In vitro cell response to differences in poly-L-lactide crystallinity

In vitro cell response to differences in poly-L-lactide crystallinity

Ann Park and Linda Griffith Cima* Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, M A 02139

Many different processing techniques are currently being used to produce tissue regeneration devices from polyesters in the polylactide/polyglycolide family. While it is generally well recognized that processing techniques influence bulk mechanical and degradation properties of these materials, the effects on surface properties are relatively less well stud- ied. We thus investigated the effects of processing conditions that are known to change bulk properties, but not composition, on the surface properties of poly-L-lactide (PLLA). Spe- cifically, we investigated the role of bulk crystallinity of PLLA substrates on several physicochemical aspects of the surface and on the attachment, morphology, and differentiated function of cultured primary hepatocytes and growth of 3T3 fibroblasts. We fabricated smooth, clear PLLA films of 13- 37% crystallinity. Glancing angle X-ray diffraction in- dicated that low crystallinity films lacked order in the first 50 A of the surface while relatively high crystallinity films had detectable order in this range. In other aspects, the surfaces of all PLLA substrates appeared identical with XPS, SEM, and

advancing contact angle analysis, but contact angle hysteresis was slightly greater for more crystalline films. Although the physicochemical properties of the surfaces appeared almost identical, we observed differences in cell behavior on less crystalline versus more crystalline films. Hepatocytes formed spheroids on all PLLA substrates, but spheroid formation was faster (24-48 h) on crystalline substrates. Quantitative image analysis was used to assess the average cell area as a function of time in culture, and our data confirm previous reports that retention of differentiated function is inversely related to cell spreading where function was assessed by P- 450 enzyme activity. In addition, the growth rate of 3T3 fibroblasts was lower on crystalline substrates than on amorphous substrates. An important conclusion from this work is that processing techniques that lead to seemingly inconse- quential changes in bulk and surface properties of these polymers may influence biological response. 0 1996 John Wiley & Sons, Inc.

INTRODUCTION

Bioresorbable polyesters in the polylactide and polyglycolide family are attractive materials for tissue regeneration scaffolds because they have a long and fa- vorable clinical record in other surgical applications and offer a wide range of physical properties and degradation rates. Devices for skin, bone, blood vessels, liver, cartilage, intestine, nerve, and esophagus made from these resorbable materials are in various stages of investigation and development as clinical products.'-8 Although the specific needs of each tissue are quite different, many of the same general problems must be resolved in adapting bioresorbable polyesters as scaffolds for these wide-ranging applications. These issues include: development of new processing techniques to make porous three-dimensional devices with controlled architectures; understanding the relationships among device composition, device structure, and the resulting degradation properties; and understand-

* To whom correspondence should be addressed; e-mail: [email protected]

ing and controlling cell-surface interactions, such as adhesion, migration, and differentiation. These issues are inextricably intertwined. Resorption behavior, for example, is governed not only by material composition but also by device dimensions?-" The degradation rate is enhanced by the presence of degradation products. Since gradients in the concentrations of these products inherently develop, creating gradients in degradation rates, device dimension is a variable in predicting long- term resorption behavior.

While the situation is complex for the amorphous polymers in the family, poly-aL-lactide and most polylactide-co-glycolides (PLGA), the situation is even more complex for the crystalline polymers, poly-L- lactide (PLLA) and polyglycolide (PGA). The degree of crystallinity of a sample strongly affects several material properties, most notably the mechanical behavior during degradation. Independent investigations have shown that PLLA samples that initially are amorphous retain mechanical strength under both in vitro and in vivo degradation conditions far longer than samples that otherwise are identical but initially ~rystalline.'~-'~

Journal of Biomedical Materials Research, Vol. 31, 117-130 (1996) 0 1996 John Wiley & Sons, Inc. CCC 0021-9304/96/010117-14

118 PARK AND CIMA

Investigations of the effects of processing on these polymers have focused primarily on bulk mechanical and degradation properties. In the original surgical applications of these materials (sutures, controlled drug delivery devices, and fracture fixation hardware) bulk properties were of prime importance. In tissue engineering applications, bulk properties remain important, but surface properties arguably play a greater role than in previous surgical applications. The process of tissue regeneration is often governed by the interactions of cells with the surface of the device. Thus, the surface properties of these materials-and how they are affected by processing-are also very important to understand.

Processing these polymers by methods that achieve desired bulk properties can alter surface properties in ways that are not anticipated and that may have nega- tive influences on biological outcome. In some cases, the effect on surface properties can be attributed to an obvious step in the processing. For example, in one recent study of bone. regeneration in mandibular defects using poly-L-lactide and polylactide-co-glycolide microbeads produced by a solvent-evaporation technique, the beads failed to induce ossification.'6 Because similar structures and others from similar materials have been reported to result in bone regeneration, this failure tentatively was attributed to the presence of polyvinyl alcohol, a surfactant used in bead manufac- ture, on the surfaces of the beads.I6 Even when no obvious surface-active agents are used during device fabrication, however, the choice of processing technique may influence final surface properties, particu- larly with crystallizable polymers and those with significant levels of residual monomer. Surface mobility of polymer chains induced by increasing the temperature to near the glass transition has been shown to influence interfacial adhesion in generalI7 and to affect protein diffusion across polymer surfaces.'* Since temperatures near glass transition affect the amorphous regions of the crystallizable polymer, it is reasonable to expect the degree of surface amorphousness (or crystallinity) to influence biological interactions with resorbable polyesters.

