Craniofacial and three-dimensional palatal analysis in cleft lip and palate patients treated in Spain.

Growth alterations have been described in patients operated on for oral clefts. The purpose of this work was to analyze the craniofacial and palate morphology and dimensions of young adults operated on for oral clefts in early childhood in Spain. Eighty-three patients from eight different hospitals were divided into four groups based on their type of cleft: cleft lip (CL, n = 6), unilateral cleft lip and palate (UCLP, n = 37), bilateral cleft lip and palate (BCLP, n = 16), and cleft palate only (CPO, n = 24). A control group was formed of 71 individuals. Three-dimensional (3D) digital models were obtained from all groups with an intraoral scanner, together with cephalometries and frontal, lateral, and submental facial photographs. Measurements were obtained and analyzed statistically. Our results showed craniofacial alterations in the BCLP, UCLP, and CPO groups with an influence on the palate, maxilla, and mandible and a direct impact on facial appearance. This effect was more severe in the BCLP group. Measurements in the CL group were similar to those in the control group. Cleft characteristics and cleft type seem to be the main determining factors of long-term craniofacial growth alterations in these patients. Prospective research is needed to clearly delineate the effects of different treatments on the craniofacial appearance of adult cleft patients.

Transforming growth factor ß3 deficiency promotes defective lipid metabolism and fibrosis in murine kidney.

Glomerulosclerosis and tubulointerstitial fibrosis are pathological features of chronic kidney disease. Transforming growth factor ß (TGFß) is a key player in the development of fibrosis. However, of the three known TGFß isoforms, only TGFß1 has an established role in fibrosis, and the pathophysiological relevance of TGFß2 and TGFß3 is unknown. Because Tgfb3 deficiency in mice results in early postnatal lethality, we analyzed the kidney phenotype of heterozygous Tgfb3-knockout mice (Tgfb3+/-) and compared it with that of matched wild-type mice. Four-month-old Tgfb3+/- mice exhibited incipient renal fibrosis with epithelial-mesenchymal transition, in addition to glomerular basement membrane thickening and podocyte foot process effacement associated with albuminuria. Also evident was insulin resistance and oxidative stress at the renal level, together with aberrant renal lipid metabolism and mitochondrial function. Omics analysis revealed toxic species, such as diacylglycerides and ceramides, and dysregulated mitochondrial metabolism in Tgfb3+/- mice. Kidneys of Tgfb3+/- mice showed morphological alterations of mitochondria and overactivation of non-canonical MAPK ERK1/2 and JNK cascades. Our study indicates that renal TGFß3 might have antifibrotic and renoprotective properties, opposing or counteracting the activity of TGFß1. This article has an associated First Person interview with the first author of the paper.

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.

Transforming Growth Factor-ß3 Regulates Adipocyte Number in Subcutaneous White Adipose Tissue.

White adipose tissue (WAT) mass is determined by adipocyte size and number. While adipocytes are continuously turned over, the mechanisms controlling fat cell number in WAT upon weight changes are unclear. Herein, prospective studies of human subcutaneous WAT demonstrate that weight gain increases both adipocyte size and number, but the latter remains unaltered after weight loss. Transcriptome analyses associate changes in adipocyte number with the expression of 79 genes. This gene set is enriched for growth factors, out of which one, transforming growth factor-ß3 (TGFß3), stimulates adipocyte progenitor proliferation, resulting in a higher number of cells undergoing differentiation in vitro. The relevance of these observations was corroborated in vivo where Tgfb3+/- mice, in comparison with wild-type littermates, display lower subcutaneous adipocyte progenitor proliferation, WAT hypertrophy, and glucose intolerance. TGFß3 is therefore a regulator of subcutaneous adipocyte number and may link WAT morphology to glucose metabolism.

Craniofacial structure alterations of foetuses from folic acid deficient pregnant mice.

