VMT/ACP/Dextran composite nanosheets against dental caries through promoting mineralization of dentin tubules, pH buffering, and antibacterial.

Dental caries is a worldwide public healthcare concern, and is closely related to the acidic environment that caused by bacterial decomposition of food. In this study, a two-step ion exchange liquid-phase stripping method was applied to strip out vermiculite (VMT) nanosheets, then amorphous calcium phosphate (ACP) and dextran were inserted between the VMT nanosheets interlayer to obtain a composite two-dimension nanosheets (VMT/ACP/Dextran). VMT/ACP/Dextran composite nanosheets exhibited excellent biocompatibility and could provide exogenous Ca2+and PO43- from ACP, provide SiO44-, Mg2+, Fe2+ and obtain buffering pH and antibacterial properties from VMT, as well as improve suspension stability and targeting Streptococcus mutans through glucan. The in vitro study showed that the composite materials could promote the mineralization and sealing of dentin tubules by releasing active ions, buffer pH 4.5 (a value close to the pH in the dental plaque environment) to pH 6.6-7.1 (values close to the pH in human saliva) through ion exchange, and exert antibacterial effects by targeting Streptococcus mutans and exerting oxidase like and peroxidase like activities to produce reactive oxygen species (ROS). The in vivo animal study showed that daily cleaning teeth using VMT/ACP/Dextran composite nanosheets could effectively reduce the incidence rate and severity of dental caries in rats. Taking together, the developed VMT/ACP/Dextran composite nanosheets, which integrated the excellent properties of VMT, ACP and dextran, can effectively prevent dental caries through a combination of factors such as buffering acids, antibacterial properties, and promoting calcification, and may be used as an active ingredient for daily oral hygiene or filling materials to prevent and treat dental caries.

Three-dimensional genome structure and function.

Linear DNA undergoes a series of compression and folding events, forming various three-dimensional (3D) structural units in mammalian cells, including chromosomal territory, compartment, topologically associating domain, and chromatin loop. These structures play crucial roles in regulating gene expression, cell differentiation, and disease progression. Deciphering the principles underlying 3D genome folding and the molecular mechanisms governing cell fate determination remains a challenge. With advancements in high-throughput sequencing and imaging techniques, the hierarchical organization and functional roles of higher-order chromatin structures have been gradually illuminated. This review systematically discussed the structural hierarchy of the 3D genome, the effects and mechanisms of cis-regulatory elements interaction in the 3D genome for regulating spatiotemporally specific gene expression, the roles and mechanisms of dynamic changes in 3D chromatin conformation during embryonic development, and the pathological mechanisms of diseases such as congenital developmental abnormalities and cancer, which are attributed to alterations in 3D genome organization and aberrations in key structural proteins. Finally, prospects were made for the research about 3D genome structure, function, and genetic intervention, and the roles in disease development, prevention, and treatment, which may offer some clues for precise diagnosis and treatment of related diseases.

Integration of 3D genome topology and local chromatin features uncovers enhancers underlying craniofacial-specific cartilage defects.

Aberrations in tissue-specific enhancers underlie many developmental defects. Disrupting a noncoding region distal from the human SOX9 gene causes the Pierre Robin sequence (PRS) characterized by the undersized lower jaw. Such a craniofacial-specific defect has been previously linked to enhancers transiently active in cranial neural crest cells (CNCCs). We demonstrate that the PRS region also strongly regulates Sox9 in CNCC-derived Meckel's cartilage (MC), but not in limb cartilages, even after decommissioning of CNCC enhancers. Such an MC-specific regulatory effect correlates with the MC-specific chromatin contacts between the PRS region and Sox9, highlighting the importance of lineage-dependent chromatin topology in instructing enhancer usage. By integrating the enhancer signatures and chromatin topology, we uncovered >10,000 enhancers that function differentially between MC and limb cartilages and demonstrated their association with human diseases. Our findings provide critical insights for understanding the choreography of gene regulation during development and interpreting the genetic basis of craniofacial pathologies.

