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#32652865   2020/07/11 To Up

The Response of Gingiva Monolayer, Spheroid and ex vivo Tissue Cultures to Collagen Membranes and Bone Substitute.

Collagen membranes and bone substitute are popular biomaterials in guided tissue regeneration for treatment of traumatized or diseased periodontal tissue. Development of these biomaterials starts in monolayer cell culture, failing to reflect in vivo tissue organization. Spheroid cultures potentially mimic in vivo tissues in structure and functionality. This study aims to compare GC monolayers and spheroids to ex vivo gingiva. Human GC monolayers, spheroids and gingiva ex vivo tissues were cultured on plastic surfaces, collagen membranes or bone substitute. Hematoxylin-eosin (HE) staining, immunohistochemistry for KI67 and caspase 3 (CASP3), resazurin-based toxicity assays, qPCR for collagen I (COL1A1), vascular endothelial growth factor (VEGF), angiogenin (ANG), interleukin (IL)6 and IL8 as well as ELISA for COL1A1, VEGF, ANG, IL6 and IL8 were performed in all cultures. Morphology was different in all culture set-ups. Staining of KI67 was positive in monolayers and staining of CASP3 was positive in spheroids. All culture set-ups were viable. COL1A1 production was modulated in monolayers and ex vivo tissues at mRNA levels, VEGF in monolayers and ex vivo tissues at mRNA levels and in spheroids at protein levels, ANG in spheroids at mRNA levels and in monolayers and spheroids at protein levels, IL6 in monolayers and spheroids at mRNA levels and in spheroids and ex vivo tissues at protein levels and IL8 in monolayers and ex vivo tissues at mRNA levels. Modulations were surface-dependent. In conclusion, each culture model is structurally and functionally different. Neither GC monolayers nor spheroids mimicked gingiva ex vivo tissue in all measured aspects.
Klara Janjić, Barbara Schädl, Oleh Andrukhov, Hermann Agis

2358 related Products with: The Response of Gingiva Monolayer, Spheroid and ex vivo Tissue Cultures to Collagen Membranes and Bone Substitute.

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#32652796   2020/07/11 To Up

Decellularised human corneal stromal cell sheet as a novel matrix for ocular surface reconstruction.

The shortage of donor corneas as well as the limitations of tissue substitutes leads to the necessity to develop alternative materials for ocular surface reconstruction. Corneal surface substitutes must fulfill specific requirements such as high transparency, low immunogenicity, and mechanical stability combined with elasticity. This in vitro study evaluates a decellularized matrix secreted from human corneal fibroblast (hCF) as an alternative material for ocular surface reconstruction. HCF from human donors were cultivated with the supplementation of Vitamin C to form a stable and thick matrix. Furthermore, due to enhanced cultivation time, a three-dimensional like multilayered construct which partly mimics the complex structure of the corneal stroma could be generated. The formed human cell-based matrices (so-called cell sheets (CS)) were subsequently decellularized. The complete cell removal, collagen content, ultrastructure and cell toxicity of the decellularised CS (DCS) as well as biomechanical properties were analyzed. Surgical feasibility was tested on enucleated porcine eyes. After decellularization and sterilization, a transparent, thick, cell free and sterile tissue substitute resulted, which allowed expansion of limbal epithelial stem cells with no signs of cytotoxicity, and good surgical feasibility. DCS seem to be a promising new corneal tissue substitute derived from human cells without the limitation of donor material; however future in vivo studies are necessary to further elucidate its potential for ocular surface reconstruction.
Sonja Mertsch, Meike Hasenzahl, Stephan Reichl, Gerd Geerling, Stefan Schrader

1421 related Products with: Decellularised human corneal stromal cell sheet as a novel matrix for ocular surface reconstruction.

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#32652570   2020/07/11 To Up

Molecular and functional characterization of urine-derived podocytes from patients with Alport syndrome.

