- Email: [email protected]
WySnJ Mouse
J Oral Maxillofac Surg 71:143-150, 2013
Vitamin B-Complex Application Promotes Secondary Palate Development in a Palate Organ Model of the A/WySnJ Mouse Konstanze Scheller, MD, DMD,* Julia Orce y Tiggers, DMD,† and Johannes Schubert, MD, DMD, PhD‡ Purpose: This study analyzed the direct influence of vitamin B-complex supplements (Polybion N,
Merck Pharma GmbH, Germany) in medium on secondary palatal development in palatal organ cultures of A/WySnJ mice. Because of positive clinical experiences with prophylactic vitamin B substitution in mothers of cleft-related families, the direct influence of the vitamin B-complex on palatal tissue was analyzed. Materials and Methods: The inbred A/WySnJ mouse strain shows a highly spontaneous, genetically determined clefting rate of 20% to 44%. One hundred seventy-seven A/WySnJ fetuses were microdissected on gestational day 14.3 before the occurrence of palatal fusion. Palatal organ cultures were prepared and incubated in chemically defined serum-free medium with different concentrations (0.1% and 1.0%) of the vitamin B-complex Polybion N for 72 hours. Palatal development was analyzed microscopically according to the 6-step visual scale that describes the approximation of palatal shelves during development. Results: At the beginning of the experiment (gestational day 14.3), the palatal development of all specimens used for in vitro organ culture showed a clear approach of the palatal shelves at stage II (2.25 ⫾ 0.78). Seventy-two hours after in vitro cultivation, the palatal shelves of the organ cultures supplemented with the vitamin B-complex showed significant growth (0.1%, P ⫽ .00017; 1.0%, P ⫽ .00078), whereas the untreated control group remained at initial developmental stage II (P ⫽ .291). Conclusions: The results of this in vitro study suggest a significant positive influence of vitamin B supplementation on palatal shelf development in organ culture. Further studies will focus on the vitamin B concentration in the amniotic fluid of dams with or without cleft in their offspring. © 2013 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 71:143-150, 2013 relatively resistant to teratogenic actions.7,8 Therefore, it is a well-established animal system for cleft studies. A/WySnJ mice allow the analysis of timedependent palatal fusion during early fetal development9,10 and provide an opportunity to analyze normal and cleft palatal genesis11 in 1 animal system. The high rate of phenotypic clefts (CL/P) in A/WySnJ mice, a strain with minimal genetic variability (http:// www.jax.org), is caused mostly by genetic factors influencing embryonic and fetal development.8 Therefore, extrinsic factors, such as food restriction during the critical period of palatal genesis (days 12 to
Cleft lip with or without cleft palate (CL/P) is one of the most common birth defects in humans.1 To understand its pathogenesis, it is necessary to study the physiologic and pathologic processes of embryonic facial and palatal development. This has been the focus of many human and animal studies for many years.1-4 The impressive similarity in fetal development in humans and other mammals in special animal models has been established over the years.2,5,6 The A/WySnJ mouse strain in vivo shows a highly spontaneous cleft rate of 20% to 44% (Fig 1) that is
Surgery, Martin Luther University Halle-Wittenberg, Ernst-Grube-strae
Received from the Martin Luther University Halle-Wittenberg, Halle, Germany. *Senior Physician, Department of Oral and Maxillofacial Surgery. †Student, Department of Oral Medicine. ‡Head of the Department of Oral and Maxillofacial Surgery. Address correspondence and reprint requests to Dr Scheller: Department of Oral and Maxillofacial Surgery and Facial Plastic
40, 06120, Halle (Saale), Germany; e-mail: konstanze.weinzierl@ medizin.uni-halle.de © 2013 American Association of Oral and Maxillofacial Surgeons
0278-2391/13/7101-0$36.00/0 http://dx.doi.org/10.1016/j.joms.2012.04.002
143
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VITAMIN B ACCELERATES PALATAL GROWTH IN VITRO
FIGURE 1. Aspects of A, B, bilateral and C, D, left-side cleft lip and palate in an A/WySnJ mouse fetus at gestational day 18. Scheller, Orce y Tiggers, and Schubert. Vitamin B Accelerates Palatal Growth in Vitro. J Oral Maxillofac Surg 2013.
13), vitamin B-complex (B1, B2, B6, folic acid) deficiency over the entire period of gestation (days 1 to 18), and total vitamin B restriction, do not produce any significant change in the cleft rate in this A/WySnJ strain. Even the preventative application of the vitamin B-complex has no influence on the clefting rate and shows the relative stability of this system. Other mouse strains without this genetic predisposition toward CL/P, such as the NMRI mouse (normal cleft rate, 0.7% to 3.8%), are clearly influenced by these teratogenic arrangements and show an embryo toxicity and cleft increase of up to 25% in their offspring.8 Many clinical investigations have been performed to decrease the risk for women with an increased risk
for clefting (familial cases, abortions in former pregnancies). In human and animal studies, folic acid supplementation (vitamin B9) during pregnancy has shown a positive effect by significantly decreasing the frequency of many fetal malformations, especially the incidence of neural tube defects.12 Therefore, folic acid supplementation during pregnancy has been recommended in many countries (United States Preventive Services Task Force, 2009). Even animal experiments have shown this positive effect of folic acid by a decrease in the teratogenic CL/P appearance,12,13 although this positive effect has not been clearly observed in humans.14 The nonspecific vitamin B supplementation of women with some form of increased
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risk for clefting during the entire period from embryogenesis to fetal development (6 to 16 days post conceptionem) has shown a clearly effective protection in up to 70%.15 The aim of this study was to analyze the direct influence of the vitamin B-complex (Polybion N, Merck GmbH, Darmstadt, Germany) on palatal shelf development in a genetically cleft-related animal model. Therefore, an established and reproducible in vitro palatal organ model16-18 was chosen to eliminate any maternal influence.
