lable at ScienceDirect

Biomaterials 29 (2008) 4195–4204

Contents lists avai

Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

Sequential growth factor delivery from complexed microspheres for bonetissue engineering

F. Buket Basmanav a, Gamze T. Kose b, Vasif Hasirci a,*

a Middle East Technical University (METU), BIOMAT, Department of Biological Sciences, Biotechnology Research Unit, 06531 Ankara, Turkeyb Yeditepe University, Faculty of Engineering and Architecture, Department of Genetics and Bioengineering, Istanbul, Turkey

a r t i c l e i n f o

Article history:Received 28 April 2008Accepted 9 July 2008Available online 8 August 2008

Keywords:Bone tissue engineeringComplex microspheresBone morphogenetic proteinsSequential delivery

* Corresponding author. Tel.: þ90 312 2105180; faxE-mail address: vhasirci@metu.edu.tr (V. Hasirci).

0142-9612/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.biomaterials.2008.07.017

a b s t r a c t

Aim of the study was to design a 3D tissue-engineering scaffold capable of sequentially delivering twobone morphogenetic proteins (BMP). The novel delivery system consisted of microspheres of poly-electrolyte complexes of poly(4-vinyl pyridine) (P4VN) and alginic acid loaded with the growth factorsBMP-2 and BMP-7 which themselves were loaded into the scaffolds constructed of PLGA. Microspherescarrying the growth factors were prepared using polyelectrolyte solutions with different concentrations(4–10%) to control the growth factor release rate. Release kinetics was studied using albumin as themodel drug and the populations that release their contents very early and very late in the release studywere selected to carry BMP-2 and BMP-7, respectively. Foam porosity changed when the microsphereswere loaded. Bone marrow derived stem cells (BMSC) from rats were seeded into these foams. Alkalinephosphatase (ALP) activities were found to be lowest and cell proliferation was highest at all time pointswith foams carrying both the microsphere populations, regardless of BMP presence. With the presentdoses used neither BMP-2 nor BMP-7 delivery had any direct effect on proliferation, however, theyenhanced osteogenic differentiation. Co-administration of BMP enhanced osteogenic differentiation toa higher degree than with their single administration.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Bone defects are caused by a variety of routes including trauma,congenital deformity or pathological deformation. The currentlyused approaches for the treatment of these defects include use ofbone substitutes and grafts [1,2]. However, certain disadvantagessuch as donor site morbidity, limited donor tissue availability forautografts and risk of donor pathogen transmission, rejection dueimmunogenic response have led the scientists to focus on theemerging interdisciplinary field of biomaterials and tissue engi-neering as an alternative treatment approach. Developingimplantable cellular constructs to enable and/or accelerate repairand regeneration of bone tissue at the defect site is a major goal ofbone tissue engineering.

Use of mesenchymal stem cells (MSC) in bone tissue engi-neering is preferred because of reduced immunoreactivity [3–6],resistance to low oxygen conditions [7], rapid proliferation and,especially, high differentiation potential under the influence ofcertain bioactive agents [8,9]. BMP are regulatory moleculesinvolved in skeletal tissue formation during embryogenesis,growth, adulthood, and healing [10] and are known to be very

: þ90 312 2101542.

All rights reserved.

important regulators of proliferation and osteogenic differentia-tion of the MSC [11] and it was demonstrated that their with-drawal during differentiation cascade can result in the loss of theosteogenic differentiation potential [12]. BMP-2 and BMP-7 aretwo critical biosignalling molecules with various roles in naturalbone regeneration cascade. The proliferative roles of BMP-2[13,14], and BMP-7 [15,16] on bone marrow derived MSC arecontroversial. However, they have been demonstrated to enhancebone regeneration in various in vitro [15,16], in vivo [17,18]and clinical studies [19]. In a specific study BMP-2 when added tothe proliferation medium increased the osteogenic activity ofMSC [20].

