Thermoresponsive Artificial Extracellular Matrix for Tissue Engineering

which is a kind of iniferter (initiator, transfer agent and terminator). The degree of ... decreased with an increase in both degree of grafting and M...
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Biomacromolecules 2001, 2, 856-863

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Thermoresponsive Artificial Extracellular Matrix for Tissue Engineering: Hyaluronic Acid Bioconjugated with Poly(N-isopropylacrylamide) Grafts Shoji Ohya,† Yasuhide Nakayama,† and Takehisa Matsuda*,‡ Department of Bioengineering, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan; and Department of Biomedical Engineering, Graduate School of Medicine, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan Received February 26, 2001; Revised Manuscript Received May 2, 2001

Thermoresponsive hyaluronans (HAs) were prepared by graft polymerization of N-isopropylacrylamide (NIPAM) on HA (number-averaged molecular weight, Mn, ca. 1.5 × 105 and 5.0 × 105) using dithiocarbamate which is a kind of iniferter (initiator, transfer agent and terminator). The degree of dithiocarbamylation (DD) as an iniferter ranged from 0.4 to 11.4% per disaccharide unit of HA. The estimated Mn of the grafted polyNIPAM (PNIPAM) ranged from approximately 5.0 × 103 to 8.4 × 104. The PNIPAM-grafted HAs (PNIPAM-HAs) were water-soluble at room temperature, while they precipitated at temperatures above approximately 34 °C in water. The temperature at the onset of precipitation (lower critical solution temperature: LCST) was independent of parameters of molecular architecture such as Mn of HA, degree of grafting of PNIPAM, and Mn of PNIPAM. Equilibrium transmittance of the aqueous solution above LCST decreased with an increase in both degree of grafting and Mn of PNIPAM. At physiological temperature, the PNIPAM-HA film cast from a cold solution was very wettable with water. A markedly reduced adhesion of endothelial cells to the film was observed, indicating that the PNIPAM-HA film may serve as a noncell-adhesive matrix. Scanning electron microscopic observation appeared to differentiate supramolecular structures between rapidly freeze-dried PNIPAM-HA and nongrafted HA:PNIPAM-HA exhibited a nonuniform fibrous network, whereas the morphology of which is markedly different from that of a nongrafted HA gel exhibited a mixture of sharp needle- and platelike structures. Introduction The extracellular matrix (ECM), such as collagen, elastin, and proteoglycans, fills the extracellular space in living tissues. These biopolymers have been utilized for wound healing technology for many years and have recently attracted great interest as basic materials for scaffolds used in cell-incorporated, tissue-engineered devices or hybrid tissues.1 Hyaluronan (HA), a naturally occurring glycosaminoglycan, is composed of the repeating disaccharide unit of D-glucuronic acid and N-acetyl-D-glucosamine. It exists in human and animal tissues as the major component of ECMs, particularly in synovial fluid and the vitreous body of the eye, and plays an important role in many biological processes such as tissue hydration or moisturizing, diffusion of ions, nutrients and oxygen, proteoglycan organization in ECMs providing mechano-compressive strength, and cell differentiation. An aqueous solution of HA has been used in opthalmic therapy due to its unique rheological properties. However, biomaterial applications such as wound healing and drug delivery matrices require insolubilization or cross* To whom correspondence should be addressed. Telephone: +81-92-642-6210. Fax: +81-92-642-6212. E-mail: matsuda@ med.kyushu-u.ac.jp. † National Cardiovascular Center Research Institute. ‡ Kyushu University.

linking of HA, exhibiting nondissolution, or water-swellable characteristics. 2-6 The hydrogelation of HA is usually performed by the chemical cross-linking method including carbodiimidepromoted coupling. On the other hand, we have developed the photoinduced hydrogelation method in which sol-to-gel transformation is driven by a photochemical reaction utilizing photodimerizable groups such as cinnamate and thymine groups.7,8 These photocurable solutions or photocured films serve as tissue adhesion prevention materials. On the other hand, using the principle of thermoresponsive phase transition, sol-to-gel phase transformation has been realized for potential biomedical applications as follows. Poly(N-isopropylacrylamide) (PNIPAM), the most popular thermally sensitive water-soluble nonionic polymer, has a lower critical solution temperature (LCST) of almost 32 °C as described below.9 PNIPAM exists as individual chains with a coil conformation at temperatures lower than about 32 °C, but undergoes a sharp coil-to-globule transition at higher temperatures to form inter- and intrachain associations, resulting in precipitation. This thermo-insolubilization method has recently been used to produce a matrix for drug delivery systems,10 a reversible cell-attachment/detachment matrix,11,12 and a hemostatic agent,13,14 which is a PNIPAM copolymer partially derivatized with a cell-adhesion peptidyl moiety containing the RGD (Arg-Gly-Asp) sequence, which is the

