# Patent

## Title
Photochemical tissue bonding

## Abstract

Photochemical tissue bonding methods 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 and corneal repair.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a continuation-in-part of U.S. application Ser. No. 09/900,504, filed Jul. 6, 2001 now abandoned, 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 which are incorporated herein 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 which are incorporated herein by reference.


STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
This work was supported by the government, in part, by grants from the Department of Defense Medical Free Electron Laser Program, Award Nos. N00014-94-1-0927 and F49620-01-1-0014 and the Office of Naval Research, award number N000149910617. The government may have certain rights to this invention.


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
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 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
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.
Possible alternatives to sutures include hemostatic adhesives, such as fibrin sealants (Henrick et al. (1987) J Cataract Refract Surg 13:551-553; Henrick 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. (1 998) 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) Surv 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).
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 often lack suturability. 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.
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/cm2 (Dahlstrom et al. (1991) Skin Transplantation 89(5): 968-72).
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 normal 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.
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 posterial collateral ligament (PCL) (Bosch et al. (1994) Acta Orthop Belg 60(suppl): 57-61).
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 offoot 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.
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 CO2 and Argon lasers, showed that thermal welding of ruptured achilles tendon in rats led to  ̃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 ( ̃30%) increase in collagen production but insignificant increase ( ̃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  ̃30% but statistically insignificant increase (Reddy et al. (1998) Med Sci Sports Exerc 30(6): 794-800).
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.
SUMMARY
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.
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-5-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.
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, 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.
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.
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, renal, occular, oral, connective, respiratory, otolaryngological, dermatological, genital, gynecological or musculoskeletal tissue.
In a 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.
In yet another embodiment, the photosensitizer agent is Rose Bengal.
In yet another embodiment, the contacting step occurs ex vivo.
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.
In yet another embodiment, the subject is other than an albino animal, e.g., other than an albino rabbit.
In yet another embodiment, the subject is a human.
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.
In yet another embodiment, the application of electromagnetic energy to the tissue-photosensitizer complex occurs without more than a 15° C. rise in temperature as measured, e.g., with an imaging thermal camera during irradiation.
In yet another embodiment, the application of electromagnetic energy to the tissue-photosensitizer complex occurs without more than a 10° C. rise in temperature as measured, e.g., with an imaging thermal camera during irradiation.
In yet another embodiment, the application of electromagnetic energy to the tissue-photosensitizer complex occurs without more than a 3° C. rise in temperature as measured, e.g., with an imaging thermal camera during irradiation.
In yet another embodiment, the application of electromagnetic energy to the tissue-photosensitizer complex occurs without more than a 2° C. rise in temperature as measured, e.g., with an imaging thermal camera during irradiation.
In yet another embodiment, the application of electromagnetic energy to the tissue-photosensitizer complex occurs without more than a 1° C. rise in temperature as measured, e.g., during irradiation with an imaging thermal camera.
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.
In one embodiment, the corneal lesion is caused by a surgical procedure.
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.
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.
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.
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.
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.
In yet another embodiment, the electromagnetic energy applied is greater than 1200 J/cm2. In another preferred embodiment, the electromagnetic energy applied is between 200 and 1200 J/cm2. In another preferred embodiment, the electromagnetic energy applied is between 200 and 800 J/cm2. In yet another preferred embodiment, the electromagnetic energy applied is between 200 and 500 J/cm2. In yet another preferred embodiment, the electromagnetic energy applied is between 300 and 600 J/cm2. In another preferred embodiment, the electromagnetic energy applied is between 350 and 550 J/cm2.
In yet another embodiment, the electromagnetic energy is applied at an irradiance less than 3.5 W/cm2.
In yet another embodiment, the electromagnetic energy is applied at an irradiance less than 1.5 W/cm2.
In yet another embodiment, the electromagnetic energy is applied at an irradiance of about 0.10 W/cm2.
In yet another embodiment, the subject is other than an albino animal, e.g., other than an albino rabbit.
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.
In one embodiment, the subject is a human.
In yet another embodiment, the corneal lesion is caused by a surgical procedure.
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.
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.
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.
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.
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.
In yet another embodiment, the subject is other than an albino animal, e.g., other than an albino rabbit.
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.
In one embodiment, the photosensitizer agent is Rose Bengal.
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.
In one embodiment, the subject is a human.
In yet another embodiment, the musculoskeletal tissue is a tendon.
In yet another embodiment, the tendon is ruptured.
In yet another embodiment, the tendon is damaged by an avulsion from the bone and tendon transection.
In yet another embodiment, the ruptured ends of the tendon are joined together.
In yet another embodiment, there is a laceration of the tendon.
In yet another embodiment, the edges of the laceration of the tendon are joined together.
In yet another embodiment, the tendon is joined to a tendon graft.
In yet another embodiment, the musculoskeletal tissue is a ligament.
In yet another embodiment, the ligament is a small ligament.
In yet another embodiment, the ligament is the anterior cruciate ligament.
In yet another embodiment, the photosensitizer agent is Rose Bengal.
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.
In yet another embodiment, the electromagnetic energy applied to the musculoskeletal tissue is greater than 750 J/cm2. In another preferred embodiment, the electromagnetic energy applied is between 50 and 750 J/cm2. In another preferred embodiment, the electromagnetic energy applied is between 250 and 750 J/cm2. In yet another preferred embodiment, the electromagnetic energy applied is between 250 and 500 J/cm2.
In yet another embodiment, the electromagnetic energy is applied at an irradiance less than 1.5 W/cm2.
In yet another embodiment, the electromagnetic energy is applied at an irradiance less than 1.0 W/cm2.
In yet another embodiment, the electromagnetic energy is applied at an irradiance of between 0.5 and 1.0 W/cm2.
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.
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.
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.
The patent file contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.



