Composition for cytocompatible, injectable, self-gelling chitosan solutions for encapsulating and delivering live cells or biologically active factors

• US 20060127873A1
• Filed: 07/16/2003
• Published: 06/15/2006
• Est. Priority Date: 07/16/2002
• Status: Abandoned Application

• Current Assignee: Piramal Healthcare Limited (Piramal Enterprises Limited)
• Original Assignee: Piramal Healthcare Limited (Piramal Enterprises Limited)
• Inventors: Sun, Jun, Buschmann, Michael, Chenite, Abdellatif, Hoemann, Caroline, Sarreqi, Alessio
• Application Number:
  US10/521,524
• Publication Number:
  US 20060127873A1
• Time in Patent Office: Days
• US Class Current: 435/2
• CPC Class Codes: A01N 1/0205 Chemical aspects
• CPC Class Codes: A01N 1/021 Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
• CPC Class Codes: A01N 1/0231 Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
• CPC Class Codes: A61K 31/717 Celluloses
• CPC Class Codes: A61K 9/0024 Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue compositions for intravenous administration, normal injectable solutions or dispersions for, e.g. subcutaneous administration A61K9/0019; brain implants A61K9/0085; (coated) prostheses, catheters or stents A61L
• CPC Class Codes: A61L 27/20 Polysaccharides
• CPC Class Codes: A61L 27/3604 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
• CPC Class Codes: A61L 27/3641 characterised by the site of application in the body materials for artificial blood vessels A61L27/507; materials for use in artificial skin A61L27/60
• CPC Class Codes: A61L 27/365 Bones
• CPC Class Codes: A61L 27/3654 Cartilage, e.g. meniscus
• CPC Class Codes: A61L 27/38 containing added animal cells organs or tissue containing native cells A61L27/36
• CPC Class Codes: A61L 27/3839 characterised by the site of application in the body materials for artificial blood vessels A61L27/507; materials for use in artificial skin A61L27/60
• CPC Class Codes: A61L 27/3847 Bones
• CPC Class Codes: A61L 27/3852 Cartilage, e.g. meniscus
• CPC Class Codes: A61L 27/50 Materials characterised by their function or physical properties , e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
• CPC Class Codes: A61L 27/52 Hydrogels or hydrocolloids
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 3/02 Nutrients, e.g. vitamins, minerals
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: C08L 5/08 : Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof

Abstract

The present invention provides compositions and methods for tissue repair using a cytocompatible self-gelling cross-linked hydrogel. The composition comprises a biocompatible mixture of chitosan, bifunctional dialdehyde, and hydroxylated polymer, which can be used to immobilize or encapsulate viable cells, or bioactive substances. The method includes the process of mixing bioactive substances, live cells, and/or extracellular matrix components with a cross-linking solution comprising a bifunctional aldehyde-treated hydroxylated polymer such as hydroxyethyl cellulose. The cross-linking solution is then mixed homogenously with a neutral isotonic chitosan solution. The chitosan becomes cross-linked by the bifunctional aldehyde, while the cells are protected from potentially nocive effects of the aldehyde cross-linker by the hydroxylated polymer. The injectable solution retains cell viability and bioactivity, and immobilizes cells at the site of injection or delivery. Depending on the particular application, mixtures of chitosan and bifunctional dialdehyde may be employed. The injectable solution also liberates bioactive substances with controlled release kinetics from the site of injection.

Claims

1. A composition for immobilizing and encapsulating viable and functional cells or bioactive substances comprising:

a) a liquid polysaccharide solution of isotonic neutral chitosan; and

b) a cross-linking solution consisting of a bifunctional or multifunctional, aldehyde or aldehyde-treated hydroxyl-containing polymer dissolved in physiological media.

2. The composition of claim 1, wherein the cross-linking solution consists of a bifunctional or multifunctional cross-linker and a hydroxylated polymer of appropriate ratio and molecular mass such as to permit the hydroxylated polymer to remain liquid in solution.

3. The composition of claim 1, where the cross-linking solution consists of glyoxal, or glyoxal-treated hydroxyethyl cellulose dissolved in a physiological medium.

4. The composition of claim 1, wherein the composition comprises:

a) 0.5 to 5.0% by weight chitosan, or chitosan derivative, or poly-amine containing polymer; and

b) 0.01 to 5.0% by weight hydroxyethyl cellulose, wherein said solution form a gel between temperatures of 4 C and 42 C, said gel providing a physiological environment for maintaining viability of cells.

5. The composition of claim 4, further comprising:

c) 0.0001-3% glyoxal.

6. The composition of claim 4, wherein the composition forms a gel within seconds to several hours after mixing (a) and (b).

