Biomacromolecules 2003, 4, 1026-1033
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In Vitro Cytotoxicity of Injectable and Biodegradable Poly(propylene fumarate)-Based Networks: Unreacted Macromers, Cross-Linked Networks, and Degradation Products Mark D. Timmer,† Heungsoo Shin,† R. Adam Horch,† Catherine G. Ambrose,‡ and Antonios G. Mikos*,† Department of Bioengineering, Rice University, MS-142, Houston, Texas 77251-1892, and Department of Orthopaedic Surgery, University of Texas Health Science Center, Houston, Texas 77030 Received February 13, 2003; Revised Manuscript Received April 14, 2003
This study evaluates the in vitro biocompatibility of an injectable and biodegradable polymeric network based on poly(propylene fumarate) (PPF) and the cross-linking agent PPF-diacrylate (PPF-DA). Using a methyl tetrazolium (MTT) assay, the effect of the concentrations of PPF and PPF-DA on the cytotoxicity of its unreacted macromers, cross-linked networks, and degradation products was examined. The influence of network structure properties on cell viability and attachment to the cross-linked material was also investigated. The unreacted macromers exhibited a time- and dose-dependent cytotoxic response that increased with more PPF-DA in the mixture. Conversely, the cross-linked networks formed with more PPF-DA did not demonstrate an adverse response because increases in conversion and cross-linking density prevented the extraction of toxic products. Fibroblast attachment was observed on the PPF/PPF-DA networks with the highest double bond conversions. The degradation products, obtained from the complete breakdown of the networks in basic conditions, displayed a dose-dependent cytotoxic response. These results show that there are concerns regarding the biocompatibility of injectable, biodegradable PPF/PPF-DA networks but also sheds light onto potential mechanisms to reduce the cytotoxic effects. Introduction Considerable research is being conducted in developing multifunctional macromers for biomedical applications. These polymers can form highly cross-linked networks in situ so that they can be used as injectable materials that can fill irregularly shaped cavities or defects. The first of these materials was based on diacrylate and dimethacrylate monomers and was found in dental restorative composites1-3 and tissue adhesives.4-6 More recently, acrylate and methacrylate modified poly(R-hydroxy esters)7,8 and poly(anhydrides)9,10 as well as unsaturated polyesters of fumaric acid11,12 have been developed as in situ forming biodegradable materials. This has expanded the use of these polymer networks as drug delivery vehicles,13,14 tissue barriers,15 cell carriers,16 bone cements,17 and tissue engineering scaffolds.18 For many years now, our laboratory has been investigating the potential of poly(propylene fumarate) (PPF) and its copolymers as an orthopaedic biomaterial. PPF networks are produced by free radical polymerization of the unsaturated groups within the repeating fumaric acid unit. Characteristically different biodegradable networks can be formed by cross-linking PPF with a variety of cross-linking agents that include N-vinyl pyrrolidinone,19 poly(ethylene glycol)dimethacrylate,20 and PPF-diacrylate (PPF-DA).21 We have * To whom correspondence should be addressed. Phone: (713) 3485355. Fax: (713) 348-4244. E-mail:
[email protected]. † Rice University. ‡ University of Texas Health Science Center.
recently been focusing on PPF/PPF-DA networks (Figure 1) because, unlike the other cross-linking agents, PPF-DA is biodegradable. The number of repeating propylene fumarate units in PPF-DA can be modulated to alter the network properties;21 however, the most common form consists of only a single unit. In addition, PPF/PPF-DA network strength and degradation can easily be tailored to desired levels by altering its composition as dictated by the double bond ratio of PPF to PPF-DA.22,23 The PPF/PPF-DA networks have demonstrated suitable physical properties as a material for orthopaedic implants,24 scaffolds for bone tissue engineering,21 and delivery vehicles for osteogenic proteins.25 The next stage in the development of the PPF/PPF-DA system is to evaluate the cytotoxicity of the biomaterial. It is desirable that these networks elicit a minimal cytotoxic response and facilitate cell attachment for their use as a tissue engineering scaffolds. In vitro cytotoxicity testing provides a convenient and reliable method to assess the biological response to a biomaterial and also serves as an initial screening process for future in vivo studies. In vitro experiments can be particularly beneficial for injectable, biodegradable polymer networks because they can individually test all three phases that occur in the material’s life cycle during implantation, namely, its unreacted macromers, the cross-linked network, and its eventual degradation products. In this study, we evaluated the in vitro cytotoxicity of these three phases of the PPF/PPF-DA system to determine how it is influenced by the double bond ratio and therefore the concentrations of PPF and PPF-DA.
