U.S. patent application number 11/006017 was filed with the patent office on 2005-05-12 for cyclic implant perfusion, cleaning and passivation process and implant produced thereby.
Invention is credited to Hanstke, Sean, Mills, C. Randal, Wironen, John F..
Application Number | 20050100862 11/006017 |
Document ID | / |
Family ID | 22704656 |
Filed Date | 2005-05-12 |
United States Patent
Application |
20050100862 |
Kind Code |
A1 |
Mills, C. Randal ; et
al. |
May 12, 2005 |
Cyclic implant perfusion, cleaning and passivation process and
implant produced thereby
Abstract
This invention is a novel method for perfusion of a porous
implant which achieves efficient interpenetration of desired
factors into and removal of undesirable factors from the pores of
the implant, cleaning of the implant, efficient passivation of the
implant (inactivation of pathogens, microorganisms, cells, viruses
and the like and reduction in antigenicity thereof), and the novel
implant produced by such treatment. The process presents a system
wherein the rate of pressure cycling, the fact of pressure cycling,
and the amplitude of pressure cycling, results in highly cleaned
tissues and other implants for implantation. Target decontamination
goals for this process include between about a one (1) to twelve
(12) log reduction in bacterial contamination, between about a one
(1) to fifteen (15) log reduction in enveloped virus contamination,
up to about a five (5) log reduction in non-enveloped virus
contamination, between about a two (2) to ten (10) fold reduction
in endotoxin, maintenance of implant or graft biologic and
biomechanical properties, absence of tissue toxicity due to
cleaning solutions used, and reduced implant antigenicity.
Inventors: |
Mills, C. Randal; (Alachua,
FL) ; Wironen, John F.; (Gainesville, FL) ;
Hanstke, Sean; (Woodbridge, CA) |
Correspondence
Address: |
MCANDREWS HELD & MALLOY, LTD
500 WEST MADISON STREET
SUITE 3400
CHICAGO
IL
60661
|
Family ID: |
22704656 |
Appl. No.: |
11/006017 |
Filed: |
December 7, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11006017 |
Dec 7, 2004 |
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10192180 |
Jul 10, 2002 |
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10192180 |
Jul 10, 2002 |
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09191232 |
Nov 13, 1998 |
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6482584 |
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Current U.S.
Class: |
433/172 |
Current CPC
Class: |
A61L 2/025 20130101;
A01N 1/0289 20130101; A61L 2/0088 20130101 |
Class at
Publication: |
433/172 |
International
Class: |
A61C 013/225 |
Claims
1-35. (canceled)
36. An implant cleaning process which comprises subjecting an
implant contained in a chamber to cyclic exposure of increased and
decreased pressure above one atmosphere or below one atmosphere or
both; the process comprising the steps of: (a) filling a chamber
with a cleaning solution or a mixture of cleaning solutions; (b)
evacuating said chamber; (c) pressurizing said chamber; and (d)
cycling between steps (b) and (c) more than 2 cycles; and wherein
said implant comprises autograft, allograft or xenograft tissue, or
combinations thereof.
37. The process of claim 36 wherein the steps are carried out
sufficient times to produce an implant that is passivated.
38. The process of claim 36 wherein the implant produced is
suitable for implantation or processing for implantation in
humans.
39. The process of claim 36 wherein said implant is entirely
enclosed in said chamber.
40. The process of claim 36 wherein the process is conducted under
computer or programmable logic circuit control.
41. The process of claim 36 wherein said cleaning solution is
selected from the group consisting of sterile water, Triton X-100,
TNBP, 3% hydrogen peroxide, a water miscible alcohol, saline
solution, providone iodine, ascorbic acid solution, aromatic
hydrocarbons, aliphatic hydrocarbons, ethers, ketone, amines, urea,
guanidine hydrochloride, esters, glycoproteins, proteins,
saccharides, enzymes, gasseous acids, gasseous peroxides, and
mixtures thereof.
42. The process of claim 36 wherein said implant comprises at least
one of the following: cortical bone, cancellous bone, fascia, whole
joints, tendons, ligaments, dura, pericardia, heart valves, veins,
neural tissue, submucosal tissue, or cartilage, or combinations
thereof.
43. The process of claim 36, further comprising placing said
implant into a sterile, sealable package.
44. The process of claim 43, further comprising performing a
surface decontamination step prior to or after sealing said
package.
45. The process of claim 36 wherein the implant is perfused or
coated with a bioactive substance.
46. The process of claim 36 wherein the steps are carried out
sufficient times to produce an implant having one or more of the
following properties: (a) between about a one to twelve log
reduction in bacterial contamination; (b) between about a one to
fifteen log reduction in enveloped virus contamination; (c) up to
about a five log reduction in non-enveloped virus contamination;
(d) between about a two to ten fold reduction in endotoxin; (e)
maintenance of implant biologic and biomechanical properties; (f)
absence of tissue toxicity due to cleaning solutions used; (g)
reduced implant antigenicity.
47. The process of claim 36 further comprising the step of
machining said implant to final dimensions.
48. The process of claim 36 wherein one or more steps are conducted
under elevated or reduced temperatures, with respect to ambient
temperature.
49. An implant cleaning process which comprises subjecting the
entire implant to cyclic exposure of increased and decreased
pressure above one atmosphere or below one atmosphere or both,
wherein said implant comprises autograft, allograft or xenograft
tissue, or combinations thereof; and wherein the decreased pressure
is a pressure below one atmosphere.
50. The process of claim 49, wherein the decreased pressure is a
pressure between about 60 and 100 torr.
51. The process of claim 49 wherein the steps are carried out
sufficient times to produce an implant that is passivated.
52. The process of claim 49 wherein the implant produced is
suitable for implantation or processing for implantation in
humans.
53. The process of claim 49 wherein said cyclic exposure is
performed in a chamber in which said implant is entirely
enclosed.
54. The process of claim 49 wherein the process is conducted under
computer or programmable logic circuit control.
55. The process of claim 49 wherein said implant comprises at least
one of the following: cortical bone, cancellous bone, fascia, whole
joints, tendons, ligaments, dura, pericardia, heart valves, veins,
neural tissue, submucosal tissue, or cartilage, or combinations
thereof.
56. The process of claim 49, further comprising placing said
implant into a sterile, sealable package.
57. The process of claim 56, further comprising performing a
surface decontamination step prior to or after sealing said
package.
58. The process of claim 49 wherein the implant is perfused or
coated with a bioactive substance.
59. The process of claim 49 wherein the steps are carried out
sufficient times to produce an implant having one or more of the
following properties: (a) between about a one to twelve log
reduction in bacterial contamination; (b) between about a one to
fifteen log reduction in enveloped virus contamination; (c) up to
about a five log reduction in non-enveloped virus contamination;
(d) between about a two to ten fold reduction in endotoxin; (e)
maintenance of implant biologic and biomechanical properties; (f)
absence of tissue toxicity due to cleaning solutions used; (g)
reduced implant antigenicity.
60. The process of claim 49 further comprising the step of
machining said implant to final dimensions.
61. The process of claim 49 wherein one or more steps are conducted
under elevated or reduced temperatures, with respect to ambient
temperature.
62. An implant cleaning process which comprises subjecting the
entire implant to cyclic exposure of increased and decreased
pressure above one atmosphere or below one atmosphere or both,
wherein said implant comprises autograft, allograft or xenograft
tissue, or combinations thereof; and wherein the increased pressure
is a pressure above one atmosphere.
63. The process of claim 62, wherein the increased pressure is a
pressure between about 6 and 10 atmospheres.
64. The process of claim 62 wherein the steps are carried out
sufficient times to produce an implant that is passivated.
65. The process of claim 62 wherein the implant produced is
suitable for implantation or processing for implantation in
humans.
66. The process of claim 62 wherein said cyclic exposure is
performed in a chamber in which said implant is entirely
enclosed.
67. The process of claim 62 wherein the process is conducted under
computer or programmable logic circuit control.
68. The process of claim 62 wherein said implant comprises at least
one of the following: cortical bone, cancellous bone, fascia, whole
joints, tendons, ligaments, dura, pericardia, heart valves, veins,
neural tissue, submucosal tissue, or cartilage, or combinations
thereof.