Alport syndrome (AS) is a genetic disorder involving mutations in the genes encoding collagen IV α3, α4 or α5 chains, resulting in the impairment of glomerular basement membrane. Podocytes are responsible for production and correct assembly of collagen IV isoforms; however, data on the phenotypic characteristics of human AS podocytes and their functional alterations are currently limited. The evident loss of viable podocytes into the urine of patients with active glomerular disease enables their isolation in a non-invasive way. We here isolated, immortalized and subcloned podocytes from the urine of three different AS patients for molecular and functional characterization. AS podocytes expressed a typical podocyte signature and showed a collagen IV profile reflecting each patient's mutation. Furthermore, RNA-sequencing analysis revealed 348 genes differentially expressed in AS podocytes compared with control podocytes. Gene Ontology analysis underlined the enrichment in genes involved in cell motility, adhesion, survival, and angiogenesis. In parallel, AS podocytes displayed reduced motility. Finally, a functional permeability assay, using a podocyte-glomerular endothelial cell co-culture system, was established and AS podocyte co-cultures showed a significantly higher permeability of albumin compared to control podocyte co-cultures, in both static and dynamic conditions under continuous perfusion. In conclusion, our data provide a molecular characterization of immortalized AS podocytes, highlighting alterations in several biological processes related to extracellular matrix remodelling. Moreover, we have established an in vitro model to reproduce the altered podocyte permeability observed in patients with AS. This article is protected by copyright. All rights reserved.
Corinne Iampietro, Linda Bellucci, Fanny O Arcolino, Maddalena Arigoni, Luca Alessandri, Yonathan Gomez, Elli Papadimitriou, Raffaele A Calogero, Enrico Cocchi, Lambertus Van Den Heuvel, Elena Levtchenko, Benedetta Bussolati

2822 related Products with: Molecular and functional characterization of urine-derived podocytes from patients with Alport syndrome.

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#32652228   2020/07/08 To Up

Biomimetic Corneal Stroma Using Electro-Compacted Collagen.

Engineering substantia propria (or stroma of cornea) that mimics the function and anatomy of natural tissue is vital for in vitro modelling and in vivo regeneration. There are, however, few examples of bioengineered biomimetic corneal stroma. Here we describe the construction of an orthogonally oriented 3D corneal stroma model (3D-CSM) using pure electro-compacted collagen (EC). EC films comprise aligned collagen fibrils and support primary human corneal stromal cells (hCSCs). Cell-laden constructs are analogous to the anatomical structure of native human cornea. The hCSCs are guided by the topographical cues provided by the aligned collagen fibrils of the EC films. Importantly, the 3D-CSM are biodegradable, highly transparent, glucose-permeable and comprise quiescent hCSCs. Gene expression analysis indicated the presence of aligned collagen fibrils is strongly coupled to downregulation of active fibroblast/myofibroblast markers α-SMA and Thy-1, with a concomitant upregulation of the dormant keratocyte marker ALDH3. The 3D-CSM represents the first example of an optimally robust biomimetic engineered corneal stroma that is constructed from pure electro-compacted collagen for cell and tissue support. The 3D-CSM is a significant advance for synthetic corneal stroma engineering, with the potential to be used for full-thickness and functional cornea replacement, as well as informing in vivo tissue regeneration.
Zhi Chen, Xiao Liu, Jingjing You, Yihui Song, Eva Tomaskovic-Crook, Gerard Sutton, Jeremy M Crook, Gordon G Wallace

1122 related Products with: Biomimetic Corneal Stroma Using Electro-Compacted Collagen.

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#32652221   2020/07/08 To Up

Multi-protease analysis of Pleistocene bone proteomes.

