Increased plaque, calculus, and tartar build-up are some of the worst consequences of braces and orthodontic treatment. Orthodontic brackets and wires act as inadvertent traps that collect food and plaque where bacteria accumulate, and subsequently cause heavy inflammation of the gums, cause pain, and generate subsequent bone loss. Current supplemental oral hygiene aids include floss threaders, “super-floss,” interproximal brushes, and most importantly reinforcing proper oral hygiene habits at every orthodontic visit. However, implementing these strategies often render ineffective due to the increased cost of additional appliances and due to patients’ non-compliance at such a young age and their unwillingness to change their oral hygiene habits.

Prior investigations within the last decade have recently identified arginine, an amino acid, as an active ingredient in oral dentifrices that help prevent dental sensitivity and dental decay. These recent investigations and advancements of amino acids in oral health care products have led to questions of whether they can also help in decreasing plaque that subsequently surround braces once orthodontic treatment starts. A recent study at UCLA (2015) has shown in-vitro bacterial plaque and biofilm that is specifically formed from an orthodontic bracket (Braces). Additionally, different combinations of amino acids were added to evaluate its efficacy in reducing this specific biofilm. Resultant data showed that a combination of the D-forms of arginine, alanine, and lysine proved to be the most effective of any combination tested towards the decrease of the bracket-derived plaque.

This previous study in-vitro study and previous notable associations of amino acids in its positive outcomes in oral health care have led us to investigate whether amino-acids (more specifically arginine, alanine, and lysine) in toothpaste can help prevent and reduce plaque in patients with braces. This will be conducted via two randomized, double-blind clinical trials and its analyses using both objective and subjective measures.

Background: Fibromodulin (FMOD) has been demonstrated to decrease scarring in mouse models. We developed an FMOD-based peptide, F06-C40, which has similar effect as FMOD while being more efficient and less costly to produce. In this study, the effect of FMOD and F06-C40 on scars was tested in porcine models for future preclinical studies.

Methods: Full-thickness incisional wounds were generated on the dorsum of Yorkshire pigs or red Duroc pigs to simulate human normal or hypertrophic scarring. FMOD, F06-C40, or PBS was injected intradermally around the wound edge. At 8 weeks post-injury, scar formation was evaluated by gross appearance, histological analysis, and tensile strength.

Results: FMOD and F06-C40 peptide significantly reduced scar formation and increased tensile strength in both Yorkshire pig and red Duroc pig models.

Conclusion: The results indicate F06-C40 has the potential to be developed as a novel therapeutic for improving wound healing in normal and hypertrophic scarring patients.

Current available growth factors for cartilage tissue engineering are flawed by significant functional heterogeneity and side effects. A novel growth factor, Nell-1 (Nel1-like molecule-1), has recently been shown to hold promise for successful cartilage regeneration. NFATc2 (Nuclear factor of Activated T-Cells, Cytoplasmic 2) is a primary response gene for Nell-1 and has been shown to play crucial role in pathogenesis of osteoarthritis in mouse model. In this study we used a Nell-1 haploinsufficient (Nell-1+/-) mouse model to examine the effect of disturbance of Nell-1/NFATc2 axis on adult articular cartilage at different postnatal stages with focus on inflammation.

The articular cartilage of wild-type and Nell-1+/- hip joints at 1, 3, 7, 10 and 12 month old were first evaluated with H&E and Alcian blue staining. Temporospatial pattern of transcription factor NFTAc2, and proinflammatory markers IL-6 and IL-17 were examined by immunohistochemistry. The data from immunohistochemistry at 1 and 3 month samples was validated by qPCR. Nell-1+/- mice showed depletion of matrix proteoglycans and alterations in articular cartilage and subchondral bone plate thicknesses. Significantly, this was associated with reduction of NFATc2 expression along with early-onset and robust expression of proinflammatory markers in adult articular cartilage and periarticular tissues. Current study shows an important mechanism of Nell-1 in regulating adult articular cartilage homeostasis, and inflammation in periarticular tissues via NFATc2 and/or inherent anti-inflammatory nature of Nell-1 molecule itself.