Because of the strong interest in PLLA and blends of PLLA as materials for tissue engineering, we investigated the effects of processing conditions that are known to change bulk properties-but not composition-on the surface of these materials. Specifically, we investigated the role of bulk crystallinity of PLLA substrates on several physicochemical aspects of the surface and on the biological response of cultured cells. We fabricated films with systematically varied bulk crystallinities and characterized the surface properties of these films by several methods. The growth, morphology, and differentiated function of primary hepatocytes were evaluated over several days in culture. Hepatocyte behavior is highly dependent on the nature

of the cell-substrate interaction^,'^,^^ and there is substantial interest in transplantation of hepatocytes for the treatment of liver disease.21 This system thus serves as a useful model for illustrating the sensitivity of the biological response to what may appear to be inconse- quential choices in processing techniques. The growth rates of 3T3 fibroblasts on amorphous and crystalline PLLA films also were evaluated.

MATERIALS AND METHODS

Polymer substrate preparation

PLLA (M, = 45,000 Da; M,/M, = 1.9) and PLGA (85 : 15 lactide : glycolide monomer ratio; M, = 140,000 Da; M,/M, = 2.1) were obtained from Boehringer In- gelheim (Germany) and Henley Chemicals, Inc. (Mont- vale, NJ) and used as received (<0.1% residual monomer). The same lots of polymer were used in all experiments. Films of 150 pm thickness were solvent cast from fresh 15% (w/v) polymer solutions in meth- ylene chloride into glass petri dishes (34 mm i.d.). Solu- tions were vortex-mixed for 30 s prior to casting. The dishes were covered during solvent evaporation at room temperature for 24 h. PLLA films were subse- quently placed under 0.010 Torr vacuum for at least 48 h and PLGA films for at least one week to remove residual solvent.

Amorphous PLLA films were produced by melting solvent-cast PLLA at 200°C for approximately 30 min in a vacuum oven (125 Torr). The oven was vented with nitrogen and the polymers were quenched by rapidly placing the covered dishes in a shallow ice water bath. The films then were placed under 0.010 Torr vacuum for at least 24 h. Crystalline PLLA films were obtained by annealing amorphous films in a vacuum oven at 70°C and 125 Torr. They then were placed under 0.010 Torr vacuum for at least 24 h. All films were used within two weeks of fabrication and steri- lized under a W lamp for 2 h immediately prior to use.

Polymer characterization

Thermal transitions of polymer films were determined with a Perkin-Elmer differential scanning calorimeter (DSC7) at a heating rate of 10"C/min. Gel permeation chromatography (GPC) analysis was performed in chloroform with a Perkin-Elmer ISS 200 system equipped with Plgel (Polymer Laboratories, Amherst, MA) columns and an LC-30 refractive index detector. The standard consisted of narrow molecular-weight distribution polystyrenes.

Wide-angle X-ray diffraction (WAXD) measurements were performed by the Bragg-Brettano method

CELL RESPONSES TO PLLA 119

with a diffractometer (Rigaku) equipped with a Cu K, (A = 1.54 A) source. Glancing angle X-ray diffraction measurements were made at an incident angle of 0.05'.

Polymer film surfaces were observed at 20 kV with a Hitachi 5-530 scanning electron microscope (SEM). Polymers were sputter-coated with gold (Denton Vac- uum Desk 11) for 65 s at 40 mA prior to examination. X- ray photoelectron spectroscopy (XPS) was performed with a Surface Science Instruments SSXlOO spectrome- ter at the joint Harvard-MIT surface analytical facility. Monochromatic A1 K, (1486.6 eV) radiation was used to probe the core levels.

Advancing and receding contact angles were measured at room temperature by the sessile drop method with MilliQ water using a Ramk-Hart 100-00 goniome- ter. Underwater contact angles were measured in an optically clear chamber using octane and air as the probing fluids.

Surface free energies were calculated using both sets of contact angle data. By combining an empirically derived equation of state with Young's equation, Neu- mann et a1.22 determined the following relationship:

(0.015 YSV - 2.00) + YLV cos e, = YLV(O.015 %x/ - 1)

where 8, is the measured advancing contact angle of the liquid with the surface, yLv is the liquid/vapor interfacial tension, and ysv is the unknown solid/vapor interfacial tension.

From the underwater air-octane contact angle data, surface free energies were calculated using Young's equation for both air and octane

YSV = YSW + YVW COSeair Y S L = Ysw + YLW C O S ~ o c t

and dividing the surface free energies into additive polar and dispersive component~,2~,~~ for example:

Ysv = Y E V + .ydsv.

Using the harmonic mean approximation for the solid- liquid interfacial tensions

4( y:vVydLv ) - 4( p Y ! v ) $v + Yfv Ysv + $v

Y S L = Ysv + yLv -

Ysw = ysv + yvw - 4 ( l 4 V r " v W ) - 4( $3; Y b + 7 4 w Ysv + Yvw '

where yvw = 72.1 erg/cm2, y& = yLw = 50.5 erg/cm2, Y$W = YLV = SV = 21.6 erg/cm2, and yEv = 0 erg/cm2 at room temperature, we solved for ygv and .ydsv.

