Biomacromolecules 2008, 9, 481–486
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Thermoreversible Hydrogels from RAFT-Synthesized BAB Triblock Copolymers: Steps toward Biomimetic Matrices for Tissue Regeneration† Stacey E. Kirkland,‡ Ryan M. Hensarling,‡ Shawn D. McConaughy,‡ Yanlin Guo,§ William L. Jarrett,‡ and Charles L. McCormick*,‡,4 Department of Polymer Science, Department of Biological Sciences, and Department of Chemistry and Biochemistry, The University of Southern Mississippi, Hattiesburg, Mississippi 39406 Received August 30, 2007; Revised Manuscript Received November 1, 2007
Narrowly dispersed, temperature-responsive BAB block copolymers capable of forming physical gels under physiological conditions were synthesized via aqueous reversible addition fragmentation chain transfer (RAFT) polymerization. The use of a difunctional trithiocarbonate facilitates the two-step synthesis of BAB copolymers with symmetrical outer blocks. The outer B blocks of the triblock copolymers consist of poly(N-isopropylacrylamide) (PNIPAM) and the inner A block consists of poly(N,N-dimethylacrylamide). The copolymers form reversible physical gels above the phase transition temperature of PNIPAM at concentrations as low as 7.5 wt % copolymer. Mechanical properties similar to collagen, a naturally occurring polypeptide used as a three-dimensional in vitro cell growth scaffold, have been achieved. Herein, we report the mechanical properties of the gels as a function of solvent, polymer concentration, and inner block length. Structural information about the gels was obtained through pulsed field gradient NMR experiments and confocal microscopy.
Introduction Research on stimuli-responsive hydrogels has been prompted by a number of potential applications in biomedical fields that include drug delivery and tissue engineering.1–3 In order for a hydrogel to serve as an in vitro three-dimensional cell growth scaffold, several design criteria must be considered, including biocompatibility, gelation mechanism, mechanical properties, pore size, and gel-cell interactions.4–7 Commercially available three-dimensional in vitro cell growth platforms are typically irreversible physical gels comprised of naturally occurring biomolecules extracted from cells or tissue.7 These materials can meet many of the above design criteria but do not allow the facile removal of cells from the matrix and therefore limit a number of advanced applications. Chemically cross-linked gels have also been studied but have the same problem regarding cell removal as the physical gels. In addition, unreacted crosslinking reagents can lead to high toxicity.4 Nanofiber networks with appropriate dimensions may be formed by electrospinning.8 However, the fiber network must be formed in situ without harming the cells.7 A three-dimensional in vitro cell growth matrix with a mechanism for cell release under nontoxic conditions would allow researchers to harvest intact cells or tissue for further studies or possible implantation. Physical gels from BAB triblock copolymers have been investigated because of the noncovalent nature of their crosslinks and the potential reversibility of these systems. As seen in Figure 1, at moderate to high concentrations of polymer, physical cross-links can form when the polymer is placed in a solvent selective for the inner block. Physical networks may also be formed from the entanglements between coronas of * To whom correspondence should be addressed. E-mail:
[email protected]. † Paper number 132 in a series entitled Water Soluble Polymers. ‡ Department of Polymer Science. § Department of Biological Sciences. 4 Department of Chemistry and Biochemistry.
Figure 1. The formation and structure of a reversible physical gel.
micelles.9,10 The formation of a physical network is dependent on the relative populations of “loops” and “bridges”.11,12 The inner block forms a bridge when the outer blocks of a polymer are incorporated into different hydrophobic domains while a loop forms when both blocks of a chain are incorporated into the same hydrophobic domain. Loop formation is limited by the entropically unfavorable conformational constraints that are imposed on the system as the inner block attempts to backfold on itself.13–16 Bridge formation may also be entropically unfavorable if the inner block of the polymer chain must adopt a stretched conformation in order to span between hydrophobic domains. Another structural possibility, dangling chain ends, is limited by the increase in interfacial free energy which occurs as the chain end is placed in an incompatible solvent.12,13 As previously reported, exchange rates between unimers and micellar structures decrease as the hydrophobicity of the lipophilic block is increased.17–19 By use of Monte Carlo simulations, the formation of loops, dangling chain ends, and bridges in symmetric BAB triblock copolymers was studied by Mattice et al.9 The results indicate that the existence of dangling chain ends in physical gels should decrease as the solvent becomes more incompatible with the outer block.9 Further
10.1021/bm700968t CCC: $40.75 2008 American Chemical Society Published on Web 12/29/2007
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Table 1. Reaction Conditions and Gel Permeation Data for the Synthesized Polymers entry no.
