Based Semi-interpenetrating Polymer Networks for Tissue

Biomacromolecules 2002, 3, 591-600

591

Poly(N-isopropylacrylamide)-Based Semi-interpenetrating Polymer Networks for Tissue Engineering Applications. 1. Effects of Linear Poly(acrylic acid) Chains on Phase Behavior Ranee A. Stile† and Kevin E. Healy*,‡ Department of Biomedical Engineering, Robert R. McCormick School of Engineering and Applied Sciences, Northwestern University, Evanston, Illinois 60208, and Departments of Bioengineering and Materials Science and Engineering, University of California at Berkeley, Berkeley, California 94720 Received September 21, 2001; Revised Manuscript Received January 21, 2002

Poly(N-isopropylacrylamide)-based [P(NIPAAm)-based] semi-interpenetrating polymer networks (semi-IPNs), consisting of P(NIPAAm)-based hydrogels and linear poly(acrylic acid) [P(AAc)] chains, were synthesized, and the effects of the P(AAc) chains on semi-IPN injectability and phase behavior were analyzed. In P(NIPAAm)- and P(NIPAAm-co-AAc)-based semi-IPN studies, numerous reaction conditions were varied, and the effects of these factors on semi-IPN injectability, transparency, phase transition, lower critical solution temperature (LCST), and volume change were examined. The P(AAc) chains did not significantly affect the LCST or volume change of the semi-IPNs, compared to control hydrogels. However, the P(AAc) chains affected the injectability, transparency, and phase transition of the matrices, and these effects were dependent on chain amount and molecular weight (MW) and on interactions between the P(AAc) chains and the solvent and/or copolymer chains in P(NIPAAm-co-AAc) hydrogels. These results can be used to design “tailored” P(NIPAAm)-based semi-IPNs that have the potential to serve as functional scaffolds in tissue engineering applications. Introduction Three-dimensional (3D) polymer matrices, or scaffolds, can act as artificial templates that mimic the in vivo environment of the native extracellular matrix (ECM). When isolated cells are seeded into these scaffolds, the 3D structures guide the cells’ organization and development into tissues and organ-like structures both in vitro and in vivo.1,2 Polymer scaffolds have been synthesized using a plethora of different materials and fabrication methods, and various scaffold implantation procedures have been reported.3-9 In addition, numerous reports have detailed the modification of scaffolds with biologically active macromolecules to promote specific responses (e.g., cell proliferation and differentiation) within the matrices, allowing the scaffolds to interact with the biological environment (i.e., cells and ECM components) at a molecular level.10-15 Previously, we reported the development of injectable thermoresponsive hydrogels for use in tissue engineering applications.16 The hydrogels, composed of loosely crosslinked networks of N-isopropylacrylamide (NIPAAm) and acrylic acid (AAc), were injectable through a small-diameter aperture at 22 °C. In designing these injectable materials, we exploited the principles of phase separation and utilized a polymer, poly(NIPAAm) [P(NIPAAm)], that phase sepa* To whom correspondence should be addressed: University of California at Berkeley, Departments of Bioengineering and Materials Science and Engineering, 465 Evans Hall #1762, Berkeley, CA 94720-1762; phone, (510) 643-3559; facsimile, (510) 642-5835; e-mail, [email protected]. † Northwestern University. ‡ University of California at Berkeley.

rates from water when the temperature is increased above the lower critical solution temperature (LCST).17-22 For P(NIPAAm) in water, the LCST is approximately 32 °C.20 Below the LCST, P(NIPAAm) chains are soluble in aqueous media, and cross-linked hydrogels swell.23-25 At the LCST, P(NIPAAm) chains precipitate out of solution, while P(NIPAAm) hydrogels demonstrate a volume-phase transition, during which they collapse considerably, expel a large fraction of pore water, and become stiff and opaque. This phase behavior is reversible25 and can be modified by polymerizing the NIPAAm monomer with more hydrophilic or more hydrophobic comonomers.16,19,22,23,26-36 Due to the unique phase behavior of P(NIPAAm) in water, the injectable P(NIPAAm-co-AAc) hydrogels demonstrated a significant increase (e.g., 10×) in complex shear modulus (G*) (i.e., rigidity) when heated from 22 °C to body temperature (i.e., 37 °C), without exhibiting a significant change in either volume or water content.16 Furthermore, the injectable P(NIPAAm-co-AAc) hydrogels supported bovine articular chondrocyte viability and promoted articular cartilage-like tissue formation in vitro.16 While the injectable P(NIPAAm-co-AAc) hydrogel results were extremely promising, the matrices could not interact with the biological environment at a molecular level. To induce these types of interactions, we functionalized the AAc groups in the hydrogels with peptides containing relevant sequences found in ECM macromolecules (e.g., bone sialoprotein).11 Rat calvarial osteoblasts (RCO) seeded into the peptide-modified hydrogels spread more and demonstrated significantly greater proliferation, as compared to RCO

10.1021/bm0101466 CCC: $22.00 © 2002 American Chemical Society Published on Web 03/30/2002

592

Biomacromolecules, Vol. 3, No. 3, 2002

seeded into control hydrogels. Although these results were very encouraging, the functionalization process adversely affected the phase behavior of the P(NIPAAm-co-AAc) hydrogels. The matrices collapsed significantly at the LCST, making the process unacceptable for use in tissue engineering applications. To circumvent this functionalization issue, we have developed injectable semi-interpenetrating polymer networks (semi-IPNs) consisting of P(NIPAAm-co-AAc) hydrogels and linear P(AAc) chains. The semi-IPNs are synthesized by simultaneously polymerizing and cross-linking NIPAAm and AAc in the presence of the linear P(AAc) chains, yielding a P(NIPAAm-co-AAc) hydrogel with P(AAc) chains physically entangled within the network. It is our long-term goal to functionalize the -COO- groups in the P(AAc) chains with relevant biomolecules prior to the synthesis of the semi-IPN. With this approach, user-defined admixtures of functionalized P(AAc) chains can be created to promote specific biological interactions, while the P(NIPAAm-coAAc) hydrogel remains unaffected by the functionalization process. Thus, the objective of the current study was to determine the effects of nonfunctionalized P(AAc) chains on the injectability and phase behavior of the semi-IPNs. An extensive analysis was performed in which both P(NIPAAm)- and P(NIPAAm-co-AAc)-based semi-IPNs were synthesized. In the P(NIPAAm)-based semi-IPN studies, the P(AAc) chain type and the solvent were varied. In the P(NIPAAm-co-AAc)-based semi-IPN studies, the molar ratio of NIPAAm:AAc in the feed, the amount (i.e., the theoretical maximum number of -COO- binding sites for functionalization) and molecular weight (MW) of the P(AAc) chains, and the solvent were varied. The injectability and transparency at 22 °C, the phase transition, the LCST, and the volume change of the semi-IPNs were examined. The linear P(AAc) chains did not significantly affect the LCST or volume change of the semi-IPNs, as compared to control hydrogels. However, the injectability, transparency, and phase transition of the matrices were altered by the presence of the P(AAc) chains, and these effects were dependent on the amount and MW of the P(AAc) chains and on interactions between the P(AAc) chains and the solvent and/or copolymer chains in the P(NIPAAm-co-AAc) hydrogels. Experimental Section Materials. N-Isopropylacrylamide (NIPAAm), acrylic acid (AAc), N,N′-methylenebisacrylamide (BIS) Chemzymes Ultrapure grade, ammonium peroxydisulfate (AP) Chemzymes Ultrapure grade, N,N,N′,N′-tetramethylethylenediamine (TEMED) Chemzymes Ultrapure grade, and linear P(AAc) chains (average MW 50 000 g/mol, acid form; average MW 450 000 g/mol, acid form; average MW 500 000 g/mol, sodium salt form) were obtained from Polysciences, Inc. (Warrington, PA). Dulbecco’s phosphate-buffered saline (PBS, 1.51 mM KH2PO4, 155 mM NaCl, and 2.7 mM Na2HPO4; without CaCl2, without MgCl2; pH ) 7.2 ( 0.1; ionic strength 0.165), Dulbecco’s Modified Eagle Medium (DMEM, high glucose, with L-glutamine, with pyridoxine hydrochloride, without sodium pyruvate), and heat-inacti-