Ancient protein analysis is providing new insights into the evolutionary relationships between hominin fossils across the Pleistocene. Protein identification commonly relies on the proteolysis of a protein extract using a single protease, trypsin. As with modern proteome studies, alternative or additional proteases have the potential to increase both proteome size and protein sequence recovery. This could enhance the phylogenetic potential recovered from ancient proteomes. Here we identify 18 novel hominin bone specimens from the Kleine Feldhofer Grotte using MALDI-TOF MS peptide mass fingerprinting of collagen type I. Next, we use one of these hominin bone specimens and three Late Pleistocene Equidae specimens identified in a similar manner and present a comparison of the bone proteome size and protein sequence recovery obtained after using nanoLC-MS/MS and parallel proteolysis using six different proteases, including trypsin. We observe that the majority of the preserved bone proteome is inaccessible to trypsin. We also observe that for proteins recovered consistently across several proteases, protein sequence coverage can be increased significantly by combining peptide identifications from two or more proteases. Our results thereby demonstrate that the proteolysis of Pleistocene proteomes by several proteases has clear advantages when addressing evolutionary questions in palaeoproteomics. SIGNIFICANCE: Maximizing proteome and protein sequence recovery of ancient skeletal proteomes is important when analyzing unique hominin fossils. As with modern proteome studies, palaeoproteomic analysis of Pleistocene bone and dentine samples has almost exclusively used trypsin as its only protease, despite the demonstrated advantages of alternative proteases to increase proteome recovery in modern proteome studies. We demonstrate that Pleistocene bone proteomes can be significantly expanded by using additional proteases beside trypsin, and that this also improves sequence coverage of individual proteins. The use of several alternative proteases beside trypsin therefore has major benefits to maximize the phylogenetic information retrieved from ancient skeletal proteomes.
Liam T Lanigan, Meaghan Mackie, Susanne Feine, Jean-Jacques Hublin, Ralf W Schmitz, Arndt Wilcke, Matthew J Collins, Enrico Cappellini, Jesper V Olsen, Alberto J Taurozzi, Frido Welker

2563 related Products with: Multi-protease analysis of Pleistocene bone proteomes.

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#32651838   2020/07/10 To Up

Why Do Falls and Lower Limb Fractures Occur More Frequently in the Diabetic Patient and How Can They Be Prevented?

Due to primarily sarcopenia and hypoglycemia but also neuropathy, hypotension, analgesics and polypharmacy, there is an increased incidence of falls and hip fractures in both the type 1 and type 2 diabetic patient. Utilization of insulin, hypotensive drugs, analgesics and perhaps canagliflozin further increases the risk. Thiazolidinedione use may increase the risk of osteoporosis and fracture. Prolonged hyperglycemia resulting in cross-linking of collagen and advanced glycosylation end products alter the microarchitecture and increase bone fragility. Higher serum vitamin D levels seem to decrease the incidence of both falls and fractures. Following a hip fracture, mortality in the diabetic patient is increased largely because of cardiovascular events and pneumonia. Prevention of sarcopenia includes dietary therapy, vitamin D and testosterone replacement when appropriate.
David S H Bell, Edison Goncalves

1031 related Products with: Why Do Falls and Lower Limb Fractures Occur More Frequently in the Diabetic Patient and How Can They Be Prevented?

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#32651825   2020/07/10 To Up

Ultrastructural Changes in the Air-Blood Barrier of Rats in Acute Intoxication with Furoplast Pyrolysis Products.

Rats were exposed to fluoroplast-4 pyrolysis products (sample weight 2.6 g, pyrolysis temperature 440-750°C, pyrolysis duration 4 min) containing perfluoroisobutylene over 15 min. Lung tissue samples for histological and electron microscopic examination were isolated in 3 and 30 min after intoxication and processed routinely. Histological examination revealed no structural changes in the lungs. In ultrathin sections of rat lungs, some changes in the structure of type I pneumocytes were detected in 3 min after the exposure: detachment of cytoplasmic processes and the appearance of transcytosis pores. These changes attested to impaired cell-cell interactions and their adhesion to the basement membrane, where structural disorganization and edema of the collagen matrix were observed. In 30 min following exposure, the signs of damage to type I pneumocytes became more pronounced. The increase in the equivalents of transcellular and paracellular permeability in the alveolar lining profile was observed. No changes in the pulmonary capillary endotheliocytes were detected, which suggest that type I pneumocytes are the primary target of the toxic effect of perfluoroisobutylene. The vulnerability of a particular cell population, in view of specific metabolism of these cells, can be the key to deciphering of the mechanisms of the toxic effect of pyrolysis products of fluorinated polymer materials.
P G Tolkach, V A Basharin, S V Chepur, A N Gorshkov, D T Sizova

1364 related Products with: Ultrastructural Changes in the Air-Blood Barrier of Rats in Acute Intoxication with Furoplast Pyrolysis Products.

