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Organogenesis logoLink to Organogenesis
. 2011 Oct 1;7(4):242–254. doi: 10.4161/org.7.4.17926

Palatogenesis

Engineering, pathways and pathologies

Benjamin Levi 1, Samantha Brugman 1, Victor W Wong 1, Monica Grova 1, Michael T Longaker 1,2, Derrick C Wan 1,
PMCID: PMC3265826  PMID: 21964245

Abstract

Cleft palate represents the second most common birth defect and carries substantial physiologic and social challenges for affected patients, as they often require multiple surgical interventions during their lifetime. A number of genes have been identified to be associated with the cleft palate phenotype, but etiology in the majority of cases remains elusive. In order to better understand cleft palate and both surgical and potential tissue engineering approaches for repair, we have performed an in-depth literature review into cleft palate development in humans and mice, as well as into molecular pathways underlying these pathologic developments. We summarize the multitude of pathways underlying cleft palate development, with the transforming growth factor β superfamily being the most commonly studied. Furthermore, while the majority of cleft palate studies are performed using a mouse model, studies focusing on tissue engineering have also focused heavily on mouse models. A paucity of human randomized controlled studies exists for cleft palate repair, and so far, tissue engineering approaches are limited. In this review, we discuss the development of the palate, explain the basic science behind normal and pathologic palate development in humans as well as mouse models and elaborate on how these studies may lead to future advances in palatal tissue engineering and cleft palate treatments.

Key words: palatal development, cleft palate, Wnt signaling, Hedgehog signaling, palate culture, cleft palate surgery, osteogenesis

Introduction

Cleft lip and palate (CLP) represents the second most common birth defect, with an incidence ranging from 1 in 500 to about 1 in 2,500 births. This suggests that susceptibility genes likely differ between races.1 Furthermore, environmental risk factors have been identified, making it difficult to isolate a single cause behind CLP. When assessing all cases, annual treatment costs exceed $100 million in direct hospital costs alone in the United States.2 Taken together, whether one is referring to CLP or isolated cleft palate, these are common birth defects, and they represent an enormous biomedical burden. Affected children with CLP require their first operation as neonates to close the cleft lip; closure of the palate happens during a second operation at approximately 6–12 months of life. Following the initial closure of the cleft palate, these children face continuing challenges with speech, facial growth, dental occlusion and hearing. Affected children often undergo intensive therapy to establish normal speech patterns, but may require further surgery if unsuccessful. In addition to speech, children endure substantial dental problems that will require orthodontic intervention. Not infrequently, affected children have associated midface hypoplasia due to the restraining effect of palatal scarring on the growing maxilla and require orthognathic surgery later in life.

Development of the primary and secondary palate have been shown to be unique entities on a genetic and embryologic level.1,3 In the majority (70%) of cleft lip and palate, these deformities occur independent from other craniofacial abnormalities and are called “isolated, non-syndromic cleft lip and palate.” Though the large growth of genetics research has uncovered numerous pathways involved with syndromic cleft lip, we lack a solid genetic and molecular understanding of the cause for these isolated cleft cases.

Previous studies indicate that the pathogenesis of cleft palate is multifactorial and likely has both genetic and environmental factors. Much of our knowledge of craniofacial clefting arises from case studies of patients and selected animal models. A number of genes have been identified to be associated with the cleft palate phenotype, but the etiology of the majority of cases remains elusive. In this review, we discuss palatal development, explore the basic science behind normal and pathologic palate development and elaborate on how these findings may lead to future advances in the treatment of cleft palate.

Cleft Palate Development

The facial region of the mammalian embryo originates mainly from the frontonasal prominence (forehead, nose, philtrum and primary palate), the maxillomandibular prominence from the first branchial arch (maxilla, mandible, lateral upper lip and secondary palate) and the lateral nasal prominences. The intermaxillary segment forms when the two medial nasal prominences fuse together at the midline, giving rise to the philtrum of the lip, four incisor teeth and the primary palate of the adult. The secondary palate forms from outgrowths of the maxillary prominences called palatal shelves or palatine processes; these palatal shelves fuse at the midline (Fig. 1A and B). The definitive palate is formed following fusion of the primary and secondary palates at the incisive foramen and with the nasal septum above (Fig. 1C).

Figure 1.

Figure 1

Human palatal development. (A) Week six of human palatal development, with the secondary palate shown vertically on each side of the tongue and a gap between the secondary palate, nasal septum and primary palate. (B) After descent of the tongue, the secondary palatal shelves elevate and orient horizontally, allowing them to come in contact and begin fusing. (C) Fusion of the primary and secondary palate and the nasal septum separating the oropharynx from the nasopharynx. Figure modified from Dixon et al.1

The palate is limited anteriorly by the incisive foramen and extends posteriorly through the structures of the hard palate, soft palate and uvula. The structures anterior to the incisive foramen are collectively referred to as the pre-palatal structures or the primary palate (upper lip and alveolus). The structures posterior to the incisive foramen are called the secondary palate. As is the case for all branchial arches, the first arch that forms the palate contains mesodermal mesenchymal cells, ectoderm-derived neural crest cells, a cranial nerve (trigeminal) and a blood supply (maxillary artery).

Development of the human hard palate occurs between weeks 5 and 12 of gestation. The lateral palatine processes gradually grow toward the midline, fusing first anteriorly by week 8 and posteriorly as far back as the uvula by week 12. Development of the secondary palate occurs from the lateral palatine processes that grow vertically and obliquely on both sides of the developing tongue (Fig. 1A). As the tongue descends, the palatine processes swing upward into a more transverse orientation (Fig. 1B). Some instances of cleft palate can be caused by failure of descent of the tongue, as in the case of Pierre Robin Syndrome where children have an underdeveloped mandible and failure of tongue descent, leading to cleft palate formation and respiratory compromise.

