Superior transfection efficiency of phagocytic astrocytes by large chitosan/DNA nanoparticles.

PURPOSE: Mechanism study of why astrocytes isolated from experimental autoimmune encephalomyelitis (EAE)-induced B6 mice or after being exposed to inflammatory factors had the highest transfection efficiency to larger-sized, but not compacted, pspCS/pDNA particles. METHODS: Phosphorylatable short peptide conjugated chitosan (pspCS) was compounded with plasmid DNA (pDNA) at different N:P ratios to form pspCS/pDNA particles of different size and zeta potentials. These pspCS/pDNA particles were used for the transfection of astrocytes isolated from either EAE induced or healthy B6 mice. Transfection efficiency and cell permeability of the particles were determined by the internalization of radio [H3]-labeled plasmid and the expression of a luciferase reporter gene respectively. Phagocytosis of EAE-astrocytes was determined by the internalization of FITC labeled dextran beads. By comparing the transfection efficiency of differently-sized pspCS/pDNA particles to normal and phagocytic astrocytes, with or without cytochalasin D, a phagocytosis inhibitor, in the presence, the contribution of phagocytosis to cell permeability and transfection efficiency was evaluated. RESULTS: In vivo EAE-induction or in vitro inflammatory factors treatment transferred normal astrocytes to be phagocytic astrocytes which underwent phagocytosis, had the highest cell permeability and transfection efficiency to larger-sized pspCS/pDNA particles formed at lower N:P ratios. When phagocytosis was inhibited by cytochalasin D, both cell permeability and transfection efficiency of phagocytic astrocytes to larger were significantly decreased. Thereafter, particle size, not zeta potential, was verified as the key factor for determining whether the particles could be phagocytosed. In addition phagocytosis was successfully induced in ARPE-19 cells as well, which also improved the transfection efficiency of larger pspCS/pDNA particles. CONCLUSION: A generally accepted concept is that the internalization of cationic polymer/pDNA particles, chitosan-DNA complex for instance, is mainly through the procedure of endocytosis of the transfected cells. More compacted particles with higher zeta potential were used to be considered had higher cell permeability and transfection efficiency. However, here we reported that phagocytosis is another important procedure for determining internalization and transfection efficiency of cationic polymer/pDNA nanoparticles, especially for advanced transfection efficiency of large pspCS/pDNA particles. Thus, for gene delivery applications, the environmental condition of the target cells should be seriously considered for selecting an appropriate gene transfer strategies.

The anatomical study of transoral atlantoaxial reduction plate internal fixation.

OBJECTIVE: To study relevant anatomical features of the structures involved in transoral atlanto-axial reduction plate (TARP) internal fixation through transoral approach for treating irreducible atlanto-axial dislocation and providing anatomical basis for the clinical application of TARP. METHODS: Ten fresh craniocervical specimens were microsurgically dissected layer by layer through transoral approach. The stratification of the posterior pharyngeal wall, the course of the vertebral artery, anatomical relationships of the adjacent structures of the atlas and axis, and the closely relevant anatomical parameters for TARP internal fixation were measured. RESULTS: The posterior pharyngeal wall consisted of two layers and two interspaces: the mucosa, prevertebral fascia, retropharyngeal space, and prevertebral space. The range from the anterior edge of the foramen magnum to C(3) could be exposed by this approach. The thickness of the posterior pharyngeal wall was (3.6+/-0.3) mm (ranging 2.9-4.3 mm) at the anterior tubercle of C1, (6.1+/-0.4) mm (ranging 5.2-7.1 mm) at the lateral mass of C(1) and (5.5+/-0.4) mm (ranging 4.3-6.5 mm) at the central part of C(2), respectively. The distance from the incisor tooth to the anterior tubercle of C(1), C(1) screw entry point, and C(2)screw entry point was (82.5+/-7.8) mm (ranging 71.4-96.2 mm), (90.1+/-3.8) mm (ranging 82.2-96.3 mm), and (89.0+/-4.1) mm (ranging 81.3-95.3 mm), respectively. The distance between the vertebral artery at the atlas and the midline was (25.2+/- 2.3) mm (ranging 20.4-29.7 mm) and that between the vertebral artery at the axis and the midline was (18.4+/- 2.6) mm (ranging 13.1-23.0 mm). The allowed width of the atlas and axis for exposure was (39.4+/-2.2) mm (ranging 36.2-42.7 mm) and (39.0+/-2.1) mm (ranging 35.8-42.3 mm), respectively. The distance (a) between the two atlas screw insertion points (center of anterior aspect of C(1) lateral mass) was (31.4+/-3.3) mm (ranging 25.4-36.6 mm). The vertical distance (b) between the line connecting the two C(1) screw entry points and that connecting the two C(2) screw entry points (at the central part of the vertebrae, namely 3-4 mm lateral to the midline of C(2) vertebrae) was (21.3+/-2.7) mm (ranging 19.4-24.3 mm), with an a/b ratio of 1.3-1.5. The screws of TARP had a lateral tilt of 12.2 degrees+/-0.4 degrees(ranging 10.2 degrees-14.6 degrees) at C(1) and a medial tilt of 7.3 degrees+/-0.3 degrees (ranging 5.1 degrees-9.4 degrees) at C(2) relative to the coronal plane. CONCLUSIONS: An atlanto-axial surgery through transoral approach is safe and feasible. This approach is suitable for an anterior TARP internal fixation, and the design of the internal fixation system should be based on the above anatomical data.

