Treatment of tissue with undifferentiated mesenchymal cells

• US 20040101959A1
• Filed: 11/21/2002
• Published: 05/27/2004
• Est. Priority Date: 11/21/2002
• Status: Abandoned Application

• Current Assignee: Isolagen Technologies Incorporated
• Original Assignee: Isolagen Technologies Incorporated
• Inventors: Marko, Olga, Boss, William K. Jr.
• Application Number:
  US10/301,058
• Publication Number:
  US 20040101959A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61L 27/3804 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
• 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: A61P 1/02 Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/48 of the pancreatic hormones
• CPC Class Codes: A61P 9/14 Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers

Abstract

The present invention provides compositions and methods for correcting cosmetic, aesthetic, and degenerative defects in the skin, soft tissue, and bone of a subject. In particular, methods of the invention involve the injection or implantation of autologous UMC, fibroblasts, and/or keratinocytes into the tissue (e.g., subcutaneous tissue) adjacent or subadjacent to a defect or at the site of a defect. The cells that are injected, as provided herein, are histocompatible with the subject (e.g., are autologous) and have been expanded by passage in a cell culture system.

Claims

1. A method for making a cellular composition for repairing tissue, said method comprising:

(a) providing a biopsy of undifferentiated mesenchymal cell (UMC)-containing tissue to obtain starting cells;

(b) separating said starting cells from said biopsy;

(c) culturing said starting cells; and

(d) harvesting a population of non-adherent derivative cells from said culture, wherein said non-adherent derivative cells comprise UMC.

2. The method of claim 1, further comprising one or more rounds of derivitization comprising repeating steps (c) and (d) utilizing the harvested population of non-adherent derivative cells from the previous round as the starting cells.

3. The method of claim 2, wherein said one or more additional rounds of derivatization comprises from one to twenty rounds.

4. The method of claim 1, wherein said UMC-containing tissue is selected from the group consisting of:

dermal tissue, adipose tissue, connective tissue, fascia, lamina propria and bone marrow.

5. The method of claim 1, further comprising culturing said non-adherent cells in the presence of acidic fibroblast growth factor.

6. A composition for repairing tissue, wherein said composition comprises autologous, passaged UMC and autologous, passaged fibroblasts, and wherein said composition is substantially free of culture medium serum-derived proteins.

7. The composition of claim 6, wherein said autologous fibroblasts are from gums, palate, skin, lamina propria, connective tissue, bone marrow, fascia, or adipose tissue of said subject.

8. The composition of claim 6, wherein said UMC are from dermal tissue, adipose tissue, connective tissue, fascia, lamina propria, or bone marrow.

9. The composition of claim 6, wherein said UMC or said fibroblasts have been treated with one or more activating compounds.

10. The composition of claim 9, wherein said one or more activating compounds is selected from the group consisting of:

ascorbyl palmitate, linoleic acid, quinic acid, a quinic acid salt, a quinic lactone, CoEnzyme Q-10, L-hydroxy acid, L-lipoic acid, calcium monophosphate, calcium triphosphate, niacin, dehydroepiandrosterone (DHEA), dimethylamino ethanol (DMAE), α

-tocopherol, a bone morphogenic protein (BMP), vitamin E, vitamin C, carotenoids, glycolic acid, carboxyalkyl esters, estriol, acetyl-L-carnitine, deprenyl, lycopene, nicotinamide adenine dinucleotide (NADH), cysteine, procysteine, pikamilon, vinpocetine, retinoic acid, antineoplastons, and a growth factor.

11. The composition of claim 6, wherein said UMC or said fibroblasts have been exposed to a low energy laser light.

12. The composition of claim 6, wherein said composition further comprises a biodegradable matrix, wherein said UMC and said fibroblasts are integrated within or on said matrix.

13. The composition of claim 12, wherein said matrix, prior to combination with said UMC and said fibroblasts, comprises one or more substances selected from the group consisting of:

collagen, glycosaminoglycans, gelatin, polyglycolic acid, cat gut, demineralized bone, hydroxyapatite, and anorganic bone.

14. The composition of claim 6, further comprising a biodegradable acellular injectable filler.

15. The composition of claim 14, wherein said biodegradable acellular injectable filler, prior to combination with said UMC, comprises one or more substances selected from the group consisting of:

(a) an injectable dispersion of autologous collagen fibers;

(b) collagen;

(c) solubilized gelatin;

(d) solubilized polyglycolic acid;

(e) solubilized cat gut;

(f) porcine gelatin powder and amino caproic acid dispersed in sodium chloride solution and an aliquot of plasma from said subject; and

(g) hyaluronic acid.

51. A method for repairing tissue that has degenerated in a subject as a result of a disease, disorder, or defect in said subject, said method comprising injecting of the composition of claim 14 into said subject at the site of said degeneration so that said tissue is repaired.