Controlled delivery is the process of delivering certain mole-cules at a determined rate achieving their prolonged availability inaddition to providing protection for the bioactive agent whichmight otherwise be rapidly metabolized. Since tissue formationand repair is a complex cascade of events in which a number ofgrowth factors are involved, controlled delivery of combinations ofgrowth factors from scaffolds appears to be a logical strategy inmimicking nature in applications such as tissue engineering. Thishas been employed by various researchers in a number of potentialtissue engineering applications such as in the case of simultaneousdelivery of insulin-like growth factor-1 (IGF-1) and transforminggrowth factor-h1 (TGF-h1) for repair of injured cartilage tissueemploying the water soluble polymer hydrogels, oligo(poly-

Table 1Foam samples and their compositions

Sample Code Composition

F 4% PLGA foamF-4 4% PLGA foamþ 5 mg 4% microspheresF-10 4% PLGA foamþ 12 mg 10% microspheresF-4&10 4% PLGA foamþ 5 mg 4% microspheresþ 12 mg 10% microspheres

F. Buket Basmanav et al. / Biomaterials 29 (2008) 4195–42044196

(ethylene glycol) fumarate) encapsulating gelatin microparticles[21]. Another is the case of sequential delivery of BMP-2 and TGF-b from glutaraldehyde crosslinked gelatin layers for the purpose oftissue engineering [22]. The two major challenges in growth factordelivery from tissue engineering scaffolds are the selection ofproper growth factor cocktails and fine control of the relationshipbetween concentration and timing. The significance of enhancedosteogenic effect with multiple growth factors has been wellestablished by a comprehensive study which demonstratedthat certain bone morphogenetic proteins with very low or noosteogenic activity exhibited strong osteogenic activity when co-expressed in stem cells [23]. In various studies enhanced boneformation upon co-administration of growth factors was reported.For instance, enhanced bone formation was observed with appli-cation of TGF-b1 and IGF-1 simultaneously [24]. Similarly, TGF-b3and BMP-2 loaded alginate scaffolds had significantly enhancedbone formation in mice in comparison to single growth factorloaded scaffolds [25]. However, as mentioned earlier, selection ofthe right combinations of growth factors is crucial and they mightnot always yield positive results; there are cases where decreasedbone formation was reported when BMP-2 and bFGF were co-administered from collagen sponges [26]. It has been suggestedthat multiple growth factor use can also be cost effective byyielding better results with reduced amounts of growth factorutilization when compared to much higher doses of singlegrowth factors required for comparable results [25,27]. Variousstrategies are being employed in the design of 3D scaffoldscapable of delivering multiple growth factors in a controlledmanner and preferentially with different release kinetics becauseit could be more advantageous in comparison to their simulta-neous delivery. This concept was demonstrated with sequentialdelivery of BMP-2 and IGF-1, respectively, from bilayered gelatincoatings which led to accelerated and enhanced osteogenicdifferentiation of BMSC in comparison to their simultaneousdelivery as well as sequential delivery in the reverse order [22].Although direct loading into the scaffold structure is a widelyused approach in multiple growth factor delivery [26,28], it doesnot provide a means to control the relative release rates of thegrowth factors for a sequential delivery. Alternative strategies toimprove this situation can, therefore, be employed such as in thecase of bilayered coatings with different release rates from thetop and bottom layers [22] or growth factor release from oli-go(poly(ethylene glycol) fumarate) hydrogels with a faster releaseof TGF-b1 directly from gel phase and a slower release of IGF-1from gelatin microparticles embedded in the gel thus enablingthe control over relative timing and concentration of the growthfactors in the healing tissue [29]. Delivery from scaffoldembedded micro or nanoparticles could overcome the disad-vantages of direct delivery from scaffolds which have poorcontrol over release rates due to the open pore structure andexposure of the growth factors to the medium leading to a loss ofgrowth factor bioactivity. It has previously been demonstratedthat release kinetics from microsphere-loaded scaffolds could beadjusted by using microspheres prepared under different condi-tions and attaining different release behaviors [30]. In the samestudy it was suggested that delivery of multiple growth factorswith different release kinetics could be achieved by preparingscaffolds which encapsulate mixed microsphere populations andthus promising precise control over relative release kinetics. Thedelivery system in the present study was based on such anapproach which to the best of our knowledge has not beenpreviously employed for sequential delivery of two differentgrowth factors. Among the emerging approaches is the manu-facture of scaffolds from fused microparticles leading to differentrelease behaviors. A recent study has reported fusion of PLGAmicrosphere populations displaying different release kinetics into

3D scaffolds which can sequentially deliver IGF-1 and TGF-b1 forcartilage tissue engineering [31].