NELL-1 is an osteogenic, secretory molecule previously shown to enhance bone regeneration in multiple rodent and ovine orthopedic defect models. Excitingly, we have recently shown that systemic delivery of NELL-1 induces robust bone formation in healthy and osteoporotic mice; however, like other cytokine therapies, NELL-1 has a relatively short half-life requiring a frequent dosing schedule (q2d). Here, we investigate the potential of PEGylation, a chemical process of adding polyethylene glycol (PEG), to improve NELL-1's pharmacokinetics, while maintaining bioactivity. Three different PEGylation patterns (5K-linear, 20K-linear, and 40K-branched) were covalently attached to NELL-1. First, thermostability and bioactivity of PEG-NELL-1 was compared to unmodified NELL-1 in vitro. Next, we evaluated the biological half-life and osteogenic potential of systemically administered PEG-NELL-1 (5K-linear, 1.25mg/kg, i.v., q4d and q7d) in healthy mice. To monitor the changes in bone mineral density (BMD) and bone turnover rate in vivo, dual-energy X-ray absorptiometry (DXA) and microPET/CT bone scans was performed weekly. Animals were sacrificed after four weeks of treatment for microCT and histological analyses. PEGylation was observed to improve thermostability and biological half-life, while retaining in vitro bioactivity of NELL-1 protein comparable to unmodified NELL-1. In vivo, systemic PEG-NELL-1 therapy significantly increased femoral and lumbar BMD as well as bone turnover rate at overall skeletal sites after four weeks of treatment. MicroCT confirmed significant increases in trabecular BMD and fractional bone volume in PEG-NELL-1-treated groups. Further, immunohistochemistry revealed an increase in osteocalcin expression, while TRAP staining showed a reduction in osteoclast number in PEG-NELL-1 groups. Collectively, PEGylation significantly extends NELL-1's half-life, while retaining osteoinductive potential. Thus, PEG-NELL-1 holds promise for non-invasive, wide clinical application for the treatment of osteopenic disorders.

Systemically delivered NELL-1, a potent pro-osteogenic protein, promotes bone formation in healthy and osteoporotic mouse models. PEGylation of NELL­1 (NELL-PEG) increases the half-life of the protein in a mouse model without compromising its osteogenic potential, thereby improving its pharmacokinetics upon systemic delivery. Weekly intravenous (IV) NELL-PEG injection significantly enhances overall bone properties. This study consists of a two-fold approach; a biodistribution test and an in vivo osteogenic potential test. The biodistribution test compared two commonly used administration methods for drug delivery other than IV – intraperitoneal (IP) and subcutaneous (SC) – to examine NELL-PEG biodistribution in mice. Compared to a single-dose SC injection (1.25 mg/kg), a single-dose IP administration yielded a higher protein uptake in the targeted bone sites. When the IP injection dose was doubled to 2.5 mg/kg, the protein remained in the femurs, tibias, and vertebrae for up to 72 hours. Next, based on the results of the biodistribution study, IP administration was selected to further investigate the in vivo osteogenic effects of weekly NELL-PEG injection (q7d). In vivo, the IP administered NELL-PEG group showed significantly greater bone mineral density (BMD), bone volume fraction (BV/TV), and trabecular bone formation in the targeted bone sites compared to the PBS control. In summary, weekly NELL-PEG injection via IP administration successfully enhanced the overall bone quality. These findings demonstrate that systemic delivery of NELL-PEG via IP administration may serve as an effective osteogenic therapy for preventing and treating osteoporosis.

Secretory protein neural EGFL like 1 (Nell-1) has been shown to exert potent osteogenic effects in multiple small and large animal models. Here, we identified and validated contactin-associated protein-like 4 (Cntnap4) as the first Nell-1 specific cell surface receptor. Cntnap4, a transmembrane neurexin superfamily protein member, has critical functions in neurodevelopment but no known function in osteogenesis. In the present study, we demonstrate that: 1) Nell-1 and Cntnap4 co-localize on the surface of osteogenic-committed cells, 2) Nell-1 and Cntnap4 exhibit high-affinity interactions, 3) Cntnap4 knockdown reduces Nell-1-responsive osteogenesis, and 4) Cntnap4 mediates Nell-1-activated MAPK and Wnt signaling in osteogenic-committed cells. Taken together, these findings and recent literature further the notion of potential neuroskeletal interplay. This unanticipated discovery connecting Cntnap4 to Nell-1 during osteogenesis provides a foundational framework for further exploration of a novel functional axis as it relates to skeletal and potential neuronal function.

