Mechanical Properties of Biomimetic Tissue Adhesive Based on the

Biomacromolecules 2004, 5, 1270-1279

1270

Mechanical Properties of Biomimetic Tissue Adhesive Based on the Microbial Transglutaminase-Catalyzed Crosslinking of Gelatin Martin K. McDermott,† Tianhong Chen,‡ Christina M. Williams,§ Kolleen M. Markley,| and Gregory F. Payne*,‡,⊥ Division of Mechanics and Materials Science, Office of Science and Technology, Food and Drug Administration, 9200 Corporate Blvd., HFZ-150, Rockville, Maryland 20850, Center for Biosystems Research, University of Maryland Biotechnology Institute, 5115 Plant Sciences Building, College Park, Maryland 20742, Department of Biomedical Engineering, Texas A&M University, College Station, Texas 77843-0100, Department of Biomedical Engineering, Marquette University, Milwaukee, Wisconsin 53201-1881, and Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250 Received December 15, 2003; Revised Manuscript Received March 11, 2004

Fibrin sealants are a type of soft tissue adhesive that employs biochemical reactions from the late stages of the blood coagulation cascade. Intrinsic to these adhesives are a structural protein and a transglutaminase crosslinking enzyme. We are investigating an alternative biomimetic adhesive based on gelatin and a calciumindependent microbial transglutaminase (mTG). Rheological measurements show that mTG catalyzes the conversion of gelatin solutions into hydrogels, and gel times are on the order of minutes depending on the gelatin type and concentration. Tensile static and dynamic loading of the adhesive hydrogels in bulk form demonstrated that the Young’s modulus ranged from 15 to 120 kPa, and these bulk properties were comparable to those reported for hydrogels obtained from fibrin-based sealants. Lap-shear adhesion tests of porcine tissue were performed using a newly published American Society for Testing and Materials (ASTM) standard for tissue adhesives. The gelatin-mTG adhesive bound the opposing tissues together with ultimate adhesive strengths of 12-23 kPa which were significantly higher than the strength observed for fibrin sealants. Even after failure, strands of the gelatin-mTG adhesive remained attached to both of the opposing tissues. These results suggest that gelatin-mTG adhesives may offer the benefits of fibrin sealants without the need for blood products. Introduction Soft tissue adhesives are required to perform a variety of functions: stop bleeding, seal leaks, bind tissue, and/or facilitate healing.1,2 Typically, these functions are met through the in situ generation of a three-dimensional polymeric network that is bonded to the tissue. There are two chemical approaches commonly used for generating such networks: the in situ polymerization of reactive monomers and the in situ crosslinking of preformed polymers. An example of the former is the cyanoacrylate-based adhesives that undergo rapid in situ polymerization upon contact with the tissue. An example of the latter is the gelatin-based adhesives that are in situ crosslinked by resorcinol and formaldehyde. A disadvantage of both of these adhesives is the toxicity of formaldehyde which is a degradation product of cyanoacrylate adhesives3,4 and a reactant in gelatin-resorcinolformaldehyde adhesives. There has been considerable recent * To whom correspondence should be addressed. Phone: (301) 4058389. Fax: (301) 314-9075. E-mail: [email protected]. † Food and Drug Administration. ‡ University of Maryland Biotechnology Institute. § Texas A&M University. | Marquette University. ⊥ University of Maryland, Baltimore County.

effort to develop soft tissue adhesives that are less toxic and address other limitations of currently marketed adhesives. These efforts include the use of alternative materials (e.g., albumin, polysaccharides, poly(ethylene oxide)s),5-10 better crosslinkingchemistries(e.g.,glutaraldehyde,carbodiimide),11-15 and more controllable polymerization reactions (e.g., photoinitiated polymerization).16-18 Biochemical crosslinking approaches could offer a more biocompatible alternative to synthetic crosslinking for the in situ generation of the three-dimensional polymeric network required for adhesion. One biochemical approach is to exploit the water-resistant adhesive that allows mussels to attach to wet and submerged surfaces (i.e. the mussel glue).19,20 Scheme 1a shows that the mussel uses an enzyme to oxidize phenolic residues (i.e., tyrosine or dihydroxyphenylalanine residues) of its adhesive protein. These oxidized residues undergo subsequent uncatalyzed crosslinking reactions to yield the three-dimensional polymeric network required for adhesion.21-24 A second biochemical crosslinking approach relies on the reactions that naturally occur during blood coagulation. Scheme 1b shows that during coagulation thrombin cleaves the blood protein fibrinogen into fibrin that undergoes subsequent crosslinking by factor XIIIa. As

10.1021/bm034529a CCC: $27.50 © 2004 American Chemical Society Published on Web 04/21/2004

Biomimetic Tissue Adhesive Scheme 1

illustrated in Scheme 1b, factor XIIIa is a calcium-dependent transglutaminase enzyme.25-27 Fibrin sealants are currently used in clinical practice28,29 and are prepared by mixing two solutions, typically one contains thrombin and calcium chloride and the second contains fibrinogen and factor XIII (factor XIII is a zymogen that is activated into factor XIIIa by thrombin and calcium27). One concern with these biochemical approaches is the source of the protein; the mussel’s adhesive protein is difficult to obtain, and components of the fibrin sealant are derived from blood. We propose an alternative adhesive that is analogous to the fibrin sealant. Like the fibrin sealant, Scheme 1c shows that this proposed adhesive exploits the same biochemical crosslinking chemistry as the blood coagulation cascade. The differences are that the proposed adhesive uses gelatin as the “structural” protein (not fibrin) and a calcium-independent microbial transglutaminase as the crosslinking catalyst (not factor XIIIa).

