Smart Hydrogels for in Situ Generated Implants - American Chemical

Biomacromolecules 2005, 6, 1168-1175

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Smart Hydrogels for in Situ Generated Implants† Daniel Cohn,* Alejandro Sosnik,‡ and Shai Garty Casali Institute of Applied Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel Received August 14, 2004; Revised Manuscript Received December 14, 2004

The objective of this study was to explore the use of reverse thermo-responsive (RTG) polymers for generating implants at their site of performance, following minimally invasive surgical procedures. Aiming at combining syringability and enhanced mechanical properties, a new family of injectable RTG-displaying polymers that exhibit improved mechanical properties was created, following two different strategies: (1) to synthesize high-molecular-weight polymers by covalenty joining poly(ethylene glycol) and poly(propylene glycol) chains using phosgene as the coupling molecule and (2) to cross-link poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-PEO triblocks after end-capping them with triethoxysilane or methacrylate reactive groups. While the methacrylates cross-linked rapidly, the triethoxysilane groups enabled the system to cross-link gradually over time. The chain-extended PEO/PPO copolymers had molecular weights in the 39 00054 000 interval and exhibited improved mechanical properties. Reverse thermo-responsive systems displaying gradually increasing mechanical properties were generated by cross-linking triethoxysilane-capped (EO)99(PO)67-(EO)99 (F127) triblocks. Over time, the ethoxysilane groups hydrolyzed and created silanol moieties that subsequently condensated. With the aim of further improving their mechanical behavior, F127 triblocks were reacted with methacryloyl chloride and the resulting dimethacrylate was subsequently cross-linked in an aqueous solution at 37 °C. The effect of the concentration of the F127 dimethacrylate on the mechanical properties and the porous structure of the cross-linked matrixes produced was assessed. Rheometric studies revealed that the cross-linked hydrogels attained remarkable mechanical properties and allowed the engineering of robust macroscopic constructs, such as large tubular structures. The microporosity of the matrixes produced was studied by scanning electron microscopy. Monolayered conduits as well as structures comprising two and three layers were engineered in vitro, and their compliance and burst strength were determined. Introduction The goal of the “in situ generated implants” strategy is to engineer biomedical systems at their site of performance, using minimally invasive surgical procedures. Various methods were pursued to this end during the past decade, such as the in situ polymerization approach1-4 and the in vivo precipitation technique developed by Dunn et al.5 Reverse thermo-responsive polymers constitute the material basis of a very promising strategy for the in situ generation of advanced polymeric systems.6-9 The unique behavior of these polymers, usually known as reverse thermal gelation (RTG), pertains to their ability to produce lowviscosity aqueous solutions at ambient temperature, creating a gel at a higher temperature. The phase transition temperature for these polymers is called lower critical solution temperature. The poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-PEO triblocks are one of the most important RTG-displaying materials.10-15 The endothermic transition displayed by this family of polymers is driven by * Corresponding author. E-mail: [email protected]. Fax: 972-2658-6312. † This paper was presented at the 43rd Microsymposium on Polymer Biomaterials: Biomimetic and Bioanalogous Systems, held in Prague, Czech Republic, July 11-15, 2004. ‡ Present address: Institute of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario M5S 3E5, Canada.

the entropy gain caused by the release of water molecules bound to the hydrophobic PPO segments in the triblock, as temperature rises.16-18 Their unique behavior has been thoroughly investigated during the past decade,19-21 with special attention being given to the PEO99-PPO67-PEO99 triblocks, known as Pluronic F127. The gels formed by these triblocks, however, proved to be not viscous and sturdy enough, displaying clearly inadequate mechanical properties and deficient stability. Thus, despite their clinical potential, these fundamental shortcomings severely restricted their use, particularly in those appplications where engineering considerations play a key role. Injectable systems can be classified into two main categories, depending on the fundamental characteristics of the biomedical system to be created: those in which their mechanical properties play a central role and those where their high water content is their essential attribute. Clearly, hydrophobic materials are required when the mechanical behavior is a major consideration, whereas water-rich systems are especially suitable for the controlled release of hydrophilic macromolecules and in the tissue engineering field. The objective of this study is to combine the advantageous attributes of both classes of injectables, by engineering mechanically robust structures using reverse thermoresponsive polymers. To this end, two key pathways were followed in our laboratory. First, high-molecular-weight

