Effect of PEGPLLA diblock copolymer on macroporous PLLA scaffolds by thermally induced phase separation

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    Keywords: Macroporous scaffold; Thermally induced phase separation (TIPS); PLLA; PEG-PLLA diblock copolymer; MC3T3-E1 cell

    from the healthy issues of a patient can be implantedback to him or her without requiring an immunoisola-

    regeneration [16,17].Poly(l-lactic acid) (PLLA) scaffolds are currently

    ARTICLE INtion system. In the system reported here, the cell cultureis related to the scaffold shape because the scaffold playsan important role in the design of the specializedtemporary substrates to be grown [69]. Biodegradableand biocompatible synthetic polymers, such aspoly(lactic acid), poly(glycolic acid), and poly(d,l-lactic

    being tested for hard-tissue cell transplantation, and arefabricated by various techniques, such as porogenleaching/salt leaching, emulsion freeze-drying, expan-sion in high-pressure gas, and phase separation [1822].Recently, thermally induced phase separation (TIPS)and freeze-drying have been used to prepare 3Dmacroporous PLLA scaffolds [2329]. The TIPS meth-*Correspondin

    E-mail addres

    0142-9612/$ - see

    doi:10.1016/j.bio1. Introduction

    The incidence of organ and tissue loss or failure isincreasing steadily, whereas the traditional surgicaltreatment of implantation of a healthy organ from adonor is limited by immune rejection and the number ofdonors. As an application of tissue engineering, the useof cell transplantation is now being investigated as analternative therapeutic strategy for tissue repair andorgan replacement [15]. Transplanted cells cultured

    acid-co-glycolic acid), have been widely utilized as three-dimensional (3D) scaffolds [1012]. Polymeric scaffoldsshould be porous enough to allow a high density of cellsto be seeded, and provide sufcient mechanical stabilityand well-dened networks of interconnected pores topermit ingrowth into the implanted structure [8,13]. Theoptimum pore size of the scaffold differs with the type ofcells or tissue; for example, close to 20 mm for theingrowth of broblasts and hepatocytes [14], 50150 mmfor skin regeneration [15], and 100150 mm for boneAbstract

    A regular and highly interconnected macroporous poly(l-lactic acid) (PLLA) scaffold was fabricated from a PLLAdioxanewater ternary system with added polyethylene glycol (PEG)PLLA diblock using thermally induced phase separation (TIPS). The

    morphology of the scaffold was investigated in detail by controlling the following TIPS parameters: quenching temperature, aging

    time, polymer concentration, molecular structure, and diblock concentration. The phase diagram was assessed visually on the basis

    of the turbidity. The cloud-point curve shifted to higher temperatures with increasing PEG content in the additives (PEGPLLA

    diblocks), due to a stronger interaction between PEG and water in solution. The addition of diblock series (0.5wt% in solution)

    stabilized interconnections of pores at a later stage without segregation or sedimentation. The pore size of the scaffold could be

    easily controlled in the range 50300mm. A macroporous PLLA scaffold was used to study an MC3T3-E1 cell (an osteoblast-likecell) culture. The cells successfully proliferated in the PLLA scaffold in the presence of added PEG-PLLA diblock for 4 weeks.

    r 2003 Elsevier Ltd. All rights reserved.Biomedical Research Center, 39-1, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, South Korea

    Received 19 June 2003; accepted 7 September 2003Biomaterials 25 (20

    Effect of PEGPLLA diblock cscaffolds by thermally

    Hyun Do Kima, Eun Hee Baeb, Ick CJae Do Nama,

    aDepartment of Polymer Science and Engineering, Center

    Suwon, KyungkibRegenbiotech, 945A, 39-1, Korea Institute of Science and Tec

    cg author. Fax: +82-31-292-8790.

    s: dslee@skku.edu (D.S. Lee).

    front matter r 2003 Elsevier Ltd. All rights reserved.