The purpose of this study was to sequentially deliver BMP-2 andBMP-7 via a controlled release system and to investigate in vitrowhether it is possible to improve osteogenic activity of MSC. TheBMP were entrapped in microspheres of polyelectrolyte complexesof alginic acid and poly(4-vinyl pyridine) (P4VN) in order to achievethe encapsulation under very mild conditions since alginic acid canbe easily crosslinked in an aqueous Caþ2 solution thus avoiding theuse of organic solvents and any other chemical treatments that mayharm and reduce bioactivity of the growth factors. The kinetics ofrelease from the microspheres was studied by microBradford usinga model protein, bovine serum albumin (BSA) to represent thegrowth factors. Growth factor loaded microspheres were thenintroduced into scaffolds of poly(lactic acid-co-glycolic acid)(PLGA), a FDA approved and widely used scaffold material inbone tissue engineering studies. The microsphere loaded foamswere then seeded with bone marrow stem cells. The proliferativeand differentiative effects of BMP-2 and BMP-7 on BMSC werestudied by Alamar Blue and Alkaline Phosphatase assays,respectively.

2. Materials and methods

2.1. In situ studies

2.1.1. Microsphere preparationMicrospheres were prepared by complexation of polyelectrolytes and cross-

linking with CaCl2 according to a modified version of an earlier study [32]. P4VN(MW 150,000–200,000, Polysciences, USA) was dissolved in 1:1 dioxane/water (4, 6,8, 10%, w/v). Alginic acid (Sigma–Aldrich Co., USA) and BSA (Fluka Biochemica,Switzerland) as the model protein for BMP were dissolved in distilled water (4, 6, 8,10%, w/v). P4VN and Alginate–BSA solutions of the same concentrations were mixedin 3:1 volume ratio, respectively, and then dropwise added to the CaCl2 solution andstirred for 1 h. The microspheres were washed three times with distilled H2O andthen were freeze dried for 8 h.

2.1.2. Encapsulation efficiencyProtein contained in the microspheres (2 mg) was determined by extraction

with 1:1 EtOH/water solution followed by Coomassie Plus� Protein Assay (Pierce,USA).

2.1.3. Protein release studiesMicrospheres (4 mg) were incubated in 1 mL of PBS (0.01 M, pH 7.4) and the

medium was changed daily. The supernatant was tested with Coomassie Plus�Protein Assay (Pierce, USA).

2.1.4. Preparation of PLGA foams loaded with microspheresThe approach used in microsphere entrapment in foams was similar to some

previously reported procedures [33,34] except that no chemical crosslinking wascarried out after particle loading. PLGA (50:50) (RG 503, Boehringer–Ingelheim,Germany) was dissolved in dioxane (4%, w/v). P4VN–Alginic acid microsphereswere placed into glass molds (1 cm diameter), PLGA solution (300 mL) was pouredon top, the suspension was frozen at �20 �C overnight and freeze dried for 8 h toyield disc shaped foams with entrapped microspheres. The pore size distributionof PLGA scaffolds was determined by mercury porosimetry (METU Central Labo-ratory). Samples prepared for characterization studies are listed in Table 1.

2.1.5. MicroscopyStereomicrographs of freeze dried microspheres and microsphere loaded PLGA

foams were obtained by Nikon SMZ 1500 (Japan). For Scanning Electron Microscopy,microspheres and microsphere loaded PLGA foams were gold coated under vacuumand SEM micrographs were obtained by a SEM (JSM 6400, JEOL, Japan).

Table 2Microsphere loaded foams used in in vitro studies

Sample code Foam type BMP content (ng)

F-4(�) F-4 –F-4 (2) F-4 BMP-2 (32)F-10(�) F-10 –F-10 (7) F-10 BMP-7 (32)F-4(�)&10(�) F-4&10 –F-(4)2&10(7) F-4&10 BMP-2 (32)þ BMP-7 (32)

Table 3Kinetic analysis of release from microspheres

Polymer concentration(%, w/v), crosslinkingduration (min), crosslinkingtemperature (�C)

Release models and release parameters (k and r2)

Zero order First order Higuchi

k0 r2 k1 r2 kH r2

4, 60, 25 0.0866 0.9106 0.1656 0.7155 0.383 0.97484, 30, 25 – – – – 0.427 0.98354, 60, 4 – – – – 0.672 0.98916, 60, 25 0.0617 0.9247 0.1291 0.7306 0.314 0.98278, 60, 25 0.0873 0.9038 0.2438 0.8191 0.312 0.955610, 60, 25 0.0404 0.9097 0.1439 0.8209 0.154 0.969810, 30, 25 – – – – 0.167 0.968710, 60, 4 – – – – 0.375 0.9922

F. Buket Basmanav et al. / Biomaterials 29 (2008) 4195–4204 4197

2.1.6. Pore size distribution analysis of foamsThe pore sizes and size distribution of PLGA scaffolds were determined by

mercury porosimetry (PoreMaster 60, Quantachrome Corporation, USA) at METUCentral Laboratory).

