U.S. patent application number 13/543137 was filed with the patent office on 2013-06-20 for photochemical tissue bonding.
This patent application is currently assigned to The General Hospital Corporation. The applicant listed for this patent is Barbara Chan, Timothy Shane Johnson, Irene E. Kochevar, Mark Randolph, Robert W. Redmond, Jonathan M. Winograd. Invention is credited to Barbara Chan, Timothy Shane Johnson, Irene E. Kochevar, Mark Randolph, Robert W. Redmond, Jonathan M. Winograd.
Application Number | 20130158342 13/543137 |
Document ID | / |
Family ID | 34139890 |
Filed Date | 2013-06-20 |
United States Patent
Application |
20130158342 |
Kind Code |
A1 |
Chan; Barbara ; et
al. |
June 20, 2013 |
PHOTOCHEMICAL TISSUE BONDING
Abstract
Photochemical tissue boding methods for bonding neural tissues
include the application of a photosensitizer to a tissue and/or
tissue graft, followed by irradiation with electromagnetic energy
to produce a tissue seal. The methods are useful for tissue
adhesion, such as in wound closure, tissue grafting, skin grafting,
musculoskeletal tissue repair, ligament or tendon repair, neural
repair, blood vessel repair and corneal repair.
Inventors: |
Chan; Barbara; (Cambridge,
MA) ; Johnson; Timothy Shane; (Hershey, PA) ;
Kochevar; Irene E.; (Charlestown, MA) ; Randolph;
Mark; (Chelmsford, MA) ; Redmond; Robert W.;
(Newton Centre, MA) ; Winograd; Jonathan M.;
(Farmingham, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chan; Barbara
Johnson; Timothy Shane
Kochevar; Irene E.
Randolph; Mark
Redmond; Robert W.
Winograd; Jonathan M. |
Cambridge
Hershey
Charlestown
Chelmsford
Newton Centre
Farmingham |
MA
PA
MA
MA
MA
MA |
US
US
US
US
US
US |
|
|
Assignee: |
The General Hospital
Corporation
Boston
MA
|
Family ID: |
34139890 |
Appl. No.: |
13/543137 |
Filed: |
July 6, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10732032 |
Dec 9, 2003 |
8215314 |
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13543137 |
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10691180 |
Oct 21, 2003 |
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10732032 |
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10094120 |
Mar 8, 2002 |
7073510 |
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10691180 |
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09900504 |
Jul 6, 2001 |
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10094120 |
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09781577 |
Feb 12, 2001 |
7331350 |
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09900504 |
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60181980 |
Feb 11, 2000 |
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Current U.S.
Class: |
600/36 ;
606/213 |
Current CPC
Class: |
A61L 24/001 20130101;
A61B 2017/00517 20130101; A61F 2009/00842 20130101; A61N 5/062
20130101; A61B 17/11 20130101; A61L 2430/32 20130101; A61F
2009/00872 20130101; A61F 9/0079 20130101; A61F 9/00821 20130101;
A61B 2017/00508 20130101; A61F 9/0081 20130101; A61F 2009/00887
20130101; A61F 2009/00853 20130101 |
Class at
Publication: |
600/36 ;
606/213 |
International
Class: |
A61B 17/11 20060101
A61B017/11 |
Goverment Interests
GOVERNMENT INTEREST
[0002] This work was supported by the Government, in part, by
grants from the Department of Defense Medical Free Electron Laser
Program, Award Nos. N0014-94-1-0927 and F49620-01-1-0014, the
Department of Defense DAMD Program, Award No. 17-02-2-0006, the
AFOSR, Award No. F49620-01-1-0014, and the Office of Naval
Research, award number N000149910617. The government has certain
rights to this invention.
Claims
1-43. (canceled)
44. A method for adhering blood vessel tissue in a subject,
comprising: contacting an outer surface of a proximal vessel and an
inner surface of a distal vessel with a photosensitizer agent;
folding the distal vessel back to form a distal cuff; apposing the
proximal vessel and the distal vessel; everting the distal cuff
over the proximal vessel to form an overlap region; and applying
electromagnetic energy to the tissue-photosensitizer complex in a
manner effective to bond the tissues, thereby creating a tissue
seal between the blood vessel tissue.
45. The method of claim 44, wherein prior to contacting the
proximal vessel with the distal vessel, the vessels are mounted
over a cylindrical support.
46. The method of claim 45, wherein the cylindrical support is a
glass rod.
47. The method of claim 44, further comprising applying at least
one suture between the vessels before applying the electromagnetic
energy.
48. The method of claim 44, wherein the electromagnetic energy is
applied externally around the circumference of the overlap
region.
49. The method of claim 44, wherein the electromagnetic energy is
applied internally around the interior of the overlap region.
50. The method of claim 44, wherein the photosensitizer agent is
selected from the group consisting of xanthene, pyridine,
phenothiazine, cyanine, flavin, and porphyrin.
51. The method of claim 44, wherein the photosensitizer agent is
Rose Bengal.
52. The method of claim 44, wherein the method occurs ex vivo.
53. The method of claim 44, wherein the method occurs in vivo in
the subject.
54. The method of claim 44, wherein the subject is a human.
55. A method for adhering blood vessel tissue in a subject,
comprising: placing a first blood vessel tissue and second blood
vessel tissue in a conduit; contacting the first blood vessel
tissue and second blood vessel tissue with at least one
photosensitizer agent to form a tissue-photosensitizer complex; and
applying electromagnetic energy to the tissue-photosensitizer
complex in a manner effective to bond the tissues, thereby creating
a tissue seal between the blood vessel tissues and the conduit.
56. The method of claim 55, wherein the conduit comprises
collagen.
57. The method of claim 55, wherein the conduit is formed from a
synthetic absorbable polymer.
58. The method of claim 55, wherein the conduit is formed from
PGA.
59. The method of claim 55, wherein the conduit is formed from
silicone.
60. The method of claim 55, wherein the electromagnetic energy is
applied externally around the circumference of the blood vessel
tissue.
61. The method of claim 55, wherein the electromagnetic energy is
applied internally around the interior of the blood vessel
tissue.
62. The method of claim 55, wherein the photosensitizer agent is
selected from the group consisting of xanthene, pyridine,
phenothiazine, cyanine, flavin, and porphyrin.
63. The method of claim 55, wherein the photosensitizer agent is
Rose Bengal.
64. The method of claim 55, wherein the method occurs ex vivo.
65. The method of claim 55, wherein the method occurs in vivo in
the subject.
66. The method of claim 55, wherein the subject is a human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser. No.
10/732,032, filed Dec. 9, 2003, now U.S. Pat. No. 8,215,314, which
is a continuation-in-part of U.S. application Ser. No. 10/691,180,
filed Oct. 21, 2003, which is a continuation-in-part of U.S.
application Ser. No. 10/094,120, filed Mar. 8, 2002, which is a
continuation-in-part of U.S. application Ser. No. 09/900,504, filed
Jul. 6, 2001, which is a continuation-in-part application of U.S.
application Ser. No. 09/781,577, filed Feb. 12, 2001, claiming
priority to U.S. Provisional Application Ser. No. 60/181,980, filed
Feb. 11, 2000, the contents of all of which are incorporated herein
in their entireties by reference. Reference is also made to PCT
application No. PCT/US01/40093, filed on Feb. 12, 2001 and
published on as PCT Publication No. WO 01/58495 on Aug. 16, 2001,
claiming priority to U.S. Provisional Application Ser. No.
60/181,980, filed Feb. 11, 2000, the contents of all which are
incorporated herein in their entireties by reference.
[0003] Each of the applications and patents cited in this text, as
well as each document or reference cited in each of the
applications and patents (including during the prosecution of each
issued patent; "application cited documents"), and each of the PCT
and foreign applications or patents corresponding to and/or
claiming priority from any of these applications and patents, and
each of the documents cited or referenced in each of the
application cited documents, are hereby expressly incorporated
herein by reference. More generally, documents or references are
cited in this text, either in a Reference List before the claims,
or in the text itself; and, each of these documents or references
("herein-cited references"), as well as each document or reference
cited in each of the herein-cited references (including any
manufacturer's specifications, instructions, etc.), is hereby
expressly incorporated herein by reference.
FIELD OF THE INVENTION
[0004] The present invention relates to methods of photochemical
tissue bonding for use in tissue adhesion, such as in wound
closure, tissue grafting, skin grafting, musculoskeletal tissue
repair, ligament or tendon repair, neural repair, blood vessel
repair and corneal repair. The present invention further relates to
methods of photochemical tissue bonding for use in tissue adhesion,
wherein a graft comprising a synthetic tissue substitute,
engineered tissue or natural biomaterial is adhered to a host
tissue.
BACKGROUND
[0005] Traditional wound closure methods, such as staples and
sutures, have numerous drawbacks, including the possible occurrence
of inflammation, irritation, infection, wound gap, and leakage. The
cosmetic results of the use of staples and sutures can also be
undesirable. In corneal applications, sutures often produce
astigmatism due to uneven suture tension. In tissue grafting
techniques, sutures can lead to a variety of complications in wound
healing, including foreign body responses that cause scarring.
Repair of injuries to tendons and ligaments involve an additional
component whereby the wound must return to functional integrity,
which would be severely compromised by the presence of scar tissue
or the failure of sutures. Traditional wound closure and/or tissue
adhesion methods suffer from a number of drawbacks that are
addressed by the present invention.
[0006] Possible alternatives to sutures include hemostatic
adhesives, such as fibrin sealants (Herrick et al. (1987) J
Cataract Refract Surg 13:551-553; Herrick et al. (1991) J Cataract
Refract Surg 17:551-555), cyanoacrylate adhesives (Shigemitsu et
al. (1997) International Ophthalmology 20:323-328), and
photodynamic tissue glue, composed of a mixture of
riboflavin-5-phosphate and fibrinogen, which has been reported to
close cataract incisions and attach donor cornea in corneal
transplants (Goins et al. (1997) J Cataract Refract Surg
23:1331-1338; Goins et al. (1998) J Cataract Refract Surg
24:1566-1570; U.S. Pat. No. 5,552,452). In addition,
temperature-controlled tissue welding has been attempted in bovine
cornea and rat intestine (Barak et al. (1997) Sury Ophthalmol 42
Supp.1:S77-81; Cilesiz et al. (1997) Lasers Surg Med 21:269-86).
Photochemical tissue welding of dura mater has also been reported,
using 1,8 naphthalimides irradiated with visible light (Judy et al.
(1993) Proc. SPIE--Int. Soc. Opt. Eng. 1876:175-179).
[0007] Tissue grafts and/or tissue substitutes (e.g., extracellular
matrix-based scaffolds, such as collagen and proteoglycan, and/or
other engineered tissue implants) are important components used in
structural tissue engineering. Although many tissue grafts and/or
substitutes are made of naturally occurring biomaterials, these
structures, once implanted, do not attach well to the target tissue
and can lack the ability to hold sutures. As a result, additional
support material, such as silicon tubing, is implanted for further
support and integration of the implant with the target tissue. The
disadvantage of this method is that a second surgical procedure is
often required for the removal of the support material once the
implants become integrated with the host tissue.
[0008] In particular, tissue grafts comprising skin grafts and/or
skin substitutes are widely used in surgical procedures such as
skin transplantation, burn and ulcer wound management and plastic
surgery. Current fixation aids for grafting mainly consist of
mechanical and adhesive means (Bass & Treat (1995) Lasers Surg
Med 17: 315-49). Surgical sutures and staples mechanically hold the
tissue in position while tissue and fibrin glues
chemically/biochemically bond the graft to the host. However, the
use of sutures and staples has low aesthetic/cosmetic value and may
lead to foreign-body reactions as well as wound complications (Bass
& Treat (1995) Lasers Surg Med 17: 315-49). The use of tissue
glues such as cyanoacrylate provides excellent binding strength but
results in persistent inflammation and foreign body giant cell
reaction (Forseth et al. (1992) J Long Term Eff Med Implants 2(4):
221-33, Toriumi et al. (1990) Arch Otololaryngol Head Neck Surg
116: 546-550). Although the use of autogenous fibrin glue
eliminates the foreign-body reactions and the associated
complications, it elicits other problems. Firstly, it is costly and
time-consuming to extract and purify autogenous fibrinogen from the
patient's blood (Dahlstrom et al. (1991) Skin Transplantation
89(5): 968-72) and secondly, the mechanical outcome is not
satisfactory since the breaking strength at the interface was less
than 0.2N/cm.sup.2 (Dahlstrom et al. (1991) Skin Transplantation
89(5): 968-72).
[0009] Tendon and ligament injuries, including Achilles tendon
rupture (Davis et al. (1999) Mil Med 164(12): 872-3; Maffulli et
al. (1999) Clin J Sport Med 9(3): 157-60; Houshian et al. (1998)
Injury 29(9): 651-4), are extremely common, and can occur in
various anatomical regions (Best & Garrett Jr (1993)
Orthopaedic Sports Medicine pp. 1-45): rupture or inflammation may
occur at the supraspinatus and subscapularis tendon of the shoulder
region, and in the biceps and triceps tendons of the upper limb;
extensive tendon and ligament injuries can occur in both hands and
fingers, disturbing the nonnal function of the appendage and making
delicate movements impossible; knee injuries often involve the
medial collateral ligament (MCL), anterior cruciate ligament (ACL)
or posterior cruciate ligament (PCL), and can also involve the
patellar tendon. Tendons are usually injured upon excessive
acceleration or deceleration, especially when the associated
muscles become fatigued. The injury often occurs as a laceration or
an avulsion from the bone and tendon transaction, which is deemed
rupture of the tendon. Such injuries are very common in individuals
who frequently engage in strenuous activities for extended periods
of time.
[0010] Although many nonsurgical treatment regimens such as
bracing, rehabilitation program, immobilization, passive controlled
movement and ultrasound have been used, surgical repair and
reconstruction of a completely torn tendon or ligament is still the
preferred treatment, in particular among young patients and those
who require an early return to normal activities (Leppilahti &
Orava (1998) Sports Med 25(2): 79-100), such as athletes and
military personnel. Typical treatments include surgical repair
using the Kessler suture procedure and the pedicle flap turn-down
procedure (Shereff (1993) Atlas of foot and ankle surgery pp.
304-11) in achilles tendon, surgical reconstruction of torn
anterior cruciate ligament (ACL) using autogenous patellar tendon
graft (Shino et al. (1993) Am J Sports Med 21(4): 609-616) and
reconstruction of ruptured posterior cruciate ligament (PCL) (Bosch
et al. (1994) Acta Orthop Belg 60(suppl): 57-61).
[0011] Despite its popularity, the current surgical management of
musculoskeletal tissues is not problem-free. The major complaint
involving surgical treatment is the high rate of complications
(Leppilahti & Orava (1998) Sports Med 25(2): 79-100). Most of
these procedures involve multiple sutures and staples, which may be
associated with wound complications such as infection and necrosis
(Shereff (1993) Atlas of foot and ankle surgery pp. 304-11; Koh
& Lim (1999) Hand Surg 4(2): 197-202). For procedures making
use of autogenous tendon or facia grafts for reinforcement or
reconstruction, additional soft tissue injuries at the donor sites
are created which make the procedure more invasive and may lead to
donor site morbidity. Recurrent rupture, skin adhesions and
excessive scarring are other surgery-associated problems.
[0012] These complications have prompted investigation into laser
tissue welding as an alternative or supplement to surgical options.