    materials.2003.09.0113192329

    olymer on macroporous PLLAuced phase separation

    n Kwonc, Ravindra Ramsurat Pala,o Sung Leea,*

    dvanced Functional Polymers, Sungkyunkwan University,

    46, South Korea

    gy, P.O. Box 131, Cheongryang, Seoul 130-650, South Korea

    PRESSod involves two thermodynamic processes: (1) nuclea-tion and growth in the metastable region of the phase

  • scaffold. If the solution is quenched whilst in the formerstate, a scaffold with irregular pore size and poorly

    genous PLLA solution was reheated to approximately10C above the expected cloud-point temperature, andthen slowly cooled in steps of 1C, equilibrating thesystem for 10min at each new temperature. The cloudpoint was determined as the temperature at which theclear solution became visually turbid. The gelation pointwas determined by inverting the vial horizontally after ithad been maintained for 10min at a constant tempera-ture, as described in a previous paper [28].

    INterialinterconnected structure will be induced, whereas ifquenching occurs in the latter state, an open, porous,and 3D structure will be induced. The open, porousmorphology can be controlled by several experimentalparameters: the quenching temperature, quenching rate,quenching period or aging time, polymer concentration,ratio of solvent to nonsolvent, molecular structure, andthe addition of porogen [2832]. Nam and Park reportedthat an open porous PLLA scaffold with a pore size of1050 mm resulted from the coarsening process in thelater stages of TIPS in a PLLAdioxanewater ternarysystem [25]. They also found that the addition of aporogen such as Pluronic surfactant (F-127) in the TIPSformulation increased the pore size and controlled themorphology.In previous papers [2830], we reported that the

    PLLAdioxanewater ternary system (dioxane:water at87:13% w/w) could be used to fabricate a highlyinterconnected macroporous scaffold with a pore sizeof 50150 mm through a liquidliquid spinodal decom-position mechanism at room temperature. However, thegelation due to partial crystallization of PLLA pre-vented the development of phase separation in the latestage, restricted the interconnections, and reduced thepore size during the coarsening process. The addition ofporogen to the PLLAdioxanewater ternary systemraised the cloud point and increased the thermodynamicdriving force, resulting nally in an increase in the poresize of the scaffold.In the present study, we investigated the effect of the

    addition of PEGPLLA diblock and PEG on themorphology of PLLA scaffolds. The morphology ofthe scaffold was investigated whilst adjusting TIPSparameters, such as quenching temperature, aging time,and additives and their concentrations. In order todetermine the optimal pore size of the PLLA scaffold,MC3T3-E1 cells were cultured in a series of PLLAscaffolds with different pore sizes, and the number ofcell was counted in a BCA protein assay.

    2. Experimental

    2.1. Materialsdiagram [23], and (2) spinodal decomposition process inthe unstable region [2429]. The thermodynamic state ofthe polymeric solution at the time that the solution isquenched determines the morphology of the resulting

    ARTICLEH.D. Kim et al. / Bioma2320PLLA (Lacty 5000) was purchased from Shimadzu.The number-average molecular weight was 218,000(PDI, Mw/Mn=1.55). 1,4-Dioxane and deionized waterwere a good solvent and nonsolvent for PLLA,respectively. Monomethoxy poly(ethylene oxide)(MPEG, Mn: 2000, 5000, Mw/Mn=1.1, Aldrich) wasdissolved in dried chloroform and then precipitated inn-hexane. l-lactide (Boehringer Ingelheim) was puriedby recrystallization from thoroughly dried ethyl acetateunder a dry nitrogen atmosphere and sublimed beforeuse.PEG-PLLA diblocks (listed in Table 1) were synthe-

    sized from MPEG and l-lactide and characterized asreported in a previous paper [33]. Brief synthesis methodis as follows. MPEO was introduced into a round-bottom, three necked ask with dried toluene. Residualwater in the solution was removed by azeotropicdistillation, resulting in half of toluene solution.Appropriate amount of l-lactide and Sn(Oct)2 wereadded and reuxed under dry nitrogen atmosphere for12 h for the synthesis of PEOPLLA diblock copolymer.After the reaction was completed, the solution wasprecipitated in diethyl ether and then dried undervacuum at ambient temperature for 24 h. Mn wasmeasured by 1H-NMR and GPC.

