ARTICLE pubs.acs.org/Biomac
Polysaccharide-Based Adhesive for Biomedical Applications: Correlation between Rheological Behavior and Adhesion Aurelie Serrero,*,†,‡ Stephane Trombotto,† Yves Bayon,‡ Philippe Gravagna,‡ Suzelei Montanari,‡ and Laurent David*,† †
Universite de Lyon, Universite Lyon 1, UMR CNRS 5223, Ingenierie des Materiaux Polymeres, Laboratoire des Materiaux Polymeres et Biomateriaux (IMP/LMPB), F-69622 Villeurbanne Cedex, France ‡ Research and Development, Covidien, F-01600 Trevoux, France ABSTRACT: Adhesion to biological tissues is a challenge especially when the adhesive is in contact with physiological fluids. Abdominal hernia is a disease that often requires the implantation of a mesh within the abdominal wall. To minimize pain and postsurgical complications, gluing the mesh appears to be a convenient method. For this purpose, a bioadhesive system based on solutions of chitosan and modified starch (oxidized maltodextrin) has been developed. Mixtures of these polysaccharides form either viscoelastic solutions or hydrogels, depending on various experimental parameters (chitosan concentration, starch degree of oxidation, molar ratio between amine and aldehyde functions, pH, etc.). An adhesion test was developed to assess the adherence of such systems under conditions similar to the intended use. The rheological behavior of each formulation was correlated to its adherence, and it was found that optimum adhesion is obtained for systems exhibiting an intermediate behavior between the viscoelastic solution and the gel.
’ INTRODUCTION Adhesion to biological tissues is a challenge because the adhesive, which is in contact with physiological fluids, has to be both efficient and biocompatible. Tissue adhesives can be used for a wide range of applications such as hemostasis and sealing or adhesion between different biological tissues or between an implanted device and biological tissues.1 Commercial adhesives for internal use can be classified into synthetic (mainly cyanoacrylates and PEG hydrogels) and biological adhesives (fibrin- or protein-based).2 Synthetic adhesives and especially cyanoacrylates have limited application for internal use because of their poor biocompatibility.2 Fibrin glue, which reproduces the last steps of natural clotting, is biocompatible, but it is expensive and suffers from specific drawbacks because of its blood-derived origin. When bovine thrombin or aprotinin are part of its composition, anaphylactic as well as hypersensitivity reactions might occur.3 In addition, although no serious viral transmission has been observed,3 this possibility cannot be excluded with certainty, and transmission of virus causing mild infection has already been reported.4 Abdominal hernia is a protrusion of viscera through an abdominal wall defect. At present, over 20 million abdominal wall hernia repairs are performed each year around the world.5 Treatment often involves the use of a reinforcement mesh fixed to the abdominal wall with staples or sutures. However, these fixation methods can be associated with postsurgical complications due to nerve irritation.6 In principle, gluing the mesh may be a milder and more convenient method. r 2011 American Chemical Society
Considering the case of several natural substances (e.g., protein from genus Notaden frog, sericin from Bombyx mori, protein/saccharide associations from echinoderms),7 adhesion can be seen as a balance between different types of molecular interactions. These are mainly hydrophilic/hydrophobic interactions, ionic interactions, and hydrogen bond interactions. Most of the time, different kinds of interactions are involved simultaneously. Therefore, to develop a new tissue adhesive, we focused on polysaccharide structures able to generate such a variety of molecular interactions. Chitosan is a linear polysaccharide composed of randomly distributed β-(1f4) linked D-glucosamine and N-acetyl-D-glucosamine residues. Chitosan can be dissolved in acid aqueous solutions in the protonated state and therefore can generate ionic interactions. This polysaccharide has drawn a lot of attention, particularly in the biomedical field, because of its biocompatibility, bioresorbability, and bioactivity as well as its bacteriostatic and fungistatic properties.8 Chitosan has been proposed for biomedical adhesion,9,10 but its implementation often requires the use of an external source of energy (e.g., laser), which complicates the implantation procedure, and is at the origin of specific drawbacks. Biomimicking adhesives based on chitosan have also been developed.11,12 This latter approach was inspired by the glue secreted by mussels and consists of chitosan crosslinked with tyrosinase-oxidized dopamine to form an adhesive Received: December 13, 2010 Revised: February 20, 2011 Published: March 16, 2011 1556
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hydrogel. Such adhesives exhibited tensile shear adhesive strength over 400 kPa (measured with glass slides). Previously, we reported on the structural characterization and linear viscoelastic behavior of a system containing chitosan and oxidized starch (OS).13 OS is a polysaccharide-based cross-linker efficient at forming chitosan hydrogels with controlled rheological properties. We obtained a wide range of rheological behaviors from viscoelastic solutions to a stiff hydrogels. Under certain conditions, the system also exhibited adhesive properties. Hoffmann et al.14 recently described a system close to ours based on chitosan and oxidized dextran for bone glue. However, they did not give insight into the rheology of their systems, nor did they vary the same parameters as we did. Here we investigated the influence of chitosan concentration, chitosan molecular weight, the starch degree of oxidation (DO), and the [NH2]/[CHO] molar ratio (MR). Moreover, we correlated the adhesion results with the rheological behavior for each formulation.