INTRODUCTION: Craniofacial development in mammals is a complex process that involves a coordinated series of molecular and morphogenetic events. Folic acid (FA) deficiency has historically been associated with congenital spinal cord malformations, but the effect that a maternal diet deficient in FA has on the development of other structures has been poorly explored. In the present study, the objective was to describe and quantify the alterations of craniofacial structures presented in mouse foetuses from dams fed a FA deficient (FAD) diet compared with controls that were given a regular maternal diet. MATERIAL AND METHODS: E17 mouse foetuses were removed from dams that were fed with a control diet or with a FAD diet for several weeks. Foetuses with maternal FAD diets were selected for the study when they showed an altered tongue or mandible. Histological sections were used to quantify the dimensions of the head, tongue, mandibular bone and masseter muscle areas using ImageJ software. The muscles of the tongue, suprahyoid muscles, lingual septum, submandibular ducts, and lingual arteries were also analysed. RESULTS: The heads of malformed foetuses were smaller than the heads of the controls, and they showed different types of malformations: microglossia with micrognathia (some of which were combined with cleft palate) and aglossia with either micrognathia or agnathia. Lingual and suprahyoid muscles were affected in different forms and degrees. We also found alterations in the lingual arteries and in the ducts of the submandibular glands. Summarised we can state that pharyngeal arches-derived structures were affected, and the main malformations observed corroborate the vulnerability of cranial neural crest cells to FA deficiency. CONCLUSION: The present study reveals alterations in the development of craniofacial structures in FAD foetuses. This study provides a new focus for the role of FA during embryological development.

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.

Folic acid deficiency induces premature hearing loss through mechanisms involving cochlear oxidative stress and impairment of homocysteine metabolism.

Nutritional imbalance is emerging as a causative factor of hearing loss. Epidemiologic studies have linked hearing loss to elevated plasma total homocysteine (tHcy) and folate deficiency, and have shown that folate supplementation lowers tHcy levels potentially ameliorating age-related hearing loss. The purpose of this study was to address the impact of folate deficiency on hearing loss and to examine the underlying mechanisms. For this purpose, 2-mo-old C57BL/6J mice (Animalia Chordata Mus musculus) were randomly divided into 2 groups (n = 65 each) that were fed folate-deficient (FD) or standard diets for 8 wk. HPLC analysis demonstrated a 7-fold decline in serum folate and a 3-fold increase in tHcy levels. FD mice exhibited severe hearing loss measured by auditory brainstem recordings and TUNEL-positive-apoptotic cochlear cells. RT-quantitative PCR and Western blotting showed reduced levels of enzymes catalyzing homocysteine (Hcy) production and recycling, together with a 30% increase in protein homocysteinylation. Redox stress was demonstrated by decreased expression of catalase, glutathione peroxidase 4, and glutathione synthetase genes, increased levels of manganese superoxide dismutase, and NADPH oxidase-complex adaptor cytochrome b-245, α-polypeptide (p22phox) proteins, and elevated concentrations of glutathione species. Altogether, our findings demonstrate, for the first time, that the relationship between hyperhomocysteinemia induced by folate deficiency and premature hearing loss involves impairment of cochlear Hcy metabolism and associated oxidative stress.

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.

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.

The context of learning anatomy: does it make a difference?