In mice, there is a significant amount of similarity in palatal development (Fig. 2A–D). Mouse facial development begins at E9.5 (corresponding to week 4 gestation in humans) with the appearance of the frontonasal process, paired maxillary and mandibular processes (late week 4 in humans). These processes have a cranial neural crest mesenchymal core surrounded by ectoderm-derived epithelium.4 The upper lip develops from the ventral frotonasal process at E10. Subsequently, the nasal placodes invaginate, and the medial and lateral nasal processes form. Growth and apposition of the medial nasal processes with each other and with the maxillary process create the intermaxillary segment, which consists of upper lip, upper two incisors and the primary palate (week 4 in human).5 By E12.5, the primary palate and upper lip development is complete (week 7 in humans). Secondary palate development starts on E11.5 (early week 7 human gestation). From E12–E14, the palatal shelves enter their active growth phase (human gestation weeks 7–8), at which time the palatal shelves are positioned vertically between the cheeks and lateral to the elevated tongue. At E14.5–E15 (week 9 in humans), the palatal shelves elevate and reorient into a horizontal position above the tongue. Subsequently, the shelves oppose and adhere along their medial edge, creating a transient medial epithelial seam. The palatal shelves then fuse anteriorly with the primary palate at the incisive foramen and dorsally with the nasal septum. The medial epithelial seam disintegrates and fusion of the palatal shelf, primary palate and vomer epithelia allow separation of the oral and nasal cavities, which is necessary for simultaneous breathing and feeding. The hard palate forms from osteogenic differentiation of palatal shelf mesenchymal cells into osteoblasts. Palatal fusion is completed by E15.5, at which point mesenchymal condensation occurs, followed by the osteogenic differentiation of the palatal mesenchyme, leading to formation of the palatine bone in the secondary palate. By E16.5, secondary palate formation is complete (10 weeks in humans).

Figure 2.

Figure 2

Correlation between human and mouse palates. (A) Normal human upper lip, hard palate and soft palate. (B) Normal mouse upper lip, hard palate and soft palate. (C) Cleft of the secondary palate in human patient. (D) Clefting of secondary palate in transgenic mouse.

Abnormal Palate Development/Types of Cleft Palate

Clefts of the palate alone or associated with cleft lip may involve either the primary or secondary palate and frequently both (complete clefts). Those involving the primary palate are associated with clefts of the lip. Palatal clefts may also be unilateral or bilateral. Isolated clefts of the secondary palate (incomplete clefts) occur in the absence of defects in either the lip or the alveolar process (Fig. 2C and D). Because palatal fusion occurs in an anterior to posterior direction, clefts of the secondary palate may involve only the soft palate or both the soft and hard palates together.6 Clinically, clefting in the secondary palate extends anteriorly from the uvula to varying degrees, often involving the hard palate.7 In complete forms, the cleft can affect the entire secondary palate, reaching the incisive foramen, leaving the nasopharynx in direct communication with the oral cavity. The vomer can thus be seen as a midline structure extending from the base of the skull.

While complete and incomplete clefts of the palate may be readily apparent on physical exam, other, more subtle forms may also exist with variable import with regard to feeding, speech development and ear infections.8 The submucous cleft palate is defined by the classic triad of a bifid uvula, palatal muscle diastasis and a midline notch in the posterior edge of the bony palate.8,9 The muscle separation results in a bluish, two-layered mucosal bridge, the zona pellucida, while the midline notch results from an abnormal development of the posterior nasal spine. Though the majority of patients with submucous clefts remain asymptomatic, approximately 15% develop velopharyngeal insufficiency with hypernasal speech.9 This occurs as the velum is often too short and thin, resulting in limited mobility and easy fatigability. Eventual failure to properly obturate the pharyngeal space develops, particularly after patients undergo adenoidectomy.

All clefts involving the secondary palate (with or without associated cleft lip), including those of the submucous variety, demonstrate abnormal morphology with respect to the levator veli palatini and tensor veli palatini muscles. The levator veli palatini muscles originate from the petrous portion of the temporal bone and medial surface of the auditory tube and normally interdigitate within the central velum to form a sling suspending the soft palate from the base of the skull.10 The tensor veli palatini originates from the scaphoid fossa and pterygoid plate, coursing around the hamulus to form an aponeurosis in the anterior third of the soft palate.10 In patients with clefts of the secondary palate, these muscles aberrantly insert into the posterior edge of the hard palate forming Veau's cleft muscle.11 As the levator veli palatini is oriented sagittaly in an anterior-posterior direction, any attempt at surgical correction must anatomically restore the levator sling to a medial-lateral course for proper vector of pull.12,13

Molecular Genetics of Cleft Palate

Gaining a solid understanding of the molecular causes of cleft palate has been complicated due to the plethora of factors that can, when mutated, result in various forms of clefting. There has been a large number of studies on a variety of pathways addressing palatal development. We performed a literature search of PubMed as an attempt to break down this plethora of pathways investigated. The following search terms were used in combination to identify appropriate studies: Cleft palate, palatogenesis, gene signaling and animal studies. The search was limited to studies published in English from 2000–2011 and included both human and animal studies (Tables 1 and 2). Extracellular signaling factors were the most commonly studied, with the transforming growth factor beta (TGFβ) superfamily as the subject of the majority of these studies (Table 2). To assess animal models currently being used to study CLP, a literature search of PubMed was performed using the following search terms: Cleft palate, palatogenesis, repair, gene signaling and animal studies. The search was limited to studies published in English from 2000 to 2011. Studies were excluded if the full text was inaccessible, or if the animal model was not clearly identified in the methods section. Clearly, the majority of studies used mice as the primary animal model (Table 3). Most in vivo models are of two varieties: transgenic mice with cleft phenotype and a teratogen-induced cleft palate.

