Thermo-Responsive Peptide-Modified Hydrogels for Tissue

Biomacromolecules 2001, 2, 185-194

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Thermo-Responsive Peptide-Modified Hydrogels for Tissue Regeneration 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 13, 2000; Revised Manuscript Received December 19, 2000

Loosely cross-linked hydrogels consisting of N-isopropylacrylamide (NIPAAm) and acrylic acid (AAc) were synthesized, characterized, and used as model scaffolds for studying cell-material interactions in three-dimensions (3D). The AAc groups were functionalized with peptides containing the -RGD- and -FHRRIKA- sequences found in bone sialoprotein. Chemical modification of the hydrogels was verified via solid-state 1H nuclear magnetic resonance spectroscopy, lower critical solution temperature studies, and volume change studies. The peptide-modified hydrogels were pliable at 22 °C and could be injected through a small-diameter aperture. Rat calvarial osteoblasts (RCO) seeded into the peptide-modified hydrogels were viable for at least 21 days of in vitro culture. The RCO spread more and demonstrated significantly greater proliferation when cultured within the peptide-modified hydrogels, as compared to control hydrogels. These peptide-modified P(NIPAAm-co-AAc) hydrogels serve as useful tools for studying cell-material interactions within 3D structures and have the potential to be used as injectable scaffolds for tissue engineering applications. Introduction Attempts to regenerate tissues, such as bone,1,2 cartilage,3,4 blood vessels,5 and peripheral nerves,6 have been reported in numerous tissue engineering initiatives. In these studies, natural and synthetic materials act as artificial threedimensional (3D) templates, or scaffolds, that foster cell proliferation and guide the cells’ organization into tissue both in vitro and in vivo.7,8 While the results have been encouraging, there are still limitations with many of these systems, including the invasiveness of the implantation procedure and the inability of the material to interact with the biological system at the molecular level. To address the invasive implantation issue common to many polymer scaffolds, we developed injectable hydrogels that supported bovine articular chondrocyte viability and cartilage-like tissue formation in vitro.9 The hydrogels were synthesized by simultaneously polymerizing and crosslinking N-isopropylacrylamide (NIPAAm) and acrylic acid (AAc). At room temperature (i.e., approximately 22 °C), these loosely cross-linked poly(NIPAAm-co-AAc) [P(NIPAAm-co-AAc)] hydrogels were injectable through a 2 mm diameter aperture and did not exhibit appreciable macroscopic fracture following injection. Furthermore, the hydrogels demonstrated a significant increase in complex * To whom correspondence should be addressed: Kevin E. Healy, Ph.D., University of California at Berkeley, Departments of Bioengineering and Materials Science and Engineering, 465 Evans Hall #1762, Berkeley, CA 94720-1762. Telephone: (510) 643-3559. Facsimile: (510) 642-5835. E-mail: [email protected]. † Northwestern University. ‡ University of California at Berkeley.

modulus (i.e., rigidity) when heated from 22 to 37 °C without exhibiting a significant change in either volume or water content. We imparted these unique characteristics to the P(NIPAAmco-AAc) hydrogels by exploiting the phase behavior of P(NIPAAm) in aqueous media. With increasing temperature, P(NIPAAm) phase-separates from water at the lower critical solution temperature (LCST), which is approximately 32 °C.10 Below the LCST, P(NIPAAm) chains are soluble in aqueous media, and cross-linked P(NIPAAm) hydrogels swell.11-14 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. The phase behavior of P(NIPAAm) chains and hydrogels is reversible13 and can be modified by polymerizing NIPAAm monomer with more hydrophilic or more hydrophobic comonomers. The addition of more hydrophilic comonomers (e.g., AAc) increases the LCST of P(NIPAAm) copolymers15,16 and copolymer hydrogels,9,11,17 decreases the extent of aggregation experienced by P(NIPAAm) copolymer chains,15 and decreases the extent of temperature-sensitive volume change exhibited by P(NIPAAm) copolymer hydrogels.9,17,18 By exploiting the phase properties of P(NIPAAm) in aqueous media, we developed injectable P(NIPAAm-coAAc) hydrogels that supported cell viability and tissue formation in vitro. In addition, the hydrogels demonstrated in situ stabilization (i.e., an increase in rigidity when heated) without exhibiting a significant decrease in volume or water content. In situ stabilization is critical from a tissue engineer-

10.1021/bm0000945 CCC: $20.00 © 2001 American Chemical Society Published on Web 02/08/2001

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ing perspective, as the scaffold must provide adequate mechanical integrity to support tissue formation. While the hydrogels would exhibit in situ stabilization in clinical applications, we eliminated the potential adverse effects of in situ polymerization, since the hydrogels would be synthesized and purified prior to being injected into the body. Furthermore, because degradation of the hydrogel may be an important characteristic in in vivo and clinical applications, the hydrogel chemistry is amenable to the incorporation of degradable cross-links. For example, we have synthesized P(NIPAAm) hydrogels using hydrolytically cleavable crosslinkers, and the matrices degraded into low-viscosity liquids (unpublished results). Thus, it is possible to incorporate degradation into the injectable P(NIPAAm-co-AAc) hydrogels for use in in vivo and clinical applications. Importantly, the synthetic strategy we have employed allows for easy control of the mechanical and chemical properties of the P(NIPAAm-co-AAc) hydrogel, allowing parametric analysis of the effects of these properties on tissue development both in vitro and in vivo. One limitation of our previous work, however, was that the P(NIPAAm-co-AAc) hydrogel did not interact with mammalian cells at a molecular level. Therefore, the hydrogel could not act as an artificial extracellular matrix (ECM) through exploitation of the natural associations between cells and the native ECM. To induce interactions between a material and a biological system, the material is commonly modified with biologically active synthetic peptides containing sequences that interact with cell-surface receptors. The amino acid sequence -Arg-Gly-Asp(-RGD-), a ubiquitous cell-binding domain found in many ECM proteins (e.g., fibronectin and vitronectin) and recognized by cell-surface receptors called integrins,19-21 has been extensively studied.22-29 The -RGD- peptide has been covalently grafted to two-dimensional (2D) substrates22-28 or within 3D networks.29 Other cell-binding domains have been investigated as well, including heparin-binding domains, such as -Phe-His-Arg-Arg-Ile-Lys-Ala- (-FHRRIKA-).30-32 A more extensive cell response (e.g., cell attachment, spreading, formation of discrete focal contacts, and organized cytoskeletal assembly) was obtained on 2D surfaces when both the -RGD- and heparin-binding domains of fibronectin were provided.30,33 To avoid the technical limitations involved with investigating ligand-receptor interactions within 3D matrices, we initially focused on studies of mammalian cell-material interactions on 2D substrates. In our previous work, we examined the behavior of bone-forming cells on 2D biomimetic surfaces containing both the integrin-binding (i.e., -RGD-) and heparin-binding (i.e., -FHRRIKA-) domains of bone sialoprotein (BSP).22,31,32 Knowledge gained from these 2D experiments has provided the foundation for examining cell behavior within 3D networks. In this paper, we describe an approach to study the biomolecular associations between bone-forming cells and P(NIPAAm-co-AAc) hydrogels modified with -RGD- and -FHRRIKA- peptides. It is important to note that this approach can be generalized to study any biomolecular interactions within the P(NIPAAm-co-AAc) hydrogels.