    2.2. Phase diagram

    The cloud-point curve of the PLLA ternary system inthe presence of PEG or PEGPLLA diblocks wasdetermined by visual turbidimetry. Weighted PLLA (1,3, 4.5, 5.5, or 7wt%) and PEG or PEGPLLA diblocks(0.2, 0.5, or 1wt% in whole solution) were added in a 4-ml-vial with a 1,4-dioxane:water mixture (87%:13%w/w) as the solvent, and then dissolved by heating at63C for 5 h whilst stirring continuously. The homo-

    PRESS

    Table 1

    Diblock copolymers used

    Copolymer Mna

    Diblock1 PEG114PLLA6 5000413

    Diblock2 PEG114PLLA40 50002848

    Diblock3 PEG114PLLA62 50004532

    Diblock4 PEG45PLLA26 20001830

    aNumber-average molecular weight as measured by 1H-NMR.

    s 25 (2004) 231923292.3. Preparation of PLLA scaffold

    PLLA (4.5 or 5.5wt%) solutions containing PEG orPEGPLLA diblocks (0.2, 0.5, or 1wt%) with a mixtureof 1,4-dioxane and water (87:13%w/w) as the solvent

  • 2.6. Morphology characterization

    The macroporous morphology of the scaffolds wasobserved using scanning electron microscopy (SEM,Hitachi S-2400). Fracture-frozen cross-sections of thescaffold were mounted on an aluminum stub coveredwith a carbon adhesive, and then coated with goldparticles.

    3. Results and discussion

    3.1. Cloud-point curve

    Cloud-point temperatures of the PLLAdioxanewater ternary system with various PEGPLLA diblocks

    IN PRESS

    ) Pure PLLA

    terials 25 (2004) 23192329 2321were prepared. Each sample was reheated to 15C abovethe measured cloud-point temperature, and then wasplaced in a water bath preheated to the quenchingtemperature. It was kept for 2, 10, 30, 60, or 120min atthe quenching temperature to observe the coarseningeffect. The annealed sample was directly immersed inliquid nitrogen to be fast frozen for 1 h, and then a smallhole was cut in the vial cap to release the solvents.Freeze-drying was performed in a freeze dryer at 77Cand 7mTorr for 3 days in order to remove solvents andobtain the macroporous scaffolds. The dry scaffoldswere cut into cubes with a surgical blade (78 78mm,thickness 2mm; 1315mg PLLA). Prior to cell seeding,3D scaffolds were prewetted with 70% ethanol for 3 h tosterilize them and enhance their water uptake. Theethanol was removed by soaking with agitation for 1 h insix changes of phosphate-buffered saline (PBS), andthen the scaffolds were left overnight in the media.Finally, the pure PLLA scaffolds were prepared byfollowing similar procedures.

    2.4. Cell culture

    MC3T3-E1 cells (osteoblast-like cells derived frommouse calvaria) were kept in a-minimum essential media(a-MEM, Gibco BRL) containing 10% fetal bovineserum (FBS, Gibco BRL). Cells were trypsinised andsuspended at the concentration of 2 105 cells per ml infresh media. Aliquots of 3-ml cell suspensions(2 105 cells/ml) were seeded onto the top of prewettedscaffolds placed in a 60-mm petri dish. The loadedscaffolds were placed in a humidied 5% CO2 incubatorat 37C, and the medium was changed every 23 days.Cell proliferation on each specimen was determinedafter 1, 3, 7, 10, 14, 21, and 28 days after seeding.