Table 4Entrapment efficiencies of microspheres

Polymer concentration (%, w/v) Entrapment efficiency (%, w/w)

Crosslinking duration (min), crosslinkingtemperature (�C)

30, 25 60, 25 60, 4

4 11.07� 0.29 9.31� 1.92 1.74� 0.216 _ 8.07� 1.80 _8 _ 5.25� 0.73 _10 7.66� 0.21 6.92� 0.75 2.53� 0.14

2.2. In vitro studies

In vitro studies were performed with 4% and 10% microsphere populationscontaining the bone growth factors rhBMP-2 (Sigma–Aldrich Co., USA) and rhBMP-7(Sigma–Aldrich Co., USA), respectively, instead of the model protein (BSA) used inthe release studies. Samples used in in vitro studies and the calculated amounts(from the BSA encapsulation efficiencies) of BMP they contain are listed in Table 2.

2.2.1. Isolation, culture and storage of stem cellsBMSC were isolated from femur and tibia of six-week-old, young adult, male,

Sprague–Dawley rats according to the procedure developed in our laboratory [35]and cultured in polystyrene tissue culture flasks (5% CO2, 37 �C) with high glucoseDMEM (Gibco, USA) containing Penicillin/Streptomycin and fetal calf serum (PAA,Austria). After confluency, cells were trypsinized, centrifuged, resuspended in fetalcalf serum (FCS), frozen at �70 �C and stored at �196 �C until use.

2.2.2. Cell seedingDisc shaped foams loaded with microspheres were added in 24-well plates and

UV sterilized for 30 min. BMSC were grown to confluency for 1 week (5% CO2, 37 �C)in high glucose DMEM (Penicillin/Streptomycin (100 unit/mL) 1%, Fetal calf serum5% and Amphotericin B 0.4%). After confluency, cells were trypsinized, centrifugedand resuspended in DMEM. The viable cells were quantified by using a Nucleo-Counter (Chemometec A/S Nucleo Counter, Denmark) and 4�104 cells were seededonto each foam. The samples were incubated at 37 �C for 1 h for attachment, and1.2 mL of high glucose DMEM (containing Penicillin/Streptomycin (100 unit/mL) 1%,Fetal calf serum 5% and Amphotericin B 0.4%) was added onto each well. Themedium was changed every three days.

2.2.3. Cell proliferation assayIn the determination of cell numbers, Alamar Blue Assay was used. The medium

in cell seeded PLGA foams was decanted and the wells were washed with colorlessDMEM medium (HyClone�, USA) containing 1% Penicillin/Streptomycin (100 unit/mL). Alamar Blue (Biosource, USA) solution (1.2 mL, 10% in colorless DMEM medium)was added onto each well and the cells were incubated for 1 h at 37 �C in a CO2

incubator, and then absorbances were measured at 595 nm and 570 nm by the ElisaPlate Reader and cell numbers were calculated with the help of a calibration curve.The wells were washed with colorless DMEM medium (1% Penicillin/Streptomycin)until the color of the Alamar Blue was removed, then 1.2 mL of medium was addedonto each well for continuity of culture. Alamar Blue test was performed on days 7,14 and 21. Unseeded foams kept under the same culture conditions were usedas blanks.

0

20

40

60

80

100

120

0 5 10Time (day

Cu

mu

lative

B

SA

R

elease (%

)

Fig. 1. Release profiles of microspheres (4, 6, 8, and 10% microspheres crossl

2.2.4. Determination of cell differentiation by ALP assayDifferentiation of BMSC into osteoblasts was studied with Alkaline phosphatase

(ALP) assay on days 7,14 and 21 [36]. At these time points, the foams were washed withPBS and transferred into 700 mL of Tris Buffer (10 mM, pH 7.5, 0.1% Triton�X-100) andstored at �20 �C until the assay. On the day of the assay, foams in the lysis buffer werethawed in the CO2 incubator at 37 �C and then re-frozen at�20 �C to ensure completelysis and this was repeated three times. Then each sample was sonicated at 25 W on icein 30 s on and 30 s off cycles, in total for 10 min (Ultrasonic Homogenizer, Cole Parmer,USA). The samples were centrifuged at 2000 rpm for 10 min, and 100 mL of supernatantwas added to 150 mL of substrate p-nitrophenyl phosphate reconstituted with MgCl2-diethanolamine buffer supplied by Randox (AP307 kit, UK). Absorbance was deter-mined at 405 nm every 2 min for a total of 14 min by Elisa Plate Reader.

