Effect of different hydroxyapatite incorporation methods on the structural and biological properties of porous collagen scaffolds for bone repair

Scaffolds which aim to provide an optimised environment to regenerate bone tissue require a balance between mechanical properties and architecture known to be conducive to enable tissue regeneration, such as a high porosity and a suitable pore size. Using freeze‐dried collagen‐based scaffolds as an analogue of native ECM, we sought to improve the mechanical properties by incorporating hydroxyapatite (HA) in different ways while maintaining a pore architecture sufficient to allow cell infiltration, vascularisation and effective bone regeneration. Specifically we sought to elucidate the effect of different hydroxyapatite incorporation methods on the mechanical, morphological, and cellular response of the resultant collagen‐HA scaffolds. The results demonstrated that incorporating either micron‐sized (CHA scaffolds) or nano‐sized HA particles (CnHA scaffolds) prior to freeze‐drying resulted in moderate increases in stiffness (2.2‐fold and 6.2‐fold, respectively, vs. collagen‐glycosaminoglycan scaffolds, P < 0.05, a scaffold known to support osteogenesis), while enabling good cell attachment, and moderate mesenchymal stem cell (MSC)‐mediated calcium production after 28 days'' culture (2.1‐fold, P < 0.05, and 1.3‐fold, respectively, vs. CG scaffolds). However, coating of collagen scaffolds with a hydroxyapatite precipitate after freeze‐drying (CpHA scaffolds) has been shown to be a highly effective method to increase the compressive modulus (26‐fold vs. CG controls, P < 0.001) of scaffolds while maintaining a high porosity (~ 98%). The coating of the ligand‐dense collagen structure results in a lower cell attachment level (P < 0.05), although it supported greater cell‐mediated calcium production (P < 0.0001) compared with other scaffold variants after 28 days'' culture. The comparatively good mechanical properties of these high porosity scaffolds is obtained partially through highly crosslinking the scaffolds with both a physical (DHT) and chemical (EDAC) crosslinking treatment. Control of scaffold microstructure was examined via alterations in freezing temperature. It was found that the addition of HA prior to freeze‐drying generally reduced the pore size and so the CpHA scaffold fabrication method offered increased control over the resulting scaffolds microstructure. These findings will help guide future design considerations for composite biomaterials and demonstrate that the method of HA incorporation can have profound effects on the resulting scaffold structural and biological response.     

The association between hypertensive arteriopathy and cerebral amyloid angiopathy in spontaneously hypertensive stroke‐prone rats

We aimed to test the hypothesis that in spontaneously hypertensive stroke‐prone rats (SHRSP), non‐amyloid cerebral small vessel disease/hypertensive arteriopathy (HA) results in vessel wall injury that may promote cerebral amyloid angiopathy (CAA). Our study comprised 21 male SHRSP (age 17–44 weeks) and 10 age‐ and sex‐matched Wistar control rats, that underwent two‐photon (2PM) imaging of the arterioles in the parietal cortex using Methoxy‐X04, Dextran and cerebral blood flow (CBF) measurements. Our data suggest that HA in SHRSP progresses in a temporal and age‐dependent manner, starting from small vessel wall damage (stage 1A), proceeding to CBF reduction (stage 1B), non‐occlusive (stage 2), and finally, occlusive thrombi (stage 3). Wistar animals also demonstrated small vessel wall damage, but were free of any of the later HA stages. Nearly half of all SHRSP additionally displayed vascular Methoxy‐X04 positivity indicative of cortical CAA. Vascular β‐amyloid deposits were found in small vessels characterized by thrombotic occlusions (stage 2 or 3). Post‐mortem analysis of the rat brains confirmed the findings derived from intravital 2PM microscopy. Our data thus overall suggest that advanced HA may play a role in CAA development with the two small vessel disease entities might be related to the same pathological spectrum of the aging brain.     