Two-dimensional nanovermiculite and polycaprolactone electrospun fibers composite scaffolds promoting diabetic wound healing.

BACKGROUND: Promoting diabetic wound healing is still a challenge, and angiogenesis is believed to be essential for diabetic wound healing. Vermiculite is a natural clay material that is very easy to obtain and exhibits excellent properties of releasing bioactive ions, buffering pH, adsorption, and heat insulation. However, there are still many unsolved difficulties in obtaining two-dimensional vermiculite and using it in the biomedical field in a suitable form. RESULTS: In this study, we present a versatile organic-inorganic composite scaffold, which was constructed by embedding two-dimensional vermiculite nanosheets in polycaprolactone electrospun fibers, for enhancing angiogenesis through activation of the HIF-1α signaling pathway and promoting diabetic wound healing both in vitro and in vivo. CONCLUSIONS: Together, the rational-designed polycaprolactone electrospun fibers-based composite scaffolds integrated with two-dimensional vermiculite nanosheets could significantly improve neo-vascularization, re-epithelialization, and collagen formation in the diabetic wound bed, thus promoting diabetic wound healing. This study provides a new strategy for constructing bioactive materials for highly efficient diabetic wound healing.

Reducing relapse and accelerating osteogenesis in rapid maxillary expansion using an injectable mesoporous bioactive glass/fibrin glue composite hydrogel.

Rapid maxillary expansion (RME), as a common treatment for craniomaxillofacial deformity, faces the challenge of high relapse rates and unsatisfactory therapeutic effects. In this study, a standardized Sprague-Dawley (SD) rat RME model was first established with a modified expander as well as retainer design and optimized anterior maxillary expanding force of 100 g which exerted the most synchronized mobility of mid-palatal suture and incisors. Via the standardized model, the high relapse rate was proven to be attributed to insufficient osteogenesis in expanded suture, requiring long-term retainer wearing in clinical situations. To reduce the relapse rate, mesoporous bioactive glass/fibrin glue (MBG/FG) composite hydrogels were developed for an in situ minimal invasive injection that enhance osteogenesis in the expanded palate. The component of 1 wt% MBG was adopted for enhanced mechanical strength, matched degradation rate and ion dissolution, excellent in vitro biocompatibility and osteoinductivity. Effects of 1%MBG/FG composite hydrogel on osteogenesis in expanded mid-palatal sutures with/without retention were evaluated in the standardized model. The results demonstrated that injection of 1%MBG/FG composite hydrogel significantly promoted bone formation within the expanded mid-palatal suture, inhibited osteoclastogenesis and benefited the balance of bone remodeling towards osteogenesis. Combination of retainer and injectable biomaterial was demonstrated as a promising treatment to reduce relapse rate and enhance osteogenesis after RME. The model establishment and the composite hydrogel development in this article might provide new insight to other craniomaxillofacial deformity treatment and design of bone-repairing biomaterials with higher regenerative efficiency.

Effect of Swell1 on regulating chondrocyte hypertrophy during the condylar osteochondral development process in mice.

Chondrocyte hypertrophy is a significant factor in cartilage development, yet the molecular mechanism for cell volume expand during the process is remains unclear. In the present study, the relationship between Swell1, a cell volume regulated anion channel, and chondrocyte hypertrophy was explored. The results reveal that the spatiotemporal expression of Swell1 was similar with the development process of hypertrophic chondrocytes in condyles. Through Col10a1 mediated knock out of Swell1 in hypertrophy chondrocytes, we found that there are less obvious boundary between different condylar cartilage layers in which increased hypertrophic chondrocytes were scattered in all three cartilage layers. The cortical bone mass and bone mineral density in the subchondral bone significantly increased. Additionally, knock out of Swell1 could increase the expression of OCN in the femur condyle. Based on the aforementioned findings, a conclusion could be drawn that Swell1 is a significant factor in chondrocyte hypertrophy during the condylar osteochondral development process, and there was some difference between the mandibular and femur condyles, which will provide some new clues for understanding the development of cartilage and related diseases.