10.1021/bm010040a CCC: $20.00 © 2001 American Chemical Society Published on Web 07/10/2001

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Artificial Extracellular Matrix Scheme 1. Synthetic Route of PNIPAM-Grafted Hyaluronan (PNIPAM-HA (4))

minimally active cell adhesive peptidyl sequence common to adhesive proteins such as fibronectin, vitronectin, and fibrinogen. In this study, thermoresponsive HA, which is partially grafted with PNIPAM, is prepared via an photoradicalpolymerization technique using dithiocarbamate15,16 which is a kind of iniferter (initiator, transfer agent, and terminator), in which the desired degree of grafting and desired length of the graft chain are obtained under appropriate reaction conditions. The effects of such parameters as the degree of grafting and chain length (or number-averaged molecular weight, Mn) of the graft on LCST in aqueous solution are examined. A preliminary study of the cell adhesion behavior on PNIPAM-HA films is reported. The potential application of the thermoresponsive HA as a functional artificial ECM in tissue engineering is discussed.

Experimental Section General Methods. All 1H NMR spectra were recorded in DMSO-d6 or CDCl3 using tetramethylsilane (0 ppm) as the internal standard with a 270 MHz NMR spectrometer (JEOL, GX-270, Tokyo, Japan) at room temperature. Gel permeation chromatographic (GPC) analyses in N,N-dimethylformamide (DMF) were carried out with HPLC-8020 instrument (Tosoh, Tokyo, Japan) (column: Tosoh TSKgel R-3000 and R-5000). The columns were calibrated with narrow weight distribution poly(ethylene glycol) standards. UV absorption spectra were measured on a Ubest-30 UV/ vis spectrometer (JASCO, Tokyo, Japan). Static contact angle with deionized water was measured with a contact angle

meter (CA-D, Kyowa Kaimen Kagaku Co., Ltd., Tokyo, Japan) at 40 °C by the sessile drop method. Materials. Sodium hyaluronan (HANa; Mn: ca. 1.5 × 105 and 5.0 × 105) was kindly supplied by Seikagaku Kogyo Co., Ltd. (Tokyo, Japan). Dicyclohexylcarbodiimide (DCC), tri-n-butylamine, 4-(chloromethyl)benzoic acid, and N-isopropylacrylamide (NIPAM) were obtained from Tokyo Chemical Industry Ltd. (Tokyo, Japan), and NIPAM was used after recrystallization from a benzene-hexane solution. N,N-(Dimethylamino)pyridine (DMAP) and sodium N,Ndiethyldithiocarbamate trihydrate were obtained from Wako Pure Chemical Industry Ltd. (Osaka, Japan). Solvents and other reagents, all of which are of special reagent grade, were purchased from Wako and used after conventional purification. Preparation of Dithiocarbamate-Derivatized Hyaluronan (DCB-HA, 3, and DCA-HA, 6). The general procedure for preparing DCB-HA (3), for which the molecular structure is shown in Scheme 1, is followed. An aqueous solution (1.3 L) of HANa (1.3 g; Mn: ca. 5.0 × 105) was subjected to ion-exchange column chromatography (Dowex 50w x 8[H+], Dow Chemicals, Midland, MI) to obtain an aqueous solution (1.9 L) of HA. To the aqueous solution was added a DMF solution (25 mL) of tri-n-butylamine (2.3 g, 12.4 mmol). After the solution was stirred at room temperature for 2 h, water was replaced with DMF by evaporation. To the obtained DMF solution (300 mL) containing HA tri-nbutylamine salt (1) (concentration: 6 g/L) were added a DMF solution of DCC (2.1 mmol, 0.4 mL) and 4-(N,N-diethyldithiocarbamylmethyl)benzoic acid (DCB, 2) (3.2 mmol, 2.4 mL) and DMAP (195 mg, 1.60 mmol). After being stirred for 1 h, the reaction mixture was poured into diethyl ether

