Neural EGFL-Like 1 Regulates Cartilage Maturation through Runt-Related Transcription Factor 3-Mediated Indian Hedgehog Signaling.

The pro-chondrogenic function of runt-related transcription factor 2 (Runx2) was previously considered to be dependent on direct binding with the promoter of Indian hedgehog (Ihh)-the major regulator of chondrocyte differentiation, proliferation, and maturation. The authors' previous studies identified neural EGFL like 1 (Nell-1) as a Runx2-responsive growth factor for chondrogenic differentiation and maturation. In this study, it was further revealed that the pro-chondrogenic activities of Nell-1 also rely on Ihh signaling, by showing: i) Nell-1 significantly elevated Ihh signal transduction; ii) Nell-1 deficiency markedly reduced Ihh activation in chondrocytes; and iii) Nell-1-stimulated chondrogenesis was significantly reduced by the specific hedgehog inhibitor cyclopamine. Importantly, the authors demonstrated that Nell-1-responsive Ihh signaling and chondrogenic differentiation extended to Runx2-/- models in vitro and in vivo. In Runx2-/- chondrocytes, Nell-1 stimulated the expression and signal transduction of Runx3, another transcription factor required for complete chondrogenic differentiation and maturation. Furthermore, knocking down Runx3 in Runx2-/- chondrocytes abolished Nell-1's stimulation of Ihh-associated molecule expression, which validates Runx3 as a major mediator of Nell-1-stimulated Ihh activation. For the first time, the Runx2→Nell-1→Runx3→Ihh signaling cascade during chondrogenic differentiation and maturation has been identified as an alternative, but critical, pathway for Runx2 to function as a pro-chondrogenic molecule via Nell-1.

Fibromodulin reduces scar size and increases scar tensile strength in normal and excessive-mechanical-loading porcine cutaneous wounds.

Hypertrophic scarring is a major postoperative complication which leads to severe disfigurement and dysfunction in patients and usually requires multiple surgical revisions due to its high recurrence rates. Excessive-mechanical-loading across wounds is an important initiator of hypertrophic scarring formation. In this study, we demonstrate that intradermal administration of a single extracellular matrix (ECM) molecule-fibromodulin (FMOD) protein-can significantly reduce scar size, increase tensile strength, and improve dermal collagen architecture organization in the normal and even excessive-mechanical-loading red Duroc pig wound models. Since pig skin is recognized by the Food and Drug Administration as the closest animal equivalent to human skin, and because red Duroc pigs show scarring that closely resembles human proliferative scarring and hypertrophic scarring, FMOD-based technologies hold high translational potential and applicability to human patients suffering from scarring-especially hypertrophic scarring.

Neural EGFL-like 1, a craniosynostosis-related osteochondrogenic molecule, strikingly associates with neurodevelopmental pathologies.

Various craniofacial syndromes cause skeletal malformations and are accompanied by neurological abnormalities at different levels, leading to tremendous biomedical, financial, social, and psychological burdens. Accumulating evidence highlights the importance of identifying and characterizing the genetic basis that synchronously modulates musculoskeletal and neurobehavioral development and function. Particularly, previous studies from different groups have suggested that neural EGFL-like-1 (Nell-1), a well-established osteochondrogenic inducer whose biopotency was initially identified in the craniofacial tissues, may also play a vital role in the central nervous system, particularly regarding neurological disorder pathologies. To provide first-hand behavior evidence if Nell-1 also has a role in central nervous system abnormalities, we compared the Nell-1-haploinsufficient (Nell-1+/6R) mice with their wild-type counterparts regarding their repetitive, social communication, anxiety-related, locomotor, sensory processing-related, motor coordination, and Pavlovian learning and memory behaviors, as well as their hippocampus transcriptional profile. Interestingly, Nell-1+/6R mice demonstrated core autism spectrum disorder-like deficits, which could be corrected by Risperidone, an FDA-approved anti-autism, anti-bipolar medicine. Besides, transcriptomic analyses identified 269 differential expressed genes, as well as significantly shifted alternative splicing of ubiquitin B pseudogene Gm1821, in the Nell-1+/6R mouse hippocampus, which confirmed that Nell-1 plays a role in neurodevelopment. Therefore, the current study verifies that Nell-1 regulates neurological development and function for the first time. Moreover, this study opens new avenues for understanding and treating craniofacial patients suffering from skeletal deformities and behavior, memory, and cognition difficulties by uncovering a novel bone-brain-crosstalk network. Furthermore, the transcriptomic analysis provides the first insight into deciphering the mechanism of Nell-1 in neurodevelopment.