Table 1.

Genetic pathways involved in cleft lip and palate

Article No. PMID Genetic Pathway (wnt, shh, BMP) Evidence
1 12223417 Fgf8 1C
2 15572143 Fgf10 1C
3 21185284 Wnt 1E
4 19631205 BMP, FGF, wnt 1A, C, E
5 15102710 BMP 1A
6 16990542 Sumo1 3
7 17846996 TBX22/Sumo1 3
8 17089422 PVRL1 4
9 12520078 EGFR 1A
10 14728799 Msx homeobox 1A
11 17601559 Wnt 1E
12 14755462 Wnt 1E
13 11359935 TGFβ 1A
14 12019011 TGFβ 1A
15 16607638 Jag2-Notch1 6
16 18413325 Wnt 1E
17 14756664 BMP, Shh 1A, D
18 14645125 MES degeneration 4
19 15361870 PDGF 1B
20 15203181 GABA 6
21 14729481 Smad2-dependent Alk-5-driven by TGFβ3 1A, 2
22 16955138 Insig 1,2 cholesterol pathway 7
23 18161794 Nat2 8
24 15831593 CDH1/E-cadherin 4
25 12376112 TGF/β3 1A
26 10932185 Ryk (RTK) 8
27 17041601 IRF 2
28 18452349 GABRB3 6
29 17849453 GAD 67 6
30 12975342 Tgf βr2 1A
31 12807959 MSX1 1A
32 16284941 Meox-2 6
33 16998816 Wnt9b 1E
34 9215640 TGFb3 1A
35 15103710 GAD67 6
36 2416245 HA and CS 5
37 12219090 IRF6 2
38 18001154 Activin signaling 1A
39 16496313 Wnt3/Wnt9b 1E
40 11420059 Col11a1 5
41 18191119 Wnt11 and Fgfr1b 1C, 1E
42 11391199 Fgfr 1/2 1C
43 17377962 SATB2 3
44 15716346 Bmp 1A
45 15731757 TGFβR1/2 1A
46 10716617 Bmp 1A
47 10753521 TGFβ3 1A
48 12673280 Col11A2 5
49 15870292 Mn1 3
50 15721140 Notch/jagged2 6
51 16723652 MSX1 1A
52 10853828 Desmosomal DSC2 4
53 17376812 Snail, slug 6
54 15275855 TGFβ3 1A
55 17452626 TGfβ3/smad 1A, 2
56 17442359 Retinoic acid 6
57 16880535 Crk 4
58 15199404 Fgf/shh 1C, 1D
59 16168717 Shh 1D
60 17360555 Fgf 1C
61 17097601 Pax9/shh in TGFβ3 regulation 1D, 1A
62 15562585 Type II collagen 5
63 12203729 Sim2 in HA synthesis 5
64 10932188 PVRL1, nectin-1 4
65 10731669 p57Kip2, CDK inhib. 8
66 12490557 PDGFRalpha 1B
67 12854656 Folbp1, Shh 1A
68 15028671 Mnt 2
69 12068957 TGFβ3 1A
70 10742093 Msx1 1A
71 16109396 MES cells/Shh 1D
72 12651933 MSX1TGFβ3 1A
73 16280635 DHCR7, cholesterol 7
74 12812790 Foxf2 2
75 15888125 TGFβ/Smad 3/dishevelled 1A, 1E, 2
76 17693063 FGF/Spry2/Shh 1C, 1D
77 17941048 PDGF-C 1B
78 15543606 PDGFR-alpha 1B
79 16780827 Irf6/TGFβ 1A, 2
80 16141225 Shox2, Fgf 1C
81 17652350 PDS5B 6
82 12163415 Msx1/Bmp 1A
83 11165488 Fgf 1C
84 15939375 IRF6/Smad 2
85 11559848 TBX22 3
86 12412015 Tbx22 3
87 15118109 Tbx10 3
88 12165566 TTF-2 2
89 19092777 PDGF-C 1B
90 12815624 TGFβ3 1A
91 12627230 FGFR1 1C
92 18264099 PDGF 1B
93 17066417 PDGF-C/RA 1B, 6
94 18278815 IRF6 2
95 19401770 IRF6 2
96 12412014 IP 3/TGF 1A
97 19282774 IRF6 2
98 19036739 IRF6 2
99 18948418 TBx22 3
100 16405370 PTCH 1D
101 14729838 TBX22 3
102 18431835 TGFβ3 1A
103 18423521 TFAP2A 9
104 19281781 TGFβ1/3 1A
105 16641627 PVRL1/Jag2 4, 6
106 14630905 MSX1 1A
107 18836445 AP-2/IRF6 2
108 11559849 PVRL1 4
109 17868388 TBX22 3
110 19249007 BMP4 1A
111 12514106 Fgfr1 1C
112 16563169 IRF6, LHX6, LHX7 2
113 19341725 Hand2 1A
114 12571099 Hand2 1A
115 12163415 Msx1à BMP, shh 1A, 1D
116 15317890 IRF6 2
117 12374769 TBX22 3
118 20652317 IRF6 2
119 21295280 FAF1 6
120 21195053 Sprouty-2/FGF 1C
121 21061289 ATRA/BMP 1A
122 21058326 Chlorcyclizine, H1 antag, Wnt, FGF, BMP 1A, 1C,1E
123 21041365 ADAMTS9/20 9
124 21034733 BMP 1A
125 20967564 TGFB3 1A
126 20945347 TGFB3 1A
127 20940229 Fz 1E
128 20920724 FGF10 1C
129 20809987 TGFB3 1A
130 20727875 BMP/Noggin 1A
131 20616530 Twist1, Snai1 and Runx2 6
132 20506229 JARID2 9
133 20451169 RBM10 9
134 20424327 P63/IRF6 2
135 20424318 P63/IRF6 2
136 20196077 IRF6 2
137 20165883 RECK, MMP2/3/9 5
138 20133659 FGFR2 1C
139 20007998 PRDM16/TGFβ 1A
140 19934017 Msx1/Shh 1A, 1D
141 19782673 Shh 1D
142 19769959 TBX2/3 3
143 19676060 p63 3
144 19653318 Hoxa2/msx1/BMP 1A
145 19648291 Tbx22 3
146 19598129 Wnt 1E
147 19415673 TGFβ3 1A
148 19394325 Shh 1D
149 19388084 Smad2/3, RA 2, 6
150 19097194 Pkdcc 8
151 18948417 Wnt 1E
152 18816854 Gli3 2
153 18697225 TGFβ/twist 1A
154 18694570 TGFβ/Smad 1A, 2
155 18483623 Pax3/BMP 1A
156 18470539 Zfhx1a/TGFβ 1A
157 18437907 RA/BMP 6, 1A
158 17967447 TGFβ 1A
159 17927973 Menin, Pax3, Wnt 6, 1D
160 17586487 Tshz1 2
161 17520166 Eh (hairy ears) Hoxc5 9
162 17337617 MYH9 6
163 17293880 GSK3β glycogen 5
164 17028893 Tbx3/BMP4 3, 1A
165 17022082 CBFB 6
166 16761282 Runx3/TGFβ/BMP 1A
167 16751186 Fidgetin/ATPase, AKAP95 4
168 16546851 Sox9 6
169 16410076 atRA/TGFb/Smad 1A, 2
170 16330189 Msx/BMP 1A
171 16313756 BMP 1A
172 16199551 PHF8 2
173 16028273 FGF twist, snail/BMP ID1 1C, 1A, 6
174 16026545 TGFβ2 1A
175 15914589 twist, snail and E-cadherin 6, 4
176 15649471 Smad/TGFβ 1A, 2
177 15282310 Sall3 2
178 15175245 Osr2, Pax9, TGFβ3 1A
179 15162512 Fgf8 1C
180 14761683 PKA pathway 8
181 14702176 iNOS, BMP 1A
182 14691138 TGFβ 1A
183 14643678 Zfhep 6
184 12666199 Decorin, biglycan 5
185 12666195 Tbx22 3
186 12606284 GABA(a) β3 6
187 12388835 PKA 8
188 12204278 Tbx22 3
189 12203737 Titf2/foxe, TTF2 fork 2
190 12175701 CRE B, phosphotase 8
191 12086327 Lhx6 2
192 11969262 RA/BMP 1A, 6
193 11795938 EGFR 1A
194 11669453 TGFβ/RA 1A
195 11354513 CREB, cAMP 8
196 11330859 Twist, Fgfr 1C
197 10789828 Hoxa-2 1A
198 10669089 TSP-2/TGFβ 1A
199 21246652 Ephrins 8
200 20333300 GABA, GAD1, Viaat 6
201 20213699 Shh 1D
202 19235875 Fgfr2 1C
203 18176224 Fgfr2 1C
204 16712891 MAPK 8
205 15543606 PDGFRα 1B
206 12011971 TGFβ, RhoA 1A
207 11238884 DIg, SAP97 6