    2.5. Lysis and cell count from BCA protein assay

    The number of cells in the scaffolds was determinedindirectly by using BCA protein assay for total cellularprotein on each alternate culture day. The culturedscaffolds were harvested every other day and washedtwice with PBS. The washed scaffolds were homoge-nized with 500 ml of lysis buffer (60mm TrisCl pH 6.8,25% glycerol, 2% SDS, 1mm PMSF, 1 mg/ml Aprotinin)for 2min on ice. An aliquot of the lysate was measuredfor total protein with a commercial BCA protein assaykit (Pierce, Rockford, IL, USA) according to themanufacturers instructions. After incubation for 2 h,absorbance readings were performed with an ELISA

    ARTICLEH.D. Kim et al. / Biomareader at a wavelength of 562 nm. The amount of totalprotein was extrapolated to cell numbers by comparisonto the total protein of known number of MC3T3-E1cells treated in exactly same conditions except beingloaded in scaffolds.Tem

    pera

    ture

    (C

    30

    40

    50and PEG are shown in Figs. 1 and 2. As described inprevious reports [2830], the cloud-point curve of thepure PLLAdioxanewater ternary system graduallyincreased with increasing polymer concentration at axed dioxane:water ratio (87%:13% w/w). The cloud-point curves of the systems containing PEG5000 ordiblock3 shifted to higher temperatures as the amountof additive increased from 0.2 to 1wt% (Fig. 1).In Fig. 2, the cloud-point curves of solution in the

    presence of various additives (at 0.5wt%) are shifted tohigher temperatures in the following order: PEG5000>diblock1>diblock2>diblock3>diblock4. It is knownthat phase separation is assisted by addition of diblockor triblock copolymer as a surfactant. This assistance isinduced by the amphiphilic effect of the addedsurfactant serving as a nucleus for phase separation. Itis affected by the molecular properties of the additives,including molecular weight, hydrophobic/hydrophilicblock ratio, and block lengths [34,35]. Obviously, an

    60

    70PEG 5000 1wt%PEG 5000 0.5wt%PEG 5000 0.2wt%

    Diblock3 1wt% Diblock3 0.5wt% Diblock3 0.2wt%PLLA Contentration (wt%)0 1 2 3 4 5 6 7 8

    20

    Fig. 1. Cloud-point curves of diblock3 and PEG5000.

  • INterialincrease in PEG content in the diblock copolymer(PEG>diblock1>diblock2>diblock3) raises the cloudpoint to a higher temperature. Increasing the PEGcontent in a diblock copolymer can enhance theinteraction between the diblock copolymer and water,because PEG exhibits stronger solubility in water thanPLLA, so that the cloud-point temperature (liquid

    ARTICLE

    PLLA Concentration (wt%)0 1 2 3 4 5 6 7 8

    Tem

    pera

    ture

    (C)

    20

    30

    40

    50

    60

    70

    Sedimentation boundary

    GelationSediment

    PEG 5000 0.5wt% Diblock1 0.5wt% Diblock2 0.5wt% Diblock3 0.5wt%Diblock4 0.5wt%

    Pure PLLAPEG 5000 0.5wt% gelation pointDiblock2 0.5wt% gelation point

    Diblock3 0.5wt% gelation point Pure PLLA

    A B

    Fig. 2. Cloud-point curves (closed symbols) and gelation-point curves

    (open symbols) of diblocks and PEG. Sedimentation line of pure

    PLLA solution (A) and with additives (B).