10.1021/bm0300150 CCC: $25.00 © 2003 American Chemical Society Published on Web 05/13/2003
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In Vitro Cytotoxicity of PPF/PPF-DA
Figure 1. Cross-linking and degradation scheme of PPF/PPF-DA networks.
In addition to altering the PPF/PPF-DA double bond ratio, we also conditioned the cross-linked networks at elevated temperatures to further manipulate the network structure.26 The macromolecular structure has been shown to influence many of the physical properties of the network,27 but it is not yet understood how it effects biocompatibility. Cytotoxicity and cell attachment to these conditioned PPF/PPFDA networks was also investigated in order to establish this relationship. Methods Polymer Characterization. PPF was synthesized as previously described.12 The molecular weight of PPF was determined relative to polystyrene standards by gel permeation chromatography. The PPF synthesized for this study had a number average molecular weight (Mn) of 1700 g/mol, a weight average molecular weight (Mw) of 2600 g/mol, and a polydispersity index of 1.6. PPF-DA was synthesized as previously described.26 The structure of a single fumarate unit with two terminal acrylate groups was confirmed by the integration ratio of acryl to fumarate protons in the 1H NMR spectrum as previously described.21 Specimen Preparation. Mixtures of PPF and PPF-DA were prepared according to the double bond ratio (Table 1). The double bond ratio (DBR) is defined as the mole fraction of fumarate bonds within the PPF chain to the acrylate bonds in PPF-DA. The polymers were combined by first dissolving each component in methylene chloride. The two solutions were added and stirred for 15 min. The mixture was then
Table 1. Formulation of PPF/PPF-DA Networks Evaluated in This Study PPF/PPF-DA double bond ratio
PPF (g)
PPF-DA (g)
0 0.5 1 2 infinity (∞)
0.00 1.00 1.00 1.00 1.00
1.00 2.08 1.04 0.52 0.00
rotary evaporated to remove the solvent. Prior to crosslinking, 0.5 wt % of the photoinitiator bis(2,4,6-trimethylbenzoyl) phenylphosphine oxide (BAPO) was administered to the polymer in a solution of acetone (0.1 g/mL) and thoroughly stirred. Test specimens were prepared by photo-cross-linking PPF/ PPF-DA in transparent silicone molds as previously described.24 Briefly, molds were formed with a two component room-temperature vulcanizing silicone rubber kit (Silicones Inc, High Point, NC) that was poured over a suspended master form. The master form was an aluminum disk with a 13 mm diameter and 1 mm thickness. Following curing of the silicone, the rubber was cut with a knife to produce the two-part flexible mold. The molds were stored under vacuum and purged with nitrogen prior to their use. PPF/PPF-DA was injected into the molds through a 5 mL syringe equipped with a 20 gauge needle. Once the cavity was filled, the molds were placed in an Ultralum (Paramount, CA) ultraviolet (UV) light box. The molds were positioned roughly 20 cm below four bulbs that provided the majority of light at 365 nm and an intensity of approximately 2 mW/ cm2. The specimens were photo-cross-linked for 30 min and then released from the molds.