[Temporal and spatial expression of Swell1 gene in the development of mouse condyle cartilage].

PURPOSE: To explore the temporal and spatial expression pattern of Swell1 (LRRC8A) gene in mouse condylar cartilage. METHODS: By obtaining condyle samples of embryos at 15.5, 16.5, 18.5 days and newborn mice, H-E staining, immunofluorescence staining and qRT-PCR were used to explore the microstructure of mouse condyles and the temporal and spatial expression changes of genes related to cartilage development and Swell1 gene. Statistical analysis was performed using SPSS 25.0 software package. RESULTS: Swell1 gene expression began in the hypertrophic chondrocyte layer during the development of the condyle from embryonic day 16.5, and then gradually increased, and continued to be expressed during mouse embryonic development until the mouse was born. CONCLUSIONS: Swell1 is mainly expressed in hypertrophic chondrocytes during the development of mouse condyles, and it may be involved in the regulation of chondrocyte hypertrophy.

Liquid Phase Concentrated Growth Factor Improves Autologous Fat Graft Survival In Vivo in Nude Mice.

OBJECTIVE: The present study aimed to explore the efficacy and safety profile of liquid phase concentrated growth factor (LPCGF) in promoting autologous fat graft survival. METHODS: LPCGF/PRP was mixed with human fat tissues at different proportions and transplanted into nude mice. Three months after transplantation, the implanted fat tissues were retrieved for analysis. H&E staining was used to quantify the neovascularization. Immunohistochemical staining was applied to quantify the CD34-positive stem cells and the fluorescence intensity of VEGF and TGF-ß. RESULTS: Addition of LPCGF to autologous fat reduced the fat absorption by 5-15%, especially at the early stage, and no complications were observed. In addition, the effect was improved with increased CGF. Liquid phase concentrated growth factor improves autologous fat graft survival, and the most suitable ratio of LPCGF/fat is 1:8. CONCLUSION: LPCGF is rich in VEGF, TGF-ß and CD34-positive stem cells, which can improve the fat transplantation effect, but the specific influence of a single component requires future evaluation. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

A novel and injectable strontium-containing hydroxyapatite bone cement for bone substitution: A systematic evaluation.

Reconstruction of bone defects is still a challenge. In this study, we developed and systematically evaluated a novel injectable strontium-containing hydroxyapatite (Sr-HA) bone cement in which Sr-HA powder included 5% Sr and was mixed with a setting liquid that included 5% potassium citrate. This Sr-HA cement was mainly composed of HA and α-tricalcium phosphate (TCP) and exhibited favorable injectability (100%), setting times (the initial setting time was 240 s and the final setting time was 420 s), compressive strength (73.4 MPa), maximal load and maximum bending stress, and excellent radiopacity. In addition, the Sr-HA cement also had excellent biocompatibility that exhibited low cytotoxicity for cell proliferation and no obvious disturbing effect on the osteogenic differentiation of periodontal ligament stem cells (DLSCs) and dental pulp stem cells (DPSCs). However, the Sr-HA cement could slightly promote the osteogenic differentiation of MC3T3 cells, which also implied that it would promote osseointegration between the cement and surrounding bone but would not obviously disturb the biological behavior of DLSCs and DPSCs. An in vivo study further confirmed that Sr-HA cement exhibited favorable osseointegration with the maxilla and tibia. All these findings implied that the novel Sr-HA cement was a suitable bone substitution for bone defects.

ITPR1 Mutation Contributes to Hemifacial Microsomia Spectrum.

Hemifacial microsomia (HM) is a craniofacial congenital defect involving the first and second branchial arch, mainly characterized by ocular, ear, maxilla-zygoma complex, mandible, and facial nerve malformation. HM follows autosomal dominant inheritance. Whole-exome sequencing of a family revealed a missense mutation in a highly conserved domain of ITPR1. ITPR1 is a calcium ion channel. By studying ITPR1's expression pattern, we found that ITPR1 participated in craniofacial development, especially the organs that corresponded to the phenotype of HM. In zebrafish, itpr1b, which is homologous to human ITPR1, is closely related to craniofacial bone formation. The knocking down of itpr1b in zebrafish could lead to a remarkable decrease in craniofacial skeleton formation. qRT-PCR suggested that knockdown of itpr1b could increase the expression of plcb4 while decreasing the mRNA level of Dlx5/6. Our findings highlighted ITPR1's role in craniofacial formation for the first time and suggested that ITPR1 mutation contributes to human HM.