    H.D. Kim et al. / Bioma2322liquid demixing) shifts to a higher temperature.A diblock copolymer having the same PEG andPLLA content but smaller size (diblock4) shows aslightly lower cloud-point temperature than diblock3in Fig. 2.As illustrated above, the PLLA solution shows

    gelation at temperatures lower than the cloud pointtemperature. This gelation resulted from the partialcrystallization of PLLA in the polymer-rich phase afterthe liquidliquid phase separation. The pure PLLAsolution having a higher concentration than thesedimentation boundary (o4wt%, dotted line A inFig. 2) shows gelation when it was cooled to below thegelation point [28]. The gelation point graduallyincreased with increasing polymer concentration. Belowthe sedimentation boundary, the polymer solution wasseparated into two layers: a polymer-rich phase and asolvent phase. Fig. 2 shows that the gelation-pointcurves of ternary system with PEG or diblocks shifted toa slightly higher temperature. The addition of diblocksor PEG has only a small effect on the gelation of PLLA,while the cloud-point curve was greatly shifted. Thesedimentation boundary is shifted to a higher polymerconcentration (o4.5wt%, solid line B in Fig. 2) withincreasing PEG content in solution, but this change inthe sedimentation boundary is smaller than the changewhen NaCl is used as an additive [29].3.2. Effect of quenching temperature

    As described above, the porous morphology of thescaffold is determined by the thermodynamic state ofthe solution. The porous morphology was formed by aphase-separation mechanism, such as binodal or spino-dal decomposition. The morphology depended onvarious processing parameters, including quenchingtemperature, polymer concentration, solvent composi-tion, and aging time. In a previous paper [28], anoptimum process condition was 4.5 wt% polymerconcentration at a dioxane:water ratio of 87%:13%(w/w), which has been found to result in a regular PLLAscaffold with a pore size of 50150 mm. On the basis ofthis nding, various PLLA scaffolds were prepared from4.5wt% PLLA in dioxane/water (87:13w/w) withadditives.Fig. 3 shows SEM micrographs of a 4.5wt% PLLA

    solution with 0.5wt% diblock2 made by quenching atvarious temperatures, as a function of aging time. Thephase diagram in Fig. 2 shows that three quenchingtemperatures (25C, 30C, and 35C) are located in theunstable region (probably the spinodal region). There-fore, the interconnected and open porous structure isobserved even at the starting period of phase separation(o10min). The pore size of the scaffold at a quenchingtemperature of 30C increases continuously with agingtime; after 30min the pore size is greater than 150 mm(Fig. 3b).When the scaffold was fabricated at a higher

    quenching temperature (35C, rather than 30C), whichhas a smaller quench depth (9C), pores developedmuch faster. The liquidliquid phase separation is fasterat a higher temperature due to the lower viscosity of thepolymer solution, even though it has a lower drivingforce due to the smaller quench depth. In this case thepore size grew to 200 mm at 30min. At a longer agingtime (120min), the walls between the pores weredestroyed. This seems to represent the nal stage ofthe coarsening process, as shown in Fig. 3c, whichmeans that the coalescence process progresses quickly atlow viscosity. In contrast, when the solution wasquenched at 25C, the pore size and variation weresmaller even though the driving force was larger due tothe larger quench depth. In this case quenching occursbelow the gelation-point temperature, so the partialcrystallization of PLLA reduces the rate of phaseseparation and restricts the growth of pores at a longeraging time (>30min).

    3.3. Effect of additives

    PRESSs 25 (2004) 23192329At the starting period of phase separation, themorphology of the scaffold is determined by the initialthermodynamic driving force which is dependent on thequench depth from the cloud-point temperature. The

  • INterialARTICLE

    10min

    100 m 100m100m

    H.D. Kim et al. / Biomacloud-point temperature is increased by the addition ofdiblocks or PEG5000, as shown in Fig. 2, whichincreases the quench depth for the same quenchingtemperature. Fig. 4 illustrates the effect of diblock1 anddiblock3 on PLLA scaffold morphology with aging timeat a quenching temperature of 30C. The addition ofdiblock1 or diblock3 increases the thermodynamicdriving force relative to that in the pure PLLA solution.This increases the size of interconnected pores for ashort aging time (o2min), but after 10min of aging thephase separation of the pure PLLA solution proceeds tothe later stage of phase separation. After 60min, anirregular macroporous structure was observed due to acoalescence process involving the combining of larger

