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INTRODUCTION

INCREASING DEMAND FOR HEALING THERAPEUTICS ad-ministered by minimally invasive surgical techniques

requires the synthesis of new injectable biomaterials. Or-thopedic clinical indications for injectable therapeuticsinclude distal radius and vertebral compression fractures,as well as treatments to enhance fracture healing.1 Ide-ally, scaffolds should support the attachment and prolif-eration of cells and ingrowth of new tissue, biodegradeat a rate in register with tissue healing, and deliver bioac-tive components that promote tissue healing. Two-com-ponent polyurethanes prepared from lysine methyl (or

ethyl) ester diisocyanate (LDI) and polyester and poly-ether polyols have been reported to degrade to non-toxicby-products and to support the migration of cells andgrowth of new tissue both in vitro2–7 and in vivo.4,8,9

The biocompatibility of poly(ester urethane) foams, aswell as the ability to inject the materials as a reactive liq-uid mixture10,11 that cures in situ, promotes their utilityas injectable scaffolds. Two-component poly(ester ure-thane) foams have been prepared for bone and cartilagetissue engineering from LDI, water, and polyester andpolyether polyols.2–7,12 However, in these studies, thesurface chemistry and, in some cases, the reactivity ofthe polyurethanes were not accurately controlled. In this

TISSUE ENGINEERINGVolume 12, Number 5, 2006© Mary Ann Liebert, Inc.

Synthesis and In Vitro Biocompatibility of InjectablePolyurethane Foam Scaffolds

SCOTT A. GUELCHER, Ph.D.,1 VISHAL PATEL, B.S.,2 KATIE M. GALLAGHER, B.S.,3SUSAN CONNOLLY, B.S.,2 JONATHAN E. DIDIER, B.S.,2 JOHN S. DOCTOR, Ph.D.,3

and JEFFREY O. HOLLINGER, Ph.D., D.D.S.2

ABSTRACT

The development of therapeutics for orthopedic clinical indications exploiting minimally invasivesurgical techniques has substantial benefits, especially for treatment of fragility fractures in the dis-tal radius of osteoporotics and vertebral compression fractures. We have designed six formulationsof injectable polyurethane foams to address these clinical indications. The polyurethanes were pre-pared by mixing two liquid components and injecting the reactive liquid mixture into a mold whereit hardens in situ. Porous polyurethane foams were synthesized from lysine methyl ester diisocyanate,a poly(�-caprolactone-co-glycolide) triol, a tertiary amine catalyst, anionic and non-ionic stabiliz-ers, and a fatty acid pore opener. The rise time of the foams varied from 8–20 min. The porositywas approximately 95% and the pores varied in size from 100–1000 �m. The polyurethane foamssupported attachment of viable (�95%) MG-63 cells under dynamic seeding conditions. We antic-ipate compelling opportunities will be available as a consequence of the favorable biological andphysical properties of the injectable polyurethane foams.

1Department of Chemical Engineering, Vanderbilt University, Nashville, Tennessee.2Bone Tissue Engineering Center, Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsyl-

vania.3Department of Biological Sciences, Duquesne University, Pittsburgh, Pennsylvania.This work has appeared in part previously in the abstract form: Guelcher, S.A., Patel, V., Gallagher, K.M., Connolly, S., Di-

dier, J.E., Doctor, J.S., and Hollinger, J.O. Synthesis of polyurethane foam scaffolds for bone tissue engineering. Presented at theAnnual Meeting of the American Institute of Chemical Engineering, Austin, Texas, 2004, Abstract 63a.

study, polyurethane foams were prepared from LDI, wa-ter, polyester polyols, stabilizers, catalysts, and poreopeners. The stabilizer, catalyst, and pore opener addi-tives were incorporated to prepare foams with fast risetimes (e.g., �20 min) and high porosity (e.g., �90%).

The chemical reactions relevant to the synthesis ofpolyurethane foams are shown in Figure 1. LDI reacts withpolyester polyol to form a crosslinked polymer (the gelling

GUELCHER ET AL.

reaction, Fig. 1A) and with water to form carbon dioxidegas, which rises through the mixture to create pores (theblowing reaction, Fig. 1B). The rates of the blowing andgelling reactions must be balanced to yield a stable foam.Triethylene diamine (TEDA), a moderately active tertiaryamine catalyst that catalyzes both the gelling and blowingreactions,11 was selected to avoid the toxicity associatedwith conventional organotin urethane catalysts. Tertiary