exptl structure (subscripts refer to DP)
time (h)
temperature (°C)
Mna
Mn innera,b
Mw/Mna
dn/dc
H910 P210 P277
NIPAM910 NIPAM455-b-DMA210-b-NIPAM455 NIPAM455-b-DMA277-b-NIPAM455
10 1 1
30 27 27
103200 124100 130700
20900 27500
1.07 1.06 1.08
0.074 0.078 0.084
a As determined by triple detection GPC conducted in 0.02 M LiBr DMF eluent. b As determined by the difference between the triblock and homopolymer molecular weight.
theoretical treatments on the mechanism and driving forces behind the physical gelation of associating polymers may be found in the literature.20–27 Triblock copolymers with switchable hydrophobicity offer a high level of sophistication because gel formation can be controlled by external stimuli. The hydrophobicity of the blocks is often altered through changes in solvent, temperature, or pH.28–33 Because of this reversible sol–gel transition, physical gels from stimuli-responsive triblock copolymers have been studied as potential cell growth platforms.1,3,13,28–34 Until recently, research in this area has been limited by the techniques available to produce triblock architectures from water-soluble monomers. The advent of controlled/“living” free radical techniques offers new routes to obtain polymers of complex architecture with a wide range of monomers.35–41 In addition, these techniques provide narrowly dispersed polymers of controlled molecular weight. The ability to control the molecular weight of individual block lengths while maintaining narrow polydispersity affords unique opportunities to study structure–property relationships of (co)polymers. For this reason, polymers synthesized via controlled/“living” free radical techniques have potential applications in numerous biomedical fields. Armes et al. have synthesized a number of BAB triblock copolymers via atom transfer radical polymerization (ATRP) that physically gel in response to changes in pH or temperature.28–32 Liu and coworkers recently reported the synthesis of ABA and BAB N-isopropylacrylamide (NIPAM) and 2-hydroxyethyl methacrylate (HEMA) triblock copolymer via ATRP.42 Several of the copolymer gels showed promise in applications such as cell growth scaffolds28 and controlled-release substrates.29 The controlled/“living” polymerization technique based on degenerative chain transfer, RAFT polymerization, is of particular significance because of its tolerance to a wide variety of solvents, temperatures, monomers, and functional groups. For further information on the RAFT polymerization technique, the reader is referred to several comprehensive reviews.35,37–41 Winnik and co-workers synthesized a series of C18-PNIPAMC18 which formed physical networks at concentrations of 20 g L-1 using reversible addition fragmentation chain transfer (RAFT) polymerization.43 Herein we report the synthesis of narrowly dispersed temperature-responsive BAB triblock copolymers via RAFT polymerization for potential three-dimensional cell growth scaffold materials. The block lengths can be varied in a facile manner to prepare reversible hydrogel, elastic networks. Poly(Nisopropylacrylamide) (PNIPAM) was chosen as the temperatureresponsive outer block because of its reversible phase transition temperature (32 °C) near physiological conditions (37 °C)44 and studies indicating its cytocompatability.45 It is well-known that the incorporation of hydrophilic monomers increases the lower critical solution temperature (LCST) of PNIPAM, and therefore, the polymer used as the inner block must be carefully selected.46,47 Block length must be controlled in order to prevent the LCST from reaching physiological temperature (37 °C). In order to minimize the elevation of the LCST, an N,N-
disubstituted acrylamido polymer, poly(N,N-dimethylacrylamide) (PDMA), was chosen as the inner block. Mechanical properties were targeted to those of collagen, a commonly used material for both two- and three-dimensional cell growth matrices. The effects of inner block length, solvent, and polymer concentration on mechanical properties were investigated. In addition, the apparent pore sizes of the gels were determined through pulsed field gradient NMR.