En-Mass Retraction of Maxillary Anterior Teeth with Severe Proclination and Root Resorption-A Case Report.

Molar distalization has been a validated method to correct dental sagittal relationships and create space to relieve mild to moderate crowding. In the current case report, an adult female patient had a mild skeletal Class III relationship and dental Class III molar relationship. Four premolars and one lower incisor were extracted during the previous two rounds of orthodontic treatments, and the maxillary anterior teeth were left with severe proclination and root resorption. Limited by the available teeth, extraction was not an option for her. Thus, molar distalization with TADs was the best option used in the treatment to address her chief complaint. In addition, a proper bite opening was performed to eliminate occlusion trauma. Utilizing the mid-palatal TADs, the maxillary central incisors were retracted 7.9 mm and retroclined 33 degrees, and the molar distalization was achieved as much as 8 mm. The cross-section slices of CBCT images confirmed the proper retraction of maxillary incisors and well-positioned roots in the alveolar bone. Moreover, the root resorption was not worsened from the treatment. Clinically, the maxillary anterior teeth were preserved esthetically and functionally. This case report illustrates that with proper diagnosis and treatment mechanics, significant tooth movement can be achieved even on extremely proclined maxillary incisors with severe root resorption.

Total maxillary arch distalization by using headgear in an adult patient.

Although headgear is rarely used in adult patients, its use in adults is mainly for anchorage control. In the current case report, a 24-year-old patient had a skeletal Class I relationship with a Class II tendency, brachyfacial pattern, significant facial asymmetry, and dental 3/4 cusp Class II molar and canine relationships on both sides. The patient declined surgery, and facial asymmetry was not his concern. The final treatment goal was to achieve a stable Class I dental relationship and normal occlusion without significantly compromising the patient's profile. The patient was compliant with the use of cervical-pull headgear after he refused the options of orthodontic-orthognathic combined treatment, maxillary premolar extraction, or temporary skeletal anchorage mini-implants. A 5-mm maxillary arch distal movement was accomplished without significant distal tipping of the molar crowns. The active treatment duration was 31 months. Proper overbite and overjet, balanced occlusion, and an acceptable facial profile were achieved. The treatment results inspire reconsideration of the possibility of using headgear in dental Class II correction in adult patients.

Neural EGFL like 1 as a potential pro-chondrogenic, anti-inflammatory dual-functional disease-modifying osteoarthritis drug.

Arthritis, an inflammatory condition that causes pain and cartilage destruction in joints, affects over 54.4 million people in the US alone. Here, for the first time, we demonstrated the emerging role of neural EGFL like 1 (NELL-1) in arthritis pathogenesis by showing that Nell-1-haploinsufficient (Nell-1+/6R) mice had accelerated and aggravated osteoarthritis (OA) progression with elevated inflammatory markers in both spontaneous primary OA and chemical-induced secondary OA models. In the chemical-induced OA model, intra-articular injection of interleukin (IL)1ß induced more severe inflammation and cartilage degradation in the knee joints of Nell-1+/6R mice than in wildtype animals. Mechanistically, in addition to its pro-chondrogenic potency, NELL-1 also effectively suppressed the expression of inflammatory cytokines and their downstream cartilage catabolic enzymes by upregulating runt-related transcription factor (RUNX)1 in mouse and human articular cartilage chondrocytes. Notably, NELL-1 significantly reduced IL1ß-stimulated inflammation and damage to articular cartilage in vivo. In particular, NELL-1 administration markedly reduced the symptoms of antalgic gait observed in IL1ß-challenged Nell-1+/6R mice. Therefore, NELL-1 is a promising pro-chondrogenic, anti-inflammatory dual-functional disease-modifying osteoarthritis drug (DMOAD) candidate for preventing and suppressing arthritis-related cartilage damage.

Correction: Cumulative inactivation of Nell-1 in Wnt1 expressing cell lineages results in craniofacial skeletal hypoplasia and postnatal hydrocephalus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cumulative inactivation of Nell-1 in Wnt1 expressing cell lineages results in craniofacial skeletal hypoplasia and postnatal hydrocephalus.

