Single-shot 20-fold expansion microscopy.

Expansion microscopy (ExM) is in increasingly widespread use throughout biology because its isotropic physical magnification enables nanoimaging on conventional microscopes. To date, ExM methods either expand specimens to a limited range (~4-10× linearly) or achieve larger expansion factors through iterating the expansion process a second time (~15-20× linearly). Here, we present an ExM protocol that achieves ~20× expansion (yielding <20-nm resolution on a conventional microscope) in a single expansion step, achieving the performance of iterative expansion with the simplicity of a single-shot protocol. This protocol, which we call 20ExM, supports postexpansion staining for brain tissue, which can facilitate biomolecular labeling. 20ExM may find utility in many areas of biological investigation requiring high-resolution imaging.

Precise control of synthetic hydrogel network structure via linear, independent synthesis-swelling relationships.

Hydrogel physical properties are tuned by altering synthesis conditions such as initial polymer concentration and polymer-cross-linker stoichiometric ratios. Traditionally, differences in hydrogel synthesis schemes, such as end-linked poly(ethylene glycol) diacrylate hydrogels and cross-linked poly(vinyl alcohol) hydrogels, limit structural comparison between hydrogels. In this study, we use generalized synthesis variables for hydrogels that emphasize how changes in formulation affect the resulting network structure. We identify two independent linear correlations between these synthesis variables and swelling behavior. Analysis through recently updated swollen polymer network models suggests that synthesis-swelling correlations can be used to make a priori predictions of the stiffness and solute diffusivity characteristics of synthetic hydrogels. The same experiments and analyses performed on methacrylamide-modified gelatin hydrogels demonstrate that complex biopolymer structures disrupt the linear synthesis-swelling correlations. These studies provide insight into the control of hydrogel physical properties through structural design and can be used to implement and optimize biomedically relevant hydrogels.

A regional Australasian experience of extended endoscopic transsphenoidal surgery for craniopharyngioma: Progression of the mentoring model.

Endoscopic endonasal transsphenoidal approaches to craniopharyngioma has become increasingly popular over the last 15 years. We present the results of our retrospective series of craniopharyngiomata resected by an endoscopic, endonasal approach at a low-volume service in Australasia. Between the years of 2009 and 2017, 11 patients underwent pure endoscopic endonasal transsphenoidal resection for a craniopharyngioma at our institutions. The medical records, histopathology, intraoperative findings and patient imaging were retrospectively assessed. 11 patients were included with 5 male and 6 female patients. The mean age was 32.0 years (range 14-68 years). Of this patient series a gross total resection of the tumour was achieved in 8 of 11 patients (73%). In the immediate postoperative phase, 10 of the 11 patients developed diabetes insipidus (91%). The pituitary stalk was formally not seen in 4 patients and all were treated with vasopressin. Of the 7 patients where the pituitary stalk was identified it was formally divided in 6 and preserved in 1 patient where the tumour was separate to the stalk. The endoscopic endonasal transsphenoidal approach for excision of craniopharyngioma, utilising and progressing the surgical mentoring model, can achieve adequate decompression of critical structures. Furthermore, our aggressive approach to divide and remove the involved pituitary stalk results in high rates of gross macroscopic resection with excellent long-term disease control with a greater risk of postoperative diabetes insipidus and panhypopituitarism.

Preoperative screening for coagulopathy in elective neurosurgical patients in Wellington Regional Hospital and survey of practice across Australia and New Zealand.

