EDC/NHS cross-linked collagen foams as scaffolds for artificial corneal stroma

In this study, a highly porous collagen-based biodegradable scaffold was developed as an alternative to synthetic, non-degradable corneal implants. The developed method involved lyophilization and subsequent stabilization through N-ethyl-N'-[3-dimethylaminopropyl] carbodiimide/N-hydroxy succinimide (EDC/NHS) cross-linking to yield longer lasting, porous scaffolds with a thickness similar to that of native cornea (500 microm). For collagen-based scaffolds, cross-linking is essential; however, it has direct effects on physical characteristics crucial for optimum cell behavior. Hence, the effect of cross-linking was studied by examining the influence of cross-linking on pore size distribution, bulk porosity and average pore size. After seeding the foam with human corneal keratocytes, cell proliferation, cell penetration into the scaffold and ECM production within the scaffold were studied. After a month of culture microscopical and immunohistochemical examinations showed that the foam structure did not undergo any significant loss of integrity, and the human corneal keratocytes populated the scaffold with cells migrating both longitudinally and laterally, and secreted some of the main constituents of the corneal ECM, namely collagen types I, V and VI. The foams had a layer of lower porosity (skin layer) both at the top and the bottom. Foams had an optimal porosity (93.6%), average pore size (67.7 microm), and chemistry for cell attachment and proliferation. They also had a sufficiently rapid degradation rate (73.6+/-1.1% in 4 weeks) and could be produced at a thickness close to that of the natural corneal stroma. Cells were seeded at the top surface of the foams and their numbers there was higher than the rest, basically due to the presence of the skin layer. This is considered to be an advantage when epithelial cells need to be seeded for the construction of hemi or full thickness cornea.     

Amyloidogenicity and clinical phenotype associated with five novel mutations in apolipoprotein A-I

The phenotype of hereditary apolipoprotein A-I amyloidosis is heterogeneous with some patients developing extensive visceral amyloid deposits and end-stage renal failure as young adults and others having only laryngeal and/or skin amyloid, which may be of little clinical consequence. Clinical management and prognosis of patients with systemic amyloidosis depend entirely on correct identification of the fibril protein, such that light chain amyloidosis (AL, previously referred to as "primary"), the most frequently diagnosed type, is treated with chemotherapy, which has absolutely no role in hereditary apolipoprotein A-I amyloidosis. We report five novel apolipoprotein A-I variants, four of which were amyloidogenic and one of which was incidental in a patient with systemic AL amyloidosis. Interestingly, only one of four patients with apolipoprotein A-I amyloidosis had a family history of similar disease. Laser microdissection and tandem mass spectrometry-based proteomics were used to confirm the amyloid fibril protein and, for the first time in apolipoprotein A-I amyloidosis, demonstrated that only mutated protein as opposed to wild-type apolipoprotein A-I was deposited as amyloid. The clinical spectrum and outcome of hereditary apolipoprotein A-I amyloidosis are reviewed in detail and support the need for sequencing of the apolipoprotein A-I gene among patients with apparent localized amyloidosis in whom IHC is nondiagnostic of the fibril protein, even in the absence of a family history of disease.