10.1021/bm0495250 CCC: $30.25 © 2005 American Chemical Society Published on Web 03/01/2005

Smart Hydrogels for in Situ Generated Implants

RTG-displaying polymers, exhibiting enhanced mechanical properties, were synthesized by covalenty binding poly(ethylene glycol) (PEG) and poly(propylene glycol) (PPG) chains, using phosgene as the coupling molecule.6,7 Aiming to improve the mechanical behavior of these systems even further, initial reverse thermo-responsiveness was combined with in situ cross-linkability. On the basis of this working concept, reverse thermo-responsive polymers were functionalized to enable them to cross-link at their location of performance, under clinically acceptable conditions. RTGdisplaying triblocks were end-capped with carbon-carbon double bonds22 or with ethoxysilane groups, aiming at allowing their immediate or gradual cross-linking, respectively.9 The objective of the “in situ generated implants” area is not only to deploy materials but also primarily to engineer structures at a precise body site, having specific geometric and mechanical characteristics. This study describes the in vitro production of RTG-based macroscopic constructs (e.g., tubular structures), capitalizing on the improved mechanical properties of the reverse thermo-responsive materials developed in our laboratory. The effect of the concentration of F127 dimethacrylate on the dynamic mechanical properties and the microporosity of the cross-linked matrixes generated was assessed. Monolayered structures as well as constructs comprising two and three layers were engineered in vitro, and their mechanical response (burst strength, compliance) was studied. Experimental Section (A) Materials. In general, the solvents used were of analytical grade and were dried adding molecular sieves 4A (BDH). Pluronic F127 (molecular weight 12 600) and F87 (molecular weight 7500) were purchased from Sigma and dried at 120 °C under a vacuum for 3 h before using. PEG and PPG chains having molecular weights of 6000 (PEG6000) and 3000 (PPG3000), respectively, were supplied by Aldrich and dried at 120 °C under a vacuum for 1 h before using. The phosgene chloroformic solution was prepared in our laboratory from 1,3,5-trioxane (Aldrich) and carbon tetrachloride (Frutarom), using aluminum chloride (Merck) as the catalyst following a previously described technique.23 Pyridine was purchased from BDH and was dried with molecular sieves 4A (BDH). (3-Isocyanatopropyl)triethoxysilane (IPTS, Fluka) and the stannous 2-ethylhexanoate (SnOct2, Sigma) catalyst were used as received. Methacryloyl chloride was obtained from Aldrich and was distilled before use. Triethylamine (TEA) and sodium metabisulfite were supplied by Aldrich and Riedel de-Haen, respectively, and were used as received. The 0.1 M buffer phosphate solutions were prepared from K2HPO4 and KH2PO4 (Merck) in Milli-Q water. (B) Synthesis of the Polymers. (I) High-MolecularWeight Poly(ether-carbonate)s. The synthesis of these random block poly(ether-carbonate)s was carried out by a one-pot reaction:24 stoichiometric amounts of PEG6000 and PPG3000 were poured in a three-necked flask, dried as described, and dissolved in 50 mL of chloroform. The