Osteoporosis affects more than 200 million people worldwide. Therapeutic approaches to osteoporotic bone loss have focused primarily on anti-resorptive agents but with significant clinical limitations. Therefore, there is pressing need to develop new agents, particularly anabolic therapies that promote bone formation. Microgravity-induced bone loss is an important medical consideration of long-duration space flight and has been measured to degrade bone at a rate up to four times faster than disuse atrophy on Earth. As such, the microgravity model of osteoporosis serves as a proxy for extreme cases of disuse osteoporosis on the ground.

NELL-1 is a potent pro-osteogenic protein most studied for its local bone forming effects. Studies have demonstrated that recombinant NELL-1 acts as a combined anabolic and anti-osteoclastic agent to protect against osteoporotic bone loss. In addition, through PEGylation, we successfully increased the circulation half-life of NELL-1 (NELL- PEG) to be up to six times longer. In this study, the therapeutic effects of NELL-PEG were further amplified by conjugation with inactivated bisphosphonate (BP) to increase bone binding affinity. We verified inactivated BP acts only as a targeting molecule to optimize NELL-PEG deposition in bone and does not exert its own anti-osteoclastic effect. This has led to my central hypothesis that systemic BP-NELL-PEG therapy will prevent bone loss and promote new bone formation in long-duration spaceflight. This study was a part of the Rodent Research 5 spaceflight mission and supported by the Center for the Advancement of Science in Space (CASIS) and the National Aeronautics and Space Administration (NASA).

I tested my hypothesis through two AIMs: (1) Pre-Flight Ground Operation to optimize the delivery route and dose of systemic BP-NELL-PEG for the reversal of ovariectomy (OVX)-induced osteoporosis in mice; (2) Flight Operation to examine the effects of optimized systemic delivery of BP-NELL-PEG for the reversal of microgravity-induced osteoporosis in mice. Results showed that systemic BP-NELL-PEG therapy successfully prevented bone loss from long-duration spaceflight by promoting new bone generation. Furthermore, our findings with inactivated BP conjugation introduce a promising platform for improving skeletal delivery of systemic therapeutics for bone diseases.

Age-related osteoporosis is commonly associated with decreased bone mass, deterioration of bone microarchitecture, and increased bone marrow adipose tissue. Various risk factors and biological changes that occur with aging exponentially increase the prevalence of osteoporosis, which affects more than 200 million people worldwide. With the staggering biomedical burden, significant efforts have been made to explore new therapies that target the primary regulator of bone metabolism, the Wnt signaling pathway, and suppress PPARγ-mediated adipogenesis to enhance therapeutic efficacy. NELL-1 is a potent pro-osteogenic protein that has been well-characterized for its local and systemic bone-forming effects. Previously, we discovered that PEGylation of NELL-1 (NELL-PEG) significantly improved NELL-1’s bioactivities and potency for local and systemic use without inducing considerable biotoxicity. The current study seeks to explore the feasibility of NELL-PEG, with the combination of PPARγ suppression, as a more scientifically sophisticated osteoporosis therapy in which bone formation is promoted and lipid accumulation in the bone marrow is suppressed. We discovered that co-treatment with NELL-PEG and PPARγ suppression significantly enhanced the expression of the Wnt pathway, which led to increased bone formation and reduced bone resorption. In addition, the dual treatment strategy also suppressed the expression of PPARγ and CEBPa, which resulted in decreased bone marrow fat formation. Moreover, the dual treatment strategy was found to significantly enhance cell proliferation and reduce cell apoptosis. These findings prove the feasibility of NELL-1 for use in combination with other molecules in order to enhance the efficacy and safety profile of the co-treatment strategy. The feasibility and readiness of the NELL-1 protein to be modified and used in combination with other molecules, without inducing undesirable side effects, is one of NELL-1’s unique features, in addition to its pro-osteogenic, anti-osteoclastic, and anti-adipogenic properties. In conclusion, optimizing the novel combination of a NELL-1-based therapy with a PPARγ suppressor could be crucial for facilitating long-term drug formulations that have improved efficacy and safety profiles. This development could serve as a novel platform that can be applied to other Wnt-ligand targeting therapies for the purpose of improving the efficacy and safety profile of current standard of care for osteoporosis.