An adhesive based on gelatin and microbial transglutaminase (mTG) should be nontoxic since gelatin is already used in various medical products while mTG is approved for food use.30 (It should be noted that mTG’s approval for food use does not necessarily indicate that it will be biocompatible in medical applications.) A gelatin-mTG adhesive should also be simple to produce since both components are commercially available and derived from sources more convenient than blood. Finally, a gelatin-mTG adhesive may be simpler to use and subject to fewer sources of variability since there are fewer components and only one reaction step (as illustrated in Scheme 1b, the fibrin sealant requires two

Biomacromolecules, Vol. 5, No. 4, 2004 1271

thrombin-catalyzed reactions and one transglutaminasecatalyzed reaction). Here we examined whether the mTG-crosslinked gelatin hydrogels offer appropriate mechanical properties for applications as a soft tissue adhesive. There are two challenges to characterizing the mechanical properties of such adhesives. First, when such an adhesive “sets”, its bulk mechanical properties undergo dramatic changes and these changes are not readily observed using a single measurement method. Typically, “setting” is initiated by mixing the structural and crosslinking components.31 As the three-dimensional polymeric network is being formed, the sample undergoes a characteristic sol-gel transition that yields an elastic gel. Rheology is commonly used to examine the sol-gel transition (i.e., to measure gel time), monitor the initial evolution of mechanical properties, and evaluate the properties of weak gels.32-36 Once the hydrogels become strong, however, rheology becomes less convenient; gels formed outside the rheometer cannot be easily loaded onto the instrument while gels formed within the instrument adhere to the parts (i.e., to the cone and plate). To characterize the mechanical properties of strong gels, standard tensile tests and dynamic mechanical analysis can be used.37 The second challenge in evaluating the mechanical properties of a tissue adhesive is the testing method for establishing its adhesive strength. Numerous ASTM methods exist for industrial adhesives. However, these methods are not appropriate for evaluating tissue adhesives because tissues are mechanically compliant, have variable moisture levels, and present chemically complex surfaces. The lack of appropriate methods coupled with an increasing interest in medical adhesives has resulted in the development of a new ASTM method for testing tissue adhesives. This method is based on traditional lap-shear methods but employs porcine skin as the model tissue.38 In the current study, we designed fixtures to create lap-shear adhesive joints that could be tested following this ASTM method. Materials and Methods Materials. Gelatin (type A from porcine) was obtained at two separate bloom values, 175 and 300 (Sigma-Aldrich, St. Louis, MO). Bloom is a standard industrial measure used to rate gelatin “quality” and is related to the shear modulus of physical gelatin gels. We selected a gelatin prepared by acid treatment (type A) and not base treatment (type B), because base treatment hydrolyzes the amide groups of glutamine residues39 and suppresses enzymatic crosslinking.40 The calcium-independent microbial transglutaminase (mTG; Activa TI) was obtained from Ajinomoto and was used without further purification. This enzyme is supplied as a proprietary formulation with a maltodextrin support and is reported by the manufacturer to have a specific activity of 100 U/gm (lot number L-04207). The skin tissue was obtained from Brennen Medical (St. Paul, MN) and was nonperforated Porcine Mediskin Xenograft (S-106) that was supplied frozen in 7 × 9 cm2 sections with a thickness of 0.032 ( 0.005 cm. The fibrin sealant, BaxTisseel Fibrin Sealant, was kindly supplied by Baxter Healthcare Corp. (Glendale, CA).

1272

Biomacromolecules, Vol. 5, No. 4, 2004

A concentrated enzyme solution was prepared for each experiment by mixing 0.5 gm of mTG per mL of deionized water. This enzyme solution was stored on ice until it was ready for use. A concentrated gelatin solution was prepared by adding 50 gm of gelatin to 100 mL of warm deionized water and mixing at 50 °C until the protein was dissolved. The pH of this concentrated gelatin solution was adjusted to 6.0 by adding small amounts of 1 M NaOH. Aliquots from this concentrated gelatin solution were then mixed with deionized water to prepare solutions with differing gelatin concentrations. The gelatin solutions were either used immediately, or stored at 4 °C for no more than 4 days. Gelatin solutions that were refrigerated were melted at 50 °C for no more than 1 h prior to use. Rheological Testing of Adhesive. Rheological tests were performed to examine the dynamics of gel formation. Gel formation was initiated by adding mTG solution to gelatin solutions to achieve a final enzyme activity of 15 U/gmgelatin.41 After mixing mTG, the solution was immediately loaded on top of the bottom plate of the rheometer, bubbles were removed from the solution using a pipet, and the rheometer’s top plate (i.e., the cone) was lowered onto the sample. Excess solution that exuded from the gap between the top cone and bottom plate was removed using a paper towel, silicon oil was placed along the edge of top plate to limit sample drying, and the rheometer test was initiated. Less than 2 min were required from the time mTG was mixed with the gelatin solution until the rheological test was initiated. Rheological tests were performed using a Thermo Haake RheoStress 1 rheometer (Thermo Electron Corporation, Waltham, MA) at a temperature of 37 ( 1 °C. A 6.0 cm diameter cone-and-plate sensor was used with an angle of 1° and a gap distance of 0.052 mm. The samples were subjected to an oscillatory stress of 0.5 Pa at a frequency of 0.1 Hz. Bulk Mechanical Testing of Adhesive. The bulk mechanical properties of the gelatin-mTG gels were characterized by tensile static and dynamic testing. The samples were prepared by mixing mTG with gelatin solutions to a final enzyme activity of 15 U/gm-gelatin. After adding mTG, the solutions were poured into a Petri dish, sealed with a dampened lid and allowed to undergo crosslinking on a level surface at 37 °C. After incubating for 90 min, the crosslinked hydrogel was lifted from the Petri dish and cut into tensile specimens having cross-sectional dimensions of 0.95 ( 0.03 cm and 0.32 ( 0.01 cm and lengths of 2 to 2.5 cm. After cutting the specimens and measuring their dimensions at room temperature (which required about 20 min), the tensile specimens were returned to the humid, 37 °C incubator. The first sample was tested 150 min after mixing the enzyme into the gelatin solution. The mechanical tests were performed using a dynamic mechanical thermal analyzer (DMTA) V Instrument (Rheometric Scientific, Inc., Piscataway, NJ). The tensile samples were removed from the incubator and placed in the tensile fixture grips that were separated by a 1 cm gap. A 0.03 cm diameter wire thermocouple was inserted into a portion of the sample that protruded above the top grip. After closing the sample chamber, 2-5 min were required for the sample

McDermott et al.

