Abstract
The pathogenesis of cleft lip and palate (CL/P) is studied in animal experiments. This study revealed significant differences in foetal secondary palate development in two strains of mice (NMRI, A/WySnJ) using a palatal organ model. Palatal shelves of 114 NMRI embryos, resistant to cleft occurrence, and 93 A/WySnJ embryos, a strain with a high spontaneous CL/P rate, were micro-dissected at 14.25 GD (gestational day), before palatal fusion takes place. After cultivation in serum-free medium, palatal development was investigated microscopically and scored in a six-step system. At death (14.25 GD) the palatal shelves of the NMRI embryos (mean 3.5) were significant more developed than those of A/WySnJ (mean 2.7; p = 0.05). After incubation, 53% (60/114) NMRI and 14% (13/93) A/WySnJ cultures had over two-thirds fusion to stage V–VI, therefore in 17% NMRI (19/114) and 1% A/WySnJ cultures (1/93) fusion was macroscopically complete. 62% of the A/WySnJ cultures showed no significant development in vitro (mean 2.84; p = 0.094). There is a significant palatal development difference between normally developed NMRI (mean 4.45, p = 0.05) and CL/P appearance in A/WySnJ mice (mean 2.84). Palatal development of both strains was significantly delayed in organ culture ( p = 0.05). The A/WySnJ strain was more susceptible to manipulation and vulnerable.
The physiological process of palatogenesis and secondary palatal development has been studied for more than 30 years . The mouse model is well-established . The development of the palatal shelves in the mouse during the early foetal period in organogenesis has been studied and this animal model allows the study of normal and cleft palatogenesis . The advantage of the mouse model is the easy transferability of the main features of palatogenesis and palatal development to human palatal development .
Normal secondary palatal development in the mouse begins on the twelfth day of gestation (12.5 GD) in the late embryonic/early foetal period (12.5–14 GD) . The palatal shelves are raised from the maxillary processes and consist mainly of mesenchymal tissue, derived from the cranioneural crest cells, and a simple layer of undifferentiated ectoderm . They initially grow vertically down the lateral aspect of the tongue . Then a downward movement of the mandible and tongue occurs (14 GD). After the initial reflex opening and closing movements of the mouth at 14 GD the palatal shelves re-orientate into a horizontal position . When the medial edges of the paired palatal shelves come into direct contact in the midline of the palate, they form a medial epithelial seam (partial fusion) . After epithelial breakdown in the contact zone of the palatal shelves and mesenchymal immigration at 15 GD fusion is complete. A cleft palate occurs when the bilateral palatal shelves fail to fuse in the midline interface . This development of the palatal shelves (palatogenesis) is mouse strain specific and may differ up to 1 day .
This study analysed the palatal development of two mouse strains (NMRI, A/WySnJ) in a palatal organ model (14.25 GD). The attributes of both strains are well known ( www.jax.org ). The A/WySnJ strain presents a high spontaneous cleft lip and palate (CL/P) rate of 38 to 50% whilst the NMRI strain has a low spontaneous palatal cleft incidence of 0.7–3.8% . These genetically different mouse strains were chosen for their palatal developmental behaviour and palatal fusion potential in organ culture.
Many aetiological, extrinsic and intrinsic factors are associated with CL/P appearance. The influence of extrinsic factors, such as the contact and approach to the palatal shelves at the time of death and the tissue culture preparation technique were minimized by using standardized preparation and cultivation protocols. The influence of intrinsic factors could therefore be analysed easily in this palatal organ model. The use of serum-free medium , an important fact for the standardization of the method, allowed improvement in the reproducibility of the results by avoiding the use of undefined serum supplements that can be different in different batches.
This in vitro study provided important landmarks for palatal development in the palatal organ culture system of two genetically different mouse strains. Future studies could analyse the direct influence of different substances on the growth of palatal shelves, cleft appearance, cleft prevention and the possibility of intra-uterine repair in this organ model.
Materials and methods
The A/WySnJ strain, an inbred strain of F268 generation has minimal genetic variance ( www.jax.org ) and develops cleft lip with or without cleft palate at a high spontaneous rate of 38–50% . The NMRI mice, an out-bred strain, were used to compare the variability of spontaneous CL/P. The NMRI have a spontaneous palatal cleft incidence of 1–4% with no genetic determination.
Female and male mice of the NMRI and A/WySnJ strains were kept in the laboratory for years under standardized housing conditions and separated by sex. The room temperature was held constant at 22 °C, the humidity at 65% and the lighting with a 12/12 h light/dark cycle had a luminosity of 70 lx. Food (standard pellet diets) and water were provided ad libitum . At 3–4 months the female mice were paired in the morning between 8 and 9.30 a.m. to determine an exact time of fertilization (±1.5 h) .
The occurrence of a vaginal plug was counted as day 0 of pregnancy. The pregnant mice were kept in groups of four animals per cage until they were killed by neck fracture (according to the Federal Office of Veterinary – Basel, directive 3.01) at the defined time of 14 days and 6 h after gestation (14.25 GD). Foetuses were delivered by laparatomy and preserved on ice in phosphate buffered saline until micro-dissection was performed.
Preparation of embryonic mice
After death (14.25 GD) the embryonic palate was prepared by micro-dissection. The palatal shelves were isolated with the surrounding tissue under direct visualization (Binocular Stemi 2000, Zeiss, Jena, Germany). The dissected tissue included the primary palate, the maxillary arch, the mandible and the supporting tissue. It was prepared from the dorso-cranial direction and cultured according to Abbott and Buckalew .