Table 2.

Summary of molecular pathways

Pathway Studied Number of Studies Percent of Studies
Extracellular signaling factors TGFβ superfamily: TGFα, TGFβ, EGFR, Msx1, BMP, Activns, Hand2 71 30.1%
Platelet derived growth factor: PDGF-C 8 3.4%
Fibroblast growth factor: FGF 21 8.9%
Sonic Hedgehog: Shh, PTCH, SMO, CBP 13 5.5%
WNT: Wnt, Dv, Fz 13 5.5%
Total: 126 53.4%
Transcription Factors FFAP2a, AP-2a, IR F6, FOXE1, TTF-2, GLI, SMAD1, Mnt, Phf8 31 13.1%
T-box Transcription Factors/Protein Modification TBX22, TBX1, TBX10, Mn1, SUMO, p63, SATB2 17 7.2%
Cell adhesion molecules PVRL1, Nectin, Integrin, E-cadherin, Desmosomal protein, Crk 10 4.2%
Extracellular Matrix Collagen, GAG, CSPG, HA, MMP 8 3.4%
Other Factors Jag, Notch, GABA, RA, Snai1, Snai2, Runx2, Meox, Pax1, Pax3, PDS5B, FAF, Sox9, MYH9, Zfhep, DIg 27 11.4%
Cholesterol Pathway Sterol, Insig 1, Insig 2 2 0.85%
Kinase Cascades NAT2, MAPK, CRE B, PKA, camp, Eph, RTK, p57Kip2 10 4.2%
Genes JARID2, ADAMTS9/20, TFAP2A, RBM10, Hoxc5 5 2.1%

Table 3.

