The effects of different preservation processes on the total protein and growth factor content in a new biological produ
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The effects of different preservation processes on the total protein and growth factor content in a new biological product developed from human amniotic membrane Alessandra Russo • Paola Bonci • Paolo Bonci
Received: 20 July 2010 / Accepted: 26 May 2011 / Published online: 18 June 2011 Ó Springer Science+Business Media B.V. 2011
Abstract The aim of this work is to quantify the total protein and growth factors content in a tissuesuspension obtained from processed human amniotic membrane (hAM). hAM was collected, frozen, freeze dried, powdered and sterilized by c-irradiation. At each step of the process, samples were characterized for the total protein amounts by a Bradford protein assay and for the growth factor concentrations by ELISA test of the tissue suspensions. Frozen-hAM samples show higher release of total proteins and specific growth factors in the tissue suspension in comparison with freeze-dried hAM. We observed that even if the protein extraction is hindered once the tissue is dried, the powdering process allows a greater release in the tissue suspension of total proteins and growth factors after tissue re-solubilization in comparison with only the freeze-drying process (?91 ± 13% for EGF, ?16 ± 4% for HGF, ?11 ± 5% for FGF, ?16 ± 9% for TGF-b1), and a greater release of EGF (85 ± 10%) in comparison with only the freezing process, because proteins become much readily solubilized in the solution. According with these results, we describe a protocol to obtain a new sterile biological product from hAM tissue, with well-known effects of thermal,
A. Russo (&) P. Bonci P. Bonci Eye Bank, Department of Ophthalmology, S. Maria della Scaletta Hospital, Via Montericco 4, Imola, Bologna, Italy e-mail: [email protected]
mechanical and physical processes on the total protein and grow factors contents. Keywords Growth factor Human amniotic membrane ELISA Freeze-drying Tissuepowdering Irradiation
Introduction The use of hAM in ocular surgery was first suggested by Sorsby (Sorsby and Symmons 1946; Sorsby et al. 1947), who examined its role in the management of ocular surface damage. Very good results were reported but, for no evident reason, its use was abandoned or went unreported until recently. The early 1990s were the starting point in tracing the modern history of the use of hAM in ophthalmic surgery, by introducing the now well-accepted method of preservation and storage, by adding considerably to the knowledge of the key components of the membrane and by developing some of the current surgical techniques (Dua et al. 2004). The functional and structural similarities among different kinds of tissues, such as hAM and cornea, could explain the beneficial effects of hAM transplants in ocular surgery. The mechanisms of action of the membrane are inferred from the composition of the membrane, rather than proven scientifically, in
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relation to its application in ocular surgery (Dua et al. 2004). Among the properties of hAM, it acts as a substrate or basement membrane for epithelial
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