Stile and Healy Scheme 1. Synthesis of the P(NIPAAm)-Based Semi-IPN

vated fetal bovine serum (FBS) were obtained from GIBCO BRL (Grand Island, NY). All materials were used as received. Synthesis of P(NIPAAm)-Based Semi-IPNs. The P(NIPAAm)-based semi-IPNs were synthesized based on methods published previously.16 A schematic representation of the semi-IPN synthesis is shown in Scheme 1. For all formulations, 2.5 g (22.1 mmol) of NIPAAm, 0.005 g (0.0325 mmol) of BIS, 0.05 g of linear P(AAc) chains, and 50 mL of solvent were bubbled with dry nitrogen gas in a two-neck flask for 15 min to remove dissolved oxygen. The P(AAc) chains and the solvent were varied as follows: (1) 0.05 g (0.00011 mmol) of 450 000 g/mol P(AAc) chains in acid form and PBS solvent; (2) 0.05 g (0.00011 mmol) of 450 000 g/mol P(AAc) chains in acid form and ultrapure water (UPW; 18 MΩ-cm; pH between 5.5 and 7) solvent; (3) 0.05 g (0.0001 mmol) of 500 000 g/mol P(AAc) chains in sodium salt form and PBS solvent; (4) 0.05 g (0.0001 mmol) of 500 000 g/mol P(AAc) chains in sodium salt form and UPW solvent. Following the nitrogen gas purge, 0.020 g (0.0876 mmol) of AP and 200 µL (1.3 mmol) of TEMED were added as the initiator and accelerator, respectively. The mixture was stirred vigorously for 15 s and allowed to polymerize at 22 °C for 19 h under regular fluorescent lighting in a 250 mL glass beaker covered with a glass plate. Following the polymerization, the P(NIPAAm)-based semiIPN was washed three times, 15-20 min each, in excess UPW to remove unreacted compounds. P(NIPAAm) hydrogels, without linear P(AAc) chains, were synthesized under identical conditions using UPW and PBS as the solvent. In separate experiments, the pH of the polymerization formulations for the P(NIPAAm)-based semi-IPNs and the P(NIPAAm) hydrogels was determined. The formulations were prepared in the absence of the cross-linker BIS to more accurately measure the pH. Prior to the start of the pH experiments, it was verified that the cross-linker BIS (0.0325 mmol) did not change the pH of the solution. The polymerization formulations were allowed to react for at least 24 h before measuring the pH, and all of the formulations tested demonstrated pH values >8.90.

Polymer Networks for Tissue Engineering Scheme 2. Synthesis of the P(NIPAAm-co-AAc)-Based Semi-IPN

Synthesis of P(NIPAAm-co-AAc)-Based Semi-IPNs. The P(NIPAAm-co-AAc)-based semi-IPNs were synthesized according to methods reported previously,16 as shown in Scheme 2. The polymerization formulations are detailed in Table 1 and were chosen based on previous work.11,16 The molar ratio of NIPAAm:AAc in the feed, the amount (i.e., the theoretical maximum number of -COO- groups for functionalization) and MW of the linear P(AAc) chains, and the solvent were varied. The P(AAc) chains used to synthesize the P(NIPAAm-co-AAc)-based semi-IPNs were in acid form, rather than in sodium salt form, since sodium ions have been shown to affect the phase behavior of P(NIPAAm-co-AAc) hydrogels.16 In a typical synthesis, NIPAAm, AAc, 0.005 g (0.0325 mmol) of BIS, linear P(AAc) chains, and 50 mL of solvent were bubbled with dry nitrogen gas in a two-neck flask for 15 min to remove dissolved oxygen. The total amount of NIPAAm and AAc in the feed was always 2.5 g/50 mL of solvent. The remainder of the synthesis followed exactly that described for the P(NIPAAm)-based semi-IPNs. P(NIPAAmco-AAc) hydrogels, without linear P(AAc) chains, were made under identical conditions using PBS as the solvent. The P(NIPAAm-co-AAc) hydrogels contained 0.005 g (0.0325 mmol) of BIS, and NIPAAm:AAc molar ratios of 96:4, 96.25:3.75, 96.5:3.5, 96.75:3.25, and 97:3 were used. The total amount of NIPAAm and AAc in the feed was also 2.5 g/50 mL of solvent. The polymerization formulations for the P(NIPAAm-coAAc)-based semi-IPNs and the P(NIPAAm-co-AAc) hydrogels were also examined in pH studies. The protocol used was identical to that described for the P(NIPAAm)-based semi-IPNs and P(NIPAAm) hydrogels. The pH values for the P(NIPAAm-co-AAc)-based semi-IPN polymerization formulations are shown in Table 1. The P(NIPAAm-co-AAc) hydrogel polymerization formulations exhibited the following pH values for the given NIPAAm:AAc molar ratio in the feed: pH 8.35 for 96:4, pH 8.40 for 96.25:3.75, pH 8.54 for 96.5:3.5, and pH 8.89 for 97:3.

Biomacromolecules, Vol. 3, No. 3, 2002 593

Injectability Studies. A 10 mL syringe with a 2 mm diameter aperture was used to determine the injectability of the hydrogels and the semi-IPNs. Samples were both collected and injected using the syringe. Injectability was determined qualitatively, based on the ease of injection and the amount of macroscopic fracture demonstrated by the sample following injection. LCST Studies. The LCST of the hydrogel and semi-IPN samples was determined using a Beckman DU-64 UV-vis spectrophotometer with a DU series 60 water-regulated single-cell holder (Fullerton, CA), as previously reported.16 The transmittance of visible light (λ ) 500 nm; path length ) 1 cm) through the samples was recorded as the sample temperature was varied. The heating rate was 0.1-0.25 °C/ min. At the start of each experiment, the spectrophotometer was calibrated with UPW. Once a plot of transmittance versus temperature was obtained, the LCST was judged to be the initial break point of the curve.37 Volume Change Studies. The volume change exhibited by the hydrogel and semi-IPN samples when heated from 22 to 37 °C in the presence of PBS or cell-culture media (i.e., DMEM with 10% (v/v) FBS) was determined according to methods published previously.16 By use of a syringe, samples at 22 °C were placed in a glass vial at a volumetric ratio of 1.67 mL of hydrogel or semi-IPN/mL of PBS or cell-culture media. The vials were placed in an incubator at 37 °C and a humidified atmosphere of 5% CO2 (95% air; 21% O2) for 6 days. The volume of the 37 °C samples was estimated by the displacement of 37 °C UPW. Since the 37 °C samples were immersed in 37 °C UPW just long enough to measure the displacement of the water (i.e., less than 1 min), we feel the time course of the UPW step was short enough to neglect substantial swelling of the matrices. The volume change when heated from 22 to 37 °C was then computed by subtracting the 22 °C volume from the 37 °C volume and dividing by the 22 °C volume. Statistical Analyses. Analysis of variance (ANOVA) tests and Newman-Keuls post-hoc analyses were performed using StatSoft Statistica 5.0 (Tulsa, OK). Statistical significance was determined at a value of p < 0.01. Results and Discussion P(NIPAAm)-Based Semi-IPNs. At 22 °C, the P(NIPAAm) hydrogels and the P(NIPAAm)-based semi-IPNs were all injectable through a 2 mm diameter aperture and did not demonstrate appreciable macroscopic fracture following injection (Table 2). All matrices were transparent at 22 °C, except for the semi-IPNs synthesized in PBS, which were opaque at 22 °C, independent of P(AAc) chain type (Table 2). This change in transparency is indicative of phase separation and also signifies incompatibility within the network.38 Practically all IPNs and related two-polymer systems (e.g., semi-IPNs) demonstrate incompatibility, which results in the formation of two immiscible phases. Thermodynamically, the phase separation is caused by the low entropy of mixing that results when two polymers are blended. In contrast to small molecules, randomness is restricted when polymer chains are mixed, leading to the