DESCRIPTION OF THE DRAWINGS
The patent file contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.
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.
FIG. 2 shows a graph of the mean IOPL values for PTB treated eyes (n=5) using 514 nm light (2.55 W/cm2) and Rose Bengal (1.5 mM) in PBS. Additional controls are incisions treated with Rose Bengal or buffer but no laser light.
FIG. 3 shows graphs of mean IOPL before and after PTB using Rose Bengal and 514 nm irradiation. Rose Bengal (10 μl, 1.5 mM) was applied to the incision surfaces then treated with the doses indicated using irradiances of: (A) 1.27 W/cm2, (B) 2.55 W/cm2 and (C) 3.82 W/cm2.
FIG. 4 shows graphs of mean IOPL before and after PTB using R-5-P and 488 nm irradiation. R-5-P (40 μl, 11 mM) was applied to the incision surfaces then treated with the doses indicated using irradiances of: (A) 1.27 W/cm2, (B) 2.55 W/cm2 and (C) 3.82 W/cm2.
FIG. 5 shows graphs of mean IOPL values before and after PTB using F1 and 488 nm irradiation. F1 (40 μl, 0.6 mM) was applied to the incision surfaces then treated with the doses indicated using irradiances of: (A) 1.27 W/cm2, (B) 2.55 W/cm2 and (C) 3.82 W/cm2.
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.
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.
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.
FIG. 9 shows a graph of the skin adherence after PTB at 0.56 (FIG. 9A) and 1.68 W/cm2 (FIG. 9B). Irradiation levels of 0.56 and 1.68 W/cm2 provided a positive dose-dependent relationship between the laser energy (fluence) and the adherence of the skin grafts.
FIG. 10 shows cell viability and collagen organization. Following irradiation levels of both 0 (control) and 0.56 W/cm2, 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.
FIG. 11 shows cell viability after PTB of skin grafts at an irradiance of 0.56 W/cm2 and various fluences.
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/cm2 (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.
FIG. 13 shows a scatter plot of the maximal surface temperature of skin grafts during photochemical tissue bonding at 0.56 W/cm2 and 1.68 W/cm2, and up to a fluence of 504 J/cm2.
FIG. 14 shows a schematic diagram of photochemical tissue bonding of an esophagus implant to a host muscle flap.
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.
FIG. 16 shows the viability of esophageal keratinocytes after photochemical tissue bonding treatment.
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.
FIG. 18 shows a typical force-deflection curve obtained by measurements from the tensiometer.
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.
FIGS. 20A and 20B show graphs of the ultimate stress and stiffness in ruptured tendons at a fixed fluence dosage of 500J/cm2, while increasing the laser power density. Ultimate stress was significantly increased, but stiffness was not.
FIGS. 21A and 21B show a graph of the ultimate stress and stiffness in ruptured tendons at increasing Rose Bengal concentrations.
FIGS. 22A and 22B show the ultimate stress and the stiffness following in vivo PTB treatment compared to the controls.
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/cm2.
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.