Upregulation of Nell-1 has been associated with craniosynostosis (CS) in humans, and validated in a mouse transgenic Nell-1 overexpression model. Global Nell-1 inactivation in mice by N-ethyl-N-nitrosourea (ENU) mutagenesis results in neonatal lethality with skeletal abnormalities including cleidocranial dysplasia (CCD)-like calvarial bone defects. This study further defines the role of Nell-1 in craniofacial skeletogenesis by investigating specific inactivation of Nell-1 in Wnt1 expressing cell lineages due to the importance of cranial neural crest cells (CNCCs) in craniofacial tissue development. Nell-1flox/flox; Wnt1-Cre (Nell-1Wnt1 KO) mice were generated for comprehensive analysis, while the relevant reporter mice were created for CNCC lineage tracing. Nell-1Wnt1 KO mice were born alive, but revealed significant frontonasal and mandibular bone defects with complete penetrance. Immunostaining demonstrated that the affected craniofacial bones exhibited decreased osteogenic and Wnt/ß-catenin markers (Osteocalcin and active-ß-catenin). Nell-1-deficient CNCCs demonstrated a significant reduction in cell proliferation and osteogenic differentiation. Active-ß-catenin levels were significantly low in Nell-1-deficient CNCCs, but were rescued along with osteogenic capacity to a level close to that of wild-type (WT) cells via exogenous Nell-1 protein. Surprisingly, 5.4% of young adult Nell-1Wnt1 KO mice developed hydrocephalus with premature ossification of the intrasphenoidal synchondrosis and widened frontal, sagittal, and coronal sutures. Furthermore, the epithelial cells of the choroid plexus and ependymal cells exhibited degenerative changes with misplaced expression of their respective markers, transthyretin and vimentin, as well as dysregulated Pit-2 expression in hydrocephalic Nell-1Wnt1 KO mice. Nell-1Wnt1 KO embryos at E9.5, 14.5, 17.5, and newborn mice did not exhibit hydrocephalic phenotypes grossly and/or histologically. Collectively, Nell-1 is a pivotal modulator of CNCCs that is essential for normal development and growth of the cranial vault and base, and mandibles partially via activating the Wnt/ß-catenin pathway. Nell-1 may also be critically involved in regulating cerebrospinal fluid homeostasis and in the pathogenesis of postnatal hydrocephalus.

CDKN2B upregulation prevents teratoma formation in multipotent fibromodulin-reprogrammed cells.

Tumorigenicity is a well-documented risk to overcome for pluripotent or multipotent cell applications in regenerative medicine. To address the emerging demand for safe cell sources in tissue regeneration, we established a novel, protein-based reprogramming method that does not require genome integration or oncogene activation to yield multipotent fibromodulin (FMOD)-reprogrammed (FReP) cells from dermal fibroblasts. When compared with induced pluripotent stem cells (iPSCs), FReP cells exhibited a superior capability for bone and skeletal muscle regeneration with markedly less tumorigenic risk. Moreover, we showed that the decreased tumorigenicity of FReP cells was directly related to an upregulation of cyclin-dependent kinase inhibitor 2B (CDKN2B) expression during the FMOD reprogramming process. Indeed, sustained suppression of CDKN2B resulted in tumorigenic, pluripotent FReP cells that formed teratomas in vivo that were indistinguishable from iPSC-derived teratomas. These results highlight the pivotal role of CDKN2B in cell fate determination and tumorigenic regulation and reveal an alternative pluripotent/multipotent cell reprogramming strategy that solely uses FMOD protein.

Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis.