594


Stile and Healy

Table 1. Polymerization Formulations for the P(NIPAAm-co-AAc)-Based Semi-IPNs formulation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

molar ratio NIPAAm:AAca 96:4 96:4 96:4 96:4 96.25:3.75 96.5:3.5 96.75:3.25 97:3 97.5:2.5 96.25:3.75 96.25:3.75 96.25:3.75 96.25:3.75 96.25:3.75 96.25:3.75 96.25:3.75 96.25:3.75

50000 g/mol P(AAc) chains in acid form

450000 g/mol P(AAc) chains in acid form

mmol)b

0.0055 g (0.00011 0.0055 g (0.00011 mmol)b 0.05 g (0.00011 mmol)c 0.05 g (0.00011 mmol)c 0.0055 g (0.00011 mmol)b 0.0055 g (0.00011 mmol)b 0.0055 g (0.00011 mmol)b 0.0055 g (0.00011 mmol)b 0.0055 g (0.00011 mmol)b 0.001 g (0.00002 mmol)d 0.013 g (0.00026 mmol)e 0.05 g (0.00097 mmol)c 0.130 g (0.0026 mmol)f 0.001 g (0.0000022 mmol)d 0.0055 g (0.000012 mmol)b 0.013 g (0.000029 mmol)e 0.05 g (0.00011 mmol)c

solvent (50 mL) UPW PBS UPW PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS

pH 8.06 7.75 6.69 7.07 8.10

8.87 8.31 7.02 6.30 8.39 8.18 7.13

a Molar ratio in feed; the total amount of NIPAAm and AAc was always 2.5 g/50 mL of solvent. b Theoretical maximum number of -COO- groups from P(AAc) chains is 4.6 × 1019. c Theoretical maximum number of -COO- groups from P(AAc) chains is 4.2 × 1020. d Theoretical maximum number of -COO- groups from P(AAc) chains is 8.4 × 1018. e Theoretical maximum number of -COO- groups from P(AAc) chains is 1.1 × 1020. f Theoretical maximum number of -COO- groups from P(AAc) chains is 1.1 × 1021.

Table 2. Injectability, Transparency, LCST, and Volume Change Results for the P(NIPAAm)-Based Hydrogels and Semi-IPNs description

solvent

appearance at 22 °C

injectable at 22 °C?

LCST (°C)

volume change (%)a

P(NIPAAm) hydrogel P(NIPAAm) hydrogel Semi-IPN with 450000 g/mol P(AAc) chains in acid formb Semi-IPN with 450000 g/mol P(AAc) chains in acid formb Semi-IPN with 500000 g/mol P(AAc) chains in sodium salt formb Semi-IPN with 500000 g/mol P(AAc) chains in sodium salt formb

UPW PBS UPW

transparent transparent transparent

yes yes yes

32.5 ( 0.7 30.0 ( 0.4c 32.4 ( 0.2

-92.0 ( 0d -90.7 ( 2.3 -86.7 ( 2.3

PBS

opaque

yes

30.9 ( 0.1c

-85.3 ( 2.3d

UPW

transparent

yes

32.2 ( 0.3

-89.3 ( 2.3

PBS

opaque

yes

30.7 ( 0.5c

-87.3 ( 1.6

a Performed in the presence of PBS. b 0.05 g (0.0001 mmol) of P(AAc) chains; theoretical maximum number of -COO- groups from P(AAc) chains is 4.2 × 1020. c Significantly different from other LCST values at p < 0.01 but not significantly different from each other. d Significantly different from each other at p < 0.01.

low entropic contribution. Furthermore, the entropy of mixing decreases as the MW of the polymer chains increases.39 The thermodynamically driven phase separation results in the formation of phases that vary in amount, size, shape, interfacial sharpness, and degree of continuity.38 These phases constitute the macroscopic and microscopic morphology of the material, and the morphology then directly influences the material properties of the system. It is interesting to note that the transparency change occurred during the polymerization of the semi-IPNs, which is similar to that observed in polymerization-induced phase separation (PIPS), a method regularly used to synthesize polymer-dispersed liquid crystal (PDLC).40 Since the P(NIPAAm)-based semi-IPNs synthesized in PBS were opaque at 22 °C, but the P(NIPAAm) hydrogels synthesized in PBS and the P(NIPAAm)-based semi-IPNs synthesized in UPW were transparent at 22 °C (Table 2), it appears that an interaction exists between the P(AAc) chains and the PBS which enhanced the incompatibility and phase separation within the matrices. It is possible that the increased ionic concentration and the presence of co-ions (e.g., Cl- and PO43-) and counterions (e.g., Na+ and K+) in the PBS, in combination with the P(AAc) chains,

affected the phase separation within the semi-IPNs (e.g., counterions may have interacted with the -COO- groups in the P(AAc) chains, creating AAc salts that were less miscible with the matrix). Similar salt effects have been reported in other multicomponent systems.41,42 It should be noted that the PBS did not contain any divalent cations, therefore ionic cross-bridging did not contribute to the change in transparency. Importantly, P(NIPAAm)-based semi-IPNs synthesized in PBS without cross-linker still demonstrated a change in transparency (data not shown). This observation further supports the thermodynamic discussion presented above. In the absence of cross-linker, the phase separation is driven by the mixing of the P(AAc) chains with the P(NIPAAm) chains, rather than the mixing of the P(AAc) chains with the P(NIPAAm) network. In previous studies of P(AAc) chain and P(NIPAAm) chain miscibility, turbidity was observed in the P(AAc)/P(NIPAAm) solutions, and the degree of complexation (i.e., H-bonding between the -COOH groups in AAc and the -CONH- groups in NIPAAm) was dependent on the P(AAc)/P(NIPAAm) ratio, the solvent, and the MW of the P(NIPAAm) chains.43 In a later study, the

Polymer Networks for Tissue Engineering

Figure 1. Transmittance versus temperature data for P(NIPAAm) hydrogels synthesized in UPW (9), P(NIPAAm) hydrogels synthesized in PBS (0, the dashed line is added for clarity), P(NIPAAm)-based semi-IPNs with 450 000 g/mol P(AAc) chains synthesized in UPW (2), P(NIPAAm)-based semi-IPNs with 500 000 g/mol P(AAc) chains synthesized in UPW (b), P(NIPAAm)-based semi-IPNs with 450 000 g/mol P(AAc) chains synthesized in PBS (4), and P(NIPAAm)-based semi-IPNs with 500 000 g/mol P(AAc) chains synthesized in PBS (O). Each line is the average of at least three experiments, and the error bars were excluded for clarity. The P(NIPAAm)-based semi-IPNs contained 0.05 g (0.0001 mmol) of P(AAc) chains.