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Network Conditioning. The PPF/PPF-DA networks were conditioned at different temperatures to alter the network structure. Specimens were placed in scintillation vials that were purged with nitrogen three times and then sealed with Parafilm. The vials were then stored in a freezer, an environmental chamber, and an oven for conditioning at -20, +37, and +60 °C, respectively, for a three week period. Network Characterization. The method to characterize the structure of PPF/PPF-DA networks is described elsewhere.27 Briefly, the networks were hydrolytically degraded in accelerated conditions (1 N NaOH and 60 °C) to release the unreacted double bonds as their corresponding unsaturated organic acids. Unreacted fumarate and acrylate bonds were released as fumaric and acrylic acid, respectively (Figure 1). The number of unreacted species within the degradation products were quantified by high performance liquid chromatography (HPLC) and compared to those obtained from the hydrolysis of the un-cross-linked linear polymers. Knowing the number of double bonds available for cross-linking (from the linear polymers) and the number of unreacted bonds within the cross-linked networks, the double bond conversion as well as the average molecular weight between cross-links (Mc) was calculated as previously described.27 The sol fraction of the PPF/PPF-DA networks was measured gravimetrically. The disk specimens were immersed in 5 mL of PBS at 37 °C for 24 h. The specimens were removed from the PBS, washed with water, and vacuum-dried. The sol fraction was calculated by the following equation: sol fraction (% ) )
W i - Wd 100 Wi
where Wi represents the initial weight and Wd corresponds the dried weight after immersion in PBS. Cell Culture. A rat fibroblast cell line (CRL1764, ATCC, Manassas, VA) was used in this study. The cell line was cultured on T-75 culture flasks using culture media (Dulbecco’s modified Eagle medium (DMEM, Gibco Life, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum (FBS, Gemini Bio-Products, Calabasas, CA) and 1% (v/v) antibiotics containing penicillin, streptomycin, and amphothericin (Gibco Life)). The cell cultures were incubated at 37 °C, 95% relative humidity, and 5% CO2. Cytotoxicity of Unreacted Monomers. The cytotoxicity of the un-cross-linked polymers was evaluated by extraction of leachable components in culture medium because the linear polymers are insoluble in water. The polymer resins, without photoinitiator, were sterilized by exposure to UV light for 3 h. Un-cross-linked PPF/PPF-DA mixtures of each double bond ratio (DBR) were immersed in culture media (0.2 g/mL) and incubated for two different extraction periods: 30 min and 24 h. Polypropylene pellets (Sun Plastics Inc., Salt Lake City, UT) and latex rubber (Microflex Corp., Sparks, NV) were used as positive (live) and negative (dead) controls, respectively. Following extraction, the media was collected and diluted by 10 and 100 times. Prior to each cytotoxicity test, the cells were harvested at 80-90%
Timmer et al.
confluency with a Trypsin/EDTA solution (2 mL/flask), resuspended at a density of 8 × 104 cells/mL, and seeded into 96 well tissue culture plates (100 µL cell suspension/ well) for a seeding density of 8 × 103 cells/well. The plates were then incubated for 24 h before testing to achieve 8090% confluency within the well. The three different concentrations of extract media for each DBR was then added to the cultured fibroblast cells in the 96 well plates (100 µL/ well), replacing the culture media. The cells were then incubated for 24 h with the extraction media. The relative cytotoxicity of the leachable products was assessed with a methyl tetrazolium (MTT) viability assay. Following the 24 h incubation, the extraction media was removed, the cells were washed with PBS, and 100 µL of MTT solution (1 mg/mL in phenol red free DMEM (Sigma, St. Louis, MO)) was added to each well. The plates were wrapped in tin foil and incubated at 37 °C, 95% relative humidity, and 5% CO2 for 3 h. The MTT solution was then removed, and 100 µL of DMSO/2-propanol 1:1 (v/v) solution was added to dissolve the formazan crystals. The solution in each well was mixed with a pipet, and the absorbance of the solution was measured at 570 nm with an absorbance microplate reader (Powerwave ×340, BIO-TEK Instruments, Winooski, VT). The cell viability was normalized to that of fibroblasts cultured in the culture media without polymer extracts. Cytotoxicity of Cross-Linked Networks. The cytotoxicity of cross-linked PPF/PPF-DA networks conditioned at the three temperatures were also evaluated by an extraction test. The circular disk specimens were washed with PBS, dried overnight, and sterilized with UV light for 3 h. The disks were soaked in culture media with a surface area to fluid volume of 3 cm2/mL and incubated at 37 °C, 95% relative humidity, and 5% CO2 for 24 h. Polypropylene pellets and latex rubber were again used as positive and negative controls, respectively. The extraction media was collected and 100 µL was added to the fibroblast cells cultured in the 96 well plates, which were then incubated for another 24 h. As described in the previous section, a MTT viability assay was utilized to evaluate toxicity. Cell viability was normalized to that of fibroblasts cultured in culture media without extracts. Cytotoxicity of Degradation Products. The cytotoxicity of the PPF/PPF-DA degradation products was carried out by completely degrading the networks and exposing them to the cultured cells. Networks were hydrolytically degraded in accelerated conditions. PPF/PPF-DA disks of DBR 0.5 (5 g) were placed in 50 mL 1 N NaOH solution and stirred at 80 °C under a nitrogen atmosphere. The networks took approximately 7 days to completely degrade. The solution was then filtered through a cellulose acetate membrane filter (0.2 µm pore diameter) and the pH was adjusted to 7.4 with 1 N HCl. The solution was filtered again for sterilization. The degradation solution was then diluted by 2, 10, 50, and 100 times with culture media. Sterile PBS was similarly diluted with media to serve as a control. The solutions were added to the cultured cells in the 96 well plates (100 µL/ well), which were then incubated at 37 °C, 95% relative humidity, and 5% CO2 for 24 h. Wells treated with 70%
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In Vitro Cytotoxicity of PPF/PPF-DA Table 2. Concentrations (mg/mL) of Degradation Components in Culture Media Evaluated in This Studya corresponding dilution of degradation solution component
2×
10×
50×
100×
fumaric acid propylene glycol acrylic acid poly(acrylic acid-co-maleic acid)
N/A 25.00 21.00 39.00
3.50 5.00 4.20 7.80
0.70 1.00 0.84 1.56
0.35 0.50 0.42 0.78
a
Concentrations based upon maximum amounts that can be released from 5 g of PPF/PPF-DA DBR 0.5 in 50 mL.