contact (Binocular Stemi 2000, Zeiss, Jena, Germany), the head was divided with 2 parallel cuts, 1 at the level of the palatal shelves and passing through the oral opening and the other at the level of the eyes. The hindbrain was removed, and the organ cultures were placed head-down on 0.4-m pore filter inserts in 6-well cell culture plates (Nunc, Langenselbold, Germany). The palatal organ culture included the primary palate, the maxillary arch, and parts of the forehead.20 The tissues were cultured in a 5% humidified CO2 atmosphere at 37°C. ORGAN CULTURING AND VITAMIN B-COMPLEX APPLICATION
Materials and Methods ANIMALS
Female and male A/WySnJ mice (Jackson Laboratory, Ben Harbor, ME) were kept under standard housing conditions in the authors’ laboratory according to the guidelines for use of laboratory animals (http:// www.apa.org/science/anguide.html). The room temperature was held constant at 22°C, with humidity at 65%. Lighting at a luminosity of 70 lx was kept at a 12-/12-hour light/dark cycle. All mice consumed food (standard pellet diet) and water ad libitum. Female and male mice were kept separate. At 3 to 4 months of age, the mice were paired from 8 to 9:30 AM to determine the exact time (⫾1.5 hr) of fertilization.19 The pregnancy was confirmed by the occurrence of a vaginal plug. The pregnant female mice were kept in groups of 4 animals per cage. At 14 days 8 hours (⫾1.5 hr) after gestation, the female animals were killed by cervical dislocation (according to directive 3.01 of the Federal Veterenary Office, Basel, Switzerland) and the fetuses were delivered by laparotomy. TISSUE PREPARATION
A serum-free culture medium was used to minimize unknown components and interactions.21 The palatal tissues were cultured in Dulbecco Minimal Essential Medium and Ham nutrient mixture F12 medium at a 1:1 ratio with 2% penicillin 10.000U/ml/streptomycin 10 mg/ml (Sigma-Aldrich, Buchs, Switzerland). The medium was changed every day. One study group had a Polybion N (Merck GmbH) concentration of 0.1% (group 1), and the other had a concentration of 1.0% (group 2) in the culture medium. The exact concentrations of the different B vitamins are listed in Table 1. Polybion N includes thiamine-HCl 5.0 mg/mL (B1), riboflavin-5=-phosphate mononatrium salt 2 mg/mL (B2), nicotinamide 20 mg/mL (B3), biotin 0.25 mg/mL (B7), pantothenic acid 3 mg/mL (B5), and pyridoxine hydrochloride 2 mg/mL (B6). Neither cyanocobalamin (B12) nor folic acid (B9), which have been recommended during pregnancy (Ministry of Agriculture and Forestry, Finland, 1998), were included in the vitamin B-complex. SCORING SYSTEM
At gestational day (GD) 14.3, the embryonic palate was prepared by microdissection. Under direct visual
All specimens were photographed and examined for palatal shelf development under 8- and 20-fold
Table 1. VITAMIN B CONCENTRATIONS IN MEDIA
Vitamin
DMEM/Ham F12 (mg/L)
Polybion N (mg/L)
0.1% Polybion N (mg/L)
Relative Concentration Increase
1% Polybion N (mg/L)
Relative Concentration Increase
B1 B2 B3 B5 B6 B7 B9 B12
2.17 0.219 2.0185 2.24 2.031 0.00365 2.65 0.68
5,000 2,000 20,000 3,000 2,000 250
7.17 2.22 22.02 5.24 4.03 0.25
3.30 10.13 10.91 2.34 1.98 69.49
52.17 20.22 202.02 32.24 22.03 2.50
24.04 92.32 100.08 14.39 10.85 685.93
Note: The exact concentration of Polybion N and the change of the different B vitamins in the medium are listed in the table. There was no separate addition of folic acid and vitamin B12. Abbreviation: DMEM, Dulbecco Minimal Essential Medium. Scheller, Orce y Tiggers, and Schubert. Vitamin B Accelerates Palatal Growth in Vitro. J Oral Maxillofac Surg 2013.
146 magnification (Binocular Stemi 2000; Nikon [Tokio, Japan] Coolpix E4500) at the time of dissection (GD 14.3) and 72 hours after in vitro cultivation. The degree of palatal fusion was measured using the visual scoring system introduced by Al-Obaidi et al.22 This system classifies the developmental process of the palatal shelves in a 6-step system (Fig 2): stage I, barely recognizable palatal shelves; stage II, a clear approach of the shelves; stage III, a stronger approach of the palatal shelves but no contact; stage IV, the first contact of the shelves; stage V, approach greater than two thirds; and stage, VI, totally fused palate with or without an epithelial seam in the fusion line in histologic sections. STATISTICAL EVALUATION
All scores were documented and analyzed for statistical significance using SPSS 12 (SPSS, Inc, Chicago, IL). The results at the time of dissection (GD 14.3) and after a 72-hour incubation (GD 17.3) were analyzed using the Wilcoxon signed-rank test and Wilcoxon rank-sum test (Mann-Whitney U test), with a significance level of ␣ ⫽ 0.05.