Knockdown of HIF‐1α impairs post‐ischemic vascular reconstruction in the brain via deficient homing and sprouting bmEPCs

Although the critical role of hypoxia inducible factor‐1α (HIF‐1α) in cerebral neovascularization after stroke has been well characterized, the details regarding the regulation of endothelial progenitor cell (EPC)‐dependent neovascularization by HIF‐1α are not completely understood. Using lentiviral shRNA to knockdown HIF‐1α, we showed that HIF‐1α plays a central role in bone marrow‐derived EPC (bmEPC) homing and sprouting in the post‐acute stage of ischemic Sprague Dawley (SD) rat brains. First, knockdown of HIF‐1α decreased the homing of both endogenous and exogenous bmEPCs to the ischemic brain. Additionally, the knockdown impaired the incorporation and sprouting of bmEPCs in the ischemic brain. In vitro, knockdown of HIF‐1α inhibited the spheroid sprouting and tube formation of bmEPCs. Mechanically, the HIF‐1α‐dependent recruitment of bmEPCs to the ischemic brain was relative to the CXCL12/CXCR4 axis and HMGB1, which were relative to astrocytes. In addition, the loss of HIF‐1α resulted in deficient expression levels of VEGF‐A, Flk‐1, NRP1, and Dll4 in the ischemic brains, bmEPCs, and astrocytes. These findings suggested that HIF‐1α implicates in bmEPC homing via CXCL12/CXCR4 and HMGB1 and that it promotes bmEPC sprouting via VEGF‐A/flk1‐NRP1/Dll4.     

A 29 Year Old Woman with Acute Mental Status Changes

Moyamoya disease is a rare cerebrovascular disease that causes occlusion of the supraclinoid internal carotid arteries and its branches within the Circle of Willis. While rare in the United States and Europe, moyamoya disease is much more common in eastern Asian populations. We present a 29 year‐old Caucasian woman who initially presented with severe mental status changes and ataxia. MRI and MRA imaging, as well as pathologic examination at autopsy, revealed occlusion of the middle cerebral and anterior cerebral arteries bilaterally with associated infarctions. This case underscores the importance considering this diagnosis as an etiologic possibility during the workup of multiple infarcts, even in non‐Asian populations     

29 Year‐Old Man with New Onset Seizures

Imaging of a 29‐year‐old man with seizures showed a frontal lobe mass with curvilinear narrow calcifications, cystic components and multiple flow‐voids. An AVM was considered. A DSA confirmed the hypervascular nature of the lesion. It was resected and microscopic examination showed an anaplastic oligodendroglioma remarkable for a diffuse and hypertrophic vasculature with areas of frank vascular proliferation. The marked vascularity seen on the MRI, the gyriform calcifications and the cystic degeneration are all features which can be encountered in an AVM. This case illustrates that highly vascular malignant gliomas can simulate vascular lesions by radiology and may require an angiogram for diagnosis.     

Form and function of the intervertebral disc in health and disease: a morphological and stain comparison study

Multiple histologic measurements are commonly used to assess degenerative changes in intervertebral disc (IVD) structure; however, there is no consensus on which stains offer the clearest visualization of specific areas within the IVD. The objective of this study was to compare multiple tinctorial stains, evaluate their ability to highlight structural features within the IVD, and investigate how they influence the capacity to implement a degeneration scoring system. Lumbar IVDs from seven human autopsy specimens were stained using six commonly used stains (Hematoxylin/Eosin, Toluidine Blue, Safranin‐O/Fast Green, Extended FAST, modified Gomori''s Trichrome, and Picrosirius Red Alcian Blue). All IVDs were evaluated by three separate graders to independently determine which stains (i) were most effective at discerning different structural features within different regions of the IVDs and (ii) allowed for the most reproducible assessment of degeneration grade, as assessed via the Rutges histological scoring system (Rutges et al. A validated new histological classification for intervertebral disc degeneration. Osteoarthritis Cartilage, 21, 2039‐47). Although Trichrome, XFAST and PR/AB stains were all effective at highlighting different regions of whole IVDs, we recommend the use of PR/AB because it had the highest degree of rater agreement on assigned degeneration grade, allowed greater resolution of degeneration grade, has an inferential relationship between color and composition, and allowed clear differentiation of the different regions and structural disruptions within the IVD. The use of a standard set of stains together with a histological grading scheme can aid in the characterization of structural changes in different regions of the IVD and may simplify comparisons across the field. This collection of human IVD histological images highlights how IVD degeneration is not a single disease but a composite of multiple processes such as aging, injury, repair, and disease, each of which are unique to the individual.     