Surgical Outcome and Prognostic Analysis of Transoral Atlantoaxial Reduction Plate System for Basilar Invagination: A Voxel-Based Morphometry Study.

BACKGROUND: The use of a transoral atlantoaxial reduction plate (TARP) system is an effective surgical approach for the treatment of basilar invagination. With the aim of improving the therapeutic efficacy of the TARP operation, we conducted a voxel-based morphometric study to quantitatively investigate the descent of the odontoid process and craniocervical volume changes. METHODS: We enrolled 20 patients with basilar invagination who underwent a TARP procedure. Craniocervical computed tomography (CT) scanning and a 3-dimensional (3-D) reconstruction of the craniocervical junction were performed. Craniocervical volumes and odontoid process descent distances were measured preoperatively and postoperatively. Individual neurological function was evaluated according to the Japanese Orthopaedic Association (JOA) scoring system for cervical disorders. Pearson correlation analysis was applied for statistical testing. RESULTS: Surgical efficacy (the JOA-score improvement rate) was significantly associated with the craniocervical volume improvement rate, the odontoid descent distance, and the absolute craniocervical volume changes (p < 0.01 for all), with correlation coefficients (r) of 0.83, 0.80, and 0.61, respectively. No significant correlation was noted between surgical efficacy and age, symptom duration, preoperative neurological function, odontoid process displacement, or change in clivus-odontoid angle (p > 0.05). The craniocervical volume improvement rate was significantly associated with the odontoid descent distance (r = 0.8; p < 0.01), but it was not associated with the odontoid displacement or the change in the clivus-odontoid angle (p > 0.05). CONCLUSIONS: We found that the odontoid descent distance predicted the craniocervical volume improvement rate following TARP procedures in patients with basilar invagination, and we believe that both can serve as predictors of surgical efficacy. We believe that planning the odontoid descent distance preoperatively may help to improve the efficacy of TARP operations. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Treatment of Basilar Invagination With Klippel-Feil Syndrome: Atlantoaxial Joint Distraction and Fixation With Transoral Atlantoaxial Reduction Plate.

BACKGROUND: Klippel-Feil syndrome (KFS) is characterized by congenital vertebral fusion of the cervical spine and a wide spectrum of associated anomalies. In patients with KFS with basilar invagination (BI), compression of the brainstem and upper cervical cord results in neurological deficits, and decompression and occipitocervical reconstruction are required. The highly varied anatomy of KFS makes a posterior occipitocervical fixation strategy challenging. For these patients, the transoral atlantoaxial reduction plate (TARP) operation is an optimal option to perform a direct anterior fixation to achieve stabilization. OBJECTIVE: To evaluate the effectiveness of TARP internal fixation for the treatment of BI with KFS. METHODS: Ten consecutive patients with BI and KFS who underwent TARP reduction and fixation from 2010 to 2012 were reviewed. Clinical assessment and image measurements were performed preoperatively and at the most recent follow-up. Nine patients (9/10) were followed for an average of 31.44 months. RESULTS: Symptoms were alleviated in 9 of 9 patients (100.00%). The odontoid process was ideally corrected with the TARP system. The mean clivus canal angle improved from 124° preoperatively to 152° postoperatively. The average preoperative and postoperative Japanese Orthopedic Association scores were 10.56 (n = 9) and 14.67 (n = 9), respectively, indicating 63.82% improvement. There was bony bridge catenation on the computed tomography scans and no evidence of hardware failure at 6 months. CONCLUSION: The TARP operation is effective and safe for treating patients with BI with KFS. The midterm clinical results were satisfactory.

