Diverse effect of BMP-2 homodimer on mesenchymal progenitors of different origin.

Bone morphogenetic protein-2 (BMP-2), is a potential factor to enhance osseointegration of dental implants. However, the appropriate cellular system to investigate the osteogenic effect of BMP-2 in vitro in a standardized manner still needs to be defined. The aim of this study was to examine the effect of BMP-2 on the cell proliferation and osteogenic differentiation of human osteogenic progenitors of various origins: dental pulp stem cells (DPSC), human osteosarcoma cell line (Saos-2) and human embryonic palatal mesenchymal cell line (HEPM). For induction of osteogenic differentiation, cell culture medium was supplemented with BMP-2 homodimer alone or in combination with conventionally used differentiation inducing agents. Differentiation was monitored for 6-18 days. To assess differentiation, proliferation rate, alkaline phosphatase activity, calcium deposition and the expression level of osteogenic differentiation marker genes (Runx2, BMP-2) were measured. BMP-2 inhibited cell proliferation in a concentration and time-dependent manner. In a concentration which caused maximal cell proliferation, BMP-2 did not induce osteogenic differentiation in any of the tested systems. However, it had a synergistic effect with the osteoinductive medium in both DPSC and Saos-2, but not in HEPM cells. We also found that the differentiation process was faster in Saos-2 than in DPSCs. Osteogenic differentiation could not be induced in the osteoblast progenitor HEPM cells. Our data suggest that in a concentration that inhibits proliferation the differentiation inducing effect of BMP-2 is evident only in the presence of permissive osteoinductive components. ß-glycerophosphate, was identified interacting with BMP-2 in a synergistic manner.

Median facial clefts in Xenopus laevis: roles of retinoic acid signaling and homeobox genes.

The upper lip and primary palate form an essential separation between the brain, nasal structures and the oral cavity. Surprisingly little is known about the development of these structures, despite the fact that abnormalities can result in various forms of orofacial clefts. We have uncovered that retinoic acid is a critical regulator of upper lip and primary palate development in Xenopus laevis. Retinoic acid synthesis enzyme, RALDH2, and retinoic acid receptor gamma (RARγ) are expressed in complementary and partially overlapping regions of the orofacial prominences that fate mapping revealed contribute to the upper lip and primary palate. Decreased RALDH2 and RARγ result in a median cleft in the upper lip and primary palate. To further understand how retinoic acid regulates upper lip and palate morphogenesis we searched for genes downregulated in response to RARγ inhibition in orofacial tissue, and uncovered homeobox genes lhx8 and msx2. These genes are both expressed in overlapping domains with RARγ, and together their loss of function also results in a median cleft in the upper lip and primary palate. Inhibition of RARγ and decreased Lhx8/Msx2 function result in decreased cell proliferation and failure of dorsal anterior cartilages to form. These results suggest a model whereby retinoic acid signaling regulates Lhx8 and Msx2, which together direct the tissue growth and differentiation necessary for the upper lip and primary palate morphogenesis. This work has the potential to better understand the complex nature of the upper lip and primary palate development which will lead to important insights into the etiology of human orofacial clefts.

Biochemical alterations in the palatal processes in fetuses of biotin-deficient mice.

To clarify the role of biotin in palatal formation, we investigated the effects of biotin deficiency on the development of palatal processes in mouse fetuses at midgestation. We also investigated protein expressions in the palatal processes. Pregnant mice were given either a biotin-deficient diet or a biotin-supplemented (control) diet from day 0 of gestation (dg 0). Some dams in the biotin-deficient group were changed to a biotin-supplemented diet on dg 12, 13 or 14. On dg 15, the palatal processes were dissected from these fetuses and their peptides were characterized using two-dimensional electrophoresis and liquid chromatography/tandem mass spectrometry (LC-MS/MS) system. Regarding Trasler's stage for the growth of the palatal processes in mouse fetuses on dg 15, the average stage of palatal development was 5.83 +/- 0.39 in the biotin-supplemented group, 5.39 +/- 0.66 in the dg 13-supplemented group, and 4.64 +/- 0.90 in the biotin-deficient group. The development of the palatal processes significantly increased in relation to the earlier day of biotin supplementation. In a protein analysis of palatal processes by isoelectro focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 19-kDa spot was confirmed around position at pI 6-7 in the biotin-supplemented group, but this protein was not present in either the biotin-deficient group or the dg 13-supplemented group. From the MS/MS database of peptides, adenosine diphosphate (ADP)-ribosylation factor 2 (arf2) and alpha-crystallin were detected in the mesenchyme of the palatal processes. It is suggested that the expression of these proteins may be downregulated by biotin deficiency, inducing the inhibited development of palatal processes.