Laser tissue welding is a developing technique with numerous
clinical applications for many surgical specialties including
orthopedics (Bass & Treat (1995) Lasers Surg Med 17: 315-49),
of which repair of tendons is a potential use. An immediate
regaining of partial strength after repair is essential because of
the large stress the tendon is subjected to post-operatively. A
previous report (Kilkelly & Choma (1996) Laser Surg Med 19:
487-91) using CO.sub.2 and Argon lasers, showed that thermal
welding of ruptured achilles tendon in rats led to .about.50-70%
tensile strength recovery at 2 weeks post-op but no immediate
tensile strength improvement. Other modes of laser tissue
interaction mechanisms such as low energy laser photostimulation
have also been studied to improve tendon repair by stimulating the
intrinsic tendon healing. These means were found to induce a
significant (.about.30%) increase in collagen production but
insignificant increase (.about.10%) in mature crosslinks in
ruptured rabbit achilles tendon (Reddy et al. (1998) Laser Surg Med
22: 281-7). In addition, the mechanical properties of the repaired
tendon, in particular stress and stiffness demonstrated a
.about.30% but statistically insignificant increase (Reddy et al.
(1998) Med Sci Sports Exerc 30(6): 794-800).
[0013] The ideal technique for tissue adhesion would be simpler,
more rapid, and prone to fewer post-operative complications than
conventional techniques. In the cornea, an ideal tissue repair or
wound closure technique would produce a watertight seal without
inducing astigmatism. In tissue grafts and/or tissue substitutes,
such as collagen-based scaffolds, the ideal technique would enhance
fixation to the surrounding host tissues. In particular, skin
grafting techniques enabling rapid and sustained adherence to the
wound surface and the ability to resist shear stress are ideal for
successful graft take. Repair of injuries to tendons and ligaments
would ideally minimize or eliminate the use of multiple sutures and
autogenous tendon grafts, minimize complications associated with
foreign-body reactions, and minimize the thermal damage to
surrounding tissue currently associated with thermal laser tissue
welding
[0014] Initial repair technique plays an important role in
determining overall outcome in peripheral nerve regeneration after
injury (de Medinaceli et al. (1982), Exp Neurol 77(3): 634-43).
[0015] Microsurgical re-approximation of severed nerve ends is
currently the most commonly used technique for repair of peripheral
nerve injuries in which a significant deficit of neural tissue does
not occur. Unfortunately, even in more favorable lesions, such as
median or ulnar nerve injuries in the forearm, only 10% of patients
who undergo microsurgical repair achieve an excellent recovery,
i.e. full muscle strength and normal two-point discrimination.
There are both biologic and technical reasons for this. Following
peripheral nerve injury, a large proportion of the lower motor
neurons and sensory neurons from the injured nerve undergo
apoptotic death, with loss of up to 30% of the neurons within the
nerve. This biologic limitation virtually ensures inadequate or
absent recovery distally for any proximal nerve injury. Although
current microsurgical techniques utilizing end-to-end repair and
nerve grafting are adequate for more distal injuries, those with
longer nerve gaps and injuries that are more proximal rarely
achieve complete recovery. Further complicating this issue, surgery
itself is traumatic to the nervous tissue with suture material
acting as a potential nidus for the formation of scar tissue, which
inhibits regeneration of proximal nerve fibers (Korff et al (1992),
Otolaryngol-Head and Neck Surg 106(4): 345-350).
[0016] In addition to standard surgical techniques, laser-based
techniques have also been used in nerve repair. However, these
methods typically result in heat absorption, which denatures
proteins. Thus, there exists in the art a need for improved methods
of nerve repair. Improved methods of regenerating nerve fibers,
ideally without the formation of scar tissue, would be highly
desirable for the treatment of many nerve injuries.
SUMMARY
[0017] The present invention is based, in part, on the discovery
that the application of a photosensitizer (e.g., Rose Bengal (RB),
riboflavin-5-phosphate (R-5-P), methylene blue (MB), or
N-hydroxypyridine-2-(1H)-thione (N-HTP)) to a tissue, followed by
photoactivation produces an adhesive tissue-tissue seal (e.g., to
repair a wound, or seal a tissue transplant) without collagen
denaturation or heat induced peripheral tissue damage. Furthermore,
the tissue-tissue seal can be produced when the photosensitizer is
applied to the tissue in the absence of an exogenously supplied
source of cross-linkable substrate, e.g., a protein such as fibrin
or fibrinogen or other protein-based tissue adhesive or glue. Such
exogenous substances are often suggested to be used to contribute
cross-linkable protein to a tissue. (Herein, a graft tissue or the
components thereof is not considered such a source of exogenously
supplied cross-linkable substrate.) This procedure is referred to
herein as photochemical tissue bonding (PTB). PTB can be used ex
vivo or in vivo in a subject, e.g., a human, or a non-human
animal.
[0018] Accordingly, in one aspect, the invention features a method
for cross-linking tissue, e.g., creating a tissue seal, such as in
tissue grafting. The method includes identifying a tissue in need
of repair, e.g., a collagenous tissue, cornea, skin, cartilage,
ligament or tendon; contacting the tissue with a photosensitizer
e.g., Rose Bengal (RB), riboflavin-5-phosphate (R-S-P), methylene
blue (MB), or N-hydroxypyridine-2-(1H)-thione (N-HTP), and
optionally contacting a second tissue, e.g. a tissue graft or
substitute comprising natural or synthetic extracellular
matrix-based scaffolds, such as collagen and proteoglycan, and/or
other engineered tissue implants, with photosensitizer, to form a
photosensitizer-tissue complex; and applying electromagnetic
energy, e.g., light, to the tissue-photosensitizer complex
sufficient to produce cross linking in the tissue or tissue graft.
The tissue is not contacted with an exogenously supplied source of
cross-linkable substrate, e.g., protein, e.g., fibrin or
fibrinogen, or protein-based adhesive or glue, which is cross
linked by the application of electromagnetic energy. (Herein, a
graft tissue or the components thereof is not considered such a
source of exogenously supplied cross-linkable substrate or
adhesive.) PTB can be used to graft tissue ex vivo or in vivo in a
subject, e.g., a human, or a non-human animal.
[0019] In one aspect, the tissue is in need of repair. This tissue
can be of any type where adhesion or wound closure is necessary,
for example a cardiovascular, neurological, gastrointestinal,
urological, vascular, renal, occular, oral, connective,
respiratory, otolaryngological, dermatological, genital,
gynecological or musculoskeletal tissue. Wound closure can comprise
the joining of cut or otherwise separated edges or surfaces of the
tissue/damaged tissue.
[0020] In one aspect, the tissue is corneal tissue. For example,
the tissue, e.g., cornea, has been subjected to trauma, a surgical
incision, LASIK flap reattachment, corneal transplant, or
correction of astigmatism. One or more elements, for instance, cut
or otherwise separated edges or surfaces, of the subject's corneal
tissue can be joined together, or to graft tissue through
photochemical tissue bonding.
[0021] In one embodiment, the tissue in need of repair is grafted
with an exogenous tissue. An exogenous tissue is one supplied from
a site other than the site of the lesion/wound. Preferably, this
tissue is skin. The exogenous tissue can be in the form of a tissue
graft, e.g. a graft or substitute comprising natural or synthetic
extracellular matrix-based scaffolds, such as collagen and
proteoglycan, and/or other engineered tissue implants. Suitable
exogenous tissue can be supplied to a variety of sites, for
example, to a wound, tear or lesion in a cardiovascular,
neurological, gastrointestinal, urological, vascular, renal,
occular, oral, connective, respiratory, otolaryngological,
dermatological, genital, gynecological or musculoskeletal
tissue.
[0022] In yet another embodiment, the photosensitizer agent is
selected from the group consisting of Rose Bengal,
riboflavin-5-phosphate, methylene blue, and
N-hydroxypyridine-2-(1H)-thione.
[0023] In yet another embodiment, the photosensitizer agent is Rose
Bengal.
[0024] In yet another embodiment, the contacting step occurs ex
vivo.
[0025] In yet another embodiment, the contacting step occurs in
vivo in a subject, e.g., a human, or an non-human animal,
preferably a non-albino animal, e.g., a non-albino rabbit.
[0026] In yet another embodiment, the subject is other than an
albino animal, e.g., other than an albino rabbit.
[0027] In yet another embodiment, the subject is a human.
[0028] In yet another embodiment, the application of
electromagnetic energy to the tissue-photosensitizer complex occurs
without substantial thermal tissue damage, e.g., shrinkage or
deformation around the wound site and thermal cell damage.
[0029] In yet another embodiment, the application of
electromagnetic energy to the tissue-photosensitizer complex occurs
without more than a 15.degree. C. rise in temperature as measured,
e.g., with an imaging thermal camera during irradiation.
[0030] In yet another embodiment, the application of
electromagnetic energy to the tissue-photosensitizer complex occurs
without more than a 10.degree. C. rise in temperature as measured,
e.g., with an imaging thermal camera during irradiation.
[0031] In yet another embodiment, the application of
electromagnetic energy to the tissue-photosensitizer complex occurs
without more than a 3.degree. C. rise in temperature as measured,
e.g., with an imaging thermal camera during irradiation.
[0032] In yet another embodiment, the application of
electromagnetic energy to the tissue-photosensitizer complex occurs
without more than a 2.degree. C. rise in temperature as measured,
e.g., with an imaging thermal camera during irradiation.
[0033] In yet another embodiment, the application of
electromagnetic energy to the tissue-photosensitizer complex occurs
without more than a 1.degree. C. rise in temperature as measured,
e.g., during irradiation with an imaging thermal camera.
[0034] In yet another aspect, the invention features, a method for
repairing a corneal lesion, e.g., a corneal incision, laceration,
or a corneal transplant, in a subject, e.g., a human, or a
non-human animal, preferably a non-albino animal. The method
includes: contacting a corneal tissue with at least one
photosensitizer agent, e.g., RB, R-5-P, MB, or N-HTP, and applying
electromagnetic energy, e.g., light, to the corneal
tissue-photosensitizer complex sufficient to produce a reactive
species, e.g., a reactive oxygen species, from the photosensitizer.
The corneal tissue is not contacted with an exogenously supplied
source of cross-linkable substrate, e.g., protein, e.g., fibrin or
fibrinogen, or protein-based tissue adhesive or glue, which is
cross-linked by the application of electromagnetic energy.
[0035] In one embodiment, the corneal lesion is caused by a
surgical procedure.
[0036] In yet another embodiment, the surgical procedure is
selected from the group consisting of corneal transplant surgery,
cataract surgery, laser surgery, keratoplasty, penetrating
keratoplasty, posterior lamellar keratoplasty, LASIK, refractive
surgery, cornea reshaping, and treatment of corneal laceration.
[0037] In yet another embodiment one or more elements, e.g., cut or
otherwise separated edges or surfaces, of the subject's corneal
tissue can be joined together, or to graft tissue.
[0038] In yet another embodiment, a subject's muscle tendon can be
joined to the subject's eye. E.g., an ocular misalignment can be
reduced, adjusted, or corrected, for instance, by joining an eye
muscle tendon to the eye.
[0039] In yet another embodiment, the cornea is in need of
correction for astigmatism. For example, PTB can be used to
correct, reduce, or decrease astigmatism, e.g., by inducing
astigmatism in the orthogonal meridian, thereby counteracting
preexisting astigmatism. In a preferred embodiment, PTB induces a
predictable degree of corrective astigmatism.
[0040] In yet another embodiment, the method further comprises
administration of an adjunctive therapy, e.g., contact lens
therapy, amniotic membrane therapy, LASIK therapy, or
administration of antibiotics.
[0041] In yet another embodiment, the electromagnetic energy
applied is greater than 1200/cm.sup.2. In another preferred
embodiment, the electromagnetic energy applied is between 200 and
1200/cm.sup.2. In another preferred embodiment, the electromagnetic
energy applied is between 200 and 800/cm.sup.2. In yet another
preferred embodiment, the electromagnetic energy applied is between
200 and 500/cm.sup.2. In yet another preferred embodiment, the
electromagnetic energy applied is between 300 and 600/cm.sup.2. In
another preferred embodiment, the electromagnetic energy applied is
between 350 and 550/cm.sup.2.
[0042] In yet another embodiment, the electromagnetic energy is
applied at an irradiance less than 3.5 W/cm.sup.2.
[0043] In yet another embodiment, the electromagnetic energy is
applied at an irradiance less than 1.5 W/cm.sup.2.
[0044] In yet another embodiment, the electromagnetic energy is
applied at an irradiance of about 0.10 W/cm.sup.2.
[0045] In yet another embodiment, the subject is other than an
albino animal, e.g., other than an albino rabbit.
[0046] In yet another aspect, the invention features, a method for
repairing a corneal lesion in vivo in a living subject, e.g., a
human, or a non-human animal, preferably a non-albino animal. The
method includes contacting a corneal tissue with Rose Bengal (RB)
to form a corneal tissue-RB complex; and applying electromagnetic
energy, e.g., light, to the corneal tissue-RB complex in a manner
effective to elicit the production of a reactive species, e.g., a
reactive oxygen species, from the RB. The corneal tissue is not
contacted with an exogenously supplied source of cross-linkable
substrate, e.g., protein, e.g., fibrin or fibrinogen, or
protein-based tissue adhesive or glue, which is cross-linked by the
application of electromagnetic energy.
[0047] In one embodiment, the subject is a human.
[0048] In yet another embodiment, the corneal lesion is caused by a
surgical procedure.
[0049] In yet another embodiment, the surgical procedure is
selected from the group consisting of corneal transplant surgery,
cataract surgery, laser surgery, keratoplasty, penetrating
keratoplasty, posterior lamellar keratoplasty, LASIK, refractive
surgery, cornea reshaping, and treatment of corneal laceration.
[0050] In yet another embodiment one or more elements, e.g., cut or
otherwise separated edges or surfaces, of the subject's corneal
tissue can be joined together, or to graft tissue.
[0051] In yet another embodiment, a subject's muscle tendon can be
joined to the subject's eye, e.g., an ocular misalignment can be
reduced, adjusted, or corrected, e.g., by joining an eye muscle
tendon to the eye.
[0052] In yet another embodiment, the cornea is in need of
correction for astigmatism. For example, PTB can be used to
correct, reduce, or decrease astigmatism, e.g., by inducing
astigmatism in the orthogonal meridian, thereby counteracting
preexisting astigmatism. In a preferred embodiment, PTB induces a
predictable degree of corrective astigmatism.
[0053] In yet another embodiment, the method further comprises
administration of an adjunctive therapy, e.g., contact lens
therapy, amniotic membrane therapy, LASIK therapy, or
administration of antibiotics.
[0054] In yet another embodiment, the subject is other than an
albino animal, e.g., other than an albino rabbit.
[0055] In yet another aspect, the invention features a kit for
repairing corneal lesions, which kit includes a photosensitizer
agent, e.g., RB, R-5-P, MB, or N-HTP, instructions for
photoactivation of the photosensitizer agent to repair the corneal
lesion, accessory tools and instructions for effective tissue edge
approximation. In a preferred embodiment the kit does not include a
source of cross-linkable substrate, e.g., protein, e.g., fibrin or
fibrinogen, or protein-based tissue adhesive or glue, for use with
the photosensitizer.
[0056] In one embodiment, the photosensitizer agent is Rose
Bengal.
[0057] In yet another aspect, the invention features a method for
repairing a musculoskeletal tissue e.g., a tendon, ligament or
cartilage, damaged by a laceration or rupture, in a subject, e.g.,
a human, or a non-human animal. The method includes: contacting
musculoskeletal tissue with at least one photosensitizer agent,
e.g., RB, R-5-P, MB, or N-HTP, and applying electromagnetic energy,
e.g., light, to the tendon tissue-photosensitizer complex
sufficient to produce a reactive species, e.g., a free radical,
from the reaction between the photosensitizer and the tissue. The
musculoskeletal tissue is not contacted with an exogenously
supplied source of cross-linkable substrate, e.g., protein, e.g.,
fibrin or fibrinogen, or protein-based tissue adhesive or glue,
which is cross-linked by the application of electromagnetic
energy.
[0058] In one embodiment, the subject is a human.
[0059] In yet another embodiment, the musculoskeletal tissue is a
tendon.
[0060] In yet another embodiment, the tendon is ruptured.
[0061] In yet another embodiment, the tendon is damaged by an
avulsion from the bone and tendon transection.
