Efficacy of Cranial Orthosis for Plagiocephaly Based on 2D and 3D Evaluation.

Background With the advent of cranial orthoses as therapeutic medical devices for the treatment of severe positional head deformities in Japan, an increasing number of patients are being treated with them. However, assessing the effectiveness of a treatment is often difficult due to the use of different metrics. This study aimed to evaluate the effectiveness of cranial orthoses for deformational plagiocephaly using two- (2D) and three-dimensional (3D) evaluation metrics. Methods We conducted a retrospective study of infant patients with deformational plagiocephaly who underwent cranial orthosis treatment. We evaluated the severity of deformational plagiocephaly using cranial asymmetry (CA) and the cranial vault asymmetry index (CVAI) as 2D metrics, and anterior and posterior symmetry ratios as 3D metrics. The patients were divided into 24 subgroups based on the initial severity of each outcome and their age at the start of treatment. We analyzed the changes in outcomes and correlations within improvements across the age and severity categories. Results Overall, 1,038 infants were included in this study. The mean CA, CVAI, and anterior and posterior symmetry ratios improved significantly after cranial orthosis treatment. The improvement in each score was greater in patients with more severe initial deformities and in those who underwent treatment at a younger age. Conclusion Cranial orthosis treatment was effective in correcting deformational plagiocephaly in infants, as demonstrated by improvements in both 2D and 3D metrics. Patients with more severe initial deformities and those who underwent treatment at a younger age showed greater improvement.

Evaluation of EV Storage Buffer for Efficient Preservation of Engineered Extracellular Vesicles.

Extracellular vesicles (EVs), detectable in all bodily fluids, mediate intercellular communication by transporting molecules between cells. The capacity of EVs to transport molecules between distant organs has drawn interest for clinical applications in diagnostics and therapeutics. Although EVs hold potential for nucleic-acid-based and other molecular therapeutics, the lack of standardized technologies, including isolation, characterization, and storage, leaves many challenges for clinical applications, potentially resulting in misinterpretation of crucial findings. Previously, several groups demonstrated the problems of commonly used storage methods that distort EV integrity. This work aims to evaluate the process to optimize the storage conditions of EVs and then characterize them according to the experimental conditions and the models used previously. Our study reports a highly efficient EV storage condition, focusing on EV capacity to protect their molecular cargo from biological, chemical, and mechanical damage. Compared with commonly used EV storage conditions, our EV storage buffer leads to less size and particle number variation at both 4 °C and -80 °C, enhancing the ability to protect EVs while maintaining targeting functionality.

Erratum: Engineering Extracellular Vesicles to Target Pancreatic Tissue In Vivo: Erratum.

[This corrects the article DOI: 10.7150/ntno.54879.].

Advances of engineered extracellular vesicles-based therapeutics strategy.

Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer membrane-bound vesicles which encapsulate bioactive molecules, such as nucleic acids, proteins, and lipids. They mediate intercellular communication through transporting internally packaged molecules, making them attractive therapeutics carriers. Over the last decades, a significant amount of research has implied the potential of EVs servings as drug delivery vehicles for nuclear acids, proteins, and small molecular drugs. However, several challenges remain unresolved before the clinical application of EV-based therapeutics, including lack of specificity, stability, biodistribution, storage, large-scale manufacturing, and the comprehensive analysis of EV composition. Technical development is essential to overcome these issues and enhance the pre-clinical therapeutic effects. In this review, we summarize the current advancements in EV engineering which demonstrate their therapeutic potential.

Design and Evaluation of Engineered Extracellular Vesicle (EV)-Based Targeting for EGFR-Overexpressing Tumor Cells Using Monobody Display.

BACKGROUND: Extracellular vesicles (EVs) are attracting interest as a new class of drug delivery vehicles due to their intrinsic nature of biomolecular transport in the body. We previously demonstrated that EV surface modification with tissue-specific molecules accomplished targeted EV-mediated DNA delivery. METHODS: Here, we describe reliable methods for (i) generating EGFR tumor-targeting EVs via the display of high-affinity monobodies and (ii) in vitro measurement of EV binding using fluorescence and bioluminescence labeling. Monobodies are a well-suited class of small (10 kDa) non-antibody scaffolds derived from the human fibronectin type III (FN3) domain. RESULTS: The recombinant protein consists of the EGFR-targeting monobody fused to the EV-binding domain of lactadherin (C1C2), enabling the monobody displayed on the surface of the EVs. In addition, the use of bioluminescence or fluorescence molecules on the EV surface allows for the assessment of EV binding to the target cells. CONCLUSIONS: In this paper, we describe methods of EV engineering to generate targeted delivery vehicles using monobodies that will have diverse applications to furnish future EV therapeutic development, including qualitative and quantitative in vitro evaluation for their binding capacity.