It is common practice to perform pre-operative coagulation screening in elective neurosurgery patients, including international normalised ratio (INR) and activated partial thromboplastin time (aPTT). We present a retrospective analysis of 1143 elective neurosurgical patients at Wellington Regional Hospital (WRH) in New Zealand between 2013 and 2017 on whom coagulation screening including INR and aPTT was performed prior to surgery. 21 patients (1.8%) had clinically significant derangements on coagulation profile defined as raised INR or prolonged aPTT. 15 (1.3%) of these patients would be expected to have derangement based on previous history and 6 (0.5%) had unexpected derangements in coagulation profile. Of the 6 patients with unexpected derangements in coagulation profile, all had raised aPTT, none had preoperative correction of coagulopathy and none had bleeding complications or mortality. The cost of coagulation screening across the duration of the study was $68,009 New Zealand Dollars (NZD). A survey of major elective neurosurgery units in Australia and New Zealand found that 85% perform routine laboratory coagulation screening. In the 15% who do not perform laboratory coagulation screening, none use a standardised questionnaire to screen for coagulopathy. We developed a structured questionnaire to assist in detection of coagulopathy in elective neurosurgery patients. Our findings suggest that there is limited value in performing indiscriminate laboratory coagulation screening in patients with no risk factors on history. Despite this, routine laboratory coagulation screening is common practice in Australia and New Zealand. We propose a structured questionnaire to guide laboratory testing and discussions with haematology colleagues.

Relationship between permeability and diffusivity in polyethylene glycol hydrogels.

The transport properties of hydrogels largely affect their performance in biomedical applications ranging from cell culture scaffolds to drug delivery systems. Solutes can move through the polymer mesh as a result of concentration gradients in the interstitial fluid or pressure gradients that move the fluid and solutes simultaneously. The relationship between the two modalities of transport in hydrogels can provide insight for the design of materials that can function effectively in the dynamic conditions experienced in vitro and in vivo, yet this correlation has not been previously elucidated. Here, fluorescence recovery after photobleaching (FRAP) is used to measure the diffusivity of dextran molecules of different size within polyethylene glycol hydrogels. Spherical indentation analyzed in a poroelastic framework is used to measure the permeability to fluid flow of the same hydrogels. It is found that while the diffusivity varies with exp(ξ -2), where ξ is the mesh size of the hydrogels, it also varies with exp(k -1), where k is the intrinsic permeability. For the same hydrogel structure, diffusive transport is affected by the solute size, while convective transport is unaffected. As spherical indentation is a reliable, quick and non-destructive testing method for hydrated soft materials, the relationship provides the means to faster assessment of the transport properties of hydrogels and, ultimately, of their effective use in biomedical applications.

Below the surface: Twenty-five years of seafloor litter monitoring in coastal seas of North West Europe (1992-2017).

Marine litter presents a global problem, with increasing quantities documented in recent decades. The distribution and abundance of marine litter on the seafloor off the United Kingdom's (UK) coasts were quantified during 39 independent scientific surveys conducted between 1992 and 2017. Widespread distribution of litter items, especially plastics, were found on the seabed of the North Sea, English Channel, Celtic Sea and Irish Sea. High variation in abundance of litter items, ranging from 0 to 1835 pieces km-2 of seafloor, was observed. Plastic tems such as bags, bottles and fishing related debris were commonly observed across all areas. Over the entire 25-year period (1992-2017), 63% of the 2461 trawls contained at least one plastic litter item. There was no significant temporal trend in the percentage of trawls containing any or total plastic litter items across the long-term datasets. Statistically significant trends, however, were observed in specific plastic litter categories only. These trends were all positive except for a negative trend in plastic bags in the Greater North Sea - suggesting that behavioural and legislative changes could reduce the problem of marine litter within decades.

Spatially graded hydrogels for preclinical testing of glioblastoma anticancer therapeutics.

While preclinical models such as orthotopic tumors generated in mice from patient-derived specimens are widely used to predict sensitivity or therapeutic interventions for cancer, such xenografts can be slow, require extensive infrastructure, and can make in situ assessment difficult. Such concerns are heightened in highly aggressive cancers, such as glioblastoma (GBM), that display genetic diversity and short mean survival. Biomimetic biomaterial technologies offer an approach to create ex vivo models that reflect biophysical features of the tumor microenvironment (TME). We describe a microfluidic templating approach to generate spatially graded hydrogels containing patient-derived GBM cells to explore drug efficacy and resistance mechanisms.

Transpetrosal approach to petro-clival meningioma.

