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Biomaterials 30 (2009) 3150–3157

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Biomaterials

journal homepage: www.elsevier .com/locate/biomater ia ls

The bioactivity of rhBMP-2 immobilized poly(lactide-co-glycolide) scaffolds

Hong Shen, Xixue Hu, Fei Yang, Jianzhong Bei, Shenguo Wang*

BNLMS, State Key Laboratory of Polymer Physics & Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China

a r t i c l e i n f o

Article history:Received 8 November 2008Accepted 2 February 2009Available online 18 February 2009

Keywords:rhBMP-2Oxygen plasma treatmentAnchorageImmobilizationPLGA scaffoldTissue engineering

* Corresponding author. Tel./fax: þ86 10 62581241E-mail address: wangsg@iccas.ac.cn (S. Wang).

0142-9612/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.biomaterials.2009.02.004

a b s t r a c t

In this study, immobilization of rhBMP-2 on polylactone-type polymer scaffolds via plasma treatmentwas investigated. To introduce proper functional groups on the surface of poly(lactide-co-glycolide)(PLGA) matrix, PLGA films were treated under different atmospheres, such as oxygen, ammonia andcarbon dioxide, respectively, and then incubated in rhBMP-2 solution of de-ionized water. The effect ofvarious plasma-treated PLGA films on binding rhBMP-2 was investigated and compared. It was foundthat PLGA binding ability to rhBMP-2 was enhanced by carbon dioxide and oxygen plasma treatment,and the binding ability of the oxygen plasma-treated PLGA (OT-PLGA) to rhBMP-2 was the strongest afteroxygen plasma treating for 10 min under a power of 50 W. The changes of surface chemistry and surfacetopography of PLGA matrix induced by oxygen plasma treatment played main roles in improving thePLGA binding ability to rhBMP-2. The stability of rhBMP-2 bound on OT-PLGA film was determined undera dynamic condition by a Parallel Plate Flow Chamber. The result showed that the rhBMP-2 had beenimmobilized on the OT-PLGA film. Mouse OCT-1 osteoblast-like cell as a model cell was cultured on therhBMP-2 bound OT-PLGA (OT-PLGA/BMP) in vitro, which showed that the bound rhBMP-2 via oxygenplasma treatment was bioactive. Depending on hydrophilicity and rich polar O-containing groups of theOT-PLGA scaffold, different amount of rhBMP-2 could be evenly immobilized on the surface of the OT-PLGA scaffold. The immobilized rhBMP-2 had stimulated differentiation of OCT-1 cell and acceleratedprocess of mineralization of OCT-1 cell in the scaffold. It revealed the rhBMP-2 immobilized PLGA scaffoldhad good cell affinity.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Tissue engineering aims at the repair, restoration or regenera-tion of damaged tissue function using cells, scaffolds and growthfactors alone or in combination [1,2]. Many results of experimentaland clinical researches show that the success of any tissue engi-neering approach mainly relies on the delicate and dynamicinterplay among these three components [3–5]. Therefore, one ofthe significant challenges for tissue engineering is to design andfabricate suitable biodegradable scaffolds which not only possessadequate biodegradation rate, mechanical strength and morpho-logical structural but also can effectively deliver specific growthfactor to actively guide and control cell attachment, migration,proliferation and differentiation. Polylactone-type biodegradablepolymers, such as poly(L-lactide) (PLLA), polyglycolide (PGA) andtheir copolymer poly(lactide-co-glycolide) (PLGA), are extensivelystudied as scaffold materials for tissue engineering [6–9], since theypossess good mechanical property, low immunogenicity, non-

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toxicity and adjustable degradation rate. However, cytocompati-bility of the polylactone-type biodegradable polymer scaffold is notgood due to the lack of cell recognition sites on their surface. So it isfar from the ideal scaffold which can bind growth factor on it andmake the scaffold supply biological signals for guiding and acceler-ating cell attachment, migration, differentiation and proliferation.

Bone morphogenetic protein (BMP) is a well used growth factorthat plays a crucial role in bone formation and repair [10,11]. BMPregulate cell growth and differentiation of a variety of cell typesincluding osteoblasts and chondrocytes [10,12]. In particular, BMP-2, as member of the BMP family, has become one of the most potentmembers of the BMP family due to the induction of bone formationin vivo by promoting the maturation of committed cells to becomemore differentiated osteoblasts [10,12,13]. Moreover, sincerecombinant human bone morphogenetic protein-2 (rhBMP-2) isavailable in large quantities and lacks risks associated with matrixextracts related to potential viral infection, BMP-2 has beenextensively studied in tissue engineering application [14–17].Recently, to improve polylactone-type biodegradable polymerscaffolds, a number of strategies including physisorption, ionicinteraction, and blending have been designed to immobilize BMP-2on the polylactone-type scaffolds [13,18–21]. The challenge is how

H. Shen et al. / Biomaterials 30 (2009) 3150–3157 3151

to tightly incorporate the water-soluble protein into the hydro-phobic polymer scaffolds evenly. BMP-2 can be incorporateddirectly into the polymer scaffolds at [20,22,23] or after fabrication[21,24,25]. However, use of organic solvent during the polymerscaffolds fabrication process will damage bioactivity of the BMP-2.On the other hand, the poor hydrophilicity and lack of functionalgroup of the polymers often results in low loading efficiency andun-tight binding of BMP-2 by solution dipping method afterfabrication. Aimed to improve binding ability of the polylactone-type polymer scaffold to the BMP-2, surface modification of thepolylactone-type polymers has been developed [10]. However,since the functional groups are absent in the backbone of thepolymers, it is difficult to modify surface property of polylactone-type polymers by common chemical method.

The present authors previously have reported an effectivemethod to immobilize basic fibroblast growth factor (bFGF) on PLGAscaffold [26]. In the method, the PLGA scaffold was pretreated bycarbon dioxide (CO2) plasma and then was anchored with bFGF.bFGF had been immobilized on the PLGA scaffold by electrostaticinteraction between the basic group of bFGF and the rich acidiccarboxylic group of the PLGA scaffold which resulted from CO2

plasma treatment of the polymer scaffold. The immobilizationmethod of CO2 plasma treatment combining with bFGF anchorage isa rapid, clean and non-solvent pollution method. Plasma treatmentcan be used to introduce some specific element or functional grouponto surface of a polymer only by selecting and applying somesuitable gas [27–29]. Surface property of a material such as wetta-bility, topography, surface charge states and biocompatibility can beeffectively modified and adjusted by controlling parameters ofplasma treatment, although bulk properties of the material will belittle changed. Since some specific functional groups such as amine,carbonyl, carboxyl, hydroxyl, as well as ether can provide specialchemical reactivity and varying physical properties of the surface, itis benefit for functionalization of the surface with bioactive mole-cules. It is considered that the plasma treatment combining withanchorage of bFGF technique is hopeful to extend to immobilizeother growth factors onto the PLGA scaffold by only choosing andapplying other suitable gases for the plasma treatment.