DETAILED DESCRIPTION
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.
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).
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.
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.
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.
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 an immediate partial regaining of strength after the repair, due to the large stress placed on the tendon during the period of recovery following surgery. The methods described herein 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.
Photoactivation and Photosensitizers
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.
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 form 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 degrees Celsius (C.), more preferably a rise of less than 10 degrees C., more preferably a rise of less than 3 degrees C., more preferably a rise of less than 2 degrees 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, chlorine6, mono-1-aspartyl derivative of chlorine6, di-1-aspartyl derivative of chlorine6, tin(IV) chlorine6, 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.
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-5-phosphate (R-5-P); methylene blue (MB); and N-hydroxypyridine-2-(1H)-thione (N-HTP).
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.
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.
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 μl of photosensitizer solution, preferably 50 μl (microliter), 100 μl, 250 μl, 500 μl, 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.
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/cm2. Even more preferably, the electromagnetic energy applied is between 100 and 500 J/cm2. 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, infra red or near ultra violet spectra. The energy can be delivered at an irradiance of about between 0.1 and 5 W/cm2, preferably between about 0.5 and 2 W/cm2. 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.
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 form 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.
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.
Uses
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.
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.
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 mm 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.
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.
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.
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 ApligrafTM comprises epidermal or dermal analogs derived from neonatal foreskin, the host-graft composite of which will become repopulated with cells from the host subject. Commercially available skin substitutes include BiobraneTM, composed of silicon, nylon and collagen, TransCyteTM, composed of silicon, collagen, fibronectin and glycosaminoglycan, and IntegraTM, composed of silicon, collagen and glycosaminoglycan. Skin substitutes can be used in applications of permanent and semi-permanent grafting. Preferably, IntegraTM is used for permanent grafting.
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.
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.
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.
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.
Kits
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.
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.
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
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.
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, MA) 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.
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 (IOPL) 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 μl 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 μM laterally into the wall of the incision.
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 dm3 mol−1 cm−1 at 514 nm; fluorescein (F1), 490 nm, 88300 dm3 mol−1 cm−1 at 488 nm; methylene blue (MB), 664 nm, 15600 dm3 mol−1 cm−1 at 661 nm; riboflavin-5-phosphate (R-5-P), 445 nm, 4330 dm3 mol−1 cm−1 at 488 nm; and N-hydroxypyridine-2-(1H)-thione (N-HPT), 314 nm, 2110 dm3 mol−cm−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 μm at the laser irradiation wavelength (with the exception of N-HPT for which the absorption was approximately a factor of 10 lower).
Irradiations employed a continuous wave (CW) argon-ion laser (Innova 100; Coherent, Inc., Palo Alto, Calif.) at 488 nm (for F1 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-dimethylaminostyryl)-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 45° 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/cm2 and the irradiances used were 0.64, 1.27, 2.55 and 3.86 W/cm2. 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 IOPL 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.
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
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/cm2. The irradiance delivered can be from about 0.2 to 3 W/cm2. The duration of irradiation is preferably from about 1 to 10 minutes.
Treatment of incisions with 1.5 mM RB and 514 nm laser light resulted in an increase in post-treatment IOPL, as measured as described in Example 1. Control experiments demonstrated that a significant increase (p<0.005) in the IOPL, following PTB treatment, occurred when both RB and laser irradiation were applied and not by either alone (FIG. 2). The mean IOPL of incisions treated with RB and 514 nm laser light was greater than 300±48 mm Hg, whereas laser irradiation alone or photosensitizer alone produced no significant increase between the pre- and post-treatment IOPL values.