7. The composition of claim 5, wherein the composition forms a gel within seconds to several hours after mixing (a), (b) and (c).

8. The composition of claim 4, wherein the solution forms a gel between temperatures of 20 C and 42 C.

9. The composition of claim 1, wherein the chitosan is dissolved in dilute acid and mixed with 1.0 to 2.5% by weight of a salt of polyol consisting of mono-phosphate dibasic salt, or mono-sulfate salt.

10. The composition of claim 9, wherein said mono-phosphate dibasic salt is mono-phosphate dibasic salt of glycerol.

11. The composition of claim 9, wherein mono-phosphate dibasic salt of glycerol is selected from the group consisting of glycerol-2-phosphate dibasic salt, sn-glycerol 3-phosphate dibasic salt and L-glycerol-3-phosphate dibasic salt.

12. The composition of claim 1, wherein chitosan is further mixed with phosphate buffer and salt.

13. The composition of claim 1, further comprising a biologically active factor.

14. The composition of claim 13, wherein the biologically active factor is selected from the group consisting of cells, a hormones, a drug, DNA, a bulking agent, a growth factors, a DNA, DNA-polymer complex, liposomes, a pharmacological agent, a metabolic factor, an antibody, a nutritive factor, an angiogenic factor, and a radioisotope.

15. The composition of claim 14, wherein said cells are live cells.

16. The composition of claim 14, wherein the cells are nucleus pulpopus, annulus fibrosis, or a mixture thereof.

17. The composition of claim 14, wherein the cells are embryonic stem cells or stem cells derived from a tissue selected from the group consisting of bone marrow, adipose, muscle, brain, skin, liver, vascular smooth muscle, endothelium, blood, or placenta.

18. The composition of claim 14, wherein the cells are primary cells, differentiated cells, genetically modified cells, hybridomas, immortalized cells, transformed cells, tissue fragment cells, organelles, or a mixture thereof, nucleated cells, enucleated cells, germ cells, platelet cells, matrix vesicles, cell vesicles, demineralized bone paste, bone chips, cartilage fragments, or cell fragments or tissue fragments.

19. The composition of claim 14, wherein the cells are autologous cells, allogeneic cells or xenogeneic cells.

20. The composition of claim 14, wherein the biologically active factor is a cell attachment factor selected from the group consisting of fibrinogen, fibrin, fibronectin, hyaluronic acid, heparin, collagen, polylysine, polyornithine, receptor-binding cyclic peptide, receptor-binding protein.

21. The composition of claim 14, wherein the biologically active factor is an enzyme, a growth-factor or a growth factor-immobilized substance.

22. The composition of claim 14, wherein the biologically active factor is a plasmid DNA in the form of liposomes, a lipid complex, a chitosan complex, a poly-lysine complex, a DEAE dextran complex.

23. The composition of claim 14, wherein the biologically active factor is a vaccine.

24. The composition of claim 23, wherein the vaccine comprises an infective viral particle.

25. The composition of claim 14, wherein the biologically active factor is a nutritive or metabolic factor.

26. The composition of claim 25, wherein the a nutritive or metabolic factor is a lipid, amino acids, and a co-factor selected from the group consisting of cholesterol, glutamin, glucosamine, ascorbic acid, pyruvate, and lactate.

27. The composition of claim 14, wherein the biologically active factor is at least one element selected from the group consisting of peripheral blood, bone blood, cord blood, a blood product, blood-borne cells, serum, platelets, platelet-rich plasma, fibrinogen, a clotting factor, and a blood-borne enzyme.

28. The composition of claim 14, wherein the biologically active factor is an osteogenic substance.

29. The composition of claim 28, wherein the osteogenic substance is a member of the bone morphogenetic protein family selected from the group consisting of TGF-p1, BMP-2, BMP-6, BMP-7, or a mixture thereof.

30. The composition in claim 1, wherein hydroxyl-containing polymer is polyvinyl alcohol, dextran, linked with a bifunctional reactive aldehyde.

41. A method for repairing soft tissue, said method comprising the step of administering the composition of claim 1 at the site of a soft tissue in need of repair of a patient.

42. A method for repairing or resurfacing a damaged cartilage, said method comprising the step of administering the composition of claim 1 in or around a cartilage in need of repair or resurfacing of a patient.

43. A method for repairing a meniscus of a patient, said method comprising the step of administering the composition of claim 1 at the site of the meniscus in need of repair.

44. The composition of claim 3, where the physiological medium comprises cell nutrients selected from the group consisting of glucose, amino acids, and vitamins, or a combination thereof, at isotonic and neutral pH.

31-40. -40. (canceled)