47‐Year‐Old Man with Left Leg Numbness

A 47‐year‐old white male with a history of uveitis, hypercalcemia and nephrolithiasis presented with acute onset partial seizure. On exam he had decreased sensation to light touch on his left lower extremity. A Brain MRI revealed a right frontal mass, which was initially thought to be a metastatic lesion or a primary brain tumor. However, biopsy of the lesion revealed it to be a non‐caseating granulomatous lesion consistent with neurosarcoidosis.     

Prognostic Relevance of Histomolecular Classification of Diffuse Adult High‐Grade Gliomas with Necrosis

Diffuse adult high‐grade gliomas (HGGs) with necrosis encompass anaplastic oligodendrogliomas (AOs) with necrosis (grade III), glioblastomas (GBM, grade IV) and glioblastomas with an oligodendroglial component (GBMO, grade IV). Here, we aimed to search for prognostic relevance of histological classification and molecular alterations of these tumors. About 210 patients were included (63 AO, 56 GBM and 91 GBMO). GBMO group was split into “anaplastic oligoastrocytoma (AOA) with necrosis grade IV/GBMO,” restricted to tumors showing intermingled astrocytic and oligodendroglial component, and “GBM/GBMO” based on tumors presenting oligodendroglial foci and features of GBM. Genomic arrays, IDH1 R132H expression analyses and IDH direct sequencing were performed. 1p/19q co‐deletion characterized AO, whereas no IDH1 R132H expression and intact 1p/19q characterized both GBM and GBM/GBMO. AOA with necrosis/GBMO mainly demonstrated IDH1 R132H expression and intact 1p/19q. Other IDH1 or IDH2 mutations were extremely rare. Both histological and molecular classifications were predictive of progression free survival (PFS) and overall survival (OS) (P < 10⁻⁴). Diffuse adult HGGs with necrosis can be split into three histomolecular groups of prognostic relevance: 1p/19q co‐deleted AO, IDH1 R132H‐GBM and 1p/19q intact IDH1 R132H+ gliomas that might be classified as IDH1 R132H+ GBM. Because of histomolecular heterogeneity, we suggest to remove the name GBMO.     

Quantitative imaging of tissue sections using infrared scanning technology

Quantification of immunohistochemically (IHC) labelled tissue sections typically yields semi‐quantitative results. Visualising infrared (IR) ‘tags’, with an appropriate scanner, provides an alternative system where the linear nature of the IR fluorophore emittance enables realistic quantitative fluorescence IHC (QFIHC). Importantly, this new technology enables entire tissue sections to be scanned, allowing accurate area and protein abundance measurements to be calculated from rapidly acquired images. Here, some of the potential benefits of using IR‐based tissue imaging are examined, and the following are demonstrated. Firstly, image capture and analysis using IR‐based scanning technology yields comparable area‐based quantification to those obtained from a modern high‐resolution digital slide scanner. Secondly, IR‐based dual target visualisation and expression‐based quantification is rapid and simple. Thirdly, IR‐based relative protein abundance QIHC measurements are an accurate reflection of tissue sample protein abundance, as demonstrated by comparison with quantitative fluorescent Western blotting data. In summary, it is proposed that IR‐based QFIHC provides an alternative method of rapid whole‐tissue section low‐resolution imaging for the production of reliable and accurate quantitative data.     