Dose response curves for IOPL are shown in FIG. 3 for RB doses delivered at irradiances of 1.27 (3A), 2.55 (3B) and 3.82 W/cm2 (3C). A dose-response relationship was observed at the lowest irradiance (1.27 W/cm2) for doses between 508 and 1270 J/cm2 (3A). No significant rise in the IOPL was observed for doses below 508 J/cm2 at any irradiance tested. PTB was most efficient at 1270 J/cm2 delivered at an irradiance of 1.27 W/cm2. All doses delivered at the two lower irradiances (1.27 and 2.55 W/cm2) gave IOPL values greater than 100 mm Hg. Treatment using irradiances of 2.55 and 3.82 W/cm2 produced no obvious dose response pattern. In general, for a selected dose the IOPL was lower at higher irradiances. For example, at 1270 J/cm2 the mean IOPL values were 274, 150 and 130 mm Hg for the irradiances 1.27 W/cm2, 2.55 W/cm2 and 3.86 W/cm2.
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 irradiance tested (1.27 W/cm2). Thermal damage could be observed at doses of 762 to 1524 J/cm2 at the highest irradiance (3.82 W/cm2) and occasionally at 2.55 W/cm2. 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
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/cm2. The irradiance delivered can be from about 0.2 to 3 W/cm2. The duration of irradiation is preferably from about 1 to 10 minutes.
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/cm2 and 1016 J/cm2, significantly increased the post PTB treatment IOPL value (p<0.05), see FIG. 4. The IOPL values observed using R-5-P are of a similar magnitude to those for RB. However, the IOPL values observed for each dye at the same irradiance and dose were not comparable. Although the treatment produces significant increases in IOPL, no simple pattern between the two dyes is observed.
Example 4
Use of N-hydroxypyridine-2-(1H)-thione (N-HTP) in PTB
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/cm2. The irradiance delivered can be from about 0.2 to 3 W/cm2. The duration of irradiation 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/cm2) at doses ranging from 127 J/cm2 to 508 J/cm2. Mean IOPL values of 60±23 mm Hg and 126±40 mm Hg were produced when using the doses of 254 J/cm2 and 508 J/cm2 respectively, lower doses than used for the other photosensitizers.
Example 5
Use of Methylene Blue (MB) in PTB
MB is a frequently used dye in ophthalmic surgery that has been reported to photosensitive 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/cm2 of 661 nm light. Doses of 508/cm2, 762 J/cm2 and 1016 J/cm2 did not increase the post-treatment, IOPL. 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
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 Medicine 19:9-16; 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 deformation produced by thermal tissue damage. When performing PTB on the cornea, heating must be avoided.
We evaluated the possibility that non-photochemical processes contribute to wound closure by comparing PTB produced by RB with that produced by fluorescein (F1), a dye with a similar structure but which is not expected to induce protein cross-links. RB and F1 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/cm2 to 1016 J/cm2 (FIG. 5). No increase in IOPL 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 IOPL values was observed with values ranging from 63±30 to 89±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 IOPL value of 116±40 mm Hg obtained using a dose of 762 J/cm2 at 3.82 W/cm2 (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/cm2) and dose (762 J/cm2) suggests that some other effect is operating, such as a thermal mechanism under these high irradiance conditions.
Example 7
PTB Versus Sutures
The IOPL 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. IOPL values with sutures were approximately 230 mm Hg. This value is similar for the incisions closed with PTB treatment.
Example 8
In Vivo PTB
PTB was performed in vivo in New Zealand rabbits to repair two types of corneal wounds.
In group I, 3.5-mm 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.
Group I wounds were healed using, e.g., 191 J/cm2, applying 1.5 mM RB. The immediate in vivo bursting pressure was 495±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±125 mmHg). At 14 days, the bursting pressure exceeded 500 mm Hg in both PTB-treated and control eyes.
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/cm2. 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±70 mm Hg for the PTB-treated eyes, compared to 250±150 mm Hg for the control eyes with sutures alone. This result indicate that PTB is useful and effective as a supplement to sutures, as well as on its own.
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
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.
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 enables one to extrapolate the therapeutic results obtained in a porcine model system to a therapeutic result in humans.
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° C. until needed (Fujita T et al. (2000) J Burn Care Rehabil 21: 304-9).
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 cm2 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.