69. The process of claim 62, further comprising placing said
implant into a sterile, sealable package.
70. The process of claim 69, further comprising performing a
surface decontamination step prior to or after sealing said
package.
71. The process of claim 62 wherein the implant is perfused or
coated with a bioactive substance.
72. The process of claim 62 wherein the steps are carried out
sufficient times to produce an implant having one or more of the
following properties: (a) between about a one to twelve log
reduction in bacterial contamination; (b) between about a one to
fifteen log reduction in enveloped virus contamination; (c) up to
about a five log reduction in non-enveloped virus contamination;
(d) between about a two to ten fold reduction in endotoxin; (e)
maintenance of implant biologic and biomechanical properties; (f)
absence of tissue toxicity due to cleaning solutions used; (g)
reduced implant antigenicity.
73. The process of claim 62 further comprising the step of
machining said implant to final dimensions.
74. The process of claim 62 wherein one or more steps are conducted
under elevated or reduced temperatures, with respect to ambient
temperature.
75. An implant cleaning process which comprises exposing an implant
to a cleaning solution for cycles of increased and decreased
pressure above one atmosphere or below one atmosphere, or both, for
sufficient cycles to render said implant passivated and to provide
a 5 to 6 log reduction in any forms of viable organisms.
76. The process of claim 75 wherein the implant produced is
suitable for implantation or processing for implantation in
humans.
77. The process of claim 75 wherein said process occurs, at least
in part, with concurrent exposure of said implant to
sonication.
78. The process of claim 75 wherein said cycles are performed in a
chamber in which said implant is entirely enclosed.
79. The process of claim 80 wherein the process is conducted under
computer or programmable logic circuit control.
80. The process of claim 75 wherein said cleaning solution is
selected from the group consisting of sterile water, Triton X-100,
TNBP, 3% hydrogen peroxide, a water miscible alcohol, saline
solution, providone iodine, ascorbic acid solution, aromatic
hydrocarbons, aliphatic hydrocarbons, ethers, ketone, amines, urea,
guanidine hydrochloride, esters, glycoproteins, proteins,
saccharides, enzymes, gasseous acids, gasseous peroxides, and
mixtures thereof.
81. The process of claim 75 wherein said implant comprises
autograft, allograft or xenograft tissue, or combinations
thereof.
82. The process of claim 75, further comprising placing said
implant into a sterile, sealable package.
83. The process of claim 82, further comprising performing a
surface decontamination step prior to or after sealing said
package.
84. The process of claim 75 wherein the implant is perfused or
coated with a bioactive substance.
85. The process of claim 75 further comprising the step of
machining said implant to final dimensions.
86. The process of claim 75 wherein one or more steps are conducted
under elevated or reduced temperatures, with respect to ambient
temperature.
87. An implant cleaning process which comprises exposing an implant
to a cleaning solution for cycles of increased and decreased
pressure above one atmosphere or below one atmosphere, or both; to
render said implant passivated.
88. The process of claim 87 wherein the implant produced is
suitable for implantation or processing for implantation in
humans.
89. The process of claim 87 wherein said process occurs, at least
in part, with concurrent exposure of said implant to
sonication.
90. The process of claim 87 wherein said cycles are performed in a
chamber in which said implant is entirely enclosed.
91. The process of claim 87 wherein the process is conducted under
computer or programmable logic circuit control.
92. The process of claim 87 wherein said cleaning solution is
selected from the group consisting of sterile water, Triton X-100,
TNBP, 3% hydrogen peroxide, a water miscible alcohol, saline
solution, providone iodine, ascorbic acid solution, aromatic
hydrocarbons, aliphatic hydrocarbons, ethers, ketone, amines, urea,
guanidine hydrochloride, esters, glycoproteins, proteins,
saccharides, enzymes, gasseous acids, gasseous peroxides, and
mixtures thereof.
93. The process of claim 87 wherein said implant comprises
autograft, allograft or xenograft tissue, or combinations
thereof.
94. The process of claim 87, further comprising placing said
implant into a sterile, sealable package.
95. The process of claim 94, further comprising performing a
surface decontamination step prior to or after sealing said
package.
96. The process of claim 87 wherein the implant is perfused or
coated with a bioactive substance.
97. The process of claim 87 wherein the steps are carried out
sufficient times to produce an implant having one or more of the
following properties: (a) between about a one to twelve log
reduction in bacterial contamination; (b) between about a one to
fifteen log reduction in enveloped virus contamination; (c) up to
about a five log reduction in non-enveloped virus contamination;
(d) between about a two to ten fold reduction in endotoxin; (e)
maintenance of implant biologic and biomechanical properties; (f)
absence of tissue toxicity due to cleaning solutions used; (g)
reduced implant antigenicity.
98. The process of claim 87 further comprising the step of
machining said implant to final dimensions.
99. The process of claim 87 wherein one or more steps are conducted
under elevated or reduced temperatures, with respect to ambient
temperature.
100. An implant cleaning process which comprises exposing an
implant to rapid cycles of increased and decreased pressure above
one atmosphere or below one atmosphere, or both; said implant being
in contact with one or more cleaning solutions during at least a
portion of said rapid cycles.
101. The process of claim 100 wherein the steps are carried out
sufficient times to produce an implant that is passivated.
102. The process of claim 100 wherein the implant produced is
suitable for implantation or processing for implantation in
humans.
103. The process of claim 100 wherein said process occurs, at least
in part, with concurrent exposure of said implant to
sonication.
104. The process of claim 100 wherein said rapid cycles are
performed in a chamber in which said implant is entirely
enclosed.
105. The process of claim 100 wherein the process is conducted
under computer or programmable logic circuit control.
106. The process of claim 100 wherein said cleaning solution is
selected from the group consisting of sterile water, Triton X-100,
TNBP, 3% hydrogen peroxide, a water miscible alcohol, saline
solution, providone iodine, ascorbic acid solution, aromatic
hydrocarbons, aliphatic hydrocarbons, ethers, ketone, amines, urea,
guanidine hydrochloride, esters, glycoproteins, proteins,
saccharides, enzymes, gasseous acids, gasseous peroxides, and
mixtures thereof.
107. The process of claim 100 wherein said implant comprises
autograft, allograft or xenograft tissue, or combinations
thereof.
108. The process of claim 100, further comprising placing said
implant into a sterile, sealable package.
109. The process of claim 108, further comprising performing a
surface decontamination step prior to or after sealing said
package.
110. The process of claim 100 wherein the implant is perfused or
coated with a bioactive substance.
111. The process of claim 100 wherein the steps are carried out
sufficient times to produce an implant having one or more of the
following properties: (a) between about a one to twelve log
reduction in bacterial contamination; (b) between about a one to
fifteen log reduction in enveloped virus contamination; (c) up to
about a five log reduction in non-enveloped virus contamination;
(d) between about a two to ten fold reduction in endotoxin; (e)
maintenance of implant biologic and biomechanical properties; (f)
absence of tissue toxicity due to cleaning solutions used; (g)
reduced implant antigenicity.
112. The process of claim 100 further comprising the step of
machining said implant to final dimensions.
113. The process of claim 100 wherein one or more steps are
conducted under elevated or reduced temperatures, with respect to
ambient temperature.
Description
2.0 BACKGROUND OF THE INVENTION
2.1 FIELD OF THE INVENTION
[0001] This invention is a novel method for perfusion of a porous
implant which achieves efficient interpenetration of desired
factors into the pores or channels of the implant, cleaning of the
implant, efficient passivation of the implant (inactivation of
pathogens, microorganisms, cells, viruses and the like and
reduction in antigenicity thereof), and the novel implant produced
by such treatment.
2.2 DESCRIPTION OF KNOWN PROCEDURES FOR IMPLANT TREATMENT
[0002] As used in this disclosure, the term "implant" refers to any
material the implantation of which into a human or an animal is
considered to be beneficial. Accordingly, the implant may be
tissue-derived material, such as bone, skin, and the like, or it
may be a metallic or synthetic material having an internal
structure that may require cleaning or sterilization. Bearing this
definition in mind, it will be apparent that many procedures have
been described in the art for treatment of implants to either clean
such implant, inactivate contaminating microorganism or cells that
may be present in or on such implant, or to infuse the implant with
desirable factors. This section of the disclosure discusses several
known methods for achieving one or more of these results, in order
to more clearly and definitively set forth that which has been
invented, and which is disclosed and claimed as novel and
inventive, as defined by the claims appended hereto.