    (a) (b)

    30min

    60min

    120min

    100m 100m

    100m

    100 m 100 m

    100 m

    Fig. 3. SEM micrographs of scaffolds prepared from a 4.5wt% PLLA dioxa

    of aging time at quenching temperatures of 25C (a), 30C (b), and 35C (cPRESS

    100m

    s 25 (2004) 23192329 2323structures at the expense of smaller structures [36] (asshown in Fig. 4a).The addition of diblocks to a ternary system appears

    to make the porous structure stable even after a longaging time, and prevents the coalescence of the porousstructure and the sedimentation of the polymer-richphase. This is particularly clear for the systems contain-ing diblocks in Figs. 3b, 4b and c. For these systems,regular, open, and well-interconnected macropores wereeasily fabricated in the size range 150200 mm after60min. These amphiphilic diblocks can act like asurfactant, lowering the interfacial tension that isimportant at the later stage of phase separation. At thisstage, the phase-separation kinetics is controlled by the

    (c)

    500 m

    100m

    100 m

    500 m

    ne:water (87%:13%w/w) solution with 0.5wt% diblock2, as a function

    ).

  • INterialARTICLE

    2min

    100m 100m

    H.D. Kim et al. / Bioma2324motion of interfaces driven by interfacial tension [34].The lowering of surface tension could decelerate thephase separation and enhance the stability of the porousstructure and its interconnections. The hydrophilic/hydrophobic ratio of the diblocks can be a major factorin changing the interfacial tension. The addition ofdiblock3 (rather than diblock1) with a low PEG/PLLAratio causes low interfacial tension, making it is easier toobtain the desired pore size and scaffold interconnec-tions. A system with a pore size of 150250 mm and ahigh degree of interconnections was obtained after120min when using diblock3 (Fig. 4).

    10min

    30min

    60min

    120min

    100 m

    100m 100m

    100m100m

    100m 100m

    100m

    (a) (b)Fig. 4. SEM micrographs of scaffolds prepared at a quenching temperature

    diblock1 (b), and 0.5wt% diblock3 (c).PRESS

    100 m

    s 25 (2004) 231923293.4. Effect of molecular structure of additives

    The morphology of scaffolds prepared from thePLLA solution with PEG and diblock4 at the samePEG/PLLA ratio but with a shorter block length areshown in Fig. 5. The scaffolds were prepared from4.5wt% PLLA solution at a quenching temperature of30C. The system with PEG added at 0.5 wt% exhibitedthe largest quench depth (18C) at 30C; it is located at1C above the gelation-point temperature. Within ashort aging time (o2min), the large thermodynamicdriving force produces a large pore size. However, after

    100 m

    100 m

    100 m

    100m

    100 m

    (c)of 30C from a 4.5wt% PLLA solution with no additives (a), 0.5wt%

  • INterialARTICLEH.D. Kim et al. / Bioma10min the pore growth gradually decelerated andstopped during the coarsening process due to gelation,as shown in Fig. 5a.In comparison with diblock3, the molecular structure

    of diblock4 is characterized by a similar hydrophobic/hydrophilic ratio but a shorter block length. As

    2min

    10min

    30min

    60min

    120min

    (a)

    100 m

    100 m

    100 m

    100 m

    100 m

    100 m

    Fig. 5. SEM micrographs of scaffolds prepared at a quenching temperature

    0.5wt% diblock4 (b).PRESSs 25 (2004) 23192329 2325mentioned above, the lower the PEG content and theshorter the block length of additives, the lower thecloud-point temperature. In this case, a smaller drivingforce is produced at the same quenching temperature.The initially small pores increase gradually over a longaging time (3060min), developing into a regular, highly

    (b)

