Micellized protein transduction domain-bone morphogenetic protein-2 accelerates bone healing in a rat tibial distraction osteogenesis model.

The clinical application of growth factors such as recombinant human bone morphogenetic protein-2 (rh-BMP-2), for functional bone regeneration remains challenging due to limited in vivo efficacy and adverse effects of previous modalities. To overcome the instability and short half-life of rh-BMP-2 in vivo, we developed a novel osteogenic supplement by fusing a protein transduction domain (PTD) with BMP-2, effectively creating a prodrug of BMP-2. In this study, we first created an improved PTD-BMP-2 formulation using lipid nanoparticle (LNP) micellization, resulting in downsizing from micrometer to nanometer scale and achieving a more even distribution. The micellized PTD-BMP-2 (mPTD-BMP-2) demonstrated improved distribution and aggregation profiles. As a prodrug of BMP-2, mPTD-BMP-2 successfully activated Smad1/5/8 and induced mineralization with osteogenic gene induction in vitro. In vivo pharmacokinetic analysis revealed that mPTD-BMP-2 had a much more stable pharmacokinetic profile than rh-BMP-2, with a 7.5-fold longer half-life. The in vivo BMP-responsive element (BRE) reporter system was also successfully activated by mPTD-BMP-2. In the in vivo rat tibia distraction osteogenesis (DO) model, micro-computed tomography (micro-CT) scan findings indicated that mPTD-BMP-2 significantly increased bone volume, bone surface, axis moment of inertia (MOI), and polar MOI. Furthermore, it increased the expression of osteogenesis-related genes, and induced bone maturation histologically. Based on these findings, mPTD-BMP-2 could be a promising candidate for the next-generation osteogenesis drug to promote new bone formation in DO surgery. STATEMENT OF SIGNIFICANCE: This study introduces micellized bone morphogenetic protein-2 (mPTD-BMP-2), a next-generation osteogenic supplement that combines protein transduction domain (PTD) and nano-sized micelle formulation technique to improve transduction efficiency and stability. The use of PTD represents a novel approach, and our results demonstrate the superiority of mPTD-BMP-2 over rh-BMP-2 in terms of in vivo pharmacokinetic profile and osteogenic potential, particularly in a rat tibial model of distraction osteogenesis. These findings have significant scientific impact and potential clinical applications in the treatment of bone defects that require distraction osteogenesis. By advancing the field of osteogenic supplements, our study has the potential to contribute to the development of more effective treatments for musculoskeletal disorders.

Glycogen Synthase Kinase-3 Interaction Domain Enhances Phosphorylation of SARS-CoV-2 Nucleocapsid Protein.

A structural protein of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), nucleocapsid (N) protein is phosphorylated by glycogen synthase kinase (GSK)-3 on the serine/arginine (SR) rich motif located in disordered regions. Although phosphorylation by GSK-3ß constitutes a critical event for viral replication, the molecular mechanism underlying N phosphorylation is not well understood. In this study, we found the putative alpha-helix L/FxxxL/AxxRL motif known as the GSK-3 interacting domain (GID), found in many endogenous GSK-3ß binding proteins, such as Axins, FRATs, WWOX, and GSKIP. Indeed, N interacts with GSK-3ß similarly to Axin, and Leu to Glu substitution of the GID abolished the interaction, with loss of N phosphorylation. The N phosphorylation is also required for its structural loading in a virus-like particle (VLP). Compared to other coronaviruses, N of Sarbecovirus lineage including bat RaTG13 harbors a CDK1-primed phosphorylation site and Gly-rich linker for enhanced phosphorylation by GSK-3ß. Furthermore, we found that the S202R mutant found in Delta and R203K/G204R mutant found in the Omicron variant allow increased abundance and hyper-phosphorylation of N. Our observations suggest that GID and mutations for increased phosphorylation in N may have contributed to the evolution of variants.

