Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1983-10-14
1985-04-16
Schain, Howard E.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
2601237, 128334R, 422243, 435 41, 514 21, 514801, C08H 106, C12P 2106
Patent
active
045116539
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a process for the industrial preparation of new insoluble and soluble collagenous materials from human placental tissues as well as to the human collagenous materials obtained and their application as biomaterials.
Collagens are the main proteinic constituents of conjunctive tissue such as the dermis, cartilage, tendons, vascular walls . . . to which they impart the mechanical properties essential to their physiological functions. At the present time, at least five main types of collagen have been isolated and characterized. They can be classified into two main groups: collagens of types I, II and III present in tissues as fibers and often termed interstitial collagens and collagens of types IV and V termed basal membrane collagens whose molecular composition and form within the tissues are still imperfectly known.
These collagens, although different with regard to their primary sequence, have a common triple helix tertiary structure which protects them against degradation by proteolytic enzymes other than collagenases.
After their intracellular synthesis, collagens are excreted in soluble form into the extracellular medium where they aggregate to form fibrils. During maturation of the tissues, these fibrils become rapidly insolubilized as a result of crosslinking of the collagen molecules through formation of interchain covalent bonds. These bonds form at the extremity of at least one of the molecules in a non helicoidal region and therefore in a region which may be cleaved by the action of proteolytic enzymes.
Two types of processes make it possible, at present, to extract substantial quantities of collagens from a tissue.
One of these processes uses tissues obtained from young mammals in which there is a substantial fraction of non crosslinked collagen and uses the solubility properties of these collagens in neutral saline solutions or in dilute acids as described, for example, in French Pat. Nos. 1 568 829 and 1 596 789 in which soluble collagen is prepared from calf skins.
These extraction processes have, however, several drawbacks: on the one hand, they require the use of animal tissues only as a base material and, on the other hand, they provide collagen of type I only.
Other types of processes use mature tissues; it then becomes indispensable to use the proteolytic activity of enzymes such as pepsin, pronase or trypsin to cut down the telopeptides of the collagens not protected by the triple helix and thus break the intermolecular bonds responsible for their insolubility.
In human tissues, the placental tissue alone is available in sufficient quantities to eventually provide for a separation of human collagens on an industrial scale.
The difficulties encountered in the preparation of purified collagens from this tissue are numerous and result essentially from:
the low collagen content of the starting tissue (10 to 15%)
the insolubility of these collagens
the possible presence of hepatitis viruses in the starting material.
It has been proposed to treat this tissue with neutral or acidic saline solutions (NaCl) (German patent application DAS No. 26 16 939) or with concentrated urea solutions (German patent application OS No. 24 62 221) in order to extract therefrom the soluble part present in low concentration. Various studies carried out in the laboratory have improved this collagen extraction from placental tissue by partial digestion with pepsin of 20 to 40% of the starting collagens (see, in particular, BIOCHEMISTRY, vol. 18, no. 14--July 10, 1979--T. F. Kresina--p. 3091) and fractionation thereof into collagens of types I, III, IV and V. The main drawbacks of these methods are as follows:
enzymatic digestion of the total tissue requires the treatment of substantial weights and volumes of tissue and extracts as well as the use of large quantities of enzymes. Indeed, the proteolytic enzyme acts at this level not only on collagen telopeptides but also on the non collagenous protein mass (60% of the starting dry weight),
it is possible to extract only 20 to 40% of the total
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Bonneau Marc
Comte Philippe
Herbage Daniel
Merieux Charles
Play Dominique
Centre Technique du Cuir
Dubno Herbert
Foundation Merieux
Ross Karl F.
Schain Howard E.
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