The grafts were irradiated using a continuous-wave (CW) argon-ion laser (Innova 100; Coherent, Palo Alto, Calif.) at 514.5 nm. 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/cm2 while the dose-dependent response of the laser fluence from 126 to 504 J/cm2 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.
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/cm2) 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.
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/cm2) provided a positive dose-dependent relationship between the laser energy (fluence) and the adherence of the skin grafts (FIGS. 9a and 9b).
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 RA 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 5 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/cm2, 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 (1.68W/cm2) as indicated in FIG. 10C.
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° 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° 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. 11). 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/cm2 was not significantly different from the control. Although there was a slight reduction in the number of viable cells in the 504 J/cm2 group to  ̃80% of the control group, one-way ANOVA showed no statistical significant difference among the groups (p=0.899).
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/cm2) 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
PTB advantageously proceeds by a non-thermal mechanism, as demonstrated by the following results.
The maximal temperature of skin grafts was recorded by a ThermaCAM infrared imaging radiometer (PM180, Inframetrics, MA). The ThermaCAM was set approximately 30° 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° 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/cm2 and 5 minutes for 1.68 W/cm2). Images were analyzed by ThermaGRAM 95 software (Inframetrics, MA), 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/cm2 (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/cm2 and 1.68 W/cm2, and up to 504 J/cm2 (FIG. 13). At 0.56 W/cm2, there was a gradual, slight increase in temperature throughout the 15 minutes of irradiation, from room temperature at 23° C., to an average of 31.2° C. after 1 minute of irradiation and then to 39.9° C. after 15 minutes of irradiation. By contrast, the maximal skin temperature reached 48.5° C. at 1 minute and then increased up to 59.5° C. by the end of a 5 minute irradiation using an irradiance of 1.68 W/cm2.
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° C., which is far lower than the optimal coagulation temperature for laser thermal welding, 65-75° 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.
To reduce the irradiation time during PTB, the irradiance was increased from 0.56 W/cm2 to 1.68 W/cm2 (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° 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° C. for a short period of heating of less than 3 minutes and  ̃50° C. for a longer period of 24 minutes (Heisterkamp, J et al. (1999) Lasers Surg Med 25(3): 257-62).
Irradiance is an important factor in photochemical tissue bonding. Maintaining the temperature below 40° 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
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.
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 1×1 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 dm3/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.
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/cm2 (100 seconds) and 100 J/cm2 (200 seconds), respectively. For both samples, the irradiance of applied laser was 0.25 W/cm2. 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.
Following irradiation, the adherence of the collagen-based esophageal implant to the host muscle flap was tested for the 50 J/cm2 sample, 100 J/cm2 sample, and the three controls, using a tensiometer (Chartillon) coupled to a force transducer (DFA2, Ametek). The adherence (N/cm2; N=force applied, cm2=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/cm2, respectively (FIG. 15). In contrast, the adherence measurements determined for the 50 and 100 J/cm2 composite samples were 0.15 and 0.13 N/cm2, 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/cm2 irradiated composite samples suggests clinical importance.
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/cm2 composite sample, and the 100 J/cm2 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.
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
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.
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.
Fresh bovine achilles tendons were obtained and stored at 40° 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.
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.
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.
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/cm2, while the fluence of laser energy applied was 125, 250, 500, or 750 J/cm2. The exposure time ranged from 1 min and 23 seconds to 25 minutes.
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.
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.
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/cm2, however, apparent dehydration in the tendon was reported in the highest dosage group.
At a fixed fluence dosage of 500 J/cm2, 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).
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.
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/cm2 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 1