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FIG. 1. Schematic of polyurethane foam gelling (a) and blowing (b) reactions.

amine blowing catalysts (e.g., ethanolamine) used previ-ously to prepare poly(ester urethane) foams were found tobe non-toxic in vivo.9 Stabilizers, including sulfated castoroil (an anionic surfactant that is a derivative of naturallipids) and Tegostab8300 (a synthetic polyethersiloxaneblock co-polymer),11,13,14 were added to emulsify the im-miscible LDI and polyol components, as well as to controlthe pore size. As the blowing and gelling reactions con-tinue, the foam rises until the chemical gel point is reached,where the polymer viscosity dramatically increases. Thewindows in the pores must rupture prior to the gel point toproduce a stable, non-shrinking foam. Calcium stearate, adivalent metal salt of a fatty acid, was added as a poreopener to promote the opening of pore windows near thechemical gel point.11,13 The foams were designed to bebiodegradable by using LDI and polyester polyol interme-diates, which have been reported to degrade in vitro and invivo to non-toxic components.4,8,9 Both the polyester polyoland LDI components are low-viscosity liquids (e.g., �700cSt) at room temperature15 and are therefore suitable forprocessing by reactive liquid molding.

MATERIALS AND METHODS

Materials

Methyl 2,6-diisocyanatohexane (lysine methyl ester di-isocyanate, LDI) was purchased from Kyowa HakkoUSA (New York, NY). Stannous octoate, sulfated castoroil, glycerol, and �-caprolactone were purchased fromAldrich (St. Louis, MO). Glycolide was purchased fromPolysciences (Warrington, PA). Tegostab8300 andTegoamin33 were obtained from the Goldschmidt Chem-ical Company (Hopewell, VA). Caspol5004 was receivedfrom Caschem (Rutherford, NJ). Glycerol was dried at10 mm Hg for 3 h at 80°C prior to use.2 �-caprolactonewas dried over anhydrous magnesium sulfate prior to use.All other materials were used as originally received.Polyurethane foams were mixed using a HauschildSpeedMixer DAC 150 FVZ-K (FlackTek, Landrum, SC).

MG-63 human osteosarcoma cells were obtained fromAmerican Type Culture Collection (Manassas, VA). Cellswere cultured in Ham’s F12 and Minimal EssentialMedium (1:1, F12/MEM) supplemented with 10% fetalbovine serum and penicillin/streptomycin, all obtainedfrom Invitrogen (Carlsbad, CA). Phosphate-buffered saline(PBS) was also purchased from Invitrogen. Spinner flasks(100 mL), magnetic stirring bars (0.8 cm diameter � 4 cmlong), and Multi Stir 4 magnetic stirrer were purchasedfrom Bellco Glass (Vineland, NJ). Silicon tubing (no. 13)and silicone stoppers (no. 13) were purchased from ColeParmer (Niles, IL). Kirschner wire (Trochar shaped tip onboth ends, 0.9 mm diameter, 19.1 cm long) was obtainedfrom surgicaltools.com (Mystic, CT). Trypan blue solution

INJECTABLE POLYURETHANE FOAM SCAFFOLDS

and FITC labeled phalloidin were purchased from Aldrich.Live/Dead Viability/Cytotoxicity Kit and CyQuant CellProliferation Assay Kit were purchased from MolecularProbes (Eugene, OR). Medpor medical-grade polyethyl-ene (Porex Surgical, Newnan, GA) and Interpore 200 hy-droxyapatite (Irvine, CA) were used as positive controlsin the cell culture experiments.

Polyurethane synthesis and characterization

Polyester polyol synthesis. A 900 Da polyester triol(P7C3G900) was synthesized from a glycerol starter anda 70/30 (w/w) mixture of �-caprolactone/glycolidemonomers using previously published techniques.15

Briefly, 5.12 g dried glycerol, 31.54 g dried �-caprolac-tone, 13.48 g glycolide, and 45 �L stannous octoate weremixed in a 100 mL flask and heated under an argon atmo-sphere with mechanical stirring to 130°C. After a reac-tion time of 24 h, the mixture was removed from the oilbath. Nuclear magnetic resonance spectroscopy (NMR)was performed with a Bruker 300 MHz NMR (Bellerica,MA) to verify the structure of the polyester triol usingdeuterated dichloromethane (DCM) as a solvent. Dy-namic viscosity was measured using a Brookfield TC500viscometer (spindle #51, Middleboro, MA).