Stile and Healy

The objective of the current study was to extend our observations from 2D surfaces to 3D injectable P(NIPAAmco-AAc) hydrogels. Synthetic peptides containing the -RGDand -FHRRIKA- sequences were covalently grafted to the AAc moieties in the hydrogel. The peptide-modified P(NIPAAm-co-AAc) hydrogels were characterized via solidstate 1H magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, LCST studies, and volume change studies in which the matrix was heated from 22 to 37 °C in an aqueous environment. To qualitatively examine the behavior of osteoblast-like cells within the peptidemodified P(NIPAAm-co-AAc) hydrogels, in situ phasecontrast images were obtained and in situ viability studies were performed. Finally, preliminary quantitative studies were performed to measure osteoblast-like cell proliferation within the peptide-modified P(NIPAAm-co-AAc) hydrogels. Experimental Section Materials. NIPAAm, AAc, N,N′-methylenebisacrylamide (BIS; Chemzymes Ultrapure grade), ammonium peroxydisulfate (AP; Chemzymes Ultrapure grade), and N,N,N′,N′tetramethylethylenediamine (TEMED; Chemzymes Ultrapure grade) were purchased from Polysciences, Inc. (Warrington, PA). Phosphate-buffered saline (PBS; pH approximately 7.0; without calcium chloride, without magnesium chloride), Dulbecco’s modified Eagle medium (DMEM), heat-inactivated fetal bovine serum (FBS), penicillin-streptomycin, fungizone, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) buffer, and sodium pyruvate were purchased from GIBCO BRL (Grand Island, NY). 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysulfosuccinimide (Sulfo-NHS), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxlyate (Sulfo-SMCC), and 2-(N-morpholino)ethanesulfonic acid, 0.9% NaCl, conjugation buffer (MES) were purchased from Pierce (Rockford, IL). Diaminopoly(ethylene glycol) [PEG(NH2)2; 3400 g/mol, Chromatographically pure] was purchased from Shearwater Polymers (Huntsville, AL). Synthetic peptides containing the integrin-binding sequence -RGD- (-RGD- peptide; AcCGGNGEPRGDTYRAY-NH2) and the heparin-binding sequence -FHRRIKA- (-FHRRIKA- peptide; Ac-CGGFHRRIKA-NH2) found in BSP were purchased from Tana Laboratories (Houston, TX). Additional -RGDpeptide was purchased from the University of Illinois at Chicago Protein Research Laboratory (Chicago, IL). To reduce the peptide end group reactivity and degradation by exoproteases, the amino terminus was acetylated (Ac) and the carboxy terminus was amidated (NH2). Deuterium oxide (D2O), ascorbic acid, ethyl alcohol, and ethylene glycol bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA) were purchased from Aldrich (Milwaukee, WI). All materials were used as received. P(NIPAAm-co-AAc) Hydrogel Synthesis. The method used to synthesize the P(NIPAAm-co-AAc) hydrogels was similar to that published previously.9 A schematic representation of the hydrogel synthesis is shown in Scheme 1. Briefly, 2.435 g (21.5 mmol) of NIPAAm, 0.065 g (0.903 mmol) of AAc, 0.005 g (0.0325 mmol) of BIS, and 50 mL of PBS

Peptide-Modified Hydrogels for Tissue Regeneration Scheme 1. Synthesis of the P(NIPAAm-co-AAc) Hydrogel

were bubbled with dry nitrogen gas in a two-neck flask for 15 min to remove dissolved oxygen. 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(NIPAAmco-AAc) hydrogel was washed three times, 15-20 min each, in excess ultrapure water (UPW; 18 MΩ cm) to remove unreacted compounds. These hydrogels contained 96% mol NIPAAm/mol total monomer, 4% mol AAc/mol total monomer, and 0.14% mol BIS/mol (total monomer + crosslinker). Peptide Functionalization. Peptide modification of the hydrogels was similar to that described previously for the functionalization of 2D surfaces.24 A schematic representation of the peptide modification is presented in Scheme 2. Forceps were used to carefully grasp a small sample from the top of a P(NIPAAm-co-AAc) hydrogel, and scissors were used to cut samples (roughly 0.5 mL) from the bulk hydrogel. The samples were placed in a 24-well polystyrene tissue-culture dish (Becton Dickinson, Lincoln Park, NJ). A PEG(NH2)2 spacer arm was grafted to the AAc groups in the hydrogel using 0.200 g/mL (0.059 mmol/mL) PEG(NH2)2, 0.400 mg/ mL (0.0021 mmol/mL) EDC, and 1.1 mg/mL (0.0051 mmol/ mL) Sulfo-NHS in 0.1 M MES buffer at a pH of 6.0. The reaction proceeded for 1 h at 22 °C on a shaker table. Following the reaction, the samples were soaked in UPW for 5 min. Buffer-control samples were immersed in MES buffer for 1 h at 22 °C and soaked in UPW for 5 min as well. The samples were then presoaked in 50 mM sodium borate buffer at a pH of 7.5, and the excess buffer was removed. Sulfo-SMCC was used as a heterobifunctional cross-linker and was added at a concentration of 0.5 mg/mL (0.0011 mmol/mL) in 50 mM sodium borate buffer at a pH