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#32651628   2020/07/10 To Up

Combined corneal cross-linking and 320° intrastromal corneal ring segments in progressive keratoconus: one-year results.

Intrastromal corneal ring segments (ICRS) attain regularization of corneal surface in keratoconus (KC), while collagen cross-linking (CXL) halts or slows its progression. The long-arc 320° rings combined with CXL may have a dual-benefit synergistic effect of surface regularity and progression halt.
Adel Galal Zaky, Mahmoud Tawfik KhalafAllah, Abdelrahman Elsebaey Sarhan

1697 related Products with: Combined corneal cross-linking and 320° intrastromal corneal ring segments in progressive keratoconus: one-year results.

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#32650855   // To Up

Desmoplastic Melanoma as a Diagnostic Pitfall.

Dear Editor, Desmoplastic melanoma (DM) is a rare histological subtype of melanoma, usually presenting as a slowly-growing, amelanotic, discoid, and/or firm lesion composed of spindle cells with abundant collagen (1). It is more common on sun-exposed areas, especially on head and neck in elderly patients (2). Regional lymph node involvement is reported to be less frequent than in other cutaneous melanomas (3). Desmoplastic melanoma can clinically mimic a wide spectrum of benign and malignant lesions, including Bowen's disease, desmoplastic nevus, basal cell carcinoma, squamous cell carcinoma, lentigo maligna, dermatofibrosarcoma protuberans, peripheral nerve sheath tumors, cysts, or hypertrophic/keloid scars (4). Regarding its appearance, at the time of diagnosis DM frequently presents as advanced lesions with deep infiltration. A 60-year-old man presented with an one-year history of an asymptomatic, erythematous, well-defined plaque in the right lumbar region (Figure 1). Dermatological examination revealed a 5×5 cm, pink/red infiltrated plaque accompanied by a 6 mm dark-brown melanocytic lesion. Dermoscopically, atypical vascular structures in the form of linear, irregular, and dotted vessels, milky-red areas, and atypical pigment network, and streaks were observed near the melanocytic lesion (Figure 2). A 4 mm punch biopsy was performed on the erythematous plaque next to the melanocytic lesion, and a dermal-based, paucicellular proliferation of atypical spindle cells without melanin in a sclerotic stroma was found histologically (Figure 3, a). Immunohistochemically, dermal spindle cells were stained with S-100 and HMB45 antibodies (Figure 3, b). The patient was histologically diagnosed with melanoma, of the desmoplastic subtype. The lesion was totally excised with 2 cm clear margins. A diagnosis of nonulcerated nodular melanoma with a Breslow thickness of 4 mm and a mitotic index 1/mm2 was established. Sentinel lymph node biopsy revealed no metastases. No systemic metastases were detected in PET-CT scanning and cranial magnetic resonance imaging. The patient remained under follow-up and has been free of any local recurrence or primary or systemic metastasis for 3 years. Dermoscopic characteristics of DM are not well known, probably due to it not being considered a melanocytic lesion. Debarbieux et al. first reported the dermoscopic features of desmoplastic melanoma in six cases (5). They found that only half of the cases presented one classical feature of a melanocytic lesion, whereas the other cases were diagnosed based on the presence of figures of regression such as white scar-like and "peppering", multiple (>4) color, and melanoma-related vascular patterns (five out of six) such as linear-irregular vessels and milky-red areas (5). In the largest DM case series, Jaime et al. reported that all DM featured at least 1 melanoma-specific structure, with atypical vascular structures being the most common (6). Similarly, in our patient dermoscopy showed an atypical pigment network and streaks, atypical vascular structures, and milky-red areas, which is predictive for melanoma. We reported this case to serve as a reminder to consider desmoplastic melanoma in the differential diagnosis of pink tumoral lesions despite its rarity and atypical localization.
Ilkin Zindanci, Ebru Zemheri, Tugba Kevser Uzuncakmak, Necmettin Akdeniz, Ayse Serap Karadag, Mukaddes Kavala, Burce Can, Zafer Turkoglu

2679 related Products with: Desmoplastic Melanoma as a Diagnostic Pitfall.

