or thrombocytopenia. RESULTS: Thirty-two of 35 patients were evaluable for safety and tolerability. No notable toxicity was observed with weekly topotecan doses < 4 mg/m(2). Additionally, there was an absence of dose-limiting myelotoxicity and thrombocytopenia with weekly topotecan. The MTD of weekly topotecan without the use of granulocyte colony-stimulating factor support was 4 mg/m(2), with grade 2 anemia, chronic fatigue, and grade 2 gastrointestinal toxicity limiting further dose escalation. Weekly topotecan also demonstrated antitumor activity at doses >2 mg/m(2). CONCLUSIONS: The establishment of a well-tolerated, weekly regimen of topotecan (4 mg/m(2), with a maximum recommended dose of 6 mg/m(2)) provides the basis for further investigation in phase II studies of single-agent and combination regimens in previously treated ovarian cancer patients.

Transient estrogen exposure of female mice during early development permanently affects osteoclastogenesis in adulthood.

Estrogens modulate bone tissue turnover in both experimental animal models and postmenopausal women. Our previous studies have shown that exposure to diethylstilbestrol (DES) during the perinatal period increases peak bone mass in female mice in adulthood. We investigated whether developmental DES exposure can influence bone mass by affecting osteoclastogenesis. Female mice were injected with 100 microg/kg body weight DES from days 9-16 of gestation or, alternatively, pups received neonatal injections of 2 microg of DES from days 1-5 of life. Animals were weaned at 21 days of age and effects of estrogen on bone cells were evaluated in adulthood. A significant increase in bone mass in female mice was already observed at 2 months, with a maximal effect in older animals. Bone sections from DES-treated animals showed a significant decrease in osteoclast number and tartrate-resistant acid phosphatase (TRAP) enzymatic activity as compared with controls. To verify the importance of the estrogen surge at puberty in this event, a group of control and DES-treated mice were ovariectomized at 17 days to prevent puberty, and potential effect on osteoclastic cells was evaluated in adulthood. As expected, ovariectomy induced an increase of TRAP-positive cells. DES treatment blunted the ovariectomized-dependent increase of the total number of osteoclastic cells, suggesting a role of developmental DES exposure in the process of bone-cell imprinting. Our data indicate, for the first time, that transient changes in estrogen levels during development modulate bone turnover and osteoclastogenesis likely participating in bone-cell imprinting during early phases of bone development, and that this effect could be induced by direct alteration of bone microenvironment.

Cisplatin, radiation, and adjuvant hysterectomy compared with radiation and adjuvant hysterectomy for bulky stage IB cervical carcinoma.

BACKGROUND: Bulky stage IB cervical cancers have a poorer prognosis than smaller stage I cervical cancers. For the Gynecologic Oncology Group, we conducted a trial to determine whether weekly infusions of cisplatin during radiotherapy improve progression-free and overall survival among patients with bulky stage IB cervical cancer. METHODS: Women with bulky stage IB cervical cancers (tumor, > or =4 cm in diameter) were randomly assigned to receive radiotherapy alone or in combination with cisplatin (40 mg per square meter of body-surface area once a week for up to six doses; maximal weekly dose, 70 mg), followed in all patients by adjuvant hysterectomy. Women with evidence of lymphadenopathy on computed tomographic scanning or lymphangiography were ineligible unless histologic analysis showed that there was no lymph-node involvement. The cumulative dose of external pelvic and intracavitary radiation was 75 Gy to point A (cervical parametrium) and 55 Gy to point B (pelvic wall). Cisplatin was given during external radiotherapy, and adjuvant hysterectomy was performed three to six weeks later. RESULTS: The relative risks of progression of disease and death among the 183 women assigned to receive radiotherapy and chemotherapy with cisplatin, as compared with the 186 women assigned to receive radiotherapy alone, were 0.51 (95 percent confidence interval, 0.34 to 0.75) and 0.54 (95 percent confidence interval, 0.34 to 0.86), respectively. The rates of both progression-free survival (P<0.001) and overall survival (P=0.008) were significantly higher in the combined-therapy group at four years. In the combined-therapy group there were higher frequencies of transient grade 3 (moderate) and grade 4 (severe) adverse hematologic effects (21 percent, vs. 2 percent in the radiotherapy group) and adverse gastrointestinal effects (14 percent vs. 5 percent). CONCLUSIONS: Adding weekly infusions of cisplatin to pelvic radiotherapy followed by hysterectomy significantly reduced the risk of disease recurrence and death in women with bulky stage IB cervical cancers.