In this study, firstly the effect of various plasma treatments onanchoring rhBMP-2 on PLGA was investigated and compared. Basedon the result that oxygen plasma treatment could provide properproperty for surface of PLGA matrix for anchoring rhBMP-2, theoxygen plasma treatment was chosen to pretreat PLGA for immo-bilizing rhBMP-2. Then the binding rhBMP-2 ability of the oxygenplasma-treated PLGA matrix was investigated in vitro by thegradient-binding experiment. Moreover, the stability of rhBMP-2bound to PLGA film was further tested under shear stress bya Parallel Plate Flow Chamber [30,31], and bioactivity of theimmobilized rhBMP-2 was evaluated by measuring ALP activity ofmouse OCT-1 osteoblast-like cell cultured on the immobilizedrhBMP-2-PLGA film. Finally, rhBMP-2 immobilized PLGA scaffoldwas prepared and the influence of immobilized rhBMP-2 on thegrowth of mouse OCT-1 cell was investigated.

2. Materials and methods

2.1. Materials

PLGA (molar ratio of lactyl/glycotyl¼ 70/30, Mw¼ 120,000) was prepared byring-opening polymerization of L-lactide (PURAC, Netherlands) and glycolide(PURAC, Netherlands) under high vacuum at 160 �C for 20 h in the presence ofstannous octoate (SIGMA, German) as catalyst (0.05 wt%) [32].

2.2. Preparation of PLGA film and scaffold

The PLGA film with dense structure was prepared by casting 5 wt% PLGA chloro-form solution into a poly (tetrafluoroethylene) (PTFE) mould. After solvent

evaporation in air at room temperature the formed film was removed from themould and performed removing residual solvent thoroughly under vacuum at roomtemperature for 48 h. Thickness of the obtained PLGA film was 0.1 mm.

The PLGA scaffold was manufactured by an improved solid–liquid phaseseparation method [33]. A certain weight of sieved NaCl granules (diameter 200–280 mm) was added into 5% (w/v) solution of the PLGA in dioxane, then the slurrywas maintained at 0 �C for over 24 h to perform solid–liquid phase separationcompletely. After the solvent was removed by freeze–drying for 3 days, theformed matrix was put into distilled water to leach the NaCl out. The distilledwater was renewed every 3 h until no chloric ion could be detected by droppingof AgNO3 aqueous solution, and then a porous structured PLGA scaffold wasfabricated. At the same time, due to the miscibility between the dioxane andwater, the residual dioxane was extracted out and replaced with water. Finally, thefabricated porous structured PLGA scaffold was dried and kept in a desiccator forusage.

2.3. Plasma treatment

Plasma treatment of the PLGA film and scaffold was carried out on Samco PlasmaDeposition (Model PD-2, 13.56 MHz) under different atmosphere, such as oxygen,ammonia and carbon dioxide, respectively. PLGA film or scaffold was placed on theelectrode in the plasma chamber. The chamber was evacuated to less than 10 Pabefore filling with gas. After gaseous pressure of the chamber was stabilized to 20 Pa,a glow discharge plasma was created by controlling the electric power at a radiofrequency of 13.56 MHz for a predetermined time. Finally, the plasma-treatedsample was further exposed to the atmosphere for another 10 min before the samplewas taken out from the chamber.

2.4. Determination of bound rhBMP-2

Both plasma-treated and untreated PLGA films were cut into disks witha diameter of 7 mm and placed in the bottom of a 96-well plate. 100 ml of rhBMP-2(PeproTech, USA) solutions with increasing concentration from 0 to 120 mg/ml wereused for incubation. After the PLGA samples were incubated in the rhBMP-2 solutionfor 1 h at room temperature on a shaker, the PLGA samples were rinsed for threetimes with phosphate buffer saline (PBS, pH 7.4) and then the binding proteins weremeasured by a modified ELISA assay method [34,35].

Briefly, after 200 ml of 5% BSA/PBS was respectively added in the PLGA samplecontained wells of the 96-well plate and incubated for 2 h on a platform shaker, thewells were washed for three times with PBS. Then another 200 ml (1:1000 dilution)anti-polyHistidine antibody (Sigma, USA) was added in the wells at room temper-ature for additional 2 h incubation. After unbound primary antibody was removedby three washes with PBS, the secondary antibody of 200 ml of ALP conjugated goat-anti-mouse IgG (1:10000 dilution, Sigma, USA) was added to each well. The ALPreaction product was developed by incubation with para-nitrophenylphosphate(pNPP) (Sigma, USA) at room temperature for 10 min and then the absorbance of thereaction product at 405 nm was determined by using plate reader (TECAN, SUNRISE,Austria).

DOD405 ¼ OD405s� OD405c

where OD405s was the absorbance derived from the experimental sample whichincubated in the solutions with concentration of rhBMP-2> 0 mg/ml, and OD405cwas the absorbance derived from the control which incubated in the solution withconcentration of rhBMP-2¼ 0 mg/ml, respectively.

In the present research, the untreated PLGA and oxygen, ammonia as well ascarbon dioxide plasma-treated PLGA were respectively abbreviated as UT-PLGA, OT-PLGA, AT-PLGA and CT-PLGA. The rhBMP-2 bound PLGA prepared under withoutplasma pretreatment and with oxygen plasma pretreatment were abbreviated asUT-PLGA/BMP and OT-PLGA/BMP, respectively.