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amount of pyridine (250% molar excess) was added, and the mixture was cooled in an ice bath. Then, the molar corresponding phosgene amount (250% molar excess) in chloroformic solution was added dropwise during 15 min. Once the dropping was completed, the temperature was allowed to rise back to room temperature and the reaction was continued for 45 min. After that, the temperature was raised to 35 °C and the reaction was continued for an additional 1 h. The polymer produced was separated from the reaction mixture by adding it to 600 mL of petroleum ether at 40-60 °C. The lower phase of the two-phase system formed was separated and dried at room temperature. Finally, the polymer was washed repeatedly with portions of petroleum ether and dried. Light yellow, brittle, and water-soluble powders were obtained. The PEG/PPG initial ratio was changed accordingly, to obtain different PEO/PPO ratios in the final molecule. (Caution! Because of phosgene’s high toxicity, the gas must be handled with extreme care and all the work was conducted under a suitable hood). (II) PEO-PPO-PEO Dimethyl Methacrylate. The reaction is shown for F127, even though it was conducted also for F87. A total of 40.1 g (3.2 mmol) of Pluronic F127 was poured in a three-neck flask and dried as described above. Then, the polymer was dissolved in 75 mL of dry chloroform and the solution was cooled to 0 °C in an ice bath. A total of 2.63 g of TEA (26.3 mmol) was added. A total of 2.65 g (26.3 mmol) of freshly distilled methacryloyl chloride was diluted in 20 mL of chloroform and added dropwise for 2 h into the cooled mixture, under a dry nitrogen flow and magnetic stirring. Finally, the reaction was allowed to proceed for 24 h at room temperature. The crude product was dried under a vacuum and was re-suspended in hot toluene (100 mL). The hot mixture was then filtered to remove the triethylammonium hydrochloride salt. The toluene solution was precipitated in 400 mL of petroleum ether at 60-80 °C. The white solid product, Pluronic F127 dimethacrylate (F127-DMA), was filtered under a vacuum, washed with several portions of petroleum ether at 40-60 °C, and dried under a vacuum at room temperature. The functionalized triblocks will be denoted F127-DMA and F87DMA, respectively. Preparation of Cross-Linked F127-DMA Gels. The crosslinking reaction is hereby exemplified for F127-DMA, even though it was conducted also for F87-DMA. The gels were prepared by dissolving 3 g of F127-dimethacrylate in 12 mL of distilled water. A total of 20 mg of ammonium persulfate (APS) was dissolved in 100 µL of water, added to the solution at a low temperature, and homogenized. Then, 20 mg of sodium metabisulfite was dissolved in 100 µL of water, added to the solution, and mixed thoroughly. Finally, the 20% (w/w) polymer system was incubated at 37 °C for 24 h. The cross-linked gels will be denoted X-F127 and X-F87. (III) Ethoxysilane-Capped Pluronic F127 (F127-DIPTS). A total of 25.2 g (0.002 mol) of Pluronic F127 was weighed in a three-necked flask and dried as described above. Then, 1.2 g (0.005 mol) of IPTS and 0.1 g (3 × 10-4 mol) of SnOct2 were added to the reaction mixture and reacted at

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75 °C for 1 h, under mechanical stirring and a dry nitrogen atmosphere. The material produced was dissolved in chloroform (30 mL), precipitated in petroleum ether at 40-60 °C (400 mL), and filtered. Finally, the F127 derivative (F127DIPTS) was washed repeatedly with portions of petroleum ether at 40-60 °C (3 × 100 mL) and dried in a vacuum at room temperature. Characterization. Gel Permeation Chromatography (GPC). The average molecular weights and polydispersity (M h w/M h n) were determined by GPC (Differential Separations Module Waters 2690 with refractometer detector Waters 410 and Millenium Chromatography Manager), using polystyrene standards between 472 and 360 000 Da. Nuclear Magnetic Resonance (NMR) Spectroscopy. 1H NMR spectra were recorded in a Bruker 300 MHz NMR (300 MHz for 1H measurements). All spectra were obtained at room temperature from 10% (w/v) CDCl3 solutions. The PEO/PPO ratio in the different poly(ether-carbonate)s was determined by the 1H NMR, using calibration curves based on PEG/PPG blends. Fourier Transform Infrared (FTIR) Spectroscopy. The characterization of the functional groups was carried out by FTIR analysis using a Nicolet Avatar 360 FTIR spectrometer. The samples were prepared by solvent casting from chloroform solutions directly on sodium chloride crystals (Aldrich). Dynamic Light Scattering (DLS). The average hydrodynamic radius of the nanostructures formed in aqueous media was measured by DLS (Zetasizer 3000 HAS, Malvern Instruments, U.K.) using an external laser (Ar laser, 488 nm, power 70 mW) in 4 mL of poly(methyl methacrylate) disposable cuvettes. The particle size was taken as the mean value of three measurements. Rheological BehaVior Study. The viscosity versus temperature behavior of the polymer aqueous systems prior to cross-linking was studied using a Brookfield viscometer DVII, with Bath/Circulator TC-500 and Wingather Software, using a T-F spindle at 0.05 rpm. The temperature was stabilized for at least 7 min before each measurement. The rheological analysis was conducted using a parallel plates HAAKE RheoScope-1 Optical rheometer. Cross-linked samples were allowed to swell in water at 37 °C until equilibrium was achieved, and then disk-shaped specimens (20 mm in diameter and 2.5 mm in thickness) were cut out. Two oscillation test types were performed: frequency sweep and temperature ramp. With the objective of identifying the optimal conditions to measure the dynamic mechanical properties of these gels, initially a sinusoidal stress was applied to the sample over a broad range of frequencies (10-2 to 20 Hz), and the storage modulus (G′), the loss modulus (G′′), and the phase angle (tan δ) were determined. It was found that a 10 Hz frequency and a 5 Pa shear stress were optimal. Then, the viscoelastic properties were measured as a function of temperature in the range of 5 to 55 °C, at a 1 °C/min heating rate and a constant angular frequency of 10 Hz. Mechanical BehaVior of Cross-Linked Systems. The different gels were analyzed by means of a penetration test, using a universal testing machine, according to a previously