structure of CTS is given in Scheme 1. Starch (Glucidex1, potato-based maltodextrin with multimodal distribution, Mw = 80 000 g/mol, PDI = 3.2) was purchased from Roquette. Fibrin glue (Beriplast) was obtained from Aventis Behring. All other chemicals were purchased from Acros Organics or Sigma Aldrich and used as received.
Preparation and Characterization of Oxidized Starch. Starch was oxidized in a water solution by sodium metaperiodate, as previously described.13 The DO of starch was determined by reacting an excess of hydroxylammonium chloride with OS. OS was characterized by 1H and 13C NMR and FTIR.13 For clarity, we will refer to an OS with a DO of 30 as OS30. The simplified chemical structure of OS is given in Scheme 1. (More complicated structures take into account the equilibrium of aldehydes with hemiacetals18.) Preparation of CTS/OS Solutions and Hydrogels. Unless otherwise stated, all concentrations are expressed in w/w. Chitosan (chemical structure in Scheme 1) was dissolved in water at 5% (w/w) with a stoichiometric amount of hydrochloric acid necessary to achieve the protonation of amine residues. The pH was then adjusted with NaOH to obtain 3% (w/w) chitosan solution at pH 4, unless otherwise stated. In every formulation, the pH of the OS and CTS were adjusted to the same value before mixing. Mixing was performed by connecting two syringes and pushing the contents back and forth during 30 s at room temperature. To obtain a given molar ratio MR = [NH2]/[CHO], we varied the concentrations of OS solutions. In the different systems, chitosan concentration was varied in the range 1 to 2.3% (7% for HMW chitosan) and MR was varied in the range 1-40. This corresponds to OS concentrations being varied in the range 0.01 to 3.3%. All combinations of CTS/OS mixes can be found in the Table 1. Rheological Measurements. Rheological experiments were performed with a controlled stress rheometer (AR 2000, TA Instruments) in parallel plate geometry (25 mm diameter) with a 0.5 mm gap. Solution and gel rheological behavior was assessed in dynamic mode. For these systems, the linear viscoelastic region corresponded to a shear stress range from 1 to 10 Pa. The test temperature remained 25 C for all experiments, and silicon oil was used to prevent evaporation during the experiment. Angular frequency sweep measurements were performed from 100 to 10-1 rad/s. Adhesion Test. Lap shear tests are commonly found in the literature to assess adhesive strength for bonding biological tissue.19-21 In our work, we chose porcine dermis and muscle as the biologic substrates (this choice is further explained in the Result and Discussion section), and the methods were adapted from ASTM F2255-03 “Standard Test Method for Strength Properties of Tissue Adhesives in Lap-Shear by Tension Loading”.22 Porcine dermal tissue was excised from the back of the animal, and all nondermal tissue was removed by blunt dissection. Porcine muscle tissue was excised from the abdominal wall and manually cleaned from all residual fatty tissue, resulting in a substrate composed of muscle covered by its aponeurosis. All tissues were frozen rapidly after harvest and cleaning and thawed prior to testing.
’ MATERIAL AND METHODS Materials. Chitosan (CTS) (degree of acetylation (DA) of 2%, Mw = 450 000 g/mol, PDI = 1.5, monomodal distribution) produced from squid pens was purchased from Mahtani Chitosan (batch 113), purified, and characterized as previously described15,16 and will be referred to as high-molecular-weight chitosan (HMW). Low-molecular-weight chitosan (LMW) (DA 2%, Mw = 120 000 g/mol, PDI = 1.3) was obtained by sonification of a high-molecular-weight chitosan with a Lixea formulator type BA (Sinaptec SA, France), as previously described.17 The chemical
Scheme 1. Chemical Structure of (a) Chitosan and (b) Oxidized Starch (Simplified Structure)a
a
DA: degree of acetylation; DO: degree of oxidation.