[0062] In yet another embodiment, the ruptured ends of the tendon
are joined together.
[0063] In yet another embodiment, there is a laceration of the
tendon.
[0064] In yet another embodiment, the edges of the laceration of
the tendon are joined together.
[0065] In yet another embodiment, the tendon is joined to a tendon
graft.
[0066] In yet another embodiment, the musculoskeletal tissue is a
ligament.
[0067] In yet another embodiment, the ligament is a small
ligament.
[0068] In yet another embodiment, the ligament is the anterior
cruciate ligament.
[0069] In yet another embodiment, the photosensitizer agent is Rose
Bengal.
[0070] In yet another embodiment, the Rose Bengal is applied to the
musculoskeletal tissue at a concentration of less than 1.0% weight
per volume in phosphate buffered saline. In another preferred
embodiment, the Rose Bengal is applied at a concentration of less
than 0.5% weight per volume in phosphate buffered saline. In
another preferred embodiment, the Rose Bengal is applied at a
concentration between 0.1 and 0.5% weight per volume in phosphate
buffered saline.
[0071] In yet another embodiment, the electromagnetic energy
applied to the musculoskeletal tissue is greater than 750/cm.sup.2.
In another preferred embodiment, the electromagnetic energy applied
is between 50 and 750/cm.sup.2. In another preferred embodiment,
the electromagnetic energy applied is between 250 and 750/cm.sup.2.
In yet another preferred embodiment, the electromagnetic energy
applied is between 250 and 500/cm.sup.2.
[0072] In yet another embodiment, the electromagnetic energy is
applied at an irradiance less than 1.5 W/cm.sup.2.
[0073] In yet another embodiment, the electromagnetic energy is
applied at an irradiance less than 1.0 W/cm.sup.2.
[0074] In yet another embodiment, the electromagnetic energy is
applied at an irradiance of between 0.5 and 1.0 W/cm.sup.2.
[0075] In yet another embodiment, immediately after joining, the
photochemically joined tendon has an ultimate stress, which is
greater than 10% of that of a healthy tendon. In a preferred
embodiment, immediately after joining, the photochemically joined
tendon has an ultimate stress, which is greater than 25% of that of
a healthy tendon. In a preferred embodiment, immediately after
joining, the photochemically joined tendon has an ultimate stress
which is greater than 50% of that of a healthy tendon.
[0076] In yet another embodiment, immediately after joining, the
photochemically joined tendon has a stiffness which is greater than
10% of that of a healthy tendon. In a preferred embodiment,
immediately after joining, the photochemically joined tendon has a
stiffness which is greater than 25% of that of a healthy tendon. In
a preferred embodiment, immediately after joining, the
photochemically joined tendon has a stiffness which is greater than
50% of that of a healthy tendon.
[0077] In another embodiment, the present invention provides
methods for adhering neural tissue, comprising contacting a first
neural tissue and second neural tissue, and at least one
photosensitizer agent to form a tissue-photosensitizer complex; and
applying electromagnetic energy to the tissue-photosensitizer
complex in a manner effective to bond the tissues, thereby creating
a tissue seal between neural tissues.
[0078] In another embodiment, the present invention provides a
method for adhering neural tissue, comprising placing a first
neural tissue and second neural tissue in a conduit; contacting the
first neural tissue and second neural tissue with at least one
photosensitizer agent to form a tissue-photosensitizer complex; and
applying electromagnetic energy to the tissue-photosensitizer
complex in a manner effective to bond the tissues, thereby creating
a tissue seal between the neural tissues and the conduit.
[0079] In another embodiment, the present invention provides a
method for adhering neural tissue, comprising retracting the
epineural tissue of a first neural segment and exposing the
endoneurium; removing the exposed endoneurium; contacting the first
and a second neural segment with at least one photosensitizer agent
to form a tissue-photosensitizer complex; joining the second neural
segment with the first neural segment and applying at least one
suture between the segments; and applying electromagnetic energy to
the tissue-photosensitizer complex in a manner effective to bond
the tissues, thereby creating a tissue seal between the neural
segments. Preferably, the first neural segment is distal and the
second neural segment is proximal to the spinal column.
[0080] In another embodiment, the present invention provides a
method for adhering blood vessel tissue, comprising placing a first
blood vessel tissue and second blood vessel tissue in a conduit;
contacting the first blood vessel tissue and second blood vessel
tissue with at least one photosensitizer agent to form a
tissue-photosensitizer complex; and applying electromagnetic energy
to the tissue-photosensitizer complex in a manner effective to bond
the tissues, thereby creating a tissue seal between the blood
vessel tissues and the conduit.
[0081] In another embodiment, the present invention provides a
method for adhering blood vessel tissue, comprising retracting a
tissue layer of a distal vessel segment; contacting the distal and
a proximal vessel segment with at least one photosensitizer agent
to form a tissue-photosensitizer complex; joining the proximal
vessel segment with the distal vessel segment and applying at least
one suture between the segments; and applying electromagnetic
energy to the tissue-photosensitizer complex in a manner effective
to bond the tissues, thereby creating a tissue seal between the
neural segments.
[0082] Other features, objects, and advantages of the invention
will be apparent from the description and drawings, and from the
claims, and are part of the invention.
DESCRIPTION OF THE DRAWINGS
[0083] FIG. 1 shows a graph of a typical trace of increasing IOP
with infusion time for a PTB treated eye, showing IOPL at 300 mm
Hg.
[0084] FIG. 2 shows a graph of the mean IOPL values for PTB treated
eyes (n=5) using 514 nm light (2.55 W/cm.sup.2) and Rose Bengal
(1.5 mM) in PBS. Additional controls are incisions treated with
Rose Bengal or buffer but no laser light.
[0085] FIG. 3 shows graphs of mean IOPL before and after PTB using
Rose Bengal and 514 nm irradiation. Rose Bengal (10 .mu.l, 1.5 mM)
was applied to the incision surfaces then treated with the doses
indicated using irradiances of: (A) 1.27 W/cm.sup.2, (B) 2.55
W//cm.sup.2 and (C) 3.82 W/cm.sup.2.
[0086] FIG. 4 shows graphs of mean IOPL before and after PTB using
R-5-P and 488 nm irradiation. R-5-P (40 .mu.l, 11 mM) was applied
to the incision surfaces then treated with the doses indicated
using irradiances of: (A) 1.27 W//cm.sup.2, (B) 2.55 W/cm.sup.2 and
(C) 3.82 W//cm.sup.2.
[0087] FIG. 5 shows graphs of mean IOPL values before and after PTB
using F1 and 488 nm irradiation. F1 (40 .mu.l, 0.6 mM) was applied
to the incision surfaces then treated with the doses indicated
using irradiances of: (A) 1.27 W/cm.sup.2, (B) 2.55 W/cm.sup.2 and
(C) 3.82 W/cm.sup.2.
[0088] FIG. 6 shows a tensiometer coupled to a force transducer.
Force was applied along the direction of the skin grafts at a
constant speed of 12.7 mm/min by pulling on the pre-attached suture
loop.
[0089] FIG. 7 shows a graph of the average penetration depth of
rose bengal based on the association between dermal uptake and time
of Rose Bengal exposure. An increase in exposure time to 10 minutes
increased the depth of dermal uptake to approximately 25
microns.
[0090] FIG. 8 shows a graph of the effect of Rose Bengal at various
concentrations on laser-induced skin graft adherence. Use of Rose
Bengal at a concentration of 0.1% (w/v) provided an optimal
increase in adherence levels.
[0091] FIG. 9 shows a graph of the skin adherence after PTB at 0.56
(FIG. 9A) and 1.68 W/cm.sup.2 (FIG. 9B). Irradiation levels of 0.56
and 1.68 W/cm.sup.2 provided a positive dose-dependent relationship
between the laser energy (fluence) and the adherence of the skin
grafts.
[0092] FIG. 10 shows cell viability and collagen organization.
Following irradiation levels of both 0 (control) and 0.56
W/cm.sup.2, skin grafts were viable, as indicated by
NADH-diaphorase activity, shown as the dark blue precipitates in
the cytoplasm contrasted by the red nuclear counterstain.
[0093] FIG. 11 shows cell viability after PTB of skin grafts at an
irradiance of 0.56 W/cm.sup.2 and various fluences.
[0094] FIG. 12 shows typical thermographs of the focused edge of a
cross-section of the skin graft during irradiation at 0.56 (FIG.
12A) and 1.68 W/cm.sup.2 (FIG. 12B). The horizontal bar represents
a distance of 0.1 inch (2.54 mm). The maximum temperature is
represented by a color scale at the left-hand side of the
thermograph.
[0095] FIG. 13 shows a scatter plot of the maximal surface
temperature of skin grafts during photochemical tissue bonding at
0.56 W/cm.sup.2 and 1.68 W/cm.sup.2, and up to a fluence of
504/cm.sup.2.
[0096] FIG. 14 shows a schematic diagram of photochemical tissue
bonding of an esophagus implant to a host muscle flap.
[0097] FIG. 15 shows the mean adherence of collagen gel on muscle
flap of the control, Rose Bengal control, Laser control and the PTB
treated groups.
[0098] FIG. 16 shows the viability of esophageal keratinocytes
after photochemical tissue bonding treatment.
[0099] FIG. 17 shows a strip of tendon attached to the fixture of
the tensiometer. Cyclic loading of the tendon was conducted by
stretching the tendon at a magnitude of 0.5 mm for 6 cycles, before
tendon was pulled to failure at a constant speed of 100 mm/min.
[0100] FIG. 18 shows a typical force-deflection curve obtained by
measurements from the tensiometer.
[0101] FIGS. 19A and 19B show a graph of the ultimate stress and
stiffness in ruptured tendons treated with laser only, Rose Bengal
only, laser and Rose Bengal or no treatment. Combinatory use of
visible light (argon laser) and Rose Bengal significantly increased
the ultimate stress and the stiffness.
[0102] FIGS. 20A and 20B show graphs of the ultimate stress and
stiffness in ruptured tendons at a fixed fluence dosage of
500/cm.sup.2, while increasing the laser power density. Ultimate
stress was significantly increased, but stiffness was not.
[0103] FIGS. 21A and 21B show a graph of the ultimate stress and
stiffness in ruptured tendons at increasing Rose Bengal
concentrations.
[0104] FIGS. 22A and 22B show the ultimate stress and the stiffness
following in vivo PTB treatment compared to the controls.
[0105] FIGS. 23A and 23B show an increase in the ultimate stress
(p=0.034) but not the stiffness (p=0.088) of the ruptured tendon in
vivo following irradiance at a fixed fluence dosage of 500
J/cm.sup.2.
[0106] FIGS. 24A and 24B show the statistically significant
dose-response in vivo in both the ultimate stress (p=0.002) and the
stiffness (p=0.004) of the photochemically bonded tendon.
[0107] FIG. 25 shows a schematic illustration of the procedure for
the formation of epineurial cuffs, as described in Example 14.
[0108] FIG. 26A shows a photograph of a sciatic nerve following
epineurial cuff and PTB treatment.
[0109] FIG. 26B shows a graph of nerve repair data, showing the
effects of epineurial cuff formation, and epineurial cuffs in
conjunction with PTB. Nerve repair was assayed using the
gastrocnemius muscle mass assay.
[0110] FIG. 27 shows a graph of total light transmittance and
diffuse reflectance of the NeuraGen.TM. collagen conduit.
Wavelengths of light ranging from 350 to 750 nm were tested
revealing greater than 50% transmittance at ideal wavelength of 532
nm, and minimal diffuse reflectance.
[0111] FIG. 28 shows a graph illustrating the tensile strength of
the bond formed between NeuraGen.TM. collagen conduit and bovine
tendon over a 0.25 cm.sup.2 surface area. The top line (marked with
squares) indicates specimens coated with Rose Bengal dye prior to
laser irradiation, whereas the lower line (marked with circles)
indicates laser irradiation alone, without dye treatment.
[0112] FIG. 29 shows a schematic illustration of the procedure for
the formation of epineural conduits, as described in Example
16.
[0113] FIG. 30 shows a schematic representation of the steps
involved using the "cuff" approach in PTB-mediated anastomosis of
blood vessels.
[0114] FIG. 31 shows a schematic representation of the steps
involved using the "conduit" approach in PTB-mediated anastomosis
of blood vessels.
[0115] FIG. 32 shows a graph illustrating the force necessary to
rupture anastomosed porcine arteries as a function of time.
[0116] FIG. 33 shows a graph illustrating the force necessary to
rupture anastomosed porcine veins as a function of time.
DETAILED DESCRIPTION
[0117] Photochemical tissue bonding (PTB), as described herein,
provides a method to create a tissue-tissue seal, e.g., to treat a
wound, e.g., a corneal wound, without collagen denaturation or
heat-induced peripheral tissue damage. PTB, as described herein,
involves the application of a photosensitizer to a wound surface
followed by photoactivation by laser irradiation to seal the wound.
The photosensitizer can be effectively applied to seal a wound, or
otherwise repair a tissue, such as by graft, in the absence of an
exogenous protein-based adhesive, such as fibrinogen.
[0118] Photochemical tissue bonding has the advantage of producing
covalent crosslinks that are theoretically stronger than the
non-covalent interactions created by the thermal welding technique,
such as those disclosed in U.S. Pat. Nos. 5,292,362 and 5,209,776.
Photochemical tissue bonding provides for an immediate return of
strength to the injured area in a manner that has considerably less
associated tissue damage (Bass & Treat (1995) Lasers Surg Med
17: 315-49; Judy et al. (1993) SPIE Proc 1882: 221-4).
[0119] Methods of the invention provide strong covalent bonding at
the tissue to tissue interface and have no requirement for an
exogenous protein (other than what may be present in a tissue
graft), e.g., fibrinogen, that must be isolated from the patient to
be treated or derived from one or more donors. Methods of the
invention do not require the use of chemical glues, e.g.,
cyanoacrylate adhesives. The methods described herein minimize
tissue thermal denaturation of proteins caused by tissue
heating.
[0120] Current methods of tissue grafting are complicated by
multiple use of sutures, low cosmetic value, wound complications
such as foreign body reactions, void and non-adherent grafts. The
present invention overcomes problems known in the art. The methods
of tissue adhesion described herein are ideal for tissues in need
of repair and/or a water-tight seal. These tissues can be of any
type where tissue adhesion such as wound closure is necessary, for
example a cardiovascular, neurological, gastrointestinal,
urological, renal, occular, oral, connective, respiratory,
otolaryngological, dermatological, genital, gynecological or
musculoskeletal tissue. Wound closure can comprise the joining of
cut or otherwise separated edges or surfaces of the damaged tissue.
Wound closure can further comprise the grafting of an exogenous
tissue on to the surface of a damaged tissue. Preferably, this
tissue is skin.
[0121] Closure of corneal wounds or corneal transplants with
sutures can be associated with neo-vascularisation, rejection of
the donor cornea, and induced post-operative astigmatism partly due
to uneven suture tension. This can occur after penetrating
keratoplasty where numerous sutures are needed to hold the graft in
place. Suturing techniques designed to evenly distribute tension
across corneal grafts may still result in significant astigmatism.
Additionally, loose or broken sutures can leave a patient
vulnerable to microbial keratitis. The sutures used are skill
intensive and are mainly performed by corneal specialists. The
methods described herein minimize the use of sutures. Although
factors such as wound healing, host graft sizing and trephination
techniques also play a role in post-operative astigmatism, the
methods described herein hold the graft with equally distributed
force and help reduce post-operative astigmatism. PTB reduces the
operating and rehabilitation time for procedures to close wounds,
e.g., to treat incisions or corneal lacerations, spot seal LASIK
flaps, perform cataract surgery, and attach donor cornea.
[0122] Surgical repair of musculoskeletal tissue, such as ruptured
tendons and ligaments, is currently plagued by complications
resulting from the use of multiple sutures and staples. The use of
these can lead to infection and necrosis, thereby negating the
potential benefits of the reparative surgery. Other complications
include recurrent rupture, skin adhesions and excessive scarring.