A 38-year-old woman had a 3-week gradual onset of right-sided weakness in the upper and lower extremities. MRI showed a large left petro-clival meningioma encasing the basilar and left superior cerebellar artery and compressing the brainstem. A posterior transpetrosal approach, with a left temporal and retrosigmoid craniotomy and mastoidectomy, was performed. The tumor was removed in a gross-total resection with questionable remnants adherent to the brainstem. Intraoperative partial iatrogenic injury to the left oculomotor nerve was repaired with fibrin glue. Postoperatively, the hemiparesis improved, and the patient was discharged to the rehabilitation center with left oculomotor and abducens palsies. A postoperative MRI scan showed complete resection of tumor with no remnants on the brainstem. A 6-month follow-up examination showed complete resolution of motor symptoms and complete recovery of cranial nerve (CN) palsies affecting CN III and CN VI. The video can be found here: https://youtu.be/vOu6YFA8uoo .

Spatial and temporal analysis of the risks posed by total petroleum hydrocarbon and trace element contaminants in coastal waters of Kuwait.

Nine trace elements including As, Cd, Cu, Fe, Hg, Ni, Pb, V and Zn, and total petroleum hydrocarbons were analysed from water samples collected from 23 stations since 1984 from Kuwaiti coastal waters. Here it was investigated whether concentrations of these determinants are at levels above Kuwaiti and internationally established assessment criteria (AC). The results indicate that Cu and Cd had the most Kuwaiti AC breaches over time. Comparing the data of the last sampled year to the least stringent international AC, then Cu and Cd showed breaches at all stations. The trends for trace metals are significantly downwards, especially for Cd and Hg. No determinant measured showed a significant upward trend, indicating that water pollution for these contaminants is not a worsening situation. However, further sampling should be carried out to confirm these findings, especially at shoreline locations, where routine monitoring ceased in 2011 to investigate any recent changes.

Cyclic tensile strain enhances human mesenchymal stem cell Smad 2/3 activation and tenogenic differentiation in anisotropic collagen-glycosaminoglycan scaffolds.

Stem cell research arose from the need to explore new therapeutic possibilities for intractable and lethal diseases. Although musculoskeletal disorders are basically nonlethal, their high prevalence and relative ease of performing clinical trials have facilitated the clinical application of stem cells in this field. However, few reliable clinical studies have been published, despite the plethora of in vitro and preclinical studies in stem cell research for regenerative medicine in the musculoskeletal system. Stem cell therapy can be applied locally for bone, cartilage and tendon regeneration. Candidate disease modalities in bone regeneration include large bone defects, nonunion of fractures, and osteonecrosis. Focal osteochondral defect and osteoarthritis are current targets for cartilage regeneration. For tendon regeneration, bone-tendon junction problems such as rotator cuff tears are hot topics in clinical research. To date, the literature supporting stem cell-based therapies comprises mostly case reports or case series. Therefore, high-quality evidence, including from randomised clinical trials, is necessary to define the role of cell-based therapies in the treatment of musculoskeletal disorders. It is imperative that clinicians who adopt stem cell treatment into their practices possess a good understanding of the natural course of the disease. It is also highly recommended that treating physicians do not thrust aside the concomitant use of established measures until stem cell therapy is evidently proved worthy in terms of efficacy and cost. The purpose of this review is to summarise on the current status of stem cell application in the orthopaedic field along with the author's view of future prospects.

Evaluation of multi-scale mineralized collagen-polycaprolactone composites for bone tissue engineering.

A particular challenge in biomaterial development for treating orthopedic injuries stems from the need to balance bioactive design criteria with the mechanical and geometric constraints governed by the physiological wound environment. Such trade-offs are of particular importance in large craniofacial bone defects which arise from both acute trauma and chronic conditions. Ongoing efforts in our laboratory have demonstrated a mineralized collagen biomaterial that can promote human mesenchymal stem cell osteogenesis in the absence of osteogenic media but that possesses suboptimal mechanical properties in regards to use in loaded wound sites. Here we demonstrate a multi-scale composite consisting of a highly bioactive mineralized collagen-glycosaminoglycan scaffold with micron-scale porosity and a polycaprolactone support frame (PCL) with millimeter-scale porosity. Fabrication of the composite was performed by impregnating the PCL support frame with the mineral scaffold precursor suspension prior to lyophilization. Here we evaluate the mechanical properties, permeability, and bioactivity of the resulting composite. Results indicated that the PCL support frame dominates the bulk mechanical response of the composite resulting in a 6000-fold increase in modulus compared to the mineral scaffold alone. Similarly, the incorporation of the mineral scaffold matrix into the composite resulted in a higher specific surface area compared to the PCL frame alone. The increased specific surface area in the collagen-PCL composite promoted increased initial attachment of porcine adipose derived stem cells versus the PCL construct.