Stimulating Factors and Origins of Precursor Cells in Traumatic Heterotopic Ossification Around the Temporomandibular Joint in Mice.

The contributing factors and the origins of precursor cells in traumatic heterotopic ossification around the temporomandibular joint (THO-TMJ), which causes obvious restriction of mouth opening and maxillofacial malformation, remain unclear. In this study, our findings demonstrated that injured chondrocytes in the condylar cartilage, but not osteoblasts in the injured subchondral bone, played definite roles in the development of THO-TMJ in mice. Injured condylar chondrocytes without articular disc reserves might secrete growth factors, such as IGF1 and TGFß2, that stimulate precursor cells, such as endothelial cells and muscle-derived cells, to differentiate into chondrocytes or osteoblasts and induce THO-TMJ. Preserved articular discs can alleviate the pressure on the injured cartilage and inhibit the development of THO-TMJ by inhibiting the secretion of these growth factors from injured chondrocytes. However, the exact molecular relationships among trauma, the injured condylar cartilage, growth factors such as TGFß2, and pressure need to be explored in detail in the future.

Evaluation of Mandibular Symmetry in Patients With Condylar Osteochondroma Who Underwent Intro-Oral Condylar Resection and Simultaneous Bimaxillary Orthognathic Surgery.

Mandibular condylar osteochondroma (OC) can result in facial asymmetry, malocclusion, and temporomandibular joint dysfunction. The authors have previously demonstrated a novel method for conservative condylectomy and simultaneous orthognathic surgery for treatment of mandibular condylar OC. The purpose of this study was to evaluate the immediate improvement and long-term stability of mandibular symmetry in the treatment of condylar OC. Fifty-six patients with unilateral mandibular condylar OC combined with secondary facial asymmetry and malocclusion were enrolled in this retrospective study. The computerized tomography (CT) scans were acquired with the mandible in centric relation (CR) before surgery, 1 week and 12 to 18 months after surgery. The images were reconstructed and processed for the analysis with ProPlan CMF 2.1 software. After defining the skeletal landmarks and the reference planes, the chin deviation, chin rotation and mandibular asymmetry index were calculated. The operations and healing were uneventful and the patients showed no signs of recurrence or temporomandibular joint ankylosis during the follow-up. Facial symmetry was greatly improved right after the surgery in the chin deviation (from 9.2 to 1.7 mm, P < 0.01), chin rotation (from 11.2 to 2.3, P < 0.01) and the asymmetry index of three mandibular landmarks (Go, MF and Sg, all P < 0.01). It also showed a stable result after 12 to 18 months follow-up. We also established a novel measurement method, which showed that the combination of conservative condylectomy via the intraoral approach based on intraoperative navigation and simultaneous orthognathic surgery is effective for improving the facial symmetry when treat the mandibular condylar OC.

Comparison of temporomandibular joint function and morphology after surgical and non-surgical treatment in adult condylar head fractures.