Transoral approach for revision surgery of os odontoideum with atlantoaxial dislocation.

Revision surgery for os odontoideum with irreducible atlantoaxial dislocation with a transoral approach is not commonly seen. Typically, management of this type of atlantoaxial dislocation is through posterior fixation and fusion or transoral decompression and posterior fusion. This report describes revision surgery in a patient with os odontoideum who was treated with a transoral approach. A 50-year-old man was diagnosed with os odontoideum and atlantoaxial dislocation in 2007 and was treated surgically with posterior occipitocervical internal fixation and fusion. In 2012, he had recurrence of neck pain and numbness of the limbs. Neurologic function was grade D according to the standard neurologic classification of spinal cord injury from the American Spinal Injury Association. Because this was a revision surgery, the internal fixation implant was removed through a posterior approach and a transoral approach was used for release, reduction, internal fixation, and fusion. Two 6-mm cages filled with autogenous bone were introduced into the lateral mass spaces for bony fusion and distraction, and 2 cervical compressive mini-frames were used for fixation. Complete atlantoaxial reduction and decompression of the spinal cord were achieved. The patient reported improvement of symptoms after surgery. Movement of the extremities increased from grade III force to grade V, and neurologic status improved from American Spinal Injury Association grade D to grade E. A transoral approach for release, reduction, bony fusion, and fixation could be an effective procedure for the treatment of os odontoideum with irreducible atlantoaxial dislocation. It provides a new option for bony fusion and internal fixation of the atlantoaxial joint.

Designing a binding interface for control of cancer cell adhesion via 3D topography and metabolic oligosaccharide engineering.

This study combines metabolic oligosaccharide engineering (MOE), a technology where the glycocalyx of living cells is endowed with chemical features not normally found in sugars, with custom-designed three-dimensional biomaterial substrates to enhance the adhesion of cancer cells and control their morphology and gene expression. Specifically, Ac(5)ManNTGc, a thiol-bearing analog of N-acetyl-d-mannosamine (ManNAc) was used to introduce thiolated sialic acids into the glycocalyx of human Jurkat T-lymphoma derived cells. In parallel 2D films and 3D electrospun nanofibrous scaffolds were prepared from polyethersulfone (PES) and (as controls) left unmodified or aminated. Alternately, the materials were malemided or gold-coated to provide bio-orthogonal binding partners for the thiol groups newly expressed on the cell surface. Cell attachment was modulated by both the topography of the substrate surface and by the chemical compatibility of the binding interface between the cell and the substrate; a substantial increase in binding for normally non-adhesive Jurkat line for 3D scaffold compared to 2D surfaces with an added degree of adhesion resulting from chemoselective binding to malemidede-derivatived or gold-coated surfaces. In addition, the morphology of the cells attached to the 3D scaffolds via MOE-mediated adhesion was dramatically altered and the expression of genes involved in cell adhesion changed in a time-dependent manner. This study showed that cell adhesion could be enhanced, gene expression modulated, and cell fate controlled by introducing the 3D topograhical cues into the growth substrate and by creating a glycoengineered binding interface where the chemistry of both the cell surface and biomaterials scaffold was controlled to facilitate a new mode of carbohydrate-mediated adhesion.

Hexosamine analogs: from metabolic glycoengineering to drug discovery.

Metabolic glycoengineering, a technique pioneered almost two decades ago wherein monosaccharide analogs are utilized to install non-natural sugars into the glycocalyx of mammalian cells, has undergone a recent flurry of advances spurred by efforts to make the methodology more efficient. This article describes the versatility of metabolic glycoengineering, which is a prime example of 'chemical glycobiology,' and gives an overview of its capability to endow complex carbohydrates in living cells and animals with interesting (and useful!) functionalities. Then an overview is provided describing how acylated monosaccharides, a class of molecules originally intended to be efficiently-used, membrane-permeable metabolic intermediates, have led to the discovery that a subset of these compounds (e.g. tributanoylated hexosamines) display unanticipated 'scaffold-dependent' activities; this finding establishes these molecules as a versatile platform for drug discovery.