Summary of animal models used for palate study

Animal Type % of studies
Mouse 156/166 = 94.0%
Chicken 4/166 = 2.4%
Zebrafish 3/166 = 1.8%
Rat 3/166 = 1.8%

Although advances have been made in identifying the genetic causes for some syndromic forms of cleft palate, etiology of the more common non-syndromic forms remains poorly characterized.1 Using various experimental approaches, researchers are now uncovering the molecules and cellular processes that can go awry in cases of palatal clefting.1 Exploring each pathway in detail is beyond the scope of this review; however, herein, we review those pathways most commonly found to be aberrant in the onset of cleft palate.

PDGF signaling.

Platelet-derived growth factor (PDGF) and its receptors (PDGFRα and -β) have specific roles in promoting tissue-tissue interactions to control cell migration, proliferation and survival during embryonic development.14 Deletion of Pdgfrα in the neural crest leads to defects in palatal fusion, nasal septation and abnormal development of several facial bones and cartilage structures in mouse models.14 Deletion of Pdgfa and Pdgfc also produce severe craniofacial phenotypes. Pdgfc-null neonates have a complete cleft of the secondary palate, accompanied by failure of the palatal bones to extend across the roof of the oronasal cavity. Compound deletions lead to more severe phenotypes, for example, Pdgfc/Pdgfa double-knockout embryos develop a cleft face with cranial bone defects.15

Wingless type (Wnt) protein signaling.

Wnt signaling regulates numerous developmental processes, including cell proliferation, differentiation and survival.16 Wnt signaling also plays an important role in the generation and migration of neural crest cells and the development and patterning of the embryonic face in various species.1719 Several members of the Wnt family, including ligands, receptors and co-receptors are expressed in the developing facial prominences.2022 Wnt pathway activity is specifically localized to facial epithelia and underlying mesenchyme in the lateral nasal, maxillary and mandibular prominences.17 The hypothesized role for Wnt activity in the facial prominences varies within different tissues. In neural crest mesenchyme, Wnts promote proliferation, thus promoting the growth of the maxillary prominences that come together to form the palate.17 In the facial epithelium, expression of multiple Wnts is essential for the fusion of facial prominences.2224 Onset of clefting is linked to disruptions in various Wnt genes,2527 and perturbations of the pathway produce mild to severe facial clefting in various animal models17,24 as well as in humans.28 For example, mutation of Wnt9b in mice leads to cleft lip and palate and the A/Wysn strain of mice, which have an insertional mutation near the Wnt9b locus, have an increased incidence of spontaneous cleft lip and palate.29 Furthermore, aberrant expression of the low density lipoprotein receptor-related protein 6, Lrp6, a Wnt pathway coreceptor, also results in cleft lip and palate.24

Our laboratory has studied cleft palates present in both the Indian Hedghehog-null mutant as well as the GSK-3β-null mutant. In both of these models, we noted an obvious cleft in the secondary hard palate, which we believe is caused by dysregulated Wnt and Hedgehog signaling. Previous studies have shown that increase in canonical Wnt30 or decrease in Hedgehog signaling31 result in inhibited ossification. These reports suggest that increased Wnt signaling, such as that observed in the GSK-3β-null embryo, act to inhibit the ossification program already in place.32

BMP signaling.

Bone Morphogenic Protein (BMP) signaling participates in the induction, formation, determination and migration of the cranial neural crest cells which give rise to most of the craniofacial structures. Subsequently, it is also important for patterning and formation of facial primordia. In mice, loss-of-function type I BMP receptor (Bmpr1a) mutations in the craniofacial primordia results in cleft lip and palate. Interestingly, deficiency of Bmp4 ligand alone resulted in cleft lip only.33 Furthermore, downstream targets within the BMP pathway also have a link to cleft palate. Mutations in the mouse homeobox gene Msx1 results in cleft palate, and this represents a potential model on which to study cleft palate development.34

TGFβ3.

TGFβ3 is a member of the TGFβ superfamily and is expressed by medial edge epithelial (MEE) cells just prior to fusion of the palatal shelves. TGFβ3 is required for palatal shelf fusion,35,36 as evidenced by homozygous null TGFβ3 newborns, which exhibit a cleft secondary palate.37 Furthermore, administration of anti-TGFβ3 antibodies prevents fusion of the palatal shelves.38 Data suggest the role for TGFβ3 in palatogenesis relates to regulation of the breakdown of epithelia that lie between the palatal shelves. In the TGFβ3-null mice, the palatal shelves appear to approximate and adhere, but the epithelial seam remains, thus preventing fusion. Further support for the role of TGFβ3 during palatal fusion comes from biochemical approaches.

FOXE1 (Forkhead box protein E1).

FOXE1 is a forkhead-containing transcription factor that is involved in embryonic pattern formation. The FOXE1 gene is expressed at the point of fusion between maxillary and nasal processes during palatogenesis.39 Positional cloning and candidate gene sequencing show a correlation between mutations in FOXE1 and the occurrence of cleft lip and palate.39 FOXE1 is expressed in the secondary palate epithelium of both mice40 and human embryos.41 Furthermore, mice with a null mutation in FOXE1 have cleft palates.42

Irf (Interferon regulatory factor).

Irf6 is a member of a large family of transcription factors that bind to specific DNA sequences and regulate gene expression. In mice, disruption of this gene results in clefting.43,44 In humans, mutations in IRF6 have been shown to cause Van Der Woude syndrome and popliteal ptyergium syndrome, two disorders that are characterized by the presence of a cleft palate.45 Furthermore, variations in IRF6 have been found to increase the risk for isolated cleft lip and palate.46 Irf6 mutant mice exhibit a hyper-proliferative epidermis that fails to undergo terminal differentiation, causing epithelial adhesions that occlude the oral cavity and result in cleft palate.43,44 Taken together, these data suggest Irf6 mutations may result in defective elevation of palatine shelves, secondary to inappropriate adhesions with oral epithelium.