PURPOSE: The aim of this study was to evaluate whether surgical treatment can distinctly improve temporomandibular joint (TMJ) function and morphology in adult patients with condylar head fractures (CHFs) when compared with conservative treatment. PATIENTS AND METHODS: A retrospective study was performed to evaluate surgical and conservative outcomes in all patients who had suffered CHFs. In this study, all patients were divided into a surgical group and a conservative group. In the surgical group patients were treated by open reduction and internal fixation (ORIF) combined disc anchorage, while in the conservative group patients were treated by a removable splint combined with intermaxillary elastics. Clinical and radiological outcomes were evaluated and functional outcomes were assessed using the Helkimo index score. Paired t-tests, Wilcoxon signed rank tests, independent t-tests and χ2 tests were used to assess inter-group differences. RESULTS: 75 TMJs in 56 patients were included in this study and were divided into a surgical group (56 TMJs in 40 patients) and a conservative group (19 TMJs in 16 patients). The results showed that the Helkimo index score for TMJ in the surgical group was better than in the conservative group (pAi = 0.032, pDi = 0.001, respectively). Ramus height in the surgical group (61.08 ± 4.04 mm) recovered more than in the conservative group (54.82 ± 3.06 mm) (p = 0.012). Discs became shorter, moved further forward, and became severely distorted in the conservative group (disc length = 7.32 ± 1.35 mm). In contrast, discs became longer, retained a normal disc-condyle relationship, and formed a normal shape in the surgical group (disc length = 11.05 ± 2.07 mm) (p = 0.003). According to the MRI images, joint effusion, retrodiscal tear, and lateral capsular tear were better cured in the surgical group (p = 0.001, p < 0.001, p = 0.012, respectively). CONCLUSION: Based on these results, it seems that surgical treatment of condylar head fractures should be the preferred approach if there are no contraindications for general anesthesia.

Does Nasal Cavity Enlargement Associated With Respiratory Function Improvement After Surgically Assisted Rapid Maxillary Expansion?

PURPOSE: To systematically evaluate the effects of surgically assisted rapid maxillary expansion (SARME) on nasal cavity and its associated respiratory function changes. MATERIAL AND METHODS: Multiple electronic databases were searched, authors were contacted as required, and reference lists of potentially relevant studies were screened. Articles that included patients older than 16 who had received SARME were considered. Data extraction and quality assessment were performed independently and in duplicate. RESULTS: Eight articles, including 161 patients, were finally selected and analyzed. Nasal cavity significantly augmentation after SARME has been recognized by 7 studies with moderate-quality evidence. Among them, the nasal cavity volume increased <20% in 4 studies, 20% to 40% in 1 study, and >40% in 2 studies. Totally, the range of increasing in nasal cavity volume was 7.6% to 99%. One study had recorded the nasal expiratory and inspiratory flow significantly increased 18.5% and 21.7%, respectively. CONCLUSION: SARME was found to produce meaningful volume augmentation in nasal cavity with adult patients. However, the improvement of nasal respiratory function has not been well elucidated; thus, SARME is not yet recommended for the purpose of improving nasal respiratory.

Thalidomide leads to mandible hypoplasia through inhibiting angiogenesis and secondary hemorrhage in the fetal craniofacial region in rabbits.

The effect of thalidomide on mandibular development is unclear. In this study, thalidomide was delivered to pregnant rabbits from the 8th to 14th day of gestation. Then, embryos were harvested for examination on the 16th day (GD16), 20th day (GD20) and 24th day (GD24) of gestation. The results showed obvious hemorrhage and hematoma on one side of the craniofacial region in 50 % of the thalidomide-treated embryos and obvious hemorrhage and hematoma on both sides of the craniofacial region in 50 % of the thalidomide-treated embryos at GD16. Histological examination showed soft tissues and mandible defects on the affected side of the maxillofacial region. The expression of Vegf-α, Ki67 and Sox9 on the affected side was significantly down-regulated in comparison to their expression on the unaffected side at GD20. There was also an obvious defect in the affected mandible, and the density of the skull and mandible was decreased compared to the unaffected side or the control group at GD24. These findings demonstrated that thalidomide may lead to hemorrhage and hematoma in the craniofacial region by inhibiting angiogenesis, resulting in the abnormal development of cranial neural crest cells that are involved in the normal development of the mandible in rabbits.

Histopathological features of condylar hyperplasia and condylar Osteochondroma: a comparison study.