Hepatocyte isolation and culture

Primary hepatocytes were obtained from adult Lewis rats (weight -200 g) by collagenase perfusion and purified by isodensity Percoll (Pharmacia Biotech

AB, Uppsala, Sweden) ~entrifugation.'~,~~ Hepatocytes were cultured on amorphous and crystalline PLLA substrates and on control 35 mm tissue culture petri dishes (Falcon 3001) coated with a monolayer (1 pg/cm2) of Type I ~ollagen.'~ The polymer films were rinsed with 2 mL phosphate buffered saline (PBS) pH 7.4 and 2 mL complete culture medium immediately prior to seeding cells on the films. The cells were seeded at a concentration of 30,000 cells/cm2. Viability at plating was at least 80%, as determined by trypan blue exclusion. Cells were cultured in supplemented, chemically defined, serum-free Williams' medium E (Gibco, Grand Island, NY ).I9 The medium was changed daily.

Cell viability in situ

A dual label live/dead viability/cytotoxicity assay (Molecular Probes, Eugene, OR) was used to determine the viability of cultured cells. The assay uses two fluorescent markers: ethidium homodimer, which pene- trates damaged membranes of nonviable cells and am- plifies red fluorescence (>600 nm) 40X upon binding to nucleic acids in the nucleus, and calcein AM, which becomes incorporated in viable cells, producing a uni- form green fluorescence (530 nm) in live cells.

Hepatocyte number determination

The number of cells in 2-3 representative dishes was determined at 24 h intervals during the culture period. The population of hepatocytes in a dish at each given time point was heterogeneous because cells detached from the polymer films as the culture progressed. De- tached hepatocytes comprised a mixture of single cells and cell aggregates, or spheroids. Each group of cells in the dish-attached and detached-was characterized with respect to number and viability. The viability assay consistently showed that almost all detached cells in spheroids and attached cells were viable whereas all detached single cells were nonviable. The total number of viable cells in each dish reported at each time point thus includes cells that had detached in spheroids in the culture medium and cells attached to the substrate.

To count the cells attached to the surface, the substrate was rinsed once with warm PBS and attached cells were removed with 0.05% trypsin-EDTA (Gibco) and counted with a hemacytometer. Cells that had detached in spheroids were counted by replating the spent medium containing unattached cells in a collagen-coated well of a 24-well tissue culture plate (Falcon 3047) filled with 0.5 mL fresh medium. After 24 h of incubation, all spheroids had attached to the surface and many cells had migrated out from the spheroids. Single cells were observed floating in the

120 PARK AND CIMA

medium. The viability assay confirmed that most cells (>90%) that readily attached to the adhesion-promot- ing collagen-coated surface were viable and that all floating single cells were nonviable. The attached cells were removed with trypsin and counted with a hemacytometer as described above.

Hepatocyte function

The rate of serum albumin secretion from cultured hepatocytes was determined by a sandwich enzyme- linked immunosorbent assay (ELISA) using polyclonal antibodies specific for rat albumin (Organon Teknika Corporation/Cappel, Durham, Samples of the medium were collected daily for analysis. Albumin secretion is reported per million viable cells, including attached and detached cells, counted on the day the sample was collected.

P-450 enzymatic activity was determined by a modified fluorescence assay.27 Cells were removed from the plate with a cell scraper, homogenized, and stored at -70°C in a microfuge tube until the day of the assay. NADPH and 7-ethoxycoumarin were added to the microfuge tube to initiate the conversion of 7- ethoxycoumarin to 7-hydroxycoumarin, a fluorescent molecule. The rate of product formation was measured with a fluorescence spectrophotometer (Perkin-Elmer 650-10M) with excitation at 390 nm and emission at 440 nm. Protein content of the cell homogenate was quantified by a bicinchoninic acid (BCA) method using bovine serum albumin (1 mg/mL) as the standard (Sigma Chemical Company, St. Louis, MO).

Quantification of hepatocyte morphology

The average area per cell attached to the culture substrate was assessed by measuring the total area of the substrate covered by cells and dividing by the total number of cells attached to the substrate. Image analysis to determine average cell area was conducted with customized LabVIEW 2 (National Instruments, Austin, TX) and CONCEPT VI (Graftek Imaging, Mystic, CT) software developed by Engineering Technology Cen- ter (Mystic, CT). Five to eight fields (0.625 mm X 0.80 mm = 0.50 mm2) were marked on 2-3 samples of each substrate. The fields were marked on the bottom of the dish so that the same fields could be viewed at each time point. The microscope (Nikon inverted microscope Diaphot-TMD), to which a video camera (Hitachi Denshi, Ltd., model KP-MlU, Research Preci- sion Instruments Co., Wayland, MA) and computer were connected, was mostly enclosed in a flexible plas- tic chamber. A heater equipped with a thermocouple maintained the microscope stage temperature at 37.5”C, and COP flowed through the chamber. The pH of medium in a 35 mm petri dish in the incubated

chamber did not change during the time required to obtain image data (approximately 20 min).

The light and dark scales of each field were adjusted to optimize contrast between the cells and the surface. The program traced the regions of the field occupied by cells and determined the area of these regions in pixel number. (Total pixels per field = 640 X 480 = 307200.) The number of attached cells was determined as described above, and the area per cell was calculated in square microns.

