Noggin Overexpression Impairs the Development of Muscles, Tendons, and Aponeurosis in Soft Palates by Disrupting BMP-Smad and Shh-Gli1 Signaling

The roles of bone morphogenetic protein (BMP) signaling in palatogenesis were well documented in the developing hard palate; however, little is known about how BMP signaling regulates the development of soft palate. In this study, we overexpressed Noggin transgene via Osr2-creKI allele to suppress BMP signaling in the developing soft palate. We found that BMP-Smad signaling was detected in the palatal muscles and surrounding mesenchyme. When BMP-Smad signaling was suppressed by the overexpressed Noggin, the soft palatal shelves were reduced in size with the hypoplastic muscles and the extroversive hypophosphatasia (HPP). The downregulated cell proliferation and survival in the Osr2-creKI;pMes-Noggin soft palates were suggested to result from the repressed Shh transcription and Gli1 activity, implicating that the BMP-Shh-Gli1 network played a similar role in soft palate development as in the hard palate. The downregulated Sox9, Tenascin-C (TnC), and Col1 expression in Osr2-creKI;pMes-Noggin soft palate indicated the impaired differentiation of the aponeurosis and tendons, which was suggested to result in the hypoplasia of palatal muscles. Intriguingly, in the Myf5-creKI;pMes-Noggin and the Myf5-creKI;Rosa26R-DTA soft palates, the hypoplastic or abrogated muscles affected little the fusion of soft palate. Although the Scx, Tnc, and Co1 transcription was significantly repressed in the tenogenic mesenchyme of the Myf5-creKI;pMes-Noggin soft palate, the Sox9 expression, and the Tnc and Col1 transcription in aponeurosis mesenchyme were almost unaffected. It implicated that the fusion of soft palate was controlled by the mesenchymal clues at the tensor veli palatini (TVP) and levator veli palatini (LVP) levels, but by the myogenic components at the palatopharyngeus (PLP) level.

Human levator veli palatini muscle: a novel source of mesenchymal stromal cells for use in the rehabilitation of patients with congenital craniofacial malformations

Background Bone reconstruction in congenital craniofacial differences, which affect about 2–3% of newborns, has long been the focus of intensive research in the field of bone tissue engineering. The possibility of using mesenchymal stromal cells in regenerative medicine protocols has opened a new field of investigation aimed at finding optimal sources of multipotent cells that can be isolated via non-invasive procedures. In this study, we analyzed whether levator veli palatini muscle fragments, which can be readily obtained in non-invasive manner during palatoplasty in cleft palate patients, represent a novel source of MSCs with osteogenic potential. Methods We obtained levator veli palatini muscle fragments (3–5 mm3), during surgical repair of cleft palate in 5 unrelated patients. Mesenchymal stromal cells were isolated from the muscle using a pre-plating technique and other standard practices. The multipotent nature of the isolated stromal cells was demonstrated via flow cytometry analysis and by induction along osteogenic, adipogenic, and chondrogenic differentiation pathways. To demonstrate the osteogenic potential of these cells in vivo, they were used to reconstruct a critical-sized full-thickness calvarial defect model in immunocompetent rats. Results Flow cytometry analysis showed that the isolated stromal cells were positive for mesenchymal stem cell antigens (CD29, CD44, CD73, CD90, and CD105) and negative for hematopoietic (CD34 and CD45) or endothelial cell markers (CD31). The cells successfully underwent osteogenic, chondrogenic, and adipogenic cell differentiation under appropriate cell culture conditions. Calvarial defects treated with CellCeram™ scaffolds seeded with the isolated levator veli palatini muscle cells showed greater bone healing compared to defects treated with acellular scaffolds. Conclusion Cells derived from levator veli palatini muscle have phenotypic characteristics similar to other mesenchymal stromal cells, both in vitro and in vivo. Our findings suggest that these cells may have clinical relevance in the surgical rehabilitation of patients with cleft palate and other craniofacial anomalies characterized by significant bone deficit.

Human levator veli palatini muscle: A novel source of mesenchymal stromal cells for use in the rehabilitation of patients with congenital craniofacial malformations