52. A method for repairing tissue that has degenerated in a subject as a result of a disease, disorder, or defect in said subject, said method comprising placing the composition of claim 12 into said subject at the site of said degeneration so that said tissue is repaired.

16. A method for making a composition for repairing tissue that has degenerated in a subject as a result of a disease, disorder, or defect in said subject, said method comprising:

(a) providing a biopsy of UMC-containing tissue from said subject;

(b) separating autologous UMC from said biopsy;

(c) culturing said autologous UMC under conditions that produce autologous UMC that are substantially free of culture medium serum-derived proteins;

(d) exposing said cultured autologous UMC to conditions that result in suspension of said UMC;

(e) providing a biopsy of fibroblast-containing tissue from said subject;

(f) separating autologous fibroblasts from said biopsy;

(g) culturing said autologous fibroblasts under conditions that produce fibroblasts that are substantially free of culture medium serum-derived proteins;

(h) exposing said cultured autologous fibroblasts to conditions that result in suspension of said fibroblasts; and

(i) mixing said UMC suspension with said fibroblast suspension.

17. The method of claim 16, wherein said UMC-containing tissue is selected from the group consisting of:

gums, palate, skin, lamina propria, connective tissue, fascia, bone marrow, and adipose tissue.

18. The method of claim 16, wherein said autologous fibroblast-containing tissue is selected from the group consisting of:

gums, palate, skin, lamina propria, connective tissue, fascia, bone marrow, and adipose tissue.

19. The method of claim 16, wherein prior to said combining, said UMC or said fibroblasts are exposed to one or more activating compounds.

20. The method of claim 19, wherein said one or more activating compounds is selected from the group consisting of:

ascorbyl palmitate, linoleic acid, quinic acid, a quinic acid salt, a quinic lactone, CoEnzyme Q-10, L-hydroxy acid, L-lipoic acid, calcium monophosphate, calcium triphosphate, niacin, DHEA, DMAE, α

-tocopherol, a BMP, vitamin E, vitamin C, carotenoids, glycolic acid, carboxyalkyl esters, estriol, acetyl-L-carnitine, deprenyl, lycopene, NADH, cysteine, procysteine, pikamilon, vinpocetine, retinoic acid, antineoplastons, and a growth factor.

21. The method of claim 16, wherein said UMC or said fibroblasts are exposed to a low energy laser light.

22. A method for making a composition for repairing tissue that has degenerated in a subject as a result of a disease, disorder, or defect in said subject, wherein said method comprises:

(a) providing autologous, passaged UMC;

(b) providing autologous, passaged fibroblasts;

(c) providing a biodegradable acellular matrix; and

(d) incubating said UMC and said fibroblasts with said biodegradable acellular matrix such that said UMC and/or fibroblasts integrate on or within said biodegradable acellular matrix, wherein said incubation results in a composition for repairing tissue, and wherein the conditions of said incubation are such that said composition is substantially free of culture medium serum-derived proteins.

23. The method of claim 22, wherein the step of providing autologous, passaged UMC comprises:

(a) providing a biopsy of UMC-containing tissue from said subject;

(b) separating autologous UMC from said biopsy;

(c) culturing said UMC; and

(d) exposing said cultured UMC to conditions that result in suspension of said UMC.

24. The method of claim 22, wherein said UMC-containing tissue is selected from the group consisting of:

gums, palate, skin, lamina propria, connective tissue, fascia, bone marrow, and adipose tissue.

25. The method of claim 22, wherein said biodegradable acellular matrix, prior to combination with said UMC and said fibroblasts, comprises one or more substances selected from the group consisting of:

collagen, glycosaminoglycans, gelatin, polyglycolic acid, cat gut, demineralized bone, hydroxyapatite, coral, and anorganic bone.

26. The method of claim 22, wherein the step of providing autologous, passaged fibroblasts comprises:

(a) providing a biopsy of fibroblast-containing tissue from said subject;

(b) separating autologous fibroblasts from said biopsy;

(c) culturing said fibroblasts; and

(d) suspending said fibroblasts.

27. The method of claim 26, wherein said fibroblast-containing tissue is selected from the group consisting of:

gums, palate, skin, lamina propria, connective tissue, fascia, bone marrow, and adipose tissue.

28. The method of claim 22, wherein prior to said incubating with said biodegradable acellular matrix, said UMC or said fibroblasts are exposed to one or more activating compounds.

29. The method of claim 28, wherein said one or more activating compounds is selected from the group consisting of:

ascorbyl palmitate, linoleic acid, quinic acid, a quinic acid salt, a quinic lactone, CoEnzyme Q-10, L-hydroxy acid, L-lipoic acid, calcium monophosphate, calcium triphosphate, niacin, DHEA, DMAE, α

-tocopherol, a BMP, vitamin E, vitamin C, carotenoids, glycolic acid, carboxyalkyl esters, estriol, acetyl-L-carnitine, deprenyl, lycopene, NADH, cysteine, procysteine, pikamilon, vinpocetine, retinoic acid, antineoplastons, and a growth factor.