Biomacromolecules, Vol. 2, No. 1, 2001 187 Scheme 2. Peptide Functionalization of the P(NIPAAm-co-AAc) Hydrogel

of 7.5. The reaction proceeded for 30 min at 22 °C on a shaker table. The samples were soaked in UPW for 5 min following the reaction. The buffer-control samples were exposed to identical conditions, without the addition of SulfoSMCC. Finally, the samples were then presoaked in 0.1 M sodium phosphate buffer at a pH of 6.6, and the excess buffer was removed. Solutions of the -RGD- peptide and the -FHRRIKA- peptide were prepared at concentrations of 0.6 mg/mL (3.6 × 10-4 mmol/mL) and 0.4 mg/mL (2.4 × 10-4 mmol/mL), respectively, in 0.1 M sodium phosphate buffer at a pH of 6.6. The hydrogel samples were exposed to a 50:50 molar ratio of the peptides for 24 h at 4 °C. During this step, the maleimide end of Sulfo-SMCC reacted with the thiol group in the cysteine residue of the peptides. Following the reaction, the samples were soaked in UPW for 10 min. The same experimental conditions were applied to the buffer-control samples, in the absence of the peptides.

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Table 1. Description of the Samples Analyzed in the LCST and Volume Change Studies P(NIPAAm-co-AAc) hydrogel buffer-control P(NIPAAm-co-AAc) hydrogel PEG(NH2)2-modified P(NIPAAm-co-AAc) hydrogel peptide-modified P(NIPAAm-co-AAc) hydrogel

P(NIPAAm-co-AAc) hydrogel (96 mol % NIPAAm and 4 mol % AAc) without exposure to the buffer steps P(NIPAAm-co-AAc) hydrogel exposed to buffer steps P(NIPAAm-co-AAc) hydrogel containing PEG(NH2)2 grafted to the AAc P(NIPAAm-co-AAc) hydrogel containing -RGD- and -FHRRIKA- peptides grafted to the PEG(NH2)2

In our previous 2D studies,24 we grafted the PEG(NH2)2 spacer arm to an interpenetrating polymer network (IPN). We used Robust Design to optimize the IPN, but we did not optimize the length of the PEG(NH2)2 spacer arm. However, in our 2D studies, the PEG(NH2)2 spacer arm presented the peptides in a way that promoted osteoblast spreading, proliferation, and phenotypic expression, and we used the same spacer arm in the 3D studies presented here. Nuclear Magnetic Resonance (NMR) Spectroscopic Studies. Buffer-control and peptide-modified P(NIPAAmco-AAc) hydrogel samples (see Table 1 for descriptions) were dried in a vacuum desiccator for 7 days to remove the hydrogel water. Once dried, the samples were immersed in excess D2O and allowed to swell for 7 days. The D2Oswollen samples were analyzed using solid-state 1H magic angle spinning (MAS) NMR spectroscopy (Spectral Data Services, Champaign, IL). The spectra were obtained on a 270 MHz spectrometer, and spin speeds of 2.1 and 2.0 kHz were used to analyze the buffer-control and peptide-modified samples, respectively. LCST Determination. A Beckman DU-64 UV-vis spectrophotometer with a DU series 60 water-regulated single-cell holder (Fullerton, CA) was used to determine the LCST of the hydrogel samples as previously reported.9 A description of the samples tested is given in Table 1. The transmittance of visible light (λ ) 500 nm; path length ) 1 cm) through the hydrogel was recorded as the hydrogel temperature was varied with a Fisher model 90 refrigerated bath (Pittsburgh, PA). A K-type thermocouple attached to an Omega HH-81 digital thermometer (Stamford, CT) was used to measure the hydrogel temperature. 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 vs temperature was obtained, the LCST was judged to be the initial break point of the curve.34 Volume Change When Heated from 22 to 37 °C. The volume change exhibited by the hydrogel samples when heated from 22 to 37 °C in the presence of PBS or cellculture media [i.e., DMEM with 10% (v/v) FBS] was determined as previously reported.9 A description of the samples tested is given in Table 1. Using a syringe, hydrogel samples were placed in a glass vial at a concentration of 1.67 mL hydrogel/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 heated samples was estimated by the displacement of 37 °C UPW. The hydrogel volume change when heated from 22 to 37 °C was computed by subtracting the 22 °C volume from the 37 °C volume and dividing by the 22 °C volume. Rat Calvarial Osteoblast (RCO) Isolation and Culture. RCO were isolated as reported previously.35 The RCO were