Applied anatomy of transoral atlantoaxial reduction plate internal fixation.

STUDY DESIGN: A C1-C2 operation by the transoral approach was simulated to study the anatomic stratification, various structures, and adjacent relationships. The anatomic parameters in relation to transoral atlantoaxial reduction plate (TARP) internal fixation were measured. OBJECTIVES: To study relevant anatomic features of the structures involved in TARP internal fixation through transoral approach for treating irreducible atlantoaxial dislocation, so as to provide anatomic basis for the clinical application of TARP. SUMMARY AND BACKGROUND DATA: Irreducible anterior atlantoaxial dislocation (IAAD) with ventral spinal cord compression is difficult for surgical correction. Despite previous description of direct plate internal fixations through the transoral approach, the problem has not been fully resolved: the Harms' plate lacked a locking mechanism while the other plates unable to achieve immediate reduction of the atlantoaxial joint. We therefore designed the TARP system with which the decompression, reduction, internal fixation, and fusion procedures could be completed in the same transoral approach. The anatomic structures and stratification involved in the transoral approach, which were seldom addressed in previous anatomic studies, need to be clarified for internal fixation with TARP system. METHODS: Twenty fresh craniocervical specimens were microsurgically dissected layer by layer according to a transoral approach. Stratification of the posterior pharyngeal wall, course of the vertebral artery, anatomic relationships of the adjacent structures of the atlas and axis, and closely relevant anatomic parameters for TARP internal fixation were measured. RESULTS: The posterior pharyngeal wall consisted of two layers and two interspaces: the mucosa, prevertebral fascia, retropharyngeal space, and prevertebral space. The range from the anterior edge of the foramen magnum to C3 could be exposed by this approach. The thickness of the posterior pharyngeal wall was 3.6 +/- 0.3 mm (range, 2.9-4.3 mm) at the anterior tubercle of C1, 6.1 +/- 0.4 mm (range, 5.2-7.1 mm) at lateral mass of C1 and 5.5 +/- 0.4 (range, 4.3-6.5 mm) at the central part of C2, respectively. The distance from the incisor tooth to the anterior tubercle of C1, C1 screw entry point, and C2screw entry point was 82.5 +/- 7.8 mm (range, 71.4-96.2 mm), 90.1 +/- 3.8 mm (range, 82.2-96.3 mm), and 89.0 +/- 4.1 mm (range, 81.3-95.3 mm), respectively. The distance between the vertebral artery at atlas and the midline was 25.2 +/- 2.3 mm (range, 20.4-29.7 mm) and that between the vertebral artery at the axis and the midline was 18.4 +/- 2.6 mm (range, 13.1-23.0 mm). The allowed width of the atlas and axis for exposure was 39.4 +/- 2.2 mm (range, 36.2-42.7 mm) and 39.0 +/- 2.1 mm (range, 35.8-42.3 mm), respectively. The distance (a) between the two atlas screw insertion points (center of anterior aspect of C1 lateral mass) was 31.4 +/- 3.3 mm (range, 25.4-36.6 mm). The vertical distance (b) between the line connecting the two C1 screw entry points and that connecting the two C2 screw entry points (at the central part of the vertebrae, namely, 3 to 4 mm lateral to the midline of C2 vertebrae) was 21.3 +/- 2.7 mm (range, 19.4-24.3 mm), with an a/b ratio of 1.3 to 1.5. The screws of TARP had a lateral tilt of 12.2 degrees +/- 0.4 degrees (range, 10.2 degrees -14.6 degrees ) at C1 and a medial tilt of 7.3 degrees +/- 0.3 degrees (range, 5.1 degrees -9.4 degrees ) at C2 relative to the coronal plane. CONCLUSION: An atlantoaxial surgery through transoral approach is safe and feasible. This approach is suitable for an anterior TARP internal fixation, and the design of the internal fixation system should be based on the above anatomic data.