2.2.5. Confocal laser scanning microscopyOn day 21 of the culture, cells were fixed in formaldehyde (Merck, Germany) (4%

in PBS) for 30 min [37], washed with PBS (0.01 M, pH 7.4) and incubated in 1% TritonX-100 for 5 min at room temperature. Samples were washed several times with PBS,labeled for 1 h at 37 �C with Phalloidin (Sigma–Aldrich Co., USA) (0.5 mg/mL FITC-labelled Phalloidin in 0.1% PBS-BSA) and examined with CLSM with the filter forexcitation set at 488 nm.

3. Results and discussion

3.1. In situ BSA release

An initial burst effect was followed by a much lower BSA releasefor all types of microspheres. The microspheres prepared from 4%

15 20s)

4% ms6% ms8% ms10% ms4°C, 4% ms4°C, 10% ms

inked at RT for 1 h, 4 and 10% microspheres crosslinked at 4 �C for 1 h).

Fig. 2. Microscopy of microspheres and microsphere-loaded foam constructs. SEM images of microspheres (a) 4% (�100), (b) 4% (�3000), (c) 10% (�100), (d) 10% (�3000), SEMimages of microsphere-loaded foam constructs, (e) foam-microsphere interface, (�45), stereomicroscope images of microsphere/foam constructs, (f) cell seeding surface, (�200),(g) cell seeding surface, (�2.25), (h) F-4&10 foam-microsphere interface, (�2.25).

F. Buket Basmanav et al. / Biomaterials 29 (2008) 4195–42044198

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

No

rm

alize

d V

olu

me

[m

L/g

]

3--5 5--10 10--20 20--50 50--100 100--200 200--250Pore Diameter Range (µm)

FF-4F-10F-4&10

Fig. 3. Pore size distribution of foams.

25

F. Buket Basmanav et al. / Biomaterials 29 (2008) 4195–4204 4199

and 6% polymer solutions released all of their content in 10 and 15days, respectively, (Fig. 1). The 8% and 10% microspheres, on theother hand released only 59% and 41% of their content, respectively,in 7 days and no further release was detected in the following 9days. This plateau could be due to high levels of retention of BSA inthe denser matrices prepared using higher polymer concentrations.Therefore, BSA released daily could be very low, even below thesensitivity limit of the microBradford assay (1 mg/mL). In the liter-ature, release from pure alginate beads is generally complete withinhours [38]. However, complexation of alginate with other poly-electrolytes, strengthens these particles, lowers their hydrophilicityand swelling, and prolongs the release [39,40]. Results obtained inthis study reveal that complexation is quite satisfactory inachieving prolonged release (up to 15 days). The analysis of therelease behavior was made by fitting the data to relations obtainedfor diffusion (Higuchi) and Zero and First Order release kinetics(Table 3). The best fit was obtained with the Higuchi model indi-cating that the release from the microspheres is diffusioncontrolled. kH values showed that, as expected, release ratedecreases with increasing polymer concentration in the micro-sphere preparation medium. Four percent microspheres were

0

50

100

150

200

250

300

350

0 10 20 30Time (days)

Cell N

um

ber (x 10

3)

F-4(-)F-4(2)F-10(-)F-10(7)F-4(-)&10(-)F-4(2)&10(7)

Fig. 4. Cell proliferation curves in BMP(þ) and BMP(�) foams.

selected to provide the early stage release of BMP-2 and the 10%microspheres were selected as the long term BMP-7 releasecomponent with the highest and lowest release rates, respectively.Effect of crosslinking duration (30 vs 60 min) and temperature(room temperature vs 4 �C) on release behavior was studied withthese two populations and the data were treated in accordancewith the Higuchi model. Doubling the crosslinking duration did nothave a significant influence on release rates (Table 3) contrary tothe expectation that increasing crosslinking duration prolongsrelease [41]. However, if the crosslinking is achieved very rapidly, itis possible that the reaction is already over at 30 min and thus, nosignificant changes could be detected upon prolonging the cross-linking duration. A significant difference was observed in therelease profiles of microspheres prepared at two different cross-linking temperatures, especially in the first 5 days, until 30–70% ofthe content was released (Fig. 1). There was an approximately 2-fold increase in release rates when crosslinking temperature wasreduced to 4 �C (Table 3). This difference can be attributed to slower

0

5

10

15

20

0 7 14 21 28Time (days)

AL

P A

ctivity (n

mo

les/m

in

/sam

ple)

F-4(-)

F-4(2)

F-10(-)

F-10(7)

F-4(-)&10(-)

F-4(2)&10(7)

Fig. 5. ALP activity in BMP(þ) and BMP(�) foams.