2.5. Stability determination of bound rhBMP-2

According to previous reports [30,31], stability of bound rhBMP-2 was deter-mined under shear stress by a circuit flow system in a Parallel Plate Flow Chamber(PPFC). Firstly, the UT-PLGA/BMP and OT-PLGA/BMP films with a diameter of10 mm were immobilized on bottom glass plate of PPFC and the top glass platewas assembled in the PPFC. A certain distance between both the parallel glassplates was kept. After all bubbles in the circuit flow system were removed care-fully, PBS solution (pH 7.4) was circularly flowed in the system initiated bya peristaltic pump. The flowing rate of the PBS solution was controlled byadjusting the peristaltic pump to keep 11.5 N/m2 of shear stress in the PPFC. At thesame time, temperature of the PPFC was also controlled by adjusting heatingvoltage to keep 37 �C. After the films had been exposed to the shear stress fora certain time, they were taken out from the glass plate and the retained rhBMP-2was assayed. Finally, the stability of the bound rhBMP-2 on UT-PLGA or OT-PLGAfilm was compared.

Fig. 1. Effect of oxygen plasma treatment on surface treatment degree of PLGA scaffolddetermined by ink dyeing method. (a) UT-PLGA scaffold; (b) OT-PLGA (50 W for10 min).

H. Shen et al. / Biomaterials 30 (2009) 3150–31573152

2.6. Preparation of cells

OCT-1 osteoblast-like cells were derived from osteocalcin promoter-driven SV-40 T-antigen transgenic mouse calvarias [36] and supplied by Institute of MolecularBiology Study of Nankai University (China). Briefly, cells were incubated at 37 �C inDMEM supplemented with 10% FBS and 100 U/cm3 each of penicillin and strepto-mycin in a 5% CO2 incubator. When the cells had grown to confluence, they weredetached by trypsin/EDTA (0.05%trypsin/0.02%EDTA) and resuspended in freshculture medium to the correct concentration for seeding onto samples.

2.7. Bioactivity assay of bound rhBMP-2

UT-PLGA, OT-PLGA, UT-PLGA/BMP and OT-PLGA/BMP films were cut into diskswith a diameter of 10 mm. The disks were respectively placed at bottom of a 48-wellplate and OCT-1 cell suspension containing 15,000 cells were poured into each well.After OCT-1 cells were cultured on various PLGA films for 1, 2, 4, 7, 10 or 14 days, thefilms were washed with phosphate buffer saline (PBS, pH 7.4) and 500 ml of 0.1%Triton X-100 was added to each well. After the OCT-1 cells on the films were frozenand then thawed repeatedly for four times, the solution was collected to measurethe Alkaline phosphatase (ALP) activity.

ALP activity was determined by using disodium phenyl phosphate as thesubstrate at pH 10. Each reaction was initiated with disodium phenyl phosphate, andallowed to proceed for 15 min at 37 �C, and then a developer potassium ferricyanidewas quickly added. The absorbance at 520 nm was measured with UV spectropho-tometer (752-type Ultraviolet grating spectrophotometer, Shanghai). The ALPactivity was normalized by total intracellular protein synthesis and thus expressedas U/g protein. Total protein content was determined at 595 nm using a protein assaykit (Nanjing Jianchen Bioengineering Institute, China) according to manufacturer’sinstruction.

2.8. rhBMP-2 immobilization procedure of PLGA scaffolds

The original rhBMP-2 solution was diluted with de-ionized water and adjustedthe concentration of rhBMP-2 to 5, 50 and 100 mg/ml. 100 ml of rhBMP-2 aqueoussolution was dropped into OT-PLGA scaffold (8.0� 8.0� 3.5 mm3) and completelyabsorbed into the OT-PLGA scaffold because the rhBMP-2 solution volume was lessthan the equilibrium swell volume of the OT-PLGA scaffold. After the rhBMP-2impregnated OT-PLGA scaffold was statically placed at room temperature for 1 h, itwas dried by freeze-drying and the rhBMP-2 immobilized PLGA scaffold wasobtained.

The rhBMP-2 solutions with concentrations of 5, 50 and 100 mg/ml were used inthe immobilization procedure, and the obtained scaffolds were respectively namedOT-PLGA/BMP-5, OT-PLGA/BMP-50 and OT-PLGA/BMP-100. All of the rhBMP-2immobilized PLGA scaffolds were with the same size of 8.0� 8.0� 3.5 mm3.

2.9. MTT assay

OT-PLGA and various OT-PLGA/BMP scaffolds were placed into a 24-well cultureplate and 100 ml of cell suspension with a cell density of 5�105 cells/ml was seededinto the scaffolds, respectively. The cell-seeded scaffolds were maintained at 37 �Cunder 5% CO2 atmosphere for 3 h and then 1.5 ml of culture medium was added toeach well. At predetermined interval, 15 ml of MTT solution (5 mg/ml in PBS) wasadded to each well, followed by incubation at 37 �C for 4 h to MTT formazanformation. Then upper medium was carefully removed and the intracellular for-mazan was dissolved by adding 800 ml of 0.04 mol/l HCl/iso-propanol to each well.The absorbance of produced formazan was measured at 570 nm with microplatereader (ZS-2, Beijing).

2.10. Alkaline phosphatase activity assay

OT-PLGA and various OT-PLGA/BMP scaffolds were placed into a 24-well cultureplate and 100 ml of cell suspension with a cell density of 5�105 cells/ml wasrespectively seeded into the scaffolds. After the OCT-1 cells were cultured on variousPLGA scaffolds for 4, 7, 10, 14 or 21 days, the scaffolds were washed with PBS and1.5 ml of 0.1% Triton X-100 was added to each well. After the OCT-1 cells on thescaffolds were frozen and then thawed repeatedly for four times, the solution wascollected to measure the Alkaline phosphatase (ALP) activity.

2.11. Observation of cell morphology

OT-PLGA and various OT-PLGA/BMP scaffolds were located in a 24-well cultureplate. Cell suspension (8� 105 cells/ml) was seeded on the scaffolds until the scaf-folds became saturated and then they were cultured for 3 h prior to addition of1.5 ml culture medium into culture plate. After being cultured for 4 weeks, thescaffolds were taken out from the culture plate and washed with PBS, then fixedwith 2.5% glutaraldehyde for 24 h at 4 �C. The scaffolds were dehydrated througha series of graded alcohols, dried and sputter-coated with gold. The cell morphologyon the scaffolds was observed by SEM (Hitachi S-530, Japan).

2.12. Statistical analysis

The data were expressed as means� standard deviations (SD) (n¼ 3 or 4).Statistical comparisons were performed by using Student’s t-test. P-values< 0.05were considered statistically significant.