Effect of chemically modified titanium surfaces on protein adsorption and osteoblast precursor cell behavior.

PURPOSE: To investigate the effects of different chemically modified titanium surfaces on protein adsorption and the osteoblastic differentiation of human embryonic palatal mesenchymal (HEPM) cells. MATERIALS AND METHODS: Three different surfaces were evaluated. The first, a machined surface (Ti-M), was considered a control. The second surface was acid etched (Ti-AE). The third surface was prepared by exposing the Ti-AE samples to sodium hydroxide (NaOH) solution (Ti-AAE). The surface characteristics of chemically modified titanium were investigated by means of scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and profilometry. To evaluate the production of biomarkers, commercial kits were utilized. RESULTS: Surface composition and morphology affected the kinetics of protein adsorption. Ti-AE surfaces manifested a greater affinity for fibronectin adsorption compared to Ti-M or Ti-AAE surfaces. It was observed that Ti-AE and Ti-AAE surfaces promoted significantly greater cell attachment compared to Ti-M surfaces. Statistically significant differences were also observed in the expression of alkaline phosphatase (ALP) activity, osteocalcin, and osteopontin on all 3 titanium surfaces. ALP activity and osteocalcin production up to day 12 suggested that differentiation of the cells into osteoblasts had occurred and that cells were expressing a bone-forming phenotype. CONCLUSIONS: It was thus concluded from this study that surface morphology and composition play a critical role in enhancing HEPM cell proliferation and differentiation into osteoblast cells.

Rotary culture enhances pre-osteoblast aggregation and mineralization.

Three-dimensional environments have been shown to enhance cell aggregation and osteoblast differentiation. Thus, we hypothesized that three-dimensional (3D) growth environments would enhance the mineralization rate of human embryonic palatal mesenchymal (HEPM) pre-osteoblasts. The objective of this study was to investigate the potential use of rotary cell culture systems (RCCS) as a means to enhance the osteogenic potential of pre-osteoblast cells. HEPM cells were cultured in a RCCS to create 3D enviroments. Tissue culture plastic (2D) cultures served as our control. 3D environments promoted three-dimensional aggregate formations. Increased calcium and phosphorus deposition was significantly enhanced three- to 18-fold (P < 0.001) in 3D cultures as compared with 2D environments. 3D cultures mineralized in 1 wk as compared with the 2D cultures, which took 4 wks, a decrease in time of nearly 75%. In conclusion, our studies demonstrated that 3D environments enhanced osteoblast cell aggregation and mineralization.

An in vitro screening system for characterizing the cleft palate-inducing potential of chemicals and underlying mechanisms.

An in vitro organ culture system with developing mouse palates was improved to characterize the cleft palate (CP)-inducing potential of chemicals and underlying mechanisms. Palatal explants collected from gestation day 12 mouse fetuses were cultured with various concentrations of teratogens and examined for palatal development after 48 and 72 h of culture to assess effects of the chemicals on growth and/or fusion of palatal shelves. When the explants were exposed to diphenylhydantoin or 5-fluorouracil, palatal growth was inhibited in a concentration-dependent manner at 48 h. Suppression of the expression of proliferative cell nuclear antigen revealed poor cell proliferation. At 72 h, the incidence of explants with CP was significantly increased in the high-dose groups, suggesting that CP induction is mainly attributable to inhibition of palatal growth. By contrast, retinoic acid and hydrocortisone significantly lowered the rates of fused palates at 72 h in all treated groups, while they exhibited no effects on palatal growth at 48 h even at the highest concentration. Because no apoptosis was found in the epithelial cells at the tip of these palates, these chemicals are suggested to inhibit palatal fusion process by preventing apoptosis.

In vivo/in vitro studies on the effects of cyclophosphamide on growth and differentiation of hamster palate.