cultured at 37 °C in a humidified atmosphere of 5% CO2 (95% air; 21% O2). The growth media, which was changed three times each week, consisted of DMEM, with 15% (v/ v) FBS, 1% (v/v) penicillin-streptomycin, 1% (v/v) fungizone, 15 mM HEPES buffer, 1 mM sodium pyruvate, and 5 µg/mL ascorbic acid. The RCO used in the study were obtained from passage 2 or 3 and were of the osteoblast phenotype based on positive staining for membrane-bound alkaline phosphatase and mineralization of the synthesized ECM. RCO Seeding into the Hydrogels. The buffer-control and peptide-modified hydrogel samples were sterilized in a 70% solution (v/v) of ethyl alcohol in water for 1 h and washed in excess PBS for up to 12 h. The washing included soaking the samples in PBS for 1-2 h after which fresh PBS was added. Following the sterilization, the samples were placed in a 24-well polystyrene tissue-culture dish (Becton Dickinson). The cells were released using EGTA, centrifuged, suspended in growth media, and counted using a hemocytometer. The cell suspension was added to a syringe containing a 20-gauge needle, and equal volumes (roughly 1.0 mL) were injected at 22 °C into the hydrogel samples. The initial cell seeding density was 20 000-50 000 cells/ mL of hydrogel. The cells were injected throughout the sample as evenly as possible (i.e., >10 injections per hydrogel), producing a relatively uniform distribution of cells. Following the cell seeding, growth media was added to the wells, and the cell-loaded samples were cultured at 37 °C in a humidified atmosphere of 5% CO2 (95% air; 21% O2). The next day, the samples were transferred to a new 24well tissue-culture dish to separate the cells that passed through the hydrogel and settled on the bottom of the dish from the cells that were retained within the hydrogel. Analysis of RCO in the Hydrogel Samples. At certain points during the in vitro culture, the RCO in the buffercontrol and peptide-modified hydrogels were viewed on a Nikon Diaphot inverted microscope (Nippon Kogaku K. K., Tokyo, Japan). Images were obtained using a Photometrics Ltd. (Tucson, AZ) cooled charged coupled device (CCD) camera. Image analysis was performed using IPLab (Scanalytics, Inc., Fairfax, VA), Adobe Photoshop 3.0 (Adobe Systems Inc., Mountain View, CA), Canvas 3.5.3 (Deneba Software, Miami, FL), and N. I.H Image (NIH, Bethesda, MD). In Situ Fluorescent Viability Study. An in situ fluorescent viability study using calcein AM and ethidium homodimer-1 (EthD-1) (Molecular Probes, Eugene, OR) was performed at various points during the in vitro culture. Intracellular esterases inside healthy cells convert the nonfluorescent cell-permeant calcein AM to fluorescent calcein (ex/em max roughly 495 nm/515 nm). EthD-1 enters cells with damaged membranes and fluoresces when bound to

Peptide-Modified Hydrogels for Tissue Regeneration

nucleic acids (ex/em max roughly 495 nm/635 nm). RCOloaded peptide-modified P(NIPAAm-co-AAc) hydrogel samples were immersed in PBS for 10 min at 22 °C, after which they were soaked in a 2 µM solution of calcein AM for 5 min and a 1 µM solution of EthD-1 for 5 min. Both solutions were prepared using PBS. Preliminary experiments were performed to determine the soaking time, which allowed adequate penetration of the probes (i.e., fluorescent cells could be seen). Live and dead cells were viewed on a Nikon Diaphot inverted microscope (Nippon Kogaku K. K.) using epi-illumination and standard Nikon filter sets for fluorescein and rhodamine. A 40× oil objective was used to view the cells. Cell Proliferation Study. Cell proliferation was quantified using CyQuant (Molecular Probes), which fluoresces when bound to nucleic acids (ex/em max roughly 480 nm/520 nm). At various points during the in vitro culture, RCO-loaded peptide-modified P(NIPAAm-co-AAc) hydrogel samples and RCO-loaded buffer-control P(NIPAAm-co-AAc) hydrogel samples were homogenized using a hand-held homogenizer. PBS was added, the mixture was centrifuged, the supernatant was removed, and CyQuant solution was added to the remaining cell pellet. The CyQuant solution consisted of 0.2 mL of cell lysis buffer, 3.8 mL of UPW, and 10 µL of CyQuant. The absorbance was recorded on a Shimadzu RF1501 spectrofluorophotometer (Kyoto, Japan). Control gels without cells were analyzed, and no fluorescence was observed. A standard curve was prepared using isolated RCO to determine the linear relationship between absorbance and cell number (intensity ) 0.0171 × cell number; r2 ) 0.99). Histological Analysis. On days 0, 4, and 8 of in vitro culture, a buffer-control sample and a peptide-modified sample were immersed in chilled 2-methylbutane. The frozen samples were sectioned longitudinally with a Reichert-Jung cryotome (Leica, Zurich, Switzerland), and the sections were stained with hematoxylin and eosin (H&E) to study the tissue structure. Statistical Analysis. Analysis of variance (ANOVA) tests and Newman-Keuls post-hoc analyses were performed using StatSoft Statistica 5.0 (Tulsa, OK). Results and Discussion 1H MAS NMR Spectroscopic Studies. The spectrum for the buffer-control sample shown in Figure 1a contains six peaks, which are assigned in Table 2. The number and location of the peaks were consistent with those reported previously for P(NIPAAm)-based hydrogels.9,36 The theoretical intensities of peaks 1, 2, 3, and 5 should be in a ratio of 6:2:1:1, respectively. The measured intensities were in a ratio of 6.1:2.0:1.1:1. Importantly, the spectrum did not contain peaks that would indicate the presence of residual monomer or cross-linker. Peak 4 at 2.449 ppm is unassigned but was observed in the spectrum for the P(NIPAAm-co-AAc) hydrogel reported previously.9 This peak was not observed in the spectra for the monomers or the cross-linker but was observed in the spectrum for P(NIPAAm-co-AAc) chains (unpublished results). Therefore, we attribute peak 4 to isomers formed during the polymerization and/or end groups formed by different termination steps.

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Figure 1. Solid-state 1H MAS NMR spectra of the (a) buffer-control P(NIPAAm-co-AAc) and (b) peptide-modified P(NIPAAm-co-AAc) hydrogels. The peak numbers correspond to the hydrogen described in the text, and the peaks are assigned in Table 2. The hydrogels contain 96 mol % NIPAAm and 4 mol % AAc.