3. Results and discussion

3.1. Determination of plasma treatment parameters

It was reported that various gases such as oxygen, ammonia,carbon dioxide, argon and air had been used in plasma treatment forsurface modification of polylactone-type polymer, but only a fewgases could effectively immobilize growth factor or other bio-macromolecules on the gas plasma-treated polymer [26,29,33,37–41]. Hence, gas selection is the most important for immobilization ofrhBMP-2. However, considering electric power, treating time, andatmosphere pressure are important parameters of plasma treat-ment, and the plasma treatment effect would depend strongly onthese parameters [28,29,41], it is necessary to decide each parameterused in the plasma treatment before the gas selection.

In the present research, PLGA scaffolds were treated by oxygen,carbon dioxide, ammonia plasma treatment under various powersfor different time, then surface treatment degree of the scaffoldswas measured by determination of color dyed depth according tothe ink dyeing method [42]. The result revealed that treated for10 min under 50 W of power and 20 Pa of atmosphere pressure allthe depth of different plasma treatment could reach to about3.5 mm of the porous scaffold. Hydrophilicity of PLGA scaffoldbefore and after oxygen plasma treatment was shown in Fig. 1. Itcould be seen that only the most outer layer was dyed for the UT-PLGA scaffold (Fig. 1a), but for the OT-PLGA scaffold not only theouter surface but also the most inner part of the porous scaffold wasdyed (Fig. 1b). Therefore, 50 W of electric power, 10 min of treatingtime and 20 Pa of atmosphere pressure were used as commonparameters for gas selection of the plasma treatment.

3.2. Gas selection for plasma treatment

At first, the PLGA films were plasma treated under the sameparameters of 50 W of power for 10 min but in different atmo-sphere of oxygen, ammonia or carbon dioxide, and followed byincubation process with similar rhBMP-2 solution. Then the boundrhBMP-2 on each plasma-treated PLGA film was determined bya modified ELISA assay method. Finally, the effect of differentplasma treatment on binding rhBMP-2 of PLGA film was compared.

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Fig. 2. The binding ability of various PLGA films to rhBMP-2. *p< 0.05 significantagainst the binding ability of UT-PLGA film to rhBMP-2; #p< 0.05 significant againstthe binding ability of AT-PLGA film to rhBMP-2; $p< 0.05 significant against thebinding ability of CT-PLGA film to rhBMP-2. Circular UT-PLGA, AT-PLGA, CT-PLGA andOT-PLGA films with a diameter of 7 mm were loaded with 100 ml rhBMP-2 solution(15 mg/ml) for 1 h.

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Fig. 3. Comparison of rhBMP-2 binding ability of OT-PLGA and UT-PLGA films.*p< 0.05; significant against the rhBMP-2 binding ability of UT-PLGA film at the cor-responding concentration. Circular UT-PLGA and OT-PLGA films with a diameter of7 mm were loaded with 100 ml rhBMP-2 solution (0–120 mg/ml) for 1 h.

H. Shen et al. / Biomaterials 30 (2009) 3150–3157 3153

It could be seen in Fig. 2 that the amount of bound rhBMP-2 onvarious plasma-treated PLGA films was different. Comparison withthe UT-PLGA film, the amount of bound rhBMP-2 on the OT-PLGAand CT-PLGA films had increased but that on the AT-PLGA film hadreduced. On the other hand, it was clear that the amount of boundrhBMP-2 on the OT-PLGA film was the highest. It revealed that thegas used in the plasma treatment had an important effect onbinding rhBMP-2 ability of the plasma-treated PLGA, and theoxygen plasma treatment could lead to the highest binding abilityof rhBMP-2 to the PLGA film. Therefore, oxygen was chosen to usein plasma treatment of the PLGA scaffold for anchorage of rhBMP-2.

3.3. Activation mechanism of different gas plasma treatment onbinding ability of rhBMP-2

Previous research has reported that the ammonia plasmatreatment can introduce N-containing electron-accepting func-tional groups, such as –N–H– and –C–Nþ, onto the surface of thepolymers, in result density of positive charge on the polymersurface has increased [37]. However oxygen plasma treatment andcarbon dioxide plasma treatment can introduce rich oxygen-con-taining functional groups, such as >C]O, –COOH or –C–O–, ontothe surface of the polylactone-type polymers [26,41]. Since theseoxygen-containing functional groups are electron-donating groups,after plasma treatment under atmosphere of oxygen or carbondioxide the native charge density on the polymer surface hasincreased. On the other hand, since rhBMP-2 with pI of w8.5 isa positively charged protein in water solution [43,44], when theoxygen or carbon dioxide plasma-treated PLGA is immersed intothe rhBMP-2 solution the strong electrostatic attraction betweenthe positively charged rhBMP-2 and the negatively charged OT-PLGA or CT-PLGA matrix will push more rhBMP-2 to be bound ontothe surface of the OT-PLGA or CT-PLGA film. In result the amount ofbound rhBMP-2 has increased. However, due to strong electrostaticrepulsion force existed between both positively charged AT-PLGAand rhBMP-2, the amount of bound rhBMP-2 has reduced, and iseven less than that of UT-PLGA.

Of course, increase of surface area of PLGA matrix was anotherreason for enhancing amount of bound rhBMP-2 since surfaceroughness of matrix was increased by the plasma treatment

[26,29]. The matrix with large surface area is of particular utility forbiomolecular recognition as well as providing enough space toimmobilize the biomolecules [45]. Compared with CT-PLGA, morerhBMP-2 binding to the OT-PLGA film might result from thedifference of ratio of various O-containing functional groups andmicro-structured surface.

3.4. Effect of oxygen plasma treatment on binding rhBMP-2 ofPLGA film

The ability of PLGA binding rhBMP-2 before and after oxygenplasma treatment under 20 Pa of pressure and 50 W of power for10 min was further tested by the gradient-binding experiment. Asshown in Fig. 3, the binding proteins on UT-PLGA and OT-PLGAfilms increased in a concentration-dependent fashion, but thebinding curves of UT-PLGA and OT-PLGA were different in shape. Inthe case of the rhBMP-2 concentration was over 3.75 mg/ml, morerhBMP-2 was bound on the OT-PLGA films at same rhBMP-2concentration. Moreover, with the rhBMP-2 concentrationincreased from 60 to 120 mg/ml, the binding of rhBMP-2 to UT-PLGAfilm had not big change. However, for the OT-PLGA film withconcentration of rhBMP-2 increased from 60 to 120 mg/ml, thebinding of rhBMP-2 still had obvious increase.