Local Injection of Hydroxyapatite Electret Ameliorated Infarct Size After Myocardial Infarction.

Background: Previous studies showed that hydroxyapatite electret (HAE) accelerates the regeneration of vascular endothelial cells and angiogenesis. This study investigated the effects of HAE in myocardial infarction (MI) model mice. Methods and Results: MI was induced in mice by ligating the left anterior descending artery. Immediately after ligation, HAE, non-polarized hydroxyapatite (HAN), or water (control) was injected into the infarct border myocardium. Functional and histological analyses were performed 2 weeks later. Echocardiography revealed that HAE injection preserved left ventricular systolic function and the wall thickness of the scar, whereas HAN-injected mice had impaired cardiac function and thinning of the wall, similar to control mice. Histological assessment showed that HAE injection significantly attenuated the length of the scar lesion. There was significant accumulation of CD31-positive cells and increased expression of vascular endothelial growth factor (Vegf), intercellular adhesion molecule-1 (Icam1), vascular cell adhesion molecule-1 (Vcam1), hypoxia-inducible factor-1α (Hif1a), and C-X-C motif chemokine ligand 12 (Cxcl12) genes in the infarct border zone of HAE-injected mice. These effects were not induced by HAN injection. Anti-VEGFR2 antibody canceled the beneficial effect of HAE. In vitro experiments in a human cardiovascular endothelial cell line showed that HAE dose-dependently increased VEGFA expression. Conclusions: Local injection of HAE attenuated infarct size and improved cardiac function after MI, probably due to angiogenesis. The electric charge of HAE may stimulate angiogenesis via HIF1α-CXCL12/VEGF signaling.

Engineering Extracellular Vesicles to Target Pancreatic Tissue In Vivo.

Extracellular vesicles (EVs) are naturally released, cell-derived vesicles that mediate intracellular communication, in part, by transferring genetic information and, thus, have the potential to be modified for use as a therapeutic gene or drug delivery vehicle. Advances in EV engineering suggest that directed delivery can be accomplished via surface alterations. Here we assess enriched delivery of engineered EVs displaying an organ targeting peptide specific to the pancreas. We first characterized the size, morphology, and surface markers of engineered EVs that were decorated with a recombinant protein specific to pancreatic ß-cells. This ß-cell-specific recombinant protein consists of the peptide p88 fused to the EV-binding domain of lactadherin (C1C2). These engineered EVs, p88-EVs, specifically bound to pancreatic ß-cells in culture and transferred encapsulated plasmid DNA (pDNA) as early as in 10 min suggesting that the internalization of peptide-bearing EVs is a rapid process. Biodistribution of p88-EVs administrated intravenously into mice showed an altered pattern of EV localization and improved DNA delivery to the pancreas relative to control EVs, as well as an accumulation of targeting EVs to the pancreas using luciferase activity as a readout. These findings demonstrate that systemic administration of engineered EVs can efficiently deliver their cargo as gene carriers to targeted organs in live animals.

Sparsely methylated mitochondrial cell free DNA released from cardiomyocytes contributes to systemic inflammatory response accompanied by atrial fibrillation.

