1.

Introduction

Collagen is the predominant stress-bearing protein of connective tissues. Covalent crosslinks and hydrophobic interactions among collagen molecules serve to strengthen the fibrilar form of collagen.^{1 2 3} When the organization of this material is disrupted due to trauma, lytic disease, or surgery, new collagen is formed at the site of injury.^{4 5 6} This is followed by maturation and crosslinking of the collagen molecules. Proper healing is dependent on the correct reformation of the intra- and intermolecular interactions that strengthen the collagen fibrils. Traditional medical intervention in wound repair has relied on sutures and other sturdy mechanical devices to maintain tissues in apposition for a specific duration. However, there are numerous medical situations that are not amenable to these time-tested techniques. Accordingly, significant research effort has been focused on the development of adhesives for various medical applications.⁷

Tissue adhesives have been defined as substances that can form a polymeric material, where “polymerization must either hold tissues together or serve as a barrier to leakage.” ⁷ In line with this definition, most research and development in this area has been focused on technologies such as fibrin sealants,^{8 9} cyanoacrylates,¹⁰ hydrogels,¹¹ and protein/aldehyde composites.¹² Another approach to tissue bonding, often called laser tissue welding, involves the thermal coagulation and/or dehydration of tissue proteins themselves via the application of laser energy.^{13 14} The addition of light-absorbing dyes has been shown to improve heat transfer to the repair site during tissue welding.¹⁵ A modification of this approach adds exogenous human proteins to the site of welding to act as a type of “protein solder.” ^{16 17} While each of these methods has certain advantages, all exhibit one or more shortcomings such as weak bond strength, excessive collateral tissue damage, and incomplete defect repair. Some have also been shown to interfere with tissue fluid diffusion or to initiate a localized inflammatory response. It remains a considerable challenge to develop bonding agents that provide strong bonds and yet do not act as physical or chemical barriers to natural wound healing.

Recognition that certain compounds efficiently crosslink soluble proteins on photochemical activation led to the realization that these compounds might also have the potential to photochemically bond tissues, especially those primarily composed of collagen (i.e., skin, cornea, and meniscus). In fact, this novel approach to tissue bonding is effective, and the application of the brominated bisnaphthalimide 1 and the hydrophilic bisnaphthalimide 2 (Fig. 1) to the ex vivo photoinduced crosslinking of cartilage, skin, and pure collagen, as well as in vivo repair of cartilage, has been reported.^{18 19 20 21 22 23} The concept of forming a tissue bond by simply catalyzing the formation of covalent bonds between the tissue surfaces is innovative, and potentially solves the “barrier” problem inherent with traditional adhesives, as no foreign material is necessarily retained within the repair.

Figure 1

Photoactive tissue-bonding naphthalimides.

To further explore this technology, to develop an understanding of the relationship between naphthalimide structure and bonding ability, and to hopefully develop improved photochemical tissue-bonding agents, we designed and synthesized a set of substituted 4-amino naphtalimides (Fig. 2). These naphthalimides absorb visible light (λ _max ∼430 nm) and efficiently crosslink soluble protein.²⁴ Results of ex vivo experiments evaluating photochemical tissue bonding of bovine meniscus using these compounds are presented here.

Figure 2

Naphthalimides used in this study.

2.

Materials and Methods

2.3.

Sample Preparation and Irradiation

Fresh bovine knee meniscus was obtained from a local slaughterhouse within a few hours of death. Meniscal specimens were dissected from the knee and sliced into 10×5×0.3 mm strips with a specially designed microtome apparatus. Each strip was further divided lengthwise into two strips, which were placed on top of a thin piece of polyurethane film on a glass slide. The two opposing surfaces of the tissue strips were each coated using approximately 30 μL of PBS (control) or PBS naphthalimide solutions [Fig. 3(a)]. The coated surfaces were pressed together wrapped in the sheet of polyurethane film. An additional glass slide was then added to cover the sample, and two spring clips were employed to hold the two slides together, providing a pressure of approximately 3 kg/cm ². The specimens were then irradiated for 6 min at room temperature [Fig. 3(b)].

Figure 3

In vitro bonding assay.

3.

Results

The relative strengths of bonds formed in meniscal tissue using the nine photoactive naphthalimides are shown in Fig. 4. The bond strengths for these compounds (under the conditions of these tests) ranged from 0.47 kg/cm ² (for compound 3) to 1.38 kg/cm ² (for compound 11), with an average background bond strength of 0.29 kg/cm ² for control samples (irradiation/PBS with no naphthalimide). In every case, the failure occurred cleanly at the interface between the strips of meniscal tissue. Although these bonds are much weaker than a simple mattress suture (10.2 kg/cm ² in human meniscus²⁶), they are comparable to those previously reported for naphthalimide-mediated photochemical bonding of cartilage^{18 22} and skin²¹ and also to bonds formed in porcine aorta (∼1.1 kg/cm ²) using an albumin/indocyanine green thermal welding technique.²⁷ A recent study²⁸ of the photosensitized adhesion of porcine skin using rose bengal reported much weaker bonding (0.06 to 0.15 kg/cm ²) .

Figure 4

Relative bond strengths.

To examine whether this bonding technology exhibits a dose/response relationship, we also examined the bonding abilities of compound 9 at four different concentrations (Fig. 5). Although this limited experimentation did not reveal a clear linear correlation between bond strength and concentration, the bonding strength appears to plateau around approximately 24 mM (similar results were seen for other compounds; data not shown).

Figure 5

Bond strength as a function of Naphthalimide concentration.

4.

Discussion

Unfortunately the photochemical tissue-bonding results presented here are associated with large standard deviations, which seem to be inherent in this particular assay. The extreme variability observed in the available meniscal tissue is certainly a significant contributor to this poor reproducibility. This includes not only variation between samples derived from different animals or legs, but even within a single meniscus. Even so, a gross trend in the structure-activity relationship of this set of compounds can still be recognized. Compounds 3 to 6 are all poor reagents for adhering meniscal tissue. Those particular compounds contain either a nitrogen or an oxygen residue attached to the the 4^′ amino group by a two-carbon linker. On the other hand, compounds 8 to 11, which have either a hydrogen or a simple alkyl group attached to the 4^′ amine, are superior photochemical tissue bonding agents. One possible rationale for this behavior may come from recent research that has demonstrated the efficient formation of charge-separated species from 4-aminonaphthalimides,²⁹ which can readily extract electrons from a beta-amino substituted 4-amino substituents, resulting in fluorescence quenching. Accordingly, the differing activities of naphthalimides in the ex vivo tissue-bonding assay might be predicted by examining the potential for this nonproductive intramolecular electron transfer. Additionally, as compounds 8 to 11 are also the most hydrophobic compounds tested [as determined by reverse-phase high-performance liquid chromatography (HPLC) retention], it is reasonable to propose that their enhanced tissue-bonding ability may reflect enhanced tissue or protein interactions related to their hydrophobicity. We continue to pursue mechanistic studies in this area, including studies aimed at explaining the role that the substituents on the 4-amino group play in tissue bonding.

Overall, the research described here has identified structural features that might be used to design new and improved photoactivated tissue-bonding agents. Further studies are focused on better identifying the molecular basis for this tissue-bonding technology, and in parallel, the development of more active naphthalimides for meniscal repair.