    100 m

    100 m

    100 m

    100 m

    100 m

    of 30C from 4.5wt% PLLA solution with 0.5wt% PEG5000 (a) and

  • interconnected macroporous structure due to thesurfactant effect (Fig. 5b). The pore size is 150300 mm, greater than that of the diblock3 system andof any other system at 30C. The cloud-point tempera-tures of the diblock3 and diblock4 systems are similar.Note that diblock4 contains twice as many molecules asdiblock3 (by comparison of the molecular weights). Itshould be noted that the scaffold pore size can be furtherincreased by additives having the same PEG/PLLAratio and smaller molecular weight. The system contain-ing diblock4 had a stable interconnected structurebetween macropores, with no tendency for segregationor sedimentation even over a long aging time.

    3.5. Effect of additives and PLLA concentration

    The effect of additives and PLLA concentration onthe scaffold morphology was investigated. Scaffoldswere prepared at a quenching temperature of 30C anda 60-min aging time (Fig. 6). As shown in Fig. 6a, thescaffold with 4.5wt% PLLA and 0.2wt% diblock3exhibited an irregular morphology with some closedpores, although it was better interconnected than the

    highly interconnected structure, the amount of diblockadded should exceed a certain critical concentration.When a 5.5wt% PLLA solution with diblock3 was

    quenched at 30C, the pore size increased only slightly(from 80 to 100 mm) with the amount of diblock3 (from0.2 to 1.0wt%; Fig. 6bd). This pore size is smaller thanthat of the 4.5wt% PLLA system (Fig. 4c) due to thehigh viscosity of the 5.5 wt% PLLA solution induced bythe high polymer concentration and partial gelation.

    3.6. Cell culture

    PLLA has been used in hard-tissue regeneration dueto its slow degradation. In this study, the scaffolds madeof PLLA with various pore sizes were investigated forthe application as substrates for osteoblast cell, which isa precursor of bone cell. MC3T3-E1 cells (mouseosteoblast-like cells) are able to proliferate and start todifferentiate to bone cells upon conuency. To verify ifMC3T3-E1 cells could adhere to and proliferate on thescaffolds, we monitored the changes in cell number at 1,3, 7, 14, 21, and 28 days after loading MC3T3-E1 cells tothe scaffolds.

    ARTICLE IN PRESS

    (b

    H.D. Kim et al. / Biomaterials 25 (2004) 231923292326pure PLLA system (Fig. 4a). In addition, the pore sizewas smaller than that of the system with 4.5wt% PLLAand 0.5wt% diblock3 (see Fig. 4c). This indicates that tofabricate scaffolds with a regular, macroporous, and

    100m

    100 m

    (a)(c) (Fig. 6. SEM micrographs of scaffolds prepared at a quenching temperature

    4.5wt% PLLA solution with 0.2wt% diblock3; (bd) 5.5wt% PLLA solutiFig. 7 shows the results of cell proliferation assayusing aforementioned scaffolds with pore sizes from lessthan 100 mm to more than 300 mm. We found that cellnumbers were increased in all ve types of scaffold until

    100 m

    100 m

    )d)of 30C from a PLLA solution with diblock3 after 60min of aging: (a)

    on with 0.2, 0.5 and 1.0wt% diblock3, respectively.

  • 21days and most abundant on scaffolds with pore size of150200 mm. To conrm these optical data, we deter-mined the amount of total protein, which is from cellcontent and extracellular matrix protein produced from

    metabolically active cells, per mg scaffold (Fig. 8). Theamounts of total protein from all ve types of scaffoldwere increased until 28days. The amount of protein wasincreased mostly when cells were cultured on scaffold

    ARTICLE IN PRESS

    3days 7days 21days

    (a)

    (b)

    (c)

    (d)

    100 m 100 m

    100 m 100 m 100 m

    100 m

    100 m100m

    100 m100 m

    100 m

    100 m

    100 m 100m 100m

    H.D. Kim et al. / Biomaterials 25 (2004) 23192329 2327(e)

