Regulatory role of primary cilia in oral and maxillofacial development and disease.

Primary cilia have versatile functions, such as receiving signals from the extracellular microenvironment, mediating signaling transduction, and transporting ciliary substances, in tissue and organ development and clinical disease pathogenesis. During early development (embryos within 10 weeks) in the oral and maxillofacial region, defects in the structure and function of primary cilia can result in severe craniofacial malformations. For example, mice with mutations in the cilia-related genes Kif3a and IFT88 exhibit midline expansion and cleft lip/palate, which occur due to abnormalities in the fusion of the single frontonasal prominence and maxillary prominences. In the subsequent development of the oral and maxillofacial region, we discussed the regulatory role of primary cilia in the development of the maxilla, mandible, Meckel cartilage, condylar cartilage, lip, tongue, and tooth, among others. Moreover, primary cilia are promising regulators in some oral and maxillofacial diseases, such as tumors and malocclusion. We also summarize the regulatory mechanisms of primary cilia in oral and maxillofacial development and related diseases, including their role in various signaling transduction pathways. For example, aplasia of submandibular glands in the Kif3a mutant mice is associated with a decrease in SHH signaling within the glands. This review summarizes the similarities and specificities of the role of primary cilia in tissue and organ development and disease progression in the oral and maxillofacial region, which is expected to contribute several ideas for the treatment of primary cilia-related diseases.

Intraflagellar transport protein 88 interacts with polycystin 2 to regulate mechanosensitive hedgehog signaling in mandibular condylar chondrocytes.

OBJECTIVE: This study aimed to explore whether intraflagellar transport protein 88 (IFT88) was associated with polycystin 2 during mechanotransduction of mandibular condylar chondrocytes. METHODS: Rat mandibular condylar chondrocytes isolated from the condylar bone-cartilage junction were subjected to cyclic tensile strain (0.1 Hz, 10% elongation). Overexpression of IFT88 was achieved by lentiviral vector-mediated transfection. Knockdown of IFT88 and polycystin 2 was achieved by small interfering RNA (siRNA). The prevalence and length of cilia were reflected by immunofluorescence staining. The activities of hedgehog signaling were evaluated by western blot analysis. The interaction between polycystin 2 and IFT88 was evaluated by conducting a co-immunoprecipitation (co-IP) assay. RESULTS: Overexpression of IFT88 increased the length of cilia. Protein levels of polycystin 2, Indian hedgehog (Ihh), Patched 1 (Ptch1), Smoothened (Smo), and Glioma-associated oncogene homolog 1 (Gli1) were elevated in IFT88-overexpressing mandibular condylar chondrocytes under cyclic tensile strain. Knockdown of the protein level of IFT88 reduced the prevalence and length of cilia, and protein levels of polycystin 2, Ihh, Ptch1, Smo, and Gli1. A co-IP assay showed that IFT88 formed a complex with polycystin 2 under cyclic tensile strain. Knockdown of polycystin 2 decreased the protein levels of IFT88, Ihh, Ptch1, Smo, and Gli1 in mandibular condylar chondrocytes following cyclic tensile strain. CONCLUSION: These findings highlight the vital role of an interaction between IFT88 and polycystin 2 in mechanosensitive hedgehog signaling in mandibular condylar chondrocytes following cyclic tensile strain, which suggest that therapies regulating polycystin 2 may be considered for the disorders of temporomandibular joints.

Piezo1 and IFT88 synergistically regulate mandibular condylar chondrocyte differentiation under cyclic tensile strain.

OBJECTIVE(S): Mandibular condyle chondrocytes (MCCs) are exposed to various mechanical environments. Primary cilia, as a carrier for ion channels, can sense mechanical signals. Intraflagellar transport protein 88 (IFT88) is crucial for the assembly and function of primary cilia. Piezo1 is a mechanically activated ion channel that mediates mechanical signal transduction. This study aimed to identify the possible synergistic effect between Piezo1 and IFT88 in MCC differentiation during mechanical conduction. MATERIALS AND METHODS: Confocal immunofluorescence staining was used to reveal the Piezo1 localization. Small interfering RNA (siRNA) technology was used to knock down the expression levels of Piezo1 and IFT88. The chondrogenic differentiation ability of MCCs was evaluated by Alcian blue staining, and the early differentiation ability was evaluated by Western blot of SOX9 and COL2A1. RESULTS: Confocal immunofluorescence results showed that Piezo1 localized in the root of primary cilia. Without cyclic tensile strain (CTS) stimuli, Alcian blue staining showed that Piezo1 knockdown had a marginal effect on the chondrogenic differentiation of MCCs, while IFT88 knockdown inhibited the chondrogenic differentiation. The protein levels of SOX9 and COL2A1 decreased significantly with CTS stimuli. However, these protein levels were restored when Piezo1 was knocked down. In addition, IFT88 knockdown decreased the protein level of Piezo1 with or without CTS. CONCLUSION: Piezo1 and IFT88 might play a synergistic role in regulating MCC differentiation under CTS stimuli.