3T3 fibroblast culture and number determination

Cells were removed from 75 mm2 tissue culture T- flasks (Corning) by incubating in 1 mL 0.05% trypsin- EDTA for 5 min and suspended in 10 mL Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% bovine calf serum (Hyclone, Logan, UT) and 1 % penicillin/streptomycin (Gibco). 3T3 cells were seeded on amorphous and crystalline PLLA substrates and control 35 mm tissue culture polystyrene (TCPS) dishes at a density of 25,000 cells/cm2. The polymer films were rinsed with 2 mL PBS and 2 mL complete culture medium immediately prior to seeding. The medium was changed daily.

The number of cells attached to 2-3 representative dishes was determined at 24 h intervals during the culture period. The substrate was rinsed once with warm PBS, and attached cells were removed with 0.05% trypsin-EDTA. Cells were counted with a particle counter (Coulter Multisizer 11) equipped with a 100 pm diameter orifice.

BAE cell culture and number determination

BAE cells were harvested from a T-flask and seeded on solvent-cast PLGA, aged PLGA substrates, and TCPS control dishes at a density of 25,000 cells/cm2, as described above. Cells were cultured in complete DMEM for five days. The number of cells attached to 2-3 representative dishes was determined at 24 h intervals with a particle counter, as described above.

RESULTS

Polymer characterization

Differential scanning calorimetry was used to quantify the degree of crystallinity of the melt-processed polymer films. Figure 1 shows a thermogram of an amorphous PLLA film that exhibits a glass transition at 56°C and an endothermic melting temperature of 175°C. In addition, it exhibits an exothermic crystallization peak near 105”C, which represents heat released


70.0

5 2 . 0 ' I i I i I I 50 .0 75.0 100.0 125.0 150.0 175.0 20

temperature ("C)

Figure 1. DCS thermogram of melt-processed PLLA

as crystals formed during the scan. The difference between the heat of crystallization and the heat of fusion of the sample divided by the estimated heat of fusion for 100% crystalline PLLA provides the degree of crystallinity of the initial sample. The heat of fusion of an infinitely large pure PLLA crystal was calculated to be approximately 87 J/g.28 The melt-processed PLLA films were 13% 2 2% crystalline. PLLA quenched at a rate of 200"C/min by the DSC exhibited 10% +- 2% crystallinity, indicating that our method of quenching was suitably rapid. These films served as the "amorphous" substrates for our studies.

To obtain crystalline PLLA films, amorphous films were annealed at 70°C. Figure 2 shows the relationship between degree of crystallinity and annealing time.

xn I I

1 0 1 ' I ' I ' I ' 1 ' 1

n in 20 30 40 50

annealing time (11)

Figure 2. films as a function of annealing time (T = 70°C).

Degree of crystallinity of melt-processed PLLA

.o

The maximum degree of crystallinity of pure PLLA was 67%, consistent with a value of 70% previously reported in the 1iterat~re.I~ Films exhibiting bulk crystallinity greater than 40% were opaque while less crystalline films were clear. Since opacity prevents the use of light microscopy to monitor cell morphology, we used clear films that were 37% crystalline (annealed 10 h) as the "crystalline" culture substrates. PLLA previously has been reported to undergo substantial degradation during thermal proce~sing,~~-~' and differences in molecular weight of PLLA substrates have been reported to affect cellular responses to these mate- r i a l ~ . ~ ~ We thus characterized annealed samples by GPC and found that PLLA did not degrade during the annealing process: the average molecular weight and the polydispersity of the polymers remained constant during annealing.

To determine if order was present near the surface of amorphous and crystalline PLLA films, we used glancing angle X-ray diffraction, a technique that takes advantage of the small penetration depth of the evanescent X-ray waves at incident angles lower than the critical angle of a materia1.33,34 Based on parameters such as the electron density of the polymer and the wavelength of the X - r a y ~ ? ~ , ~ ~ the critical angle for PLLA was calculated to be about 0.15". Measurements were made at an incident angle of 0.05", which corsesponds to a penetration depth of approximately 50 A. Figure 3 shows that amorphous PLLA has a low degree of order in the bulk that disappears near the surface. Crystalline PLLA exhibits a greater degree of order in the bulk, as expected. More important, however, this order is retained in the top 50 A of the film surface.

The surfaces of amorphous and crystalline PLLA films were characterized by several additional mor-

122

(a) amorphous PLLA

PARK AND CIMA

5 i - t - - ~ . - c - 3 - * / - - - - t - l -C-r*h---+---*-t--+--4..----,

10 15 5 l o 15 20 25 30 35 L - I ' : L . . . . ~ . ! . . . , , . -

20 2 5 10 15 40

bulk surface

(b) crystalline PLLA

i: i- . . . . ' . . . . : . . . . . , . . . : - . < . . . . ' . . . i . . . . ~ . . . . : . . . . :----I I 10 15 20 25 30 I S 40 5 t o 15 20 25 30 15 .I c

bulk surface

Figure 3. Bulk and surface (50A) X-ray diffraction of (a) amorphous and (b) crystalline PLLA films.

