Dataset Information

Midpalatal Suture: Single-Cell RNA-Seq Reveals Intramembrane Ossification and Piezo2 Chondrogenic Mesenchymal Cell Involvement.

ABSTRACT: The midpalatal suture is mainly responsible for the growth and development of the maxillary and resistance to rapid maxillary expansion (RME). It is essential for clinical researchers to explore the intramembrane ossification and to elucidate the underlying mechanism of the maturation and ossification process of the midpalatal suture to help identify the optimum time and force of RME. However, mechanistic studies associated with the midpalatal suture are rare. The aim of this present study is to create an intramembrane osteogenesis model for the midpalatal suture region of mice. Interestingly, we discovered a type of chondrogenic mesenchymal cell expressing Piezo2, which might be related to the detection of mechanical and external stimuli. This result provides a potential molecular and cellular mechanism that explains why the midpalatal suture is not closed until adulthood. We depict a landscape of mesenchymal cells that might play an important role in the intramembrane osteogenesis of the midpalatal suture and provide new perspectives on midpalate suture maturation and ossification, which might lead to further possibilities for clinical operations.

SUBMITTER: Gao L

PROVIDER: S-EPMC9688242 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Midpalatal Suture: Single-Cell RNA-Seq Reveals Intramembrane Ossification and <i>Piezo2</i> Chondrogenic Mesenchymal Cell Involvement.

Gao Lu L Xu Tiansong T Zhang Liqi L Li Yuchen Y Yan Tianxing T Yu Guoxia G Chen Feng F

Cells 20221112 22

The midpalatal suture is mainly responsible for the growth and development of the maxillary and resistance to rapid maxillary expansion (RME). It is essential for clinical researchers to explore the intramembrane ossification and to elucidate the underlying mechanism of the maturation and ossification process of the midpalatal suture to help identify the optimum time and force of RME. However, mechanistic studies associated with the midpalatal suture are rare. The aim of this present study is to ...[more]

PMID: 36429014

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Project description:MLL4, also known as KMT2D, is a histone methyltransferase that acts as an important epigenetic regulator in various organogenesis programs. Mutations in the MLL4 gene are the major cause of Kabuki syndrome, a human developmental disorder that involves craniofacial birth defects, including anomalies in the palate. This study aimed to investigate the role of MLL4 and the underlying mechanisms in the development and growth of the palate. We generated a novel conditional knockout (cKO) mouse model with tissue-specific deletion of Mll4 in the palatal mesenchyme. Using micro-computed tomography (CT), histological analysis, cell mechanism assays, and gene expression profiling, we examined palate development and growth in the Mll4-cKO mice. Gross craniofacial examination at adult stages revealed mild midfacial hypoplasia and midline defects of the palate in Mll4-cKO mice, including a widened midpalatal suture and disrupted midline rugae pattern. Micro-CT-based time-course skeletal analysis during postnatal palatogenesis through adulthood demonstrated a transverse growth deficit in overall palate width in Mll4-cKO mice. Whole-mount and histological staining at perinatal stages identified that the midline defects in the Mll4-cKO mice emerged as early as one day prior to birth, presenting as a widened midpalatal suture, accompanied by increased cell apoptosis in the suture mesenchyme. Genome-wide mRNA expression analysis of the midpalatal suture tissue revealed that MLL4 is essential for the timely expression of major cartilage development genes, such as Col2a1 and Acan, at birth.Immunofluorescence staining for osteochondral differentiation markers demonstrated a marked decrease in the chondrogenic marker COL2A1, while the expression of the osteogenic marker RUNX2 remained unchanged, in the Mll4-cKO midpalatal suture. Additionally, SOX9, a master regulator of chondrogenesis, exhibited a significant decrease in protein expression. Indeed, time-course histological analysis during postnatal palate growth revealed retardation in the development of the suture cartilage in Mll4-cKO mice. Taken together, our results demonstrate that MLL4 is essential for orchestrating key cellular and molecular events that ensure proper midpalatal suture development and palate growth.