The adhesive heterogeneity of different compartments of oral mucosal rete ridges.

Rete ridges play a critical role in maintaining epidermal structure and mechanical properties. Notably, rete ridges can be divided into three compartments: the base, slope and tip. The present study aims to explore whether these three compartments have distinct adhesive functions. We collected 28 normal masticatory mucosae to prepare paraffin-embedded sections. Immunohistochemistry and immunofluorescent staining were used to analyse the expression pattern of integrin α6 and ß4 in different compartments of the rete ridges. To observe whether the different compartments had distinct adhesive forces, dermal-epidermal junction separation experiments were performed by peeling the oral epithelium from the lamina propria after treatment with cold saline for 72 h. The results showed that integrin α6 and ß4 prefer the basal layer keratinocytes closely adjacent to the base compartment of the rete ridges. The oral mucosal epithelium separated from the underlying lamina propria at the tip of rete ridges when they were peeled after the cold saline treatment. In conclusion, the adhesive force of the basal layer keratinocytes at the base of the rete ridges is stronger than at the tip.

Regulatory mechanism of oral mucosal rete peg formation.

Rete pegs are finger-like structures that are formed during the development and wound healing process of the skin and oral mucosa, and they provide better mechanical resistance and nutritional supply between the epithelium and dermis. An increasing number of studies have shown that rete pegs have physiological functions, such as resisting bacterial invasion, body fluid loss, and other harmful changes, which indicate that rete pegs are important structures in natural skin and oral mucosa. Although a great deal of progress has been made in scaffold materials and construction methods for tissue-engineered skin and oral mucosa in recent years, construction of the oral mucosa with functional rete pegs remains a major challenge. In this review, we summarized current research on the progress on formation of rete pegs in human oral mucosa as well as its molecular basis and regulatory mechanism, which might provide new ideas for functional construction of tissue-engineered skin and oral mucosa.

Podosome formation in the murine palatal mucosae: Its proteolytic role in rete peg formation.

BACKGROUND: Basement membrane remodeling is an indispensable factor for oral mucosal rete peg formation, but how the basement membrane is remodeled remains unclear. Our previous study indicated that keratinocyte growth factor induces the assembly of podosomes, which are dynamic organelles critical for matrix remodeling in human immortalized oral epithelial cells. This study explores podosome formation and its role in basement membrane remodeling during murine oral mucosal rete peg formation. METHODS: Perinatal murine palatal tissue slices were obtained from embryonic day 17.5 (E 17.5) to postnatal day 10.5 (P 10.5) BALB/c mice. Rete peg formation was observed by hematoxylin and eosin (HE) staining. Proteolysis of the basement membrane was detected by immunofluorescence staining. The assembly of podosomes and their correlation with basement membrane proteolysis were investigated by laser scanning confocal microscopy. RESULTS: The shape of basal layer keratinocytes at the sites of emerging rete pegs changed from typically polygonal to spindle-shaped. Basement membrane proteolysis, indicated by decreased type IV collagen (Col IV) staining, was detected during rete peg formation. Classical markers for podosomes, including cortactin/Tks5, WASP, and matrix metalloproteinase foci, were easily observed at the spindle-shaped cells. Podosomes were visible in regions where there was a significant decrease in Col IV staining. CONCLUSIONS: These observations indicated that podosomes form at the front of the emerging rete peg and may play a pivotal role in basement membrane remodeling during rete peg formation.

In vitro and in vivo synergistic anti-tumor effect of LIN28 inhibitor and metformin in oral squamous cell carcinoma.