Table 1. Summary of All Solutions/Hydrogels Presented in This Articlea [CTS] (% w/w)
[OS4] (% w/w) MR20
MR20
1.4
0.7
7.0 2.3 1.9
[OS8] (% w/w)
[OS15] MR1
MR5
MR10
MR20 1.2
0.6
1.5
0.8
0.4
0.2
2.1 0.2
0.3
1.5 1.0
[OS30] (% w/w) MR40
MR20
[OS65] (% w/w) MR20
0.01
0.15
0.2 3.3
0.7
0.4
0.15
Concentration of OS is fixed by both CTS concentration (which is connected to a given an amount of NH2) and MR. For example, for a CTS concentration of 2.3% (w/w) that is to be mixed with a OS30 at a MR of 20, the concentration of OS30 is 0.2%. a
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Biomacromolecules The first test was performed with porcine dermis as the substrate. The dermis was cut in 2.5 2.5 cm square pieces that were stapled on a PET film. The adhesive (200 μL) was applied to one piece of dermis, and the other piece was overlapped with the first piece. For the second test, one piece of dermis was replaced by a polyethylene terephthalate (PET) mesh (Parietex mesh, Sofradim) cut in 2.5 cm 10 cm strips. The last test (Scheme 2) involved a different biological substrate (porcine abdominal wall muscle covered with aponeurosis) and the PET mesh. Porcine muscle square were cut (3 cm 3 cm) and stapled on a PET film. We spread 200 μL (unless otherwise stated) of the adhesive on the mesh. The mesh was then laid on the porcine muscle. The apparent contact area between the muscle and the mesh was then 2.5 cm 2.5 cm. All samples were kept moisturized by wrapping in soaked compresses and were tested 30 min after adhesive application. Adhesion was assessed by a lap shear test performed on an Adamel-Lhomargy DY22 tensile testing machine equipped with a 50 N load cell. The crosshead speed was 10 mm s-1. For each test, the load versus displacement was measured, and the shear stress at break (ultimate adhesion strength or adhesion strength at break) is used to characterize adhesion for each formulation. Each test was repeated at least seven times.
’ RESULTS AND DISCUSSION Choice of the Biological Substrate for the Lap Shear Test. Several adhesion tests for biological adhesives have been developed with nonbiological substrates.23 Although easy to implement, using nonbiological substrates might not be representative of real adhesive application. Therefore, skin is often used to test biological adhesives. For example, porcine skin is proposed in a standard test method to assess biological adhesion properties.22 Although dermis is a biological substrate, it is significantly different from the one involved in our intended application, that is, abdominal wall tissue. Our application requires mesh to adhere to the abdominal wall, more precisely to abdominal wall muscle covered with aponeurosis. Therefore, we developed a lap shear test using both muscle and a hernia repair mesh (as in Scheme 2) and made a comparison with standard dermis/dermis system and dermis/mesh systems.
