1. A system for removing a boiler tube stub comprising,
a means for removing material from an inner surface of the boiler tube stub for a removal length leaving a shell of a removal length with a new inner diameter larger than an original inner diameter of the boiler tube stub and a remaining length of the boiler tube stub, from which no material has been removed from the inner surface, of the original inner diameter, such that there is a shoulder at the intersection of the shell and remaining length of the boiler tube stub,
a plug with a plug body of a length greater than the removal length of the shell, said plug body having a first side, a second side and a diameter larger than the original inner diameter of the tube stub and smaller than the new inner diameter of the shell, and
a plug extension extending from the first side of the plug body having a diameter smaller than the original diameter of the boiler tube stub.
2. A method for removing a boiler tube stub comprising,
removing material from an inner surface of the boiler tube stub for a removal length, leaving a shell of a removal length with a new inner diameter larger than an original inner diameter of the boiler tube stub and a remaining length of the boiler tube stub, from which no material has been removed from the inner surface, of the original inner diameter, such that there is a shoulder at the intersection of the shell and the remaining length of the boiler tube stub,
selecting a plug with
a plug body of a length greater than the removal length of the shell, said plug body having a first side, a second side and a diameter larger than the original inner diameter of the tube stub and smaller than the new inner diameter of the shell, and
a plug extension extending from the first side of the plug body having a diameter smaller than the original diameter of the boiler tube stub.
inserting the plug into the shell such that the plug extension enters the remaining length of the boiler tube stub and the first side of the plug rests on the shoulder at the intersection of the shell and the remaining length of the boiler tube stub, and
exerting sufficient force on the second side of the plug body to punch out the boiler tube stub.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. A biomatrix composition comprising collagen I, collagen IV, laminin, entactin, and tenascin.
2. The biomatrix composition of claim 1 further comprising fibronectin and proteoglycans.
3. The composition of claim 1 where collagen I is present in a concentration greater than a concentration of collagen IV, laminin, entactin and tenascin.
4. The composition of claim 1 where the two most abundant components are collagen I and collagen IV are present in a concentration greater than a concentration of collagen IV, laminin, entactin, and tenascin.
5. The composition of claim 2 where the collagen I is present in a range of 3546% by weight, collagen IV is present in a range of 19-33% by weight, laminin is present in a range of 16-21% by weight, entacin is present in a range of 7.5 to 14.5% by weight, tenascin is present in a range of 3.5-7.0% by weight, fibronectin is present in a range of 0-2.0% by weight and proteoglycans are present in a range of 0 to 1.3%.
6. The composition of claim 2 where the collagen I is present at a concentration of 36% by weight, collagen IV is present at a concentration 24% by weight, laminin is present at a concentration 20% by weight, entacin is present at a concentration of 12.5% by weight, tenascin is present at a concentration 5% by weight, fibronectin is present at a concentration 1% by weight and proteoglycans are present at a concentration 1.0% by weight.
7. The composition of claim 2 where said proteoglycan is a heparin sulfate proteoglycan.
8. The composition of claim 1 where said composition is a native composition derived from a human starting material.
9. The composition of claim 1 where said composition is a native composition derived from a human basement membrane tissue.
10. The composition of claim 1 where said composition is a native composition derived from a human placental amnion.
11. The composition of claim 1 where the composition is essentially free of endogenous growth stimulants and proteolytic enzymes.
12. The composition of claim 11 where the growth factors are selected from the group consisting of EGF, NGF, FGF, bFGF, VEGF, HGF, BDNF, and GDNF.
13. The composition of claim 11 where the proteolytic enzyme is collagenase.
14. The composition of claim 1 further comprising a growth stimulant or a growth inhibitor.
15. The composition of claim 14 where the growth stimulant is a growth factor, a cytokine, a peptide factor or an organic compound.