Acceleration of bone formation by octacalcium phosphate composite in a rat tibia critical-sized defect.

Background: The osteogenic capabilities and biodegradability of octacalcium phosphate (OCP) composites make them unique. Despite the excellent characteristics of OCP, their use is limited due to handling difficulties. In this study, we aimed to evaluate and compare three types of OCPs (cemented OCP (C-OCP), C-OCP with collagen (OCP/Col), and synthetic OCP (S-OCP) with alginate (OCP/Alg)) versus commercially available ß-tricalcium phosphate (ß-TCP) regarding their potential to accelerate bone formation in defective rat tibias. Methods: The specimens with OCP composite were manufactured into 5 â€‹mm cubes and inserted into the segmental defects of rat tibias fixed with an external fixator. In addition, 3 â€‹mm-hole defects in rat tibias were evaluated to compare the graft material properties in different clinical situations. Serial X-ray studies were evaluated weekly and the tibias were harvested at postoperative 6 weeks or 8 weeks for radiologic evaluation. Histological and histomorphometric analyses were performed to evaluate the acceleration of bone formation. Results: In the critical-defect model, OCP/Alg showed bone bridges between segmentally resected bone ends that were comparable to those of ß-TCP. However, differences were observed in the residual graft materials. Most ß-TCP was maintained until 8 weeks postoperatively; however, OCP/Alg was more biodegradable. In addition calcification in the ß-TCP occurred at the directly contacted area between graft particles and bony ingrowth was observed in the region adjacent resected surface of tibia. In contrast, no direct bony ingrowth was observed in OCP-based materials, but osteogenesis induced from resected surface of tibia was more active. In the hole-defect model, OCP/Col accelerated bone formation. ß-TCP and OCP/Alg showed similar patterns with relatively higher biodegradability. In histology, among the OCP-based materials, directly contacted new bone was formed only in OCP/Alg group. The new bone formation in the periphery area of graft materials was much more active in the OCP-based materials, and the newly formed bone showed a thicker trabecular and more mature appearance than the ß-TCP group. Conclusions: In this study, OCP/Alg was equivalent to ß-TCP in the acceleration of bone formation with better biodegradability appropriate for clinical situations in different circumstances. Our OCP/Col composite showed fast degradation, which makes it unsuitable for use in mechanical stress conditions in clinical orthopedic settings. The Translational Potential of this Article: In our research, we compared our various manufactured OCP composites to commercially available ß-TCP in critical-defect rat tibia model. OCP/Col showed acceleration in hole-defect model as previous studies in dental field but in our critical-sized defect model it resorbed fast without acceleration of bony union. OCP/Alg showed matched results compared to ß-TCP and relatively fast resorption so we showed market value in special clinical indication depending on treatment strategy. This is the first OCP composite study in orthopaedics with animal critical-sized tibia bone study and further study should be considered for clinical application based on this study.

Micellized Protein Transduction Domain-Bone Morphogenetic Protein-7 Efficiently Blocks Renal Fibrosis Via Inhibition of Transforming Growth Factor-Beta-Mediated Epithelial-Mesenchymal Transition.

Tubulointerstitial renal fibrosis is a chronic disease process affecting chronic kidney disease (CKD). While the etiological role of transforming growth factor-beta (TGF-ß) is well known for epithelial-mesenchymal transition (EMT) in chronic kidney disease, effective therapeutics for renal fibrosis are largely limited. As a member of the TGF-ß superfamily, bone morphogenetic protein-7 (BMP-7) plays an important role as an endogenous antagonist of TGF-ß, inhibiting fibrotic progression in many organs. However, soluble rhBMP-7 is hardly available for therapeutics due to its limited pharmacodynamic profile and rapid clearance in clinical settings. In this study, we have developed a novel therapeutic approach with protein transduction domain (PTD) fused BMP-7 in micelle (mPTD-BMP-7) for long-range signaling in vivo. Contrary to rhBMP-7 targeting its cognate receptors, the nano-sized mPTD-BMP-7 is transduced into cells through an endosomal pathway and secreted to the exosome having active BMP-7. Further, transduced mPTD-BMP-7 successfully activates SMAD1/5/8 and inhibits the TGF-ß-mediated epithelial-mesenchymal transition process in vitro and in an in vivo unilateral ureter obstruction model. To determine the clinical relevance of our strategy, we also developed an intra-arterial administration of mPTD-BMP-7 through renal artery in pigs. Interestingly, mPTD-BMP-7 through renal artery intervention effectively delivered into Bowman's space and inhibits unilateral ureter obstruction-induced renal fibrosis in pigs. Our results provide a novel therapeutic targeting TGF-ß-mediated renal fibrosis and other organs as well as a clinically available approach for kidney.