Scheme 2. Representation of the Third Lap Shear Test (PET Mesh: Parietex)
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We used 200 μL of a 3% (w/w) HMW chitosan solution for the three tests. Shown in Figure 1 is the adhesive strength of the 3% CTS solution as a function of substrate identity. The adherence was higher with the dermis/dermis system (10.3 kPa). When one piece of dermis was replaced by a PET mesh, the adherence decreased to 4.3 kPa. This was partially due to the macroscopic physical structure of the mesh and to lower interactions between chitosan and the PET mesh than with the dermis. Indeed, chitosan has a better affinity with dermis constituted of collagen than with PET. Chitosan is known to interact with proteins like collagen through electrostatic interaction or hydrogen bonding,24 whereas interactions with PET are more limited. When dermis was further replaced by muscle, the adherence dropped again (to 0.5 kPa). The choice of the substrate influenced greatly the adherence of the 3% (w/w) chitosan solution. Artzi et al.25 also showed that adhesion was tissuespecific, and consequently, what is observed for one biological substrate cannot be directly transposed to another substrate. For the rest of the adhesion tests, only the muscle/mesh system was retained because it better mimics our target application. Despite good wetting properties, a 3% (w/w) chitosan solution exhibits poor adhesion (Figure 1), especially for the muscle/mesh system. We observed that the rupture was cohesive: chitosan was found on both sides of the test article. Therefore, a pure chitosan solution contacted well with the substrates but lacked cohesion and was not able to generate sufficient adhesion. It was thus necessary to increase the cohesion of the joint, for example by forming a hydrogel in situ. On the basis of previous research from our group,13 we showed hydrogel formation with chitosan by adding a multifunctional low toxicity cross-linker based on a modified polysaccharide (i.e., OS).13 Moreover, because such macromolecules bear aldehyde groups, they can promote adhesion by reacting directly with the tissue through amines present in collagen.25 The balance of this report details our work to characterize and explain the effect of several CTS/OS solution variables (chitosan concentration, pH, starch DO, and MR of the reactive functions) on the viscoelastic behavior of the hydrogels formed. Table 2. Complex Viscosities of Chitosan Solutions at Different Concentrations (Extrapolation at Low Angular Frequency) chitosan solution
complex viscosity
concentration (w/w) (%)
(Pa 3 s) ( 10%
1.0%
3
1.5%
30
1.9%
60
2.3%
160
Figure 1. Lap shear test with different biological substrate, 3% (w/w) chitosan solution. The error bars represent the standard deviation of the data. 1558
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Figure 2. Influence of the HMW chitosan concentration on the ultimate adhesion strength of a chitosan/OS15 system. All solutions were adjusted to pH 4. The ratio [NH2]/[CHO] was fixed to 20. In the final system, chitosan concentrations (w/w) of 1, 1.5, 1.9, and 2.3% corresponded to oxidized starch solutions OS15 (w/w) of 0.15, 0.2, 0.3, and 0.4%, respectively (lap shear test, muscle/mesh).
Figure 3. Influence of the starch degree of oxidation on the ultimate adhesion strength of the chitosan/oxidized starch system. All solutions were adjusted to pH 4. The ratio [NH2]/[CHO] was fixed at 20. In the final system, chitosan concentration was fixed at 2.3% (w/w), which corresponded to concentrations of oxidized starch (w/w) of 1.4% for OS4, 0.7% for OS8, 0.4% for OS15, 0.2% for OS30, and 0.01% for OS65 (lap shear test, muscle/ mesh).
Influence of Chitosan Concentration on Adhesion. Chitosan in solution exhibits a viscoelastic behavior, characterized by the value of the norm of the complex viscosity |η*|. The viscosity of a chitosan solution increases with the chitosan concentration, as shown in Table 2 (complex viscosities can be extrapolated at low frequency and can be assimilated to η0, the low shear Newtonian steady-state viscosity, according to the Cox-Merz rule26). To form an adhesive with greater adhesive properties than a pure CTS solution, we mixed a solution of chitosan with a solution of OS. First, the chitosan concentration was varied and the other parameters kept constant (pH 4, [NH2]/[CHO] = 20, OS15). The influence of the concentration of CTS on the adherence of a CTS-OS system is shown in Figure 2 (lap shear test, muscle/mesh). In all cases, a cohesive break was observed within the CTS/OS system. For the CTS concentration range studied here [1.0 to 2.3%], the adhesion strength increased with CTS concentration from 2 to 5.6 kPa. The 1% (w/w) chitosanbased system exhibited the lowest adherence: again, this was due to a low cohesiveness of the system (cohesive fracture of the joint) due to the low viscosity of the precursor chitosan solution. Above a concentration of 1.5% (w/w), the adherences seemed to reach a plateau, and even though the 2.3% (w/w) chitosan system led to the best adherence, this value could be comprised within the experimental error. Concentration >2.3% resulted in
solutions that were too viscous to be easily handled in practical cases, so the concentration range 2.3 to 3% was not investigated. Influence of the Degree of Oxidation on Adhesion. The systems formed are versatile, in particular, because of the possibility of varying the amount of reactive functions on the starch chain. The influence of the DO on the lap shear adhesion strength is represented in Figure 3. As shown above, chitosan alone did not provide any significant adhesion (Figure 1). A system comprising chitosan and unoxidized starch (OS0) resulted in an adhesive with similar properties to a pure CTS system (0.4 kPa). Oxidation of starch was thus necessary to promote adhesion. The [NH2]/[CHO] ratio was fixed to 20, and the pH was maintained at 4. It is noticeable from Figure 3 that there was an optimal DO (15%) that led to the optimal adherence (5.7 kPa), comparable with fibrin glue (5.4 kPa). Results presented in Figure 3 show a considerable variability in the adhesion strength, depending on the formulation. This observation is consistent with literature, where variability is common for adhesion test, especially when biological substrates are used.19 Fibrin-based adhesives were reported to give variable adhesion results, generally between 1 and 6 kPa10,19,27,28 but sometimes up to 27 kPa.29 As stated before, this variability is due to differences in the experimental procedures19 (different testing procedure, different biological substrate). Moreover, all fibrin 1559
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Figure 4. Rheological behavior of different adhesive systems: (a) chitosan 2.3% (w/w) and chitosan-starch; (b) chitosan-oxidized starch 4%; (c) chitosan-oxidized starch 8%; (d) chitosan-oxidized starch 15%; (e) chitosan-oxidized starch 30%; (f) chitosan-oxidized starch 65%. All solutions were adjusted to pH 4, and MR = [NH2]/[CHO] was fixed at 20.