Experimental Section Materials. All chemicals were purchased from Fischer or Aldrich at the highest available purity and used as received unless otherwise noted. N-Isopropylacrylamide (NIPAM) was recrystallized from hexane. N,N-Dimethylacrylamide (DMA) was vacuum distilled. 2,2′-Azobis[2(2-imidozolin-2-yl)propane] dihydrochloride (VA-044) was used as the initiator (gifts from Wako chemicals USA, Inc.). The difunctional chain transfer agent (CTA) 2-(1-carboxy-1-methylethylsulfanylthiocarbonylsulfanyl)-2-methylpropionic acid (CMP) was used without further purification (gift from Noveon, Inc.). Type 1 collagen from rat tails (high concentration, 8.14 mg/mL) was purchased from BD Biosciences. Synthesis of NIPAM MacroCTA. On the basis of previous kinetic studies,48 polymerization conditions were chosen to produce a DP ) 900 NIPAM macroCTA at 90% conversion. The polymerization was conducted for 10 h at 30 °C in aqueous media at pH 5.0 with an initial monomer concentration ([M]0) of 0.5 M. To synthesize the macroCTA, the following components were added to a round-bottom flask: VA044 (10.2 mg, 31.5 µmol), NIPAM (9.97 g, 88.1 mmol), CMP (25.1 mg, 88.9 µmol), and deionized water (177 mL). The round-bottom flask was sealed with a septum and purged with nitrogen for 30 min. After 10 h in a 30 °C water bath the polymerization was terminated by exposure to air. The polymer was purified via dialysis against an acidic solution at 4 °C followed by lyophilization. Chain extension of NIPAM macroCTA. Polymerization conditions were selected to produce inner block DPs of 200 and 400 at 50% conversion. The polymerizations were conducted at 27 °C in aqueous media with an initial monomer concentration ([M]0) of 0.5 M. To chain extend the macroCTA, the following components were added to a round-bottom flask: VA-044 (3.16 mg, 9.78 µmol), NIPAM macroCTA (1.01 g, 9.78 µmol), DMA (0.88 g, 8.9 mmol or 0.44 g, 4.5 mmol), and deionized water (17.8 or 8.9 mL). The round-bottom flasks were sealed with a septa and purged with nitrogen for 30 min. After 1 h in a 27 °C water bath, the polymerization was terminated by exposure to air. The polymer was purified via dialysis against an acidic solution at 4 °C followed by lyophilization. Gel Permeation Chromatography. Size exclusion chromatography (SEC) was used to determine Mn, Mw, and polydispersity indices (PDIs) for (co)polymers (0.02 M LiBr DMF eluent, 1.0 mL/min, 60 °C, Viscotek I-Series Mixed Bed low-MW and mid-MW columns, Viscotek-TDA (302 nm RI, viscosity, 7 mW 90° and 7° true low angle light scattering detectors (670 nm). The dn/dc of each polymer is listed in Table 1 and was determined at 632.8 nm in DMF at 60 °C using a Viscotek refractometer and Omnisec software. The Mn of the inner blocks was determined by taking the difference between the Mn of the triblock copolymer and homopolymer. Rheometric Studies. Rheological measurements were performed with a Rheometrics SR-5000 controlled stress rheometer to determine the gel point, storage modulus (G′), and loss modulus (G′′) of the
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Scheme 1. Synthetic Route for the Preparation of BAB Triblock Copolymers via Aqueous RAFT Polymerization
temperature-responsive polymers. All measurements were performed using a 25 mm cone and plate geometry with an angle of 0.1 rad. An insulated ring was placed around the geometry to prevent water evaporation. Before measurements were performed, the polymers were allowed to dissolve overnight in either deionized water or 140 mM NaCl/20 mM phosphate buffer. Collagen gels were prepared according to the alternate gelation procedure provided on the product specification sheet. Copolymer solutions were presheared for 2 min at a constant applied shear stress of 5 Pa before dynamic temperature ramp measurements were taken. A frequency of 1 rad/s was used. Materials were heated with a Peltier plate at a rate of 0.5 °C min-1. Pulsed Field Gradient Nuclear Magnetic Resonance (PFG NMR) Studies. The time-dependent diffusion coefficient of water (Dapp) was determined using PFG NMR methods. All spectral data presented herein were obtained on a Varian UNITYInova 500 MHz spectrometer using a standard 5 mm two-channel probe equipped with gradients. Self-diffusion coefficients were determined from the negative slope of a log-attenuation plot (log Ψ vs γ2g2δ2(∆ - δ/3)), where Ψ is the echo attenuation, γ is the proton gyromagnetic ratio, δ is the width of the gradient pulse, g is the magnitude of the applied field gradient, and ∆ is the total diffusion time. The standard Stejskal-Tanner