Compositions: coating or plastic – Coating or plastic compositions – Pore forming
Reexamination Certificate
2000-05-12
2001-10-30
Foelak, Morton (Department: 1711)
Compositions: coating or plastic
Coating or plastic compositions
Pore forming
C521S084100, C424S078310, C536S056000, C536S057000
Reexamination Certificate
active
06309454
ABSTRACT:
FIELD
The present invention relates to freeze-dried pads comprising a major fraction of a mixture of collagen and oxidized regenerated cellulose (ORC), and to processes for the production of such pads.
BACKGROUND
WO98/00180 describes the use of freeze-dried sponges of collagen admixed with oxidized regenerated cellulose (ORC) for the treatment of chronic wounds. Such sponges must in practice meet stringent requirements of purity, sterility and non-antigenicity.
It has not hitherto been possible to provide sponges of collagen/ORC mixtures having high reproducibility and high tensile strength both when wet and when dry. In particular, collagen is prone to denaturation when it is sterilized by gamma-irradiation. Furthermore, collagen sponges tend to disintegrate rather rapidly in wound fluid, especially in the presence of collagenase enzymes. Whilst this problem can be diminished by chemical cross-linking of the collagen sponge using cross-linking agents such as glutaldehyde, the use of such cross-linking agents can give rise to problems of toxicity and antigenicity.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide physiologically acceptable, sterile sponge pads based on collagen/ORC mixtures that exhibit high tensile strength.
It is a further object of the present invention to provide physiologically acceptable, sterile sponge pads based on collagen/ORC mixtures that have very high purity, sterility and low bioburden.
It is a further object of the present invention to provide physiologically acceptable, sterile sponge pads based on collagen/ORC mixtures that have high uniformity.
It is a further object of the present invention to provide physiologically acceptable, sterile sponge pads based on collagen/ORC mixtures that exhibit reduced resorption rates under simulated physiological conditions.
It is a further object of the present invention to provide physiologically acceptable, sterile sponge pads based on collagen/ORC mixtures that exhibit high mechanical strength and long resorption times without chemical cross-linking.
The present invention provides a sterile freeze-dried sponge, wherein at least 80% by weight of the sponge consists of a mixture of collagen and oxidized regenerated cellulose in the weight ratio 60:40 to 40:60, and wherein the sponge has a dry tensile strength as herein defined of more than 3N.
The freeze-dried sponge pad is sterile. Preferably, the sterility assurance level is better than 10
−6
. Preferably, the sponge has been sterilized by gamma-irradiation.
The sponge comprises at least 80% by weight of a mixture of collagen and ORC in the rate ratio 60:40 to 40:60. Preferably, the weight ratio contains a small excess of collagen, in a range 50:50 to 40:60 ORC:collagen. Preferably, the freeze-dried sponge consists essentially of collagen, ORC, water and up to 5% of one or more therapeutically active substances such as growth factors. Preferably, the freeze-dried sponge contains no more than 1% by weight of constituents other than collagen, ORC and water.
The collagen content is determined by hydrolysing the collagen into its constituent amino acids and analyzing for hydroxyproline as detailed below. The collagen content is calculated to be 7.19 times the hydroxyproline content. The ORC content is determined by hydrolyzing it to its constituent monosaccharides and analyzing for glucuronic acid as detailed further below.
Preferably, the freeze-dried sponge has a pH, measured as hereinafter described, of from 2.3 to 4.0, preferably from 2.5 to 3.0.
Preferably, the sterile freeze-dried sponges according to the present invention have a degree of collagen denaturation, measured as hereinafter described, of less than 15%, preferably less than 10%, and more preferably less than 5%. It is a particularly advantageous feature of the freeze-dried sponges according to the present invention that the collagen is stabilized against denaturation by the gamma-irradiation used in the sterilizing. The degree of denaturation of the collagen is determined by treatment with trypsin to dissolve the denatured collagen (trypsin does not dissolve native collagen), followed by filtration and quantitation of the hydroxyproline in the filtrate, as detailed further below.
The sterile freeze-dried sponges according to the present invention preferably have a dry tensile strength (maximum load measured as hereinafter described) greater than 3N, preferably greater than 4N. Preferably, the dry tensile load at 20% extension, measured as hereinafter described, is greater than 2.5N, preferably greater than 3.5N. Preferably, the dry extension at break, measured as hereinafter described, is from 15 to 30%, preferably from 20 to 25%.
The tensile strength characteristics of the sponges according to the present invention are further characterised by wet strength measurements on samples that have been soaked for 15 minutes in PBS prior to testing. The resulting wet strength maximum load is preferably greater than 1N, more preferably greater than 1.25N. The wet load at 20% extension is greater than 0.1N, preferably greater than 0.2N, most preferably 0.2-0.3N. The wet extension at break is preferably 75-100% more preferably 80-90%.
Preferably, the sterile freeze-dried sponges according to the present invention are not chemically cross-linked. They have may some dehydrothermal cross-linking as a result of the freeze-drying process, but preferably there is no chemical cross-linking by glutaldehyde or the like. This reduces the antigenicity and processing costs of the sponges. The present invention achieves satisfactory physical properties of the sponges and sufficiently long resorption times in vivo by very careful control of the composition and manufacturing conditions of the sponges. In particular, the sponges preferably contain ORC fibers, wherein a volume fraction of at least 80% of the fibers have lengths in the range of 20 &mgr;m to 100 &mgr;m. Such a size distribution can be achieved, for example, by milling an ORC cloth, followed sieving the milled powder to remove fibers outside the range. Preferably, the average (mean by volume) length of the ORC fibers is in the range 250 &mgr;m to 450 &mgr;m.
The selection of ORC fiber lengths in this range results in easy mixing of the ORC and collagen and highly homogeneous products. The ORC is more thoroughly complexed with the collagen, which results in enhanced therapeutic properties of the sponge. Furthermore, the ORC is more effective to reduce denaturation of the collagen by gamma-radiation during sterilization. Surprisingly, these advantages can be achieved while maintaining the tensile strength of the sponge despite the small size of the ORC fibers.
The desired physicochemical properties of the freeze-dried sponges according to the present invention are further achieved by the use of collagen that has undergone sequential alkali and acid treatment steps to purify the collagen substantially without denaturing the collagen fibers. Preferably, the bioburden (TVC) of the freeze-dried sponge according to the present invention is less than 100 cfu/g, more preferably less than 10 cfu/g, and most preferably less than 1 cfu/g.
The sterile freeze-dried sponges according to the present invention have high and uniform porosity, and a high liquid absorption capacity. The measured absorption of the uncompressed pads in 0.9% saline is preferably greater than 12 g/100 cm
2
, more preferably greater than 15 g/100 cm
2
.
Preferably, the sterile freeze-dried sponge according to the present invetnion has a resorption time under simulated physiological conditions as described in more detail below of more than 48 hours.
The present invention further provides a method of manufacture of a freeze-dried sponge pad comprising the steps of:
providing an acidified paste of purified collagen fibers, wherein the collagen is less than 10% denatured;
providing oxidized regenerated cellulose fibers, wherein at least 80% of said fibers have lengths in the range of 20 &mgr;m to 1000 &mgr;m;
combining said collagen and said ORC fibers in
Aitken Will
Harvey Wil
Hyland Tom
Leeuwen Peter Van
Foelak Morton
Johnson & Johnson Medical Limited
Shatynski T. J.
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