complexation between the P(AAc) chains and the P(NIPAAm) chains decreased when NaOH was added to the solution.44 As the P(AAc) chains were neutralized, a certain fraction of -COOH groups was converted to -COO- groups, and the H-bonding between the P(AAc) chains and the P(NIPAAm) chains decreased. Similar results have been observed with P(NIPAAm-co-AAc) microgels45 and P(NIPAAm)/P(AAc) graft copolymers.34 Since the P(NIPAAm)-based semi-IPNs in the present study demonstrated pH values >8, independent of the solvent used, most of the -COOH groups in the P(AAc) chains existed as -COO- moieties, so the transparency results were probably not affected by H-bonding between the P(AAc) chains and the NIPAAm amide groups. The phase transitions demonstrated by all of the P(NIPAAm)-based semi-IPNs were different from those exhibited by the P(NIPAAm) hydrogels (Figure 1). The P(NIPAAm) hydrogels, whether synthesized in UPW or PBS, demonstrated abrupt and well-defined transitions, which spanned approximately 0.4 °C. This sharp transition is consistent with previously published results21,46 and is attributed to the hydrophobic/hydrophilic balance of the side groups on the polymer chain which leads to rapid dehydration of the polymer as the temperature is increased above the LCST. The transitions demonstrated by the semi-IPNs were not as abrupt as those demonstrated by the P(NIPAAm) hydrogels (Figure 1), suggesting that the linear hydrophilic P(AAc) chains interfered with the rapid dehydration of the NIPAAm segments within the P(NIPAAm) hydrogel, perhaps via simple physical obstruction. As seen in Figure 1, the transitions demonstrated by the P(NIPAAm)-based semiIPNs synthesized in UPW spanned approximately 3 °C. The transitions exhibited by the P(NIPAAm)-based semi-IPNs synthesized in PBS were different from all other transitions. As noted previously (Table 2), the semi-IPNs synthesized in PBS were opaque at 22 °C, which is signified by the low percent transmittance in Figure 1 (i.e., 40-50%). As the temperature increased, the opacity also increased, and a


transition spanning approximately 1 °C was observed. Similar to the transparency results, these results suggest that there are interactions between the P(AAc) chains and the PBS which cause changes in the phase transition beyond those observed with the P(NIPAAm)-based semi-IPNs synthesized in UPW. Interactions between the counterions in PBS and the P(AAc) chains may decrease the miscibility of the chains within the matrix, thus increasing the opacity of these semiIPNs, compared to their counterparts synthesized in UPW. The increase in opacity with increasing temperature may be explained thermodynamically, as the free energy of mixing (∆GM) is directly proportional to temperature.39 Since the P(NIPAAm)-based semi-IPNs synthesized in PBS were opaque at 22 °C, ∆GM was positive at 22 °C. Increasing the temperature, then, while leaving everything else constant, would cause ∆GM to become more positive, leading to increased phase separation. Despite the different phase transitions demonstrated by the P(NIPAAm)-based semi-IPNs, the P(AAc) chains did not significantly affect the LCST of the matrices, as compared to P(NIPAAm) hydrogels (Table 2). These results are in agreement with P(NIPAAm)-based IPN and semi-IPN work published previously.22,36,47-49 Since copolymerization of NIPAAm with more hydrophilic monomers (e.g., AAc) increases the LCST of copolymer hydrogels, compared to P(NIPAAm) homopolymer hydrogels,16,23,31,32,36 one might have expected the high MW hydrophilic P(AAc) chains to increase the LCST of the semi-IPNs. However, since the P(AAc) chains were chemically independent from the P(NIPAAm) hydrogel, this was not observed. Only the solvent used during synthesis significantly affected the LCST, as the P(NIPAAm) hydrogels and P(NIPAAm)-based semiIPNs synthesized in PBS demonstrated significantly lower LCST values than those exhibited by their counterparts synthesized in UPW (p < 0.01; Table 2). This result is consistent with the addition of salts to P(NIPAAm)-based systems.21,37 The high MW hydrophilic linear P(AAc) chains did not hinder the collapse of the P(NIPAAm) hydrogels, since all of the P(NIPAAm)-based samples collapsed >85% of their original volume (Table 2). These results are consistent with other P(NIPAAm)-based semi-IPN observations, in which P(NIPAAm)-based semi-IPNs demonstrated volume-phase transitions that were similar to those exhibited by P(NIPAAm) hydrogels.36 While it is possible that a minute fraction of the P(AAc) chains diffused out of the semi-IPNs during the 6-day immersion in PBS, reptation theory would predict otherwise.50 Thus, we are in the process of characterizing the semi-IPNs to determine the amount of P(AAc) chains present within the matrices following the 6-day volume change tests. Even though such a significant decrease in volume (i.e., >85%) is commonly observed with P(NIPAAm) hydrogels,16,23-25 we have discussed previously that extensive collapse of the matrix would be an undesirable characteristic of a scaffold for tissue repair.11,16 P(NIPAAm-co-AAc)-Based Semi-IPNs. The P(NIPAAmco-AAc)-based semi-IPN data are categorized into three separate groups according to the variables that were examined. On the basis of the formulations detailed in Table 1,

596


Stile and Healy

Table 3. Injectability, Transparency, LCST, and Volume Change Results for the P(NIPAAm-co-AAc)-Based Hydrogels and Semi-IPNs description

solvent

appearance at 22 °C

injectable at 22 °C?

LCST (°C)

volume change (%)a

P(NIPAAm-co-AAc) hydrogel (1)b semi-IPN with 50000 g/mol P(AAc) chains (2)b semi-IPN with 50000 g/mol P(AAc) chains (3)b semi-IPN with 450000 g/mol P(AAc) chains (4)b semi-IPN with 450000 g/mol P(AAc) chains

PBS UPW PBS UPW PBS

transparent transparent transparent opaque opaque

yes yes yes yes no

34.8 ( 0.2 38.0 ( 0.5c 34.8 ( 0.2 36.8 ( 0.5c 34.0 ( 0.4

+50.7 ( 4.6 +62.0 ( 7.5d +38.7 ( 6.1 +65.3 ( 3.3d +37.3 ( 8.3

a Performed in the presence of PBS. b Formulation number in parentheses; formulations detailed in Table 1. c Significantly different from all other LCSTs at p < 0.01. d Significantly different from other volume change results at p < 0.01 but not significantly different from each other.