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described technique.25,26 Briefly, a cylindrical probe (12 mm in diameter) was forced into the gel formed in a cylindrical container (30 mm in diameter) at 37 °C, at a cross-head displacement rate of 0.1 mm/min. The force measured and the penetration distance (0.3 mm) were used to determine the compression modulus, Ec. A minimum of five specimens were tested for each sample. By performing a simple uniaxial compression test and using the Mooney-Rivlin equation (eq 1), the elastic response of the hydrogels can be determined in a rather straightforward way.27 σ)

(

)

F 1 )G λ- 2 A λ

(1)

where σ is the true stress and λ is the deformation expressed as the deformed length divided by the initial length (l/l0). The modulus of elasticity G is 1/3 of the Young’s compression modulus, Ec. Swelling Test. Cylindrical specimens (28 mm in diameter and 5 mm in thickness) were prepared by cross-linking F127DMA solutions, dried until constant weight, and then incubated in deionized water at different temperatures for various periods of time. The swelling degree (%) was calculated from eq 2: swelling (%) )

Ws - Wd × 100 Wd

(2)

where Ws is the sample weight after equilibrium swelling and Wd is the weight of the dry sample. High-Resolution Scanning Electron Microscopy (HRSEM). Hydrogel specimens were allowed to reach equilibrium in water at different temperatures, deep frozen in liquid nitrogen for 4 h and lyophilized at -50 °C for 4 days until full dehydration. Afterward, the morphology of uncoated samples was studied by means of HR-SEM using a Sirion HR-SEM (1-5 kV). Results and Discussion [i] High-Molecular-Weight Poly(ether-carbonate)s. Whereas in other studies various materials were synthesized, the present study focused on polymers consisting of PEO6000 and PPO3000 segments, coupled using phosgene (ClCOCl), via a one-step reaction whereby poly(ether-carbonate) chains were created. By tightly controlling the reaction parameters, various polymers were synthesized, with the PEO content covering the 62-81 wt % range. GPC measurements revealed that the molecular weight of the polymers produced falls in the 38 700-54 000 range and that they had a rather narrow distribution (1.2-1.6). As shown in Figure 1, the gels formed by these poly(ethercarbonate)s attained markedly higher viscosities when compared to F127. While a 20% (w/w) F127 gel reached a rather modest maximum viscosity value (22 000 Pa‚s), the gels formed by the poly(ether-carbonate)s achieved viscosities 10 times higher. It is also worth stressing that these polymers generated much larger aggregates than the micelles formed by F127.


Smart Hydrogels for in Situ Generated Implants Scheme 1. Cross-Linking F127-DIPTS by Hydrolysis and Subsequent Condensation

Table 1. Hydrodynamic Radius of the Aggregates Formed by Poly(ether-carbonate)s Differing in Their PEO Contents and in Their Concentrations size [nm] PEO [wt %]

0.2 wt %

0.4 wt %

0.6 wt %

0.8 wt %

1.0 wt %

71 77 81

110 114 87

131 123 95

157 128 100

171 141 105

187 149 101

While the latter created micelles having a hydrodynamic radius of around 8-9 nm, the former generated much larger aggregates, in some instances as large as 300 nm. Table 1 shows the size of the aggregates formed in aqueous media by three poly(ether-carbonate)s, differing in their PEO/PPO ratio, as a function of concentration. As anticipated, the more hydrophobic the copolymer, the larger the aggregates it forms. An even more hydrophobic copolymer (62% PEO) not only formed especially large aggregates but also did so at significantly lower concentrations (at 0.01%, 150 nm large structures were created). In accordance with the trend apparent from the data shown in Table 1, the hydrodynamic radius measured for a 0.2 wt % solution of this copolymer was 315 nm. This family of reverse thermo-sensitive polymers was generated by copolymerizing segments which, separately, do not display the RTG phenomenon. The high molecular weight of these polymers, their multiblock nature, and the size of the aggregates they generate in aqueous media seem, therefore, to be responsible for their improved reverse