Results The study was performed in 186 A/WySnJ mouse fetuses carried by 37 dams. One hundred seventyseven of the 186 specimens (95.2%) were included in the statistical evaluation. Nine specimens (4.8%) were excluded because these could not be ranked in the visual scoring system at GD 14.3. At the time of dissection (GD 14.3), the development of all palatal shelves was at a mean of stage II (2.25 ⫾ 0.78), showing a clear approach of the palatal shelves (n ⫽ 177, 100%). A complete fusion of the palatal shelves was not seen in any specimen at this time (0/177). The 2 groups receiving the vitamin B-complex (0.1% and 1.0%) showed statistically significant (0.1%, P ⫽ .0003; 1.0%, P ⫽ .0018) palatal development during incubation (Fig 3A, B). Two specimens, 1 in each vitamin B group (0.1%, n ⫽ 62; 1.0%, n ⫽ 61), developed a macroscopically completely fused palate (stage V) at the end of the 72-hour incubation period (1.6%; Table 1). The histologic coronal sections (hematoxylin and eosin stain) of these fused palates showed only “partially” fused palates with an epithelial seam in the midline.23 Altogether, 54.8% (24/62) of the organ cultures in the 0.1% vitamin B group (group 1) grew during in vitro cultivation: 38.7% (24/61) improved by 1 score point and 16.1% (10/62) by 2 score points. Three organ cultures (3/62) regressed by 1 score point (4.8%) and 1 (1/62) by 2 score points (1.6%). The positive palatal development in the 0.1% organ cultures was significant
VITAMIN B ACCELERATES PALATAL GROWTH IN VITRO
(0.1%, P ⫽ .00017). Twenty-four organ cultures (24/ 62, 14.8%) neither improved nor degraded during incubation. In the second group with 1.0% B-complex in the medium, 42.6% (26/61) of the palatal organ cultures showed palatal shelf development: 21.3% improved by 1 score point (13/61), 19.7% improved by 2 score points (12/61), and 1.6% improved by 3 score points (1/61). Only 13.1% (8/61) regressed and 44.3% (27/61) remained at the initial value (Fig 3A). The control group (no vitamin B-complex addition, n ⫽ 54) showed a group-specific, distinct regression of palatal development from a mean score of 2.35 ⫾ 0.85 to 2.25 ⫾ 0.89 (Fig 1A). There was no significant palatal development in this group (P ⫽ .58). In the control group, 70.4% (38/54) showed no palatal growth, 14.8% (8/54) showed a decrease of 1 score point, and 3.7% (2/54) showed a decrease of 2 score points. Only 6 of 54 specimens (11.1%) grew any further and showed any noticeable approximation of the palatal shelves: 5 (9.2%) improved by 1 score point and 1 improved by 2 score points (1.9%). In summary, there was significant palatal shelf development in all organ cultures (Fig 2B) that received the vitamin B-complex in the growth medium (Fig 3B). The difference in palatal shelf growth in the vitamin B groups (0.1% and 1.0% supplementation) at the beginning of the incubation (P ⫽ .14) and after 72 hours of incubation was not significant (P ⫽ .50). Therefore, 0.1% supplementation seemed to be as effective as 1.0% supplementation.
Discussion Many different studies in animals13,24 and humans14,25 have been performed to identify a feasible method to prevent CL/P in children from families with a history of CL/P.15,26 In contrast to neural tube defects, there is no international consensus regarding preventative measures or prophylactic vitamin substitutions for pregnant women in these cleft-related families. However, it has been shown that consistent dietary vitamin supplementation, including folic acid, can decrease the frequency of cleft appearance in these families.14,26 How prophylactic vitamin and folic acid supplements influence embryonic facial development remains unknown. In this study, a palatal organ model6,18 of the A/WySnJ mouse (http://www.jax.org), a strain genetically predisposed to CL/P, was used to evaluate the direct influence of the vitamin B-complex on palatal shelf development. This organ model was chosen because multifactorial extrinsic effects that can cause facial clefts27 can be minimized. These extrinsic effects can be controlled by the elimination of any influence of the maternal organism on the organ model during the critical period of palatal genesis. However,
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FIGURE 2. The 6 different stages (I to VI) of palatal shelf development according to Al-Obaidi et al22: A, stage I, barely recognizable palatal shelves; B, stage II, a clear approach of the palatal shelves; C, stage III, stronger approach of the palatal shelves but no direct contact; D, stage VI, first contact of shelves; E, stage V, approach greater than two thirds; F, stage VI, totally fused palate with or without any epithelial seam in the fusion line in histologic sections. Scheller, Orce y Tiggers, and Schubert. Vitamin B Accelerates Palatal Growth in Vitro. J Oral Maxillofac Surg 2013.
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VITAMIN B ACCELERATES PALATAL GROWTH IN VITRO
Palatal development 4
palatal fusion score
3,5 3
Control (p=0.58)
2,5
0.1% Polybion®N (p=0.003)
2
1.0% Polybion®N
1,5
(p=0.018)
1 0,5 0
14.3 GD
17.3 GD
14.3 GD 17.3 GD
14.3 GD
17.3 GD
PolybionN supplementation groups
FIGURE 3. Palatal development in the organ culture over a 72-hour cultivation period (n ⫽ 117). In the 2 vitamin B-supplemented groups (0.1% and 1.0% Polybion N), there was significant palatal growth (P ⬍ .05). At the beginning of the cultivation period, all groups started at the same developmental stage. The developmental stage of the 2 supplemented groups did not differ significantly after the incubation period (P ⫽ .50). A positive influence of the vitamin B-complex on palatal shelf development can be assumed. GD, gestational day; p.c., post conceptionem. Scheller, Orce y Tiggers, and Schubert. Vitamin B Accelerates Palatal Growth in Vitro. J Oral Maxillofac Surg 2013.