Since the surface of the UT-PLGA films contained some O-con-taining functional groups (–C–O– and >C]O), which also leda negativelycharge surface of UT-PLGA, at low rhBMP-2 concentrationthe binding ability of UT-PLGA and OT-PLGA to the rhBMP-2 had notstatistic difference. However, the polar O-containing groups on UT-PLGA film were less. In result it could not provide enough sites forbinding rhBMP-2 even the concentration of rhBMP-2 increases. Thus,under 60 mg/ml of rhBMP-2 concentration the proteins reachedsaturation on the UT-PLGA film. On the other hand, the enriched polarO-containing groups on the surface of OT-PLGA film could providemany sites to catch rhBMP-2 byelectrostatic interaction and hydrogenbonding. In addition, nano-structured surface topography of OT-PLGAmay increase surface/volume ratio [29], which could provide morebinding sites for rhBMP-2 and thus enhance the binding.

3.5. Stability of bound rhBMP-2

The binding stability of rhBMP-2 on the OT-PLGA and UT-PLGAfilms was determined and compared under a shear stress condition

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Fig. 4. The retained rhBMP-2 on the OT-PLGA/BMP and UT-PLGA/BMP films afterexposed to a shear stress of 11.5 N/m2 for a certain time. *p< 0.05; significant againstthe retained rhBMP-2 on the UT-PLGA/BMP at the corresponding concentration.Circular UT-PLGA and OT-PLGA films with a diameter of 10 mm were loaded with200 ml rhBMP-2 solution (30 mg/ml) for 1 h.

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Fig. 6. MTT-tetrazolium assay of OCT-1 cells cultured on various PLGA scaffolds withindifferent period. *p< 0.05; significant against the proliferation and viability of OCT-1cells on OT-PLGA and OT-PLGA/BMP-5 scaffolds at the corresponding day.

H. Shen et al. / Biomaterials 30 (2009) 3150–31573154

by using a Parallel Plate Flow Chamber [30,31]. After the OT-PLGA/BMP and UT-PLGA/BMP films had been exposed to a shear stress of11.5 N/m2 for different time, the retained rhBMP-2 on the OT-PLGA/BMP and UT-PLGA/BMP films was determined as shown in Fig. 4. Itcould be seen that after the films had exposed to shear stress for3 min the bound rhBMP-2 had hardly been detected from the UT-PLGA film, but the rhBMP-2 could be easily detected from the OT-PLGA film. Moreover, some rhBMP-2 even could be detected fromthe OT-PLGA film after the OT-PLGA/BMP film had been exposed tothe shear stress of 11.5 N/m2 for 7 min. It meant the bindingstrength of rhBMP-2 on the OT-PLGA film obviously enhanced bythe oxygen plasma treatment. So, although the OT-PLGA/BMP hadexposed in shear stress for a longer period, many rhBMP-2 couldstill retain on the OT-PLGA film.

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Fig. 5. ALP activity assay of OCT-1 cells cultured on various PLGA films after culturedfor a certain period. ALP activity was determined as the enzyme activity unit (U) pergram of protein. *p< 0.05; significant against the ALP activity of OCT-1 on UT-PLGA andOT-PLGA films at the corresponding day. #p< 0.05; significant against the ALP activityof OCT-1 on UT-PLGA/BMP films at the corresponding day. Circular UT-PLGA and OT-PLGA films with a diameter of 10 mm were loaded with 200 ml rhBMP-2 solution(7.5 mg/ml) for 1 h.

Due to lack of the polar O-containing group on the UT-PLGAsurface and a weak interaction between rhBMP-2 and UT-PLGA film,only a few rhBMP-2 could be adsorbed on the UT-PLGA film. So, afterthe UT-PLGA/BMP film was exposed in the shear stress field, therhBMP-2 easily fell and was hardly to be detected. However, sincethe strong electrostatic interaction between rhBMP-2 and OT-PLGA,much more amount of rhBMP-2 could be bound on the OT-PLGAsurface and the bound rhBMP-2 could resist the shear stress andremain on the OT-PLGA surface even after exposed in the shear stressfield for a longer period. Therefore, the stability of rhBMP-2 boundonto the PLGA film was greatly improved by oxygen plasma treat-ment combining with rhBMP-2 anchorage.

3.6. Bioactivity assay of bound rhBMP-2

The biological activity of rhBMP-2 bound to PLGA films wasevaluated by measuring ALP activity of OCT-1 cells cultured on the

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Time (days)

OT-PLGAOT-PLGA/BMP-5OT-PLGA/BMP-50OT-PLGA/BMP-100

Fig. 7. ALP activity assay of OCT-1 cells cultured on various PLGA scaffolds aftercultured for different period. ALP activity was determined as the enzyme activity unit(U) per gram of protein. *p< 0.05; significant against the ALP activity of OCT-1 on OT-PLGA scaffold at the corresponding day. #p< 0.05; significant against the ALP activityof OCT-1 on OT-PLGA/BMP-5 scaffolds at the corresponding day. $p< 0.05; significantagainst the ALP activity of OCT-1 on OT-PLGA/BMP-50 scaffolds at the correspondingday.

Fig. 8. Scanning electron micrographs of OCT-1 cells cultured on various PLGA scaffolds for 4 weeks (the left was� 600, and the right was� 15,000). (a, b) OT-PLGA; (c, d) OT-PLGA/BMP-5; (e, f) OT-PLGA/BMP-50; (g, h) OT-PLGA/BMP-100, where the arrows indicated the morphology of cells (Macroscopic) and the globular accretions (Microscopic).

H. Shen et al. / Biomaterials 30 (2009) 3150–3157 3155

PLGA films. The ALP activity of OCT-1 cells cultured on various PLGAfilms for predetermined time was shown in Fig. 5. It could be seenthat the ALP activity of cells on the UT-PLGA/BMP and OT-PLGA/BMP films was higher than that on the OT-PLGA and UT-PLGA films.

Moreover, in the first two days the ALP activity of cells on the UT-PLGA/BMP and OT-PLGA/BMP films was not different. However,after cultured for more than two days the ALP activity of OCT-1 cellson the OT-PLGA/BMP film was higher than that on the UT-PLGA/

H. Shen et al. / Biomaterials 30 (2009) 3150–31573156

BMP film and cultured for 10 days the ALP activity of OCT-1 cellsalmost reached the maximum. This result indicated that all thebound rhBMP-2 on the UT-PLGA and OT-PLGA films was bioactive,but only the rhBMP-2 bound on the OT-PLGA films could sustainand preserve its bioactivity.