In addition, it is preferable that patients who undergo surgery
experience and immediate partial regaining of strength after the
repair, due the large stress placed on during the period of
recovery following surgery, The methods described in do not require
the use of sutures or staples, thereby eliminating the possibility
of foreign body reactions to them. In addition, the methods
described herein result in an immediate increase in the mechanical
properties of the tendon following the procedure, reducing the
possibility of complications including recurrent rupture, and
enhancing the subsequent healing process in tendons.
[0123] Photoactivation and Photosensitizers
[0124] The methods to create a tissue-tissue seal described herein
include treating a tissue with a photosensitizer agent, e.g., RB,
R-5-P, MB, or N-HTP, preferably in the absence of an exogenous
protein, e.g., a protein based adhesive, e.g., fibrin or
fibrinogen, and photoactivating the photosensitizer agent with
electromagnetic radiation, e.g., light.
[0125] Photoactivation is used to describe the process by which
energy in the form of electromagnetic radiation is absorbed by a
compound, e.g., a photosensitizer, thus "exciting" the compound,
which then becomes capable of converting the energy to another form
of energy, preferably chemical energy. The electromagnetic
radiation can include energy, e.g., light, having a wavelength in
the visible range or portion of the electromagnetic spectrum, or
the ultra violet and infra red regions of the spectrum. The
chemical energy can be in the foam of reactive species, e.g., a
singlet oxygen, superoxide anion, hydroxyl radical, the excited
state of the photosensitizer, photosensitizer free radical or
substrate free radical species. The photoactivation process
described herein preferably involves insubstantial transfer of the
absorbed energy into heat energy. Preferably, photoactivation
occurs with a rise in temperature of less than 15.degree. Celsius
(C), more preferably a rise of less than 10.degree. C., more
preferably a rise of less than 3.degree. C., more preferably a rise
of less than 2.degree. C. and even more preferably, a rise in
temperature of less than 1 degree C. as measured, e.g., by an
imaging thermal camera that looks at the tissue during irradiation.
The camera can be focused in the area of original dye deposit,
e.g., the wound area, or on an area immediately adjacent the wound
area, to which dye will diffuse. As used herein, a
"photosensitizer" is a chemical compound that produces a biological
effect upon photoactivation or a biological precursor of a compound
that produces a biological effect upon photoactivation. Preferred
photosensitizers are those that absorb electromagnetic energy, such
as light. While not wishing to be bound by theory, the
photosensitizer may act by producing an excited photosensitizer or
derived species that interacts with tissue, e.g., collagenous
tissue, to form a bond, e.g., a covalent bond or crosslink.
Photosensitizers typically have chemical structures that include
multiple conjugated rings that allow for light absorption and
photoactivation. Examples of photosensitive compounds include
various light-sensitive dyes and biological molecules such as, for
example, Photofrin.RTM., synthetic diporphyrins and dichlorins,
phthalocyanines with or without metal substituents, chloroaluminum
phthalocyanine with or without varying substituents, O-substituted
tetraphenyl porphyrins, 3,1-meso tetrakis (o-propionamido phenyl)
porphyrin, verdins, purpurins, tin and zinc derivatives of
octaethylpurpurin, etiopurpurin, hydroporphyrins, bacteriochlorins
of the tetra(hydroxyphenyl) porphyrin series (e.g., protoporphyrin
I through protoporphyrin IX, coproporphyrins, uroporphyrins,
mesoporphyrins, hematoporphyrins and sapphyrins), chlorins, chlorin
e6, mono-1-aspartyl derivative of chlorin e6, di-1-aspartyl
derivative of chlorin e6, tin(IV) chlorin e6,
meta-tetrahydroxphenylchlorin, benzoporphyrin derivatives,
benzoporphyrin monoacid derivatives, tetracyanoethylene adducts of
benzoporphyrin, dimethyl acetylenedicarboxylate adducts of
benzoporphyrin, Diels-Adler adducts, monoacid ring "a" derivative
of benzoporphyrin, sulfonated aluminum PC, sulfonated AlPc,
disulfonated, tetrasulfonated derivative, sulfonated aluminum
naphthalocyanines, naphthalocyanines with or without metal
substituents and with or without varying substituents,
chlorophylis, bacteriochlorophyll A, anthracenediones,
anthrapyrazoles, aminoanthraquinone, phenoxazine dyes, thiazines,
methylene blue, phenothiazine derivatives, chalcogenapyrylium dyes,
cationic selena and tellurapyrylium derivatives, ring-substituted
cationic PC, pheophorbide derivative, naturally occurring
porphyrins, hematoporphyrin, ALA-induced protoporphyrin IX,
endogenous metabolic precursors, 5-aminolevulinic acid,
benzonaphthoporphyrazines, cationic imminium salts, tetracyclines,
lutetium texaphyrin, texaphyrin, tin-etio-purpurin, porphycenes,
benzophenothiazinium, xanthenes, rose bengal, eosin, erythrosin,
cyanines, merocyanine 540, selenium substitued cyanines, flavins,
riboflavin, proflavin, quinones, anthraquinones, benzoquinones,
naphthaldiimides, naphthalimides, victoria blue, toluidine blue,
dianthroquinones (e.g., hypericin), fullerenes, rhodamines and
photosensitive derivatives thereof.
[0126] Preferred photosensitizers for use in the methods described
herein are compounds capable of causing a photochemical reaction
capable of producing a reactive intermediate when exposed to light,
and which do not release a substantial amount of heat energy.
Preferred photosensitizers are also water soluble. Preferred
photosensitizers include Rose Bengal (RB); riboflavin-S-phosphate
(R-5-P); methylene blue (MB); and N-hydroxypyridine-2-(1H)-thione
(N-HTP).
[0127] Without wanting to be bound by theory, it is believed that
the chemical energy, e.g., a reactive oxygen species, produced by
photoactivation of the photosensitizer agent with which the tissue
to be repaired is contacted, binds and causes structural changes in
the amino acids of the proteins of the tissue, resulting in the
formation of covalent bonds, polymerization, or cross-links between
amino acids of the tissue, thus creating a proteinaceous framework
that serves to seal, repair, heal, or close the tissue lesion or
wound. For example, as a result of PTB treatment, strong covalent
cross-links are believed to form between collagen molecules on
opposing surfaces of a corneal lesion to produce a tight tissue
seal.
[0128] The photosensitizer agent, e.g., RB, R-5-P, MB, or N-HTP,
can be dissolved in a biocompatible buffer or solution, e.g.,
saline solution, and used at a concentration of from about 0.1 mM
to 10 mM, preferably from about 0.5 mM to 5 mM, more preferably
from about 1 mM to 3 mM.
[0129] The photosensitizer agent can be administered to the tissue
by, e.g., injection into the tissue, or application onto the
surface of the tissue. An amount of photosensitizer sufficient to
stain, e.g., to cover the walls of, the lesion or wound to be
repaired, can be applied. For example, at least 10.mu.l of
photosensitizer solution, preferably 50.mu.l (microliter),
100.mu.l, 250.mu.l, 500.mu.l, or 1 ml, or more, of photosensitizer
solution can be applied to a tissue, e.g., a cornea. Preferably,
the photosensitizer has a binding efficiency, e.g., a collagen
binding efficiency, such that the dye is predominantly bound to the
surface of the incision.
[0130] The electromagnetic radiation, e.g., light, is applied to
the tissue at an appropriate wavelength, energy, and duration, to
cause the photosensitizer to undergo a reaction to affect the
structure of the amino acids in the tissue, e.g., to cross-link a
tissue protein, thereby creating a tissue seal. The wavelength of
light can be chosen so that it corresponds to or encompasses the
absorption of the photosensitizer, and reaches the area of the
tissue that has been contacted with the photosensitizer, e.g.,
penetrates into the region where the photosensitizer presents.
Preferably, the electromagnetic energy applied is less than 2000
J/cm.sup.2. Even more preferably, the electromagnetic energy
applied is between 100 and 500 J/cm.sup.2. The electromagnetic
radiation, e.g., light, necessary to achieve photoactivation of the
photosensitizer agent can have a wavelength from about 350 nm to
about 800 nm, preferably from about 400 to 700 nm and can be within
the visible, infrared or near ultraviolet spectra. The energy can
be delivered at an irradiance of about between 0.1 and 5
W/cm.sup.2, preferably between about 0.5 and 2 W/cm.sup.2. The
duration of irradiation can be sufficient to allow cross-linking of
one or more proteins of the tissue, e.g., of a tissue collagen. For
example, in corneal tissue, the duration of irradiation can be from
about 30 seconds to 30 minutes, preferably from about 1 to 5
minutes. The duration of irradiation to deliver the required dose
to a skin or tendon wound can be from about one minute to 60
minutes, preferably between 1 and 15 minutes. The duration of
irradiation can be substantially longer where power is lower.
[0131] Suitable sources of electromagnetic energy include
commercially available lasers, lamps, light emitting diodes, or
other sources of electromagnetic radiation. Light radiation can be
supplied in the faun of a monochromatic laser beam, e.g., an argon
laser beam or diode-pumped solid state laser beam. Light can also
be supplied to a non-external surface tissue through an optical
fiber device, e.g., the light can be delivered by optical fibers
threaded through a small gauge hypodermic needle or an arthroscope.
Light can also be transmitted by percutaneous instrumentation using
optical fibers or cannulated waveguides.
[0132] The choice of energy source will generally be made in
conjunction with the choice of photosensitizer employed in the
method. For example, an argon laser is a preferred energy source
suitable for use with RB or R-5-P because these dyes are optimally
excited at wavelengths corresponding to the wavelength of the
radiation emitted by the argon laser. Other suitable combinations
of lasers and photosensitizers will be known to those of skill in
the art. Tunable dye lasers can also be used with the methods
described herein.
[0133] Uses
[0134] The methods described herein are suitable for use in a
variety of applications, including in vitro laboratory
applications, ex vivo tissue treatments, but especially in in vivo
surgical procedures on living subjects, e.g., humans, and
non-surgical wound healing.
[0135] The methods described herein are particularly useful for
surgical applications, e.g., to seal, close, or otherwise join, two
or more portions of tissue, e.g., to perform a tissue transplant
and/or grafting operation, or to heal damaged tissue, e.g., a
corneal incision, or to prevent leakage from tissue. The methods
described herein can be used in surgical applications where precise
adhesion is necessary, and/or where the application of sutures,
staples, or protein sealants is inconvenient or undesirable. For
example, in corneal transplants and other eye operations, surgical
complications such as inflammation, irritation, infection, wound
gap, leakage, and epithelial ingrowth, often arise from the use of
sutures. The photochemical tissue bonding methods described herein
are particularly suitable for use in surgery or microsurgery, for
example, in surgical operations or maneuvers of the eye, e.g., in
the repair of corneal wounds or incisions, in refractive surgery
(the correction of irregularities or defects in the cornea by
"shaving" an even layer off the cornea), in keratoplasty, in
corneal transplants, and in correction of astigmatism, e.g., by
inducing astigmatism designed to counteract preexisting
astigmatism, e.g., in the orthogonal meridian.
[0136] As another example, sutures cannot be satisfactorily used on
bone joint cartilage because of their mechanical interference with
the mutual sliding of cartilage surfaces required for joint motion.
Neither can sutures be used to seal surfaces of small blood vessels
with diameters 1-2 min or less, as sutures impinge upon the vessel
lumen, compromising blood flow. Further, in skin grafting, sutures
can induce foreign body responses that lead to scarring and
therefore reduce cosmetic value. Thus, the methods described herein
are also useful in surgical interventions of vascular tissue, joint
cartilage, skin, gastrointestinal tract, nerve sheaths, urological
tissue, small ducts (urethra, ureter, bile ducts, thoracic duct),
oral tissue or even tissues of the middle or inner ear. Other
procedures where sutures or staples are not indicated or desirable,
and where the photochemical tissue bonding methods described herein
are useful, include procedures involving laparoscopic operations or
interventions such as laparoscopic (LP) thoracic procedures, LP
appendectomy, LP hernia repairs, LP tubal ligations and LP orbital
surgeries.
[0137] Photochemical tissue bonding methods as described herein are
optimal for the repair of musculoskeletal tissues such as tendons,
ligaments, extracellular matrix and cartilage. For example, these
methods are particularly suitable for repair of lacerations or
ruptures of tendons such that the healing of the tendon in the
patient may benefit from an immediate recovery in the strength of
the injured site following repair, and such that the recovery is
not hindered by infection of foreign-body reactions that may occur
following the use of multiple staples or sutures. In addition, use
of these methods may reduce the surgery time, may help prevent a
future recurrent rupture of the site, and may reduce
hospitalization and immobilization time during the rehabilitation
period. Photochemical tissue bonding methods as described herein
are optimal for use in sports medicine.
[0138] The photochemical tissue bonding methods described herein
can also be used in tissue grafting. Exogenous grafts can be, for
example, autografts, allografts or xenografts. In one embodiment,
an exogenous tissue graft comprising tissue such as skin, muscle,
vasculature, stomach, esophagus, colon or intestine, can be placed
over the surface of the wound, impregnated with the photosensitizer
agent described herein, and photoactivated with a visible light
source, e.g., an incandescent, fluorescent or mercury vapor light
source, e.g., a xenon arc lamp, or a laser light source, e.g.
argon-ion laser. Preferably, the photochemical bond enables rapid
and sustained adherence of the graft to the wound surface and the
ability to resist shear stress. Sources of grafted tissue can be
any known in the art, including exogenous grafts obtained from
non-injured tissues in a subject. Sources of grafted tissue can
also comprise extracellular matrix-based scaffolds, such as
collagen and proteoglycan, and/or other engineered tissue
implants.
[0139] Exogenous grafts can likewise be synthetic, e.g. skin
substitutes. Synthetic materials suitable for use in grafting
include, but are not limited to, silicon, polyurethane, polyvinyl
and nylon. Skin substitutes can be any known in the art, including
those comprising culture derivatives and cellular or acellular
collagen membranes. Culture derived substitutes give rise to
bilayer human tissue, for example Apligraf.TM. comprises epidermal
or dermal analogs derived from neonatal foreskin, the host-graft
composite of which will become repopulated with cells from the host
subject.
[0140] Commercially available skin substitutes include Biobrane,
composed of silicon, nylon and collagen, TransCyte.TM., composed of
silicon, collagen, fibronectin and glycosaminoglycan, and
Integra.TM., composed of silicon, collagen and glycosaminoglycan.
Skin substitutes can be used in applications of permanent and
semi-permanent grafting. Preferably, Integra.TM. is used for
permanent grafting.
[0141] In grafting tissues, the surface of the graft is aligned to
the lesion site through a process known in the art as
"approximation." Approximation of the graft to the lesion site can
be carried out according to methods known in the art. For instance,
a graft can be placed on top of the lesion site and aligned so that
the dye-stained dermal sides are in close approximation. Molecular
contact between the graft and the lesion site is achieved by close
approximation, which can be performed through pressing and
smoothing the dermal-to-dermal composite with several layers of
tissue paper, which are then removed without disturbing the graft
interface. The approximated graft-lesion site composite is then
ready for irradiation.
[0142] The photochemical tissue bonding methods described herein
can also be used to supplement the use of sutures, e.g., to
reinforce sutured anastomosis. Sutures leave a tract behind which
can allow for leakage of fluids and organisms. The problem of
leakage is especially critical in vascular anastomoses or for any
anastomoses of a fluid-containing structure (aorta, ureter, GI
tract, eye, etc.) where the fluid or contents inside can leak out
through the suture hole. In one embodiment, a wound can be sutured
according to general procedures and then treated with the
photochemical tissue bonding methods described herein, thereby
making the healing wound water tight, and impermeable to
bacteria.