[0003] European Patent Application No. EP 0 424 159
(Osteotech)--"Aseptic Processing of Allograft Bone and Tissue,"
(published Apr. 24, 1991, based on a U.S. Priority application
filed Oct. 19, 1989), is an extremely general disclosure relating
to aseptic processing of allograft bone and tissue. It appears that
the intent of this application was to establish an early priority
date in an effort to foreclose the entire field of aseptic
processing of allograft bone and tissue. However, the disclosure is
so general that it does not appear to contain an enabling
disclosure of any protectible allograft sterilization method. No
U.S. or European patent related to this extremely general published
application appears to have ever issued.
[0004] In U.S. Pat. No. 5,333,626 (Cryolife)--"Preparation of Bone
for Transplantation", (issued on Aug. 2, 1994, based on an
application filed on Dec. 31, 1991), relates to a method of
preparing bone for transplantation by maintaining the internal
matrix of the bone to be implanted, preferably at high pressure, in
the presence of a decontaminating agent, preferably polyvinyl
pyrrolidine-iodine (PVP-I) optionally in the presence of a
detergent, in solution. The "high pressure" feature of this patent
is described at column 5, lines 10-31: "High pressure washing
conditions should provide a force sufficient to drive the cleaning
solution into internal matrix of the bone. Such high pressure
washing conditions include, for example, vigorous agitation, such
as with a paint can shaker, or high pressure lavage such as with a
high pressure liquid jet stream. The pressure of the liquid jet
stream is preferably 100 to 3,000 psi and most preferably 500 to
1,500 psi." However, the patent does not disclose or suggest
exposure of an implant to an oscillating atmospheric pressure, the
referenced patent requires pressures significantly higher than
those required according to the present invention, and it is only
applicable to bone, while the present invention is applicable to
bone or soft tissue. In addition, the claimed process requires
approximately 1-2 days to complete.
[0005] In U.S. Pat. No. 5,513,662 (Osteotech)--"Preparation of Bone
for Transplantation" (this patent issued on May 7, 1996 as a
continuation-in-part of the application that issued as the U.S.
Pat. No. 5,333,626, based on an application filed on Jan. 21, 1994,
and claiming priority to the Dec. 31, 1991 filing date of the
application on which the U.S. Pat. No. 5,333,626 is based), relates
to a method of preparing bone for transplantation in which the
internal matrix of the bone is maintained at a pressure below one
atmosphere. It is disclosed (column 10, lines 13-19) that "optimum
times for maintaining pressure below ambient are generally in the
range of 30 to 60 minutes but can be determined for each
application by monitoring progress of blood and lipid extraction
(see Example 10)." It is further disclosed that generally use of
gas pressure below ambient for less than two minutes will be
ineffective and use for longer than five hours will confer no
further benefit. Thus, the '662 patent requires that the bone be
maintained for substantial periods of time at pressures below one
atmosphere. There is no disclosure or suggestion of rapidly cycling
between elevated and decreased pressures, even though it is
suggested that the bone might first be treated at an elevated
pressure, followed by a treatment step at a pressure below
atmospheric pressure (see, for example, claim 3, column 15). The
present invention discloses a process wherein transient and
cyclical exposure of an implant material to a given pressure
achieves the desired result of implant cleaning, perfusion or
passivation.
[0006] In U.S. Pat. No. 5,556,379 (LifeNet Research
Foundation)--"Process for Cleaning Large Bone Grafts and Bone
Grafts Produced Thereby," (issued on Sep. 17, 1996 based on an
application filed on Feb. 27, 1995, and claiming priority of an
earlier, abandoned application, filed Aug. 19, 1994), describes the
"Allowash.TM." process. The patent is explicitly directed to the
removal of "bone marrow from the luminal and cancellous bone spaces
in large, essentially whole, bone grafts." (See Summary of the
Invention). Accordingly, the referenced patent is directed only to
treatment of bone, which has to be largely intact. The stated
intent in applying the process to essentially whole bone grafts is
to reduce the load of potentially virus carrying bone marrow to
facilitate preparation of smaller bone grafts therefrom. The
process involves applying a vacuum to the bone graft to draw
solution capable of solubilizing bone marrow through articulating
cartilaginous surfaces and through the intact bone's intramedullary
canal or other bone cavity. The patent neither discloses nor
suggests a method in which oscillating pressures are used to clean
a bone graft.
[0007] U.S. Pat. No. 5,380,826 (Aphios Corporation)--"Supercritical
Fluid Disruption of and Extraction from Microbial Cells, (issued on
Jan. 10, 1995, based on an application filed on Sep. 29, 1992),
relates to a method for harvesting intracellular components by
exposing cells to an elevated pressure in the presence of a
solvent, and then rapidly and suddenly releasing the pressure to
effect disruption of the cells. The patent also discloses an
apparatus for carrying out this process continuously. However, this
patent neither discloses nor suggests applying the cell disruption
method to allograft bone.
[0008] U.S. Pat. No. 5,288,462 (Stephen D. Carter)--"Sterilization
Apparatus and Method" (issued on Feb. 22, 1994, based on an
application filed on May 18, 1992), describes a chamber for
receiving a material to be sterilized by repeatedly subjecting the
chamber to elevated pressures, followed by sudden release of the
pressure, i.e. "explosive decompression." The patent requires that
the chamber be pressurized to at least 1000 psi. The patent neither
discloses, suggests, nor claims application of this method or
chamber to sterilization of bone materials. There is no disclosure
of cleaning solutions used in connection with the described
apparatus that would be effective in sterilizing the matrix of a
bone. There is no disclosure that would allow one skilled in the
art to determine, without undue experimentation, that bone could be
sterilized in this apparatus. In addition, there is no disclosure
nor suggestion that an implant could be sterilized without use of
such highly elevated pressures, but merely by oscillation of lower
absolute pressures.
[0009] U.S. Pat. No. 5,725,579 (Bioland)--"Process for Treating
Bone Tissue and corresponding Implantable Biomaterials", (issued
Mar. 10, 1998, based on a priority French application filed Dec.
21, 1992 and an earlier U.S. priority filing of Dec. 9, 1993), is
directed to a method of cleaning bone by exposing the bone to a
supercritical fluid. As best as can be understood from this patent,
this involves exposing bone to carbon dioxide at elevated
pressures, in order to solubilize lipids.
[0010] Tissue sterilization methods known in the art have
undesirable attributes. Gamma irradiation, in order to ensure
destruction of pathogens, such as the human immunodeficiency virus
(HIV), has to be used at doses that result in tissue destruction
(e.g. 3.5 Mrad; see, for example, Rasmussen, et al., J.
Arthroscopic and Related Surgery, 10(2):188-197, (1994); Goertzen,
et al., British Soc. of Bone and Joint Surg., 77:204-211 (1005);
Loty, et al., International Orthopaedics, 14:237-242, (1990)). Use
of ethylene oxide has been found to result in implants that produce
inflammatory responses (Kudryk, et al., J. Biomedical Materials,
26:1477-1438, (1992); Thoren, et al., Clin. Orthopaedics,
318:259-263, (1995); Simonian, et al., Clin. Orthopaedics,
302:290-296, (1994); Jackson, et al., Am. J. Sports Medicine,
18:1-9, (1990)). Standard chemical solution treatments, while
effective in sterilizing surfaces with which the solutions are
brought into contact, have the major disadvantage of being
insufficiently penetrating to reach the interstices of tissues,
where potentially pathogenic organisms may reside. In view of these
shortcomings, there remains a long-felt-need for an optimized
tissue sterilization process, which would incorporate some or all
of the following features: Effective removal or inactivation of a
wide range of bacterial and viral pathogens; absence of graft
toxicity; retention of desirable tissue characteristics, such as
biomechanical strength or growth-inducing properties; effectiveness
across a wide range of operating modifications and for a wide
variety of tissue types; ability to conclude the process in a final
implant tissue container, to ensure sterile packaging and delivery
for implantation.