16. The composition of claim 15 where the growth factor is selected from the group consisting of: EGF, PDGF, bFGF, FGF, VEGF, NGF, KGF, HGF, BDNF, neurotropin-3, neurotropin-4, CNF, GGF, GMF, GDNF, and a combination of any of the foregoing.
17. The composition of claim 1 further comprising an addition of at least one protein found in an extracellular matrix.
18. The composition of claim 17 where said protein is selected from the group consisting of: fibrin, vitronectin, collagen II, collagen III, collagen V, collagen VI, collagen VII, collagen VIII, osteopontin and a combination of any of the foregoing.
19. The composition of claim 1 where said composition is capable of supporting growth of a non-transformed target cell type without inducing differentiation.
20. The composition of claim 19 where the target cell type is selected from the group consisting of: epithelial cells, stem cells, endothelial cells, transgenic cells, liver cells and neural cells.
21. The composition of claim 20 where the stem cells are totipotent or pluripotent.
22. The composition of claim 1 where said composition is capable of supporting the controlled growth of transformed cell types.
23. The composition of claim 1 where said composition can be used for the study of physiological and pathological processes.
24. The composition of claim 23 where the physiological processes are selected from the group consisting of: tissue growth, tissue development, bone remodeling, wound healing, angiogenesis, reproduction, and aging.
25. The composition of claim 23 where the pathological processes are selected from the group consisting of: tumorigenesis, metastasis, angiogenesis, vascular dysfunction, arthritis and atherosclerosis.
26. The composition of claim 1 where said composition forms a gel over a temperature range of 4 degrees C. to 37 degrees C.
27. A biomatrix composition comprising collagen I, collagen IV, and laminin.
28. The composition of claim 27 where the collagen I is present in a range of 35-46% by weight, collagen IV is present in a range of 19-33% by weight, and laminin is present in a range of 16-21% by weight.
29. The composition of claim 27 further comprising entacin and tenascin.
30. The composition of claim 29 where the collagen I is present in a range of 35-46% by weight, collagen IV is present in a range of 19-33% by weight, and laminin is present in a range of 16-21% by weight, entacin is present in a range of 7.5 to 14.5% by weight, and tenascin is present in a range of 3.5-7.0% by weight.
31. The composition of claim 29 further comprising fibronectin and proteoglycans.
32. The composition of claim 31 where the collagen I is present in a range of 35-46% by weight, collagen IV is present in a range of 19-33% by weight, and laminin is present in a range of 16-21% by weight, entacin is present in a range of 7.5 to 14.5% by weight, tenascin is present in a range of 3.5-7.0% by weight, fibronectin is present in a range of 0-2.0% by weight and proteoglycans are present in a range of 0 to 1.3%.
33. A biomatrix composition for promoting growth and differentiation of a target cell, said composition being derived from a plurality of placental amnions by a process comprising the sequential steps of:
a. processing a plurality of placental amnions by cleaning said amnions to remove an undesirable material, rinsing said amnions at least 1 time in PBS, rinsing said amnions in a first buffer comprising an ammonium hydroxide solution, and rinsing said amnions at least one time in PBS and drying said amnions.
b. solubilizing and homogenizing said amnions by dissecting said amnions into a plurality of pieces in an ice cold second buffer comprising 0.5M acetic acid, homogenizing said amnions in said second buffer comprising 0.5M acetic acid to produce a first homogenate uniform in appearance, raising the pH of the first homogenate to a pH of 2.0, and digesting the first homogenate with a proteolytic enzyme and incubating the proteolytic enzyme with the first homogenate at 4 degrees C. for at least 12 hours;
c. centrifuging said first homogenate to isolate a first supernatant and adjusting said first supernatant to a pH of 7.8;
d. extracting the first supematant by adjusting said first homogenate to a Tris-base concentration of 50 mM and a salt concentration of 4 M and incubating the first supernatant at 4 degree C. for at least 12 hours;
e. centrifuging said first supernatant to isolate a first pellet and resuspending said first pellet in a third buffer comprising 0.5M acetic acid to produce a first suspension and adjusting said first suspension to a pH of 7.8;
f. extracting the first suspension by adjusting said first suspension to a Tris-base concentration of 50 mM and a salt concentration of 4 M and incubating the first suspension at 4 degree C. for at least 3 hours;
g. centrifuging said first suspension to isolate a second pellet and resuspending said second pellet in a dialysis buffer comprising 0.5M acetic acid to produce a second and incubating said second suspension at 4 degree C. for at least 12 hours;
h. placing the second suspension in a dialysis bag and dialyzing said second suspension against a suitable dialysis buffer with multiple changes of the dialysis buffer at 4 degree C. for at least 24 hours to produce a dialysate; and
i. recovering the dialysate from step (h) and concentrating the dialysate by ultrafiltration, said final dialysate comprising collagen, collagen IV, laminin, entacin, and tenascin.