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(1 L). The precipitate obtained by filtration was dissolved in water (1 L), dialyzed (dialysis membrane, size 36, Wako) and freeze-dried. The yield of DCB-HA (3) was 1.2 g (99%). The degree of dithiocarbamylation (DD) was 0.4% per disaccharide unit, which was determined by ultraviolet (UV) spectroscopy at 280 nm. HAs with different DD were obtained at the different feed ratios of 2 vs 1 (in the text). (N,N-Diethyldithiocarbamylmethyl)acetic acid-derivatized HA (DCA-HA, 6) was obtained using a procedure similar to that used to obtain 3. Briefly, a DMF solution (4 mL) of DCC (0.9 g, 4.4 mmol) and (N,N-diethyldithiocarbamylmethyl)acetic acid (DCA, 5) (1.4 g, 6.7 mmol), which was prepared by dithiocarbamylation of chloromethyl acetic acid, was stirred at 0 °C for 1 h. The reaction mixture and DMAP (403 mg, 3.3 mmol) were added to a DMF solution (125 mL; concentration: 6.0 g/L) of 1 and then stirred at room temperature for 1 h. DCA-HA (6) was obtained by precipitation and subsequently purified by dialysis. Yield was 503 mg (96%). The DD was 5.3% per disaccharide unit. Preparation of PNIPAM-HA (4 and 7). An aqueous solution (20 mL) of NIPAM with 3 or 6 was placed in a quartz cell. A stream of dry nitrogen was introduced through a gas inlet to sweep the cell for 5 min or more. The solution was irradiated by a 250 W Hg lamp (SPOT CURE, USHIO, Tokyo, Japan) in a nitrogen atmosphere (light intensity: 0.5 mW/cm2). After dialysis for 24 h and subsequent freezedrying, PNIPAM-HA (4 from 3; 7 from 6) was obtained as a white solid. PNIPAM-HAs with different graft chain lengths were obtained by changing the concentration of NIPAM and the irradiation time (in the text). Preparation of Monodithiocarbamyl Poly(ethylene glycol) (DCB-PEG). To a 1,2-dichloroethane solution (100 mL) of poly(ethylene glycol) monomethyl ether (Mn ca. 350, 10 g, ca. 29 mmol), 4-(chloromethyl) benzoyl chloride (11.0 g, 58 mmol) was added dropwise at 0 °C, and then 5 mL of pyridine was added. After being stirred at room temperature for 24 h, the reaction mixture was filtered and concentrated under reduced pressure. The residue was dissolved in ethanol (50 mL). After this solution was added dropwise to an ethanol solution (100 mL) of sodium N,N-diethyldithiocarbamate trihydrate (15.7 g, 70 mmol) at 0 °C, the mixture was stirred at room temperature for 22 h. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (elution with chloroform). The yield was 9.3 g (64%). 1H NMR (DMSO-d6): δ 7.72 (d, 2H, J ) 8.24 Hz, C6H4), 7.52 (d, 2H, J ) 8.57 Hz, C6H4), 4.62 (s, 2H, CH2S), 4.38 (t, 2H, C(O)OCH2), 4.00 (q, 2H, J ) 14.0 Hz, CH2CH3), 3.75 (m, 2H, CH2CH3), 3.60-3.40 (m, 50H, (CH2CH2O)n), 3.23 (s, 3H, CH3O), 1.20 (t, 6H, J ) 13.7 Hz, CH2CH3). FT-IR(KBr): 2871.8 cm-1 (-CH2-), 1716.5 (-C(O)C6H4-), 1610.5 (-C6H4-), 1488.9, 1207.4 (-C(S)N-), 1110.8 (-CH2OCH2-). Measurement of Graft Chain Length. PNIPAM-HA (7) (20 mg) was hydrolyzed in 0.25 N NaOH aqueous solution (20 mL) for 15 h. After the solution was neutralized with 1 N HCl aqueous solution, the reaction mixture was concentrated to about 2 mL under reduced pressure. PNIPAM