Contactin-associated protein-like 4 (Cntnap4) is a member of the neurexin superfamily of transmembrane molecules that have critical functions in neuronal cell communication. Cntnap4 knockout mice display decreased presynaptic gamma-aminobutyric acid (GABA) and increased dopamine release that is associated with severe, highly penetrant, repetitive, and perseverative movements commonly found in human autism spectrum disorder patients. However, no known function of Cntnap4 has been revealed besides the nervous system. Meanwhile, secretory protein neural EGFL-like 1 (Nell-1) is known to exert potent osteogenic effects in multiple small and large animal models without the off-target effects commonly found with bone morphogenetic protein 2. In this study, while searching for a Nell-1-specific cell surface receptor during osteogenesis, we identified and validated a ligand/receptor-like interaction between Nell-1 and Cntnap4 by demonstrating: 1) Nell-1 and Cntnap4 colocalization on the surface of osteogenic-committed cells; 2) high-affinity interaction between Nell-1 and Cntnap4; 3) abrogation of Nell-1-responsive Wnt and MAPK signaling transduction, as well as osteogenic effects, via Cntnap4 knockdown; and 4) replication of calvarial cleidocranial dysplasias-like defects observed in Nell-1-deficient mice in Wnt1-Cre-mediated Cntnap4-knockout transgenic mice. In aggregate, these findings indicate that Cntnap4 plays a critical role in Nell-1-responsive osteogenesis. Further, this is the first functional annotation for Cntnap4 in the musculoskeletal system. Intriguingly, Nell-1 and Cntnap4 also colocalize on the surface of human hippocampal interneurons, implicating Nell-1 as a potential novel ligand for Cntnap4 in the nervous system. This unexpected characterization of the ligand/receptor-like interaction between Nell-1 and Cntnap4 indicates a novel biological functional axis for Nell-1 and Cntnap4 in osteogenesis and, potentially, in neural development and function. © 2018 American Society for Bone and Mineral Research.

Carbon Nanotube Reinforced Collagen/Hydroxyapatite Scaffolds Improve Bone Tissue Formation In Vitro and In Vivo.

Current bone regeneration strategies faced major challenges in fabricating the bionic scaffolds with nano-structure, constituents and mechanical features of native bone. In this study, we developed a new porous scaffold by adding the multi-walled carbon nanotube (MWCNT) into collagen (Col)/hydroxyapatite (HA) composites. Data showed that 0.5%CNT/Col/HA (0.5%CNT) group was approximately tenfolds stiffer than Col-HA, and it was superior in promoting bone marrow mesenchymal stem proliferation and spreading, mRNA and protein expressions of bone sialoprotein (BSP) and osteocalcin (OCN) than Col-HA group. Moreover, we utilized 0.5%CNT composite to repair the rat calvarial defects (8 mm diameter) in vivo, and observed the new bone formation by 3D reconstruction of micro CT, HE and Masson staining, and BSP, OCN by immunohistochemical analysis. Results showed that newly formed bone in 0.5%CNT group was significantly higher than that in Col-HA group at 12 weeks. These findings highlighted a promising strategy in healing of large area bone defect with MWCNT added into the Col-HA scaffold as they possessed the combined effects of mechanical strength and osteogenicity.

Fibromodulin reduces scar formation in adult cutaneous wounds by eliciting a fetal-like phenotype.

Blocking transforming growth factor (TGF)ß1 signal transduction has been a central strategy for scar reduction; however, this approach appears to be minimally effective. Here, we show that fibromodulin (FMOD), a 59-kD small leucine-rich proteoglycan critical for normal collagen fibrillogenesis, significantly reduces scar formation while simultaneously increasing scar strength in both adult rodent models and porcine wounds, which simulate human cutaneous scar repair. Mechanistically, FMOD uncouples pro-migration/contraction cellular signals from pro-fibrotic signaling by selectively enhancing SMAD3-mediated signal transduction, while reducing AP-1-mediated TGFß1 auto-induction and fibrotic extracellular matrix accumulation. Consequently, FMOD accelerates TGFß1-responsive adult fibroblast migration, myofibroblast conversion, and function. Furthermore, our findings strongly indicate that, by delicately orchestrating TGFß1 activities rather than indiscriminately blocking TGFß1, FMOD elicits fetal-like cellular and molecular phenotypes in adult dermal fibroblasts in vitro and adult cutaneous wounds in vivo, which is a unique response of living system undescribed previously. Taken together, this study illuminates the signal modulating activities of FMOD beyond its structural support functions, and highlights the potential for FMOD-based therapies to be used in cutaneous wound repair.

Factors Affecting the Clinical Success Rate of Miniscrew Implants for Orthodontic Treatment.