VAX1.

VAX1 is a member of the Emx/Not gene family and encodes a transcriptional regulator with a DNA-binding homeobox domain. Single nucleotide polymorphisms in VAX1 have been found to be overrepresented in patients with cleft lip and palate, suggesting that variants in VAX1 itself may contribute to development of clefting (reviewed in Dixon et al. 2011). Mouse knockouts for Vax1 show cleft palates, and this gene is expressed widely in developing craniofacial structures.47 Therefore, variants in VAX1 are strong candidates in the etiopathogenesis of cleft lip and palate.

Teratogen-induced cleft palate.

Along with pathway manipulation, scientists have also exposed genetically susceptible mouse strains such as C57B/L in utero to teratogens such as phenytoin, corticosteroids and retinoic acid.48 Such manipulations are thought to create a cleft due to alterations in mucopolysaccharides and glycosaminoglycans in the developing mesenchyme.

Alternative models.

Though transgenic models offer significant insight, it is difficult to manipulate the development of these mice in utero. Thus, palate organ cultures represent another promising methodology that allows direct manipulation of the palatal mesenchyme. In vitro chick palate models have been previously reported, and our laboratory has utilized a similar in vitro model using palates from E13.5 mice (Fig. 3). In our model, palate cultures are maintained for up to 96 h, and fusion is seen as early as 72 h. This model allows us to assess the critical distance needed for palates to be separated before clefting occurs. In addition, effects of various protein ligands on palatal gene expression can be studied.48,49 The disadvantage, of course, is that in vitro conditions do not completely mimic in vivo correlates, and thus palate cultures are better for analyzing pathways than morphogenesis.

Figure 3.

Figure 3

Palate cultures. (A) Schematic of palate culture using 6-well plate and insert with 0.4 µM pores allowing cytokines but not cells to pass through. (B) Schematic of paired palatal shelves in palate culture. (C) H&E stain of palatal fusion after 72 h of 2 palatal shelves in culture while in contact with each other.

Clinical Implications and Potential Therapies

While research continues on the molecular genetics of cleft formation, surgery remains the mainstay for treatment of palatal defects. The first report of a cleft palate repair is attributed to LeMonnier, who incised the cleft edges and placed sutures leading to suppuration and then healing across the defect.6,50 Von Langenbeck later introduced the use of mucoperiosteal flaps to close clefts involving the hard palate (Fig. 4).9,50

Figure 4.

Figure 4

Von Langenbeck palatal repair. (A) Secondary cleft palate palate with dotted red lines demonstrating incisions. (B) Mucuoperiosteal flap elevation with orange demonstrating opening of the incisions. (C) Midline nasal closure of the defect with the nasal layer in orange. (D) Closure of the midline incision with the oral mucosa over the nasal layer closure.

A literature search of PubMed was performed to assess practice patterns of current surgical repair of cleft palates. The following search terms were used in combination to identify appropriate studies: Cleft palate, surgery, treatment and repair. The search for repair type was limited to studies published in English from 2000 to 2011 with a focus on patients within the age group 0–23 mo. Studies were excluded if the full text was inaccessible or surgical repair type was not clearly identified in the methods section. We found a wide variety of approaches used with a paucity of randomized controlled studies for any technique (Tables 4 and 5).

Table 4.

Procedure used for palate repair

Article No. PMID Repair Type Level Evidence
1 11879069 Furlow double opposing Z-plasty 2
2 18827646 Bardach two-flap 3
3 16816695 Bardach two-flap 4
4 19443049 Millard and two-flap 3
5 12172182 Two-flap 4
6 15145666 Furlow 4
7 18594410 Furlow w/vs. w/out islandization 3
8 16799309 Furlow 4
9 16154317 Vomer flap with push back 3
10 18991170 Secondary gingivoalveoloplasty 3
11 12733955 Furlow 4
12 12142649 Modified Langenbeck, Ernst, Veau and Axhausen technique 3
13 14697077 Wave-line technique 2
14 18177196 Square-flap method 4
15 14663803 Musculomucosal buccal flap 4
16 17105319 Mucoso-periosteal flap palatoplasty 4
17 17105319 Marginal musculo-mucosal flap 2
18 16876078 Modified z-plasty 5
19 16077326 Two-flap, V-Y, and dorrance with new adhesive 5
20 11711920 Von Langenbeck, Wardill-Kilner, 4
21 16929186 Furlow 3
22 10029477 Furlow 4
23 15988251 Veau-Wardill-Kilner 4
24 15213528 Furlow 4
25 19165004 Modified 2 flap 2
26 10626963 Push-back palatoplasty 5
27 10839414 Modified Furlow 3
28 15896890 Furlow 4
29 16681406 Vomerine flap and Von Langenbeck incisions 2
30 20842387 Vomer flap 2
31 20413269 Distraction osteogenesis (DO) vs. conventional orthognathic (CO) 2
32 20335871 Millard, Pfeifer, Afroze 1
33 20299247 CO vs. DO 2?
34 20227244 Collagen membrane 4
35 20163243 Collagen matrix w/rhBMP-2 2
36 20048619 No tensor transection, tensor transection alone and tensor tenopexy 3
37 19816334 Tennison, periosteoplasy, pushback 4
38 17652050 Von Langenbeck and Furlow 3
39 17440366 Wardill-Kilner vs. Kriens 2
40 17328643 Vomerine and Von Langenbeck 2
41 17105327 Von Langenbeck 1
42 15861051 Vomer, pushback, Von Langenbeck 3
43 14577817 Millard, Von Langenbeck 2
44 12498603 Modified Von Langenbeck 2
45 11141029 Millard, Von Langenbeck 2
46 12846601 Zurich, Von Langenbeck 2
47 12846598 Millard, Von Langenbeck 2

Level 1, Prospective multi-center double blinded randomized control study; Level 2, Prospective randomized control study; Level 3, Retrospective analysis, case control study or systematic review of studies; Level 4, Case series; Level 5, Expert opinion, case report or clinical example.