Preliminary comparison of mineralizing multilayer cultures formed by primary fetal bovine mandibular osteoblasts grown on titanium, hydroxyapatite, and glass substrates.

Bone formation at implant surfaces may be directly influenced by effects of the implant material on osteoblast behavior. Cell culture models of osteoblast physiology may be used to investigate the interaction of osteoblastic cells with various surfaces. In this study, primary cultured fetal bovine mandibular osteoblastic cells were cultured on titanium, ceramic hydroxyapatite, and glass coverslip surfaces to allow for the comparison of the mineralizing matrix elaborated by osteoblasts grown on different implant material surfaces. Morphologic and immunohistochemical analysis revealed the similar formation of multilayered, mineralizing cultures on these three surfaces. The qualitative similarity of the matrix formed on these culture surfaces may reflect similar qualitative in vivo responses of bone to titanium and hydroxyapatite implants.

An aortic aneurysm model for the evaluation of endovascular exclusion prostheses.

PURPOSE: The purpose of this study was to develop an aortic aneurysm (AA) model with a predictable tendency for rupture for the evaluation of the efficacy of endovascular prostheses in preventing rupture and their long-term outcome after implantation. METHODS: An infrarenal AA measuring two to three times the diameter of the proximal aorta was created in 18 dogs with a full-thickness patch of jejunum. Seven dogs were allowed to survive without aneurysm exclusion. In 11 dogs the aneurysm was immediately excluded with a stented 8 mm Dacron graft mounted in a 14F delivery system introduced through the femoral artery with aortographic guidance. The pressure differential between the aorta and the excluded aneurysm was measured, and angiography, necropsy, and histologic examination were performed at 3- and 6-month survival. RESULTS: All animals survived aneurysm implantation. Without aneurysm exclusion, six dogs died of rupture within 1 to 6 days of surgery. In three dogs the exclusion failed because of graft-to-aorta size mismatch or misplacement demonstrated on angiography and by a low pressure differential between the aorta and the aneurysm (< 5 mm Hg); all three dogs died of rupture within 4 days. In eight dogs the aneurysm was successfully excluded on the basis of angiography results, with a mean aorta-to-aneurysm pressure differential of 51 mm Hg. Two dogs were killed at 1 and 6 days after surgery because of paraplegia produced by graft thrombosis because of kinking but without evidence of aneurysm rupture. Six dogs survived on a long-term basis, and angiography and necropsy performed at 3 and 6 months revealed patent grafts without migration, reduction in aneurysm size, no flow in the excluded lumbar arteries in five of six animals, and complete incorporation of Dacron graft and stents. No evidence of graft infection was found in any animal. The survival rate was significantly better (p < 0.023) in dogs with successfully excluded aneurysms (n = 6) compared with that in dogs without exclusion or with failed aneurysm exclusion (n = 7). CONCLUSION: This aneurysm model demonstrates that without effective aneurysm exclusion all animals die of rupture and that successfully placed endovascular prostheses can prevent AA rupture with long-term graft patency and stability. Endovascular aortic Dacron grafts in dogs undergo complete incorporation at 3 months from implantation. This aneurysm model is useful for the evaluation of endovascular devices designed for the treatment of AAs.

Cell volume alterations induced by platinum-thymine in sarcoma-180 ascites cells: ultrastructural study.