Reinforcement of Mono- and Bi-layer Poly(Ethylene Glycol) Hydrogels with a Fibrous Collagen Scaffold.

Biomaterial-based tissue engineering strategies hold great promise for osteochondral tissue repair. Yet significant challenges remain in joining highly dissimilar materials to achieve a biomimetic, mechanically robust design for repairing interfaces between soft tissue and bone. This study sought to improve interfacial properties and function in a bi-layer hydrogel interpenetrated with a fibrous collagen scaffold. 'Soft' 10% (w/w) and 'stiff' 30% (w/w) PEGDM was formed into mono- or bi-layer hydrogels possessing a sharp diffusional interface. Hydrogels were evaluated as single-(hydrogel only) or multi-phase (hydrogel + fibrous scaffold penetrating throughout the stiff layer and extending >500 µm into the soft layer). Including a fibrous scaffold into both soft and stiff mono-layer hydrogels significantly increased tangent modulus and toughness and decreased lateral expansion under compressive loading. Finite element simulations predicted substantially reduced stress and strain gradients across the soft-stiff hydrogel interface in multi-phase, bilayer hydrogels. When combining two low moduli constituent materials, composites theory poorly predicts the observed, large modulus increases. These results suggest material structure associated with the fibrous scaffold penetrating within the PEG hydrogel as the major contributor to improved properties and function-the hydrogel bore compressive loads and the 3D fibrous scaffold was loaded in tension thus resisting lateral expansion.

The impact of discrete compartments of a multi-compartment collagen-GAG scaffold on overall construct biophysical properties.

Orthopedic interfaces such as the tendon-bone junction (TBJ) present unique challenges for biomaterials development. Here we describe a multi-compartment collagen-GAG scaffold fabricated via lyophilization that contains discrete mineralized (CGCaP) and non-mineralized (CG) regions joined by a continuous interface. Modifying CGCaP preparation approaches, we demonstrated scaffold variants of increasing mineral content (40 vs. 80wt% CaP). We report the impact of fabrication parameters on microstructure, composition, elastic modulus, and permeability of the entire multi-compartment scaffold as well as discrete mineralized and non-mineralized compartments. Notably, individual mineralized and non-mineralized compartments differentially impacted the global properties of the multi-compartment composite. Of particular interest for the development of mechanically-loaded multi-compartment composites, the elastic modulus and permeability of the entire construct were governed primarily by the non-mineralized and mineralized compartments, respectively. Based on these results we hypothesize spatial variations in scaffold structural, compositional, and mechanical properties may be an important design parameter in orthopedic interface repair.

Impact of the biophysical features of a 3D gelatin microenvironment on glioblastoma malignancy.

Three-dimensional tissue engineered constructs provide a platform to examine how the local extracellular matrix (ECM) contributes to the malignancy of cancers such as human glioblastoma multiforme. Improved resolution of how local matrix biophysical features impact glioma proliferation, genomic and signal transduction paths, as well as phenotypic malignancy markers would complement recent improvements in our understanding of molecular mechanisms associated with enhanced malignancy. Here, we report the use of a gelatin methacrylate (GelMA) platform to create libraries of three-dimensional biomaterials to identify combinations of biophysical features that promote malignant phenotypes of human U87MG glioma cells. We noted key biophysical properties, namely matrix density, crosslinking density, and biodegradability, that significantly impact glioma cell morphology, proliferation, and motility. Gene expression profiles and secreted markers of increased malignancy, notably VEGF, MMP-2, MMP-9, HIF-1, and the ECM protein fibronectin, were also significantly impacted by the local biophysical environment as well as matrix-induced deficits in diffusion-mediated oxygen and nutrient biotransport. Overall, this biomaterial system provides a flexible platform to explore the role biophysical factors play in the etiology, growth, and subsequent invasive spreading of gliomas.