Polyurethane foam synthesis. Polyurethane foamswere prepared by mixing the components in a 10 g cupusing a Hauschild SpeedMixer DAC 150 FVZ-K vortexmixer and casting the reactive liquid mixture into a 120mL paper cup at atmospheric pressure. First, the hard-ener comprising polyol(s), water, catalyst, stabilizer, andpore opener was mixed in a 10 g cup at 3350 rpm for 30s. The appropriate amount of LDI was added and the twocomponents mixed at 3350 rpm for 15 s. The mixturewas then poured into a 120 mL paper cup where it wasallowed to rise. The time required for the mixture tocream (i.e., the time required for the mixture to form agel) was about 45 s after the cup was removed from themixer.

The index (targeted value � 120) is the ratio of iso-cyanate (NCO) equivalents (qNCO) to hydroxyl (OH)equivalents (qOH):

INDEX � 100 �

� 100 � (1)

The hardener comprises all the reactants with hydroxylfunctionality. The total number of hydroxyl equivalentsqOH in the hardener is given by:

qOH � �i

qOH,i � �i

�wpi

i� (2)

qNCO��

i

qOH,i

number of NCO equivalents���number of OH equivalents

1249

where wi is the equivalent weight (g/eq) and pi is the partsper hundred parts (pphp) of component i. The number ofNCO equivalents is given by:

qNCO � � �i

qOH,i (3)

The pphp of LDI (pLDI) are given by:

pLDI � wLDIqNCO (4)

where wLDI � 107.14 g/eq (calculated from manufac-turer’s measurement of free NCO content).

The mass of each component j in the foam formula-tion is given by:

mj � mT (5)

where mT � 3 g is the batch size.Preliminary feasibility experiments established the

ranges for each component. Foams with catalyst (TEDA)concentrations �2 pphp rose slowly and were unstabledue to vent collapse. The pore opener (calcium stearate)was essential for the synthesis of stable foams; when cal-cium stearate was omitted from the formulation, thefoams exhibited extreme (�25%) volumetric shrinkage.Caspol5004 polyol (w � 173 Da/eq), a castor oil-derivedmodifier polyol designed to improve the properties ofurethane coatings, was incorporated in two foam formu-lations to increase the hardness of the foam. However,foams with more than 2 pphp Caspol5004 contained largevoids and coarse (�5 mm) pores.

Polyurethane physical properties. The shape of thefoams, as shown in Figure 2, was a frustum of a right cir-

pj��

j

pj

INDEX�

100

GUELCHER ET AL.

cular cone capped by a spherical segment. The height ofthe truncated cone, hC, was measured versus time to gen-erate the rise profile. The temperature profile was ob-tained by measuring the temperature of the center of thefoam versus time using a digital thermometer. The bulkdensity �B of the foam was calculated from the measuredmass and the volume calculated from the dimensions ofthe foam after 18 h for the geometry shown in Figure 2.16

The bulk porosity �B is related to the bulk density17:

�B � 1 � � � (6)

where �P � 1200 kg m�3 is the specific gravity of thepolyurethane18 and �A � 1.29 kg m�3 is the specificgravity of air.19

Polyurethane foams are susceptible to a phenomenonknown as relaxation, wherein the foam rises to a maxi-mum height and then settles back.20 Foam relaxation Ris defined as:

R � 1 � (7)

where VB(t) is the volume of the foam at time t and VB,max

is the maximum foam volume.

Compression testing. Foams B, D, and E were selectedfor compression testing experiments. Foam C was char-acterized by coarse pores at the bottom of the foam anda gradient in pore size from the bottom to the top of thematerial. Foams A and F were characterized by an ex-cessively broad pore size distribution with many poreshaving a diameter greater than 5 mm. Due to the fact thatsuitable void-free samples (as required by the ASTMstandard) could not be cut from foams A, C, and F, thesematerials were not tested.

Triplicate rectangular cubic samples (�15 mm thick �25 mm wide � 25 mm long) were cut from the foams byremoving the skins at the sides, bottom, and top with asharp razor blade. The core density �C was calculated fromthe measured mass and volume of the cubic foam samplesin accordance with ASTM D3574–01 Test A. The stressrequired to create a deflection of 50% was measured ac-cording to ASTM D3574–01 Test C (compression forcedeflection test).21 The compressive stress at 50% deflec-tion is a measure of the compliance of the material; themore compliant the material, the lower the stress requiredto achieve 50% deflection. After the specimen was con-tacted with the platens, it was compressed to 50% of itsinitial thickness, which varied from 15–19 mm, at whichpoint the displacement was stopped. The force was mea-sured 60 s after the displacement of the upper platen wasstopped. The compressive stress was calculated from themeasured force and cross-sectional area of the sample.Three specimens were tested for foams B, D, and E in ac-cordance with the ASTM standard.