Figure 1. Viscosity versus temperature curves for a [PEO6000OCOO-PPO3000]3 poly(ether-carbonate) in aqueous solutions at different concentrations (% w/w): 10% (rhombi), 15% (squares), and 20% (triangles). A 20% F127 solution (circles) is also shown for comparison purposes.

thermo-responsive behavior. A similar behavior was encountered during the very early stages of the cross-linking of functionalized PEO-PPO-PEO triblocks, as decribed below. The response of the different gels to compression loading was studied by means of a penetration test25 and compared to that of F127. Even though the poly(ether-carbonate) gels were noticeably more robust than F127 and displayed a compression modulus almost two times higher (285 kPa as opposed to 149 kPa), this improvement was considered to be insufficient, and efforts were allocated to engineer sturdier RTG-displaying systems. [ii] Cross-Linked Matrixes. RTG-displaying in situ crosslinkable gels were produced by functionalizing the PEOPPO-PEO triblocks, aiming at combining their reverse thermo-responsive behavior with the much improved mechanical properties of cross-linked hydrogels. Two different synthetic avenues resulted in reverse thermo-responsive materials that were able to cross-link immediately or over time. In the former, the triblocks were end-capped with carbon-carbon double bonds that allowed their almost instantaneous cross-linking, whereas the latter gradually cross-linked by capitalizing on the functionalization of F127 with ethoxysilane groups. F127-DIPTS Hydrogels. A family of reverse thermoresponsive polymers was created by cross-linking triethoxysilane end-capped PEO-PPO-PEO triblocks, obtained by reacting their terminal hydroxyl groups with IPTS. Once in contact with water, the ethoxysilane moieties hydrolyzed, generating silanol groups that then condensated, whereby a cross-linked matrix developed as a function of time, gradually improving their mechanical properties (see Scheme 1).

Figure 2. Compression modulus (Ec) increase as a function of time, for an F127-DIPTS 30% solution.

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Figure 3. Compression modulus (Ec) of F127 and cross-linked X-F127 and X-F87 20% gels. A minimum of three specimens per sample were tested.

Figure 4. Equilibrium water uptake attained by the X-F127 hydrogels, at 6 °C and 37 °C.

The basic reverse thermo-responsive behavior of F127 was only marginally affected by the presence of the triethoxysilane end groups. For example, the minimal concentration required for the material to display RTG (Ci) increased from 14.6% for F127 to 15.0% for the DIPTS derivative. Because the Ci values were determined by varying the concentration

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by 0.1% steps, undoubtedly the difference above is significant. As anticipated, the rheological properties of the hydrogels increased concomitantly with the cross-linking process. Expectedly, the reaction was faster and the gel formed was studied, as the concentration of the triblock increased, with the 20 and 25% gels losing their ability to revert to their sol state after 5 and 3 days, respectively. It was especially interesting to study the behavior of F127DIPTS solutions at concentrations below their Ci (15.0%) lacking, therefore, any RTG behavior. As time evolved, though, these solutions progressively acquired reverse thermoresponsiveness, as their molecular weight started growing, due to the gradual cross-linking of the matrix. For example, at time zero, a 13% F127-DIPTS solution had no RTG behavior, but after 2 days it started gelling as the temperature increased, attaining 29 000 and 43 400 Pa‚s after 5 and 7 days, respectively. Even a diluted 10% solution became reverse thermo-responsive, albeit rather modestly, after 5 days. This behavior can be attributed to the fact that, at the very early stages of the process, the incipient cross-linking reaction results in an increase of the average molecular weight, while the degree of cross-linking is low enough so as to allow the system to retain its basic RTG behavior. Because Ci decreases substantially as the length of the chains increases,7,28 as their molecular weight raises, the actual Ci value eventually drops below the intial concentration, enabling the system to exhibit the RTG phenomenon. It is only as the reaction proceeds further and the matrix becomes substantially cross-linked that the polymer loses its ability to revert to its sol state and behaves as a thermoset material. These ethoxysilane-capped PEO-PPO-PEO triblocks attained enhanced mechanical properties, as the degree of cross-linking increased with time. This process is illustrated in Figure 2, which plots the compression modulus (Ec) of a 30% F127-DIPTS aqueous solution, which achieved 3.0 MPa already after 2 days, reaching a remarkable 4.0 MPa level after 9 days.