A study was undertaken to examine the effects of cyclophosphamide (CP) on growth and differentiation of palatal tissues. An in vivo/in vitro approach was designed to analyze (1) whether the damage caused by in vivo administration of CP in the developing palate can be altered in vitro, and (2) to determine the effects of CP on the synthesis of collagen and glycosaminoglycan (GAG), which are essential for proper palate development. In addition, effects of vitamin B1 and/or B6 on in vivo modulation of CP teratogenicity was evaluated. Pregnant hamsters were given 30 mg/kg CP or 1 ml saline on day 10 of gestation. Control and CP-treated embryonic palates were dissected on day 11 of gestation and incubated in vitro in the presence or absence of CP. In order to allow metabolic activation of CP in vitro, either a slice of hamster liver or microsomal S9 fraction of liver was added to the culture medium. To study collagen and GAG synthesis, palates were obtained between days 10 and 13 of gestation, and incubated in growth medium supplemented with [14C]proline or [3H]glucosamine, as appropriate. The rates of collagen and GAG synthesis were determined. The results showed that, in the controls, the presence of a liver slice or S9 fraction in the culture medium had no effects on in vitro closure of palate. In vivo CP exposed palates did not fuse in vitro. When drug was given in vitro, or both in vivo and in vitro, palatal closure did not occur. CP reduced synthesis of both collagen and GAG in the vertically developing palate. The drug-treated shelves reoriented only after the rates of collagen and GAG synthesis were restored to the levels comparable to the control counterparts. Co-administration of vitamin B1 and B6 did not interfere with the teratogenicity of CP. It was suggested that CP treatment affected DNA synthesis and injured growing cells, which in turn reduced the synthesis of GAG and collagen and affected the expansion of shelf volume to delay the reorientation of the palatal shelves. Furthermore, it appears that in vivo treatment with CP changes the programming of palatal tissues to prevent the fusion process in vivo, which could not be altered in vitro.

Effects of 5-fluorouracil on collagen synthesis in the developing palate of hamster.

A study was undertaken to examine the effect of 5-fluorouracil (5-Fu) on collagen synthesis in the developing secondary palate. Pregnant hamsters were given 81 mg/kg 5-FU intramuscularly or 1 ml saline on day 11 of gestation. Control and treated embryonic palates, dissected from hamsters between days 11 and 13 of gestation, were incubated in a growth medium supplemented with [14C]proline. The rate of collagen synthesis, total protein and collagen isotypes were determined. The data showed that in control hamster palate the collagen synthesis peaked between days 12:00 (12 day: 0 h) and 12:04 of gestation, which is the period of shelf reorientation. In 5-FU-exposed hamster palates, the rate of collagen synthesis was lower than controls until day 12:04 of gestation followed by a rise on day 12:12 of gestation. In 5-FU-treated embryos palatal shelf reorientation took place between days 12:16 and 13:00 of gestation. Electrophoresis showed that only type I collagen was synthesized during palate development in both the control and 5-FU-treated hamster embryos. It was suggested that collagen synthesis may play a critical role in shelf reorientation in hamster since (i) new collagen was synthesized in both the control and 5-FU-treated hamster embryos prior to and during the period of normal reorientation, and (ii) in 5-FU-treated hamster embryos, a recovery in collagen synthesis precedes reorientation. Inhibition of collagen synthesis during abnormal development is only a step in the cascade of events of 5-FU-induced effects on protein synthesis.

Effects of 5-fluorouracil on collagen synthesis during quail secondary palate development.

A study was undertaken to examine the involvement of collagen synthesis during palate development in quail where mammalian-type shelf reorientation is absent. Teratological observation showed that 100 micrograms 5-fluorouracil (FU) on day 4 of incubation increased the gap between the palatal shelves. Light microscopic observation indicated that the quail palatal shelves develop as horizontal ridges on day 5 and approximate on day 8 of incubation but never fuse. FU treatment affected the approximation of palatal shelves. In separate experiments, both the control and FU-treated quail embryonic palates, which were dissected between days 5 and 10 of incubation, were incubated in a growth medium supplemented with 14C-proline. The rate of collagen synthesis, total protein, hydroxyproline (HYP) levels, and collagen isotype were determined. The result showed that in control palates the rate of collagen synthesis increased fivefold between days 6 and 8 of incubation but dropped thereafter. In FU-exposed palates, the rate of collagen synthesis was lower than that in controls. It increased threefold between days 7 and 8 of incubation. High performance liquid chromatographic measurement of HYP levels indicated that, in comparison to controls, HYP accumulation in FU-treated palates was reduced by 50% on day 6 and 75% on day 8 of incubation. Total protein content in FU-treated palate were also 50-70% lower than those in their control counterparts between days 5 and 10 of incubation. Gel electrophoresis showed that only type I collagen was synthesized in the developing palate of both the control and FU-treated quail embryo. An analysis of results of the present study, along with the data from literature on mammals, corroborate the proposition that an increased collagen synthesis may contribute to the acquisition of volume of vertebrate's secondary palatal shelf for its continuing morphogenesis.