Figure 1. Lap-shear adhesive test. Schematics illustrate how the adhesive joints were formed and tested. Photographs of an adhesive joint alone and after mounting in the mechanical analyzer.

to reequilibrate to 37 ( 1 °C. The sample was then loaded at a strain rate of 1%/sec to a strain of 20% and then unloaded back to 0% strain. Initial studies showed that the stressstrain curves were different for the first and second loading/ unloading cycles. Such differences are commonly observed with soft, biological materials.42 The stress-strain curves for subsequent loading/unloading cycles resulted in stress-strain curves that were similar to that for the second cycle (we examined 4 loading/unloading cycles in our initial studies). Thus, we used the stress-strain curve from the sample’s second loading/unloading cycle to determine the Young’s modulus (E). E was calculated as the slope of the linear regression line for data between 0 and 4% strain. Immediately after completing the tensile static test, the sample was dynamically loaded at a peak strain of 1.5% and frequency of 0.5 Hz. These dynamic tests allowed the determination of the complex modulus (E*), storage modulus (E′), loss modulus (E′′), and loss tangent (tan δ ) E′′/E′). Lap-Shear Testing of Adhesive Strength. Lap-shear testing was performed following the procedures in ASTM F2255-03 (“Test Method for Strength Properties of Tissue Adhesives in Lap-Shear by Tension Loading”). Figure 1 shows that 0.90 ( 0.04 cm2 porcine skin tissue samples were attached to the aluminum backing using ethyl cyanoacrylate glue (Krazy Glue, Toagosel Co., Ltd, Japan). As illustrated in Figure 1, about 0.15 mL of gelatin-mTG adhesive was applied to one tissue surface, and then the two surfaces were lapped together. A 47.5 ( 0.3 gm lead weight was placed on the adhesive joint directly over the tissues. The adhesive joint with the weight were incubated at 37 ( 1 °C in enough water to cover the adhesive joint (but not the weight). After 120 min, the adhesive joint was removed from the water and attached to the tension fixture of the DMTA. After the sample temperature was equilibrated to 37 ( 1 °C in 1-2 min, the upper and lower grips were separated at a speed of 5 mm/min to measure the adhesive shear strength.

Biomimetic Tissue Adhesive

Figure 2. Evolution of mechanical properties for gels formed by the mTG-catalyzed crosslinking of gelatin. Elastic (G′) and viscous (G′′) moduli for hydrogels formed from (a) 175 bloom gelatin and (b) 300 bloom gelatin. (c) Complex viscosity (η*) of these two samples. Gels were prepared by incubating gelatin (16.5%) with mTG (15 U/gmgelatin) at 37 °C while these samples were subjected to oscillatory stresses (0.5 Pa at 0.1 Hz).

Results and Discussion Formation of Adhesive Hydrogel. Rheological methods were used to characterize the dynamics of gel formation. In these experiments, a gelatin solution was mixed with transglutaminase and immediately loaded onto the rheometer where the samples were subjected to oscillatory stresses. Figure 2 shows typical results for samples prepared with gelatins of 175 and 300 bloom. Figure 2a shows that immediately after mixing mTG with the 175 bloom gelatin, the sample behaved as a solution with the elastic modulus (G′) being less than the viscous modulus (G′′). Over time, Figure 2a shows that both moduli increased, but G′ increased more rapidly and became equal to G′′ after 28 min. The point at which G′ becomes equal to G′′ is typically used as a rheological measure of the gel-point.32,36 Figure 2b shows


Figure 3. Effect of gelatin bloom and concentration on gel formation. (a) Gel time is the time where the elastic and viscous moduli cross (i.e., G′ ) G′′). (b) Complex viscosity (η*) evaluated at the gel point. (c) Elastic moduli evaluated at the gel point. Tests were performed in triplicate.

results for a sample prepared with a 300 bloom gelatin. As expected, the higher bloom gelatin had higher initial moduli and gelled more rapidly compared to the lower bloom gelatin of Figure 2a. A convenient comparison of the two samples is summarized in Figure 2c that shows the complex viscosity (η*) increases more rapidly for the higher bloom gelatin. The results in Figure 2 show that transglutaminase catalyzed the conversion of gelatin solutions into gels (gelatin solutions lacking transglutaminase do not form gels at 37° C). These gels formed as a result of the covalent crosslinking of the gelatin chains and differ from the thermally reversible physical gelatin gels that are induced by cooling.40,41,43 A series of rheological tests were performed with samples containing different gelatin concentrations. Figure 3a shows that the gel time depends on the gelatin bloom with lower bloom gelatin taking longer to gel. Depending on the gelatin concentration, we observed gel times to vary between 10 and 29 min for the 175 bloom gelatin, and 2.7 to 5.1 min for the 300 bloom gelatin. These time frames are convenient