some critical points and potential sources of error must be considered.20 The time of sacrifice of the dam is very important, because it determines the initial developmental stage of the palatal shelves in the organ cultures. With respect to the predisposition for a generally delayed development in the A/WySnJ strain,10,28 the highest potency for any expected palatal fusion is 14 to 15 days after fertilization.28-30 Therefore, the palatal organ cultures were prepared 14 days 8 hours after fertilization. This period corresponds to the greatest potential for any possible palatal fusion.11,29 With respect to previous studies of palatal development and palatal fusion potential in the organ model, it must be mentioned that palatal development is generally delayed and the palatal fusion rate is decreased (13%) in this in vitro organ model16,17 compared with a palatal fusion rate of 65% to 50% in vivo.27,29 This may be caused by the traumatic proce-
dures during palatal organ preparation, resulting in a negative effect on palatal shelf development in the organ model. In the present investigation, the control group showed no significant palatal development during in vitro cultivation, which accorded exactly to previous results.16 In contrast, the 2 vitamin B groups (0.1% and 1.0%) showed a significant positive development of the palatal shelves during in vitro cultivation. The 0.1% and 1.0% supplementations showed no significant difference (P ⫽ .50). This effect seemed to be caused directly by the positive influence of the vitamin B-complex in the medium on secondary palatal development; however, its direct mechanism of action cannot yet be explained. In light of these findings, a good clinical option may be the intrauterine reparation of clefts during
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fetal development. An animal study by Schubert et al9 showed the potent reparative effect of B vitamins on dexamethasone-induced clefts in A/WySnJ mice when given to their dams after teratogenic dexamethasone application. In an animal experiment, Bienengräber et al13 described a clear reparative effect of folic acid (B9), indicated by a decrease in total procarbazine-induced palatal clefts, applied to persistent hard palate clefts after the teratogenic action. For normal secondary palatal development, growing from the hard palate anteriorly to the soft palate posteriorly, the fused soft palate in the presence of a hard palate cleft might be caused by reparation mechanisms in utero. Another in vitro palatal organ model study of the A/J strain showed a positive efficiency of folic acid on palatal shelf development.18 All in all, it is difficult to explain how far a teratogenic mechanism is similar to the relatively stable genetic mechanism that causes the CL/P appearance in the A/WySnJ strain.31,32 For an effective prevention of teratogenic-induced clefts, 1 mechanism to prevent facial clefting may be accelerated embryonic development.15,29 If there is any possibility to promote regular palatal shelf development in the A/WySnJ strain by the application of a pharmaceutical or over-the-counter drug in the late embryonic/early fetal period (GDs 12.5 to 14), there might be a chance for the possible reparation and prevention of CL/P in cases with a strong genetic tendency. The present in vitro study showed a significant influence of the direct application of the vitamin B-complex on palatal development in this special organ model. The inability to effectively process the vitamin B supplement by the A/WySnJ dams in vivo would explain the difference the lack of a decrease in cleft palate rates when given to the pregnant dams.8 This might result from drug and receptor interactions with a saturation or failure of binding capacity, resulting in a disruption to vitamin use or transport in the amniotic fluid. Unfortunately, there are no data on the vitamin B concentrations in the blood and amniotic fluid of this mouse strain that could explain the present findings. In conclusion, the developmental stage of the palatal organ at the time of vitamin B-complex supplementation is one of the most important factors in all experimental models in vivo and in vitro.17 From these findings, the authors conclude that vitamin B-complex supplements should be applied in the early developmental stage of the fetus for the clinical prevention of cleft palate. Further studies should analyze the different vitamin B concentrations in the blood and amniotic fluid of this mouse
strain and the receptor status for the different B vitamins.
References 1. Tolarová MM, Cervenka J: Classification and birth prevalence of orofacial clefts. Am J Med Genet 75:126, 1998 2. Ferguson MW: Palate development. Development 103:41, 1988 (suppl) 3. Kalter H: The history of the A family of inbred mice and the biology of its congenital malformations. Teratology 20:313, 1979 4. Kreybig T: Tierexperimentelle Untersuchungen zur Wirkung von Teratogenen auf die kritischen Phasen der vorgeburtlichen Entwicklung des Gesichtsschädels. Eur Arch Otorhinolaryngol 196:270, 1970 5. Bedell MA, Jenkins NA, Copeland NG: Mouse models of human disease. Part I: Techniques and resources for genetic analysis in mice. Genes Dev 11:1, 1997 6. Bedell MA, Largaespada DA, Jenkins NA, et al: Mouse models of human disease. Part II: Recent progress and future directions. Genes Dev 11:11, 1997 7. Wang KY, Juriloff DM, Diewert VM: Deficient and delayed primary palatal fusion and mesenchymal bridge formation in cleft lip-liable strains of mice. J Craniofac Genet Dev Biol 15:99, 1995 8. Schubert J: Prevention of experimentally induced cleft palate in mice. Cleft Palate J 19:83, 1982 9. Schubert J, Schmidt R, Syska E: B group vitamins and cleft lip and cleft palate. Int J Oral Maxillofac Surg 31:410, 2002 10. Ciriani D, Diewert VM: A comparative study of development during primary palate formation in A/WySn, C57BL/6, and their F1 crosses. J Craniofac Genet Dev Biol 6:369, 1986 11. Juriloff DM, Harris MJ, Brown CJ: Unravelling the complex genetics of cleft lip in the mouse model. Mamm Genome 12:426, 2001 12. Paros A, Beck SL: Folinic acid reduces cleft lip [CL(P)] in A/WySn mice. Teratology 60:344, 1999 13. Bienengräber V, Malek FA, Möritz KU, , et al: Is it possible to prevent cleft palate by prenatal administration of folic acid? An experimental study. Cleft Palate Craniofac J 38:393, 2001 14. Czeizel AE, Tímár L, Sárközi A, et al: Dose-dependent effect of folic acid on the prevention of orofacial clefts. Pediatrics 104:1, 1999 15. Schubert J, Schmidt R, Raupach HW: New findings explaining the mode of action in prevention of facial clefting and first clinical experience. J Craniomaxillofac Surg 18:343, 1990 16. Scheller K, Schubert A, Schubert J: In vitro investigation of the secondary palate development in two strains of mice. Int J Oral Maxillofac Surg 40:737, 2011 17. Erfani S, Maldonado TS, Crisera CA, et al: An in vitro mouse model of cleft palate: Defining a critical intershelf distance necessary for palatal clefting. Plast Reconstr Surg 108:403, 2001 18. Natsume N, Nagatsu Y, Kawai T: Direct effect of vitamins at the time of palatal fusion. Plast Reconstr Surg 102:2512, 1998 19. Schmidt R: [A modified mating regimen for the laboratory mouse]. Z Versuchstierkd 27:206, 1985 20. Abbott BD, Buckalew AR: Embryonic palatal responses to teratogens in serum-free organ-culture. Teratology 45:369, 1992 21. Abbott BD, Logsdon TR, Wilke TS: Effects of methanol on embryonic mouse palate in serum-free organ culture. Teratology 49:122, 1994 22. Al-Obaidi N, Kastner U, Merker HJ, et al: Development of a suspension organ culture of the fetal rat palate. Arch Toxicol 69:472, 1995 23. Vargas VI: Palatal fusion in vitro in the mouse. Arch Oral Biol 12:1283, 1967 24. Gundlach KK, Abou Tara N, von Kreybig T: [Animal experimental results on the etiology and prevention of facial clefts
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25. 26. 27. 28.