sequence,49 illustrated in Figure S-2, was utilized using an acquisition time of 0.5 s, a recycle delay of 5 s, and gradient pulses of 0.8–1.0 ms. The total diffusion time was varied from 20 to 900 ms, and gradient amplitude ranged from 20 to 80 G/cm to ensure the signal was attenuated ∼80%. The spectral width was 50 kHz, and the number of scans for each spectrum ranged from 8 to 16. Exponential line broadening was applied prior to Fourier transformation of the free induction decays. Gradient calibration was performed using a deionized water standard prior to data collection. Samples consisted of 10 wt % polymer dissolved in 140 mM NaCl/20 mM phosphate buffer pH 7.4. Confocal Microscopy. For confocal microscopy experiments, a buffer solution of 10 wt % polymer and 2.5 × 10-4 M 8-anilino-1naphthalenesulfonic acid (ANS) was used. To prepare the samples, a stock solution of ANS in acetone was prepared. An appropriate volume of the stock solution was transferred to a scintillation vial to give 2.5 × 10-7 mol of ANS. The acetone was evaporated under a stream of nitrogen. The triblock copolymer (10 mg) was added, and subsequently 1 mL of 140 mM NaCl/20 mM phosphate buffer pH 7.4. The polymer was allowed to dissolve overnight in the dark at 5 °C. A LSM510 Meta confocal microscope with an excitation wavelength of 488 nm and a LP505 filter was used. A heating block was used as a temperature controller for the gel solutions.
Results and Discussion The PNIPAM macroCTA and triblock copolymers were synthesized in water at mild temperatures utilizing the hydro-
lytically stable CTA, CMP50 (see Scheme 1). The use of a difunctional CTA allows BAB structures with symmetrical outer blocks to be synthesized in two steps.48 After dialysis and lyophilization, the polymers were characterized by triple detection DMF SEC (see Table 1 and Supporting Information). The dn/dc of each polymer, listed in Table 1, was determined using Omnisec software. The triblock copolymers have well-defined molecular architectures with polydispersities below 1.10. A large contribution to mechanical strength of the thermoreversible gels arises from the hydrophobic interactions of PNIPAM. Hence, a long outer block length was selected to increase the number of hydrophobic associations. Moderate inner block lengths were targeted to facilitate intermicellar bridging at low concentrations while maintaining phase transition temperatures below 37 °C. The NIPAM910 macroCTA (H910) and triblock copolymer NIPAM455-b-DMA210-b-NIPAM455 (P210) closely approach the respective targeted structures of NIPAM900 and NIPAM455-bDMA200-b-NIPAM455. The effects of inner block length, polymer concentration, and solvent on the storage modulus (G′), and loss modulus (G′′), and gel point (Tgel) of the temperature-responsive polymers were determined through a series of dynamic temperature ramp tests (Figure 2 and Figure S-3). The dynamic temperature ramps of P210 dissolved in deionized water and 140 mM NaCl/20 mM phosphate buffer at different weight percents are shown in Figure 2. At the specific frequency and stress used in the dynamic temperature ramp experiments, the polymer solutions respond elastically (G′ > G′′) at temperatures below the phase transition of the PNIPAM blocks. Though G′ is larger than G′′ at room temperature, the materials are not deemed gels because of their overall low modulus and ability to flow under their own weight. As can be observed in Figure 2, no significant changes in moduli occur for the 2.5 wt % solution dissolved in deionized (DI) water. The 5.0 wt % solution exhibits a slight increase in G′′ but no significant changes in G′ (see Figure S-3). According to Semenov, the formation of intermicellar bridges does not occur until a critical concentration of flower micelles is reached.21 In addition, the decrease of G′ to its initial value at 45 °C suggests negligible bridging at these temperatures. At 7.5 wt %, G′ increases as the temperature is raised from 37 to 42 °C but falls back to its initial value at higher temperatures indicating that interconnected flower micelles may form between these intermediate temperatures; however, bridging is not sufficient for the formation of a gel network. Previous reports indicate G′ is related to the lifetime of the junctions and the stability of the
484 Biomacromolecules, Vol. 9, No. 2, 2008
Figure 2. Storage (G′) and loss (G′′) moduli plotted against temperature for different concentrations of P210 in (A) DI water and (B) 140 mM NaCl/20 mM phosphate buffer pH 7.4. Closed and open symbols represent G′ and G′′, respectively.