BACKGROUND: Both mandibular condylar hyperplasia and condylar osteochondroma can lead to maxillofacial skeletal asymmetry and malocclusion, although they exhibit different biological behavior. This study attempted to compare the histological features of mandibular condylar hyperplasia and condylar osteochondroma using hematoxylin-and-eosin (H&E) staining, and immunohistochemistry staining of PCNA and EXT1 with quantitative analysis method. RESULTS: The H&E staining showed that condylar hyperplasia and condylar osteochondroma could be divided into four histological types and exhibited features of different endochondral ossification stages. There was evidence of a thicker cartilage cap in condylar osteochondroma as compared condylar hyperplasia (P = 0.018). The percentage of bone formation in condylar osteochondroma was larger than was found in condylar hyperplasia (P = 0.04). Immunohistochemical staining showed that PCNA was mainly located in the undifferentiated mesenchymal layer and the hypertrophic cartilage layer, and there were more PCNA positive cells in the condylar osteochondroma (P = 0.007). EXT1 was mainly expressed in the cartilage layer, and there was also a higher positive rate of EXT1 in condylar osteochondroma (P = 0.0366). The thicker cartilage cap, higher bone formation rate and higher PCNA positive rate indicated a higher rate of proliferative activity in condylar osteochondroma. The more significant positive rate of EXT1 in condylar osteochondroma implied differential biological characteristic as compared to condylar hyperplasia. CONCLUSIONS: These features might be useful in histopathologically distinguishing condylar hyperplasia and osteochondroma.

A genetic-phenotypic classification for syndromic micrognathia.

Micrognathia is a common craniofacial deformity which represents hypoplastic development of the mandible, accompanied by retrognathia and consequent airway problems. Usually, micrognathia is accompanied by multiple systematic defects, known as syndromic micrognathia, and is in close association with genetic factors. Now, large quantities of pathogenic genes of syndromic micrognathia have been revealed. However, how these different pathogenic genes could lead to similar phenotypes, and whether there are some common characteristics among these pathogenic genes are still unknown. In this study, we proposed a genetic-phenotypic classification of syndromic micrognathia based on pathogenic genes information obtained from Phenolyzer, DAVID, OMIM, and PubMed database. Pathogenic genes of syndromic micrognathia could be divided into four groups based on gene function, including cellular processes and structures, cell metabolism, cartilage and bone development, and neuromuscular function. In addition, these four groups exhibited various clinical characteristics, and the affected systems, such as central nervous system, skeletal system, cardiovascular system, oral and dental system, respiratory system and muscle, were different in these four groups. This classification could provide meaningful insights into the pathogenesis of syndromic micrognathia, and offer some clues for understanding the molecular mechanism, as well as guiding precise clinical diagnosis and treatment for syndromic micrognathia.

Clinical presentation and genetic profiles of Chinese patients with velocardiofacial syndrome in a large referral centre.

Diagnosis and treatment of velocardiofacial syndrome (VCFS) with variable genotypes and phenotypes are considered to be very complicated. Establishing an exact correlation between the phenotypes and genotypes of VCFS is still a challenging. In this paper, 88 Chinese VCFS patients were divided into five groups based on palatal anomalies and one or two of other four common phenotypes, and copy number variations (CNVs) were detected using multiplex ligation-dependent probe amplification (MLPA), array comparative genomic hybridization (aCGH) and quantitative polymerase chain reaction. The findings showed that palatal anomalies and characteristic malformation of face were important indicators for 22q11.2 microdeletion, and there was difference inthe phenotypic spectrum between the duplication and deletion of 22q11.2. MLPA was a highly cost-effective, sensitive and preferred method for patients with 22q11.2 deletion or duplication. Our results also firstly reported that all three patients who simultaneously exhibited palatal anomalies and cognitive disorder, without other phenotypes, have Top3b duplication, which strongly suggested that Top3b may be a pathogenic gene for these patients. Further, the findings showed that patients with palatal anomalies and congenital heart disease or immune deficiency, with or without other uncommon phenotypes, exhibited heterogeneity in CNVs, including 4q34.1-qter, 6q25.3, 4q23, Xp11.4, 13q21.1, 17q23.2, 7p21.3, 2p11.2, 11q24.3 and 16q23.3, and some possible pathogenic genes, including BCOR, PRR20A, TBX2, SMYD1, KLKB1 and TULP4 have been suggested. For these patients, aCGH, whole genomic sequencing,combined with references and phenomics database to find pathogenic gene,may be choices of priority. Taking these findings together, we offered an alternative method for diagnosis of Chinese VCFS patients based on this phenotypic strategy.