and other craniofacial anomalies]. Fortschr Kieferorthop 47: 356, 1986 Itikala PR, Watkins ML, Mulinare J, et al: Maternal multivitamin use and orofacial clefts in offspring. Teratology 63:79, 2001 Tolarova M, Harris J: Reduced recurrence of orofacial clefts after periconceptional supplementation with high-dose folic acid and multivitamins. Teratology 51:71, 1995 Hallgrímsson B, Dorval CJ, Zelditch ML, et al: Craniofacial variability and morphological integration in mice susceptible to cleft lip and palate. J Anat 205:501, 2004 Wang KY, Diewert VM: A morphometric analysis of craniofacial growth in cleft lip and noncleft mice. J Craniofac Genet Dev Biol 12:141, 1992
VITAMIN B ACCELERATES PALATAL GROWTH IN VITRO 29. Syska E, Schmidt R, Schubert J: The time of palatal fusion in mice: A factor of strain susceptibility to teratogens. J Craniomaxillofac Surg 32:2, 2004 30. Diewert VM, Wang KY: Recent advances in primary palate and midface morphogenesis research. Crit Rev Oral Biol Med 4:111, 1992 31. Shimizu N, Aoyama H, Hatakenaka N, et al: An in vitro screening system for characterizing the cleft palate-inducing potential of chemicals and underlying mechanisms. Reprod Toxicol 15:665, 2001 32. Datson GP: Advances in understanding mechanisms of toxicity and implications risk assessment. Reprod Toxicol 11: 388, 1997
Vitamin B-Complex Application Promotes Secondary Palate Development in a Palate Organ Model of the A/WySnJ Mouse Konstanze Scheller, MD, DMD,* Julia Orce y Tiggers, DMD,† and Johannes Schubert, MD, DMD, PhD‡ Purpose: This study analyzed the direct influence of vitamin B-complex supplements (Polybion N,
Merck Pharma GmbH, Germany) in medium on secondary palatal development in palatal organ cultures of A/WySnJ mice. Because of positive clinical experiences with prophylactic vitamin B substitution in mothers of cleft-related families, the direct influence of the vitamin B-complex on palatal tissue was analyzed. Materials and Methods: The inbred A/WySnJ mouse strain shows a highly spontaneous, genetically determined clefting rate of 20% to 44%. One hundred seventy-seven A/WySnJ fetuses were microdissected on gestational day 14.3 before the occurrence of palatal fusion. Palatal organ cultures were prepared and incubated in chemically defined serum-free medium with different concentrations (0.1% and 1.0%) of the vitamin B-complex Polybion N for 72 hours. Palatal development was analyzed microscopically according to the 6-step visual scale that describes the approximation of palatal shelves during development. Results: At the beginning of the experiment (gestational day 14.3), the palatal development of all specimens used for in vitro organ culture showed a clear approach of the palatal shelves at stage II (2.25 ⫾ 0.78). Seventy-two hours after in vitro cultivation, the palatal shelves of the organ cultures supplemented with the vitamin B-complex showed significant growth (0.1%, P ⫽ .00017; 1.0%, P ⫽ .00078), whereas the untreated control group remained at initial developmental stage II (P ⫽ .291). Conclusions: The results of this in vitro study suggest a significant positive influence of vitamin B supplementation on palatal shelf development in organ culture. Further studies will focus on the vitamin B concentration in the amniotic fluid of dams with or without cleft in their offspring. © 2013 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 71:143-150, 2013 relatively resistant to teratogenic actions.7,8 Therefore, it is a well-established animal system for cleft studies. A/WySnJ mice allow the analysis of timedependent palatal fusion during early fetal development9,10 and provide an opportunity to analyze normal and cleft palatal genesis11 in 1 animal system. The high rate of phenotypic clefts (CL/P) in A/WySnJ mice, a strain with minimal genetic variability (http:// www.jax.org), is caused mostly by genetic factors influencing embryonic and fetal development.8 Therefore, extrinsic factors, such as food restriction during the critical period of palatal genesis (days 12 to
Cleft lip with or without cleft palate (CL/P) is one of the most common birth defects in humans.1 To understand its pathogenesis, it is necessary to study the physiologic and pathologic processes of embryonic facial and palatal development. This has been the focus of many human and animal studies for many years.1-4 The impressive similarity in fetal development in humans and other mammals in special animal models has been established over the years.2,5,6 The A/WySnJ mouse strain in vivo shows a highly spontaneous cleft rate of 20% to 44% (Fig 1) that is
Surgery, Martin Luther University Halle-Wittenberg, Ernst-Grube-strae
Received from the Martin Luther University Halle-Wittenberg, Halle, Germany. *Senior Physician, Department of Oral and Maxillofacial Surgery. †Student, Department of Oral Medicine. ‡Head of the Department of Oral and Maxillofacial Surgery. Address correspondence and reprint requests to Dr Scheller: Department of Oral and Maxillofacial Surgery and Facial Plastic
40, 06120, Halle (Saale), Germany; e-mail: konstanze.weinzierl@ medizin.uni-halle.de © 2013 American Association of Oral and Maxillofacial Surgeons
0278-2391/13/7101-0$36.00/0 http://dx.doi.org/10.1016/j.joms.2012.04.002
143
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VITAMIN B ACCELERATES PALATAL GROWTH IN VITRO
FIGURE 1. Aspects of A, B, bilateral and C, D, left-side cleft lip and palate in an A/WySnJ mouse fetus at gestational day 18. Scheller, Orce y Tiggers, and Schubert. Vitamin B Accelerates Palatal Growth in Vitro. J Oral Maxillofac Surg 2013.
