Maternal folic acid supplementation reduces the severity of cleft palate in Tgf-ß3 null mutant mice.

BACKGROUND: Cleft palate (CP) constitutes the most frequently seen orofacial cleft and is often associated with low folate status. Folate plays an essential role in the human body as a major coenzyme in one-carbon metabolism, including DNA synthesis, repair, and methylation. Whether the administration of isolated folic acid (FA) supplements prevents the CP caused by genetic mutations is unknown, as is its effect on the mechanisms leading to palate fusion. METHODS: FA was administered to females from two different strains of transforming growth factor ß3 heterozygous mice. Null mutant progeny of these mice exhibit CP in 100% of cases of varying severity. We measured cleft length, height of palatal shelf adhesion, and the number of proliferating mesenchymal cells. Immunohistochemistry was also carried for collagen IV, laminin, fibronectin, cytokeratin-17, and EGF. RESULTS: FA supplementation significantly reduced CP severity and improved palatal shelf adhesion in both strains both in vivo and in vitro. Medial edge epithelium proliferation increased, and its differentiation was normalized as indicated by the presence and disposition of collagen IV, laminin, fibronectin, and cytokeratin-17. CONCLUSIONS: A maternal FA supplementation reduces the CP appearance by improving the mechanisms leading to palatal shelf adhesion.

Epidermal growth factor impairs palatal shelf adhesion and fusion in the Tgf-ß 3 null mutant.

The cleft palate presented by transforming growth factor-ß3 (Tgf-ß3) null mutant mice is caused by altered palatal shelf adhesion, cell proliferation, epithelial-to-mesenchymal transformation and cell death. The expression of epidermal growth factor (EGF), transforming growth factor-ß1 (Tgf-ß1) and muscle segment homeobox-1 (Msx-1) is modified in the palates of these knockout mice, and the cell proliferation defect is caused by the change in EGF expression. In this study, we aimed to determine whether this change in EGF expression has any effect on the other mechanisms altered in Tgf-ß3 knockout mouse palates. We tested the effect of inhibiting EGF activity in vitro in the knockout palates via the addition of Tyrphostin AG 1478. We also investigated possible interactions between EGF, Tgf-ß1 and Msx-1 in Tgf-ß3 null mouse palate cultures. The results show that the inhibition of EGF activity in Tgf-ß3 null mouse palate cultures improves palatal shelf adhesion and fusion, with a particular effect on cell death, and restores the normal distribution pattern of Msx-1 in the palatal mesenchyme. Inhibition of TGF-ß1 does not affect either EGF or Msx-1 expression.

Maternal folic acid-deficient diet causes congenital malformations in the mouse eye.

BACKGROUND: The eye is a very complex structure derived from the neural tube, surface ectoderm, and migratory mesenchyme from a neural crest origin. Because structures that evolve from the neural tube may be affected by a folate/folic acid (FA) deficiency, the aim of this work was to investigate whether a maternal folic acid-deficient diet may cause developmental alterations in the mouse eye. METHODS: Female C57BL/6J mice (8 weeks old) were assigned into two different folic acid groups for periods ranging between 2 and 16 weeks. Animals were killed at gestation day 17. Hepatic folate was analyzed, and the eyes from 287 fetuses were macroscopically studied, sectioned and immunolabeled with anti-transforming growth factor (TGF)-ß2 and anti-TGF-ßRII. RESULTS: Mice exposed to a FA-deficient diet exhibited numerous eye macroscopic anomalies, such as anophthalmia and microphthalmia. Microscopically, the eye was the most affected organ (43.7% of the fetuses). The highest incidence of malformations occurred from the 8th week onward. A statistically significant linear association between the number of maternal weeks on the FA-deficient diet and embryonic microscopic eye malformations was observed. The optic cup derivatives and structures forming the eye anterior segment showed severe abnormalities. In addition, TGF-ß2 and TGF-ßRII expression in the eye was also altered. CONCLUSION: This study suggests that an adequate folic acid/folate status plays a key role in the formation of ocular tissues and structures, whereas a vitamin deficiency is negatively associated with a normal eye development even after a short-term exposure.

Injection and adhesion palatoplasty: a preliminary study in a canine model.