1274


in the sense that it allows the gelatin and mTG to be thoroughly mixed before being applied to the tissue. If necessary, it should be possible to adjust the gel time by adjusting the mTG activity or adding other ingredients (not investigated here). For instance, previous studies showed that the gel time is significantly reduced by the inclusion of a high molecular weight polysaccharide (i.e. chitosan) to the solution.41 Figure 3, parts b and c, shows the complex viscosity (η*) and elastic moduli at the gel point (G′ ) G′′) for samples prepared at differing gelatin concentrations. These parameters provide a measure of the flow properties and gel strength at a single time, the gel-point, and provide an indication of the ease of mixing and applying the adhesive to the tissue surface.31 If an adhesive is too weak or is applied before it has begun to gel, then it will have low viscosities and elasticities. In this instance, the adhesive will be able to flow into and mechanically “interlock” with tissue to improve adhesive-tissue bonding. However, a lower viscosity adhesive may lack sufficient cohesive strength to be retained where it is applied and it may flow away. This problem can be particularly important if the adhesive must be applied to wet tissue. In contrast, stronger gels or gels that have been allowed to set longer have greater cohesive strength (i.e., larger G′), retain their shape, but may not effectively penetrate and interlock with tissue. Thus, the adhesive’s flow properties and gel strength are practically important and the desired values will depend on the specific situation (e.g., wet or dry tissue). The adhesive’s flow properties and gel strength can be controlled by the enzyme activity, the gelatin bloom, and the gelatin concentration, whereas the results in Figure 2 show that these properties are rapidly changing during crosslinking. Bulk Mechanical Properties of Adhesive Hydrogel. The bulk mechanical properties of the mTG-catalyzed gelatin gels were characterized by static and dynamic tensile loading. For these studies, mTG was mixed with a gelatin solution and incubated in a Petri dish to form hydrogels that were subsequently cut into tensile specimens. Approximately 2.5-3 h after initiating the reaction, the specimens were mounted in tensile fixtures for analyses. In the first study, the specimen was loaded in tension at a rate of 1%/s (0.01 cm/s) to 20% strain and then unloaded back to 0% strain. Loading and unloading were performed for two cycles, and results from the second cycle were analyzed. The typical stress-strain curve of Figure 4a shows that little hysteresis was observed between loading and unloading which indicates that the sample underwent little plastic deformation. This behavior is expected for a crosslinked elastic network. The Young’s modulus (E) was calculated from Figure 4a using the slope of the linear regression line for strains between 0 and 4%. The value of E determined for the sample of Figure 4a is 61 kPa. Tensile static tests were performed for samples prepared at differing gelatin concentrations. Figure 4b shows that the magnitude of E was between 15 and 120 kPa. This range is similar to that reported for fibrin-based adhesives containing high fibrin content (15 and 30 kPa for sealants prepared with 30 and 70 mg/mL fibrinogen, respectively).44 Figure 4b also

McDermott et al.

Figure 4. Tensile static testing of mTG-catalyzed gelatin-based gels. Samples were prepared by reacting gelatin with mTG (15 U/gmgelatin) in a Petri dish to form thin hydrogels. After reaction, the gels were cut into tensile specimens that were mounted in tensile fixtures, loaded to 20% strain and then unloaded. (a) Typical stress-strain curve for a sample prepared with 300 bloom gelatin (16.5%) during its second loading/unloading cycle. (b) Young’s modulus (E) for gelatin-mTG gels prepared with differing gelatin blooms and concentrations. Tests were performed with 5-10 replicates. Table 1. Bulk Mechanical Properties of Hydrogels Formed by mTG-Catalyzed Crosslinking of Gelatin gelatin bloom 175 175 300 300 300 300

(%)

E (kPa)

E* (kPa)

E′ (kPa)

16.5 15 (3 8.5 ( 3.2 7.8 ( 3.3 26.5 40 ( 7 32 ( 7 32 ( 7 16.5 60 ( 5 60 ( 5 60 ( 5 19.9 77 ( 12 72 ( 14 72 ( 14 23.1 91 ( 13 99 ( 24 99 ( 24 26.5 120 ( 22 130 ( 20 130 ( 20

E′′ (kPa)

tan δ

2.8 ( 1.7 2.5 ( 0.9 1.9 ( 0.3 1.9 ( 0.2 6.8 ( 5.1 6.4 ( 4.2

0.39 ( 0.22 0.08 ( 0.04 0.03 ( 0.01 0.03 ( 0.01 0.06 ( 0.04 0.05 ( 0.03

shows that E tended to increase with increasing gelatin concentration which is also consistent with results from fibrin-based adhesives.44 Finally, Figure 4b shows that bulk properties of the gelatin-mTG adhesive were significantly dependent on the gelatin bloom with higher bloom gelatin yielding higher values for E. After completing the tensile static tests, each sample was then dynamically loaded at a peak strain of 1.5% and a frequency of 0.5 Hz. These measurements allow the following properties to be determined; complex modulus (E*), storage modulus (E′), loss modulus (E′′), and tanδ ()E′′/ E′). Results from these studies are summarized in Table 1. For each of the samples prepared with the 300 bloom gelatin, the values for E* and E′ are nearly the same, whereas the values for tan δ are substantially less than 0.1. These results indicate that these materials behave as elastic solids. Consistent with the results in Figure 4b, Table 1 shows that samples prepared from the 175 bloom gelatin had lower E* and E′ values compared to samples prepared from 300 bloom



Figure 5. Alignment fixture to assemble adhesive joints. Schematics and photographs show adhesive joints in various stages of formation in the alignment fixture. Seven aluminum backings with tissue were placed in the fixture separated by removable spacer bars. Adhesive (≈0.15 mL) was applied to the upper tissue surfaces and the two tissue surfaces were contacted to create the adhesive joint. The spacer bar separating two finished adhesive joints was removed as shown, leaving the adhesive joints aligned and exposed to the environment. The completed adhesive joints in the alignment fixture were immersed in water and allowed to cure at 37 °C.

gelatin. Also, Table 1 shows good agreement in results obtained from tensile static testing and dynamic loading of the samples (i.e., E ≈ E*). It is desirable to relate the observed mechanical properties for the mTG-crosslinked gelatin gel to the underlying network structure. A strict chemical approach to assess network structure (i.e., the types and number of crosslinks) is complicated by the facts that gelatin is not a simple monodisperse starting material, and the crosslinks are difficult to detect/quantify because the linkage is not unique (i.e., an amide crosslink replaces an amide side chain). An alternative (although approximate) approach to estimate network structure is to employ simplified physical models and use averaged values for gelatin. As outlined in the Appendix, we employed an idealized model for rubber elasticity as suggested by Sperling.45 Also, we used an average amino acid composition for type A gelatin obtained from Rose39 and an average molecular weight for our gelatin provided by the supplier. This method indicates that the crosslink density for our gels reaches 8 mole crosslink/m3 and 2 crosslinks per gelatin chain, but only about 20% of the glutamine and lysine residues undergo crosslinking. Strength of Gelatin-mTG Adhesive. The testing of tissue adhesives generally results in high variability for numerous reasons: the starting tissue can have varying properties (e.g., moisture levels); alignment of the tissue surfaces prior to contact can be challenging; and the adhesive joints can be damaged in sample handling. To minimize these problems, we constructed the alignment fixture shown in Figure 5 that allows seven adhesive joints to be formed rapidly and reproducibly. Initially, skin tissue was bonded to aluminum backings (using cyanoacrylate adhesive) and the tissue was kept moist by covering it with gauze that had been soaked in PBS buffer. Seven backings with tissues attached were placed onto the alignment fixture to create the lower tissue surfaces of the seven adhesive joints. As