13), vitamin B-complex (B1, B2, B6, folic acid) deficiency over the entire period of gestation (days 1 to 18), and total vitamin B restriction, do not produce any significant change in the cleft rate in this A/WySnJ strain. Even the preventative application of the vitamin B-complex has no influence on the clefting rate and shows the relative stability of this system. Other mouse strains without this genetic predisposition toward CL/P, such as the NMRI mouse (normal cleft rate, 0.7% to 3.8%), are clearly influenced by these teratogenic arrangements and show an embryo toxicity and cleft increase of up to 25% in their offspring.8 Many clinical investigations have been performed to decrease the risk for women with an increased risk
for clefting (familial cases, abortions in former pregnancies). In human and animal studies, folic acid supplementation (vitamin B9) during pregnancy has shown a positive effect by significantly decreasing the frequency of many fetal malformations, especially the incidence of neural tube defects.12 Therefore, folic acid supplementation during pregnancy has been recommended in many countries (United States Preventive Services Task Force, 2009). Even animal experiments have shown this positive effect of folic acid by a decrease in the teratogenic CL/P appearance,12,13 although this positive effect has not been clearly observed in humans.14 The nonspecific vitamin B supplementation of women with some form of increased
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risk for clefting during the entire period from embryogenesis to fetal development (6 to 16 days post conceptionem) has shown a clearly effective protection in up to 70%.15 The aim of this study was to analyze the direct influence of the vitamin B-complex (Polybion N, Merck GmbH, Darmstadt, Germany) on palatal shelf development in a genetically cleft-related animal model. Therefore, an established and reproducible in vitro palatal organ model16-18 was chosen to eliminate any maternal influence.
contact (Binocular Stemi 2000, Zeiss, Jena, Germany), the head was divided with 2 parallel cuts, 1 at the level of the palatal shelves and passing through the oral opening and the other at the level of the eyes. The hindbrain was removed, and the organ cultures were placed head-down on 0.4-m pore filter inserts in 6-well cell culture plates (Nunc, Langenselbold, Germany). The palatal organ culture included the primary palate, the maxillary arch, and parts of the forehead.20 The tissues were cultured in a 5% humidified CO2 atmosphere at 37°C. ORGAN CULTURING AND VITAMIN B-COMPLEX APPLICATION
Materials and Methods ANIMALS
Female and male A/WySnJ mice (Jackson Laboratory, Ben Harbor, ME) were kept under standard housing conditions in the authors’ laboratory according to the guidelines for use of laboratory animals (http:// www.apa.org/science/anguide.html). The room temperature was held constant at 22°C, with humidity at 65%. Lighting at a luminosity of 70 lx was kept at a 12-/12-hour light/dark cycle. All mice consumed food (standard pellet diet) and water ad libitum. Female and male mice were kept separate. At 3 to 4 months of age, the mice were paired from 8 to 9:30 AM to determine the exact time (⫾1.5 hr) of fertilization.19 The pregnancy was confirmed by the occurrence of a vaginal plug. The pregnant female mice were kept in groups of 4 animals per cage. At 14 days 8 hours (⫾1.5 hr) after gestation, the female animals were killed by cervical dislocation (according to directive 3.01 of the Federal Veterenary Office, Basel, Switzerland) and the fetuses were delivered by laparotomy. TISSUE PREPARATION
A serum-free culture medium was used to minimize unknown components and interactions.21 The palatal tissues were cultured in Dulbecco Minimal Essential Medium and Ham nutrient mixture F12 medium at a 1:1 ratio with 2% penicillin 10.000U/ml/streptomycin 10 mg/ml (Sigma-Aldrich, Buchs, Switzerland). The medium was changed every day. One study group had a Polybion N (Merck GmbH) concentration of 0.1% (group 1), and the other had a concentration of 1.0% (group 2) in the culture medium. The exact concentrations of the different B vitamins are listed in Table 1. Polybion N includes thiamine-HCl 5.0 mg/mL (B1), riboflavin-5=-phosphate mononatrium salt 2 mg/mL (B2), nicotinamide 20 mg/mL (B3), biotin 0.25 mg/mL (B7), pantothenic acid 3 mg/mL (B5), and pyridoxine hydrochloride 2 mg/mL (B6). Neither cyanocobalamin (B12) nor folic acid (B9), which have been recommended during pregnancy (Ministry of Agriculture and Forestry, Finland, 1998), were included in the vitamin B-complex. SCORING SYSTEM
At gestational day (GD) 14.3, the embryonic palate was prepared by microdissection. Under direct visual
All specimens were photographed and examined for palatal shelf development under 8- and 20-fold
Table 1. VITAMIN B CONCENTRATIONS IN MEDIA
Vitamin
DMEM/Ham F12 (mg/L)
Polybion N (mg/L)
0.1% Polybion N (mg/L)
Relative Concentration Increase
1% Polybion N (mg/L)
Relative Concentration Increase
B1 B2 B3 B5 B6 B7 B9 B12
2.17 0.219 2.0185 2.24 2.031 0.00365 2.65 0.68
5,000 2,000 20,000 3,000 2,000 250
7.17 2.22 22.02 5.24 4.03 0.25
3.30 10.13 10.91 2.34 1.98 69.49
52.17 20.22 202.02 32.24 22.03 2.50
24.04 92.32 100.08 14.39 10.85 685.93
Note: The exact concentration of Polybion N and the change of the different B vitamins in the medium are listed in the table. There was no separate addition of folic acid and vitamin B12. Abbreviation: DMEM, Dulbecco Minimal Essential Medium. Scheller, Orce y Tiggers, and Schubert. Vitamin B Accelerates Palatal Growth in Vitro. J Oral Maxillofac Surg 2013.