micelle core.10,11 PNIPAM dangling chain ends incur a larger enthalpic penalty at elevated temperatures because of poor outer block solubility. The increased enthalpic penalty leads to a shorter lifetime of the dangling chain ends, an increase in the lifetime of the junctions, and hence an overall higher modulus. As seen in Figure 2B, the addition of salt results in a twophase transition (most notably for solutions e7.5 wt %). Previously, Bergbreiter, Cremer, and co-workers reported on the two-step phase transition of PNIPAM which occurs in the presence of kosmotropes (water-structure makers) above a certain concentration.51,52 The LCST of PNIPAM was reported to have a dependence on both anion concentration and anion type.51–53 The two-step transition was also affected by the molecular weight and concentration of the polymer.52 Hence, the two transitions seen in Figure 2B may be explained by the presence of two kinds of anions, Cl– and PO4-, as well as the two-step phase transition described in the aforementioned literature. Similar trends in the dynamic temperature ramps were seen for entry P277 (Supporting Information). The gelation temperature (Tgel) of these materials was defined as the crossover between G′ and G′′. As seen in Figures 2 and 3A, Tgel decreases as the polymer concentration increases. The concentration dependence of Tgel corresponds to the depression in the phase transition temperature of PNIPAM with increasing concentration.44,54,55 Because entries P210 and P277 have the same PNIPAM block length, the similarity of Tgel at various concentrations is not surprising. It should be noted that at 7.5
Kirkland et al.
Figure 3. (A) Gelation temperature (Tgel) and storage modulus at the gelation temperature (G′Tgel) plotted against the solution concentration of the triblock copolymer. Closed and open symbols represent polymers dissolved in DI water and 140 mM NaCl/20 mM phosphate buffer pH 7.4, respectively. (B) Storage modulus at 37 °C (G′37 °C) plotted against the solution concentration of the triblock copolymer.
wt % P277 forms a gel whereas P210 does not. Referring back to Semenov’s theory, the loops on P277 should extend further into solution than P210, resulting in overlap and therefore gelation at lower polymer concentrations.21 A series of steady stress sweep tests indicate that the triblock copolymers begin overlapping at approximately 7.5-10 wt % polymer. The addition of 140 mM NaCl/20 mM phosphate buffer uniformly depresses Tgel, resulting in systems that gel at temperatures below 37 °C. The depression of Tgel can be attributed to the disruption of the hydrophobic water shells solvating the isopropyl groups of PNIPAM.56 As seen in Figure 3, the storage modulus at the gelation temperature (G′Tgel) increases with polymer concentration. This increase can be attributed to the increased number of bridges at higher concentrations as well as an increased number of hydrophobic interactions. When dissolved in DI water, P277 exhibits a slightly larger G′Tgel than P210 over all tested concentrations, suggesting P277 has more bridges connecting hydrophobic domains. The addition of salts to the solvent results in slight increases in G′Tgel for P210 and P277. In general, kosmotrope salts disrupt the ordered water shell which solvates the isopropyl group and therefore decreases the solubility of PNIPAM. As stated previously, a decrease in solvent quality should increase G′ by decreasing the lifetime of dangling chain ends.
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Figure 4. The apparent diffusion coefficient of water at 37 °C plotted against the observation time (∆) for 10 wt % polymer in 140 mM NaCl/ 20 mM phosphate buffer pH 7.4. Closed and open symbols represent P210 and P277, respectively.