illustrated in Figure 5, the adhesive (≈0.15 mL) was spread onto each upper tissue surface, and then the upper and lower surfaces were contacted to create an adhesive joint. A weight was attached to the upper backing to apply a consistent force to each adhesive joint while it was curing. The sliding spacer bars shown in Figure 5 ensured that the upper and lower tissue surfaces were aligned when they were being contacted. These spacer bars were removed immediately after the tissue surfaces were contacted and the adhesive joint was formed. Removing the spacer bars avoids the possibility that any adhesive that is exuded from between the tissue surfaces can form a bond with the surface of the spacer. Any bonds formed between the adhesive joint and other surfaces can make it difficult to remove the adhesive joint from the alignment fixture and risks damage to the adhesive bond formed between the two tissue surfaces. Approximately 3.5 min were required to complete the formation of all seven adhesive joints in the alignment fixture. After forming the seven adhesive joints, the alignment fixture was submerged in a water bath at 37 °C for 2 h while the adhesive was curing. When incubation was complete, the adhesive joints were removed from the alignment fixture, mounted on the DMTA and tested by applying loads to separate the surfaces at a rate of 5 mm/min. Figure 6a shows a typical force versus deflection curve for this lap-shear test in which failure was defined as the maximum load achieved (the maximum load for the sample in Figure 6a was 1.6 N). When the sample was strained past failure, the force resisting further deflection diminished slowly indicating that failure was not brittle. The adhesive’s toughness results because strands of gel remain connected to the two skin surfaces even after failure. In fact, Figure 6b shows that some strands remain even after the resisting force diminished to zero and the samples were removed from the DMTA grips.

1276


McDermott et al.

Figure 6. Lap-shear testing of tissue adhesive. (a) Typical force versus deflection curve for an adhesive joint formed with 300 bloom gelatin (16.5%). Failure is the maximum load achieved (1.6 N for this sample). (b) Photograph showing that strands of adhesive remain attached to the upper and lower tissue surfaces even after the sample failed and was removed from the DMTA grips.

We prepared gelatin-mTG adhesives at differing gelatin concentrations to determine how this affects the adhesive’s shear strength. The strength was determined by dividing the maximum load by the sample’s surface area. Each gelatin concentration was tested 11-14 times, and the results are shown in Figure 7a. The first observation from Figure 7a is that there is considerable variability in the results despite the use of well-controlled experimental conditions and numerous replicates. Variability is common when testing adhesives and even greater variability is expected when biological specimens are tested. The second observation from Figure 7a is that the gelatin concentration did not appear to affect the adhesive’s strength. This observation differs somewhat from results that show the adhesive strength of the fibrin sealant increases with fibrin content.38 To further consider why the gelatin concentration does not affect the adhesive strength, we plotted the adhesive strength (measured in the lap-shear test) versus the Young’s modulus (E) of the gelatin-mTG hydrogel (measured in the tensile static test). E is a bulk property of the gelatin-mTG hydrogel and correlates with the gel’s cohesive strength. Figure 7b shows that the adhesive’s shear strength is not improved by using adhesives with increased cohesive strength. This result suggests that adhesives made from stiffer hydrogels may not effectively penetrate the tissue. In this case, tissue-adhesive bonding may be weak, and the sample may be prone to adhesive failure. This explanation is consistent with observations that fibrin sealants prepared with high concentrations of fibrin had high E values and failed half the time through an adhesive mode. When the fibrin sealants were prepared with lower fibrin levels, they had low E values and failed primarily through a cohesive mode (71% of the time).44 Table 2 shows a summary of the results for the gelatinmTG adhesive prepared in this study and compares this material with other tissue adhesives. The first entry in Table

Figure 7. Adhesive strength of the gelatin-mTG adhesive as determined from the lap-shear test with porcine tissue. (a) Adhesive lap-shear strength versus concentration of gelatin (300 bloom). Lapshear tests were performed with 11-14 replicates. (b) Cross-plot between the adhesive strength (measured using lap-shear) and the Young’s modulus (measured for the bulk hydrogel) showing that the adhesive’s strength is not significantly altered by changes in the hydrogel’s cohesive strength. Table 2. Comparison of Adhesive Strengths type of adhesive

adhesive strength (kPa)

gelatin-mTG (16.5 to 26.5% gelatin) fibrin sealant (this study) fibrin-based sealant16 fibrin-based sealant6 fibrin-based sealant38 control: tissue alone control: tissue and mTG (no gelatin) control: tissue and gelatin (no mTG)

12-23 0.7 ( 0.2a 1.5 2.5 ( 0.6 up to 27 ( 8 NDb NDb NDb

a Most adhesive joints failed when they were removed from the alignment fixture and a strength could not be measured. Value reported is for three samples that could be measured. b Could not be measured because the adhesive joints failed when they were removed from the alignment fixture.

2 shows results for gelatin-mTG samples prepared with different gelatin concentrations (these are the same values reported in Figure 7). The adhesive strengths for these samples ranged from 12 to 23 kPa. These values are within 1 order of magnitude of values reported for tissue adhesives prepared from gelatin (12,16 15,14 and 20 kPa15), chitosan (3 kPa6), and cyanoacrylate (68 kPa16). We prepared several samples using a fibrin sealant. Under our conditions, we observed that most (85%) of the samples were so weak that the adhesive joints separated before they could be analyzed (i.e., it was not possible to mount them in the DMTA grips). Table 2 shows that the three samples that we were able to analyze had a lap-shear strength of 0.7 ( 0.2 kPa. As illustrated in Table 2, this value is comparable to values reported by others.6,16 In contrast, Sierra et al.38