phological and chemical techniques. SEM revealed no obvious features on the surfaces of the amorphous and crystalline films at up to 6000X magnification, indicating that the surfaces were smooth on the scale of about 100 nm. Higher magnification causes charging and distortion of polymer samples. Additionally, the gold coating on the polymer may mask roughness on the order of about 10 nm, the approximate thickness of the gold film. XPS, which probes 50-100 A into the sample showed that there was no difference

in chemical composition between the amorphous and crystalline films (62% C, 38% 0), indicating that no contamination (e.g., Si or C1) or change in surface composition resulted from annealing the polymer. Finally, advancing contact angles, 8,, on the amorphous and crystalline PLLA films were found to be identical; however, receding contact angles, 8,, on the two substrates were statistically different (Student's f test for the equality of two means at significance level a = 0.05) (Table I). The crystalline films exhibited slightly greater


TABLE I Advancing and Receding Contact Angles on Amorphous

and Crystalline PLLA Films*

Advancing Angle Receding Angle ~~

Amorphous PLLA 74" 2 3 64" t 4 Crystalline PLLA 74" -c 3 61" t 4

"Data represent the average of 20 measurements made on at least six different polymer films (mean 5 standard deviation).

hysteresis (Oa-Or). Hysteresis may stem from surface energy heterogeneities or from surface roughne~s.~~-~O Experimental and theoretical analysis of contact angle hysteresis on heterogeneous surfaces shows that hysteresis increases as the size of the heterogeneity increases and decreases as the size of the heterogeneity de~reases,4~,~~ disappearing as the size of the heterogeneity becomes smaller than 1 The surface of amorphous PLLA is fairly homogeneous, consisting mostly of amorphous regions but also of some crystalline regions. As amorphous PLLA is annealed, new crystals nucleate and grow, and existing crystals grow larger. Thus the increase in contact angle hysteresis we observe with increasing crystallinity may be due to larger surface inhomogeneities. The crystal microstruc- tures commonly formed by PLLA, spherulites, are typi- cally hundreds of microns in and thus substantially greater than the 1 pm transition where hysteresis effects become important. Surface roughness on the scale of 0.5 p also results in contact angle hysteresis;37,42 however, roughness of this scale was not observed in our samples.

Cell interactions

Hepatocyte attachment and morphology

Hepatocytes were cultured on amorphous and crystalline PLLA films and control collagen-coated dishes. The attachment efficiency of cells 24 h after seeding ranged from 70-80% on all substrates, which is consistent with results reported in the l i terat~re . '~ ,~~ The morphology of hepatocytes on all PLLA surfaces under- went dramatic evolution during the five-day culture period in contrast to cells maintained on collagen- coated polystyrene controls. The general pattern of morphological evolution was as follows. Twenty four hours after seeding, the attached cells adopted a morphology similar to that of cells cultured on tissue- culture polystyrene [Fig. 4(a,b)l. The cells then began to spread out and form networks, or webs, of connected cells in a monolayer [Fig. 4(c)l. Again, this morphology was similar to that observed on collagen-coated polystyrene controls. After 48 h, cells gradually began to lose their adhesivity for PLLA, allowing adhesive inter-

actions between the cells to dominate [Fig. 4(d-e)]. They began to contract and eventually formed hem- ispheroidal structures with diameters of 50-200 pm [Fig. 4(f,g)]. A fraction of these then detached from the surface and floated around as viable spheroids or spheroid aggregates in the culture medium.

Cell morphology was observed to evolve significantly faster (by 24-48 h) on the crystalline polymers than on the amorphous polymers. Spheroids, the ulti- mate morphological structure of hepatocytes on these polymers, formed at a higher rate on crystalline films than on amorphous films. The percent of total viable cells that were present in spheroids on days 3 through 5 in each dish containing PLLA surfaces varied from experiment to experiment (1-33%); however, the percentage was always greater for cells cultured on crystalline films, compared to amorphous films, within any particular experiment. This difference in rate of morphological development was qualitatively very obvious and consistent. To quantify these differences in morphology, we used image analysis to determine the average area per attached cell throughout the five-day culture period.

The quantitative results, shown in Figure 5, corre- lated well with the qualitative morphological observations represented by Figure 4(a-g). Twenty-four hours after seeding, cells on both polymers have similar mor- phologies and also similar areas [Fig. 4(a,b)l. Between days 1 and 2, the cells begin to spread into networks, thus resulting in an increase in area per cell on both substrates. Between days 2 and 3, cells on the amorphous films continue spreading [Fig. 4(c)] and, indeed, their area increases. Cells on the crystalline films, however, appear to begin to contract and lose adhesivity for the substrate [Fig. 4(d,e)l, and the area per cell decreases slightly. Between days 3 and 4, the area per cell decreases dramatically for cells cultured on both substrates as the cells contract and form hemispheroi- dal structures. Finally, on day 5, most attached cells are in hemispheroids [Fig. 4(f,g)l, so the average area per cell is not statistically different for cells cultured on the amorphous or the crystalline films. The final cell area of about 800 pm2 is approximately the projected area of rounded hepatocytes in suspension.

These morphological changes are consistent with the changes in the number of cells attached to each substrate over the 5-day culture period (Fig. 6). Note that Figure 6 shows the number of cells attached to the substrate and does not include floating cells, which were removed daily. Cells cultured on control collagen- coated dishes increased slightly in number, as previously reported under these culture condition~.",~~ However, the number of cells attached to the PLLA films decreased with culture time. This number was significantly lower (a = 0.05) for cells cultured on the crystalline films on the third day of culture. Since a greater percentage of viable cells are in spheroids (i.e.,

124 PARK AND CIMA

Figure 4. (c-e), and 120 h (f,g) (phase contrast, original mag. 1OOX).