PURPOSE: The purpose of this study was to evaluate the various factors that influence the success rate of miniscrew implants used as orthodontic anchorage. MATERIALS AND METHODS: Potential confounding variables examined were sex, age, vertical (FMA) and sagittal (ANB) skeletal facial pattern, site of placement (labial and buccal, palatal, and retromandibular triangle), arch of placement (maxilla and mandible), placement soft tissue type, oral hygiene, diameter and length of miniscrew implants, insertion method (predrilled or drill-free), angle of placement, onset and strength of force application, and clinical purpose. The correlations between success rate and overall variables were investigated by logistic regression analysis, and the effect of each variable on the success rate was utilized by variance analysis. RESULTS: One hundred fourteen patients were included with a total of 253 miniscrew implants. The overall success rate was 88.54% with an average loading period of 9.5 months in successful cases. Age, oral hygiene, vertical skeletal facial pattern (FMA), and general placement sites (maxillary and mandibular) presented significant differences in success rates both by logistic regression analysis and variance analysis (P < .05). CONCLUSION: To minimize the failure of miniscrew implants, proper oral hygiene instruction and effective supervision should be given for patients, especially young (< 12 years) high-angle patients with miniscrew implants placed in the mandible.

Incorporation of aligned PCL-PEG nanofibers into porous chitosan scaffolds improved the orientation of collagen fibers in regenerated periodontium.

The periodontal ligament (PDL) is a group of highly aligned and organized connective tissue fibers that intervenes between the root surface and the alveolar bone. The unique architecture is essential for the specific physiological functionalities of periodontium. The regeneration of periodontium has been extensively studied by researchers, but very few of them pay attention to the alignment of PDL fibers as well as its functionalities. In this study, we fabricated a three-dimensional multilayered scaffold by embedding highly aligned biodegradable poly (ε-caprolactone)-poly(ethylene glycol) (PCE) copolymer electrospun nanofibrous mats into porous chitosan (CHI) to provide topographic cues and guide the oriented regeneration of periodontal tissue. In vitro, compared with random group and porous control, aligned nanofibers embedded scaffold could guide oriented arrangement and elongation of cells with promoted infiltration, viability and increased periodontal ligament-related genes expression. In vivo, aligned nanofibers embedded scaffold showed more organized arrangement of regenerated PDL nearly perpendicular against the root surface with more extensive formation of mature collagen fibers than random group and porous control. Moreover, higher expression level of periostin and more significant formation of tooth-supporting mineralized tissue were presented in the regenerated periodontium of aligned scaffold group. Incorporation of aligned PCE nanofibers into porous CHI proved to be applicable for oriented regeneration of periodontium, which might be further utilized in regeneration of a wide variety of human tissues with a specialized direction. STATEMENT OF SIGNIFICANCE: The regeneration of periodontium has been extensively studied by researchers, but very few of them give attention to the alignment of periodontal ligament (PDL) fibers as well as its functionalities. The key issue is to provide guidance to the orientation of cells with aligned arrangement of collagen fibers perpendicular against the root surface. This study aimed to promote oriented regeneration of periodontium by structural mimicking of scaffolds. The in vitro and in vivo performances of the scaffolds were further evaluated to test the topographic-guiding and periodontium healing potentials. We also think our research may provide ideas in regeneration of a wide variety of human tissues with a specialized direction.

Microcomputed tomographic and biomechanical analysis of orthodontic microscrew stability under continuous or intermittent force.

PURPOSE: To evaluate and compare the influence of continuous and intermittent forces on stability of titanium microscrews. MATERIALS AND METHODS: One hundred forty-four microscrews were inserted bilaterally in the intraradicular zones of the maxillary first molar and second premolar in 36 beagles. Loads were delivered consecutively in the continuous group (n = 12), in cycles of 12 hours on/paused for 12 hours in intermittent group A (n = 12), and in cycles of 24 hours on/paused for 24 hours in intermittent group B (n = 12). The on/off cycles were repeated for 1, 3, 5, or 7 weeks, after which the animals were sacrificed, and microcomputed tomography (micro-CT) and pull-out testing were performed. RESULTS: The micro-CT parameters of the microscrews in all three groups increased gradually with loading time. The value of peak load at extraction (Fmax) increased and reached a peak at week 5 but dropped slightly at week 7. In the continuous group, all measurements were lower than those in the intermittent groups at all times examined. All values in intermittent group A were higher than those in intermittent group B. CONCLUSION: An intermittent loading regimen appears to be more favorable for obtaining stability than continuous loading, and a 12-hour/12-hour on/off loading cycle is superior to a 24-hour/24-hour on/off protocol in promoting bone-implant contact.