This study set out to ascertain whether the context in which anatomy is learnt made a difference to students' perceptions of learning. An Approach to Learning Inventory (ASSIST) and a 31-item Anatomy Learning Experience Questionnaire (ALE) were administered to 224 students (77 dental, 132 medical and 19 speech and language) as a multi-site study. Results revealed that 45% adopted a strategic, 39% a deep and 14% a surface approach. Trends between professions are similar for a deep or strategic approach (both ~ 40%). However, a surface approach differed between professions (7% dentistry, 16% medicine, 26% speech and language science). Dental students responded more to being able to use their knowledge than did other groups (P = 0.0001). Medical students found the dissecting environment an intimidating one and subsequently reported finding online resources helpful (P = 0.015 and P = 0.003, respectively). Speech and language science students reported that they experienced greater difficulties with learning anatomy; they reported finding the amount to learn daunting (P = 0.007), struggled to remember what they did last semester (P = 0.032) and were not confident in their knowledge base (P = 0.0001). All students responded strongly to the statement 'I feel that working with cadaveric material is an important part of becoming a doctor/dentist/health care professional'. A strong response to this statement was associated with students adopting a deep approach (P = 0.0001). This study has elucidated that local curriculum factors are important in creating an enabling learning environment. There are also a number of generic issues that can be identified as being inherent in the learning of anatomy as a discipline and are experienced across courses, different student groups and institutions.

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.

Osteoinduction in the palatal submucosa by injecting BMP-2 on 2 different carriers.

In this work, we investigated the ability of injected recombinant human bone morphogenetic protein 2 (rhBMP-2) on brushite cement (a ß-tricalcium phosphate-based biomaterial) and collagen gel as carriers to induce osteogenic differentiation in the palatal submucosa of 10-day-old rats. This was part of a broader study aiming to create bone in the palatal submucosa at cleft palate edges in the search for a minimally invasive treatment. Thirteen treated animals, 7 with rhBMP-2/brushite cement and 6 with rhBMP-2/collagen gel, were injected with 5 to 10 µL of each biomaterial in the right palatal submucosa at the level between the second and third rugae. The contralateral site was uninjected and served as the control. Six weeks after injection, both brushite cement and collagen gel were histologically unrecognizable in all treated animals. New bone structures such as ossicles of woven bone were not detected. However, an augmentation in the thickness of the palatal fibromucosa was observed at the injection site of all palates. In addition, immunolabeling for osteopontin, proliferating cell nuclear antigen, and TUNEL revealed intense osteogenic induction at the injection site with both constructs, which was negative in the control site from the same specimens; no differences regarding cell proliferation and death were observed. The present study confirms the feasibility of generating osteogenic cells in the palatal submucosa by injecting low doses of rhBMP-2 in these 2 biomaterials, together with their inability to form bone.

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.

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.

Interactions between TGF-beta1 and TGF-beta3 and their role in medial edge epithelium cell death and palatal fusion in vitro.

In recent decades, studies have shown that both TGF-beta(1) and TGF-beta(3) play an important role in the induction of medial edge epithelium (MEE) cell death and palatal fusion. Many of these experiments involved the addition or blockage of one of these growth factors in wild-type (WT) mouse palate cultures, where both TGF-beta(1) and TGF-beta(3) are present. Few studies have addressed the existence of interactions between TGF-beta(1) and TGF-beta(3), which could modify their individual roles in MEE cell death during palatal fusion. We carried out several experiments to test this possibility, and to investigate how this could influence TGF-beta(1) and TGF-beta(3) actions on MEE cell death and palatal shelf fusion. We double-immunolabelled developing mouse palates with anti-TGF-beta(1) or anti-TGF-beta(3) antibodies and TUNEL, added rhTGF-beta(1) or rhTGF-beta(3) or blocked the TGF-beta(1) and TGF-beta(3) action at different concentrations to WT or Tgf-beta(3) null mutant palate cultures, performed in situ hybridizations with Tgf-beta(1) or Tgf-beta(3) riboprobes, and measured the presence of TUNEL-positive midline epithelial seam (MES) cells and MES disappearance (palatal shelf fusion) in the different in vitro conditions. By combining all these experiments, we demonstrate great interaction between TGF-beta(1) and TGF-beta(3) in the developing palate and confirm that TGF-beta(3) has a more active role in MES cell death than TGF-beta(1), although both are major inductors of MES disappearance. Finally, the co-localization of TGF-beta(1), but not TGF-beta(3), with TUNEL in the MES allows us to suggest a possible role for TGF-beta(1) in MES apoptotic clearance.