Since most rhBMP-2 was adsorbed on the UT-PLGA films bya weak interaction due to lack of the polar O-containing groups onUT-PLGA film, the bound rhBMP-2 easily diffused from the films toculture medium. In result effective role time of the rhBMP-2 boundon the UT-PLGA films was short. However, since the rhBMP-2 couldbe anchored on the OT-PLGA film by strong electrostatic interac-tion, not only the amount of the bound rhBMP-2 increased but alsobioactivity of the rhBMP-2 could preserve for a longer time.

3.7. Effect of immobilized rhBMP-2 on OCT-1 cell growth

Based on the above experimental results, it had been demon-strated that rhBMP-2 could be immobilized effectively on the PLGAfilms by the method of oxygen plasma treatment combining withrhBMP-2 anchorage. According to tissue engineering application,porous PLGA scaffold was also fabricated and then immobilizedrhBMP-2 by the method of oxygen plasma treatment combiningwith rhBMP-2 anchorage. Since the solvent (dioxane) and waterwere miscible, and large amount of water was used for leachingNaCl out, as well as the water was renewed for many times, all theresidual solvent could be removed thoroughly in the PLGA scaffoldand effect of solvent on the bioactivity of rhBMP-2 and cell growthcould be avoided and ignored. So in the research only the effects ofthe immobilized rhBMP-2 on proliferation, ALP activity andmorphology of OCT-1 cell on the OT-PLGA/BMP scaffold wereexamined and compared with that on the control OT-PLGA scaffold.

3.7.1. Effect of immobilized rhBMP-2 on OCT-1 cell proliferationand viability

Proliferation and viability of OCT-1 cells cultured on variousPLGA scaffolds were determined by MTT assay after cultured for 4, 7and 10 days, as shown in Fig. 6. It could be seen that the prolifer-ation and viability of cell on the OT-PLGA and OT-PLGA/BMP-5 werealmost equal. After 4 and 7 days of culture, the proliferation andviability of cell on the OT-PLGA/BMP-50 and OT-PLGA/BMP-100were significantly higher than that on the OT-PLGA/BMP-5 and OT-PLGA scaffolds. Moreover, no clear difference in proliferation andviability between the OT-PLGA/BMP-50 and OT-PLGA/BMP-100scaffolds was observed. After culturing 10 days, the proliferation ofcells on all PLGA scaffold was not obviously different. The resultshowed the proliferation and viability of cells on the OT-PLGA/BMPscaffolds were enhanced in the early culture time with increasingrhBMP-2 from 0.5 to 5 mg. However, further increasing rhBMP-2immobilized on PLGA scaffolds to 10 mg the influence of the rhBMP-2 on the cell proliferation and viability had not big change.

3.7.2. Effect of immobilized rhBMP-2 on OCT-1 cell differentiationThe differentiated function of OCT-1 cells on various PLGA

scaffolds was evaluated by monitoring their ALP activity as shownin Fig. 7. It could be seen that ALP was expressed at low levels forthe cell on the OT-PLGA scaffolds. However, a significant increase inALP activity was observed after rhBMP-2 was immobilized on theOT-PLGA scaffold. The ALP activity reached its maximum after 14days of culture on the OT-PLGA, OT-PLGA/BMP-5 and OT-PLGA/BMP-50 scaffolds, but it almost reached its maximum only after 10days of culture on the OT-PLGA/BMP-100 scaffolds. After 21 days ofculture no difference in ALP activity of cells on all OT-PLGA/BMPscaffolds was seen. The results revealed that rhBMP-2 immobilizedon the PLGA scaffold via oxygen plasma treatment stimulated theearly expression of ALP activity of OCT-1 cell on the PLGA scaffold,

and the ALP activity was enhanced with the rhBMP-2 immobilizedon the PLGA increasing from 0.5 to 10 mg.

3.7.3. Effect of immobilized rhBMP-2 on morphology of OCT-1 cellFig. 8 showed morphology of OCT-1 cells on various PLGA

scaffolds after cultured for four weeks. It could be seen that the cellsin all scaffolds (Fig. 8a, c, e and g) spread and grew along pore wall.Furthermore, the high magnification SEM micrographs (Fig. 8b, d, fand h) revealed that globular accretions associated with ECM ofOCT-1 cell cultured for four weeks on all PLGA scaffolds had beenproduced. However, the size and amount of globular accretions onvarious PLGA scaffolds were different. It was obvious that theglobular accretions on OT-PLGA scaffold were least (Fig. 8b). Afterimmobilized rhBMP-2 on the OT-PLGA scaffold the amount and sizeof globular accretions increased with rhBMP-2 increase (Fig. 8d, f,and h). The globular accretions were indication of calcification [46].Therefore, the immobilized rhBMP-2 on the PLGA scaffold viaoxygen plasma treatment accelerated the calcification ofosteoblasts.

The MTT test, ALP activity, SEM analyses revealed that therhBMP-2 immobilized on the OT-PLGA scaffold had influenced theproliferation and differentiation of OCT-1 cell and exerted its effectin a dose-dependent manner. This behavior could be explained bythe binding affinity of rhBMP-2 for the PLGA scaffold and its slowrelease characteristics. In this study, the modified ELISA assay (Figs.3 and 4) demonstrated that the OT-PLGA had good binding affinityof rhBMP-2 and the bound rhBMP-2 kept its bioactivity (Fig. 5). Theresult was just well agreed with our previous study whichdemonstrated that the bFGF release profiles from CO2 plasma-treated PLGA matrices in vitro exhibited a continuous releasepattern after a moderate burst release, unlike the rapid releaseobserved in UT-PLGA [26]. So, although the in vitro release kineticsof rhBMP-2 from the OT-PLGA matrix was not investigated in thisstudy, it could be hypothesized that the rhBMP-2 existed in twokinds of release. The initial burst rhBMP-2 release was causedmainly by a quick diffusion of the weakly bound rhBMP-2 fromsurface of the OT-PLGA/BMP and the subsequent sustained rhBMP-2 release was controlled by a thermodynamic equilibrium betweenthe rhBMP-2-PLGA complexes and free rhBMP-2 in the culturemedium. Therefore, the rhBMP-2 immobilized on the scaffoldproduced effect on the OCT-1 cell mainly by two ways. One is therhBMP-2 immobilized on the scaffold directly produced effect oncells attached on the scaffolds by contacting each other. Another isthe rhBMP-2 immobilized on the scaffold was released intomedium and thus produced effects on the osteoblasts. In addition,due to the concentration-dependent of growth factor effect [47],the immobilized rhBMP-2 on the PLGA scaffolds via oxygen plasmatreatment exerted its effect in a dose-dependent manner.