reported adhesive strengths as high as 27 ( 8 kPa for fibrinbased sealants. Presumably, these differences reflect differences in testing materials and protocols. In summary, the results in Table 2 indicate that the gelatin-mTG adhesive offers strengths that are greater than, or comparable to, those reported for fibrin-based sealants. The final three entries in Table 2 are for controls prepared by deleting one or both components from the adhesive. For all three of these controls, the adhesion was negligible and the samples separated before they could be mounted on the DMTA. In the first control, neither gelatin nor mTG were placed on the tissue and no adhesion was observed. This control demonstrated that there was no other mechanism acting to cause the tissues to adhere to any significant extent. The second control was prepared by adding mTG but not gelatin. The lack of adhesion in this control indicates that mTG by itself cannot catalyze bonding of the porcine skin specimens. In the third control, gelatin was added without mTG and the result indicates that un-crosslinked gelatin lacks adhesive strength under the moist, 37 °C conditions used. Results from this third control also indicate that endogenous transglutaminases are either absent from the porcine skin, or are unable to catalyze sufficient crosslinking of the added gelatin. The conclusions from these three controls demonstrate that both gelatin and mTG are required for adhesion in our system. Conclusions Fibrin sealants exploit the biochemistry of the blood coagulation cascade and have become a standard adhesive for a variety of medical procedures. The coagulation cascade is mimicked here by the use of gelatin as a structural protein and the calcium-independent microbial transglutaminase (mTG) as a crosslinking catalyst. Compared to synthetic adhesives, the use of a transglutaminase (either mTG or factor XIIIa in the fibrin sealants) to catalyze crosslinking may offer benefits since low molecular weight reagents (monomer, crosslinkers, or initiators) are not required. Further, the resulting crosslinked protein network should be resorbable as a result of normal proteolytic processes. The results from this study demonstrate that gelatin-mTG hydrogels are formed over the course of a few minutes depending on the conditions used. This gel time is convenient as it allows the components to be thoroughly mixed prior to being applied to the tissue. Additionally, the results demonstrate that the mTG-catalyzed crosslinking of gelatin yields gels with bulk properties and adhesive strengths comparable to, or better than, those reported for the fibrin sealant. Thus, the results from this in vitro study indicate that the proposed gelatin-mTG adhesive offers appropriate mechanical properties for applications as a soft tissue adhesive. In addition to their use in soft tissue adhesives, transglutaminases appear to offer broader opportunities for the construction of functional biomaterials.46 Transglutaminases catalyze reactions under mild conditions with amino acid residue specificity (reactions occur between glutamine and lysine residues). These capabilities are being actively studied for the construction of multifunctional biomaterials (e.g., bi-


domain proteins and supramolecular protein networks).47-57 The availability of a calcium-independent microbial transglutaminase provides an additional, and potentially moreconvenient, means of generating functional biomaterials.40,41 To date, mTG has been examined for immobilizing enzymes,58 conjugating cyclodexdrins to an enzyme,59 and entrapping cells.60 Acknowledgment. We thank Bruce Fleharty and Randolph Bidinger at the FDA Machine Shop for manufacturing our fixtures, Baxter, Inc. for providing samples of fibrin sealant and the fibrin sealant mixing system, and Drs. Steidl and Janjua for their helpful discussions. Financial support was provided by the U.S. National Science Foundation (BES0114790), Department of Agriculture (2001-35504-10667), and Food and Drug Administration. Appendix. Estimation of Crosslinking Using idealized models for rubber elasticity, it is possible to estimate the crosslink density (µ) from the observed Young’s Modulus (E). According to Sperling,45 when crosslinking results in four elastically active chain segments E ) 6µRT The 300 bloom gelatin sample prepared with 26.5% gelatin was observed to have an E of 120 kPa (Table 1). According to the above equation, this measured modulus corresponds to a network with a crosslink density of µ ) 7.8 mol crosslink/m3. Assuming the average molecular weight for 300 bloom gelatin is 75 000 (estimate provided by the manufacturer), then this 26.5% gelatin solution has a gelatin concentration of approximately 3.5 mol/m3. Since we did not visually observe syneresis during gel formation, we assume the gelatin concentration in the crosslinked gel is also 3.5 mol/m3. From these numbers, we can estimate that there are 2.2 crosslinks per gelatin chain in the transglutaminase-catalyzed gelatin gel. The amino acid composition of type A gelatin indicates that 15 glutamine residues and 16 lysine residues would be present in a 600-residue chain (the approximate number of residues for a gelatin chain with molecular weight of 75 000) (see Table 3 of Rose39). Since one lysine and one glutamine residue participates in a crosslink, then the above calculation that there are 2.2 crosslinks per gelatin chain suggests that only about 20% of the lysine and glutamine residues are consumed to form active crosslinks. References and Notes (1) Smith, D. C. Adhesives and sealants. In Biomaterials science: an introduction to materials in medicine; Ratner, B. D., Hoffman, A. S., Schoen, F. J., Lemons, J. E., Eds.; Academic Press: San Diego, CA, 1996; pp 319-328. (2) Donkerwolcke, M.; Burny, F.; Muster, D. Tissues and bone adhesivess historical aspects. Biomaterials 1998, 19, 1461-6. (3) Tseng, Y. C.; Tabata, Y.; Hyon, S. H.; Ikada, Y. In vitro toxicity test of 2-cyanoacrylate polymers by cell culture method. J. Biomed. Mater. Res. 1990, 24, 1355-67. (4) Tseng, Y. C.; Hyon, S. H.; Ikada, Y.; Shimizu, Y.; Tamura, K.; Hitomi, S. In vivo evaluation of 2-cyanoacrylates as surgical adhesives. J. Appl. Biomater. 1990, 1, 111-9.