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isolated by hydrolysis was precipitated at 40 °C, and its Mn was measured by GPC. Thermoresponsivness of PNIPAM-HA. The thermoresponse phase transition of an aqueous solution of PNIPAMHA (concentration: 0.063-0.5 wt %) was measured with a UV/vis spectrometer by monitoring the transmittance of a 600 nm light beam through an aqueous solution of PNIPAMHA. The samples were heated at a rate of 0.5 °C/min from 25 to 40 °C. The temperature at onset of decrease in transmittance, which was determined with an accuracy of 0.1 °C by a thermosensor that was directly immersed into a solution, was defined as the lower critical solution temperature (LCST).17 Cell Adhesion on PNIPAM-HA Film. PNIPAM-HA film was prepared by solution casting on a circular cover glass (diameter: 14.5 mm) at room temperature. Bovine endothelial cells (ECs) were seeded on the PNIPAM-HA film at a seeding density of 2 × 104 cells/well (diameter of the well: 15 mm) at 37 °C and then incubated in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Laboratories Inc., Grand Island, NY) supplemented with 15% fetal bovine serum (Gibco Laboratories Inc., Grand Island, NY) at 37 °C in a humidified atmosphere of 5% CO2. The EC-seeded surfaces were observed under a phase contrast microscope with a Diaphoto system (Nikon Co., Ltd., Tokyo, Japan). Structural Observation of PNIPAM-HA Precipitate. PNIPAM-HA, coated with an aqueous solution (50 µL, 6.3 wt %) on a cover glass (diameter: 15 mm) and precipitated at 40 °C, was immediately frozen by immersion into liquid nitrogen. After freeze-drying, the sample was osmium-coated with a plasma multicoater (APC-120, Meiwa Co., Ltd., Osaka, Japan) and observed under a scanning electron microscope (JSM-6301F, JEOL, Tokyo, Japan). Results Synthesis of PNIPAM-HA. Synthesis of PNIPAM-HA was achieved via a two-step process: the derivatization of the hydroxymethyl groups of HA tri-n-butylamine salt (1; Mn: ca. 1.5 × 105 and 5.0 × 105) with 4-(N,Ndiethyldithiocarbamylmethyl)benzoic acid (DCB, 2) or (N,Ndiethyldithiocarbamylmethyl)acetic acid (DCA, 5) as an iniferter (DCB-HA or DCA-HA) and the subsequent graft copolymerization of NIPAM via the iniferter-based quasiliving photoradical-polymerization technique. The overall reaction pathway is shown in Schemes 1 and 2. The first reaction was conducted in the presence of dicyclohexylcarbodiimide (DCC) at different feed ratios of 2 or 5 to 1 in DMF solution. Table 1 summarizes the preparation conditions and the DD of DCB-HAs (3). The DD determined by UV absorption spectral analysis ranged from 0.4 to 11.4% per disaccharide unit, depending on the feed molar ratio of 2 to 1 and the reaction time. To determine the optimal conditions for the quasi-living polymerization of an aqueous solution of NIPAM, the following preliminary experiment was designed using poly(ethylene glycol) (PEG) end-capped with a benzyl N,Ndiethyldithiocarbamyl group (DCB-PEG; Mn: ca. 600) as a water-soluble model iniferter. Figure 1 shows the relation-

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Figure 1. (A) Irradiation time and (B) NIPAM concentration dependency on number-averaged molecular weight of the produced PNIPAMgrafted PEG. Polymerization conditions: [initiator] ) 0.01 mmol L-1; light intensity ) 0.5 mW/cm2; [NIPAM] ) 10 mmol L-1 for part A; irradiation time 30 min for part B. Mn (b); polydispersity, Mw/Mn (O). Scheme 2. Synthetic Route of PNIPAM-HA in Which HA and PNIPAM Molecules Are Connected via an Ester Bond

Table 1. Preparation of DCB-HA (3)a DCB-HA (3) run

Mn of HA (×105)

feed ratiob

time (h)

yield (%)

DD (%)c

1 2 3 4 5

1.5 1.5 5.0 5.0 5.0

4.0 6.0 1.0 4.8 6.6

1 18 1 4 4

90 91 99 80 98

1.8 11.4 0.4 3.1 7.5

a

Reaction was conducted at room temperature. b Feed molar ratio of 4-(N,N-diethyldithiocarbamyl methyl) benzoic acid (DCB, 2) against the hydroxymethyl group of HA, (2):DCC:DMAP ) 1:0.67:0.5 (molar ratio). c Degree of dithiocarbamylation per disaccharide unit.