adhesives might have different compositions, which also accounts also for the variable results found in literature. The results showed a strong influence of the DO on the adhesion strength. Besides the specific interactions between the adhesive constituent and the substrate, the cohesion of the joint plays an important role. To assess this property, the viscoelastic behavior of each system was studied (Figure 4) to relate it to the adhesion results. Figure 4a shows the rheological behavior of a 2.3% (w/w) chitosan solution. Its behavior was characteristic of a viscoelastic fluid (G0 (storage modulus) > G00 (loss modulus) at high frequencies and G0 < G00 at low frequencies with G00 /G0 ≈ 1/ω). Tan δ = G00 /G0 was also plotted, which can also be used to characterize the viscoelastic state of a material. For example, in this case, tan δ increases as frequency decreases, and tan δ values are well above 1 at low frequencies (1) are characteristic of the solution behavior. When unoxidized starch was added (1.4% in final system), the behavior remained identical. Indeed, unoxidized starch did not interact enough with chitosan to change its rheological behavior. This was consistent with the same adhesion strength measured for chitosan and chitosan/unoxidized starch (Figure 3). To make the joint more cohesive, OS was added.
Although a 4% (w/w) OS slightly increased the adherence, the rheological behavior of the system remained unchanged (Figure 4b). This shows that interactions between chitosan and OS4 were low. When the DO was increased to 8%, adherence increased to 4.9 kPa. Storage modulus G0 and loss modulus G00 were higher, which corresponded to a higher viscosity of the system (Figure 4c). However, G0 , G00 and tan δ were still characteristic of a viscoelastic solution. As previously mentioned, a higher DO (15%) gave the best adherence. The rheological behavior of such a system was different compared with previous rheograms (Figure 4d). In this case, G0 > G00 in the entire angular frequency range, but the difference between G0 and G00 is only slightly decreased at low frequencies, resulting in tan δ values close to 1 at low frequencies Moreover, the increase in tan δ is far less marked than that for characteristic solutions. When the DO was further increased (30 and 65%), the adherence decreased (4.3 and 2.7 kPa, respectively). Both rheograms exhibited higher moduli than for the 15% OS and were characteristic of a gel because (i) G0 > G00 in the entire investigated frequency range and (ii) the difference between the two moduli (in log scales) was more or less constant. This can also be deduced from the constant value of tan δ < 1 (Figure 4e,f). In summary, the 1560
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Table 3. Comparison of Chitosan-Based Adhesive Hydrogels on Various Soft Tissues type of adhesive
biological substrate
adhesion strength (kPa)
photo-cross-linked chitosan hydrogel27
mice skin
3.1 ( 0.5
photo-cross-linked chitosan hydrogel10
ham
4.3 ( 1
polyampholytic chitosan-based hydrogel30
porcine dermis
4.1 ( 0.5
Figure 5. Influence of the MR = [NH2]/[CHO] on the ultimate adhesion strength of chitosan/oxidized starch OS15. All solutions were adjusted to pH 4. In the final system, chitosan concentration was 2.3% (w/w), which corresponded to concentrations of oxidized starch of (a) 1.5% for MR5, (b) 0.8% for MR10, and (c) 0.4% for MR20. The adhesive volume was 250 μL (lap shear test, muscle/mesh). 9: tan δ values.