[0011] In view of the foregoing review of the known art relating to
implant treatment and sterilization methods, it is believed that
the present invention provides a long needed improvement in that no
absolute temperatures or pressures are required to achieve
efficient implant cleaning, perfusion, or passivation. In addition,
the instant method does not require drilling of holes in implant
materials or any other manipulation or modification in order to
achieve efficient implant cleaning and sterilization. Furthermore,
the present method permits safe pooling of donor tissue for implant
production at economies of scale, without at the same time
diminishing the desirable biological properties of the pooled
implant materials. The instant process includes a number of
methodologies, the additive effect of which is the production of
highly cleansed, sterilized (passivated) tissues, which may be
implanted, without causing toxicity to the recipient. Various
embodiments of the method of this invention includes all of the
above listed features, namely: effective removal or inactivation of
a wide range of bacterial and viral pathogens; absence of graft
toxicity; retention of desirable tissue characteristics, such as
biomechanical strength or growth-inducing properties; effectiveness
across a wide range of operating modifications and for a wide
variety of tissue types; ability to conclude the process in a final
implant tissue container, to ensure sterile packaging and delivery
for implantation.
3.0 SUMMARY OF THE INVENTION
[0012] This invention provides a process wherein an oscillation of
pressure is created in a chamber containing an implant material in
the presence of various cleaning solutions (0.5%
tri(n-butyl)phosphate, TNBP; hydrogen peroxide and the like). The
process essentially comprises the following steps, assuming a
metallic or synthetic material having an internal matrix or space,
or manually cleaned (debrided) graft material, which may or may not
have undergone initial machining, is used as the starting
material:
[0013] 1. Rapidly evacuate the chamber containing the implant,
autograf, allograft or xenograft material;
[0014] 2. Rapidly backfill the chamber with cleaning
solutions--e.g. H.sub.2O.sub.2 /TritonX-100/TNBP/Betadine
mixtures;
[0015] 3. Pressurize chamber;
[0016] 4. Rapidly cycle between steps (1) and (3), for between
about 1-150 cycles, maintaining a temperature of between about
35-40 degrees centigrade, with optional application of ultrasonic
energy;
[0017] 5. Machine the product as desired if not previously
machined;
[0018] 6. Repeat steps (1)-(4) using the same or a different
cleaning compositions, optionally under elevated or reduced
temperature; and
[0019] 7. Optionally perform a surface decontamination step,
preferably in the final packaging, as in exposure to vapor phase
H.sub.2O.sub.2 or like surface decontamination treatments known in
the art.
[0020] The absolute pressures of the system do not appear to be
extremely critical to achieving deep, penetrating cleaning of the
implant or graft materials. Rather, it is the rate of pressure
cycling, the fact of cycling, and possibly the amplitude of
pressure cycling, that appears to be critical to the success of
this method. Accordingly, the entire process may be successfully
conducted at pressures above or below one atmosphere. Evacuation
pressures of 25 inches of mercury to the vapor pressure of the
solutions in the chamber are adequate. Backfill pressures of
between about 40 and 100 PSI are also adequate. Preferably, the
entire process is conducted in a chamber which permits for
sonication of the contents throughout or at particular stages of
the process. In addition, preferably, the entire process is
conducted in a progrmable system under computer or programmable
logic circuit control, so that manual processing is minimized and
reproducibility of the process is maximized. Where the processed
tissue is a bone implant or any form of allograft or xenograft
tissue, election of appropriate solvents, such as urea (preferably
about 6 M), or other chaotropic reagents, (e.g. 4 M guanidine
hydrochloride, or the like), has the additional advantage of
producing a processed tissue of even lower antigenicity than if
such treatment were not included. Target decontamination goals for
this process include:
[0021] Between about a one (1) to twelve (12) log reduction in
bacterial contamination
[0022] Between about a one (1) to fifteen (15) log reduction in
enveloped virus contamination
[0023] Up to about a five (5) log reduction in non-enveloped virus
contamination
[0024] Between about a two (2) to ten (10) fold reduction in
endotoxin
[0025] Maintenance of implant or graft biologic and biomechanical
properties
[0026] absence of tissue toxicity due to cleaning solutions
used
[0027] reduced implant antigenicity
[0028] Accordingly, it is an object of this invention to provide a
method for production of safe and effective allograft, autograf,
xenograf, metallic or synthetic implants in an efficient,
economical manner.
[0029] It is a further object of this invention to permit safe
pooling of tissue donor sources for implant production, while
mininimal the risk that any single contaminated donor will
contaminate any other donor tissue or any recipients of the pooled
tissue processed according to the method of this invention.
[0030] Another object of this invention is to provide a method for
cleaning, perfusing or passivating implant materials without at the
same time compromising the desirable biological properties of the
stating implant materials.
[0031] A further object of this invention is to produce implant
materials of reduced antigenicity.
[0032] Further objects and advantages of this invention will become
apparent from a review of the complete disclosure, including the
claims which follow.
4.0 BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1A provides a schematic in which the cyclic perfusion
passivation process of the invention through seven cycles is shown,
while FIG. 1B shows the cyclic pressure and fluid exposure to
implant materials treated according to the method of this
invention.
[0034] FIG. 2 shows a schematic of one embodiment of an apparatus
that may be employed to effect the method according to this
invention.
[0035] FIG. 3 shows a schematic representation of a further
embodiment of an apparatus layout for conducting the method
according to this invention.
[0036] FIG. 4 provides an overall flow-chart of the various stages
of processing an implant according to the cyclic perfusion
passivation process of this invention from donor tissue acquisition
through final sterile product packaging.
[0037] FIG. 5 provides one embodiment of a detailed processing
containment layout for conducting the method according to this
invention.
[0038] FIG. 6 is a photograph of a whole humerus after being
treated according to the method of this invention; a post-cleaning
coronal section through the head of the humerus reveals the
cleanliness of the inner bone matrix.
[0039] FIG. 7 is a photograph of an intact knee, including proximal
tibia, distal femur and patella, along with articulating tendons
and ligaments, before treatment according to the method of this
invention.
[0040] FIG. 8 is a photograph of the intact knee shown in FIG. 7,
after treatment according to the method of this invention, showing
cleanliness of the implant, and preservation of the articulating
tendons and ligaments.
[0041] FIG. 9 is a photograph of an anterior aspect of a coronal
section through the proximal femur prior to treatment according to
the method of this invention.
[0042] FIG. 10 is a photograph of the posterior aspect of the
coronal section through the proximal femur shown in FIG. 9, after
treatment according to the method of this invention.
[0043] FIG. 11 is a photograph of the sections shown in FIGS. 9 and
10, side-by-side, demonstrating the effectiveness of the treatment
according to this invention for removal of endogenous
substances.
[0044] FIG. 12 is a photomicrograph of an osteon from cortical bone
without fluoroisothiocyanate (FITC) fluorescent dye treatment
(400.times. magnification).
[0045] FIG. 13 is a photomicrograph of an osteon from cortical bone
after inclusion of FITC in one of the cleaning solutions of this
invention, demonstrating deep interpenetration of the dye into the
smallest of bone interstices--bright green areas indicating
structures containing FTC, including the large haversian canal
(right margin) and smaller satellite lacunae (central area;
400.times. magnification).
5.0 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0046] As used herein, the term "passivate" is intended to refer to
the elimination of potentially pathogenic organisms and immunogenic
substances from an implant. Thus, both sterility and reduced
antigenicity is intended by this term, although elimination of
beneficial biological properties of the implant, such as osteogenic
properties (osteoconduction or osteoinduction; bone fusion),
natural tissue functionality, and desirable structural strength of
an implant are not intended by this term. The term "passivation" is
preferred to the term "sterilize" because, while sterilization is a
goal, that term has an absolute connotation which can rarely, if
ever, be completely achieved without attendant tissue destruction.
In addition, while the implants produced according to the method of
this invention may not be completely devoid of any antigenicity or
pyrogenicity, these undesirable aspects are greatly reduced, and
this too is intended by the term "passivation," as used herein.
[0047] The terms "perfused" or "perfusion," as used herein, are
intended to imply efficient interpenetration of cleaning solutions
into and through the channels and crevices of materials intended
for implantation into a recipient.
[0048] As used herein, the terms "rapid" or "rapidly" as they are
applied to the process of pressure cycling according to this
invention mean time frames on the order of seconds to minutes,
rather than hours or days.