Systemic inflammation is assumed to be the consequence and the cause of atrial fibrillation (AF); however, the underlying mechanism remains unclear. We aimed to evaluate the level of cell-free DNA (cfDNA) in patients with AF and AF mimicking models, and to illuminate its impact on inflammation. Peripheral blood was obtained from 54 patients with AF and 104 non-AF controls, and cfDNA was extracted. We extracted total cfDNA from conditioned medium after rapid pacing to HL-1 cells. Nuclear and mitochondrial DNA were separately extracted and fragmented to simulate nuclear-cfDNA (n-cfDNA) and mitochondrial-cfDNA (mt-cfDNA). The AF group showed higher cfDNA concentration than the non-AF group (12.6 [9.0-17.1] vs. 8.1 [5.3-10.8] [ng/mL], p < 0.001). The copy numbers of n-cfDNA and mt-cfDNA were higher in AF groups than in non-AF groups; the difference of mt-cfDNA was particularly apparent (p = 0.011 and p < 0.001, respectively). Administration of total cfDNA and mt-cfDNA to macrophages significantly promoted IL-1ß and IL-6 expression through TLR9, whereas n-cfDNA did not. Induction of cytokine expression by methylated mt-cfDNA was lower than that by unmethylated mt-cfDNA. Collectively, AF was associated with an increased cfDNA level, especially mt-cfDNA. Sparsely methylated mt-cfDNA released from cardiomyocytes may be involved in sterile systemic inflammation accompanied by AF.

Cardiomyocyte uptake mechanism of a hydroxyapatite nanoparticle mediated gene delivery system.

Gene therapy has been explored as a future alternative for treating heart disease. Among several gene delivery systems aimed at penetrating specific target cells, we focused on safe and non-viral gene delivery materials with a high transfection efficiency. Although various techniques have been developed, the mechanisms underlying the cellular uptake of gene delivery materials have not yet been sufficiently studied in cardiomyocytes. The aim of this study was to determine how hydroxyapatite (HAp) nanoparticles contribute to the delivery of plasmid DNA (pDNA) into cardiomyocytes. We fabricated HAp nanoparticles using the water-in-oil (W/O) emulsion method and used these nanoparticles as the delivery vector for transfecting cardiomyocyte-derived HL-1 cells. HAp exhibited particles on the nanoscale and with a low cytotoxicity in HL-1 cells. The transfection assay performed with several endocytosis inhibitors suggested that the HAp/pDNA complexes were internalized by HL-1 cells through macropinocytosis. Furthermore, this HL-1 cell uptake was generated in response to HAp stimulation. Thus, HAp is a positive regulator of macropinocytosis in HL-1 cells and a good system for gene delivery in cardiomyocytes.

A Comprehensive Review of Cancer MicroRNA Therapeutic Delivery Strategies.

In the field of molecular oncology, microRNAs (miRNAs) and their role in regulating physiological processes and cancer pathogenesis have been a revolutionary discovery over the last decade. It is now considered that miRNA dysregulation influences critical molecular pathways involved in tumor progression, invasion, angiogenesis and metastasis in a wide range of cancer types. Hence, altering miRNA levels in cancer cells has promising potential as a therapeutic intervention, which is discussed in many other articles in this Special Issue. Some of the most significant hurdles in therapeutic miRNA usage are the stability and the delivery system. In this review, we cover a comprehensive update on the challenges and strategies for the development of therapeutic miRNA delivery systems that includes virus-based delivery, non-viral delivery (artificial lipid-based vesicles, polymer-based or chemical structures), and recently emerged extracellular vesicle (EV)-based delivery systems.

A case of secretory breast cancer in a 6 year-old girl: is it possible to make a correct preoperative diagnosis?

Secretory breast carcinoma constitutes the majority of breast cancers in children and young people less than 20 years of age. Noninvasive examination is particularly necessary for the diagnosis of breast carcinoma in children. Herein, we report a case of secretory breast carcinoma in a 6-year-old girl with psychomotor retardation. She was referred to our outpatient clinic for evaluation of a palpable mass in her left breast. A hard mass, rather than the increase in size typical of premature thelarche, was palpated. An excision biopsy was performed. Pathological findings revealed an invasive secretory breast carcinoma. We performed a retrospective review of the preoperative findings of this case, and compared it to the pathological diagnosis. Elastography, which can be performed without deep sedation or general anesthesia and without causing pain, resulted in a stiffness score of 4; however, the distinction between benign and malignant tumors on elastography, which is important to decide the intra-operative procedures, was not sufficient according to the Japanese breast cancer society clinical guidelines. This is the first report of secretory breast carcinoma in a child with a stiffness score determined by tissue elasticity imaging. A breast mass in a child with a high stiffness score of more than 4 on elastography should be referred for invasive diagnostic procedures, such as fine needle aspiration or excisional biopsy. According to our experience, an accurate preoperative diagnosis could be possible for malignant breast tumors in children. Such parameters as stiffness score on elastography are practical, noninvasive, and objective diagnostic tools for the accurate preoperative diagnosis of breast tumors in children.