0

2

4

6

8

10

12

14

0 7 14 21 28Time (days)

Sp

ecific A

LP

A

ctivity

(n

mo

les/m

in

/cell (x10

-5))

F-4(-)F-4(2)F-10(-)F-10(7)F-4(-)&10(-)F-(4)2&10(7)

Fig. 7. Specific ALP activity in BMP(þ) and BMP(�) foams.

-40-20

020406080

100120140160

7 14 21

Time (days)

Differen

ce in

A

LP

A

ctivity (%

)

F-4F-10F-4&10

Fig. 6. ALP activity difference between BMP(þ) and BMP(�) foams.

F. Buket Basmanav et al. / Biomaterials 29 (2008) 4195–42044200

crosslinking at lower temperatures. Another possibility is thatentrapment is less efficient, due to macromolecules (P4VN andalginate) being less extended and less mobile at the lowertemperature, leading to a less crosslinked, looser structure anda faster loss of BSA. The optimum crosslinking duration andtemperature for the rest of the studies were set as 60 min and 25 �C.

3.2. Entrapment efficiency

The entrapment efficiencies were in the range of of 5–10% for alltypes of microspheres (Table 4) and were low when compared tosome studies which involved polyelectrolyte complexes [42], butcomparable with chitosan–alginate microspheres with insulinentrapment efficiency of 11% [43] or fluorouracil entrapment effi-ciency of 3.5–11.4% for alginate beads prepared by internal gelation[44]. The main disadvantage of using internal gelation is theleaching of proteins during crosslinking [39] and the followingwash step. It has been reported that a protein loss of up to 35% [45]or almost 85% [46] can occur during this step. Another reason forthe low entrapment efficiency values could be the inability tocompletely extract the entrapped protein. Entrapment efficienciesof the microspheres subjected to two different crosslinking dura-tions indicate no significant difference (Table 4). Thus, an expectedincrease in entrapment efficiency does not result when the dura-tion is increased implying that crosslinking is complete within thefirst 30 min. The entrapment efficiencies for both types of micro-spheres decreased significantly when crosslinking temperaturewas decreased to 4 �C (Table 4). This could be due to slower, and,therefore, insufficient crosslinking of microspheres at lowertemperatures which would consequently lead to the escape of BSAduring crosslinking.

3.3. Microscopy of microsphere and foam populations

Stereomicrographs and SEM images (Fig. 2) of 4% and 10%microspheres were obtained following freeze drying. The micro-sphere size was observed to increase upon increase in polymerconcentration (Fig. 2a,c), probably a result of increased viscosity.The spherical shapes of microspheres with smooth surfaces weredistorted following freeze-drying (Fig. 2a,c). Shape loss of Caþ2

crosslinked alginate-polycation beads upon drying has previouslybeen reported [47]. Low polymer concentration microspheres wereobserved to have higher decrease in pore sizes probably due tohigher shrinkage (Fig. 2b,d). Disk shaped foams had a diameter of1 cm and a thickness of about 0.4 cm. The microspheres wereobserved to be located on the upper surface of the foams (Fig. 2e,h),due to their low density. The cells were seeded on the lower surfaceof the foams, the side where microspheres are not found (Fig. 2f,g).The porous structure of both sides was confirmed with the SEMimages.

3.4. Pore size distribution analysis of foams with microspheres

Pore size distribution analysis of the foams F, F-4, F-10 and F-4&10 (see Table 1 for properties) was performed by mercuryporosimetry (Fig. 3). In general, the highest fraction of the poreshad a diameter around 20 mm. A significant change in the pore sizedistribution took place upon entrapment of the microspheres in thefoam. Pores with diameters larger than 100 mm constituteda significant fraction in the unloaded foams and this fraction wasreduced significantly upon introduction of microspheres. Amongmicrosphere loaded foams, F-4 had the highest fraction of poreswith diameters above 100 mm, followed by F-10 and F-4&10. On theother hand, F-4&10 had the highest fraction of pores with diame-ters between 10–50 mm and below 10 mm, followed by F-10 and F-4.The upper surface of F-4&10 was totally covered with microspheres

(Fig. 2h) whereas the microspheres were concentrated around theperiphery of the top surface in F-4 and F-10 (data not shown). Thiswas expected to lead to major differences between double andsingle population loaded foams in terms of physical characteristicsand it was indeed confirmed by the pore size distribution profiles.