ships of Mn with irradiation time and monomer concentration. Upon UV light irradiation of an aqueous NIPAM solution at a fixed monomer concentration, Mn of the produced polymer rapidly increased at an early stage, followed by a gradual increase, indicating that the chain length of PNIPAM increases with irradiation time (Figure 1A). The polydisper-

sity (defined as the ratio of weight-averaged to numberaveraged molecular weight; Mw/Mn) was at 1.2-1.8 during polymerization. On the other hand, Mn increased linearly with an increase in NIPAM concentration under a fixed irradiation time of 30 min (Figure 1B). From these experiments, Mn of PNIPAM ranging from approximately 5.0 × 103 to 1.1 × 105 was obtained by adjusting the reaction conditions such as irradiation time and NIPAM concentration. On the basis of these results, PNIPAM-HAs (7) with different Mn of PNIPAMs were prepared using DCA-HA (6: DD is 5.3% per disaccharide unit). Under alkaline conditions, PNIPAM graft chains were removed from HA molecules due to hydrolysis of the ester bond that connected HA and PNIPAM molecules, as schematically shown in Scheme 2. Table 2 summarizes the preparation conditions and Mn of PNIPAM in comparison with the data obtained for polymerization initiated by DCB-PEG. Mn of the PNIPAM, calculated by GPC measurements, ranged from

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Figure 2. Thermoresponsive character of an aqueous solution of PNIPAM-HA (4). (A) Influence of Mn of PNIPAM (degree of grafting of PNIPAM is 0.4% per disaccharide unit). Mn of PNIPAM is 5.0 × 103 (0), 1.1 × 104(3), 4.6 × 104 (b), or 8.4 × 104 (2), and PNIPAM homopolymer (O) (B) Influence of degree of grafting (Mn of PNIPAM is about 4.6 × 104). Degree of grafting of PNIPAM is 0.4% (0), 3.1% (2), or 7.5% (O) per disaccharide unit. Table 2. Polymerization Conditionsa and Mn of Produced PNIPAM

Mn of PNIPAM (×104)b NIPAM concentration (mmol L-1)

irradiation time (min)

PNIPAM-PEGc

PNIPAM-HA (7)d

10 10 50 100

5 30 30 20

0.6 1.0 5.0 10.0

0.5 1.1 4.6 8.4

a [initiator] ) 0.01 mmol L-1; light intensity ) 0.5 mW/cm2. b Determined by GPC measurements. c Mn was calculated from the difference in Mn before and after photopolymerization. d PNIPAM was obtained by precipitation at 40 °C after hydrolysis of PNIPAM-HA (7) connected with ester bond between HA and PNIPAM molecules.

approximately 5.0 × 103 to 8.4 × 104. Table 2 shows that the Mn of PNIPAM grafted to HA was almost the same as that obtained for the model iniferter under identical polymerization conditions (fixed iniferter concentration, monomer concentration, and irradiation time). Following the procedure for the preparation of PNIPAM-HAs (7) initiated with (6), PNIPAM-HAs (4) were initiated with (3) with the degree of grafting of PNIPAM ranging from 0.4 to 11.4% per disaccharide unit and Mn of PNIPAM ranging from approximately 5.0 × 103 to 8.4 × 104. Thermoprecipitation of PNIPAM-HA. All PNIPAMHAs (4) were soluble in water at room temperature. The temperature-dependent optical transmittance changes of the aqueous solutions (concentration: 0.5 wt %) of PNIPAMHA (4) with different Mn of PNIPAM and the PNIPAM homopolymer are shown in Figure 2A. PNIPAM itself exhibited sharp thermoprecipitation at 32 °C, and complete precipitation occurred within the very narrow temperature range. On the other hand, irrespective of Mn of PNIPAM, aqueous PNIPAM-HA solutions exhibited turbidity over approximately 34 °C, but the degree of turbidity remained around 75-95%, depending on the parameters of graft architecture, as shown in Figure 2A. The turbid solution became transparent by cooling below 34 °C. Solution properties of all PNIPAM-HAs (4) are summarized in Table 3. Short PNIPAM chain (Mn: 5.0 × 103) and low degree of grafting of PNIPAM in HA appear to be insufficient to drastically change the solubility of the polymer. With an increase in Mn of the PNIPAM graft chain, a sharp drop of equilibrium transmittance was desired. Although there is the