Project description:MLL4, also known as KMT2D, is a histone methyltransferase that acts as an important epigenetic regulator in various organogenesis programs. Mutations in the MLL4 gene are the major cause of Kabuki syndrome, a human developmental disorder that involves craniofacial birth defects, including anomalies in the palate. This study aimed to investigate the role of MLL4 and the underlying mechanisms in the development and growth of the palate. We generated a novel conditional knockout (cKO) mouse model with tissue-specific deletion of Mll4 in the palatal mesenchyme. Using micro-computed tomography (CT), histological analysis, cell mechanism assays, and gene expression profiling, we examined palate development and growth in the Mll4-cKO mice. Gross craniofacial examination at adult stages revealed mild midfacial hypoplasia and midline defects of the palate in Mll4-cKO mice, including a widened midpalatal suture and disrupted midline rugae pattern. Micro-CT-based time-course skeletal analysis during postnatal palatogenesis through adulthood demonstrated a transverse growth deficit in overall palate width in Mll4-cKO mice. Whole-mount and histological staining at perinatal stages identified that the midline defects in the Mll4-cKO mice emerged as early as 1 day prior to birth, presenting as a widened midpalatal suture, accompanied by increased cell apoptosis in the suture mesenchyme. Genome-wide mRNA expression analysis of the midpalatal suture tissue revealed that MLL4 is essential for the timely expression of major cartilage development genes, such as Col2a1 and Acan, at birth. Immunofluorescence staining for osteochondral differentiation markers demonstrated a marked decrease in the chondrogenic marker COL2A1, while the expression of the osteogenic marker RUNX2 remained unchanged, in the Mll4-cKO midpalatal suture. Additionally, SOX9, a master regulator of chondrogenesis, exhibited a significant decrease in protein expression. Indeed, time-course histological analysis during postnatal palate growth revealed retardation in the development of the suture cartilage in Mll4-cKO mice. Taken together, our results demonstrate that MLL4 is essential for orchestrating key cellular and molecular events that ensure proper midpalatal suture development and palate growth.

Project description:BackgroundBone morphogenetic protein 2 (BMP2) is a promising chondrogenic growth factor for cartilage tissue-engineering, but it also induces robust endochondral ossification. Human synovial-derived mesenchymal stromal cells (hSMSCs) have attracted great interest due to their poor potential for differentiation into osteogenic lineages. Smad7 plays a significant in the endochondral ossification. In this study, we explored a new method to amplify the BMP2-induced chondrogenic differentiation of hSMSCs by downregulating Smad7 and applying a cellular scaffold.MethodshSMSCs were isolated from human knee joint synovium from 3 donors through adhesion growth. In vitro and in vivo models of the chondrogenic differentiation of hSMSCs were established. Transgenic expression of BMP2 and silencing of Smad7 and Smad7 was achieved by adenoviral vectors. The osteogenic differentiation was detected by alkaline phosphatase staining, alizarin red staining, and RT-PCR analysis of the osteogenic genes RUNX2, Osterix, and Osteocalcin. The chondrogenic differentiation was detected by Alcian blue staining and RT-PCR analysis of the chondrogenic genes SOX9, COL2, and aggrecan. Hypertrophic differentiation was detected by the markers COL10 and MMP13. A subcutaneous stem cell implantation model was established with polyethylene glycol citrate-co-N-isopropylacrylamide (PPCN) scaffolds and athymic nude mice (3/group, 4-6 week-old female) and evaluated by micro-CT, H&E staining, and Alcian blue staining. An immunohistochemistry assay was used to detected COL1 and COL2, and an immunofluorescence assay was used to detect COL10 and MMP13.ResultsThese hSMSCs identified by flow cytometry. These hSMSCs exhibited lower osteo-differentiation potential than iMads and C3H10T1/2-cells. When Smad7 was silenced in BMP2-induced hSMSCs, the chondrogenic differentiation genes SOX9, COL2, and aggrecan were enhanced in vitro. Additionally, it silencing Smad7 led to a decrease in the hypertrophic differentiation genes COL10 and MMP13. In subcutaneous stem cell implantation assays, immunofluorescence and immunohistochemical staining demonstrated that silencing Smad7 increased the number of COL2-positive cells and decreased the expression of COL1, COL10, and MMP13.ConclusionThis study suggests that the application of hSMSCs, cell scaffolds, and silencing Smad7 can potentiate BMP2-induced chondrogenic differentiation and inhibit endochondral ossification. Thus, inhibiting the expression of Smad7 in BMP2-induced hSMSC differentiation may be a new strategy for cartilage tissue-engineering.

Dataset Information

Midpalatal Suture: Single-Cell RNA-Seq Reveals Intramembrane Ossification and Piezo2 Chondrogenic Mesenchymal Cell Involvement.

Publications

Midpalatal Suture: Single-Cell RNA-Seq Reveals Intramembrane Ossification and <i>Piezo2</i> Chondrogenic Mesenchymal Cell Involvement.

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