Background. Bone reconstruction in congenital craniofacial differences, which affect about 2-3% of newborns, has long been the focus of intensive research in the field of bone tissue engineering. The possibility of using mesenchymal stromal cells in regenerative medicine protocols has opened a new field of investigation aimed at finding optimal sources of multipotent cells that can be isolated via non-invasive procedures. In this study we analysed whether levator veli palatini muscle fragments, which can be readily obtained in non-invasive manner during palatoplasty in cleft palate p­­atients, represent a novel source of MSCs with osteogenic potential. Methods . We obtained levator veli palatini muscle fragments (3-5 mm 3 ), during surgical repair of cleft palate in 5 unrelated patients. Mesenchymal stromal cells were isolated from the muscle using a pre-plating technique and other standard practices. The multipotent nature of the isolated stromal cells was demonstrated via flow cytometry analysis and by induction along osteogenic, adipogenic and chondrogenic differentiation pathways. To demonstrate the osteogenic potential of these cells in vivo, they were used to reconstruct a critical-sized full-thickness calvarial defect model was in immunocompentent rats. Results. Flow cytometry analysis showed that the isolated stromal cells were positive for mesenchymal stem cell antigens (CD29, CD44, CD73, CD90 and CD105) and negative for hematopoietic (CD34 and CD45) or endothelial cell markers (CD31). The cells successfully underwent osteogenic, chondrogenic and adipogenic cell differentiation under appropriate cell culture conditions. Calvarial defects treated with CellCeram TM scaffolds seeded with the isolated levator veli palatini muscle cells showed greater bone healing compared to defects treated with acellular scaffolds. Conclusion. Cells derived from levator veli palatini muscle have phenotypic characteristics similar to other mesenchymal stromal cells, both in vitro and in vivo . Our findings suggest that these cells may have clinical relevance in the surgical rehabilitation of patients with cleft palate and other craniofacial anomalies characterized by significant bone deficit.

Evaluation of the Symmetry of the Levator Veli Palatini Muscle and Velopharyngeal Closure Among a Noncleft Adult Population

Purpose: The goal of this study is to determine the typical range of asymmetry between the length and thickness of the levator veli palatini muscle and to explore the impact of the observed asymmetry on velopharyngeal closure. A second objective is to report normative length and thickness of the levator veli palatini muscle among adults with typical velopharyngeal anatomy. Method: Magnetic resonance imaging (MRI) data and Amira 5.5 Visualization software were used to evaluate the levator veli palatini muscle among 89 participants with typical velopharyngeal anatomy. Flexible nasopharyngoscopy was used to determine the function of velopharyngeal closure among 39 of the 89 participants with typical velopharyngeal anatomy to examine the functional impact of observed asymmetry. Results: Matched paired t tests demonstrated a nonsignificant difference between the length and thickness of the right and left levator muscle. The mean difference between the right and left length of the levator muscle was 2.28 mm but ranged from 0.09 mm to 10.37 mm. In all cases where individuals displayed asymmetry in the levator muscle through MRI, there was no observed impact on the symmetry of velopharyngeal closure. Discussion: This study suggest that differences in the right and left levator veli palatini muscle are not significant among individuals without cleft palate. However, among individual cases where asymmetry was sizeable, there was no direct impact on the closure pattern. This may suggest there are multiple factors that contribute to asymmetrical velopharyngeal closure that are beyond the level of the levator veli palatini muscle.

Velopharyngeal Muscle Morphology in Children With Unrepaired Submucous Cleft Palate: An Imaging Study

Objective: To identify quantitative and qualitative differences in the velopharyngeal musculature and surrounding structures between children with submucous cleft palate (SMCP) and velopharyngeal insufficiency (VPI) and noncleft controls with normal anatomy and normal speech. Methods: Magnetic resonance imaging was used to evaluate the velopharyngeal mechanism in 20 children between 4 and 9 years of age; 5 with unrepaired SMCP and VPI. Quantitative and qualitative measures of the velum and levator veli palatini in participants with symptomatic SMCP were compared to noncleft controls with normal velopharyngeal anatomy and normal speech. Results: Analysis of covariance revealed that children with symptomatic SMCP demonstrated increased velar genu angle (15.6°, P = .004), decreased α angle (13.2°, P = .37), and longer (5.1 mm, P = .32) and thinner (4 mm, P = .005) levator veli palatini muscles compared to noncleft controls. Qualitative comparisons revealed discontinuity of the levator muscle through the velar midline and absence of a musculus uvulae in children with symptomatic SMCP compared to noncleft controls. Conclusions: The levator veli palatini muscle is longer, thinner, and discontinuous through the velar midline, and the musculus uvulae is absent in children with SMCP and VPI compared to noncleft controls. The overall velar configuration in children with SMCP and VPI is disadvantageous for achieving adequate velopharyngeal closure necessary for nonnasal speech compared to noncleft controls. These findings add to the body of literature documenting levator muscle, musculus uvulae, and velar and craniometric parameters in children with SMCP.

The Behaviors of the Levator Veli Palatine Muscle Between 2 Surgical Conditions in Dog: The Comparison of Furlow and Pushback Methods

Objective: This study was conducted to compare the velopharyngeal function among the beagle dogs which the levator veli palatini (LVP) muscles construction has been experimentally changed. Methods: Four groups of LVP muscle reconstruction were made (normal LVP, cut LVP, end-to-end sutured LVP, and overlapped-sutured LVP at the midline). Levator veli palatini contraction was induced by electrical stimulation or a hypercapnia condition to analyze the strength of the velopharyngeal closure using balloon with a blood pressure meter, and the electromyogram in those operated beagle dogs. Results: Under a hypercapnia condition, the velopharyngeal function did not differ significantly among the 4 groups in the terms of velopharyngeal pressure. The strongest closure was shown at the overlapped sutured LVP group by electrical stimulation. Conclusion: The reconstruction of overlapped sutured LVP showed the most effective closure. This study suggested that the palatoplasty should be conducted along the overlapped LVP (like Furlow method).