The spectrum for the peptide-modified sample shown in Figure 1b contains seven peaks, which are assigned in Table 2. The number and locations of the peaks were consistent with those observed in the buffer-control samples and the injectable P(NIPAAm-co-AAc) hydrogel developed previously.9 The theoretical intensities of peaks 1, 2, 3, and 6 should be in a ratio of 6:2:1:1, respectively. The measured intensities were in a ratio of 6.0:1.9:1:1.1. Similar to the buffer-control P(NIPAAm-co-AAc) hydrogel spectrum, there were no peaks that would indicate the presence of unreacted monomer or cross-linker, and we attribute peak 4 to isomers and/or end groups present in the hydrogel. Peak 5 at 3.520 ppm represents the -[CH2-CH2-O]- protons in PEG(NH2)2. Even though the protons in the PEG(NH2)2 spacer arm could be observed, the protons in the peptides were not observed, presumably due to the low amount and molecular weight of the peptides vs the PEG(NH2)2. The detection limit in NMR spectroscopy is 1% (w/v). The P(NIPAAm-co-AAc) hydrogel contained a theoretical maximum of 0.13% (w/v) AAc moieties for grafting. Because of the low amount of AAc in the P(NIPAAm-co-AAc) hydrogels, the AAc was not detected in solid-state 1H MAS NMR spectroscopic studies (i.e., when D2O was not used; unpublished results). Since we were unable to detect the AAc in the hydrogels, it was unlikely that we would be able to detect the PEG(NH2)2 or the peptides, since our previous study using 2D surfaces has shown that the number of sites for peptide coupling decreases an order of magnitude with each step of the modification.32 So, the amount of grafted PEG(NH2)2 was

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Table 2. Peak Assignments for 1H MAS NMR Spectra

a

protons

P(NIPAAm-co-AAc) hydrogel (ppm)ab

buffer-control hydrogel (ppm)b

peptide-modified hydrogel (ppm)b

-CH3 protons in NIPAAm -CH2- protons in polymer backbone -CH- proton in polymer backbone isopropyl -CH- in NIPAAm residual protons in D2O unassigned -[CH2-CH2-O]- protons in PEG(NH2)2

0.921 1.350 1.781 3.658 4.547 2.4 na

0.969 1.403 1.833 3.724 4.574 2.449 na

0.969 1.404 1.842 3.708 4.581 2.435 3.520

From ref 9. b 96 mol % NIPAAm and 4 mol % AAc.

Table 3. LCST Data sample description hydrogelb

P(NIPAAm-co-AAc) buffer-control P(NIPAAm-co-AAc) hydrogelb PEG(NH2)2-modified P(NIPAAm-co-AAc) hydrogelb peptide-modified P(NIPAAm-co-AAc) hydrogelb

LCST (°C)a 34.8 ( 0.2c 35.0 ( 0.4c 35.8 ( 0.4e 34.3 ( 0.2d

a LCST studies were performed at a pH of approximately 7.0 b 96 mol % NIPAAm and 4 mol % AAc c Not significantly different from each other but significantly different from all others at p < 0.03 d Significantly different from all others at p < 0.03 e Significantly different from all others at p < 0.001

Figure 2. Transmittance vs temperature data for P(NIPAAm-co-AAc) hydrogels (triangles), buffer-control P(NIPAAm-co-AAc) hydrogels (diamonds), PEG(NH2)2-modified P(NIPAAm-co-AAc) hydrogels (squares), and peptide-modified P(NIPAAm-co-AAc) hydrogels (circles). Each line is the average of three to five experiments. The error bars were excluded for clarity. The hydrogels contain 96 mol % NIPAAm and 4 mol % AAc.

assumed to be an order of magnitude less than the amount of AAc in the P(NIPAAm-co-AAc) hydrogel, and the amount of grafted peptide was assumed to be an order of magnitude less than the amount of grafted PEG(NH2)2. Therefore, the amounts of grafted PEG(NH2)2 and peptide were most likely below the detection limit of the spectrometer. However, we were still able to detect the PEG(NH2)2 spacer arm due to the high molecular weight of the molecule and the high number of -[CH2-CH2-O]- groups per AAc site. It is important to note that detection of the PEG(NH2)2 and/or peptides using 1H NMR spectroscopy does not verify the chemical conjugation of the components, but merely indicates their presence within the P(NIPAAm-co-AAc) hydrogel. Even though we thoroughly washed the hydrogels following peptide conjugation, it is possible that ungrafted PEG(NH2)2 and/or peptide remained within the matrix. Therefore, we must rely on other, more definitive tests to verify the chemical conjugation of the PEG(NH2)2 and peptides, such as LCST and volume change studies (see the LCST Studies section below). LCST Studies. Transmittance vs temperature curves are presented in Figure 2. Each line represents the average of three to five experiments. The error bars were removed so the lines could be seen clearly. A description of the samples tested is given in Table 1, and the LCSTs are tabulated in Table 3. The LCSTs of all of the P(NIPAAm-co-AAc) hydrogel samples listed in Table 1 were significantly higher than the LCST of the P(NIPAAm) hydrogel reported previously9 (p < 0.001), due to the presence of AAc in the copolymer hydrogels.9,13,14,17,37 The LCST of the buffer-

control P(NIPAAm-co-AAc) hydrogel was not significantly different from the LCST of the P(NIPAAm-co-AAc) hydrogel. This result, which is consistent with work published previously,9 indicates that exposure to the conjugation buffers does not significantly affect the LCST. The LCST of the PEG(NH2)2-modified P(NIPAAm-co-AAc) hydrogel was significantly higher than the LCSTs of all the other samples (p < 0.001), due to the presence of the grafted hydrophilic PEG chains. These results are in agreement with other11 published work.9,11,17 The LCST of the peptide-modified P(NIPAAm-co-AAc) hydrogels was significantly lower than the LCST of the PEG(NH2)2-modified P(NIPAAm-co-AAc) hydrogel (p < 0.001) and the LCSTs of the P(NIPAAmco-AAc) hydrogel and the buffer-control P(NIPAAm-coAAc) hydrogel (p < 0.03), due to the presence of the relatively hydrophobic amino acids in the peptides (e.g., Phe, Tyr, and Pro). These results are also consistent with studies published previously.38 These LCST data indicate that the PEG(NH2)2 and the peptides were grafted to the P(NIPAAm-co-AAc) hydrogels and were not merely interpenetrated within the matrix. It is well-known that random copolymerization of NIPAAm with more hydrophilic or more hydrophobic components significantly alters the LCST and temperature-sensitive volume change demonstrated by the copolymer hydrogels.9,11,14-18,38 It has also been shown that when blocks of P(NIPAAm) chains are grafted to locally distributed polymer chains [e.g., P(AAc)], the P(NIPAAm) copolymer chains and hydrogels demonstrate phase behavior similar to that exhibited by P(NIPAAm) homopolymer chains and hydrogels.37,39 Furthermore, it has been established that P(NIPAAm)-based IPNs and semi-IPNs do not exhibit significantly different LCSTs, as compared to P(NIPAAm)-based hydrogels, because the P(NIPAAm) hydrogel is not chemically modified.38-40 Since the second network (in the case of an IPN) and the polymer chains (in the case of a semi-IPN) are chemically independent from the network, the phase behavior of the P(NIPAAm) hydrogel is unaffected. In addition,