Tracheal cartilage growth promotion by intra-tracheal administration of basic FGF.

PURPOSE: This study aimed to investigate whether intra-tracheal administration of basic fibroblast growth factor (b-FGF) promotes the growth of tracheal cartilage. METHODS: Trachea of 4-week old mice were intubated and 2.5 µg b-FGF administered (Group 4) for periods from 1 to 5 days. Cervical tracheal outer diameter and tracheal ring length were compared in Group 1 (no intervention), Group 2 (tracheal intubation), Group 3 (intra-tracheal administration of distilled water) and Group 4, at 8 weeks of age. Outer diameter and tracheal ring length in Group 4 were also compared with that in Group 1 at 12 and 16 weeks of age. RESULTS: At 8 weeks of age, tracheal ring length with b-FGF administration for more than 4 days in Group 4 was significantly increased over that following 1-day administration. At 8 weeks of age, mean outer diameter and the mean tracheal ring length in Group 4 were significantly greater than in the other groups. Mean outer diameter and mean tracheal ring length were significantly greater in Group 4 than in Group 1 at 12 and 16 weeks of age. CONCLUSION: This study has shown that intra-tracheal administration of b-FGF enlarges the tracheal lumen.

An oriented hydroxyapatite film with arrayed plate-like particles enhance chondrogenic differentiation of ATDC5 cells.

Osteochondral defects of articular cartilage cannot regenerate spontaneously. For its surgical treatment, advancements in cartilage tissue engineering have particularly focused on subchondral bone lesions that tend to delay healing. Therefore, it is important to understand interactions between subchondral bone and chondrocytes. This study aimed to investigate the behavior of chondrogenic ATDC5 cells on oriented hydroxyapatite (HAp) films that mimic bone surfaces. HAp nanoparticles prepared herein were needle like and plate like. HAp films were formed through self-organization of the nanoparticles and had 2D structures regularly arrayed with the particles. Both films prominently comprised a-plane orientation surfaces but differed in the degree of hydrophilicity because of the patterns of particle self-assembly. ATDC5 cells cultured on the HAp film with plate-like particles could adhered in a shorter period but could not spread. The adhesive force of cells was weaker with the hydrophilic surface than with other surfaces, as determined using a trypsin-based cell detachment assay. In addition, ATDC5 cells displayed enhanced proliferation and chondrogenic differentiation. Our results suggest that the oriented HAp film formed using plate-like particles provided chondrogenic cells with a desired scaffold as that of subchondral bone to increase cell proliferation and differentiation.

Fabrication of an anatomy-mimicking BIO-AIR-TUBE with engineered cartilage.

INTRODUCTION: We devised a strategy for the fabrication of an 'anatomy-mimicking' cylinder-type engineered trachea combined with cartilage engineering. The engineered BIOTUBEs are used to support the architecture of the body tissue, for long-segment trachea (>5 cm) with carinal reconstruction. The aim of the present study was to fabricate an anatomy-mimicking cylinder-type regenerative airway, and investigate its applicability in a rabbit model. METHODS: Collagen sponge rings (diameter: 6 mm) were arranged on a silicon tube (diameter: 6 mm) at 2-mm intervals. Chondrocytes from the auricular cartilage were seeded onto collagen sponges immediately prior to implantation in an autologous manner. These constructs were embedded in dorsal subcutaneous pouches of rabbits. One month after implantation, the constructs were retrieved for histological examination. In addition, cervical tracheal sleeve resection was performed, and these engineered constructs were implanted into defective airways through end-to-end anastomosis. RESULTS: One month after implantation, the engineered constructs exhibited similar rigidity and flexibility to those observed with the native trachea. Through histological examination, the constructs showed an anatomy-mimicking tracheal architecture. In addition, the engineered constructs could be anastomosed to the native trachea without air leakage. CONCLUSION: The present study provides the possibility of generating anatomy-mimicking cylinder-type airways, termed BIO-AIR-TUBEs, that engineer cartilage in an in-vivo culture system. This approach involves the use of BIOTUBEs formed via in-body tissue architecture technology. Therefore, the BIO-AIR-TUBE may be useful as the basic architecture of artificial airways.