[0049] The terms "sonicate" or "sonication" as used herein mean the
application of sonic or ultrasonic energy via a container of an
implant undergoing processing according to the method of this
invention under conditions that permit efficient transfer of the
sonic energy to the implant Those skilled in the art are familiar
with the process of sonication and conditions whereby sonic energy
may be transferred through a fluid to a workpiece such that
efficient cleaning and bacterial or cellular disruption is
achieved, without resulting in gross, ultra ctural damage to the
workpiece.
[0050] This invention provides a novel method for processing
implant materials including, but not limited to, metallic implants,
synthetic implants, ceramic implants, autograft, allograft or
xenograft materials, including bone and soft tissue. In particular,
soft tissue or allograft bone materials treated according to the
method of this invention permit soft tissue or debrided allograft,
autograf or xenograft bone to be thoroughly cleaned, machined,
sterilized, packaged and then implanted at economies of scale
heretofore not possible. In the past, tissue banks have attempted,
as much as possible, to process tissue from single donors, without
permitting contact between tissue derived from different donors.
The concern has been that any given donor tissue may contaminate
other donor tissue. Due to the extreme value of any donor's tissue,
the risk of a large batch of donor tissues being found to be
contaminated has been considered an unreasonable risk. However,
according to the method of the present invention, even if heavily
contaminated donor tissue is included in a batch of pooled donor
tissue, the resultant graft material available for implantation is
safe for implantation.
[0051] Methods for minimizing the risk that donor tissue will be
harvested and processed by a tissue bank, referred to herein as
"donor qualification", are known in the art. Accordingly, thorough
donor screening, and tissue testing by enzymatic, immunological,
biochemical and molecular biological techniques are applied to
minimize the risk that tissue carrying pathogens (viruses,
bacteria, and the like) will be included in the materials processed
and made available for implantation. Testing for contamination by
human immunodeficiency virus, HIV, hepatitis B virus, HBV,
hepatitis C virus, HCV, has now become routine in the art. Known
screening and qualification methods are desirably included as an
initial step preceding processing of the implant material according
to the present method. Due to the highly efficient implant
cleaning, permeation and passivation process encompassed by the
instant invention, it is further expected that as yet unidentified
potentially pathogenic organisms or organisms for which routine
testing has yet to be developed will, in any event, be removed from
implant materials by virtue of the instant implant treatment
process. Redundancy in the level of implant cleaning that is built
into the instant pressure cycling permeation and passivation
process ensures inactivation of such organisms while at the same
time permitting efficient implant processing.
[0052] For purposes of the following description, allograft bone is
referred to as an exemplary tissue that may be processed according
to the present method. However, those skilled in the art will
recognize that other tissues, including but not limited to
autograft bone, xenograft bone, other porous tissues, synthetic
porous materials, and various soft tissues, may be processed
according to the principles defined herein, without departing from
the spirit of the invention exemplified herein by reference to
allograft bone material.
[0053] According to this invention, allograft bone material from
qualified donors is first treated by various known bioburden
reducing methods, as in cleaning by debriding adventitious tissue
according to methods known in the art. Manual dissection may be
employed for removal from the bone surfaces of ligaments, tendons,
skin, fat, muscle, loose bone marrow, and any other non-bone
tissue. Alternatively, automated or semi-automated methods known in
the art, (see, for example, the methods disclosed in U.S. Pat. Nos.
5,333,626; 5,513,662; 5,725,579, and the like, herein incorporated
by reference for this purpose), may be employed for initial
cleaning of the donor bone material.
[0054] At this stage of the process, the cleaned allograft
materials from individual donors may be pooled and further cleaned
as described below. Alternatively, the allograft bone may be
machined to the final implant dimensions, followed by pooling with
a batch of similarly processed, dimensioned implants for further
cleaning as described below. For tracking purposes, while
individual donors would have been tracked up to this stage, upon
pooling, a batch number is defined for further tracking, with
records being maintained of all of the donors that have contributed
to the batch. In yet a further alternative, and to ensure
redundancy in the level of cleaning and potentially pathogenic
contaminant inactivation, implant materials from individual donors
may first be treated as described below, prior to pooling with
implant materials from different donors. In this event, the implant
material form individual donors may be further cleaned whole or
first machined to desired final dimensions.
[0055] When applied to bone, subsequent to initial bioburden
reduction and surface cleaning, the method of this invention
provides for further processing whereby bone marrow, blood,
proteins, and particulate matter is efficiently removed, such that
what remains is essentially a mineralized collagen matrix, in which
about a 5 to 6 log reduction in any form of viable organisms
(viruses, bacteria, amoebae, rickettsia, fungi) is achieved. As
described in greater detail below, this is achieved by a process of
pressure cycling or oscillation, employing a variety of cleaning
and sterilization solutions which are caused to efficiently
interpenetrate the matrix. By repeated cycling and changing of the
cleaning solvents, the channels of essentially any porous matrix
are unclogged, and cleansed. A pre-defined, pre-programmed cycle of
washes is employed, preferably with concurrent ultrasonic
bombardment, to achieve penetrating sterilization of the implant.
We have found that the combination of oscillating fluid pressure
and ultrasonic energy accelerates solution interpenetration and
endogenous substance removal.
[0056] In view of the foregoing description, it will be appreciated
that in one embodiment, the invention is a method which comprises
the following steps:
[0057] 1. Rapidly evacuate a chamber containing the implant such as
porous metallic or synthetic materials, autograft, allograft or
xenograft;
[0058] 2. Rapidly backfill the chamber with cleaning
solutions--e.g. H.sub.2O.sub.2 /TritonX-100/TNBP/Betadine
mixtures;
[0059] 3. Pressurize chamber;
[0060] 4. Rapidly cycle between steps (1) and (3), for between
about 1-150 cycles, maintaining a temperature of between about
35-40 degrees centigrade, with optional application of ultrasonic
energy;
[0061] 5. Machine the product as desired if not previously
machined;
[0062] 6. Repeat steps (1)-(4) using the same or a different
cleaning compositions, optionally under elevated or reduced
temperature; and
[0063] 7. Optionally perform a surface decontamination step,
preferably in the final packaging, as in exposure to vapor phase
H.sub.2O.sub.2 or like surface decontamination treatments known in
the art.
[0064] The process of perfusion passivation is further defined with
reference to FIG. 1A. This schematic shows an implant 100
comprising solid structural constituents 110, channels 120, and
adventitious materials 130 embedded within the channels 120. The
structural constituents 110 may be synthetic materials, as in
man-made polymeric material, (e.g. poly-L-lactic acid, acrylic
acids, and the like), metallic structural materials, or natural
materials, such as a mineralized or demineralized collagen matrix.
The channels 120 may be man-made channels, defined by the
polymerization, molding, melting or other manufacturing process, or
may be natural channels, such as those found in mineralized or
demineralized cancellous or cortical bone matrices. The
adventitious materials 130 may be cellular debris, bone marrow,
cells, lipids, carbohydrates, proteins, viruses, bacteria,
rickettsia, amoebae, fungi and the like. In FIG. 1A, panels (1) and
(2) relate to the first step described above. In panel (1), the
channels 120 are primed for back-filling with cleaning solutions by
exposing the tissue to decreased pressures. In panel (2), the
cleared channels 120 are shown to be substantially clear of
adventitious materials 130. Panel (3) relates to steps 2 and 3,
wherein molecules of cleaning solution 140 are introduced into a
sealed chamber and are driven into the channels 120 by elevated
pressures. Panel (4) relates to the fourth step described above,
wherein decreased pressure removes remaining cellular debris,
cleaning solution 140, and other remaining adventitious materials
from the channels 120, and again primes the matrix for deep
penetration, now possible due to the clarity of the channels 120.
In panels (5)-(7), a one cycle repeat according to the fourth step
described above is shown, whereby upon repressurizing with clean
solvents, full interpenetration of the solvents into the implant
matrix is achieved. In panel (6), reduced pressure draws the
remaining solution from the implant, which may then be dried, as
shown in panel (7), prior to further processing (e.g. machining
according to step 5 above, ether cleaning, according to step 6
above), and final packaging of the cleaned tissue. The cycle
depicted in FIG. 1A may be repeated as many times as desired to
ensure complete internal cleaning of the matrix interior. In FIG.