Table 4. Volume Change Data sample description hydrogelb

P(NIPAAm-co-AAc) buffer-control P(NIPAAm-co-AAc) hydrogelb PEG(NH2)2-modified P(NIPAAm-co-AAc) hydrogelb peptide-modified P(NIPAAm-co-AAc) hydrogelb

vol change in PBS (%)a

vol change in media (%)a

+50.7 ( -29.3 ( 18.1g,j +62.7 ( 18.6l -29.3 ( 11.5c,f

+28.0 ( 4.0d,f,h,j,k -57.3 ( 16.7h,i -17.3 ( 25.0kl -27.3 ( 17.2d,e

4.6c,e,g,i

a Volume change studies were performed at a pH of approximately 7.0 b 96 mol % NIPAAm and 4 mol % AAc significant differences at p < 0.006 (e.g., c is significantly different from c, d is significantly different from d, etc.).

P(NIPAAm)-based semi-IPNs do not demonstrate significantly different temperature-dependent volume changes, as compared to P(NIPAAm)-based hydrogels.39,40 Therefore, significant changes in the LCST or volume change data verify the chemical modification of the P(NIPAAm) hydrogels, and if the chemical components were merely absorbed into and/or physically entangled within the network, the LCST and volume change results would not be affected. Bae et al.38 have shown that IPNs of P(NIPAAm) and poly(tetramethylene ether glycol) (PTMEG) did not exhibit significantly different LCSTs as compared to P(NIPAAm) hydrogels.38 They concluded that the PTMEG network was chemically independent and thus did not affect the phase behavior of the NIPAAm components, even though the PTMEG network was in close physical contact with the P(NIPAAm) network. In addition, Kokufuta et al.39 reported that the volume-phase transition temperature (Tv) of semiIPNs consisting of P(NIPAAm) hydrogels and P(AAc) chains (450 000 g/mol) were similar to both the Tv of P(NIPAAm) hydrogels and the LCST of P(NIPAAm) solutions. Furthermore, experiments performed in our laboratory with semiIPNs consisting of P(NIPAAm-co-AAc) hydrogels and high molecular weight (50 000 and 450 000 g/mol) linear P(AAc) chains have confirmed these results.40 Therefore, since the LCSTs of the PEG(NH2)2-modified and peptide-modified P(NIPAAm-co-AAc) hydrogels were significantly different from the LCST of the P(NIPAAm-co-AAc) hydrogels, the PEG(NH2)2 and peptides were chemically grafted to the matrix. Volume Change Studies. The volume change data obtained when the samples were heated from 22 to 37 °C for 6 days in the presence of either PBS or cell-culture media are presented in Table 4. A description of the samples tested is given in Table 1. The buffer-control and peptide-modified P(NIPAAm-co-AAc) hydrogels, which were swollen in buffer for almost 2 days, collapsed significantly when heated in the presence of PBS and cell-culture media, as compared to the P(NIPAAm-co-AAc) hydrogels (p < 0.001). This result was not completely unexpected since P(NIPAAm-coAAc) hydrogels swollen in PBS have been shown to collapse significantly, due to the shielding of the -COO- groups by ions present in the PBS.9 The PEG(NH2)2-modified P(NIPAAm-co-AAc) hydrogels swelled to the same extent as the P(NIPAAm-co-AAc) hydrogels in the presence of PBS. However, the PEG(NH2)2-modified P(NIPAAm-co-AAc) hydrogels collapsed significantly in the presence of cellculture media, as compared to the P(NIPAAm-co-AAc) hydrogels when tested in the presence of cell-culture media (p < 0.006). Since the PEG(NH2)2 conjugation reaction proceeded for only 1 h and the samples did not collapse in the presence of PBS, it is unlikely that the collapse in cell-

c

-lThe letters designate statistically

Figure 3. In situ phase-contrast image of RCO within the peptidemodified P(NIPAAm-co-AAc) hydrogel after 1 day of in vitro culture. The RCO were seeded according to the methods described in the text.

culture media was due to -COO- shielding of the hydrogels by ions present in the conjugation buffer. Large highly charged proteins present in the cell-culture media most likely shielded the hydrophilic character of the PEG chain and/or the -NH2 group at the end of the PEG chain, causing the matrices to collapse. As discussed in the LCST Studies section, changes in the LCST and volume change data indicate chemical modification of the P(NIPAAm-co-AAc) hydrogels. Unfortunately, due to the duration of the peptide conjugation process, the volume change data for the peptide-modified P(NIPAAmco-AAc) hydrogels could not be used to verify conjugation, since swelling in buffer altered the volume change results. However, the LCST results did confirm peptide grafting to the P(NIPAAm-co-AAc) hydrogels. The volume change data do indicate, though, that the PEG(NH2)2 was grafted to the P(NIPAAm-co-AAc) hydrogels because the PEG(NH2)2modified P(NIPAAm-co-AAc) hydrogel volume change results are different from the P(NIPAAm-co-AAc)-based semi-IPN data obtained in our laboratory.40 We studied semiIPNs consisting of P(NIPAAm-co-AAc) hydrogels and high molecular weight (50 000 and 450 000 g/mol) linear P(AAc) chains. The volume changes demonstrated by the P(NIPAAmco-AAc)-based semi-IPNs in the presence of PBS were not statistically different from the volume changes exhibited by P(NIPAAm-co-AAc) hydrogels in the presence of PBS (p > 0.01).40 However, the volume changes demonstrated by the P(NIPAAm-co-AAc)-based hydrogels and semi-IPNs in the presence of cell-culture media were affected by the NIPAAm:AAc molar ratio in the P(NIPAAm-co-AAc) network. P(NIPAAm-co-AAc)-based semi-IPNs with the same NIPAAm:AAc molar ratio as the PEG(NH2)2-modified

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Stile and Healy

Figure 4. In situ phase-contrast image of RCO within the control P(NIPAAm-co-AAc) hydrogels after (a) 1 day and (b) 8 days of in vitro culture and within the peptide-modified P(NIPAAm-co-AAc) hydrogel after (c) 1 day and (d) 8 days of in vitro culture. The RCO were seeded according to the methods described in the text.