formulations 1-4 were used to study solvent effects, formulations 2 and 5-9 were used to study NIPAAm:AAc molar ratio effects, and formulations 5 and 10-17 were used to study the effects of the amount and MW of the P(AAc) chains. SolVent Effects. The solvent used during synthesis altered the injectability of the P(NIPAAm-co-AAc)-based semiIPNs, as the semi-IPNs with 450 000 g/mol P(AAc) chains synthesized in UPW were injectable at 22 °C, but the semiIPNs with 450 000 g/mol P(AAc) chains synthesized in PBS were not injectable (Table 3). However, since the P(NIPAAmco-AAc)-based semi-IPNs with 50 000 g/mol P(AAc) chains were injectable at 22 °C (Table 3), independent of the solvent used, and the P(NIPAAm)-based semi-IPNs with 450 000 g/mol P(AAc) chains synthesized in PBS were also injectable (Table 2), it appears that a complex interaction exists between the PBS, the high MW P(AAc) chains, and the AAc groups in the P(NIPAAm-co-AAc) hydrogel. This result can be explained in part by the fact that the entropy of mixing decreases with increasing polymer MW, as discussed previously. As the entropy of mixing decreases, the compatibility within the system decreases, and consequently, the morphology and the material properties of the matrix are affected (e.g., the injectability decreases). In addition, it is possible that there is decreased compatibility within the P(NIPAAmco-AAc)-based system, as compared to that within the P(NIPAAm)-based system, due to the presence of the NIPAAm-AAc copolymer chains. The solvent used during synthesis also affected the phase transitions demonstrated by the P(NIPAAm-co-AAc)-based semi-IPNs, as compared to the phase transition exhibited by the P(NIPAAm-co-AAc) hydrogel (Figure 2). Only the P(NIPAAm-co-AAc)-based semi-IPN with 50 000 g/mol P(AAc) chains synthesized in PBS demonstrated a phase transition which was similar to the P(NIPAAm-co-AAc) hydrogel phase transition (Figure 2). Since the only difference between the P(NIPAAm-co-AAc)-based semi-IPN with 50 000 g/mol P(AAc) chains synthesized in PBS and the P(NIPAAm-co-AAc) hydrogel was the presence of the chains (i.e., the hydrogel was also synthesized in PBS), the 50 000 g/mol P(AAc) chains did not affect the phase transition. The transition exhibited by the P(NIPAAm-co-AAc)-based semiIPN with 50 000 g/mol P(AAc) chains synthesized in UPW was broader, presumably due to the decreased number of ions in UPW. In the absence of ions and at a pH of approximately 7, the solubility and repulsion of the AAc groups (i.e., the -COO- groups) in P(NIPAAm-co-AAc) hydrogels counteract the aggregation of the hydrophobic NIPAAm components.16,31,34 Furthermore, the hydrophilic

Figure 2. Transmittance versus temperature data for P(NIPAAmco-AAc) hydrogels synthesized in PBS (0), P(NIPAAm-co-AAc)-based semi-IPNs with 50 000 g/mol P(AAc) chains synthesized in UPW (b), P(NIPAAm-co-AAc)-based semi-IPNs with 450 000 g/mol P(AAc) chains synthesized in UPW (2), P(NIPAAm-co-AAc)-based semi-IPNs with 50 000 g/mol P(AAc) chains synthesized in PBS (O), and P(NIPAAm-co-AAc)-based semi-IPNs with 450 000 g/mol P(AAc) chains synthesized in PBS (4). Each line is the average of at least three experiments, and the error bars were excluded for clarity. The P(NIPAAm-co-AAc)-based hydrogels and semi-IPNs contained a NIPAAm:AAc molar ratio of 96:4, and the semi-IPNs contained 0.00011 mmol of P(AAc) chains.

-COO- groups hinder the dehydration of the polymer chains, expanding the collapsed structure. As ions are introduced into P(NIPAAm-co-AAc)-based systems, ionic shielding of the -COO- groups occurs. This ionic shielding disrupts the solubility and repulsion of the -COO- groups (e.g., increasing the ionic strength of the solvent decreases the Debye screening length, which decreases the repulsive electrostatic interactions between -COO- groups45). As more and more ions are added, the hydrophilic -COO- groups can no longer impede the aggregation and dehydration of the NIPAAm components. As a result, the phase transition demonstrated by P(NIPAAm-co-AAc) hydrogels synthesized in UPW is typically broader than that exhibited by P(NIPAAm-co-AAc) hydrogels synthesized in PBS. The P(NIPAAm-co-AAc)-based semi-IPNs with 450 000 g/mol P(AAc) chains demonstrated transitions in which the opacity increased with increasing temperature, independent of the solvent used. These unusual transitions were similar to those exhibited by the P(NIPAAm)-based semi-IPNs synthesized in PBS (Figure 1); however, the transitions demonstrated by the P(NIPAAm-co-AAc)-based semi-IPNs were broader. This is a common result, as P(NIPAAm)-based copolymer hydrogels typically exhibit broader transitions than those demonstrated by P(NIPAAm) homopolymer hydrogels.16,31,35 It should be noted that the P(NIPAAm)-based semi-IPNs with 450 000 g/mol P(AAc) chains synthesized in UPW did not


exhibit such an unusual phase transition (Figure 1), but the P(NIPAAm-co-AAc)-based semi-IPNs synthesized in UPW did (Figure 2). As discussed previously, it is possible that the presence of the NIPAAm-AAc copolymer chains in the P(NIPAAm-co-AAc)-based semi-IPNs decreased the compatibility within the system, compared to that within the P(NIPAAm)-based system. In addition to changing the phase transitions, the solvent used during synthesis significantly affected the LCST of the P(NIPAAm-co-AAc)-based semi-IPNs, compared to that of the P(NIPAAm-co-AAc) hydrogel (Table 3). The LCSTs of the P(NIPAAm-co-AAc)-based semi-IPNs synthesized in UPW were significantly higher than the LCSTs of the P(NIPAAm-co-AAc) hydrogel and the semi-IPNs synthesized in PBS, independent of P(AAc) chain length (p < 0.01; Table 3). As discussed previously, these results are presumably due to the decreased number of ions present in the matrices synthesized in UPW, as compared to the matrices synthesized in PBS. In the absence of ions, the solubility and repulsion of the AAc groups typically increase the LCST of P(NIPAAm-co-AAc) hydrogels, compared to the LCST of P(NIPAAm) hydrogels.16,23,31,32,36 As ions are introduced into P(NIPAAm-co-AAc)-based systems, ionic shielding occurs, and the LCST decreases. Since the P(NIPAAm-coAAc)-based semi-IPNs synthesized in UPW presumably contained fewer ions than the semi-IPNs synthesized in PBS, ionic shielding occurred to a lesser degree, and the LCST was higher. Similar to the LCST results, the solvent significantly affected the volume changes demonstrated by the matrices (Table 3). The semi-IPNs synthesized in UPW exhibited significantly higher volume changes (i.e., more swelling) than those demonstrated by matrices synthesized in PBS, independent of chain length (p < 0.01; Table 3). As discussed above, since the semi-IPNs synthesized in UPW presumably contained fewer ions than the semi-IPNs synthesized in PBS, the -COO- groups were better able to counteract the dehydration and aggregation of the NIPAAm components, leading to larger volume changes. In our previous study,16 we used a NIPAAm:AAc molar ratio of 96:4 in the feed to synthesize the injectable P(NIPAAm-co-AAc) hydrogels. However, the volume change results presented in that study were significantly lower than those presented in the current study (+5.3 ( 6.1% in ref 16 vs +50.7 ( 4.6% in Table 3; p < 0.01; tests performed in the presence of PBS). We believe the difference is due to lot variations in the AAc. In the current study, P(NIPAAmco-AAc) hydrogels with a NIPAAm:AAc molar ratio of 96.5: 3.5 in the feed demonstrated volume changes that were not statistically different from the volume changes exhibited by the injectable P(NIPAAm-co-AAc) hydrogels reported previously (+9.3 ( 8.3% in Figure 3B vs +5.3 ( 6.1%; p > 0.01). NIPAAm:AAc Molar Ratio Effects. The NIPAAm:AAc molar ratio in the feed did not affect the transparency, injectability, or phase transition of the P(NIPAAm-co-AAc)based semi-IPNs with 50 000 g/mol P(AAc) chains (formulations 2 and 5-9; Table 1), as compared to P(NIPAAmco-AAc) hydrogels (data not shown). All samples were