Table 3. Influence of Degree of Grafting and Mn of PNIPAM on LCSTa and Equilibrium Transmittance degree of grafting of PNIPAM (%)b equilibrium Mn of LCST (°C) transmittance (%) PNIPAM (×104) 0.4c 1.8d 3.1c 7.5c 11.4d 0.4c 1.8d 3.1c 7.5c 11.4d 0.5 1.1 4.6 8.4

34.0 33.8 33.8 33.8

34.0 34.0 33.8 33.8

34.0 34.0 34.0 33.6

34.4 84.1 41.7 69.3 54.2 34.0 17.3 16.2 14.3 9.6 34.0 23.4 2.3 13.1 7.3 33.5 33.8 1.0 1.7 0.6 0.7

0.5

a LCST is defined as the temperature of onset of decrease in transmittance. b Per disaccharide unit. Mn of HA: c 5.0 × 105, d 1.5 × 105.

same tendency in which the degree of grafting of PNIPAM appeared to contribute to reduce the equilibrium transmittance, marked appreciable change was not observed within the degree of grafting of PNIPAM under study. The equilibrium transmittance of an aqueous PNIPAM-HA solution above LCST decreased with increasing Mn of PNIPAM. On the other hand, an increase in the degree of grafting of PNIPAM resulted in a decrease in equilibrium transmittance, while all LCSTs were almost the same (approximately 34 °C). The polymer size of PNIPAM-HA (4) in an aqueous medium at low concentration (0.5 wt %) was estimated by GPC measurements at three different temperatures (Figure 3). At 29 °C, the aqueous PNIPAM-HA solution was clear and transparent, and the polymer was eluted at about 33 min. At 33 °C, although the transmittance was almost the same as that at 29 °C, the elution of the polymer was slightly delayed to about 34 min, indicating that polymer size was slightly reduced probably due to dehydration resulting from intramolecular association. On the other hand, at 35 °C, an extremely short elution time (around 23 min) was observed. The peak of PNIPAM-HA, observed at 33 °C, disappeared. This indicates that intermolecular interaction of PNIPAMHAs occurred at temperatures higher than LCST to form large macromolecular aggregates. Thus, it is estimated that the dehydration of graft chain in the HA occurred at temperatures above approximately 32 °C in the same manner as the PNIPAM homopolymer, and at temperatures above approximately 34 °C, PNIPAM-HAs were precipitated due to intermolecular aggregation. Cell Adhesion on PNIPAM-HA Film. The surface wettability of the transparent PNIPAM-HA-coated films is

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nontreated HA hydrogel. The density and thick of fibers increased in PNIPAM-HA concentration. Discussion

Figure 3. Temperature dependence of elution time of PNIPAM-HA (4) by GPC (eluent: DMF). Mn of HA and PNIPAM were approximately 5.0 × 105 and 5.0 × 103, respectively. Degree of grafting of PNIPAM is 0.4% per disaccharide unit. Table 4. Static Water Contact Angles of PNIPAM-HA (4)-Coated Glasses surface glass PNIPAM PNIPAM-HAa

Mn of PNIPAM (×104) 48 0.5 1.1 4.6 8.4

water contact angle (deg) advancing

receding

34.3 ( 2.4 57.4 ( 1.3

17.6 ( 2.3 20.7 ( 1.5

24.0 ( 1.3 21.4 ( 1.7 20.4 ( 1.3 25.0 ( 1.3

8.7 ( 0.5 9.5 ( 0.9 7.3 ( 0.6 7.9 ( 0.6

a M of HA is 5.0 × 105. Degree of grafting of PNIPAM is 0.4% per n disaccharide unit.

tabulated in Table 4. Both advancing and receding contact angles of PNIPAM-HA-coated films with deionized water at 40 °C were lower than that of nontreated glass. There was little difference in the angles among PNIPAM-HAs with different PNIPAM chain lengths. This indicates that the outermost layer of PNIPAM-HA-coated films was almost completely covered with HA units. When the coated films were immersed into water at 40 °C, in only PNIPAM-HA (8) with the longest PNIPAM chain (Mn: 8.4 × 104) film shape was maintained, while 8s with shorter PNIPAM chain were all dissolved in water. Cell adhesion behavior on the film of 8 was observed using a phase contrast microscope (Figure 4). Markedly reduced adhesion of endothelial cells was noted on the PNIPAMHA film, probably due to the highly hydrophilic property of the hyaluronic acid chain. Structural Observation of PNIPAM-HA Precipitate. The microstructure of the precipitated PNIPAM-HA, which was prepared by incubation at 40 °C and subsequently rapid freezing following by freeze-drying, was examined by a scanning electron microscope (Figure 5). A random network of PNIPAM-HA fibrous structure was formed, whereas a freeze-dried nontreated HA hydrogel exhibited a mixture of needle- and platelike structures. PNIPAM-HA appeared to produce a larger number of fine fibrous structures than the