4. Conclusions

Oxygen plasma treatment can offer suitable functional groupsand charge on the surface of PLGA matrix. Under the condition of50 W of power, 20 Pa of oxygen pressure and 10 min of oxygenplasma treating time the rhBMP-2 could be effectively bound ontosurface of the PLGA matrix. Furthermore, the rhBMP-2 immobilizedon OT-PLGA scaffold could maintain its bioactivity. The amount ofimmobilized rhBMP-2 closely depended on the extent of theimproved hydrophilicity and rich polar O-containing groups of theOT-PLGA scaffold. The effect of immobilized rhBMP-2 on prolifer-ation of the OCT-1 cell was only present during initial stage anddisappeared at longer culture times. In addition, the immobilizedrhBMP-2 stimulated the differentiation of OCT-1 cell and acceler-ated the process of mineralization of OCT-1 cell in a dose-depen-dent manner. Thus, the rhBMP-2 immobilized PLGA scaffold by the

H. Shen et al. / Biomaterials 30 (2009) 3150–3157 3157

oxygen plasma treatment combining with rhBMP-2 anchoragemethod could be utilized as a promising scaffold for bone tissueengineering.

Acknowledgements

The authors would like to thank Prof. Dai Jianwu and Dr. ChenBing (Key Laboratory of Molecular and Developmental Biology,Institute of Genetics and Developmental Biology, China) forsupplying part rhBMP-2 and their help in ELISA. This research wassupported by a grant from Major State Basic Science Research andDevelopment Program of China (973, No.2005CB5227074) andHigh Technology Research and Development Program of China.

References

[1] Lavik E, Langer R. Tissue engineering: current state and perspectives. ApplMicrobiol Biotechnol 2004;65:1–8.

[2] LaurencinCT, Attawia MA, Lu LQ, Borden MD, Lu HH, Gorum WJ, et al. Poly(lactide-co-glycolide)/hydroxyapatite delivery of BMP-2-producing cells: a regional genetherapy approach to bone regeneration. Biomaterials 2001;12:1271–7.

[3] Biondi M, Ungaro F, Quaglia F, Netti PA. Controlled drug delivery in tissueengineering. Adv Drug Deliv Rev 2008;60:229–42.

[4] Niklason LE, Langer R. Prospects for organ and tissue replacement. JAMA2001;285:573–6.

[5] Goldberg M, Langer R, Jia X. Nanostructured materials for applications in drugdelivery and tissue engineering. J Biomater Sci Polym Ed 2007;18:241–68.

[6] Hu XX, Shen H, Yang F, Bei JZ, Wang SG. Preparation and cell affinity ofmicrotubular orientation-structured PLGA(70/30) blood vessel scaffold.Biomaterials 2008;29:3128–36.

[7] Shin KC, Kim BS, Kim JH, Park TG, Do Nam J, Lee DS. A facile preparation ofhighly interconnected macroporous PLGA scaffolds by liquid–liquid phaseseparation II. Polymer 2005;46:3801–8.

[8] Wake MC, Gupta PK, Mikos AG. Fabrication of pliable biodegradable polymerfoams to engineer soft tissues. Cell Transplant 1996;5:465–73.

[9] Cao Y, Mitchell G, Messina A, Price L, Thompson E, Penington A, et al. Theinfluence of architecture on degradation and tissue ingrowth into three-dimensional poly(lactic-co-glycolic acid) scaffolds in vitro and in vivo.Biomaterials 2006;27:2854–64.

[10] Jeon O, Song SJ, Kang SW, Putnam AJ, Kim BS. Enhancement of ectopic boneformation by bone morphogenetic protein-2 released from a heparin-conju-gated poly(L-lactic-co-glycolic acid) scaffold. Biomaterials 2007;28:2763–71.

[11] Kirker-head CA. Potential applications and delivery strategies for bonemorphogenetic proteins. Adv Drug Deliver Rev 2000;43:65–92.

[12] van den Doldera J, de Ruijtera AJE, Spauwenb PHM, Jansen JA. Observations onthe effect of BMP-2 on rat bone marrow cells cultured on titanium substratesof different roughness. Biomaterials 2003;24:1853–60.

[13] Jeon O, Song SJ, Yang HS, Bhang SH, Kang SW, Sung MA, et al. Long-term deliveryenhances in vivo osteogenic efficacy of bone morphogenetic protein-2compared to short-term delivery. Biochem Biophys Res Co 2008;369:774–80.

[14] Kato M, Namikawa T, Terai H, Hoshino M, Miyamoto S, Takaoka K. Ectopic boneformation in mice associated with a lactic acid/dioxanone/ethylene glycolcopolymer–tricalcium phosphate composite with added recombinant humanbone morphogenetic protein-2. Biomaterials 2006;27:3927–33.

[15] Bessho K, Carnes DL, Cavin R, Ong JL. Experimental studies onbone induction usinglow-molecular-weight poly (DL-lactide-co-glycolide) as a carrier for recombinanthuman bone morphogenetic protein-2. J Biomed Mater Res 2002;61:61–5.

[16] Kempen DHR, Lu L, Hefferan TE, Creemers LB, Maran A, Classic KL, et al.Retention of in vitro and in vivo BMP-2 bioactivities in sustained deliveryvehicles for bone tissue engineering. Biomaterials 2008;29:3245–52.

[17] Hosseinkhani H, Yamamoto M, Inatsugu Y, Hiraoka Y, Inoue S, Shimokawa H,et al. Enhanced ectopic bone formation using a combination of plasmid DNAimpregnation into 3-D scaffold and bioreactor perfusion culture. Biomaterials2006;27:1387–98.

[18] Chung YI, Ahn KM, Jeon SH, Lee SY, Lee JH, Tae G. Enhanced bone regenerationwith BMP-2 loaded functional nanoparticle–hydrogel complex. J ControlRelease 2007;121:91–9.

[19] Nagao H, Tachikawa N, Miki T, Oda M, Mori M, Takahashi K, et al. Effect ofrecombinant human bone morphogenetic protein-2 on bone formation inalveolar ridge defects in dogs. Int J Oral Maxillofac Surg 2002;31:66–72.

