Enhancement of osteogenic potential of bone grafts

Patent metadata

Field	Value
Publication number	US-9925304-B2
Application number	US-201615063317-A
Country	US
Kind code	B2
Filing date	Mar 7, 2016
Priority date	Jul 16, 2013
Publication date	Mar 27, 2018
Grant date	Mar 27, 2018

Abstract

The present invention concerns the enhancement of the osteogenic potential of bone graft by ex vivo treatment with a Wnt polypeptide, such as a liposomal Wnt polypeptide.

First claim

What is claimed is:

1. An effective dose of an isolated enhanced mammalian bone graft material comprising enhanced cells wherein the enhanced cells have an increased level of expression for biomarkers comprising Axin2 and either Tcf4 or Lef1 after exposure ex vivo with a liposome comprising a Wnt protein, wherein the increased level of expression for the biomarkers is relative to equivalent cells from isolated mammalian bone graft material unexposed to a liposome comprising a Wnt protein.

2. The effective dose of claim 1 , wherein the enhanced cells further have an increased level of expression for one or more biomarkers comprising Runx2, Osterix, Osteocalcin, or alkaline phosphatase.

3. The effective dose of claim 1 , wherein the increased level of expression is increased level of gene expression.

4. The effective dose of claim 1 , wherein the increased level of expression is increased level of protein expression.

5. The effective dose of claim 1 , wherein the increased level of expression is up to 9× relative to the equivalent cells from isolated mammalian bone graft material.

6. The effective dose of claim 1 , wherein the Wnt protein is Wnt3a protein.

7. The effective dose of claim 1 , wherein the enhanced cells comprise bone marrow stem cells or bone marrow progenitor cells.

8. The effective dose of claim 1 , wherein the enhanced cells comprise mesenchymal stem cells or osteocytes.

9. The effective dose of claim 1 , wherein the isolated enhanced mammalian bone graft material comprises enhanced cells with reduced apoptosis after exposure ex vivo with a liposome comprising a Wnt protein, wherein the reduced apoptosis is relative to equivalent cells from isolated mammalian bone graft material unexposed to a liposome comprising a Wnt protein.

10. The effective dose of claim 9 , wherein the reduced apoptosis in the enhanced cells is measured as reduced caspase activity relative to equivalent cells from isolated mammalian bone graft material unexposed to a liposome comprising a Wnt protein.

11. The effective dose of claim 1 , wherein the isolated enhanced mammalian bone graft material comprises enhanced cells with increased cell proliferation after exposure ex vivo with a liposome comprising a Wnt protein, wherein the increased cell proliferation is relative to equivalent cells from isolated mammalian bone graft material unexposed to a liposome comprising a Wnt protein.

12. The effective dose of claim 11 , wherein the increased cell proliferation in the enhanced cells is measured as increased mitotic activity relative to equivalent cells from isolated mammalian bone graft material unexposed to a liposome comprising a Wnt protein.

13. The effective dose of claim 1 , wherein the isolated enhanced mammalian bone graft material is isolated enhanced human bone graft material.

14. The effective dose of claim 1 , wherein the isolated enhanced human bone graft material is obtained from a human subject at least 50 years of age or older.

15. The effective dose of claim 1 , wherein the isolated enhanced mammalian bone graft material is obtained from an allogeneic donor.

16. An ex vivo method of enhancing cell survival in a bone graft in a subject in need thereof, comprising:
a) incubating a sample comprising isolated mammalian bone graft material comprising cells ex-vivo with a liposome comprising a Wnt protein to produce enhanced cells comprising an increased level of expression for biomarkers comprising Axin2 and either Tcf4 or Lef1; and
b) transplanting the enhanced cells into a target site on bone.

17. The ex vivo method of claim 16 , wherein the enhanced cells further have an increased level of expression for one or more biomarkers comprising Runx2, Osterix, Osteocalcin, or alkaline phosphatase.

18. The ex vivo method of claim 16 , wherein the incubating temperature is at room temperature or at about 37° C.

19. The ex vivo method of claim 16 , wherein the incubating time is from about 1 hour to about 36 hours.

20. The ex vivo method of claim 16 , wherein the isolated mammalian bone graft material further comprises enhanced osteogenic capacity compared to unexposed mammalian bone graft material.

21. The ex vivo method of claim 20 , wherein the enhanced osteogenic capacity comprises increased volume of bone growth of about 1.5-fold, about 2-fold, about 3-fold, or more compared to the volume of bone growth of unexposed mammalian bone marrow cells.

22. The ex vivo method of claim 16 , wherein the increased level of expression of the enhanced cells is relative to equivalent cells from isolated mammalian bone graft material without incubation with a liposome comprising a Wnt protein.

23. The ex vivo method of claim 16 , wherein the increased level of expression is increased level of gene expression.

24. The ex vivo method of claim 16 , wherein the increased level of expression is increased level of protein expression.

25. The ex vivo method of claim 16 , wherein the increased level of expression is up to 9× relative to the equivalent cells from isolated mammalian bone graft material.

26. The ex vivo method of claim 16 , wherein the enhanced cells comprise bone marrow stem cells or bone marrow progenitor cells.

27. The ex vivo method of claim 16 , wherein the enhanced cells comprise mesenchymal stem cells or osteocytes.

28. The ex vivo method of claim 16 , wherein the isolated mammalian bone graft material is isolated human bone graft material.

29. The ex vivo method of claim 16 , wherein the isolated mammalian bone graft material is obtained from a human subject at least 50 years of age or older.

30. The ex vivo method of claim 16 , wherein the isolated mammalian bone graft material is obtained from an allogeneic donor.

31. An ex vivo method of enhancing cell survival in a bone graft in a subject in need thereof, comprising:
a) isolating a bone graft sample comprising cells from a human subject,
b) incubating the bone graft sample ex-vivo with a liposome comprising a Wnt3a protein for a sufficient time to produce enhanced cells comprising an increased level of expression for biomarkers comprising Axin2 and either Tcf4 or Left relative to equivalent cells from bone graft material not incubated with a liposome comprising a Wnt3a protein; and
c) transplanting the incubated enhanced cells into a target site on bone.

32. The ex vivo method of claim 31 , wherein the enhanced cells further have an increased level of expression for one or more biomarkers comprising Runx2, Osterix, Osteocalcin, or alkaline phosphatase.

Assignees

• Univ Leland Stanford Junior
• The Board Of Trustees Of The Leland Stanford Junior Univesity

Inventors

• Helms Jill
• Dhamdhere Girija

Classifications

• A61P19/08

  for bone diseases, e.g. rachitism, Paget's disease · CPC title

• A61K38/1709 Primary

  from mammals · CPC title

• A61L27/3687 Primary

  characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents · CPC title

• A61L27/365

  Bones · CPC title

• A61K35/28 Primary

  Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells · CPC title