Harnessing cell dynamics to engineer materials

• US 9,297,005 B2
• Filed: 04/13/2010
• Issued: 03/29/2016
• Est. Priority Date: 04/13/2009
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Huebsch, Nathaniel D., Mooney, David J.
• Primary Examiner(s): Lankford, Blaine
• Assistant Examiner(s): Visone, Thomas J
• Application Number:
  US13/264,243
• Publication Number:
  US 20120122218A1
• Time in Patent Office: 2,177 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 11/04 entrapped within the carrier, e.g. gel or hollow fibres
• CPC Class Codes: C12N 2533/50 Proteins
• CPC Class Codes: C12N 2533/74 Alginate
• CPC Class Codes: C12N 5/0654 Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth

Abstract

The invention features synthetic materials and methods for inducing cell behavior. Matrix materials induce cell differentiation and cell manipulation based on mechanical and physical characteristics of the materials rather than chemical characteristics.

Claims

1. A method of inducing differentiation of a population of stem cells into an osteogenic lineage comprising

contacting said stem cells with a 3-dimensional material, wherein the 3-dimensional material comprises alginate of a stiffness in the range of 22-45 kPa, and a density of adhesion molecules presented by the alginate of 150 nmol/L to 200 μ

mol/L, wherein said stem cells are encapsulated in the 3-dimensional material,

wherein said density and said stiffness induce the formation of sufficient bonds between said stem cells and said adhesion molecules, and wherein the number of such bonds formed between said stem cells and said adhesion molecules determines a cell type into which said stem cells differentiate; and

allowing said stem cells to bind to, and mechanically reorganize, said adhesion molecules, thereby inducing differentiation of said stem cells into an osteogenic lineage.

2. The method of claim 1, wherein the stem cells comprise mesenchymal stem cells (MSC).

3. The method of claim 1, wherein the adhesion molecule interacts with an integrin protein on the stem cells.

4. The method of claim 3, wherein the integrin protein comprises a₅-integrin, a_v-integrin, or both a₅-integrin and a_v-integrin.

5. The method of claim 1, wherein the adhesion molecule comprises a peptide comprising the amino acid sequence of RGD or G₄RGDDSSKY.

6. The method of claim 5, wherein the adhesion molecule comprises a peptide comprising the amino acid sequence of RGD, PHSRN, or both RGD and PHSRN.

7. The method of claim 1, wherein the adhesion molecule is present at a concentration of 200 μ

mol/L.

8. The method of claim 1, wherein the differentiated stem cells comprise a higher level of OCN, Runx2, or both OCN and Runx2 compared to undifferentiated stem cells.

9. The method of claim 1, wherein the 3-dimensional material comprises a stiffness of 45 kPA.

10. The method of claim 1, wherein the 3-dimensional material comprises a stiffness of 22 kPA.

11. The method of claim 1, wherein the contacting step is performed in vivo.

12. The method of claim 1, wherein the contacting step is performed in vitro.

13. The method of claim 1, wherein the 3-dimensional material comprises an alginate hydrogel.

14. The method of claim 13, wherein the alginate hydrogel is present at a concentration of 1-5 g alginate/100 mL.

15. The method of claim 13, wherein the alginate hydrogel comprises alginate polymers, and wherein the alginate polymers are crosslinked.

16. The method of claim 15, wherein the alginate polymers are calcium crosslinked or covalently crosslinked.

17. The method of claim 1, further comprising implanting the 3-dimensional material into a subject in need thereof.

18. The method of claim 17, wherein the contacting step occurs before the implanting step.

19. The method of claim 17, wherein the implanting step occurs before the contacting step.

20. The method of claim 19, wherein the 3-dimensional material selectively interacts with stem cells compared to other cell types.

21. The method of claim 17, wherein the 3-dimensional material is placed over a tissue defect in the subject and mediates tissue regeneration.

22. The method of claim 21, wherein the tissue is selected from the group consisting of skin, kidney, bone, liver, and nerve tissue.

23. The method of claim 21, wherein the tissue comprises an articulating joint.

24. The method of claim 23, wherein the articulating joint is selected from the group consisting of a knee joint, a hip joint, a shoulder joint, and an elbow joint.

25. The method of claim 17, wherein the 3-dimensional material deflects cells that cause fibrosis or scar formation.

26. The method of claim 17, wherein the 3-dimensional material promotes healing of a bone injury, promotes nerve regeneration, reduces or minimizes dermatological scar formation associated with cosmetic surgery, minimizes scar formation associated with liver regeneration, minimizes scar formation associated with kidney surgery, or minimizes scar formation associated with a joint implant.

27. The method of claim 17, wherein the 3-dimensional material is coated onto or part of an orthopedic device or implant.

28. The method of claim 17, wherein the 3-dimensional material is coated onto or part of a dentistry device.

29. The method of claim 27, wherein the orthopedic device or implant comprises a joint implant, and wherein the joint implant is selected from the group consisting of a knee implant, a hip implant, a shoulder implant, and an elbow implant.

30. The method of claim 17, wherein the 3-dimensional material is coated onto or part of a stent.

31. The method of claim 17, wherein the 3-dimensional material comprises 2×

10⁷ cells/mL of stem cells prior to implantation.

32. The method of claim 1, further comprising assessing the expression level of a marker of osteogenesis.

33. The method of claim 32, wherein the marker of osteogenesis is selected from the group consisting of ALP, OCN and Runx2.

34. The method of claim 32, wherein the expression level of a marker of osteogenesis is assessed by a method selected from the group consisting of immunofluorescence, RT-PCR, and Western blot analysis.