VB(t)�VB,max

�P � �A�P/�B���P � �A

�B��P

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FIG. 2. Geometry of molded polyurethane foams.

Infrared spectroscopy was performed on solvent-castpolyurethane films with a Mattson ATI Affinity 60 ARFTIR-NIR (Madison, WI). The films were prepared bycasting a 1% solution of the polymer in dimethylfor-mamide on a calcium fluoride window at 50°C under vac-uum.

Scanning electron microscopy. SEM was performedto determine the morphology of pores in the foam. Thesamples were cut into cubes (�1 cm3 in volume), at-tached to an SEM stub with double-sided tape, and coatedin gold at three different angles using a Pelco SC-6 Sput-ter Coater (Redding, CA). Samples were examined witha Hitachi 2460N scanning electron microscope (San Jose,CA). Digital images were obtained using Quartz PCI im-age management software (Vancouver, Canada).

Cell culture system and assays

All aspects of cell culture system assays were carriedout using standard aseptic tissue culture techniques.

MG-63 cells were seeded dynamically in 100 mL spin-ner flasks with a magnetic stir bar at 70 rpm and fitted witha size 13 rubber stopper.22 Six Miltex Kirschner wires withtrocar point (11.6 cm long � 0.9 mm diameter) were se-cured in the bottom of each stopper. Polyurethane foams,Medpor polyethylene implant, and Interpore 200 hydroxy-apatite implant were cut into 2 � 5 � 8 mm pieces andsterilized in 70% ethanol followed by overnight drying.Samples were positioned on the Kirschner wires and se-cured with masterflex silicone tubing.

MG-63 cells were added to flasks at a concentrationof 5.5 � 106 cell/flask in 142 mL medium. All mediumwas removed from the spinner flasks on day 1 and sam-ples were rinsed with PBS prior to the addition of freshmedium.

Cell proliferation was determined with the CyQuantCell Proliferation Assay Kit according to the manufac-turer’s instructions, using triplicate samples on days 1

INJECTABLE POLYURETHANE FOAM SCAFFOLDS

and 4 of culture. Fluorescence was measured with aPerkin Elmer HTS 7000 BioAssay Reader and HT SoftV. 2.00 software (excitation 492 nm and emission 535nm; Gilroy, CA). After the assay, samples were rinsedwith distilled water, dried, and weighed.

The Live/Dead Viability/Cytotoxicity Kit was used todetermine cell viability of attached cells on days 1 and 4of culture. Three random areas from one piece of eachfoam and control material were assessed for cell viabil-ity by calculating the percentage of live cells relative tototal cells. To document cell viability, fluorescent imagesusing a Nikon Microphot-SA microscope equipped witha Kodak DC290 digital camera (Rochester, NY) weremade. These images were scored blindly by four inde-pendent observers for the number of cellular aggregatesper field of view, which were defined as clusters of cellswhere not all cells made direct contact with the foam sur-face.

Statistical analysis

ANOVA and the Bonferroni post-hoc test (GraphPadPrism software, San Diego, CA) were used for statisticalanalyses with significance set at p � 0.05. Cell culturedata (i.e., cell numbers and live/dead staining data) weretested for significance based on time (days 1 and 4) andmaterial type interactions between the periods (days 1and 4) and across materials. Cell aggregate data weretested for significance across materials. The compressivestress data were analyzed to determine significance acrossmaterials.

RESULTS

Polyester polyol synthesis and characterization

The number average equivalent weight w–n of the poly-ester triol P7C3G900 was calculated from the composi-tion of the reaction mixture,8 assuming essentially com-

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TABLE 1. POLYURETHANE FORMULATIONS FOR FOAMS A–F

Parts per hundred parts polyol (pphp)Component A B C D E F

P7C3G900 100.0 100.0 98.8 99.3 100.0 100.0Caspol5004 —. —. 1.2 0.7 —. —.Water 3.0 2.5 3.1 3.1 2.9 3.0Tegoamin33 2.9 3.1 3.1 3.0 2.9 2.9Turkey red oil 2.5 2.5 2.5 2.4 —. —.Tegostab8300 —. —. —. —. 1.6 1.0Calcium stearate 3.0 3.5 3.2 3.2 3.0 3.0Index 119 122 120 119 121 121LDI 84.7 79.5 86.7 86.8 85.5 86.4

Dash (—) indicates this component was absent in the formulation. The index was 120 for all six foams.

plete conversion of monomers, which was verified byNMR analysis. The dynamic viscosity of the polyestertriol P7C3G900 was measured to be 590–600 cP at shearrates, varying from 1.2–3.8 s�1.