[0143] In addition, the methods described herein can be used in
non-surgical wound healing applications, e.g., a photochemical
adhesive can be used for wound healing in addition to, or in place
of, a conventional bandage, optionally in combination with another
beneficial material for wound healing In one embodiment, a
biocompatible substrate, e.g., a conventional bandage material,
e.g., a strip of fiber, can be impregnated with the photosensitizer
agent described herein, applied to a wound, and photoactivated with
a visible light source, e.g., an incandescent, fluorescent or
mercury vapor light source, e.g., a xenon arc lamp, or a laser
light source. The photosensitizer-impregnated bandage can contain
another beneficial material for wound healing, e.g., an antibiotic.
In some embodiments, the photosensitizer-impregnated bandage,
and/or the light source, can be supplied to a subject in a kit,
e.g., a kit for use by a health care practitioner, or a kit for
household use, which kits can contain instructions for use. The
photochemical adhesive described herein can be left on the wound,
or can be replaced as necessary. Such an adhesive can be used ex
vivo, on a tissue removed from the body, or in situ on a subject,
e.g., a human subject. For example, a photochemical adhesive
described herein can be used as an "artificial skin" or covering
agent to cover large, oozing surfaces inside or outside the
body.
[0144] The methods described herein can also be used to cross-link
proteins for use in laboratory applications, e.g., to fix proteins
for microscopy; to immobilize antibodies or other protein reagents
to a substrate for diagnosis or purification; or to cross link
proteins or peptides to a solid matrix for use in chromatographic
or immunological applications.
[0145] In another embodiment, the present invention provides
methods for adhering neural tissue, comprising contacting a first
neural tissue and second neural tissue, and at least one
photosensitizer agent to form a tissue-photosensitizer complex; and
applying electromagnetic energy to the tissue-photosensitizer
complex in a manner effective to bond the tissues, thereby creating
a tissue seal between neural tissues.
[0146] The neural tissues can be any neural tissues in need of
repair. For example, the first and second neural tissues can
comprise two exposed nerve segments, or "stumps," at the proximal
and distal sides a nerve lesion. The neural tissues can comprise,
for example, epineurium, perineurium and dura. Preferably, the
neural tissues comprise epineurium.
[0147] The nerve lesion can be one that completely severs or
transects the nerve, or can be any other form of localized damage
to a nerve. Methods of the present invention are especially
suitable for shortened nerves (i.e., having severed and shortened
axonal segments). For shortened nerves, adhering neural tissue
comprising the epineurium allows the axonal segments to grow and
rejoin within the shell of the bonded epineurium. In a preferred
embodiment the neural tissue to be repaired comprises peripheral
nerves, such for example, the sciatic nerve.
[0148] The two neural tissues are treated with a suitable
photosensitizer agent. Any of the photosensitizer agents describe
herein can be used. In a preferred embodiment, the photosensitizer
agent is rose bengal. Either or both of the neural tissues can be
treated with the photosensitizer agent.
[0149] In a preferred embodiment, treatment of the neural tissues
with a photosensitizer agent is performed before the two neural
tissues are brought into close physical contact with one another.
In alternative embodiments, the photosensitizer agent is applied to
the two neural tissues at the same time, or after the two neural
tissues are brought into close physical contact with one
another.
[0150] In one embodiment, any suitable source, wavelength, and
energy of electromagnetic radiation can be used to illuminate the
tissue:photosensitizer complex, provided that it is sufficient to
activate the photosensitizer agent. Suitable illumination
parameters for various photosensitizer agents are provided herein.
Additionally, one of skill in the art could readily determine
suitable illumination parameters without undue experimentation.
[0151] In embodiments where rose bengal is used as the
photosensitizer agent, it is preferred that wavelengths in the
range of 350-750 nm are used, preferably 532 nm is used.
[0152] In another embodiment, the present invention provides a
method for adhering neural tissue, comprising placing a first
neural tissue and second neural tissue in a conduit; contacting the
first neural tissue and second neural tissue with at least one
photosensitizer agent to form a tissue-photosensitizer complex; and
applying electromagnetic energy to the tissue-photosensitizer
complex in a manner effective to bond the tissues, thereby creating
a tissue seal between the neural tissues and the conduit.
[0153] In another embodiment, the present invention provides a
method for adhering blood vessel tissue, comprising placing a first
blood vessel tissue and second blood vessel tissue in a conduit;
contacting the first blood vessel tissue and second blood vessel
tissue with at least one photosensitizer agent to form a
tissue-photosensitizer complex; and applying electromagnetic energy
to the tissue-photosensitizer complex in a manner effective to bond
the tissues, thereby creating a tissue seal between the blood
vessel tissues and the conduit.
[0154] A conduit of any suitable biocompatible material can be
used, such as collagen. The conduit is formed from a tissue
including, but not limited to, blood vessel, acellular muscle and
nerve.
[0155] The conduit can be formed from a polymer selected from the
group consisting of polylactic acid (PLA), poly-L-lactic acid
(PLLA), poly-D-lactic acid (PDLA), polyglycolide, polyglycolic acid
(PGA), polylactide-co-glycolide (PLGA), polydioxanone,
polygluconate, polylactic acid-polyethylene oxide copolymers,
modified cellulose, collagen, polyhydroxybutyrate,
polyhydroxpriopionic acid, polyphosphoester, polyalpha-hydroxy
acid), polycaprolactone, polycarbonates, polyamides,
polyanhydrides, polyamino acids, polyorthoesters, polyacetals,
polycyanoacrylates, degradable urethanes, aliphatic
polyesterspolyacrylates, polymethacrylate, acyl substituted
cellulose acetates, non-degradable polyurethanes, polystyrenes,
polyvinyl chloride, polyvinyl flouride, polyvinyl imidazole,
chlorosulphonated polyolifins, polyethylene oxide, polyvinyl
alcohol, teflon.RTM., nylon silicon, and shape memory materials,
such as poly(styrene-block-butadiene), polynorbomene, hydrogels,
metallic alloys, and oligo(.epsilon.-caprolacto-ne)diol as
switching segment/oligo(p-dioxyanone)diol as physical crosslink.
Other suitable polymers can be obtained by reference to The Polymer
Handbook, 3rd edition (Wiley, N.Y., 1989). The conduit can also be
formed from silicone.
[0156] Preferably, the conduit is a collagen conduit, and a
circumferential, watertight seal is created. Where neural tissue is
joined, it is preferable that the seal is effective to maintain the
intraneural neurotrophic environment.
[0157] In another embodiment, the present invention provides a
method for adhering neural tissue, comprising retracting the
epineural tissue of a first neural segment and exposing the
endoneurium; removing the exposed endoneurium; contacting the first
and a second neural segment with at least one photosensitizer agent
to form a tissue-photosensitizer complex; joining the second neural
segment with the first neural segment and applying at least one
suture between the segments; and applying electromagnetic energy to
the tissue-photosensitizer complex in a manner effective to bond
the tissues, thereby creating a tissue seal between the neural
segments.
[0158] This method is referred to herein as the "cuff" method, due
to the retracting of the outer layer into a "cuff." As used herein,
the term "retracting" refers to the folding back of at least one
tissue layer of a segment (i.e., nerve or vessel segment) such that
the inner surface of the layer becomes exposed.
[0159] Preferably, the first neural segment can be contacted with
at least one photosensitizer agent on the inner surface of the
retracted epineurium, and the second neural segment can be
contacted with at least one photosensitizer agent on the outer
surface of the epineurium prior to joining.
[0160] The second neural segment can be joined to the first neural
segment by placing the second neural segment within the epineurium
of the first neural segment. Preferably, the first neural segment
is distal and the second neural segment is proximal to the spinal
column. When joined in this orientation, the nerve fibers are most
effectively retained within the confines of the sealed neural
tissue.
[0161] The "cuff" method can be used to adhere blood vessels (e.g.,
an artery or a vein) as well. Thus, in another embodiment, the
present invention provides a method for adhering blood vessel
tissue, comprising retracting a tissue layer of a distal vessel
segment; contacting the distal and a proximal vessel segment with
at least one photosensitizer agent to form a tissue-photosensitizer
complex; joining the proximal vessel segment with the distal vessel
segment and applying at least one suture between the segments; and
applying electromagnetic energy to the tissue-photosensitizer
complex in a manner effective to bond the tissues, thereby creating
a tissue seal between the neural segments.
[0162] Proximal to distal is determined according to the direction
of blood flow in the vessel. The proximal vessel segment is
upstream and the distal vessel segment is downstream of the blood
flow. Preferably, the proximal vessel segment is joined to the
distal vessel segment by placing the proximal vessel segment within
the distal vessel segment. Prior to joining, the distal vessel
segment is corntacted with at least one photosensitizer agent on an
inner surface, and the proximal vessel segment is contacted with at
least one photosensitizer agent on an outer surface. Irradiation
can be provided externally, around the circumference of the vessel,
or internally, by illuminating the inner channel of the vessel
(e.g., illuminating the interior of the vessel such that the inner
surface is bonded).
[0163] Kits
[0164] The invention also includes kits for use in photochemical
tissue bonding. Such kits can be used for laboratory or for
clinical applications. Such kits include a photosensitizer agent,
e.g., a photosensitizer described herein, and instructions for
applying and irradiating the photosensitizer to cross-link at least
one protein reagent for laboratory use, or to bond, repair, or heal
an animal tissue, e.g., a human tissue, particularly in a human
patient. The kits can include a container for storage, e.g., a
light-protected and/or refrigerated container for storage of the
photosensitizer agent. A photosensitizer included in the kits can
be provided in various forms, e.g., in powdered, lyophilized,
crystal, or liquid form. Optionally, a kit can include an
additional agent for use in a tissue bonding, wound repair, or
ocular therapy application, e.g., an antibiotic or a contact
lens.
[0165] The kits described herein can also include a means to apply
the photosensitizer agent to a tissue, for example, a syringe or
syringe-like device, a dropper, a powder, an aerosol container,
sponge applicator, and/or a bandage material. Kits can further
include accessory tools for tissue approximation e.g. clips,
standard weights, aspiration apparatus, and compression gauges.
[0166] Kits can include instructions for use, e.g., instructions
for use in the absence of an exogenously supplied source of
cross-linkable substrate, e.g., protein, e.g., fibrin or
fibrinogen.
EXAMPLES
Example 1
Assessment of PTB in Repair of Corneal Incisions
[0167] PTB can be used to seal or repair a tissue, e.g., a wound,
e.g., a corneal wound. This example illustrates the experimental
procedure designed to test the efficacy of PTB, as described
herein, using mammalian corneas ex vivo. Experiments were performed
according to the following procedure.
[0168] Rabbit eyes were received on ice (Pel-Freez Biologicals)
approximately 17-24 hours after sacrifice and enucleation. The eyes
were kept on ice and used the same day. The eye to be studied was
mounted on a plastic-covered polystyrene block and fixed in
position by needles inserted through the extraocular muscles into
the polystyrene. The eye was then placed under a dissecting
microscope (Reichert Scientific Instruments, IL) allowing
visualization of the treated area during the entire procedure. A 27
G needle was inserted parallel to the iris, 2 mm anterior to the
limbus into clear cornea, and positioned above the lens in the
anterior chamber. The needle was connected to both a blood pressure
transducer (Harvard Apparatus, Mass.) and a mini-infuser 400 (Bard
Harvard) via a T coupler. The pressure transducer consists of a
transducer element that is hard wired to an amplifier box and uses
a semi-disposable dome with an integral silicone rubber membrane.
Pressure inside the dome is transmitted through the membrane to a
plastic button whose motion is translated to a voltage. The voltage
generated by the transducer amplifier combination is proportional
to the lower limit of intraocular pressure (IOP). Signals from the
transducer amplifier were recorded using a Macintosh G3 Power book
equipped with a PCMICA (DAQCARD--1200) data acquisition card
(National Instruments, TX). Data acquisition was controlled using
programs written using the LabView 4 software package (National
Instruments, TX). The voltage from the transducer and amplifier was
converted to pressure by calibrating with a standing manometer.
[0169] Experiments on individual eyes were initiated by increasing
the IOP to 30-40 mm Hg, using water infusion at a rate of 1 mL per
minute. An incision was made in the cornea, 1 mm from the limbus
and parallel to the iris, using a 3.5 mm angled keratome (Becton
Dickinson Co.). For each eye the IOP required to produce fluid
leakage from the incision (IOP.sub.L) was recorded pre- and
post-PTB treatment. A photosensitizer, dissolved in phosphate
buffer solution (PBS, pH 7.2, Gibco BRL) was applied to the walls
of the incision using a Gastight, 50.mu.l syringe (Hamilton Co.)
with a 27 G needle. Confocal fluorescence spectroscopy confirmed
the location of photosensitizer, e.g., rose Bengal, on the incision
walls and indicated that the photosensitizer penetrated only
approximately 100.mu.M laterally into the wall of the incision.
[0170] The photosensitizers, their absorption maxima, and their
absorption coefficients at the laser wavelength used in this
Example were, e.g., rose bengal (RB), 550 nm, 33000 dm.sup.3
mol.sup.-1 cm.sup.-1 at 514 nm; fluorescein (Fl), 490 nm, 88300
dm.sup.3 mol.sup.-1 cm.sup.-1 at 488 nm; methylene blue (MB), 664
nm, 15600 dm.sup.3 mol.sup.-1 cm.sup.-1 at 661 nm;
riboflavin-5-phosphate (R-5-P), 445 nm, 4330 dm.sup.3 mol.sup.-1
cm.sup.-1 at 488 nm; and N-hydroxypyridine-2-(1H)-thione (N-HPT),
314 nm, 2110 dm.sup.3 mol.sup.-1 cm.sup.-1 at 351 nm. The
photosensitizers were used as received with the exception of N-HPT
which was recrystallized twice from aqueous ethanol before use. The
concentrations of the photosensitizers were adjusted so that all
the solutions had an absorbance of approximately 1.0 in a path
length of 200.mu.m at the laser irradiation wavelength (with the
exception of N-HPT for which the absorption was approximately a
factor of 10 lower).
[0171] Irradiations employed a continuous wave (CW) argon-ion laser
(Innova 100; Coherent, Inc., Palo Alto, Calif.) at 488 nm (for Fl
and R-5-P), 514.5 nm (for RB) or 351 nm (for NHPT). An
argon-ion-pumped dye laser (CR-599; Coherent) with
4-dicyanomethylene-2-methyl-6-(p-dimethylam-inostyryl)-4H-pyran dye
(Exciton, Inc., Dayton, Ohio) was used for irradiation at 661 nm
(for MB). Laser light was coupled into a 1 mm diameter quartz fiber
and a 1 cm diameter spot on the tissue was created by using a
combination of 1 and 2 inch focal length, S1-UV grade fused silica,
biconvex lenses (Esco Products), mounted in a SM1 series cage
assembly (Thorlabs, N.J.). The 1 cm diameter circular spot was
sufficient to cover the entire incision and the optics were
adjusted so that the laser light was incident on the cornea at an
angle approximately 450 to the plane of the incision. Dose response
curves were obtained by varying the duration of the irradiation at
a constant irradiance. In separate experiments the effects of laser
irradiance were investigated by comparison of the same delivered
dose using different irradiances. The doses used ranged from 124 to
1524 J/cm.sup.2 and the irradiances used were 0.64, 1.27, 2.55 and
3.86 W/cm.sup.2. The laser exposure time varied from 33 seconds for
the lowest dose using the highest irradiance to 26 minutes, 27
seconds for the highest dose using the lowest irradiance. The
IOP.sub.L was recorded immediately following treatment. Infusion
was started (1 mL per minute) and the IOP increased until a maximum
followed by a sharp decrease occurred, corresponding to the opening
of the incision and leakage of fluid from the anterior chamber. A
typical trace showing the changes in IOP with infusion time is
shown in FIG. 1. Five to 10 rabbit eyes were tested for each
condition of dose and irradiance.