ethanol and concurrently cultured with the other samples were utilized as a negative (dead) control. Cell viability was assessed utilizing the MTT assay as described in the previous sections and was normalized to the viability of fibroblasts cultured in culture media without the degradation products. In addition to evaluating the degradation solution, the cytotoxicity of the individual degradation components (Figure 1) were assessed as well. Solutions of fumaric acid (Aldrich, Milwaukee, WI), propylene glycol (Acros, Pittsburgh, PA), acrylic acid (Aldrich), and poly(acrylic acid-co-maleic acid) (Mw ) 3000, Aldrich) in culture media were prepared with the concentrations listed in Table 2. These concentrations are based on the maximum amounts of each component that can be released from 5 g of a PPF/PPF-DA DBR 0.5 network and diluted similarly as was done with the degradation solution. Poly(acrylic acid-co-maleic acid), the isomer of poly(acrylic acid-co-fumaric acid), was used because the latter was not commercially available. The highest concentration of fumaric acid was 3.5 mg/mL because it was limited by its solubility in culture media. The solutions were filtered through a cellulose acetate membrane for sterilization. The cytotoxicity of the component solutions was tested similarly as the degradation solutions. Cell Attachment. The PPF/PPF-DA disk specimens were placed in 24 well plates, washed with PBS, and sterilized under UV light for 3 h. Autoclaved stainless steel weight rings (1.885 cm diameter, 1.6 cm height) were placed on top of the disks to ensure that the cell suspension would only have contact with the confined area of the surface.28 Fibroblasts in the T-75 culture flasks were trypsinized and resuspended at a concentration of 132 000 cells/mL. A volume of 250 µL of the suspension was added within the rings onto the polymer surface for a seeding density of 40 000 cells/cm2. The cell suspension deposited in an empty well with a weight ring served as a control. After 12 h incubation at 37 °C, 95% relative humidity, and 5% CO2, the rings were removed and the media was aspirated. The PPF/PPF-DA disks were washed with PBS twice to remove any unattached cells. Adherent cells were enzymatically lifted with 0.5 mL trypsin which was neutralized with 0.5 mL culture media. The suspension was collected and the number of cells was quantified with a Coulter Multisizer (Coulter Electronics, Hialeah, FL). Cell attachment was visualized by staining with toludine blue-O dye. The polymer disks were immersed in 10% neutral buffered formalin solution (Sigma) for 15 min to fixate the cells and then treated with the dye solution (1% w/v). The morphology of the attached cells was visualized
Table 3. Double Bond Conversion of the Conditioned PPF/ PPF-DA Networksa conditioning temperature
PPF/PPF-DA double bond ratio
-20 °C
+37 °C
+60 °C
0 0.5 1 2 ∞
82 ( 2% 65 ( 1% 58 ( 2% 52 ( 1% 22 ( 2%
84 ( 1% 69 ( 1% 63 ( 0% 58 ( 1% 35 ( 1%
86 ( 0% 72 ( 1% 67 ( 1% 61 ( 1% 39 ( 2%
a
Data represent means ( standard deviation for n ) 3.
Table 4. Average Molecular Weight between Crosslinks (Mc) in g/mol for the Conditioned PPF/PPF-DA Networksa conditioning temperature
PPF/PPF-DA double bond ratio
-20 °C
+37 °C
+60 °C
0 0.5 1 2 ∞
218 ( 6 311 ( 7 369 ( 12 443 ( 12 1028 ( 105
214 ( 4 293 ( 3 335 ( 3 393 ( 10 643 ( 15
204 ( 1 271 ( 2 311 ( 7 368 ( 10 569 ( 25
a
Data represent means ( standard deviation for n ) 3.