Figure 5. Frequency dependence of G′ and G′′ for 15, 20, 25, and 30% cross-linked F127-DMA.


Figure 6. Temperature dependence of G′, G′′, and tan(δ) for the 25% cross-linked F127-DMA gel.

Figure 7. Storage modulus of different hydrogel concentrations

F127-DMA Hydrogels. In contrast to the gradual crosslinking reaction of the triethoxysilane-capped triblocks, PEO-PPO-PEO dimethacrylate derivatives obtained by the reaction of the native triblocks with methacryloyl chloride, cross-linked almost immediately. The occurrence of the reaction was proven by 1H NMR analysis and FTIR


spectroscopy. Rheological data demonstrated that the presence of the two methacrylate terminal groups affected F127’s behavior only slightly, with the native F127 triblock and the un-cross-linked F127-DMA showing very similar viscosity versus temperature curves. F127-DMA was then cross-linked in aqueous solutions, using the APS/sodium metabisulfite redox catalytic system, following Sawheny et al.29 Figure 3 compares the compression modulus of two cross-linked gels, X-F127 and X-F87, and that of non-cross-linked F127. As is apparent from the data presented, the un-cross-linked F127 triblock exhibited a compression modulus remarkably lower (149 kPa) than those measured for the cross-linked matrixes (628 and 922 kPa, for X-F127 and X-F87, respectively). In accordance to expectations, these findings demonstrate that the cross-linking density plays an important role, as reflected by the fact that X-F87 (molecular weight 7500) generates a significantly stiffer gel, when compared to X-F127 (molecular weight 12 600). Evidently, the covalent bonds present in the cross-linked matrix prevent it from reverting to its solution state, once the temperature is reduced below the sol-gel transition. It is worth stressing, though, that the reverse thermo-responsiveness of these materials is important only at insertion time, when their enhanced injectability and full conformability play a key role. Once the solution has been deployed to the site of performance and has gelled, as it heats to body temperature, the RTG behavior completes its initial clinical function. After this initial stage, therefore, the loss of the reverse thermo-responsiveness of these matrixes is immaterial. Having said that, the relatively high molecular weight between cross-link junctions allowed the hydrogel to retain, albeit to a very limited extent, a remainder of its RTG response. This is clearly shown by the equilibrium water uptake attained by the cross-linked matrixes, below and above the sol-gel transition of the non-cross-linked triblock, respectively (see Figure 4). Reminiscent of the basic reverse thermo-responsiveness of native F127, the cross-linked sample attained at 37 °C an equilibrium swelling

Figure 8. Micrographs of cross-linked F127-DMA matrixes (A, 5%; B, 10%; C, 25%; and D, 30%).

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Figure 9. Tubular structure of a 20% X-F127 hydrogel.

degree of approximately 500%, increasing markedly to around 1500% when the temperature decreased to 6 °C. Aiming at optimizing the mechanical properties of these hydrogels, the concentration of F127-DMA was varied between 5 and 30%. First, the dynamic storage modulus (G′) and loss modulus (G′′) were measured over the whole range

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of frequencies studied, at 37 °C. While the 5 and 6% systems were too diluted to generate measurable data, the 7, 8, and 10% systems displayed only incipient elasticity. Figure 5 plots G′ and G′′ versus frequency curves, for 15, 20, 25, and 30% F127-DMA cross-linked gels. The primarily elastic response exhibited by all four gels is apparent, as revealed by the fact that G′ raised only slightly as the frequency increased, with G′′ being substantially lower than G′ over the entire frequency range. Figure 6 presents the temperature dependence of G′, G′′, and tan(δ) at a constant frequency (10 Hz), for the 25% F127DMA cross-linked hydrogel, with the other cross-linked matrixes exhibiting a similar behavior. As is apparent from the plot presented, G′ was substantially higher than G′′ over the entire temperature range, this behavior being in agreement with a typical gel behavior.30,31 The large increase in the storage modulus shown by these cross-linked hydrogels as temperature increased is due of the reverse thermo-responsiveness of their F127 building blocks. In contrast to non-cross-linked RTG-displaying gels,

Figure 10. Stress-strain curve of 20% X-F127 tubes, with wall thicknesses of 1.5 and 2.5 mm.