The mechanical properties of collagen, a commonly used in vitro cell growth platform, were tested at 37 °C (see Supporting Information). As seen in Figure 3B, mechanical properties similar to those of collagen can be achieved with the synthesized triblock copolymers. When the polymers are dissolved in DI water, G′37 °C increases linearly on the semilog plot once a critical concentration is reached. A dramatic increase in G′37 °C occurs when the polymer is dissolved in 140 mM NaCl/20 mM phosphate buffer. One may explain this trend by referring back to Semenov’s theory and basic thermodynamics. Once a critical concentration of micelles is reached, bridging occurs between hydrophobic domains. The lifetime of dangling chain ends is decreased because of the increase in interfacial free energy which occurs as the PNIPAM block dangles in the incompatible salt solution. The decreased lifetime of the dangling chain end results in a stronger network. The dependence of Dapp on total diffusion time ∆ was determined using PFG NMR spectroscopy. For restricted geometries (such as bridged micelles), the diffusion coefficient varies with ∆: At short ∆ values, Dapp will be approximately the same as that for bulk water, Dfree. As ∆ increases, H2O molecules will begin to encounter boundaries, thereby making Dapp < Dfree. At a sufficiently long ∆, Dapp will attain a limiting value.57 The onset of this limiting time (td) can be used to describe geometric aspects of the material. For example, the root-mean-square (rms) end-to-end distance, r, can be determined using
Dapp ) (1 ⁄ 6)td-1r2
(1)
where Dapp is the apparent diffusion coefficient, td is the time at which the diffusion coefficient exists, and r is the root-meansquare end-to-end distance.58 PFG NMR studies were conducted on 10 wt% P210 and P277 in buffer solution. These concentrations were chosen because their mechanical properties at 37 °C closely resemble those of collagen. As seen in Figure 4, the triblock copolymers exhibit similar trends. After 40 ms of diffusion time the diffusion coefficient of water begins decreasing rapidly until a short plateau region is reached at approximately 400–600 ms. The plateau region suggests that all diffusing species are experiencing boundaries at this time scale. With eq 1, the root-mean-square end-to-end distance can be calculated to approximately 75 and
68 µm for the initial plateau region of P210 and P277, respectively. To properly support cell growth, the surface features of a scaffold should be smaller than the dimensions of a cell (tens of micrometers).59 The decrease in Dapp at ∆ > 700 ms may be due to background gradients caused by imperfect shimming and the magnetic susceptibility of the sample. These effects have a more significant impact on measurements taken at long ∆.60,61 In order to qualitatively investigate the structure of these gels, confocal microscopy experiments were conducted. The fluorescent probe, ANS, was chosen because of its increased emission intensity in nonpolar environments.62 Therefore, fluorescence should intensify at elevated temperatures because of the existence of hydrophobic domains. As can be seen in Figure S-6, the fluorescence intensity increased significantly for P277 when the temperature was increased to 40 °C. The fluorescent image contains large hydrophobic domains separated by tens of micrometers in the X-Y plane. The distance between hydrophobic domains observed in these experiments may correlate to the rms distances calculated in the PFG studies. In addition, a distinct texture can be seen in the differential interference contrast image of P277 at elevated temperatures indicating morphological changes. Though morphological changes were observed for P210, these were less significant than those observed for P277. In summary, narrowly dispersed, temperature-responsive BAB block copolymers capable of forming physical gels under physiological conditions were synthesized via aqueous RAFT polymerization. Rheological properties similar to the commonly used cell growth scaffolding material, collagen, were achieved. The gelation temperature and mechanical properties of the gels were dependent on polymer concentration, inner block length, and solvent. The ability of P210 and P277 to serve as an in vitro cell growth scaffold is currently being investigated. Additionally, investigations on triblock copolymers with peptidemodified domains and varying outer block lengths are underway. Acknowledgment. The authors thank the MRSEC program of the National Science Foundation (DMR-0213883), the Department of Energy (DE-FC26-01BC15317), the Robert M. Hearin Foundation, and Eastman Chemical Company for financial support. The authors acknowledge the NSF Division of Materials Research/Major Research Instrumentation award 0079450 for the purchase of the Varian UnityInova 500 MHz NMR spectrometer and the Mississippi Functional Genomics Network for the use of the facility (confocal microscope). We also thank Wako Chemicals and Noveon for their gifts of VA044 and the trithiocarbonate chain transfer agent, respectively. Supporting Information Available. The standard pulse sequence used in the pulsed field gradient NMR experiments, SEC traces for (co)polymers, dynamic temperature ramp data for P210 and P277, dynamic frequency sweep of collagen, zero shear specific viscosity of P210 and P277, and confocal microscopy images of P210 and P277. This material is available free of charge via the Internet at http://pubs.acs.org.
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