1B, a representation of the pressure and fluid oscillation
throughout the various steps of the above described process is
represented.
[0065] After being medically released, (i.e. passing a battery of
risk factor and biochemical assays, including, for example,
HIV-specific PCR, and the like), donor tissue is cleaned of any
extraneous or adventitious tissue. The thus-cleaned tissue is
loaded into a sealable reaction chamber. A preferably
pre-programmed tissue cleaning process is then initiated comprising
a plurality of wash steps. Deep tissue interpenetration by cleaning
solutions is achieved by oscillating the pressure in the chamber
while adding and removing various cleaning solvents. Ultrasonic
energy is applied at various stages of the cleaning process to
accelerate solution penetration and removal of unwanted
contaminants or endogenous substances, including blood, lipid, and
non-structural or undesired proteins. In one preferred cleaning
cycle according to this invention, a steps (1-4) of the claimed
process are carried out according to a protocol similar to that
defined in the following table to remove blood, fat, bacterial,
viral, fungal or other contamination:
1TABLE I Duration Step Pressure Fluids* Sonication (min) Purpose 0
Atmospheric None Off NA Load tissue into chamber 1 Negative None
Off 2 Prime tissue matrix, remove included (60-100 torr) air and
loose debris 2 Negative B, C, D, E, On 1 De-gas cleaning fluids
(60-100 torr) mixtures 3 Positive (5-8 B, C, D, E, On 1 Force
fluids into tissue matrix atmospheres) mixtures 4 Negative/ B, C,
D, E, On (1 .times. n) Remove debris loosened by fluids, Positive
mixtures pressure oscillation and sonication *Fluids: B = Triton
X-100/TNBP, a solvent/detergent to remove debris and kill viruses
and bacteria; C = 3% hydrogen peroxide, to remove cellular debris,
inactivate viruses and bacteria; D = mixture of B and C; E =
water-miscible alcohol, such as ethanol or isopropanol; mixtures =
B, C, D, E in any desirable proportions.
[0066] According to Table I, in step 0, under atmospheric pressure,
and no fluid or sonication, a pressurizable chamber in which the
process may be conducted, is loaded with metallic, synthetic or
other man-made implant materials, allograft bone or soft tissue,
xenograft bone or soft tissue, from an individual qualified donor.
Where the implant is a tissue, the tissue is preferably first
cleaned of surface adventitious tissue, prior to initiating the
steps shown in table I. In step 1, under negative pressure
(vacuum), for a period of about two minutes, the matrix of the
implant or implants is primed (i.e. see FIG. 1, step 1, to remove
trapped air, cellular and other loose debris by vacuum). In step 2,
under negative pressure, cleaning fluid is introduced with
sonication, to aid in penetration of the fluid and to ensure gas is
removed from the introduced fluid. In step 3, under positive
pressure, and in the presence of an appropriate cleaning solvent
and sonication, solvent is forced into the matrix of the implant.
Thereafter follows a series of "n" cycles of positive and negative
pressure in the presence of solvent and sonication, during which
the matrix channels are backfiled and emptied of solution and
debris. The number of times this step is cycled may be from once to
about 150 times (i.e. n=1-150; preferably n is about 10-50
times).
[0067] After step 4 in Table I, the cleaning fluid is removed to
waste under positive pressure, the tissue is dried under negative
pressure, and is rinsed several times under oscillating positive
and negative pressure using sterile water or physiological saline
(e.g. phosphate buffered saline, PBS), with or without accompanying
sonication. The number of rinse cycles may be from 1-150 times, and
is preferably about 1-50 times. The rinse solution is drained under
positive pressure, and the tissue is again dried under negative
pressure.
[0068] After removal of the gross contamination according to the
steps outlined above, the tissue in-process may be machined into
dimensionally finished grafts if such processing has not previously
been accomplished, (step 5 of the instant process, as defined
above), and then loaded into a reaction chamber, same or different
than that used to carry out the steps according to Table I. A
deep-penetrating cleaning, passivation or sterilization cycle,
preferably under programmable logic control, is then conducted
according to a protocol similar to that defined in Table II (see
step 6 defined above, which represent a repeat of steps 1-4 of
Table I, optionally using different cleaning solvents; these steps
are distinguished by indicating the steps as 0'-4'):
2TABLE II Duration Step Pressure Fluids* Sonication (min) Purpose
0' Atmospheric None Off NA Load tissue into chamber 1' Negative
None Off 2 Prime tissue matrix, remove included (60-100 torr) air
and loose debris 2' Negative F, G, H, I, J, On 1 De-gas cleaning
fluids (60-100 torr) mixtures 3' Positive F, G, H, I, J, On 1 Force
fluids into tissue matrix (8-10 mixtures atmospheres) 4' Negative/
F, G, H, I, J, On (1 .times. n) Remove debris loosened by fluids,
Positive mixtures pressure oscillation and sonication *Fluids: F =
6M urea or other chaotropic agents, e.g. 4 M guanidine HCl, to
reduce implant antigenicity; G = 1% sodium hypochlorite, to
inactivate viruses, bacteria, fungi or other residual contaminants;
H = 1 N sodium hydroxide, to inactivate viruses and bacteria; I =
6% hydrogen peroxide, as a sterilant; J = hexane, ether,
diethanolamine (DEA), toluene, xylene, butane, CO.sub.2
(supercritical), isobutane, propane, acetone, isopropanol,
methanol, ketones, ethers, aliphatic or aromatic hydrocarbons, HCl,
gasseous HCl. mixtures = F, G, H, I, J in any desirable
proportions.
[0069] After step 4' in Table II, the cleaning fluid is preferably
retained in a positively pressurized reaction chamber for an
extended period to ensure complete killing of any residual
contaminating pathogens or other organisms. A period of from one to
sixty minutes, and preferably about ten minutes, is sufficient for
this purpose. The cleaning fluid is then removed to waste under
positive pressure, the tissue is dried under negative pressure, and
is rinsed several times under oscillating positive and negative
pressure using sterile water or physiological saline (e.g.
phosphate buffered saline, PBS, or the like), with or without
accompanying sonication. The rinse solution is drained under
positive pressure, and the implant is again dried under negative
pressure.
[0070] Those skilled in the art will appreciate that the specifics
of the process outlined according to Tables I and II above may be
modified, without departing from the essence of the present
invention. Essentially, other cleaning solvents or concentrations
than those suggested herein may be used, the number of oscillations
between elevated and reduced pressure, and the cycling times,
pressurization and depressurization levels and periods may be
altered, according to the requirements for a given tissue. However,
the conditions specified in Tables I and II result in deeply
penetrating cleaning, as evidenced by the ability to force dyes
deep into tissue matrices, to remove dyes that have been allowed to
soak deep into tissue matrices, and the ability to remove or kill
endogenous or added biological contaminants, including a wide
variety of bacteria, viruses and fungi. Tissues cleaned according
to this procedure include, but are not limited to: cortical bone,
cancellous bone, fascia, whole joints, tendons, ligaments, dura,
pericardia, heart valves, veins, neural tissue, submucoal tissue,
(e.g. intestinal tissue), and cartilage. Bone treated according to
this method and subsequently tested for retained biomechanical
strength and ability to induce new bone formation (osteoconduction
and osteoinduction, collectively referred to as osteogenic
activity) retains good biomechanical strength and is expected to
retain osteogenic activity. Furthermore, bone treated according to
one embodiment of this method and implanted as a xenograf was found
to induce little or no adverse immunological reactivity, indicating
reduction in antigenicity of the material. This is particularly
true where urea or other chaotropic agents (e.g. guanidine
hydrochloride), is used as one of the cleaning fluids or is
included in a mixture of cleaning fluids.
[0071] The method disclosed herein will suggest to those skilled in
the art a number of possible devices to achieve the programmed
steps defined above. Thus, for example, in one embodiment according
to this invention a device such as that shown schematically in FIG.