Human levator veli palatini muscle: A novel source of mesenchymal stem cells for use in the rehabilitation of patients with congenital craniofacial malformations

Background. Bone reconstruction in congenital craniofacial differences, which affect about 2-3% of newborns, has long been the focus of intensive research in the field of bone tissue engineering. The possibility of using mesenchymal stem cells in regenerative medicine protocols has opened a new field of investigation aimed at finding optimal sources of multipotent stem cells that can be isolated via non-invasive procedures. Here we analysed whether levator veli palatini muscle fragments, which can be readily obtained in non-invasive manner during surgical rehabilitation of cleft p­­atients during palatoplasty, represent a novel source of MSCs with osteogenic potential. Methods. We obtained levator veli palatini muscle fragments, in non-invasive procedure during surgical rehabilitation of 5 unrelated cleft palate patients (palatoplasty surgery). The levator veli palatini muscle fragments was used to obtain the mesenchymal cells using pre-plating technique in a clean rooms infrastructure and all procedures were performed at good practices of manipulation conditions. To prove that levator veli palatini muscle are mesenchymal stem cells they were induced to flow cytometry analysis and to differentiation into bone, cartilage, fat and muscle. To demonstrate the osteogenic potential of these cells in vivo a bilateral full thickness calvarial defect model was made in immunocompentent rats.Results. Flow cytometry analysis showed that the cells were positive for mesenchymal stem cell antigens (CD29, CD73, CD90), while negative for hematopoietic (CD45) or endothelial cell markers (CD31). Moreover, these cells were capable of undergoing chondrogenic, adipogenic, osteogenic and skeletal muscle cell differentiation under appropriate cell culture conditions characterizing them as mesenchymal stem cell. Defects treated with CellCeramTM scaffolds seeded with levator veli palatini muscle cells showed significantly greater bone healing compared to defects treated with acellular scaffolds. Conclusion. We have demonstrated that cells derived from levator veli palatini muscle have phenotypic characteristics similar to other mesenchymal stem cells, both in vitro and in vivo. Our findings suggest that these cells may have clinical relevance in the rehabilitation of patients with cleft palate and other craniofacial anomalies characterized by significant bone deficit.

Identifying Predictors of Levator Veli Palatini Muscle Contraction During Speech Using Dynamic Magnetic Resonance Imaging

Purpose The purpose of this study was to identify predictors of levator veli palatini (LVP) muscle shortening and maximum contraction velocity in adults with normal anatomy. Method Twenty-two Caucasian English-speaking adults with normal speech and resonance were recruited. Participants included 11 men and 11 women ( M = 22.8 years, SD = 4.1) with normal anatomy. Static magnetic resonance images were obtained using a three-dimensional static imaging protocol. Midsagittal and oblique coronal planes were established for visualization of the velum and LVP muscle at rest. Dynamic magnetic resonance images were obtained in the oblique coronal plane during production of “ansa.” Amira 6.0.1 Visualization and Volume Modeling Software and MATLAB were used to analyze images and calculate LVP shortening and maximum contraction velocity. Results Significant predictors ( p < .05) of maximum LVP shortening during velopharyngeal closure included mean extravelar length, LVP origin-to-origin distance, velar thickness, pharyngeal depth, and velopharyngeal ratio. Significant predictors ( p < .05) of maximum contraction velocity during velopharyngeal closure included mean extravelar length, intravelar length, LVP origin-to-origin distance, and velar thickness. Conclusions This study identified six velopharyngeal variables that predict LVP muscle function during real-time speech. These predictors should be considered among children and individuals with repaired cleft palate in future studies.

Contracted Extravelar Segments of the Levator Veli Palatini Muscle: A Magnetic Resonance Imaging Morphometric Study

Objectives: To provide detailed descriptions of contraction-induced morphometric changes in the extravelar segments of the levator veli palatini (LVP) muscle using 3-dimensional (3-D) magnetic resonance imaging (MRI). Design: Three-dimensional MRI data were acquired at rest and during “silent /i/” from 4 singers. During silent /i/, participants voluntarily sustained velar elevation while breathing orally for the entire scan time. Focusing on the extravelar segments, LVP length, angle of the muscle origin, and cross-sectional area (CSA), measurements were obtained and compared between tasks. Results: Three of the 4 participants exhibited the expected patterns of change following concentric contraction of the LVP muscle. Consistent changes from the resting to the contracted state included reductions in LVP length by 13.5% and angle of the muscle origin by 9.8%, as well as increases in CSAs by 22.1%, on average. Conclusions: This study presented high-resolution data of the LVP muscle behavior with the first in vivo 3-D measurements of the contracted LVP muscle, which can be useful for the validation of computational models that aim at describing biomechanical properties of the LVP muscle in future research. The active behavior of the extravelar LVP muscle also provides some insight on optimal LVP muscle geometry to consider during cleft palate repair.