Organic culture
Only the palatal shelve pairs with the surrounding supporting tissue were seeded on 0.4 μm pore-sized filter inserts in 6-well plates (Nunc, Langenselbold, Germany). Dulbecco minimal essential medium/HAM’s F12 growth medium (1:1) supplemented with 300 μg/ml glutamate (Invitrogen, Karlsruhe, Germany) was changed every day. The tissue cultures were placed nasal side down on the filter inserts and incubated at 37 °C in a 5% CO 2 environment. The palatal shelves were cultured for exactly 72 h.
4 h after the initial placement of the paired palatal shelves in the culture plates, specimens were examined microscopically to detect any premature migration or change in orientation. The photographic documentation of the palatal shelves was obtained at the time of death (14.25 GD) and 72 h (17.25 GD) after cultivation.
Assessment of palatal fusion
After completing organic cell culture, all organ cultures were examined grossly for palatal shelf fusion under 8- and 20-fold magnification (Binocular Stemi 2000) and photo documented. Specimens were deemed to be grossly adherent or nonadherent using inverted microscopy. Adherent specimens lacked any discernible midline interface between ‘fusing’ palatal shelves, whilst nonadherent specimens demonstrated obvious gaps between the shelves.
The fixed (4% paraformaldehyde) organ cultures were prepared for histological light microscopy examination. After dehydration with graded concentrations of ethanol they were embedded in paraffin. Serial frontal sections (coronal) of 10 μm thickness were prepared along the cranio-caudal axis of the specimens and stained with haematoxylin and eosin ( Fig. 1 ). Palatal fusion was considered ‘complete’ when there was a mesenchymal continuity and disappearance of the medial epithelial seam over more than two-thirds of the fused palate. The palatal shelves were ‘partially’ closed (or epithelial) when the fused epithelial laminae of the shelves formed an unperforated septum ( Fig. 1 C) of more than two-thirds of the epithelial fusion along the midline.
Photo documentation and scoring system
Digital photo documentation (Dimage xt, Konica Minolta, Langenhagen, Germany) was carried out at 8-fold and 20-fold (Binocular Stemi 2000, Zeiss, Jena, Germany) magnification. For detailed palatal fusion determination the score system introduced by Al-Obaidi et al. was used ( Table 1 and Fig. 1 ).
Statistical evaluation
All documented scores (NMRI, A/WySnJ) were analysed statistically (SPSS, version 12) using the Wilcoxon signed-rank test and the Wilcoxon rank-sum test (Mann–Whitney U -test) . The palatal fusion scores at the two defined observation times (14.25 GD, 17.25 GD) were compared on a statistical significance level of α = 0.05 ranking the results at the time of dissection and after incubation mouse-strain specific.
Results
The in vitro embryonic palatal development of 114 NMRI mice embryos, originating from 17 mothers (mean 6.7 embryos/mother), and 93 A/WySnJ mice from 30 mothers (mean 3.1 embryos/mother) was analysed in a palatal organ model.
At the time of death (14.25 GD), 3 of 114 palatal shelves of the NMRI strain were closed completely (score VI, 3%). The mean score for palatal development in the NMRI group at 14.25 GD was 3.51 (median 4). After in vitro cultivation (72 h), fusion score I was not seen anymore (0.0%) and 19/114 specimens (17%) showed macroscopically totally fused palatal shelves ( Table 2 and Fig. 2 a ) with 14/19 (74%) histological ‘completely’ fused palatal organs. 32% (36/114) NMRI tissue cultures did not show any change in palatal development ( Fig. 3 ), whilst 43/114 showed an upgrade of 1 score point (38%), 19/114 an upgrade of 2 score points (17%), 7/114 an upgrade of 3 score points (6%) and 2/114 an upgrade of 4 score points (2%) during the in vitro cultivation ( Fig. 3 ). A positive development was seen in about 62% of the NMRI organ cultures. A regressive development of 5%, resulting in a downgrade of 1 score point was seen in only 6/114 tissue cultures (5%) and only one tissue culture (1/114) showed a downgrade of 2 score points ( Fig. 3 ). A positive development was seen in 62%. The Wilcoxon test revealed a significant palatal development ( p = 0.05) in the organ culture of the NMRI strain ( Figs. 2 and 3 ) to a mean palatal fusion score of 4.45 (median 5).
| Score | Palatal development of NMRI and A/WySnJ organ culture | |||
|---|---|---|---|---|
| 14 days + 6 h NMRI ( n = 114) |
72 h incubation NMRI ( n = 114) |
14 days + 6 h A/WySnJ ( n = 93) |
72 h incubation A/WySnJ ( n = 93) |
|
| I | 6 (5.3%) | 0 (0%) | 11 (11.8%) | 9 (9.7%) |
| II | 18 (15.8%) | 11 (9.6%) | 37 (39.8%) | 41 (44.1%) |
| III | 24 (21.1%) | 9 (7.9%) | 24 (19.4%) | 15 (16.1%) |
| IV | 47 (41.2%) | 34 (30%) | 18 (19.4%) | 15 (16.1%) |
| V | 16 (14%) | 41 (36%) | 5 (5.4%) | 10 (10.8%) |
| VI | 3 (2.6%) | 19 (16.7%) | 1 (1.1%) | 3 (3.2%) |
| VI partial | 5/19 (26.3%) | 3/3 (100%) | ||
| VI complete | 14/19 (73.7%) | 0/3 (0%) | ||
| Mean | 3.51 | 4.45 | 2.7 | 2.84 |
| Median | 4 | 5 | 2 | 2 |
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