P(NIPAAm-co-AAc) hydrogels synthesized in the current study (i.e., 96:4) did not collapse when tested in the presence of cell-culture media, but swelled (+13.3 ( 6.1%).40 If the PEG(NH2)2 chains were simply entangled within the P(NIPAAm-co-AAc) hydrogel, then one would not expect the matrix to collapse in the presence of cell-culture media. Furthermore, we also showed in our semi-IPN work that as the molar amount of AAc in the P(NIPAAm-co-AAc) network decreased below 4%, the semi-IPNs eventually collapsed when tested in the presence of PBS or cell-culture media.40 The solubility, repulsion, and hydrogen bonding of the hydrophilic -COO- groups in the AAc counteract the aggregation and dehydration of the hydrophobic NIPAAm components.9,17,37 As the number of AAc groups decreases, the hydrophobic/hydrophilic balance within the network is shifted, and the -COO- groups are unable to effectively counteract the hydrophobic NIPAAm interactions. As a result, the matrices collapse to a greater extent. The collapse demonstrated by the PEG(NH2)2-modified P(NIPAAm-coAAc) hydrogels with a NIPAAm:AAc molar ratio of 96:4 when tested in the presence of cell-culture media is consistent

with data obtained using semi-IPNs with less than 4 mol % of AAc in the network. These results suggest that the PEG(NH2)2 was grafted to the AAc groups, diminishing the number of -COO- groups available to counteract the aggregation and dehydration of the NIPAAm components. Therefore, since the volume change of the PEG(NH2)2modified P(NIPAAm-co-AAc) hydrogel in the presence of cell-culture media was different from the volume change of the P(NIPAAm-co-AAc) semi-IPNs in the presence of cellculture media, the PEG(NH2)2 was grafted to the P(NIPAAmco-AAc) hydrogel. The volume change data do point out one limitation of the reported functionalization process. The peptide-modified P(NIPAAm-co-AAc) hydrogels were swollen in buffer for almost 2 days, which caused the samples to collapse considerably (approximately 30%) whether heated in PBS or cell-culture media. From a tissue engineering perspective, this property is undesirable. If the P(NIPAAm-co-AAc) hydrogel scaffold collapsed extensively after being injected into a tissue defect, the marginal contact between the matrix and the tissue would be lost and the probability of complete



Figure 5. Quantitative analysis of RCO proliferation within control P(NIPAAm-co-AAc) hydrogels (triangles) and peptide-modified P(NIPAAm-co-AAc) hydrogels (circles). The RCO were seeded according to the methods described in the text.

Figure 6. In situ fluorescent viability study of RCO within the peptidemodified P(NIPAAm-co-AAc) hydrogel after 7 days of in vitro culture. The RCO were seeded according to the methods described in the text.

Figure 7. RCO-seeded peptide-modified P(NIPAAm-co-AAc) hydrogels stained with H&E. The sections were obtained at (a) 0 days and (b) 8 days of in vitro culture. The RCO were seeded according to the methods described in the text.

tissue regeneration would be low. The collapse of the hydrogel is primarily a concern in clinical applications, and we are in the process of modifying the conjugation method to circumvent this issue. However, even with the volume change demonstrated by the matrices, these peptide-modified P(NIPAAm-co-AAc) hydrogels are useful tools for studying cell-material interactions within 3D structures. Analysis of RCO within the Peptide-Modified P(NIPAAm-co-AAc) Hydrogels. In situ phase-contrast images of RCO cultured in vitro within the peptide-modified P(NIPAAm-co-AAc) hydrogels are shown in Figures 3 and 4. After 1 day of in vitro culture, RCO could be seen spreading in the peptide-modified hydrogels (Figure 3). Spread cells were not seen in the buffer-control P(NIPAAmco-AAc) hydrogels after 1 day of in vitro culture (data not shown). With increased time in culture, a considerable amount of cell proliferation was qualitatively observed in the peptide-modified P(NIPAAm-co-AAc) hydrogels, and this proliferation was qualitatively greater than that observed in control P(NIPAAm-co-AAc) hydrogels (Figure 4). Quantitative analyses using CyQuant supported the observation

of cell proliferation within these hydrogels (Figure 5). In situ fluorescent studies confirmed the viability of the RCO within the peptide-modified P(NIPAAm-co-AAc) hydrogels for at least 21 days of in vitro culture (Figure 6). Histological sections of RCO-loaded peptide-modified P(NIPAAm-coAAc) hydrogels stained with H&E are presented in Figure 7. One clear difference between the images is the shape of the nuclei. When the isolated RCO were seeded into the hydrogels, the cells were round, as demonstrated by the round nuclei shown in Figure 7a. However, after 8 days of in vitro culture, the nuclei were clearly flattened (Figure 7b), which may be indicative of cell-surface receptors (i.e., integrins) engaging with the peptides conjugated to the P(NIPAAmco-AAc) hydrogel. As discussed previously, one limitation of the reported functionalization process is the amount of swelling required. Since the hydrogel cross-link density is purposefully low to allow for injectability, the samples swell considerably when immersed in buffer for extended periods of time at 22 °C. As a result, the highly swollen peptide-modified networks are weaker than the P(NIPAAm-co-AAc) hydrogels. It is