2 may be employed for semi-manual implementation of the cyclic
perfusion passivation process of this invention. According to this
embodiment of the invention, a chamber 200 comprising a lid 210 and
a trough 220 is adapted for cyclic perfusion passivation of
implants. A series of posts 230, onto which a series of bolts 240
may be tightened are provided for securing the lid 210 to the
trough 220. A grating 250 is provided inside the chamber 200 for
receiving implant material to be treated. Through the lid 210 is
provided a series of access ports 260, 261, 262, 263. Access port
260 is a sterile water input line. Access port 261 is an input line
for other fluids. Access port 262 is a vacuum line. Access port 263
is a line for pressure input. In addition, a port 264 is provided
for insertion of a temperature probe. Port 265 is a port for
supplying power to a sonicator built into the walls 225 of the
chamber 200. Port 266 is a drain. Accordingly, a device such as
that shown in FIG. 2 could be used carrying out the cyclic
perfusion passivation process according to this invention.
[0072] With reference to FIG. 3, an automated or semi-automated
apparatus 300 may be defined for carrying out the instant process.
Per this disclosure, programmable logic controllers activate or
deactivate valves or solenoids 301a-h at pre-determined times in
the cleaning cycle. An implant is placed in a reaction chamber 310
which is sealed. An atmospheric vent 320 is provided to permit
entrance and removal of waste and filtered air. Cleaning fluids are
introduced into reaction chamber 310 from a chemical mixing tank
330 which has a filtered vent to atmosphere 325, to avoid formation
of a vacuum in the tank 330. Chemical feed lines 340 lead from
fluid reservoirs 341 to the chemical mixing tank 330 via a common
conduit 345. A programmably controlled pump 350 is operated to pump
appropriately mixed fluids from the tank 330 into the reaction
vessel 310. Vacuum or negative pressure is applied to the reaction
vessel 310 by means of a vacuum receiver tank 360, in which a
source of negative pressure is created by vacuum pump 365. The
inclusion of a vacuum reservoir 360 is desirable so that
essentially instantaneous vacuum of known dimensions may be applied
to the reaction chamber 310, without the need for a vacuum pump
such as 365 having to gradually develop the negative pressure.
Vacuum receiver tank 360 may be evacuated by pump 365 while
reaction tank 310 is underpositive pressure. A source of sterile
water, physiological saline, or like aqueous solution is provided
in storage tank 370, which has a filtered vent 375 to prevent
formation of a vacuum in tank 370. Pump 376 provides for rapid
infusion of aqueous solution into chemical mixing tank 330 for
introduction into the reaction chamber 310. Those skilled in the
art will appreciate that the water from tank 370 may also be
directly introduced into reaction tank 310, without having to first
be introduced into chemical mixing tank 330. Positive pressure is
stored in pressure tank 380 which is pressurized by a compressor of
filtered gas, to retain sterility in the reaction tank 310. In
practice, an appropriately programmed computer or programmable
logic controllers permit venting of the reaction chamber 310, to
permit loading of tissue. The chamber is then sealed, evacuated,
pressurized, and fluid is introduced and removed, as outlined, for
example, in Table I and Table II above, to complete the implant
cleaning process.
[0073] Manual or automated perfusion of cleaning and sterilizing
fluids, as outlined above, results in reduction of the bioburden of
implant material from individual donors, prior to pooling with
implant materials from other donors for batch processing. Initial
bioburden reduction may be achieved according to a protocol such as
that outlined in Table I, to reduce the potential for contamination
of an uncontaminated implant by contact with a contaminated
implant. However, those skilled in the art will recognize that the
penetrating passivation process of this invention is so efficient
that for certain types of implants in which the initial prospect of
encountering a contaminated implant is sufficiently low, it may be
possible to simply batch process implant materials according to
Table I and Table II, rather than first cleaning implants from an
individual donor according to the Table I program, prior to
combining such implant materials from different donors and
processing the pooled implants according to the Table II
program.
[0074] Where an initial bioburden reducing step for implant
materials derived from individual donors is considered prudent,
individual donor tissues are processed according to the Table I
program, and are then quarantined until all quality control
criteria are passed. Only the individual donor tissues that pass
such quality control after initial bioburden reduction are pooled
for processing according to the Table II protocol. As an initial
bioburden reduction program, a combination of TritonX-100 and TNBP
may be used as a first solvent to remove debris and to inactivate
bacteria and viruses. A second solvent may be a 3% hydrogen
peroxide solution to remove cellular debris and to further reduce
bioburden. A third solvent may be povidone iodine solution to yet
further reduce bioburden. Finally, ascorbic acid solution may be
employed to decolorize the implant or remove any residual iodine.
These solutions may be employed in a different order, and indeed,
different solutions may be used to similar effect. The particular
solutions listed are preferred, however, due to their low toxicity,
and our discovery that the defined combination of solutions results
in efficient reduction in bioburden, implant cleaning, passivation
and interpenetration. The solutions of Table I are typically
employed in a cycle such as that shown in Table I, steps 0-4.
[0075] At this stage of the process, cleaned allograft or xenograft
tissue from individual donors or previously pooled donors is
optionally pooled and further cleaned as described below.
Alternatively, the tissue is first dimensioned by machining,
truning and the like, to achieve the final implant dimensions. The
dimensioned tissue is further processed individually or is pooled
with a batch of similarly or differently processed, dimensioned
implants for further cleaning as described below. For tracking
purposes, while individual donors would have been tracked up to
this stage, upon pooling, a batch number is defined for further
tracking, with records being maintained of all of the donors that
have contributed to a given batch.
[0076] In Table II, a set of solutions is described for achieving
penetrating sterilization of individual tissues or tissues pooled
from different donors which have already been treated according to
the program outlined in Table I. Thus, a first solution of 6%
hydrogen-peroxide, followed by a second solution of 1% sodium
hypochlorite, followed by a solution of 1 N sodium hydroxide, may
be used to achieve sterilization. A 70% solution of isopropanol may
be used as a broad spectrum germicide. Thus, the solutions of Table
I and Table II may be employed according to the program shown, or
modified as needed. Those skilled in the art will appreciate that
different penetrating sterilants may be employed or that mixtures
of the described sterilants may be possible. In any event, at the
conclusion of this stage of the process, the individual or pooled
batch of implants has been thoroughly cleaned, passivated (if not
sterilized), and interpenetrated by cleaning solutions. Reductions
in enveloped virus, vegetative bacteria, and fungal contamination
of up to twelve logs or higher and of non-enveloped viruses and
spores of up to about five logs are achieved according to the
process described herein. In addition, about a two to ten-fold
reduction in endotoxin levels is achieved, along with significant
elimination of blood, lipid, nucleic acid, and non-structural
protein. Furthermore, this process retains the beneficial
structural and other desirable biological properties of the implant
material. Significant enhancements in production yields, through
the ability to batch process implant from pooled donors, are also
achieved.
[0077] Subsequent to penetrating passivation of the implant
materials, the implant materials are placed in their final packing.
Preferably, this is achieved in a sterile environment to avoid
introduction of any adventitious biobburden. To ensure sterile
packaging, with the final machined grafts in their final, unsealed
packages, the implants are exposed to a vapor-phase hydrogen
peroxide/peracetic acid or like vapor-phase sterilizing
environment. The packages are then closed to ensure that no
contamination may occur upon removal of the implants from the
sterile field for storage or shipment to surgeons. The sealed
packages may then, optionally, be subjected to levels of gamma or
other types of irradiation known to not adversely affect tissue
properties (e.g. below about 3.0 Mrad, or for short periods of time
to effect surface sterilization, and to ensure internal destruction
of any residual large-genome organisms; however, such internal
treatment is generally not required, deep sterilization having been
achieved according to the cleaning protocol described herein).
Other surface and redundant internal sterilization methods,
including exposure to electron beams, exposure to ethylene oxide,
and the like, may also be conducted at this stage, so long as
toxicity or diminishment of desirable biological activities is not
thereby effected.
[0078] As a further enhancement to the process defined herein is
the ability to produce implant materials with perfusion of
desirable bioactivities. Accordingly, in the final rinse steps
after steps 0-4 or Table I or steps 0'-4' of Table II, a solution
containing desired antibiotics, anti-inflammatory drugs or other
biologically active agents may be employed to infuse antibiotic or
other desired bioactive substances into the cleaned, passivated
tissues. Alternatively or in addition, growth factors, such as bone
morphogenetic proteins, cartilage derived growth factors, tissue
growth factors, natural or recombinant, and the like known in the
art may be perfused into the implant.