With an increase in the number of annual craniomaxillofacial orthopaedic procedures being performed, and an understanding of the drawbacks of autologous bone grafting, the bone graft substitute market is burgeoning. Several bone graft alternatives, including, FDA-approved osteogenic molecule, Bone Morphogenetic Protein-2 have shown promise in promoting large volumes of de novo bone formation, however are ladened with several adverse effects, hampering their clinical application. Perivascular Stem Cells (PSC) are a prospectively purified mesenchymal stem cell population isolated from adipose tissue by fluorescent-activated cell sorting (FACS), and have previously been shown to induce robust bone formation in small and large animal models. Herein, by utilizing species-specific MHC immunohistochemistry, we demonstrated that PSC elicit their osteogenic effect primarily via a paracrine-mediate mechanism. Subsequently, after resolving that PSC mediate host mesenchymal stem cells, via the secretion of pro-osteogenic molecules, RNA sequencing was performed to compare PSC with their unpurified predecessor, stromal vascular fraction (SVF). It was observed that Wnt-related secretory molecules, WISP-1 and Wnt16 were upregulated in PSC by 72- and 21- fold, respectively, when compared to SVF. Gain-of-function assays for these two secretory molecules revealed a significant increase in osteogenic markers, whereby a significant decrease in osteogenic markers was observed when WISP-1 or Wnt16 were knocked-down in PSC. Wnt16 was observed to indeed be a mixed ligand and demonstrate activation of both the canonical and noncanonical Wnt pathways. In aggregate, these data demonstrate that PSC promote osteogenesis via a primarily paracrine-mediate secretion of pro-osteogenic molecules, of which WISP-1 and Wnt16 are highly upregulated and play a functional and necessary role in PSC osteogenesis.

Introduction: Orthodontic treatment time on average lasts 24 months. The long duration of the treatment is one of the most frequent complaints of patients. Moreover, the risks associated include caries, root resorption, decalcification, periodontal disease, and others. For this reason, there is pressing need to develop new methods to accelerate orthodontic tooth movement. The Corticopuncture (CP), Photobiomodulation (PBM) and the combination of both methods have shown in prior studies significant results of increasing tooth displacement. Based on these data, we hypothesize that CP, PBM and CP+PBM can enhance alveolar bone remodeling, rate of tooth movement and minimize root resorption by stimulating metabolic changes and cellular differentiation. I tested my hypothesis through four aims: (1) to assess the rate of tooth displacement on days 1,3 and 7; (2) to investigate the expression levels on days 1,3 and 7; (3) to analyze the changes on bone remodeling on days 1,3 and 7; and (4) to asses root resorption crater volume after 7 days. Methods: Orthodontic tooth movement was induced in 27 male Wistar rats. CP procedure included 3 perforations. GaAlAs diode laser was performed every other day for 7 days (810 nm, 100 mW, 15 s). Gingival tissue was collected and qPCR to isolate and quantify mRNA levels. The tooth displacements were measured directly from rat’s mouth. Bone responses at the tension and compression sites and root resorption were evaluated from micro-CT images. Hemi-maxillae from all groups were dissected and prepared for histological and immunohistochemistry analysis. Results and conclusions: Our findings suggest that all (1) ATM groups showed increase tooth displacement compared to control after 7 days (30% PBM, 45% CP and 58% CP+PBM); (2) all ATM groups showed less RR volume compared to the control group; (4) PBM showed a more significant role in increasing bone formation and CP showed a more significant role in catabolic activity; (5) CP+PBM illustrated synergic effects; (6) higher expression of VEGF, Prx1 and GLUT1 found in all methods; (7) and that PBM can enhance tooth movement by stimulating the RANK/RANKL/OPG system.