Ultrastructural studies of the effects of the chemotherapeutic agent platinum-thymine, on the morphology of sarcoma-180 ascites cells were studied to elucidate the cancer cells immediate response to therapy and the possible mode of cancer cell regression. Sarcoma-180 ascites cells treated with platinum-thymine in vitro at concentrations of 60 micrograms/ml at varying time intervals demonstrated that the drug is capable of killing cancer cells. The cells exhibited drastic nuclear and cytoplasmic alterations with few lipid spherules in the cytoplasm. The cell volume increased tremendously, with the nucleus relatively larger than the cytoplasm as time of treatment increased. Degradative features of the nucleus, and especially the nucleolus, inhibited metabolic processes. Nucleolar and cytoplasmic necrosis with increased cell volume and vacuolation are potential signals for cancer cells response to the drug and a possible mode of causing the eventual demise of the cancer cells.

Advanced cervical cancer therapy: concurrent radiation therapy and cisplatin chemotherapy for advanced cervical cancer--a toxicity report.

It is estimated that in 1988 there will be 12,900 cancers of the uterine cervix, representing 2.6% of cancers in women. Radiation therapy has been the primary mode of therapy/palliation; for the past 15-20 years survival results achieved with radiotherapy have plateaued. Attempts have been made to find agents to use with radiation aimed at decreasing recurrence and increasing survival. Phase II studies suggest cisplatin may be an excellent agent to combine with radiotherapy. This study was performed to evaluate the toxicity of this combination. Between December 10, 1980, and August 29, 1986, nine patients with advanced cervical cancer and poor prognosis and one patient with recurrent disease were enrolled. The Gynecologic Oncology Group (GOG) criteria for adverse effects were used in this study. Hematologic, gastrointestinal, genitourinary, and skin parameters were examined. Most adverse criteria had a score of 2 or less. Grade 2 nausea/vomiting was the most frequent problem. Anemia was the next most frequent and was the most serious problem encountered. Overall, the toxicity was acceptable; therefore it seems appropriate to proceed to larger studies to evaluate efficacy.

Malignant mixed mesodermal tumors of the ovary. A report of 13 cases.

The surgical pathology files at Thomas Jefferson University Hospital (TJUH) were reviewed for the period 1973-1984. Thirteen cases of primary ovarian malignant mixed mesodermal tumor (MMT) were found; however, material from only seven of these cases could be obtained for repeat review. No conclusions can be drawn regarding any impact on survival based on the histology. Treatment was highly varied reflecting the mix of physicians rendering treatment and the range of modalities used. Overall, survival was miserable. Six of the 13 patients (46.2%) survived 6 months or less, 10 of the 13 (76.9%) survived 12 months or less. No particular treatment modality seemed to offer enhanced survival unless aggressive cytoreductive surgery was performed. A plea is made for national cooperative groups to develop treatment protocols for this aggressive ovarian cancer.

Apical migration of oral epithelium in experimental dehiscence wounds.

The purpose of the present investigation was to determine if the placement of free mucosal grafts would delay the apical migration of oral epithelium into surgically created dehiscence wounds. Dehiscence wounds, measuring 8 x 6 mm, were surgically created on the mandibular canines of 5 beagle dogs. The exposed root surface was then curetted and horizontal grooves were made, one at a point just below the gingival sulcus and the other at the apical border of the dehiscence. Experimental teeth received free alveolar mucosal grafts while the contralateral teeth served as controls. The grafts were placed with the epithelial side against the tooth surface to bridge the dehiscence at the level of the coronal notch and were sutured in place. The flaps were then repositioned (over the mucosal grafts) and sutured. Apical migration of the oral epithelium, after 10 days, was assessed histologically using the coronal and apical grooves as points of reference. The oral epithelium was detected in the coronal one half of the dehiscence, in both the control and experimental teeth. There were no significant differences observed between the two, suggesting that the placement of a mucosal graft, as described here, provides little benefit in delaying apical migration of oral epithelium. The fact that the epithelium failed to reach the apical half of the dehiscence may indicate that features of this wound model may help our understanding of epithelial cellular kinetics operative in periodontal wound healing.

Periodontal wound healing in the absence of periodontal ligament cells.