[0172] Control experiments included: (1) irradiation with no
photosensitizer application, (2) photosensitizer application only
and (3) no photosensitizer or laser irradiation. In the experiments
using no photosensitizer, PBS was applied to the incision walls,
using the same method as described for the photosensitizers. In
control experiments with no laser irradiation the eye was allowed
to stand for the same period of time as the laser-treated
samples.
Example 2
Use of Rose Bengal (RB) in PTB
[0173] In the cornea, RB can be used in PTB at a concentration of
about 0.5 mM to 5 mM, preferably about 1 mM to 3 mM. The wavelength
of irradiation for RB is preferably about 450-600 nm, more
preferably about 500 to 560 nm. The dose of irradiation can be from
about 0.5 to 2 kJ/cm.sup.2. The irradiance delivered can be from
about 0.2 to 3 W/cm.sup.2. The duration of irradiation is
preferably from about 1 to 10 minutes.
[0174] Treatment of incisions with 1.5 mM RB and 514 nm laser light
resulted in an increase in post-treatment IOP.sub.L, as measured as
described in Example 1. Control experiments demonstrated that a
significant increase (p<0.005) in the IOP.sub.L, following PTB
treatment, occurred when both RB and laser irradiation were applied
and not by either alone (FIG. 2). The mean IOP.sub.L of incisions
treated with RB and 514 nm laser light was greater than 300.+-0.48
mm Hg, whereas laser irradiation alone or photosensitizer alone
produced no significant increase between the pre- and
post-treatment IOP.sub.L values.
[0175] Dose response curves for IOP.sub.L are shown in FIG. 3 for
RB doses delivered at irradiances of 1.27 (3A), 2.55 (3B) and 3.82
W/cm.sup.2 (3C). A dose-response relationship was observed at the
lowest irradiance (1.27 W/cm) for doses between 508 and 1270
J/cm.sup.2 (3A). No significant rise in the IOP.sub.L was observed
for doses below 508 J/cm.sup.2 at any irradiance tested. PTB was
most efficient at 1270 J/cm.sup.2 delivered at an irradiance of
1.27 W/cm.sup.2. All doses delivered at the two lower irradiances
(1.27 and 2.55 W/cm.sup.2) gave IOP.sub.L values greater than 100
mm Hg. Treatment using irradiances of 2.55 and 3.82 W/cm.sup.2
produced no obvious dose response pattern. In general, for a
selected dose the IOP.sub.L was lower at higher irradiances. For
example, at 1270 J/cm.sup.2 the mean IOP.sub.L values were 274, 150
and 130 mm Hg for the irradiances 1.27 W/cm.sup.2, 2.55 W/cm.sup.2
and 3.86 W/cm.sup.2.
[0176] Post-treatment, the eyes were examined for the presence of
thermal damage. Tissue shrinkage and deformation around the wound
site were taken as signs of thermal damage. Thermal damage to the
cornea was not observed at the lowest irradia nce tested (1.27
W/cm.sup.2). Thermal damage could be observed at doses of 762 to
1524 J/cm.sup.2 at the highest irradiance (3.82 W/cm.sup.2) and
occasionally at 2.55 W/cm.sup.2. Thermal effects produced using
high irradiances may produce collagen contraction resulting in
distortion of the patient's vision.
Example 3
Use of Riboflavin-5-Phosphate (R-5-P) in PTB
[0177] In the cornea, R-5-P can be applied for PTB at a
concentration of about 1 mM to 30 mM, preferably about 10 mM to 20
mM. The wavelength of irradiation for R-5-P is preferably about
400-600 nm, more preferably about 450 to 550 nm. The dose of
irradiation can be from about 0.5 to 2 kJ/cm.sup.2. The irradiance
delivered can be from about 0.2 to 3 W/cm.sup.2. The duration of
irradiation is preferably from about 1 to 10 minutes.
[0178] The effect of R-5-P PTB was assessed as described in Example
1. The application of 11 mM R-5-P and irradiation using 488 nm
light, at the same irradiances used for RB, and doses of 762
J/cm.sup.2 and 1016 J/cm.sup.2, significantly increased the post
PTB treatment IOP.sub.L value (p<0.05), see FIG. 4. The
IOP.sub.L values observed using R-5-P are of a similar magnitude to
those for RB. However, the IOP.sub.L values observed for each dye
at the same irradiance and dose were not comparable. Although the
treatment produces significant increases in IOP.sub.L, no simple
pattern between the two dyes is observed.
Example 4
Use of N-hydroxypyridine-2-(1H)-thione (N-HTP) in PTB
[0179] In the cornea, N-HTP can be applied at a concentration of
about 0.5 mM to 10 mM, preferably about 3 mM to 6 mM. The
wavelength of irradiation for N-HTP is preferably about 330-400 nm.
The dose of irradiation can be from about 0.5 to 2 kJ/cm.sup.2. The
irradiance delivered can be from about 0.2 to 3 W/cm.sup.2. The
duration of irra/cm.sup.2diation is preferably from about 1 to 10
minutes. A 4.5 mM solution of NHPT was applied to the walls of the
incision, as described in Example 1, and irradiated using 351 nm
light (0.64 W/cm.sup.2) at doses ranging from 127 J/cm.sup.2 to 508
J/cm.sup.2. Mean IOP.sub.L values of 60.+-0.23 mm Hg and 126.+-0.40
mm Hg were produced when using the doses of 254 J/cm.sup.2 and 508
J/cm.sup.2 respectively, lower doses than used for the other
photosensitizers.
Example 5
Use of Methylene Blue (MB) in PTB
[0180] MB is a frequently used dye in ophthalmic surgery that has
been reported to photosensitize collagen cross-links in rat tail
tendon (Ramshaw et al. (1994) Biochim Biophys Acta 1206:225-230).
Our previous studies showed that MB and 355 nm light did not
produce efficient cross-linking of soluble collagen. MB was
therefore used as a control in these ex vivo studies. MB (3 mM) was
applied to the walls of the incision, as described in Example 1,
and irradiated with 0.64 W/cm.sup.2 of 661 nm light. Doses of
508/cm.sup.2, 762 J/cm.sup.2 and 1016 J/cm.sup.2 did not increase
the post-treatment, IOP.sub.L However, it was observed that MB did
not stain the corneal tissue efficiently, which perhaps explains
its low efficiency for PTB.
Example 6
Assessment of Thermal Contribution to PTB
[0181] Laser activated tissue welding has been studied in a variety
of tissues (Abergel et al. (1986) J Am Acad Dermatol. 14: 810-814;
Cilesiz et al., supra; Massicotte et al. (1998) Lasers in Surgery
and Medicine 23:18-24; Oz et al. (1990) J Vasc Surg. 11:718-725;
Poppas et al. (1996) Lasers in Surgery and Medicine 18:335-344;
Poppas et al. (1996) Lasers in Surgery and Medicine 19: 360-368;
Stewart et al. (1996) Lasers in Surgery and Medicinel 9:9-16; Wider
et al. (1991) Plastic Reconstr Surg 88:1018-1025). In tissue
welding, the laser radiation is used to heat the tissue to
temperatures at which collagen denatures and, upon cooling, the
collagen molecules intertwine to form a `weld`. Additionally,
dye-enhanced thermal welding has been investigated (Bass &
Treat (1995) Lasers Surg and Med 17: 315-349; Chuck et al. (1989)
Lasers Surg and Med 9:471-477). In this method the dye selectively
absorbs the laser energy and then releases heat to the desired
area, reducing peripheral tissue damage. These methods, however,
are not appropriate for the cornea due to the potential reduction
in visual acuity that would result from the corneal defonnation
produced by thermal tissue damage. When performing PTB on the
cornea, heating must be avoided.
[0182] We evaluated the possibility that non-photochemical
processes contribute to wound closure by comparing PTB produced by
RB with that produced by fluorescein (Fl), a dye with a similar
structure but which is not expected to induce protein cross-links.
RB and Fl are both xanthene dyes. However, RB is halogenated (4
iodines and 4 chlorines) and the presence of these heavy atoms
causes RB to be photochemically active (Lessing et al. (1982) J Mol
Struct 84:281-292). F1 has a high quantum yield of fluorescence and
lower quantum yield of triplet state formation than RB (Fleming et
al. (1977) JACS 99:4306-4311) and will, therefore, produce a lower
proportion of active species with the potential to produce collagen
cross-links. A solution of 0.6 mM F1 was applied and irradiated
using 488 nm laser light at the same range of irradiances used for
RB and at doses from 508 J/cm.sup.2 to 1016 J/cm.sup.2 (FIG. 5). No
increase in IOP.sub.L was observed for the incisions treated with
the two lowest doses using the two lowest irradiances studied.
However, at the highest dose for all irradiances an increase
IOP.sub.L values was observed with values ranging from 63.+-0.30 to
89.+-0.42 mm Hg although this is much less efficient than RB
(compare FIGS. 3 and 5). These results suggest that PTB is indeed
produced by photochemical processes. The IOP.sub.L value of
116.+-0.40 mm Hg obtained using a dose of 762 J/cm.sup.2 at 3.82
W/cm.sup.2 (laser exposure time of 3 min, 10 sec) is considerably
higher than any other observed using F1. The sealing observed at
the highest irradiance (3.82 W/cm.sup.2) and dose (762 J/cm.sup.2)
suggests that some other effect is operating, such as a thermal
mechanism under these high irradiance conditions.
Example 7
PTB Versus Sutures
[0183] The IOP.sub.L following PTB treatment, as described in
Example 1, was compared to that obtained using sutures. Two
interrupted radial sutures of black monofilament 10-0 nylon
(Ethilon Inc.) were used to close the keratome incision. The
sutures were placed in a radial fashion at approximately 90%
corneal depth. IOP.sub.L values with sutures were approximately 230
mm Hg. This value is similar for the incisions closed with PTB
treatment.
Example 8
In Vivo PTB
[0184] PTB was performed in vivo in New Zealand rabbits to repair
two types of corneal wounds.
[0185] In group 1,3,5-nun incisions were performed in 20 rabbit
(New Zealand White) corneas. Dose and laser irradiance were varied
in subgroups of five or more eyes for each condition and
appropriate control eyes. Photoactivation was performed with a 514
nm Argon Laser. Wound leak and incisional bursting pressure of the
treated and untreated rabbit eyes was determined in vivo, with the
animals under anesthetic.
[0186] Group I wounds were healed using, e.g., 191 J/cm.sup.2,
applying 1.5 mM RB. The immediate in vivo bursting pressure was
495.+-0.10 mm Hg for PTB treated eyes. Under the same conditions
the values of the busting pressure in the control eye varied from
15 to 60 mm Hg. One day after surgery, the bursting pressure was
the same for PTB treated eyes and control eyes (approximately
450.+-0.125 mmHg). At 14 days, the bursting pressure exceeded 500
mm Hg in both PTB-treated and control eyes.
[0187] In Group II, 6-mm Penetrating Keratoplasy (PK) incisions
anchored by 4-16 sutures were performed in 16 rabbit corneas. Half
of the corneas in each group underwent PTB where 1% Rose Bengal dye
was applied to the wound edges followed by laser irradiation at
fluence of 191 J/cm.sup.2. Photoactivation was performed with a 514
nm Argon Laser and a 532-nm CW Nd: YAG laser. Wound leak and
incisional bursting pressure were determined in vivo in the
immediate postoperative period. PTB-treated eyes showed an
immediate bursting pressure of 410.+-0.70 mm Hg for the PTB-treated
eyes, compared to 250.+-0.150 mm Hg for the control eyes with
sutures alone. This result indicates that PTB is useful and
effective as a supplement to sutures, as well as on its own.
[0188] The results described herein show that PTB is effective to
seal, close, or heal a tissue, e.g., a corneal incision, in vivo,
in a subject, e.g, an animal, or a human. The presence of a
protein, e.g., a protein based sealant, e.g., fibrinogen, is not
necessary to obtain a good tissue seal. PTB may be used instead of,
or in addition to, other wound healing techniques, e.g.,
sutures.
Example 9
Assessment of PTB in Adhesion of Skin Grafts
[0189] Skin grafts and/or skin substitutes are widely used in
surgical procedures such as skin transplantation, burn and ulcer
wound management and plastic surgery. The primary qualities of
successful skin grafts include rapid and sustained adherence to the
wound surface and the ability to resist shear stress in order to be
void-free and adherent.
[0190] To test the ability of PTB to quickly and effectively bond
skin grafts to a wound site, an ex vivo model utilizing mini pigs
was developed. The use of porcine models in wound healing is well
known by those skilled in the art. The similarities between porcine
wound healing and that of humans enable one to extrapolate the
therapeutic results obtained in a porcine model system to a
therapeutic result in humans.
[0191] Partial thickness skin grafts of approximately 0.020 inch
(corresponding to the thick partial-thickness grafts used in
clinical situation) were harvested from mini pigs (2 to 7 months
old, weight 15 to 43 kgs) after euthanasia. The grafts were
temporarily stored by wrapping the graft around gauze, which was
soaked in phosphate buffered saline (PBS). The grafts were then
immersed in vitrification fluid and cryo-preserved at -80.degree.
C. until needed (Fujita T et al. (2000) J Burn Care Rehabil 21:
304-9).
[0192] The skin grafts were freshly thawed on the day of the
experiment and were washed with PBS before being cut into either
square biopsies of 1 cm.sup.2 area or into round biopsies of 0.6 cm
diameter. The photosensitizer used was rose bengal (RB) (Sigma),
which has the absorption maximum and absorption coefficient of 550
nm and 33,000 dm3/mol/cm at 514 nm, respectively. The RB was
dissolved in PBS at concentrations of 0, 0.01, 0.1, and 1%
(weight/volume) and kept in darkness before being applied liberally
onto the dermal side of the skin grafts for time periods of 30 sec,
1, 2, 5, or 10 minutes, after which time the excess fluid was
removed by aspiration and blotting. The round graft was attached to
a suture loop while the square graft was secured onto a flat
surface with sutures to prepare for the adherence test. The round
layer was placed on top of the flat layer with the dye-stained
dermal sides in approximation. Excess dye and air at the interface
were removed by pressing and rubbing the graft surface over several
layers of paper tissue, which was then removed without disturbing
the graft interface.
[0193] The grafts were irradiated using a continuous-wave (CW)
argon-ion laser (Irmova 100; Coherent, Palo Alto, Calif.) at
514.5.mu.m. The approximated grafts were irradiated at a spot size
of 0.6 cm diameter, transmitted through a 1 mm diameter quartz
fiber. The irradiance of laser applied was 0.56 and 1.68 W/cm.sup.2
while the dose-dependent response of the laser fluence from 126 to
504 J/cm.sup.2 was determined. As a result, the exposure time
ranged from 2.5 to 15 minutes. During this time the skin grafts
were sprayed with PBS at regular intervals in order to prevent
dessication.
[0194] Following the irradiation, the adherence of the skin grafts
was tested utilizing a tensiometer (Chartillon) coupled to a force
transducer (DFA2, Ametek). The applied force versus the
displacement of the force plate was acquired through the transducer
and recorded by computer software (Labview 4.0, National
Instrument). A force was applied along the direction of the skin
grafts at a constant speed of 12.7 mm/min by pulling on the
pre-attached suture loop (FIG. 6). Force-deflection plots were
obtained, and the average force needed to separate the skin grafts
was calculated and normalized by the size of the upper (round)
graft (stress n/cm.sup.2) using KaleidaGragh software. This
measurement was used to compare the effects of the variations in
concentration of RB, dose-response to the change in fluence level
as well as differences between the two irradiance levels.
[0195] Frozen sections of tissue were analyzed using an eye-piece
grid on a light microscope to determine the correlation between the
time of exposure to RB and the distance of diffusion/penetration of
RB. Exposure times of 5 minutes or less resulted in a dermal
diffusion/penetration to a depth of approximately 10 microns. When
exposure time was increased to 10 minutes, the depth of dermal
diffusion/penetration increased to approximately 25 microns (FIG.