Polyurethane foam synthesis and characterization

Foam rise. Six polyurethane foam formulations arelisted in Table 1. Foam rise is plotted as the height of thefoam (hC, Fig. 2) versus time in Figure 3. The rise time,defined as the time required for the foam to reach 90%of its final height, was 8 min for the sulfated castor oil-stabilized foams and 20 min for the polyethersiloxane-stabilized foams. The rise times of all the foams arehigher than those for conventional polyurethane foamsmade from aromatic diisocyanates, which typically risein less than 3 min.20 This observation is consistent withthe higher reactivity (about 8 times faster) of aromaticdisocyanates, such as MDI and TDI, and the higher activity of organotin catalysts used in conventionalpolyurethane foam formulations.23 The appropriate risetime for clinical applications depends on the specific in-dication and is expected to be less than 10–15 min.

Isocyanates react vigorously with polyols, resulting ina significant exotherm. In conventional MDI- and TDI-polyurethane foams, the maximum temperature can reach175°C.20 The temperature profile of foam A is plottedversus time in Figure 4. The maximum temperaturereached is 38°C (corresponding to a maximum tempera-ture rise of 15°C), which occurs after the foam hasreached approximately 80% of its final height. The tem-perature then decreases steadily until reaching room tem-perature after the foam has reached its final height andthe reaction is essentially complete. The relatively low

GUELCHER ET AL.

temperature rise of foam A compared to conventionalpolyurethane foams is consistent with the use of less re-active aliphatic diisocyanates and the absence of organ-otin catalysts.

Pore structure. SEM images of void-free sections offoams A-F are shown in Figure 5. The pores range in sizefrom 100 � 1000 �m. All the materials, except foam C,were generally characterized by a uniform pore size dis-tribution from the bottom to the top of the material. FoamC, which contained 1.2 pphp Caspol 5004 castor oilpolyol, exhibited a pore-size gradient characterized bythe presence of coarse pores larger than 10 mm near thebottom surface of the foam.

Occasionally the foams contained large voids as shownin Figure 6. These voids typically formed near the cen-ter of the foams and were characterized by a hairy andstringy appearance similar to that of voids formed by ex-trusion collapse in conventional molded polyurethanefoams.20 Foams A and F exhibited a large number ofvoids and coarse pores having a diameter �5 mm dis-tributed throughout the material. These materials ap-peared to be poorly stabilized, which is believed to re-sult from the low stabilizer concentration relative to thewater concentration. Consistent with the ASTM standard,density and compression tests were performed on void-free sections of the foam. These tests were not performedfor foams A, C, and F, because void-free samples couldnot be obtained.

Density, porosity, and relaxation. The bulk and coredensities, calculated bulk porosity, and relaxation offoams A–F after an 18 h cure (at room temperature) arelisted in Table 2. The bulk densities calculated from Eq6 range from 62–67 kg m�3, and the agreement betweenthe bulk and core densities is reasonable. The Bonferronipost-hoc test indicates no statistically significant differ-

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FIG. 3. Rise profile of polyurethane foams A–F plotted as thepercentage of the final height versus time. Key to symbols: A,filled circles; B, open circles; C, filled triangles; D, open tri-angles; E, filled squares; F, open squares (see Table 1 for for-mulations of foams).

FIG. 4. Profile of temperature (left axis, solid circles) and rise(right axis, solid triangles) for polyurethane foam A.

ences in core density among foams B, D, and E. The cal-culated bulk porosities of the foams ranged from 9–95vol%, which is consistent with the open porous structuredemonstrated by SEM (Fig. 5). The foams reached theirmaximum height after a period of 1–4 h, after which theheight decreased slightly and reached a constant value af-

INJECTABLE POLYURETHANE FOAM SCAFFOLDS

ter 4–8 h. The relaxation of foams A–F after 18 h rangedfrom 4.0–7.5% (Table 2, calculated from Eq 7).