behavior of the optimal formulation (CTS concentration 2.3% (w/w); starch DO 15%; OS15 concentration 0.4% (w/w); MR = 20; pH 4) was comprised between the viscoelastic fluid (clearly exhibiting flow) and the gel (which is dominated by the elastic response). An optimal DO (DO15) led to the optimal adherence, with values comparable to what is obtained with fibrin glue (Figure 3). Materials with less adhesive properties were either too liquid (OS4, OS8) or too rigid (OS30, OS65). Optimal adhesion of CTS/OS system corresponded to a balance between the cohesiveness of the joint and the macromolecular mobility (chain segment diffusion) that is necessary for the formation of specific interaction, that is, adhesion with the substrate.
As previously observed,13 a key parameter is the repartition of the aldehyde functions along the OS chains. Increasing the DO of starch from 4 to 30% resulted in a higher density of aldehyde-amine cross-links and a more cohesive network (Figure 4a-e). Above this value (65% Figure 4f), the system does not gain any more cohesiveness (same rheological behavior as 30%). This happens because there are too many aldehydes on the chains and not all of them can react with chitosan. In conclusion, rheological measurements can provide an easy determination of optimum of adhesion, at least for systems with sufficient chitosan concentration and with an excess of amine functions. The optimal adherence is obtained when tan δ converges to values close to unity at low frequencies (ω < 10-1 rad/s). 1561
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Figure 6. Influence of the MR = [NH2]/[CHO] on the ultimate adhesion strength of chitosan/oxidized starch OS15. All solutions were adjusted to pH 4. In the final system, chitosan concentration was 1.9% (w/w), which corresponded to concentrations of oxidized starch of (a) 6.2% for MR1, (b) 0.3% for MR2, and (c) 0.15% for MR40 (lap shear test, muscle/mesh). 9: tan δ values.
To go a step further, we compared our optimal CTS/OS system with other chitosan-based adhesives reported in literature, also tested on soft tissues. (See Table 3.) We found that our system offers greater or comparable adhesion strength (5.7 vs 3.1 to 4.3 kPa). Influence of the Molar Ratio on Adhesion. We showed previously that the molar ratio MR = [NH2]/[CHO] had a strong influence on the viscoelastic properties of the systems.13 It can be reasonably assumed that there is also an influence of the cross-link density on the adhesion. The influence of the MR on a 2.3% CTS/OS system was investigated at three different MR values (5, 10, and 20) (Figure 5). Experimentally, this ratio was adjusted by modifying the OS concentration. For all formulations, OS 15 was used, and pH was maintained at 4. For this test, the amount of adhesive was 250 versus 200 μL for all other tests. Each formulation was also assessed rheologically (Figure 5a-c). There was no significant difference in terms of adhesion strength between different MRs (5, 10, or 20), and all formulations exhibited an adherence around 7 kPa. (A statistical analysis based on ANOVA methods yields a p value close to 0.7.) However, we could observe a larger difference in the rheological behavior of
these formulations. The more cohesive gel was found at the MR ratio closer to stoichiometry. We observed a similar increase in cohesion when the DO was increased at constant MR = 20. However in this previous case, higher DOs led to a decrease in the adhesion strength. It is therefore concluded that in the range of lower MR ratios, a larger fraction of aldehyde moieties contributes to interfacial adhesion and preserves the adhesion strength with stiffer joint gels. In fact, only a small amount of aldehyde (MR 20) was needed to promote adhesion under the investigated physicochemical conditions, which is in contradiction with common aldehyde-based adhesives development strategies.31,32 Indeed, a 2.3% (w/w) chitosan solution is already highly viscous (entangled cationic polyelectrolyte solution), so only a small amount of crosslinker is needed to modify the rheological behavior and increase adhesion through the formation of a chemical and physical network, implying chitosan/chitosan interaction and chitosan/ starch interactions.13 This result is consistent with the effect of a low density of cross-linking leading to a slight restriction of chitosan chain mobility for chemical reaction but increases the disentanglement relaxation time drastically.33 1562
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Figure 7. Influence of the MR = [NH2]/[CHO] on the ultimate adhesion strength of chitosan/oxidized starch OS15. All solutions were adjusted to pH 4. In the final system, chitosan concentration was 1% (w/w), which corresponded to concentrations of oxidized starch of (a) 3.3% for MR1, (b) 0.7% for MR5, and (c) 0.16% for MR20 (lap shear test, muscle/mesh). 9: tan δ values.