7). The depth of penetration of the dye will be minimized in order
to prevent photochemical damage and intrinsic toxicity of the dye
if any. Use of RB at a concentration of 0.1% (w/v) provided an
optimal increase in adherence levels (FIG. 8). In addition, both
irradiation levels (0.56 and 1.68 W/cm.sup.2) provided a positive
dose-dependent relationship between the laser energy (fluence) and
the adherence of the skin grafts (FIGS. 9a and 9b).
[0196] To test the viability of the cells after the irradiation,
nicotinamide adenine dinucleotide (NADH-diaphorase) staining was
carried out based on the methods described by Heisterkamp J et al.
(1999) Lasers Sug Med 25(3): 257-62 and Neumann R A et al. (1991) J
Am Acad Dermatol 25: 991-8. The skin grafts were exposed overnight
to an incubation solution consisting of nicotinamide adenine
dinuleotide (NADH) and nitroblue tetrazolium chloride (NBT)
(Sigma). A water-insoluble blue precipitate formed at the points
where NADH-diaphorase activity was present, indicating the
viability of the cell. The grafts were washed in PBS and prepared
in paraffin sections with a thickness of microns. The sections were
counterstained with nuclear fast red to show the cell nuclei.
Following irradiation levels of both 0 (control) and 0.56
W/cm.sup.2, the skin grafts and the cells were viable, as indicated
by the dark blue precipitates in the cytoplasm of the cells
contrasted by the red nuclear counterstain (FIGS. 10A and B).
However, signs of thermal damage such as less intensive dark blue
staining and disorganization of collagen bundles were found at high
irradiance (1.68 W/cm.sup.2) as indicated in FIG. 10C.
[0197] The cell viability in the skin grafts was further assessed
quantitatively by trypan blue exclusion assay. The upper grafts
were chosen to be examined because they absorbed more light, and
therefore would be damaged to a greater extent, if any, than the
lower ones. Following irradiation, the upper skin grafts were
incubated with 1 mg/ml dispase (Life Technologies, Rockville, Md.)
in PBS overnight at 4.degree. C. The epidermis was gently peeled
off from the dermis and washed twice in PBS. Epidermal cells were
released by incubating the epidermis with 0.25% trypsin-EDTA
(Cellgro) at 37.degree. C. for 15 minutes, accompanied by gentle
agitation. Fetal calf serum (FCS) was added to inhibit trypsin
activity and the cell suspension was then centrifuged at 2500 rpm
for 10 mins. The cell pellet was washed once and re-suspended in
PBS. The trypan blue exclusion assay was performed after mixing
equal volumes of cell suspension and 0.2% (w/v) trypan blue and
loading onto a hemocytometer. The number of viable cells released
from each skin graft was calculated. After preparing a single cell
suspension from intact epidermis, the cell viability was assessed
(FIG. 1). The data for the number of viable cells per skin graft in
different fluence groups is shown in FIG. 11. The number of viable
cells per skin graft after irradiation (0.1% RB) at 126, 252 and
504 J/cm.sup.2 was not significantly different from the control.
Although there was a slight reduction in the number of viable cells
in the 504 J/cm.sup.2 group to 80% of the control group, one-way
ANOVA showed no statistical significant difference among the groups
(p=0.899).
[0198] This example indicates that the utilization of low
concentration RB, e.g., 0.1% and non-thermal irradiation (e.g.,
between approximately 0.1 and 1 W/cm.sup.2) causes immediate skin
graft adherence which maintains graft viability. As a non-thermal
bonding method, this type of procedure minimizes or eliminates the
tissue damage caused by thermal bonding methods. The procedures can
be varied to incorporate various photosensitizers, appropriate
laser light sources, concentrations of RB and amounts of
irradiation. This type of treatment is beneficial in humans and
would advantageously eliminate the need for or minimize the use of
staples, sutures, glues, and other adhesives.
Example 10
Non-Thermal Adhesion of Skin Grafts
[0199] PTB advantageously proceeds by a non-thermal mechanism, as
demonstrated by the following results.
[0200] The maximal temperature of skin grafts was recorded by a
ThermaCAM infrared imaging radiometer (PM180, Inframetrics, Mass.).
The ThermaCAM was set approximately 30.degree. above the plane
perpendicular to the direction of laser irradiation. The focus was
set at one edge of the skin grafts before irradiation. Room
temperature at 23.degree. C. was set as the reference. Real time
thermal images of the cross-sectional view of the focused edge of
the skin grafts were captured at 30 second intervals throughout the
irradiation (15 minutes for 0.56 W/cm.sup.2 and 5 minutes for 1.68
W/cm.sup.2). Images were analyzed by ThermaGRAM 95 software
(Inframetrics, Mass.), which converted the values of the
color-scale to temperatures. Typical thermographs of the
cross-sectional view of the focused edge of the skin graft during
irradiation at both 0.56 (FIG. 12A) and 1.68 W/cm.sup.2 (FIG. 12B).
The color images were interpreted using the color scale on the
figure. The maximal temperature of the cross-sectional image of the
skin grafts during the course of photochemical tissue bonding was
recorded at 0.56 W/cm.sup.2 and 1.68 W/cm.sup.2, and up to 504
J/cm.sup.2 (FIG. 13). At 0.56 W/cm.sup.2, there was a gradual,
slight increase in temperature throughout the 15 minutes of
irradiation, from room temperature at 23.degree. C., to an average
of 31.2.degree. C. after 1 minute of irradiation and then to
39.9.degree. C. after 15 minutes of irradiation. By contrast, the
maximal skin temperature reached 48.5.degree. C. at 1 minute and
then increased up to 59.5.degree. C. by the end of a 5 minute
irradiation using an irradiance of 1.68 W/cm.sup.2.
[0201] The results demonstrate that PTB proceeds by a non-thermal
tissue bonding procedure. The average of the maximal temperature of
the skin grafts during the whole course of irradiation, 15 minutes,
ranges from 31.2 to 39.9.degree. C., which is far lower than the
optimal coagulation temperature for laser thermal welding,
65-75.degree. C. (Fung, L. C., et al., (1999) Lasers Surg Med 25:
285-290). This temperature change is expected to be even lower in
vivo due to the thermal regulation in highly perfused skin.
[0202] To reduce the irradiation time during PTB, the irradiance
was increased from 0.56 W/cm.sup.2 to 1.68 W/cm.sup.2 (a
photochemical effect generally does not depend on the rate that the
photons are absorbed). The high irradiance induced a greater
temperature increase ranging from 48.5 to 59.5.degree. C. during
the 5 minute-long irradiation (FIG. 13). This increase in
temperature also produced a decrease in cell viability and collagen
organization as shown by NADH-diaphorase staining and the eosin
counter staining, respectively (FIG. 10C). This is consistent with
a previous report in which the critical temperature for cell death
was 50-60.degree. C. for a short period of heating of less than 3
minutes and .about.50.degree. C. for a longer period of 24 minutes
(Heisterkamp, J et al. (1999) Lasers Surg Med 25(3): 257-62).
[0203] Irradiance is an important factor in photochemical tissue
bonding. Maintaining the temperature below 40.degree. C. is
desirable for minimizing side effects. Temperature can be
maintained by controlling irradiance conditions alone, or by
including cooling agents such as spray coolants (Zenzie, H. H. et
al. (2000) Lasers Surg Med 26: 130-44). In particular, surface
coolants can be applied to protect the epidermis from thermal
injury.
Example 11
Use of PTB to Enhance Bonding of Collagen Implants to Host Muscle
Flap
[0204] An ex vivo experimental system was developed to investigate
the effect of PTB on the bonding of an engineered, collagen-based,
esophagus implant to host muscle flap tissue without the need for
silicon tubing.
[0205] FIG. 14 provides an overview of the experimental methods.
Esophageal keratinocytes were seeded on uncontracted collagen gel
(ICN Biomedicals) for about one week until confluence was reached.
A 1.times.1 cm piece of seeded collagen gel was removed (implant)
from the culture dish and a suture loop was laced through at its
edge. An equivalently-sized section of rat latissimus dorsi muscle
flap was secured onto a flat plastic surface with sutures. Rose
bengal (RB) at 0.1% (w/v), a photosensitizer having an absorption
maximum and absorption coefficient of 550 nm and 33,000
dm.sup.3/mo/cm at 514 nm, respectively, was applied to the collagen
side of the implant and the surface of the muscle flap and
incubated for about two minutes. The esophagus implant and the
muscle flap were approximated with the dye-treated surfaces facing
each other by gently pressing over the esophagus implants without
damaging the keratinocyte layer. Excess dye was removed by
aspiration and blotting.
[0206] The approximated composite was photoactivated by irradiation
with an optic fiber argon laser (514 nm), having a spot diameter of
1 cm. Two distinct irradiation conditions were used for separate
samples in which the fluence of applied laser energy was 50
J/cm.sup.2 (100 seconds) and 100 J/cm.sup.2 (200 seconds),
respectively. For both samples, the irradiance of applied laser was
0.25 W/cm.sup.2. The composites were sprayed with PBS at 1 minute
intervals to prevent desiccation. As controls, an RB untreated and
nonirradiated composite (negative control), an RB treated and
nonirradiated composite (RB control), and a RB untreated and
irradiated composite (laser control) were prepared
concurrently.
[0207] Following irradiation, the adherence of the collagen-based
esophageal implant to the host muscle flap was tested for the 50
J/cm.sup.2 sample, 100 J/cm.sup.2 sample, and the three controls,
using a tensiometer (Chartillon) coupled to a force transducer
(DFA2, Ametek). The adherence (N/cm.sup.2; N=force applied,
cm.sup.2=area of collagen implant) of the collagen implant on the
muscle flap was determined from the force-deflection curve,
generated by plotting the amount of peeling force applied to the
collagen implant to the force plate displacement level of the force
transducer. It was observe that the mean adherence measurements
determined for the negative, RB, and laser controls were 0.05,
0.06, and 0.07 N/cm.sup.2, respectively (FIG. 15). In contrast, the
adherence measurements determined for the 50 and 100 J/cm.sup.2
composite samples were 0.15 and 0.13 N/cm.sup.2, respectively (FIG.
15). Statistical analysis using the Kruskal Wallist test showed a
statistically significant result at a significance level of 0.1
(p=0.09). The increase in adherence of the 50 and 100 J/cm
irradiated composite samples suggests clinical importance.
[0208] The viability of the photoactivated cells was also assessed.
Nicotinamide adenine dinucleotide (NADH-diaphorase) staining was
carried out based on the methods described by Heisterkamp J et al.
(1999) Lasers Sug Med 25(3): 257-62 and Neumann RA et al. (1991) J
Am Acad Dermatol 25: 991-8. Following irradiation, the keratinocyte
layer was gently peeled away from the collagen gel and was exposed
overnight to an incubation solution consisting of nicotinamide
adenine dinucleotide (NADH) and nitroblue tetrazolium chloride
(NBT) (Sigma). The viability of the cells was indicated by the
formation of a water-insoluble blue precipitate (diformazan), which
corresponded to areas with NADH-diaphorase activity. The insoluble
blue precipitate was dissolved in DMSO (an organic solvent) and
quantified by measuring the optical density (OD) at 550 nm against
a commercially available standard. The negative control, RB
control, and the laser control composites were also tested using
identical conditions. It was determined for the negative control,
RB control, laser control, 50 J/cm.sup.2 composite sample, and the
100 J/cm.sup.2 composite sample that the levels of corresponding
blue precipitate were 143, 151, 158, 173 and 158 diformazan/nM
after photochemical tissue bonding treatment (FIG. 16). It was
determined by non-parametric Kruskal Wallis test that the
differences between measurements was not significant (p=0.596),
indicating that viability was the same between treated and
non-treated controls.
[0209] The current ex vivo study demonstrates that photochemical
tissue bonding is able to increase the adherence of collagen-based
esophagus implants to the host muscle flap tissue. The mean
adherence measurements of the negative, RB, and laser controls were
less than the mean adherence measurements of the PTB treated
groups. The bonding strength produced reduces or even eliminates
the need for surgical supporting aids, such as silicon tubing,
during integration of collagen-based scaffold tissue implants.
Viability assessment of the top keratinocyte layer after PTB
treatment was shown to be the same as the controls, indicating that
PTB is a safe procedure that retains high cell viability.
Example 12
Use of PTB in Ruptured Tendons
[0210] Repair of ruptured or torn tendons and ligaments is often
accomplished by surgically reattaching the tissue or closing the
laceration. In addition to the physiological repair, the wound must
return to functional integrity.
[0211] To test the ability of PTB to quickly and effectively bond
ruptured tendons, an in vitro model utilizing bovine tendons was
developed. The use of bovine models in wound healing is well known
by those skilled in the art. The similarities between bovine wound
healing and that of humans enables one to extrapolate the
therapeutic results obtained in a bovine model system to a
therapeutic result in humans.
[0212] Fresh bovine achilles tendons were obtained and stored at
40.degree. C. until use. The tendon was cut into dumbbell-shaped 5
cm strips with the central 3 cm strip at an approximate
cross-sectional area of 1 mm by 1 mm, and were irrigated constantly
with phosphate buffered saline (PBS) to prevent desiccation.
[0213] Rupture of the tendon was induced by weakening the central 1
cm of the tendon with a surgical blade and mechanically pulling it
apart. A solution of Rose Bengal at various concentrations (0.1,
0.5 and 1.0% weight per volume in PBS) was applied to the ends of
the ruptured tendons for two minutes, after which the ruptured ends
were approximated and the excess dye was removed.
[0214] The ruptured ends of the tendon were crossed over each other
with an overlap of approximately 1 cm before being wrapped by
several layers of tissue paper. The tissue wrapped tendon was
rolled and pressed in order to approximate the ruptured ends and
remove the excess dye. The tissue paper was carefully removed
before the tendon was placed between two glass slides that were
then clipped together.
[0215] The center of the approximated tendon was irradiated with an
argon laser (514 nm), having a spot size of 1 cm in diameter, which
was transmitted through an optic fiber. The laser was applied with
an irradiance of 0.5, 1.0, or 1.5 W/cm.sup.2, while the fluence of
laser energy applied was 125, 250, 500, or 750 J/cm.sup.2. The
exposure time ranged from 1 min and 23 seconds to 25 minutes.
[0216] Control tendons were ruptured in the same manner, however
treatments consisted of application of Rose Bengal and
approximation of the tendon ends without laser irradation,
approximation of the tendon ends with laser irradiation but no
exposure to rose bengal, and approximation of the ends with no
further treatment.
[0217] To test the effectiveness of the photochemical tissue
bonding, immediately after photochemical tissue bonding, the tendon
strips were attached to the fixture of a tensiometer, which was
coupled to a force transducer (FIG. 17). The tendon was held at a
straight and vertical position, and cyclic loading of the tendon
was conducted by stretching the tendon for a magnitude of 0.5 mm
for 6 cycles. The tendon was brought back to a zero deflection
position before being pulled to failure at a constant speed of 100
mm/min. The peak force needed to rupture the tendon was recorded,
and a typical force-deflection curve was obtained (FIG. 18). The
ultimate stress and stiffness (peak force for tendon rupture
normalized by the cross-sectional area, and slope of the linear
region of the force-deflection curve, respectively) were calculated
from the force-deflection curve.
[0218] The ruptured tendons treated with visible light (argon
laser) and a photoactivated dye (Rose Bengal) showed a significant
increase in the ultimate stress and stiffness compared to the
controls (FIGS. 19A and 19B). The fluence dose response was studied
up to 750 J/cm.sup.2, however, apparent dehydration in the tendon
was reported in the highest dosage group.
[0219] At a fixed fluence dosage of 500 J/cm.sup.2, increasing the
power density (irradiance) significantly increased the ultimate
stress (p=0.034), but not the stiffness (p=0.088 of the ruptured
tendon (FIGS. 20A and 20B). Dehydration of the tendon was noticed
at the highest irradiance (1.5 W/cm2).