Embryonic palatal responses to teratogens in serum-free organ culture.

This study examines development of rat, mouse, and human embryonic palates in submerged, serum-free organ culture. The concentration-response profiles for retinoic acid (RA), triamcinolone (TRI), hydrocortisone (HC), dexamethasone (DEX), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were examined and the mechanisms of clefting in vitro were compared to observed in vivo responses. Craniofacial regions were dissected on gestational day (GD) 12 for mice and GD 14 for rat, and cultured for 3-4 days in Bigger's BGJb medium in flasks flushed with 50% O2, 45% N2, 5% CO2. Growth and fusion of secondary palates were scored under a dissecting microscope. In serum-free control medium, mouse and rat palatal fusion occurred within the 4-day culture period. Supplementing with fetal bovine serum (FBS) in excess of 1% interfered with growth and fusion in control medium. RA significantly inhibited fusion of mouse and rat palates at 5 x 10(-9) and 1 x 10(-10) M, respectively, with RA-induced clefting related to abnormal proliferation and differentiation of medial epithelia. In contrast, glucocorticoid-induced clefting was due to concentration-dependent inhibition of shelf growth. TRI significantly inhibited fusion at 4 x 10(-5) M, and 1 x 10(-4) M DEX or HC, inhibited fusion of 19 and 42% of shelves, respectively. The response rate for DEX in the presence of 1% FBS was increased (42% unfused). TCDD clefting was due to altered medial epithelial differentiation and 1 x 10(-8) M TCDD affected 36% of CD-1 mouse, 23% of C57BL/6N mouse, and 47% of F344 rat palates. When the medium was supplemented with 1% FBS, selenium, transferrin, and additional glutamine, the response of C57BL/6N embryos increased to 75%. This rate is similar to that reported for Trowell's-type cultures with IMEM:F12 medium and 1% FBS. The increased responsiveness to DEX or TCDD in the presence of serum suggests that an unknown factor in serum may be required for full activity. Three human embryonic palatal explants (GD 52 or 53) were cultured for 3-6 days and fused during culture. The present study demonstrates that serum-free organ culture supports development of mouse, rat, and human palatal explants. The present study demonstrates the capacity of this organ culture system to model palatogenesis for several species, and to distinguish between various mechanisms of clefting as presented through selected model compounds. This model should be useful for exploring mechanisms of activity at a cellular and molecular level.

Heterogeneous distribution of the precursor of type I and type III collagen and fibronectin in the rough endoplasmic reticulum of palatal mesenchymal cells of the mouse embryo cultured in ascorbate-depleted medium.

In order to examine the intracellular distribution of precursors of type I and type III collagen and fibronectin in the palatal mesenchymal (MEPM) cells of the mouse embryo cultured under ascorbate-deficient conditions, immuno-electron-microscopic studies were carried out by use of affinity purified antibodies for these proteins. MEPM cells were obtained from the palatal shelves of 14-day-old mouse fetuses and cultured for 3-7 days in medium, either with or without 50 ng/dish/day ascorbic acid. Results obtained were as follows: (1) Although the rough endoplasmic reticulum (rER) of MEPM cells cultured for 5 days in ascorbate-supplemented medium was flattened, that in cells cultured in ascorbate-deficient medium had a distended or vesicular appearance. (2) Vesicular or distended rER showed heterogeneous staining for both type I and type III collagen, namely, some parts of rER showed positive staining for both types of collagen, while others showed negative staining. (3) Both type I and type III collagen showed codistribution in the same vesicular rER. (4) Vesicular rER showed negative or very faint labelling for fibronectin. These results may suggest regional differences in the function of rER.

Etiology of retinoic acid-induced cleft palate varies with the embryonic stage.