As a consequence of this interpretation, if the MR is further decreased (MR < 5), then both an increase in the stiffness of the gel and a decrease in the adherence are expected to occur. Similarly, for MR > 20, the system would lose adherence and tend to a viscoelastic solution (MR = ¥ is representative of a pure chitosan solution). To better assess this optimum, we had to decrease the concentration of chitosan. Indeed, these systems are obtained by diluting a 3% (w/w) chitosan solution with OS, so obtaining a stoichiometric system (MR1) with 2.3% (w/w) chitosan in the final solution would require a 32% (w/w) oxidized starch solution (OS15). At such high concentration, the solution is not stable and OS precipitates. Therefore, it was necessary to decrease the chitosan concentration to increase the MR range investigated. The influence of the MR on the adhesion and the rheology of a 1.9% (w/w) chitosan-based system was investigated (Figure 6) with a 1-40 MR range. Here again the pH was fixed at 4 and OS 15 was used. As expected, the MR1 formulation exhibited a gellike behavior, whereas at MR 20 and 40, rheological behavior is close to solution properties. The MR 20 formulation, although it
is still a viscoelastic solution, can be seen as an intermediate between the rheological behavior of MR40 and MR1. The change in rheological behavior between each formulation was more drastic than in Figure 5 because of a wider range of MR investigated. When comparing the changes in the rheological behavior to the variations in the adherence, the best adherences are obtained for MR1 (4.1 kPa, tan δ = 0.5) and MR20 (4.7 kPa, tan δ = 1.6), that is, for tan δ ≈ 1. It is also clear from Figure 6 that only a low amount of cross-links is needed to achieve a good adhesion (MR20), consistent with the case of more concentrated solutions at 2.3% (w/w). The influence of MR was further extended to lower concentrations of chitosan and thus initial solutions with lower viscosities. The influence of the MR on the adhesion of a 1% (w/w) chitosan-based system was investigated (Figure 7). The pH was fixed at 4, and OS15 was used. Here again, a maximum of adherence (3.6 kPa) was observed for a critical value of the MR of 5. As a result, when the chitosan concentration was decreased, the MR to obtain optimal adhesion was lower (from 20 for 2.3% (w/w) chitosan-based system to 5 for 1% (w/w) system); that is, 1563
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Figure 8. Influence of the MR = [NH2]/[CHO] on the ultimate adhesion strength of chitosan/oxidized starch OS15. All solutions were adjusted to pH 5. In the final system, chitosan concentration was 1% (w/w), which corresponded to concentrations of oxidized starch of (a) 0.7% (w/w) for MR5, (b) 0.4% for MR 10, and (c) 0.16% for MR20 (lap shear test, muscle/mesh). 9: tan δ values.
a higher number of cross-links was required for increasing the strength of the gel. At MR = 20 the system is not cohesive anymore and exhibits the solution behavior (flow) (Figure 7c). On the contrary, for a 2.3% (w/w) based system, we observed at MR 20 an “intermediate state” (Figure 5c). Therefore, we can conclude that optimal MR value is intrinsically dependent on the polymer concentration because rheological and adhesion criteria both rely on the formation of the network resulting from the complex interactions between chitosan and OS. The pH is an important kinetic parameter for the formation of the hydrogels, that is, for the formation of covalent cross-links between chitosan and aldehyde groups.13,34 Whereas at pH 4, a 1.9% (w/w) chitosan-based system remained a viscoelastic solution, at pH 5, it became a hydrogel with much higher moduli (at room temperature and 24 h after mixing). This is due to the higher amount of free amine functions that are present at pH 5 (in comparison with pH 4) and can react with aldehyde moieties. Therefore, another way to shift the MR of the optimal formulation (optimal adhesion) at a given concentration would consist of varying the pH. Figure 8 shows the influence of the MR (in the range between 5 and 20) on a 1% (w/w) chitosan-based system
at pH 5. The optimal adherence (2.4 kPa) occurred for an MR of 10 (vs 5 at pH 4), even if the system with MR 20 exhibits a value of tan δ closest to 1. Such a result illustrates that a unique rheological criterion based on the value of tan δ and which is also able to predict optimal adhesion strength is difficult to find in every case. In particular, the MR = 20 system had the lowest adhesion strength, probably because the moduli are too low to ensure a cohesive joint. Therefore, a condition of minimal moduli should be considered in addition to the value of tan δ to determine the more adhesive system in a wide range of formulations. As far as adhesion is concerned, results were overall lower at pH 5 than at pH 4 (1.3 to 2.4 kPa vs 2.1 to 3.6 kPa). This result could be explained by faster kinetics of the gel formation at high pH, yielding less diffusing chain portions able to interact with the substrate. In addition, at pH 4, a higher fraction of aldehyde moieties remains free to react with amine from tissue proteins, contributing to equilibrium between cohesive and interfacial strengths. This thorough study of the influence of the MR on the adhesion and the rheology showed that the optimal cross-link density was governed by the chitosan concentration (and density of entanglements). In addition, for higher chitosan concentrations, 1564