30. The method of claim 22, wherein said UMC or said fibroblasts are exposed to a low energy laser light.

31. A method for making a composition for repairing tissue that has degenerated in a subject as a result of a disease, disorder, or defect in said subject, wherein said method comprises:

(a) providing autologous, passaged UMC, wherein said UMC are substantially free of culture medium serum-derived proteins;

(b) providing autologous, passaged fibroblasts, wherein said autologous, passaged fibroblasts are substantially free of culture medium serum-derived proteins;

(c) providing a biodegradable acellular filler; and

(d) combining said autologous, passaged UMC, said autologous, passaged fibroblasts, and said biodegradable acellular filler.

32. The method of claim 31, wherein the step of providing autologous, passaged UMC comprises:

(a) providing a biopsy of UMC-containing tissue from said subject;

(b) separating autologous UMC from said biopsy;

(c) culturing said autologous UMC under conditions that result in UMC that are substantially free of culture medium serum-derived proteins; and

(d) exposing said cultured UMC to conditions that result in suspension of said UMC.

33. The method of claim 32, wherein said UMC-containing tissue is selected from the group consisting of:

gums, palate, skin, lamina propria, connective tissue, fascia, bone marrow, and adipose tissue.

34. The method of claim 31, wherein said biodegradable acellular filler, prior to combination with said UMC and said fibroblasts, comprises one or more substances selected from the group consisting of:

(a) an injectable dispersion of autologous collagen fibers;

(b) collagen;

(c) solubilized gelatin;

(d) solubilized polyglycolic acid;

(e) solubilized cat gut;

(f) porcine gelatin powder and amino caproic acid dispersed in sodium chloride solution and an aliquot of plasma from said subject; and

(g) hyaluronic acid.

35. The method of claim 32, wherein the step of providing autologous, passaged fibroblasts comprises:

(a) providing a biopsy of fibroblast-containing tissue from said subject;

(b) separating autologous fibroblasts from said biopsy;

(c) culturing said autologous fibroblasts under conditions that result in fibroblasts that are substantially free of culture medium serum-derived proteins; and

(d) exposing said incubated autologous fibroblasts to conditions that result in suspension of said fibroblasts.

36. The method of claim 35, wherein said fibroblast-containing tissue is selected from the group consisting of:

gums, palate, skin, lamina propria, connective tissue, fascia, bone marrow, and adipose tissue.

37. The method of claim 31, wherein prior to said combining with said biodegradable acellular filler, said UMC or said fibroblasts are exposed to one or more activating compounds.

38. The method of claim 37, wherein said one or more activating compounds is selected from the group consisting of:

ascorbyl palmitate, linoleic acid, quinic acid, a quinic acid salt, a quinic lactone, CoEnzyme Q-10, L-hydroxy acid, L-lipoic acid, calcium monophosphate, calcium triphosphate, niacin, DHEA, DMAE, α

-tocopherol, a BMP, vitamin E, vitamin C, carotenoids, glycolic acid, carboxyalkyl esters, estriol, acetyl-L-carnitine, deprenyl, lycopene, NADH, cysteine, procysteine, pikamilon, vinpocetine, retinoic acid, antineoplastons, and a growth factor.

39. The method of claim 31, wherein said UMC or said fibroblasts are exposed to a low energy laser light.

40. A method for repairing tissue in a subject, wherein said method comprises:

(a) providing a composition comprising autologous, passaged UMC, wherein said composition is substantially free of culture medium serum-derived proteins;

(b) identifying a site of tissue defect or tissue degeneration in said subject; and

(c) placing said composition at said site so that said tissue defect or degeneration is repaired;

wherein said tissue defect or degeneration comprises a soft tissue defect, a defect of an oral mucosa, trauma to an oral mucosa, periodontal disease, diabetes, a cutaneous ulcer, venous stasis, or a cosmetic defect of the skin.

41. The method of claim 40, wherein said soft tissue defect is selected from the group consisting of a facial depression, underdevelopment of the breast, absence of breast, a vocal cord defect, velopharyngeal incompetence, Poland'"'"'s syndrome, underdevelopment of the penis, hypoplasia of the lips, and a subcutaneous atrophy or muscular atrophy.

42. The method of claim 40, wherein said trauma to an oral mucosa is an extraction socket resulting from an extraction of a tooth.

43. The method of claim 40, wherein said periodontal disease comprises periodontal degeneration, gingivitis, or non-healing wounds of a palatal mucosa or a gingival mucosa.