Table 5.

Summary of procedures used for palate repair

Level % of studies at that level
1 2/47 = 4.3%
2 16/47 = 34.0%
3 11/47 = 23.4%
4 15/47 = 31.9%
5 3/47 = 6.4%

Overall, the most widely used techniques include Von Langenbeck's, the Vaeu-Wardill-Kilner and the two-flap repair described by Bardach.6,51 While many modifications of each exist, the main principles across all cleft palate repairs include tension-free closure of the oral and nasal layers, dissection of muscles from the posterior edge of the hard palate and construction of a horizontally oriented palatal sling to restore normal velar function.51

Repair of the cleft palate begins with an incision along the cleft margin at the junction between oral and nasal mucosa. The incision is carried anteriorly along the gingiva, allowing elevation of mucoperiosteal flaps off the hard palate.10,51 With exposure of the greater palatine neurovascular bundle, mobilization can be performed by gentle stretching using scissors. The tendon of the tensor veli palatini can also be divided medial to the hamulus to facilitate medialization of the levator muscle.9 The nasal mucoperiosteum is then widely mobilized from the undersurface of the hard palate. Posteriorly, an intravelar veloplasty is typically performed, with separation of the oral, muscle and nasal linings and release of the muscles from their abnormal attachment to the posterior edge of the bony palate. Closure of the defect is performed in three layers (nasal mucosa, velar muscle and oral mucosa), with horizontal reorientation of the levator veli palatini establishing proper orientation of the sling. In the region of the hard palate, a two-layer repair is performed, with the nasal layer sometimes requiring a vomerine flap.6

In patients with either clefting of the soft palate or a submucous cleft, a Furlow palatoplasty can also be performed. Double opposing z-plasties are fashioned on the velum, with release of the levator muscle from the posterior edge of the hard palate.52 Transposition of the flaps yields retropositioning of the muscle to a more medial-lateral position.53 With this technique, simultaneous palatal lengthening and reconstruction of the levator sling is established along with additional narrowing of the nasopharyngeal aperture.11 Velopharyngeal competence allowing development of normal speech is one of the most critical outcomes in cleft surgery, and the Furlow technique has been associated with some of the lowest rates of persistent velopharyngeal insufficiency following primary repair.5255

While techniques for cleft palate repair have become well-established, postoperative development of oronasal fistulas still remains a significant problem. Reports have noted an incidence ranging from 11% to 23%, with the most likely site being the junction of the hard and soft palates.5659 Depending on size, fistulas may contribute to hypernasal speech, nasal regurgitation and food trapping. Several retrospective studies have identified the extent of cleft to be a significant factor, as patients with bilateral clefts were found to have a 2- to 3-fold higher incidence of postoperative fistula development compared with unilateral clefts.51,60 Operator experience has also been shown to play a role.58,60 Recent investigations have evaluated the utility of the buccal fat pad as adjunctive tissue for use in both primary palatal cleft repair and treatment of postoperative fistulas. Used in a pedicled fashion with overlying mucosa, the buccal fat pad has been shown to successfully treat wide oroantral and oronasal clefts.61,62 More recently, buccal fat has also been employed to cover laterally exposed bone adjacent to gingival mucosa following medialization of the mucoperiosteal flaps.63 As this was found to re-epithelialize within two weeks, use of the buccal fat pad may result in an eventual reduction of palatal scarring, which may limit subsequent growth restriction of the maxilla. And in similar fashion to AlloDerm, the buccal fat pad has also been shown to be effective as an interpositional layer between oral and nasal lining in the repair of postoperative fistulas.63 Fuimora et al. have even evaluated the utility of combining the two, with the successful treatment of oronasal fistulas using pedicled buccal fat covered with lyophilized dermis in adult patients.64

Palatal Tissue Engineering

The use of autogenous grafted material is now the standard of care, but tissue engineering is an attractive alternative that could greatly reduce the morbidity of surgery and potentially enhance the healing process. An exhaustive literature search only yielded 36 non-review studies discussing tissue engineering of the palate (Table 6). Of these 36 studies, 20 discussed engineering of the mucosa (Table 7). Regarding mucosal repair, cultured epithelial grafts, dermal substitutes and a combination of the two, called mucosa equivalents, are commonly used to provide extra tissue and aide in wound healing after cleft palate repair. Cultured epithelial grafts can provide coverage for large areas while being derived from only a small biopsy, but they are prone to infection and fail to reduce scarring or contraction in full-thickness wounds due to absence of a dermal component.65 Such grafts can be either allogenic or autologous. Allogenic grafts have the advantage of being readily available but have a low take rate and are generally only used for temporary coverage, while autologous grafts take require extra time to culture but have a higher take rate.

Table 6.