Primary rat hepatocytes cultured on amorphous (a, c, f) or crystalline (b, d, e, g) PLLA films for 24 h (a,b), 72 h


120 -

I00 -

80 -

60 -

40 -

20 -

,":"F , , , , , 1 0

0 I 2 3 4 5 6

day

Figure 5. Average projected area of hepatocytes attached to amorphous and crystalline PLLA films as a function of culture time. Each point represents the average of 27-34 fields observed on 6 different polymer films in 2 separate experiments (i.e., 2 different cell isolations). The difference in average area of cells cultured on the two polymers was statistically significant on days 3-4 (Student's t test for the equality of two means at significance level a! = 0.05)

not attached to the substrate) on crystalline films than on amorphous films at any time, lower cell numbers on the crystalline films are expected. However, the reduction in percent initial cell number is due not only to spheroid formation but also partly to cell death. Our

O j , , , , , , , ~, , , I 0 I 2 3 4 5 6

day

- - t - + amorphous PLLA

d- cry?talline PLLA

collagen control

Figure 6. Percent of initial cells attached to PLLA films and control collagen-coated polystyrene petri dishes as a function of culture time. Each point represents the average of 2-3 plates for all culture substrates from 2-4 experiments (mean 2 standard deviation). The difference in cell attachment to the two polymers was statistically different on day 3 of culture (Student's t test for the equality of two means at significance level a! = 0.05).

data show that a greater number of cells detach from and die on the crystalline films.

Spheroids form when anchorage-dependent cells preferentially anchor to themselves rather than to the surface. For example, hepatocytes have been found to form spheroids when cultured on nonadherent surfaces, such as positively charged polystyrene (Prima- ria, Falcon)46 and polyhydroxyethyl metha~rylate,4~,~~ or when cultured in suspension on a rotating plat- form.49 The fact that spheroids tended to form at a greater rate on the crystalline films indicates that cells lose affinity for the crystalline films more rapidly than for the amorphous films. If a cell losing affinity for its substrate is adjacent to other cells, it may survive by anchoring to them to form tight cell-cell aggregates, or spheroids. Production of an extracellular matrix, required for cell survival, has been demonstrated within spheroid^.^^,^' However, if the cell is relatively isolated and loses affinity for the substrate, it may simply detach and die via apoptosis.

Apoptosis, or programmed self-destruction of cells, recently has been linked to integrin occupancy and ~ i g n a l i n g . ~ ~ , ~ ~ Death of anchorage-dependent primary epithelial and endothelial cells cultured on nonadherent surfaces has been attributed to a p o p t o s i ~ . ~ ~ , ~ ~ Tumor cells also have been reported to undergo apoptosis when seeded at low density because of inhibited cell- cell contacts.55 Although we have not determined that cell death on PLLA films is occurring by apoptosis, we consistently observe that isolated, detached cells are nonviable while aggregated, detached cells are viable. These results are consistent with the general patterns of apoptosis observed in many cell types.

Hepatocyte function

Through integrin occupancy, signaling, and cy- toskeleton organization, cell shape is thought to play an important part in the expression of differentiated function of cells. Cell shape has been found to correlate with hepatocyte function under some culture conditions.20 Cells that are more spread tend to lose tissue- specific function whereas cells that are rounded are able to retain function. Indeed, hepatocytes cultured in spheroids have been shown to exhibit enhanced and prolonged differentiated function compared to cells cultured in mono layer^.^^-^* Because of their superior retention of function, spheroids recently have been investigated for use in extracorporeal bioartificial livers.%

We investigated two markers of hepatocyte differentiated function, albumin secretion and P-450 enzymatic activity, and obtained results consistent with these previously reported observations. The rate of albumin secretion was measured for each day of culture and nor- malized for cell number. The highly spread cells on collagen showed loss of albumin secretion over the five-day culture period whereas the less-spread cells

126

2500000 -

3T3 fibroblast growth

I

PARK AND CIMA

120 -

100 -

80 -

60 -

40 -

20 -

- - t - + amorphous PLLA

+ crystalline PLLA

collaycn control

Figure 7. Albumin secretion rate per million cells cultured on PLLA films and collagen-coated polystyrene petri dishes. Each point represents the average of 3 plates for all culture substrates from 2-4 experiments (mean 2 standard deviation). Cells cultured on the amorphous and crystalline polymer films showed no statistical difference in albumin secretion (Student's t test for the equality of two means at significance level (Y = 0.05).

on the PLLA films showed high levels of albumin secretion. Similar secretion rates were found for cells on the crystalline films and on the amorphous films (Fig. 7).

Cytochrome P-450 enzymatic activity also was used to evaluate liver cell function. P-450 activity is ex- tremely labile in ~ u l t u r e ~ ~ ~ ~ ~ and generally is regarded as a more sensitive measure of cell function than is albumin secretion. Because of the prohibitively large cell numbers required for this assay, P-450 activity was measured only on day 3 of the culture, when the greatest differences in cell morphology were observed on the amorphous and crystalline polymers. Cells cultured on the crystalline polymers, which had a lower average spread area, exhibited greater activity than cells cultured on the amorphous films (Table 11).

TABLE I1 P-450 Enzymatic Activity of Hepatocytes on PLLA Films

72 h after Seeding,

Avg Area P-450 Activity Per Cell (pmol product/hr/mg Attached (pm? protein) Cell #

Amorphous 1666 1040 ? 66 158000 PLLA

Crystalline 1261 PLLA

1797 t 173 8500

*Data represent the average activity of cells from three plates of each polymer (mean 2 standard deviation).