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possible that these swollen matrices do not demonstrate adequate mechanical integrity to support extensive cell spreading and proliferation. In future work, the conjugation process will be modified to reduce the swelling. In addition, the cross-link density of the hydrogel will be altered to examine the effects of stiffness (e.g., G*) on cell proliferation. Conclusions Injectable P(NIPAAm-co-AAc) hydrogels were grafted with -RGD- and -FHRRIKA- peptides, thus successfully extending our work on 2D surfaces to 3D networks. Chemical modification of the hydrogels was confirmed via 1H MAS NMR spectroscopic, LCST, and volume change studies. These hydrogels supported mammalian cell viability, spreading, and proliferation during in vitro culture. While there are some limitations with the functionalization process from a clinical perspective, the data are compelling and suggest the potential for peptide-modified P(NIPAAm-coAAc) hydrogels to be used as model networks for studying cell-material interactions in 3D. Acknowledgment. We wish to thank G. Tarjan for graciously developing the histological methods and performing the histological studies. 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. are greatly appreciated. References and Notes (1) 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. (2) Breitbart, A. S.; Grande, D. A.; Kessler, R.; Ryaby, J. T.; Fitzsimmons, R. J.; Grant, R. T. Plast. Reconstr. Surg. 1998, 101, 567. (3) Paige, K. T.; Cima, L. G.; Yaremchuk, M. J.; Vacanti, J. P.; Vacanti, C. A. Plast. Reconstr. Surg. 1995, 96, 1390. (4) Kawamura, S.; Wakitani, S.; Kimura, T.; Maeda, A.; Caplan, A. I.; Shino, K.; Ochi, T. Acta Orthop. Scand. 1998, 69, 56. (5) Shinoka, T.; Shum-Tim, D.; Ma, P. X.; Tanel, R. E.; Isogai, N.; Langer, R.; Vacanti, J. P.; Mayer Jr., J. E. J. Thoracic CardioVas. Surg. 1998, 115, 536. (6) Seckel, B. R.; Jones, D.; Hekimian, K. J.; Wang, K.-K.; Chakalis, D. P.; Costas, P. D. J. Neurosci. Res. 1995, 40, 318. (7) Cima, L. G.; Vacanti, J. P.; Vacanti, C.; Ingber, D.; Mooney, D.; Langer, R. J. Biomech. Eng. 1991, 113, 143.

Stile and Healy (8) Hubbell, J. A.; Langer, R. Chem. Eng. News 1995, March 13, 42. (9) Stile, R. A.; Burghardt, W. R.; Healy, K. E. Macromolecules 1999, 32, 7370. (10) Heskins, M.; Guillet, J. E. J. Macromol. Sci., Chem. Ed. 1968, A2, 1441. (11) Hoffman, A. S.; Afrassiabi, A.; Dong, L. C. J. Controlled Release 1986, 4, 213. (12) Hirokawa, Y.; Tanaka, T. J. Chem. Phys. 1984, 81, 6379. (13) Hoffman, A. S. MRS Bull. 1991, September, 42. (14) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163. (15) Vernon, B.; Gutowska, A.; Kim, S. W.; Bae, Y. H. Macromol. Symp. 1996, 109, 155. (16) Taylor, L. D.; Cerankowski, L. D. J. Polym. Sci., Polym. Chem. Ed. 1975, 13, 2551. (17) Yoshida, R.; Sakai, K.; Okano, T.; Sakurai, Y. J. Biomater. Sci., Polym. Ed. 1994, 6, 585. (18) Vakkalanka, S. K.; Brazel, C. S.; Peppas, N. A. J. Biomater. Sci., Polym. Ed. 1996, 8, 119. (19) Pierschbacher, M. D.; Ruoslahti, E. Nature (London) 1984, 309, 30. (20) Ruoslahti, E.; Pierschbacher, M. D. Cell 1986, 44, 517. (21) Albelda, S. M.; Buck, C. A. FASEB J. 1990, 4, 2868. (22) Rezania, A.; Healy, K. E. J. Ortho. Res. 1999, 17, 615. (23) Rezania, A.; Thomas, C. H.; Branger, A. B.; Waters, C. M.; Healy, K. E. J. Biomed. Mater. Res. 1997, 37, 9. (24) Bearinger, J. P.; Castner, D. G.; Healy, K. E. J. Biomater. Sci., Polym. Ed. 1998, 9, 629. (25) Massia, S. P.; Hubbell, J. A. Anal. Biochem. 1990, 187, 292. (26) Drumheller, P. D.; Hubbell, J. A. Anal. Biochem. 1994, 222, 380. (27) Drumheller, P. D.; Elbert, D. L.; Hubbell, J. A. Biotech. Bioeng. 1994, 43, 772. (28) Rowley, J. A.; Madlambayan, G.; Mooney, D. J. Biomaterials 1999, 20, 45. (29) Moghaddam, M. J.; Matsuda, T. J. Polym. Sci., Polym. Chem. Ed. 1993, 31, 1589. (30) Dalton, B. A.; McFarland, C. D.; Underwood, P. A.; Steele, J. G. J. Cell Sci. 1995, 108, 2083. (31) Rezania, A.; Healy, K. E. Biotechnol. Prog. 1999, 15, 19. (32) Rezania, A.; Johnson, R.; Lefkow, A. R.; Healy, K. E. Langmuir 1999, 15, 6931. (33) Woods, A.; Couchman, J. R.; Johansson, S.; Hook, M. EMBO J. 1986, 5, 665. (34) Schild, H. G.; Tirrell, D. A. J. Phys. Chem. 1990, 94, 4352. (35) Healy, K. E.; Thomas, C. H.; Rezania, A.; Kim, J. E.; McKeown, P. J.; Lom, B.; Hockberger, P. E. Biomaterials 1996, 17, 195. (36) Tokuhiro, T.; Takayuki, A.; Mamada, A.; Tanaka, T. Macromolecules 1991, 24, 2936. (37) Chen, G.; Hoffman, A. S. Nature 1995, 373, 49. (38) Bae, Y. H.; Okano, T.; Kim, S. W. Pharm. Res. 1991, 8, 531. (39) Kokufuta, E.; Wang, B.; Yoshida, R.; Khokhlov, A. R.; Hirata, M. Macromolecules 1998, 31, 6878. (40) Stile, R. A.; Healy, K. E. Macromolecules 2001, in preparation.

BM0000945

Thermo-Responsive Peptide-Modified Hydrogels for Tissue

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