Severe white matter astrocytopathy in CADASIL

ObjectivesCerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is characterized by strategic white matter (WM) hyperintensities on MRI. Pathological features include WM degeneration, arteriolosclerosis, lacunar infarcts, and the deposition of granular osmiophilic material. Based on the hypothesis that the gliovascular unit is compromised, we assessed the nature of astrocyte damage in the deep WM of CADASIL subjects.MethodsWe evaluated post‐mortem brains from CADASIL, cerebral small vessel disease, similar age cognitively normal and older control subjects. Standard immunohistochemical, immunofluorescent, and unbiased stereological methods were used to evaluate the distribution of astrocytes, microvessels, and autophagy markers in five different brain regions.ResultsCompared to the controls, the deep WM of CADASIL subjects overall showed increased numbers of glial fibrillary acidic protein (GFAP)‐positive clasmatodendritic astrocytes (P=0.037) and a decrease in the percentage of normal appearing astrocytes (P=0.025). In accord with confluent WM hyperintensities, the anterior temporal pole contained abundant clasmatodendritic astrocytes with displaced aquaporin 4 immunoreactivity. Remarkably, we also found strong evidence for the immunolocalization of autophagy markers including microtubule‐associated protein 1, light chain 3 (LC3), and sequestosome 1/p62 and Caspase‐3 in GFAP‐positive clasmatodendritic cells, particularly within perivascular regions of the deep WM. LC3 was co‐localized in more than 90% of the GFAP‐positive clasmatodendrocytes.ConclusionsOur novel findings show astrocytes undergo autophagy‐like cell death in CADASIL, with the anterior temporal pole being highly vulnerable. We propose astrocytes transform from normal appearing type A to hypertrophic type B and eventually to clasmatodendritic type C cells. These observations also suggest the gliovascular unit of the deep WM is severely impaired in CADASIL.     

Investigating the effect of hypoxic culture on the endothelial differentiation of human amniotic fluid‐derived stem cells

Amniotic fluid‐derived stem cells (AFSCs) are a unique stem cell source that may have great potential for use in tissue engineering (TE) due to their pluripotentiality. AFSCs have previously shown angiogenic potential and may present an alternative cell source for endothelial‐like cells that could be used in range of applications, including the pre‐vascularisation of TE constructs and the treatment of ischaemic diseases. This study investigated the ability of these cells to differentiate down an endothelial lineage with the aim of producing an endothelial‐like cell suitable for use in pre‐vascularisation. As hypoxia and the associated HIF‐1 pathway have been implicated in the induction of angiogenesis in a number of biological processes, it was hypothesised that culture in hypoxic conditions could enhance the endothelial differentiation of AFSCs. The cells were cultured in endothelial cell media supplemented with 50 ng mL⁻¹ of VEGF, maintained in normoxia, intermittent hypoxia or continuous hypoxia and assessed for markers of endothelial differentiation at day 7 and 14. The results demonstrated that AFSCs subjected to these culture conditions display an endothelial gene expression profile and adopted functional endothelial cell characteristics indicative of early endothelial differentiation. Culture in continuous hypoxia enhanced endothelial gene expression but did not enhance functional endothelial cell characteristics. Overall, AFSCs subjected to endothelial stimuli demonstrated a less mature endothelial gene expression profile and phenotype when compared with HUVECs, the endothelial cell control. However, this study is the first time that the positive effect of an extended period of continuous hypoxic culture on endothelial differentiation in AFSCs has been demonstrated.     