Subcutaneous emphysema related to dental procedures.

OBJECTIVES: The objective of this study was to analyze 11 cases of subcutaneous emphysema associated with dental procedures from a single hospital and discuss approaches for accurate diagnosis and treatment of the condition. MATERIALS AND METHODS: The medical records of 11 patients who were treated for subcutaneous emphysema related to dental procedures between January 2009 and April 2017 were analyzed retrospectively. Patients with subcutaneous emphysema within the facial area or that spread to the neck and beyond, including the facial region, were assigned to two groups and compared in terms of age, sex, and durations of antibiotic use, hospitalization, and follow-up until improvement. The correlation between location of the origin tooth and range of emphysema spread was analyzed. RESULTS: The average durations of antibiotic use during conservative treatment and follow-up until improvement were 8.55 days (standard deviation [SD], 4.46 days) and 1.82 weeks (SD, 1.19 weeks), respectively. There was no intergroup difference in duration of antibiotic use (P=0.329) or follow-up (P=0.931). Subcutaneous emphysema was more common after dental procedures involving the maxilla or posterior region than after those involving the mandible or anterior region. There was no significant difference in air distribution according to location of the air orifice (maxilla, mandible, or both; P=0.106). CONCLUSION: Upon adequate conservative treatment accompanied by prophylactic antibiotic treatment considering the risk of infection, patients showed signs of improvement within a few days or weeks. There was no significant difference in treatment period between patients with subcutaneous emphysema localized to the facial region and those with subcutaneous emphysema spreading to the neck or beyond. These findings need to be confirmed by analysis of additional cases.

Three-dimensional analysis of the anterior loop of the inferior alveolar nerve in relation to the growth pattern of the mandibular functional subunit.

BACKGROUND: The purpose of the present study was to investigate the differences in the position and shape of the anterior loop of the inferior alveolar nerve (ALIAN) in relation to the growth pattern of the mandibular functional subunit. METHODS: The study was conducted on 56 patients among those who had undergone orthognathic surgery at the Gangnam Severance Hospital between January 2010 and December 2015. Preoperative computerized tomography (CT) images were analyzed using the Simplant OMS software (ver.14.0 Materialise Medical, Leuven, Belgium). The anterior and inferior lengths of ALIAN (dAnt and dInf) and each length of the mandibular functional subunits were measured. The relationship between dAnt, dInf, and the growth pattern of the mandibular subunits was analyzed. RESULTS: The length of the anterior portion of ALIAN (dAnt) reached 3.34 ± 1.59 mm in prognathism and 1.00 ± 0.97 mm in retrognathism. The length of the inferior portion of ALIAN (dInf) reached 6.81 ± 1.33 mm in prognathism and 5.56 ± 1.34 mm in retrognathism. The analysis of Pearson's correlation coefficiency on all samples showed that the lengths of functional subunits were positively correlated with the loop depth. The length of the symphysis area in prognathic patients was positively correlated with the anterior loop depth (p = 0.005). CONCLUSIONS: Both the anterior and inferior length of ALIAN are longer in prognathic patients. Especially, it seems to be associated with the growth of the symphysis area.