Cancer stem cell therapy is becoming a focal point for oral squamous cell carcinoma (OSCC). They can be regulated by tumor glucose metabolism, whereas the regulation is not fully investigated in OSCC. Herein, we studied the synergistic anti-tumor effect of a LIN28 inhibitor C1632 and hypoglycemic medication metformin in OSCC. In this study, OSCC cell lines SCC9 and CAL27 were treated with C1632 and metformin respectively or synergistically. First, western blotting was performed to detect the expression level of LIN28 and its downstream molecule HMGA2. Second, MTT assay was conducted to assess cell proliferation. Next, wound healing assay and transwell assay were applied to evaluate cell migration. Then, xenograft mouse experiment was done to explore anti-tumor effect in vivo. Finally, western blotting was used to investigate the pharmacological mechanisms of the synergistic effect oft he two medication. Results showed that LIN28 and HMGA2 expression decreased significantly in SCC9 and CAL27 cells under 240 µM C1632 treatment for 72 h. These effects were synergized under combined treatment for 24 h. Cell proliferation ability and migration ability of both cell lines decreased significantly under respective and combined treatment. In xenograft mouse experiment, tumor weights decreased by 48% under 40 mg/kg/3d C1632 treatment, 53% under 250 mg/kg/d metformin treatment and 91% under combined treatment for 18 days. Tumor volumes decreased by 32%, 57% and 47% under C1632, metformin and combined treatment respectively. These results indicated that C1632 and metformin exerts synergistic anti-tumor effects in OSCC cell lines SCC9 and CAL27, and also inhibits xenograft tumor growth in vivo.

Obvious morphologic changes in the mandible and condylar cartilage after triple botulinum toxin injections into the bilateral masseter.

INTRODUCTION: Nonsurgical treatments that can prevent or reduce the extent of the mandibular excess at an early stage are desirable. A single botulinum toxin (BTX) injection into the unilateral and bilateral masseter can regulate mandibular contour and condylar cartilage. However, BTX injection is frequency dependent when used in facelifts. This study aimed to evaluate the effect of BTX injection into the bilateral masseter at different frequencies on the mandibular contour and condylar cartilage. METHODS: In the present study, 24 female Sprague Dawley rats (4 weeks old) were divided into 3 groups: control, single injection, and triple injection. Contour measurement of the mandible was carried out by radiographic imaging. Microcomputerized tomography was performed to determine the change in bone volume in the subchondral bone. Hematoxylin and eosin staining was used to observe the morphologic changes of condylar cartilage. Immunohistochemistry was performed to detect the expression level of biomechanically sensitive factors, including transforming growth factor-ß1, parathyroid hormone-related protein, SRY-box 9, and type II collagen. RESULTS: Bone volume and/or total volume, trabecular number, and trabecular thickness of the mineralized cartilage and subchondral bone significantly decreased in the triple injection group when compared with the single injection group. Mandibular contour also diminished after increased BTX injection frequencies. Chondrocyte proliferation ability and the expression levels of transforming growth factor-ß1, parathyroid hormone-related protein, SRY-box 9, and type II collagen significantly decreased in all BTX injection groups and more in the triple injection group. CONCLUSIONS: Morphologic changes of the mandible and condylar cartilage become more obvious after increased BTX injection frequencies, suggesting that multiple BTX injections into the masseter of patients may relieve the severity of mandibular deformity at an early stage.

Keratinocyte growth factor (KGF) induces podosome formation via integrin-Erk1/2 signaling in human immortalized oral epithelial cells.

Recent study established the role of integrins in keratinocyte growth factor (KGF)-induced oral epithelial adhesion and rete peg elongation. However, how extracellular matrix (ECM) remodeling cooperates with the increased epithelial adhesion during rete peg elongation has yet to be determined. Podosomes are cell-matrix contact structures that combine several abilities, including adhesion and matrix degradation. In the present study, we identified podosome formation at the ventral side of human immortalized oral epithelial cells (HIOECs) upon KGF treatment. Moreover, podosomal components including integrin α6，ß4，α3，ß1 and MMP14 colocalized with the F-actin-cortactin complex and matrix degradation assays demonstrated the ability of the F-actin-cortactin complex to degrade matrix. Inhibition both of integrin subunits ß4 and ß1 with specific blocking antibodies and inhibition of Erk1/2 abrogated the KGF-induced podosome formation. Notably, knockdown of integrin subunits ß4 and ß1 with specific small interfering RNA (siRNA) downregulated the phosphorylation levels of Erk1/2. In contrast, inhibition of both Erk1/2 could upregulate the expression of integrin subunits ß4 and ß1. These results demonstrate that KGF induces podosome formation via integrin-Erk1/2 signaling in HIOECs, suggesting a novel mechanism by which integrins enhance oral epithelial adhesion and rete peg elongation.