BACKGROUND: Raising mucoperiosteal flaps in traditional palatoplasty impairs mid-facial growth. Hyaluronic acid-based hydrogels have been successfully tested for minimally invasive craniofacial bone generation in vivo as carriers of bone morphogenetic protein-2 (BMP-2). We aimed to develop a novel flapless technique for cleft palate repair by injecting a BMP-2 containing hydrogel. MATERIAL AND METHODS: Dog pups with congenital cleft palate were either non-treated (n=4) or treated with two-flap palatoplasty (n=6) or with the proposed injection/adhesion technique (n=5). The experimental approach was to inject a hyaluronic acid-based hydrogel containing hydroxyapatite and BMP-2 subperiosteally at the cleft palate margins of pups aged six weeks. At week ten, a thin strip of the medial edge mucosa was removed and the margins were closed directly. Occlusal photographs and computed tomography (CT) scans were obtained up to week 20. RESULTS: Four weeks after the gel injection the cleft palate margins had reached the midline and engineered bone had enlarged the palatal bones. Removal of the medial edge mucosa and suturing allowed complete closure of the cleft. Compared to traditional palatoplasty, the injection/adhesion technique was easier, and the post-surgical recovery was faster. CT on week 20 revealed some overlapping or "bending" of palatal shelves in the two-flap repair group, which was not observed in the experimental nor control groups. CONCLUSION: A minimally invasive technique for cleft palate repair upon injectable scaffolds in a dog model of congenital cleft palate is feasible. Results suggest better growth of palatal bones. This represents an attractive clinical alternative to traditional palatoplasty for cleft palate patients.

Occurrence of cleft-palate and alteration of Tgf-ß(3) expression and the mechanisms leading to palatal fusion in mice following dietary folic-acid deficiency.

Folic acid (FA) is essential for numerous bodily functions. Its decrease during pregnancy has been associated with an increased risk of congenital malformations in the progeny. The relationship between FA deficiency and the appearance of cleft palate (CP) is controversial, and little information exists on a possible effect of FA on palate development. We investigated the effect of a 2-8 weeks' induced FA deficiency in female mice on the development of CP in their progeny as well as the mechanisms leading to palatal fusion, i.e. cell proliferation, cell death, and palatal-shelf adhesion and fusion. We showed that an 8 weeks' maternal FA deficiency caused complete CP in the fetuses although a 2 weeks' maternal FA deficiency was enough to alter all the mechanisms analyzed. Since transforming growth factor-ß(3) (TGF-ß(3)) is crucial for palatal fusion and since most of the mechanisms impaired by FA deficiency were also observed in the palates of Tgf-ß(3)null mutant mice, we investigated the presence of TGF-ß(3) mRNA, its protein and phospho-SMAD2 in FA-deficient (FAD) mouse palates. Our results evidenced a large reduction in Tgf-ß(3) expression in palates of embryos of dams fed an FAD diet for 8 weeks; Tgf-ß(3) expression was less reduced in palates of embryos of dams fed an FAD diet for 2 weeks. Addition of TGF-ß(3) to palatal-shelf cultures of embryos of dams fed an FAD diet for 2 weeks normalized all the altered mechanisms. Thus, an insufficient folate status may be a risk factor for the development of CP in mice, and exogenous TGF-ß(3) compensates this deficit in vitro.

Tongue Abnormalities Are Associated to a Maternal Folic Acid Deficient Diet in Mice.

It is widely accepted that maternal folic acid (FA) deficiency during pregnancy is a risk factor for abnormal development. The tongue, with multiple genes working together in a coordinated cascade in time and place, has emerged as a target organ for testing the effect of FA during development. A FA-deficient (FAD) diet was administered to eight-week-old C57/BL/6J mouse females for 2-16 weeks. Pregnant dams were sacrificed at gestational day 17 (E17). The tongues and heads of 15 control and 210 experimental fetuses were studied. In the tongues, the maximum width, base width, height and area were compared with width, height and area of the head. All measurements decreased from 10% to 38% with increasing number of weeks on maternal FAD diet. Decreased head and tongue areas showed a harmonic reduction (Spearman nonparametric correlation, Rho = 0.802) with respect to weeks on a maternal FAD diet. Tongue congenital abnormalities showed a 10.9% prevalence, divided in aglossia (3.3%) and microglossia (7.6%), always accompanied by agnathia (5.6%) or micrognathia (5.2%). This is the first time that tongue alterations have been related experimentally to maternal FAD diet in mice. We propose that the tongue should be included in the list of FA-sensitive birth defect organs due to its relevance in several key food and nutrition processes.

Efficacy of supraspinatus tendon repair using mesenchymal stem cells along with a collagen I scaffold.