The major components of ECMs in living tissues are proteins such as collagen and elastin, both of which have structural functions to maintain tissue architecture, and glycosaminoglycans such as HA and chondroitin sulfate, both of which are highly water-soluble and specifically bound to side chains of proteins to form complexes termed proteoglycans. These ECM molecules exist as a supramolecular assembled complex which is often insoluble in water, thus maintaining the structural integrity of tissues. Among the family of collagens, type I has been popularly used as a matrix or platform for cell adhesion and proliferation since collagen forms a fibrous network made up of collagen fibers upon incubation of its aqueous solution at physiological pH and temperature. This thermoinduced gelation allows us to prepare hybrid collagenous tissue into which cells are inoculated. Examples of such are hybrid skin and hybrid vascular graft.18,19 On the other hand, the denatured collagen, gelatin, does not have such thermoinduced gelation characteristics at physiological conditions. Our approaches to chemical insolubilization or structuring of gelatin are based on photochemically20 or thermally21,22 induced sol-to-gel phase transition. The latter method enables in situ hydrogelation utilizing PNIPAM, a thermoresponsive synthetic polymer. For example, our designed thermoresponsive, cell-adhesive macromolecules are PNIPAM partially derivatized with a cell-adhesive core peptide on the side chain, and PNIPAM-grafted gelatin, both of which precipitated at 37 °C and served as thermoresponsive cell-adhesive two-dimensional (2D) matrices when cells are cultured on polymer-coated dishes, and as 3D hybrid tissues when a buffered solution of these polymers is mixed with cells and incubated at 37 °C.21,22 In this study, our interest was extended to the development of a thermoresponsive HA, which behaves as an extended coil under physiological conditions and providing stability and space-occupying structure to the extracellular matrix of all tissues. HA was partially graft-polymerized with PNIPAM. Graft polymerization was achieved by the photoiniferter technique using N,N-diethyldithiocarbamate as the photochemically radical-producing group.15,16 The unique feature of this iniferter-based polymerization technique is that polymerization proceeds in a quasi-living fashion, resulting in the control of chain length. The parameters of the designed molecule, which may influence the thermoresponsive characteristics of this molecule in aqueous solution, include (1) Mn of HA, (2) the degree of grafting of PNIPAM (or graft density on HA molecule), and (3) Mn of PNIPAM. Regarding the DD, it is carried out by condensation between a hydroxyl group of HA and a carboxyl group of a dithiocarbamate derivative (3 or 6) in the presence of a condensation agent, DCC. When excess DCC was employed, intramolecular and intermolecular cross-linking of HA occurred. Therefore, the molar amount of DCC was set to two-thirds of that of the carboxyl group of HA. A higher concentration of 3 and a longer reaction time resulted in a higher DD (Table 1).

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Figure 4. Phase contrast micrographs of bovine ECs on PNIPAM-HA (4) film surface (right) and glass surface (left) after 3 h of incubation (Degree of grafting of PNIPAM is 0.4% per disaccharide unit and Mn of HA and PNIPAM are 5.0 × 105 and 8.4 × 104, respectively).

Figure 5. Scanning electron microscopic images of the precipitate of PNIPAM-HA (4) (right) and freeze-dried, nontreated HANa (left). (Degree of grafting of PNIPAM is 7.5% per disaccharide unit and Mn of HA and PNIPAM are 5.0 × 105 and 8.4 × 104, respectively).