Mean change in ultimate
Mean change in stiffness



Group
stress % (95% CI)
% (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









This example indicates that the utilization of low concentration RB and non-thermal irradiation (e.g. between approximately 0.5 and 1 W/cm2) 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
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.
PTB treatment significantly increased the ultimate stress (p=0.004), 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).
At a fixed fluence dosage of 500 J/cm 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).
At a fixed irradiance of 1.0 w/cm2, 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).
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.

## Claims

1. A method for repairing a skin lesion in vivo in a subject, comprising:
contacting a skin lesion, a tissue comprising a skin graft and Rose Bengal (RB) at a concentration of less than 1.0% weight per volume to form a skin tissue-RB complex; and 
applying electromagnetic energy having a wavelength of at least 488 nm to the skin tissue-RB complex in a manner effective to bond the complex, 
wherein the skin lesion and/or graft is not contacted with an exogenous cross-linkable substrate, 
thereby promoting a partial or complete repair of the skin lesion.

2. The method of claim 1, wherein the subject is a human.

3. The method of claim 1, wherein the skin lesion is caused by a laceration, burn, surgical procedure or ulceration.

4. The method of claim 1, wherein repair of the lesion comprises a seal that is resistant to shear stress.

5. The method of claim 1, wherein the application of electromagnetic energy to the tissue-photosensitizer complex occurs without more than a 15° C. rise in temperature.

6. The method of claim 1, wherein the application of electromagnetic energy to the tissue-photosensitizer complex occurs without more than a 10° C. rise in temperature.

7. The method of claim 1, wherein the application of electromagnetic energy to the tissue-photosensitizer complex occurs without more than a 3° C. rise in temperature.

8. The method of claim 1, wherein the electromagnetic energy applied is less than 2000 J/cm2.

9. The method of claim 1, wherein the electromagnetic energy is applied at an irradiance less than 1.5 W/cm2.

10. The method of claim 1, wherein the electromagnetic energy is applied at an irradiance of about 0.60 W/cm2.

11. A method for bonding musculoskeletal tissue with a tissue seal, comprising:
contacting a first musculoskeletal tissue, a second musculoskeletal tissue, and rose bengal at a concentration of less than 1.0% weight per volume to form a musculoskeletal tissue-photosensitizer complex; and 
applying electromagnetic energy having a wavelength of at least 488 nm to the musculoskeletal tissue-photosensitizer complex in a manner effective to bond the musculoskeletal tissues, 
wherein the musculoskeletal tissues are not contacted with an exogenous cross-linkable substrate, 
thereby creating a tissue seal between the first and second tissues.

12. The method of claim 11, wherein the first and second musculoskeletal tissues are independently selected from the group consisting of tendon, ligament and cartilage.

13. The method of claim 11, wherein the contacting step occurs ex vivo.

14. The method of claim 11, wherein the contacting step occurs in vivo in a subject.

15. The method of claim 14, wherein the subject is a human.

16. The method of claim 11, wherein the application of electromagnetic energy to the musculoskeletal tissue-photosensitizer complex occurs without substantial thermal tissue damage.

17. The method of claim 11, wherein the application of electromagnetic energy to the musculoskeletal tissue-photosensitizer complex occurs without more than a 15° C. rise in temperature.

18. The method of claim 11, wherein the application of electromagnetic energy to the musculoskeletal tissue-photosensitizer complex occurs without more than a 10° C. rise in temperature.

19. The method of claim 11, wherein the application of electromagnetic energy to the musculoskeletal tissue-photosensitizer complex occurs without more than a 3° C. rise in temperature.

20. The method of claim 11, wherein the electromagnetic energy applied is less than 2000 J/cm2.

21. The method of claim 11, wherein the electromagnetic energy applied is between 125 and 750 J/cm2.

22. The method of claim 11, wherein the electromagnetic energy applied is between 250 and 750 J/cm2.

23. The method of claim 11, wherein the electromagnetic energy applied is between 250 and 500 J/cm2.

24. The method of claim 11, wherein the electromagnetic energy is applied at an irradiance less than 1.5 W/cm2.

25. The method of claim 11, wherein the electromagnetic energy is applied at an irradiance less than 1.0 W/cm2.

26. The method of claim 11, wherein the electromagnetic energy is applied at an irradiance of between 0.1 and 1.0 W/cm2.

27. The method of claim 11, wherein the two musculoskeletal tissues comprise the ends of a ruptured tendon.

28. The method of claim 1 or 11, wherein the rose bengal is applied at a concentration of less than 0.5% weight per volume in phosphate buffered saline.

29. The method of claim 1 or 11, wherein the rose bengal is applied at a concentration between 0.1 and 0.5% weight per volume in phosphate buffered saline.

30. The method of claim 11, wherein immediately after joining, the bonded tendon has a tensile strength which is greater than an untreated tendon.