34. The composition of claim 33 further comprising fibronectin and proteoglycans.
35. The composition of claim 34 where the collagen I is present in a range of 35-46% by weight, collagen IV is present in a range of 19-33% by weight, and laminin is present in a range of 16-21% by weight, entacin is present in a range of 7.5 to 14.5% by weight, tenascin is present in a range of 3.5-7.0% by weight, fibronectin is present in a range of 0-2.0% by weight and proteoglycans are present in a range of 0 to 1.3%.
36. The composition of claim 33 where said amnions are frozen at \u221220 degrees C. prior to said homogenization step.
37. The composition of claim 33 where the proteolytic enzyme is pepsin and said incubating the pepsin with said first homogenate occurs for at least 24 hours.
38. The composition of claim 33 where step (d) is repeated at least one time.
39. The composition of claim 33 where the dialysis buffer comprises 5 mM acetic acid, 5 mM KCl and 135 mM NaCl.
40. The composition of claim 33 where step (h) further comprises a sterilizing amount of a sterilization reagent.
41. The composition of claim 40 where the sterilization reagent is chloroform.
42. The composition of claim 33 where the first buffer is PBS and said ammonium hydroxide is present at a concentration of at least 0.1N.
43. The composition of claim 33 where the second buffer and the third buffer are PBS.
44. A method for analyzing a physiological process of interest by culturing a population of target cells relevant to said physiological process cells in three dimensions, said method comprising:
a. layering on a substrate a sufficient concentration of a biologically active biomatrix composition to produce a biomatrix coated disk, said biomatrix comprising collagen L collagen IV, laminin, entacin, and tenascin;
b. incubating said biomatrix coated substrate with a quantity of target cells in a buffered cell-culture medium to produce a target cell culture;
c. culturing said target cell culture under defined conditions; and
d. observing at least one characteristic relevant to said physiological process of said target cell culture over time.
45. The method of claim 44 where the biomatrix further comprises fibronectin and proteoglycans.
46. The method of claim 45 where the collagen I is present in a range of 35-46% by weight, collagen IV is present in a range of 19-33% by weight, and laminin is present in a range of 16-21% by weight, entacin is present in a range of 7.5 to 14.5% by weight, tenascin is present in a range of 3.5-7.0% by weight, fibronectin is present in a range of 0-2.0% by weight and proteoglycans are present in a range of 0 to 1.3%.
47. The method of claim 44 where the physiological process is tissue growth, tissue development, bone remodeling, wound healing, angiogenesis, reproduction, or aging.
48. The method of claim 44 where the target cells are transformed cells or non-transformed cells.
49. The method of claim 44 where the target cells are epithelial cells, stem cells, endothelial cells, liver cells, transgenic cells or neural cells.
50. The method of claim 49 where the stem cells are totipotent or pluripotent
51. The method of claim 44 where the substrate is a microbead or a disc.
52. The method of claim 51 where said disk has a porosity and said biomatrix composition is present at a concentration of 3 to 5 mgml.
53. The method of claim 51 where said biomatrix composition is present at a concentration of 25 to 125 \u03bcgbead and said microbeads are incubated under gravity free conditions.