    Fig. 7. SEM micrographs of cell proliferation in scaffolds prepared from 4

    scaffold, quenching temperature of 20C, after 30min of aging (pore sizeB130min of aging (pore size 100150mm). (c) With 0.5wt% diblock1, quenchi(d) With 0.5wt% diblock3, quenching temperature of 30C, after 60min of

    temperature of 30C, after 60min of aging (pore size 200300mm)..5 wt% PLLA solution as a function of culture days: (a) Pure PLLA

    00mm). (b) Pure PLLA scaffold, quenching temperature of 30C, afterng temperature of 30C, after 60min of aging (pore size 150200mm).aging (pore size 200300mm). (e) With 0.5wt% diblock4, quenching

  • IN

    4Num

    terialwith pore size of 150200 mm and secondly on scaffoldwith pore size of 100150 mm. The amount of proteinwas increased least on scaffold with pore size of 100 mm.These results suggested that the optimum pore size tosupport growth of metabolically active MC3T3-E1 cellsis around 150 mm. We could speculate that this pore sizeis the maximum to hold enough number of cellsinducing cellcell interaction and the minimum tofacilitate material transfer to provide nutrients to cells.

    0 5 10 15 20 25 30

    0

    2Cel

    l

    Culture days

    Fig. 8. Cell proliferation curves from a BCA protein assay.ARTICLE

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    8

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    20Diblock1Pure 30 CDiblock3Diblock4Pure 20 C

    ber

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    4 / P

    LLA

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    H.D. Kim et al. / Bioma2328MC3T3-E1 cells have morphology of adherent bro-blast-like cells when growing as monolayer. However,when reached to conuency with sufcient cell-cellinteraction, they start to differentiate to bone cells.There is high possibility that the scaffold with pore sizeof 150200 mm could be used to induce the differentia-tion of MC3T3-E1 cells to bone. To clear this issue,culturing beyond 28days and assay for differentiationmarker will be performed.

    4. Conclusions

    The addition of amphiphilic diblocks to a PLLAdioxanewater ternary system could provide a newmethod for preparing macroporous scaffolds by TIPS.Using this technique it is possible to fabricate a regularand highly interconnected and macroporous PLLAscaffold without segregation or sedimentation over along aging time (>30min). The pore size of PLLAscaffold was easily controllable from 50 to 300 mm byadjusting the quenching temperature, aging time, poly-mer concentration, and composition and block length ofthe diblocks.Cell proliferation prole was characterized by BCA

    protein assay, PLLA scaffold with size of 150200 mm,

    attachment and transplantation. J Biomed Mater Res 1993;

    Press; 1991.

    [11] Kaplan DL, Wiley BJ, Mayer JM. Biosynthetic polysaccharides.In: Biomedical polymers: designed-to-degrade systems. Munich,

    Germany: Carl Hanser Verlag; 1994.

    [12] Freed LE, Marquis JC, Nohria A, Emmanual J, Mikos AG,

    Langer R. Neocartilage formation in vitro and in vivo using cells

    cultured on synthetic biodegradable polymers. J Biomed Mater

    Res 1993;27:1123.

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    group [2830,37], provides a promising method forcontrolling the pore size of open, regular, and well-interconnected macroporous scaffolds for the growthand culture of cells, when the scaffolds are prepared byTIPS.

    Acknowledgements

    This work was supported by a grant from the KoreaResearch Foundation (no. KRF-2001-005-E00006).

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    Effect of PEG-PLLA diblock copolymer on macroporous PLLA scaffolds by thermally induced phase separationIntroductionExperimentalMaterialsPhase diagramPreparation of PLLA scaffoldCell cultureLysis and cell count from BCA protein assayMorphology characterization

    Results and discussionCloud-point curveEffect of quenching temperatureEffect of additivesEffect of molecular structure of additivesEffect of additives and PLLA concentrationCell culture

    ConclusionsAcknowledgementsReferences

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