Figure 3. (A) LCST values for P(NIPAAm-co-AAc) hydrogels (solid line, squares) and P(NIPAAm-co-AAc)-based semi-IPNs with 50 000 g/mol P(AAc) chains (dashed line, circles). Both the hydrogels and the semi-IPNs were synthesized in PBS. The P(NIPAAm-co-AAc)based semi-IPNs contained 0.0055 g (0.00011 mmol) of P(AAc) chains. The LCST of the hydrogel with a NIPAAm:AAc molar ratio of 96:4 was significantly different from the LCST of the hydrogel with a NIPAAm:AAc molar ratio of 97:3 at p < 0.01, as designated by a. The LCST of the semi-IPN with a NIPAAm:AAc molar ratio of 96:4 was significantly different than the LCST of the semi-IPN with a NIPAAm:AAc molar ratio of 97.5:2.5 at p < 0.01, as designated by b. (B) Volume changes for the P(NIPAAm-co-AAc) hydrogels tested in the presence of PBS (solid line, squares) or in the presence of cell-culture media (dashed line, squares) and for the P(NIPAAm-coAAc)-based semi-IPNs with 50 000 g/mol P(AAc) chains tested in the presence of PBS (solid line, circles) or in the presence of cell-culture media (dashed line, circles). Both the hydrogels and the semi-IPNs were synthesized in PBS. The P(NIPAAm-co-AAc)-based semi-IPNs contained 0.0055 g (0.00011 mmol) of P(AAc) chains.

transparent and injectable at 22 °C, and the phase transitions demonstrated by the P(NIPAAm-co-AAc)-based semi-IPNs with 50 000 g/mol P(AAc) chains synthesized in PBS were similar to those exhibited by the P(NIPAAm-co-AAc) hydrogels (data not shown). The LCSTs of the semi-IPNs were not significantly different from the LCSTs of the P(NIPAAm-co-AAc) hydrogels, for a given molar ratio of NIPAAm:AAc (p >0.01; Figure 3A), indicating that the P(AAc) chains did not affect the phase transition temperature. These results also suggest that the pH of the matrix did not affect the LCST, for a given NIPAAm:AAc molar ratio. For example, with a NIPAAm: AAc molar ratio of 96:4, the pH of the P(NIPAAm-co-AAc) hydrogel was 8.36, while the pH of the P(NIPAAm-co-AAc)based semi-IPN was 7.75. Despite the difference in pH, the LCSTs were not significantly different. However, the LCSTs

598


Stile and Healy

Table 4. Injectability, Transparency, LCST, and Volume Change Results for the P(NIPAAm-co-AAc)-Based Hydrogels and Semi-IPNs 1.1 × 1020 sitesa

4.6 × 1019 sitesa

50000 g/molc 450000 g/mold

transparent transparent

transparent opaque

Appearance at 22 °C transparent opaque opaque opaque

opaque na

transparent

50000 g/molc 450000 g/mold

yes yes

yes yes

Injectable at 22 °C? yes yes no no

no na

yes

50000 g/molc 450000 g/mold

34.8 ( 0.3 34.8 ( 0.2

34.7 ( 0.1 34.8 ( 0.1

34.5 ( 0.2 na

34.5 ( 0.1

50000 g/molc 450000 g/mold

+6.0 ( 8.3f +13.3 ( 4.6

Volume Change in the Presence of PBS (%) +18.0 ( 6.1 +10.3 ( 9.3 +17.3 ( 11.5 +14.0 ( 3.3 +6.7 ( 6.5f +14.7 ( 7.0

+9.8 ( 9.0 na

+25.3 ( 4.6

50000 g/molc 450000 g/mold

+2.2 ( 5.7 +9.6 ( 5.9

LCST (°C) 34.8 ( 0.1 34.5 ( 0.1

4.2 × 1020 sitesa

34.5 ( 0.4 33.8 ( 0.1e

1.1 × 1021 sitesa

P(NIPAAm-co-AAc) hydrogelb

8.4 × 1018 sitesa

Volume Change in the Presence of Cell-Culture Media (%) +3.7 ( 3.7 -2.7 ( 10.6 +9.8 ( 9.0 +29.6 ( 6.9g +8.3 ( 5.6 -4.4 ( 9.0 +12.0 ( 12.1 na

+4.0 ( 6.9

a Theoretical maximum number of -COO- groups for functionalization on P(AAc) chains. b NIPAAm:AAc molar ratio in feed was 96.25:3.75; PBS solvent used. c From left to right, the formulation numbers are 10, 5, 11, 12, and 13; formulations are detailed in Table 1. d From left to right, the formulation numbers are 14, 15, 16, and 17; formulations are detailed in Table 1. e Significantly different from all others at p < 0.01. f Significantly different from P(NIPAAm-co-AAc) hydrogel at p < 0.01 but not different from each other. g Significantly different from all others at p < 0.01.

significantly decreased with decreasing molar amounts of AAc in the P(NIPAAm-co-AAc)-based hydrogels and semiIPNs (p < 0.01; Figure 3A). Again, this result does not appear to be due to a pH effect. For example, a P(NIPAAmco-AAc) hydrogel synthesized using a NIPAAm:AAc molar ratio of 96:4 had a significantly higher LCST than a hydrogel synthesized using a NIPAAm:AAc molar ratio of 97:3. The pH of the matrices was 8.36 and 8.89, respectively. The hydrogel with the higher pH presumably contained a higher percentage of ionized groups, yet it demonstrated a significantly lower LCST, due to the decreased amount of AAc in the network. As the number of AAc groups decreases, the hydrophobic/hydrophilic balance in the P(NIPAAm-co-AAc) network is shifted, and the -COO- groups are unable to effectively counteract the hydrophobic NIPAAm interactions. As a result, the LCST decreases. The volume changes demonstrated by the P(NIPAAmco-AAc) hydrogels and the P(NIPAAm-co-AAc)-based semiIPNs in the presence of PBS were, for the most part, not statistically different from each other, for a given molar ratio of NIPAAm:AAc (p > 0.01; Figure 3B). Only when the matrices were synthesized using a NIPAAm:AAc molar ratio of 96:4 in the feed did the P(AAc) chains affect the volume change results (i.e., the semi-IPNs swelled less than the hydrogels; p < 0.01). It is possible that the physical presence of the chains hindered the swelling of the semi-IPNs. Similar results were observed when the volume change studies were performed in the presence of cell-culture media. The volume changes exhibited by the hydrogels and semi-IPNs were not significantly different from each other (p > 0.01; Figure 3B), except when the matrices were synthesized using a NIPAAm: AAc molar ratio of 96:4 in the feed. Again, the semi-IPNs swelled less than the hydrogels (p < 0.01; Figure 3B). It should be noted that the solvent did not have a consistent effect on the volume change results. Amount and MW of P(AAc) Chains. The amount and MW of the P(AAc) chains affected both the transparency and