Clusterin contributes to early stage of Alzheimer's disease pathogenesis

While clusterin is reportedly involved in Alzheimer''s disease (AD) pathogenesis, how clusterin interacts with amyloid‐β (Aß) to cause Aß neurotoxicity remains unclear in vivo. Using 5×FAD transgenic mice, which develop robust AD pathology and memory deficits when very young, we detected interactions between clusterin and Aß in the mouse brains. The two proteins were concurrently upregulated and bound or colocalized with each other in the same complexes or in amyloid plaques. Neuropathology and cognitive performance were assessed in the progeny of clusterin‐null mice crossed with 5×FAD mice, yielding clu ^−/−;5×FAD and clu ^+/+;5×FAD. We found far less of the various pools of Aß proteins, most strikingly soluble Aß oligomers and amyloid plaques in clu ^−/−;5×FAD mice at 5 months of age. At that age, those mice also had higher levels of neuronal and synaptic proteins and better motor coordination, spatial learning and memory than age‐matched clu ^+/+;5×FAD mice. However, at 10 months of age, these differences disappeared, with Aß and plaque deposition, neuronal and synaptic proteins and impairment of behavioral and cognitive performance similar in both groups. These findings demonstrate that clusterin is necessarily involved in early stages of AD pathogenesis by enhancing toxic Aß pools to cause Aß‐directed neurodegeneration and behavioral and cognitive impairments, but not in late stage.     

Exploring the heterogeneity of MS lesions using positron emission tomography: a reappraisal of their contribution to disability

The biological mechanisms driving disability worsening in multiple sclerosis (MS) are only partly understood. Monitoring changes in lesion load on MRI has a limited predictive value on the progression of clinical disability, and there is an essential need for novel imaging markers specific for the main candidate mechanisms underlying neurodegeneration which include failing myelin repair, innate immune cell activation and gray matter neuronal damage. Positron Emission Tomography (PET) is an imaging technology based on the injection of radiotracers directed against specific molecular targets, which has recently allowed the selective quantification in‐vivo of the key biological mechanisms relevant to MS pathophysiology. Pilot PET studies performed in patients with all forms of MS allowed to revisit the contribution of MS lesions to disability worsening and showed that the evolution of lesions toward chronic activation, together with their remyelination profile were relevant predictors of disability worsening. PET offers the opportunity to bridge a critical gap between neuropathology and in‐vivo imaging. This technique provides an original approach to disentangle some of the most relevant pathological components driving MS progression, to follow‐up their temporal evolution, to investigate their clinical relevance and to evaluate novel therapeutics aimed to prevent disease progression.     

Myelin water imaging to detect demyelination and remyelination and its validation in pathology

Damage to myelin is a key feature of multiple sclerosis (MS) pathology. Magnetic resonance imaging (MRI) has revolutionized our ability to detect and monitor MS pathology in vivo. Proton density, T₁ and T₂ can provide qualitative contrast weightings that yield superb in vivo visualization of central nervous system tissue and have proved invaluable as diagnostic and patient management tools in MS. However, standard clinical MR methods are not specific to the types of tissue damage they visualize, and they cannot detect subtle abnormalities in tissue that appears otherwise normal on conventional MRIs. Myelin water imaging is an MR method that provides in vivo measurement of myelin. Histological validation work in both human brain and spinal cord tissue demonstrates a strong correlation between myelin water and staining for myelin, validating myelin water as a marker for myelin. Myelin water varies throughout the brain and spinal cord in healthy controls, and shows good intra‐ and inter‐site reproducibility. MS plaques show variably decreased myelin water fraction, with older lesions demonstrating the greatest myelin loss. Longitudinal study of myelin water can provide insights into the dynamics of demyelination and remyelination in plaques. Normal appearing brain and spinal cord tissues show reduced myelin water, an abnormality which becomes progressively more evident over a timescale of years. Diffusely abnormal white matter, which is evident in 20%–25% of MS patients, also shows reduced myelin water both in vivo and postmortem, and appears to originate from a primary lipid abnormality with relative preservation of myelin proteins. Active research is ongoing in the quest to refine our ability to image myelin and its perturbations in MS and other disorders of the myelin sheath.     