146 magnification (Binocular Stemi 2000; Nikon [Tokio, Japan] Coolpix E4500) at the time of dissection (GD 14.3) and 72 hours after in vitro cultivation. The degree of palatal fusion was measured using the visual scoring system introduced by Al-Obaidi et al.22 This system classifies the developmental process of the palatal shelves in a 6-step system (Fig 2): stage I, barely recognizable palatal shelves; stage II, a clear approach of the shelves; stage III, a stronger approach of the palatal shelves but no contact; stage IV, the first contact of the shelves; stage V, approach greater than two thirds; and stage, VI, totally fused palate with or without an epithelial seam in the fusion line in histologic sections. STATISTICAL EVALUATION
All scores were documented and analyzed for statistical significance using SPSS 12 (SPSS, Inc, Chicago, IL). The results at the time of dissection (GD 14.3) and after a 72-hour incubation (GD 17.3) were analyzed using the Wilcoxon signed-rank test and Wilcoxon rank-sum test (Mann-Whitney U test), with a significance level of ␣ ⫽ 0.05.
Results The study was performed in 186 A/WySnJ mouse fetuses carried by 37 dams. One hundred seventyseven of the 186 specimens (95.2%) were included in the statistical evaluation. Nine specimens (4.8%) were excluded because these could not be ranked in the visual scoring system at GD 14.3. At the time of dissection (GD 14.3), the development of all palatal shelves was at a mean of stage II (2.25 ⫾ 0.78), showing a clear approach of the palatal shelves (n ⫽ 177, 100%). A complete fusion of the palatal shelves was not seen in any specimen at this time (0/177). The 2 groups receiving the vitamin B-complex (0.1% and 1.0%) showed statistically significant (0.1%, P ⫽ .0003; 1.0%, P ⫽ .0018) palatal development during incubation (Fig 3A, B). Two specimens, 1 in each vitamin B group (0.1%, n ⫽ 62; 1.0%, n ⫽ 61), developed a macroscopically completely fused palate (stage V) at the end of the 72-hour incubation period (1.6%; Table 1). The histologic coronal sections (hematoxylin and eosin stain) of these fused palates showed only “partially” fused palates with an epithelial seam in the midline.23 Altogether, 54.8% (24/62) of the organ cultures in the 0.1% vitamin B group (group 1) grew during in vitro cultivation: 38.7% (24/61) improved by 1 score point and 16.1% (10/62) by 2 score points. Three organ cultures (3/62) regressed by 1 score point (4.8%) and 1 (1/62) by 2 score points (1.6%). The positive palatal development in the 0.1% organ cultures was significant
VITAMIN B ACCELERATES PALATAL GROWTH IN VITRO
(0.1%, P ⫽ .00017). Twenty-four organ cultures (24/ 62, 14.8%) neither improved nor degraded during incubation. In the second group with 1.0% B-complex in the medium, 42.6% (26/61) of the palatal organ cultures showed palatal shelf development: 21.3% improved by 1 score point (13/61), 19.7% improved by 2 score points (12/61), and 1.6% improved by 3 score points (1/61). Only 13.1% (8/61) regressed and 44.3% (27/61) remained at the initial value (Fig 3A). The control group (no vitamin B-complex addition, n ⫽ 54) showed a group-specific, distinct regression of palatal development from a mean score of 2.35 ⫾ 0.85 to 2.25 ⫾ 0.89 (Fig 1A). There was no significant palatal development in this group (P ⫽ .58). In the control group, 70.4% (38/54) showed no palatal growth, 14.8% (8/54) showed a decrease of 1 score point, and 3.7% (2/54) showed a decrease of 2 score points. Only 6 of 54 specimens (11.1%) grew any further and showed any noticeable approximation of the palatal shelves: 5 (9.2%) improved by 1 score point and 1 improved by 2 score points (1.9%). In summary, there was significant palatal shelf development in all organ cultures (Fig 2B) that received the vitamin B-complex in the growth medium (Fig 3B). The difference in palatal shelf growth in the vitamin B groups (0.1% and 1.0% supplementation) at the beginning of the incubation (P ⫽ .14) and after 72 hours of incubation was not significant (P ⫽ .50). Therefore, 0.1% supplementation seemed to be as effective as 1.0% supplementation.
Discussion Many different studies in animals13,24 and humans14,25 have been performed to identify a feasible method to prevent CL/P in children from families with a history of CL/P.15,26 In contrast to neural tube defects, there is no international consensus regarding preventative measures or prophylactic vitamin substitutions for pregnant women in these cleft-related families. However, it has been shown that consistent dietary vitamin supplementation, including folic acid, can decrease the frequency of cleft appearance in these families.14,26 How prophylactic vitamin and folic acid supplements influence embryonic facial development remains unknown. In this study, a palatal organ model6,18 of the A/WySnJ mouse (http://www.jax.org), a strain genetically predisposed to CL/P, was used to evaluate the direct influence of the vitamin B-complex on palatal shelf development. This organ model was chosen because multifactorial extrinsic effects that can cause facial clefts27 can be minimized. These extrinsic effects can be controlled by the elimination of any influence of the maternal organism on the organ model during the critical period of palatal genesis. However,
SCHELLER, ORCE Y TIGGERS, AND SCHUBERT
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FIGURE 2. The 6 different stages (I to VI) of palatal shelf development according to Al-Obaidi et al22: A, stage I, barely recognizable palatal shelves; B, stage II, a clear approach of the palatal shelves; C, stage III, stronger approach of the palatal shelves but no direct contact; D, stage VI, first contact of shelves; E, stage V, approach greater than two thirds; F, stage VI, totally fused palate with or without any epithelial seam in the fusion line in histologic sections. Scheller, Orce y Tiggers, and Schubert. Vitamin B Accelerates Palatal Growth in Vitro. J Oral Maxillofac Surg 2013.