1278


(5) Mo, X.; Iwata, H.; Matsuda, S.; Ikada, Y. Soft tissue adhesive composed of modified gelatin and polysaccharides. J. Biomater. Sci. Polym. Ed. 2000, 11, 341-51. (6) Ishihara, M.; Nakanishi, K.; Ono, K.; Sato, M.; Kikuchi, M.; Saito, Y.; Yura, H.; Matsui, T.; Hattori, H.; Uenoyama, M.; Kurita, A. Photo-crosslinkable chitosan as a dressing for wound occlusion and accelerator in healing process. Biomaterials 2002, 23, 833-40. (7) White, J. K.; Titus, J. S.; Tanabe, H.; Aretz, H. T.; Torchiana, D. F. The use of a novel tissue sealant as a hemostatic adjunct in cardiac surgery. Heart Surg. Forum 2000, 3, 56-61. (8) Margalit, E.; Fujii, G. Y.; Lai, J. C.; Gupta, P.; Chen, S. J.; Shyu, J. S.; Piyathaisere, D. V.; Weiland, J. D.; De Juan, E., Jr.; Humayun, M. S. Bioadhesives for intraocular use. Retina 2000, 20, 469-77. (9) Hill, A.; Estridge, T. D.; Maroney, M.; Monnet, E.; Egbert, B.; Cruise, G.; Coker, G. T. Treatment of suture line bleeding with a novel synthetic surgical sealant in a canine iliac PTFE graft model. J. Biomed. Mater. Res. 2001, 58, 308-12. (10) Wallace, D. G.; Cruise, G. M.; Rhee, W. M.; Schroeder, J. A.; Prior, J. J.; Ju, J.; Maroney, M.; Duronio, J.; Ngo, M. H.; Estridge, T.; Coker, G. C. A tissue sealant based on reactive multifunctional poly(ethylene glycol). J. Biomed. Mater. Res. 2001, 58, 545-55. (11) Ennker, I. C.; Ennker, J.; Schoon, D.; Schoon, H. A.; Rimpler, M.; Hetzer, R. Formaldehyde-free collagen glue in experimental lung gluing. Ann. Thorac. Surg. 1994, 57, 1622-7. (12) Kobayashi, H.; Sekine, T.; Nakamura, T.; Shimizu, Y. In vivo evaluation of a new sealant material on a rat lung air leak model. J. Biomed. Mater. Res. 2001, 58, 658-65. (13) Matsuda, S.; Iwata, H.; Se, N.; Ikada, Y. Bioadhesion of gelatin films crosslinked with glutaraldehyde. J. Biomed. Mater. Res. 1999, 45, 20-7. (14) Sung, H. W.; Huang, D. M.; Chang, W. H.; Huang, R. N.; Hsu, J. C. Evaluation of gelatin hydrogel crosslinked with various crosslinking agents as bioadhesives: in vitro study. J. Biomed. Mater. Res. 1999, 46, 520-30. (15) Otani, Y.; Tabata, Y.; Ikada, Y. A new biological glue from gelatin and poly(L-glutamic acid). J. Biomed. Mater. Res. 1996, 31, 157166. (16) Nakayama, Y.; Matsuda, T. Photocurable surgical tissue adhesive glues composed of photoreactive gelatin and poly(ethylene glycol) diacrylate. J. Biomed. Mater. Res. 1999, 48, 511-21. (17) Li, C.; Sajiki, T.; Nakayama, Y.; Fukui, M.; Matsuda, T. Novel visible-light-induced photocurable tissue adhesive composed of multiply styrene-derivatized gelatin and poly(ethylene glycol) diacrylate. J. Biomed. Mater. Res. 2003, 66B, 439-46. (18) Ono, K.; Saito, Y.; Yura, H.; Ishikawa, K.; Kurita, A.; Akaike, T.; Ishihara, M. Photo-crosslinkable chitosan as a biological adhesive. J. Biomed. Mater. Res. 2000, 49, 289-95. (19) Strausberg, R. L.; Link, R. P. Protein-based medical adhesives. Trends Biotechnol. 1990, 8, 53-7. (20) Olivieri, M. P.; Baier, R. E.; Loomis, R. E. Surface properties of mussel adhesive protein component films. Biomaterials 1992, 13, 1000-8. (21) Burzio, L. A.; Waite, J. H. Cross-linking in adhesive quinoproteins: studies with model decapeptides. Biochemistry 2000, 39, 1114753. (22) Waite, J. H. Marine adhesive proteins: natural composite thermosets. Int. J. Biol. Macromolec. 1990, 12, 139-144. (23) Waite, J. H.; Tanzer, M. L. Polyphenolic substance of Mytilus edulis: novel adhesive containing L-DOPA and hydroxyproline. Science 1981, 212, 1038-1040. (24) McDowell, L. M.; Burzio, L. A.; Waite, J. H.; Schaefer, J. Rotational echo double resonance detection of cross-links formed in mussel byssus under high-flow stress. J. Biol. Chem. 1999, 274, 20293-5. (25) Greenberg, C. S.; Birckbichler, P. J.; Rice, R. H. Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues. FASEB J. 1991, 5, 3071-3077. (26) Adany, R.; Bardos, H. Factor XIII subunit A as an intracellular transglutaminase. Cell Mol. Life Sci. 2003, 60, 1049-60. (27) Lorand, L. Factor XIII: structure, activation, and interactions with fibrinogen and fibrin. Ann. N.Y. Acad. Sci. 2001, 936, 291-311. (28) Spotnitz, W. D. Commercial fibrin sealants in surgical care. Am. J. Surg. 2001, 182, 8S-14S. (29) Jackson, M. R. Fibrin sealants in surgical practice: An overview. Am. J. Surg. 2001, 182, 1S-7S. (30) Motoki, M.; Seguro, K. Transglutaminase and its use for food processing. Trends Food Sci. Technol. 1998, 9, 204-210.