To determine if other cell types were sensitive to differences in PLLA crystallinity, 3T3 fibroblasts were cultured on amorphous and crystalline PLLA films and control TCPS dishes for five days. Fibroblasts showed similar morphology on all substrates but showed a slower rate of growth when cultured on crystalline PLLA films, as shown in Figure 8. The doubling times of fibroblasts on TCPS, amorphous PLLA films, and crystalline PLLA films were calculated from exponen- tial fits of the data to be 16.2 h, 16.7 h, and 18.2 h, respec- tively.

DISCUSSION

Despite the widespread interest in degradable polyesters as materials for tissue regeneration devices, relatively few systematic studies of how changes in bulk properties affect the surface-sensitive biological response have been performed. Several studies have il- lustrated that chemical composition of polymer (i.e., amount and stereospecificity of each lactide and glycolide monomer) affects cellular response in vitro1y,30,54 although differences in cell response were attributed to differences in composition without specific characterization of surface properties, such as texture and chemical makeup.

We found that the biological responses of two very different cell types to PLLA are sensitive to differences in bulk structure that are not associated with measurable differences in bulk or surface chemical composition. These differences in cell response are thus appar-

20 40 60 80 100 120

h r h i n culture

- -0 - - t inue d t u i e poly\tylene & aniorphou\ PLLA & cry\lalline PLLA

Figure 8. Number of 3T3 cells attached to PLLA films and TCPS dishes. Each point represents the average cell number on 2-3 plates for all culture substrates from 2 experiments (mean 5 standard deviation).


ently due to changes in surface structure that occur in the annealing process concomitant with the increase in bulk crystallinity. The effect of annealing on surface structure at the scale relevant to cell interaction (-1 nm) is difficult to ascertain because most surface analysis techniques probe more deeply into the material. Indeed, we found no differences by XPS or SEM. The differences in crystallinity we found near the surface (50 A) by glancing angle X-ray diffraction do not rule out possible enrichment at the top 5 A of the surface by the high-energy amorphous phases, but they suggest at least some additional order in the crystalline films in this range. The most sensitive surface method we used was contact angle goniometry. Al- though the advancing contact angles on PLLA substrates were independent of bulk crystallinity, hysteresis was greater for annealed substrates, indicating that the heterogeneity of the PLLA surfaces increases with increasing bulk ~rystal l ini ty .~~,~~ This increased heterogeneity arises from the reorganization of polymer molecules, which may be associated with the crystallization of the polymer molecules upon annealing; however, the precise nature of the change in heterogeneity is unknown. Atomic force microscopy (AFM) may yield insights about the surface structure, but results for amorphous polymer surfaces can be difficult to interpret. A recent AFM study of degradable polymers, which focused on the surface morphology during degradation of poly(sebacic anhydride) (PSA) and PSA/PLLA blends, reports resolution comparable to that of vacuum-based SEM for polymers.55 The advantage of using AFM in such studies is that it can be conducted on aqueous samples during degradation. PSA and PSA/PLLA blends were solvent cast and exhibited a significant degree of spherulitic microstructure at the surface. We also have observed spherulitic microstructure on the surface of solvent-cast PLLA films using SEM. The surfaces of the melt-processed "crystalline" films used in this study were featureless at high resolution, and because of their relatively low crystallinity (37%), there was not a clear motivation for an AFM study of these surfaces.

One possible explanation for the differences in observed behavior is that the crystalline and amorphous substrates release degradation products at different rates, and that these degradation products affect cell behavior. We feel this is unlikely because several pub- lished studies of PLLA degradation report no loss of mass (and little or no change in M,) over a week and even longer in aqueous s o l ~ t i o n . ' ~ ~ ' ~ ~ ~ ~ Furthermore, the pH of the culture medium remained constant during our experiments, and no morphological changes in the substrates were observed during the culture period.

Sensitivity of cell behavior to the crystallinity of the substrate has been observed in other systems. Adhe- sion and spreading of A6 kidney cells on calcium (R,R)- tartrate tetrahydrate crystals were markedly different

on the two different faces of the crystal: cells attached to the (011) faces within 10 min but required more than 24 h to attach to the (101) face^.^^,^^ The faces were chemically identical and differed only in structure and stereochemical organization, for example, orientation and distribution of water molecules and charge. Fur- thermore, some cell lines showed different adhesion and spreading behavior on the (011) face of the (R,R) crystal and the corresponding face of the 6,s) enanti- omer, which are mirror images of each other, indicating that cells were sensitive to the atomic level structural difference of the chirality of the substrate^.^^

The measurable differences in cell behavior on substrates with little measurable difference in surface properties (i.e., slight difference in contact angle hysteresis) may be interpreted in the context of previous studies of cell spreading and growth as a function of the substrate surface free energy." Although caution must be exercised in predicting any type of cell behavior based on one physicochemical property of the material surface, the relative degrees of cell spreading on a range of nonhydrogel polymer substrates and on glass exhibited a sigmoidal relationship with respect to the substrate surface free energy, with a steep transition from round to spread as surface free energy increased in the range of 53-57 erg/cm2. This relationship was found to hold in the presence and absence of serum." We calculated the surface free energy of PLLA to be 50-51 erg/cm2 (Table 1111, placing these materials near the steep transition region of the curve. Because the transition occurs over such a small range of surface free energies, we might expect to see very different cell spreading and morphological patterns on surfaces that have relatively similar free energies.