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VITAMIN B ACCELERATES PALATAL GROWTH IN VITRO
Palatal development 4
palatal fusion score
3,5 3
Control (p=0.58)
2,5
0.1% Polybion®N (p=0.003)
2
1.0% Polybion®N
1,5
(p=0.018)
1 0,5 0
14.3 GD
17.3 GD
14.3 GD 17.3 GD
14.3 GD
17.3 GD
PolybionN supplementation groups
FIGURE 3. Palatal development in the organ culture over a 72-hour cultivation period (n ⫽ 117). In the 2 vitamin B-supplemented groups (0.1% and 1.0% Polybion N), there was significant palatal growth (P ⬍ .05). At the beginning of the cultivation period, all groups started at the same developmental stage. The developmental stage of the 2 supplemented groups did not differ significantly after the incubation period (P ⫽ .50). A positive influence of the vitamin B-complex on palatal shelf development can be assumed. GD, gestational day; p.c., post conceptionem. Scheller, Orce y Tiggers, and Schubert. Vitamin B Accelerates Palatal Growth in Vitro. J Oral Maxillofac Surg 2013.
some critical points and potential sources of error must be considered.20 The time of sacrifice of the dam is very important, because it determines the initial developmental stage of the palatal shelves in the organ cultures. With respect to the predisposition for a generally delayed development in the A/WySnJ strain,10,28 the highest potency for any expected palatal fusion is 14 to 15 days after fertilization.28-30 Therefore, the palatal organ cultures were prepared 14 days 8 hours after fertilization. This period corresponds to the greatest potential for any possible palatal fusion.11,29 With respect to previous studies of palatal development and palatal fusion potential in the organ model, it must be mentioned that palatal development is generally delayed and the palatal fusion rate is decreased (13%) in this in vitro organ model16,17 compared with a palatal fusion rate of 65% to 50% in vivo.27,29 This may be caused by the traumatic proce-
dures during palatal organ preparation, resulting in a negative effect on palatal shelf development in the organ model. In the present investigation, the control group showed no significant palatal development during in vitro cultivation, which accorded exactly to previous results.16 In contrast, the 2 vitamin B groups (0.1% and 1.0%) showed a significant positive development of the palatal shelves during in vitro cultivation. The 0.1% and 1.0% supplementations showed no significant difference (P ⫽ .50). This effect seemed to be caused directly by the positive influence of the vitamin B-complex in the medium on secondary palatal development; however, its direct mechanism of action cannot yet be explained. In light of these findings, a good clinical option may be the intrauterine reparation of clefts during
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fetal development. An animal study by Schubert et al9 showed the potent reparative effect of B vitamins on dexamethasone-induced clefts in A/WySnJ mice when given to their dams after teratogenic dexamethasone application. In an animal experiment, Bienengräber et al13 described a clear reparative effect of folic acid (B9), indicated by a decrease in total procarbazine-induced palatal clefts, applied to persistent hard palate clefts after the teratogenic action. For normal secondary palatal development, growing from the hard palate anteriorly to the soft palate posteriorly, the fused soft palate in the presence of a hard palate cleft might be caused by reparation mechanisms in utero. Another in vitro palatal organ model study of the A/J strain showed a positive efficiency of folic acid on palatal shelf development.18 All in all, it is difficult to explain how far a teratogenic mechanism is similar to the relatively stable genetic mechanism that causes the CL/P appearance in the A/WySnJ strain.31,32 For an effective prevention of teratogenic-induced clefts, 1 mechanism to prevent facial clefting may be accelerated embryonic development.15,29 If there is any possibility to promote regular palatal shelf development in the A/WySnJ strain by the application of a pharmaceutical or over-the-counter drug in the late embryonic/early fetal period (GDs 12.5 to 14), there might be a chance for the possible reparation and prevention of CL/P in cases with a strong genetic tendency. The present in vitro study showed a significant influence of the direct application of the vitamin B-complex on palatal development in this special organ model. The inability to effectively process the vitamin B supplement by the A/WySnJ dams in vivo would explain the difference the lack of a decrease in cleft palate rates when given to the pregnant dams.8 This might result from drug and receptor interactions with a saturation or failure of binding capacity, resulting in a disruption to vitamin use or transport in the amniotic fluid. Unfortunately, there are no data on the vitamin B concentrations in the blood and amniotic fluid of this mouse strain that could explain the present findings. In conclusion, the developmental stage of the palatal organ at the time of vitamin B-complex supplementation is one of the most important factors in all experimental models in vivo and in vitro.17 From these findings, the authors conclude that vitamin B-complex supplements should be applied in the early developmental stage of the fetus for the clinical prevention of cleft palate. Further studies should analyze the different vitamin B concentrations in the blood and amniotic fluid of this mouse
strain and the receptor status for the different B vitamins.
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VITAMIN B ACCELERATES PALATAL GROWTH IN VITRO 29. Syska E, Schmidt R, Schubert J: The time of palatal fusion in mice: A factor of strain susceptibility to teratogens. J Craniomaxillofac Surg 32:2, 2004 30. Diewert VM, Wang KY: Recent advances in primary palate and midface morphogenesis research. Crit Rev Oral Biol Med 4:111, 1992 31. Shimizu N, Aoyama H, Hatakenaka N, et al: An in vitro screening system for characterizing the cleft palate-inducing potential of chemicals and underlying mechanisms. Reprod Toxicol 15:665, 2001 32. Datson GP: Advances in understanding mechanisms of toxicity and implications risk assessment. Reprod Toxicol 11: 388, 1997