Biologically active collagen-based scaffolds: advances in processing and characterization.

A small number of type I collagen-glycosaminoglycan scaffolds (collagen-GAG scaffolds; CGSs) have unusual biological activity consisting primarily in inducing partial regeneration of organs in the adult mammal. Two of these are currently in use in a variety of clinical settings. CGSs appear to induce regeneration by blocking the adult healing response, following trauma, consisting of wound contraction and scar formation. Several structural determinants of biological activity have been identified, including ligands for binding of fibroblasts to the collagen surface, the mean pore size (which affects ligand density) and the degradation rate (which affects the duration of the wound contraction-blocking activity by the scaffold). Processing variables that affect these determinants include the kinetics of swelling of collagen fibres in acetic acid, freezing of the collagen-GAG suspension and cross-linking of the freeze-dried scaffold. Recent developments in the processing of CGSs include fabrication of scaffolds that are paucidisperse in pore size, scaffolds with gradients in physicochemical properties (and therefore biological activity) and scaffolds that incorporate a mineral component. Advances in the characterization of the pore structure of CGSs have been made using confocal and nonlinear optical microscopy (NLOM). The mechanical behaviour of CGSs, as well as the resistance to degradative enzymes, have been studied. Following seeding with cells (typically fibroblasts), contractile forces in the range 26-450 nN per cell are generated by the cells, leading to buckling of scaffold struts. Ongoing studies of cell-seeded CGSs with NLOM have shown an advantage over the use of confocal microscopy due to the ability of the former method to image the CGS surfaces without staining (which alters its surface ligands), reduced cell photodamage, reduced fluorophore photobleaching and the ability to image deeper inside the scaffold.

Bone density comparison of selected carpal and tarsal bones: validation for their use in compression fracture fixation studies of scaphoid screws.

INTRODUCTION: To determine if trabecular, total and cortical bone densities of the capitate, navicular, cuboid, and first cuneiform were equivalent to those of the scaphoid, such that these bones could be used in place of the scaphoid in evaluating new headless scaphoid compression screws. METHODS: Fifty scaphoids, capitates, naviculars, cuboids, and first cuneiforms were harvested from fresh frozen cadavers. The trabecular, total and cortical bone densities were measured using pQCT technology and statistically compared. RESULTS: A paired t comparison between paired scaphoids and capitates showed no difference between the trabecular bone densities. However, their total bone and cortical densities were found to be different. An independent measures ANOVA comparison of the five bones, showed no significant difference in mean trabecular density between the capitates, naviculars and first cuneiforms when compared to the scaphoids. However, the mean total and cortical densities of the first cuneiforms were less than the scaphoids and the mean trabecular, total and cortical bone densities of the cuboids were all less than the scaphoids. DISCUSSION: Compression fracture fixation studies of headless compression screws could be conducted using the capitate, navicular, and first cuneiform as models of the scaphoid when the supply of scaphoids is limited.

The effect of pore size on cell adhesion in collagen-GAG scaffolds.

The biological activity of scaffolds used in tissue engineering applications hypothetically depends on the density of available ligands, scaffold sites at which specific cell binding occurs. Ligand density is characterized by the composition of the scaffold, which defines the surface density of ligands, and by the specific surface area of the scaffold, which defines the total surface of the structure exposed to the cells. It has been previously shown that collagen-glycosaminoglycan (CG) scaffolds used for studies of skin regeneration were inactive when the mean pore size was either lower than 20 microm or higher than 120 microm (Proc. Natl. Acad. Sci., USA 86(3) (1989) 933). To study the relationship between cell attachment and viability in scaffolds and the scaffold structure, CG scaffolds with a constant composition and solid volume fraction (0.005), but with four different pore sizes corresponding to four levels of specific surface area were manufactured using a lyophilization technique. MC3T3-E1 mouse clonal osteogenic cells were seeded onto the four scaffold types and maintained in culture. At the experimental end point (24 or 48 h), the remaining viable cells were counted to determine the percent cell attachment. A significant difference in viable cell attachment was observed in scaffolds with different mean pore sizes after 24 and 48 h; however, there was no significant change in cell attachment between 24 and 48 h for any group. The fraction of viable cells attached to the CG scaffold decreased with increasing mean pore size, increasing linearly (R2 = 0.95, 0.91 at 24 and 48 h, respectively) with the specific surface area of the scaffold. The strong correlation between the scaffold specific surface area and cell attachment indicates that cell attachment and viability are primarily influenced by scaffold specific surface area over this range (95.9-150.5 microm) of pore sizes for MC3T3 cells.