44. The method of claim 40, wherein said cosmetic defect is selected from the group consisting of:

a rhytid, a depressed scar, a cutaneous depression of non-traumatic origin, a laugh line, a stretch mark, a wrinkle, a wound scar, an acne scar, and subcutaneous atrophy.

45. The method of claim 40, wherein said tissue defect is hypoplasia of a lip or a lip fold.

46. The method of claim 40, wherein said autologous UMC are from gums, palate, skin, lamina propria, connective tissue, fascia, bone marrow, or adipose tissue of said subject.

47. The method of claim 40, wherein said composition further comprises a biodegradable matrix, wherein said UMC are integrated within or on said matrix.

48. The method of claim 40, wherein said composition further comprises a biodegradable acellular filler.

49. A method for repairing a tissue defect in a subject, wherein said method comprises:

(a) providing a composition comprising autologous, passaged UMC and autologous, passaged fibroblasts, wherein said composition is substantially free of culture medium serum-derived proteins;

(b) identifying a site of tissue defect or tissue degeneration in said subject; and

(c) placing said composition at said site so that said tissue defect or degeneration is repaired.

50. A method for repairing a tissue defect in a subject, wherein said method comprises:

(a) providing a pharmaceutical composition comprising;

(1) autologous, passaged UMC, (2) autologous, passaged fibroblasts, and (3) a pharmaceutically acceptable carrier thereof;

wherein said pharmaceutical composition is substantially free of culture medium serum-derived proteins;

(b) identifying in said subject a site of tissue defect or tissue degeneration selected from the group consisting of;

a dental or periodontal defect, a cosmetic defect of the skin, and a bone defect; and

(c) injecting a therapeutically effective amount of said pharmaceutical composition adjacent to said site of tissue defect or degeneration, wherein said injecting results in repair of said tissue defect or degeneration.

53. A method for repairing tissue that has degenerated in a subject as a result of a disease, disorder, or defect in said subject, said method comprising the steps of:

(a) injecting autologous, passaged UMC into said subject at a site of tissue degeneration, wherein said UMC are substantially free of culture medium serum-derived proteins;

(b) injecting autologous, passaged fibroblasts into said subject at a site of a tissue defect or desired tissue augmentation, wherein said fibroblasts are substantially free of culture medium serum-derived proteins; and

(c) injecting a biodegradable, acellular filler into the site.

54. A method for repairing a burn wound in a subject, said method comprising providing a suspension of autologous, passaged keratinocytes and autologous, passaged fibroblasts, and injecting said suspension into said burn wound.

55. The method of claim 54, wherein said providing a suspension of autologous, passaged keratinocytes and autologous, passaged fibroblasts comprises:

(a) providing a biopsy of keratinocyte-containing tissue from said subject;

(b) separating autologous keratinocytes from said biopsy;

(c) culturing said autologous keratinocytes under conditions that produce autologous keratinocytes that are substantially free of culture medium serum-derived proteins;

(d) exposing said keratinocytes to conditions that result in suspension of said keratinocytes;

(e) providing a biopsy of fibroblast-containing tissue from said subject;

(f) separating autologous fibroblasts from said biopsy;

(g) culturing said autologous fibroblasts under conditions that produce autologous fibroblasts that are substantially free of culture medium serum-derived proteins;

(h) exposing said fibroblasts to conditions that result in suspension of said fibroblasts; and

(i) combining said suspended keratinocytes and said suspended fibroblasts.

56. The method of claim 54, wherein said keratinocyte-containing tissue is selected from the group consisting of:

skin, oral mucosa, gums, buccal tissue, esophageal tissue, exocervical tissue, and conjunctival tissue.

57. The method of claim 54, wherein said fibroblast-containing tissue is selected from the group consisting of:

gums, palate, skin, lamina propria, connective tissue, fascia, bone marrow, and adipose tissue.

58. The method of claim 54, wherein prior to combining with said keratinocytes, said fibroblasts are exposed to one or more activating compounds.

59. The method of claim 58, wherein said one or more activating compounds is selected from the group consisting of:

ascorbyl palmitate, linoleic acid, quinic acid, a quinic acid salt, a quinic lactone, CoEnzyme Q-10, L-hydroxy acid, L-lipoic acid, calcium monophosphate, calcium triphosphate, niacin, DHEA, DMAE, α

-tocopherol, a BMP, vitamin E, vitamin C, carotenoids, glycolic acid, carboxyalkyl esters, estriol, acetyl-L-carnitine, deprenyl, lycopene, NADH, cysteine, procysteine, pikamilon, vinpocetine, retinoic acid, antineoplastons, and a growth factor.

60. The method of claim 54, wherein said fibroblasts are exposed to a low energy laser light.

61. The method of claim 54, wherein said suspension further comprises autologous, passaged UMC.

62. The method of claim 54, further comprising injecting an enhancing compound into said wound.