54. The method of claim 44 where the buffered cell culture medium further comprises a growth stimulant.
55. The method of claim 54 growth stimulant is a growth factor, a cytokine, a peptide factor or an organic compound.
56. The method of claim 44 where the biomatrix composition further comprises a growth stimulant.
57. The method of claim 56 growth stimulant is a growth factor, a cytokine, a peptide factor or an organic compound.
58. The method of claim 44 further comprising adding a compound capable of a positive regulation of said physiological process or a negative regulation of said physiological process and determining the effect of said compound on at least one characteristic of said physiological process exhibited by said endothelial cell population.
59. The method of claim 44 further comprising the addition of a transformed cell type for coculture with said target cell.
60. The method of claim 44 where said method is used to study a pathological process.
61. The method of claim 60 where said pathological process is selected from the group consisting of: tumorigenesis, metastasis, angiogenesis, vascular dysfunction, arthritis and atherosclerosis.
62. A method for analyzing angiogenesis by culturing a population of endothelial cells in three dimensions, said method comprising:
a. layering on a substrate a sufficient concentration of a biologically active biomatrix composition to produce a biomatrix coated microbead, said biomatrix comprising collagen I, collagen IV, laminin, entacin, and tenascin;
b. incubating said biomatrix coated substrate with a quantity of endothelial cells in a buffered cell-culture medium to produce a endothelial cell culture;
c. culturing said endothelial cell culture under defined conditions;
d. observing at least one angiogenic characteristic of said endothelial cell culture over time.
63. The method of claim 62 where the biomatrix further comprises fibronectin and proteoglycans.
64. The method of claim 63 where the collagen I is present in a range of 35-46% by weight, collagen IV is present in a range of 19-33% by weight, and laminin is present in a range of 16-21% by weight, entacin is present in a range of 7.5 to 14.5% by weight, tenascin is present in a range of 3.5-7.0% by weight, fibronectin is present in a range of 0-2.0% by weight and proteoglycans are present in a range of 0 to 1.3%.
65. The method of claim 62 where said at least one angiogenic characteristic of said endothelial cell population is selected from the group consisting of: cell activation, sprout formation, capillary formation, cell migration, cell morphology, cell differentiation and any combination of the foregoing.
66. The method of claim 62 where the substrate is a microbead or a disc.
67. The method of claim 66 where said disk has a porosity and said biomatrix composition is present at a concentration of 3 to 5 mgml.
68. The method of claim 66 where said biomatrix composition is present at a concentration of 25 to 125 \u03bcgbead microbeads and said microbeads are incubated under gravity free conditions.
69. The method of claim 62 where the endothelial cell population comprises a plurality of human umbilical vein endothelial cells.
70. The method of claim 62 where the buffered cell culture medium further comprises a growth stimulant.
71. The method of claim 70 growth stimulant is a growth factor, a cytokine, a peptide factor or an organic compound.
72. The method of claim 62 where the biomatrix further comprises a growth stimulant
73. The method of claim 72 growth stimulant is a growth factor, a cytokine, a peptide factor or an organic compound.
74. The method of claim 62 further comprising adding a compound capable of promoting or inhibiting angiogenesis and determining the effect of said compound on at least one angiogenic characteristic of said endothelial cell population.
75. The method of claim 74 where said at least one angiogenic characteristic of said endothelial cell population is selected from the group consisting of: cell activation, sprout formation, capillary formation, cell migration, cell morphology, cell differentiation and any combination of the foregoing.
76. A method of analyzing neurogenesis by culturing a population of cells of neural origin in three dimensions, said method comprising:
a. layering on a substrate a sufficient concentration of a biologically active biomatrix composition to produce a biomatrix coated disk, said biomatrix comprising collagen I, collagen IV, laminin, entacin, and tenascin;
b. incubating said biomatrix coated substrate with a quantity of cells of neural origin in a buffered cell-culture medium to produce a neural cell culture;
c. culturing said neural cell culture under defined conditions; and
d. observing at least one neurological characteristic of said neural cell culture over time.