The effect of glucose modification of hydroxyapatite nanoparticles on gene delivery.

Surface modification techniques have been employed for the use of biocompatible and bioresorbable hydroxyapatite (HAp) nanoparticles in cell biology and medicine for the delivery of bioactive molecules. We demonstrated the effects of glucose modification of HAp (GlcHAp) on the transfection efficiency in endothelial cells. After preparing homogeneous HAp nanoparticles with a microemulsion technique, the particles with or without glucose modification and plasmid DNA (pDNA) complexes were transfected into endothelial cells. The transfection efficiency of GlcHAp/pDNA was higher than that of HAp/pDNA. To elucidate the mechanism underlying the improvement in the transfection efficiency following glucose modification, the uptake route into the cells and the inhibition of DNA degradation were investigated. GlcHAp/pDNA enhanced the transfection efficiency after interacting with the glucose transporter 1, as observed by the selective inhibitor assay. In addition, GlcHAp/pDNA was more stable than HAp/pDNA in the DNA degradation assay. Our results suggest that the glucose modification could promote the uptake of HAp nanoparticles by cells and protect the internalized DNA; properties essential for non-viral transfection carriers. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 61-66, 2019.

Long-term follow-up of tracheal cartilage growth promotion by intratracheal injection of basic fibroblast growth factor.

BACKGROUND: Intratracheal injection of basic fibroblast growth factor (b-FGF) has been shown to enlarge the tracheal lumen 4 weeks after treatment. The objective of this study was to investigate the long-term effect of tracheal cartilage growth promotion by intratracheal injection of b-FGF. MATERIALS AND METHODS: New Zealand white rabbits were classified into four groups to receive either distilled water alone (Group 1; n = 16; control), 40 µg (Group 2; n = 10), 100 µg (Group 3; n = 13), or 200 µg (Group 4; n = 16) of b-FGF dissolved in water. The treatment was injected into the posterior wall of the cervical trachea using a tracheoscope. The animals were sacrificed 4 or 12 weeks later. RESULTS: Four weeks after treatment, the mean luminal areas of tracheas for Groups 1, 2, 3, and 4 were 27.2, 25.6, 32.2, and 36.2 mm2, respectively. At 12 weeks, these were 29.3, 37.9, 42.5, and 56.0 mm2, respectively. The levels of glycosaminoglycan at 12 weeks were 93.9, 152.5, 123.2, and 210.6 µg/mg, respectively. At 12 weeks, the levels of type II collagen were 77.2, 133.1, 99.2, and 148.9 µg/mg, respectively. CONCLUSION: Twelve weeks after a single injection of b-FGF, the mean luminal area of the trachea continued to increase.

Tracheal cartilage growth by intratracheal injection of basic fibroblast growth factor.

BACKGROUND/PURPOSE: We have previously shown that intratracheal injection of slowly released (in gelatin) basic fibroblast growth factor (bFGF) significantly enlarged the tracheal lumen by a slight margin. This study aimed to investigate differences in tracheal cartilage growth by the intratracheal injection of bFGF doses in a rabbit model. METHODS: Water (group 1; n=7; control) or 100µg (group 2; n=8) or 200µg (group 3; n=8) of bFGF dissolved in water was injected into the posterior wall of the cervical trachea of New Zealand white rabbits using a tracheoscope. All animals were sacrificed four weeks later. RESULTS: The mean circumferences of cervical tracheas for groups 1, 2, and 3 were 18.8±0.83, 21.1±2.0, and 22.1±1.3mm, respectively. A significant difference was found between groups 1 and 2 (P=0.034) and groups 1 and 3 (P=0.004). The mean luminal areas of cervical tracheas for groups 1, 2, and 3 were 27.0±2.1, 32.2±4.8, and 36.3±4.6mm2, respectively. A significant difference was found between groups 1 and 3 (P=0.001). CONCLUSION: Intratracheal injection of bFGF in the dose range used significantly promoted the growth of tracheal cartilage in a rabbit model. LEVELS OF EVIDENCE: Level II at treatment study (animal experiment).

Patch tracheoplasty in body tissue engineering using collagenous connective tissue membranes (biosheets).