McDermott et al. (31) Sierra, D. H.; O’Grady, K.; Toriumi, D. M.; Foresman, P. A.; Rodeheaver, G. T.; Eberhardt, A.; Feldman, D. S.; Lemons, J. E. Modulation of mechanical properties in multiple-component tissue adhesives. J. Biomed. Mater. Res. 2000, 52, 534-42. (32) Clark, A. H.; Ross-Murphy, S. B. Structural and mechanical properties of biopolymer gels. AdV. Polym. Sci. 1987, 83, 58-192. (33) Kavanagh, G. M.; Ross-Murphy, S. B. Rheological characterization of polymer gels. Prog. Polym. Sci. 1998, 23, 533-562. (34) Gilsenan, P. M.; Ross-Murphy, S. B. Viscoelasticity of thermoreversible gelatin gels from mammalian and piscine collagens. J. Rheol. 2000, 44, 871-883. (35) Hsu, S.-H.; Jamieson, A. M. Viscoelastic behaviour at the thermal sol-gel transition of gelatin. Polymer 1993, 34, 2602-2608. (36) Winter, H. H.; Mours, M. Rheology of polymers near liquid-solid line. AdV. Polym. Sci. 1997, 134, 165-234. (37) Anseth, K. S.; Bowman, C. N.; Brannon-Peppas, L. Mechanical properties of hydrogels and their experimental determination. Biomaterials 1996, 17, 1647-57. (38) Sierra, D. H.; Feldman, D. S.; Saltz, R.; Huang, S. A method to determine shear adhesive strength of fibrin sealants. J. Appl. Biomater. 1992, 3, 147-151. (39) Rose, P. Gelatin. In Encyclopedia of Polymer Science and Engineering; Mark, H., Bikales, N., Overberger, C., Menges, G., Eds.; WileyInterscience: New York, 1987; pp 488-513. (40) Crescenzi, V.; Francescangeli, A.; Taglienti, A. New Gelatin-Based Hydrogels via Enzymatic Networking. Biomacromolecules 2002, 3, 1384-1391. (41) Chen, T.; Embree, H. D.; Brown, E. M.; Taylor, M. M.; Payne, G. F. Enzyme-catalyzed Gel-formation of Gelatin and Chitosan. Potential for In Situ Applications. Biomaterials 2003, 24, 2831-2841. (42) Fung, Y. In Biomechanics. Mechanical Properties of LiVing Tissue; Springer-Verlag: New York, 1993; pp 261-262. (43) Babin, H.; Dickinson, E. Influence of transglutaminase treatment on the thermoreversible gelation of gelatin. Food Hydrocolloids 2001, 15, 271-276. (44) Sierra, D. H.; Eberhardt, A. W.; Lemons, J. E. Failure characteristics of multiple-component fibrin-based adhesives. J. Biomed. Mater. Res. 2002, 59, 1-11. (45) Sperling, L. Appendix 9.1. Gelatin as a physically crosslinked elastomer. In Introduction to Physical Polymer Science; WileyInterscience: New York, 2001; pp 424-428. (46) Sperinde, J.; Griffith, L. Synthesis and Characterization of Enzymatically-Cross-Linked Poly(ethylene glycol) Hydrogels. Macromolecules 1997, 30, 5255-5264. (47) Zisch, A. H.; Schenk, U.; Schense, J. C.; Sakiyama-Elbert, S. E.; Hubbell, J. A. Covalently conjugated VEGF- -fibrin matrixes for endothelialization. J. Controlled Release 2001, 72, 101-13. (48) Sakiyama-Elbert, S. E.; Hubbell, J. A. Controlled release of nerve growth factor from a heparin-containing fibrin-based cell ingrowth matrix. J. Controlled Release 2000, 69, 149-58. (49) Schense, J. C.; Bloch, J.; Aebischer, P.; Hubbell, J. A. Enzymatic incorporation of bioactive peptides into fibrin matrixes enhances neurite extension. Nat. Biotechnol. 2000, 18, 415-9. (50) Sakiyama-Elbert, S. E.; Hubbell, J. A. Development of fibrin derivatives for controlled release of heparin-binding growth factors. J. Controlled Release 2000, 65, 389-402. (51) Schense, J. C.; Hubbell, J. A. Three-dimensional migration of neurites is mediated by adhesion site density and affinity. J. Biol. Chem. 2000, 275, 6813-8. (52) Sakiyama, S. E.; Schense, J. C.; Hubbell, J. A. Incorporation of heparin-binding peptides into fibrin gels enhances neurite extension: an example of designer matrixes in tissue engineering. FASEB J. 1999, 13, 2214-24. (53) Schense, J. C.; Hubbell, J. A. Cross-linking exogenous bifunctional peptides into fibrin gels with factor XIIIa. Bioconjug. Chem. 1999, 10, 75-81. (54) Westhaus, E.; Messersmith, P. B. Triggered release of calcium from lipid vesicles: a bioinspired strategy for rapid gelation of polysaccharide and protein hydrogels. Biomaterials 2001, 22, 453-62. (55) Zhang, Z. Y.; Shum, P.; Yates, M.; Messersmith, P. B.; Thompson, D. H. Formation of fibrinogen-based hydrogels using phototriggerable diplasmalogen liposomes. Bioconjug. Chem. 2002, 13, 640-6. (56) Sanborn, T. J.; Messersmith, P. B.; Barron, A. E. In situ crosslinking of a biomimetic peptide-PEG hydrogel via thermally triggered activation of factor XIII. Biomaterials 2002, 23, 2703-2710. (57) Collier, J. H.; Messersmith, P. B. Enzymatic modification of selfassembled peptide structures with tissue transglutaminase. Bioconjug. Chem. 2003, 14, 748-55.

Biomimetic Tissue Adhesive (58) Fuchsbauer, H. L.; Gerber, U.; Engelmann, J.; Seeger, T.; Sinks, C.; Hecht, T. Influence of gelatin matrices cross-linked with transglutaminase on the properties of an enclosed bioactive material using beta-galactosidase as model system. Biomaterials 1996, 17, 14811488. (59) Villalonga, R.; Fernandez, M.; Fragoso, A.; Cao, R.; Di Pierro, P.; Mariniello, L.; Porta, R. Transglutaminase-catalyzed synthesis of

Biomacromolecules, Vol. 5, No. 4, 2004 1279 trypsin-cyclodextrin conjugates: kinetics and stability properties. Biotechnol. Bioeng. 2003, 81, 732-7. (60) Chen, T.; Small, D. A.; McDermott, M. K.; Bentley, W. E.; Payne, G. F. Enzymatic Methods for In situ Cell Entrapment and Cell Release. Biomacromolecules 2003, 4, 1558-1563.

BM034529A

Mechanical Properties of Biomimetic Tissue Adhesive Based on the

Recommend Documents