77. The method of claim 76 where the biomatrix further comprises fibronectin and proteoglycans.
78. The method of claim 77 where the collagen I is present in a range of 35-46% by weight, collagen IV is present in a range of 19-33% by weight, and laminin is present in a range of 16-21% by weight, entacin is present in a range of 7.5 to 14.5% by weight, tenascin is present in a range of 3.5-7.0% by weight, fibronectin is present in a range of 0-2.0% by weight and proteoglycans are present in a range of 0 to 1.3%.
79. The method of claim 76 where said at least one neurological characteristic of said endothelial cell population is selected from the group consisting of: axon formation, neurite formation, cell activation, cell morphology, cell migration, cell differentiation and any combination of the foregoing.
80. The method of claim 76 where the substrate is a microbead or a disc.
81. The method of claim 80 where said disk has a porosity and said biomatrix composition is present at a concentration of 3 to 5 mgml.
82. The method of claim 80 where said biomatrix composition is present at a concentration of 25 to 125 \u03bcgbead and said microbeads are incubated under gravity free conditions.
83. The method of claim 76 where the cells of neural origin are selected from the group consisting of: stem cells, neurospheres, neurons, and a combination of any of the foregoing.
84. The method of claim 76 where the buffered cell culture medium further comprises a growth stimulant.
85. The method of claim 84 growth stimulant is a growth factor, a cytokine, a peptide factor or an organic compound.
86. The method of claim 76 where the biomatrix further comprises a growth stimulant.
87. The method of claim 86 growth stimulant is a growth factor, a cytokine, a peptide factor or an organic compound.
88. The method of claim 76 further comprising adding a compound capable of promoting or inhibiting neurogenesis and determining the effect of said compound on at least one neurological characteristic of said endothelial cell population.
89. The method of claim 88 where said at least one angiogenic characteristic of said endothelial cell population is selected from the group consisting of: axon formation, neurite formation, cell activation, cell morphology, cell migration, cell differentiation and any combination of the foregoing.
90. A method for treating spinal cord injury in an subject in need of such treatment, said method comprising:
a. Preparing a three-dimensional biomatrix scaffold, said biomatrix scaffold containing neural precursor and said biomatrix scaffold comprising collagen I, collagen IV, laminin, entacin, and tenascin; and
b. Introducing said biomatrix scaffold into a subject in need of such treatment.
91. The method of claim 90 where the biomatrix further comprises fibronectin and proteoglycans.
92. The method of claim 91 where the collagen I is present in a range of 35-46% by weight, collagen IV is present in a range of 19-33% by weight, and laminin is present in a range of 16-21% by weight, entacin is present in a range of 7.5 to 14.5% by weight, tenascin is present in a range of 3.5-7.0% by weight, fibronectin is present in a range of 0-2.0% by weight and proteoglycans are present in a range of 0 to 1.3%.
93. The method of claim 90 where the subject is a mammal.
94. The method of claim 90 where the subject is a human.
95. The method of claim 90 where the neural progenitor cells are neural stem cells.
96. The method of claim 95 where said stem cells are totipotent or pluripotent.
97. The method of claim 90 where said neural progenitor cells are neurospheres.
98. The method of claim 90 where said biomatrix scaffold is present at a concentration of 15 to 300 ug.
99. The method of claim 90 where said introducing is accomplished by an injection at the site of said spinal cord injury.
100. The method of claim 90 where the biomatrix scaffold further comprises a growth stimulant.
101. The method of claim 100 growth stimulant is a growth factor, a cytokine, a peptide factor or an organic compound.
102. The method of claim 90 where the biomatrix scaffold further comprises a plurality of an accessory cell type.
103. The method of claim 102 where the accessory cell type is a Schwann cell or a mesenchymal cell.