by phase contrast microscopy (Eclipse E600, Nikon, Melville, NY) equipped with CCD video camera (DXC-950P, Sony). Statistical Analysis. All experiments were carried out in triplicate (n ) 3), and the results are expressed as means ( standard deviation. Data analysis was carried out using JMP v.4 statistical software (SAS Institute, Cary, NC). Single factor analysis of variance (ANOVA) was conducted to assess statistical significance within the data set. Should ANOVA detect significance, a Tukey’s Honestly Significantly Different (HSD) multiple comparison test was used to establish treatment effects. Both tests were carried out with 95% confidence intervals (p < 0.05). Results Network Characterization. The double bond conversion of the conditioned PPF/PPF-DA networks is presented in Table 3. The conversion increased with decreasing PPF/PPFDA double bond ratio (p < 0.05). The conversion also increased with the higher conditioning temperature (p < 0.05) at every double bond ratio. The greatest conversion occurred with those specimens stored at 60 °C for three weeks. The average molecular weight between cross-links (Mc) of the PPF/PPF-DA networks is shown in Table 4. It should be noted that the Mc calculated from the analysis of the degradation products is an underestimate of the true value because it is based upon an ideal network where it is assumed that every polymer chain exists between two distinct networks.27 Mc decreased with decreasing PPF/PPF-DA double bond ratio (p < 0.05) and showed a further reduction with increasing temperature conditioning (p < 0.05). The sol fraction of the conditioned PPF/PPF-DA networks is presented in Table 5. The specimens showed limited weight loss after immersion in PBS for 24 h. The DBR ∞ networks demonstrated a significantly greater sol fraction then the other networks (p < 0.05). Cytotoxicity of Unreacted Macromers. The cytotoxic effects of the leachable products from un-cross-linked PPF/
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Table 5. Sol Fraction (%) of the Conditioned PPF/PPF-DA Networksa conditioning temperature
PPF/PPF-DA double bond ratio
-20 °C
+37 °C
+60 °C
0 0.5 1 2 ∞
0.25 ( 0.22 0.18 ( 0.10 0.34 ( 0.07 0.28 ( 0.07 1.17 ( 0.06
0.30 ( 0.20 0.29 ( 0.19 0.24 ( 0.22 0.13 ( 0.06 0.95 ( 0.33
0.14 ( 0.12 0.09 ( 0.10 0.22 ( 0.23 0.11 ( 0.07 0.58 ( 0.26
a
Data represent means ( standard deviation for n ) 3.
Figure 3. Cell viability after exposure to extract of PPF/PPF-DA networks of varying double bond ratios and temperature conditioning. Error bars represent means ( standard deviation for n ) 3. The symbol “*” represents a statistically significant difference in cell viability between PPF/PPF-DA DBR 2 networks conditioned at -20, +37, and +60 °C.
Figure 2. Cell viability after 24 h exposure to various concentrations of un-cross-linked PPF/PPF-DA polymer mixture extract obtained from 30 min (A) and 24 h (B) extraction periods. Error bars represent means ( standard deviation for n ) 3.
PPF-DA polymer mixtures are shown in Figure 2. For the 30 min extraction period, all double bond ratios showed a dose-dependent effect (Figure 2A). Regardless of the PPF/ PPF-DA double bond ratio, the original (not diluted) extraction medium showed a low cell viability (92% cell viability). The intermediate dilution (10×) exhibited no cytotoxicity for DBR 2 and ∞, whereas it increased for the lower DBR values (p < 0.05). Cytotoxicity increased with the 24 h extraction period (Figure 2B). Both 1 and 10 times dilutions of the extraction
media for all double bond ratios exhibited an acute cytotoxicity with very little cell viability except for the 10× dilution of DBR ∞, which showed 16 ( 1% viability. For the 100× dilution, cell viability ranged from 65 ( 5% to 103 ( 4% with a general trend of decreased viability for the lower double bond ratios. Cytotoxicity of Cross-Linked Networks. The cytotoxic effects to the extracted products from conditioned PPF/PPFDA networks are presented in Figure 3. All conditioned networks for DBR 0, 0.5, and 1 showed no adverse effects on the viability of the fibroblasts, where the minimum viability was 88 ( 1%. The PPF/PPF-DA DBR 2 networks showed a decrease in cytotoxicity with increasing conditioning temperatures (p < 0.05). Those conditioned at the lowest temperature (-20 °C) exhibited a cell viability of 4 ( 3%, which rose to 60 ( 1% when conditioned at 37 °C, and increased further to 70 ( 4% when conditioned at 60 °C. Networks of DBR ∞ demonstrated a high cytotoxicity (