Recent studies of palatal tissue engineering

Article no. PMID Tissue Engineered Experimental model Review? Species
1 bone/mucosa mouse/rat/human/in vitro
2 21465220 bone ALL review ALL
3 21282641 mucosa mouse ALL
4 20863943 bone human ALL
5 20676486 bone rat ALL
6 20646804 bone in vitro (rat bone marrow) ALL
7 20524191 mucosa human ALL
8 20078790 mucosa ALL review ALL
9 19615638 bone human ALL
10 19473448 mucosa rat ALL
11 18834238 mucosa human Dog
12 19387150 bone in vitro Dog
13 19184287 bone rat Dog
14 19043190 bone human human
15 19018060 both human review human
16 18774917 both ALL review human
17 18650554 mucosa in vitro human
18 18520371 mucosa rat human
19 18427300 both ALL review human
20 18427291 both ALL review human
21 18319673 both ALL review human
22 18269029 mucosa rat human
23 18206405 bone human human
24 18093570 mucosa rat human
25 18041711 mucosa in vitro human
26 18022477 both ALL review human
27 17971350 mucosa Dog in vitro
28 17764402 mucosa Dog in vitro
29 17667684 bone human in vitro
30 17465023 mucosa in vitro in vitro
31 17393689 bone Dog in vitro
32 17346560 both ALL review in vitro
33 16525276 bone rat in vitro
34 15948680 mucosa human in vitro
35 15927149 bone human mouse
36 15894371 mucosa in vitro mouse
37 15348479 bone rat rat
38 14619596 mucosa rat rat
39 14513576 mucosa rat rat
40 12544232 bone mouse rat
41 12109699 mucosa in vitro rat
42 11896993 mucosa in vitro rat
43 11213985 mucosa human rat
44 11203575 bone human rat
45 10845305 both ALL review rat
46 9821920 mucosa human rat

Table 7.

Summary of tissues described in tissue engineering of the palate

Mucosa = 20/36 0.555 = 55.5%
Bone = 16/36 0.44 = 44%

Dermal substitutes made from polymers, purified collagen or de-epidermized dermis (DED or AlloDerm) provide additional physical support that is often lacking in epithelial grafts. However, some require a secondary procedure to apply a split-thickness skin graft or cultured epithelia. In recent years, repair of palatal fistulas have begun to employ acellularized dermal matrix (AlloDerm) with promising results. Using AlloDerm as an interpositional layer between nasal and oral mucoperiosteum has been shown to significantly reduce fistula recurrence rates in multiple series.66,67 Furthermore, recent studies have also demonstrated the utility of AlloDerm as an adjunctive measure in the primary repair of wide clefts. In a series of seven patients with palatal clefts wider than 15 mm, placement of AlloDerm between the muscle and oral lining was found to result in the development of no postoperative fistulas.68 Even in two patients with oral dehiscence and exposure of the AlloDerm, uneventful healing proceeded, with remucosalization occurring over a 4-week follow-up.68

Cultured mucosa equivalents provide an epithelial and dermal substitute in a one-step process, which seem to be the optimal replacement for mucosa, since this provides material for repair with properties closest to the original tissue. However, long-term evaluation of their clinical efficacy is still lacking. Tissue engineering using epithelial and mesenchymal stem cells could greatly enhance these options for repair once characterization and isolation of true stem cells can be routinely achieved.

The other tissue discussed in 20 of the 36 palatal tissue engineering articles was palatal bone (Table 7). In cleft palate repair, one of the major challenges lies in reconstructing the bony hard palatal and alveolar defects. Surgical repair with autogenous bone grafts is the current standard of care. Bone is most commonly harvested from the iliac crest but can be taken from the rib, tibia, calvarium or mandibular symphysis. This often requires multiple operations and extensive healing time and is associated with high donor site morbidity, including postoperative pain, altered sensation, scarring and infection. In addition, bone graft harvest ultimately yields a very limited quantity of bone for reconstruction. Furthermore, this bone often does not fully integrate into the host site and can undergo some resorption. Bony repair needs to be very strong to support tooth eruption and to withstand physical stress from muscles of mastication. There are also allogeneic and synthetic material available for grafting, and while these solve the problem of donor site morbidity, there is still the risk of infection, elicitation of an immune response and problems with structural integrity and contour.69

The use of tissue engineering could avoid many disadvantages of autogenous grafting, such as donor site morbidity, and could potentially decrease the number of surgeries needed while providing improved outcomes. Only a limited number of studies currently exist exploring palatal tissue engineering, and of these studies, only 36% pertain to human subjects, making it an attractive avenue for future research endeavors (Table 8).

Table 8.

Summary of animal models in studies discussing tissue engineering of the palate

Dog = 8.3%
Human = 36%
In Vitro = 22%
Mouse = 5.5%
Rat = 27.8%

Conclusion

Craniofacial clefts, specifically clefting of the lip and palate, remain a significant biomedical burden. Therefore, understanding normal palate development as well as aberrant pathways involved in abnormal palate development is crucial to allow us to better develop therapeutic modalities to treat these patients. Clearly, despite an improvement in surgical outcomes, there is a paucity of randomized controlled studies (level I data), leading surgeons to depend on clinical reports and non-randomized trials, which are often misleading. The current gold standard for treating cleft palate patients involves a team approach between plastic surgeons, pediatricians, otolaryngologists, speech pathologists, orthodontists and geneticists, underscoring the myriad of complications caused by CLP beyond just the tissue deficit. Thus, identifying the major pathways involved and manipulating those pathways prior to birth would represent a monumental step to prevent the primary and many secondary complications caused by CLP. Tissue engineering approaches also remain an exciting and potentially profitable direction of investigation as the field of regenerative medicine advances.

Acknowledgments

This study was supported by National Institutes of Health, National Institute of Dental and Craniofacial Research grant 1R21DE018727-01, the Oak Foundation and Hagey Laboratory for Pediatric Regenerative Medicine and the National Endowment for Plastic Surgery to M.T.L. B.L. was supported by the National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases grant 1F32AR057302.

Financial Disclosure

None of the authors has a financial interest in any of the products, devices or drugs mentioned in this manuscript.

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