Composition. A representative IR spectrum for foamD measured after 24 h is shown in Figure 7. The absenceof an NCO peak at 2250–2270 cm�1 indicates that there

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FIG. 5. SEM images of polyurethane foams A–F.

is a negligible amount of free NCO.24 The peak at 1739cm�1 corresponds to C � O stretching of non-hydrogenbonded urethane carbonyl groups (1730 cm�1)25 and isconsistent with that previously reported (1723 cm�1)4 forthe adduct of glucose and LDI. There is no peak at1710–1705 cm�1, which implies that hydrogen-bondedurethane groups are absent.25 The asymmetric andbranched structure of LDI is conjectured to hinder hy-drogen bonding. The peaks at 2947 cm�1 and 2860 cm�1

correspond to stretching of asymmetric and symmetricC–H bonds, respectively.25 The broad peak at 3300–3500cm�1 corresponds to stretching of the N–H bond.24 TheIR data suggest that the polyurethane foams have com-pletely cured after 24 h at room temperature.

Compressive stress. Compressive stress data forfoams B, D, and E are shown in Figure 8. According tothe ASTM standard, the foams were compressed to 50%deflection and the stress measured 60 s after the dis-placement was stopped. The compressive stress rangesfrom 2–4 kPa, implying that the foams are soft and weak.

GUELCHER ET AL.

The IR spectrum (Fig. 7) indicates the absence of hy-drogen-bonded urethane groups, implying that there areminimal physical crosslinks imparting stiffness to thefoam. The relatively low compressive stresses (e.g., �10kPa) of the foams are partially attributed to the absenceof hard segment interactions, as well as the high poros-ity (�94%) of the materials.20 The Bonferroni post-hoctest indicates that the compressive stress measured forfoam B differs significantly from those measured forfoams D and E. There is no significant difference in thecompressive stress between foams D and E. Consideringthat there are no statistically significant differences incore density among foams B, D, and E, the differencesin compressive stresses cannot be attributed to differ-ences in density.

Cell attachment and viability

Adherent MG-63 cells on each foam and the InterPorehydroxyapatite and Medpor polyethylene controls werequantitated 1 and 4 days after seeding and cell attach-ment and proliferation were determined (Fig. 9). Ap-proximately 4000-8000 cell/mg material were attached tothe foams 1 day post-seeding and several statistically sig-nificant differences were observed among foam samplesusing one-way ANOVA with Bonferroni post-hoc test.Considering that all foams supported a cell viability ofgreater than 95% (Table 3) on both days 1 and 4 as re-vealed by live/dead viability staining and fluorescent mi-croscopy (Fig. 10), differences in cell numbers on day 1were interpreted as differences in cell attachment. FoamF had greater cell attachment (p � 0.05) than foams B,C, D, E and the controls, but there was no difference incell attachment between foams A and F. Furthermore, nodifference in cell attachment was observed among foamsB, C, D, and E. No correlations between chemical com-position and cell attachment were observed. For exam-

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TABLE 2. DENSITY AND SHRINKAGE OF POLYURETHANE FOAMS

Bulk density Core density Bulk porosity RelaxationMaterial (kg m�3) (kg m�3) (vol%) after 18 h (%)

A 63.9 — 94.8 6.7B 64.0 61.3 � 5.60 94.8 6.1C 62.6 — 94.9 7.5D 66.8 44.2 � 6.80 94.5 6.1E 64.8 69.4 � 13.8 94.7 6.4F 64.4 — 94.7 4.0Interpore 1465 — 53.4 —Medpor 602 — 29.2 —

The bulk density was calculated for each foam from the measured mass and calculated volume. The core density (foams B, D, andE) was calculated from the measured mass and volume of three samples cut from the core of the foams cast for compression testing.The porosities of the hydroxyapatite and polyethylene controls were calculated from Eq 6 assuming bulk densities of polyethylene33

� 850 kg m�3 and hydroxyapatite34 � 3140 kg m�3. The Bonferroni post-hoc test indicated no statistically significant differencesin core density between materials B, D, and E.

FIG. 6. Image of a void in a sample from polyurethane foam D.

ple, foams A and B are similar in composition but dem-onstrated significant differences in cell attachment.

A statistically significant increase (p � 0.05) in thenumber of MG-63 cells on day 4 compared to day 1 wasobserved for foams B and C using two-way ANOVA withBonferroni post-hoc test, which is consistent with cellproliferation (Fig. 9). No statistically significant changeswere observed for the other foams. No correlation wasobserved between chemical composition and cell prolif-eration for foams A, B, C, and D.