Retinoic acid (RA) has been shown to be teratogenic in many species, and 13-cis-RA is teratogenic in humans. Exposure to RA during embryonic morphogenesis produced a variety of malformations including limb defects and cleft palate. The type and severity of malformation depended on the stage of development exposed. The purpose of this study was to compare the effects of RA exposure in vivo on different stages of palate development. These results were compared to effects observed after exposure in organ culture. The vehicle used in RA dosing was also shown to be a major factor in the incidence of RA-induced cleft palate. For the in vivo studies, RA (100 mg/kg) in 10 ml corn oil/kg was given p.o. on gestation day (GD) 10 or 12, and the embryos were examined on GD 14 and 16. Exposure to RA in an oil:DMSO vehicle resulted in much higher incidences of cleft palate than were observed after dosing with RA in oil only. After exposure on GD 10, to RA, small palatal shelves formed which did not make contact and fuse on GD 14. The medial cells did not undergo programmed cell death. Instead, the medial cells differentiated into a stratified, squamous, oral-like epithelium. The RA-exposed medial cells did not incorporate 3H-TdR on GD 14 or 16, but the cells expressed EGF receptors and bound 125I-EGF. In contrast, RA-induced clefting after exposure on GD 12 did not involve growth inhibition. Shelves of normal size formed and made contact, but because of altered medial cell differentiation did not fuse. Medial cells differentiated into a pseudostratified, ciliated, nasal-like epithelium. This response was produced in vivo at exposure levels which produced cleft palate, and after exposure of palatal shelves to RA in vitro from GD 12-15. The medial cells exposed on GD 12 incorporated 3H-TdR on GD 14, expressed EGF receptors, and bound 125I-EGF. The responses to RA which lead to cleft palate differed after exposure on GD 10 or 12, and the pathways of differentiation which the medial cells followed depended on the developmental stage exposed.

Epithelial bridging of the primary palate: I. Characterization of sub-cultured epithelial cells.

Primary palatogenesis involves an intricate array of events. Cell migration, proliferation, differentiation, programmed death, and fusion occur. Prior to fusion, the morphology of the epithelium undergoes marked changes. Epithelial projections form and extend across the fusion site attaching by filopodia to the opposite prominence. By appearance, the epithelium plays a critical role in facial development. In order to monitor epithelial activities, a study was done to isolate and characterize epithelial cells derived from the primary palate. The primary palate was microdissected from day 13 Sprague-Dawley rat embryos, and the epithelium and mesenchyme were separated by enzymatic digestion with a 3% trypsin-pancreatin solution (3:1). All explants were cultured in Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium (1:1) supplemented with 10% fetal calf serum (FCS), 20 ng/ml epidermal growth factor (EGF), and antibiotics. Explant cells were gathered by trypsin harvesting and sub-cultured. These sub-cultured cells were further characterized. Transmission and scanning electron microscopy showed that the cells retained many morphological features observed in vivo. In passaged cells, type IV collagen, laminin, and cytokeratins were visualized by immunocytochemistry. Gel electrophoresis analysis of the water-insoluble extracts demonstrated major bands of proteins of 50 kD and 44 kD that were synthesized by the epithelial cells but not by the mesenchymal cells. These cytokeratin types are suggestive of a simple undifferentiated embryonic epithelium. The effect of all-trans retinoic acid (RA) on cell number and [3H]-proline incorporation was assessed. At [10(-4)M] and [10(-6)M] retinoic acid resulted in significant inhibition in cell proliferation and amount of proline incorporated, with the greater inhibition occurring in the mesenchymal cells. In the concentrations studied, retinoic acid has an inhibitory effect on the two differently derived cell types. This study established that sub-cultured epithelial cells maintain their phenotype and can be used to study fusion processes. Part 2 will demonstrate how the morphology of the epithelial cells can be modified to produce the changes that are observed during fusion of the primary palate.

Teratological research using in vitro systems. III. Embryonic organs in culture.

A search of literature published through the spring of 1986 yielded approximately 95 citations for the following embryonic organs in culture: kidney, pancreas, skin, palate, craniofacial tissue, tooth, lens, bones, digits, and liver. However, only the in vitro organ culture of the palate and tooth are reviewed in this paper. The other organ culture systems were not reported as teratogenic screens. Although some organs may have the potential for such use, many are currently used for evaluation of the pathogenesis associated with congenital abnormalities, cancer, or transplacental carcinogenesis, or in studies of the structure and function of differentiating organs, and will not be included in this review.

Prescreening for environmental teratogens using cultured mesenchymal cells from the human embryonic palate.

Mesenchymal cells from the prefusion human embryonic palate have been established in culture and can be grown in either a serum-free hormone-supplemented medium or a serum-containing medium. The growth of these cells is quite rapid in culture and inhibited in a dose-dependent manner by most teratogens thus far tested, such as dexamethasone. These cells are highly sensitive to a variety of DNA synthetic and mitotic inhibitors. The responses of these cells are complementary to the ovarian tumor cell attachment assay of Braun et al [1, and in this volume]. When used in conjunction with the tumor cells, the overall reliability is greater than 90% with only one false-negative, allopurinol.