Granted Patent B2

US 10,434,217 · App. 15/082,586 · Granted Oct 8, 2019

Abstract

Claims (42)

1. A method of preparing an osteogenic composition comprising:

obtaining a biological sample, said biological sample comprising cells and one or more osteogenic agents, wherein the biological sample comprises a bone marrow aspirate;

at least partially lysing the biological sample to produce a lysate, wherein lysing the biological sample comprises acidifying the biological sample by using 1.5N HCL to lyse the bone marrow aspirate, thereby forming an acidified lysate, said lysate comprising solid material and one or more osteogenic agents;

neutralizing the acidified lysate;

separating the lysate into a first fraction and a second fraction, said first fraction substantially comprising the one or more osteogenic agents, and said second fraction substantially comprising the solid material; and

collecting said first fraction, thereby obtaining an osteogenic composition.

2. The method of claim 1 , wherein the biological sample further comprises whole blood in combination with the bone marrow aspirate.

3. The method of claim 2 , further comprising passing the bone marrow aspirate through a three-way valve system one or more times prior to lyse the bone marrow aspirate.

4. The method of claim 1 , wherein neutralizing the acidified lysate comprises contacting the lysate with an alkaline composition.

5. The method of claim 1 , wherein neutralizing the acidified lysate comprises contacting the lysate with a phosphate buffer.

6. The method of claim 1 , wherein separating the lysate comprises applying the lysate to a tangential flow hollow fiber membrane filtration system such that the filtrate substantially comprises the first fraction and the retentate substantially comprises the second fraction.

7. The method of claim 1 , wherein separating the lysate comprises centrifuging the lysate so that the supernatant substantially comprises the first fraction and the pellet substantially comprises the second fraction.

8. The method of claim 1 , further comprising concentrating the first fraction.

9. The method of claim 8 , wherein concentrating the first fraction comprises applying the first fraction to a hollow fiber membrane system.

10. The method of claim 9 , wherein a molecular weight cut off of the membrane is about 10-20 kDa.

11. The method of claim 1 , further comprising contacting the first fraction with a pharmaceutical carrier medium suitable for use in bone repair surgeries.

12. The method of claim 1 , wherein the first fraction is an osteogenic composition enriched for osteogenic factors, including one or more of BMP-2, VEGF, PDGF and TGF-β, which is applied to an appropriate pharmaceutical carrier suitable for use in bone and soft tissue reparative therapies.

13. The method of claim 12 , wherein the pharmaceutical carrier is in the form of a block, paste, cement, powder, granule, putty, liquid, gel, or a combination thereof, which is comprised of a ceramic, a polymer, a metal, allograft bone particles, autograft bone particles, demineralized bone matrix particles, or a mixture thereof.

14. A method of preparing an osteogenic composition from bone marrow comprising:

obtaining a bone marrow aspirate from a subject;

dispersing the bone marrow aspirate;

lysing the cells in the dispersed bone marrow aspirate, wherein lysing the cells comprises acidifying the dispersed bone marrow aspirate by using 1.5 N HCL to lyse the cells, thereby forming an acidified lysate;

neutralizing the acidified lysate;

separating the lysate into a first fraction and a second fraction, said first fraction comprising one or more osteogenic agents present in the bone marrow, and said second fraction comprising solid material; and

collecting said first fraction, thereby obtaining an osteogenic composition.

15. The method of claim 14 , wherein the bone marrow comprises an autologous bone marrow aspirate obtained from a patient.

16. The method of claim 14 , wherein dispersing the bone marrow aspirate comprises passing the bone marrow aspirate through a three-way valve system.

17. The method of claim 14 , wherein neutralizing the acidified lysate comprises contacting the lysate with an alkaline composition.

18. The method of claim 14 , wherein neutralizing the acidified lysate comprises contacting the lysate with a phosphate buffer.

19. The method of claim 14 , wherein separating the lysate comprises passing the lysate through a hollow fiber membrane system such that the filtrate substantially comprises the first fraction and the retentate substantially comprises the second fraction.

20. The method of claim 14 , wherein separating the lysate comprises centrifuging the lysate such that supernatant substantially comprises the first fraction and the pellet substantially comprises the second fraction.

21. The method of claim 14 , further comprising concentrating the first fraction.

22. The method of claim 21 , wherein concentrating the first fraction comprises applying the first fraction to a hollow fiber membrane system.

23. The method of claim 14 , further comprising contacting the first fraction with a pharmaceutical carrier suitable for use in bone repair surgeries.

24. A method of preparing an autologous osteogenic composition from bone marrow comprising:

obtaining a bone marrow aspirate from a subject;

dispersing the bone marrow aspirate;

collecting at least a portion of the bone marrow cells in the dispersed bone marrow aspirate, and dividing the bone marrow cells into at least a first portion and a second portion;

lysing the cells in the first portion to produce a lysate, wherein lysing the cells comprises acidifying the bone marrow cells in the first portion by using 1.5 N HCL to lyse the cells, thereby forming an acidified lysate;

neutralizing the acidified lysate;

separating the lysate into a first fraction and a second fraction, said first fraction comprising one or more osteogenic agents present in the bone marrow, and said second fraction comprising solid material; and

collecting said first fraction, thereby obtaining an autologous osteogenic composition.

Assignments (10)