Fluorescent micrographs taken on day 1 of cultureshow regions of individual cell attachment and negligi-ble cell aggregate formation for all of the foams (data notshown). On day 4 of culture, differences in MG-63 cellmorphology were observed among foam samples, as de-picted in the fluorescent micrographs of Figure 10.Specifically, foams E and F, both of which incorporatedpolyethersiloxane stabilizers (see Table 1), were ob-served to contain fewer areas of individual cell attach-ment and more cellular aggregates than the other foams.The mean number of cellular aggregates per field ofview � tandard error of the mean was determined foreach material: foam A, 6 � .4; foam B, 5 � 0.5; foam C,4 � 0.5; foam D, 7 � 0.5; foam E, 13 � 0.6; and foamF, 12 � 1.0. The number of cellular aggregates per fieldof view for foams E and F was found to be significantlyhigher (p � 0.001) than that for foams A, B, C, and Dusing one-way ANOVA with Bonferroni post-hoc test.The higher number of cellular aggregates on foams E andF provides a partial explanation of the lack of prolifera-tion observed for these materials.

DISCUSSION

The polyurethane foams of the present study haveporosities greater than 94%, pore sizes ranging from 100

INJECTABLE POLYURETHANE FOAM SCAFFOLDS

to 1000 �m, and, with the exception of foams A, C, andF, a uniform pore size distribution. However, the foamsoccasionally exhibited voids (Fig. 6). The appearance ofvoids in free-rise foams has been attributed to an imbal-ance in the rates of the blowing and gelling reactions,20

causing thinning of the struts and subsequent pore col-lapse prior to gelation of the polymer.

Polyurethane foams of the present study also demon-strated a moderate degree of relaxation, ranging from 4to 7.5%. Excessive relaxation can cause shrinkage of thescaffold after injection, thereby creating gaps in thewound site that compromise healing. A gelling reactionthat is too slow relative to the blowing reaction has beenreported as a source of foam relaxation.20 Addition of anon-toxic catalyst with high selectivity toward the gellingreaction, such as bismuth compounds (e.g., Coscat 83),is anticipated to eliminate the occasional presence ofvoids and to reduce the degree of relaxation.

The in vitro biocompatibility of polyurethane foamswas evaluated using a dynamic culture system in orderto facilitate an even seeding distribution of cells on allsurfaces of the foams.22,26 Polyurethane foams A–F dem-onstrated no signs of cytotoxicity and supported the at-tachment of MG-63 cells in vitro, which is consistent withthe observations of previous researchers.2,4,8,9 Similarly,high cell viabilities were obtained using MC-3T3 cells,a mouse preosteoblast cell line, in the evaluation of foamsof similar chemical composition (unpublished results).This suggests that the high percent viability of cells at-tached to polyurethane foams observed in the presentstudy is not MG-63 cell-specific.

Our results also indicate that the use of polyether-siloxane stabilizer (foams E and F) does not support in-dividual cell attachment and proliferation of MG-63 cellsthrough 4 days in culture. The representative fluorescentmicrographs in Figure 10 illustrate the higher number of

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FIG. 7. Representative IR spectrum of polyurethane foam D.

FIG. 8. Compressive stress measured at 50% deflection forpolyurethane foams B, D, and E.

cellular aggregates and fewer areas of individually at-tached cells on foams E and F as compared to the otherfoams. Fluorescent images taken on day 1 of culture(data not shown) show regions of single cells and neg-ligible cellular aggregation for all of the foams, im-plying that the cellular aggregates seen on day 4 of cul-ture form after cells have attached to the foams. Wesuggest that the incorporation of Tegostab B8300 sta-bilizer (which contains polyethersiloxanes) into thepolyurethane foams results in the formation of a poly-mer surface that is incompatible with long-term cellu-lar attachment, thus leading to decreased regions of in-dividually attached cells and the formation of cellularaggregates. Polyethersiloxanes are block copolymerscomprising polysiloxane and polyoxyalkylene blocks.These molecules adsorb to the surface of the poly(es-ter urethane) to form surfactant monolayers that dom-inate the physicochemical properties of the surface.11,27

Such a change in the chemical composition of the sur-face of the foam could hinder cell proliferation, as wasobserved for foams E and F that were prepared fromTegostab B8300 (Fig. 9).

The number of attached cells per mass of polyurethanefoam is about two orders of magnitude greater than that

GUELCHER ET AL.

attached to either the Medpor polyethylene or Interpore200 hydroxyapatite control materials, which have beenreported to support cell attachment and proliferation.28–32

The higher cell counts observed for the polyurethanefoams are conjectured to result in part from the higherporosity of the polyurethanes (e.g., �95 vol%) relativeto that of the Medpor (29 vol%) or Interpore 200 (53vol%) materials. Differences in the morphology and in-terconnectivity of the pores, as well as the chemical com-position of the materials, could also promote differencesin cell attachment.