OBJECTIVES: Our main objective was to biologically improve rotator cuff healing in an elderly rat model using mesenchymal stem cells (MSCs) in combination with a collagen membrane and compared against other current techniques. METHODS: A chronic rotator cuff tear injury model was developed by unilaterally detaching the supraspinatus (SP) tendons of Sprague-Dawley rats. At 1 month postinjury, the tears were repaired using one of the following techniques: (a) classical surgery using sutures (n = 12), (b) type I collagen membranes (n = 15), and (c) type I collagen membranes + 1 × 106 allogeneic MSCs (n = 14). Lesion restoration was evaluated at 1, 2, and 3 months postinjury based on biomechanical criteria. Continuous variables were described using mean and standard deviation (SD). To analyse the effect of the different surgical treatments in the repaired tendons' biomechanical capabilities (maximum load, stiffness, and deformity), a two-way ANOVA model was used, introducing an interaction between such factor and time (1, 2, and 3 months postinjury). RESULTS: With regard to maximum load, we observed an almost significant interaction between treatment and time (F = 2.62, df = 4, p = 0.053). When we analysed how this biomechanical capability changed with time for each treatment, we observed that repair with OrthADAPT and MSCs was associated with a significant increase in maximum load (p = 0.04) between months 1 and 3. On the other hand, when we compared the different treatments among themselves at different time points, we observed that the repair with OrthADAPT and MSCs has associated with a significant higher maximum load, when compared with the use of suture, but only at 3 months (p = 0.014). With regard to stiffness and deformity, no significant interaction was observed (F = 1.68, df = 4, p = 0.18; F = 0.40, df = 4, p = 0.81; respectively). CONCLUSIONS: The implantation of MSCs along with a collagen I scaffold into surgically created tendon defects is safe and effective. MSCs improved the tendon's maximum load over time, indicating that MSCs could help facilitate the dynamic process of tendon repair.

Maxillary growth in a congenital cleft palate canine model for surgical research.

We have recently presented the Old Spanish Pointer dog, with a 15-20% spontaneous congenital cleft palate rate, as a unique experimental model of this disease. This study aimed to describe the cleft palate of these dogs for surgical research purposes and to determine whether congenital cleft palate influences maxillofacial growth. Seven newborn Old Spanish Pointer dogs of both sexes, comprising a cleft palate group (n = 4) and a normal palate group (n = 3), were fed using the same technique. Macroscopic photographs and plaster casts from the palate, lateral radiographs and computer tomograms of the skull were taken sequentially over 41 weeks, starting at week 5. The cleft morphology, the size and the tissue characteristics in these dogs resembled the human cleft better than current available animal models. During growth, the cleft width varies. Most of the transverse and longitudinal measures of the palate were statistically lower in the cleft palate group. The cleft palate group showed hypoplasia of the naso-maxillary complex. This model of congenital cleft palate seems suitable for surgical research purposes. A reduced maxillofacial pre- and post-natal development is associated to the congenital cleft palate in the Old Spanish Pointer dog.

A new technique for feeding dogs with a congenital cleft palate for surgical research.

In humans, cleft palate (CP) is one of the most common malformations. Although surgeons use palatoplasty to close CP defects in children, its consequences for subsequent facial growth have prompted investigations into other novel surgical alternatives. The animal models of CP used to evaluate new surgical treatments are frequently obtained by creating surgically induced clefts in adult dogs. This procedure has been ethically criticized due to its severity and questionable value as an animal model for human CP. Dogs born with a congenital CP would be much better for this purpose, provided they developed CP at a sufficient rate and could be fed. Up until now, feeding these pups carried the risk of aspiration pneumonia, while impeding normal suckling and chewing, and thus compromising orofacial growth. We developed a technique for feeding dog pups with CP from birth to the time of surgery using two old Spanish pointer dog pups bearing a complete CP. This dog strain develops CP in 15-20% of the offspring spontaneously. Custom-made feeding teats and palatal prostheses adapted to the pups' palates were made from thermoplastic plates. This feeding technique allowed lactation, eating and drinking in the pups with CP, with only sporadic rhinitis. To determine whether the use of this palatal prosthesis interferes with palatal growth, the palates of three littermate German shorthaired pointer pups without CP, either wearing or not wearing (controls) the prosthesis, were measured. The results showed that the permanent use of this prosthesis does not impede palatal growth in the pups.