Optimal degradation rate for collagen chambers used for regeneration of peripheral nerves over long gaps.

The experimental study of peripheral nerve regeneration has depended heavily on the use of a nerve chamber in which the stumps of the transected nerve are inserted. A large variety of chamber fillings and chamber types have been used in an effort to induce a higher quality of regeneration across the gap initially separating the two stumps. In this study we studied the morphology of nerves regenerated across a 15 mm gap following implantation of a series of five chambers. The chambers were fabricated from type I collagen and possessed identical pore volume fractions as well as average pore diameters, but differed in cross-link density continuously along the series. The residual mass of the implanted chambers at 9 weeks was observed to increase continuously with increasing cross-link density along the series, indicating a continuous decrease in degradation rate. The quality of regenerated nerves, determined by the number of large diameter fibers (A-fibers) per nerve, the average diameter of all axons and the ratio of area occupied by axons (N-Ratio), was superior at an intermediate level of chamber degradation rate. The maximal quality of peripheral nerve regeneration corresponded to an optimal degradation rate with an estimated chamber half-life of approximately 2-3 weeks following implantation. A speculative mechanistic explanation of the observed optimum focuses on the hypothetical role of cell and cytokine traffic that may take place through holes in the chamber generated by the degradation process. The data show the presence of a hitherto unreported optimal chamber degradation rate that leads to regenerated nerves of maximum quality.

Performance of a rapid phage-based test, FASTPlaqueTB, to diagnose pulmonary tuberculosis from sputum specimens in South Africa.

SETTING: Twelve primary health care clinics in the South Peninsula Administration, Cape Town, Western Cape Province, South Africa. OBJECTIVE: To evaluate the performance of FAST-PlaqueTB, a new phage-based test, for the rapid diagnosis of TB in individuals with no previous history of TB treatment presenting at primary health care clinics in Cape Town, South Africa. DESIGN: A comparative study of FASTPlaqueTB, auramine smear microscopy and Lowenstein-Jensen culture of 1692 decontaminated sputum specimens from 853 patients suspected of having TB. Resolution of discrepant results was undertaken by review of clinical information, chest X-ray and follow-up of treatment outcomes. RESULTS: FASTPlaqueTB detected TB in 75.2% of culture-confirmed cases and 70.3% of all cases with a clinical diagnosis of TB, with a specificity of 98.7% and 99.0%, respectively. The performance parameters of FASTPlaqueTB were significantly superior to those of concentrated auramine smear microscopy (63.4% and 61.3% sensitivity, and 97.4% and 97.3% specificity in culture-confirmed and all cases, respectively). Of those patients with two negative sputum smears, FAST-PlaqueTB detected TB in 54.1% of TB cases confirmed by culture and 48.8% of all cases with a clinical diagnosis of TB. A combination of smear microscopy and FASTPlaqueTB enabled 81.2% of culture-confirmed cases and 78.4% of total TB cases to be detected within 2 days of presentation. CONCLUSION: FASTPlaqueTBTM is a rapid, manual test for the diagnosis of TB. The test has significantly higher sensitivity overall compared with auramine sputum smear microscopy in individuals with no previous history of TB treatment, although smear microscopy did detect the most infectious of the TB cases. The FAST-PlaqueTB test is easy to perform, requires no dedicated equipment, and results are read by eye within 48 hours. This test can be useful for the diagnosis of TB in developing countries with a high burden of TB where other rapid diagnostic tests may not be appropriate. The test shows promising performance, particularly in the diagnosis of smear-negative disease, and could be used in conjunction with smear microscopy to aid in the diagnosis of additional cases of TB.