Growth-stimulating material derived from porcine bone therefor and a manufacturing process

Claim 2

A composition which comprises: I an active ingredient prepared by (a) cooling in liquid nitrogen fat-free bone material of metacarpalia from pigs; (b) comminuting the bone material at the temperature of liquid nitrogen, to bone meal of a particle size in the range from 0.5 to 2 mm; (c) demineralizing the bone particles in an aqueous solution; and separating from the resulting solution, the active ingredient having the following features: (a) the active ingredient causes a dose-dependent growth stimulation of fibroblasts, osteoblast precursor cells and chondrocytes and embryonic tissue of chickens in monolayer cell cultures; (b) the active ingredient causes a dose-dependent growth of embryonic tubular bones of chickens in organ culture in vitro, due to a proliferation of chondrocytes in the growth zone; (c) the active ingredient does not cause any increased incorporation of 3 H-thymidine into DNA of calvaria cells in the microtitre plate test system; (d) the feature (a) or (b) above are retained for at least 15 minutes at a temperature of from 55° to 75° C.; (e) the features (a) or (b) above are retained for at least 20 minutes at a pH of approximately 3 and of approximately 10; (f) the active ingredient can be eluted on a gel filtration column under high salt conditions in a molecular weight range of 24±8 kD; (g) the active ingredient runs through a copper sulphate-chelate column without being adsorbed, it being possible for the specific activity to be concentrated by a factor of from 5 to 20; (h) the active ingredient can be bound to a reversed phase column in high pressure liquid chromatography and eluted with an acetonitrile/water mixture having an acetonitrile content in the range of from 10 to 45% calculated by volume; (i) 100 to 200 pg of the active ingredient forms more than 5 mm 3 bone mass growth; and (ii) the active ingredient can be dialysed against 25 mM phosphate buffer; and II a solid carrier to bind the active ingredient to the site of application.

Claim 3

A composition according to claim 1 wherein the carrier is a partially to fully absorbable carrier.

Claim 4

A composition according to claim 1 having a ratio of active ingredient material:solid carrier of 1:1000 to 1:200,000 (volume:weight).

Claim 5

A process for obtaining the active ingredient according to claim 1, which comprises; cooling in liquid nitrogen fat-free bone material of metacarpalia from pigs; comminuting the bone material to bone meal of a particle size in the range of from 0.5 to 2 mm at the temperature of liquid nitrogen; demineralizing the bone meal particles with an aqueous solution; dialysing the resulting aqueous suspension to extract the active ingredient; acidifying the supernatent to an acid pH; heating the acidified supernatent to a temperature sufficient to precipitate protein; and centrifuging off insoluble components.

Claim 6

The process according to claim 4, wherein, the aqueous supernatant obtained after centrifugation is applied to a chromatography column of hydroxyapatite and eluted with guanidinium hydrochloride.

Claim 7

The composition of claim 1 wherein the solid carrier is selected from the group consisting of hydroxyapatite, tricalcium phosphate, and collagen.

Claim 8

The composition of claim 1 wherein separation of the active ingredient comprises dialysis, acidification of the dialysis supernatent, heating of the supernatent and centrifugation to remove insolubles.

Claim 9

The composition of claim 7 wherein the separated active ingredient is applied to a hydroxyapatite column and eluted with guanidinium hydrochloride.

Claim 10

The composition of claim 8 wherein the eluate is applied to a gel filtration column and eluted under high salt conditions in a molecular weight range of approximately 24±8 kD.