Magnetic and Folate Functionalization Enables Rapid Isolation and Enhanced Tumor-Targeting of Cell-Derived Microvesicles.

Cell-derived microvesicles (MVs), which are biogenic nanosized membrane-bound vesicles that convey bioactive molecules between cells, have recently received attention for use as natural therapeutic platforms. However, the medical applications of MV-based delivery platforms are limited by the lack of effective methods for the efficient isolation of MVs and the convenient tuning of their targeting properties. Herein, we report the development of magnetic and folate (FA)-modified MVs based on a donor cell-assisted membrane modification strategy. MVs inherit the membrane properties of their donor cells, which allows them to be modified with the biotin and FA on their own membrane. By conjugating with streptavidin-modified iron oxide nanoparticles (SA-IONPs), the MVs can be conveniently, efficiently, and rapidly isolated from the supernatant of their donor cells using magnetic activated sorting. Moreover, the conjugated magnetic nanoparticles and FA confer magnetic and ligand targeting activities on the MVs. Then, the MVs were transformed into antitumor delivery platforms by directly loading doxorubicin via electroporation. The modified MVs exhibited significantly enhanced antitumor efficacy both in vitro and in vivo. Taken together, this study provides an efficient and convenient strategy for the simultaneous isolation of cell-derived MVs and transformation into targeted drug delivery nanovectors, thus facilitating the development of natural therapeutic nanoplatforms.

Expression of fibrosis-related molecules in the oral mucosa of six animal species: A reference for selecting animal models.

Several animal models have been used in studies associated with oral submucous fibrosis (OSF); however, an appropriate model based on the histopathological characteristics of OSF is still needed. This study aimed to provide histological references for selecting a potential model. The expression intensities of collagen type I (Col I), type III (Col III), type IV (Col IV), fibronectin (FN), transforming growth factors ß (TGF-ß), and connective tissue growth factor (CCN2) in the oral mucosa of the human and six non-human animal species were measured by immunohistochemistry. There was little variation in the expression intensity of Col I while the expression of Col III, Col IV, and FN showed differences. The expression intensities of TGF-ß in dog, rat, sheep, and pig oral mucosae, and those of CCN2 in dog, minipig, rat, and buffalo oral mucosae were equivalent to the expression intensities in human mucosa. The expression of fibrosis-related molecules in the dog oral mucosa optimally mimics the human condition, suggesting its suitability with regard to histopathology as an animal model for the study of OSF.

Histological features of oral epithelium in seven animal species: As a reference for selecting animal models.

Several animals have been used as models for basic and clinical research on oral mucosa. Few studies have focused on the selection of an appropriate animal model. This study aimed to provide histological references for selecting a potential model. Histological features were assessed by exploring 6 morphological characteristics and 2 immunohistochemical markers. The morphological characteristics included keratinization, basal membrane appearance, epithelial thickness, rete ridge length, adjacent rete ridge distance, and regional variation; the immunohistochemical markers included Ki67 (a proliferative marker) and Cytokeratin 19 (CK19; a stemness marker). The histological similarity of each species compared to humans was calculated according to the designated scoring criteria. The results showed that the buccal mucosae from dog and pig were non-keratinized, with similar rete ridge length and distance, compared to that of humans. The dog, rat, and cavy mucosae had analogous gross appearances in the basal membrane. The dog oral mucosae shared similar epithelial thickness with human oral mucosae. Compared to the human mucosa, the dog, pig, rat, and rabbit mucosae exhibited corresponding regional variations. The Ki67-positive cells in human and canine mucosae were predominantly localized in the suprabasal layers, whereas most of the proliferative cells were in the basal layer in other species. CK19 immunoreactivities were detected only in human and canine mucosae. The canine mucosae gained the highest point value (14), whereas the scores for the pig, rat, rabbit, cavy, sheep, and buffalo mucosae were 8, 6, 5, 5, 5, and 2, respectively. The histological variations in the oral epithelium of diverse animal species are considerable; the mucosae from dogs are most similar to human mucosae, implicating its histological basis as an animal model.