Immunohistochemical localization of fibrillin-1 protein in the cells of chick corneal and conjunctival epithelia during pre- and postnatal development.

Fibrillin-1 protein is a microfibrillar glycoprotein component of the extracellular matrix, widely distributed in ocular connective tissues. In this work, we show for the first time the expression pattern of fibrillin-1 protein in the corneal and conjunctival epithelia and in stromal keratocytes during embryo development. After hatching, protein expression was maintained in the corneal epithelium cells and nonsecreting epithelium cells of the conjunctiva and disappeared in the stromal keratocytes. In the limbus region, the basal cells were negative, while superficial cells were positive for the antibody. The expression in corneal epithelial cells suggests a role for fibrillin in development and disease. Therefore, some basal cells of the limbus region do not show fibrillin-1 immunolocalization, and this may be correlated with stem cell or stem-like properties.

Alteration of medial-edge epithelium cell adhesion in two Tgf-beta3 null mouse strains.

Although palatal shelf adhesion is a crucial event during palate development, little work has been carried out to determine which molecules are responsible for this process. Furthermore, whether altered palatal shelf adhesion causes the cleft palate presented by Tgf-beta3 null mutant mice has not yet been clarified. Here, we study the presence/distribution of some extracellular matrix and cell adhesion molecules at the time of the contact of palatal shelves in both wild-type and Tgf-beta3 null mutant palates of two strains of mice (C57/BL/6J (C57), and MF1) that develop cleft palates of different severity. We have performed immunohistochemistry with antibodies against collagens IV and IX, laminin, fibronectin, the alpha5- and beta1-integrins, and ICAM-1; in situ hybridization with a Nectin-1 riboprobe; and palatal shelf cultures treated or untreated with TGF-beta3 or neutralizing antibodies against fibronectin or the alpha5-integrin. Our results show the location of these molecules in the wild-type mouse medial edge epithelium (MEE) of both strains at the time of the contact of palatal shelves; the heavier (C57) and milder (MF1) alteration of their presence in the Tgf-beta3 null mutants; the importance of TGF-beta3 to restore their normal pattern of expression; and the crucial role of fibronectin and the alpha5-integrin in palatal shelf adhesion. We thus provide insight into the molecular bases of this important process and the cleft palate presented by Tgf-beta3 null mutant mice.

In vitro manipulation of cleft palate connective tissue: setting the bases of a proposed new treatment.

BACKGROUND: Palatoplasty has the undesired side effect of impaired mid-facial growth. To avoid this problem, we propose an alternative to palatoplasty. We hypothesize that if BMP-2 is injected together with a carrier into the periosteum of the cleft palate borders, border volume will increase and connective tissue cells will be activated to produce extra bone. Once these borders supported by bone reach the midline, extraction of their covering epithelia with trypsin will permit adhesion of the underlying tissues. We investigated in vitro the ability of cleft palate connective tissue cells to produce extra bone in the presence of BMP-2 and the possibility of using trypsin to remove the epithelium covering the cleft palate borders without impairing the underlying tissues' ability to adhere. MATERIALS AND METHODS: We used the cleft palate presented by tgf-beta(3) null mice and small fragments of human cleft palate mucoperiosteum as models. Immunolabeling BMP-2-treated or untreated cultures with TUNEL and anti-osteocalcin or PCNA antibodies was performed. The epithelium of the cleft palate borders was removed with a trypsin solution, and the de-epithelialized tissues were cultured in apposition. RESULTS: BMP-2 induces differentiation toward bone on cleft palate connective tissue cells without producing cell death or proliferation. Trypsin removal of the cleft palate margins' epithelium does not impair the underlying tissues' adhesion. CONCLUSION: It is possible to generate extra bone at the cleft palate margins and to chemically eliminate their covering epithelia without damaging the underlying tissues, which allows further investigation in vivo of this new approach for cleft palate closure.