[0079] As can be appreciated from the foregoing detailed
disclosure, the process of the present invention may be carried out
at any stage of implant production, and it does not require special
preparations such as removal of cartilage, or potentially implant
damaging steps such as drilling of holes.
[0080] As a means of providing an overall concept of the flow of
the method according to the present invention, the schematic
provided according to FIG. 4 is described. In stage 1, donor
materials are introduced into the donor tissue processing facility
and are held in quarantine until the donor from which the tissue
was derived is qualified. In stage 2, released donor materials are
surface cleaned by debridement. In stage 3, surface cleaned tissue
is machined to produce implants of the desired final dimensions,
and are introduced into an automated cyclic perfusion passivation
chamber according to the present invention. In stage 4, implants
that have been passivated are introduced into their final packing
containers and are terminally sterilized by gamma irradiation,
vapor-phase exposure to decontaminants, and the like. Finally, in
stage 5, the passivated and packaged grafts are stored and released
after verification of the sterilization cycles.
[0081] In a further embodiment of this invention, a process layout
similar to that shown schematically in FIG. 5 may be employed.
According to this layout, a processing facility 500 shows three
parallel and identical tissue processing facilities A-C. Starting
in debridement chambers 510A-C, tissue to be treated according to
this invention is cleaned and debrided of gross, adventitious and
unwanted tissues. The cleaned tissue is then introduced, via
sealable port 515A-C into a reaction chamber 310A-C, to which are
connected all of the process control and input/output devices shown
in FIG. 3. Upon completion of a cleaning cycle such as that defined
according to Table I, tissue is removed via sealable port 516A-C.
The cleaned tissue is sorted and stored in quarantine freezers
520A-C, until quality control demonstrates that the tissue is fit
for further processing. The released tissues are then transferred
to graft-production rooms 530A-C, where final implant dimensioning
and machining is conducted. Following production of the finally
dimensioned implants, the thus processed tissues are loaded into
reaction chambers 310'A-C via sealable port 535. Not shown but
connected to reaction chamber 310'A-C are all the process control
and input/output devices shown in FIG. 3. Following further
cleaning, such as that defined according to Table II, the deeply
sterilized tissues are removed from sealable port 536A-C, and are
placed in final packaging. Terminal sterilization is conducted at
stations 540A-C, and the terminally sterilized tissues are sealed
in the final packaging. The sealed packages of terminally
sterilized tissues are quarantined in freezers 545 until final
quality control testing permits tissue release to surgeons.
[0082] It will be appreciated that while the process layout
provided in FIG. 5 is preferred, it is suggestive only, and the
process according to the instant invention may be conducted in
other layout formats. Further, it will be appreciated that
according to the layout shown according to FIG. 5, it is desirable
for the level of ambient particulates to be reduced as tissue is
processed through the various stages shown. Thus, while it is
adequate for the chamber 510 to be of class 100,000 (100,000
particles per billon), it is desirable for areas 520 and 530 to be
class 10,000 or lower. The final packaging area 540 is preferably
about a class 1000 area
[0083] Having generally and in detail described this invention,
including its best mode, the following specific examples are
provided to further exemplify, but not to limit, the disclosed
invention, the scope of which should be reviewed by reference to
the claims appended hereto and the equivalents thereof.
EXAMPLES
Example 1
[0084] Specific Cleaning Protocol for Bone:
[0085] In one preferred embodiment of this invention, an intact or
machined bone implant is cleaned by treatment sequentially with
povidone-I, water, ascorbic acid, TNBP/hydrogen peroxide, water,
diethanolamine, water, 6 M urea, water. The sequence of sonication,
and pressure fluctuations is conducted according to the sequence
defined in Table I or Table II. It will be appreciated from this
disclosure, however, that a wide variety of different cleaning
solutions and combinations thereof may be employed according to the
method of this invention. For example, the cleaning solutions may
include: sterile water, Triton X-100, TNBP, 3% hydrogen peroxide, a
water-miscible alcohol, saline solution, povidone iodine, ascorbic
acid solution, aromatic or aliphatic hydrocarbons, ethers, ketones,
amines, urea, guanidine hydrochloride, esters, glycoproteins,
proteins, saccharides, enzymes such as proteases (trypsin, pepsin,
subtilisin), lipases, sachrases, and the like, gasseous acids or
peroxides, and mixtures thereof. The process is conducted at
ambient temperatures, elevated temperatures (eighty degrees
centigrade) or decreased temperatures. Thus, cleaning of implants
in a liquid nitrogen phase (negative eighty degrees centigrade) is
contemplated by this invention.
Example 2
[0086] Effectiveness of Process for Implant Cleaning:
[0087] FIG. 6 is a photograph of a whole humerus after being
treated according to the method of this invention; a coronal
section through the head of the humerus reveals the cleanliness of
the inner bone matrix.
Example 3
[0088] Effectiveness of Process for Cleaning of Hard Tissue and
Soft Tissue Implants:
[0089] FIG. 7 is a photograph of an intact knee, including proximal
tibia, distal femur and patella, along with articulating tendons
and ligaments, before treatment according to the method of this
invention.
[0090] FIG. 8 is a photograph of the intact knee shown in FIG. 7,
after treatment according to the method of this invention, showing
cleanliness of the implant, and preservation of the articulating
tendons and ligaments.
[0091] In light of these results, it will be apparent that implant
materials and tissues that may be effectively cleaned according to
this procedure include, but are not limited to metallic implants,
synthetic implants, ceramic implants, allograft, autograft or
xenograft tissues. Such tissues may be selected from tissues
comprising: cortical bone, cancellous bone, fascia, whole joints,
tendons, ligaments, dura, pericardia, heart valves, veins, neural
tissue, submucoal tissue, (e.g. intestinal tissue), and cartilage.
Essentially any implantable material having an internal matrix that
is required to be cleaned may be treated to advantage according to
the method of this invention.
Example 4
[0092] Effectiveness of the Process of this Invention for Deep
Cleaning of Implants:
[0093] FIG. 9 is a photograph of an anterior aspect of a coronal
section through the proximal femur prior to treatment according to
the method of this invention.
[0094] FIG. 10 is a photograph of the posterior aspect of the
coronal section through the proximal femur shown in FIG. 9, after
treatment according to the method of this invention.
[0095] FIG. 11 is a photograph of the sections shown in FIGS. 9 and
10, side-by-side, demonstrating the effectiveness of the treatment
according to this invention for removal of endogenous substances
and deep, penetrating implant cleaning.
Example 5
[0096] Demonstration of the Ability of the Process of this
Invention to Achieve Deep Interpenetration of Cleaning Substances
and Impregnation of Implants with Desirable Biologically Active
Substances:
[0097] FIG. 12 is a photomicrograph of an osteon from cortical bone
without fluoroisothiocyanate (FITC) fluorescent dye treatment
(100.times. magnification).
[0098] FIG. 13 is a photomicrograph of an osteon from cortical bone
after inclusion of FITC in one of the cleaning solutions of this
invention, demonstrating deep interpenetration of the dye into the
smallest of bone interstices--bright green areas indicating
structures containing FITC, including the large haversian canal
(right margin) and smaller satellite lacunae (central area
100.times. magnification).
[0099] These photomicrographs demonstrate that the FITC dye is
forced into the smallest implant interstices, thereby revealing the
ability to achieve deep penetrating cleaning. In addition, these
photomicrographs demonstrate that biologically active substances,
such as antibiotics, antiviral compounds, anti-inflammatory
compounds, growth factors, osteo-inductive substances (e.g. bone
morphogenetic protein, cartilage derived morphogenetic protein,
natural or recombinant, and the like), when included in solutions
employed according to the method of this invention, may be
effectively imbedded deeply into implant materials. Thus,
biologically active substances for permeation into implants,
according to the method of this invention are selected from the
group consisting of bone morphogenetic protein, tissue growth
factor beta or member of the tissue growth factor beta family of
growth factors, cartilage derived morphogenetic proteins I or II or
both, and any related cartilage derived growth factors, angiogenic
factors, platelet derived growth factor. Any of the proteins
selected for permeation into implants may be natural or recombinant
proteins.
* * * * *