Overexpression of Dlx2 enhances osteogenic differentiation of BMSCs and MC3T3-E1 cells via direct upregulation of Osteocalcin and Alp.

Genetic studies have revealed a critical role of Distal-homeobox (Dlx) genes in bone formation, and our previous study showed that Dlx2 overexpressing in neural crest cells leads to profound abnormalities of the craniofacial tissues. The aim of this study was to investigate the role and the underlying molecular mechanisms of Dlx2 in osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) and pre-osteoblast MC3T3-E1 cells. Initially, we observed upregulation of Dlx2 during the early osteogenesis in BMSCs and MC3T3-E1 cells. Moreover, Dlx2 overexpression enhanced alkaline phosphatase (ALP) activity and extracellular matrix mineralization in BMSCs and MC3T3-E1 cell line. In addition, micro-CT of implanted tissues in nude mice confirmed that Dlx2 overexpression in BMSCs promoted bone formation in vivo. Unexpectedly, Dlx2 overexpression had little impact on the expression level of the pivotal osteogenic transcription factors Runx2, Dlx5, Msx2, and Osterix, but led to upregulation of Alp and Osteocalcin (OCN), both of which play critical roles in promoting osteoblast maturation. Importantly, luciferase analysis showed that Dlx2 overexpression stimulated both OCN and Alp promoter activity. Through chromatin-immunoprecipitation assay and site-directed mutagenesis analysis, we provide molecular evidence that Dlx2 transactivates OCN and Alp expression by directly binding to the Dlx2-response cis-acting elements in the promoter of the two genes. Based on these findings, we demonstrate that Dlx2 overexpression enhances osteogenic differentiation in vitro and accelerates bone formation in vivo via direct upregulation of the OCN and Alp gene, suggesting that Dlx2 plays a crucial role in osteogenic differentiation and bone formation.

Development process of traumatic heterotopic ossification of the temporomandibular joint in mice.

PURPOSE: The exact development process underlying traumatic heterotopic ossification of the temporomandibular joint (THO-TMJ) is largely unclear. In this study, we try to explore the histological development process of THO-TMJ. MATERIALS AND METHODS: Condylar cartilage of one-month-old male mice was partially removed from the left joint with small scissors to induce THO-TMJ. The phenotypes were observed using gross observation, microcomputed tomography (micro-CT) scans and histological examination from one month to six months after surgery. RESULTS: The micro-CT examination results showed that the injured condyle integrated with ectopic bone tissue to form an osteophyte and that the volume and density of the osteophyte grew exponentially with time. Hematoxylin and eosin (H&E), safranin O and fast green staining of the THO-TMJ specimens revealed that the ectopic bone tissue was mainly nonmineralized fibrous tissue 1 month after surgery. This tissue gradually transformed into cartilage 3 months after surgery. Finally, the tissues transformed into mature bone tissue 6 months after surgery. Immunofluorescence staining showed VEGF-α expression in the heterotopic tissue 1 month after surgery, and the expression of Sox9 in the heterotopic tissue was obvious 3 months after surgery. Furthermore, OCN expression was evident in most of the heterotopic tissue 6 months after surgery. The results also showed clear hypoxia-inducible factor 1-alpha (Hif-1α) expression in the injured chondrocytes of the condyle, especially in the articular proliferative zone and fibrocartilaginous zone. CONCLUSIONS: The THO-TMJ imaging characteristics indicated an exponential change with time. Histologically, the development process of THO-TMJ is an endochondral ossification process and includes three stages, fibroproliferative, chondrogenic and osteogenic stage. In addition, Hif-1α, which was expressed in some of the injured chondrocytes, may play an essential role in the initial THO-TMJ.