The preliminary experiment using DCB-PEG determined the reaction conditions to realize adequate “quasi-living” polymerization behavior (Figure 1). Under the determined experimental conditions, PNIPAM-HAs, initiated from the derivatized DCA iniferter with an easily hydrolyzable ester bond (acetate group) between the HA molecule and the PNIPAM grafts, were prepared at different monomer concentrations. Mn of the graft chain cleaved from the HA molecule increased linearly with an increase in monomer concentration (Figure 1), suggesting that “quasi-living” polymerization appears to proceed under the appropriate experimental conditions. Regarding graft chain control, in general, the concentrations of the iniferter and the monomer and the photoirradiation time greatly affect the characteristics of photoiniferter polymerization. There was little decrease in Mn of HA molecules upon photoirradiation and also during photopolymerization (data not shown), indicating that little radical-induced or photoinduced degradation occurred within these experimental conditions. As a result, we prepared thermoresponsive PNIPAM-HAs with different degrees of grafting (0.4-11.4% per disaccharide unit) and Mn (ca. 5.0 × 103 to 8.4 × 104) of PNIPAM. PNIPAM-HA was easily purified by dialysis at room temperature. All of the obtained PNIPAM-HAs were soluble in water to give transparent solutions at room temperature. PNIPAM-HA precipitated by heating to about 34 °C and became transparent by cooling, indicating reversible thermoresponsive character. Irrespective of the molecular structural parameters of PNIPAM-HA, such as Mn of HA, the degree of grafting of PNIPAM, and Mn of PNIPAM, LCST was about 34 °C under the present experimental conditions. However, GPC analysis strongly suggests that PNIPAM graft chains tend to aggregate to form a smaller conformation within the narrow temperature range of 32 (LCST of PNIPAM) to 34 °C (LCST of PNIPAM-HA) (Figure 3).

As compared with PNIPAM, a small shift of LCST of PNIPAM-HA to higher temperatures may be due to the swellability of HA. As can be seen in Figure 4, PNIPAM-HA-coated film was non-cell adhesive. PNIPAM-HA could be applied as a tissue adhesion prevention material. Thus, thermoresponsive HA, which is a bioconjugate of biomacromolecules and thermoresponsive synthetic polymers, was developed for use as a non-cell-adhesive matrix. Soon, construction of functional hybrid artificial tissues using a mixture of thermoresponsive HA and thermoresponsive gelatin will be attempted. Acknowledgment. The authors are grateful to Seikagaku Kogyo Co., Ltd. for supply of hyaluronan. This study was financially supported by the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (OPSR) under Grant No. 97-15. References and Notes (1) Krauss, M. C. Semin. Cutaneous Med. Surg. 1999, 18, 119. (2) Balazs, E. A.; Leshchiner, E. A. Proceedings of the Nisshinbo International Conference on Cellulosics Utilization in the Near Future; Elsevier Applied Science Publ.: New York, 1989; p 233. (3) Balazs, E. A.: Bland, P. A.; Denlinger, J. L.; Goldman, A. I.; Larsen, N. E.; Leshchiner, E. A.; Morales, B. Blood Coagulation Fibrinolysis 1991, 2, 173. (4) Giusti, P.; Lazzeri, L.; Lelli, L. Trends Polym. Sci. (Cambridge, U.K.) 1993, 1, 261. (5) Campoccia, D.; Doherty, P.; Radice, M.; Brun, P.; Abatangelo, G.; Williams, D. F. Biomaterials 1998, 19, 2101. (6) Vercruysse, K. P.; Prestwich, G. D. Crit. ReV. Ther. Drug Carrier Syst. 1998, 15, 513. (7) Matsuda, T.; Moghaddam, M. J.; Miwa, H.; Sakurai, K.; Iida, F. ASAIO 1992, 38, M154. (8) Matsuda, T.; Miwa, H.; Moghaddam, M. J.; Iida, F. ASAIO 1993, 39, M327. (9) Heskins, M.; Guillet, J. E. J. Makromol. Sci. Chem. 1968, A2, 1441. (10) Dong, L.; Hoffman, A. S. J. Controlled Release 1991, 15, 141. (11) Yamada, N.; Okano, T.; Sakai, H.; Karikusa, F.; Sawasaki, Y.; Sakurai, Y. Makromol. Chem., Rapid Commun. 1990, 11, 571.

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Biomacromolecules, Vol. 2, No. 3, 2001 863 (18) Weinberg, C. B.; Bell, E. Science 1986, 231, 397. (19) Kobashi, T.; Matsuda, T. Cell Transplantation 2000, 9, 93. (20) Nakayama, Y.; Matsuda, T. J. Biomed. Mater. Res. (Appl. Biomater.) 1999, 48, 511. (21) Matsuda, T. Jpn. J. Artif. Organs 1999, 28, 242. (22) Morikawa, N.; Matsuda, T. Manuscript in contribution.

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