Figure 11. (a) Tubular two-layered hydrogel: inner layer X-F127-DMA 20%, outer layer X-F127-DMA 15%. (b). Tubular three-layered hydrogel: inner layer X-F127-DMA 15%, midlayer 20%, and outer layer X-F127-DMA 25%.


where the increase in viscosity is rather sharp, the limited chain mobility of the cross-linked systems resulted in a more gradual increase of the storage modulus, over a broader temperature interval. The glass transition temperature, defined as the temperature at which tan(δ) reaches its maximum value, appeared around 27 °C and was only marginally affected by the cross-linking density. Figure 7 graphically displays the change in the storage modulus of four different hydrogels, as the temperature monotonically increased. The relatively diluted 15% system showed a rather modest response, exhibiting 16 and 40 kPa G′ values at 22 and 37 °C, respectively. The significantly more cross-linked 30% F127-DMA hydrogel, on the other hand, displayed a sizable elastic response already at 6 °C, with a storage modulus of 283 kPa that steadily increased with temperature, attaining a remarkable 650 kPa value at physiological temperature. The micrographs shown in Figure 8 reveal the porous structure of the different materials produced, as a function of the concentration of F127-DMA. Expectedly, the porosity of the matrix decreased as the concentration of F127-DMA increased. It should be kept in mind, though, that the possibility of artifacts being caused by the preparation technique cannot be completely ruled out. Having said that, it is apparent that over the eight different concentrations studied (only four are shown in Figure 8), there is a gradual and consistent change in the microstructure generated, from the highly porous matrix formed by the 5% X-127 hydrogel down to the limited porosity shown by the 30% X-127 matrix. [iii] Macroscopic Structures. With the aim of determining the basic feasibility of creating macroscopic constructs using reverse thermo-responsive polymers, tubular structures were engineered, as exemplified in Figure 9 for a 20% X-F127 system. The F127-DMA solution containing the catalytic redox system was syringed into a cylindrical glass mold, and then the temperature was raised to 37 °C, resulting in the polymer rapidly gelling and subsequently cross-linking. The mechanical characterization of these tubes was conducted, as shown in Figure 10. Expectedly, the burst strength increased with the wall thickness, raising from 16 to 22 kPa as the wall thickness rose from 1.5 to 2.5 mm. It is also worth stressing that these significantly compliant conduits failed at rather large strain values, increasing from 65 to 475% as the wall thickness decreased. With the aim of developing more advanced structures, systems comprising two and also three layers were engineered, as shown in Figure 11a,b. The working concept behind the engineering of the three-layered conduit was to crudely generate a scaffold that mimics the trilayered structure of the arterial wall. Conclusions Two different strategies were pursued to engineer constructs having enhanced mechanical properties, using new reverse thermo-responsive polymeric systems developed in our laboratory: (i) the generation of RTG-displaying highmolecular-weight polymers by covalently binding PEO and