injectability of the P(NIPAAm-co-AAc)-based semi-IPNs (Table 4). All of the matrices listed in Table 4 were synthesized using the same NIPAAm:AAc molar ratio in the feed (96.25:3.75) and the same solvent (PBS), but the amount (i.e., the theoretical maximum number of -COO- binding sites) and MW of the P(AAc) chains were varied. As the P(AAc) chain amount increased, the semi-IPNs became opaque, and the injectability decreased (Table 4). This result is consistent with the thermodynamic description given previously. As more P(AAc) chains are added to the semiIPN, the entropy of mixing decreases, leading to decreased compatibility within the system. This decreased compatibility results in increased phase separation (i.e., opacity), which affects the morphology and, consequently, the material properties of the matrix (i.e., decreased injectability). As seen in Table 4, a lower P(AAc) chain amount was needed to change the transparency and injectability of the semi-IPNs with 450 000 g/mol P(AAc) chains, compared to the semiIPNs with 50 000 g/mol P(AAc) chains. Again, these results make sense from a thermodynamic perspective, since the entropy of mixing decreases with increasing polymer MW.39 The amount and MW of the P(AAc) chains also affected the phase transitions of the P(NIPAAm-co-AAc)-based semiIPNs (Figure 4). With lower amounts of P(AAc) chains, the phase transitions demonstrated by the P(NIPAAm-co-AAc)based semi-IPNs were similar to the transition exhibited by the P(NIPAAm-co-AAc) hydrogel (Figure 4). As the P(AAc) chain amount increased, the transitions changed considerably, but the transitions demonstrated by the semi-IPNs with 450 000 g/mol P(AAc) chains were altered at a lower number of -COO- binding sites. These results are consistent with the decrease in the entropy of mixing which occurs in systems with higher MW polymers. Despite the different phase transitions demonstrated by the P(NIPAAm-co-AAc)-based semi-IPNs, the amount and MW of the P(AAc) chains did not significantly affect the LCSTs of the semi-IPNs, as compared to the P(NIPAAm-co-AAc)



change exhibited by the P(NIPAAm-co-AAc) hydrogel in the presence of cell-culture media (+29.6 ( 6.9% vs +4.0 ( 6.9%; p < 0.01). Interestingly, the polymerization formulation for this semi-IPN demonstrated the lowest pH (6.30; Table 1), since it contained the highest amount of P(AAc) chains. However, even at a pH of 6.30, a large amount of the -COOH groups should exist as -COOgroups. Since this formulation presumably contained the greatest number of -COO- groups, it is possible that the dissociation created an osmotic effect, which caused these semi-IPNs to swell. Relevance of Results to Applications in Tissue Engineering

Figure 4. (A) Transmittance versus temperature data for P(NIPAAmco-AAc) hydrogels synthesized in PBS (9) and P(NIPAAm-co-AAc)based semi-IPNs with 450 000 g/mol P(AAc) chains synthesized in PBS containing 8.4 × 1018 -COO- groups (+), 4.6 × 1019 -COOgroups (2), 1.1 × 1020 -COO- groups ([), and 4.2 × 1020 -COOgroups (b). Both the hydrogels and the semi-IPNs were synthesized using a NIPAAm:AAc molar ratio of 96.25:3.75. Each line is the average of at least three experiments, and the error bars were excluded for clarity. (B) Transmittance versus temperature data for P(NIPAAm-co-AAc) hydrogels synthesized in PBS (9) and P(NIPAAmco-AAc)-based semi-IPNs with 50 000 g/mol P(AAc) chains synthesized in PBS containing 8.4 × 1018 -COO- groups (+), 4.6 × 1019 -COO- groups (2), 1.1 × 1020 -COO- groups ([), 4.2 × 1020 -COO- groups (b), and 1.1 × 1021 -COO- groups (×). Both the hydrogels and the semi-IPNs were synthesized using a NIPAAm:AAc molar ratio of 96.25:3.75. Each line is the average of at least three experiments, and the error bars were excluded for clarity.

hydrogels (p > 0.01; Table 4). Only the P(NIPAAm-coAAc)-based semi-IPN with 450 000 g/mol P(AAc) chains and 4.2 × 1020 -COO- binding sites demonstrated an LCST that was significantly lower than the LCST of the P(NIPAAmco-AAc) hydrogel (p < 0.01; Table 4). The amount and MW of the P(AAc) chains did not significantly affect the volume changes demonstrated by the P(NIPAAm-co-AAc)-based semi-IPNs, as compared to the volume change demonstrated by the P(NIPAAm-co-AAc) hydrogel (p > 0.01; Table 4). Only two P(NIPAAm-coAAc)-based semi-IPN formulations (formulations 10 and 16) demonstrated significantly lower volume changes in the presence of PBS, as compared to the volume change exhibited by the P(NIPAAm-co-AAc) hydrogel in the presence of PBS (p < 0.01). Furthermore, only the P(NIPAAm-co-AAc)-based semi-IPN with 50 000 g/mol P(AAc) chains and 1.1 × 1021 -COO- binding sites demonstrated a volume change in the presence of cell-culture media that was significantly different from the volume

This analysis of the effects of various reaction conditions on the injectability and phase behavior of P(NIPAAm)- and P(NIPAAm-co-AAc)-based semi-IPNs can be used to develop tailored matrices that serve as functional scaffolds in tissue engineering applications. At first glance, the data may suggest that lower MW P(AAc) chains would be preferred, due to their increased miscibility and improved injectability; however, the appropriate choice of MW depends on the specific application and the extent to which the P(AAc) chains are required to diffuse within or out of the network. For example, if adhesion between a polymer matrix and the native ECM is desired (e.g., in articular cartilage regeneration), then lower MW P(AAc) chains would be preferred because they may better promote polymer-polymer interdiffusion. In contrast, higher MW P(AAc) chains may be preferred when the chains serve as an integral part of the semi-IPN, such as in bone regeneration, where the matrices would function as bioactive scaffolds to promote tissue formation. Obviously, a combination of chains with various MWs could allow for multiple effects (e.g., polymerpolymer interdiffusion and bioactivity). To maintain the injectability of a semi-IPN containing high MW chains, the amount of the chains can be reduced (Table 4). In addition to injectability, the LCST and associated changes in the mechanical properties and the volume change of the semiIPN are other important characteristics to consider in tissue engineering applications. Since these attributes are largely defined by the molar ratio of NIPAAm:AAc in the P(NIPAAm-co-AAc) network, the MW of the linear P(AAc) chains can be modulated, as described above, without affecting the material properties of the matrix. Thus, these environmentally sensitive P(NIPAAm)- and P(NIPAAm-coAAc)-based semi-IPNs can be tuned to incorporate native functions of the ECM and may serve as ideal materials to test hypotheses regarding the effects of the physical and biological properties of the matrix on the regulation of tissue regeneration. Summary P(NIPAAm)- and P(NIPAAm-co-AAc)-based semi-IPNs, consisting of P(NIPAAm) and P(NIPAAm-co-AAc) hydrogels and linear P(AAc) chains, were synthesized, and the effect of the P(AAc) chains on the injectability and phase