[20] Yan YN, Xiong Z, Hu YY, Wang SG, Zhang RJ, Zhang C. Layered manufacturing of tissueengineering scaffolds via multi-nozzle deposition. Mater Lett 2003;57:2623–8.

[21] Xiong Z, Yan YN, Wang SG, Zhang RJ, Zhang C. Fabrication of porous scaffoldsfor bone tissue engineering via low-temperature deposition. Scripta Mater2002;46:771–6.

[22] Nie H, Soh BW, Fu YC, Wang CH. Three-dimensional fibrous PLGA/HApcomposite scaffold for BMP-2 delivery. Biotechnol Bioeng 2007;99:223–34.

[23] Whang K, Tsai DC, Nam EK, Aitken M, Sprague SM, Patel PK, et al. Ectopic boneformation via rhBMP-2 delivery from porous bioabsorbable polymer scaffolds.J Biomed Mater Res 1998;42:491–9.

[24] Jeon O, Rhie JW, Kwon IK, Kim JH, Kim BS, Lee SH. In vivo bone formationfollowing transplantation of human adipose-derived stromal cells that are notdifferentiated osteogenically. Tissue Eng: Part A 2008;14:1285–94.

[25] Cowan CM, Aghaloo T, Chou YF, Walder B, Zhang XL, Soo C, et al. MicroCTevaluation of three-dimensional mineralization in response to BMP-2 doses invitro and in critical sized rat calvarial defects. Tissue Eng 2007;13:501–12.

[26] Shen H, Hu XX, Bei JZ, Wang SG. The immobilization of basic fibroblast growthfactor on plasma-treated poly(lactide-co-glycolide). Biomaterials 2008;29:2388–99.

[27] Favia P, D’agostino R. Plasma treatments and plasma deposition of polymersfor biomedical applications. Surf Coat Technol 1998;98:1102–6.

[28] Wang MJ, Chang YI, Poncin-Epaillard F. Acid and basic functionalities ofnitrogen and carbon dioxide plasma-treated polystyrene. Surf Interface Anal2005;37:348–55.

[29] Wan YQ, Qu X, Lu J, Zhu CF, Wan LJ, Yang JL, et al. Characterization of surfaceproperty of poly(lactide-co-glycolide) after oxygen plasma treatment.Biomaterials 2004;25:4777–83.

[30] Yang J, Wan WQ, Yang YL, Bei JZ, Wang SG. Plasma-treated, collagen-anchoredpolylactone: its cell affinity evaluation under shear or shear-free conditions.J Biomed Mater Res 2003;67A:1139–47.

[31] Wan YQ, Yang J, Yang JL, Bei JZ, Wang SG. Cell adhesion on gaseous plasmamodified poly-(L-lactide) surface under shear stress field. Biomaterials2003;24:3757–64.

[32] Gilding DK, Reed AM. Biodegradable polymers for use in surgery-polyglycolic/polyactic acid homo and copolymers. Polymer 1979;20:1459–64.

[33] Shen H, Hu XX, Yang F, Bei JZ, Wang SG. Combining oxygen plasma treatmentwith anchorage of cationized gelatin for enhancing cell affinity of poly(lactide-co-glycolide). Biomaterials 2007;28:4219–30.

[34] Chen B, Lin H, Wang JH, Zhao YN, Wang B, Zhao WX, et al. Homogeneousosteogenesis and bone regeneration by demineralized bone matrix loadingwith collagen-targeting bone morphogenetic protein-2. Biomaterials2007;28:1027–35.

[35] Han B, Perelman N, Tang B, Hall F, Shors EC, Nimni ME. Collagen-targetedBMP3 fusion proteins arrayed on collagen matrices or porous ceramicsimpregnated with type I collagen enhance osteogenesis in a rat cranial defectmodel. J Orthop Res 2002;20:747–55.

[36] Chen D, Chen H, Feng JQ, Windle JJ, Koop BA, Harris HA, et al. Osteoblastic celllines derived from a transgenic mouse containing the osteocalcin promoterdriving the SV-40 T-antigen. Mol Cell Diff 1995;3:193–212.

[37] Yang J, Bei JZ, Wang SG. Enhanced cell affinity of poly (D, L-lactide) bycombining plasma treatment with collagen anchorage. Biomaterials2002;23:2607–14.

[38] Nakagawa M, Teraoka F, Fujimoto S, Hamada Y, Kibayashi H, Takahashi J.Improvement of cell adhesion on poly(L-lactide) by atmospheric plasmatreatment. J Biomed Mater Res 2006;77A:112–8.

[39] Inagaki N, Narushima K, Tsutsui Y, Ohyama Y. Surface modification anddegradation of poly(lactic acid) films by Ar-plasma. J Adhesion Sci Technol2002;16:1041–54.

[40] Ho MH, Hou LT, Tu CY, Hsieh HJ, Lai JY, Chen WJ, et al. Promotion of cell affinityof porous PLLA scaffolds by immobilization of RGD peptides via plasmatreatment. Macromol Biosci 2006;6:90–8.

[41] Qu X, Cui WJ, Yang F, Min CC, Shen H, Bei JZ, et al. The effect of oxygen plasmapretreatment and incubation in modified simulated body fluids on theformation of bone-like apatite on poly(lactide-co-glycolide)(70/30). Biomate-rials 2007;28:9–18.

[42] Wan YQ, Tu CF, Yang J, Bei JZ, Wang SG. Influences of ammonia plasmatreatment on modifying depth and degradation of poly(L-lactide) scaffolds.Biomaterials 2006;27:2699–704.

[43] Saito T, Kobayashi F, Fujii T, Bessho K. Effect of phosphophoryn on rhBMP-2-induced bone formation. Arch Oral Biol 2004;49:239–43.

[44] Uludag H, D’Augusta D, Golden J, Li J, Timony G, Riedel R, et al. Implantation ofrecombinant human bone morphogenetic proteins with biomaterial carriers:a correlation between protein pharmacokinetics and osteoinduction in the ratectopic model. J Biomed Mater Res 2000;50:227–38.

[45] You CC, Chompoosor A, Rotello V. The biomacromolecule-nanoparticle inter-face. Nano Today 2007;2(3):34–43.

[46] Li C, Vepari C, Jin HJ, Kim HJ, Kaplan DL. Electrospun silk-BMP-2 scaffolds forbone tissue engineering. Biomaterials 2006;27:3115–24.

[47] Babensee JE, Mcintire LV, Mikos AG. Growth factor delivery for tissue engi-neering. Pharm Res 2000;17:497–504.