This experiment was designed to study the ability of mucoperiosteal flap connective tissue to form new attachment on partially demineralized roots as well as nondemineralized roots in a wound-model where periodontal ligament (PDL) cells are absent, and ingrowth of bone granulation tissue is controlled. After reflecting mucoperiosteal flaps, bone was removed circumferentially around the distal root of mandibular second premolars and the mesial root of mandibular third premolars in five beagle dogs. The roots were completely denuded from the cementoenamel junction to the apex and curetted to remove PDL and cementum. Following root canal filling, the denuded roots on one side were conditioned with citric acid while the contralateral roots served as controls. To prevent bone-derived granulation tissue from contacting denuded roots, sheets of Nuclepore membrane were placed over the exposed bone margins and secured in place with steel ligatures. The flaps were coronally positioned and sutured. Histologic examination was made after three months of healing accompanied by regular plaque control. Both the experimental (acid conditioned) and control roots showed epithelium extending into the apical third of denuded roots and root resorption confined to the root apices. Some experimental specimens showed connective tissue adhesion in the apical third of roots without any new cementum or oriented fibers. Inflammatory cell infiltrate was seen extending to the apical third of denuded roots in both groups. In this model, the flap connective tissue cells failed to form new cementum and inserting fibers. Whether this was the result of inflammation and epithelial downgrowth or the inability of flap connective tissues to form new attachment could not be concluded from the study.

Guided tissue regeneration. An experimental procedure in beagle dogs.

This preliminary study examined the healing following an experimental procedure designed to facilitate coronal migration of progenitor cells from the periodontal ligament circumferentially on roots of premolar teeth in beagle dogs. Mucoperiosteal flaps were reflected on the buccal and lingual aspects of premolars in six beagle dogs with periodontal disease. Following root preparation, pieces of orthodontic wire were placed interproximally on the crowns to bridge the spaces between teeth. Biobrane, a synthetic membrane bonded to a knitted nylon fabric and coated with collagen, was placed as a physical barrier between the roots and the flaps to be replaced. The membrane extended as a single piece from the cementoenamel junction (CEJ) to overlap the crest of alveolar bone by 3 to 4 mm on both the buccal and lingual surfaces of the three premolars in each quadrant. The membrane was attached to the crowns at the CEJ with resin. The flaps were replaced and sutured. Postoperative care included plaque control and the membranes were removed after 5 weeks. The dogs were sacrificed to provide observation periods of 8 and 16 weeks after placement of membranes. Histologic examination revealed new connective tissue attachment in the apical part of the 8- and 16-week experimental specimens. Some experimental specimens showed new attachment up to 2.94 mm while others showed a long junctional epithelium (JE). Root resorption was also seen in some specimens. These preliminary findings suggest that placement of physical barriers between root surface and flaps may be beneficial in facilitating coronal migration of progenitor cells from the periodontal ligament.

In vivo differentiation of progenitor cells of the periodontal ligament. An experimental study using physical barriers.

The present study was designed to test the hypothesis that during regeneration of cementum, the progenitor cells from periodontal ligament must come in contact with root dentin in order to differentiate into cementoblasts. After reflecting mucoperiosteal flaps, fenestration wounds were made in the buccal cortical plates of mandibular canines in 6 beagle dogs. The exposed root surfaces were curretted to remove all cementum. The exposed root surface on one side was demineralized with citric acid while the contralateral wound had saline treatment. The exposed root surfaces were then dried and pieces of Nuclepore membrane (pore size 0.1 mu) were attached to part of the exposed root surface to prevent contact of progenitor cells with root dentin. The fenestration wounds were then covered with Millipore filter to facilitate the population of wounds by progenitor cells from the periodontal ligament. Histologic analysis was performed after 3 months of healing. In specimens where the Nuclepore membrane had remained attached to root dentin, no new cementum was seen over the membrane. At the borders of the wounds and in specimens where the Nuclepore membrane had detached from root dentin, new connective tissue attachment was consistently seen. Also, root resorption was very rarely observed in both the acid-treated and control specimens. The present findings suggest that contact with root dentin may be necessary for progenitor cell differentiation into formative cells like cementoblasts.