600


behavior of the matrices was determined. Numerous reaction conditions were varied, and the effects of these factors on semi-IPN injectability and transparency at 22 °C, phase transition, LCST, and volume change were examined. Overall, the linear P(AAc) chains did not significantly affect the LCST or volume-phase transition of the semi-IPNs, as compared to P(NIPAAm) and P(NIPAAm-co-AAc) hydrogels. The P(AAc) chains did affect the injectability and transparency of the semi-IPNs at 22 °C, as well as the phase transition, and these effects were dependent on the chain amount and MW and on interactions with the solvent and/ or copolymer chains in the P(NIPAAm-co-AAc) hydrogels. These results can be used to design “tailored” P(NIPAAm)based semi-IPNs (e.g., matrices with specific injectability, LCST, and volume change characteristics) that have the potential to serve as functional scaffolds in tissue engineering applications. Acknowledgment. This work was funded by a grant from the N.I.H. National Institute of Arthritis and Musculoskeletal and Skin Diseases AR47304 and a Whitaker Foundation Graduate Student Fellowship awarded to R.A.S. References and Notes (1) Cima, L. G.; Vacanti, J. P.; Vacanti, C.; Ingber, D.; Mooney, D.; Langer, R. J. Biomech. Eng. 1991, 113, 143. (2) Hubbell, J. A.; Langer, R. Chem. Eng. News 1995, March 13, 42. (3) Whang, K.; Tsai, D. C.; Nam, E. K.; Aitken, M.; Sprague, S. M.; Patel, P. K.; Healy, K. E. J. Biomed. Mater. Res. 1998, 42, 491. (4) Paige, K. T.; Cima, L. G.; Yaremchuk, M. J.; Vacanti, J. P.; Vacanti, C. A. Plast. Reconstr. Surg. 1995, 96, 1390. (5) Kawamura, S.; Wakitani, S.; Kimura, T.; Maeda, A.; Caplan, A. I.; Shino, K.; Ochi, T. Acta Orthop. Scand. 1998, 69, 56. (6) Vernon, B.; Kim, S. W.; Bae, Y. H. J. Biomater. Sci., Polym. Ed. 2000, 10, 183. (7) Freed, L. E.; Grande, D. A.; Lingbin, Z.; Emmanual, J.; Marquis, J. C.; Langer, R. J. Biomed. Mater. Res. 1994, 28, 891. (8) Seckel, B. R.; Jones, D.; Hekimian, K. J.; Wang, K.-K.; Chakalis, D. P.; Costas, P. D. J. Neurosci. Res. 1995, 40, 318. (9) Shinoka, T.; Shum-Tim, D.; Ma, P. X.; Tanel, R. E.; Isogai, N.; Langer, R.; Vacanti, J. P.; Mayer, J. E., Jr. J. Thorac. CardioVasc. Surg. 1998, 115, 536. (10) Moghaddam, M. J.; Matsuda, T. J. Polym. Sci., Polym. Chem. Ed. 1993, 31, 1589. (11) Stile, R. A.; Healy, K. E. Biomacromolecules 2001, 2, 185. (12) Yu, X.; Dillon, G. P.; Bellamkonda, R. B. Tissue Eng. 1999, 5, 291. (13) Suzuki, Y.; Tanihara, M.; Suzuki, K.; Saitou, A.; Sufan, W.; Nishimura, Y. J. Biomed. Mater. Res. 2000, 50, 405. (14) Bellamkonda, R.; Ranieri, J. P.; Aebischer, P. J. Neurosci. Res. 1995, 41, 501. (15) Mann, B. K.; Schmedlen, R. H.; West, J. L. Biomaterials 2001, 22, 439. (16) Stile, R. A.; Burghardt, W. R.; Healy, K. E. Macromolecules 1999, 32, 7370.

Stile and Healy (17) Casassa, E. F. In Fractionation of Synthetic Polymers: Principles and Practices; Tung, L. H., Ed.; Marcel Dekker: New York, 1977; p 3. (18) Kamide, K. Thermodynamics of Polymer Solutions: Phase Equilibria and Critical Phenomena; Elsevier: New York, 1990. (19) Taylor, L. D.; Cerankowski, L. D. J. Polym. Sci., Polym. Chem. Ed. 1975, 13, 2551. (20) Heskins, M.; Guillet, J. E. J. Macromol. Sci., Chem. Ed. 1968, A2, 1441. (21) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163. (22) Bae, Y. H.; Okano, T.; Kim, S. W. Pharm. Res. 1991, 8, 531. (23) Hoffman, A. S.; Afrassiabi, A.; Dong, L. C. J. Controlled Release 1986, 4, 213. (24) Hirokawa, Y.; Tanaka, T. J. Chem. Phys. 1984, 81, 6379. (25) Hoffman, A. S. MRS Bull. 1991, 16, 42. (26) Vernon, B.; Gutowska, A.; Kim, S. W.; Bae, Y. H. Macromol. Symp. 1996, 109, 155. (27) Priest, J. H.; Murray, S. L.; Nelson, R. J.; Hoffman, A. S. ACS Symp. Ser. 1987, No. 350, 255. (28) Hoffman, A. S.; Chen, G. H.; Kaang, S. Y.; Ding, Z. L.; Randeri, K.; Kabra, B. In AdVanced Biomaterials in Biomedical Engineering and Drug DeliVery Systems; Ogata, N., Kim, S. W., Feijen, J., Okano, T., Eds.; Springer-Verlag: Tokyo, 1996; p 62. (29) Aoyagi, T.; Ebara, M.; Sakai, K.; Sakurai, Y.; Okano, T. J. Biomater. Sci., Polym. Ed. 2000, 11, 101. (30) Vernon, B.; Kim, S. W.; Bae, Y. H. J. Biomed. Mater. Res. 2000, 51, 69. (31) Yoshida, R.; Sakai, K.; Okano, T.; Sakurai, Y. J. Biomater. Sci., Polym. Ed. 1994, 6, 585. (32) Dong, L. C.; Hoffman, A. S. J. Controlled Release 1986, 4, 223. (33) Feil, H.; Bae, Y. H.; Feijen, J.; Kim, S. W. J. Membr. Sci. 1991, 64, 283. (34) Chen, G.; Hoffman, A. S. Nature 1995, 373, 49. (35) Vakkalanka, S. K.; Brazel, C. S.; Peppas, N. A. J. Biomater. Sci., Polym. Ed. 1996, 8, 119. (36) Kokufuta, E.; Wang, B.; Yoshida, R.; Khokhlov, A. R.; Hirata, M. Macromolecules 1998, 31, 6878. (37) Schild, H. G.; Tirrell, D. A. J. Phys. Chem. 1990, 94, 4352. (38) Sperling, L. H. Interpenetrating Polymer Networks and Related Materials; Plenum Press: New York, 1981. (39) Sperling, L. H. Introduction to Physical Polymer Science; John Wiley & Sons: New York, 1992. (40) Lee, J. C. Phys. ReV. E 1999, 60, 1930. (41) Wang, H.; Li, W.; Lu, Y.; Wang, Z. J. Appl. Polym. Sci. 1997, 65, 1445. (42) Zhang, L.; Eisenberg, A. Macromolecules 1996, 29, 8805. (43) Garay, M. T.; Llamas, M. C.; Iglesias, E. Polymer 1997, 38, 5091. (44) Garay, M. T.; Alava, C.; Rodriguez, M. Polymer 2000, 41, 5799. (45) Kratz, K.; Hellweg, T.; Eimer, W. Colloids Surf., A 2000, 170, 137. (46) Bae, Y. H.; Okano, T.; Kim, S. W. J. Polym. Sci., Polym. Phys. Ed. 1990, 28, 923. (47) Okano, T.; Bae, Y. H.; Jacobs, H.; Kim, S. W. J. Controlled Release 1990, 11, 255. (48) Gutowska, A.; Bae, Y. H.; Jacobs, H.; Feijen, J.; Kim, S. W. Macromolecules 1994, 27, 4167. (49) Park, T. G.; Choi, H. K. Macromol. Rapid Commun. 1998, 19, 167. (50) de Gennes, P. G. J. Chem. Phys. 1971, 55, 572.

BM0101466

Based Semi-interpenetrating Polymer Networks for Tissue

Recommend Documents