Claim 11

A composition useful as a mitogenic factor, which is prepared by; (a) cooling in liquid nitrogen fat-free bone material of metacrapalia from pigs; (b) comminuting the bone material at the temperature of liquid nitrogen, to bone meal of a particle size in the range from 0.5 to 2 mm; (c) demineralizing the bone particles in an aqueous solution; (d) dialyzing the aqueous solution against a 10 percent (weight/volume) solution of N,N,N',N',-ethylenediaminetetraacetic acid; (e) heating the supernatent at a temperature of 75° C. for 15 minutes to precipitate protein; and (f) acidifying the solution to a pH of 3.0; and (g) centrifuging to remove precipitated protein; said composition having the following features: (a) a dose-dependent growth stimulation of fibroblasts, osteoblast precursor cells and chondrocytes and embryonic tissue of chickens in monolayer cell cultures; (b) a dose-dependent growth of embryonic tubular bones of chickens in organ culture in vitro, due to a proliferation of chondrocytes in the growth zone; (c) do not cause any increased incorporation of 3 H-thymidine into DNA of calvaria cells in the microtitre plate test system; (d) the feature (a) and (b) above are retained for at least 15 minutes at a temperature of from 55° to 75° C.; (e) the features (a) or (b) above are retained for at least 20 minutes as a pH of approximately 3 and of approximately 10; (f) the composition can be eluted on a gel filtration column under high salt conditions in a molecular weight range of 24±8 kD; (g) the composition run through a copper (11) sulphate-chelate column without being adsorbed, it being possible for the specific activity to be concentrated by a factor of from 5 to 20; (h) the composition can be bound to a reversed phase column in high pressure liquid chromatography and eluted with an acetonitrile/water mixture having an acetonitrile content in the range of from 10 to 45%; (i) 100 to 200 μg of the active ingredient forms more than 5 mm 3 bone mass growth; and (j) the active ingredient can be dialysed against 25 mM phosphate buffer; and II a solid carrier to bind the active ingredient to the site of application.

Claim 12

A composition useful as a mitogenic factor, which is prepared by; (a) cooling in liquid nitrogen fat-free bone material of metacrapalia from pigs; (b) comminuting the bone material at the temperature of liquid nitrogen, to bone meal of a particle size in the range from 0.5 to 2 mm; (c) demineralizing the bone particles in an aqueous solution; (d) dialyzing the aqueous solution against a 10 percent (weight/volume) solution of N,N,N',N',-ethylenediaminetetraacetic acid; (e) heating the supernatent at a temperature of 75° C. for 15 minutes to precipitate protein; and (f) acidifying the solution to a pH of 3.0; and (g) centrifuging to remove precipitated protein; said composition having the following features: (a) a dose-dependent growth stimulation of fibroblasts, osteoblast precursor cells and chondrocytes and embryonic tissue of chickens in monolayer cell cultures; (b) a dose-dependent growth of embryonic tubular bones of chickens in organ culture in vitro, due to a proliferation of chondrocytes in the growth zone; (c) do not cause any increased incorporation of 3 H-thymidine into DNA of calvaria cells in the microtitre plate test system; (d) the feature (a) and (b) above are retained for at least 15 minutes at a temperature of from 55° to 75° C.; (e) the features (a) or (b) above are retained for at least 20 minutes as a pH of approximately 3 and of approximately 10; (f) the composition can be eluted on a gel filtration column under high salt conditions in a molecular weight range of 24±8 kD; (g) the composition run through a copper (11) sulphate-chelate column without being adsorbed, it being possible for the specific activity to be concentrated by a factor of from 5 to 20; (h) the composition can be bound to a reversed phase column in high pressure liquid chromatography and eluted with an acetonitrile/water mixture having an acetonitrile content in the range of from 10 to 45%; (i) 100 to 200 μg of the active ingredient forms more than 5 mm 3 bone mass growth; and (j) the active ingredient can be dialysed against 25 mM phosphate buffer; and II a solid carrier to bind the active ingredient to the site of application.

Claim 13

The composition of claim 10 wherein after centrifuging, the supernatent is applied to a chromatography column of hydroxyapatite and eluted with guanidinium hydrochloride and the eluate is applied to a gel filtration column and eluted with 4M guanidinium hydrochloride in a molecular weight range of approximately 24±8 kD.