[0220] The concentration of Rose Bengal was varied to determine its
influence on the photochemical tissue bonding. Dosages of 0.0, 0.1,
0.5 and 1.0% (w/v in PBS) were tested, with the results showing a
significant increase in both the ultimate stress and the stiffness
at higher Rose Bengal concentrations (FIGS. 21A and 21B). When 1.0%
Rose Bengal was tested, dehydration of the tendon was noticed.
[0221] Healthy tendons were also placed in the tensiometer, and
were pulled to the point of failure. Following failure, they were
treated with photochemical tissue bonding at 500 J/cm.sup.2 fluence
and again pulled to failure to study the percentage of recovery of
mechanical properties. Immediately after the photochemical tissue
bonding, the ultimate stress and the stiffness returned to
approximately 59.66% and 52.75% of the healthy tendon, respectively
(Table 1).
TABLE-US-00001 TABLE 1 Mean change in ultimate Mean change in Group
stress %(95% CI) stiffness %(95% CI) n Laser control 17.07 (4.19,
29.95) 35.00 (0.75, 69.25) 6 Dye control 24.18 (6.57, 41.79) 20.65
(10.16, 31.14) 6 Laser + Dye 59.66 (8.71, 110.61) 52.75 (24.05,
81.45) 7
[0222] This example indicates that the utilization of low
concentration RB and non-thermal irradiation (e.g. between
approximately 0.5 and 1 W/cm.sup.2) causes immediate tendon bonding
which enhanced the mechanical properties, including the ultimate
stress and the stiffness, of the ruptured tendon immediately after
the bonding procedure. As a non-thermal bonding method, this type
of procedure eliminates the tissue damage caused by thermal bonding
methods. The procedures can be varied to incorporate various
photosensitizers, appropriate laser light sources, concentrations
of RB and amounts of irradiation. This type of treatment is
beneficial in humans and would advantageously eliminate the need
for or minimize the use of staples, sutures, glues, and other
adhesives.
Example 13
Use of PTB in Ruptured Tendons In Vivo
[0223] The tensile strength was tested one week after in vivo
bonding of rat achilles tendon. The bonding was carried out in
vivo. The tendon was removed and tested ex vivo. The peak force
needed to break the tendon was normalized by the cross sectional
area of the tendon. The elasticity of the tendon was also
calculated from a stress-strain curve obtained during the tensile
strength test.
[0224] PTB treatment significantly increased the ultimate stress
(v0.004), which is the peak force for tendon rupture normalized by
the cross-sectional area and the stiffness (p=0.006), which is the
slope of the linear region of the force-deflection curve, of
ruptured tendons (FIGS. 22A and 22B).
[0225] At a fixed fluence dosage of 500 J/cm.sup.2, increasing the
laser power density, or the irradiance, significantly increased the
ultimate stress (p=0.034) but not the stiffness (p=0.088) of the
ruptured tendon (FIGS. 23A and 23B).
[0226] At a fixed irradiance of 1.0 W/cm.sup.2, increasing the
fluence dosage induced a significant increase in both the ultimate
stress (p=0.002) and the stiffness (p=0.004) of the photochemically
bonded tendon (FIGS. 24A and 24B).
Example 14
Use of PTB in an In Vivo Rat Sciatic Nerve Repair Model
[0227] Methods of the present invention can be used to effectively
repair neural tissue.
[0228] Presently, the goal in peripheral nerve repair is to
maximize axonal regeneration while minimizing scar/neuroma
formation and operative time. In addition to creating scar tissue,
gaps created by suture-based repair compromise the nerve's
environment by allowing the entrance of inflammatory cells, such as
macrophages, as well as the escape of regenerating axons and
essential neurotrophic and neurotropic factors. The methods
described herein are designed to overcome these obstacles.
[0229] Photochemical tissue bonding was performed on a rat sciatic
nerve model of peripheral nerve injury. Various experimental and
control surgical protocols were tested, each being tested on a
group of 10 rats (n=10).
[0230] Adult, male, 300 g Fischer rats (NCI) were anesthetized with
pentobarbital (40 mg/kg IP) and weighed. The left hind limb was
shaved with clippers and prepared with povidone-iodine solution.
Under semi-sterile conditions using a double-headed operative
microscope, a longitudinal incision along the lateral aspect of the
left hind limb was made to expose the sciatic nerve via a muscle
splitting incision. A 1 cm segment of sciatic nerve was dissected
free from all surrounding tissues and then sharply transected using
microsurgical scissors approximately 1 cm from the pelvic hiatus.
Animals were then randomly assigned to one of the four treatment
groups shown below in Table 2.
TABLE-US-00002 TABLE 2 Experimental Surgical Repair PTB with Number
of Group Procedure Rose Bengal Animals A Standard Suture No 10 B No
Surgical Repair No 10 C Epineurial Cuff No 10 D Epineurial Cuff Yes
10
[0231] In Experimental group A, following transection the proximal
and distal segments of the sciatic nerve were microsurgically
repaired in an end-to-end fashion, by placing multiple epineurial
10-0 nylon sutures.
[0232] In experimental group B, where the sciatic nerve was
transected but not repaired, the distal sciatic nerve segment was
buried in the thigh musculature and anchored using a 10-0 nylon
suture.
[0233] In experimental group C, "epineurial cuff" procedure was
performed, as illustrated in FIG. 25, to produce a protrusion or
"cuff" of the epineurial tissue extending beyond the inner neural
tissue. Following transection, the epineurial cuff of the distal
segment was gently retracted and approximately 2 mm of endoneurium
was sharply removed. A single 10-0 nylon suture was used as an aid
to draw the proximal sciatic segment into the distal sciatic cuff,
as illustrated in FIG. 25. The suture was then gently removed. This
"cuffing" procedure served to maximize the area of surface contact
between the proximal and distal stumps of the transected nerve.
[0234] In experimental group D, the transected nerves of the rats
were treated as for group C, except that PTB treatment was
performed by applying 25 .mu.l of 0.1% RB dye to the exposed
epineurial surfaces of the sciatic nerve and left in place for 30
seconds prior to alignment of the nerve segments. Endoneurium
exposed to the dye was removed. The repair site was then exposed to
green laser light at 514 nm. The nerve was then rotated 180.degree.
and the irradiation was repeated.
[0235] After completion of the above surgical and/or PTB
procedures, and after insuring adequate hemostasis, all wounds were
closed in a subcuticular fashion with 4-0 absorbable, braided
sutures. Once the animals were awakened from anesthesia, only those
animals moving comfortably and tolerating food and liquids were
used in subsequent experiments. All animals received Buprenex (0.03
mg/kg IM) postoperatively and then every 12 hrs for 3 days.
[0236] Initially, the left lower leg of all rats was
non-functional, as is to be expected after sciatic nerve
transection.
[0237] Functional assays for sciatic nerve repair were performed as
follows. At 90 days following surgery, animals were sacrificed. A
"gastrocnemius muscle mass assay" was used to assess functional
nerve repair. This assay makes us of the phenomenon whereby
denervated muscles waste away and lose mass, over time. The weight
of the gastrocnemius muscle of the treated hind limb of each rat
was compared to that in the untreated hind limb, which acted as an
internal control.
[0238] The results of this study are shown in FIG. 26. The methods
of neural repair described herein provide an enhancement of
function approaching that of conventional suture repair.
[0239] Thus, methods of the present invention provide a useful
alternative to conventional surgical methods for nerve repair.
Photochemical bonds can be formed that are atraumatic, instant and
watertight thus minimizing any loss of neurotropic and neurotrophic
factors. In addition such methods can restore the blood-nerve
barrier, and minimize or eliminate any foreign-body responses and
excessive scarring.
Example 15
Alternative Assays for Nerve Repair
[0240] In Example 14, a gastrocnemius muscle mass assay was used to
assess nerve repair following the various treatments. Alternative
assays that can also be used to assess nerve repair include tensile
strength assays, histological assays, electrophysiological, and
walking assays.
[0241] Tensile strength assays can be performed at post-operative
days 30, 60 and 90. For example, a minimum of 4 cm of repaired
sciatic nerve is removed form each animal, and a 5-0 silk suture is
attached to both ends of the nerve segment immediately after
dissection. The proximal nerve stump is hooked to the upper fixture
of the tensile strength testing apparatus through the pre-attached
suture, while the suture attached to the distal end is clamped to
the lower fixture of the tensile strength testing apparatus. The
nerve is adjusted to a vertical position with zero-tension before
vertical tension is applied. The tensile strength testing
apparatus, and the methods for testing tensile strength are the
same as those previously described for skin and tendon tensile
strength measurements. The force transducer records the peak force,
time and the amount of deflection during the tensile test. The
ultimate tensile strength of the nerve is calculated after dividing
the peak force that the nerve can withstand by the cross-sectional
area. Higher tensile strength measurements will typically a more
effective physical repair of the nerve.
[0242] Functional analysis in the form of walking assays, as
described by De Medinaceli et al. (1982), and Bain et al. (1989)
can also be performed. For example, walking track analysis is
performed pre-operatively and every ten days post-operatively to
gauge functional recovery in the rat sciatic nerve. The animals'
hind feet are dipped in dilute india ink after which they are
allowed to walk down a 10 cm.times.40 cm corridor into a darkened
box. The floor of the corridor is covered with a removable piece of
paper used to record the animals' paw print. Measurements based on
the print-length, toe-spread (the distance between the first and
fifth toes), and the intermediary toe-spread (the distance between
second and fourth toes) are used to formulate a sciatic function
index (sfi). The sfi values are compared between experimental and
control animals. The non-operative (right) side paw print is used
to normalize SFI values between different animals.
[0243] At 90 days post-operatively, animals can be re-anesthetized
and electrophysiological studies performed. The sciatic nerve is
re-exposed and recording leads are placed both proximal and distal
to the repair site. Stimulation and ground leads are placed and
mean nerve conduction velocities are measured, giving an indication
of functional nerve repair.
[0244] Histological assays to determine nerve repair can be
performed also. Animals are sacrificed at a chosen day
post-operatively and the muscle and neural tissues are removed.
Nerve segments to be analyzed are fixed in 3% glutaraldehyde,
embedded, and sectioned on a microtome. The sections are stained
with hematoxylin/eosin and osmium tetroxide, and histomorphometric
analysis is performed using computer-assisted quantification of
digitized images from histologic slides.
Example 16
Use of Conduit Technology in Nerve Repair
[0245] Conduits consisting of sleeves of 100% collagen have been
used to assist nerve repair in various animal models (Archibald et
al, 1991; Li et al, 1992). The PTB methods of the present invention
can be used in conjunction with collagen conduits to produce
suture-less bonds. Serving as a sleeve, collagen conduits can be
used to join the collagen rich epineurium of the proximal and
distal ends of transected or damaged nerves. The immediate,
circumferential seal formed by such methods serves to enhance the
confinement of neurotrophic factors, such as nerve growth factor
(NGF), and will also maximize axonal capture. Additionally
inflammation and scar tissue formation is minimized.
[0246] Before performing nerve repair studies, the optical
suitability of 100% collagen conduits (NeuraGen.TM., Integra
Neurosciences) for use in the PTB methods of the present invention,
was assessed. Total transmittance and diffuse reflectance of light
from the outside to the inside of the conduit was measured. It was
found that the conduit effectively transmitted laser light at
wavelengths appropriate for activation of rose bengal (RB) dye
(around 532 nm). As shown in FIG. 27, at ideal wavelengths, greater
than 50% of the light is transmitted through the conduit. These
optical properties are similar to those of other tissues in which
PTB has successfully been used in PTB with RB
[0247] Additional experiments were also performed to determine the
effectiveness of PTB bonding between the NeuraGen.TM. conduits and
an animal tissue, namely bovine tendon tissue. To do this, the
tensile strength of a PTB bond formed between the bovine tendon and
a segment of Neuregen.TM. conduit was tested, using various
different photoactive dyes. Both the tendon surface and the inner
surface of a piece of conduit were coated with RB dye and allowed
to sit in the dark for 60 seconds. The two surfaces were then
thoroughly dried and pressed together to form a tendon/conduit
interface of approximately 0.25 cm.sup.2. The construct was
irradiated at 532 nm through the conduit toward the tendon/conduit
interface at various fluence levels (Joules). Constructs were then
pulled into tension using a tensiometer until the bond between the
tendon and the conduit broke. The breaking force was measured in
Newtons. A tensile strength curve based on fluence both with and
without Rose Bengal dye was generated (see FIG. 28) demonstrating
that an effective bond was formed at a fluence of 150-300
Joules.
[0248] Having thus demonstrated the suitability of the NeuraGen.TM.
conduit for PTB method, PTB nerve repair experiments can be
performed. The sciatic nerve is transected 10 mm from the pelvic
hiatus as described in the above examples. Both the proximal and
the distal ends of the severed nerve are coated with a 0.1%
solution of Rose Bengal dye and allowed to dry for 60 seconds.
Excess dye is removed and the nerve ends are drawn into a 10 mm
segment of NeuraGen.TM. conduit using 10-0 non-absorbable nylon
monofilament suture (See FIG. 29). An argon laser with a fixed
wavelength of 532 nm is used for bonding. After 1 min of
application of the dye, the nerve-conduit complex is irradiated
with the argon laser for 3 minutes; the nerve is then rotated 180
degrees and the opposite side is irradiated for another 3 min. The
sutures are then removed from the nerve-conduit complex.
Example 17
Use of PTB in an Ex Vivo Porcine Blood Vessel Repair Model
[0249] Laser welding of blood vessels has attracted major attention
in the past, however laser welding induces thermal damage in
tissues causing protein denaturation. Methods of the invention
avoid thermal damage while maintaining the structural integrity of
the collagen, forming stronger chemical bonds.
[0250] In adhering segments of vascular tissue according to methods
of the invention, two pieces of blood vessel are joined and
photochemically bonded together to create an intimate, watertight
seal. Two methods were considered to accomplish anastomosis. The
"cuff" approach, which entails administration of the photoactive
dye to the outer surface of the proximal vessel and the inside
surface of the distal stump of the vessel. The distal stump is
folded back on itself and the proximal stump brought into contact.
The distal cuff is then placed over the proximal vessel to form a
cuff. The overlap region in the cuff is then irradiated with
visible light to bond the ends of the vessel together. FIG. 30
represents a schematic overview of the cuff approach.
[0251] FIG. 31 demonstrates the conduit approach, wherein the
photoactive dye is administered to the outer surfaces of both
proximal and distal vessel stumps and the inside of a tubular
conduit with an inner diameter equal to the outer diameter of the
blood vessel. This conduit can be of numerous materials including
collagen. The conduit is placed over the approximated stumps and
bonded in place using visible light.
[0252] The cuff approach was used with pig vein and arteries ex
vivo. The vessels were sharply transected using a scalpel. Rose
bengal at a concentration of 0.1% v/v in saline was applied to the
outside surface of the proximal stump of the vessel and the inside
surface of the distal vessel. The vessels were then mounted over a
cylindrical glass rod that acts as support for the hollow vessels.
A distal cuff was formed by folding the end of the distal end back
on itself, as described above and in FIG. 30. Following apposition,
the cuff was everted over the proximal vessel and the cuff region
was then irradiated using 532 rim green light from a CW Nd/YAG
laser (Coherent) at 0.5 W/cm.sup.2 for 2.5 minutes.
[0253] Following illumination, the glass support was removed and
the anastomosed vessel was mounted on a strain gauge for
measurement of the tensile strength of the anastomosed bond.
Sutures were placed at each end of the vessel and were used to
attach to the strain gauge attachments. FIG. 32 shows the force
withstood by a pig artery with an inner diameter of around 3 mm
before rupture took place at the site of the seal. FIG. 33 shows
data collected in the same way following re-anastomosis of a pig
vein of similar dimensions treated in the same way.
[0254] Very strong bonds were formed and the vessels were stretched
to 3-4 times their normal length before rupture was achieved. In
some cases, the bond was strong enough to withstand the force and
the suture actually tore out of the end of the vessel before
rupture of the seal could be achieved.
[0255] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
* * * * *