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Figure 9. Influence of the molecular weight of chitosan on the ultimate adhesion strength of the chitosan/oxidized starch system for a lowmolecular-weight chitosan. All solutions were adjusted to pH 4, and the [MR]/[CHO] ratio was fixed at 20. For LMW chitosan, chitosan concentration was 7% (w/w), which corresponded to oxidized starch concentration of 2.1% (w/w) for OS8, 1.2% for OS15, and 0.6% for OS30. LMW (120 000 g 3 mol-1) systems at 7% were compared with an HMW (450 000 g 3 mol-1) system at 1.9% because both chitosan solutions exhibit the same viscosity (lap shear test, muscle/mesh).
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behavior of the different formulations. It was found that the optimally adhesive systems had an intermediate rheological behavior between the solution and the gel, as long as the systems were viscous enough. Indeed, a polysaccharide solution will not generate sufficient adhesion. On the contrary, in stiffer gels, polymer chains are entrapped in the network and lack mobility to interact with the biological substrate. In addition to the viscoelastic criteria for the prediction of adherence, it was shown that interfacial adhesion should also be taken into account and could result from covalent bonding between aldehyde functions and amines from the proteins of biological tissues. Finally, the adhesion of our optimal systems (e.g., CTS HMW 2.3% (w/w); OS15; MR20; pH 4) lies in the same range as the fibrin glue. The next step is to assess the biological response of our systems. Such in vivo biological evaluations, which are not presented in this Article, are underway.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected],
[email protected]. only a small amount of cross-linker is required to form an optimal adhesive system. This is particularly interesting because these systems are intended for biomedical uses where cross-linkers generally decrease the biocompatibility of hydrogels.25 Even though we choose a macromolecular cross-linker with minimal toxicity, that is, a polysaccharide with different amounts of oxidized functions, it is essential to minimize the cross-linker amount to preserve the specific biocompatibility of chitosan hydrogels.8 Therefore, a key point in this study is the assessment of an optimal adhesion system with a large excess of amine groups over aldehyde moieties. Influence of Chitosan Molecular Weight on Adhesion. The last investigated parameter was the chitosan molecular weight. Decreasing chitosan molecular weight might increase the mobility of chitosan chains in solution and consequently have an impact on the adhesion. The influence of the DO (OS8-OS30 range) on the adherence of a LMW chitosan (Mw = 120 000 g/mol) (Figure 9) was investigated. With LMW chitosan, the adherence did not seem to vary much with the DO; that is, all adherences lay between 6.2 and 6.8 kPa. These values are in the same range as for fibrin glue. The adherences of LMW-based systems were overall higher than with HMW systems (6.8 vs 5.7 kPa for 2.3% (w/w) system or 4.7 kPa for 1.9% (w/w) system, all with OS15). This was not explained by a difference in viscosity because both 7% (w/w) LMW chitosan solution and HMW 1.9% (w/w) solution exhibited a similar viscosity (∼60 Pa 3 s). The impact of molecular weight on the adherence must be related to a higher mobility of LMW chitosan chains in comparison with HMW chains. Such mobility is likely to promote covalent and physical bonding with the biological substrate and therefore could increase the interfacial adhesion.
’ CONCLUSIONS Multipolysaccharide systems composed of chitosan and OS are relevant for the design of bioadhesives for tailor-made biological applications. The precise control of experimental physicochemical parameters (chitosan concentration, DO, MR, pH, chitosan MW) is necessary to find optimal adhesion. An ex vivo adhesion test was specifically developed and enabled the evaluation of biofunctionality (adhesion to biological substrates). It is also possible to link the adhesion with the viscoelastic
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