Annealing amorphous PLLA increases its crystallinity but also promotes physical ageing of the glassy regions of the polymer, which results in increased density and brittleness of the polymer.65 Physical ageing has been observed in quenched amorphous PLLA polymers at room temperature66 as manifested by an endothermic peak near the glass transition temperature of the polymer, which arises as a result of relaxation effects. Physical ageing is a function of the

TABLE I11 Surface Free Energies of PLLA Films Calculated from

Underwater Contact Angle Measurements*

YEV Y4v Ysv 8 Air 8 Octane (erg/ (erg/ (erg/

(Degrees) (Degrees) cm2) cm2) cm2)

Amorphous 68 5 4 97? 4 14 37 51

Crystalline 69 2 3 98 2 3 14 37 51 PLLA

PLLA

*Data represent the average contact angles of at least 12 measurements from at least 3 different films (mean 2 standard deviation).

128 PARK AND CIMA

molecular weight of the polymer, ageing time, and temperature, and can occur below the glass transition temperature because short-range motions result in molecular rearrangements that try to drive the system toward thermodynamic equilibrium. In our system, the amorphous and crystalline films were fabricated from the same polymer, and the ageing times were identical. However, the ageing temperatures were different during the annealing period of 10 h (25” for amorphous PLLA, 70” for crystalline PLLA). This difference in temperature may result in immeasurable differences in the structure of the amorphous regions of the amorphous and crystalline polymers, which may have an influence on cell behavior since the surface contains mostly amorphous regions. To determine if ageing of amorphous regions of the polymer had a measurable effect on cell response, we compared BAE cell growth rates on solvent-cast PLGA films and on films that had been thermally aged for 10 h at fifteen degrees above the glass transition temperature (65”C, 125 Torr) to mimic the PLLA annealing conditions. We used PLGA because it is inherently amorphous and thus allowed us to exclude any effects of crystallinity on cell response. The attachment efficiency and growth rates of cells on aged and unaged PLGA films were identical, indicating that in this system, ageing the polymer for 10 h above the glass transition temperature did not influence cell behavior.

Finally, our studies confirm and extend previous observations*’ that the level of expression of specific hepatocellular functions in vitro correlates inversely with the degree of cell spreading for cells anchored to substrates. The level of cytochrome P-450 activity was 75% greater in cells cultured on crystalline versus amorphous substrates (measured on the third day of culture) when the average area per cell on crystalline substrates was 25% lower. Although the rate of albumin secretion per cell (Fig. 7) also appears to follow this trend (i.e., greater function on crystalline substrates on which cells are more rounded), the differences seen in Figure 7 are not statistically different. This is not surprising because albumin secretion is only a gross indicator of biological response of hepatocytes; drastic changes in cell-substrate interactions are required to effect a change in albumin secretion rates. In one study, Mooney” coated dishes with varying densities of ECM molecules (e.g., laminin) and determined projected hepatocyte areas and albumin secretion. For an increase in projected area of hepatocytes of 170%, cells showed a 20% loss of albumin secretion. In our experiments, the morphological differences were not as great (cells on amorphous substrates were at most 140% more spread); hence, the difference in albumin secretion might be expected to be less than 20%, a difference that would not be statistically distinguishable based on the error of the albumin assay itself. For this reason, we quantified P-450 enzymatic activity, a much more

sensitive indicator of hepatocyte response. As with many aspects of cell behavior, the influence of the substrate is often exerted through changes in cell shape. Significant differences were noted in the spreading of cells on the amorphous and crystalline substrates at certain time points, and this was reflected by a significant difference in the activity of P-450 enzymes. Bind- ing of ECM adhesion receptors to ECM proteins is known to activate intracellular signaling and this may contribute to the results observed previously.20 In this study, no exogenous ECM proteins were added to the system (although hepatocytes are known to secrete ECM proteins in culture). This system thus offers independent confirmation of the relationship between cell shape and cell function.

CONCLUSIONS

We have demonstrated that primary hepatocytes and 3T3 fibroblasts are sensitive to differences in the bulk crystallinity of PLLA substrates. Although it is difficult to establish the precise nature of the differences in surface properties between the amorphous and crystalline substrates with standard surface analytical techniques, the greater contact angle hysteresis of crystalline films may reflect differences in surface free energies of the amorphous and crystalline films. A general conclusion from this work is that processing techniques that lead to seemingly small changes in bulk and surface properties of these polymers may influence cell behavior; thus, processing variables must be carefully considered and reported when designing devices for tissue regeneration with these materials.

This work was supported by an NSF Presidential Investi- gator Award to L.G.C., and by the Procter and Gamble Com- pany. We thank Professors Robert Cohen and Gregory Rut- ledge for helpful discussions about polymer surface analysis, Drs. Kaoru Sano and Matthias Kaufmann for assistance with hepatocyte isolations, Mioko Hirakata and So0 Young Kim for technical assistance, and Professors Paul Laibinis and Robert Langer for use of their analytical facilities.

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Received February 24, 1995 Accepted September 9, 1995

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In vitro cell response to differences in poly-L-lactide crystallinity