3.5. Cell proliferation

Cell numbers were determined on days 7, 14 and 21 of culture.Overall, there was an increase in cell numbers during the 21 dayculture period, except for F-4(�)&10(�) which showed a significantdecrease between days 14 and 21 probably due to overpopulationby day 14 (Fig. 4). Proliferation of cells on foams which containedboth microsphere populations (with or without BMP) was highestat all times. This can be attributed to differences in physical prop-erties of foam constructs such as pore size distribution andexplained by higher microporosity (pores with diameters between10–50 mm and below 10 mm) of the F-4&10 foams (Fig. 3), which isreported to be an important stimulus for proliferation [48]. Inter-estingly, after day 14, all BMP-free foams had more cells than theirBMP-2 and/or BMP-7 loaded counterparts (Fig. 4). This impliesa suppression of proliferation by BMP. It is known that osteogenicactivity increases with down regulation of cell proliferation [49],therefore, lower proliferation in BMP loaded foams can be relatedto an increased osteogenic activity starting from day 14 due to BMPsupply. However, obtaining enhanced proliferation in the early

F. Buket Basmanav et al. / Biomaterials 29 (2008) 4195–4204 4201

stages and then enhanced osteogenic differentiation in later stagesdue to sequential delivery from scaffolds was the main goal of thisstudy. Previous studies have reported increased cell growth fol-lowed by enhanced chondrogenic [50] and osteogenic differentia-tion [51] when culture mediums were sequentially supplementedwith different exogenous growth factors. This was not observed inthe present study. However, the effect of free and carrier entrappedgrowth factors may not be same because time–concentrationrelations are altered. An example is the loss of proliferative effect ofexogenous soluble BMP-2 on pluripotent C3HT101/2 cell line whenit was delivered from gelatin scaffolds [22]. Another reason couldbe the dose dependence of proliferative effects of BMP-2 andBMP-7 as reported in a very recent study which demonstrated thatBMP-2 enhanced proliferation of osteoblasts significantly at

Fig. 8. Confocal laser scanning microscopy of phalloidin stained cells, (a) top view distributicells inside F-10(7), (c) top view distribution and penetration of cells inside F-4(2)&10(7), (d)(f) cross section of F-4(2)&10(7) (Z-axis direction). Magnification: �20.

a concentration of 10 ng/mL whereas it lost its effect whenconcentration was increased to 100 ng/mL [52]. The situation wasthe opposite for BMP-7. The results discussed in the followingsection suggest that BMP-2 and/or BMP-7 when delivered in thepresented fashion and doses exerted their effect on ALP activitiesrather than on proliferation. A direct effect of increased osteogenicactivity is the down regulation of proliferation and this could be theother reason for the observed inactivity.

3.6. ALP activity

ALP activity was determined on days 7, 14 and 21 of culture. ALPactivity in single BMP loaded foams (F-4(2) and F-10(7)) wasobserved to be higher in comparison to their corresponding BMP

on and penetration of cells inside F-10(�), (b) top view distribution and penetration ofcross section of F-10(�) (Z-axis direction), (e) cross-section of F-10(7) (Z-axis direction),