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#32650853   // To Up

Unusual Case of Granuloma Annulare Associated with Diabetes Mellitus.

Dear Editor, Granuloma annulare (GA) is an asymptomatic, chronic, and relatively common granulomatous skin condition which presents with annular papules usually slowly progressing into plaques on the extremities and the trunk. It usually presents with non-scaly, erythematous, annular plaques on the distal extremity (1,2). The pathogenesis of GA is still unknown, although a variety of possible factors contributing the disease have been reported, including drugs (3), insect bites, sun exposure, trauma, vaccinations, and viral infections (e.g. hepatitis B, hepatitis C, HIV, Epstein-Barr virus) (1). Several cases in which GA developed on residual skin changes from herpes zoster have also been reported (4). A 47-year-old woman presented with erythematous-livid plaques on the dorsa of her hands and linear and circular lesions on her neck, gradually spreading for the last 4 months prior to admission at our Department (Figure 1a and Figure 1b). She reported excessive thirst and sweating in the last 30 days, but did not consider it significant since it was summer. The patient was otherwise healthy and was not taking any medications. Mycological swabs taken from the dorsal parts of both hands and the neck were negative. Biopsy of the skin changes was consistent with GA, showing palisading granulomatous inflammation which surrounded degenerated collagen within the dermis. A routine laboratory check revealed increased levels of glucose (23 mmol/L) and HgbA1C, while lipid and thyroid hormone levels were normal. Fasting blood sugar lever was 17 mmol/L. Therapy with topical corticosteroid (betamethasone cream) for skin lesions was initiated and applied two times daily for 2 weeks. The patient was immediately referred to an endocrinologist and insulin therapy was initiated due to diabetes mellitus. Complete remission of the skin changes was observed on the follow-up visit after 3 months. There are many clinical variants of GA such as localized, generalized, disseminated, subcutaneous, arcuate dermal erythema, and perforating GA (1). The localized form of GA is most common with annular plaques on the distal extremities. In addition to the typical lesions on the dorsal side of both hands, our patient also presented with atypical, circular lesions around her neck. The relationship between GA and systemic diseases such as diabetes mellitus, thyroid disorders, dyslipidemia, and malignancies remains unclear (5). It is also uncertain whether genetic factors influence susceptibility to GA. Familial cases have been documented, but studies investigating the association between the disease and human leukocyte antigen (HLA) genes have yielded inconsistent results (6). Increased frequency of HLA-B35 in patients with the generalized form has been reported in a few studies (7). GA mostly affects children and young adults, mostly women. Many cases of GA resolve spontaneously within 2 years, but relapses occur in many patients. Treatment is divided into localized skin therapies and systemic therapies (1). High potency topical corticosteroids along with intralesional corticosteroids are the most common localized treatments (8). Systemic therapy includes corticosteroids, chloroquine, dapsone, and isotretinoin (1,9). Cryotherapy and UV-therapy can also be used, although with limited efficacy (10). GA is a common idiopathic disorder of the dermis and subcutaneous tissue that can be associated with a variety of underlying conditions such as diabetes mellitus. The relationship between GA and diabetes mellitus is still unknown. Since skin lesions preceded the diagnosis of DM in our patient and complete remission of skin changes occurred with induction of insulin therapy, it is important to perform routine laboratory test in every patient.
Stjepan Patrun, Suzana Ljubojević Hadžavdić

1583 related Products with: Unusual Case of Granuloma Annulare Associated with Diabetes Mellitus.

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