PPO segments, using phosgene as the chain extender, and (ii) the use of functionalized PEO-PPO-PEO triblocks that combine initial syringability with the capacity to cross-link in situ. Even though the poly(ether-carbonate)s displayed significantly improved mechanical properties when compared to F127, they were considered to be unsuitable for a broad range of biomedical systems. The hydrogels formed by in situ cross-linking reverse thermo-responsive polymers, particularly F127 dimethacrylate, attained rather remarkable mechanical properties and allowed the engineering of robust macroscopic constructs, such as large tubular structures. Monolayered conduits as well as structures comprising two and three layers were formed in vitro, and their compliance and burst strength were determined. The broad range of mechanical properties covered by these cross-linked hydrogels may allow the in situ generation of implants that mimic those of the host soft tissue. References and Notes (1) Hubbell, J. A.; Pathak, C. P.; Sawhney, A. S.; Desai, N. P.; Hill, J. L. U.S. Patent 5,410,016, 1995. (2) Temenoff, J. S.; Mikos, A. G. Biomaterials 2000, 21, 2405. (3) Elisseeff, J.; McIntosh, W.; Fu, K.; Blunk, T.; Langer, R. J. Orthop. Res. 2001, 19 (6), 1098. (4) Scopelianos, A. G.; Bezwada, R. S.; Arnold, S. C. U.S. Patent 5,824,333, 1998. (5) Dunn, R. L.; English, J. P.; Cowsar, D. R.; Vanderbilt, D. P. U.S. Patent 4,938,763, 1990. (6) Cohn, D.; Sosnik, A.; Levy, A. Biomaterials 2003, 24 (21), 3707. (7) Sosnik, A.; Cohn, D. Biomaterials 2005, 26 (4), 349. (8) Cohn, D.; Sosnik, A.; Kheyfetz, M. U.S. Patent 2003/0082235, 2003. (9) Sosnik, A.; Cohn, D. Biomaterials 2004, 25 (14), 2851. (10) Peppas, N. A.; Bures, P.; Leobandungm, W.; Ichikawa, H. Eur. J. Pharm. Biopharm. 2000, 50, 27. (11) Alexandridis, P.; Hatton, T. A. Colloids Surf., A 1995, 96, 1. (12) Hoffman, A. AdV. Drug DeliVery ReV. 2002, 54, 3. (13) Bromberg, L. E.; Ron, E. S. AdV. Drug DeliVery ReV. 1998, 31 (3), 197. (14) Bromberg, L. Polymer 1998, 39 (23), 5663. (15) Zana, R.; Kamenka, N.; Burgaud, L.; Lindman, B. J. Phys. Chem. 1994, 98, 6785. (16) Vadnere, M.; Amidon, G. L.; Lindenbaum, S.; Haslam, J. L. Int. J. Pharm. 1984, 22, 207. (17) Alexandridis, P.; Andersson, K. J. Colloid Interface Sci. 1997, 194 (1), 166. (18) Mortensen, K. Colloids Surf., A 2001, 183, 277. (19) Deng, Y.; Yu, G.; Price, C.; Booth, C. J. Chem. Soc., Faraday Trans. 1992, 88, 1441. (20) Yang, L.; Alexandridis, P.; Steyler, D. C.; Kositza, M. J.; Holzwarth, J. F. Langmuir 2000, 16, 8555. (21) Kabanov, A. V.; Batrakova, E. V.; Melik-Nubarov, N. S.; Fedoseev, N. A.; Dorodnich, T. Y. J. Controlled Release 1992, 22, 141. (22) Sosnik, A.; Cohn, D.; San Roman, J.; Abraham, G. A. J. Biomater. Sci., Polym. Ed. 2003, 14 (3), 227. (23) Kheyfets, N. V.; Lopyrev, V. A.; Eisenschtadt, I. N. Zhurnal prikladnoi khimii 1968, 21, 1380. (24) Yu, C.; Kohn, J. Biomaterials 1999, 20, 253. (25) Oakenfull, D. G., Parker, N. S., Tanner, R. I., Phillips, G. O., Williams, P. A., Wedlock, D. J., Eds.; Gums and Stabilisers for Food Industry 4; IRL Press: Oxford, 1988; p 231. (26) Gregson, C. M.; Hill, S. E.; Mitchell, J. R.; Smewing, J. Carbohydr. Polym. 1999, 38, 255. (27) Guenet, J. M., Ed.; ThermoreVersible gelation of polymers and biopolymers Academic Press: London, 1992. (28) Cohn, D.; Sosnik, A. J. Mater. Sci.: Mater. Med. 2003, 14 (2), 175. (29) Sawhney, A.; Melanson, D. A.; Pathak, C. P.; Hubbell, J. A.; Avila, L. Z.; Kieras, M. T.; Goodrich, S. D.; Barman, S. P.; Coury, A. J.; Rudowsky, R. S.; Weaver, D. J. K. U.S. Patent 5,844,016, 1998. (30) Kim, M. R.; Park, T. G. J. Controlled Release 2002, 80, 69. (31) Nijenhuis, K. AdV. Polym. Sci. 1997, 130, 8.

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Smart Hydrogels for in Situ Generated Implants - American Chemical

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