CONCLUSIONS

Dimensionally stable (4–8% shrinkage), porous (94–95%) polyurethane foams were synthesized from lysinemethyl ester diisocyanate and polyester polyols. A ter-tiary amine catalyst was added to balance the blowingand gelling reactions, sulfated castor oil and polyether-siloxane stabilizers were added to control the surfacechemistry, and a calcium stearate pore opener was addedto promote dimensional stability and open pores. The risetime was 8–20 min. The size of the pores ranged from100 to1000 �m. The polyurethane foams supported at-tachment of viable (�95%) MG-63 cells under dynamicseeding conditions, and the use of a polyethersiloxanestabilizer was found to be associated with decreased re-gions of individually attached cells and increased cell ag-gregate formation. Polyurethanes reported in this studycan be cast as a two-component reactive liquid mixturethat cures in situ. Considering these desirable properties,polyurethane foams may provide compelling new op-portunities as injectable delivery systems for bone tissue-engineering applications.

1256

TABLE 3. HIGH VIABILITY OF MG-63 CELLS

ON ALL EXPERIMENTAL MATERIALS

Mean % viability � SDMaterial Day 1 Day 4

A 100 � 0.0 99 � 0.4B 98 � 1.5 99 � 0.9C 100 � 0.0 99 � 0.5D 99 � 1.8 99 � 0.5E 99 � 1.1 99 � 0.6F 98 � 3.3 99 � 0.6Interpore 95 � 2.6 96 � 2.0Medpor 100 � 0.0 98 � 1.2

Viability assessed by live/dead viability staining after 4 daysin culture. Mean percentage � one standard deviation of viablecells relative to total cells from two (Medpor, Interpore 200) orthree (foams A–F) random field of view. 200–500 cells werecounted in each field of view.

FIG. 9. Adherent MG-63 cells on each foam and the Inter-pore hydroxyapatite (IP) and Medpor polyethylene (MP) con-trols were quantitated 1 and 4 days after seeding by CyQuantassay. Foams and controls were seeded and cultured under dy-namic conditions in spinner flasks as described in the Methodssection. Cells/mg material (dry weight) were determined byCyQuant assay at day 1 (black bars) and day 4 (gray bars). Dataare the mean of cell number/mg � one standard error of themean for triplicate samples from one experiment. Statisticallysignificant differences (p � 0.05) of attached cells on day 1 forthe foams and controls are indicated by the lowercase letters.A statistically significant increase (*p � 0.05) in the number ofMG-63 cells on day 4 compared to day 1 was observed forfoams B and C. Data were analyzed using one- or two-wayANOVA with Bonferroni post-hoc test.

INJECTABLE POLYURETHANE FOAM SCAFFOLDS 1257

FIG. 10. MG-63 cells show high viability on foams A-F. Live/dead fluorescent staining of MG-63 cells after 4 days in culturereveals viability of �95%. The mean number of cellular aggregates per field of view � standard error of the mean was deter-mined for each material: Foam A, 6 � 0.4; Foam B, 5 � 0.5; Foam C, 4 � 0.5; Foam D, 7 � 0.5; Foam E, 13 � 0.6; Foam F,12 � 1.0. Foams E and F contain a significantly higher (p � 0.001) number of cellular aggregates per field of view as comparedto the other foam materials as determined by one-way ANOVA with Bonferroni post-hoc test. Several cell aggregates are indi-cated by arrows in foams E and F. Scale bars, 125 �m.

ACKNOWLEDGMENTS

This work was funded by the National Institutes of Health (NIH/NIBIB Grant T32EB00424 and NIH/NIGMS Grant R25 GM066943), the Bone Tissue Engi-neering Center at Carnegie Mellon University, andDuquesne University. The NMR spectrometers of the De-partment of Chemistry NMR Facility at Carnegie Mel-lon University were purchased in part with funds fromthe National Science Foundation (CHE-0130903). KMGis supported by research and teaching assistantships fromthe Bayer School of Natural and Environmental Sciences(Duquesne University).

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Address reprint requests to:Scott A. Guelcher, Ph.D.

Department of Chemical EngineeringVU Station B#351604

2301 Vanderbilt PlaceNashville, TN 37235

E-mail: scott.guelcher@vanderbilt.edu

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