Magnetic resonance imaging correlates of neuro‐axonal pathology in the MS spinal cord

In people with multiple sclerosis (MS), the spinal cord is the structure most commonly affected by clinically detectable pathology at presentation, and a key part of the central nervous system involved in chronic disease deterioration. Indices, such as the spinal cord cross‐sectional area at the level C₂ have been developed as tools to predict future disability, and—by inference—axonal loss. However, this and other histo‐pathological correlates of spinal cord magnetic resonance imaging (MRI) changes in MS remain incompletely understood. In recent years, there has been a surge of interest in developing quantitative MRI tools to measure specific tissue features, including axonal density, myelin content, neurite density, and orientation, among others, with an emphasis on the spinal cord. Quantitative MRI techniques including T₁ and T₂, magnetization transfer and a number of diffusion‐derived indices have all been applied to MS spinal cord. Particularly diffusion‐based MRI techniques combined with microscopic resolution achievable using high magnetic field scanners enable a new level of anatomical detail and quantification of indices that are clinically meaningful.     

48‐Year‐Old Woman with A Cerebellopontine Angle Mass

Intracranial chondromas are rare benign neoplasms with an estimated incidence rate of 0.2–0.3%. They usually occur at the skull base, or arise from the dura and leptomeninges. Rarely, they have been reported at the cerebellopontine (CP) angle. Here we describe an interesting case of CP angle chondroma which was clinically and radiologically diagnosed as schwannoma and meningioma.     

A 62‐Year‐Old Female With an Intranasal Mass Extending into the Lamina Cribrosa

Malignant tumors with neuroendocrine differentiation of the nasal cavity include neuroendocrine carcinoma, small cell undifferentiated carcinoma, olfactory neuroblastoma (esthesioneuroblastoma), and sinonasal undifferentiated carcinoma (SNUC). The rarity of these tumors and overlapping clinical, histological and immunohistochemical findings make the correct diagnosis difficult. However accurate diagnosis is critical for therapy and the outcome differs significantly between these entities. Here we describe the case of a 62‐year‐old woman with an intranasal tumor extending into the adjacent anatomical compartments and with infiltration of the meninges. The suspected preoperative diagnosis was olfactory neuroblastoma. Histological and immunohistochemical analysis showed a lobulated tumor with neuroendocrine differentiation and we ultimately diagnosed the process as a SNUC. In this report, we discuss the differential diagnosis at hand and present histological findings found in the specimen indicating that the process originated from local glands present in the nasal mucosa.     

Extracellular matrix production by nucleus pulposus and bone marrow stem cells in response to altered oxygen and glucose microenvironments

Bone marrow (BM) stem cells may be an ideal source of cells for intervertebral disc (IVD) regeneration. However, the harsh biochemical microenvironment of the IVD may significantly influence the biological and metabolic vitality of injected stem cells and impair their repair potential. This study investigated the viability and production of key matrix proteins by nucleus pulposus (NP) and BM stem cells cultured in the typical biochemical microenvironment of the IVD consisting of altered oxygen and glucose concentrations. Culture‐expanded NP cells and BM stem cells were encapsulated in 1.5% alginate and ionically crosslinked to form cylindrical hydrogel constructs. Hydrogel constructs were maintained under different glucose concentrations (1, 5 and 25 mM) and external oxygen concentrations (5 and 20%). Cell viability was measured using the Live/Dead® assay and the production of sulphated glycosaminoglycans (sGAG), and collagen was quantified biochemically and histologically. For BM stem cells, IVD‐like micro‐environmental conditions (5 mM glucose and 5% oxygen) increased the accumulation of sGAG and collagen. In contrast, low glucose conditions (1 mM glucose) combined with 5% external oxygen concentration promoted cell death, inhibiting proliferation and the accumulation of sGAG and collagen. NP‐encapsulated alginate constructs were relatively insensitive to oxygen concentration or glucose condition in that they accumulated similar amounts of sGAG under all conditions. Under IVD‐like microenvironmental conditions, NP cells were found to have a lower glucose consumption rate compared with BM cells and may in fact be more suitable to adapt and sustain the harsh microenvironmental conditions. Considering the highly specialised microenvironment of the central NP, these results indicate that IVD‐like concentrations of low glucose and low oxygen are critical and influential for the survival and biological behaviour of stem cells. Such findings may promote and accelerate the translational research of stem cells for the treatment of IVD degeneration.