F. Buket Basmanav et al. / Biomaterials 29 (2008) 4195–42044202

free controls (F-4(�) and F-10(7)) only on the 21st day of culture(Fig. 5). However, higher ALP activity in double BMP loaded foam(F-4(2)&10(7)) in comparison to its corresponding BMP free control(F-4(�)&10(�)) was detected both on days 14 and 21. This datasuggested that sequentially delivered BMP-2 and BMP-7 acceler-ated osteogenic differentiation in comparison to single delivery ofeither factor. When the figure is examined as a whole it is observedthat ALP activity was significantly lower at all times for constructscontaining both microsphere populations than the single pop-ulation loaded constructs (regardless of BMP presence). Thissuggests that physical characteristics of foams (porosity, pore sizedistribution) had a very significant effect on osteogenic differenti-ation as also was reported in the literature [48]. In order to isolateand compare the effect of growth factors on differentiation the ALPactivities of the BMP-free constructs were subtracted from theirBMP loaded counterparts (Fig. 6). In this figure an increase indifferentiation due to BMP delivery can be seen clearly at the end of21 days. Enhancement due to sequential BMP delivery on days 7, 14and 21 were 20%, 51% and 144%, respectively. Enhancement due tosingle BMP-7 delivery during the same period were 7%, 11% and57%, respectively. For the single BMP-2 delivery, however, it was14% only on day 21. This data clearly demonstrates that combineddelivery of BMP-2 and BMP-7 created a synergistic effect and wasmuch more effective than single delivery of either BMP. In theliterature there are a number of studies which support our findings.For example, in a similar study higher ALP activities were inducedby sequentially delivered BMP-2 and IGF-I which led to highermatrix mineralization levels [22]. Also increased osteogenic effecton osteoblasts was observed with free BMP-2 and BMP-7 as shownby higher osteocalcin levels when compared to cultures supple-mented with only BMP-2 or BMP-7 [52]. The comparison ofdifferentiation and proliferation yields the expected result. In Fig. 7F-2 is seen to have the highest positive influence on differentiationfollowed by F-7 and F-2&7, respectively. This order is the reverse ofthe order for positive proliferative influence (Fig. 4). Specific ALPactivity in BMP-free constructs did not significantly differ betweendays 7 and 21 indicating that osteogenic differentiation ceases toincrease in the absence of BMP. In contrast, there was a significantincrease in specific ALP activities in all of the BMP loaded foams atthe end of 21 days and activities were higher in comparison tocorresponding BMP-free controls. These confirmed the positiveeffect of all BMP loaded constructs in enhancing differentiation. Insummary, provision of two growth factors has a stronger effect ondifferentiation than single growth factors or growth factor-freesystems. Foam properties were also effective on differentiation.Single population was more effective than two populations loadedonto the scaffolds. It is a well-established fact that in vivo might not

Fig. 9. SEM images of cell seeded and unseeded F-10 after 21 da

yield the same results as in vitro [29] and, therefore, the synergisticeffects of growth factors may not always correlate well with the invivo. It is, therefore, necessary that this study should be extended toin vivo after more comprehensive characterization studies areperformed.

3.7. Confocal laser scanning microscopy

CSLM images of phalloidin-stained constructs revealed signifi-cant difference in cell morphologies and spreading between BMPloaded and BMP-free foams (Fig. 8). On the BMP-free foams, cellswere in clumps (Fig. 8a) whereas well spread cells with osteoblast-like morphology and interconnecting extensions were observed onBMP loaded foams (Fig. 8b,c). This was also confirmed with themicrographs of the cross sections; there are gaps between cells inBMP-free foams (Fig. 8d) while there are continuous layers oftightly connected cells in BMP loaded foams (Fig. 8e,f). It wasalso observed that cell layer was tighter in dual loaded foam(F-4(2)&10(7)) than F-10(7) (Fig. 8e,f).

3.8. Scanning electron microscopy

SEMs of cell loaded and unloaded were obtained after 21 days ofculture. Fig. 9 shows the cell seeding side (reverse of microsphereloaded side) of the foams. Comparison of unseeded (Fig. 9a) andseeded F-10 (Fig. 9b) foams clearly reveals that the cell seededfoams are highly populated with cells with their extensions span-ning over voids after 21 days of culture. Fig. 10 shows the micro-sphere loaded side of the foams and the microspheres. This figurealso reveals spreading cells and the contact of the extensions ofcells as was on the other side (Fig. 10a). The interconnected cellswere observed to spread over microspheres entrapped in the foamindicating that cells also adhere to the slightly hydrophilic P4VN-alginate complexes (Fig. 10b). The SEMs also reveal signs of osteo-genic activity of the cells; mineral formation in the form of tubularcrystals (observed but not chemically analyzed), indicate osteo-genic activity (Fig. 10c).

4. Conclusion

There was a difference in the pore size distribution profiles of theconstructs loaded with single and double microsphere populations.Cell proliferation was highest and total ALP activities were lowest atall time points for foams containing both types of microspheresregardless of BMP presence or absence. When BMP-2 and/or BMP-7were included in the constructs, proliferation decreased anddifferentiation increased in comparison to their corresponding

ys of culture. (a) unseeded, (b) seeded. Magnification: �250.

Fig. 10. SEM images of cell seeded F-4&10 after 21 days of culture. Magnification (a) �500, (b) �1000, (c) �1000. Arrow heads indicate formed crystals.

F. Buket Basmanav et al. / Biomaterials 29 (2008) 4195–4204 4203

BMP-free controls. When the contribution of the foam propertieswas subtracted it was observed that sequential provision of BMP-2and BMP-7 created a synergy and enhanced the differentiationmore than single BMP constructs and also accelerated the process.This study suggests that sequentially delivered BMP-2 and BMP-7plays a significant role in enhancing differentiation.

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