BACKGROUND: Collagenous connective tissue membranes (biosheets) are useful for engineering cardiovascular tissue in tissue engineering. The aim was to evaluate the use of biosheets as a potential tracheal substitute material in vivo in a rabbit model. METHODS: Group 1: Rectangular-shaped Gore-Tex (4×7mm) was implanted into a 3×6mm defect created in the midventral portion of the cervical trachea. Group 2: Rectangular-shaped dermis was implanted into a tracheotomy of similar size. Group 3: Biosheets were prepared by embedding silicone moulds in dorsal subcutaneous pouches in rabbits for 1month. Rectangular-shaped biosheets were implanted into a tracheotomy of similar size in an autologous fashion. All groups (each containing 10 animals) were sacrificed 4weeks after implantation. MAIN RESULTS: All materials maintained airway structure for up to 4weeks after implantation. Regenerative cartilage in implanted Biosheets in group 3 was confirmed by histological analysis. Tracheal epithelial regeneration occurred in the internal lumen of group 3. There were significant differences in the amounts of collagen type II and glycosaminoglycan between group 3 and group 1 or 2. CONCLUSION: We confirm that cartilage can self-regenerate onto an airway patch using Biosheets.

Promotion of muscle regeneration by myoblast transplantation combined with the controlled and sustained release of bFGFcpr.

Although myoblast transplantation is an attractive method for muscle regeneration, its efficiency remains limited. The efficacy of myoblast transplantation in combination with the controlled and sustained delivery of basic fibroblast growth factor (bFGF) was investigated. Defects of thigh muscle in Sprague-Dawley (SD) rats were created, and GFP-positive myoblasts were subsequently transplanted. The rats were divided into three groups. In control group 1 (C1) only myoblasts were transplanted, while in control group 2 (C2) myoblasts were introduced along with empty gelatin hydrogel microspheres. In the experimental group (Ex), myoblasts were transplanted along with bFGF incorporated into gelatin hydrogel microspheres. Four weeks after transplantation, GFP-positive myoblasts were found to be integrated into the recipient muscle and to contribute to muscle fibre regeneration in all groups. A significantly higher expression level of GFP in the Ex group demonstrated that the survival rate of transplanted myoblasts in Ex was remarkably improved compared with that in C1 and C2. Furthermore, myofibre regeneration, characterized by centralization of the nuclei, was markedly accelerated in Ex. The expression level of CD31 in Ex was higher than that in both C1 and C2, but the differences were not statistically significant. A significantly higher expression level of Myogenin and a lower expression level of MyoD1 were both observed in Ex after 4 weeks, suggesting the promotion of differentiation to myotubes. Our findings suggest that the controlled and sustained release of bFGF from gelatin hydrogel microspheres improves the survival rate of transplanted myoblasts and promotes muscle regeneration by facilitating myogenesis rather than angiogenesis.

Tailor-made treatment combined with proton beam therapy for children with genitourinary/pelvic rhabdomyosarcoma.

BACKGROUND: Rhabdomyosarcoma (RMS) is one of the most common soft tissue sarcomas among children. Patients who developed genitourinary/pelvic rhabdomyosarcoma (GU/P-RMS) have a higher complication ratio and relatively poorer event free survival, with local therapy being very important. While proton beam therapy (PBT) is expected to reduce co-morbidity, especially for children, this lacks firm evidence and analysis. We analyzed GU/P-RMS children who had undergone multimodal therapy combined with PBT at a single institution. METHOD: We retrospectively reviewed charts of children with GU/P-RMS treated from January 2007 to May 2013 at the University of Tsukuba Hospital who had undergone multimodal therapy with PBT. RESULTS: There were 5 children and their median age at diagnosis was 2.8 years (0.6-4.4 years). Primary sites were the bladder (2) and the prostate (3). All received neo-adjuvant chemotherapy and 3 underwent chemotherapy during PBT (Group Cx). All patients of Group Cx developed leukocytopenia (WBC <1000/µL). The median dose of PBT was 47.7 GyE (41.4-50.4 GyE). All patients survived by their last hospital visit (median, 36 months). CONCLUSIONS: We analyzed multimodal treatment combined with PBT applied for GU/P-RMS. PBT was well tolerated and could be a plausible choice instead of photon therapy for this population.